Archive for the ‘Pharmacotherapy and Cell Activity’ Category

Alzheimer’s Disease – tau art thou, or amyloid

Larry H. Bernstein, MD, FCAP, Curator



Alzheimer’s Disease and Tau  


Pathogenic Mechanisms and Therapeutic Approaches

Organizers: Robert Martone (St. Jude Children’s Research Hospital) and Sonya Dougal (The New York Academy of Sciences)Presented by the Brain Dysfunction Discussion Group

Reported by Caitlin McOmish | Posted February 2, 2016




Microtubule-associated protein tau helps maintain the stability and flexibility of microtubules in neuronal axons. Alternative splicing of the tau gene, MAPT, produces 6 isoforms of tau in the brain and many more in the peripheral nervous system. Tau can be phosphorylated at over 30 sites, and it undergoes many posttranslational modifications to operate as a substrate for multiple enzymes. However, tau also mediates pathological functions including neuroinflammatory response, seizure, and amyloid-β (Aβ) toxicity, and tau pathology is a hallmark of conditions including frontotemporal dementia, traumatic brain injury (TBI), Down syndrome, focal cortical dysplasia, and Alzheimer’s disease (AD), as well as some tumors and infections. On September 18, 2015, speakers at the Brain Dysfunction Discussion Group’s Alzheimer’s Disease and Tau: Pathogenic Mechanisms and Therapeutic Approaches symposium discussed the mechanisms by which tau becomes pathological and how the pathology spreads. They also described emerging therapeutic strategies for AD focused on tau.



Microtubule-associated protein tau has a complex biology, including multiple splice variants and phosphorylation sites. Tau is a key component of microtubules, which contribute to neuronal stability. In AD, tau changes, causing microtubules to collapse, and tau proteins clump together to form neurofibrillary tangles. (Image presented by Robert Martone courtesy of the National Institute on Aging)


Tau is ubiquitous in the brain, with widespread effects, but has historically been overlooked as a driving force in AD. In his introduction to the symposium, Robert Martone from St. Jude Children’s Research Hospital highlighted tau’s activity and emergence as a treatment target for this devastating disorder. Hyperphosphorylated tau (p-tau) has long been recognized as a principle component of neurofibrillary tangles in AD; tau monomers are misfolded into oligomers that form tau filaments. As Hartmuth Kolb from Johnson & Johnson explained, the development in 2012 of a tau-specific positron emission tomography (PET) tracer led to important insights into the presence and spread of tau pathology over the course of tauopathies, including AD, in humans. Notably, researchers demonstrated that tau pathology propagates through the brain in a predictable pattern, corresponding to the Braak stages of AD.


Tau pathology spreads through the brain in a predictable pattern. Abnormal tau protein is first observed in the transentorhinal region (stages I and II) and spreads to the limbic regions in stages III and IV, when early signs of AD begin to be observed. Pathology subsequently extends throughout the neocortex, driving fully developed AD. This staging was first described by Braak and Braak in 1991. (Image courtesy of Hartmuth Kolb)


It is likely that the symptoms of AD are produced by the combined effects of tau and Aβ pathologies. George Bloom from the University of Virginia described how Aβ and tau interact to cause mature neurons to reenter the cell cycle, leading to cell death. In a healthy brain, insulin acts as a gatekeeper that maintains adult neurons in the G0 phase after the cells permanently exit the cell cycle. In AD, amyloid oligomers sequester neuronal insulin receptors, causing insulin resistance. In parallel, tau phosphorylation at key sites—pY18 (fyn site), pS409 (PKA site), pS416 (CAM Kinase site), and pS262—drives mTOR signaling at the plasma membrane but not at the lysosome, resulting in cell cycle reentry. In a normal cell, activation of mTOR at the lysosome overrides the cell cycle reentry signal—creating an important regulatory mechanism for maintaining healthy neurons. However, lysosomal activation of mTOR is insulin dependent and thus affected by Aβ-induced insulin insensitivity. Amyloid oligomers, via insulin regulation, release the brakes on a cascade of events driven by p-tau that leads to cell cycle reentry and cell death.


Hallmark dysfunction produced by Aβ is dependent on tau. Pathological Aβ drives the formation of p-tau in the brain, resulting in synaptic dysfunction, cell death, and broad neurocognitive symptoms. This process can be influenced by a range of factors including genetic predisposition, environmental risk factors, and biochemical signaling pathways. (Image courtesy of George Bloom)


Khalid Iqbal from the New York State Institute for Basic Research in Developmental Disabilities described research showing that p-tau spreads through the brain in a rodent model, well beyond the injection site, in a prion-like manner, and that the spread of pathology can be mitigated by the addition of PP2A—a phosphatase known to be decreased in gray and white matter in AD. PP2A regulation is affected in AD, stroke, and brain acidosis, providing a link between these disorders and tau pathology.

Discussion of the pathophysiology of AD commonly focuses on Aβ plaques and neurofibrillary tangles (NFTs) composed of misassembled hyperphosphorylated tau; it has generally been thought that these plaques and tangles are the primary causes of symptoms. However, recent evidence indicates that oligomeric variants of tau are actually far more toxic than the form of tau present in NFTs. Michael Hutton from Eli Lilly and Company studies the properties needed for tau to become pathological. He used animal models to show that the abnormal p-tau “seed,” from which a prion-like spread develops, must be of a high molecular weight (with at minimum three tau units) and highly phosphorylated to induce healthy tau to become pathological. These characteristics are necessary but not sufficient for effective seeding. There is also evidence that tau pathology propagates via an autocatalytic cycle of seeded aggregation and fragmentation.

Propagation, in addition to requiring a large number of p-tau units in aggregates, may be affected by the isomerization of those monomers. Kun Ping Lu from Harvard Medical School provided data suggesting that cis but not trans pT231-tau is a precursor of tauopathy, linking TBI to the later development of neurodegenerative diseases such as chronic traumatic encephalopathy and AD. He demonstrated a role for Pin1, a phosphorylation-specific prolyl isomerase, in this process using animal models of TBI and AD. Pin1, which is regulated in response to stress, prevents the accumulation of toxic cis p-tau by converting it to the trans isoform, but this process is inhibited in AD and TBI. Lu showed that cis p-tau’s ability to cause and spread neurodegeneration can be blocked by a cis p-tau monoclonal antibody in vitro and in animal models, pointing to the therapeutic potential of targetingcis p-tau for treatment of TBI and AD.

Culturing p-tau seeds in vitro produces a broad array of tau aggregate structures. Marc Diamond from the University of Texas Southwestern Medical Center discussed the diverse structures produced by different tau seeds, which his team has studied in a series of experiments using in vitro models, animal models, and human postmortem analyses. His lab showed that distinct conformations of aggregate seeds propagate stably, infecting normal cells and leading them to acquire abnormal tau aggregates with distinct, reproducible structures and different biochemical properties. In another study, the team showed that the morphology of the p-tau aggregates was related to diagnosis. Seeds sourced from postmortem human tissue produced reliable phenotypes in culture, which tracked with different diagnoses, retroactively predicting biological outcome. Thus, the characteristics of the p-tau seed have a large influence on the biological outcome, providing a new prospect for presymptomatic diagnosis.




Tau seeds obtained from postmortem brain tissue from AD, argyrophilic grain disease (AGD), corticobasal degeneration (CBD), Pick’s disease (PiD), and progressive supranuclear palsy (PSP) produce unique aggregate pathologies in cell culture, including toxic, mosaic, ordered, disordered, and speckled. AD-derived seeds largely produce the speckled phenotype. (Images courtesy of Marc Diamond)


With the mechanisms by which p-tau forms, converts healthy tau, and seeds dysfunction established, the question of how p-tau exits the cell and moves through the brain arises. The pattern of spread and the speed with which the pathology progresses suggests that p-tau propagates trans-synaptically. Nicole Leclerc from the University of Montreal provided evidence to support this view. It is likely, her lab has shown, that tau is secreted and taken up by neurons in an active process, in response to neuronal activity. Tau secretion in vitro increases under conditions such as starvation and lysosomal dysfunction, phenomena found in the early stages of AD. Moreover, hyperphosphorylation appears to increase the targeting of tau to the secretory pathway, potentially accelerating the spread of p-tau. Intriguingly, however, the extracellular tau is hypophosphorylated, suggesting large-scale dephosphorylation during the secretory process. This hypo-tau may activate muscarinic acetylcholine receptors, increasing intracellular Ca2+ and promoting cell death.

These findings suggest that the synapse plays a critical role in the development of AD; the extrasynaptic environment is known to be exquisitely regulated by microglia. The focus of studies into neurodegenerative disorders is often neurons, but genetic studies have repeatedly identified changes in expression of microglial genes in AD, including in one of the leading AD candidate genes, TREM2, demonstrating a fundamental contribution of these cells to AD. Richard Ransohoff of Biogen discussed the importance of this cell type. Microglia enter the brain at around embryonic day (E) 9.5 in rodents and are crucially involved in maintaining brain health. During development the cells play a major role in large-scale synaptic pruning required for effective neural maturation. They are also highly responsive to the environment, and stress in adulthood can reengage microglial synaptic pruning—a process that is adaptive during development but maladaptive in adulthood. The process is regulated by complement system cascades. TGF-β expressed by astrocytes drives neurons to express C1q presynaptically, initiating complement elements to accumulate at the site, ultimately activating microglia to prune the synaptic connection. In AD, inappropriate activation of this cascade may lead to the removal of otherwise healthy connections. Ransohoff described a role for CXCR3, the fractalkine receptor, in regulating reactivity of microglia, and thus mitigating pruning of adult synapses. Regulation of microglia reactivity is driven by epigenetically induced changes in inflammatory response genes. Correspondingly, in the absence of CXCR3, tau pathology is aggravated in htau mice (which express human tau isoforms), suggesting a protective effect of the CXCR3 pathway. Ransohoff closed with the caveat that microglia are not intrinsically helpful or harmful; their properties are context dependent and must be unraveled by empirical observations in appropriate models.

Peter Davies from the Feinstein Institute for Medical Research discussed the need to better incorporate current knowledge into research model design, particularly to develop monoclonal antibodies for the treatment of AD. Monoclonal antibodies are a promising strategy, but translating preclinical findings into successful clinical outcomes will require careful consideration of the context of the early research. Most transgenic animal models for AD express p-tau in all neurons, but such extensive p-tau spread is not found in human AD brains. There are several hurdles to determine the drugs’ efficacy and safety in humans; it is difficult to assess specificity and find appropriate dosages. In a series of studies with a focus on external reproducibility, Davies presented evidence from animal models showing that immunotherapy can block the spread of p-tau but cannot undo pathology already present in the brain. In the htau mouse model several putative antibodies lacked efficacy and in some cases appeared to worsen pathology. These findings underscore the need for both better models and improved understanding of mechanisms of action before moving drugs to the clinic.


The New York Academy of Sciences. Alzheimer’s Disease and Tau: Pathogenic Mechanisms and Therapeutic Approaches. Academy eBriefings. 2015. Available at: www.nyas.org/Tau2015-eB


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Breakup of amyloid plaques

Larry H. Bernstein, MD, FCAP, Curator




Small Molecule EPPS Breaks Up Amyloid Plaques

Alzheimers Plaque Therapy, Alzheimers small molecule, amyloid plaque treatment

One of the hallmarks of Alzheimer’s disease has been the generation of Amyloid-β (Aβ) oligomers, fibrils, and ultimately plaques. It is currently contended whether these plaques are a cause of Alzheimer’s disease and related mental deficits, or merely an effect. Researchers at the Korea Institute of Science and Technology have demonstrated in vivo formation and disaggregation of Aβ plaques. They previously reported small ionic molecules which could accelerate the formation of Aβ plaques. Six small molecules which inhibited aggregate formation were discovered at the same time. One of these molecules, 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS), works as a therapeutic in a Alzheimer’s mouse model. EPPS was found to be both orally available and cross the blood brain barrier where it directly binds to Aβ plaques. Double transgenic mice , APPswe/PS1-dE9 (amyloid precursor protein/presenilin protein 1) mice were administered EPPS in their drinking water for 3.5 months and compared to non-treated transgenic controls. EPPS treated mice both improved from their baseline and out-performed transgenic controls in both the Morris water maze and contextual fear response tests. Immunofluorescent staining of matched brain regions demonstrated elimination of Aβ plaques in the hippocampus of EPPS treated mice. Further study is required to completely understand the mechanism by which EPPS disaggregates the Aβ plaques. This study demonstrates the cause and effects Aβ plaque generation, and subsequent removal, has on Alzheimer’s disease related cognitive function. Should the effect transfer to humans, this could prove a significant discovery for the treatment of Alzheimer’s disease.


Kim, et al. (October, 2015) EPPS rescues hippocampus-dependent cognitive deficits in APP/PS1 ice by disaggregation of amyloid-b oligomers and plaques Nature Communications


EPPS  rescues hippocampus-dependent cognitive deficits in APP/PS1 mice by disaggregation of amyloid-β oligomers and plaques

Hye Yun KimHyunjin Vincent KimSeonmi JoC. Justin LeeSeon Young ChoiDong Jin Kim & YoungSoo Kim

Nature Communications 2016; 6(8997)     http://dx.doi.org:/10.1038/ncomms9997

Alzheimer’s disease (AD) is characterized by the transition of amyloid-β (Aβ) monomers into toxic oligomers and plaques. Given that Aβ abnormality typically precedes the development of clinical symptoms, an agent capable of disaggregating existing Aβ aggregates may be advantageous. Here we report that a small molecule, 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS), binds to Aβ aggregates and converts them into monomers. The oral administration of EPPS substantially reduces hippocampus-dependent behavioural deficits, brain Aβ oligomer and plaque deposits, glial γ-aminobutyric acid (GABA) release and brain inflammation in an Aβ-overexpressing, APP/PS1 transgenic mouse model when initiated after the development of severe AD-like phenotypes. The ability of EPPS to rescue Aβ aggregation and behavioural deficits provides strong support for the view that the accumulation of Aβ is an important mechanism underlying AD.


During Alzheimer’s disease (AD) pathogenesis, amyloid-β (Aβ) monomers aberrantly aggregate into toxic oligomers, fibrils and eventually plaques. The concentration of misfolded Aβ species highly correlates with the severity of neurotoxicity and inflammation that leads to neurodegeneration in AD1, 2, 3. Accordingly, substantial efforts have been devoted to reducing Aβ levels, including methods to prevent the production and aggregation of Aβ4, 5, 6, 7. Although these approaches effectively prevent the de novo formation of Aβ aggregates, existing Aβ oligomers and plaques will still remain in the patient’s brain8, 9, 10. Thus, the desirable effects of Aβ inhibitors may be expected when administered before a patient develops toxic Aβ deposits5, 6, 7. However, in AD patients with mild-to-moderate symptoms, anti-amyloidogenic agents have not yielded expected outcomes, which may be due to the incomplete removal of pre-existing Aβ aggregates11. As Aβ typically begins to aggregate long before the onset of AD symptoms, interventions specifically aimed at disaggregating existing plaques and oligomers may constitute a useful approach to AD treatment, perhaps in parallel with agents aimed at inhibiting aggregate formation8, 9, 10, 11, 12.


Result highlights  

EPPS reduces Aβ-aggregate-induced memory deficits in mice

Figure 1: EPPS ameliorates Aβ-induced memory deficits in mice.


EPPS ameliorates A[beta]-induced memory deficits in mice.

(a) Time course of the experiments. (b) Intracerebroventricular (i.c.v.) injection site brain schematic diagram. (c) Pretreated effects of EPPS on Aβ-aggregate-induced memory deficits observed by the % alternation on the Y-maze. EPPS, 0 (n=10), 30 (n=9) or 100mgkg−1 per day (n=10), was orally given to 8.5-week-old ICR male mice for 1 week; then, vehicle (10% DMSO in PBS, n=10) or Aβ aggregates (50pmol per 10% DMSO in PBS; Supplementary Fig. 1A) were injected into the intracerebroventricular region (P=0.022). (d) Co-treated effects of EPPS on Aβ-aggregate-induced memory deficits observed by the % alternation on the Y-maze. Male, 8.5-week-old ICR mice received an injection of vehicle (n=9) or Aβ aggregates into the intracerebroventricular region, and then EPPS, 0 (n=10), 30 (n=10) or 100mgkg−1 per day (n=10), was orally given to these mice for 5 days. From the top, P=0.003, 0.006, 0.015. The error bars represent the s.e.m. One-way analysis of variance followed by Bonferroni’s post-hoc comparisons tests were performed in all statistical analyses. (*P<0.05, **P<0.01, ***P<0.001; other comparisons were not significant).


EPPS is orally safe and penetrates the blood–brain barrier

Orally administered EPPS rescues cognitive deficits in APP/PS1 mice


Figure 2: EPPS rescues hippocampus-dependent cognitive deficits.



Figure 3: EPPS does not affect synaptic plasticity in mice.



Figure 4: EPPS disaggregates Aβ plaques and oligomers in APP/PS1 mice.

EPPS disaggregates A[beta] plaques and oligomers in APP/PS1 mice.

APP/PS1 mice and WTs from the aforementioned behavioural tests were killed and subjected to brain analyses. EPPS, 0 (TG(), male, n=15), 10 (TG(+), male, n=11) or 30mgkg-1 per day (TG(++), male,n=8), was orally given to 10.5-month-old APP/PS1 for 3.5 months and their brains were compared with age-matched WT brains (WT(), male, n=16). (a) ThS-stained Aβ plaques in whole brains (scale bars, 1mm) and the hippocampal region (scale bars, 200μm) of each group. The mouse brain schematic diagram was created by authors (green and red boxes: regions of brain images, a and f, respectively). (b) Number or area of plaques normalized (%) to the level in 10.5-month-old TG mice. Plaque number: P-values compared with TG (male, 10.5-month-old) are all <0.0001 (#). P-values compared with TG() (male, 14-month-old) are all <0.0001 (*). Plaque area: P-values compared with TG (male, 10.5-month-old) are all <0.0001 (#). P-values compared with TG() (male, 14-month-old) are all <0.0001 (*). (ce) Aβ-insoluble and -soluble fractions analyses from brain lysates. (c) Sandwich ELISA of Aβ-insoluble fractions. Hippocampus: all P<0.0001; cortex: P=0.004, 0.046. (d) Sandwich ELISA of Aβ-soluble fractions. (e) Dot blotting of the total Aβ (anti-Aβ: 6E10, also recognizes APP) and oligomers (anti-amyloidogenic protein oligomer: A11). (f) Histochemical analyses of Aβ deposition. Aβs were stained with the 6E10 antibody and ThS. Aβ plaques (first row): green; all Aβs (second row): red; 4,6-diamidino-2-phenylindole (DAPI): blue (as a location indicator). The third and bottom rows show merged images of plaques and Aβs, and plaques and Aβs with DAPI staining. Scale bars, 50μm. (g) Western blotting analyses of APP expression in hippocampal and cortical lysates (detected at ~100kDa by 6E10 antibody). Densitometry (see Supplementary Fig. 3A). Full version (see Supplementary Fig. 7). The error bars represent the s.e.m. One-way analysis of variance followed by Bonferroni’s post-hoc comparisons tests were performed in all statistical analyses (*P<0.05, **P<0.01, ***P<0.001, #P<0.05, ##P<0.01,###P<0.001; other comparisons were not significant).


EPPS removes Aβ plaques and oligomers in APP/PS1 mice

Collectively, these results indicate that EPPS rescues hippocampus-dependent cognitive deficits when orally administered to aged, symptomatic APP/PS1 TG mice.

Collectively, these results indicate that orally administered EPPS effectively decreases Aβ plaques and oligomers in APP/PS1 model mouse brains.


EPPS lowers Aβ-dependent inflammation and glial GABA release

Figure 5: EPPS lowers inflammation and glial GABA release.

EPPS disaggregates Aβ oligomers and fibrils by direct interaction and reduces cytotoxicity

Figure 6: EPPS disaggregates Aβ aggregates by selective binding.


(1) a small molecule, EPPS, converts neurotoxic oligomers and plaques into non-toxic monomers by directly binding to Aβ aggregates;

(2) orally administered EPPS produces a dose-dependent reduction of Aβ plaque deposits and behavioural deficits in APP/PS1 TG mice, even when administration was delayed until after the pathology was well established;

(3) the beneficial effect of EPPS probably operates through an Aβ-related mechanism rather by facilitating cognitive processes; and

(4) large doses of EPPS appeared to be well tolerated in initial toxicity studies6, 7, 33.

Dr. T. Ronald Theodore
Email rtheodore@integratedbiologics.com
URL http://www.integratedbiologics.com
In Response To Breakup of amyloid plaques
Submitted on 2016/05/18 at 3:33 am
Comment Re: “EPPS rescues hippocampus-dependent cognitive deficits in APP/PS1 mice by disaggregation of amyloid-β oligomers and plaques” Kim et al, Nature Communications 8 December 2015
HEPES, Zwitterions, and the “Good” Buffers as Biological Response Modifiers

In reference to the article “EPPS rescues hippocampus-dependent cognitive deficits in APP/PS1 mice by disaggregation of amyloid-β oligomers and plaques” Kim et al, Nature Communications 8 December 2015, we note some important omissions.

Kim et al state specific effects of EPPS affecting Alzheimer’s disease. We would point out that EPPS is also referenced as HEPPS.1 HEPPS has been accepted as a “Good” buffer and a zwitterion. The authors attribute the effects of EPPS to anti-inflammatory action. The authors omit reference that EPPS (HEPPS) is a listed “Good” buffer and a zwitterion.1 The anti-inflammatory effects of zwitterions and “Good” buffers have been previously described.3,4 The effects of these zwitterions as biological response modifiers with effects on neurological diseases including Alzheimer’s have been previously noted.4,5 ( HEPES has been used preferentially based on Good’s original data showing HEPES has the highest ability to increase the rate of mitochondrial oxidative phosphorylation). Kim et al attribute the effects of EPPS to anti-inflammatory actions. The anti-inflammatory effects of the buffers are well known.3,4 We would suggest that anti-inflammatory effects of the buffers may be singular, synergistic or combined effects of other biological responses that have been noted including mitochondrial and other actions.4,5,6,7 Prior literature and data would certainly anticipate the findings of Kim et al. It is noted that all these zwitterionic buffers have effects on the neurological system.

What is important is that further research to determine the effects of these zwitterionic buffers as biological response modifiers on neurological diseases including Alzheimer’s is continued. The ability of the zwitterionic buffers on brain and other organ injury are currently under review.

T. Ronald Theodore
Integrated Biologics, LLC

1. Merck Index, 15th Edition, Feb 2015.
2. Norman E. Good et al., Hydrogen Ion Buffers for Biological Research, Biochemistry vol.5, No. 2, Feb. 1966.
3. “Effects of In-vivo Administration of Taurine and HEPES on the Inflammatory Response in Rats” Pharmacy and Pharmacology, vol. 46, No. 9, Sept. 1994.
4. Theodore et al., Zwitterionic Compositions and Methods as Biological Response Modifiers, US Patent No. 6,071,919.
5. Garvey et al., Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer’s Aβ peptide, Biochemical and Biophysical Research Communications, 06/2011; 409(3):385-8. DOI: 10.1016/j.bbrc.2011.04.141.
6. Theodore et al., Pilot Ascending Dose Tolerance Study of Parenterally Administered 4-(2 Hydroxyethyl)-l-piperazine Ethane Sulfonic Acid (TVZ-7) in Dogs, Cancer Biotherapy & Radiopharmaceuticals, Volume 12, Number 5, 1997.
7. Theodore et al., Preliminary Evaluation of a Fixed Dose of Zwitterionic Piperazine (TVZ-7) in Clinical Cancer, Cancer Biotherapy and Radiopharmaceuticals, Volume 12, Number 5, 1997.


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Beyond tau and amyloid

Larry H. Bernstein, MD, FCAP, Curator






Neurovascular pathways to neurodegeneration in Alzheimer’s disease and other disorders.

Berislav V. Zlokovic

Nature Reviews Neuroscience 12, 723-738 (December 2011) |   http:dx.doi.org:/10.1038/nrn3114

The neurovascular unit (NVU) comprises brain endothelial cells, pericytes or vascular smooth muscle cells, glia and neurons. The NVU controls blood–brain barrier (BBB) permeability and cerebral blood flow, and maintains the chemical composition of the neuronal ‘milieu’, which is required for proper functioning of neuronal circuits. Recent evidence indicates that BBB dysfunction is associated with the accumulation of several vasculotoxic and neurotoxic molecules within brain parenchyma, a reduction in cerebral blood flow, and hypoxia. Together, these vascular-derived insults might initiate and/or contribute to neuronal degeneration. This article examines mechanisms of BBB dysfunction in neurodegenerative disorders, notably Alzheimer’s disease, and highlights therapeutic opportunities relating to these neurovascular deficits.



The neurovascular unit comprises vascular cells (endothelial cells, pericytes and vascular smooth muscle cells (VSMCs)), glial cells (astrocytes, microglia and oliogodendroglia) and neurons.
Neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) are associated with microvascular dysfunction and/or degeneration in the brain, neurovascular disintegration, defective blood–brain barrier (BBB) function and/or vascular factors.
The interactions between endothelial cells and pericytes are crucial for the formation and maintenance of the BBB. Indeed, pericyte deficiency leads to BBB breakdown and extravasation of multiple vasculotoxic and neurotoxic circulating macromolecules, which can contribute to neuronal dysfunction, cognitive decline and neurodegenerative changes.
Alterations in cerebrovascular metabolic functions can also lead to the secretion of multiple neurotoxic and inflammatory factors.
BBB dysfunction and/or breakdown and cerebral blood flow (CBF) reductions and/or dysregulation may occur in sporadic Alzheimer’s disease and experimental models of this disease before cognitive decline, amyloid-β deposition and brain atrophy. In patients with ALS and in some experimental models of ALS, CBF dysregulation, blood–spinal cord barrier breakdown and spinal cord hypoperfusion have been reported prior to motor neuron cell death.
Several studies in animal models of Alzheimer’s disease and, more recently, in patients with this disorder have shown diminished amyloid-β clearance from brain tissue. The recognition of amyloid-β clearance pathways opens exciting new therapeutic opportunities for this disease.
‘Multiple-target, multiple-action’ agents will stand a better chance of controlling the complex disease mechanisms that mediate neurodegeneration in disorders such as Alzheimer’s disease than will agents that have only one target. According to the vasculo-neuronal-inflammatory triad model of neurodegenerative disorders, in addition to neurons, brain endothelium, VSMCs, pericytes, astrocytes and activated microglia all represent important therapeutic targets.


Neurons depend on blood vessels for their oxygen and nutrient supplies, and for the removal of carbon dioxide and other potentially toxic metabolites from the brain’s interstitial fluid (ISF). The importance of the circulatory system to the human brain is highlighted by the fact that although the brain comprises ~2% of total body mass, it receives up to 20% of cardiac output and is responsible for ~20% and ~25% of the body’s oxygen consumption and glucose consumption, respectively1. To underline this point, when cerebral blood flow (CBF) stops, brain functions end within seconds and damage to neurons occurs within minutes2.

Neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) are associated with microvascular dysfunction and/or degeneration in the brain, neurovascular disintegration, defective blood–brain barrier (BBB) function and/or vascular factors1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Microvascular deficits diminish CBF and, consequently, the brain’s supply of oxygen, energy substrates and nutrients. Moreover, such deficits impair the clearance of neurotoxic molecules that accumulate and/or are deposited in the ISF, non-neuronal cells and neurons. Recent evidence suggests that vascular dysfunction leads to neuronal dysfunction and neurodegeneration, and that it might contribute to the development of proteinaceous brain and cerebrovascular ‘storage’ disorders. Such disorders include cerebral β-amyloidosis and cerebral amyloid angiopathy (CAA), which are caused by accumulation of the peptide amyloid-β in the brain and the vessel wall, respectively, and are features of Alzheimer’s disease1.

In this Review, I will discuss neurovascular pathways to neurodegeneration, placing a focus on Alzheimer’s disease because more is known about neurovascular dysfunction in this disease than in other neurodegenerative disorders. The article first examines transport mechanisms for molecules to cross the BBB, before exploring the processes that are involved in BBB breakdown at the molecular and cellular levels, and the consequences of BBB breakdown, hypoperfusion, and hypoxia and endothelial metabolic dysfunction for neuronal function. Next, the article reviews evidence for neurovascular changes during normal ageing and neurovascular BBB dysfunction in various neurodegenerative diseases, including evidence suggesting that vascular defects precede neuronal changes. Finally, the article considers specific mechanisms that are associated with BBB dysfunction in Alzheimer’s disease and ALS, and therapeutic opportunities relating to these neurovascular deficits.

The neurovascular unit

The neurovascular unit (NVU) comprises vascular cells (that is, endothelium, pericytes and vascular smooth muscle cells (VSMCs)), glial cells (that is, astrocytes, microglia and oliogodendroglia) and neurons1,2, 13 (Fig. 1). In the NVU, the endothelial cells together form a highly specialized membrane around blood vessels. This membrane underlies the BBB and limits the entry of plasma components, red blood cells (RBCs) and leukocytes into the brain. The BBB also regulates the delivery into the CNS of circulating energy metabolites and essential nutrients that are required for proper neuronal and synaptic function. Non-neuronal cells and neurons act in concert to control BBB permeability and CBF. Vascular cells and glia are primarily responsible for maintenance of the constant ‘chemical’ composition of the ISF, and the BBB and the blood–spinal cord barrier (BSCB) work together with pericytes to prevent various potentially neurotoxic and vasculotoxic macromolecules in the blood from entering the CNS, and to promote clearance of these substances from the CNS1.

In the brain, pial arteries run through the subarachnoid space (SAS), which contains the cerebrospinal fluid (CSF). These vessels give rise to intracerebral arteries, which penetrate into brain parenchyma. Intracerebral arteries are separated from brain parenchyma by a single, interrupted layer of elongated fibroblast-like cells of the pia and the astrocyte-derived glia limitans membrane that forms the outer wall of the perivascular Virchow–Robin space. These arteries branch into smaller arteries and subsequently arterioles, which lose support from the glia limitans and give rise to pre-capillary arterioles and brain capillaries. In an intracerebral artery, the vascular smooth muscle cell (VSMC) layer occupies most of the vessel wall. At the brain capillary level, vascular endothelial cells and pericytes are attached to the basement membrane. Pericyte processes encase most of the capillary wall, and they communicate with endothelial cells directly through synapse-like contacts containing connexins and N-cadherin. Astrocyte end-foot processes encase the capillary wall, which is composed of endothelium and pericytes. Resting microglia have a ‘ramified’ shape and can sense neuronal injury.

Figure 2 | Blood–brain barrier transport mechanisms.

