Faculty Bibliography

Endocytic system dysfunction is one of the earliest disturbances that occur in Alzheimer's disease (AD), and may underlie the selective vulnerability of cholinergic basal forebrain (CBF) neurons during the progression of dementia. Herein we report that genes regulating early and late endosomes are selectively upregulated within CBF neurons in mild cognitive impairment (MCI) and AD. Specifically, upregulation of rab4, rab5, rab7, and rab27 was observed in CBF neurons microdissected from postmortem brains of individuals with MCI and AD compared to age-matched control subjects with no cognitive impairment (NCI). Upregulated expression of rab4, rab5, rab7, and rab27 correlated with antemortem measures of cognitive decline in individuals with MCI and AD. qPCR validated upregulation of these select rab GTPases within microdissected samples of the basal forebrain. Moreover, quantitative immunoblot analysis demonstrated upregulation of rab5 protein expression in the basal forebrain of subjects with MCI and AD. The elevation of rab4, rab5, and rab7 expression is consistent with our recent observations in CA1 pyramidal neurons in MCI and AD. These findings provide further support that endosomal pathology accelerates endocytosis and endosome recycling, which may promote aberrant endosomal signaling and neurodegeneration throughout the progression of AD

The proteolytic machinery comprising metalloproteases and gamma-secretase, an intramembrane aspartyl protease involved in Alzheimer's disease, cleaves several substrates in addition to the extensively studied amyloid precursor protein. Some of these substrates, such as N-cadherin, are synaptic proteins involved in synapse remodeling and maintenance. Here we show, in rats and mice, that metalloproteases and gamma-secretase are physiologic regulators of synapses. Both proteases are synaptic, with gamma-secretase tethered at the synapse by delta-catenin, a synaptic scaffolding protein that also binds to N-cadherin and, through scaffolds, to AMPA receptor and a metalloprotease. Activity-dependent proteolysis by metalloproteases and gamma-secretase takes place at both sides of the synapse, with the metalloprotease cleavage being NMDA receptor-dependent. This proteolysis decreases levels of synaptic proteins and diminishes synaptic transmission. Our results suggest that activity-dependent substrate cleavage by synaptic metalloproteases and gamma-secretase modifies synaptic transmission, providing a novel form of synaptic autoregulation

It has recently become clear that proteins associated with neurodegenerative disorders can be selectively incorporated into intraluminal vesicles of multivesicular bodies and subsequently released within exosomes. Multiple lines of research support a neuroprotective role for cystatin C in Alzheimer's disease (AD). Herein we demonstrate that cystatin C, a protein targeted to the classical secretory pathway by its signal peptide sequence, is also secreted by mouse primary neurons in association with exosomes. Immunoproteomic analysis using SELDI-TOF MS revealed the presence in exosomes of at least 9 different cystatin C glycoforms. Moreover, the over-expression of familial AD-associated presenilin 2 mutations (PS2 M239I and PS2 T122R) resulted in reduced levels of all cystatin C forms (native and glycosylated) and of amyloid-β precursor protein (APP) metabolites within exosomes. A better understanding of the mechanisms involved in exosomal processing and release may have important implications for the fight against AD and other neurodegenerative diseases.

Autophagy, the major degradative pathway for organelles and long-lived proteins, is essential for the survival of neurons. Mounting evidence has implicated defective autophagy in the pathogenesis of several major neurodegenerative diseases, particularly Alzheimer's disease (AD). A continuum of abnormalities of the lysosomal system has been identified in neurons of the AD brain, including pathological endocytic pathway responses at the very earliest disease stage and a progressive disruption of autophagy leading to the massive buildup of incompletely digested substrates within dystrophic axons and dendrites. In this review, we examine research on autophagy in AD and evaluate evidence addressing the specific step or steps along the autophagy pathway that may be defective. Current evidence strongly points to disruption of substrate proteolysis within autolysosomes for the principal mechanism underlying autophagy failure in AD. In the most common form of familial early onset AD, mutant presenilin 1 disrupts autophagy directly by impeding lysosomal proteolysis while, in other forms of AD, autophagy impairments may involve different genetic or environmental factors. Attempts to restore more normal lysosomal proteolysis and autophagy efficiency in mouse models of AD pathology have yielded promising therapeutic effects on neuronal function and cognitive performance, demonstrating the relevance of autophagy failure to the pathogenesis of AD and the potential of autophagy modulation as a therapeutic strategy

The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect: in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-beta peptide (Abeta) accumulation, extracellular beta-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Abeta, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Abeta40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Abeta clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD

Background: Although the hippocampus is selectively vulnerable to neurodegeneration during the early stages of Alzheimer's disease (AD), the molecular and cellular mechanisms underlying this dysfunction are poorly understood. Nerve growth factor, itscognate receptors and downstreamevents aswell as endosomal- lysosomal systemimpairmentmayunderlie hippocampal dysfunction during the progression of AD.Methods: To explore the role of thesemolecular factors in hippocampal degeneration during the progression of AD we performed single population gene expression array analysis and quantitative immunoblotting on tissues obtained from subjects who died with an antemortem clinical diagnosis of nocognitive impairment (NCI), mild cognitive impairment (MCI), or AD. Expression profiling observations were validated with real-time qPCR and immunocytochemistry. Results: In the present investigation, we found an increase in hippocampal protein levels of proNGF (p = 0.027) and to a lesser extent phopho-JNK (Ser 473, p = 0.066) in AD compared to NCI andMCI. Hippocampal p75^NTR remained stable across the three clinical groups while TrkA levels were reduced w60% in MCI compared to NCI orAD. No differences were found in sortilin, NRH2, phospho-AKT, phospho- ERK1/2 orAKT, ERK1/2 and JNK in the hippocampus across groups. ProNGF levels were positively correlated with phospho-JNK and to a lesser extent to phospho-AKT, suggesting activation of downstream cell survival and stress activation signals. Increased proNGF and phospho-JNK levels were associated with lower MMSE scores but not Braak neuropathology. Phospho-AKT and phosphor-ERK1/2levels were not associated with MMSE or Braak stage. On the other hand, single cell expression profiling of hippocampal CA1 neurons indicate a significant upregulation of early endosome effectors rab4 (AD>MCI&NCI) and rab5 (AD>MCI>NCI), late endosome constituent rab7 (AD &MCI > NCI), and the trafficking molecule rab24 (AD > MCI & NCI). Down regulation of thesynaptic-related marker r!

