Posts Tagged ‘amyloid beta’

Alzheimer Disease Developments – Spring 2015

Larry H. Bernstein, MD, FCAP, Curator




Cognitive Stimulation Modulates Platelet Total Phospholipases A2 Activity in Subjects with Mild Cognitive Impairment


JNK: A Putative Link Between Insulin Signaling and VGLUT1 in Alzheimer’s Disease

Omega-3 Fatty Acid Status Enhances the Prevention of Cognitive Decline by B Vitamins in Mild Cognitive ImpairmentOpenly Available
Oulhaj, Abderrahim | Jernerén, Fredrik | Refsum, Helga | Smith, A. David | de Jager, Celeste A.

Preliminary Study of Plasma Exosomal Tau as a Potential Biomarker for Chronic Traumatic EncephalopathyOpenly Available
Stern, Robert A. | Tripodis, Yorghos | Baugh, Christine M. | Fritts, Nathan G. | Martin, Brett M. | Chaisson, Christine | Cantu, Robert C. | Joyce, James A. | Shah, Sahil | Ikezu, Tsuneya | Zhang, Jing | Gercel-Taylor, Cicek | Taylor, Douglas D

AZD3293: A Novel, Orally Active BACE1 Inhibitor with High Potency and Permeability and Markedly Slow Off-Rate KineticsOpenly Available
Eketjäll, Susanna | Janson, Juliette | Kaspersson, Karin | Bogstedt, Anna | Jeppsson, Fredrik | Fälting, Johanna | Haeberlein, Samantha Budd | Kugler, Alan R. | Alexander, Robert C. | Cebers, Gvido

Predictive Value of Cerebrospinal Fluid Visinin-Like Protein-1 Levels for Alzheimer’s Disease Early Detection and Differential Diagnosis in Patients with Mild Cognitive Impairment
Babić Leko, Mirjana | Borovečki, Fran | Dejanović, Nenad | Hof, Patrick R. | Šimić, Goran

Plasma Phospholipid and Sphingolipid Alterations in Presenilin1 Mutation Carriers: A Pilot Study
Chatterjee, Pratishtha | Lim, Wei L.F. | Shui, Guanghou | Gupta, Veer B. | James, Ian | …… | Wenk, Marcus R. | Bateman, Randall J. | Morris, John C. | Martins, Ralph N.

Cognitive reserve in ageing and Alzheimer’s disease / Stern Y / Lancet Neurol. 2012 Nov; 11(11):1006-12. PMID: 23079557.

A mutation in APP protects against Alzheimer’s disease and age-related cognitive decline/ Jonsson T, Atwal JK, Steinberg S, Snaedal J, Jonsson PV, Bjornsson S, Stefansson H, Sulem P, Gudbjartsson D, Maloney J, et al. / Nature. 2012 Aug 2; 488(7409):96-9. PMID: 22801501.

 Propagation of tau pathology in a model of early Alzheimer’s disease / de Calignon A, Polydoro M, Suárez-Calvet M, William C, Adamowicz DH, Kopeikina KJ, Pitstick R, Sahara N, Ashe KH, Carlson GA, et al. / Neuron. 2012 Feb 23; 73(4):685-97. PMID: 22365544.

Stages of the pathologic process in Alzheimer disease: age categories from 1 to 100 years/ Braak H, Thal DR, Ghebremedhin E, Del Tredici K / J Neuropathol Exp Neurol. 2011 Nov; 70(11):960-9. PMID: 22002422.

Neuroinflammation in Alzheimer’s disease and mild cognitive impairment: a field in its infancy / McGeer EG, McGeer PL / J Alzheimers Dis. 2010; 19(1):355-61. PMID: 20061650.

Metallothioneins in Prion- and Amyloid-Related Diseases


Microglia are the immune cells of the CNS and account for approximately 10% of the CNS cellpopulation, with regional variation in density [27, 28]. During embryonic development, microglia originate from yolk sac progenitor cells that migrate into the developing CNS during early embryogenesis [29,30].Following construction of the blood-brain barrier (BBB), microglia are renewed by local turnover [31]. In the healthy brain, microglia actively support neurons through the release of insulin-like growth factor 1, nerve growth factor, ciliary neurotrophic factor, and brain-derived neurotrophic factor (BDNF) [32–34]. Microglia also provide indirect support to neurons by clearance of debris to maintain the extracellular environment, and phagocytosis of apoptotic cells to facilitate neurogenesis [35, 36]. In the adult brain, microglia coordinate much of their activity with astrocytes and activate in response to similar stimuli [37, 38]. Dysfunctional signaling between microglia and astrocytes often results in chronic inflammation, a characteristic of many neurodegenerative diseases [39, 40].

Historically, it has been thought that microglia ‘rest’ when not responding to inflammatory stimuli or damage [41, 42]. However, this notion is being increasingly recognized as inaccurate [43]. When not involved in active inflammatory signaling, microglia constantly patrol the neuropil by extension and retraction of their finely branched processes [44]. Microglial activation is often broadly classified into two states; pro-inflammatory (M1) or anti-inflammatory (M2) [36, 45], based on similar phenotypes in peripheral macrophages [46]. M1 activated microglia are characterized by increased expression of pro-inflammatory mediators and cytokines, including inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β, often under the control of the transcription factor nuclear factor-κB [45]. Pro-inflammatory microglia rapidly retract their processes and adopt an amoeboid morphology and often migrate closer to the site of injury [47]. Anti-inflammatory M2 activation of microglia, often referred to as alternative activation, represents the other side of microglial behavior. Anti-inflammatory activation is characterized by increased expression of cytokines including arginase 1 and interleukin-10, and is associated with increased ramification of processes [45]. The polarization of microglia into M1 or M2 throughout the brain is well characterized, especially in neurodegenerative diseases [48]. In the AD brain, microglia expressing markers of M1 activation are typically localized to brain regions such as the hippocampus that are most heavily affected in the disease [49]. However, it is important to note that M1 and M2 classifications of microglia may over-simplify microglial phenotypes and may only represent the extremes of microglial activation [50]. It has been more recently proposed that microglia likely occupy a continuum between these phenotypes [39, 51].

Do microglia have multiple roles in AD?

Classical pro-inflammatory activation of microglia has long been associated with AD [39, 49]. Samples taken from late-stage AD brains contain characteristic signs of inflammation, including amoeboid morphology of microglia, high levels of pro-inflammatory cytokines in the cerebrospinal fluid, and evidence of neuronal damage due to chronic exposure to pro-inflammatory cytokines and oxidative stress [52, 53]. The cause of this inflammation may be in response to direct toxicity of Aβ to neurons resulting in activation of nearby microglia and astrocytes [53, 54]. However, Aβ may also induce inflammatory activation of microglia and astrocytes. Activated immune cells are typically present surrounding amyloid plaques [55–57], with such peri-plaque cells exhibiting strong evidence of pro-inflammatory activation [56, 58–60]. The presence of undigested Aβ particles within these activated microglia may suggest that the Aβ peptide itself is a pro-inflammatory signal for microglia [61–64]. In vitro experiments provide supporting evidence for the in vivo studies, with Aβ promoting pro-inflammatory microglial activation [65, 66], and also acting as a potent chemotactic signal [67].

However, it is important to note that although widespread inflammation is characteristic of late-stage AD, it remains unclear what role inflammation could play in early stages of the disease. Some evidence suggests that reducing inflammation through the long-term use of some non-steroidal anti-inflammatory drugs (NSAIDs) can reduce the risk of AD [68]. However, these findings have not yet been verified in clinical trials [69, 70]. Little is understood about how NSAIDs and related compounds affect the delicate balance of pro- versus anti-inflammatory microglial activity within the brain. Although there is considerable evidence to suggest that chronic inflammation may contribute to pathology in the later stages of AD, it is important to note that inflammation normally only represents a small aspect of microglial function. The non-inflammatory functions of microglia may play a more important role in early disease; specifically, microglial functions relating to maintenance of the CNS.

Phagocytosis: A vital role of microglia that may be lost in AD    


Recently, a new function has been proposed for microglia. A number of studies have provided evidence that microglia prune synapses throughout life. Microglia are known to remove extraneous synapses during development to ensure that only meaningful connections remain [43]. It was, however, thought that differentiated astrocytes performed the majority of synaptic pruning in the adult brain [91]. The discovery that microglial processes are constantly active within the brain and are often positioned near synapses raised the question of whether microglial synaptic pruning continued throughout life [44, 47, 92–94]. This question was answered in 2014 in a study that demonstrated that microglia do prune synapses into adulthood, and that this activity is important for normal brain function [95]. These findings supported those found a year earlier in a study reporting that ablation of microglia from brain slices increases synapse density and results in abnormal firing of hippocampalneurons [96].