Small lipophilic drugs, oxygen and carbon dioxide diffuse across the blood–brain barrier (BBB), whereas ions require ATP-dependent transporters such as the (Na++K+)ATPase. Transporters for nutrients include the glucose transporter 1 (GLUT1; also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)), the lactate transporter monocarboxylate transporter 1 (MCT1) and the L1 and y+ transporters for large neutral and cationic essential amino acids, respectively. These four transporters are expressed at both the luminal and albuminal membranes. Non-essential amino acid transporters (the alanine, serine and cysteine preferring system (ASC), and the alanine preferring system (A)) and excitatory amino acid transporter 1 (EAAT1), EAAT2 and EAAT3 are located at the abluminal side. The ATP-binding cassette (ABC) efflux transporters that are found in the endothelial cells include multidrug resistance protein 1 (ABCB1; also known as ATP-binding cassette subfamily B member 1) and solute carrier organic anion transporter family member 1C1 (OATP1C1). Finally, transporters for peptides or proteins include the endothelial protein C receptor (EPCR) for activated protein C (APC); the insulin receptors (IRs) and the transferrin receptors (TFRs), which are associated with caveolin 1 (CAV1); low-density lipoprotein receptor-related protein 1 (LRP1) for amyloid-β, peptide transport system 1 (PTS1) for encephalins; and the PTS2 and PTS4–vasopressin V1a receptor (V1AR) for arginine vasopressin.


Transport across the blood–brain barrier. The endothelial cells that form the BBB are connected by tight and adherens junctions, and it is the tight junctions that confer the low paracellular permeability of the BBB1. Small lipophilic molecules, oxygen and carbon dioxide diffuse freely across the endothelial cells, and hence the BBB, but normal brain endothelium lacks fenestrae and has limited vesicular transport.

The high number of mitochondria in endothelial cells reflects a high energy demand for active ATP-dependent transport, conferred by transporters such as the sodium pump ((Na++K+)ATPase) and the ATP-binding cassette (ABC) efflux transporters. Sodium influx and potassium efflux across the abluminal side of the BBB is controlled by (Na++K+)ATPase (Fig. 2). Changes in sodium and potassium levels in the ISF influence the generation of action potentials in neurons and thus directly affect neuronal and synaptic functions1, 12.

Brain endothelial cells express transporters that facilitate the transport of nutrients down their concentration gradients, as described in detail elsewhere1, 14 (Fig. 2). Glucose transporter 1 (GLUT1; also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)) — the BBB-specific glucose transporter — is of special importance because glucose is a key energy source for the brain.

Monocarboxylate transporter 1 (MCT1), which transports lactate, and the L1 and y+ amino acid transporters are expressed at the luminal and abluminal membranes12, 14. Sodium-dependent excitatory amino acid transporter 1 (EAAT1), EAAT2 and EAAT3 are expressed at the abluminal side of the BBB15 and enable removal of glutamate, an excitatory neurotransmitter, from the brain (Fig. 2). Glutamate clearance at the BBB is essential for protecting neurons from overstimulation of glutaminergic receptors, which is neurotoxic16.

ABC transporters limit the penetration of many drugs into the brain17. For example, multidrug resistance protein 1 (ABCB1; also known as ATP-binding cassette subfamily B member 1) controls the rapid removal of ingested toxic lipophilic metabolites17 (Fig. 2). Some ABC transporters also mediate the efflux of nutrients from the endothelium into the ISF. For example, solute carrier organic anion transporter family member 1C1 (OATP1C1) transports thyroid hormones into the brain. MCT8 mediates influx of thyroid hormones from blood into the endothelium18 (Fig. 2).

The transport of circulating peptides across the BBB into the brain is restricted or slow compared with the transport of nutrients19. Carrier-mediated transport of neuroactive peptides controls their low levels in the ISF20, 21, 22, 23, 24 (Fig. 2). Some proteins, including transferrin, insulin, insulin-like growth factor 1 (IGF1), leptin25, 26, 27 and activatedprotein C (APC)28, cross the BBB by receptor-mediated transcytosis (Fig. 2).

Circumventricular organs. Several small neuronal structures that surround brain ventricles lack the BBB and sense chemical changes in blood or the cerebrospinal fluid (CSF) directly. These brain areas are known as circumventricular organs (CVOs). CVOs have important roles in multiple endocrine and autonomic functions, including the control of feeding behaviour as well as regulation of water and salt metabolism29. For example, the subfornical organ is one of the CVOs that are capable of sensing extracellular sodium using astrocyte-derived lactate as a signal for local neurons to initiate neural, hormonal and behavioural responses underlying sodium homeostasis30. Excessive sodium accumulation is detrimental, and increases in plasma sodium above a narrow range are incompatible with life, leading to cerebral oedema (swelling), seizures and death29.

Vascular-mediated pathophysiology

The key pathways of vascular dysfunction that are linked to neurodegenerative diseases include BBB breakdown, hypoperfusion–hypoxia and endothelial metabolic dysfunction (Fig. 3). This section examines processes that are involved in BBB breakdown at the molecular and cellular levels, and explores the consequences of all three pathways for neuronal function and viability.

Figure 3 | Vascular-mediated neuronal damage and neurodegeneration.

a | Blood–brain barrier (BBB) breakdown that is caused by pericyte detachment leads to leakage of serum proteins and focal microhaemorrhages, with extravasation of red blood cells (RBCs). RBCs release haemoglobin, which is a source of iron. In turn, this metal catalyses the formation of toxic reactive oxygen species (ROS) that mediate neuronal injury. Albumin promotes the development of vasogenic oedema, contributing to hypoperfusion and hypoxia of the nervous tissue, which aggravates neuronal injury. A defective BBB allows several potentially vasculotoxic and neurotoxic proteins (for example, thrombin, fibrin and plasmin) to enter the brain. b | Progressive reductions in cerebral blood flow (CBF) lead to increasing neuronal dysfunction. Mild hypoperfusion, oligaemia, leads to a decrease in protein synthesis, whereas more-severe reductions in CBF, leading to hypoxia, cause an array of detrimental effects.

Blood–brain barrier breakdown. Disruption to tight and adherens junctions, an increase in bulk-flow fluid transcytosis, and/or enzymatic degradation of the capillary basement membrane cause physical breakdown of the BBB.

The levels of many tight junction proteins, their adaptor molecules and adherens junction proteins decrease in Alzheimer’s disease and other diseases that cause dementia1, 9, ALS31, multiple sclerosis32 and various animal models of neurological disease8, 33. These decreases might be partly explained by the fact that vascular-associated matrix metalloproteinase (MMP) activity rises in many neurodegenerative disorders and after ischaemic CNS injury34, 35; tight junction proteins and basement membrane extracellular matrix proteins are substrates for these enzymes34. Lowered expression of messenger RNAs that encode several key tight junction proteins, however, has also been reported in some neurodegenerative disorders, such as ALS31.

Endothelial cell–pericyte interactions are crucial for the formation36, 37and maintenance of the BBB33, 38. Pericyte deficiency can lead to a reduction in expression of certain tight junction proteins, including occludin, claudin 5 and ZO1 (Ref. 33), and to an increase in bulk-flow transcytosis across the BBB, causing BBB breakdown38. Both processes can lead to extravasation of multiple small and large circulating macromolecules (up to 500 kDa) into the brain parenchyma33, 38. Moreover, in mice, an age-dependent progressive loss of pericytes can lead to BBB disruption and microvasular degeneration and, subsequently, neuronal dysfunction, cognitive decline and neurodegenerative changes33. In their lysosomes, pericytes concentrate and degrade multiple circulating exogenous39 and endogenous proteins, including serum immunoglobulins and fibrin33, which amplify BBB breakdown in cases of pericyte deficiency.

BBB breakdown typically leads to an accumulation of various molecules in the brain. The build up of serum proteins such as immunoglobulins and albumin can cause brain oedema and suppression of capillary blood flow8, 33, whereas high concentrations of thrombin lead to neurotoxicity and memory impairment40, and accelerate vascular damage and BBB disruption41. The accumulation of plasmin (derived from circulating plasminogen) can catalyse the degradation of neuronal laminin and, hence, promote neuronal injury42, and high fibrin levels accelerate neurovascular damage6. Finally, an increase in the number of RBCs causes deposition of haemoglobin-derived neurotoxic products including iron, which generates neurotoxic reactive oxygen species (ROS)8, 43(Fig. 3a). In addition to protein-mediated vasogenic oedema, local tissue ischaemia–hypoxia depletes ATP stores, causing (Na++K+)ATPase pumps and Na+-dependent ion channels to stop working and, consequently, the endothelium and astrocytes to swell (known as cytotoxic oedema)44. Upregulation of aquaporin 4 water channels in response to ischaemia facilitates the development of cytotoxic oedema in astrocytes45.

Hypoperfusion and hypoxia. CBF is regulated by local neuronal activity and metabolism, known as neurovascular coupling46. The pial and intracerebral arteries control the local increase in CBF that occurs during brain activation, which is termed ‘functional hyperaemia’. Neurovascular coupling requires intact pial circulation, and for VSMCs and pericytes to respond normally to vasoactive stimuli33, 46, 47. In addition to VSMC-mediated constriction and vasodilation of cerebral arteries, recent studies have shown that pericytes modulate brain capillary diameter through constriction of the vessel wall47, which obstructs capillary flow during ischaemia48. Astrocytes regulate the contractility of intracerebral arteries49, 50.

Progressive CBF reductions have increasingly serious consequences for neurons (Fig. 3b). Briefly, mild hypoperfusion — termed oligaemia — affects protein synthesis, which is required for the synaptic plasticity mediating learning and memory46. Moderate to severe CBF reductions and hypoxia affect ATP synthesis, diminishing (Na++K+)ATPase activity and the ability of neurons to generate action potentials9. In addition, such reductions can lower or increase pH, and alter electrolyte balances and water gradients, leading to the development of oedema and white matter lesions, and the accumulation of glutamate and proteinaceous toxins (for example, amyloid-β and hyperphopshorylated tau) in the brain. A reduction of greater than 80% in CBF results in neuronal death2.

The effect of CBF reductions has been extensively studied at the molecular and cellular levels in relation to Alzheimer’s disease. Reduced CBF and/or CBF dysregulation occurs in elderly individuals at high risk of Alzheimer’s disease before cognitive decline, brain atrophy and amyloid-β accumulation10, 46, 51, 52, 53, 54. In animal models, hypoperfusion can induce or amplify Alzheimer’s disease-like neuronal dysfunction and/or neuropathological changes. For example, bilateral carotid occlusion in rats causes memory impairment, neuronal dysfunction, synaptic changes and amyloid-β oligomerization55, leading to accumulation of neurotoxic amyloid-β oligomers56. In a mouse model of Alzheimer’s disease, oligaemia increases neuronal amyloid-β levels and neuronal tau phosphophorylation at an epitope that is associated with Alzheimer’s disease-type paired helical filaments57. In rodents, ischaemia leads to the accumulation of hyperphosphorylated tau in neurons and the formation of filaments that resemble those present in human neurodegenerative tauopathies and Alzheimer’s disease58. Mice expressing amyloid-β precursor protein (APP) and transforming growth factor β1 (TGFβ1) develop deficient neurovascular coupling, cholinergic denervation, enhanced cerebral and cerebrovascular amyloid-β deposition, and age-dependent cognitive decline59.

Recent studies have shown that ischaemia–hypoxia influences amyloidogenic APP processing through mechanisms that increase the activity of two key enzymes that are necessary for amyloid-β production; that is, β-secretase and γ-secretase60, 61, 62, 63. Hypoxia-inducible factor 1α (HIF1α) mediates transcriptional increase in β-secretase expression61. Hypoxia also promotes phosphorylation of tau through the mitogen-activated protein kinase (MAPK; also known as extracellular signal-regulated kinase (ERK)) pathway64, downregulates neprilysin — an amyloid-β-degrading enzyme65 — and leads to alterations in the expression of vascular-specific genes, including a reduction in the expression of the homeobox protein MOX2 gene mesenchyme homeobox 2 (MEOX2) in brain endothelial cells5 and an increase in the expression of the myocardin gene (MYOCD) in VSMCs66. In patients with Alzheimer’s disease and in models of this disorder, these changes cause vessel regression, hypoperfusion and amyloid-β accumulation resulting from the loss of the key amyloid-β clearance lipoprotein receptor (see below). In addition, hypoxia facilitates alternative splicing of Eaat2 mRNA in Alzheimer’s disease transgenic mice before amyloid-β deposition67 and suppresses glutamate reuptake by astrocytes independently of amyloid formation68, resulting in glutamate-mediated neuronal injury that is independent of amyloid-β.

In response to hypoxia, mitochondria release ROS that mediate oxidative damage to the vascular endothelium and to the selective population of neurons that has high metabolic activity. Such damage has been suggested to occur before neuronal degeneration and amyloid-β deposition in Alzheimer’s disease69, 70. Although the exact triggers of hypoxia-mediated neurodegeneration and the role of HIF1α in neurodegeneration versus preconditioning-mediated neuroprotection remain topics of debate, mitochondria-generated ROS seem to have a primary role in the regulation of the HIF1α-mediated transcriptional switch that can activate an array of responses, ranging from mechanisms that increase cell survival and adaptation to mechanisms inducing cell cycle arrest and death71. Whether inhibition of hypoxia-mediated pathogenic pathways will delay onset and/or control progression in neurodegenerative conditions such as Alzheimer’s disease remains to be determined.

When comparing the contributions of BBB breakdown and hypoperfusion to neuronal injury, it is interesting to consider Meox2+/− mice. Such animals have normal pericyte coverage and an intact BBB but a substantial perfusion deficit5 that is comparable to that found in pericyte-deficient mice that develop BBB breakdown33 Notably, however, Meox2+/− mice show less pronounced neurodegenerative changes than pericyte-deficient mice, indicating that chronic hypoperfusion–hypoxia alone can cause neuronal injury, but not to the same extent as hypoperfusion–hypoxia combined with BBB breakdown.

Endothelial neurotoxic and inflammatory factors. Alterations in cerebrovascular metabolic functions can lead to the secretion of multiple neurotoxic and inflammatory factors72, 73. For example, brain microvessels that have been isolated from individuals with Alzheimer’s disease (but not from neurologically normal age-matched and young individuals) and brain microvessels that have been treated with inflammatory proteins release neurotoxic factors that kill neurons74, 75. These factors include thrombin, the levels of which increase with the onset of Alzheimer’s disease76. Thrombin can injure neurons directly40and indirectly by activating microglia and astrocytes73. Compared with those from age-matched controls, brain microvessels from individuals with Alzheimer’s disease secrete increased levels of multiple inflammatory mediators, such as nitric oxide, cytokines (for example, tumour necrosis factor (TNF), TGFβ1, interleukin-1β (IL-1β) and IL-6), chemokines (for example, CC-chemokine ligand 2 (CCL2; also known as monocyte chemoattractant protein 1 (MCP1)) and IL-8), prostaglandins, MMPs and leukocyte adhesion molecules73. Endothelium-derived neurotoxic and inflammatory factors together provide a molecular link between vascular metabolic dysfunction, neuronal injury and inflammation in Alzheimer’s disease and, possibly, in other neurodegenerative disorders.

Neurovascular changes

This section examines evidence for neurovascular changes during normal ageing and for neurovascular and/or BBB dysfunction in various neurodegenerative diseases, as well as the possibility that vascular defects can precede neuronal changes.

Age-associated neurovascular changes. Normal ageing diminishes brain circulatory functions, including a detectable decay of CBF in the limbic and association cortices that has been suggested to underlie age-related cognitive changes77. Alterations in the cerebral microvasculature, but not changes in neural activity, have been shown to lead to age-dependent reductions in functional hyperaemia in the visual system in cats78 and in the sensorimotor cortex in pericyte-deficient mice33. Importantly, a recent longitudinal CBF study in neurologically normal individuals revealed that people bearing the apolipoprotein E (APOE) ɛ4allele — the major genetic risk factor for late-onset Alzheimer’s disease79, 80, 81 — showed greater regional CBF decline in brain regions that are particularly vulnerable to pathological changes in Alzheimer’s disease than did people without this allele82.

A meta-analysis of BBB permeability in 1,953 individuals showed that neurologically healthy humans had an age-dependent increase in vascular permeability83. Moreover, patients with vascular or Alzheimer’s disease-type dementia and leucoaraiosis — a small-vessel disease of the cerebral white matter — had an even greater age-dependent increase in vascular permeability83. Interestingly, an increase in BBB permeability in brain areas with normal white matter in patients with leukoaraiosis has been suggested to play a causal part in disease and the development of lacunar strokes84. Age-related changes in the permeability of the blood–CSF barrier and the choroid plexus have been reported in sheep85.

Vascular pathology. Patients with Alzheimer’s disease or other dementia-causing diseases frequently show focal changes in brain microcirculation. These changes include the appearance of string vessels (collapsed and acellular membrane tubes), a reduction in capillary density, a rise in endothelial pinocytosis, a decrease in mitochondrial content, accumulation of collagen and perlecans in the basement membrane, loss of tight junctions and/or adherens junctions3, 4, 5, 6, 9,46, 86, and BBB breakdown with leakage of blood-borne molecules4, 6,7, 9. The time course of these vascular alterations and how they relate to dementia and Alzheimer’s disease pathology remain unclear, as no protocol that allows the development of the diverse brain vascular pathology to be scored, and hence to be tracked with ageing, has so far been developed and widely validated87. Interestingly, a recent study involving 500 individuals who died between the ages of 69 and 103 years showed that small-vessel disease, infarcts and the presence of more than one vascular pathological change were associated with Alzheimer’s disease-type pathological lesions and dementia in people aged 75 years of age87. These associations were, however, less pronounced in individuals aged 95 years of age, mainly because of a marked ageing-related reduction in Alzheimer’s disease neuropathology relative to a moderate but insignificant ageing-related reduction in vascular pathology87.

Accumulation of amyloid-β and amyloid deposition in pial and intracerebral arteries results in CAA, which is present in over 80% of Alzheimer’s disease cases88. In patients who have Alzheimer’s disease with established CAA in small arteries and arterioles, the VSMC layer frequently shows atrophy, which causes a rupture of the vessel wall and intracerebral bleeding in about 30% of these patients89, 90. These intracerebral bleedings contribute to, and aggravate, dementia. Patients with hereditary cerebral β-amyloidosis and CAA of the Dutch, Iowa, Arctic, Flemish, Italian or Piedmont L34V type have accelerated VSMC degeneration resulting in haemorrhagic strokes and dementia91. Duplication of the gene encoding APP causes early-onset Alzheimer’s disease dementia with CAA and intracerebral haemorrhage92.

Early studies of serum immunoglobulin leakage reported that patients with ALS had BSCB breakdown and BBB breakdown in the motor cortex93. Microhaemorrhages and BSCB breakdown have been shown in the spinal cord of transgenic mice expressing mutant variants of human superoxide dismutase 1 (SOD1), which in mice cause an ALS-like disease8, 94, 95. In mice with ALS-like disease and in patients with ALS, BSCB breakdown has been shown to occur before motor neuron degeneration or brain atrophy8, 11, 95.

BBB breakdown in the substantia nigra and the striatum has been detected in murine models of Parkinson’s disease that are induced by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)96, 97, 98. However, the temporal relationship between BBB breakdown and neurodegeneration in Parkinson’s disease is currently unknown. Notably, the prevalence of CAA and vascular lesions increases in Parkinson’s disease99, 100. Vascular lesions in the striatum and lacunar infarcts can cause vascular parkinsonism syndrome101. A recent study reported BBB breakdown in a rat model of Huntington’s disease that is induced with the toxin 3-nitropropionic acid102.

Several studies have established disruption of BBB with a loss of tight junction proteins during neuroinflammatory conditions such as multiple sclerosis and its murine model, experimental allergic encephalitis. Such disruption facilitates leukocyte infiltration, leading to oliogodendrocyte death, axonal damage, demyelination and lesion development32.

Functional changes in the vasculature. In individuals with Alzheimer’s disease, GLUT1 expression at the BBB decreases103, suggesting a shortage in necessary metabolic substrates. Studies using18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) have identified reductions in glucose uptake in asymptomatic individuals with a high risk of dementia104, 105. Several studies have suggested that reduced glucose uptake across the BBB, as seen by FDG PET, precedes brain atrophy104, 105, 106, 107, 108.

Amyloid-β constricts cerebral arteries109. In a mouse model of Alzheimer’s disease, impairment of endothelium-dependent regulation of neocortical microcirculation110, 111 occurs before amyloid-β accumulation. Recent studies have shown that CD36, a scavenger receptor that binds amyloid-β, is essential for the vascular oxidative stress and diminished functional hyperaemia that occurs in response to amyloid-β exposure112. Neuroimaging studies in patients with Alzheimer’s disease have shown that neurovascular uncoupling occurs before neurodegenerative changes10, 51, 52, 53. Moreover, cognitively normal APOE ɛ4 carriers at risk of Alzheimer’s disease show impaired CBF responses to brain activation in the absence of neurodegenerative changes or amyloid-β accumulation54. Recently, patients with Alzheimer’s disease as well as mouse models of this disease with high cerebrovascular levels of serum response factor (SRF) and MYOCD, the two transcription factors that control VSMC differentiation, have been shown to develop a hypercontractile arterial phenotype resulting in brain hypoperfusion, diminished functional hyperaemia and CAA66, 113. More work is needed to establish the exact role of SRF and MYOCD in the vascular dysfunction that results in the Alzheimer’s disease phenotype and CAA.

PET studies with 11C-verapamil, an ABCB1 substrate, have indicated that the function of ABCB1, which removes multiple drugs and toxins from the brain, decreases with ageing114 and is particularly compromised in the midbrain of patients with Parkinson’s disease, progressive supranuclear palsy or multiple system atrophy115. More work is needed to establish the exact roles of ABC BBB transporters in neurodegeneration and whether their failure precedes the loss of dopaminergic neurons that occurs in Parkinson’s disease.

In mice with ALS-like disease and in patients with ALS, hypoperfusion and/or dysregulated CBF have been shown to occur before motor neuron degeneration or brain atrophy8, 116. Reduced regional CBF in basal ganglia and reduced blood volume have been reported in pre-symptomatic gene-tested individuals at risk for Huntington’s disease117. Patients with Huntington’s disease display a reduction in vasomotor activity in the cerebral anterior artery during motor activation118.

Vascular and neuronal common growth factors. Blood vessels and neurons share common growth factors and molecular pathways that regulate their development and maintenance119, 120. Angioneurins are growth factors that exert both vasculotrophic and neurotrophic activities121. The best studied angioneurin is vascular endothelial growth factor (VEGF). VEGF regulates vessel formation, axonal growth and neuronal survival120. Ephrins, semaphorins, slits and netrins are axon guidance factors that also regulate the development of the vascular system121. During embryonic development of the neural tube, blood vessels and choroid plexus secrete IGF2 into the CSF, which regulates the proliferation of neuronal progenitor cells122. Genetic and pharmacological manipulations of angioneurin activity yielded various vascular and cerebral phenotypes121. Given the dual nature of angioneurin action, these studies have not been able to address whether neuronal dysfunction results from a primary insult to neurons and/or whether it is secondary to vascular dysfunction.

Increased levels of VEGF, a hypoxia-inducible angiogenic factor, were found in the walls of intraparenchymal vessels, perivascular deposits, astrocytes and intrathecal space of patients with Alzheimer’s disease, and were consistent with the chronic cerebral hypoperfusion and hypoxia that were observed in these individuals73. In addition to VEGF, brain microvessels in Alzheimer’s disease release several molecules that can influence angiogenesis, including IL-1β, IL-6, IL-8, TNF, TGFβ, MCP1, thrombin, angiopoietin 2, αVβ3 and αVβ5 integrins, and HIF1α73. However, evidence for increased vascularity in Alzheimer’s disease is lacking. On the contrary, several studies have reported that focal vascular regression and diminished microvascular density occur in Alzheimer’s disease4, 5, 73 and in Alzheimer’s disease transgenic mice123. The reason for this discrepancy is not clear. The anti-angiogenic activity of amyloid-β, which accumulates in the brains of individuals with Alzheimer’s disease and Alzheimer’s disease models, may contribute to hypovascularity123. Conversely, genome-wide transcriptional profiling of brain endothelial cells from patients with Alzheimer’s disease revealed that extremely low expression of vascular-restricted MEOX2 mediates aberrant angiogenic responses to VEGF and hypoxia, leading to capillary death5. This finding raises the interesting question of whether capillary degeneration in Alzheimer’s disease results from unsuccessful vascular repair and/or remodelling. Moreover, mice that lack one Meox2 allele have been shown to develop a primary cerebral endothelial hypoplasia with chronic brain hypoperfusion5, resulting in secondary neurodegenerative changes33.

Does vascular dysfunction cause neuronal dysfunction? In summary, the evidence that is discussed above clearly indicates that vascular dysfunction is tightly linked to neuronal dysfunction. There are many examples to illustrate that primary vascular deficits lead to secondary neurodegeneration, including CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts), an hereditary small-vessel brain disease resulting in multiple small ischaemic lesions, neurodegeneration and dementia124; mutations in SLC2A1 that cause dysfunction of the BBB-specific GLUT1 transporter in humans resulting in seizures; cognitive impairment and microcephaly125; microcephaly and epileptiform discharges in mice with genetic deletion of a single Slc2a1allele126; and neurodegeneration mediated by a single Meox2 homebox gene deletion restricted to the vascular system33. Patients with hereditary cerebral β-amyloidosis and CAA of the Dutch, Iowa, Arctic, Flemish, Italian or Piedmont L34V type provide another example showing that primary vascular dysfunction — which in this case is caused by deposition of vasculotropic amyloid-β mutants in the arterial vessel wall — leads to dementia and intracerebral bleeding. Moreover, as reviewed in the previous sections, recent evidence suggests that BBB dysfunction and/or breakdown, and CBF reductions and/or dysregulation may occur in sporadic Alzheimer’s disease and experimental models of this disease before cognitive decline, amyloid-β deposition and brain atrophy. In patients with ALS and in some experimental models of ALS, CBF dysregulation, BSCB breakdown and spinal cord hypoperfusion have been reported to occur before motor neuron cell death. Whether neurological changes follow or precede vascular dysfunction in Parkinson’s disease, Huntington’s disease and multiple sclerosis remains less clear. However, there is little doubt that vascular injury mediates, amplifies and/or lowers the threshold for neuronal dysfunction and loss in several neurological disorders.

Disease-specific considerations

This section examines how amyloid-β levels are kept low in the brain, a process in which the BBB has a central role, and how faulty BBB-mediated clearance mechanisms go awry in Alzheimer’s disease. On the basis of this evidence and the findings discussed elsewhere in the Review, a new hypothesis for the pathogenesis of Alzheimer’s disease that incorporates the vascular evidence is presented. ALS-specific disease mechanisms relating to the BBB are then examined.

Alzheimer’s disease. Amyloid-β clearance from the brain by the BBB is the best studied example of clearance of a proteinaceous toxin from the CNS. Multiple pathways regulate brain amyloid-β levels, including its production and clearance (Fig. 4). Recent studies127, 128, 129 have confirmed earlier findings in multiple rodent and non-human primate models demonstrating that peripheral amyloid-β is an important precursor of brain amyloid-β130, 131, 132, 133, 134, 135, 136. Moreover, peripheral amyloid-β sequestering agents such as soluble LRP1 (ref.137), anti-amyloid-β antibodies138, 139, 140, gelsolin and the ganglioside GM1 (Ref. 141), or systemic expression of neprilysin142, 143have been shown to reduce the amyloid burden in Alzheimer’s disease mice by eliminating contributions of the peripheral amyloid-β pool to the total brain pool of this peptide.

Figure 4 | The role of blood–brain barrier transport in brain homeostasis of amyloid-β.

Amyloid-β (Aβ) is produced from the amyloid-β precursor protein (APP), both in the brain and in peripheral tissues. Clearance of amyloid-β from the brain normally maintains its low levels in the brain. This peptide is cleared across the blood–brain barrier (BBB) by the low-density lipoprotein receptor-related protein 1 (LRP1). LRP1 mediates rapid efflux of a free, unbound form of amyloid-β and of amyloid-β bound to apolipoprotein E2 (APOE2), APOE3 or α2-macroglobulin (not shown) from the brain’s interstitial fluid into the blood, and APOE4 inhibits such transport. LRP2 eliminates amyloid-β that is bound to clusterin (CLU; also known as apolipoprotein J (APOJ)) by transport across the BBB, and shows a preference for the 42-amino-acid form of this peptide. ATP-binding cassette subfamily A member 1 (ABCA1; also known as cholesterol efflux regulatory protein) mediates amyloid-β efflux from the brain endothelium to blood across the luminal side of the BBB (not shown). Cerebral endothelial cells, pericytes, vascular smooth muscle cells, astrocytes, microglia and neurons express different amyloid-β-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme (IDE), tissue plasminogen activator (tPA) and matrix metalloproteinases (MMPs), which contribute to amyloid-β clearance. In the circulation, amyloid-β is bound mainly to soluble LRP1 (sLRP1), which normally prevents its entry into the brain. Systemic clearance of amyloid-β is mediated by its removal by the liver and kidneys. The receptor for advanced glycation end products (RAGE) provides the key mechanism for influx of peripheral amyloid-β into the brain across the BBB either as a free, unbound plasma-derived peptide and/or by amyloid-β-laden monocytes. Faulty vascular clearance of amyloid-β from the brain and/or an increased re-entry of peripheral amyloid-β across the blood vessels into the brain can elevate amyloid-β levels in the brain parenchyma and around cerebral blood vessels. At pathophysiological concentrations, amyloid-β forms neurotoxic oligomers and also self-aggregates, which leads to the development of cerebral β-amyloidosis and cerebral amyloid angiopathy.

The receptor for advanced glycation end products (RAGE) mediates amyloid-β transport in brain and the propagation of its toxicity. RAGE expression in brain endothelium provides a mechanism for influx of amyloid-β144, 145 and amyloid-β-laden monocytes146 across the BBB, as shown in Alzheimer’s disease models (Fig. 4). The amyloid-β-rich environment in Alzheimer’s disease and models of this disorder increases RAGE expression at the BBB and in neurons147, 148, amplifying amyloid-β-mediated pathogenic responses. Blockade of amyloid-β–RAGE signalling in Alzheimer’s disease is a promising strategy to control self-propagation of amyloid-β-mediated injury.

Several studies in animal models of Alzheimer’s disease and, more recently, in patients with this disorder149 have shown that diminished amyloid-β clearance occurs in brain tissue in this disease. LRP1 plays an important part in the three-step serial clearance of this peptide from brain and the rest of the body150 (Fig. 4). In step one, LRP1 in brain endothelium binds brain-derived amyloid-β at the abluminal side of the BBB, initiating its clearance to blood, as shown in many animal models151, 152, 153, 154, 155, 156 and BBB models in vitro151, 157,158. The vasculotropic mutants of amyloid-β that have low binding affinity for LRP1 are poorly cleared from the brain or CSF151, 159, 160. APOE4, but not APOE3 or APOE2, blocks LRP1-mediated amyloid-β clearance from the brain and, hence, promotes its retention161, whereas clusterin (also known as apolipoprotein J (APOJ)) mediates amyloid-β clearance across the BBB via LRP2 (Ref. 153). APOE and clusterin influence amyloid-β aggregation162, 163. Reduced LRP1 levels in brain microvessels, perhaps in addition to altered levels of ABCB1, are associated with amyloid-β cerebrovascular and brain accumulation during ageing in rodents, non-human primates, humans, Alzheimer’s disease mice and patients with Alzheimer’s disease66, 151, 152, 164, 165, 166. Moreover, recent work has shown that brain LRP1 is oxidized in Alzheimer’s disease167, and may contribute to amyloid-β retention in brain because the oxidized form cannot bind and/or transport amyloid-β137. LRP1 also mediates the removal of amyloid-β from the choroid plexus168.

