Pathobiology of the autophagy-lysosomal pathway in the Huntington's disease brain.

Accumulated levels of mutant huntingtin protein (mHTT) and its fragments are considered contributors to the pathogenesis of Huntington's disease (HD). Although lowering mHTT by stimulating autophagy has been considered a possible therapeutic strategy, the role and competence of autophagy-lysosomal pathway (ALP) during HD progression in the human disease remains largely unknown. Here, we used multiplex confocal and ultrastructural immunocytochemical analyses of ALP functional markers in relation to mHTT aggresome pathology in striatum and the less affected cortex of HD brains staged from HD2 to HD4 by Vonsattel neuropathological criteria compared to controls. Immunolabeling revealed the localization of HTT/mHTT in ALP vesicular compartments labeled by autophagy-related adaptor proteins p62/SQSTM1 and ubiquitin, and cathepsin D (CTSD) as well as HTT-positive inclusions. Although comparatively normal at HD2, neurons at later HD stages exhibited progressive enlargement and clustering of CTSD-immunoreactive autolysosomes/lysosomes and, ultrastructurally, autophagic vacuole/lipofuscin granules accumulated progressively, more prominently in striatum than cortex. These changes were accompanied by rises in levels of HTT/mHTT and p62/SQSTM1, particularly their fragments, in striatum but not in the cortex, and by increases of LAMP1 and LAMP2 RNA and LAMP1 protein. Importantly, no blockage in autophagosome formation and autophagosome-lysosome fusion was detected, thus pinpointing autophagy substrate clearance deficits as a basis for autophagic flux declines. The findings collectively suggest that upregulated lysosomal biogenesis and preserved proteolysis maintain autophagic clearance in early-stage HD, but failure at advanced stages contributes to progressive HTT build-up and potential neurotoxicity. These findings support the prospect that ALP stimulation applied at early disease stages, when clearance machinery is fully competent, may have therapeutic benefits in HD patients.

Faulty autolysosome acidification in Alzheimer's disease mouse models induces autophagic build-up of Aß in neurons, yielding senile plaques.

Autophagy is markedly impaired in Alzheimer's disease (AD). Here we reveal unique autophagy dysregulation within neurons in five AD mouse models in vivo and identify its basis using a neuron-specific transgenic mRFP-eGFP-LC3 probe of autophagy and pH, multiplex confocal imaging and correlative light electron microscopy. Autolysosome acidification declines in neurons well before extracellular amyloid deposition, associated with markedly lowered vATPase activity and build-up of Aß/APP-ßCTF selectively within enlarged de-acidified autolysosomes. In more compromised yet still intact neurons, profuse Aß-positive autophagic vacuoles (AVs) pack into large membrane blebs forming flower-like perikaryal rosettes. This unique pattern, termed PANTHOS (poisonous anthos (flower)), is also present in AD brains. Additional AVs coalesce into peri-nuclear networks of membrane tubules where fibrillar ß-amyloid accumulates intraluminally. Lysosomal membrane permeabilization, cathepsin release and lysosomal cell death ensue, accompanied by microglial invasion. Quantitative analyses confirm that individual neurons exhibiting PANTHOS are the principal source of senile plaques in amyloid precursor protein AD models.

Axonal transport of late endosomes and amphisomes is selectively modulated by local Ca2+ efflux and disrupted by PSEN1 loss of function.

Dysfunction and mistrafficking of organelles in autophagy- and endosomal-lysosomal pathways are implicated in neurodegenerative diseases. Here, we reveal selective vulnerability of maturing degradative organelles (late endosomes/amphisomes) to disease-relevant local calcium dysregulation. These organelles undergo exclusive retrograde transport in axons, with occasional pauses triggered by regulated calcium efflux from agonist-evoked transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1) channels-an effect greatly exaggerated by exogenous agonist mucolipin synthetic agonist 1 (ML-SA1). Deacidification of degradative organelles, as seen after Presenilin 1 (PSEN1) loss of function, induced pathological constitutive "inside-out" TRPML1 hyperactivation, slowing their transport comparably to ML-SA1 and causing accumulation in dystrophic axons. The mechanism involved calcium-mediated c-Jun N-terminal kinase (JNK) activation, which hyperphosphorylated dynein intermediate chain (DIC), reducing dynein activity. Blocking TRPML1 activation, JNK activity, or DIC1B serine-80 phosphorylation reversed transport deficits in PSEN1 knockout neurons. Our results, including features demonstrated in Alzheimer-mutant PSEN1 knockin mice, define a mechanism linking dysfunction and mistrafficking in lysosomal pathways to neuritic dystrophy under neurodegenerative conditions.