Altered microglial behavior may underlie altered neuronal firing in AD  

Altered neuronal activity is an early phenomenon in AD

The cause of DMN hypoactivity in AD is not yet clear; however studies performed in cohorts that are genetically predisposed to AD suggest that DMN hypoactivity is preceded by a period of hyperactivity and increased functional connectivity [123, 136], often manifesting as an absence of normal DMN deactivation during external tasks [137–140]. DMN hyperactivity may interfere with hippocampal memory encoding, leading to the memory deficits that are present in mild cognitive impairment [141, 142]. It has been proposed that hippocampal hyperexcitability in AD may develop as a protective mechanism against increased input from the DMN [142–144]. As AD progresses, the initial hyperexcitability of the DMN and hippocampus may result in hypoactivity due to exhaustion of compensatory mechanisms [123, 136]. Evidence from both transgenic AD mice and longitudinal human studies supports an exhaustion model of hyperactivation leading to later hypoactivation [143, 145–147]. Interestingly, a number of studies report a lower incidence of AD among those who regularly practice meditation which specifically ‘calms’ the DMN [148].

Our understanding of AD as a disease is changing. Historically considered to be primarily a disease of neuronal degeneration, this neurocentric view has widened to encompass non-neuronal cells such as astrocytes into our understanding of the disease process and pathogenesis. A proposed model for microglia in AD is shown in Fig. 2. Microglia perform a wide range of functions in the CNS and although this includes induction of an inflammatory reaction in response to damage, they also have critical roles for maintaining normal function in the brain. Recent evidence shows that microglia regulate neuronal activity through synaptic pruning throughout life as an extension on their normal phagocytosis behavior. The discovery of a large number of AD risk genes associated with reduced immune cell function suggests that perturbed microglial phagocytosis could lead to AD. In our model, altered microglial phagocytosis of synapses results in network dysfunction and onset of AD, occurring downstream of Aβ.

The immune system and microglia represent a novel target for intervention in AD. Importantly, a large number of anti-inflammatory drugs are already in use for other conditions. What is important to know at this stage is exactly how to best target immune cell function. The studies outlined here provide evidence that an indiscriminate dampening down of all microglial activity may result in a worse outcome for individuals by suppressing normal microglial regulatory functions. We currently do not know whether future microglial-based therapies should focus on reducing chronic inflammation or conversely, whether they should be aimed at boosting microglial phagocytosis. It is also likely that future treatment strategies may use a combination of approaches to target Aβ, immune cell phagocytosis and network activity. An increasing view in the AD field is that any drug or therapy needs to be provided very early in the disease process to maximize its beneficial effects. Although we are currently unable to effectively target those at risk of AD at such an early stage, advances in neuroimaging for subtle changes in network activity, or in assays for immune cell function, may provide new avenues for identification of early damage and risk of disease.



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Late-Onset Metachromatic Leukodystrophy with Early Onset Dementia Associated with a Novel Missense Mutation in the Arylsulfatase A Gene

Microbes and Alzheimer’s DiseaseOpenly Available
Itzhaki, Ruth F. | Lathe, Richard | Balin, Brian J. | Ball, Melvyn J. | Bearer, Elaine L. | Braak, Heiko | Bullido, Maria J. | Carter, Chris | Clerici, Mario | Cosby, S. Louise | Del Tredici, Kelly | Field, Hugh | Fulop, Tamas | Grassi, Claudio | Griffin, W. Sue T. | Haas, Jürgen | Hudson, Alan P. | Kamer, Angela R. | Kell, Douglas B. | Licastro, Federico | Letenneur, Luc | Lövheim, Hugo | Mancuso, Roberta | Miklossy, Judith | Otth, Carola | Palamara, Anna Teresa | Perry, George | Preston, Christopher | Pretorius, Etheresia | Strandberg, Timo | Tabet, Naji | Taylor-Robinson, Simon D. | Whittum-Hudson, Judith A.

Longitudinal Relationships between Caloric Expenditure and Gray Matter in the Cardiovascular Health StudyOpenly Available
Raji, Cyrus A. | Merrill, David A. | Eyre, Harris | Mallam, Sravya | Torosyan, Nare | Erickson, Kirk I. | Lopez, Oscar L. | Becker, James T. | Carmichael, Owen T. | Gach, H. Michael | Thompson, Paul M. | Longstreth Jr., W.T. | Kuller, Lewis H.

Preliminary Study of Plasma Exosomal Tau as a Potential Biomarker for Chronic Traumatic EncephalopathyOpenly Available
Stern, Robert A. | Tripodis, Yorghos | Baugh, Christine M. | Fritts, Nathan G. | Martin, Brett M. | Chaisson, Christine | Cantu, Robert C. | Joyce, James A. | Shah, Sahil | Ikezu, Tsuneya | Zhang, Jing | Gercel-Taylor, Cicek | Taylor, Douglas D.

Unraveling Alzheimer’s: Making Sense of the Relationship between Diabetes and Alzheimer’s Disease1Openly Available
Schilling, Melissa A.

Pain Assessment in Elderly with Behavioral and Psychological Symptoms of DementiaOpenly Available
Malara, Alba | De Biase, Giuseppe Andrea | Bettarini, Francesco | Ceravolo, Francesco | Di Cello, Serena | Garo, Michele | Praino, Francesco | Settembrini, Vincenzo | Sgrò, Giovanni | Spadea, Fausto | Rispoli, Vincenzo

Editor’s Choice from Volume 50, Number 4 / 2016

Post Hoc Analyses of ApoE Genotype-Defined Subgroups in Clinical Trials
Kennedy, Richard E. | Cutter, Gary R. | Wang, Guoqiao | Schneider, Lon S.

Protective Effect of Amyloid-β Peptides Against Herpes Simplex Virus-1 Infection in a Neuronal Cell Culture Model
Bourgade, Karine | Le Page, Aurélie | Bocti, Christian | Witkowski, Jacek M. | Dupuis, Gilles | Frost, Eric H. | Fülöp, Tamás

Association Between Serum Ceruloplasmin Specific Activity and Risk of Alzheimer’s Disease
Siotto, Mariacristina | Simonelli, Ilaria | Pasqualetti, Patrizio | Mariani, Stefania | Caprara, Deborah | Bucossi, Serena | Ventriglia, Mariacarla | Molinario, Rossana | Antenucci, Mirca | Rongioletti, Mauro | Rossini, Paolo Maria | Squitti, Rosanna

Effects of Hypertension and Anti-Hypertensive Treatment on Amyloid-β (Aβ) Plaque Load and Aβ-Synthesizing and Aβ-Degrading Enzymes in Frontal Cortex
Ashby, Emma L. | Miners, James S. | Kehoe , Patrick G. | Love, Seth

AZD3293: A Novel, Orally Active BACE1 Inhibitor with High Potency and Permeability and Markedly Slow Off-Rate KineticsOpenly Available
Eketjäll, Susanna | Janson, Juliette | Kaspersson, Karin | Bogstedt, Anna | Jeppsson, Fredrik | Fälting, Johannad | Haeberlein, Samantha Budd | Kugler, Alan R. | Alexander, Robert C. | Cebers, Gvido

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Role of infectious agent in Alzheimer’s Disease?

Larry H. Bernstein, MD, FCAP, Curator



Role of Infection in Alzheimer’s Ignored, Experts Say

Nancy A. Melville   http://www.medscape.com/viewarticle/860615

The potentially critical role of infection in the etiology of Alzheimer’s disease is largely neglected, despite decades of robust evidence from hundreds of human studies, as well as the possible therapeutic implications, experts say.

“Despite all the supportive evidence, the topic [of linking infections to Alzheimer’s disease] is often dismissed as ‘controversial,’ ” the authors of an editorial, signed by an international group of 33 researchers and clinicians, write.

The editorial was published online March 8 in theJournal of Alzheimer’s Disease.

Antiviral Treatment

“One recalls the widespread opposition initially to data showing that viruses cause some types of cancer, and that a bacterium causes stomach ulcers,” the authors write.

The implications could be just as important with regard to Alzheimer’s disease, coauthor Ruth F. Itzhaki, PhD, of the Faculty of Life Sciences at the University of Manchester, United Kingdom, toldMedscape Medical News.

“The implications are that patients could be treated with antiviral agents. These would not cure them, but might slow or even stop the progression of the disease,” she said.

The evidence points to herpes simplex virus type 1 (HSV1), Chlamydia pneumoniae, and several types of spirochetes, which make their way into the central nervous system (CNS), where they can remain in latent form indefinitely, the authors note.

The link with HSV1 is supported by as many as 100 studies. Only two studies oppose the association; both were published more than a decade ago, the authors state.

Under the prevailing theory, agents such as HSV1 undergo reactivation in the brain during aging and with the decline of the immune system, as well as when persons are under stress.

“The consequent neuronal damage ― caused by direct viral action and by virus-induced inflammation ― occurs recurrently, leading to (or acting as a cofactor for) progressive synaptic dysfunction, neuronal loss, and ultimately AD [Alzheimer’s disease],” the authors write

Importantly, that damage includes the induction of amyloid-β (Aβ) peptide deposits, considered a hallmark of Alzheimer’s disease, which initially appears to be only a defense mechanism, the authors add.

Causative Role?