In step two, circulating soluble LRP1 binds more than 70% of plasma amyloid-β in neurologically normal humans137. In patients with Alzheimer’s disease or mild cognitive impairment (MCI), and in Alzheimer’s disease mice, amyloid-β binding to soluble LRP1 is compromised due to oxidative changes137, 169, resulting in elevated plasma levels of free amyloid-β isoforms comprising 40 or 42 amino acids (amyloid-β1–40 and amyloid-β1–42). These peptides can then re-enter the brain, as has been shown in a mouse model of Alzheimer’s disease137. Rapid systemic removal of amyloid-β by the liver is also mediated by LRP1 and comprises step three of the clearance process170.

In brain, amyloid-β is enzymatically degraded by neprilysin171, insulin-degrading enzyme172, tissue plasminogen activator173 and MMPs173,174 in various cell types, including endothelial cells, pericytes, astrocytes, neurons and microglia. Cellular clearance of this peptide by astrocytes and VSMCs is mediated by LRP1 and/or another lipoprotein receptor66, 175. Clearance of amyloid-β aggregates by microglia has an important role in amyloid-β-directed immunotherapy176 and reduction of the amyloid load in brain177. Passive ISF–CSF bulk flow and subsequent clearance through the CSF might contribute to 10–15% of total amyloid-β removal152, 153, 178. In the injured human brain, increasing soluble amyloid-β concentrations in the ISF correlated with improvements in neurological status, suggesting that neuronal activity might regulate extracellular amyloid-β levels179.

The role of BBB dysfunction in amyloid-β accumulation, as discussed above, underlies the contribution of vascular dysfunction to Alzheimer’s disease (see Fig. 5 for a model of vascular damage in Alzheimer’s disease). The amyloid hypothesis for the pathogenesis of Alzheimer’s disease maintains that this peptide initiates a cascade of events leading to neuronal injury and loss and, eventually, dementia180, 181. Here, I present an alternative hypothesis — the two-hit vascular hypothesis of Alzheimer’s disease — that incorporates the vascular contribution to this disease, as discussed in this Review (Box 1). This hypothesis states that primary damage to brain microcirculation (hit one) initiates a non-amyloidogenic pathway of vascular-mediated neuronal dysfunction and injury, which is mediated by BBB dysfunction and is associated with leakage and secretion of multiple neurotoxic molecules and/or diminished brain capillary flow that causes multiple focal ischaemic or hypoxic microinjuries. BBB dysfunction also leads to impairment of amyloid-β clearance, and oligaemia leads to increased amyloid-β generation. Both processes contribute to accumulation of amyloid-β species in the brain (hit two), where these peptides exert vasculotoxic and neurotoxic effects. According to the two-hit vascular hypothesis of Alzheimer’s disease, tau pathology develops secondary to vascular and/or amyloid-β injury.

Figure 5 | A model of vascular damage in Alzheimer’s disease.

a | In the early stages of Alzheimer’s disease, small pial and intracerebral arteries develop a hypercontractile phenotype that underlies dysregulated cerebral blood flow (CBF). This phenotype is accompanied by diminished amyloid-β clearance by the vascular smooth muscle cells (VSMCs). In the later phases of Alzheimer’s disease, amyloid deposition in the walls of intracerebral arteries leads to cerebral amyloid angiopathy (CAA), pronounced reductions in CBF, atrophy of the VSMC layer and rupture of the vessels causing microbleeds. b | At the level of capillaries in the early stages of Alzheimer’s disease, blood–brain barrier (BBB) dysfunction leads to a faulty amyloid-β clearance and accumulation of neurotoxic amyloid-β oligomers in the interstitial fluid (ISF), microhaemorrhages and accumulation of toxic blood-derived molecules (that is, thrombin and fibrin), which affect synaptic and neuronal function. Hyperphosphorylated tau (p-tau) accumulates in neurons in response to hypoperfusion and/or rising amyloid-β levels. At this point, microglia begin to sense neuronal injury. In the later stages of the disease in brain capillaries, microvascular degeneration leads to increased deposition of basement membrane proteins and perivascular amyloid. The deposited proteins and amyloid obstruct capillary blood flow, resulting in failure of the efflux pumps, accumulation of metabolic waste products, changes in pH and electrolyte composition and, subsequently, synaptic and neuronal dysfunction. Neurofibrillary tangles (NFTs) accumulate in response to ischaemic injury and rising amyloid-β levels. Activation of microglia and astrocytes is associated with a pronounced inflammatory response. ROS, reactive oxygen species.

Amyotrophic lateral sclerosis. The cause of sporadic ALS, a fatal adult-onset motor neuron neurodegenerative disease, is not known182. In a relatively small number of patients with inherited SOD1 mutations, the disease is caused by toxic properties of mutant SOD1 (Ref. 183). Mutations in the genes encoding ataxin 2 and TAR DNA-binding protein 43 (TDP43) that cause these proteins to aggregate have been associated with ALS182, 184. Some studies have suggested that abnormal SOD1 species accumulate in sporadic ALS185. Interestingly, studies in ALS transgenic mice expressing a mutant version of human SOD1 in neurons, and in non-neuronal cells neighbouring these neurons, have shown that deletion of this gene from neurons does not influence disease progression186, whereas deletion of this gene from microglia186 or astrocytes187 substantially increases an animal’s lifespan. According to an emerging hypothesis of ALS that is based on studies in SOD1 mutant mice, the toxicity that is derived from non-neuronal neighbouring cells, particularly microglia and astrocytes, contributes to disease progression and motor neuron degeneration186, 187, 188, 189, 190, whereas BBB dysfunction might be critical for disease initiation8, 43, 94, 95. More work is needed to determine whether this concept of disease initiation and progression may also apply to cases of sporadic ALS.

Human data support a role for angiogenic factors and vessels in the pathogenesis of ALS. For example, the presence of VEGF variations (which were identified in large meta-analysis studies) has been linked to ALS191. Angiogenin is another pro-angiogenic gene that is implicated in ALS because heterozygous missense mutations in angiogenin cause familial and sporadic ALS192. Moreover, mice with a mutation that eliminates hypoxia-responsive induction of the Vegf gene (Vegfδ/δ mice) develop late-onset motor neuron degeneration193. Spinal cord ischaemia worsens motor neuron degeneration and functional outcome in Vegfδ/δmice, whereas the absence of hypoxic induction of VEGF in mice that develop motor neuron disease from expression of ALS-linked mutant SOD1G93A results in substantially reduced survival191.

Therapeutic opportunities

Many investigators believe that primary neuronal dysfunction resulting from an intrinsic neuronal disorder is the key underlying event in human neurodegenerative diseases. Thus, most therapeutic efforts for neurodegenerative diseases have so far been directed at the development of so-called ‘single-target, single-action’ agents to target neuronal cells directly and reverse neuronal dysfunction and/or protect neurons from injurious insults. However, most preclinical and clinical studies have shown that such drugs are unable to cure or control human neurological disorders2, 181, 183, 194, 195. For example, although pathological overstimulation of glutaminergic NMDA receptors (NMDARs) has been shown to lead to neuronal injury and death in several disorders, including stroke, Alzheimer’s disease, ALS and Huntington’s disease16, NMDAR antagonists have failed to show a therapeutic benefit in the above-mentioned human neurological disorders.

Recently, my colleagues and I coined the term vasculo-neuronal-inflammatory triad195 to indicate that vascular damage, neuronal injury and/or neurodegeneration, and neuroinflammation comprise a common pathological triad that occurs in multiple neurological disorders. In line with this idea, it is conceivable that ‘multiple-target, multiple-action’ agents (that is, drugs that have more than one target and thus have more than one action) will have a better chance of controlling the complex disease mechanisms that mediate neurodegeneration than agents that have only one target (for example, neurons). According to the vasculo-neuronal-inflammatory triad model, in addition to neurons, brain endothelium, VSMCs, pericytes, astrocytes and activated microglia are all important therapeutic targets.

Here, I will briefly discuss a few therapeutic strategies based on the vasculo-neuronal-inflammatory triad model. VEGF and other angioneurins may have multiple targets, and thus multiple actions, in the CNS120. For example, preclinical studies have shown that treatment of SOD1G93A rats with intracerebroventricular VEGF196 or intramuscular administration of a VEGF-expressing lentiviral vector that is transported retrogradely to motor neurons in SOD1G93A mice197 reduced pathology and extended survival, probably by promoting angiogenesis and increasing the blood flow through the spinal cord as well as through direct neuronal protective effects of VEGF on motor neurons. On the basis of these and other studies, a phase I–II clinical trial has been initiated to evaluate the safety of intracerebroventricular infusion of VEGF in patients with ALS198. Treatment with angiogenin also slowed down disease progression in a mouse model of ALS199.

IGF1 delivery has been shown to promote amyloid-β vascular clearance and to improve learning and memory in a mouse model of Alzheimer’s disease200. Local intracerebral implantation of VEGF-secreting cells in a mouse model of Alzheimer’s disease has been shown to enhance vascular repair, reduce amyloid burden and improve learning and memory201. In contrast to VEGF, which can increase BBB permeability, TGFβ, hepatocyte growth factor and fibroblast growth factor 2 promote BBB integrity by upregulating the expression of endothelial junction proteins121 in a similar way to APC43. However, VEGF and most growth factors do not cross the BBB, so the development of delivery strategies such as Trojan horses is required for their systemic use25.

A recent experimental approach with APC provides an example of a neurovascular medicine that has been shown to favourably regulate multiple pathways in non-neuronal cells and neurons, resulting in vasculoprotection, stabilization of the BBB, neuroprotection and anti-inflammation in several acute and chronic models of the CNS disorders195 (Box 2).

The recognition of amyloid-β clearance pathways (Fig. 4), as discussed above, opens exciting new therapeutic opportunities for Alzheimer’s disease. Amyloid-β clearance pathways are promising therapeutic targets for the future development of neurovascular medicines because it has been shown both in animal models of Alzheimer’s disease1 and in patients with sporadic Alzheimer’s disease149 that faulty clearance from brain and across the BBB primarily determines amyloid-β retention in brain, causing the formation of neurotoxic amyloid-β oligomers56 and the promotion of brain and cerebrovascular amyloidosis3. The targeting of clearance mechanisms might also be beneficial in other diseases; for example, the clearance of extracellular mutant SOD1 in familial ALS, the prion protein in prion disorders and α-synuclein in Parkinson’s disease might all prove beneficial. However, the clearance mechanisms for these proteins in these diseases are not yet understood.

Conclusions and perspectives

Currently, no effective disease-modifying drugs are available to treat the major neurodegenerative disorders202, 203, 204. This fact leads to a question: are we close to solving the mystery of neurodegeneration? The probable answer is yes, because the field has recently begun to recognize that, first, damage to neuronal cells is not the sole contributor to disease initiation and progression, and that, second, correcting disease pathways in vascular and glial cells may offer an array of new approaches to control neuronal degeneration that do not involve targeting neurons directly. These realizations constitute an important shift in paradigm that should bring us closer to a cure for neurodegenerative diseases. Here, I raise some issues concerning the existing models of neurodegeneration and the new neurovascular paradigm.

The discovery of genetic abnormalities and associations by linkage analysis or DNA sequencing has broadened our understanding of neurodegeneration204. However, insufficient effort has been made to link genetic findings with disease biology. Another concern for neurodegenerative research is how we should interpret findings from animal models202. Genetically engineered models of human neurodegenerative disorders in Drosophila melanogaster andCaenorhabditis elegans have been useful for dissecting basic disease mechanisms and screening compounds. However, in addition to having much simpler nervous systems, insects and avascular species do not have cerebrovascular and immune systems that are comparable to humans and, therefore, are unlikely to replicate the complex disease pathology that is found in people.

For most neurodegenerative disorders, early steps in the disease processes remain unclear, and biomarkers for these stages have yet to be identified. Thus, it is difficult to predict whether mammalian models expressing human genes and proteins that we know are implicated in the intermediate or later stages of disease pathophysiology, such as amyloid-β or tau in Alzheimer’s disease7, 181, will help us to discover therapies for the early stages of disease and for disease prevention, because the exact role of these pathological accumulations during disease onset remains uncertain. According to the two-hit vascular hypothesis of Alzheimer’s disease, incorporating vascular factors that are associated with Alzheimer’s disease into current models of this disease may more faithfully replicate dementia events in humans. Alternatively, by focusing on the comorbidities and the initial cellular and molecular mechanisms underlying early neurovascular dysfunction that are associated with Alzheimer’s disease, new models of dementia and neurodegeneration may be developed that do not require the genetic manipulation of amyloid-β or tau expression.

The proposed neurovascular triad model of neurodegenerative diseases challenges the traditional neurocentric view of such disorders. At the same time, this model raises a set of new important issues that require further study. For example, the molecular basis of the neurovascular link with neurodegenerative disorders is poorly understood, in terms of the adhesion molecules that keep the physical association of various cell types together, the molecular crosstalk between different cell types (including endothelial cells, pericytes and astrocytes) and how these cellular interactions influence neuronal activity. Addressing these issues promises to create new opportunities not only to better understand the molecular basis of the neurovascular link with neurodegeneration but also to develop novel neurovascular-based medicines.

The construction of a human BBB molecular atlas will be an important step towards understanding the role of the BBB and neurovascular interactions in health and disease. Achievement of this goal will require identifying new BBB transporters by using genomic and proteomic tools, and by cloning some of the transporters that are already known. Better knowledge of transporters at the human BBB will help us to better understand their potential as therapeutic targets for disease.

Development of higher-resolution imaging methods to evaluate BBB integrity, key transporters’ functions and CBF responses in the microregions of interest (for example, in the entorhinal region of the hippocampus) will help us to understand how BBB dysfunction correlates with cognitive outcomes and neurodegenerative processes in MCI, Alzheimer’s disease and related disorders.

The question persists: are we missing important therapeutic targets by studying the nervous system in isolation from the influence of the vascular system? The probable answer is yes. However, the current exciting and novel research that is based on the neurovascular model has already begun to change the way that we think about neurodegeneration, and will continue to provide further insights in the future, leading to the development of new neurovascular therapies.


  1. Zlokovic, B. V. The blood–brain barrier in health and chronic neurodegenerative disorders. Neuron 57, 178–201 (2008).

  2. Moskowitz, M. A., Lo, E. H. & Iadecola, C. The science of stroke: mechanisms in search of treatments. Neuron 67, 181–198 (2010).
    A comprehensive review describing mechanisms of ischaemic injury to the neurovascular unit.

  3. Zlokovic, B. V. Neurovascular mechanisms of Alzheimer’s neurodegeneration. Trends Neurosci. 28, 202–208 (2005).

  4. Brown, W. R. & Thore, C. R. Review: cerebral microvascular pathology in ageing and neurodegeneration. Neuropathol. Appl. Neurobiol. 37, 56–74 (2011).

  5. Wu, Z. et al. Role of the MEOX2 homeobox gene in neurovascular dysfunction in Alzheimer disease. Nature Med. 11, 959–965 (2005).
    A study demonstrating that low expression of MEOX2 in brain endothelium leads to aberrant angiogenesis and vascular regression in Alzheimer’s disease.

  6. Paul, J., Strickland, S. & Melchor, J. P. Fibrin deposition accelerates neurovascular damage and neuroinflammation in mouse models of Alzheimer’s disease. J. Exp. Med. 204, 1999–2008 (2007).
    A study showing BBB breakdown in models of Alzheimer’s disease.

  7. Zipser, B. D. et al. Microvascular injury and blood–brain barrier leakage in Alzheimer’s disease. Neurobiol. Aging 28, 977–986 (2007).

  8. Zhong, Z. et al. ALS-causing SOD1 mutants generate vascular changes prior to motor neuron degeneration. Nature Neurosci. 11, 420–422 (2008).
    A study demonstrating that BSCB defects precede motor neuron degeneration in mice that develop an ALS-like disease.

  9. Kalaria, R. N. Vascular basis for brain degeneration: faltering controls and risk factors for dementia. Nutr. Rev. 68, S74–S87 (2010).

  10. Knopman, D. S. & Roberts, R. Vascular risk factors: imaging and neuropathologic correlates. J. Alzheimers Dis. 20, 699–709 (2010).

  11. Miyazaki, K. et al. Disruption of neurovascular unit prior to motor neuron degeneration in amyotrophic lateral sclerosis. J. Neurosci. Res. 89, 718–728 (2011).

  12. Neuwelt, E. A. et al. Engaging neuroscience to advance translational research in brain barrier biology. Nature Rev. Neurosci. 12, 169–182 (2011).

  13. Guo, S. & Lo, E. H. Dysfunctional cell–cell signaling in the neurovascular unit as a paradigm for central nervous system disease.Stroke 40, S4–S7 (2009).

  14. Redzic, Z. Molecular biology of the blood–brain and the blood–cerebrospinal fluid barriers: similarities and differences. Fluids Barriers CNS 8, 3 (2011).

  15. O’Kane, R. L., Martinez-Lopez, I., DeJoseph, M. R., Vina, J. R. & Hawkins, R. A. Na+-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood–brain barrier. A mechanism for glutamate removal. J. Biol. Chem. 274, 31891–31895 (1999).

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Author affiliations

  1. Department of Physiology and Biophysics, and Center for Neurodegeneration and Regeneration at the Zilkha Neurogenetic Institute, University of Southern California, Keck School of Medicine, 1501 San Pablo Street, Los Angeles, California 90089, USA.
    Email: bzlokovi@usc.edu


Retromer in Alzheimer disease, Parkinson disease and other neurological disorders.

Scott A. Small and Gregory A. Petsko

Nature Reviews Neuroscience  2015; 16:126-132.   http://dx.doi.org:/10.1038/nrn3896


Retromer is a protein assembly that has a central role in endosomal trafficking, and retromer dysfunction has been linked to a growing number of neurological disorders. First linked to Alzheimer disease, retromer dysfunction causes a range of pathophysiological consequences that have been shown to contribute to the core pathological features of the disease. Genetic studies have established that retromer dysfunction is also pathogenically linked to Parkinson disease, although the biological mechanisms that mediate this link are only now being elucidated. Most recently, studies have shown that retromer is a tractable target in drug discovery for these and other disorders of the nervous system.

Yeast has proved to be an informative model organism in cell biology and has provided early insight into much of the molecular machinery that mediates the intracellular transport of proteins1,2. Indeed, the term ‘retromer’ was first introduced in a yeast study in 1998 (Ref. 3). In this study, retromer was referred to as a complex of proteins that was dedicated to transporting cargo in a retrograde direction, from the yeast endosome back to the Golgi.

By 2004, a handful of studies had identified the molecular4 and the functional5, 6 homologies of the mammalian retromer, and in 2005 retromer was linked to its first human disorder, Alzheimer disease (AD)7. At the time, the available evidence suggested that the mammalian retromer might match the simplicity of its yeast homologue. Since then, a dramatic and exponential rise in research focusing on retromer has led to more than 300 publications. These studies have revealed the complexity of the mammalian retromer and its functional diversity in endosomal transport, and have implicated retromer in a growing number of neurological disorders.

New evidence indicates that retromer is a ‘master conductor’ of endosomal sorting and trafficking8. Synaptic function heavily depends on endosomal trafficking, as it contributes to the presynaptic release of neurotransmitters and regulates receptor density in the postsynaptic membrane, a process that is crucial for neuronal plasticity9. Therefore, it is not surprising that a growing number of studies are showing that retromer has an important role in synaptic biology10, 11, 12, 13. These observations may account for why the nervous system seems particularly sensitive to genetic and other defects in retromer. In this Progress article, we briefly review the molecular organization and the functional role of retromer, before discussing studies that have linked retromer dysfunction to several neurological diseases — notably, AD and Parkinson disease (PD).

Function and organization

The endosome is considered a hub for intracellular transport. From the endosome, transmembrane proteins can be actively sorted and trafficked to various intracellular sites via distinct transport routes (Fig. 1a). Studies have shown that the mammalian retromer mediates two of the three transport routes out of endosomes. First, retromer is involved in the retrieval of cargos from endosomes and in their delivery, in a retrograde direction, to the trans-Golgi network (TGN)5,6. Retrograde transport has many cellular functions but, as we describe, it is particularly important for the normal delivery of hydrolases and proteases to the endosomal–lysosomal system. The second transport route in which retromer functions is the recycling of cargos from endosomes back to the cell surface14, 15 (Fig. 1a). It is this transport route that is particularly important for neurons, as it mediates the normal delivery of glutamate and other receptors to the plasma membrane during synaptic remodelling and plasticity10, 11, 12, 13.

Figure 1: Retromer’s endosomal transport function and molecular organization.
Retromer's endosomal transport function and molecular organization.

a | Retromer mediates two transport routes out of endosomes via tubules that extend out of endosomal membranes. The first is the retrograde pathway in which cargo is retrieved from the endosome and trafficked to the trans-Golgi network (TGN). The second is the recycling pathway in which cargo is trafficked back from the endosome to the cell surface. The degradation pathway, which is not mediated by retromer, involves the trafficking of cargo from endosomes to lysosomes for degradation. b | The retromer assembly of proteins can be organized into distinct functional modules, all of which work together as part of retromer’s transport role. The ‘cargo-recognition core’ is the central module of the retromer assembly and comprises a trimer of proteins, in which vacuolar protein sorting-associated protein 26 (VPS26) and VPS29 bind VPS35. The ‘tubulation’ module includes protein complexes that bind the cargo-recognition core and aid in the formation and stabilization of tubules that extend out of endosomes, directing the transport of cargos towards their final destinations. The ‘membrane-recruiting’ proteins recruit the cargo-recognition core to the endosomal membrane. The WAS protein family homologue (WASH) complex of proteins also binds the cargo-recognition core and is involved in endosomal ‘actin remodelling’ to form actin patches, which are important for directing cargos towards retromer’s transport pathways. Retromer cargos includes a range of receptors — which bind the cargo-recognition core — and their ligands. PtdIns3P, phosphatidylinositol-3-phosphate.

As well as extending the endosomal transport routes, recent studies have considerably expanded the number of molecular constituents and what is known about the functional organization of the mammalian retromer. Following this expansion in knowledge of the molecular diversity and organizational complexity, retromer might be best described as a multimodular protein assembly. The protein or group of proteins that make up each module can vary, but each module is defined by its distinct function, and the modules work in unison in support of retromer’s transport role.

Two modules are considered central to the retromer assembly. First and foremost is a trimeric complex that functions as a ‘cargo-recognition core’, which selects and binds to the transmembrane proteins that need to be transported and that reside in endosomal membranes5, 6. This trimeric core comprises vacuolar protein sorting-associated protein 26 (VPS26), VPS29 and VPS35; VPS35 functions as the core’s backbone to which the other two proteins bind16. VPS26 is the only member of the core that has been found to have two paralogues, VPS26a and VPS26b17,18, and studies suggest that VPS26b might be differentially expressed in the brain19, 20. Some studies suggest that VPS26a and VPS26b are functionally redundant21, whereas others suggest that they might form distinct cargo-recognition cores20, 22.

The second central module of the retromer assembly is the ‘tubulation’ module, which is made up of proteins that work together in the formation and the stabilization of tubules that extend out of endosomes and that direct the transport of cargo towards its final destination (Fig. 1b). The proteins in this module, which directly binds the cargo-recognition core, are members of the subgroup of the sorting nexin (SNX) family that are characterized by the inclusion of a carboxy-terminal BIN–amphiphysin–RVS (BAR) domain23. These members include SNX1, SNX2, SNX5 and SNX6 (Refs 24,25). As part of the tubulation module, these SNX-BAR proteins exist in different dimeric combinations, but typically SNX1 interacts with SNX5 or SNX6, and SNX2 interacts with SNX5 or SNX6 (Refs 26,27). The EPS15-homology domain 1 (EHD1) protein can be included in this module, as it is involved in stabilizing the tubules formed by the SNX-BAR proteins28.

A third module of the retromer assembly functions to recruit the cargo-recognition core to endosomal membranes and to stabilize the core once it is there (Fig. 1b). Proteins that are part of this ‘membrane-recruiting’ module include SNX3 (Ref. 29), the RAS-related protein RAB7A30, 31,32 and TBC1 domain family member 5 (TBC1D5), which is a member of the TRE2–BUB2–CDC16 (TBC) family of RAB GTPase-activating proteins (GAPs)28. In addition, the lipid phosphatidylinositol-3-phosphate (PtdIns3P), which is found on endosomal membranes, contributes to recruiting most of the retromer-related SNXs through their phox homology domains33. Interestingly, another SNX with a phox homology domain, SNX27, was recently linked to retromer and its function15, 34. SNX27 functions as an adaptor for binding to PDZ ligand-containing cargos that are destined for transport to the cell surface via the recycling pathway. Thus, according to the functional organization of the retromer assembly, SNX27 belongs to the module that engages in cargo recognition and selection.

Recent studies have identified a fourth module of the retromer assembly. The five proteins in this module — WAS protein family homologue 1 (WASH1), FAM21, strumpellin, coiled-coil domain-containing protein 53 (CCDC53) and KIAA1033 (also known as WASH complex subunit 7) — form the WASH complex and function as an ‘actin-remodelling’ module28, 35, 36 (Fig. 1b). Specifically, the WASH complex functions in the rapid polymerization of actin to create patches of actin filaments on endosomal membranes. The complex is recruited to endosomal membranes by binding VPS35 (Ref. 28), and together they divert cargo towards retromer transport pathways and away from the degradation pathway.

The cargos that are transported by retromer include the receptors that directly bind the cargo-recognition core and the ligands of these receptors that are co-transported with the receptors. The receptors that are transported by retromer that have so far been identified to be the most relevant to neurological diseases are the family of VPS10 domain-containing receptors (including sortilin-related receptor 1 (SORL1; also known as SORLA), sortilin, and SORCS1, SORCS2 and SORCS3)7; the cation-independent mannose-6-phosphate receptor (CIM6PR)6, 5; glutamate receptors10; and phagocytic receptors that mediate the clearing function of microglia37. The most disease-relevant ligand to be identified that is trafficked as retromer cargo is the β-amyloid precursor protein (APP)7, 38, 39, 40, 41, which binds SORL1 and perhaps other VPS10 domain-containing receptors42 at the endosomal membrane.

Retromer dysfunction

Guided by retromer’s established function, and on the basis of empirical evidence, there are three well-defined pathophysiological consequences of retromer dysfunction that have proven to be relevant to AD and nervous system disorders. First, retromer dysfunction can cause cargos that typically transit rapidly through the endosome to reside in the endosome for longer than normal durations, such that they can be pathogenically processed into neurotoxic fragments (for example, APP, when stalled in the endosome, is more likely to be processed into amyloid-β, which is implicated in AD43 (Fig. 2a)). Second, by reducing endosomal outflow via impairment of the recycling pathway, retromer dysfunction can lead to a reduction in the number of cell surface receptors that are important for brain health (for example, microglia phagocytic receptors37 (Fig. 2b)).

Figure 2: The pathophysiology of retromer dysfunction.
The pathophysiology of retromer dysfunction.

Retromer dysfunction has three established pathophysiological consequences. In the examples shown, the left graphic represents a cell with normal retromer function and the right graphic represents a cell with a deficit in retromer function. a | Retromer dysfunction causes increased levels of cargo to reside in endosomes. For example, in primary neurons, retromer transports the β-amyloid precursor protein (APP) out of endosomes. Accordingly, retromer dysfunction increases APP levels in endosomes, leading to accelerated APP processing, resulting in an accumulation of neurotoxic fragments of APP (namely, β-carboxy-terminal fragment (βCTF) and amyloid-β) that are pathogenic in Alzheimer disease. b | Retromer dysfunction causes decreased cargo levels at the cell surface. For example, in microglia, retromer mediates the transport of phagocytic receptors to the cell surface and retromer dysfunction results in a decrease in the delivery of these receptors. Studies suggest that this cellular phenotype might have a pathogenic role in Alzheimer disease. c | Retromer dysfunction causes decreased delivery of proteases to the endosome. Retromer is required for the normal retrograde transport of the cation-independent mannose-6-phosphate receptor (CIM6PR) from the endosome back to the trans-Golgi network (TGN). It is in the TGN that this receptor binds cathepsin D and other proteases, and transports them to the endosome, to support the normal function of the endosomal–lysosomal system. By impairing the retrograde transport of the receptor, retromer dysfunction ultimately leads to reduced delivery of cathepsin D to this system. Cathepsin D deficiency has been shown to disrupt the endosomal–lysosomal system and to trigger tau pathology either within endosomes or secondarily in the cytosol.

The third consequence (Fig. 2c) is a result of the established role that retromer has in the retrograde transport of receptors, such as CIM6PR5, 6 or sortilin44, after these receptors transport proteases from the TGN to the endosome. Once at the endosome, the proteases disengage from the receptors, are released into endosomes and migrate to lysosomes. These proteases function in the endosomal–lysosomal system to degrade proteins, protein oligomers and aggregates45. Retromer functions to transfer the ‘naked’ receptor from the endosome back to the TGN via the retrograde pathway5, 6, allowing the receptors to continue in additional rounds of protease delivery. Accordingly, by reducing the normal retrograde transport of these receptors, retromer dysfunction has been shown to reduce the proper delivery of proteases to the endosomal–lysosomal system5,6, which, as discussed below, is a pathophysiological state linked to several brain disorders.

Although requiring further validation, recent studies suggest that retromer dysfunction might be involved in two other mechanisms that have a role in neurological disease. One study suggested that retromer might be involved in trafficking the transmembrane protein autophagy-related protein 9A (ATG9A) to recycling endosomes, from where it can then be trafficked to autophagosome precursors — a trafficking step that is crucial in the formation and the function of autophagosomes46. Autophagy is an important mechanism by which neurons clear neurotoxic aggregates that accumulate in numerous neurodegenerative diseases47. A second study has suggested that retromer dysfunction might enhance the seeding and the cell-to-cell spread of intracellular neurotoxic aggregates48, which have emerged as novel pathophysiological mechanisms that are relevant to AD49, PD50 and other neurodegenerative diseases.

Alzheimer disease

Retromer was first implicated in AD in a molecular profiling study that relied on functional imaging observations in patients and animal models to guide its molecular analysis7. Collectively, neuroimaging studies confirmed that the entorhinal cortex is the region of the hippocampal circuit that is affected first in AD, even in preclinical stages, and suggested that this effect was independent of ageing (as reviewed in Ref. 51). At the same time, neuroimaging studies identified a neighbouring hippocampal region, the dentate gyrus, that is relatively unaffected in AD52. Guided by this information, a study was carried out in which the two regions of the brain were harvested post mortem from patients with AD and from healthy individuals, intentionally covering a broad range of ages. A statistical analysis was applied to the determined molecular profiles of the regions that was designed to address the following question: among the thousands of profiled molecules, which are the ones that are differentially affected in the entorhinal cortex versus the dentate gyrus, in patients versus controls, but that are not affected by age? The final results led to the determination that the brains of patients with AD are deficient in two core retromer proteins — VPS26 and VPS35 (Ref. 7).

