Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations.

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.

Peripherin is a subunit of peripheral nerve neurofilaments: implications for differential vulnerability of CNS and peripheral nervous system axons.

Peripherin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the neurofilament triplet proteins [neurofilament light (NFL), medium (NFM), and heavy (NFH) chain] but has an unknown function. The earlier peak expression of peripherin than the triplet during brain development and its ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, have supported a widely held view that peripherin and neurofilament triplets form separate filament systems. However, here, we demonstrate that, despite a postnatal decline in expression, peripherin is as abundant as the triplet in the adult PNS and exists in a relatively fixed stoichiometry with these subunits. Peripherin exhibits a distribution pattern identical to those of triplet proteins in sciatic axons and colocalizes with NFL on single neurofilaments by immunogold electron microscopy. Peripherin also coassembles into a single network of filaments containing NFL, NFM, and NFH with and without α-internexin in quadruple- or quintuple-transfected SW13vim(-) cells. Genetically deleting NFL in mice dramatically reduces peripherin content in sciatic axons. Moreover, peripherin mutations has been shown to disrupt the neurofilament network in transfected SW13vim(-) cells. These data show that peripherin and the neurofilament proteins are functionally interdependent. The results strongly support the view that, rather than forming an independent structure, peripherin is a subunit of neurofilaments in the adult PNS. Our findings provide a basis for its close relationship with neurofilaments in PNS diseases associated with neurofilament accumulation.

Alzheimer's-related endosome dysfunction in Down syndrome is Abeta-independent but requires APP and is reversed by BACE-1 inhibition.

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.

Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer's disease ameliorates amyloid pathologies and memory deficits.

Autophagy, a major degradative pathway for proteins and organelles, is essential for survival of mature neurons. Extensive autophagic-lysosomal pathology in Alzheimer's disease brain contributes to Alzheimer's disease pathogenesis, although the underlying mechanisms are not well understood. Here, we identified and characterized marked intraneuronal amyloid-ß peptide/amyloid and lysosomal system pathology in the Alzheimer's disease mouse model TgCRND8 similar to that previously described in Alzheimer's disease brains. We further establish that the basis for these pathologies involves defective proteolytic clearance of neuronal autophagic substrates including amyloid-ß peptide. To establish the pathogenic significance of these abnormalities, we enhanced lysosomal cathepsin activities and rates of autophagic protein turnover in TgCRND8 mice by genetically deleting cystatin B, an endogenous inhibitor of lysosomal cysteine proteases. Cystatin B deletion rescued autophagic-lysosomal pathology, reduced abnormal accumulations of amyloid-ß peptide, ubiquitinated proteins and other autophagic substrates within autolysosomes/lysosomes and reduced intraneuronal amyloid-ß peptide. The amelioration of lysosomal function in TgCRND8 markedly decreased extracellular amyloid deposition and total brain amyloid-ß peptide 40 and 42 levels, and prevented the development of deficits of learning and memory in fear conditioning and olfactory habituation tests. Our findings support the pathogenic significance of autophagic-lysosomal dysfunction in Alzheimer's disease and indicate the potential value of restoring normal autophagy as an innovative therapeutic strategy for Alzheimer's disease.

The contributions of myelin and axonal caliber to transverse relaxation time in shiverer and neurofilament-deficient mouse models.