In outlining some of the strongest evidence behind the theory, the authors note that although viruses and other microbes are common in the elderly brain and are usually dormant, influences such as stress and immunosuppression can cause reactivation.

“For example, HSV1 DNA is amplified in the brain of immunosuppressed patients,” they write.

In addition, herpes simplex encephalitis is known to damage regions of the CNS linked to the limbic system, and therefore to memory as well as cognitive and affective processes, the same regions affected in Alzheimer’s disease.

HSV infection is known to be significantly associated with the development of Alzheimer’s, and the disease is known to have a strong inflammatory component that is characteristic of infection, the authors say.

On a genetic level, research has shown that polymorphisms in the apolipoprotein E gene (APOE) that are linked to the risk for Alzheimer’s also control immune function and susceptibility to infectious disease.

In terms of evidence of a causative role of infection in Alzheimer’s disease, the authors cite studies indicating that brain infection, such as HIV or herpes virus, is linked to pathology similar to Alzheimer’s.

Notably, infection with HSV1 or bacteria in mice and cell culture studies has been shown to result in Aβ deposition and tau abnormalities typical of Alzheimer’s disease.

In addition, the olfactory dysfunction that is an early symptom of Alzheimer’s disease is consonant with a role of infection: The olfactory nerve leads to the lateral entorhinal cortex, where Alzheimer’s pathology spreads through the brain, and it is the likely portal of entry of HSV1 and other viruses into the brain, the authors note.

“Further, brainstem areas that harbor latent HSV directly irrigate these brain regions: brainstem virus reactivation would thus disrupt the same tissues as those affected in Alzheimer’s disease,” they write.

In terms of mechanisms, the authors cite mounting evidence that virus infection selectively upregulates the gene encoding cholesterol 25-hydroxylase (CH25H), and innate antiviral immunity is induced by its enzymatic product 25-hydroxycholesterol (25OHC).

The human CH25H polymorphisms control susceptibility to Alzheimer’s as well as Aβ deposition.

Consequently, “Aβ induction is likely to be among the targets of 25OHC, providing a potential mechanistic link between infection and Aβ production,” the authors write.

Considering the devastating toll Alzheimer’s disease takes on individual lives and society, the need to reconsider the collective evidence of a role for infection is pressing, the authors note.

“Alzheimer’s disease causes great emotional and physical harm to sufferers and their carers, as well as having enormously damaging economic consequences,” they write.

“Given the failure of the 413 trials of other types of therapy for Alzheimer’s disease carried out in the period 2002-2012, antiviral/antimicrobial treatment of Alzheimer’s disease patients, notably those who areAPOE ɛ4 carriers, could rectify the ‘no drug works’ impasse.

“We propose that further research on the role of infectious agents in Alzheimer’s disease causation, including prospective trials of antimicrobial therapy, is now justified.”

Chicken or the Egg?

Commenting on the editorial for Medscape Medical News, Richard B. Lipton, MD, Edwin S. Lowe Professor, vice chair of neurology, and director of the Division of Cognitive Aging and Dementia at Albert Einstein College of Medicine in New York City, applauded the effort to raise awareness of the issue.

“The authors are to be commended for reminding us of the hypothesis that infection may contribute to Alzheimer’s disease,” he told Medscape Medical News.

He noted the variety of genetic and environmental factors that can influence onset and progression of complex disorders such as Alzheimer’s disease.

“For Alzheimer’s disease, most people would agree that cardiovascular risk factors, traumatic brain injury, and stress increase risk of disease,” he said.

“It is entirely plausible that infectious agents may be one of many factors that contribute to the development of Alzheimer’s disease. Infectious agents could operate through several mechanisms.”

The evidence does not necessarily prove a causative role, he added.

“Temporality means that infection precedes disease,” he said. “The studies showing infectious and inflammatory markers in the Alzheimer’s brain don’t tell us which came first. Alzheimer’s disease could be a state which predisposes to infection.”

The editorialists’ financial disclosures are available online. Dr Lipton has disclosed no relevant financial relationships.

Microbes and Alzheimer’s Disease


  • Herpes simplex virus 1 (HSV-1) encephalitis predominantly involves the orbital surface of the frontal lobes and medial surface of the temporal lobes, resulting in areas of increased T2 signal on MRI
  • Herpes simplex virus 2 (HSV-2) is the primary cause of recurrent meningitis
  • After varicella, the varicella zoster virus (VZV) becomes latent in ganglia along the entire neuraxis; its reactivation can lead to herpes zoster, vasculopathy, myelitis, necrotizing retinitis or zoster sine herpete
  • The neurological complications of Epstein–Barr virus are diverse, and include meningitis, encephalitis, myelitis, radiculoneuropathy, and even autonomic neuropathy
  • The most common neurological complication of cytomegalovirus (CMV) is poly-radiculoneuropathy in immunocompromised individuals
  • Virological confirmation of neurological disease relies on the detection of herpesvirus-specific DNA in bodily fluids or tissues, herpesvirus-specific IgM in blood, or herpesvirus-specific IgM or IgG antibody in cerebrospinal fluid
  • HSV-1, HSV-2, VZV and CMV are the most treatable herpesviruses

Most HHVs can cause serious neurological disease of the PNS and CNS through primary infection or following virus reactivation from latently infected human ganglia or lymphoid tissue. The neurological complications include meningitis, encephalitis, myelitis, vasculopathy, acute and chronic radiculoneuritis, and various inflammatory diseases of the eye. Disease can be monophasic, recurrent or chronic.


The researchers also add that a gene mutation – APOEe4 – which appears to makes some of the population more susceptible to Alzheimer’s disease, could also increase these people’s susceptibility to infectious diseases.

 As a counter view, Professor John Hardy, Teacher of Neuroscience, UCL, told the website Journal Focus he was doubtful about the claims: “This is a minority sight in Alzheimer research study. There had actually been no convincing evidence of infections triggering Alzheimer disease. We require constantly to maintain an open mind however this editorial does not show exactly what many scientists think of Alzheimer disease.”

However, another of the researchers, Resia Pretorius of the University of Pretoria, told Bioscience Technology: “The microbial presence in blood may also play a fundamental role as causative agent of systemic inflammation, which is a characteristic of Alzheimer’s disease. Furthermore, there is ample evidence that this can cause neuroinflammation and amyloid-β plaque formation.”

The possibility of transfer has been reported to the journal Nature. The paper is titled “Evidence for human transmission of amyloid-β pathology and cerebral amyloid angiopathy.”

The report explains that during the period from 1958 to 1985, 30,000 people worldwide — mainly children — were administered injections of human growth hormone. This was designed to treat short stature. The hormone was extracted from thousands of human pituitary glands, with the source material being recently deceased people.

It now appears, The Economist summarizes, that some of these hormonal extracts contained prions. Around one in 16 of the children developed the brain disorder Creutzfeldt-Jakob disease (CJD). The concern with CJD centered on prions.

Read more: http://www.digitaljournal.com/science/alzheimer-s-and-parkinson-s-diseases-may-be-transmissible/article/444338#ixzz43Y

Chain reaction

Evidence emerges that Alzheimer’s disease, and other neurodegenerative disorders such as Parkinson’s, may be transmissible



Reporting from the frontiers of health and medicine

A rare disease killed her mother. Can this scientist save herself?


CAMBRIDGE, Mass. — Five years ago, after watching her 51-year-old mother descend quickly into dementia, disability, and then death, Sonia Vallabh learned she was destined for the same fate. They both shared an extremely rare genetic mutation that leads a protein in the brain to turn toxic.

Vallabh, then a recent Harvard Law School graduate working as a consultant, decided to quit her job to spend time learning more about the mutation and nascent efforts to understand and treat it.

Now, she and her husband, Eric Minikel, a former transportation planner, are first authors on a paper about so-called prion diseases. Published Wednesday in Science Translational Medicine, the paper found that not all prion gene mutations are an early death sentence — though Vallabh’s variation is.

The husband-and-wife team, now both PhD students working in the same lab at the Broad Institute, also found that people can survive with only one copy of the prion gene, suggesting that a treatment to block the mutated version can be delivered safely.

Prion diseases were made famous by “mad cow disease,” outbreaks of which have led to mass killings of cattle. Eating sick cows can cause the fatal neurodegenerative illness known as Creutzfeldt-Jakob disease. But there are genetic versions of prion diseases that account for about 15 percent of cases. They come from mutations to the prion protein gene PRNP, which causes a protein in the brain to fold the wrong way, forming toxic clumps. Once these proteins get a foothold in the brain, they can cause extremely rapid damage.

Vallabh’s mother, who seemed completely normal at Christmastime in 2009, showed the first symptoms of disease in January 2010 and was demented and unable to speak clearly by March. She last recognized her daughter in May, Vallabh said, and died two days before Christmas that year, shortly after doctors finally identified the cause of her bizarre symptoms.

Vallabh, Minikel, and their coauthors compared a data set — painstakingly collected over decades — of gene sequences from 16,000 prion disease patients from all over the world, with two data sets of sequences from healthy people: more than 60,000 collected by the Broad-led Exome Aggregation Consortium and 530,000 from 23andMe, a consumer genetics company that invites clients to volunteer their gene sequences for research.