Little was known about the receptors of the neuronal retromer, so to understand how retromer deficiency might be mechanistically linked to AD, an analysis was carried out on the molecular data set that looked for transmembrane molecules for which expression levels correlated with VPS35 expression. The top ‘hit’ was the transcript encoding the transmembrane protein SORL1 (Ref. 43). As SORL1 belongs to the family of VPS10-containing receptors and as VPS10 is the main retromer receptor in yeast3, it was postulated that SORL1 and the family of other VPS10-containing proteins (sortillin, SORCS1, SORCS2 and SORCS3) might function as retromer receptors in neurons7. In addition, SORL1 had recently been reported to bind APP53, so if SORL1 was assumed to be a receptor that is trafficked by retromer, then APP might be the cargo that is co-trafficked by retromer. This led to a model in which retromer traffics APP out of endosomes7, which are the organelles in which APP is most likely to be cleaved by βAPP-cleaving enzyme 1 (BACE1; also known as β-secretase 1)43; this is the initial enzymatic step in the pathogenic processing of APP.

Subsequent studies were required to further establish the pathogenic link between retromer and AD, and to test the proposed model. The pathogenic link was further supported by human genetic studies. First, a genetic study investigating the association between AD, the genes encoding the components of the retromer cargo-recognition core and the family of VPS10-containing receptors found that variants of SORL1 increase the risk of developing AD38. This finding was confirmed by numerous studies, including a recent large-scale AD genome-wide association study54. Other genetic studies identified AD-associated variants in genes encoding proteins that are linked to nearly all modules of the retromer assembly55, including genes encoding proteins of the retromer tubulation module (SNX1), genes encoding proteins of the retromer membrane-recruiting module (SNX3 and RAB7A) and genes encoding proteins of the retromer actin-remodelling module (KIAA1033). In addition, nearly all of the genes encoding the family of VPS10-containing retromer receptors have been found to have variants that associate with AD56. Finally, a study found that brain regions that are differentially affected in AD are deficient in PtdIns3P, which is the phospholipid required for recruiting many sorting nexins to endosomal membranes57. Thus, together with the observation that the brains of patients with AD are deficient in VPS26a and VPS35 (Refs 7,37), all modules in the retromer assembly are implicated in AD.

Studies in mice39, 58, 59, flies39 and cells in culture34, 40, 41, 60, 61 have investigated how retromer dysfunction leads to the pathogenic processing of APP. Although rare discrepancies have been observed among these studies62, when viewed in total, the most consistent findings are that retromer dysfunction causes increased pathogenic processing of APP by increasing the time that APP resides in endosomes. Moreover, these studies have confirmed that SORL1 and other VPS10-containing proteins function as APP receptors that mediate APP trafficking out of endosomes.

Retromer has unexpectedly been linked to microglial abnormalities37 — another core feature of AD — which, on the basis of recent genetic findings, seem to have an upstream role in disease pathogenesis54, 63. A recent study found that microglia harvested from the brains of individuals with AD are deficient in VPS35 and provided evidence suggesting that retromer’s recycling pathway regulates the normal delivery of various phagocytic receptors to the cell surface of microglia37, including the phagocytic receptor triggering receptor expressed on myeloid cells 2 (TREM2) (Fig. 2b). Mutations in TREM2 have been linked to AD63, and a recent study indicates that these mutations cause a reduction in its cell surface delivery and accelerate TREM2 degradation, which suggests that the mutations are linked to a recycling defect64. While they are located at the microglial cell surface, these phagocytic receptors function in the clearance of extracellular proteins and other molecules from the extracellular space65. Taken together, these recent studies suggest that defects in the retromer’s recycling pathway can, at least in part, account for the microglial defects observed in the disease.

The microtubule-associated protein tau is the key element of neurofibrillary tangles, which are the other hallmark histological features of AD. Although a firm link between retromer dysfunction and tau toxicity remains to be established, recent insight into tau biology suggests several plausible mechanisms that are worth considering. Tau is a cytosolic protein, but nonetheless, through mechanisms that are still undetermined, it is released into the extracellular space from where it gains access to neuronal endosomes via endocytosis66, 67. In fact, recent studies suggest that the pathogenic processing of tau is triggered after it is endocytosed into neurons and while it resides in endosomes67. Of note, it still remains unknown which specific tau processing step — its phosphorylation, cleavage or aggregation — is an obligate step towards tau-related neurotoxicity. Accordingly, if defects in microglia or in other phagocytic cells reduce their capacity to clear extracellular tau, this would accelerate tau endocytosis in neurons and its pathogenic processing.

A second possibility comes from the established role retromer has in the proper delivery of cathepsin D and other proteases to the endosomal–lysosomal system via CIM6PR or sortilin (Fig. 2c). Studies in sheep, mice and flies68 have shown that cathepsin D deficiency can enhance tau toxicity and that this is mediated by a defective endosomal–lysosomal system68. Whether this mechanism leads to abnormal processing of tau within endosomes or in the cytosol via caspase activation68 remains unclear. As discussed above, retromer dysfunction will lead to a decrease in the normal delivery of cathepsin D to the endosome and will result in endosomal–lysosomal system defects. Retromer dysfunction can therefore be considered as a functional phenocopy of cathepsin D deficiency, which suggests a plausible link between retromer dysfunction and tau toxicity. Nevertheless, although these recent insights establish plausibility and support further investigation into the link between retromer and tau toxicity, whether this link exists and how it may be mediated remain open and outstanding questions.

Parkinson disease

The pathogenic link between retromer and PD is singular and straightforward: exome sequencing has identified autosomal-dominant mutations in VPS35 that cause late-onset PD69, 70, one of a handful of genetic causes of late-onset disease. However, the precise mechanism by which these mutations cause the disease is less clear.

Among a group of recent studies, all46, 48, 71, 72, 73, 74, 75, 76 but one77 strongly suggest that these mutations cause a loss of retromer function. At the molecular level, the mutations do not seem to disrupt mutant VPS35 from interacting normally with VPS26 and VPS29, and from forming the cargo-recognition core. Rather, two studies suggest that the mutations have a restricted effect on the retromer assembly but reduce the ability of VPS35 to associate with the WASH complex46, 75. Studies disagree about the pathophysiological consequences of the mutations. Four studies suggest that the mutations affect the normal retrograde transport of CIM6PR71, 73, 75, 76 from the endosome back to the TGN (Fig. 2c). In this scenario, the normal delivery of cathepsin D to the endosomal–lysosomal system should be reduced and this has been empirically shown73. Cathepsin D has been shown to be the dominant endosomal–lysosomal protease for the normal processing of α-synuclein76, and mutations could therefore lead to abnormal α-synuclein processing and to the formation of α-synuclein aggregates, which are thought to have a key pathogenic role in PD.

A separate study suggested that the mutation might cause a mistrafficking of ATG9, and thereby, as discussed above, reduce the formation and the function of autophagosomes46. Autophagosomes have also been implicated as an intracellular site in which α-synuclein aggregates are cleared. Thus, although future studies are needed to resolve these discrepant findings (which may in fact not be mutually exclusive), these studies are generally in agreement that retromer defects will probably increase the neurotoxic levels of α-synuclein aggregates48.

Several studies in flies71, 74 and in rat neuronal cultures71 provide strong evidence that increasing retromer function by overexpressing VPS35 rescues the neurotoxic effects of the most common PD-causing mutations in leucine-rich repeat kinase 2 (LRRK2). Moreover, a separate study has shown that increasing retromer levels rescues the neurotoxic effect of α-synuclein aggregates in a mouse model48. These findings have immediate therapeutic implications for drugs that increase VPS35 and retromer function, as discussed in the next section, but they also offer mechanistic insight. LRRK2 mutations were found to phenocopy the transport defects caused either by theVPS35 mutations or by knocking down VPS35 (Ref. 71). Together, this and other studies78suggest that LRRK2 might have a role in retromer-dependent transport, but future studies are required to clarify this role.

Other neurological disorders

Besides AD and PD, in which a convergence of findings has established a strong pathogenic link, retromer is being implicated in an increasing number of other neurological disorders. Below, we briefly review three disorders for which the evidence of the involvement of retromer in their pathophysiology is currently the most compelling.

The first of these disorders is Down syndrome (DS), which is caused by an additional copy of chromosome 21. Given the hundreds of genes that are duplicated in DS, it has been difficult to identify which ones drive the intellectual impairments that characterize this condition. A recent elegant study provides strong evidence that a deficiency in the retromer cargo-selection protein SNX27 might be a primary driver for some of these impairments79. This study found that the brains of individuals with DS were deficient in SNX27 and that this deficiency may be caused by an extra copy of a microRNA (miRNA) encoded by human chromosome 21 (the miRNA is produced at elevated levels and thereby decreases SNX27 expression). Consistent with the known role of SNX27 in retromer function, decreased expression of this protein in mice disrupted glutamate receptor recycling in the hippocampus and led to dendritic dysfunction. Importantly, overexpression of SNX27 rescued cognitive and other defects in animal models79, which not only strengthens the causal link between retromer dysfunction and cognitive impairment in DS but also has important therapeutic implications.

Hereditary spastic paraplegia (HSP) is another disorder linked to retromer. HSP is caused by genetic mutations that affect upper motor neurons and is characterized by progressive lower limb spasticity and weakness. Although there are numerous mutations that cause HSP, most are unified by their effects on intracellular transport80. One HSP-associated gene in particular encodes strumpellin81, which is a member of the WASH complex.

The third disorder linked to retromer is neuronal ceroid lipofuscinosis (NCL). NCL is a young-onset neurodegenerative disorder that is part of a larger family of lysosomal storage diseases and is caused by mutations in one of ten identified genes — nine neuronal ceroid lipofuscinosis (CLN) genes and the gene encoding cathepsin D82. Besides cathepsin D, for which the link to retromer has been discussed above, CLN3 seems to function in the normal trafficking of CIM6PR83. However, the most direct link to retromer has been recently described for CLN5, which seems to function, at least in part, as a retromer membrane-recruiting protein84.

Retromer as a therapeutic target

As suggested by the first study implicating retromer in AD7, and in several subsequent studies71,85, increasing the levels of retromer’s cargo-recognition core enhances retromer’s transport function. Motivated by this observation and after a decade-long search86, we identified a novel class of ‘retromer pharmacological chaperones’ that can bind and stabilize retromer’s cargo-recognition core and increase retromer levels in neurons61.

Validating the motivating hypothesis, the chaperones were found to enhance retromer function, as shown by the increased transport of APP out of endosomes and a reduction in the accumulation of APP-derived neurotoxic fragments61. Although there are numerous other pharmacological approaches for enhancing retromer function, this success provides the proof-of-principle that retromer is a tractable therapeutic target.

As retromer functions in all cells, a general concern is whether enhancing its function will have toxic adverse effects. However, studies have found that in stark contrast to even mild retromer deficiencies, increasing retromer levels has no obvious negative consequences in yeast, neuronal cultures, flies or mice40, 48, 61, 71. This might make sense because unlike drugs that, for example, function as inhibitors, simply increasing the normal flow of transport through the endosome might not be cytotoxic.

If retromer drugs are safe and can effectively enhance retromer function in the nervous system — which are still outstanding issues — there are two general indications for considering their clinical application. One rests on the idea that these agents will only be efficacious in patients who have predetermined evidence of retromer dysfunction. The most immediate example is that of individuals with PD that is caused by LRRK2 mutations. As discussed above, several ‘preclinical’ studies in flies and neuronal cultures have already established that increasing retromer levels71, 74can reverse the neurotoxic effects of such mutations and, thus, if this approach is proven to be safe, LRRK2-linked PD might be an appropriate indication for clinical trials.

Alternatively, the pathophysiology of a disease might be such that retromer-enhancing drugs would be efficacious regardless of whether there is documented evidence of retromer dysfunction. AD illustrates this point. As reviewed above, current evidence suggests that retromer-enhancing drugs will, at the very least, decrease pathogenic processing of APP in neurons and enhance microglial function, even if there are no pre-existing defects in retromer.

More generally, histological studies comparing the entorhinal cortex of patients with sporadic AD to age-matched controls have documented that enlarged endosomes are a defining cellular abnormality in AD87, 88. Importantly, enlarged endosomes are uniformly observed in a broad range of patients with sporadic AD, which suggests that enlarged endosomes reflect an intracellular site at which molecular aetiologies converge87. In addition, because they are observed in early stages of the disease in regions of the brain without evidence of amyloid pathology87, enlarged endosomes are thought to be an upstream event. Mechanistically, the most likely cause of enlarged endosomes is either too much cargo flowing into endosomes — as occurs, for example, with apolipoprotein E4 (APOE4), which has been shown to accelerate endocytosis89, 90 — or too little cargo flowing out, as observed in retromer dysfunction40, 61 and related transport defects57. By any mechanism, retromer-enhancing drugs might correct this unifying cellular defect and might be expected to be beneficial regardless of the specific aetiology.


The fact that retromer defects, including those derived from bona fide genetic mutations, seem to differentially target the nervous system suggests that the nervous system is differentially dependent on retromer for its normal function. We think that this reflects the unique cellular properties of neurons and how synaptic biology heavily depends on endosomal transport and trafficking. Although plausible, future studies are required to confirm and to test the details of this hypothesis.

However, currently, it is the clinical rather than the basic neuroscience of retromer that is much better understood, with the established pathophysiological consequences of retromer dysfunction providing a mechanistic link to the disorders in which retromer has been implicated. Nevertheless, many questions remain. The two most interesting questions, which are in fact inversions of each other, relate to regional vulnerability in the nervous system. First, why does retromer dysfunction target specific neuronal populations? Second, how can retromer dysfunction cause diseases that target different regions of the nervous system? Recent evidence hints at answers to both questions, which must somehow be rooted in the functional and molecular diversity of retromer.

The type and the extent of retromer defects linked to different disorders might provide pathophysiological clues as well as reasons for differential vulnerability. As discussed, in AD there seem to be across-the-board defects in retromer, such that each module of the retromer assembly as well as multiple retromer cargos have been pathogenically implicated. By contrast, the profile of retromer defects in PD seems to be more circumscribed, involving selective disruption of the interaction between VPS35 and the WASH complex. These insights might agree with histological87, 88 and large-scale genetic studies54 that suggest that endosomal dysfunction is a unifying focal point in the cellular pathogenesis of AD. In contrast, genetics and other studies91suggest that the cellular pathobiology of PD is more distributed, implicating the endosome but other organelles as well, in particular the mitochondria.

Interestingly, studies suggest that the entorhinal cortex — a region that is differentially vulnerable to AD — has unique dendritic structure and function92, which are highly dependent on endosomal transport. We speculate that it is the unique synaptic biology of the entorhinal cortex that can account for why it might be particularly sensitive to defects in endosomal transport in general and retromer dysfunction in particular, and for why this region is the early site of disease. Future studies are required to investigate this hypothesis, as well as to understand why the substantia nigra or other regions that are differentially vulnerable to PD would be particularly sensitive to the more circumscribed defect in retromer.

Perhaps the most important observation for clinical neuroscience is the now well-established fact that increasing levels of retromer proteins enhances retromer function and has already proved capable of reversing defects associated with AD, PD and DS in either cell culture or in animal models. The relationships between protein levels and function are not always simple, but emerging pharmaceutical technologies that selectively and safely increase protein levels are now a tractable goal in drug discovery93. With the evidence mounting that retromer has a pathogenic role in two of the most common neurodegenerative diseases, we think that targeting retromer to increase its functional activity is an important goal that has strong therapeutic promise.


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……. 93


Taub Institute for Research on Alzheimer’s Disease and the Ageing Brain, Departments of Neurology, Radiology, and Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.

Scott A. Small

Helen and Robert Appel Alzheimer’s Disease Research Institute, Department of Neurology and Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, New York 10065, USA.

Gregory A. Petsko


See also:

Neurobiol Aging. 2011 Nov;32(11):2109.e1-14. doi: 10.1016/j.neurobiolaging.2011.05.025.
Altered intrinsic neuronal excitability and reduced Na+ currents in a mouse model of Alzheimer’s disease.
Brown JT, Chin J, Leiser SC, Pangalos MN, Randall AD.

Trends Neurosci. 2013 Jun;36(6):325-35. doi: 10.1016/j.tins.2013.03.002.
Why size matters – balancing mitochondrial dynamics in Alzheimer’s disease.
DuBoff B, Feany M, Götz J.

Neuron. 2014 Dec 3;84(5):1023-33. doi: 10.1016/j.neuron.2014.10.024.
Dendritic structural degeneration is functionally linked to cellular hyperexcitability in a mouse model of Alzheimer’s disease.
Šišková Z, Justus D, Kaneko H, Friedrichs D, Henneberg N, Beutel T, Pitsch J, Schoch S, Becker A, von der Kammer H, Remy S.



Video: How can we tease out the role of other toxic insults in AD pathogenesis?




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Kurzweill Reports in Medical Science I

Curator: Larry H. Bernstein, MD, FCAP




E-coli bacteria found in some China farms and patients cannot be killed with antiobiotic drug of last resort

“One of the most serious global threats to human health in the 21st century” — could spread around the world, requiring “urgent coordinated global action”
November 20, 2015


Colistin antibiotic overused in farm animals in China apparently caused E-coli bacteria to become completely resistant to treatment; E-coli strain has already spread to Laos and Malaysia (credit: Yi-Yun Liu et al./Lancet Infect Dis)

Widespread E-coli bacteria that cannot be killed with the antiobiotic drug of last resort — colistin — have been found in samples taken from farm pigs, meat products, and a small number of patients in south China, including bacterial strains with epidemic potential, an international team of scientists revealed in a paper published Thursday Nov. 19 in the journal The Lancet Infectious Diseases.

The scientists in China, England, and the U.S. found a new gene, MCR-1, carried in E-coli bacteria strain SHP45. MCR-1 enables bacteria to be highly resistant to colistin and other polymyxins drugs.

“The emergence of the MCR-1 gene in China heralds a disturbing breach of the last group of antibiotics — polymixins — and an end to our last line of defense against infection,” said Professor Timothy Walsh, from the Cardiff University School of Medicine, who collaborated on this research with scientists from South China Agricultural University.

Walsh, an expert in antibiotic resistance, is best known for his discovery in 2011 of the NDM-1 disease-causing antibiotic-resistant superbug in New Delhi’s drinking water supply. “The rapid spread of similar antibiotic-resistant genes such as NDM-1 suggests that all antibiotics will soon be futile in the face of previously treatable gram-negative bacterial infections such as E.coli and salmonella,” he said.

Likely to spread worldwide; already found in Laos and Malaysia

The MCR-1 gene was found on plasmids — mobile DNA that can be easily copied and transferred between different bacteria, suggesting an alarming potential to spread and diversify between different bacterial populations.

Structure of plasmid pHNSHP45 carrying MCR-1 from Escherichia coli strain SHP45 (credit: Yi-Yun Liu et al./Lancet Infect Dis)

“We now have evidence to suggest that MCR-1-positive E.coli has spread beyond China, to Laos and Malaysia, which is deeply concerning,” said Walsh.  “The potential for MCR-1 to become a global issue will depend on the continued use of polymixin antibiotics, such as colistin, on animals, both in and outside China; the ability of MCR-1 to spread through human strains of E.coli; and the movement of people across China’s borders.”

“MCR-1 is likely to spread to the rest of the world at an alarming rate unless we take a globally coordinated approach to combat it. In the absence of new antibiotics against resistant gram-negative pathogens, the effect on human health posed by this new gene cannot be underestimated.”

“Of the top ten largest producers of colistin for veterinary use, one is Indian, one is Danish, and eight are Chinese,” The Lancet Infectious Diseases notes. “Asia (including China) makes up 73·1% of colistin production with 28·7% for export including to Europe.29 In 2015, the European Union and North America imported 480 tonnes and 700 tonnes, respectively, of colistin from China.”

Urgent need for coordinated global action

“Our findings highlight the urgent need for coordinated global action in the fight against extensively resistant and pan-resistant gram-negative bacteria,” the journal paper concludes.

“The implications of this finding are enormous,” an associated editorial comment to the The Lancet Infectious Diseases paper stated. “We must all reiterate these appeals and take them to the highest levels of government or face increasing numbers of patients for whom we will need to say, ‘Sorry, there is nothing I can do to cure your infection.’”

Margaret Chan, MD, Director-General of the World Health Organization, warned in 2011 that “the world is heading towards a post-antibiotic era, in which many common infections will no longer have a cure and, once again, kill unabated.”

“Although in its 2012 World Health Organization Advisory Group on Integrated Surveillance of Antimicrobial Resistance (AGISAR) report the WHO concluded that colistin should be listed under those antibiotics of critical importance, it is regrettable that in the 2014 Global Report on Surveillance, the WHO did not to list any colistin-resistant bacteria as part of their ‘selected bacteria of international concern,’” The Lancet Infectious Diseases paper says, reflecting WHO’s inaction in Ebola-stricken African countries, as noted last September by the international medical humanitarian organization Médecins Sans Frontières.

Funding for the E-coli bacteria study was provided by the Ministry of Science and Technology of China and National Natural Science Foundation of China.

Abstract of Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study

Until now, polymyxin resistance has involved chromosomal mutations but has never been reported via
horizontal gene transfer. During a routine surveillance project on antimicrobial resistance in commensal Escherichia coli from food animals in China, a major increase of colistin resistance was observed. When an E coli strain, SHP45, possessing colistin resistance that could be transferred to another strain, was isolated from a pig, we conducted further analysis of possible plasmid-mediated polymyxin resistance. Herein, we report the emergence of the first plasmid-mediated polymyxin resistance mechanism, MCR-1, in Enterobacteriaceae.

The mcr-1 gene in E coli strain SHP45 was identified by whole plasmid sequencing and subcloning. MCR-1 mechanistic studies were done with sequence comparisons, homology modelling, and electrospray ionisation mass spectrometry. The prevalence of mcr-1 was investigated in E coli and Klebsiella pneumoniae strains collected from five provinces between April, 2011, and November, 2014. The ability of MCR-1 to confer polymyxin resistance in vivo was examined in a murine thigh model.

Polymyxin resistance was shown to be singularly due to the plasmid-mediated mcr-1 gene. The plasmid carrying mcr-1 was mobilised to an E coli recipient at a frequency of 10−1 to 10−3 cells per recipient cell by conjugation, and maintained in K pneumoniae and Pseudomonas aeruginosa. In an in-vivo model, production of MCR-1 negated the efficacy of colistin. MCR-1 is a member of the phosphoethanolamine transferase enzyme family, with expression in E coli resulting in the addition of phosphoethanolamine to lipid A. We observed mcr-1 carriage in E coli isolates collected from 78 (15%) of 523 samples of raw meat and 166 (21%) of 804 animals during 2011–14, and 16 (1%) of 1322 samples from inpatients with infection.

The emergence of MCR-1 heralds the breach of the last group of antibiotics, polymyxins, by plasmid-mediated resistance. Although currently confined to China, MCR-1 is likely to emulate other global resistance mechanisms such as NDM-1. Our findings emphasise the urgent need for coordinated global action in the fight against pan-drug-resistant Gram-negative bacteria.


Researchers discover signaling molecule that helps neurons find their way in the developing brain

November 20, 2015


This image shows a section of the spinal cord of a mouse embryo. Neurons appear green. Commissural axons (which connect the two sides of the brain) appear as long, u-shaped threads, and the bottom, yellow segment of the structure represents the midline (between brain hemispheres). (credit: Laboratory of Brain Development and Repair/ The Rockefeller University)

Rockefeller University researchers have discovered a molecule secreted by cells in the spinal cord that helps guide axons (neuron extensions) during a critical stage of central nervous system development in the embryo. The finding helps solve the mystery: how do the billions of neurons in the embryo nimbly reposition themselves within the brain and spinal cord, and connect branches to form neural circuits?

Working in mice, the researchers identified an axon guidance factor, NELL2, and explained how it makes commissural axons (which connect the two sides of the brain).

The findings could help scientists understand what goes wrong in a rare disease called horizontal gaze palsy with progressive scoliosis. People affected by the condition often suffer from abnormal spine curvature, and are unable to move their eyes horizontally from side to side. The study was published Thursday Nov. 19 in the journal Science.

Abstract of Operational redundancy in axon guidance through the multifunctional receptor Robo3 and its ligand NELL2

Axon pathfinding is orchestrated by numerous guidance cues, including Slits and their Robo receptors, but it remains unclear how information from multiple cues is integrated or filtered. Robo3, a Robo family member, allows commissural axons to reach and cross the spinal cord midline by antagonizing Robo1/2–mediated repulsion from midline-expressed Slits and potentiating deleted in colorectal cancer (DCC)–mediated midline attraction to Netrin-1, but without binding either Slits or Netrins. We identified a secreted Robo3 ligand, neural epidermal growth factor-like-like 2 (NELL2), which repels mouse commissural axons through Robo3 and helps steer them to the midline. These findings identify NELL2 as an axon guidance cue and establish Robo3 as a multifunctional regulator of pathfinding that simultaneously mediates NELL2 repulsion, inhibits Slit repulsion, and facilitates Netrin attraction to achieve a common guidance purpose.

A sensory illusion that makes yeast cells self-destruct

A possible tactic for cancer therapeutics
November 20, 2015



Effects of osmotic changes on yeast cell growth. (A) Schematic of the flow chamber used to create osmotic level oscillations for different periods of time. (B) Cell growth for these periods. The graphs show the average number of progeny cells (blue) before and after applying stress for different periods (gray shows orginal “no stress” line). The inset shows representative images of cells for two periods. (credit: Amir Mitchell et al./Science)

UC San Francisco researchers have discovered that even brainless single-celled yeast have “sensory biases” that can be hacked by a carefully engineered illusion — a finding that could be used to develop new approaches to fighting diseases such as cancer.

In the new study, published online Thursday November 19 in Science Express, Wendell Lim, PhD, the study’s senior author*, and his team discovered that yeast cells falsely perceive a pattern of osmotic levels (by applying potassium chloride) that alternate in eight minute intervals as massive, continuously increasing stress. In response, the microbes over-respond and kill themselves. (In their natural environment, salt stress normally gradually increases.)

The results, Lim says, suggest a whole new way of looking at the perceptual abilities of simple cells and this power of illusion could even be used to develop new approaches to fighting cancer and other diseases.

“Our results may also be relevant for cellular signaling in disease, as mutations affecting cellular signaling are common in cancer, autoimmune disease, and diabetes,” the researchers conclude in the paper. “These mutations may rewire the native network, and thus could modify its activation and adaptation dynamics. Such network rewiring in disease may lead to changes that can be most clearly revealed by simulation with oscillatory inputs or other ‘non-natural’ patterns.

“The changes in network response behaviors could be exploited for diagnosis and functional profiling of disease cells, or potentially taken advantage of as an Achilles’ heel to selectively target cells bearing the diseased network.”

UC San Francisco (UCSF) | Sensory Illusion Causes Cells to Self-Destruct

* Chair of the Department of Cellular and Molecular Pharmacology at UCSF, director of the UCSF Center for Systems and Synthetic Biology, and a Howard Hughes Medical Institute (HHMI) investigator.

** Normally, sensor molecules in a yeast cell detect changes in salt concentration and instruct the cell to respond by producing a protective chemical. The researchers found that the cells were perfectly capable of adapting when they flipped the salt stress on and off every minute or every 32 minutes. But to their surprise, when they tried an eight-minute oscillation of precisely the same salt level the cells quickly stopped growing and began to die off.

Abstract of Oscillatory stress stimulation uncovers an Achilles’ heel of the yeast MAPK signaling network

Cells must interpret environmental information that often changes over time. We systematically monitored growth of yeast cells under various frequencies of oscillating osmotic stress. Growth was severely inhibited at a particular resonance frequency, at which cells show hyperactivated transcriptional stress responses. This behavior represents a sensory misperception—the cells incorrectly interpret oscillations as a staircase of ever-increasing osmolarity. The misperception results from the capacity of the osmolarity-sensing kinase network to retrigger with sequential osmotic stresses. Although this feature is critical for coping with natural challenges—like continually increasing osmolarity—it results in a tradeoff of fragility to non-natural oscillatory inputs that match the retriggering time. These findings demonstrate the value of non-natural dynamic perturbations in exposing hidden sensitivities of cellular regulatory networks.

Google Glass helps cardiologists complete difficult coronary artery blockage surgery

November 20, 2015



Google Glass allowed the surgeons to clearly visualize the distal coronary vessel and verify the direction of the guide wire advancement relative to the course of the occluded vessel segment. (credit: Maksymilian P. Opolski et al./Canadian Journal of Cardiology

Cardiologists from the Institute of Cardiology, Warsaw, Poland have used Google Glass in a challenging surgical procedure, successfully clearing a blockage in the right coronary artery of a 49-year-old male patient and restoring blood flow, reports the Canadian Journal of Cardiology.

Chronic total occlusion, a complete blockage of the coronary artery, sometimes referred to as the “final frontier in interventional cardiology,” represents a major challenge for catheter-based percutaneous coronary intervention (PCI), according to the cardiologists.

That’s because of the difficulty of recanalizing (forming new blood vessels through an obstruction) combined with poor visualization of the occluded coronary arteries.

Coronary computed tomography angiography (CTA) is increasingly used to provide physicians with guidance when performing PCI for this procedure. The 3-D CTA data can be projected on monitors, but this technique is expensive and technically difficult, the cardiologists say.

So a team of physicists from the Interdisciplinary Centre for Mathematical and Computational Modelling of theUniversity of Warsaw developed a way to use Google Glass to clearly visualize the distal coronary vessel and verify the direction of the guide-wire advancement relative to the course of the blocked vessel segment.

Three-dimensional reconstructions displayed on Google Glass revealed the exact trajectory of the distal right coronary artery (credit: Maksymilian P. Opolski et al./Canadian Journal of Cardiology)

The procedure was completed successfully, including implantation of two drug-eluting stents.

“This case demonstrates the novel application of wearable devices for display of CTA data sets in the catheterization laboratory that can be used for better planning and guidance of interventional procedures, and provides proof of concept that wearable devices can improve operator comfort and procedure efficiency in interventional cardiology,” said lead investigatorMaksymilian P. Opolski, MD, PhD, of the Department of Interventional Cardiology and Angiology at the Institute of Cardiology, Warsaw, Poland.

“We believe wearable computers have a great potential to optimize percutaneous revascularization, and thus favorably affect interventional cardiologists in their daily clinical activities,” he said. He also advised that “wearable devices might be potentially equipped with filter lenses that provide protection against X-radiation.

Abstract of First-in-Man Computed Tomography-Guided Percutaneous Revascularization of Coronary Chronic Total Occlusion Using a Wearable Computer: Proof of Concept

We report a case of successful computed tomography-guided percutaneous revascularization of a chronically occluded right coronary artery using a wearable, hands-free computer with a head-mounted display worn by interventional cardiologists in the catheterization laboratory. The projection of 3-dimensional computed tomographic reconstructions onto the screen of virtual reality glass allowed the operators to clearly visualize the distal coronary vessel, and verify the direction of the guide wire advancement relative to the course of the occluded vessel segment. This case provides proof of concept that wearable computers can improve operator comfort and procedure efficiency in interventional cardiology.

