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Archive for the ‘Small Molecules in Development of Therapeutic Drugs’ Category


Beyond tau and amyloid

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

BEYOND AΒ AND TAU: OTHER TOXIC INSULTS AND AD PATHOLOGY

 

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.

 

Summary

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.

References

  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.

Conclusions

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

Affiliations   

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?

https://neuroalzheimerscommunity.nature.com/videos/3896-other-toxic-insults/download.mp4

 

 

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Variability of Gene Expression and Drug Resistance

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

New Data Suggest Extreme Genetic Diversity of Tumors May Impart Drug Resistance

NEW YORK (GenomeWeb) – Researchers from the University of Chicago and the Beijing Institute of Genomics have undertaken one of the most extensive analyses of the genome of a single tumor and found far greater genetic diversity than anticipated. Such variation, they said, may enable even small tumors to resist treatment.

“With 100 million mutations, each capable of altering a protein in some way, there is a high probability that a significant minority of tumor cells will survive, even after aggressive treatment,” Chung-I Wu, a University of Chicago researcher and senior author of the study, said in a statement. “In a setting with so much diversity, those cells could multiply to form new tumors, which would be resistant to standard treatments.”

 

Extremely high genetic diversity in a single tumor points to prevalence of non-Darwinian cell evolution

Shaoping Linga,1Zheng Hua,1Zuyu Yanga,1Fang Yanga,1Yawei LiaPei LinbKe ChenaLili DongaLihua CaoaYong TaoaLingtong HaoaQingjian ChenbQiang Gonga, et al.

Shaoping Ling,  PNAS   http://dx.doi.org:/10.1073/pnas.1519556112      http://www.pnas.org/content/early/2015/11/11/1519556112

A tumor comprising many cells can be compared to a natural population with many individuals. The amount of genetic diversity reflects how it has evolved and can influence its future evolution. We evaluated a single tumor by sequencing or genotyping nearly 300 regions from the tumor. When the data were analyzed by modern population genetic theory, we estimated more than 100 million coding region mutations in this unexceptional tumor. The extreme genetic diversity implies evolution under the non-Darwinian mode. In contrast, under the prevailing view of Darwinian selection, the genetic diversity would be orders of magnitude lower. Because genetic diversity accrues rapidly, a high probability of drug resistance should be heeded, even in the treatment of microscopic tumors.

The prevailing view that the evolution of cells in a tumor is driven by Darwinian selection has never been rigorously tested. Because selection greatly affects the level of intratumor genetic diversity, it is important to assess whether intratumor evolution follows the Darwinian or the non-Darwinian mode of evolution. To provide the statistical power, many regions in a single tumor need to be sampled and analyzed much more extensively than has been attempted in previous intratumor studies. Here, from a hepatocellular carcinoma (HCC) tumor, we evaluated multiregional samples from the tumor, using either whole-exome sequencing (WES) (n = 23 samples) or genotyping (n = 286) under both the infinite-site and infinite-allele models of population genetics. In addition to the many single-nucleotide variations (SNVs) present in all samples, there were 35 “polymorphic” SNVs among samples. High genetic diversity was evident as the 23 WES samples defined 20 unique cell clones. With all 286 samples genotyped, clonal diversity agreed well with the non-Darwinian model with no evidence of positive Darwinian selection. Under the non-Darwinian model,MALL (the number of coding region mutations in the entire tumor) was estimated to be greater than 100 million in this tumor. DNA sequences reveal local diversities in small patches of cells and validate the estimation. In contrast, the genetic diversity under a Darwinian model would generally be orders of magnitude smaller. Because the level of genetic diversity will have implications on therapeutic resistance, non-Darwinian evolution should be heeded in cancer treatments even for microscopic tumors.

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The findings, which appeared in the Proceedings of the National Academy of Sciences this week, also call into question the widely held view that evolution at the cellular level is driven by Darwinian selection, revealing a level of rapid and extensive genetic diversity beyond what would be expected under this model.

In the study, the researchers focused on a single hepatocellular carcinoma tumor, roughly the size of a ping pong ball. They sampled 286 regions from a single slice of the tumor, studying each one with either whole-exome sequencing or genotyping under both the infinite-site and infinite-allele models of population genetics.

Based on their analyses, the team estimated more than 100 million coding region mutations in what they called an “unexceptional” tumor — more mutations than would ordinarily be expected by orders of magnitude, according to Wu.

This extreme genetic diversity, the study’s authors wrote, implies evolution under the non-Darwinian mode, which is driven by random mutations largely unaffected by natural selection. It also raises the question of why there is so little apparent Darwinian selection in the tumor.

The scientists speculated that in solid tumors, cells remain together and do not migrate, “so that when an advantageous mutation indeed emerges, cells carrying it are competing mostly with themselves. These mutations may confer advantages in fighting for space or extracting nutrients, but they are stifled by their own advantages,” they wrote.

Beneficial mutations may emerge on occasion, but in solid tumors the cell populations are “so structured that selection may often be blunted,” they stated. “The physiological effect has to be very strong to overcome those constraints.” Cancer drugs could remove those constraints, loosening up a cell population and allowing competition to occur, the investigators added.

Wu and his colleagues see the presence of so many mutations in a tumor as creating problems when it comes to treatment. “It almost guarantees that some cells will be resistant,” study co-author and University of Chicago oncologist Daniel Catenacci said in the statement. “But it also suggests that aggressive treatment could push tumor cells into a more Darwinian mode.”

Overall, the findings highlight the need to consider non-Darwinian evolution and the vast genetic diversity it can confer as factors when developing treatment strategies, even for small tumors, the researchers concluded.

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Neural Networks in Alzheimer’s

Larry H. Bernstein, MD, FCAP, Curator

LPBI

SfN 2015 Recap: The Role of Synapses, Neural Networks in Alzheimer’s

Stephanie Guzowski, Editor

http://www.dddmag.com/articles/2015/11/sfn-2015-recap-role-synapses-neural-networks-alzheimers

http://www.dddmag.com/sites/dddmag.com/files/perineuronal%20nets_SfN.jpg

Perineuronal nets, shown in green, in three regions of the mouse brain. Credit: S.F. Palida et al.

Cognition and behavior rely on communication between individual neurons and extensive interactions between neural networks. But when synaptic dysfunction occurs, the results can be dire, leading to neurodegenerative symptoms in Alzheimer’s disease.

“The brain is the seed of our personal identity,” said Valina Dawson, Ph.D., director of neurogeneration and stem cell programs at Johns Hopkins University in Baltimore, Maryland. “It allows us to interact with our world but when things go wrong in the brain, it’s disastrous for the individual as well as the family.

“Our ability to treat these diseases is limited at the moment. We need new insight into what goes wrong.”

A lesser-known protein

Researchers, for years, have targeted amyloid beta (Aβ) in attempts to halt the progression of Alzheimer’s disease, and have recently, shown increased interest in the protein, tau.

But Paula Pousinha, Ph.D., at the French National Centre for Scientific Research, has focused her research on a lesser-known protein fragment: amyloid precursor protein intracellular domain (AICD). AICD is a fragment of amyloid precursor protein (APP), which is formed at the same time as Aβ in the brain. New evidence suggests that in addition to Aβ, AICD also disrupts communication between neurons during the progression of Alzheimer’s disease. Pousinha presented thesepublished findings at this year’s Society for Neuroscience (SfN) conference, which took place from October 17 to 21 in Chicago.

“Although AICD has been known for more than 10 years, it has been poorly studied,” said Pousinha.

Pousinha’s research team demonstrated that overexpressing AICD levels with AAV vector in rats’ brains “perturbs neuronal communication in the hippocampus,” a key structure necessary in forming memories and an area earliest affected in Alzheimer’s disease.

“In normal animals, if we apply to these neurons a high-frequency stimulation, afterward the neurons are stronger,” said Pousinha. “Neurons where we overexpressed AICD failed to have this potentization.”

Pousinha doesn’t negate the importance of Aβ in the development of neurodegenerative diseases. “Our study doesn’t exclude the pathological effects of Aβ,” she said. “We believe that Alzheimer’s disease is much more complex and has more than one candidate that has implications.

“It’s very important for the scientific community to understand the role of all these APP fragments of neuroinflammation — different pieces of the puzzle of how we can stop the disease progression.”

How do memories persist in the brain long term?

New research, also presented at this year’s SfN, has implications for understanding memory to develop treatments for Alzheimer’s disease and dementias. Sakina Palida, a graduate student at the University of California, San Diego found that localized modifications in the perineuronal net (PNN) at synapses could be a mechanism by which information is stably encoded and preserved in the brain over time.

“We still don’t understand how we stably encode and store memories in our brains for up to our entire lifetimes,” said Palida. The prevailing idea on how memories are maintained over time generally focus on postsynaptic proteins, said Palida. “But the problem with looking at intracellular synaptic proteins is that the majority turn over rapidly, of hours to at most a few days. So they’re very unstable.”

So, Palida and her team identified PNN as an ideal substrate for long-term memory. “Kind of like how you carve into stone — stone is a stable substrate — you retain the information regardless of what comes and goes over it.” They demonstrated that individual PNN proteins are highly stable, and that the PNN is locally degraded when synapses are strengthened.

And the team also demonstrated that mice lacking enzymes that degrade the PNN have deficient long-term, but not short-term, memory. “Which is a really exciting new result,” said Palida.

To track the PNN in live animals, Palida and her team fused a fluorescent protein to a small link protein in the PNN to allow tracking of PNN dynamics in real time. They also monitored PNN degradation in live cells after stimulating neurons with brain-derived neurotrophic factor (BDNF), a chemical secreted in the nervous system to enhance signaling — and observed localized degradation of the PNN at some newly formed synapses.

Crtl 1-Venus. Fusion of a fluorescent protein to small link proteins in the PNN allows tracking of PNN dynamics over time. Credit: S.F. Palida et al. Crtl1-Venus Neurons. Tracking PNN dynamics in live cells, in mouse brain tissue. Credit: S.F. Palida et al.

What’s next? “We’re currently making transgenic animals to express this protein, which would allow us to monitor PNN dynamics simultaneously with synaptic dynamics in a live animal brain, and really investigate this hypothesis further,” said Palida.

Increased APP intracellular domain (AICD) production perturbs synaptic signal integration via increased NMDAR function

*Paula A Pousinha1PubmedElisabeth Raymond1PubmedXavier Mouska1PubmedMichael Willem2PubmedHélène Marie1Pubmed

1660 Route de Lucioles, CNRS IPMC UMR 7275, Valbonne, France2Ludwig-Maximilians-University Munich, Munich, Germany

Alzheimer’s disease (AD) is a neurodegenerative disease that begins as mild short-term memory deficits and culminates in total loss of cognition and executive functions. The main culprit of the disease, resulting from Amyloid-Precursor Protein (APP) processing, has been thought to be amyloid-b peptide (Ab). However, despite the genetic and cell biological evidence that supports the amyloid cascade hypothesis, it is becoming clear that AD etiology is complex and that Ab alone is unable to account for all aspects of AD [Pimplikar et al. J Neurosci.30: 14946. 2010]. Gamma-secretase not only liberates Ab, but also its C-terminal intracellular counterpart called APP intracellular domain (AICD) [Passer. et al. JAlzheimers Dis.2: 289-301. 2000], which is known to also accumulate in AD patient’s brain [Ghosal et al. PNAS.106:18367. 2009], but surprisingly little is known about its functions in the hippocampus. To address this crucial issue, we increased AICD production in vivo in adult CA1 pyramidal neurons, mimicking the human pathological condition. Different ex-vivo electrophysiological and pharmacological approaches, including double- patch of neighbor neurons were used. We clearly demonstrate that in vivo AICD production increases synaptic NMDA receptor currents. This causes a frequency-dependent disruption of synaptic signal integration, leading to impaired long-term potentiation, which we were able to rescue by different pharmacological approaches. Our results provide convincing and entirely novel evidence that increased in vivo production of AICD is enough, per se, to cause synaptic dysfunction in CA1 hippocampal neurons.