The size of the data sets allowed the researchers to draw conclusions even with a condition as rare as prion disease. Doctors had previously only known about 63 possible mutations in people with disease, so they had thought that all the mutations necessarily caused problems. But the researchers found 141 healthy people in the 23andMe dataset who had mutations to the PRNP gene — a rate far higher than the incidence of prion disease. That means some of the mutations must be harmless or at least not always cause disease, said J. Fah Sathirapongsasuti, a computational biologist at 23andMe and a study coauthor.

Out of 16 mutations for which there was evidence in the larger populations, they concluded that three were likely benign, three caused somewhat increased risk of disease, and four others, including Vallabh’s mutation, definitely do cause the fatal illness, they found.

They also discovered three older, healthy people who carried only one functional copy of the PRNP gene. That means that knocking out the mutated version of PRNP with gene therapy, or tamping down its activity with drugs, should be an effective way to eliminate the risk of disease without causing life-threatening problems.

Their paper has already helped at least one person, according to Dr. Robert Green, a medical geneticist at Brigham and Women’s Hospital, who cowrote an opinion piece published alongside the new study.

One of Green’s patients, whose mother died of prion disease, had been told her mom’s mutation — which she didn’t inherit, but her sister did — was always fatal. After seeing the new study, Green was able to inform the sister that her mutation was most likely harmless.


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Long Term Memory and Prions

Larry H. Bernstein, MD, FCAP, Curator



updated 12/12/2015


Possible biochemical mechanism underlying long-term memories identified

Why is a prion-like molecular state necessary for persistence of memory? Could a transient memory be made permanent with a “Limitless” NZT-type neurotropic drug — or permanently forgotten?

It’s a nagging question: why do some of our memories fade away, while others last forever? Now scientists at the Stowers Institute for Medical Research have identified a possible biochemical mechanism: a specific synaptic protein called Orb2 can either block or maintain neural synapses (connections between neurons), which create and maintain long-term memories.

So for a memory to persist, the synaptic connections must be kept strong. But how? The researchers previously identified a synaptic protein called CPEB that is responsible for maintaining the strength of such connections in the sea slug (a model organism used in memory research). Recently, they identified a similar protein, called Orb2, in the fruit fly.

Now, using a fruit fly model system, they found that the synaptic connections are kept strong by the transformation of Orb2 from one molecular state to another. And that transformation causes Orb2 molecules to solidify and strengthen the memory connections in the brain.

The authors conclude their paper, published in the current issue of the journal Cell, with several questions. How and what triggers this transformation, how long does it persist? Is the continued presence of a prion-like state necessary for the persistence of memory, and is it correlated with or predictive of long-lasting memory? And most interestingly: can a transient memory about to be forgotten be stabilized by artificial recruitment of the prion-like state (perhaps by a neurotropic compound)?

And what about that ironic link with prions, associated with neurodegenerative disorders? Are prions some twisted form of memory that could one day even have value? We’ll be keeping an eye on where this fascinating research leads.

Technical details: the memory switch

In their latest study, the researchers determined that Orb2 exists in two distinct physical states: monomeric (a single molecule that can bind to other molecules) and oligomeric (a molecular complex).

Like CPEB, oligomeric Orb2 is prion-like — that is, it’s a self-copying cluster. (But unlike prions, oligomeric Orb2 and CPEB are not toxic.) Monomeric Orb2 represses, and oligomeric Orb2 activates a crucial step in the complex cellular process that leads to protein synthesis.

During this crucial step, messenger RNA (mRNA), which is an RNA copy of a gene’s recipe for a protein, is translated by the cell’s ribosome into the sequence of amino acids that will make up a newly synthesized protein. The monomeric form of Orb2 binds to the target mRNA, keeping it in a repressed state.

The Stowers scientists also determined that prion-like Orb2 not only activates translation into amino acids but imparts its translational state to nearby monomer forms of Orb2. As a result, monomeric Orb2 is transformed into prion-like Orb2, so its role in translation switches from repression to activation.

Self-sustaining activation maintains synaptic activity

Stowers Associate Investigator Kausik Si, Ph.D. thinks this switch is the possible mechanism by which fleeting experiences create an enduring memory. “Because of the self-sustaining nature of the prion-like state, this creates a local and self-sustaining translation activation of Orb2-target mRNA, which maintains the changed state of synaptic activity over time,” says Si.

The discovery that the two distinct states of Orb2 have opposing roles in the translation process provides “for the first time a biochemical mechanism of synapse-specific persistent translation and long-lasting memory,” he states.

“To our knowledge, this is the first example of a prion-based protein switch that turns a repressor into an activator,” Si adds. “The recruitment of distinct protein complexes at the non-prion and prion-like forms to create altered activity states indicates the prion-like behavior is in essence a protein conformation-based switch.

“Through this switch, a protein can lose or gain a function that can be maintained over time in the absence of the original stimuli. Although such a possibility has been anticipated prior to this study, there was no direct evidence.”

The research builds upon previous studies by Si and Eric Kandel, M.D., of Columbia University and other scientists. These studies revealed that both short-term and long-term memories are created in synapses.


Abstract of Amyloidogenic Oligomerization Transforms Drosophila Orb2 from a Translation Repressor to an Activator

Memories are thought to be formed in response to transient experiences, in part through changes in local protein synthesis at synapses. In Drosophila, the amyloidogenic (prion-like) state of the RNA binding protein Orb2 has been implicated in long-term memory, but how conformational conversion of Orb2 promotes memory formation is unclear. Combining in vitro and in vivo studies, we find that the monomeric form of Orb2 represses translation and removes mRNA poly(A) tails, while the oligomeric form enhances translation and elongates the poly(A) tails and imparts its translational state to the monomer. The CG13928 protein, which binds only to monomeric Orb2, promotes deadenylation, whereas the putative poly(A) binding protein CG4612 promotes oligomeric Orb2-dependent translation. Our data support a model in which monomeric Orb2 keeps target mRNA in a translationally dormant state and experience-dependent conversion to the amyloidogenic state activates translation, resulting in persistent alteration of synaptic activity and stabilization of memory.


New Finding on Synapse Destruction May Open Path to Alzheimer’s Therapy


A team led by scientists at the University of New South Wales in Australia say they have discovered how connections between brain cells are destroyed in the early stages of Alzheimer’s disease. They believe their work opens up a new avenue for research on possible treatments for the degenerative brain condition.

“One of the first signs of Alzheimer’s disease is the loss of synapses—the structures that connect neurons in the brain,” noted study leader, Vladimir Sytnyk, Ph.D., of the UNSW School of Biotechnology and Biomolecular Sciences. “Synapses are required for all brain functions, and particularly for learning and forming memories. In Alzheimer’s disease, this loss of synapses occurs very early on, when people still only have mild cognitive impairment, and long before the nerve cells themselves die. We have identified a new molecular mechanism which directly contributes to this synapse loss, a discovery we hope could eventually lead to earlier diagnosis of the disease and new treatments.”

The team studied a specific protein in the brain, neural cell adhesion molecule 2 (NCAM2), one of a family of molecules that physically connects the membranes of synapses and help stabilize these long lasting synaptic contacts between neurons. The researchers paper (“Aβ-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer’s disease”) is published in Nature Communications.

Using post-mortem brain tissue from people with and without the condition, they discovered that synaptic NCAM2 levels in the part of the brain known as the hippocampus were low in those with Alzheimer’s disease. They also showed in mice studies and in the laboratory that NCAM2 was broken down by beta-amyloid, which is the main component of the plaques that build up in the brains of people with the disease.

“Our research shows the loss of synapses is linked to the loss of NCAM2 as a result of the toxic effects of beta-amyloid,” pointed out Dr. Sytnyk. “It opens up a new avenue for research on possible treatments that can prevent the destruction of NCAM2 in the brain.”


Aβ-dependent reduction of NCAM2-mediated synaptic adhesion contributes to synapse loss in Alzheimer’s disease

Iryna Leshchyns’kaHeng Tai LiewClaire ShepherdGlenda M. HallidayClaire H. StevensYazi D. KeLars M. Ittner & Vladimir Sytnyk
Nature Communications Nov 2015; 6(8836)        doi:10.1038/ncomms9836

Alzheimer’s disease (AD) is characterized by synapse loss due to mechanisms that remain poorly understood. We show that the neural cell adhesion molecule 2 (NCAM2) is enriched in synapses in the human hippocampus. This enrichment is abolished in the hippocampus of AD patients and in brains of mice overexpressing the human amyloid-β (Aβ) precursor protein carrying the pathogenic Swedish mutation. Aβ binds to NCAM2 at the cell surface of cultured hippocampal neurons and induces removal of NCAM2 from synapses. In AD hippocampus, cleavage of the membrane proximal external region of NCAM2 is increased and soluble extracellular fragments of NCAM2 (NCAM2-ED) accumulate. Knockdown of NCAM2 expression or incubation with NCAM2-ED induces disassembly of GluR1-containing glutamatergic synapses in cultured hippocampal neurons. Aβ-dependent disassembly of GluR1-containing synapses is inhibited in neurons overexpressing a cleavage-resistant mutant of NCAM2. Our data indicate that Aβ-dependent disruption of NCAM2 functions in AD hippocampus contributes to synapse loss.