Modulating brain’s stress circuity might prevent Alzheimer’s disease

Drug significantly prevented onset of cognitive and cellular effects in mice
November 17, 2015



Effect of drug treatment on AD mice in control group (left) or drug (right) on Ab plaque load. (credit: Cheng Zhang et al./Alzheimer’s & Dementia: The Journal of the Alzheimer’s Association)

In a novel animal study design that mimicked human clinical trials, researchers at University of California, San Diego School of Medicine report that long-term treatment using a small-molecule drug that reduces activity of  the brain’s stress circuitry significantly reduces Alzheimer’s disease (AD) neuropathology and prevents onset of cognitive impairment in a mouse model of the neurodegenerative condition.

The findings are described in the current online issue of the journal Alzheimer’s & Dementia: The Journal of the Alzheimer’s Association.

Previous research has shown a link between the brain’s stress signaling pathways and AD. Specifically, the release of a stress-coping hormone called corticotropin-releasing factor (CRF), which is widely found in the brain and acts as a neurotransmitter/neuromodulator, is dysregulated in AD and is associated with impaired cognition and with detrimental changes in tau protein and increased production of amyloid-beta protein fragments that clump together and trigger the neurodegeneration characteristic of AD.

“Our work and that of our colleagues on stress and CRF have been mechanistically implicated in Alzheimer’s disease, but agents that impact CRF signaling have not been carefully tested for therapeutic efficacy or long-term safety in animal models,” said the study’s principal investigator and corresponding author Robert Rissman, PhD, assistant professor in the Department of Neurosciences and Biomarker Core Director for the Alzheimer’s Disease Cooperative Study (ADCS).

The researchers determined that modulating the mouse brain’s stress circuitry mitigated generation and accumulation of amyloid plaques widely attributed with causing neuronal damage and death. As a consequence, behavioral indicators of AD were prevented and cellular damage was reduced.  The mice began treatment at 30-days-old — before any pathological or cognitive signs of AD were present — and continued until six months of age.

One particular challenge, Rissman noted, is limiting exposure of the drug to the brain so that it does not impact the body’s ability to respond to stress. “This can be accomplished because one advantage of these types of small molecule drugs is that they readily cross the blood-brain barrier and actually prefer to act in the brain,” Rissman said.

“Rissman’s prior work demonstrated that CRF and its receptors are integrally involved in changes in another AD hallmark, tau phosphorylation,” said William Mobley, MD, PhD, chair of the Department of Neurosciences and interim co-director of the Alzheimer’s Disease Cooperative Study at UC San Diego. “This new study extends those original mechanistic findings to the amyloid pathway and preservation of cellular and synaptic connections.  Work like this is an excellent example of UC San Diego’s bench-to-bedside legacy, whereby we can quickly move our basic science findings into the clinic for testing,” said Mobley.

Rissman said R121919 was well-tolerated by AD mice (no significant adverse effects) and deemed safe, suggesting CRF-antagonism is a viable, disease-modifying therapy for AD. Drugs like R121919 were originally designed to treat generalized anxiety disorder, irritable bowel syndrome and other diseases, but failed to be effective in treating those disorders.

Rissman noted that repurposing R121919 for human use was likely not possible at this point. He and colleagues are collaborating with the Sanford Burnham Prebys Medical Discovery Institute to design new assays to discover the next generation of CRF receptor-1 antagonists for testing in early phase human safety trials.

“More work remains to be done, but this is the kind of basic research that is fundamental to ultimately finding a way to cure — or even prevent —Alzheimer’s disease,” said David Brenner, MD, vice chancellor, UC San Diego Health Sciences and dean of UC San Diego School of Medicine. “These findings by Dr. Rissman and his colleagues at UC San Diego and at collaborating institutions on the Mesa suggest we are on the cusp of creating truly effective therapies.”

Abstract of Corticotropin-releasing factor receptor-1 antagonism mitigates beta amyloid pathology and cognitive and synaptic deficits in a mouse model of Alzheimer’s disease

Introduction: Stress and corticotropin-releasing factor (CRF) have been implicated as mechanistically involved in Alzheimer’s disease (AD), but agents that impact CRF signaling have not been carefully tested for therapeutic efficacy or long-term safety in animal models.

Methods: To test whether antagonism of the type-1 corticotropin-releasing factor receptor (CRFR1) could be used as a disease-modifying treatment for AD, we used a preclinical prevention paradigm and treated 30-day-old AD transgenic mice with the small-molecule, CRFR1-selective antagonist, R121919, for 5 months, and examined AD pathologic and behavioral end points.

Results: R121919 significantly prevented the onset of cognitive impairment in female mice and reduced cellular and synaptic deficits and beta amyloid and C-terminal fragment-β levels in both genders. We observed no tolerability or toxicity issues in mice treated with R121919.

Discussion: CRFR1 antagonism presents a viable disease-modifying therapy for AD, recommending its advancement to early-phase human safety trials.

Allen Institute researchers decode patterns that make our brains human
Conserved gene patterning across human brains provide insights into health and disease
November 17, 2015



Percentage of known neuron-, astrocyte- and oligodendrocyte-enriched genes in 32 modules, ordered by proportion of neuron-enriched gene membership. (credit: Michael Hawrylycz et al./Nature Neuroscience)

Allen Institute researchers have identified a surprisingly small set of just 32 gene-expression patterns for all 20,000 genes across 132 functionally distinct human brain regions, and these patterns appear to be common to all individuals.

In research published this month in Nature Neuroscience, the researchers used data for six brains from the publicly available Allen Human Brain Atlas. They believe the study is important because it could provide a baseline from which deviations in individuals may be measured and associated with diseases, and could also provide key insights into the core of the genetic code that makes our brains distinctly human.

While many of these patterns were similar in human and mouse, many genes showed different patterns in human. Surprisingly, genes associated with neurons were most conserved (consistent) across species, while those for the supporting glial cells showed larger differences. The most highly stable genes (the genes that were most consistent across all brains) include those associated with diseases and disorders like autism and Alzheimer’s, and these genes include many existing drug targets.

These patterns provide insights into what makes the human brain distinct and raise new opportunities to target therapeutics for treating disease.

The researchers also found that the pattern of gene expression in cerebral cortex is correlated with “functional connectivity” as revealed by neuroimaging data from the Human Connectome Project.

“The human brain is phenomenally complex, so it is quite surprising that a small number of patterns can explain most of the gene variability across the brain,” says Christof Koch, Ph.D., President and Chief Scientific Officer at the Allen Institute for Brain Science. “There could easily have been thousands of patterns, or none at all. This gives us an exciting way to look further at the functional activity that underlies the uniquely human brain.”

Abstract of Canonical genetic signatures of the adult human brain

The structure and function of the human brain are highly stereotyped, implying a conserved molecular program responsible for its development, cellular structure and function. We applied a correlation-based metric called differential stability to assess reproducibility of gene expression patterning across 132 structures in six individual brains, revealing mesoscale genetic organization. The genes with the highest differential stability are highly biologically relevant, with enrichment for brain-related annotations, disease associations, drug targets and literature citations. Using genes with high differential stability, we identified 32 anatomically diverse and reproducible gene expression signatures, which represent distinct cell types, intracellular components and/or associations with neurodevelopmental and neurodegenerative disorders. Genes in neuron-associated compared to non-neuronal networks showed higher preservation between human and mouse; however, many diversely patterned genes displayed marked shifts in regulation between species. Finally, highly consistent transcriptional architecture in neocortex is correlated with resting state functional connectivity, suggesting a link between conserved gene expression and functionally relevant circuitry.

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Important Lead in Alzheimer’s Disease Model

Larry H. Bernstein, MD, FCAP, Curator




UCSD team targeting new stress pathway in Alzheimer’s program

By John Carroll

has long been one of the most frustrating targets in R&D. Despite repeated assurances from rival camps that toxic loads of amyloid beta and tau are likely causes of the diseases, no one is quite sure what is going on and clinical failures are routine. But investigators at UC San Diego School of Medicine say they have been garnering some preclinical clues that would suggest there could be a new pathway to follow in the clinic.

Following the idea that the brain’s stress signaling circuitry may play a role in the development of the disease, the UCSD group centered on a hormone called corticotropin-releasing factor. CRF is a neuropeptide that triggers the behavioral and biologic responses to stress, which UC says has been associated with worsening cognition as well as the alteration of tau and the creation of a-beta.

The team found a way to block the CRF receptor in mouse models for the disease with an anti-anxiety and IBS drug called R121919. Cellular damage was reduced, the scientists say, while the behavioral changes associated with the disease were also avoided in the mice.

“The novelty of this study is two-fold: We used a preclinical prevention paradigm of a CRF-antagonist (a drug that blocks the CRF receptor in brain cells) called R121919 in a well-established AD model–and we did so in a way that draws upon our experience in human trials,” said Robert Rissman, an assistant professor in the Department of Neurosciences and Biomarker Core Director for the Alzheimer’s Disease Cooperative Study, in a release. “We found that R121919 antagonism of CRF-receptor-1 prevented onset of cognitive impairment and synaptic/dendritic loss in AD mice.”

The group followed up by saying that R121919 appeared to be a safe way to hit the stress pathway, but that it was unlikely that they could repurpose the drug specifically for Alzheimer’s. Now the team plans to search for new drugs that can do the same thing, with an eye to getting into the clinic.

“Rissman’s prior work demonstrated that CRF and its receptors are integrally involved in changes in another AD hallmark, tau phosphorylation,” said Dr. William Mobley, chair of the Department of Neurosciences and interim co-director of the Alzheimer’s Disease Cooperative Study at UC San Diego, in the release. “This new study extends those original mechanistic findings to the amyloid pathway and preservation of cellular and synaptic connections. Work like this is an excellent example of UC San Diego’s bench-to-bedside legacy, whereby we can quickly move our basic science findings into the clinic for testing.”


Corticotropin-releasing factor receptor-1 antagonism mitigates beta amyloid pathology and cognitive and synaptic deficits in a mouse model of Alzheimer’s disease

Cheng Zhang, Ching-Chang Kuo, Setareh H. Moghadam, Louise Monte, Shannon N. Campbell, Kenner C. Rice, Paul E. Sawchenko, Eliezer Masliah, Robert A. Rissman
Introduction   Stress and corticotropin-releasing factor (CRF) have been implicated as mechanistically involved in Alzheimer’s disease (AD), but agents that impact CRF signaling have not been carefully tested for therapeutic efficacy or long-term safety in animal models.

Methods   To test whether antagonism of the type-1 corticotropin-releasing factor receptor (CRFR1) could be used as a disease-modifying treatment for AD, we used a preclinical prevention paradigm and treated 30-day-old AD transgenic mice with the small-molecule, CRFR1-selective antagonist, R121919, for 5 months, and examined AD pathologic and behavioral end points.

Results   R121919 significantly prevented the onset of cognitive impairment in female mice and reduced cellular and synaptic deficits and beta amyloid and C-terminal fragment-β levels in both genders. We observed no tolerability or toxicity issues in mice treated with R121919.

Discussion   CRFR1 antagonism presents a viable disease-modifying therapy for AD, recommending its advancement to early-phase human safety trials.


Preclinical study points to GPR3 as a potential target for Alzheimer’s


The role of G protein-coupled receptors in the pathology of Alzheimer’s disease
Amantha Thathiah and Bart De Strooper
Nature Reviews Feb 2011; 12: 73-87

Abstract | G protein-coupled receptors (GPCRs) are involved in numerous key neurotransmitter systems in the brain that are disrupted in Alzheimer’s disease (AD). GPCRs also directly influence the amyloid cascade through modulation of the α-, β- and γ-secretases, proteolysis of the amyloid precursor protein (APP), and regulation of amyloid-β degradation. Additionally, amyloid-β has been shown to perturb GPCR function. Emerging insights into the mechanistic link between GPCRs and AD highlight the potential of this class of receptors as a therapeutic target for AD.


Figure 1 | Modulation of APP processing by GPcrs. Cleavage of amyloid precursor protein (APP) by α-secretase generates the soluble amino-terminal ectodomain of APP (sAPPα) and the carboxy-terminal fragment C83. Subsequent cleavage of C83 by the γ-secretase complex yields the APP intracellular domain (AICD) and a short fragment termed p3. Several G protein-coupled receptors (GPCRs), including muscarinic, metabotropic and serotonergic receptors modulate α-secretase-mediated proteolysis. Alternatively, cleavage of APP by β-secretase generates sAPPβ and the C-terminal fragment C99. Subsequent cleavage of C99 by the γ-secretase complex yields the AICD and the amyloid-β peptide. Of the GPCRs that regulate this processing, the δ-opioid receptor (DOR) and the adensoine A2A receptor (A2AR) have been shown to modulate β-secretase-mediated cleavage of APP, whereas the β2 adrenergic receptor (β2-AR), G protein-coupled receptor 3 (GPR3), and CXC-chemokine receptor 2 (CXCR2) have been shown to modulate γ-secretasemediated cleavage of C99 or C83. Aβ, amyloid-β; ADAM, a disintegrin and metalloproteinase; BACE1, β-site APP-converting enzyme 1; CRHR1, corticotrophinreleasing hormone (CRH) receptor type I; 5-HT, 5-hydroxytryptamine (serotonin); mAChR, muscarinic acetylcholine receptor; mGluR, metabotropic glutamate receptor; PAC1R, pituitary adenylate cyclase 1 receptor.


Box 1 | The cholinergic and amyloid cascade hypotheses
The amyloid cascade hypothesis The amyloid cascade hypothesis postulates that gradual changes in the metabolism and aggregation of amyloid-β initiates a cascade of neuronal and inflammatory injury that culminates in extensive neuronal dysfunction and cell death associated with neurotransmitter deficits and dementia145,146. The cholinergic hypothesis The cholinergic hypothesis posits that a dysfunction in acetylcholine (ACh)-containing neurons substantially contributes to the cognitive decline observed in Alzheimer’s disease (AD)147. This is based on the observation that cholinergic transmission has a fundamental role in cognition and is disrupted in patients with AD148,149. convergence of the amyloid cascade and cholinergic hypotheses ACh is a key neurotransmitter involved in learning and memory150 that binds to distinct receptor subtypes in the brain: nicotinic ACh receptors (nAChRs) and muscarinic ACh receptors (mAChRs). Nicotinic neurotransmission is implicated in the pathogenesis of AD (TABle 1). Additional evidence suggests that the major mAChR subtypes involved in AD are the postsynaptic M1 mAChRs, which mediate the effects of ACh, and the presynaptic M2 mAChRs, which inhibit ACh release151, 152. Amyloid-β deposition may contribute to the cholinergic dysfunction in AD by decreasing the release of presynaptic ACh and impairing the coupling of postsynaptic M1 mAChRs with G proteins. This leads to decreased signal transduction, impairments in cognition, a reduction in the levels of amyloid precursor protein (APP), the generation of more neurotoxic amyloid-β and a further decrease in ACh release111. Genetic ablation of the M1 mAChR in a transgenic mouse model of AD decreases the production of the soluble amino-terminal ectodomain of APP (sAPPα), increases amyloid-β generation and exacerbates the amyloid plaque pathology28, supporting the development of M1-selective agonists. In addition, M1 mAChR activation reduces tau phosphorylation27,153 and alleviates hippocampus-dependent memory impairments27, making M1 mAChRs a compelling therapeutic target for AD. Furthermore, receptor subtype specificity will be of key importance as M2 and M4 mAChRs seem to inhibit sAPPα release and potentially aggravate amyloid-β generation28,30, and activation of nAChRs exacerbates the tau pathology154.


Figure 2 | GPcr signalling and the α-secretase pathway. G protein-coupled receptors (GPCRs) exert their multiple functions through a complex network of intracellular signalling pathways. Ligand-bound GPCRs activate heterotrimeric G proteins, inducing the exchange of GDP for GTP and the formation of a GTP-bound Gα subunit and the release of a Gβγ dimer. The G protein subunits then activate specific secondary effector molecules, such as adenylyl cyclase (AC), phospholipase C (PLC) and phospholipase A2 (PLA2), leading to the generation of secondary messengers and activation of extracellular signal-regulated kinase 1/2 (ERK1/2), Janus kinase (JAK) and phophoinositide 3-kinase (PI3K), and modulation of the α-secretase pathway. In the case of the M1 muscarinic acetylcholine receptor (M1 mAChR), the group I metabotropic glutamate receptors (mGluRs) and the 5-hydroxytryptamine receptors 5-HT2A/2CR and 5-HT4R, agonist stimulation leads to an increase in soluble amyloid precursor protein (sAPP) release, a decrease in amyloid-β (Aβ) generation, a decrease in tau phosphorylation and/or an alleviation of the cognitive deficits in a mouse model of Alzheimer’s disease (AD). Conversely, agonist stimulation of the Group II mGluRs leads to an increase in amyloid-β42 generation, tau phosphorylation and an exacerbation of the cognitive deficits in an AD mouse model. In the case of the 5-HT6 receptor (5-HT6R), antagonism of the receptor leads to an improvement in cognition. Solid arrows represent direct signalling pathways and dashed arrows represent signalling via intermediates that are not shown. ACh, acetylcholine; ADAM, a disintegrin and metalloproteinase; cAMP, cyclic AMP; GSK3β, glycogen synthase kinase 3β; NMDAR, NMDA receptor; PKC, protein kinase C; sAPPα, soluble amino-terminal ectodomain of APP; STAT, signal transducer and activator of transcription.


Pituitary adenylate cyclase 1 receptor. The pituitary adenylate cyclase 1 receptor (PAC1R) is a GPCR that is stimulated by the neuropeptide pituitary adenylate cyclase­activating polypeptide (PACAP). The receptor is primarily localized to the hypothalamus but is also expressed in the cerebral cortex and hippocampus72, areas of the human brain affected by AD. The major form of PACAP, composed of 38 amino acids (PACAP38), has been show to improve memory in rats73. Together with a C­terminal truncated form, PACAP27, it stimulates an increase in sAPPα release74. This effect is blocked by a broad­spectrum metalloprotease inhibitor and by an ADAM10­specific inhibitor, GI254023X74. Thus, stimulation of PAC1R enhances α­secretase activity. Although the molecular mechanism of this effect has not been elucidated, neuropeptide hormones such as PACAP27 and PACAP38 display a high flux rate across the blood–brain barrier (bbb)75, which should permit the in vivo examination of the effect of PACAP in a transgenic mouse model of AD.
Regulation of b-secretase The β­secretase bACE1 (β­site APP­converting enzyme 1), is a type I transmembrane aspartyl protease that is active at low pH and is predominantly localized in acidic intracellular compartments, such as endosomes and the trans­Golgi network. Cleavage of APP by bACE1 generates a soluble n­terminal ectodomain of APP (sAPPβ) and the n terminus of amyloid­β. Subsequent cleavage of the membrane­bound C­terminal fragment C99 by the γ­secretase liberates the amyloid­β peptide species (FIG. 1). bACE1 is abundantly expressed in neurons in the brain. Bace1–/– mice are viable and fertile, facilitating the study of the role of this enzyme in AD. bACE1 deficiency in an AD mouse model abrogates amyloid­β generation, amyloid pathology, electrophysiological dysfunction and cognitive deficits, implying that therapeutic inhibition of bACE1 would decrease generation of all amyloid­β species. However, Bace1–/– mice display phenotypic abnormalities that are related to the processing of additional proteins by bACE1, suggesting that therapeutic inhibition of bACE1 could have adverse side effects (reviewed in ReFS 76,77). nevertheless, bACE1 is arguably the primary therapeutic target to deter amyloid­β generation. Detailed structural analysis of bACE1 has led to the discovery of many transition state­based inhibitors with activity in the low nanomolar range, although the in vivo efficacy of these compounds is limited because most of them do not penetrate the bbb or are actively exported from the brain by P­glycoprotein. Recent evidence suggests that GPCRs such as the δ­opioid receptor (DOR)78 could provide a therapeutic opportunity to modulate bACE1 and amyloid­β generation .
δ‑ and μ‑opioid receptors. The opioid receptors, which play important parts in learning and memory, are deregulated in specific regions of the AD brain79. There is evidence to suggest that the DOR, together with the β2 adrenergic receptor (β2­AR), promotes the γ­secretasemediated cleavage of the APP C­terminal fragment after its generation by β­secretase80. A more recent study by the same group suggested that activation of the DOR promotes the translocalization of a complex consisting of the DOR, β­secretase and γ­secretase from the cell surface to the late endosomes and lysosomes (LEL), which results in enhanced β­ and γ­secretase proteolysis of APP78. In a mouse model of AD, administration of natrindole, a selective DOR antagonist, improved spatial learning and reference memory, and reduced the amyloid plaque burden78. Similarly, in vivo knock down of the DOR reduced amyloid­β40 accumulation in the hippoc ampus of an AD mouse model. However, there was no effect on the more hydrophobic (and therefore more toxic) amyloid­β42 (ReF. 78). by contrast, administration of a μ­opioid receptor (MOR) antagonist had no effect on amyloid­β generation or amyloid plaque formation and was unable to reverse the learning and memory deficiency of the AD mouse model78, although another group reported improved spatial memory retention in this transgenic AD mouse model81. DOR binding is decreased in the amygdala and ventral putamen, and MOR binding is decreased in the hippocampus and subiculum79 of post­mortem brain samples from patients with AD. Elevated hippocampal levels of enkephalin, the ligand for these receptors, have been detected in AD transgenic mice and in the human AD brain81,82. Excessive stimulation by enkephalin may uncouple the opioid receptors from G proteins, resulting in receptor internalization83,84 and reduced receptor binding in patients with AD79,85. These adaptive changes in opioid receptor expression in response to increased enkephalin levels might limit the efficacy of opioid receptor antagonists in AD and could explain the variable effects of different DOR antagonists on amyloid­β generation in AD transgenic mouse models.
Regulation of g-secretase The γ-­secretase complex is composed of four integral membrane proteins: the catalytic component presenilin 1 (PS1) or PS2 and the essential cofactors nicastrin, anterior pharynx defective 1 (APH1) and presenilin enhancer 2 (PEn2)86. Proteolysis of the α­ cleavage product C83 by the γ­secretase complex generates a short p3 fragment, which precludes formation of amyloid­β. by contrast, proteolysis of the β­secretase product C99 by the γ­secretase complex generates the amyloid­β peptide, which ranges in length from 35 to 43 residues (FIG. 1). The majority of amyloid­β produced is 40 amino acids in length (amyloid­β40), whereas a small proportion (~10%) is the 42­residue variant (amyloid­β42). Several γ­secretase inhibitors have been developed but they have limited clinical efficacy owing to the severe side effects associated with inhibition of the notch receptor, which is a substrate for γ­secretase proteolysis. Therefore, determination of the cellular mechanisms that specifically regulate amyloid­β generation by γ­secretase is of crucial importance for understanding the factors that cause AD and could highlight new therapeutic targets.


b 2‑adrenergic receptor. Stimulation of β2­AR increases amyloid­β generation in vitro, independently of an elevation in cAMP levels80. In an AD transgenic mouse model, treatment with a β2­AR agonist or antagonist respectively increased and decreased the amyloid plaque burden80. It has been suggested that the β2­AR constitutively associates with PS1 at the plasma membrane and undergoes clathrin­mediated endocytosis together with the γ­secretase complex following agonist stimulation80. This proposed localization of the γ­secretase in LEL compartments, which is supported by other studies87,88, could promote cleavage of C99 and thereby the generation of amyloid­β80. As a therapeutic application, it will be important to determine whether β2­AR activation also modulates cleavage of the notch receptor, given the adverse side effects of targeting γ­secretase discussed above. Importantly, the β2­AR is expressed in the hippocampus and the cortex in humans89, and polymorphisms in the gene encoding the β2­AR are associated with an increased risk of developing sporadic lateonset AD90, providing support for the potential clinical relevance of the in vitro and AD mouse model findings.
G protein‑coupled receptor 3. G protein­coupled receptor 3 (GPR3) is an orphan GPCR with a putative ligand91 that has not been validated92,93. The receptor was identified as a modulator of amyloid­β generation in a high­throughput functional genomics screen designed to identify potential therapeutic targets for AD92. GPR3 is strongly expressed in neurons in the hippocampus, amygdala, cortex, entorhinal cortex and thalamus in the normal human brain94,95, and its expression is increased in a subset of patients with sporadic AD92. Several lines of evidence support the involvement of GPR3 in the generation of amyloid­β. In vitro models of AD suggest that this effect is independent of its ability to stimulate the production of cAMP92. In an AD transgenic mouse model96, hippocampal overexpression of GPR3 enhanced amyloid­β40 and amyloid­β42 generation in the absence of an effect on γ­secretase expression92. Genetic ablation of Gpr3 in these mice dramatically reduced amyloid­β40 and amyloid­β42 levels92, demonstrating that endogenous GPR3 is involved in amyloid­β generation. Further in vitro studies suggested that GPR3 promotes increased association of the individual γ­secretase complex components within detergent­resistant membrane domains and stabilizes the mature γ­secretase complex92. Thus, similar to the β2­AR, the effect of GPR3 signalling on amyloid­β generation is not mediated through an elevation in cAMP levels. Rather, both GPCRs modulate the trafficking and/or localization of the γ­secretase complex to membrane domains where it can more efficiently process the β­secretase product C99. Importantly, the in vitro effect of GPR3 expression on amyloid­β generation occurs in the absence of an effect on notch processing, suggesting that GPR3 can selectively target specific γ­secretase pathways.
CXC‑chemokine receptor 2. The CXC­chemokine receptor type 2 (CXCR2) is abundantly expressed in neurons and is strongly upregulated in a subpopulation of neuritic plaques in the post­mortem human AD brain97,98. In an AD transgenic mouse model, treatment with the CXCR2 antagonist Sb­225002 reduces amyloid­β40 levels99 and is accompanied by a reduction in PS1–C­terminal fragment (CTF) levels, resulting in a probable decrease in the proteolytically active mature γ­secretase complex99. Crossing the Cxcr2­deficient mouse with an AD transgenic mouse also results in a decrease in amyloid­β40 and amyloid­β42 generation, and γ­secretase complex expression100. In vitro evidence suggests that antagonism of CXCR2 reduces expression levels of other γ­secretase complex components, inhibiting generation of both the AICD and the notch intracellular domain. Whether CXCR2 is involved in enhanced turnover, degradation or stabilization of the PS1–CTF has not been determined. However, inhibition of Jun n­terminal kinase (JnK) activity, which is involved in signalling downstream of CXCR2, correlates with reduced phosphorylation and stability of the PS1–CTF101,102. Given that antagonism of CXCR2 leads to general changes in γ­secretase expression and activity, it will be challenging to therapeutically target CXCR2.
GPCRs and amyloid-b toxicity One of the most puzzling aspects of the amyloid cascade hypothesis is why amyloid­β exerts a neurotoxic effect on cells. There is no clear correlation between exposure of the brain to amyloid­β plaques and neurodegeneration and, in cell culture models, the toxicity associated with amyloid­β is variable and poorly understood. Small oligomeric structures of amyloid­β, known as amyloidβ­derived diffusible ligands (ADDLs)103, cause synaptotoxicity, interfering with glutamate signalling at several levels, including direct and indirect effects on Ca2+ levels, endocytosis, and possibly membrane damage and clustering of various membrane proteins. A further complication is that a component of the toxicity associated with amyloid­β might be the consequence of a general mechanism such as interaction with the plasma membrane, which could affect multiple GPCRs. Moreover, several GPCRs are involved in neuro inflammation, with beneficial or detrimental effects on amyloid­β­mediated toxicity depending on the model under investigation. Thus, it remains unclear how the involvement of GPCRs in amyloid­-β ­mediated toxicity can be clinically exploited. Studies on the angiotensin type 2 receptor (AT2R), the adenosine A2A receptor (A2AR) and CC­chemokine receptor 2 (CCR2) provide insight into this complicated matter.


Figure 3 | Amyloid-β toxicity and deregulation of AT2r and M1 mAchr signalling . Oxidative stress and amyloid-β (Aβ) accumulation leads to an increase in reactive oxygen species (ROS) generation and dimerization of angiotensin type 2 receptors (AT2R). An increase in levels of the protein-crosslinking enzyme transglutaminase, as occurs in Alzheimer’s disease, and further Aβ deposition trigger crosslinking and subsequent oligomerization of AT2R dimers. The AT2R oligomers sequester Gαq/11 and thereby inhibit Gαq/11 from coupling to M1 muscarinic acetylcholine receptors (M1 mAChRs). Sequestration of Gαq/11 results in tau phosphorylation, neuronal degeneration and Alzheimer’s disease progression. PKC, protein kinase C. Figure is reproduced, with permission, from REF. 111 © (2009) American Association for the Advancement of Science.


GPCRs and amyloid-b degradation Promoting amyloid­β clearance from the brain is an alternative therapeutic strategy to inhibition of amyloid­β generation. Such an approach is the basis for the passive and active immunotherapy with amyloid­βspecific antibodies. However, stimulation of GPCRs, in particular the somatostatin receptor, could represent an interesting alternative approach to promoting amyloid­β clearance, as these GPCRs induce expression of amyloidβ­degrading enzymes, such as neprilysin, in the brain. A combination of memory enhancement, neuroprotection and anti­amyloid­β activity makes this an attractive therapeutic approach for AD.
Somatostatin receptors. Somatostatin (also known as somatotropin release­ inhibiting factor, SRIF) is a regulatory peptide with two bioactive forms, SRIF14 and SRIF28, which are widely expressed throughout the CnS and function in neurotransmission, protein secretion and cell proliferation133,134. Expression of the two most abundant SRIF receptors in the brain, somatostatin receptor type 2 (SSTR2) and SSTR4, is reduced in the cortex of human patients with AD135. Interestingly, intracerebroventricular injection of amyloid­β25–35 results in a selective decrease in SSTR2 mRnA and protein levels in the temporal cortex of rats, whereas cognitive deficits correlate with reduced SRIF concentrations in the CSF136 or middle front gyrus (brodmann area 9)137. SRIF levels are also reduced in the CSF136, cortex135 and hippocampus138 of patients with AD. Compelling evidence suggests that SRIF is a modulator of neprilysin activity in the brain139. neprilysin, one of the main amyloid­β­degrading enzymes, regulates the steady state levels of amyloid­β40 and amyloid­β42 in vivo140. SRIF has been shown to significantly elevate neprilysin levels in primary murine cortical neuronal cultures, which accompanies a reduction in amyloid­β42 levels139. Conversely, neprilysin activity and localization are altered in the hippocampus of SRIF­deficient mice, with a corresponding increase in amyloid­β42 levels139. There are conflicting results from AD transgenic mouse models, which show either an increase141 or a decrease in SRIF levels142. Further work is necessary to clarify the cause of the changes in SRIF levels in these AD models.

Figure 4 | Adenosine A2A receptor and amyloid-β-mediated toxicity. a | Amyloid-β (Aβ) deposition has been shown to activate the p38 mitogen-activated protein kinase (MAPK) signalling pathway, which leads to Aβ-induced neurotoxicity. Pharmacological blockade of the adenosine A2A receptor (A2AR) with the compound SCH 58261 reduces Aβ-induced p38 MAPK phosphorylation, synaptotoxicity and cognitive impairment. b | Similarly, caffeine, an A2AR antagonist, is also protective against Aβ-mediated toxicity and may regulate the expression levels of the β-secretase, via the cRaf-1/nuclear factor-κB pathway and presenilin 1, which leads to a decrease in Aβ40 and Aβ42 deposition and is protective against cognitive impairment in an Alzheimer’s disease mouse model. Solid arrows represent direct signalling pathways and dashed arrows represent signalling via intermediates that are not shown. JNK, Jun N-terminal kinase.