White matter disorders can involve injury to myelin or axons but the respective contribution of each to clinical course is difficult to evaluate non-invasively. Here, to develop a paradigm for further investigations of axonal pathology by MRI, we compared two genetic mouse models exhibiting relatively selective axonal or myelin deficits using quantitative MRI relaxography of the transverse relaxation times (T2) in vivo and ultrastructural morphometry. In HM-DKO mice, which lack genes encoding the heavy (NF-H) and medium (NF-M) subunits of neurofilaments, neurofilament content of large myelinated axons of the central nervous system (CNS) is markedly reduced in the absence of changes in myelin thickness and volume. In shiverer mutant mice, which lack functional myelin basic protein, CNS myelin sheath formation is markedly reduced but neurofilament content is normal. We observed increases in T2 in nearly all white matter in shiverer mice compared to their wild type, while more subtle increases in T2 were observed in HM-DKO in the corpus callosum. White matter T2 was generally greater in shiverer mice than HM-DKO mice. Ultrastructural morphometry of the corpus callosum, which exhibited the greatest T2 differences, confirmed that total cross-sectional area occupied by axons was similar in the two mouse models and that the major ultrastructural differences, determined by morphometry, were an absence of myelin and larger unmyelinated axons in shiverer mice and absence of neurofilaments in HM-DKO mice. Our findings indicate that T2 is strongly influenced by myelination state and axonal volume, while neurofilament structure within the intra-axonal compartment has a lesser effect upon single compartment T2 estimates.

Macroautophagy--a novel Beta-amyloid peptide-generating pathway activated in Alzheimer's disease.

Macroautophagy, which is a lysosomal pathway for the turnover of organelles and long-lived proteins, is a key determinant of cell survival and longevity. In this study, we show that neuronal macroautophagy is induced early in Alzheimer's disease (AD) and before beta-amyloid (Abeta) deposits extracellularly in the presenilin (PS) 1/Abeta precursor protein (APP) mouse model of beta-amyloidosis. Subsequently, autophagosomes and late autophagic vacuoles (AVs) accumulate markedly in dystrophic dendrites, implying an impaired maturation of AVs to lysosomes. Immunolabeling identifies AVs in the brain as a major reservoir of intracellular Abeta. Purified AVs contain APP and beta-cleaved APP and are highly enriched in PS1, nicastrin, and PS-dependent gamma-secretase activity. Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by modulating mammalian target of rapamycin kinase elicits parallel changes in AV proliferation and Abeta production. Our results, therefore, link beta-amyloidogenic and cell survival pathways through macroautophagy, which is activated and is abnormal in AD.

Marked calpastatin (CAST) depletion in Alzheimer's disease accelerates cytoskeleton disruption and neurodegeneration: neuroprotection by CAST overexpression.

Increased activity of calpains is implicated in synaptic dysfunction and neurodegeneration in Alzheimer's disease (AD). The molecular mechanisms responsible for increased calpain activity in AD are not known. Here, we demonstrate that disease progression is propelled by a marked depletion of the endogenous calpain inhibitor, calpastatin (CAST), from AD neurons, which is mediated by caspase-1, caspase-3, and calpains. Initial CAST depletion focally along dendrites coincides topographically with calpain II and ERK 1/2 activation, tau cleavage by caspase-3, and tau and neurofilament hyperphosphorylation. These same changes, together with cytoskeletal proteolysis and neuronal cell death, accompany CAST depletion after intrahippocampal kainic acid administration to mice, and are substantially reduced in mice overexpressing human CAST. Moreover, CAST reduction by shRNA in neuronal cells causes calpain-mediated death at levels of calcium-induced injury that are sublethal to cells normally expressing CAST. Our results strongly support a novel hypothesis that CAST depletion by multiple abnormally activated proteases accelerates calpain dysregulation in AD leading to cytoskeleton disruption and neurodegeneration. CAST mimetics may, therefore, be neuroprotective in AD.

Down syndrome fibroblast model of Alzheimer-related endosome pathology: accelerated endocytosis promotes late endocytic defects.