Learning and memory processes depend on the number and correct functioning of synapses in the brain. Cell adhesion molecules are enriched in the pre- and postsynaptic membranes. These molecules physically connect synaptic membranes, providing mechanical stabilization of synaptic contacts1, 2, 3, are necessary for the formation of new synapses during neuronal development4, 5, and maintain and regulate synaptic plasticity in adults6, 7, 8, 9, 10.

Alzheimer’s disease (AD) is a neurodegenerative brain condition predominantly of the aging population. One of the earliest signs of AD is the loss of synapses11, which can at least partially be linked to the toxicity mediated by Aβ12, 13, 14, a peptide that accumulates in the brains of AD patients. The impact of AD on synaptic adhesion and the role of synaptic cell adhesion molecules in the progression of the disease remains poorly understood.

The neural cell adhesion molecule 2 (NCAM2), sometimes designated OCAM, belongs to the immunoglobulin superfamily of cell adhesion molecules. NCAM2 participates in homophilic trans-interactions15, 16. During human embryonic development, NCAM2 is expressed in several tissues, including lung, liver, and kidney with the highest expression in the brain17. The expression level of NCAM2 peaks around postnatal day 21 and remains high during adulthood15, suggesting that the protein is necessary both during development and in adult brains. Accordingly, studies with cultured neurons and in NCAM2 deficient mice show that NCAM2 is important for the development of the brain, and the olfactory system in particular18, 19.

The NCAM2 gene is located on chromosome 21 in humans and NCAM2 overexpression has been suggested to be one of the factors contributing to the symptoms of Down syndrome17, which presents with early-onset AD pathology. Single-nucleotide polymorphisms in the NCAM2 gene have been reported as a risk factor related to the progression of AD in the Japanese population20. A recent genome-wide association study has found an association between single-nucleotide polymorphisms in the NCAM2 gene and levels of Aβ in the cerebrospinal fluid in humans, suggesting that NCAM2 is involved in the pathogenic pathway to the senile plaques that concentrate in AD brains21. Since genetic association studies indicate a link between NCAM2 and AD, we have analysed whether AD pathology influences levels of NCAM2 in synapses. Our results indicate that the synaptic adhesion mediated by NCAM2 is highly susceptible to Aβ toxicity and that proteolytic fragments of NCAM2 generated in an Aβ-dependent manner can directly contribute to the induction of synapse disassembly.


Synaptic NCAM2 is reduced in the hippocampus in AD

To analyse whether functions of NCAM2 are affected in AD, frozen post-mortem brain tissue of AD patients and non-affected controls (n=10 each) was analysed by western blot with antibodies against NCAM2. The detailed demographic data for the subjects analysed are presented inSupplementary Table 1. Total levels of NCAM2 were slightly increased in the hippocampus, but not significantly affected in the cerebellum or superior temporal cortex in AD (Supplementary Fig. 1). In contrast, levels of VGLUT1, a presynaptic marker-protein of excitatory synapses, were reduced in AD hippocampus (Supplementary Fig. 1), indicating a loss of excitatory synapses. Levels of VGAT, a presynaptic marker-protein of inhibitory synapses, were not significantly affected in any brain region analysed (Supplementary Fig. 1).

Changes in the protein levels in brain homogenates do not necessarily reflect changes in the protein levels in synapses. To analyse whether the synaptic function of NCAM2 is affected in AD, we compared the enrichment of NCAM2 in synaptosomes isolated from the brain tissue of individuals with AD and non-affected controls by western blot analysis of synaptosomes and total homogenates of the brains used for synaptosome preparations. Equal total protein amounts from each probe were applied to the gels to compensate for any possible differences in the yield of synaptosomes because of the synapse loss observed in AD. Western blot analysis with antibodies against actin, VGLUT1, VGAT, synaptophysin (a general presynaptic marker-protein), and PSD95 (a postsynaptic marker-protein), showed that these proteins were enriched to similar levels in synaptosomes from AD and control brains, indicating similar purities of intact synaptosome isolations (Fig. 1a). Western blot analysis showed that in control individuals NCAM2 was highly enriched in synaptosomes from the hippocampus and to a lower degree in synaptosomes from the temporal cortex and cerebellum (Fig. 1a,b). This synaptic enrichment of NCAM2 was significantly reduced in synaptosomes from AD hippocampi (Fig. 1a,b). The synaptic enrichment of NCAM2 was slightly lower in the AD versus control cerebellum, however the difference was not statistically significant (Fig. 1a,b).


Figure 1: Synaptic accumulation of NCAM2 is reduced in the hippocampus of AD-affected individuals.

Figure 2: Cleavage of the membrane-adjacent extracellular fragment of NCAM2 is increased in AD brains.

Figure 3: The extracellular domain of NCAM2 binds to Aβ.

Cleavage of NCAM2aa682-701 is increased in AD brains

NCAM2 binds to Aβ in vitro

Figure 4: NCAM2 accumulates in excitatory synapses of cultured hippocampal neurons.

NCAM2 accumulates in excitatory synapses of cultured hippocampal neurons.


(a) Low-magnification image of a cultured hippocampal neuron labelled by indirect immunofluorescence with antibodies against NCAM2, synaptophysin and MAP2. Note expression of NCAM2 along MAP2 positive dendrites. NCAM2 is also expressed in astrocytes (marked a) which are present in these cultures. Scale bar, 20μm. (b) High-magnification image of dendrites of neurons co-labelled with antibodies against NCAM2, synaptophysin and MAP2. Arrows show clusters of NCAM2 partially overlapping with synaptophysin accumulations. NCAM2-negative synapses are also observed (arrowheads). Scale bar, 10μm. (c) High-magnification image of a dendrite of a cultured hippocampal neuron labelled with antibodies against NCAM2, synaptophysin and PSD95. NCAM2 clusters partially overlap with accumulations of PSD95 and synaptophysin (arrows). Scale bar, 10μm. Three-dimensional analysis of the co-localization within the outlined area is on the right. Z-stack has been acquired with 0.15μm steps. The xz and yz sections along the dashed lines on the xy image are shown. Note co-localization of the NCAM2 cluster with synaptic markers. (d) Negative control, that is, labelling performed without primary antibodies, is shown. Scale bar, 10μm.


Figure 5: Aβ1–42 oligomers bind to NCAM2 at the cell surface of neurons.

Figure 6: Levels of NCAM2 are reduced in synaptosomes of cultured hippocampal neurons treated with Aβ1-42 oligomers.

Figure 7: NCAM2 co-localizes with Aβ1-42 in brains of APP23 transgenic mice.

Figure 8: NCAM2 binds to Aβ and its synaptic accumulation is reduced in the hippocampus of APP23 transgenic mice.

Aβ removes NCAM2 from synapses of hippocampal neurons

Western blot analysis showed that levels of soluble NCAM2 with the molecular weight of ~100kDa were significantly increased in culture medium from Aβ1-42-treated hippocampal neurons (Fig. 6b), further indicating that Aβ1-42 induces removal of NCAM2 off the neuronal cell surface. In contrast, levels of the soluble proteolytic products of CHL1, another synaptic cell adhesion molecule of the immunoglobulin superfamily26, 27, were not changed in the culture medium from Aβ1-42-treated hippocampal neurons (Fig. 6b). Incubation with Aβ1-42 did not increase levels of soluble NCAM2 in the culture medium from cortical neurons (Fig. 6b), suggesting that cortical neurons are more resistant to Aβ1-42-dependent NCAM2 proteolysis.

Aβ binds to and removes NCAM2 from synapses in APP23 mice

Disruption of NCAM2 adhesion promotes synapse disassembly

Figure 9: Disruption of NCAM2 functions at the neuronal cell surface promotes glutamatergic synapse disassembly.

Disruption of NCAM2 functions at the neuronal cell surface promotes glutamatergic synapse disassembly.