Box 2 | GPCRs, diabetes and Alzheimer’s disease Glucagon-like peptide 1 receptor Type 2 diabetes (T2D) has been identified as a risk factor for Alzheimer’s disease (AD)155, and insulin signalling has a role in learning and memory156-158, which potentially links insulin resistance to AD dementia. Indeed, deregulated insulin signalling has been observed in brains of patients with AD and may contribute to the development of AD159. The combination of insulin with other antidiabetic medications is also associated with lower amyloid plaque density and a diminution of the cognitive decline associated with AD160,161. Strategies have therefore been developed to normalize insulin signalling in the brain to deter the progression of AD162. One promising intervention is the use of the incretin hormone glucagon-like peptide 1 (GLP1) as a treatment for neurodegenerative diseases163. In vivo administration of GLP1 or exendin-4, a more stable analogue of GLP1, reduces endogenous levels of amyloid-β40 in the mouse brain and protects against cell death164. In addition, GLP1 and the stable analogue (Val8)GLP1 enhance long-term potentiation (LTP) and reverse the LTP impairment induced by amyloid-β25-35 administration in rodents, which might underlie an improvement in cognitive function165. Most recently, (Val8)GLP1 also prevented amyloid-β40-induced impairment in late-phase LTP, and spatial learning and memory in rodents166. Some evidence also suggests that the desensitization of insulin receptors that occurs in AD can be reversed by activation of GLP1 receptors (GLP1Rs)167. GLP1 binds to GLP1R, which activates diverse signalling pathways, including cyclic AMP, protein kinase A, phospholipase C, phosphatidylinositol 3-kinase, protein kinase C and mitogen-activated protein kinase168–171. GLP1R-deficient mice display an impairment in synaptic plasticity163 and a decrease in the acquisition of contextual learning, a learning deficit that can be reversed following hippocampal gene transfer of Glp1r172. By contrast, overexpression of GLP1R through hippocampal gene transfer markedly enhanced learning and memory in rodents172. Taken together, these studies suggest that the GLP1R represents a novel and promising therapeutic target for AD. Amylin receptor Amylin (also known as islet amyloid polypeptide) is a peptide that was first isolated from amyloid deposits from the pancreatic islets of Langerhans of patients with type 2 diabetes173. Interestingly, human amylin, which acts through the G protein-coupled amylin receptor, possesses amyloidogenic and neurotoxic properties similar to amyloid-β174. Accordingly, treatment of rat neuronal cultures with an amylin receptor antagonist, AC187, attenuates amyloid-β42- and amylin-induced neurotoxicity by blocking caspase activation175. It would be interesting to determine whether treatment with GLP1 could alleviate the cognitive deficits, and to determine the expression levels of GLP1R in this diabetic AD mouse model. Most recently, studies conducted by crossing two T2D mouse models with an AD mouse model have provided further mechanistic insight into the relationship between diabetes and AD, demonstrating that the onset of diabetes exacerbates cognitive dysfuntion in the absence of an elevation in amyloid-β levels and leads to increased cerebrovascular inflammation and amyloid angiopathy176. Conversely, the diabetic AD mice display an accelerated diabetic phenotype relative to the diabetic mouse model alone, suggesting that the amyloid pathology may adversely affect the T2D and vice versa.


Concluding remarks numerous drug discovery efforts target the inhibition of amyloid-­β production, the prevention of amyloid­β aggregation and the enhancement of amyloid-­β clearance. Although these may seem to be straightforward biochemical pathways, several feedback loops enhance not only amyloid­β deposition but also its toxicity, clearance and overall impact on memory function and neuronal health. Such feedback loops also imply that a monotherapy will not be sufficient to prevent the progression of AD. based on the discussion above, it is clear that several GPCRs are involved at many stages of AD disease progression (TABle 1). There also seems to be a pathologically reinforcing loop between type 2 diabetes and AD, with GPCRs providing an avenue for therapeutic intervention for both diseases (BOX 2). Drugs that target GPCRs could diversify the symptomatic therapeutic portfolio for AD and potentially provide disease­modifying treatments. In this sense, they complement the current areas of investigation, which are primarily focused on secretase inhibitors77 and amyloid immunotherapy144. Given that the current anti­amyloidogenic therapy under development is considered to be most effective as a preventative measure or in early stages of AD, additional drugs that preferentially enhance cognition will become a necessary complement to treatment, especially as the disease progresses to more advanced stages. In this regard, GPCRs represent the largest therapeutic target in the pharmaceutical industry and provide ample opportunities for AD­related drug development. nevertheless, progress in the field is hampered by the difficulty in developing highly receptor­specific ligands and the adverse side effects of currently available drugs. Recent advances in the GPCR field suggest that a more functional approach towards the classification of GPCRs, which are now organized according to structural similarity, might enhance the therapeutic potential of GPCRs and assist in the development of selective GPCR candidate drugs for AD and many other diseases.


Related Articles:

Alzheimer’s: Investigators spotlight a pathway for amyloid beta clearance

Eta-amyloid discovered in Alzheimer’s adds new piece to the complex puzzle

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Amyloid-Targeting Immunotherapy

Curator: Larry H. Bernstein, MD, FCAP

Possible Reasons Found for Failure of Alzheimer’s Treatment

By Staff Editor


(HealthNewsDigest.com) – Agglutinated proteins in the brain, known as amyloid-β plaques, are a key characteristic of Alzheimer’s. One treatment option uses special antibodies to break down these plaques. This approach yielded good results in the animal model, but for reasons that are not yet clear, it has so far been unsuccessful in patient studies. Scientists at the Technical University of Munich (TUM) have now discovered one possible cause: they noticed that, in mice that received one antibody treatment, nerve cell disorders did not improve and were even exacerbated.

Immunotherapies with antibodies that target amyloid-β were long considered promising for treating Alzheimer’s. Experiments with animals showed that they reduced plaques and reversed memory loss. In clinical studies on patients, however, it has not yet been possible to confirm these results. A team of researchers working with Dr. Dr. Marc Aurel Busche, a scientist at the TUM hospital Klinikum rechts der Isar Klinik und Poliklinik für Psychiatrie und Psychotherapie and at the TUM Institute of Neuroscience, and Prof. Arthur Konnerth from the Institute of Neuroscience has now clarified one possible reason for this. The findings were published in Nature Neuroscience.

Immunotherapy Increases Number of Hyperactive Nerve Cells

The researchers used Alzheimer’s mice models for their study. These animals carry a transgene for the amyloid-β precursor protein, which, as in humans, leads to the formation of amyloid-β plaques in the brain and causes memory disorders. The scientists treated the animals with immunotherapy antibodies and then analyzed nerve cell activity using high-resolution two-photon microscopy. They found that, while the plaques disappeared, the number of abnormally hyperactive neurons rose sharply.

“Hyperactive neurons can no longer perform their normal functions and, after some time, wear themselves out. They then fall silent and, later, possibly die off,” says Busche, explaining the significance of their discovery. “This could explain why patients who received the immunotherapy experienced no real improvement in their condition despite the decrease in plaques,” he adds.

Released Oligomers Potential Reason for Hyperactivity

Even in young Alzheimer’s mice, when no plaques were yet detectable in the brain, the antibody treatment led to increased development of hyperactive nerve cells. “Looking at these findings, even using the examined immunotherapies at an early stage, before the plaques appear, would offer little chance of success. As the scientist explains, the treatment already exhibits these side effects here, too.

“We suspect that the mechanism is as follows: The antibodies used in treatment release increasing numbers of soluble oligomers. These are precursors of the plaques and have been considered problematic for some time now. This could cause the increase in hyperactivity,” says Busche.

The work was funded by an Advanced ERC grant to Prof. Arthur Konnerth, the EU FP7 program (Project Corticonic) and the Deutsche Forschungsgemeinschaft (IRTG 1373 and SFB870). Marc Aurel Busche was supported by the Hans und Klementia Langmatz Stiftung.

Marc Aurel Busche, Christine Grienberger, Aylin D. Keskin, Beomjong Song, Ulf Neumann, Matthias Staufenbiel, Hans Förstl and Arthur Konnerth, Decreased amyloid-β and increased neuronal hyperactivity by immunotherapy in Alzheimer’s models, Nature Neuroscience, November 9, 2015.
DOI: 10.1038/nn.4163

Amyloid-Targeting Immunotherapy Disrupts Neuronal Function

Some antibodies designed to eliminate the plaques prominent in Alzheimer’s disease can aggravate neuronal hyperactivity in mice.

By Karen Zusi | November 9, 2015  http://www.the-scientist.com//?articles.view/articleNo/44435/title/Amyloid-Targeting-Immunotherapy-Disrupts-Neuronal-Function/


Removing built-up plaques of amyloid-β in the brain is a long-sought therapy for patients with Alzheimer’s disease, but for a variety of reasons, few treatments have succeeded in alleviating symptoms once they reach clinical trials. In a study published today (November 9) in Nature Neuroscience, an international team examined the effects of two amyloid-β antibodies on neuronal activity in a mouse model, finding that the antibodies in fact led to an increase in neuronal dysfunction.

Decreased amyloid-β and increased neuronal hyperactivity by immunotherapy in Alzheimer’s models

Marc Aurel BuscheChristine GrienbergerAylin D KeskinBeomjong SongUlf NeumannMatthias StaufenbielHans Förstl & Arthur Konnerth
Nature Neuroscience (2015)

Among the most promising approaches for treating Alzheimer´s disease is immunotherapy with amyloid-β (Aβ)-targeting antibodies. Using in vivo two-photon imaging in mouse models, we found that two different antibodies to Aβ used for treatment were ineffective at repairing neuronal dysfunction and caused an increase in cortical hyperactivity. This unexpected finding provides a possible cellular explanation for the lack of cognitive improvement by immunotherapy in human studies.

Marc Busche, a psychiatrist at Technical University of Munich in Germany, and others had previously found that neuronal hyperactivity is common in mouse models of Alzheimer’s disease. The chronically rapid-firing neurons can interfere with normal brain function in mice. “There’s evidence from human fMRI [functional magnetic resonance imaging] studies that humans will show hyperactivation early in the disease, followed by hypoactivation later on,” Busche told The Scientist. “It’s an early stage of neuronal dysfunction that can later turn into neural silencing.”

To investigate whether certain antibodies would alleviate this Alzheimer’s disease-associated phenotype, Busche and his colleagues first turned to bapineuzumab—a human monoclonal antibody that initially showed promise in treating mice modeling Alzheimer’s disease, but failed in human clinical trials. The dominant hypothesis for bapineuzumab’s failure is that it was administered too late in the disease progression, said Busche. “But it’s still a hypothesis,” he added. “There’s no real explanation for why these antibodies failed.”

The team’s latest experimenters used mice with a genetic mutation that caused them to overexpress the human amyloid-β protein; these engineered mice also displayed neuronal hyperactivity. The researchers injected 3D6, the mouse version of bapineuzumab, into the engineered mice, as well as into wild-type mice that had normal expression levels of the mouse amyloid-β protein. The team observed the effects using two-photon calcium imaging in a blinded study.

As expected, 3D6 decreased the amount of amyloid-β plaques in the engineered mice, while the control mice displayed no reaction to the injected antibodies. However, the mice engineered to overexpress human amyloid-β showed increased neuronal hyperactivity in response to the antibody, regardless of what stage of plaque development they were in. Even mice too young to have developed plaques showed aggravated hyperactive neurons. The team observed the same phenomenon when it tested a second antibody, β1, which went through early stages of drug development but was never used in human clinical trials.

As expected, 3D6 decreased the amount of amyloid-β plaques in the engineered mice, while the control mice displayed no reaction to the injected antibodies. However, the mice engineered to overexpress human amyloid-β showed increased neuronal hyperactivity in response to the antibody, regardless of what stage of plaque development they were in. Even mice too young to have developed plaques showed aggravated hyperactive neurons. The team observed the same phenomenon when it tested a second antibody, β1, which went through early stages of drug development but was never used in human clinical trials.

The results surprised Busche. “When it turned out that the antibody group was worse than the control group, it was unbelievable. But we checked many times and there was no mistake,” he said. “We don’t see this effect in wild-type mice so it must be dependent on the interaction between the antibody and amyloid-β.”

Busche was quick to point out that the mouse model is not the same as a human Alzheimer’s patient. However, he said, “it gives a sense that we don’t understand the antibody’s action, and this might go on in the human brain as well.”

“I fully believe in their results, but I have some hesitation in saying that this result explains the failed clinical trials for amyloid-β immunotherapy,” said Cynthia Lemere, a neurologist and Alzheimer’s disease researcher at the Brigham and Women’s Hospital in Boston. “I think the major reason for clinical trials failing for immunotherapy is that up until now, they’ve been done in people with moderate-to-severe Alzheimer’s disease, and then mild-to-moderate. Now the studies are going further to include people with very early stages of clinical symptoms—and to my knowledge, they haven’t been stopped because patients are getting worse.”

Thomas Wisniewski, a cognitive neurologist at New York University, voiced a similar perspective. “I don’t think this is an explanation for why immunotherapy isn’t working—I think there are other more plausible reasons for that,” he said, citing clinical trials that treated patients during later stages of Alzheimer’s disease progression, as well as those that haven’t addressed tau-related pathologies, or didn’t target the key types of amyloid-β. “[The neuronal hyperactivity] is an interesting phenomenon to be studied,” he added, “but I think it’s a separate issue.”

M.A. Busche et al., “Decreased amyloid-β and increased neuronal hyperactivity by immunotherapy in Alzheimer’s models,” Nature Neuroscience, doi:10.1038/nn.4163, 2015.

Figure 2: Worsening of neuronal dysfunction by anti-Aβ antibodies can occur independently of the effects on Aβ pathology.

Worsening of neuronal dysfunction by anti-A[beta] antibodies can occur independently of the effects on A[beta] pathology.

(a) Top, representative in vivo activity maps in WT (left) as well as isotype-treated (middle) and β1-treated (right) Tg2576 mice. Bottom, Ca2+ transients of neurons indicated above. The further aggravation of neuronal hyperactivity (mi…


Anti-Aβ treatment aggravates abnormal brain activity in a mouse model of Alzheimer’s disease

Nature Neuroscience   Nov 10, 2015


Therapies that reduce deposits of amyloid-β (Aβ) in the brain are ineffective at repairing neuronal impairment in mice and actually increase it, finds a study published online in Nature Neuroscience. Aβ deposits aggregate into clumps in the brain which are a pathological hallmark of Alzheimer’s disease.

Expression of mutant human amyloid protein in animals results in deposits of Aβ plaques that induce abnormal increases in neuronal activity and impair the normal function of neuronal circuits.

Arthur Konnerth, Marc Busche and colleagues explored whether they could reverse these impairments by treating mice that overexpress the human mutant amyloid precursor protein with either of two different antibodies targeting Aβ (14 mice) or a control antibody (19 mice). They found that, although treatment with the Aβ targeting antibodies reduced the amount of plaques in the animals’ brains, it also increased the amount of hyperactive neurons.

This was true whether the treatment was given to older mice (14 treated, 19 control) or younger mice in which the accumulation of Aβ had yet to occur (10 treated, 13 control). The same therapies had no effect on neuronal activity in a group of normal mice (5 treated, 3 control), suggesting that the observed exacerbation in mutant mice is dependent on the presence of Aβ and cannot be explained by incidental effects of inflammation in response to the antibodies.

The authors note that, although other research has shown that anti-Aβ treatment can prevent the weakening of neuronal connections and memory impairments in animal models of Alzheimer’s disease, these benefits are not enough to repair neuronal dysfunction.

They suggest that their findings provide a cellular mechanism that may explain, in part, why treatments targeting Aβ in human clinical trials have failed to improve cognitive deficits. However, the authors point out that future studies are needed to determine whether the increase in abnormal neural activity seen in their animal models is related to the poor efficacy of Aβ therapy in patients.


ANAVEX™ 2-73

ANAVEX™ 2-73 is an orally available drug candidate developed to potentially modify Alzheimer’s disease rather than temporarily address its symptoms. It has a clean Phase 1 data profile and shows reversal of memory loss (anti-amnesic properties) and neuroprotection in several models of Alzheimer’s disease.

Successful Phase 1 Clinical Trial

A Phase 1 single ascending dose human clinical trial of ANAVEX 2-73 was successfully completed in healthy human volunteers. It was a randomized, placebo-controlled study. Healthy male volunteers aged 18 to 55 received single, ascending oral doses over the course of the trial. The trial objectives were to define the maximum tolerated dose, assess pharmacokinetics (PK), clinical and lab safety.


  • Dosing from 1-60 mg.
  • Maximum tolerated dose 55-60 mg; above the equivalent dose shown to have positive effects in mouse models of Alzheimer’s disease.
  • Well tolerated below the 55-60 mg dose with only mild adverse events in some volunteers.
  • Observed adverse events at doses above the maximum tolerated single dose included headache and dizziness, which were moderate in severity and reversible. These side effects are often seen with drugs that target central nervous system (CNS) conditions, including Alzheimer’s disease.
  • No significant changes in blood safety measurements.
  • No changes in ECG.
  • Favorable PK profile.
    • Rapid absorption into blood.
    • Dose proportional kinetics.

The trial was conducted in Germany by ABX-CRO in collaboration with the Technical University of Dresden. ABX-CRO and the Technical University of Dresden are well regarded for their experience with clinical trials and CNS compounds.


ANAVEX 2-73,

Clinical-stage biopharmaceutical company Anavex Life Sciences Corp. is working on an investigational oral treatment for Alzheimer’s disease called ANAVEX 2-73, with full PART A data and preliminary PART B data from its ongoing Phase 2a clinical trial to be presented during the Clinical Trials on Alzheimer’s Disease (CTAD) conference, November 5 and 7 in Barcelona, Spain.

The trial’s Principal Investigator, Stephen Macfarlane, who also serves as director and associate professor at Aged Psychiatry, Caulfield Hospital in Melbourne, Australia, will represent the company and host a late-breaking oral session entitled “New Exploratory Alzheimer’s Drug ANAVEX 2-73: Assessment of Safety and Cognitive Performance in a Phase 2a Study in mild-to-moderate Alzheimer’s Patients.” During the presentation, which will take place Saturday, November 7, at 9:45 a.m. CET, at the Gran Hotel Princesa Sofia, in Barcelona, Macfarlane will focus on the the multicenter Phase 2a clinical trial of ANAVEX 2-73. The study includes two separate phases and includes 32 mild-to-moderate Alzheimer’s patients. While PART A is a simple randomized, open-label, two-period, cross-over, adaptive trial of up to 36 days, PART B is an open-label extension trial for an additional 52 weeks.

The research intends to assess the maximum dose of treatment tolerated by patients, and to explore cognitive efficacy using mini-mental state examination score (MMSE), dose response, bioavailability, Cogstate and electroencephalographic (EEG) activity, including event-related potentials (EEG/ERP), as well as the preformance of ANAVEX 2-73 as an add-on therapy to donepezil (Aricept).

ANAVEX 2-73 is Anavex’s lead investigational treatment for Alzheimer’s disease, in line with the company’s goal of finding effective therapies for Alzheimer’s disease, other central nervous system (CNS) diseases, pain, and various types of cancer. The novel drug targets sigma-1 and muscarinic receptors, which are thought to decrease the amount of protein misfolding, beta amyloid tau and inflammation through upstream actions.

Last November, the biopharmaceutical company presented encouraging results from their phase 1 clinical trial for Anavex 2-73, during the CNS Summit 2014 in Boca Raton, Florida. The phase 1 study demonstrated that the treatment is safe and well tolerated, suggesting a favorable pharmacokinetics profile. During the randomized, double-blind, placebo-controlled study no severe adverse events were registered, while the adverse events reported included moderate and reversible headache and dizziness, which are common symptoms associated with drugs that target central nervous system (CNS) conditions, such as Alzheimer’s.

New Exploratory Alzheimer’s Drug ANAVEX 2-73: Assessment of Safety and Cognitive Performance in a Phase 2a Study in mild-to-moderate Alzheimer’s Patients

Steve Macfarlane, MD1 , Paul Maruff, PhD2 , Marco Cecchi, PhD3 , Dennis Moore, PhD3 , Anastasios Zografidis, PhD4 , Christopher Missling, PhD4 (1)

Caulfield Hospital, Melbourne, Australia (2), Cogstate, Melbourne, Australia (3), Neuronetrix, KY, USA (4), Anavex Life Sciences, Corp., New York, NY, USA

Background: Despite major efforts aimed at finding a treatment for Alzheimer’s disease (AD), progress in developing compounds that can relieve cognitive deficits associated with the disease has been slow. ANAVEX 2-73 is a sigma-1 and muscarinic receptor agonist that in preclinical studies has shown memory-preserving and neuroprotective effects. In our ongoing phase 2a clinical study we are assessing ANAVEX 2-73 safety in subjects with mild-to-moderated AD, and measuring drug effects on MMSE, EEG and Event Related Potentials (ERP) cognitive measures, and Cogstate test batteries to optimize dosing.

Methods: Thirty-two subjects that meet NINCDS-ADRDA criteria for probable AD are being recruited at up to seven clinical sites in Melbourne, Australia. Subjects are between 55 and 85 years of age, and have an MMSE of 16 to 28. In PART A of the study, participants are administered ANAVEX 2-73 orally and IV in an open-label, 2-period, cross-over trial with adaptive study design lasting up to 36 days for each participant. In PART B of the study, all participants are administered ANAVEX 2-73 daily orally. MMSE, EEG/ERP (P300) and Cogstate tests are performed at baseline and subsequently at weeks 12, 26, 38 and 52 of the PART B open label extension.

Results: The primary outcome of the study is safety, and ANAVEX 2-73 was well tolerated. In the secondary outcome endpoints preliminary analysis of data from subjects shows an average improvement of the MMSE score at week 5. A majority of all patients tested so far improved their respective MMSE score. The average EEG/ERP (P300 amplitude) signal also improved and also the average Cogstate test improved across the test batteries.

Conclusions: Data collected so far indicate that ANAVEX 2-73 is safe and well tolerated. Interim results also show improved cognitive performance after drug administration in subjects with mild-to-moderate AD. The current results seem to justify a prospective comparison with current standard of care in a larger clinical trial study. A more complete set of results will be available at the time of the conference.

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The Vibrant Philly Biotech Scene: Focus on KannaLife Sciences and the Discipline and Potential of Pharmacognosy

Curator and Interviewer: Stephen J. Williams, Ph.D.


philly2nightThis post is the third in a series of posts highlighting interviews with Philadelphia area biotech startup CEO’s and show how a vibrant biotech startup scene is evolving in the city as well as the Delaware Valley area. Philadelphia has been home to some of the nation’s oldest biotechs including Cephalon, Centocor, hundreds of spinouts from a multitude of universities as well as home of the first cloned animal (a frog), the first transgenic mouse, and Nobel laureates in the field of molecular biology and genetics. Although some recent disheartening news about the fall in rankings of Philadelphia as a biotech hub and recent remarks by CEO’s of former area companies has dominated the news, biotech incubators like the University City Science Center and Bucks County Biotechnology Center as well as a reinvigorated investment community (like PCCI and MABA) are bringing Philadelphia back. And although much work is needed to bring the Philadelphia area back to its former glory days (including political will at the state level) there are many bright spots such as the innovative young companies as outlined in these posts.

In today’s post, I had the opportunity to talk with both Dr. William Kinney, Chief Scientific Officer and Thoma Kikis, Founder/CMO of KannaLife Sciences based in the Pennsylvania Biotech Center of Bucks County.   KannaLifeSciences, although highlighted in national media reports and Headline news (HLN TV)for their work on cannabis-derived compounds, is a phyto-medical company focused on the discipline surrounding pharmacognosy, the branch of pharmacology dealing with natural drugs and their constituents.

Below is the interview with Dr. Kinney and Mr. Kikis of KannaLife Sciences and Leaders in Pharmaceutical Business Intelligence (LPBI)


PA Biotech Questions answered by Dr. William Kinney, Chief Scientific Officer of KannaLife Sciences



LPBI: Your parent company   is based in New York. Why did you choose the Bucks County Pennsylvania Biotechnology Center?


Dr. Kinney: The Bucks County Pennsylvania Biotechnology Center has several aspects that were attractive to us.  They have a rich talent pool of pharmaceutically trained medicinal chemists, an NIH trained CNS pharmacologist,  a scientific focus on liver disease, and a premier natural product collection.


LBPI: The Blumberg Institute and Natural Products Discovery Institute has acquired a massive phytochemical library. How does this resource benefit the present and future plans for KannaLife?


Dr. Kinney: KannaLife is actively mining this collection for new sources of neuroprotective agents and is in the process of characterizing the active components of a specific biologically active plant extract.  Jason Clement of the NPDI has taken a lead on these scientific studies and is on our Advisory Board. 


LPBI: Was the state of Pennsylvania and local industry groups support KannaLife’s move into the Doylestown incubator?


Dr. Kinney: The move was not State influenced by state or industry groups. 


LPBI: Has the partnership with Ben Franklin Partners and the Center provided you with investment opportunities?


Dr. Kinney: Ben Franklin Partners has not yet been consulted as a source of capital.


LPBI: The discipline of pharmacognosy, although over a century old, has relied on individual investigators and mainly academic laboratories to make initial discoveries on medicinal uses of natural products. Although there have been many great successes (taxol, many antibiotics, glycosides, etc.) many big pharmaceutical companies have abandoned this strategy considering it a slow, innefective process. Given the access you have to the chemical library there at Buck County Technology Center, the potential you had identified with cannabanoids in diseases related to oxidative stress, how can KannaLife enhance the efficiency of finding therapeutic and potential preventive uses for natural products?


Dr. Kinney: KannaLife has the opportunity to improve upon natural molecules that have shown medically uses, but have limitations related to safety and bioavailability. By applying industry standard medicinal chemistry optimization and assay methods, progress is being made in improving upon nature.  In addition KannaLife has access to one of the most commercially successful natural products scientists and collections in the industry.


LPBI: How does the clinical & regulatory experience in the Philadelphia area help a company like Kannalife?


Dr. Kinney: Within the region, KannaLife has access to professionals in all areas of drug development either by hiring displaced professionals or partnering with regional contract research organizations.


LPBI  You are focusing on an interesting mechanism of action (oxidative stress) and find your direction appealing (find compounds to reverse this, determine relevant disease states {like HCE} then screen these compounds in those disease models {in hippocampal slices}).  As oxidative stress is related to many diseases are you trying to develop your natural products as preventative strategies, even though those type of clinical trials usually require massive numbers of trial participants or are you looking to partner with a larger company to do this?


Dr. Kinney: Our strategy is to initially pursue Hepatic Encephalophy (HE) as the lead orphan disease indication and then partner with other organizations to broaden into other areas that would benefit from a neuroprotective agent.  It is expected the HE will be responsive to an acute treatment regimen.   We are pursuing both natural products and new chemical entities for this development path.



General Questions answered by Thoma Kikis, Founder/CMO of KannaLife Sciences


LPBI: How did KannaLife get the patent from the National Institutes of Health?


My name is Thoma Kikis I’m the co-founder of KannaLife Sciences. In 2010, my partner Dean Petkanas and I founded KannaLife and we set course applying for the exclusive license of the ‘507 patent held by the US Government Health and Human Services and National Institutes of Health (NIH). We spent close to 2 years working on acquiring an exclusive license from NIH to commercially develop Patent 6,630,507 “Cannabinoids as Antioxidants and Neuroprotectants.” In 2012, we were granted exclusivity from NIH to develop a treatment for a disease called Hepatic Encephalopathy (HE), a brain liver disease that stems from cirrhosis.


Cannabinoids are the chemicals that compose the Cannabis plant. There are over 85 known isolated Cannabinoids in Cannabis. The cannabis plant is a repository for chemicals, there are over 400 chemicals in the entire plant. We are currently working on non-psychoactive cannabinoids, cannabidiol being at the forefront.


As we started our work on HE and saw promising results in the area of neuroprotection we sought out another license from the NIH on the same patent to treat CTE (Chronic Traumatic Encephalopathy), in August of 2014 we were granted the additional license. CTE is a concussion related traumatic brain disease with long term effects mostly suffered by contact sports players including football, hockey, soccer, lacrosse, boxing and active military soldiers.


To date we are the only license holders of the US Government held patent on cannabinoids.



LPBI: How long has this project been going on?


We have been working on the overall project since 2010. We first started work on early research for CTE in early-2013.



LPBI: Tell me about the project. What are the goals?


Our focus has always been on treating diseases that effect the Brain. Currently we are looking for solutions in therapeutic agents designed to reduce oxidative stress, and act as immuno-modulators and neuroprotectants.


KannaLife has an overall commitment to discover and understand new phytochemicals. This diversification of scientific and commercial interests strongly indicates a balanced and thoughtful approach to our goals of providing standardized, safer and more effective medicines in a socially responsible way.


Currently our research has focused on the non-psychoactive cannabidiol (CBD). Exploring the appropriate uses and limitations and improving its safety and Metered Dosing. CBD has a limited therapeutic window and poor bioavailability upon oral dosing, making delivery of a consistent therapeutic dose challenging. We are also developing new CBD-like molecules to overcome these limitations and evaluating new phytochemicals from non-regulated plants.


KannaLife’s research is led by experienced pharmaceutically trained professionals; Our Scientific team out of the Pennsylvania Biotechnology Center is led by Dr. William Kinney and Dr. Douglas Brenneman both with decades of experience in pharmaceutical R&D.



LPBI: How do cannabinoids help neurological damage? -What sort of neurological damage do they help?


Cannabinoids and specifically cannabidiol work to relieve oxidative stress, and act as immuno-modulators and neuroprotectants.


So far our pre-clinical results show that cannabidiol is a good candidate as a neuroprotectant as the patent attests to. Our current studies have been to protect neuronal cells from toxicity. For HE we have been looking specifically at ammonia and ethanol toxicity.



– How did it go from treating general neurological damage to treating CTE? Is there any proof yet that cannabinoids can help prevent CTE? What proof?


We started examining toxicity first with ammonia and ethanol in HE and then posed the question; If CBD is a neuroprotectant against toxicity then we need to examine what it can do for other toxins. We looked at CTE and the toxin that causes it, tau. We just acquired the license in August from the NIH for CTE and are beginning our pre-clinical work in the area of CTE now with Dr. Ron Tuma and Dr. Sara Jane Ward at Temple University in Philadelphia.



LPBI: How long until a treatment could be ready? What’s the timeline?


We will have research findings in the coming year. We plan on filing an IND (Investigational New Drug application) with the FDA for CBD and our molecules in 2015 for HE and file for CTE once our studies are done.



LPBI: What other groups are you working with regarding CTE?


We are getting good support from former NFL players who want solutions to the problem of concussions and CTE. This is a very frightening topic for many players, especially with the controversy and lawsuits surrounding it. I have personally spoken to several former NFL players, some who have CTE and many are frightened at what the future holds.


We enrolled a former player, Marvin Washington. Marvin was an 11 year NFL vet with NY Jets, SF 49ers and won a SuperBowl on the 1998 Denver Broncos. He has been leading the charge on KannaLife’s behalf to raise awareness to the potential solution for CTE.


