Faculty Bibliography

Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here, we show that macroautophagy requires the Alzheimer's disease (AD)-related protein presenilin-1 (PS1). In PS1 null blastocysts, neurons from mice hypomorphic for PS1 or conditionally depleted of PS1, substrate proteolysis and autophagosome clearance during macroautophagy are prevented as a result of a selective impairment of autolysosome acidification and cathepsin activation. These deficits are caused by failed PS1-dependent targeting of the v-ATPase V0a1 subunit to lysosomes. N-glycosylation of the V0a1 subunit, essential for its efficient ER-to-lysosome delivery, requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the Sec61alpha/oligosaccharyltransferase complex. PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests previously unidentified therapeutic targets

Cystatin C (CysC) expression in the brain is elevated in human patients with epilepsy, in animal models of neurodegenerative conditions, and in response to injury, but whether up-regulated CysC expression is a manifestation of neurodegeneration or a cellular repair response is not understood. This study demonstrates that human CysC is neuroprotective in cultures exposed to cytotoxic challenges, including nutritional-deprivation, colchicine, staurosporine, and oxidative stress. While CysC is a cysteine protease inhibitor, cathepsin B inhibition was not required for the neuroprotective action of CysC. Cells responded to CysC by inducing fully functional autophagy via the mTOR pathway, leading to enhanced proteolytic clearance of autophagy substrates by lysosomes. Neuroprotective effects of CysC were prevented by inhibiting autophagy with beclin 1 siRNA or 3-methyladenine. Our findings show that CysC plays a protective role under conditions of neuronal challenge by inducing autophagy via mTOR inhibition and are consistent with CysC being neuroprotective in neurodegenerative diseases. Thus, modulation of CysC expression has therapeutic implications for stroke, Alzheimer's disease, and other neurodegenerative disorders

An additional copy of the beta-amyloid precursor protein (APP) gene causes early-onset Alzheimer's disease (AD) in trisomy 21 (DS). Endosome dysfunction develops very early in DS and AD and has been implicated in the mechanism of neurodegeneration. Here, we show that morphological and functional endocytic abnormalities in fibroblasts from individuals with DS are reversed by lowering the expression of APP or beta-APP-cleaving enzyme 1 (BACE-1) using short hairpin RNA constructs. By contrast, endosomal pathology can be induced in normal disomic (2N) fibroblasts by overexpressing APP or the C-terminal APP fragment generated by BACE-1 (betaCTF), all of which elevate the levels of betaCTFs. Expression of a mutant form of APP that cannot undergo beta-cleavage had no effect on endosomes. Pharmacological inhibition of APP gamma-secretase, which markedly reduced Abeta production but raised betaCTF levels, also induced AD-like endosome dysfunction in 2N fibroblasts and worsened this pathology in DS fibroblasts. These findings strongly implicate APP and the betaCTF of APP, and exclude Abeta and the alphaCTF, as the cause of endocytic pathway dysfunction in DS and AD, underscoring the potential multifaceted value of BACE-1 inhibition in AD therapeutics

Alzheimer's disease often results in impaired olfactory perceptual acuity-a potential biomarker of the disorder. However, the usefulness of olfactory screens to serve as informative indicators of Alzheimer's is precluded by a lack of knowledge regarding why the disease impacts olfaction. We addressed this question by assaying olfactory perception and amyloid-beta (Abeta) deposition throughout the olfactory system in mice that overexpress a mutated form of the human amyloid-beta precursor protein. Such mice displayed progressive olfactory deficits that mimic those observed clinically-some evident at 3 months of age. Also, at 3 months of age, we observed nonfibrillar Abeta deposition within the olfactory bulb-earlier than deposition within any other brain region. There was also a correlation between olfactory deficits and the spatial-temporal pattern of Abeta deposition. Therefore, nonfibrillar, versus fibrillar, Abeta-related mechanisms likely contribute to early olfactory perceptual loss in Alzheimer's disease. Furthermore, these results present the odor cross-habituation test as a powerful behavioral assay, which reflects Abeta deposition and thus may serve to monitor the efficacy of therapies aimed at reducing Abeta