(ae) Cultured hippocampal neurons were either mock-treated or incubated with the recombinant soluble extracellular domains of NCAM2 (NCAM2-ED), antibodies against the extracellular domain of NCAM2 (NCAM2mAb), or Aβ1-42 oligomers. In a,b, neurons were labelled with antibodies against the extracellular domain of GluR1 before permeabilization of membranes with detergent, and co-labelled with antibodies against synaptophysin after permeabilization of membranes with detergent. Representative images of dendrites are shown (a). Note co-localization of cell surface GluR1 accumulations with synaptophysin clusters in mock-treated neurons, and increased levels of non-synaptic cell surface GluR1 accumulations in neurons treated with NCAM2-ED, NCAM2mAb or Aβ1-42. Graphs (b) show the percentage of synaptic and non-synaptic GluR1 clusters relative to total number of GluR1 clusters along dendrites and numbers of synaptophysin accumulations per dendrite length (mean+s.e.m.). *P<0.0001 (analysis of variance with Dunnett’s multiple comparison test, n>80 dendrites from 20 neurons were analysed in each group). In c, neurons were labelled with antibodies against the extracellular domain of NR1 before permeabilization of membranes with detergent, and co-labelled with antibodies against synaptophysin after permeabilization of membranes with detergent. Graphs show the percentage of synaptic and non-synaptic NR1 clusters relative to total number of NR1 clusters along dendrites (mean+s.e.m.). *P<0.0001 (analysis of variance with Dunnett’s multiple comparison test, n>85 dendrites from 20 neurons were analysed). In d,e, neurons were co-labelled with fluorescent phalloidin and synaptophysin antibodies. Representative images of dendrites are shown in d. Note higher labelling intensity and co-localization with synaptophysin of the phalloidin-labelled polymerized actin accumulations in control neurons versus neurons treated with Aβ1-42, NCAM2-ED or NCAM2mAb. Note increased numbers of filopodia and lamellipodia in neurons treated with Aβ1-42, NCAM2-ED or NCAM2 mAb. Graphs (e) show ratio of the dendrite area-to-length and phalloidin labelling intensity of dendrites of neurons. Mean values+s.e.m. are shown. *P<0.0001 (analysis of variance with Dunnett’s multiple comparison test, n=50 dendrites from 20 neurons were analysed in each group). Scale bar, 10μm (in a,d).

Cleavage-resistant NCAM2 reduces Aβ-dependent synapse loss

Figure 10: Aβ1-42 reduces the number of GluR1-containing synapses in the NCAM2-dependent manner.


(a) Representative images of dendrites of cultured hippocampal neurons transfected either with control negative miRNA (negative miR) or NCAM2miR and either mock-treated or incubated with Aβ1-42. Transfected neurons were identified by fluorescence of GFP, which is co-expressed together with miRNA. Neurons were co-labelled with antibodies against cell surface GluR1 and synaptophysin. Note that the number of synaptic GluR1 clusters is reduced and the number of non-synaptic GluR1 clusters is increased in neurons transfected with NCAM2miR. Scale bar, 10μm. (b,c) Graphs show mean+s.e.m. percentage of synaptic and non-synaptic GluR1 clusters relative to the total number of GluR1 clusters along dendrites (b) and numbers of synaptophysin accumulations per dendrite length normalized to the mean number in mock-treated neurons (c) for neurons described in (a). (df) Graphs show mean+s.e.m. percentage of synaptic and non-synaptic GluR1 clusters relative to the total number of GluR1 clusters along dendrites (d), number of synaptophysin accumulations per dendrite length normalized to the mean number in mock-treated neurons (e), and area/length ratio (f) in cultured hippocampal neurons transfected either with GFP alone or co-transfected with GFP and non-mutated NCAM2 (NCAM2WT) or NCAM2D693A mutant and either mock-treated or incubated with Aβ1-42. (g,h) Graphs show mean+s.e.m. percentage of non-synaptic GluR1 clusters relative to the total number of GluR1 clusters along dendrites (g) and area/length ratio (h) in cultured hippocampal neurons co-transfected with NCAM2 miR and either GFP, non-mutated NCAM2 (WT) or NCAM2D693A mutant (D693A) and either mock-treated or incubated with Aβ1-42. In bh, *P<0.01 (compared as indicated), ˆP<0.01 (compared with mock-treated neurons transfected with negative miR (b), GFP (df) or co-transfected with NCAM2miR and GFP (gh)), analysis of variance with Tukey’s multiple comparison test, n>50 dendrites from 20 neurons were analysed in each group.


Taken together, our results indicate that Aβ affects the numbers of GluR1-containing glutamatergic synapses in a NCAM2-dependent manner.

Alzheimer’s disease is characterized by loss of synapses, which is the strongest correlate of cognitive decline11, 29, 30, 31, 32 and possibly one of the earliest events in AD pathogenesis30, 33. Synapses are long lasting contacts between neurons, which are stabilized by a number of cell adhesion molecules that concentrate in pre- and postsynaptic membranes2, 5. Cell adhesion molecules play an essential role in maintaining synapse functionality and stability. Although cell adhesion molecules of many families are required for the synapse integrity8, 10, elimination of even one type of synaptic cell adhesion molecule is often sufficient to induce abnormalities in synapse ultrastructure and protein composition6, 7. In the present study, we show that levels of the synaptic cell adhesion molecule NCAM2 are markedly reduced in hippocampal synapses in AD brains and Aβ-forming APP23 mice. Our observations that disruption of NCAM2 interactions at the cell surface, knockdown of NCAM2 expression and Aβ exposure result in reduced numbers of glutamatergic synapses in hippocampal neurons suggest that abnormalities in NCAM2-mediated synaptic adhesion contribute to synapse loss in AD.

Although the mechanisms of synapse disassembly in AD remain poorly understood, previous studies indicated that synapse loss can be linked to Aβ-induced toxicity12, 34, 35. Our observations showing that synaptic levels of NCAM2 are similarly reduced in APP23 mice and in cultured hippocampal neurons from wild-type mice exposed to Aβ argue in favour of Aβ-dependent mechanisms in the disruption of NCAM2-mediated synaptic adhesion. We however do not exclude that other factors, such as disrupted trafficking of NCAM2 to synapses, may also contribute to the reduction of NCAM2 levels at synapses. Strikingly, the effects of Aβ on synaptic targeting of NCAM2 were particularly strong in hippocampal but not cortical or cerebellar neurons. The enhanced susceptibility of synaptic NCAM2 to Aβ-dependent proteolysis may therefore contribute to selective vulnerability of the hippocampus to AD.

Our observations that NCAM2 directly interacts with synthetic Aβ1-42, that Aβ1-42 forms a molecular complex with NCAM2 at the neuronal cell surface and that complexes of NCAM2 and oligomers of Aβ can be isolated from APP23 mouse brains, indicate that NCAM2 may function as a previously unrecognized receptor for Aβ at the neuronal cell surface. Previous studies have shown that Aβ can also bind to other cell adhesion molecules at the neuronal cell surface, among which are the prion protein36 and L137. In addition, a number of cell adhesion molecules have been shown to interact with APP, including the neural cell adhesion molecule 1 (NCAM1)38 and TAG1 (ref. 39). It remains to be investigated whether the NCAM2/Aβ complex comprises other adhesion molecules and cell surface proteins. Interestingly, NCAM1, a homologue of NCAM2, binds to prion protein40 and L1 (ref. 41). However, in spite of homology to NCAM2, NCAM1 binds to a region of APP which is different to the Aβ-containing region38.


Taken together, we show that Aβ induces synaptic loss and proteolysis of NCAM2 in cell culture and APP transgenic mouse models, providing a mechanistic explanation for synaptic NCAM2 changes in AD brains. The detrimental effects of proteolyically cleaved extracellular NCAM2 on synapses may augment the Aβ toxicity in the pathogenesis of AD. The exact molecular mechanisms underlying Aβ-induced NCAM2 changes, and to which degree it contributes to onset and progression of disease remains to be established. Nevertheless, our data reveal a new role of NCAM2 in AD that warrants further investigation.

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Larry H Bernstein, MD, FCAP, Reporter

A Pot[age] to Die For

A Pot[age] to Die For (Photo credit: jazzijava)

Neurodegerative Disease
Tumeric-Derived Compound Curcumin May Treat Alzheimer’s
Curry chemical shows promise for treating the memory-robbing disease
By Lauren K. Wolf
Department: Science & Technology
News Channels: Biological SCENE
Keywords: alternative medicine, dietary supplements, curcumin, tumeric, Alzheimer’s disease

Curcumin, derived from the rootstalk of the turmeric plant, not only gives Indian dishes their color but might treat Alzheimer’s.
Credit: Shutterstock
More than 5 million people in the U.S. currently live with Alzheimer’s disease. And according to the Alz­heimer’s Association, the situation is only going to get worse.
By 2050, the nonprofit estimates, up to 16 million Americans will have the memory-robbing disease. It will cost the U.S. $1.1 trillion annually to care for them unless a successful therapy is found.
Pharmaceutical companies have invested heavily in developing Alzheimer’s drugs, many of which target amyloid-β, a peptide that misfolds and clumps in the brains of patients. But so far, no amyloid-β-targeted medications have been successful. Expectation for the most advanced drugs—bapineu­zumab from Pfizer and Johnson & Johnson and solanezumab from Eli Lilly & Co.—are low on the basis of lackluster data from midstage clinical trials. That sentiment was reinforced last week when bapineuzumab was reported to have failed the first of four Phase III studies.
Even if these late-stage hopefuls do somehow work, they won’t come cheap, says Gregory M. Cole, a neuroscientist at the University of California, Los Angeles. These drugs “would cost patients tens of thousands of dollars per year,” he estimates. That hefty price tag stems from bapineuzumab and solanezumab being costly-to-manufacture monoclonal antibodies against amyloid-β.
“There’s a great need for inexpensive Alzheimer’s treatments,” as well as a backup plan if pharma fails, says Larry W. Baum, a professor in the School of Pharmacy at the Chinese University of Hong Kong. As a result, he says, a great many researchers have turned their attention to less pricy alternatives, such as compounds from plants and other natural sources.
Curcumin, a spice compound derived from the rootstalk of the turmeric plant (Curcuma longa), has stood out among some of the more promising naturally derived candidates.