We tried approaching the NFL in 2013 but they didn’t want to meet. I can understand that they don’t want to take a position. But ultimately, they’re going to have to make a decision and look into different research to treat concussions. They have already given the NIH $30 Million for research into football related injuries and we hold a license with the NIH, so we wanted to have a discussion. But currently cannabinoids are part of their substance abuse policy connected to marijuana. Our message to the NFL is that they need to lead the science, not follow it.


Can you imagine the NFL’s stance on marijuana treating concussions and CTE? These are topics they don’t want to touch but will have to at some point.


LPBI: Thank you both Dr. Kinney and Mr. Kikis.


Please look for future posts in this series on the Philly Biotech Scene on this site

Also, if you would like your Philadelphia biotech startup to be highlighted in this series please contact me or

http://pharmaceuticalintelligence.com at:

sjwilliamspa@comcast.net or @StephenJWillia2  or @pharma_BI.

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Other posts on this site in this VIBRANT PHILLY BIOTECH SCENE SERIES OR referring to PHILADELPHIA BIOTECH include:

The Vibrant Philly Biotech Scene: Focus on Computer-Aided Drug Design and Gfree Bio, LLC

RAbD Biotech Presents at 1st Pitch Life Sciences-Philadelphia

The Vibrant Philly Biotech Scene: Focus on Vaccines and Philimmune, LLC

What VCs Think about Your Pitch? Panel Summary of 1st Pitch Life Science Philly

1st Pitch Life Science- Philadelphia- What VCs Really Think of your Pitch

LytPhage Presents at 1st Pitch Life Sciences-Philadelphia

Hastke Inc. Presents at 1st Pitch Life Sciences-Philadelphia

PCCI’s 7th Annual Roundtable “Crowdfunding for Life Sciences: A Bridge Over Troubled Waters?” May 12 2014 Embassy Suites Hotel, Chesterbrook PA 6:00-9:30 PM

Pfizer Cambridge Collaborative Innovation Events: ‘The Role of Innovation Districts in Metropolitan Areas to Drive the Global an | Basecamp Business

Mapping the Universe of Pharmaceutical Business Intelligence: The Model developed by LPBI and the Model of Best Practices LLC



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More Complexity in Protein Evolution

Author and Curator: Larry H. Bernstein, MD, FCAP 

Lactate dehydrogenase like crystallin: a potentially protective shield for Indian spiny-tailed lizard (Uromastix ltardwickit) lens against environmental stress?
A Atta, A Ilyas, Z Hashim, A Ahmed and S Zarina
The Protein Journal 2014; 33(2), p. 128-34.

Taxon specific lens crystallins in ve1iebrates are either similar or identical with various metabolic enzymes. These bifunctional crystallins serve as structural protein in lens along with their catalytic role. In the present study, we have partially purified and characterized lens crystallin from Indian spiny-tailed lizard (Uroma stix hardwickii). We have found lactate dehydrogenase (LDH) activity in lens indicating presence of an enzyme crystallin with dual functions. Taxon specific lens crystallins are product of gene sharing or gene duplication phenomenon where a pre-existing enzyme is recruited as lens crystallin in addition to structural role. In lens, same gene adopts refractive role in lens without modification or loss of pre-existing function during gene sharing phenomenon. Apart from conventional role of structural protein, LDH activity containing crystallin in Uromastix hardwickii lens is likely to have adaptive characteristics to offer protection against toxic effects of oxidative stress and ultraviolet light, hence justifying its recruitment. Taxon specific crystallins may serve as good models to understand structure-function relationship of these proteins.

αB-Crystallin and 27-kd Heat Shock Protein Are Regulated by Stress Conditions in the Central Nervous System and Accumulate in Rosenthal Fibers
T Iwaki, A Iwaki, J Tateishi, Y Sakaki, and JE Goldmant
Ameri J Pathol  1993; 143(2):487-495.

To understand the significance of the accumulation of αB-crystallin in Rosenthal fibers within astrocytes, the expression and metabolism of αB-crystallin in glioma cell lines were examined under the conditions of heat and oxidative stress. αB-crystallin mRNA was increased after both stresses, and αB-crystallin protein moved from a detergent-soluble to a detergent-insoluble form. In addition, Western blotting of Alexander’s  disease brain homogenates revealed that the 27-kd heat shock protein (HSP27), which is related to αB-crystallin, accumulates along with αB-crystallin. The presence of HSP27 in Rosenthal fibers was directly demonstrated by immunohistochemistry. Our results suggest that astrocytes in Alexander’s disease may be involved in an as yet unknown kind of stress reaction that causes the accumulation of αB-ccystallin and HSP27 and results in Rosenthal fiber formation.

α-Crystallin can function as a molecular chaperone
Joseph Horwitz
Proc. Nadl. Acad. Sci. USA Nov 1992; 89: 10449-10453. Biochemistry

The α-crystallins (αA and αB) are major lens structural proteins of the vertebrate eye that are related to the small heat shock protein family. In addition, crystallins (especially αB) are found in many cells organs outside the lens, and aα is overexpressed in several neurological disorders and in cell lines under stress conditions. Here I show that α-crystallin can function as a molecular chaperone. Stoichiometric amounts of αA and αB suppress thermally induced aggregation of various enzymes. In particular, α-crystalln is very efficient in suppressing the thermally induced aggregation of β- and y-crystallins, the two other major mammalian stuctural lens proteins. α-Crystallin was also effective in preventing aggregation and in refolding guanidine hydrochloride-denatured y-crystallin, as judged by circular dichroism spectroscopy. My results thus indicate that α-crystallin refracts light and protects proteins from aggregation in the transparent eye lens and that in nonlens cells α-crystallin may have other functions in addition to its capacity to suppress aggregation of proteins.

Gene sharing by δ-crystallin and argininosuccinate Iyase
J Piatigorsky, WE O’Brient, BL Norman, K Kalumuckt, GJ Wistow, T Borras, et al.
Proc. Natl. Acad. Sci. USA  May 1988; 85: 3479-3483. Evolution.

The lens structural protein δ-crystallin and the metabolic enzyme argininosuccinate lyase (ASL; Largininosuccinate argine-lyase, EC have striking sequence similarity. We have demonstrated that duck δ-crystallin has enormously high ASL activity, while chicken δ-crystallin has lower but significant activity. The lenses of these birds had much greater ASL activity than other tissues, suggesting that ASL is being expressed at unusually high levels as a structural component. In Southern blots of human genomic DNA, chicken δ1-crystallin cDNA hybridized only to the human ASL gene; moreover, the two chicken δ-crystallin genes accounted for all the sequences in the chicken genome able to cross-hybridize with a human ASL cDNA, with preferential hybridization to the δ2 gene. Correlations of enzymatic activity and recent data on mRNA levels in the chicken lens suggest that ASL activity depends on expression of the δ2-crystallin gene. The data indicate that the same gene, at least in ducks, encodes two different functions, an enzyme (ASL) and a structural protein (δ-crystallin), although in chickens specialization and separation of functions may have occurred.

Gecko i-crystallin: How cellular retinol-binding protein became an eye lens ultraviolet filter
PJ L Werten, Beate Roll, DMF van Aalten, and WW de Jong
PNAS Mar 2000; 97(7): 3282–3287 http://pnas.org/cgi/doi/10.1073ypnas.050500597

Eye lenses of various diurnal geckos contain up to 12% i-crystallin. This protein is related to cellular retinol-binding protein type I (CRBP I) but has 3,4-didehydroretinol, rather than retinol, as a ligand. The 3,4-didehydroretinol gives the lens a yellow color, thus protecting the retina by absorbing short-wave radiation. i-Crystallin could be either the gecko’s housekeeping CRBP I, recruited for an additional function in the lens, or the specialized product of a duplicated CRBP I gene. The finding of the same CRBP I-like sequence in lens and liver cDNA of the gecko Lygodactylus picturatus now supports the former option. Comparison with i-crystallin of a distantly related gecko, Gonatodes vittatus, and with mammalian CRBP I, suggests that acquiring the additional lens function is associated with increased amino acid changes. Compared with the rat CRBP I structure, the i-crystallin model shows reduced negative surface charge, which might facilitate the required tight protein packing in the lens. Other changes may provide increased stability, advantageous for a long-living lens protein, without frustrating its role as retinol transporter outside the lens. Despite a number of replacements in the ligand pocket, recombinant i-crystallin binds 3,4-didehydroretinol and retinol with similar and high affinity (1.6 nM). Availability of ligand thus determines whether it binds 3,4-didehydroretinol, as in the lens, or retinol, in other tissues. i-Crystallin presents a striking example of exploiting the potential of an existing gene without prior duplication.

Expression of βA3/A1-crystallin in the developing and adult rat eye
G Parthasarathy, Bo Ma, C Zhang, C Gongora, JS Zigler, MK Duncan, D Sinha
J Molec Histol 2011; 42(1): 59-69. http://dx.doi.org:/10.1007/s10735-010-9307-1

Crystallins are very abundant structural proteins of the lens and are also expressed in other tissues. We have previously reported a spontaneous mutation in the rat βA3/A1-crystallin gene, termed Nuc1, which has a novel, complex, ocular phenotype. The current study was undertaken to compare the expression pattern of this gene during eye development in wild type and Nuc1 rats by in situ hybridization (ISH) and immunohistochemistry (IHC).
βA3/A1-crystallin expression was first detected in the eyes of both wild type and Nuc1 rats at embryonic (E) day 12.5 in the posterior portion of the lens vesicle, and remained limited to the lens fibers throughout fetal life.
After birth, βA3/A1-crystallin expression was also detected in the neural retina (specifically in the astrocytes and ganglion cells) and in the retinal pigmented epithelium (RPE).
This suggested that βA3/A1-crystallin is not only a structural protein of the lens, but has cellular function(s) in other ocular tissues.
In summary, expression of βA3/A1-crystallin is controlled differentially in various eye tissues with lens being the site of greatest expression.
Similar staining patterns, detected by ISH and IHC, in wild type and Nuc1 animals suggest that functional differences in the protein, rather than changes in mRNA/protein level of expression likely account for developmental abnormalities in Nuc1.

βA3/A1Crystallin controls anoikis-mediated cell death in astrocytes by modulating PI3K/AKT/mTOR and ERK survival pathways through the PKD/Bit1-signaling axis
B Ma, T Sen, L Asnaghi, M Valapala, F Yang, S Hose, D S McLeod, Y Lu, et la.
Cell Death and Disease 2011; 2(10). http://dx.doi.org:/10.1038/cddis.2011.100

During eye development, apoptosis is vital to the maturation of highly specialized structures such as the lens and retina. Several forms of apoptosis have been described, including anoikis, a form of apoptosis triggered by inadequate or inappropriate cell–matrix contacts. The anoikis regulators, Bit1 (Bcl-2 inhibitor of transcription-1) and protein kinase-D (PKD), are expressed in developing lens when the organelles are present in lens fibers, but are downregulated as active denucleation is initiated.
We have previously shown that in rats with a spontaneous mutation in the Cryba1 gene, coding for βA3/A1-crystallin, normal denucleation of lens fibers is inhibited. In rats with this mutation (Nuc1), both Bit1 and PKD remain abnormally high in lens fiber cells. To determine whether βA3/A1-crystallin has a role in anoikis, we induced anoikis in vitro and conducted mechanistic studies on astrocytes, cells known to express βA3/A1-crystallin.
The expression pattern of Bit1 in retina correlates temporally with the development of astrocytes. Our data also indicate that loss of βA3/A1-crystallin in astrocytes results in a failure of Bit1 to be trafficked to the Golgi, thereby suppressing anoikis. This loss of βA3/A1-crystallin also induces insulin-like growth factor-II, which increases cell survival and growth by modulating the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR and extracellular signal-regulated kinase pathways. We propose that βA3/A1-crystallin is a novel regulator of both life and death decisions in ocular astrocytes.

βA3/A1-crystallin in astroglial cells regulates retinal vascular remodeling during development
D Sinha, A Klise, Y Sergeev, S Hose, IA Bhutto, L Hackler Jr., T Malpic-llanos, et al.
Molec Cell Neurosci 2008; 37(1): 85-95.


Vascular remodeling is a complex process critical to development of the mature vascular system. Astrocytes are known to be indispensable for initial formation of the retinal vasculature; our studies with the Nuc1 rat provide novel evidence that these cells are also essential in the retinal vascular remodeling process.
Nuc1 is a spontaneous mutation in the Sprague–Dawley rat originally characterized by nuclear cataracts in the heterozygote and microphthalmia in the homozygote. We report here that the Nuc1 allele results from mutation of the βA3/A1-crystallin gene, which in the neural retina is expressed only in astrocytes. We demonstrate striking structural abnormalities in Nuc1 astrocytes with profound effects on the organization of intermediate filaments. While vessels form in the Nuc1 retina, the subsequent remodeling process required to provide a mature vascular network is deficient. Our data implicate βA3/A1-crystallin as an important regulatory factor mediating vascular patterning and remodeling in the retina.

A developmental defect in astrocytes inhibits programmed regression of the hyaloid vasculature in the mammalian eye
C Zhang, L Asnaghi, C Gongora, B Patek, S Hose, Bo Ma, MA Fard, L Brako, et al.
Eur J Cell Biol 2011; 90(5): 440-448.

Previously we reported the novel observation that astrocytes ensheath the persistent hyaloid artery, both in the Nuc1 spontaneous mutant rat, and in human PFV (persistent fetal vasculature) disease (Developmental Dynamics 234:36–47, 2005). We now show that astrocytes isolated from both the optic nerve and retina of Nuc1 rats migrate faster than wild type astrocytes. Aquaporin 4 (AQP4), the major water channel in astrocytes, has been shown to be important in astrocyte migration. We demonstrate that AQP4 expression is elevated in the astrocytes in PFV conditions, and we hypothesize that this causes the cells to migrate abnormally into the vitreous where they ensheath the hyaloid artery. This abnormal association of astrocytes with the hyaloid artery may impede the normal macrophage-mediated remodeling and regression of the hyaloid system.

βA3/A1-crystallin is required for proper astrocyte template formation and vascular remodeling in the retina.
D Sinha; WJ Stark; M Valapala; IA Bhutto; M Cano; S Hose; GA Lutty; et al.  Transgenic research 2012; 21(5):1033-42.

Nuc1 is a spontaneous rat mutant resulting from a mutation in the Cryba1 gene, coding for βA3/A1-crystallin. Our earlier studies with Nuc1 provided novel evidence that astrocytes, which express βA3/A1-crystallin, have a pivotal role in retinal remodeling. The role of astrocytes in the retina is only beginning to be explored. One of the limitations in the field is the lack of appropriate animal models to better investigate the function of astrocytes in retinal health and disease. We have now established transgenic mice that overexpress the Nuc1 mutant form of Cryba1, specifically in astrocytes. Astrocytes in wild type mice show normal compact stellate structure, producing a honeycomb-like network. In contrast, in transgenics over-expressing the mutant (Nuc1) Cryba1 in astrocytes, bundle-like structures with abnormal patterns and morphology were observed. In the nerve fiber layer of the transgenic mice, an additional layer of astrocytes adjacent to the vitreous is evident. This abnormal organization of astrocytes affects both the superficial and deep retinal vascular density and remodeling. Fluorescein angiography showed increased venous dilation and tortuosity of branches in the transgenic retina, as compared to wild type. Moreover, there appear to be fewer interactions between astrocytes and endothelial cells in the transgenic retina than in normal mouse retina. Further, astrocytes overexpressing the mutant βA3/A1-crystallin migrate into the vitreous, and ensheath the hyaloid artery, in a manner similar to that seen in the Nuc1 rat. Together, these data demonstrate that developmental abnormalities of astrocytes can affect the normal remodeling process of both fetal and retinal vessels of the eye and that βA3/A1-crystallin is essential for normal astrocyte function in the retina.

Ontogeny of oxytocin and vasopressin receptor binding in the lateral septum in prairie and montane voles
Z. Wang, L.J. Young
Developmental Brain Research 1997; 104:191–195.

Adult prairie (Microtus ochrogaster). and montane voles (M. montanus). differ in the distribution of oxytocin OT. and vasopressin AVP receptor binding in the brain. The present study examined the ontogenetic pattern of these receptor bindings in the lateral septum in both species to determine whether adult differences in the receptor binding are derived from a common pattern in development. In both species, OT and AVP receptor binding in the lateral septum were detected neonatally, increased during development, and reached the adult level at weaning third week. The progression of OT and AVP receptor differed, as OT receptor binding increased continually until weaning while AVP receptor binding did not change in the first week, increased rapidly in the second week, and was sustained thereafter. For both receptors, the binding increased more rapidly in montane than in prairie voles, resulting in species differences in receptor binding at weaning and in adulthood. Together, these data indicate that OT and AVP could affect the brain during development in a peptide- and species-specific manner in voles.

Evolution of the vasopressin/oxytocin superfamily: Characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis
RE Van Kesteren, AB Smit, RW Dirksi, ND De With, WPM Geraerts, and J Joosse
Proc. Nadl. Acad. Sci. USA May 1992; 89: 4593-4597. Neurobiology

Although the nonapeptide hormones vasopressin, oxytocin, and related peptides from vertebrates and some nonapeptides from invertebrates share similarities in amino acid sequence, their evolutionary relationships are not dear. To investigate this issue, we doned a cDNA encoding a vasopressin-related peptide, Lys-conopressin, produced in the central nervous system of the gastropod mollusc Lymnaea stagnalis. The predicted preproconopressin has the overall architecture of vertebrate preprovasopressins, with a signal peptide, Lys-conopressin, that is flanked at the C terminus by an amidation signal and a pair of basic residues, followed by a neurophysin domain. The Lymnaea neurophysin and the vertebrate neurophysins share high sequence identity, which includes the conservation of all 14 cysteine residues. In addition, the Lymnaea neurophysin possesses unique structural characteristics. It contains a putative N-linked glycosylation site at a position in the vertebrate neurophysins where a strictly conserved tyrosine residue, which plays an essential role in binding of the nonapptide hormones, is found. The C-terminal copeptin homologous extension of the Lymnaea neurophysin has low sequence identity with the vertebrate counterparts and is probably not cleaved from the prohormone, as are the mammalin copeptins. The conopressin gene is expressed in only a few neurons in both pedal ganglia of the central nervous system. The conopressin transcript is present in two sizes, due to alternative use of polyadenylylation signals. The data presented here demonstrate that the typical organization of the prohormones of the vasopressin/oxytocin superfamily must have been present in the common ancestors of vertebrates and invertebrates.

A common allele in the oxytocin receptor gene (OXTR) impacts prosocial temperament and human hypothalamic-limbic structure and function
H Tosta, B Kolachanaa, S Hakimia, H Lemaitrea, BA Verchinskia, et al.
PNAS Aug 3, 2010; 107(31): 13936–13941

The evolutionarily highly conserved neuropeptide oxytocin is a key mediator of social and emotional behavior in mammals, including humans. A common variant (rs53576) in the oxytocin receptor gene (OXTR) has been implicated in social-behavioral phenotypes, such as maternal sensitivity and empathy, and with neuropsychiatric disorders associated with social impairment, but the intermediate neural mechanisms are unknown. Here, we used multimodal neuroimaging in a large sample of healthy human subjects to identify structural and functional alterations in OXTR risk allele carriers and their link to temperament. Activation and interregional coupling of the amygdala during the processing of emotionally salient social cues was significantly affected by genotype. In addition, evidence for structural alterations in key oxytocinergic regions emerged, particularly in the hypothalamus. These neural characteristics predicted lower levels of reward dependence, specifically in male risk allele carriers. Our findings identify sex-dependent mechanisms impacting the structure and function of hypothalamic-limbic circuits that are of potential clinical and translational significance.
Test of Association Between 10 SNPs in the Oxytocin Receptor Gene and Conduct Disorder
JT Sakai, TJ Crowley, MC Stallings, M McQueen, JK Hewitt, C Hopfer, et al.
Psychiatr Genet. 2012 Apr; 22(2): 99–102. http://dx.doi.org:/10.1097/YPG.0b013e32834c0cb2

Animal and human studies have implicated oxytocin (OXT) in affiliative and prosocial behaviors. We tested whether genetic variation in the OXT receptor (OXTR) gene is associated with conduct disorder (CD).
Utilizing a family-based sample of adolescent probands recruited from an adolescent substance abuse treatment program, control probands and their families (total sample n=1,750), we conducted three tests of association with CD and 10 SNPs (single nucleotide polymorphisms) in the OXTR gene: (1) family-based comparison utilizing the entire sample; (2) within-Whites, case control comparison of adolescent patients with CD and controls without CD; and (3) within-Whites case-control comparison of parents of patients and parents of controls.
Family-based association tests failed to show significant results (no results p<0.05). While strictly correcting for the number of tests (α=0.002), adolescent patients with CD did not differ significantly from adolescent controls in genotype frequency for the OXTR SNPs tested; similarly, comparison of OXTR genotype frequencies for parents failed to differentiate patient and control family type, except a trend association for rs237889 (p=0.004). In this sample, 10 SNPs in the OXTR gene were not significantly associated with CD.

Leu55Pro transthyretin accelerates subunit exchange and leads to rapid formation of hybrid tetramers
CA Keetch, EHC Bromley, MG McCammon, N Wang, J Christodoulou, CV Robinson
JBC  Oct 11, 2005 M508753200. http://jbc.org/cgi/doi/10.1074/jbc.M508753200

Transthyretin is a tetrameric protein associated with the commonest form of

systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry we compared subunit exchange in wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, Leu55Pro. Wild-type subunit exchange occurs via both monomers and dimers , while exchange via dimers is the dominant mechanism for the Leu55Pro variant. Since patients with the Leu55Pro mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and Leu55Pro tetramers . We found that hybrid tetramers containing two or three Leu55Pro subunits dominate in the early stages of the reaction. Surprisingly we also found that in the presence of Leu55Pro transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidos is.

Beyond Genetic Factors in Familial Amyloidotic Polyneuropathy: Protein Glycation and the Loss of Fibrinogen’s Chaperone Activity
G da Costa, RA Gomes, A Guerreiro, E Mateus, E Monteiro, et al.
PLoS ONE 2011; 6(10): e24850. http://dx.doi.org:/10.1371/journal.pone.0024850

Familial amyloidotic polyneuropathy (FAP) is a systemic conformational disease characterized by extracellular amyloid fibril formation from plasma transthyretin (TTR). This is a crippling, fatal disease for which liver transplantation is the only effective therapy. More than 80 TTR point mutations are associated with amyloidotic diseases and the most widely accepted disease model relates TTR tetramer instability with TTR point mutations. However, this model fails to explain two observations. First, native TTR also forms amyloid in systemic senile amyloidosis, a geriatric disease. Second, age at disease onset varies by decades for patients bearing the same mutation and some mutation carrier individuals are asymptomatic throughout their lives. Hence, mutations only accelerate the process and non-genetic factors must play a key role in the molecular mechanisms of disease. One of these factors is protein glycation, previously associated with conformational diseases like Alzheimer’s and Parkinson’s. The glycation hypothesis in FAP is supported by our previous discovery of methylglyoxal-derived glycation of amyloid fibrils in FAP patients. Here we show that plasma proteins are differentially glycated by methylglyoxal in FAP patients and that fibrinogen is the main glycation target. Moreover, we also found that fibrinogen interacts with TTR in plasma. Fibrinogen has chaperone activity which is compromised upon glycation by methylglyoxal. Hence, we propose that methylglyoxal glycation hampers the chaperone activity of fibrinogen, rendering TTR more prone to aggregation, amyloid formation and ultimately, disease.

Aromatic Sulfonyl Fluorides Covalently Kinetically Stabilize Transthyretin to Prevent Amyloidogenesis while Affording a Fluorescent Conjugate
NP Grimster, S Connelly, A Baranczak, J Dong, …, JW Kelly
J Am Chem Soc. 2013 Apr 17; 135(15): 5656–5668. http://dx.doi.org:/10.1021/ja311729d

Molecules that bind selectively to a given protein and then undergo a rapid chemoselective reaction to form a covalent conjugate have utility in drug development. Herein a library of 1,3,4-oxadiazoles substituted at the 2 position with an aryl sulfonyl fluoride and at the 5 position with a substituted aryl known to have high affinity for the inner thyroxine binding subsite of transthyretin (TTR) were conceived of by structure-based design principles and were chemically synthesized. When bound in the thyroxine binding site, most of the aryl sulfonyl fluorides react rapidly and chemoselectively with the pKa-perturbed K15 residue, kinetically stabilizing TTR and thus preventing amyloid fibril formation, known to cause polyneuropathy. Conjugation t50s range from 1 to 4 min, ~ 1400 times faster than the hydrolysis reaction outside the thyroxine binding site. Xray crystallography confirms the anticipated binding orientation and sheds light on the sulfonyl fluoride activation leading to the sulfonamide linkage to TTR. A few of the aryl sulfonyl fluorides efficiently form conjugates with TTR in plasma. A few of the TTR covalent kinetic stabilizers synthesized exhibit fluorescence upon conjugation and therefore could have imaging applications as a consequence of the environment sensitive fluorescence of the chromophore.

Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis
A Lim, T Prokaeva, ME Mccomb, LH Connors, M Skinner, and CE Costello
Protein Science 2003; 12:1775–1786.

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild-type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease. Previously, we have developed a mass spectrometry-based approach, in combination with direct DNA sequence analysis, to fully identify TTR variants. Our methodology uses immunoprecipitation to isolate TTR from serum, and electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry (MS) peptide mapping to identify TTR variants and posttranslational modifications. Unambiguous identification of the amino acid substitution is performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis. The MS and MS/MS analyses also yield information about posttranslational modifications. Using this approach, we have recently identified a novel pathologic TTR variant. This variant has an amino acid substitution (Phe — Cys) at position 33. In addition, like the Cys10 present in the wild type and in this variant, the Cys33 residue was both S-sulfonated and S-thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.

Evolutionary relationships of lactate dehydrogenases (LDHs) from mammals, birds, an amphibian, fish, barley, and bacteria: LDH cDNA sequences from Xenopus, pig, and rat
S Tsuji, MA Qureshi, EW Hou, WM Fitch, and S S.-L. Li
Proc. Natl. Acad. Sci. USA Sep 1994; 91: 9392-9396. Evolution

The nucleotide sequences of the cDNAs encoding LDH (EC subunits LDH-A (muscle), LDH-B (liver), and LDH-C (oocyte) from Xenopus laevis, LDH-A (muscle) and LDH-B (heart) from pig, and LDH-B (heart) and LDH-C (testis) from rat were determined. These seven newly deduced amino acid sequences and 22 other published LDH sequences, and three unpublished fish LDH-A sequences kindly provided by G. N. Somero and D. A. Powers, were used to construct the most parsimonious phylogenetic tree of these 32 LDH subunits from mammals, birds, an amphibian, fish, barley, and bacteria. There have been at least six LDH gene duplications among the vertebrates. The Xenopus LDH-A, LDH-B, and LDH-C subunits are most closely related to each other and then are more closely related to vertebrate LDH-B than LDH-A. Three fish LDH-As, as well as a single LDH of lamprey, also seem to be more related to vertebrate LDH-B than to land vertebrate LDH-A. The mammalian LDH-C (testis) subunit appears to have diverged very early, prior to the divergence of vertebrate LDH-A and LDH-B subunits, as reported previously.

Evidence for neutral and selective processes in the recruitment of enzyme-crystallins in avian lenses
Graeme Wistow, Andrea Anderson, and Joram Piatigorsky
Proc. Natl. Acad. Sci. USA Aug 1990; 87: 6277-6280, Evolution

In apparent contrast to most other tissues, the ocular lenses in vertebrates show striking differences in protein composition between taxa, most notably in the recruitment of different enzymes as major structural proteins. This variability appears to be the result of at least partially neutral evolutionary processes, although there is also evidence for selective modification in molecular structure. Here we describe a bird, the chimney swift (Chaetura pelagica), that lacks δ-crystallin/ argininosuccinate lyase, usually the major crystallin of avian lenses. Clearly, δ-crystallin is not specifically required for a functionally effective avian lens. Furthermore the lens composition of the swift is more similar to that of the related hummingbirds than to that of the barn swallow (Hirundo rustica), suggesting that phylogeny is more important than environmental selection in the recruitment of crystallins. However differences in ε-crystallin/lactate dehydrogenase-B sequence between swift and hummingbird and other avian and reptilian species suggest that selective pressures may also be working at the molecular level. These differences also confirm the close relationship between swifts and hummingbirds.

Enzyme/crystallins and extremely high pyridine nucleotide levels in the eye lens.
Zigler, J. S., Jr.; Rao, P. V.
FASEB J. 1991; 3: 223-225.

Taxon-specific crystallins are proteins present in high abundance in the lens of phylogenetically restricted groups of animals. Recently it has been found that these proteins are actually enzymes which the lens has apparently adopted to serve as structural proteins. Most of these proteins have been shown to be identical to, or related to, oxidoreductases. In guinea pig lens, which contains zeta-crystallin, a protein with an NADPH dependent oxidoreductase activity, the levels of both NADPH and NADP* are extremely high and correlate with the concentration of zeta-crystallin. We report here nucleotide assays on lenses from vertebrates containing other enzyme/crystallins. In each case where the enzyme/crystallin is a pyridine nucleotide-binding protein the level of that particular nucleotide is extremely high in the lens. The presence of an enzyme/crystallin does not affect the lenticular concentrations of those nucleotides which are not specifically bound. The possibility that nucleotide binding may be a factor in the selection of some enzymes to serve as enzyme/crystallins is considered.

Comparison of stability properties of lactate dehydrogenase B4/ε-crystallin from different species
CEM Voorter, LTM Wintjes, PWH Heinstra, H Bloemendal and WW De Jong
Eur. J. Biochem. 1993; 211: 643-648

ε-Crystallin occurs as an abundant lens protein in many birds and in crocodiles and has been identified as heart-type lactate dehydrogenase (LDH-B4). Lens proteins have, due to their longevity and environmental conditions, extraordinary requirements for structural stability. To study lens protein stability, we compared various parameters of LDH-B4/ε-crystallin from lens and/or heart of duck, which has abundant amounts of this enzyme in its lenses, and of chicken and pig, which have no λ-crystallin. Measuring the thermostability of LDH-B4 from the different sources, the t50 values (temperature at which 50% of the enzyme activity remains after a 20-min period) for LDH-B4 from duck heart, duck lens and chicken heart were all found to be around 76°C whereas pig heart LDHB4 was less thermostable, having a t50 value of 625°C. A similar tendency was found with urea inactivation studies. Plotting the first-order rate constants obtained from inactivation kinetic plots against urea concentration, it was clear that LDH-B4 from pig heart was less stable in urea than the homologous enzymes from duck heart, chicken heart and duck lens. The duck and chicken enzymes were also much more resistant against proteolysis than the porcine enzyme. Therefore, it is concluded that avian LDH-B4 is structurally more stable than the homologous enzyme in mammals. This greater stability might make it suitable to function as a ε-crystallin, as in duck, but is not necessarily associated with high lens expression, as in chicken.