Endosomal Dysfunction Induced by Directly Overactivating Rab5 Recapitulates Prodromal and Neurodegenerative Features of Alzheimer's Disease.

Neuronal endosomal dysfunction, the earliest known pathobiology specific to Alzheimer's disease (AD), is mediated by the aberrant activation of Rab5 triggered by APP-ß secretase cleaved C-terminal fragment (APP-ßCTF). To distinguish pathophysiological consequences specific to overactivated Rab5 itself, we activate Rab5 independently from APP-ßCTF in the PA-Rab5 mouse model. We report that Rab5 overactivation alone recapitulates diverse prodromal and degenerative features of AD. Modest neuron-specific transgenic Rab5 expression inducing hyperactivation of Rab5 comparable to that in AD brain reproduces AD-related Rab5-endosomal enlargement and mistrafficking, hippocampal synaptic plasticity deficits via accelerated AMPAR endocytosis and dendritic spine loss, and tau hyperphosphorylation via activated glycogen synthase kinase-3ß. Importantly, Rab5-mediated endosomal dysfunction induces progressive cholinergic neurodegeneration and impairs hippocampal-dependent memory. Aberrant neuronal Rab5-endosome signaling, therefore, drives a pathogenic cascade distinct from ß-amyloid-related neurotoxicity, which includes prodromal and neurodegenerative features of AD, and suggests Rab5 overactivation as a potential therapeutic target.

mTOR hyperactivation in Down Syndrome underlies deficits in autophagy induction, autophagosome formation, and mitophagy.

Down syndrome (DS), a complex genetic disorder caused by chromosome 21 trisomy, is associated with mitochondrial dysfunction leading to the accumulation of damaged mitochondria. Here we report that mitophagy, a form of selective autophagy activated to clear damaged mitochondria is deficient in primary human fibroblasts derived from individuals with DS leading to accumulation of damaged mitochondria with consequent increases in oxidative stress. We identified two molecular bases for this mitophagy deficiency: PINK1/PARKIN impairment and abnormal suppression of macroautophagy. First, strongly downregulated PARKIN and the mitophagic adaptor protein SQSTM1/p62 delays PINK1 activation to impair mitophagy induction after mitochondrial depolarization by CCCP or antimycin A plus oligomycin. Secondly, mTOR is strongly hyper-activated, which globally suppresses macroautophagy induction and the transcriptional expression of proteins critical for autophagosome formation such as ATG7, ATG3 and FOXO1. Notably, inhibition of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) using AZD8055 (AZD) restores autophagy flux, PARKIN/PINK initiation of mitophagy, and the clearance of damaged mitochondria by mitophagy. These results recommend mTORC1-mTORC2 inhibition as a promising candidate therapeutic strategy for Down Syndrome.

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.

BACE and gamma-secretase characterization and their sorting as therapeutic targets to reduce amyloidogenesis.

Secretases are named for enzymes processing amyloid precursor protein (APP), a prototypic type-1 membrane protein. This led directly to discovery of novel Aspartyl proteases (beta-secretases or BACE), a tetramer complex gamma-secretase (gamma-SC) containing presenilins, nicastrin, aph-1 and pen-2, and a new role for metalloprotease(s) of the ADAM family as a alpha-secretases. Recent advances in defining pathways that mediate endosomal-lysosomal-autophagic-exosomal trafficking now provide targets for new drugs to attenuate abnormal production of fibril forming products characteristic of AD. A key to success includes not only characterization of relevant secretases but mechanisms for sorting and transport of key metabolites to abnormal vesicles or sites for assembly of fibrils. New developments we highlight include an important role for an 'early recycling endosome' coated in retromer complex containing lipoprotein receptor LRP-II (SorLA) for switching APP to a non-amyloidogenic pathway for alpha-secretases processing, or to shuttle APP to a 'late endosome compartment' to form Abeta or AICD. LRP11 (SorLA) is of particular importance since it decreases in sporadic AD whose etiology otherwise is unknown.

Glucosylceramide synthase decrease in frontal cortex of Alzheimer brain correlates with abnormal increase in endogenous ceramides: consequences to morphology and viability on enzyme suppression in cultured primary neurons.