When administered to mice that develop Alzheimer’s symptoms, curcumin decreases inflammation and reactive oxygen species in the rodents’ brains, researchers have found. The compound also inhibits the aggregation of troublesome amyloid-β strands among the animals’ nerve cells. But the development of curcumin as an Alzheimer’s drug has been stymied, scientists say, both by its low uptake in the body and a lack of funds for effective clinical trials—obstacles researchers are now trying to overcome.
In addition to contributing to curry dishes’ yellow color and pungent flavor, curcumin has been a medicine in India for thousands of years. Doctors practicing traditional Hindu medicine admire turmeric’s active ingredient for its anti-inflammatory properties and have used it to treat patients for ailments including digestive disorders and joint pain.
Only in the 1970s did Western researchers catch up with Eastern practices and confirm curcumin’s anti-inflammatory properties in the laboratory. Scientists also eventually determined that the polyphenolic compound is an antioxidant and has chemotherapeutic activity.

Bharat B. Aggarwal, a professor at the University of Texas M. D. Anderson Cancer Center, says curcumin is an example of a pleiotropic agent: It has a number of different effects and interacts with many targets and biochemical pathways in the body. He and his group have discovered that one important molecule targeted and subsequently suppressed by curcumin is NF-κB, a transcription factor that switches on the body’s inflammatory response when activated (J. Biol. Chem., DOI: 10.1074/jbc.270.42.24995).
Aside from NF-κB, curcumin seems to interact with several other molecules in the inflammatory pathway, a biological activity that Aggarwal thinks is advantageous. “All chronic diseases are caused by dysregulation of multiple targets,” he says. “Chemists don’t yet know how to design a drug that hits multiple targets.” With curcumin, “Mother Nature has already provided a compound that does so.”
Curcumin’s pleiotropy also brought it to the attention of UCLA’s Cole during the early 1990s while he was searching for possible Alzheimer’s therapeutics. “That was before we knew about amyloid-β” and its full role in Alzheimer’s, he says. “We were working on the disease from an oxidative damage and inflammation point of view—two processes implicated in aging.”
When Cole and his wife, Sally A. Frautschy, also at UCLA, searched the literature for compounds that could tackle both of these age-related processes, curcumin jumped out at them. It also didn’t hurt that the incidence of Alz­heimer’s in India, where large amounts of curcumin are consumed regularly, is lower than in other parts of the developing world (Lancet Neurol., DOI:10.1016/s1474-4422(08)70169-8).

In 2001, Cole, Frautschy, and colleagues published the first papers that demonstrated curcumin’s potential to treat neurodegenerative disease (Neurobiol. Aging, DOI: 10.1016/s0197-4580(01)00300-1; J. Neurosci.2001, 8370). The researchers studied the effects of curcumin on rats that had amyloid-β injected into their brains, as well as mice engineered to develop amyloid brain plaques. In both cases, curcumin suppressed oxidative tissue damage and reduced amyloid-β deposits.
Those results, Cole says, “turned us into curcuminologists.”
Although the UCLA team observed that curcumin decreased amyloid plaques in animal models, at the time, the researchers weren’t sure of the molecular mechanism involved.
Soon after the team’s first results were published, Cole recalls, a colleague brought to his attention the structural similarity between curcumin and the dyes used to stain amyloid plaques in diseased brain tissue. When Cole and Frautschy tested the spice compound, they saw that it, too, could stick to aggregated amyloid-β. “We thought, ‘Wow, not only is curcumin an antioxidant and an anti-inflammatory, but it also might be an anti-amyloid drug,’ ” he says.
In 2004, a group in Japan demonstrated that submicromolar concentrations of curcumin in solution could inhibit aggregation of amyloid-β and break up preformed fibrils of the stuff (J. Neurosci. Res., DOI: 10.1002/jnr.20025). Shortly after that, the UCLA team demonstrated the same (J. Biol. Chem., DOI: 10.1074/jbc.m404751200).
As an Alzheimer’s drug, however, it’s unclear how important it is that the spice compound inhibits amyloid-β aggregation, Cole says. “When you have something that’s so pleiotropic,” he adds, “it’s hard to know” which of its modes of action is most effective.
Having multiple targets may be what helps curcumin have such beneficial, neuroprotective effects, says David R. Schubert, a neurobiologist at the Salk Institute for Biological Studies, in La Jolla, Calif. But its pleiotropy can also be a detriment, he contends.
The pharmaceutical world, Schubert says, focuses on designing drugs aimed at hitting single-target molecules with high affinity. “But we don’t really know what ‘the’ target for curcumin is,” he says, “and we get knocked for it on grant requests.”
Another problem with curcumin is poor bioavailability. When ingested, UCLA’s Cole says, the compound gets converted into other molecular forms, such as curcumin glucuronide or curcumin sulfate. It also gets hydrolyzed at the alkaline and neutral pHs present in many areas of the body. Not much of the curcumin gets into the bloodstream, let alone past the blood-brain barrier, in its pure, active form, he adds.

Unfortunately, neither Cole nor Baum at the Chinese University of Hong Kong realized the poor bioavailability until they had each launched a clinical trial of curcumin. So the studies showed no significant difference between Alzheimer’s patients taking the spice compound and those taking a placebo (J. Clin. Psychopharma­col., DOI: 10.1097/jcp.0b013e318160862c).
“But we did show curcumin was safe for patients,” Baum says, finding a silver lining to the blunder. “We didn’t see any adverse effects even at high doses.”

Some researchers, such as Salk’s Schubert, are tackling curcumin’s low bioavailability by modifying the compound to improve its properties. Schubert and his group have come up with a molecule, called J147, that’s a hybrid of curcumin and cyclohexyl-bisphenol A. Like Cole and coworkers, they also came upon the compound not by initially screening for the ability to interact with amyloid-β, but by screening for the ability to alleviate age-related symptoms.

The researchers hit upon J147 by exposing cultured Alzheimer’s nerve cells to a library of compounds and then measuring changes to levels of biomarkers for oxidative stress, inflammation, and nerve growth. J147 performed well in all categories. And when given to mice engineered to accumulate amyloid-β clumps in their brains, the hybrid molecule prevented memory loss and reduced formation of amyloid plaques over time (PLoS One, DOI: 10.1371/journal.pone.0027865).

Other researchers have tackled curcumin’s poor bioavailability by reformulating it. Both Baum and Cole have encapsulated curcumin in nanospheres coated with either polymers or lipids to protect the compound from modification after ingestion. Cole tells C&EN that by packaging the curcumin in this way, he and his group have gotten micromolar quantities of it into the bloodstream of humans. The researchers are now preparing for a small clinical trial to test the formulation on patients with mild cognitive impairment, who are at an increased risk of developing Alzheimer’s.

An early-intervention human study such as this one comes with its own set of challenges, Cole says. People with mild cognitive impairment “have good days and bad days,” he says. A large trial over a long period would be the best way to get any meaningful data, he adds.  Such a trial can cost up to $100 million, a budget big pharma might be able to scrape together but that is far out of reach for academics funded by grants, Cole says. “If you’re down at the level of what an individual investigator can do, you’re running a small trial,” he says, “and even if the result is positive, it might be inconclusive” because of its small size or short duration. That’s one of the reasons the curcumin work is slow-going, Cole contends.
NIH-Funded Research Provides New Clues on How ApoE4 Affects Alzheimer’s Risk
Published: Tuesday, October 30, 2012
Last Updated: Tuesday, October 30, 2012

Researchers found that ApoE4 triggers an inflammatory reaction that weakens the blood-brain barrier.
Common variants of the ApoE gene are strongly associated with the risk of developing late-onset Alzheimer’s disease, but the gene’s role in the disease has been unclear.

Now, researchers funded by the National Institutes of Health have found that in mice, having the most risky variant of ApoE damages the blood vessels that feed the brain.

The researchers found that the high-risk variant, ApoE4, triggers an inflammatory reaction that weakens the blood-brain barrier, a network of cells and other components that lines brain’s brain vessels.

Normally, this barrier allows nutrients into the brain and keeps harmful substances out.

The study appears in Nature, and was led by Berislav Zlokovic, M.D., Ph.D., director of the Center for Neurodegeneration and Regeneration at the Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles.

“Understanding the role of ApoE4 in Alzheimer’s disease may be one of the most important avenues to a new therapy,” Dr. Zlokovic said. “Our study shows that ApoE4 triggers a cascade of events that damages the brain’s vascular system,” he said, referring to the system of blood vessels that supply the brain.