Duck lens ε-crystallin and lactate dehydrogenase B4 are identical: A single-copy gene product with two distinct functions
W Hendriks, JWM Mulders, MA Bibby, C Slingsby, H Bloemendal, and WW De Jong
Proc. Natl. Acad. Sci. USA Oct 1988; 85: 7114-7118. Biochemistry

To investigate whether or not duck lens ε-crystaliin and duck heart lactate dehydrogenase (LDH) B4 are the product of the same gene, we have isolated and sequenced cDNA clones of duck ε-crystallin. By using these clones we demonstrate that there is a single-copy Ldh-B gene in duck and in chicken. In the duck lens this gene is overexpressed, and its product is subject to posttranslational modification. Reconstruction of the evolutionary history of the LDH protein family reveals that the mammalian Ldh-C gene most probably originated from an ancestral Ldh-A gene and that the amino acid replacement rate in LDH-C is approximately 4 times the rate in LDH-A. Molecular modeling of LDH-B sequences shows that the increased thermostability of the avian tetramer might be explained by mutations that increase the number of ion pairs. Furthermore, the replacement of bulky side chains by glycines on the corners of the duck protein suggests an adaptation to facilitate close packing in the lens.

Lactate Dehydrogenase A as a Highly Abundant Eye Lens Protein in Platypus (Ornithorhynchus anatinus): Upsilon (υ)-Crystallin
T van Rheede,  R Amons, N Stewart, and WW de Jong
Mol. Biol. Evol. 2003; 20(06):994–998. http://dx.doi.org:/10.1093/molbev/msg116

Vertebrate eye lenses mostly contain two abundant types of proteins, the α-crystallins and the β/λ-crystallins. In addition, certain housekeeping enzymes are highly expressed as crystallins in various taxa. We now observed an unusual approximately 41-kd protein that makes up 16% to 18% of the total protein in the platypus eye lens. Its cDNA sequence was determined, which identified the protein as muscle-type lactate dehydrogenase A (LDH-A). It is the first observation of LDH-A as a crystallin, and we designate it upsilon (υ)-crystallin. Interestingly, the related heart-type LDH-B occurs as an abundant lens protein, known as ε-crystallin, in many birds and crocodiles. Thus, two members of the ldh gene family have independently been recruited as crystallins in different higher vertebrate lineages, suggesting that they are particularly suited for this purpose in terms of gene regulatory or protein structural properties. To establish whether platypus LDH-A/υ-crystallin has been under different selective constraints as compared with other vertebrate LDH-A sequences, we reconstructed the vertebrate Ldh-A gene phylogeny. No conspicuous rate deviations or amino acid replacements were observed.

Isozymes, moonlighting proteins and promiscous enzymes
M Nath Gupta, M Kapoor, AB Majumder and V Singh
Current Science Apr 2011; 100(8): 1152-1162.

The structures of isoenzymes differ and yet these catalyse the same type of reaction. These structures evolved to suit the physiological needs and are located in different parts of cells or tissues. Moonlighting proteins represent the same structure performing very different biological functions. Biological promiscuity reveals that the same active sites can catalyse different types of reactions. These three different phenomena, all illustrate similar evolutionary strategies. Viewed together, it emerges that biologists need to take a hard look at the ‘structure–function’ paradigm as well as the notions of biological specificity. Meanwhile, biotechnologists  continue to exploit the opportunities which ‘nonspecificity’ offers.

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Plant-based Nutrition, Neutraceuticals and Alternative Medicine: Article Compilation the Journal PharmaceuticalIntelligence.com

Curator: Larry H. Bernstein, MD, FCAP


  1. Green tea polyphenols alleviate early BBB damage
  2. What do you know about Plants and Neutraceuticals?

Author and Curator, Larry H. Bernstein, MD, FCAP


  1. The Final Considerations of the Role of Platelets and Platelet Endothelial Reactions in Atherosclerosis and Novel Treatments

Author and Curator: Larry H Bernstein, MD, FCAP


  1. Endothelial Function and Cardiovascular Disease

Author and Curator: Larry H Bernstein, MD, FCAP


  1. NO Nutritional remedies for hypertension and atherosclerosis. It’s 12 am: do you know where your electrons are?

Author and Reporter: Meg Baker, Ph.D., Registered Patent Agent


  1. Cocoa and Heart Health

Reporter: Larry H Bernstein, MD, FCAP


  1. Metabolomics: its applications in food and nutrition research

Reporter and Curator: Dr. Sudipta Saha, Ph.D.


  1. Japanese knotweed extract (Polygonum cuspidatum) Resveratrol 98%

Reporter: Larry H Bernstein, MD, FCAP   Stanford Lee, Shanghai Natural Bio-engineering Co., Ltd
Key products: resveratrol, curcumin,artemisinin,artemether,artesunate,dihydroartemisinin,Lumefantrine,etc


  1. Antimicrobial resistance
    Reporter: Larry H Bernstein, MD, FCAP   
  2. Macrocycles in new drug discovery
    Reporter: Larry H Bernstein, MD, FCAP     Jamie MallinsonIan Collins
    Future Medicinal Chemistry, Jul 2012, Vol. 4, No. 11, Pages 1409-1438.

Natural product macrocycles and their synthetic derivatives


  1. Lipid Metabolism

ALA and LA, LCPUFAs (EPA, DHA, and AA), eicosanoids, delta-3-desaturase, prostaglandins, leukotrienes

Ginseng fights fatigue in cancer patients, Mayo Clinic-led study finds https://pharmaceuticalintelligence.com/2014/08/15/lipid-metabolism/

  1. Ginseng fights fatigue in cancer patients, Mayo Clinic-led study finds

Reporter: Larry H Bernstein, MD, FCAP


  1. Scientists develop new cancer-killing compound from salad plant / 1,200 times more specific in killing certain kinds of cancer cells than currently available drugs
    Reporter: Larry H Bernstein, MD, FCAP
  2. Protein heals wounds, boosts immunity and protects from cancer – Lactoferrin
    Reporter: Larry H Bernstein, MD, FCAP
  3. Inula helenium ( elecampane ) 100% Effective against MRSA in vitro, 200 Strains
    Reporter: Larry H Bernstein, MD, FCAP
  4. Thymoquinone, an extract of nigella sativa seed oil, blocked pancreatic cancer cell growth and killed the cells by enhancing the process of programmed cell death.
    Reporter: Larry H Bernstein, MD, FCAP
  5. Cinnamon is lethal weapon against E. coli O157:H7
    Reporter: Larry H Bernstein, MD, FCAP
  6. Garlic compound fights source of food-borne illness better than antibiotics (100 times more effective than two popular antibiotics )

Reporter: Larry H Bernstein, MD, FCAP


  1. Reference Genes in the Human Gut Microbiome: The BGI Catalogue

Reporter: Aviva Lev-Ari, PhD, RN


  1. Study suggests consuming whey protein before meals could help improve blood glucose control in people with diabetes
    Reporter: Larry H Bernstein, MD, FCAP
  2. Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease
    Larry H. Bernstein, MD, FCAP, Curator
  3. Health benefit of anthocyanins from apples and berries noted for men
    Larry H. Bernstein, MD, FCAP, Curator
  4. Carrots Cut Men’s Prostate Cancer Risk by 50%
    Larry H. Bernstein, MD, FCAP, Reporter
  5. A Recipe To Make Cannabis Oil For A Chemotherapy Alternative
    Larry H. Bernstein, MD, FCAP, Reporter
  6. Plant flavonoid found to reduce inflammatory response in the brain: luteolin
    Larry H. Bernstein, MD, FCAP, Reporter
  7. Omega-3 fatty acids protect eyes against retinopathy, study finds
    Larry H. Bernstein, MD, FCAP, Reporter
  8. Scientists identify new pathogenic and protective microbes associated with severe diarrhea
    Larry H. Bernstein, MD, FCAP, Reporter
  9. 2,000-year-old herb regulates autoimmunity and inflammation / Chang Shan, from a type of hydrangea that grows in Tibet and Nepal
    Larry H. Bernstein, MD, FCAP, Reporter
  10. Turmeric-based drug effective on Alzheimer flies
    Larry H. Bernstein, MD, FCAP, Reporter
  11. Plant flavonoid luteolin blocks cell signaling pathways in colon cancer cells
    Larry H. Bernstein, MD, FCAP, Reporter
  12. Study Finds Shu Gan Liang Xue Herbal Formula Has Breast Cancer Anti Tumor Effect
    Larry H. Bernstein, MD, FCAP, Reporter
  13. HMPC Q&A Documents on Herbal Medicinal Products published
    Larry H. Bernstein, MD, FCAP, Reporter
  14. Garden Cress Extract Kills 97% of Breast Cancer Cells in Vitro
    Larry H. Bernstein, MD, FCAP, Reporter
  15. Moringa Oleifera Kills 97% of Pancreatic Cancer Cells in Vitro
    Larry H. Bernstein, MD, FCAP, Reporter

16. The Discovery and Properties of Avemar – Fermented Wheat Germ Extract: Carcinogenesis Suppressor
Larry H. Bernstein, MD, FCAP, Author and Curator



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Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation: a Compilation of Articles in the Journal http://pharmaceuticalintelligence.com

Compilation of References by Leaders in Pharmaceutical Business Intelligence in the Journal http://pharmaceuticalintelligence.com about
Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation

Curator: Larry H Bernstein, MD, FCAP


  1. The Human Proteome Map Completed

Reporter and Curator: Larry H. Bernstein, MD, FCAP


  1. Proteomics – The Pathway to Understanding and Decision-making in Medicine

Author and Curator, Larry H Bernstein, MD, FCAP


3. Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/10/22/advances-in-separations-technology-for-the-omics-and-clarification-         of-therapeutic-targets/

  1. Expanding the Genetic Alphabet and Linking the Genome to the Metabolome

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-                metabolome/

5. Genomics, Proteomics and standards

Larry H Bernstein, MD, FCAP, Author and Curator


6. Proteins and cellular adaptation to stress

Larry H Bernstein, MD, FCAP, Author and Curator




  1. Extracellular evaluation of intracellular flux in yeast cells

Larry H. Bernstein, MD, FCAP, Reviewer and Curator


  1. Metabolomic analysis of two leukemia cell lines. I.

Larry H. Bernstein, MD, FCAP, Reviewer and Curator


  1. Metabolomic analysis of two leukemia cell lines. II.

Larry H. Bernstein, MD, FCAP, Reviewer and Curator


  1. Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics

Reviewer and Curator, Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/22/metabolomics-metabonomics-and-functional-nutrition-the-next-step-          in-nutritional-metabolism-and-biotherapeutics/

  1. Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation

Larry H. Bernstein, MD, FCAP, Reviewer and curator

https://pharmaceuticalintelligence.com/2014/08/27/buffering-of-genetic-modules-involved-in-tricarboxylic-acid-cycle-              metabolism-provides-homeomeostatic-regulation/

Metabolic Pathways

  1. Pentose Shunt, Electron Transfer, Galactose, more Lipids in brief

Reviewer and Curator: Larry H. Bernstein, MD, FCAP


  1. Mitochondria: More than just the “powerhouse of the cell”

Ritu Saxena, PhD


  1. Mitochondrial fission and fusion: potential therapeutic targets?

Ritu saxena


4.  Mitochondrial mutation analysis might be “1-step” away

Ritu Saxena


  1. Selected References to Signaling and Metabolic Pathways in PharmaceuticalIntelligence.com

Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/14/selected-references-to-signaling-and-metabolic-pathways-in-                     leaders-in-pharmaceutical-intelligence/

  1. Metabolic drivers in aggressive brain tumors

Prabodh Kandal, PhD


  1. Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes

Writer and Curator, Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/10/22/metabolite-identification-combining-genetic-and-metabolic-                        information-genetic-association-links-unknown-metabolites-to-functionally-related-genes/

  1. Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation

Larry H Bernstein, MD, FCAP, author and curator

https://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-            glycolysis-metabolic-adaptation/

  1. Therapeutic Targets for Diabetes and Related Metabolic Disorders

Reporter, Aviva Lev-Ari, PhD, RD


10.  Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeomeostatic regulation

Larry H. Bernstein, MD, FCAP, Reviewer and curator

https://pharmaceuticalintelligence.com/2014/08/27/buffering-of-genetic-modules-involved-in-tricarboxylic-acid-cycle-              metabolism-provides-homeomeostatic-regulation/

11. The multi-step transfer of phosphate bond and hydrogen exchange energy

Larry H. Bernstein, MD, FCAP, Curator:

https://pharmaceuticalintelligence.com/2014/08/19/the-multi-step-transfer-of-phosphate-bond-and-hydrogen-                          exchange-energy/

12. Studies of Respiration Lead to Acetyl CoA


13. Lipid Metabolism

Author and Curator: Larry H. Bernstein, MD, FCAP


14. Carbohydrate Metabolism

Author and Curator: Larry H. Bernstein, MD, FCAP


15. Update on mitochondrial function, respiration, and associated disorders

Larry H. Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/08/update-on-mitochondrial-function-respiration-and-associated-                   disorders/

16. Prologue to Cancer – e-book Volume One – Where are we in this journey?

Author and Curator: Larry H. Bernstein, MD, FCAP


17. Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/04/04/introduction-the-evolution-of-cancer-therapy-and-cancer-research-          how-we-got-here/

18. Inhibition of the Cardiomyocyte-Specific Kinase TNNI3K

Author and Curator: Larry H. Bernstein, MD, FCAP


19. The Binding of Oligonucleotides in DNA and 3-D Lattice Structures

Author and Curator: Larry H. Bernstein, MD, FCAP


20. Mitochondrial Metabolism and Cardiac Function

Author and Curator: Larry H. Bernstein, MD, FCAP


21. How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia

Curator: Larry H. Bernstein, MD, FCAP


22. AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo

Author and Curator: Stephen J. Williams, PhD

https://pharmaceuticalintelligence.com/2013/03/12/ampk-is-a-negative-regulator-of-the-warburg-effect-and-suppresses-         tumor-growth-in-vivo/

23. A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

Author and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/12/03/a-second-look-at-the-transthyretin-nutrition-inflammatory-                         conundrum/

24. Mitochondrial Damage and Repair under Oxidative Stress

Author and Curator: Larry H. Bernstein, MD, FCAP


25. Nitric Oxide and Immune Responses: Part 2

Author and Curator: Aviral Vatsa, PhD, MBBS


26. Overview of Posttranslational Modification (PTM)

Writer and Curator: Larry H. Bernstein, MD, FCAP


27. Malnutrition in India, high newborn death rate and stunting of children age under five years

Writer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/15/malnutrition-in-india-high-newborn-death-rate-and-stunting-of-                   children-age-under-five-years/

28. Update on mitochondrial function, respiration, and associated disorders

Writer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/08/update-on-mitochondrial-function-respiration-and-associated-                  disorders/

29. Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease

Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2014/07/06/omega-3-fatty-acids-depleting-the-source-and-protein-insufficiency-         in-renal-disease/

30. Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine

Larry H. Bernstein, MD, FCAP, writer, and Aviva Lev- Ari, PhD, RN

https://pharmaceuticalintelligence.com/2014/04/27/larryhbernintroduction_to_cardiovascular_diseases-                                  translational_medicine-part_2/

31. Epilogue: Envisioning New Insights in Cancer Translational Biology
Series C: e-Books on Cancer & Oncology

Author & Curator: Larry H. Bernstein, MD, FCAP, Series C Content Consultant


32. Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone                         and Neurotransmitter

Writer and Curator: Larry H Bernstein, MD, FCAP and
Curator and Content Editor: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-                    hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocy

33. Cardiac Contractility & Myocardial Performance: Therapeutic Implications of Ryanopathy (Calcium Release-                           related Contractile Dysfunction) and Catecholamine Responses

Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC
Author and Curator: Larry H Bernstein, MD, FCAP
and Article Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-      and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-                    contractile/

34. Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Author and Curator: Larry H Bernstein, MD, FCAP Author: Stephen Williams, PhD, and Curator: Aviva Lev-Ari, PhD, RN


35. Identification of Biomarkers that are Related to the Actin Cytoskeleton

Larry H Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-                           cytoskeleton/

36. Advanced Topics in Sepsis and the Cardiovascular System at its End Stage

Author: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/08/18/advanced-topics-in-Sepsis-and-the-Cardiovascular-System-at-its-              End-Stage/

37. The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology

Demet Sag, PhD, Author and Curator

https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-               immunology/

38. IDO for Commitment of a Life Time: The Origins and Mechanisms of IDO, indolamine 2, 3-dioxygenase

Demet Sag, PhD, Author and Curator

https://pharmaceuticalintelligence.com/2013/08/04/ido-for-commitment-of-a-life-time-the-origins-and-mechanisms-of-             ido-indolamine-2-3-dioxygenase/

39. Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Homeostasis of Immune Responses for Good and Bad

Curator: Demet Sag, PhD, CRA, GCP

https://pharmaceuticalintelligence.com/2013/07/31/confined-indolamine-2-3-dehydrogenase-controls-the-hemostasis-           of-immune-responses-for-good-and-bad/

40. Signaling Pathway that Makes Young Neurons Connect was discovered @ Scripps Research Institute

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/06/26/signaling-pathway-that-makes-young-neurons-connect-was-                     discovered-scripps-research-institute/

41. Naked Mole Rats Cancer-Free

Writer and Curator: Larry H. Bernstein, MD, FCAP


42. Late Onset of Alzheimer’s Disease and One-carbon Metabolism

Reporter and Curator: Dr. Sudipta Saha, Ph.D.


43. Problems of vegetarianism

Reporter and Curator: Dr. Sudipta Saha, Ph.D.


44.  Amyloidosis with Cardiomyopathy

Writer and Curator: Larry H. Bernstein, MD, FCAP


45. Liver endoplasmic reticulum stress and hepatosteatosis

Larry H Bernstein, MD, FACP


46. The Molecular Biology of Renal Disorders: Nitric Oxide – Part III

Curator and Author: Larry H Bernstein, MD, FACP


47. Nitric Oxide Function in Coagulation – Part II

Curator and Author: Larry H. Bernstein, MD, FCAP


48. Nitric Oxide, Platelets, Endothelium and Hemostasis

Curator and Author: Larry H Bernstein, MD, FACP


49. Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Curator and Author: Larry H Bernstein, MD, FACP


50. Nitric Oxide and Immune Responses: Part 1

Curator and Author:  Aviral Vatsa PhD, MBBS


51. Nitric Oxide and Immune Responses: Part 2

Curator and Author:  Aviral Vatsa PhD, MBBS


52. Mitochondrial Damage and Repair under Oxidative Stress

Curator and Author: Larry H Bernstein, MD, FACP


53. Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/17/is-the-warburg-effect-the-cause-or-the-effect-of-cancer-a-21st-                 century-view/

54. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-                  proteolysis-and-cell-apoptosis/

55. Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-                   proteolysis-and-cell-apoptosis-reconsidered/

56. Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II

Curator and Author: Larry H Bernstein, MD, FACP


57. New Insights on Nitric Oxide donors – Part IV

Curator and Author: Larry H Bernstein, MD, FACP


58. Crucial role of Nitric Oxide in Cancer

Curator and Author: Ritu Saxena, Ph.D.


59. Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-         a-concomitant-influence-on-mitochondrial-function/

60. Targeting Mitochondrial-bound Hexokinase for Cancer Therapy

Curator and Author: Ziv Raviv, PhD, RN 04/06/2013


61. Biochemistry of the Coagulation Cascade and Platelet Aggregation – Part I

Curator and Author: Larry H Bernstein, MD, FACP


Genomics, Transcriptomics, and Epigenetics

  1. What is the meaning of so many RNAs?

Writer and Curator: Larry H. Bernstein, MD, FCAP


  1. RNA and the transcription the genetic code

Larry H. Bernstein, MD, FCAP, Writer and Curator


  1. A Primer on DNA and DNA Replication

Writer and Curator: Larry H. Bernstein, MD, FCAP


4. Synthesizing Synthetic Biology: PLOS Collections

Reporter: Aviva Lev-Ari


5. Pathology Emergence in the 21st Century

Author and Curator: Larry Bernstein, MD, FCAP


6. RNA and the transcription the genetic code

Writer and Curator, Larry H. Bernstein, MD, FCAP


7. A Great University engaged in Drug Discovery: University of Pittsburgh

Larry H. Bernstein, MD, FCAP, Reporter and Curator


8. microRNA called miRNA-142 involved in the process by which the immature cells in the bone  marrow give                              rise to all the types of blood cells, including immune cells and the oxygen-bearing red blood cells

Aviva Lev-Ari, PhD, RN, Author and Curator

https://pharmaceuticalintelligence.com/2014/07/24/microrna-called-mir-142-involved-in-the-process-by-which-the-                   immature-cells-in-the-bone-marrow-give-rise-to-all-the-types-of-blood-cells-including-immune-cells-and-the-oxygen-             bearing-red-blood-cells/

9. Genes, proteomes, and their interaction

Larry H. Bernstein, MD, FCAP, Writer and Curator


10. Regulation of somatic stem cell Function

Larry H. Bernstein, MD, FCAP, Writer and Curator    Aviva Lev-Ari, PhD, RN, Curator


11. Scientists discover that pluripotency factor NANOG is also active in adult organisms

Larry H. Bernstein, MD, FCAP, Reporter

https://pharmaceuticalintelligence.com/2014/07/10/scientists-discover-that-pluripotency-factor-nanog-is-also-active-in-           adult-organisms/

12. Bzzz! Are fruitflies like us?

Larry H Bernstein, MD, FCAP, Author and Curator


13. Long Non-coding RNAs Can Encode Proteins After All

Larry H Bernstein, MD, FCAP, Reporter


14. Michael Snyder @Stanford University sequenced the lymphoblastoid transcriptomes and developed an
allele-specific full-length transcriptome

Aviva Lev-Ari, PhD, RN, Author and Curator

https://pharmaceuticalintelligence.com/014/06/23/michael-snyder-stanford-university-sequenced-the-lymphoblastoid-            transcriptomes-and-developed-an-allele-specific-full-length-transcriptome/

15. Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: Views by Larry H                                     Bernstein, MD, FCAP

Author: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/07/16/commentary-on-biomarkers-for-genetics-and-genomics-of-                        cardiovascular-disease-views-by-larry-h-bernstein-md-fcap/

16. Observations on Finding the Genetic Links in Common Disease: Whole Genomic Sequencing Studies

Author an curator: Larry H Bernstein, MD, FCAP


17. Silencing Cancers with Synthetic siRNAs

Larry H. Bernstein, MD, FCAP, Reviewer and Curator


18. Cardiometabolic Syndrome and the Genetics of Hypertension: The Neuroendocrine Transcriptome Control Points

Reporter: Aviva Lev-Ari, PhD, RN


19. Developments in the Genomics and Proteomics of Type 2 Diabetes Mellitus and Treatment Targets

Larry H. Bernstein, MD, FCAP, Reviewer and Curator

https://pharmaceuticalintelligence.com/2013/12/08/developments-in-the-genomics-and-proteomics-of-type-2-diabetes-           mellitus-and-treatment-targets/

20. Loss of normal growth regulation

Larry H Bernstein, MD, FCAP, Curator


21. CT Angiography & TrueVision™ Metabolomics (Genomic Phenotyping) for new Therapeutic Targets to Atherosclerosis

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/11/15/ct-angiography-truevision-metabolomics-genomic-phenotyping-for-           new-therapeutic-targets-to-atherosclerosis/

22.  CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics

Genomics Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/08/30/cracking-the-code-of-human-life-the-birth-of-bioinformatics-                      computational-genomics/

23. Big Data in Genomic Medicine

Author and Curator, Larry H Bernstein, MD, FCAP


24. From Genomics of Microorganisms to Translational Medicine

Author and Curator: Demet Sag, PhD

https://pharmaceuticalintelligence.com/2014/03/20/without-the-past-no-future-but-learn-and-move-genomics-of-                      microorganisms-to-translational-medicine/

25. Summary of Genomics and Medicine: Role in Cardiovascular Diseases

Author and Curator, Larry H Bernstein, MD, FCAP


 26. Genomic Promise for Neurodegenerative Diseases, Dementias, Autism Spectrum, Schizophrenia, and Serious                      Depression

Author and Curator, Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/02/19/genomic-promise-for-neurodegenerative-diseases-dementias-autism-        spectrum-schizophrenia-and-serious-depression/

 27.  BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair

Sudipta Saha, PhD

https://pharmaceuticalintelligence.com/2012/12/04/brca1-a-tumour-suppressor-in-breast-and-ovarian-cancer-functions-         in-transcription-ubiquitination-and-dna-repair/

28. Personalized medicine gearing up to tackle cancer

Ritu Saxena, PhD


29. Differentiation Therapy – Epigenetics Tackles Solid Tumors

Stephen J Williams, PhD


30. Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment

     Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/17/mechanism-involved-in-breast-cancer-cell-growth-function-in-early-          detection-treatment/

31. The Molecular pathology of Breast Cancer Progression

Tilde Barliya, PhD


32. Gastric Cancer: Whole-genome reconstruction and mutational signatures

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/12/24/gastric-cancer-whole-genome-reconstruction-and-mutational-                   signatures-2/

33. Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine –                                                       Part 1 (pharmaceuticalintelligence.com)

Aviva  Lev-Ari, PhD, RN


34. LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer                                         Personalized Treatment: Part 2

A Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/13/leaders-in-genome-sequencing-of-genetic-mutations-for-therapeutic-       drug-selection-in-cancer-personalized-treatment-part-2/

35. Personalized Medicine: An Institute Profile – Coriell Institute for Medical Research: Part 3

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/13/personalized-medicine-an-institute-profile-coriell-institute-for-medical-        research-part-3/

36. Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of                           Cancer Scientific Leaders @http://pharmaceuticalintelligence.com

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/13/7000/Harnessing_Personalized_Medicine_for_ Cancer_Management-      Prospects_of_Prevention_and_Cure/

37.  GSK for Personalized Medicine using Cancer Drugs needs Alacris systems biology model to determine the in silico
effect of the inhibitor in its “virtual clinical trial”

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/11/14/gsk-for-personalized-medicine-using-cancer-drugs-needs-alacris-             systems-biology-model-to-determine-the-in-silico-effect-of-the-inhibitor-in-its-virtual-clinical-trial/

38. Personalized medicine-based cure for cancer might not be far away

Ritu Saxena, PhD


39. Human Variome Project: encyclopedic catalog of sequence variants indexed to the human genome sequence

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/11/24/human-variome-project-encyclopedic-catalog-of-sequence-variants-         indexed-to-the-human-genome-sequence/

40. Inspiration From Dr. Maureen Cronin’s Achievements in Applying Genomic Sequencing to Cancer Diagnostics

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/10/inspiration-from-dr-maureen-cronins-achievements-in-applying-                genomic-sequencing-to-cancer-diagnostics/

41. The “Cancer establishments” examined by James Watson, co-discoverer of DNA w/Crick, 4/1953

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/09/the-cancer-establishments-examined-by-james-watson-co-discover-         of-dna-wcrick-41953/

42. What can we expect of tumor therapeutic response?

Author and curator: Larry H Bernstein, MD, FACP


43. Directions for genomics in personalized medicine

Author and Curator: Larry H. Bernstein, MD, FCAP


44. How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis.

Stephen J Williams, PhD

https://pharmaceuticalintelligence.com/2012/10/31/how-mobile-elements-in-junk-dna-prote-cancer-part1-transposon-            mediated-tumorigenesis/

45. mRNA interference with cancer expression

Author and Curator, Larry H. Bernstein, MD, FCAP


46. Expanding the Genetic Alphabet and linking the genome to the metabolome

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-               metabolome/

47. Breast Cancer, drug resistance, and biopharmaceutical targets

Author and Curator: Larry H Bernstein, MD, FCAP


48.  Breast Cancer: Genomic profiling to predict Survival: Combination of Histopathology and Gene Expression                            Analysis

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/12/24/breast-cancer-genomic-profiling-to-predict-survival-combination-of-           histopathology-and-gene-expression-analysis

49. Gastric Cancer: Whole-genome reconstruction and mutational signatures

Aviva  Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/12/24/gastric-cancer-whole-genome-reconstruction-and-mutational-                   signatures-2/

50. Genomic Analysis: FLUIDIGM Technology in the Life Science and Agricultural Biotechnology

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/2012/08/22/genomic-analysis-fluidigm-technology-in-the-life-science-and-                   agricultural-biotechnology/

51. 2013 Genomics: The Era Beyond the Sequencing Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

Aviva Lev-Ari, PhD, RD


52. Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1

Aviva Lev-Ari, PhD, RD

https://pharmaceuticalintelligence.com/Paradigm Shift in Human Genomics_/

Signaling Pathways

  1. Proteins and cellular adaptation to stress

Larry H Bernstein, MD, FCAP, Curator


  1. A Synthesis of the Beauty and Complexity of How We View Cancer:
    Cancer Volume One – Summary

Author and Curator: Larry H. Bernstein, MD, FCAP


  1. Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in
    serous endometrial tumors

Sudipta Saha, PhD

https://pharmaceuticalintelligence.com/2012/11/19/recurrent-somatic-mutations-in-chromatin-remodeling-ad-ubiquitin-           ligase-complex-genes-in-serous-endometrial-tumors/

4.  Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition

Stephen J Williams, PhD

https://pharmaceuticalintelligence.com/2012/11/30/histone-deacetylase-inhibitors-induce-epithelial-to-mesenchymal-              transition-in-prostate-cancer-cells/

5. Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis

Author and Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-                   proteolysis-and-cell-apoptosis/

6. Signaling and Signaling Pathways

Larry H. Bernstein, MD, FCAP, Reporter and Curator


7.  Leptin signaling in mediating the cardiac hypertrophy associated with obesity

Larry H. Bernstein, MD, FCAP, Reporter and Curator

https://pharmaceuticalintelligence.com/2013/11/03/leptin-signaling-in-mediating-the-cardiac-hypertrophy-associated-            with-obesity/

  1. Sensors and Signaling in Oxidative Stress

Larry H. Bernstein, MD, FCAP, Reporter and Curator


  1. The Final Considerations of the Role of Platelets and Platelet Endothelial Reactions in Atherosclerosis and Novel

Larry H. Bernstein, MD, FCAP, Reporter and Curator

https://pharmaceuticalintelligence.com/2013/10/15/the-final-considerations-of-the-role-of-platelets-and-platelet-                      endothelial-reactions-in-atherosclerosis-and-novel-treatments

10.   Platelets in Translational Research – Part 1

Larry H. Bernstein, MD, FCAP, Reporter and Curator


11.  Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and
Cardiovascular Calcium Signaling Mechanism

Author and Curator: Larry H Bernstein, MD, FCAP, Author, and Content Consultant to e-SERIES A:
Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-             smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

12. The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and
Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia,
Similarities and Differences, and Pharmaceutical Targets

     Author and Curator: Larry H Bernstein, MD, FCAP, Author, and Content Consultant to
e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and
Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-       kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-           differen/

13.  Nitric Oxide Signalling Pathways

Aviral Vatsa, PhD, MBBS


14. Immune activation, immunity, antibacterial activity

Larry H. Bernstein, MD, FCAP, Curator


15.  Regulation of somatic stem cell Function

Larry H. Bernstein, MD, FCAP, Writer and Curator    Aviva Lev-Ari, PhD, RN, Curator


16. Scientists discover that pluripotency factor NANOG is also active in adult organisms

Larry H. Bernstein, MD, FCAP, Reporter


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