131.21P2X2R-FE65 interaction induces synaptic failure and neuronal dyshomeostasis after treatments with soluble oligomers of amyloid beta peptide

300.15Early synaptic deficits in Alzheimer’s disease involve neuronal adenosine A2A receptors

215.08Homeostatic coupling between surface trafficking and cleavage of amyloid precursor protein

280.11A novel mechanism for lowering Abeta

383.22Impact of intracellular soluble oligomers of amyloid-β peptide on glutamatergic synaptic transmission

Society for Neuroscience Annual Meeting Showcases Strides in Brain Research

10/23/2015 – Stephanie Guzowski, Editor

CHICAGO – Nearly 30,000 researchers from more than 80 countries gathered this week at the annual Society for Neuroscience (SfN) meeting, the world’s largest conference focused on scientific discovery related to the brain and nervous system.

The 45th annual SfN meeting at McCormick Place convention center showcased more than 15,000 scientific presentations on advances in technologies and new research about brain structure, disease and treatments, and 517 exhibitors, according to event organizers.

Presentations covered a wide variety of topics including new technologies to study the brain, the science behind addiction, potential treatments for spinal cord injuries, and the role of synapses in neurological conditions.

Of particular focus was the Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative, the large collaborative quest to develop technologies for a dynamic view of the brain. In early October, the National Institutes of Health announced its second round of funding to support goals, bringing the NIH investment to $85 million in fiscal year 2015.

Toxic Tau Could be Key to Alzheimer’s Treatment

01/06/2015 – Stephanie Guzowski, Editor

http://www.dddmag.com/articles/2015/01/toxic-tau-could-be-key-alzheimers-treatment

http://www.dddmag.com/sites/dddmag.com/files/tangles_Alz2.jpg

“But now, we know that tau is not simply a bystander but also a player,” Li said. “Both proteins work together to damage cell functions as the disease unfolds.”

Targeting tau

In the healthy brain, tau protein helps with the building and functioning of neurons. But when tau malfunctions, it creates abnormal clumps of protein fibers—neurofibrillary tangles—which spread rapidly throughout the brain. This highly toxic and altered form of the brain protein tau is called “tau oligomer.”

“There’s growing evidence that tau oligomers, not tau protein in general, are responsible for the development of neurodegenerative diseases, like Alzheimer’s,” said Julia Gerson, a graduate student in neuroscience at the University of Texas Medical Branch.

In Gerson’s research, which she presented at this year’s Society for Neuroscience meeting in Washington, D.C., Gerson and her team injected tau oligomers from people with Alzheimer’s into the brains of healthy mice. Subsequent testing revealed that the mice had developed memory loss.

“When we inject mice with tau oligomers, we see that they spend the same amount of time exploring a familiar object as an unfamiliar object,” said Gerson. “So they’re incapable of remembering that they’ve already seen this familiar object.”

What’s more, the molecules had multiplied throughout the animals’ brains. “This suggests that tau oligomers may spread from the injection site to other unaffected regions,” said Gerson.

Future treatments

Understanding tau’s connection to Alzheimer’s could have implications for potential therapies. “If we can stop the spread of these toxic tau oligomers, we may be capable of either preventing, or reversing, symptoms,” said Gerson. Gerson’s lab is currently investigating antibodies, which specifically fight tau oligomers.

Click to Enlarge. Normal brain vs. Alzheimer’s brain (Credit: Garrondo)

Erik Roberson, M.D., Ph.D., at the University of Alabama at Birmingham, and colleagues looked at how boosting the function of a specific type of neurotransmitter receptor, the NMDA receptor, provided benefit to people with the second most common type of dementia: frontotemporal dementia (FTD), a disease in which people experience rapid and dramatic changes in behavior, personality and social skills. People often quickly deteriorate and usually die about three years after diagnosis; there is also no effective treatment for FTD.

Since mutated tau impairs synapses—the connections between neurons—by reducing the size of NMDA receptors, “boosting the function of remaining NMDA receptors may help restore synaptic firing, and reverse behavioral abnormalities,” said Roberson.

Roberson’s, along with others’ work presented at the Society of Neuroscience meeting, focused on using animal models that mimic developing tau pathology. “These new mouse models, which contain both tau tangles and amyloid plaques” said Dr. Li, “offer the possibility of more accurately testing therapies directed at delaying the onset of amyloid beta plaques, tau accumulation and neuronal loss, all characteristic features of Alzheimer’s.”

Are clinical trials next?

Potentially, yes. “This arena of academic research has been ongoing for several years—it’s a younger area in terms of involvement of drug discovery,” said Sangram Sisodia, Ph.D., director of the Center for Molecular Neurobiology at the University of Chicago. “But I believe there is growing interest in pharma companies about targeting tau.

“The tau protein plays an incredibly complex role in the development of Alzheimer’s and other neurodegenerative diseases,” said Sisodia. “We are in the early stages of understanding that role, which will be crucial for developing effective preventions or treatments.”

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antibody-like proteins to awaken and destroy HIV holdouts

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Two-Faced Proteins May Tackle HIV Reservoirs

Researchers design antibody-like proteins to awaken and destroy HIV holdouts.

By Amanda B. Keener | October 21, 2015

http://www.the-scientist.com//?articles.view/articleNo/44293/title/Two-Faced-Proteins-May-Tackle-HIV-Reservoirs/#.

For the millions of people living with HIV worldwide, a life-long commitment to antiretroviral drugs is a must. Without these drugs, reservoirs of HIV hiding within resting T cells throughout the body can easily resurge and cause disease. In a study published yesterday (October 20) in Nature Communications, researchers from the National Institute of Allergy and Infectious Diseases (NIAID) in Bethesda, Maryland, described a bispecific antibody-like protein that attacks those reservoirs by coaxing HIV out of hiding and targeting infected cells for destruction.

“In order to kill the [infected] cell, the cell has to be activated,” said study coauthor John Mascola, director of NIAID’s Vaccine Research Center. This is because HIV has a way of hiding out inside inactive CD4+ T cells where the virus adopts a dormant-like state known as latency. In this state, the virus is impervious to antiretroviral drugs as well as antibodies that might otherwise alert other immune cells to the virus’ presence inside an infected cell. “By definition, latently-infected cells don’t express virus proteins,” Oliver Schwartz, the head of the virus and immunity laboratory at the Pasteur Institute in Paris, who was not involved in the work, told The Scientist.

Mascola and his colleagues designed a protein that activates latently-infected T cells by targeting a protein found on the surface of all T cells called CD3. Engagement of CD3 signals infected CD4+ T cells to start dividing, which revamps HIV’s replication machinery causing the virus to make proteins that appear on the surface of the infected cell. Mascola’s team tested the protein, called VRC07-αCD3, on T cells donated by HIV patients on antiretroviral therapies. The researchers found that VRC07-αCD3 caused the T cells to display Env, indicating that latent virus had become reactivated.

In recent years, researchers have come up with a handful of approaches to activate latent HIV, such ashistone deacetylase inhibitors, which increase viral gene expression. VRC07-αCD3, however, doesn’t just activate latent HIV—it also binds Env on the surface of infected CD4+ T cells and tags the cells for killing by another sort of cell called CD8+ killer T cells. Using T cells in culture, Mascola’s team demonstrated that the CD3-binding region of the protein triggers killer CD8+ T cells to lyse CD4+ T cells expressing Env.

The NIAID study was preceded by one in The Journal of Clinical Investigation that described a similar protein with dual specificities for CD3 and Env, but with a slightly different structure. Both designs share features that allow the proteins to activate latent cells, tag Env, and activate and bring killer CD8+ T cells into close proximity of their infected targets.

The dual specificity for CD3 and Env also provides a layer of safety: it ensured the killer CD8+ T cells only acted in full force when HIV was present. “That was an encouraging part of the data,” Mascola said.

Schwartz said this feature potentially addresses the concern that nonspecific activation of large numbers of T cells could elicit a dangerous overactivation of the immune system called a cytokine storm.

Mascola and his colleagues tested the safety of VRC07-αCD3 in five HIV-infected Rhesus macaques by giving the animals six doses over the course of three weeks. The monkeys were also on antiretroviral drugs, and the virus remained undetectable throughout the treatment. Although the monkeys tolerated the drug well, VRC07-αCD3 did activate T cells and caused serum cytokine levels to increase. “So this type of treatment is not risk-free,” Caltech virologist Pamela Bjorkman, who was not involved in the work, wrote in an email to The Scientist.

Mascola said his group plans to continue testing VRC07-αCD3 in macaques and in humanized mice to work out a balance between T cell activation and HIV killing. However, neither model “really tells you what’s going to happen in people,” he said. “We’ll have to proceed slowly in the clinic.”

A. Pegu et al., “Activation and lysis of human CD4 cells latently infected with HIV-1,”Nature Communications, 6:8447 doi:10.1038/ncomms9447, 2015.

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New Topoisomerase Inhibitors in Clinical Trials

Curator: Stephen J. Williams, Ph.D.

Below is a great review of topoisomerase in cancer, approved inhibitors as well as some in clinical trials.

Biomolecules 2015, 5, 1652-1670; doi:10.3390/biom5031652

OPEN ACCESS

biomolecules

ISSN 2218-273X

www.mdpi.com/journal/biomolecules/

Review

Inhibition of Topoisomerase (DNA) I (TOP1): DNA Damage Repair and Anticancer Therapy

Yang Xu and Chengtao Her *

School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Mail Drop 64-7520, Pullman, WA 99164, USA; E-Mail: davidxy22@vetmed.wsu.edu

* Author to whom correspondence should be addressed; E-Mail: cher@wsu.edu; Tel.: +1-509-335-7537; Fax: +1-509-335-4159.

Academic Editors: Wolf-Dietrich Heyer, Thomas Helleday and Fumio Hanaoka Received: 22 May 2015 / Accepted: 14 July 2015 / Published: 22 July 2015

Abstract: Most chemotherapy regimens contain at least one DNA-damaging agent that preferentially affects the growth of cancer cells. This strategy takes advantage of the differences in cell proliferation between normal and cancer cells. Chemotherapeutic drugs are usually designed to target rapid-dividing cells because sustained proliferation is a common feature of cancer [1,2]. Rapid DNA replication is essential for highly proliferative cells, thus blocking of DNA replication will create numerous mutations and/or chromosome rearrangements—ultimately triggering cell death [3]. Along these lines, DNA topoisomerase inhibitors are of great interest because they help to maintain strand breaks generated by topoisomerases during replication. In this article, we discuss the characteristics of topoisomerase (DNA) I (TOP1) and its inhibitors, as well as the underlying DNA repair pathways and the use of TOP1 inhibitors in cancer therapy.

Biomolecules 2015, 5                                                                                                                           1653

  1. Type IB Topoisomerases and Inhibitors
    1.1. TOP1

DNA topoisomerases resolve topological constraints that may arise from DNA strand separation and are therefore important for transcription and replication [4]. There are six topoisomerases in humans, classified as Type IA, IB and IIA. Type IA topoisomerases TOP3a and TOP3b cleave one DNA strand to relax only negative supercoiling. In addition, TOP3a forms the BTR complex with BLM and RMI1/2, which plays a role in the dissolution of double-Holliday junctions [5]. Type IIA topoisomerases TOP2a and TOP2b generate double-strand breaks on one DNA molecule to allow the passing of other DNA strands [6]. Topoisomerases are attractive drug targets in cancer therapy. For example, the commonly used anticancer agents doxorubicin and etoposide (VP-16) are TOP2 inhibitors [7]. Type IB topoisomerases include the nuclear TOP1 and mitochondrial TOP1mt [4]. TOP1 initiates the DNA relaxation by nicking one DNA strand. It then forms a TOP1-DNA cleavage complex (TOP1cc) by covalently linked to the 3′-phosphate end via its tyrosine residue Y723 (3′-P-Y). Following the resolution of topological entanglements and the removal of TOP1, the 5′-hydroxyl end is realigned with the 3′-end for religation. Each nicking-closing cycle enables the relaxation of one DNA supercoiling (Figure 1).