Progressive cortical pathology is common to several neurodegenerative and psychiatric disorders. The entorhinal cortex (EC) and frontal cortex (FC) are particularly vulnerable, and neurotrophins have been implicated because they appear to be protective. A downstream signal transducer of neurotrophins, the ankyrin repeat-rich membrane spanning scaffold protein/Kidins 220 (ARMS) is expressed in the cortex, where it could play an important role in trophic support. To test this hypothesis, we evaluated mice with a heterozygous deletion of ARMS (ARMS(+/-) mice). Remarkably, the EC and FC were the regions that demonstrated the greatest defects. Many EC and FC neurons became pyknotic in ARMS(+/-) mice, so that large areas of the EC and FC were affected by 12 months of age. Areas with pyknosis in the EC and FC of ARMS(+/-) mice were also characterized by a loss of immunoreactivity to a neuronal antigen, NeuN, which has been reported after insult or injury to cortical neurons. Electron microscopy showed that there were defects in mitochondria, myelination, and multilamellar bodies in the EC and FC of ARMS(+/-) mice. Although primarily restricted to the EC and FC, pathology appeared to be sufficient to cause functional impairments, because ARMS(+/-) mice performed worse than wild-type on the Morris water maze. Comparisons of males and females showed that female mice were the affected sex in all comparisons. Taken together, the results suggest that the expression of a prominent neurotrophin receptor substrate normally protects the EC and FC, and that ARMS may be particularly important in females

This review critically examines progress in understanding the link between Alzheimer's disease (AD) molecular pathogenesis and behavior, with an emphasis on the impact of amyloid-beta. We present the argument that the AD research field requires more multifaceted analyses into the impacts of Alzheimer's pathogenesis which combine simultaneous molecular-, circuit-, and behavior-level approaches. Supporting this argument is a review of particular research utilizing similar, 'systems-level' methods in mouse models of AD. Related to this, a critique of common physiological and behavioral models is made-highlighting the likely usefulness of more refined and specific tools in understanding the relationship between candidate molecular pathologies and behavioral dysfunction. Finally, we propose challenges for future research which, if met, may greatly extend our current understanding of how AD molecular pathology impacts neural network function and behavior and possibly may lead to refinements in disease therapeutics

In the hallmark neuritic dystrophy of Alzheimer's disease (AD), autophagic vacuoles containing incompletely digested proteins selectively accumulate in focal axonal swellings, reflecting defects in both axonal transport and autophagy. Here, we investigated the possibility that impaired lysosomal proteolysis could be a basis for both of these defects leading to neuritic dystrophy. In living primary mouse cortical neurons expressing fluorescence-tagged markers, LC3-positive autophagosomes forming in axons rapidly acquired the endo-lysosomal markers Rab7 and LAMP1 and underwent exclusive retrograde movement. Proteolytic clearance of these transported autophagic vacuoles was initiated after fusion with bidirectionally moving lysosomes that increase in number at more proximal axon levels and in the perikaryon. Disrupting lysosomal proteolysis by either inhibiting cathepsins directly or by suppressing lysosomal acidification slowed the axonal transport of autolysosomes, late endosomes, and lysosomes and caused their selective accumulation within dystrophic axonal swellings. Mitochondria and other organelles lacking cathepsins moved normally under these conditions, indicating that the general functioning of the axonal transport system was preserved. Dystrophic swellings induced by lysosomal proteolysis inhibition resembled in composition those in several mouse models of AD and also acquired other AD-like features, including immunopositivity for ubiquitin, amyloid precursor protein, and hyperphosphorylated neurofilament proteins. Restoration of lysosomal proteolysis reversed the affected movements of proteolytic Rab7 vesicles, which in turn essentially cleared autophagic substrates and reversed the axonal dystrophy. These studies identify the AD-associated defects in neuronal lysosomal proteolysis as a possible basis for the selective transport abnormalities and highly characteristic pattern of neuritic dystrophy associated with AD

Alzheimer's disease and fronto- temporal dementia are characterized by a continuous loss of neurons that are not replaced and the cause of neu- ronal death in affected brain regions is still a matter of discussion. It has recently become clear that proteins associated with neurodegenerative disorders can be selectively incorporated into intraluminal vesicles of multivesicular bodies and subsequently released within exosomes. We demonstrated that long-term depletion of neurotrophic factors (pro- granulin and cystatin c) might play a key role in the molecular cascade leading to neurodegeneration. In human primary fibroblast from subjects carrying pathogenic mutations as well as in disease cellular models, we observed an impaired vesicular trafficking of these disease-associated proteins and of their glycosyl- ated forms. A wide kind of factors (genetic or environmental) seem to influence - changing exosomes sorting and/or composition - the fate of those aging neurons forced to use this nano-compartment for their reciprocal communication. A better understanding of the mechanisms involved in exosomal processing and release may have important implications for the fight against neurodegenerative diseases