The ApoE gene encodes a protein that helps regulate the levels and distribution of cholesterol and other lipids in the body. The gene exists in three varieties.

ApoE2 is thought to play a protective role against both Alzheimer’s and heart disease, ApoE3 is believed to be neutral, and ApoE4 confers a higher risk for both conditions.

Outside the brain, the ApoE4 protein appears to be less effective than other versions at clearing away cholesterol; however, inside the brain, exactly how ApoE4 contributes to Alzheimer’s disease has been a mystery.

Dr. Zlokovic and his team studied several lines of genetically engineered mice, including one that lacks the ApoE gene and three other lines that produce only human ApoE2, ApoE3 or ApoE4. Mice normally have only a single version of ApoE.

The researchers found that mice whose bodies made only ApoE4, or made no ApoE at all, had a leaky blood-brain barrier. With the barrier compromised, harmful proteins in the blood made their way into the mice’s brains, and after several weeks, the researchers were able to detect loss of small blood vessels, changes in brain function, and a loss of connections between brain cells.

“The study demonstrates that damage to the brain’s vascular system may play a key role in Alzheimer’s disease, and highlights growing recognition of potential links between stroke and Alzheimer’s-type dementia,” said Roderick Corriveau, Ph.D., a program director at NIH’s National Institute of Neurological Disorders and Stroke (NINDS), which helped fund the research. “It also suggests that we might be able to decrease the risk of Alzheimer’s disease among ApoE4 carriers by improving their vascular health.”

The researchers also found that ApoE2 and ApoE3 help control the levels of an inflammatory molecule called cyclophilin A (CypA), but ApoE4 does not. Levels of CypA were raised about five-fold in blood vessels of mice that produce only ApoE4.

The excess CypA then activated an enzyme, called MMP-9, which destroys protein components of the blood-brain barrier. Treatment with the immunosuppressant drug cyclosporine A, which inhibits CypA, preserved the integrity of the blood-brain barrier and lessened damage to the brain.

An inhibitor of the MMP-9 enzyme had similar beneficial effects. In prior studies, inhibitors of this enzyme have been shown to reduce brain damage after stroke in animal models.

“These findings point to cyclophilin A as a potential new drug target for Alzheimer’s disease,” said Suzana Petanceska, Ph.D., a program director at NIH’s National Institute on Aging (NIA), which also funded Dr. Zlokovic’s study.

“Many population studies have shown an association between vascular risk factors in mid-life, such as high blood pressure and diabetes, and the risk for Alzheimer’s in late-life. We need more research aimed at deepening our understanding of the mechanisms involved and to test whether treatments that reduce vascular risk factors may be helpful against Alzheimer’s.”

Alzheimer’s disease is the most common cause of dementia in older adults, and affects more than 5 million Americans. A hallmark of the disease is a toxic protein fragment called beta-amyloid that accumulates in clumps, or plaques, within the brain.

Gene variations that cause higher levels of beta-amyloid are associated with a rare type of Alzheimer’s that appears early in life, between age 30 and 60.

However, it is the ApoE4 gene variant that is most strongly tied to the more common, late-onset type of Alzheimer’s disease. Inheriting a single copy of ApoE4 from a parent increases the risk of Alzheimer’s disease by about three-fold. Inheriting two copies, one from each parent, increases the risk by about 12-fold.

Dr. Zlokovic’s study and others point to a complex interplay between beta-amyloid and ApoE4. On the one hand, beta-amyloid is known to build up in and damage blood vessels and cause bleeding into the brain.

On the other hand, Dr. Zlokovic’s data suggest that ApoE4 can damage the vascular system independently of beta-amyloid. He theorizes that this damage makes it harder to clear beta-amyloid from the brain.

Some therapies under investigation for Alzheimer’s focus on destroying amyloid plaques, but therapies designed to compensate for ApoE4 might help prevent the plaques from forming, he said.

Compound Could Become Alzheimer’s Treatment
Thu, 10/11/2012 – 1:29pm
A new molecule designed to treat Alzheimer’s disease has significant promise and is potentially the safest to date, according to researchers.

Purdue University professor Arun Ghosh designed the molecule, which is a highly potent beta-secretase inhibitor with unique features that ensure it goes only to its target and does not affect healthy physiological processes, he said.

“This molecule maintains the disease-fighting properties of earlier beta-secretase inhibitors, but is much less likely to cause harmful side effects,” said Ghosh, the Ian P. Rothwell Distinguished Professor of Chemistry and Medicinal Chemistry and Molecular Pharmacology. “The selectivity we achieved is unprecedented, which gives it great promise for the long-term medication required to treat Alzheimer’s. Each time a treatment misses its disease target and instead interacts with a healthy cell or molecule, damage is done that we call toxicity. Even low levels of this toxicity could build up over years and years of treatment, and an Alzheimer’s patient would need to be treated for the rest of his or her life.”

The new molecule shows a 7,000-fold selectivity for its target enzyme, which far surpasses the benchmark of a 1,000-fold selectivity for a viable treatment molecule, and dwarfs the selectivity values in the hundreds for past beta-secretase inhibitors, he said. A paper detailing the work will be published in an upcoming Alzheimer’s research issue of the Journal of Medicinal Chemistry and is currently available online. The National Institutes of Health funded the research.

Beta-secretase inhibitors, which could allow for intervention in the early stages of Alzheimer’s disease, have promise as a potential treatment. Several drugs based on this molecular target have made it to clinical trials, including one based on a molecule Ghosh designed previously. These molecules prevent the first step in a chain of events that leads to the formation of amyloid plaque in the brain, fibrous clumps of toxic proteins that are believed to cause the disease’s devastating symptoms.

The National Institute on Aging estimates that 5.1 million Americans suffer from Alzheimer’s disease, which leads to dementia by affecting parts of the brain that control thought, memory and language.

“Alzheimer’s is a progressive disease that destroys the brain and also destroys the quality of life for those who suffer from it,” Ghosh said. “It eventually robs people of their ability to recognize their own spouse or child and to complete basic tasks necessary for independence, like getting dressed. It is a truly devastating disease for those who suffer from it and for their friends and loved ones.”

Earlier versions of the beta-secretase inhibitor were able to stop and even reverse the progression of amyloid plaques in tests on mice, but potency and selectivity are only two of the three pillars of a viable Alzheimer’s treatment, Ghosh said. It has yet to be shown whether this molecule possesses the third pillar, the ability to be turned into an easily administered drug that passes through the blood-brain barrier.

Ghosh collaborates with Jordan Tang, the J.G. Puterbaugh Chair in Medical Research at the Oklahoma Medical Research Foundation, who in 2000 identified beta-secretase and its role in the progression of Alzheimer’s. Later that year Ghosh designed his first molecule that bound to and inhibited the activity of the enzyme. He has strived to create the needed improvements ever since.

Ghosh bypasses the usual lengthy process of trial and error in finding useful inhibitor molecules by using a structure-based design strategy. He uses the structures of the inhibitor bound to the enzyme as a guide to what molecular features are important for desirable and undesirable characteristics. Then he removes, replaces and adds molecular groups to amplify the desirable and eliminate the undesirable.

“I believe structure-based design is vital to the development of new and improved medicine,” said Ghosh, who also is a member of the Purdue University Center for Cancer Research. “These strategies have the potential to eliminate enormous costs and time needed in traditional random screening protocols for drug development. Structure-based strategies allow us to design molecules that do precisely what we need them to do with fewer undesirable side effects.”

Tang performed the X-ray crystallography and captured the crystal structures to reveal important insights and serve as a guide for Ghosh’s designs.

“Developing inhibitors into clinically useful drugs is an evolutionary process,” Tang said. “We learn what works and what doesn’t along the way, and the knowledge permits us to do better in the next step. The miracles of modern medicine are built on top of excellent scientific findings. We try to do good science and know that the consequence will be a better chance for conquering diseases and improving lives.”

Beta-secretase belongs to a class of enzymes called aspartyl proteases. Research into beta-secretase inhibitors faced setbacks when other aspartyl proteases similar in structure, called memapsin 1 and cathepsin D, were discovered and found to be involved in many important physiological processes. Earlier designed beta-secretase inhibitors were found also to work against the biologically necessary enzymes.

Ghosh’s team focused on developing ways to make the inhibitor more selective so that it would avoid these other, physiologically important enzymes. They compared the structures of beta-secretase and memapsin 1 as they interacted with the inhibitor to find an active area unique only to beta-secretase. Then they added a functional molecular feature that targets and interacts with the unique area, making the inhibitor more attractive to beta-secretase and less attractive to the other enzymes.

“The added feature serves as a bait on the inhibitor molecule that entices beta-secretase and also grabs onto it tightly, greatly enhancing its selectivity,” he said. “This is a fundamental insight into the origins of selectivity and ways to increase it.”
Ghosh said this work highlights an important purpose of academic research.

“Academic research lays out and shares the fundamentals to advance drug discovery,” he said. “Advances in treatment are built upon the basic research happening at universities.”

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