Figure 1. A schematic representation of strand passages catalyzed by three types of topoisomerases (adapted from ref. [8]).

fig1topto

TOP1 is essential for embryonic development in mammals [9]. Although TOP1 plays an important role in the deconvolution of supercoils arising amid DNA replication, the precise steps involved with

Biomolecules 2015, 5                                                                                                                         1654

the recruitment of TOP1 to topological constraints remains to be revealed. It appears that in yeast TOP1 travels at a distance of 600 bp ahead of the replication fork [10] and remains associated with the GINS-MCM complex [11]. However, the yeast TOP1 is distinct from its human counterpart in that it has little effect on fork progression or the firing of replication origin [12]. In humans, TOP1 binds to the regions of the pre-replicative complex in cells during the M, early G1, and G1/S phases of the cell cycle to control the firing of replication origins [12]. This difference may explain why yeast cells are viable in the absence of TOP1. In addition, TOP1 also has functions in transcription that are independent of its role in resolving DNA topological entanglements. First, TOP1 is known to repress transcription by binding to TFIID [13]. Second, inhibition of TOP1 can cause the induction of c-Jun in leukemia cells, suggesting its additional role in the control of transcription [14]. Furthermore, TOP1 interacts with the splicing factor ASF/SF2 by which it promotes the maturation of RNA—through suppressing the formation of R-loops (RNA-DNA hybrids)—and prevents collision between transcription bubble and replication fork [15,16]. It appears that the levels of TOP1 have to be dynamically regulated. In B cells, TOP1 is reduced by activation-induced cytidine deaminase (AID) to facilitate class-switch recombination (CSR) and somatic hypermutation (SHM) [17,18]. Although TOP1mt is important for mitochondrial integrity and metabolism, mice lacking mitochondrial TOP1mt are viable and fertile but they are associated with increased negative supercoiling of mtDNA [19,20].

1.2. TOP1 Inhibitors

Stabilization of TOP1cc by topoisomerase poison is detrimental to cells due to the disruption of DNA uncoiling, increased strand breaks, and unstable RNA transcripts as well as incomplete DNA replication [21]. The TOP1 inhibitor camptothecin (CPT), first isolated from the Chinese tree Camptotheca acuminate, was clinically used for cancer treatment long before it was identified as a TOP1 inhibitor [22]. Due to side effects, CPT is no longer used clinically and it has been replaced by more effective and safer TOP1 inhibitors [23]. Currently, CPT derivatives topotecan (trade name: Hycamtin) and irinotecan (CPT-11, trade name: Camptosar) are routinely used to treat colorectal, ovarian and lung cancers, while a few other TOP1 inhibitors are being tested in clinical trials.

CPT is a 5-ring alkaloid that is active in its closed E-ring (lactone) form but it is inactive with an open E-ring (carboxylate) at physiological and alkaline pH [24]. Therefore, CPT is not effective for inhibiting TOP1mt due to a higher pH mitochondrial environment. The inactive form of CPT tends to bind to serum albumin, which might be a reason for its side effects. CPT is highly specific for TOP1 and the binding is of relatively low affinity and can be reversed after drug removal. These features make the action of CPT controllable [24], and in fact CPT is widely used in studies of replication-associated DNA damage response. There are a few CPT derivatives and non-CPT TOP1 inhibitors [4,8,24]. For example, CPT derivatives Diflomotecan and S39625 were designed to stabilize the E-ring. Irinotecan has the bis-piperidine side chain to increase its water solubility, but it also contributes to some side effects. Non-CPTs—such as indolocarbazoles, phenanthrolines (e.g., ARC-111) and indenoisoquinolines—refer to drugs that have no typical CPT E-ring structures but they can still specifically target TOP1 and bind irreversibly to TOP1cc. Some of the CPT derivatives (i.e., Gimatecan and Belotecan) and non-CPTs (i.e., NSC 725776 and NSC 724998) are presently tested in clinical trials [23].

Biomolecules 2015, 5                                                                                                                           1655

How does CPT trap TOP1cc? Analysis of the crystal structure and modeling suggest that CPT-TOP1-DNA forms a ternary complex to prevent the two DNA ends from religation [25–27]. Although it is still controversial on how CPT is intercalated into DNA, it seems that CPT traps TOP1cc with a thymine (T) at the -1 position and a guanine (G) at the +1 position on the scissile strand, and it is therefore sequence-specific [28]. Three amino acid residues of the TOP1 enzyme, R364, D533 and N722, combined with DNA bases, contribute to the stabilization of the ternary complex by forming hydrogen bonds and hydrophobic interactions. It is of note that several point mutations, including N722S, in Camptotheca acuminata TOP1 confer resistance to CPT [29]. Interestingly, the same amino acids also contribute to the inhibition of TOP1 by non-CPT drugs [24].

  1. Repair of TOP1 Poison-Induced DNA Lesions

As aforementioned, CPT-induced trapping of TOP1cc creates a single strand break with a free 5′-hydroxyl group, whereas the 3′-phosphate is connected to Y723 of TOP1 (3′-P-Y). At least two pathways contribute to the repair of DNA lesions created by TOP1 poison [30]. The tyrosyl-DNA-phosphodiesterase (TDP1) pathway starts with the ubiquitination and proteasome-mediated degradation of TOP1 in the CPT-TOP1-DNA complex to generate a 3′-P end linked to a short peptide [31]. TDP1 then cleaves the P-Y bond to release the 3′-P end; however, the 3′-P end cannot be directly ligated to the 5′-OH end because of the requirements of DNA ligases. The human polynucleotide kinase (PNKP) can process the DNA ends by functioning as both a 3′-phosphatase and a kinase to generate the required 3′-OH and 5′-P termini for direct ligation. The rest of the repair events can be best described by the single-strand break (SSB) repair pathway, which will be discussed below. Indeed, TDP1 and PNKP are tightly associated with the SSB repair machinery [32,33].

The endonuclease pathway requires multiple endonucleases to excise the DNA—usually at a few nucleotides away from the 3′-P-TOP1 end – on the scissile strand to release the DNA-TOP1 complex [30]. Initial studies were carried out to identify genes that functioned in CPT repair in the absence of TDP1 in yeast [34,35]. These studies led to the identification of RAD1-RAD10, SLX1-SLX4, MUS81-MMS4, MRE11-SAE2 as well as genes involved in recombination. The RAD1-RAD10 (human XPF/ERCC4-ERCC1) complex is a DNA structure-specific endonuclease that can act on 5′ overhang structures [36]. Interestingly, the cleavage site of XPF-ERCC1 is in the non-protruding DNA strand, about 3–4 nucleotides away from the 3′ end [36]. Therefore, trapped TOP1ccs can be removed by this endonuclease activity. Likewise, MUS81-MMS4 (human MUS81-EME1) can also cleave nicked duplex at the 5′ of the nick [37]. The SLX1-SLX4 endonuclease, although not tested on nicked duplexes, is able to process 3′ flap and other DNA structures [38,39]. In human cells, SLX4 also associates with XPF-ERCC1 and MUS81-EME1 endonucleases to process specific DNA intermediates [39,40]. Moreover, MRE11-RAD50 cleaves the 3′-P-Y bond and resects DNA to produce a 3′-OH end [41]. A direct role of SAE2 (human CtIP) in processing 3′-P-TOP1 is unknown, and its endonuclease activity appears to be limited to the 5′ flap or DNA “hairpin” structures [42,43]. Nonetheless, the endonuclease activity of CtIP is essential for processing CPT adducts [42]. In addition, like CtIP, the 5′ flap endonuclease RAD27 (human FEN1) seems to be unable to directly process 3′-P-TOP1 ends [44]. However, the gap endonuclease activity of FEN1 is important for processing stalled replication forks and CPT-induced adducts [45]. The role of FEN1 in SSB repair will be discussed further in the next section.

Biomolecules 2015, 5                                                                                                                           1656

During DNA replication, SSBs created by CPT are most likely converted to double-strand breaks (DSBs) by replication fork runoff. This conversion appears to be dependent on the proteolysis of TOP1 [46]. The repair of one-ended DSBs, as will be discussed in the next section, is largely dependent on homologous recombination (HR). However, low doses of CPT may also induce PARP1 and/or RAD51 dependent replication fork regression—generating no or few DSBs [47,48]. The regressed fork leads to the formation of a “chicken foot” DNA structure by newly synthesized strands [3,49,50]. The formation of regressed fork can be largely suppressed by ATR, EXO1, and DNA2 [51–53]. However, fork reversal can also be beneficial as it provides time for the repair of TOP1-induced DNA lesions by TDP1, thereby preventing DSB formation and the activation of error-prone non-homologous end-joining (NHEJ) [30].

  1. Pathways Involved in the Repair of CPT-Induced DNA Lesions

Normal cells use DNA damage response (DDR) pathways to maintain genomic stability [54]. As aforementioned, SSB and DSB repair mechanisms are the two major DDR pathways that repair TOP1-induced DNA lesions. Paradoxically, cancer cells exploit DDR pathways to accumulate necessary genomic alterations for promoting proliferation. Furthermore, altered DDR and apoptotic responses in cancer cells are the major obstacles to successful chemotherapy. Thus, the delineation of TOP1-related SSB and DSB repair mechanisms is of great importance for identifying drug targets that can selectively affect cancer cell survival.

3.1. Single-Strand Break (SSB) Repair

Trapping of TOP1cc results in a 3′-P-TOP1 end and a 5′-OH terminus. Because the two ends cannot be directly religated, the persisting SSB is likely to be detected by PARP1 in which activated PARP1 catalyzes the synthesis of poly(ADP-ribose) (PAR) chains for recruiting repair proteins [55]. This reaction can be rapidly reversed by PARG, which hydrolyzes the PAR chains. The PAR chains at the SSB sites are important for the recruitment of XRCC1 that functions as a loading dock for other SSB repair proteins including TDP1 and PNKP. TDP1 generates 3′-P and PNKP converts 3′-P to 3′-OH, and PNKP also converts 5′-OH to 5′-P, making ends compatible for religation with no base loss. The rejoining of the 3′-OH and 5′-P ends is mainly mediated by LIG3, in which XRCC1 mediates the recruitment of LIG3.

If the trapped TOP1cc intermediates are processed by endonucleases, the initial SSBs will be converted to 3′-OH and 5′-OH ends with a gap over a few nucleotides (in the case of XPF-ERCC1, the loss is in the range of 3–4 nt), leading to the activation of PARP1 and XRCC1 recruitment. Consequentially, Pol3 recruited by XRCC1 can catalyze the gap filling, and PCNA-Polö/E also plays a role in this process [55]. If the 5′-OH is not processed by PNKP, the 5′-flap resulted from gap filling is likely to be removed by FEN1, which explains why FEN1 deficiency also leads to an increased CPT sensitivity. The final ligation is catalyzed by LIG1 because of the presence of PCNA.

Biomolecules 2015, 5                                                                                                                           1657

3.2. Double-Strand Break (DSB) Repair

Successful DSB repair requires concerted actions of proteins involved in DNA damage signaling and repair [54]. To repair TOP1 poison-induced DNA lesions, ATR signaling is required due to the runoff of replication fork and the presence of long single-strand DNA (ssDNA) [56]. The full activation of ATR follows a “two-man” rule—the ssDNA-ATRIP-dependent recruitment of ATR kinase and the RAD17 clamp loader/9-1-1/TOPBP1 mediator loading at the ssDNA-dsDNA junction. ATR phosphorylates CHEK1 to harness cell cycle arrest. If one-ended DSB is formed, ATM will be activated through the action of the MRE11-RAD50-NBS1 (MRN) complex. ATM mainly phosphorylates CHEK2 to mediate cell cycle arrest. Both ATM and ATR are able to phosphorylate hundreds of proteins in response to DSB formation [57]. One remarkable substrate is the histone H2AX, which can be phosphorylated by both kinases to yield g-H2AX. It is conceived that the propagation of g-H2AX signaling along the chromatin facilitates MDC1 recruitment and BRCA1 signaling via the MDC1-RNF8-RNF168-RAP80 ubiquitin cascade—events that are essential for HR [58].