Abnormal increase in native long-chain ceramides (lcCer) in AD implicates roles in neuronal atrophy and cognitive dysfunction especially in view of divergent roles this second messenger plays in cell function. Since clearance is mediated by glucosylceramide synthase (GCS, EC 2.4.1.80) levels of the enzyme were compared for 18 samples of AD Brodmann area 9/10 frontal cortex with 11 age-matched controls. Western analysis for (ir)GCS showed a significant decrease in AD brain (p<0.01) consistent with the hypothesis that enzyme dysfunction contributes to neuronal decay. To examine kinetics and consequences to morphology, cerebellar granule cells were treated in vitro with d-threo-P4 (P4). This potent inhibitor of GCS induced a time- and concentration-dependent increase in lcCer parallel to loss of viability and dramatic changes in neuron/neurite morphology via caspase-independent pathways distinct from those of apoptosis or necrosis. Fluorescent labeling with NBD-sphingolipids or immunostaining with anti-synaptic or cytoskeletal markers showed unusual formation of globular swellings along neurites rich in synaptophysin that may resemble formation of dystrophic neurites in AD. Effects of the inhibitor were verified by changes in lcCer mass and turnover of (14)[C]-acetate and -galactose or NBD-labeled anabolic products. Addition of a panel of inhibitors of other pathways confirms GCS as the major route for clearance in the present model. Pretreatment with GM(1) whose turnover is compromised was protective and pointed to useful therapeutic applications by supplementing existing membrane stores prior to GSC dysfunction.

Neurosecretases provide strategies to treat sporadic and familial Alzheimer disorders.

Recent discoveries on neurosecretases and their trafficking to release fibril-forming neuropeptides or other products, are of interest to pathology, cell signaling and drug discovery. Nomenclature arose from the use of amyloid precursor protein (APP) as a prototypic type-1 substrate leading to the isolation of beta-secretase (BACE), multimeric complexes (gamma-secretase, gamma-SC) for intramembranal cleavage, and attributing a new function to well-characterized metalloproteases of the ADAM family (alpha-secretase) for normal APP turnover. While purified alpha/beta-secretases facilitate drug discovery, gamma-SC presents greater challenges for characterization and mechanisms of catalysis. The review comments on links between mutation or polymorphisms in relation to enzyme mechanisms and disease. The association between lipoprotein receptor LRP11 variants and sporadic Alzheimer's disease (SAD) offers scope to integrate components of pre- and post-Golgi membranes, or brain clathrin-coated vesicles within pathways for trafficking as targets for intervention. The presence of APP and metabolites in brain clathrin-coated vesicles as significant cargo with lipoproteins and adaptors focuses attention as targets for therapeutic intervention. This overview emphasizes the importance to develop new therapies targeting neurosecretases to treat a major neurological disorder that has vast economic and social implications.

APP processing enzymes (secretases) as therapeutic targets: insights from the use of transgenics (Tgs) and transfected cells.

Secretases degrade amyloid precursor protein (APP) releasing fragments (beta-peptides A beta, A beta x) that assemble to form hallmark extracellular deposits in Alzheimer's disease (AD) correlating with disease severity. As such, secretases supply targets for therapeutic intervention and form the focus of this overview. Progress in elucidating secretases and their modes of catalysis come from exploiting the use of transgenics or transfected cells. In addition to A beta x, secretases also release C-terminal fragments with putative signaling properties (amyloid intracellular domain, AICD) similar in concept to those available for conversion of the Notch-r to release the nuclear transactivator NICD. The review considers lingering questions on APP fragmentation by secretase action, ancillary proteins such as presenilins (PS1/2), nicastrin, XII, or proteases (caspases), and the influence of familial mutations (mAPP, mPS) in terms of fibrillogenesis.

Brain damage results in down-regulation of N-acetylaspartate as a neuronal osmolyte.

N-acetyl-L-aspartate (NAA) is present in the vertebrate brain, where its concentration is one of the highest of all free amino acids. Although NAA is synthesized and stored primarily in neurons, it is not hydrolyzed in these cells. However, after its regulated release into extracellular fluid, neuronal NAA is hydrolyzed by amidohydrolase II that is present in oligodendrocytes. About 30% of neurons do not contain appreciable amounts of NAA, but its prominence in 1H nuclear magnetic resonance spectroscopic (MRS) studies has led to its wide use as a neuronal marker in diagnostic human medicine as both an indicator of brain pathology, and of disease progression in a variety of central nervous system (CNS) diseases. Loss of NAA has been interpreted as indicating either loss of neurons, or loss of neuron viability. In this investigation, the upregulation of NAA in early stages of construction of the CNS, and its downregulation in experimentally induced damage models of the CNS is reported. The results of this study indicate that the buildup of NAA is not required for viability of neurons in monocellular cultures, and that NAA is lost from multicellular cultured brain slice explants that contain viable neurons. Thus, loss of NAA does not necessarily indicate either loss of neurons or their function. The NAA system, when present in the brain, appears to reflect a high degree of cellular integration, and therefore may be a unique metabolic construct of the intact vertebrate brain.