Endocytic alterations are one of the earliest changes to occur in Alzheimer's disease (AD), and are hypothesized to be involved in the selective vulnerability of specific neuronal populations during the progression of AD. Previous microarray and real-time quantitative PCR experiments revealed an upregulation of the early endosomal effector rab5 and the late endosome constituent rab7 in the hippocampus of people with mild cognitive impairment (MCI) and AD. To assess whether these select rab GTPase gene expression changes are reflected in protein levels within selectively vulnerable brain regions (basal forebrain, frontal cortex, and hippocampus) and relatively spared areas (cerebellum and striatum), we performed immunoblot analysis using antibodies directed against rab5 and rab7 on postmortem human brain tissue harvested from cases with a premortem clinical diagnosis of no cognitive impairment (NCI), MCI, and AD. Results indicate selective upregulation of both rab5 and rab7 levels within basal forebrain, frontal cortex, and hippocampus in MCI and AD, which also correlated with Braak staging. In contrast, no differences in protein levels were found in the less vulnerable cerebellum and striatum. These regional immunoblot assays are consistent with single cell gene expression data, and provide protein-based evidence for endosomal markers contributing to the vulnerability of cell types within selective brain regions during the progression of AD

The ultrastructural view of the axonal cytoskeleton as an extensively cross-linked network of neurofilaments (NFs) and other cytoskeletal polymers contrasts with the dynamic view suggested by axonal transport studies on cytoskeletal elements. Here we reconcile these perspectives by showing that neurons form a large NF network along axons which is unequivocally stationary, metabolically stable, and maintained by NFs and nonfilamentous subunit assemblies undergoing slow transport by intermittent rapid movements and pauses. In mouse primary cortical neurons transfected with EGFP-NFL, formation of this stationary NF network requires a critical level of NFs, which explains its absence in NF-poor developing neurons studied previously. Most NFs at proximal axon regions were in a stationary structure coexisting with a smaller pool of moving EGFP-NFL assemblies that were mainly nonfilamentous. Distally along the same axon, EGFP-labeled NFL was much less abundant, and we detected only short filaments moving bidirectionally by slow transport (rapid movements and pauses) as previously described. In living mice, >25% of radiolabeled newly synthesized NFs remained in optic axons after slowly transported NFs had exited. Retained NF remained fixed over several months in a nonuniform distribution and exhibited exceptionally slow turnover (t(1/2) >2.5 months), implying that, at steady state, >90% of NFs in mature optic axons comprise the stationary cytoskeleton and <10% are undergoing slow transport. These findings reconcile in vitro and in vivo axonal transport observations, showing that slowly transported NFs or subunit oligomers are precursors to a highly stable stationary cytoskeletal network that supports mature axons

Individuals with Down syndrome develop beta-amyloid deposition characteristic of early-onset Alzheimer's disease (AD) in mid-life, presumably because of an extra copy of the chromosome 21-located amyloid precursor protein (App) gene. App mRNA and APP metabolite levels were assessed in the brains of Ts65Dn mice, a mouse model of Down syndrome, using quantitative PCR, western blot analysis, immunoprecipitation, and ELISAs. In spite of the additional App gene copy, App mRNA, APP holoprotein, and all APP metabolite levels in the brains of 4-month-old trisomic mice were not increased compared with the levels seen in diploid littermate controls. However starting at 10 months of age, brain APP levels were increased proportional to the App gene dosage imbalance reflecting increased App message levels in Ts65Dn mice. Similar to APP levels, soluble amino-terminal fragments of APP (sAPPalpha and sAPPbeta) were increased in Ts65Dn mice compared with diploid mice at 12 months but not at 4 months of age. Brain levels of both Abeta40 and Abeta42 were not increased in Ts65Dn mice compared with diploid mice at all ages examined. Therefore, multiple mechanisms contribute to the regulation towards diploid levels of APP metabolites in the Ts65Dn mouse brain