The repair of TOP1 poison-induced DNA lesions is in essence the repair of one-ended DSBs, which facilitates the restoration of replication forks to restart DNA replication. It is important to note that one-ended DSB repair occurs in the S phase and relies on HR rather than NHEJ [59]. The first step in HR is end resection to generate a 3′-overhang for homology searching. A TOP1 cleavage in the leading strand may require end resection by the MRN-CtIP-BRCA1 and BLM-EXO1-DNA2 complexes [60], whereas a cleavage in the lagging strand automatically forms a 3′-overhang. Rad51 then associates with the 3′-ssDNA to form a nucleofilament for strand invasion, which leads to the formation of a D-loop structure [61]. This process continues with DNA synthesis, branch migration and the resolution of Holliday junction structures to reconstitute a functional replication fork [62]. TOP1 poisons can also lead to the formation of two-ended DSB if two replication forks collide into each other at the site of SSB. The repair of this type of DSBs is not aimed for fork restoration and can be accomplished by the classical DSB repair mechanisms [61].

3.3. Genes Involved in CPT-Induced Damage Repair

A long list of genes, in which mutations confer sensitivity to CPT in yeast, chicken or mammalian cells, has been compiled [24,30,63]. With no surprise, many genes involved in SSB and DSB repair are on the list, such as PARP1, XRCC1, PNKP, TDP1 for SSB repair; MRN, ATM-CHK2, ATR-CHK1 for DSB signaling; BRCA1/2, XRCC2, XRCC3 for HR. Most recently, the hMSH5-FANCJ complex has also been implicated to play a role in CPT-induced DNA damage response and repair [64]. Mutations in the binding partners of these repair factors are also likely to sensitize cells to CPT treatment. For example, depletion of the MRN-binding partner hnRNPUL increases the sensitivity to CPT [65]; and deficiencies in ZRANB3 and SPIDR, binding partners of PCNA and RAD51, cause CPT hypersensitivity in cancer cells [66–68]. In addition, the two DNA helicases BLM and WRN have also been implicated in the repair of CPT-induced DNA lesions [69,70]. Early studies revealed that chicken BLM knockout cells and human BLM-deficient fibroblasts showed increased sensitivity to CPT [71,72]. On the contrary, mouse BLM knockout embryonic stem cells showed mild resistance to

Biomolecules 2015, 5                                                                                                                           1658

CPT [73]. This discrepancy is likely attributable to the complexity of CPT-induced DNA lesion repair as well as different treatment conditions and experimental systems.

Interstrand crosslinks (ICLs) resemble CPT-induced lesions in that they block both replication and transcription [74]. They may induce replication fork reversal and fork collapse, which require DNA incision for lesion processing and HR for repair. ICL repair is accomplished by the coordinated actions of 17 Fanconi anemia (FA) genes whose mutations contribute to FA in patients [75]. Depletion of FANCP/SLX4 or FANCQ/XPF causes cellular sensitivity to CPT because they form an endonuclease complex involved in the repair of trapped TOP1cc [38]. Likewise, depletion of FANCS/BRCA1, FANCD1/BRCA2, FANCN/PALB2 or FANCO/RAD51C sensitizes cells to CPT because of their involvement in HR [76]. Accordingly, depletion of the FA core complex except FANCM—involved in fork reversal—is not expected to increase CPT sensitivity because they are unable to recognize the trapped TOP1cc [76]. However, the roles of FANCI, D2, J and FAN1 in the process are elusive due to conflicting reports presumably reflecting different experimental systems [76–78]. For example, in a multicolor competition assay, loss of FANCI or FAN1 rendered cells sensitive to CPT treatment [77]. However, this observation could not be recapitulated in studies performed with FANCI-deficient lymphoblasts and FAN1-depleted HEK293 cells [76,79], indicating that the involvement of these two genes in CTP sensitivity might be cell type specific.

It is interesting to note that the MMS22L-TONSL complex plays a prominent role in mediating CPT sensitivity [80–83]. Depletion of this complex impairs RAD51 foci formation and triggers G2/M arrest, indicating that the MMS22L-TONSL complex participates in HR repair. Furthermore, this complex associates with MCM, FACT, ASF1 and histones. FACT and ASF1 are histone chaperones that function in H2A/H2B and H3/H4 chromatin assembly and disassembly, respectively [84]. They recycle parental histones from old DNA strands unwound by MCM and incorporate them into newly synthesized DNA strands. FACT and ASF1 also function in checkpoint signaling; therefore the involvement of MMS22L-TONSL in CPT response implies the existence of a close association between HR, DNA damage signaling and replication restart.

  1. TOP1 Inhibition in Cancer Treatment

The understanding of the function of TOP1 and the cellular effects of TOP1 inhibition has been a stepping-stone for the development of effective CPT derivatives in cancer therapy. Since TOP1 functions in normal and cancer cells, the use of low doses of TOP1 inhibitors are actively sought to treat cancers that heavily rely on the function of TOP1 for survival (e.g., highly malignant, rapid-dividing tumor cells). In fact, the FDA-approved CPT derivatives topotecan and irinotecan are currently used to treat ovarian and colorectal cancers, respectively [24].

Furthermore, the promising results from a Phase I trial have warranted further evaluation of the CPT derivative Diflomotecan in Phase II trials [85]. Other derivatives like Gimatecan, Lurtotecan and Exatecan are also being tested in clinical trials (Table 1). The non-CPT indolocarbazole BMS-250749 showed great anti-tumor activity against preclinical xenograft models [86], but no further evaluation beyond Phase I trials is presently available (Table 2). Another indolocarbazole compound Edotecarin has shown promising anti-tumor activity in xenograft models and it is now advanced to Phase II studies of patients with advanced solid tumors [87]. By contrast, Phenanthroline ARC-111 (topovale)

Biomolecules 2015, 5                                                                                                                             1659

was potently against human tumor xenografts and displayed anti-cancer activity in colon and Wilms’ tumors [88]; however, no result from Phase I clinical trials is available owing to profound bone marrow toxicity [89]. To date, indenoisoquinolines are the most promising non-CPT inhibitors in clinical trials. LMP400 (NSC 743400, indotecan) and LMP776 (NSC 725776, indimitecan) show significant anti-tumor activities in animal models and both are being evaluated in Phase I clinical trials for relapsed solid tumors and lymphomas [8,90].

Table 1. CPT derivatives in clinical trials [91].

Name                            Structure                     Clinical Trial            Malignancy        Reference

Biomolecules 2015, 5                                                                                                                           1660

Given the observation that CPT-mediated TOP1 inhibition provokes DNA repair activities, a synergistic effect is then anticipated on cancer cells by inhibition of TOP1 and downregulation of DNA repair activities. The rationale for this approach is to accelerate the accumulation of DNA breaks and trigger cellular apoptosis, probably through mitotic catastrophe [92]. Which DNA repair pathways can we exploit? Currently, the major interests are in SSB and DSB repair mechanisms. Indeed, PARP inhibitors can enhance the cytotoxicity of TOP1 inhibitors in cancer cell lines as well as in mouse models [93–96]. Phase I studies of combination therapy using PARP inhibitors veliparib or olaparib (FDA-approved) together with topotecan were carried out in patients with advanced solid tumors but showed some dose-dependent side effects [97,98]. TDP1 can be another potential target because it functions directly downstream of PARP1 in the repair of TOP1 poison-induced DNA lesions [99]. TDP1 inhibitors sensitize cells to CPT treatment in vitro [100,101], however in vivo evaluation is presently unavailable due to unsuitable properties of the compounds [102].

Table 2. Non-CPT derivatives in preclinical and clinical trials [91].

Name                       Structure               Clinical Trial            Malignancy             Reference

Indolocarbazoles
(Edotecarin,
BMS-250749)
Phase II

(Edotecarin, Pfizer)

Stomach, breast
neoplasms
Preclinical
(BMS-250749)
Anti-tumor activity
in preclinical
xenograft models
[86,87,103]
Phenanthridines
(ARC-111/topovale)
Anti-tumor activity

Preclinical                    in preclinical            [88,89,103]
xenograft models

Indenoisoquinolines
(LMP400, LMP776)
Phase I                              Lymphomas             [8,90,103]

DSB repair can be targeted by either inhibition of DSB signaling or inhibition of HR. ATM and ATR inhibitors can largely increase the sensitivity to CPT in cancer cells [104,105]. This can be explained by the fact that abrogation of the cell cycle arrest will allow cells with unreplicated or unrepaired chromosomes to enter mitosis thereby triggering mitotic catastrophe and cell death. Similarly, CHEK1 and CHEK2 inhibitors are tested in Phase I studies in combination with irinotecan [106,107]. Inhibitors that can directly block HR proteins are very limited [108]. This is partially attributed to the fact that HR genes are often mutated in cancer cells, thus diminishing the enthusiasm for developing HR inhibitors. One diterpenoid compound, however, was found to be able to inhibit the function of BRCA1 and render cytotoxicity in human prostate cancer cells [109]. Several RAD51 inhibitors have also been

Biomolecules 2015, 5                                                                                                                           1661

identified but have not been tested in cell lines [110]. Inhibition of BRCA1 and RAD51 can be also achieved indirectly by harnessing corresponding kinases [106]. Clearly, defective hMRE11 sensitizes colon cancer cells to CPT treatment [111]. Although MRE11-deficeint tumor xenografts failed to display significant growth inhibition by irinotecan alone, combining thymidine with irinotecan caused a dramatic growth delay [112].

TOP1 inhibitors might be also useful for treating cancers with BRCA1/2 mutations. The successful use of PARP inhibitors in treating BRCA1/2-deficient tumors has ignited a broad interest in searching for synthetic lethality among DNA damage response and repair genes [113,114]. In the PARP-BRCA1/2 example, the accumulation of SSBs by PARP inhibition would lead to the formation of DSBs during replication. In HR-deficient cells, DSBs can only be repaired by illegitimate (toxic) NHEJ—joining one-ended DSBs from different locations—leading to cell death [115,116]. However, resistance to PARP inhibitors can arise in BRCA1-deficient tumors during treatment from either genetic reversion of BRCA1 mutations or the loss of NHEJ [117–122]. Therefore, it would be beneficial to explore the possibility of developing a similar synthetic lethal strategy to use TOP1 inhibitors in the treatment of BRCA1/2-deficient tumors.

Figure 2. An overview of the effects of TOP1 inhibition is provided. Inhibitors and key DNA repair factors are highlighted.

Biomolecules 2015, 5                                                                                                                         1662

  1. Conclusions

Trapping of TOP1 by inhibitors generates SSBs and DSBs that are repaired by their corresponding repair pathways (Figure 2). Therefore, developing effective TOP1 inhibitors not only provides powerful tools to study DNA replication and repair but also establishes a foundation to devise new synthetic lethal strategies for efficient cancer treatments. The accumulation of DNA strand breaks (SSBs and DSBs) by TOP1 inhibition in HR-deficient tumor cells is expected to enhance cytotoxicity. However, increased DNA repair activities in cancer cells can make TOP1 inhibitors less effective, so silencing of repair pathways in conjunction with the use of TOP1 inhibitors offers an attractive new means for cancer control. Since each tumor is unique, it would be advantageous to identify the individualities of DNA repair pathways or biomarkers reflecting the changes of DNA repair activities in tumor cells [92,123]. This will make it possible to achieve better and predictable prognosis through tailored therapeutic regimens. Given that TOP1 is essential for transcription and DNA replication, future design of novel TOP1 inhibitors and combinational therapy strategies should aim to increase therapeutic efficacy of the inhibitors, thus reducing side effects.

Acknowledgments

The work in the Her laboratory is supported by the NIH grant GM084353.

Author Contributions

Yang Xu and Chengtao Her wrote and revised the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest with the contents of this article.