Endocytic dysfunction is an early pathological change in Alzheimer's disease (AD) and Down's syndrome (DS). Using primary fibroblasts from DS individuals, we explored the interactions among endocytic compartments that are altered in AD and assessed their functional consequences in AD pathogenesis. We found that, like neurons in both AD and DS brains, DS fibroblasts exhibit increased endocytic uptake, fusion, and recycling, and trafficking of lysosomal hydrolases to rab5-positive early endosomes. Moreover, late endosomes identified using antibodies to rab7 and lysobisphosphatidic acid increased in number and appeared as enlarged, perinuclear vacuoles, resembling those in neurons of both AD and DS brains. In control fibroblasts, similar enlargement of rab5-, rab7-, and lysobisphosphatidic acid-positive endosomes was induced when endocytosis and endosomal fusion were increased by expression of either a rab5 or an active rab5 mutant, suggesting that persistent endocytic activation results in late endocytic dysfunction. Conversely, expression of a rab5 mutant that inhibits endocytic uptake reversed early and late endosomal abnormalities in DS fibroblasts. Our results indicate that DS fibroblasts recapitulate the neuronal endocytic dysfunction of AD and DS, suggesting that increased trafficking from early endosomes can account, in part, for downstream endocytic perturbations that occur in neurons in both AD and DS brains.

Autophagy flux in CA1 neurons of Alzheimer hippocampus: Increased induction overburdens failing lysosomes to propel neuritic dystrophy.

Defective autophagy contributes to Alzheimer disease (AD) pathogenesis although evidence is conflicting on whether multiple stages are impaired. Here, for the first time, we have comprehensively evaluated the entire autophagic process specifically in CA1 pyramidal neurons of hippocampus from early and late-stage AD subjects and nondemented controls. CA1 neurons aspirated by laser capture microdissection were analyzed using a custom-designed microarray comprising 578 neuropathology- and neuroscience-associated genes. Striking upregulation of autophagy-related genes, exceeding that of other gene ontology groups, reflected increases in autophagosome formation and lysosomal biogenesis beginning at early AD stages. Upregulated autophagosome formation was further indicated by elevated gene and protein expression levels for autophagosome components and increased LC3-positive puncta. Increased lysosomal biogenesis was evidenced by activation of MiTF/TFE family transcriptional regulators, particularly TFE3 (transcription factor binding to IGHM enhancer 3) and by elevated expression of their target genes and encoded proteins. Notably, TFEB (transcription factor EB) activation was associated more strongly with glia than neurons. These findings establish that autophagic sequestration is both competent and upregulated in AD. Autophagosome-lysosome fusion is not evidently altered. Despite this early disease response, however, autophagy flux is progressively impeded due to deficient substrate clearance, as reflected by autolysosomal accumulation of LC3-II and SQSTM1/p62 and expansion of autolysosomal size and total area. We propose that sustained induction of autophagy in the face of progressively declining lysosomal clearance of substrates explains the uncommonly robust autophagic pathology and neuritic dystrophy implicated in AD pathogenesis.

Partial BACE1 reduction in a Down syndrome mouse model blocks Alzheimer-related endosomal anomalies and cholinergic neurodegeneration: role of APP-CTF.

ß-amyloid precursor protein (APP) and amyloid beta peptide (Aß) are strongly implicated in Alzheimer's disease (AD) pathogenesis, although recent evidence has linked APP-ßCTF generated by BACE1 (ß-APP cleaving enzyme 1) to the development of endocytic abnormalities and cholinergic neurodegeneration in early AD. We show that partial BACE1 genetic reduction prevents these AD-related pathological features in the Ts2 mouse model of Down syndrome. Partially reducing BACE1 by deleting one BACE1 allele blocked development of age-related endosome enlargement in the medial septal nucleus, cerebral cortex, and hippocampus and loss of choline acetyltransferase (ChAT)-positive medial septal nucleus neurons. BACE1 reduction normalized APP-ßCTF elevation but did not alter Aß40 and Aß42 peptide levels in brain, supporting a critical role in vivo for APP-ßCTF in the development of these abnormalities. Although ameliorative effects of BACE1 inhibition on ß-amyloidosis and synaptic proteins levels have been previously noted in AD mouse models, our results highlight the additional potential value of BACE1 modulation in therapeutic targeting of endocytic dysfunction and cholinergic neurodegeneration in Down syndrome and AD.

App gene dosage modulates endosomal abnormalities of Alzheimer's disease in a segmental trisomy 16 mouse model of down syndrome.