Please see the following file for the referencesReferences for top paper

From a 2015 Clinical Cancer Research paper:

Phase 1 clinical pharmacology study of F14512, a new polyamine-vectorized anti-cancer drug, in naturally occurring canine lymphoma

Dominique Tierny1, Francois Serres1, Zacharie Segaoula1, Ingrid Bemelmans1, Emmanuel Bouchaert1,

Aurelie Petain2, Viviane Brel3, Stephane Couffin4, Thierry Marchal5, Laurent Nguyen6, Xavier Thuru7,

Pierre Ferre2, Nicolas Guilbaud8, and Bruno Gomes9,*

Abstract

Purpose: F14512 is a new topoisomerase II inhibitor containing a spermine moiety that facilitates selective uptake by tumor cells and increases topoisomerase II poisoning. F14512 is currently in Phase I/II clinical trial in patients with acute myeloid leukemia. The aim of this study was to investigate F14512 potential in a new clinical indication. Because of the many similarities between human and dog lymphomas, we sought to determine the tolerance, efficacy, PK/PD relationship of F14512 in this indication, and potential biomarkers that could be translated into human trials. Experimental design: Twenty-three dogs with stage III-IV naturally occurring lymphomas were enrolled in the Phase 1 dose-escalation trial which consisted of three cycles of F14512 intravenous injections. Endpoints included safety and therapeutic efficacy. Serial blood samples and tumor biopsies were obtained for PK/PD and biomarker studies. Results: Five dose levels were evaluated in order to determine the recommended dose. F14512 was well tolerated, with the expected dose-dependent hematological toxicity. F14512 induced an early decrease of tumoral lymph node cells, and a high response rate of 91% (21/23) with 10 complete responses, 11 partial responses, 1 stable disease and 1 progressive disease. Phosphorylation of histone H2AX was studied as a potential pharmacodynamic biomarker of F14512. Conclusions: This trial demonstrated that F14512 can be safely administered to dogs with lymphoma resulting in strong therapeutic efficacy. Additional evaluation of F14512 is needed to compare its efficacy with standards of care in dogs, and to translate biomarker and efficacy findings into clinical trials in humans.

AND From ASCO 2015 Annual Meeting

Survival impact of switching to different topoisomerase I or II inhibitors-based regimens (topo-I or topo-II) in extensive-disease small cell lung cancer (ED-SCLC): supplemental analysis from JCOG0509.

Abstract:

Background: The J0509 (phase III study for chemotherapy-naive ED-SCLC) demonstrated amrubicin plus cisplatin (AP) was inferior to irinotecan plus cisplatin (IP). However, median overall survival (OS) of both AP and IP (15 and 17 mo) was more favorable than those of previous trials (9-12 mo), probably because switching to different topo-I or topo-II in the second-line therapy, especially the use of topo-II in IP arm, was frequent. This analysis aimed to investigate whether observed survival benefit of IP arm can be explained by the treatment switching, and how post-protocol chemotherapy affected the result of J0509. Methods: Two analysis sets from J0509 were used: all randomized 283 pts and 250 pts who received post-protocol chemotherapy. One pt without initiation date of second-line therapy was excluded. A rank-preserving structural failure time (RPSFT) model was used to estimate “causal survival benefit” that would have been observed if all pts had been followed with the same type of regimen as randomized throughout the follow-up period. Additionally, to assess the survival impact of second-line use of topo-II, OS after initiating second-line therapy (OS2) was analyzed by multivariate Cox models. Results: %treatment switching in IP arm and AP arm was 65.2% (92/141) and 43.7% (62/142). By RPSFT model, estimated OS excluding the effect of the treatment switching was 2.7-fold longer in IP (topo-I) arm than AP (topo-II) arm. This causal survival benefit was stronger than the original report of J0509 (nearly 1.4-fold extension by Cox model), indicating that re-challenging topo-I in IP arm appeared beneficial. The multivariate Cox analysis for OS2 (n = 250) revealed second-line use of topo-II was detrimental (hazard ratio, 1.5; 95%CI, 1.1-2.1). Among sensitive relapsed pts in IP arm, OS2 was favorable in the following order: irinotecan-based regimen > the other topo-I > topo-II. Conclusions: IP remains the standard therapy. Re-challenging topo-I, especially irinotecan-based topo-I, seemed beneficial for IP-sensitive pts. This result should be confirmed in further investigations with large sample size. Clinical trial information: 000000720.

 

 

 

 

Below is actively recruiting clinical trials evaluating topoisomerase inhibitors. Shown are only a few trials for a complete list from CancerTrials.gov please see this link:

https://clinicaltrials.gov/ct2/results?term=topoisomerase+inhibitor&recr=Open#wrapper

A service of the U.S. National Institutes of Health

897 studies found for:    topoisomerase inhibitor | Open Studies

Include only open studies Exclude studies with Unknown status

Status Study
Recruiting A Study of Standard Treatment +/- Enoxaparin in Small Cell Lung Cancer

Condition: Small Cell Lung Cancer
Interventions: Drug: cisplatinum or carboplatin and e.g.etoposide.;   Drug: cisplatinum or carboplatin and e.g.etoposide+enoxaparin
Recruiting A Phase I Study of Indenoisoquinolines LMP400 and LMP776 in Adults With Relapsed Solid Tumors and Lymphomas

Conditions: Neoplasms;   Lymphoma
Interventions: Drug: LMP 400;   Drug: LMP 776
Recruiting A Dose-Ranging Study Evaluating the Efficacy, Safety, and Tolerability of GSK2140944 in the Treatment of Uncomplicated Urogenital Gonorrhea Caused by Neisseria Gonorrhoeae

Condition: Gonorrhea
Intervention: Drug: GSK2140944
Recruiting Selinexor in Combination With Irinotecan in Adenocarcinoma of Stomach and Distal Esophagus

Conditions: Esophageal Cancer;   Gastric Cancer
Interventions: Drug: Selinexor;   Drug: Irinotecan
Recruiting Multimodal Molecular Targeted Therapy to Treat Relapsed or Refractory High-risk Neuroblastoma

Condition: Neuroblastoma Recurrent
Interventions: Drug: Dasatinib;   Drug: Rapamycin;   Drug: Irinotecan;   Drug: Temozolomide
Unknown  Study of the Farnesyl Transferase Inhibitor, R115777, in Combination With Topotecan (NYU 99-32)

Condition: Cancer
Interventions: Drug: R115777 (farnesyl transferase inhibitor);   Drug: Topotecan
Recruiting Pegylated Irinotecan NKTR 102 in Treating Patients With Relapsed Small Cell Lung Cancer

Condition: Recurrent Small Cell Lung Carcinoma
Interventions: Other: Laboratory Biomarker Analysis;   Drug: Pegylated Irinotecan;   Other: Pharmacological Study
Recruiting Selinexor and Chemotherapy in Treating Patients With Relapsed or Refractory Acute Myeloid Leukemia

Conditions: Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities;   Adult Acute Myeloid Leukemia With Del(5q);   Adult Acute Myeloid Leukemia With Inv(16)(p13;q22);   Adult Acute Myeloid Leukemia With t(15;17)(q22;q12);   Adult Acute Myeloid Leukemia With t(16;16)(p13;q22);   Adult Acute Myeloid Leukemia With t(8;21)(q22;q22);   Recurrent Adult Acute Myeloid Leukemia;   Secondary Acute Myeloid Leukemia
Interventions: Drug: mitoxantrone hydrochloride;   Drug: etoposide;   Drug: cytarabine;   Drug: selinexor;   Other: laboratory biomarker analysis;   Other: pharmacological study
Recruiting WEE1 Inhibitor MK-1775 and Irinotecan Hydrochloride in Treating Younger Patients With Relapsed or Refractory Solid Tumors

Conditions: Childhood Solid Neoplasm;   Recurrent Childhood Medulloblastoma;   Recurrent Childhood Supratentorial Primitive Neuroectodermal Tumor;   Recurrent Neuroblastoma
Interventions: Drug: Irinotecan Hydrochloride;   Other: Laboratory Biomarker Analysis;   Other: Pharmacological Study;   Drug: WEE1 Inhibitor AZD1775
Recruiting PARP Inhibitor BMN-673 and Temozolomide or Irinotecan Hydrochloride in Treating Patients With Locally Advanced or Metastatic Solid Tumors

Conditions: Metastatic Cancer;   Unspecified Adult Solid Tumor
Interventions: Drug: PARP inhibitor BMN-673;   Drug: temozolomide;   Drug: irinotecan hydrochloride;   Other: pharmacological study;   Other: laboratory biomarker analysis
Recruiting A Phase II Multicenter, Randomized, Placebo Controlled, Double Blinded Clinical Study of KD018 as a Modulator of Irinotecan Chemotherapy in Patients With Metastatic Colorectal Cancer

Condition: Colorectal Neoplasms
Interventions: Drug: KD018;   Drug: Irinotecan;   Drug: Placebo
Recruiting The Efficacy of the 7 Days Tailored Therapy as 2nd Rescue Therapy for Eradication of H. Pylori Infection

Condition: Helicobacter Infection
Interventions: Procedure: H. pylori culture and antimicrobial susceptibility testing;   Drug: 14 days empirical bismuth quadruple therapy (Proton pump inhibitor);   Drug: Metronidazole;   Drug: Tetracycline;   Drug: tripotassium dicitrate bismuthate;   Drug: 7 days tailored therapy Proton Pump Inhibitor;   Drug: Moxifloxacin;   Drug: Amoxicillin
Recruiting G1T28 (CDK 4/6 Inhibitor) in Combination With Etoposide and Carboplatin in Extensive Stage Small Cell Lung Cancer (SCLC)

Condition: Small Cell Lung Cancer
Interventions: Drug: G1T28 + carboplatin/ etoposide;   Drug: Placebo + carboplatin/ etoposide
Recruiting Trial of Topotecan With VX-970, an ATR Kinase Inhibitor, in Small Cell Lung Cancer

Conditions: Carcinoma, Non-Small -Cell Lung;   Ovarian Neoplasms;   Small Cell Lung Carcinoma;   Uterine Cervical Neoplasms;   Carcinoma, Neuroendocrine
Interventions: Drug: Topotecan;   Drug: VX-970
Recruiting Prospective Analysis of UGT1A1 Promoter Polymorphism for Irinotecan Dose Escalation in Metastatic Colorectal Cancer Patients Treated With Bevacizumab Combined With FOLFIRI as the First-line Setting

Condition: Metastatic Colorectal Cancer
Interventions: Genetic: UGT1A1 genotyping (6,6);   Genetic: UGTIA1 genotyping (6,7);   Genetic: UGTIA1 genotyping (7,7);   Genetic: UGT1A1 non-genotyping;   Drug: bevacizumab (Avastin);   Drug: irinotecan;   Drug: Leucovorin;   Drug: 5-FU
Recruiting A Study of the Bruton’s Tyrosine Kinase Inhibitor, PCI-32765 (Ibrutinib), in Combination With Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone in Patients With Newly Diagnosed Non-Germinal Center B-Cell Subtype of Diffuse Large B-Cell Lymphoma

Condition: Lymphoma
Interventions: Drug: Ibrutinib;   Drug: Placebo;   Drug: Rituximab;   Drug: Cyclophosphamide;   Drug: Doxorubicin;   Drug: Vincristine;   Drug: Prednisone (or equivalent)
Recruiting Irinotecan Combination Chemotherapy for Refractory or Relapsed Brain Tumor in Children and Adolescents

Condition: Brain Tumor
Intervention: Drug: Irinotecan combination chemotherapy
Recruiting A Study To Evaluate PF-04449913 With Chemotherapy In Patients With Acute Myeloid Leukemia or Myelodysplastic Syndrome

Condition: Acute Myeloid Leukemia
Interventions: Drug: PF-04449913;   Drug: Low dose ARA-C (LDAC);   Drug: Decitabine;   Drug: Daunorubicin;   Drug: Cytarabine
Recruiting Veliparib and Pegylated Liposomal Doxorubicin Hydrochloride in Treating Patients With Recurrent Ovarian Cancer, Fallopian Tube Cancer, or Primary Peritoneal Cancer or Metastatic Breast Cancer

Conditions: Estrogen Receptor Negative;   HER2/Neu Negative;   Male Breast Carcinoma;   Progesterone Receptor Negative;   Recurrent Breast Carcinoma;   Recurrent Fallopian Tube Carcinoma;   Recurrent Ovarian Carcinoma;   Recurrent Primary Peritoneal Carcinoma;   Stage IV Breast Cancer;   Triple-Negative Breast Carcinoma
Interventions: Other: Laboratory Biomarker Analysis;   Drug: Pegylated Liposomal Doxorubicin Hydrochloride;   Other: Pharmacological Study;   Drug: Veliparib
Recruiting A Study To Evaluate Ara-C and Idarubicin in Combination With the Selective Inhibitor Of Nuclear Export (SINE) Selinexor (KPT-330) in Patients With Relapsed Or Refractory AML

Condition: Acute Myeloid Leukemia (Relapsed/Refractory)
Interventions: Drug: Selinexor;   Drug: Idarubcin;   Drug: Cytarabine

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Targeted gene modification

Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2: 7.8

Mario R. Capecchi won a 2007 Nobel Prize for his work on targeted gene modification.

Born in Italy in 1937, scientist Mario R. Capecchi emigrated to the United States after World War II and later became a geneticist and professor. His groundbreaking work on targeted gene modification won him a Nobel Prize in 2007.

The Making of a Scientist II

In 1996, as a Kyoto Prize laureate, I was asked to write an autobiographical sketch of my early upbringing. Through this exercise, shared by all of the laureates, the hope was to uncover potential influences or experiences that may have been key to fostering the creative spirit within us. In my own case, what I saw was that, despite the complete absence of an early nurturing environment, the intrinsic drive to make a difference in our world is not easily quenched and that given an opportunity, early handicaps can be overcome and dreams achieved. This was intended as a message of hope for those who have struggled early in their lives. As I have previously noted, our ability to identify the genetic and environmental factors that contribute to talents such as creativity are too complex for us to currently predict. In the absence of such wisdom our only recourse is to provide all children with the opportunities to pursue their passions and dreams. Our understanding of human development is too meager to allow us to predict the next Beethoven, Modigliani, or Martin Luther King.

The content of the autobiographical sketch was based on my own memories, on conversations with my aunt and uncle, who raised me once I arrived in the United States, and on conversations with my mother. Because of the added exposure resulting from the winning of the Nobel Prize, I have received letters from people who knew me in Italy during those formative early years. In addition members of the press have taken an interest in my story and have sought independent corroboration. An amazing and wonderful surprise is that they have discovered a half-sister of whom I was completely unaware. She is two years younger than I, and was given up for adoption before she was one year old. Most recently I had the opportunity to meet my half-sister. She was a very nice person, as a sister should be. I am grateful for all of these new sources of information and revelation. Where appropriate, I will weave the new information into this retelling of my story.

Autobiographical Sketch
I was born in Verona, Italy on October 6, 1937. Fascism, Nazism, and Communism were raging through the country. My mother, Lucy Ramberg, was a poet; my father, Luciano Capecchi, an officer in the Italian Air Force. This was a time of extremes, turmoil and juxtapositions of opposites. They had a passionate love affair, and my mother wisely chose not to marry him. This took a great deal of courage on her part. It embittered my father.

Capecchi's mother
Figure 1. A photograph of my mother, Lucy Ramberg, at age 19.

I have only a few pictures of my mother. She was a beautiful woman with a passion for languages and a flair for the dramatic (see Figure 1). This picture was taken when she was 19. She grew up, with her two brothers, in a villa in Florence, Italy. There were magnificent gardens, a nanny, gardeners, cooks, house cleaners, and private tutors for languages, literature, history, and the sciences. She was fluent in half a dozen languages. Her father, Walter Ramberg, was an archeologist specializing in Greek antiquities, born and trained in Germany. Her mother was a painter born and raised in Oregon, USA. In her late teens, my grandmother, Lucy Dodd, packed up her steamer trunks and sailed with her mother from Oregon to Florence, Italy, where they settled. My grandmother was determined to become a painter. This occurred near the end of the 19th century, a time when young women were not expected to set off on their own with strong ambitions of developing their own careers.

Painting
Figure 2. A painting done by my grandmother, Lucy Dodd Ramberg, of her three children, left to right, Edward, Lucy, and Walter. It was painted at their villa in Florence, Italy in 1913.
Painting
Figure 3. A painting by Lucy Dodd Ramberg of my mother, Lucy, and uncle Edward having tea at the villa in Florence, Italy (1913).

My grandmother became a very gifted painter. Let me share with you a couple of her paintings, which also illustrate the young lives of her children. These paintings are very large, approximately seven feet by five feet. The first painting (Figure 2) is the center panel of a triptych depicting my mother and her two brothers Walter and Edward (both of whom became physicists) surrounded by olive trees at the villa in Florence. The influence of the French impressionist painters is evident. The second painting (Figure 3) is of my mother, age 8, and her younger brother Edward, age 6, having a tea party, again at the villa in Florence. Their father, the German archeologist, was killed as a young man in World War I. My grandmother finished raising her three children on her own by painting, mostly portraits, and by converting the family villa into a finishing school for young women, primarily from the United States.

Chalet in Italy
Figure 4. A photograph of the chalet where my mother and I lived in Wolfgrübben just north of Bolzano, Italy. In the foreground is my mother, Lucy.

My mother’s love and passion was poetry. She published in German. She received her university training at the Sorbonne in Paris and was a lecturer at that university in literature and languages. At that time, she joined with a group of poets, known as the Bohemians, who were prominent for their open opposition to Fascism and Nazism. In 1937, my mother moved to the Tyrol, the Italian Alps. Figure 4 shows the chalet north of Bolzano, in Wolfgrübben, with my mother in the foreground. We lived in this chalet until I was 3½ years old. In the spring of 1941, German officers came to our chalet and arrested my mother. This is one of my earliest memories. My mother had taught me to speak both Italian and German, and I was quite aware of what was happening. I sensed that I would not see my mother again for many years, if ever. She was incarcerated as a political prisoner in Germany.

I have believed that her place of incarceration was Dachau. This was based on conversations with my uncle Edward, my mother’s younger brother. During World War II, my uncle lived in the United States. Throughout these war years, he made many attempts to locate where my mother was being held. The most reliable information indicated that the location was near Munich. Dachau is located near Munich and was built to hold political prisoners. My mother survived her captivity, but after the war, despite my prodding, she refused to talk about her war experiences.

Reporters from the Associated Press (AP) have found records that my mother was indeed a prisoner during the war in Germany. In fact, they have found records of German interest in my mother’s political activities preceding 1939. In that year, they had her arrested by the Italian authorities and jailed in Perugia and subsequently released. However, the AP reporters did not find records indicating that my mother was incarcerated in Dachau. Though Germans were noted for their meticulous record keeping, it would be difficult now to evaluate the accuracy of the existing war records, particularly for cases where data is missing. It is clear, however, that exactly where in Germany my mother was held has not yet been determined. Regardless of which prison camp was involved, her experiences were undoubtedly more horrific than mine. She had aged beyond recognition during those five years of internment. Following her release, though she lived until she was 82 years old, she never psychologically recovered from her wartime experiences.

My mother had anticipated her arrest by German authorities. Prior to their arrival, she had sold most of her possessions and gave the proceeds to an Italian peasant family in the Tyrol so that they could take care of me. I lived on their farm for one year. It was a very simple life. They grew their own wheat, harvested it, and took it to the miller to be ground. From the flour they made bread which they took to the baker to be baked. During this time, I spent most of my time with the women of the farm. In the late fall, the grapes were harvested by hand and put into enormous wooden vats. The children, including me, stripped, jumped into the vats and mashed the grapes with our feet. We became squealing masses of purple energy. I still remember the pungent odor and taste of the fresh grapes. Most recently, members of the Dolomiten Press have located this farm and I had the opportunity to visit it. It is still owned by the same family that occupied it when I was there. The old farm house has been taken down and a new one erected. However, the pictures of the old farm house, as well as the surrounding land are remarkably consistent with my memories.

World War II was now fully under way. The American and British forces had landed in Southern Italy and were proceeding northward. Bombings of northern Italian cities were a daily occurrence. As constant reminders of the war, curfews and blackouts were in effect every night; no lights were permitted. In the night we could hear the drone of presumed American and British reconnaissance planes which we nicknamed “Pepe.” One hot afternoon, American planes swooped down from the sky and began machine gunning the peasants in the fields. A senseless exercise. A bullet grazed my leg, fortunately not breaking any bones. I still have the scar, which, many years later my daughter proudly had me display to her third-grade class in Utah.

For reasons that have never been clear to me, my mother’s money ran out after one year and, at age 4½, I set off on my own. I headed south, sometimes living in the streets, sometimes joining gangs of other homeless children, sometimes living in orphanages, and most of the time being hungry. My recollections of those four years are vivid but not continuous, rather like a series of snapshots. Some of them are brutal beyond description, others more palatable.

There are records in the archives of Ritten, a region of the Southern Alps of Italy, that I left Bozen to go to Reggio Emilia on July 18, 1942. AP reporters exploring this history have suggested that my father came to the farm, picked me up, and that we went together to Reggio Emilia where he was living. I have no memory of his coming to the farm, nor of having travelled with him to Reggio Emilia. I have recently received a letter from a man who remembers me as the youngest member of his street gang operating in Bolzano, which is on the way to Reggio Emilia.

I did end up in Reggio Emilia, which is approximately 160 miles south of Bolzano. I knew that my father lived in Reggio Emilia and I have previously noted that I had lived with him a couple of times from 1942-1946, for a total period of approximately three weeks. The question has been raised why I didn’t live with him for a much longer period. The reason was that he was extremely abusive. Amidst all of the horrors of war, perhaps the most difficult for me to accept as a child was having a father who was brutal to me.

Recently, I have also received a very nice letter from the priest in Reggio Emilia who ran the orphanage in which I was eventually placed. I remember him because he was one of the very few men I encountered in Reggio Emilia who showed compassion for the children and took an interest in me. I am surprised, but pleased, that after all these years he still remembers me among the thousands of children he was responsible for over the years. Further, I believe I was at that orphanage for only several months, the first time in the fall of 1945, after which I ran away, followed by a second period, in the same orphanage, in the spring of 1946. But his memory is genuine, for he recounts incidents consistent with my memories that could only have been known through our common experience.

In the spring of 1945, Munich was liberated by the American troops. My mother had survived her captivity and set out to find me. In October 1946, she succeeded. As an example of her flair for the dramatic, she found me on my ninth birthday, and I am sure that this was by design. I did not recognize her. In five years she had aged a lifetime. I was in a hospital when she found me. All of the children in this hospital were there for the same reasons: malnutrition, typhoid, or both. The prospects for most of those children ever leaving that hospital were slim because they had no nourishing food. Our daily diet consisted of a bowl of chicory coffee and a small crust of old bread. I had been in that hospital in Reggio Emilia for what seemed like a year. Scores of beds lined the rooms and corridors of the hospital, one bed touching the next. There were no sheets or blankets. It was easier to clean without them. Our symptoms were monotonously the same. In the morning we awoke fairly lucid. The nurse, Sister Maria, would take our temperature. She promised me that if I could go through one day without a high fever, I could leave the hospital. She knew that without any clothes I was not likely to run away. By late morning, the high, burning fever would return and we would pass into oblivion. Consistent with the diagnosis of typhoid, many years later I received a typhoid/paratyphoid shot, went into shock, and passed out.

Edward Ramberg
Figure 5. A photograph of my uncle Edward Ramberg working in his laboratory at RCA Princeton, New Jersey.

The same day that my mother arrived at the hospital, she bought me a full set of new clothes, a Tyrolean outfit complete with a small cap with a feather in it. I still have the hat. We went to Rome to process papers, where I had my first bath in six years, and then on to Naples. My mother’s younger brother, Edward, had sent her money to buy two boat tickets to America. I was expecting to see roads paved with gold in America. As it turned out, I found much more: opportunities.

On arriving in America, my mother and I lived with my uncle and aunt, Edward and Sarah Ramberg. Edward, my mother’s younger brother was a brilliant physicist. He was a Ph.D. student in quantum mechanics with Arnold Sommerfeld and translated one of Sommerfeld’s major texts into English. Among Edward’s many contributions was his discovery of how to focus electrons, knowledge which he used in helping to build the first electron microscope at RCA. Edward’s books on electron optics have been published in many languages. During my visit to Japan to celebrate the Kyoto Prize, several Japanese physicists approached me to express how grateful they were for my uncle’s texts from which they learned electron optics. Another achievement, of which he was less proud was being a principal contributor to the development of both black and white and color television. While I grew up in his home, television was not allowed. Figure 5 shows a photograph of my uncle working in his laboratory.