Altered neuronal endocytosis is the earliest known pathology in sporadic Alzheimer's disease (AD) and Down syndrome (DS) brain and has been linked to increased Abeta production. Here, we show that a genetic model of DS (trisomy 21), the segmental trisomy 16 mouse Ts65Dn, develops enlarged neuronal early endosomes, increased immunoreactivity for markers of endosome fusion (rab5, early endosomal antigen 1, and rabaptin5), and endosome recycling (rab4) similar to those in AD and DS individuals. These abnormalities are most prominent in neurons of the basal forebrain, which later develop aging-related atrophy and degenerative changes, as in AD and DS. We also show that App, one of the triplicated genes in Ts65Dn mice and human DS, is critical to the development of these endocytic abnormalities. Selectively deleting one copy of App or a small portion of the chromosome 16 segment containing App from Ts65Dn mice eliminated the endosomal phenotype. Overexpressing App at high levels in mice did not alter early endosomes, implying that one or more additional genes on the triplicated segment of chromosome 16 are also required for the Ts65Dn endosomal phenotype. These results identify an essential role for App gene triplication in causing AD-related endosomal abnormalities and further establish the pathogenic significance of endosomal dysfunction in AD.

Presenilin mutations in familial Alzheimer disease and transgenic mouse models accelerate neuronal lysosomal pathology.

The neuronal lysosomal system is a major degradative pathway, induced by cell stress and closely linked to Alzheimer disease (AD) and other neurodegenerative diseases. Here, we show that mutations of presenilin (PS) 1 and 2, which cause familial early-onset AD (FAD), induce more severe lysosomal system neuropathology in humans than does sporadic AD (SAD). Cathepsin D and B levels were higher in PS-FAD neocortex than in SAD and, unlike neurons in SAD, expressed higher levels of the cation-independent mannose-6-phosphate receptor. Lysosomal pathology was also evident in more populations of neurons in PS-FAD brains, including the less vulnerable neurons in laminae II and IV and affected neurons contained high numbers of hydrolase-positive vesicular compartments with a broader range of abnormal morphology. In transgenic mice expressing mutant amyloid precursor protein (APPswe), introducing mutant PSI significantly upregulated the lysosomal system in neocortical and hippocampal neurons. This upregulation, though milder in severity, resembled that seen in human PS-FAD. Accumulation of hydrolases in dystrophic neurites in senile plaques was particularly strong, suggesting that amyloid deposition may be a stimulus for local mobilization of the lysosomal system. PS1 mice lacking the APPswe transgene also had a mild lysosomal response in some neuronal populations, which was not seen in the APPswe mice. Our findings suggest that presenilin mutations have amyloid-independent effects on the lysosomal system, which are synergistic with the lysosomal system pathology that is associated with beta-amyloid.

Abeta localization in abnormal endosomes: association with earliest Abeta elevations in AD and Down syndrome.

Early endosomes are a major site of amyloid precursor protein (APP) processing and a convergence point for molecules of pathologic relevance to Alzheimer's disease (AD). Neuronal endosome enlargement, reflecting altered endocytic function, is a disease-specific response that develops years before the earliest stage of AD and Down syndrome (DS). We examined how endocytic dysfunction is related to Abeta accumulation and distribution in early stage AD and DS. We found by ELISA and immunocytochemistry that the appearance of enlarged endosomes coincided with an initial rise in soluble Abeta40 and Abeta42 peptides, which preceded amyloid deposition. Double-immunofluorescence using numerous Abeta antibodies showed that intracellular Abeta localized principally to rab5-positive endosomes in neurons from AD brains and was prominent in enlarged endosomes. Abeta was not detectable in neurons from normal controls and was diminished after amyloid deposition in neuropathologically confirmed AD. These studies support growing evidence that endosomal pathology contributes significantly to Abeta overproduction and accumulation in sporadic AD and in AD associated with DS and may signify earlier disease-relevant disturbances of the signaling functions of endosomes.

Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis.

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-ß peptide (Aß) accumulation, extracellular ß-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 Aß, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aß40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aß 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.