My aunt and uncle were Quakers and they did not support violence as solutions to political problems anywhere in the world. During World War II, my uncle did alternative service rather than bear arms. He worked in a mental institution in New Hampshire, cleared swamps in the south, and was a guinea pig for the development of vaccines against tropical diseases. After the war he settled in a commune in Pennsylvania, called Bryn Gweled, which he helped found. People of all races and religious affiliations were welcomed in this community. It was a marvelous place for children: it contained thick woods for exploration and had communal activities of all kinds – painting, dance, theater, sports, electronics, and many sessions devoted to the discussion of the major religious philosophies of the world. Every week there were communal work parties, putting in roads, phone lines, and electrical lines, building a community center and so on.

The contrast between living primarily alone in the streets of Italy and living in an intensely cooperative and supportive community in Pennsylvania was enormous. Time was needed for healing and for erasing the images of war from my mind. I remember that for many years after coming to the United States I would go to sleep tossing and turning with such force that by morning the sheets were torn and the bed frame broken. This activity disturbed my aunt and uncle to the extent that Sarah would take me from one child psychologist or psychiatrist, to another. These professionals were not very helpful, but the support of the community was. The nightly activity eventually subsided. There may be lessons to be learned from such experiences for the treatment of the children from Darfur, the Congo, and now Kenya.

Sarah and Edward took on the challenge of converting me into a productive human being. This, I am sure, was a very formidable task. I had received little or no formal education or training for living in a social environment. Quakers do not believe in frills, but rather in a life of service. My aunt and uncle taught me by example. I was given few material goods, but every opportunity to develop my mind and soul. What I made of myself would be entirely up to me. The day after I arrived in America, I went to school. I started in the third grade in the Southampton public school system. Sarah also took on the task of teaching me to read, starting from the very beginning.

The first task was to learn English. I had a marvelous third grade teacher. She was patient and encouraging. The class was studying Holland, so I started participation in class functions by painting a huge mural on butcher block paper with tulips, windmills, children ice skating, children in Dutch costumes, and ships. It was a collage of activities and colors. This did not require verbal communication.

I was a good, but not serious, student in grade school and high school. Academics came easily to me. I attended an outstanding high school, George School, a Quaker school north of Philadelphia. The teachers were superb, challenging, enthusiastic, competent, and caring. They enjoyed teaching. The campus was also magnificent, particularly in the spring when the cherry and dogwood trees were bursting with blossoms. An emphasis on Quaker beliefs permeated all of the academic and sports programs. A favorite period for many, including me, was Quaker meeting, a time set aside for silent meditation, and taking stock of where we were going. My wife and I sent our daughter to George School for her own last two years in high school so that she might also benefit from the personal virtues it promotes, and we think she has.

Sports were very important to me at George School, and physical activity has remained an important activity for me to this day. I played varsity football, soccer, and baseball, and wrestled. I was particularly proficient at wrestling. I enjoyed the drama of a single opponent, as well as the physical and psychological challenges of the sport. After George School, I went to Antioch, a small liberal arts college in Ohio.

At Antioch College I became a serious student, converting to academics all of the energy I had previously devoted to sports. Coming from George School, I carried the charge of making this a better, more equitable world for all people. Most of the problems appeared to be political, so I started out at Antioch majoring in political science. However, I soon became disillusioned with political science since there appeared to be little science to this discipline, so I switched to the physical sciences – physics and chemistry. I found great pleasure in the simplicity and elegance of mathematics and classical physics. I took almost every mathematics, physics, and chemistry course offered at Antioch, including Boolean algebra and topology, electrodynamics, and physical chemistry.

Although I found physics and mathematics intellectually satisfying, it was becoming apparent that what I was learning came from the past. The newest physics that was taught at Antioch was quantum mechanics, a revolution that had occurred in the 1920’s and earlier. Also, many frontiers of experimental physics, particularly experimental particle physics, were requiring the use of larger and larger accelerators, which involved bigger and bigger teams of scientists and support groups to execute the experiments. I was looking for a science in which the individual investigator had a more intimate, hands-on involvement with the experiments. Fortunately, Antioch had an outstanding work-study program; one quarter we studied on campus, the next was spent working on jobs related to our fields of interest. The jobs, in my case laboratory jobs, were maintained all over the country, and every three months we packed up our bags and set off for a new city and a new work experience. So one quarter off I went to Boston and the Massachusetts Institute of Technology (MIT).

There I encountered molecular biology as the field was being born (late 1950’s). This was a new breed of science and scientist. Everything was new. There were no limitations. Enthusiasm permeated this field. Devotees from physics, chemistry, genetics, and biology joined its ranks. The common premises were that the most complex biological phenomena could, with persistence, be understood in molecular terms and that biological phenomena observed in simple organisms, such as viruses and bacteria, were mirrored in more complex ones. Implicit corollaries to this premise were that whatever was learned in one organism was likely to be directly relevant to others and that similar approaches could be used to study biological phenomena in many organisms. Genetics, along with molecular biology, became the principal means for dissecting complex biological phenomena into workable subunits. Soon all organisms came under the scrutiny of these approaches.

I became a product of the molecular biology revolution. The next generation. As an Antioch college undergraduate, I worked several quarters in Alex Rich’s laboratory at MIT. He was an x-ray crystallographer, with very broad interests in molecular biology. While at MIT I was also fortunate to be influenced bySalvador Luria, Cyrus Leventhal and Boris Magasanik, through courses, seminars, and personal discussions. At that time Sheldon Penman and Jim Darnell were also working in Alex Rich’s laboratory. When placed in the same room, these two were particularly boisterous, providing comic relief to the fast moving era.

After Antioch, I set off for what I perceived as the “Mecca” of molecular biology, Harvard University. I had interviewed with Professor James D. Watson, of “Watson and Crick” fame, and asked him where should I do my graduate studies. His reply was curt and to the point: “Here. You would be fucking crazy to go anywhere else.” The simplicity of the message was very persuasive.

James D. Watson
Figure 6. A photograph of James D. Watson.

James D. Watson had a profound influence on my career (see Figure 6). He was my mentor. He did not teach me how to do molecular biology; because of my Antioch job experiences, I had already become a proficient experimenter. Jim instead taught me the process of science – how to extract the questions in a field that are critical to it and at the same time approachable through current technology. As an individual, he personified molecular biology, and, as his students, we were its eager practitioners. His bravado encouraged self-confidence in those around him. His stark honesty made our quest for truth uncompromising. His sense of justice encouraged compassion. He taught us not to bother with small questions, for such pursuits were likely to produce small answers. At a critical time, when I was contemplating leaving Harvard as a faculty member and going to Utah, he, being familiar with my self-sufficiency, counseled me that I could do good science anywhere. The move turned out to be a good decision. In Utah I had the luxury to pursue long-term projects that were not readily possible at Harvard, which, in too many cases had become a bastion of short-term gratification.

Doing science in Jim’s laboratory was exhilarating. As a graduate student, I was provided with what appeared to be limitless resources. I could not be kept out of the laboratory. Ninety-hour weeks were common. The lab was filled with talented students, each working on his or her own set of projects. Represented was a mixture of genetics, molecular biology, and biochemistry. We were cracking the genetic code, determining how proteins were synthesized, and isolating and characterizing the enzymatic machinery required for transcription. At this time, Walter Gilbert was also working in Jim’s laboratory. He was then a member of the physics department, but had also been bitten by the molecular biology bug. Jim and Wally complemented each other brilliantly, because they approached science from very different perspectives. Jim was intuitive, biological; Wally quantitative, with a physicist’s perspective. They were both very competitive. As students, we received the benefit of both, but also their scrutiny. They were merciless, but fair. You had to have a tough hide, but you learned rigor, both with respect to your science and your presentations. Once you made it through Jim’s laboratory, the rest of the world seemed a piece of cake. It was excellent training. Despite the toughness, which at times was hard, Jim was extremely supportive. He also made sure that you, the student, received full credit for your work. Despite the fact that Jim was responsible for all of the resources needed to run his laboratory, if you did all of the work for a given paper, then you were the sole author on that paper. Among all of the laboratory heads in the world, I believe that Jim Watson was among very few in implementing this policy.

The summer before I started graduate school, Marshall Nirenberg had announced that polyU directs the synthesis of polyphenylalanine in a cell free protein synthesizing extract. That paper was a bombshell! I decided I would generate a cell-free extract capable of synthesizing real, functional proteins. Jim’s laboratory had started working on the RNA bacteriophage, R17. Its genome also served as messenger RNA to direct the synthesis of its viral proteins. That would be my message. The cell-free protein synthesizing extract worked beautifully. Authentic viral coat protein and replicase were shown to be synthesized in the extract1. Further, the coat protein was functional, it bound to a specific sequence of the R17 genome, thereby modulating the synthesis of the replicase. To this day, the high affinity of the viral coat protein for this RNA sequence is exploited as a general reporter system to track RNA trafficking within living cells and neuronal axons. In collaboration with Gary Gussin, also a graduate student in Jim’s laboratory, this system was used to determine the molecular mechanism of genetic suppression of nonsense mutations2. In collaboration with Jerry Adams, another graduate student in Jim’s laboratory, the system was also used to determine that initiation of the synthesis of all proteins in bacteria proceeded through the use of formyl-methionine-tRNA3,4. A similar mechanism is involved in the initiation of protein synthesis in all eukaryotic organisms. Finally, I used the same in vitro system to show that termination of protein synthesis unexpectedly utilized protein factors, rather than tRNA, to accomplish this end5,6. Jim Watson would later offer the very complimentary comment “that Capecchi accomplished more as a graduate student than most scientists accomplish in a lifetime.” It was, indeed, a productive time, but it wasn’t work; it was sheer joy.

While a graduate student in Jim’s laboratory, I was invited to become a junior fellow of the Society of Fellows at Harvard. Being a junior fellow was very special. The society’s membership, junior and senior fellows, represented a broad spectrum of disciplines; all the members were talented, and most of them were much more verbal than I. Social discourse centered around meals, prepared by an exquisite French chef and ending with fine brandy and Cuban cigars. Frequent guests at these dinners were the likes of Leonard Bernstein. Surreal maybe, but also very special.

Karl G. Lark
Figure 7. A photograph of Karl G. Lark.

From Jim’s laboratory, I joined the faculty in the Department of Biochemistry at Harvard Medical School, across the river in Boston. During my four years at Harvard Medical School I quickly rose through the ranks, but then, I unexpectedly decided to go to Utah. I was looking for something different. There were excellent scientists in the department I was in at Harvard Medical School, but the department was not built with synergy in mind. Each research group was an island onto itself. At that time, they were also unwilling to hire additional young faculty and thereby provide the department with a more youthful, energetic character. At the University of Utah, I would be joining a newly formed department that was being assembled by a very talented scientist and administrator, Karl G. Lark (Figure 7). He had excellent taste in scientists and a vision of assembling a faculty that would enjoy working together and striving together for excellence. I could be a participant in the growth of that department and help shape its character. Furthermore, the University’s administration, led then by President David P. Gardner, was in synchrony with this vision and a strong supporter. Gordon had already attracted Baldomero (Toto) Olivera, Martin Rechsteiner, Sandy Parkinson, and Larry Okun to Utah. After I arrived at Utah, we were able to bring to Utah such outstanding scientists as Ray Gesteland, John Roth, and Mary Beckerle. Utah also provided wide open space, an entirely new canvas upon which to create a new career (see Figures 8). These are views from one of the homes in Utah which I have shared with my wife, Laurie Fraser, and daughter, Misha. The air is clean, and I can look for long distances. The elements of nature are all around us. What a place to begin a new life!

Views
Figure 8. Views from one of our homes in Utah and a photograph of my wife, Laurie Fraser, and daughter, Misha, just after she was born. Misha is now graduating from the University of California, Santa Cruz as an arts major.
References
1. Capecchi, M. R. (1966). Cell-free protein synthesis programmed with R17 RNA: Identification of two phage proteins. J. Mol. Bol. 21:173–193.
2. Capecchi, M. R. and Gussin, G. N. (1965). Suppression in vitro: Identification of a serine-tRNA as a “Nonsense Suppressor.” Science 149:417–422.
3. Adams, J. M. and Capecchi, M. R. (1966). N-formylmethionine-tRNA as the initiator of protein syntheses. Proc. Natl. Acad. Sci. USA 55:147–155.
4. Capecchi, M. R. (1966). Initiation of E. coli proteins. Proc. Natl. Acad. Sci. USA 55:1517–1524.
5. Capecchi, M. R. (1967). Polypeptide chain termination in vitro: Isolation of a release factor. Proc. Natl. Acad. Sci. USA 58:1144–1151.
6. Capecchi, M. R. and Klein, H. A. (1970). Release factors mediating termination of complete proteins. Nature 26:1029–1033.

From Les Prix Nobel. The Nobel Prizes 2007, Editor Karl Grandin, [Nobel Foundation], Stockholm, 2008

This autobiography/biography was written at the time of the award and later published in the book series Les Prix Nobel/ Nobel Lectures/The Nobel Prizes. The information is sometimes updated with an addendum submitted by the Laureate.

Copyright © The Nobel Foundation 2007

Dr. Capecchi is a member of the National Academy of Sciences (1991) and the European Academy of Sciences (2002). He has won numerous awards, including the Bristol-Myers Squibb Award for Distinguished Achievement in Neuroscience Research (1992), the Gairdner Foundation International Award for Achievements in Medical Sciences (1993), the General Motors Corporation’s Alfred P. Sloan Jr. Prize for Outstanding Basic Science Contributions to Cancer Research (1994), the German Molecular Bioanalytics Prize, (1996), the Kyoto Prize in Basic Sciences (1996), the Franklin Medal for Advancing Our Knowledge of the Physical Sciences (1997), the Feodor Lynen Lectureship (1998), the Rosenblatt Prize for Excellence (1998), the Baxter Award for Distinguished Research in the Biomedical Sciences (1998), the Helen Lowe Bamberger Colby and John E. Bamberger Presidential Endowed Chair in the University of Utah Health Sciences Center (1999), lectureship in the Life Sciences for the Collège de France (2000), the Horace Mann Distinguished Alumni Award, Antioch College (2000), the Italian Premio Phoenix-Anni Verdi for Genetics Research Award (2000), the Spanish Jiménez-Diáz Prize (2001), the Pioneers of Progress Award (2001), the Albert Lasker Award for Basic Medical Research (2001), the National Medal of Science (2001), the John Scott Medal Award (2002), the Massry Prize (2002), the Pezcoller Foundation-AACR International Award for Cancer Research (2003), the Wolf Prize in Medicine (2002/03), the March of Dimes Prize in Developmental Biology (2005),and the Nobel Prize in Physiology and Medicine (2007) with Oliver Smithies and Martin Evans.

Research interests include: the molecular genetic analysis of early mouse development, neural development in mammals, production of murine models of human genetic diseases, gene therapy, homologous recombination and programmed genomic rearrangements in the mouse.

http://www.hhmi.org/news/making-scientist

Mario Capecchi received a Kyoto Prize from the Inamori Foundation in 1996. The lecture he delivered when he accepted the prize in Japan in November 1996 tells the story of his remarkable life. The text of the lecture has been edited for length.

Radoslav Bozov commented on Targeted gene modification

Targeted gene modification Larry H Bernstein, MD, FCAP, Curator Leaders in Pharmaceutical Intelligence Series E. 2: …

Larry, same thing, data redundancy of data mining issues, of what data is in reality of physics beyond nano space in time! Working on something that does not exits in space and time, but computable mass of ‘designed’ energy formulated systems: Hox gene does not exist: It is a piece of time we percive through some kind of imagination +1,

The data generated through m/z methods is space-time unaccurate! Guess what is double R doing here instead of double Y, wonder why miRNA are obejctive to polymer degradation process ??!! what are we really seeing is not what is really in there!

Score Expect Method Identities Positives Gaps
19.2 bits(38) 0.076 Composition-based stats. 8/17(47%) 10/17(58%) 0/17(0%)

Query 511 LTEDRRAFAARMAEIGE 527
LT DRR AR+ + E
Sbjct 38 LTRDRRYEVARLLNLTE 54

Breaking news about genomic engineering, T2DM and cancer treatments

Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2015/09/28/breaking-news-about-genomic-engineering-t2dm-and-cancer-treatments/

Read Full Post »


To understand what happens in the brain to cause mental illness

Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 5.10

https://bbrfoundation.org/research/basic-research

Fernando Sampaio Goes, M.D., a 2008 NARSAD Young Investigator at Johns Hopkins School of Medicine, and his colleagues took an alternative approach to the ongoing genome-wide association studies (GWAS) that hunt for these factors by scouring the complete genomes of tens of thousands of individuals. The team––which included 2005 Young InvestigatorDimitrios Avramopoulos, M.D., Ph.D.; 2000 Young Investigator, 2008 Independent Investigator, and BBRF Scientific Council member James B. Potash, M.D., M.P.H.; and 2004 Young Investigator Peter P. Zandi, Ph.D.––conceived the study to detect rare genetic variations that GWAS are not designed to find.

Rather than scanning entire genomes for depression-associated variations, Goes’s team narrowed its search to a set of genes in which they already suspected alterations might contribute to depression: those that encode proteins found at or near the junctions between neurons, where cell-to-cell communication takes place. Based on previous surveys of these synaptic proteins, the scientists chose 1,742 genes to include in their analysis.

They compared the protein-coding sequences of that set of genes in 259 people with major depression to the same set in 334 unaffected individuals. To increase the chance of finding relevant genetic factors, all the patients with depression were selected based on the criterion of early-onset, recurrent depression, which is suspected by some to be a more heritable form of the illness. (An important component of depression causation is environmental, and reflects the particular life circumstances of those affected, who may or may not be naturally resilient when faced with stress and other environmental factors.)

The team’s analysis pointed to two sets of genes in which variations were linked with major depression. One includes genes that control the growth of dendritic spines (tiny knob-like protrusions from a neuron’s surface that receive inputs from other neurons). Other research has suggested that the size, density, and shape of these structures may be involved in mood disorders and other mental illnesses. The second gene set includes genes linked with the entry of calcium into neurons, which regulates a variety of processes, including the release of message-propagating neurotransmitters. Variations within this gene set have also been linked to autism and epilepsy.

Researchers have identified unique characteristics of emotional processing in young people with post-traumatic stress disorder (PTSD), showing for the first time how that processing might be disrupted at different ages.

Publishing their findings online August 5th in Neuropsychopharmacology, were 2012 NARSAD Young Investigator grantee Ryan J. Herringa, M.D., Ph.D., of the University of Wisconsin School of Medicine and Public Health and Richard C. Wolf, a Ph.D. candidate at the university. Together they  looked at brain activity during an emotion-related task in children aged 8 to 18, both with and without PTSD. The children with PTSD had experienced trauma such as sexual abuse, the death of a loved one, a physical accident, or witnessing violence.

The children viewed emotionally “threatening” and “neutral” pictures. During this task, the researchers used imaging to measure activity in brain regions associated in PTSD with an increased fear response and sense of threat. These regions include the amygdala, important for processing emotions; the dorsal anterior cingulate cortex (dACC), which helps to gauge threat levels; and the medial portion of the prefrontal cortex (PFC), crucial for dialing back fear responses and putting perceived threats in context.

The researchers found higher threat-related dACC activity in youths with PTSD, as well as weaker connections between the amygdala and mPFC. The findings suggest these brain regions contribute to the difficulty young people with PTSD have in assessing perceptions of threat.  The study also found that amygdala-PFC connections followed a different developmental path for youths with PTSD. Whereas those connections were stronger at older ages in those without PTSD, the same connections grew weaker for children with PTSD as they aged. This may reflect a progressive weakening in the ability of the PFC to reduce fear.

In research reported June 17th in the journal Neuron, scientists have shown that a protein called CPEB3 is critical for the stabilization and storage of long-term memories in mice. Three-timeNARSAD Distinguished Investigator and BBRF Scientific Council member Eric Kandel, M.D., led the research. Also on the team is 2013 NARSAD Young Investigator Pierre Trifilieff, Ph.D.

CPEB3 is a “prion-like” protein. Prions––infectious, misshapen proteins best known for the devastation they cause––clump together and lead to brain damage in people and animals with mad cow disease and related conditions. Similar protein-clumping mechanisms may also contribute to neurodegenerative diseases including Alzheimer’sParkinson’s, and amyotrophic lateral sclerosis. (Curiously, certain proteins with prion-like properties have an important role in the healthy brain.)

The new finding extends previous work showing that prion-like proteins are vital for the stabilization of long-term term memory in sea slugs and fruit flies. Although further work is needed to understand whether the same mechanism is at work in humans, humans do produce a protein similar to the mouse protein CPEB3.

Memories are stored in the connections between neurons, and proteins play a role in the long-term storage of the information. But since proteins degrade over time, scientists had wondered how a memory can persist long after a new experience triggers neurons to make memory-specific proteins. Prion-like proteins, which are self-perpetuating because they can convert normal proteins to their own misshapen form, appear to be the answer.

Genetic studies have recently yielded large numbers of “hits” for genes that subtly increase or decrease risk for disorders, including for schizophrenia. However, there have been no hits for major depression, perhaps because the studies are not yet large enough or because depression is less heritable. Estimates put the heritability of major depression at around 50%, with the remaining contribution coming from environmental and experiential causes.

However, a new approach has paid off: a study published online July 15th at the journal Natureidentifies two genomic regions that harbor genes that increase risk of major depression. A multinational collaboration employed a strategy of narrowing the pool of subjects to women in China with the most severe and stubborn form of depression, with the hope that a more homogenous population would yield results.

In an accompanying News and Views, 2014 Lieber Prizewinner for Outstanding Achievement in Schizophrenia Research,Patrick Sullivan, M.D., of the University of North Carolina, writes, “This first identification of replicable, significant genome-wide associations for MDD is exceptional.”

Qi Xu, Ph.D., of Peking Union Medical College and Jun Wang, Ph.D., of BGI-Shenzhen, who led the China components of the study, along with Foundation Scientific Council Member Kenneth Kendler, M.D., of Virginia Commonwealth University and 2007 NARSAD Distinguished Investigator Grantee Jonathan Flint, M.D., of the University of Oxford in the United Kingdom focused exclusively on women with severe, recurrent depression (an average of more than five episodes), building a sample of 5,303 cases and 5,337 controls. The results were replicated in a separate group of 3,231 Chinese women with major depression and 3,186 mixed male/female controls.

“I think this paper is groundbreaking because it really demonstrates that we can make progress in reducing genetic heterogeneity by paying attention to key clinical indicators,” said three-time NARSAD Grantee, Francis McMahon, M.D., of the National Institute of Mental Health (NIMH), who was not an author on the paper.

A team based at the University of Edinburgh analyzed data from thousands of Scottish adults to see whether they had genetic mutations either linked with obesity or major depressive disorder. They tested for relationships between those genetic profiles, the presence of depression or other psychological distress, and body mass index, a measure used to determine obesity. A genetic predisposition for obesity more strongly predicted actual obesity among those adults who were also depressed.

The findings were reported June 30th in Translational Psychiatry by a team including 2008NARSAD Distinguished Investigator Grantee David J. Porteous, Ph.D., and 2010 Independent Investigator Andrew M. McIntosh, Ph.D.

The study results show people becoming obese in part because of their depression, rather than becoming depressed because they are already obese. The experience of depression may drive disordered eating habits. It may also trigger chemical responses in the body (such as the release of the stress hormone cortisol) that promote weight gain, the researchers hypothesize.

The team also found some degree of association between a genetic profile linked to obesity and current psychological distress, even among individuals who were not obese. Obesity-linked genes also more closely predicted actual obesity among people experiencing distress even if they were not diagnosed with depression. This indicates that psychological strain, and not just depression per se, contributes to obesity.

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