Autophagy-lysosomal-associated neuronal death in neurodegenerative disease.

Autophagy, the major lysosomal pathway for degrading damaged or obsolete constituents, protects neurons by eliminating toxic organelles and peptides, restoring nutrient and energy homeostasis, and inhibiting apoptosis. These functions are especially vital in neurons, which are postmitotic and must survive for many decades while confronting mounting challenges of cell aging. Autophagy failure, especially related to the declining lysosomal ("phagy") functions, heightens the neuron's vulnerability to genetic and environmental factors underlying Alzheimer's disease (AD) and other late-age onset neurodegenerative diseases. Components of the global autophagy-lysosomal pathway and the closely integrated endolysosomal system are increasingly implicated as primary targets of these disorders. In AD, an imbalance between heightened autophagy induction and diminished lysosomal function in highly vulnerable pyramidal neuron populations yields an intracellular lysosomal build-up of undegraded substrates, including APP-ßCTF, an inhibitor of lysosomal acidification, and membrane-damaging Aß peptide. In the most compromised of these neurons, ß-amyloid accumulates intraneuronally in plaque-like aggregates that become extracellular senile plaques when these neurons die, reflecting an "inside-out" origin of amyloid plaques seen in human AD brain and in mouse models of AD pathology. In this review, the author describes the importance of lysosomal-dependent neuronal cell death in AD associated with uniquely extreme autophagy pathology (PANTHOS) which is described as triggered by lysosomal membrane permeability during the earliest "intraneuronal" stage of AD. Effectors of other cell death cascades, notably calcium-activated calpains and protein kinases, contribute to lysosomal injury that induces leakage of cathepsins and activation of additional death cascades. Subsequent events in AD, such as microglial invasion and neuroinflammation, induce further cytotoxicity. In major neurodegenerative disease models, neuronal death and ensuing neuropathologies are substantially remediable by reversing underlying primary lysosomal deficits, thus implicating lysosomal failure and autophagy dysfunction as primary triggers of lysosomal-dependent cell death and AD pathogenesis and as promising therapeutic targets.

Pathophysiologic abnormalities in transgenic mice carrying the Alzheimer disease PSEN1 Δ440 mutation.

Mutations in PSEN1 were first discovered as a cause of Alzheimer's disease (AD) in 1995, yet the mechanism(s) by which the mutations cause disease still remains unknown. The generation of novel mouse models assessing the effects of different mutations could aid in this endeavor. Here we report on transgenic mouse lines made with the Δ440 PSEN1 mutation that causes AD with parkinsonism:- two expressing the un-tagged human protein and two expressing a HA-tagged version. Detailed characterization of these lines showed that Line 305 in particular, which expresses the untagged protein, develops age-dependent memory deficits and pathologic features, many of which are consistent with features found in AD. Key behavioral and physiological alterations found in the novel 305 line included an age-dependent deficit in spontaneous alternations in the Y-maze, a decrease in exploration of the center of an open field box, a decrease in the latency to fall on a rotarod, a reduction in synaptic strength and pair-pulse facilitation by electrophysiology, and profound alterations to cerebral blood flow regulation. The pathologic alterations found in the line included, significant neuronal loss in the hippocampus and cortex, astrogliosis, and changes in several proteins involved in synaptic and mitochondrial function, Ca2+ regulation, and autophagy. Taken together, these findings suggest that the transgenic lines will be useful for the investigation of AD pathogenesis.

Mechanisms of autophagy-lysosome dysfunction in neurodegenerative diseases.

Autophagy is a lysosome-based degradative process used to recycle obsolete cellular constituents and eliminate damaged organelles and aggregate-prone proteins. Their postmitotic nature and extremely polarized morphologies make neurons particularly vulnerable to disruptions caused by autophagy-lysosomal defects, especially as the brain ages. Consequently, mutations in genes regulating autophagy and lysosomal functions cause a wide range of neurodegenerative diseases. Here, we review the role of autophagy and lysosomes in neurodegenerative diseases such as Alzheimer disease, Parkinson disease and frontotemporal dementia. We also consider the strong impact of cellular ageing on lysosomes and autophagy as a tipping point for the late-age emergence of related neurodegenerative disorders. Many of these diseases have primary defects in autophagy, for example affecting autophagosome formation, and in lysosomal functions, especially pH regulation and calcium homeostasis. We have aimed to provide an integrative framework for understanding the central importance of autophagic-lysosomal function in neuronal health and disease.

mTOR inhibition in Q175 Huntington's disease model mice facilitates neuronal autophagy and mutant huntingtin clearance.

Huntington's disease (HD) is caused by expansion of the polyglutamine stretch in huntingtin protein (HTT) resulting in hallmark aggresomes/inclusion bodies (IBs) composed of mutant huntingtin protein (mHTT) and its fragments. Stimulating autophagy to enhance mHTT clearance is considered a potential therapeutic strategy for HD. Our recent evaluation of the autophagic-lysosomal pathway (ALP) in human HD brain reveals upregulated lysosomal biogenesis and relatively normal autophagy flux in early Vonsattel grade brains, but impaired autolysosome clearance in late grade brains, suggesting that autophagy stimulation could have therapeutic benefits as an earlier clinical intervention. Here, we tested this hypothesis by crossing the Q175 HD knock-in model with our autophagy reporter mouse TRGL ( T hy-1- R FP- G FP- L C3) to investigate in vivo neuronal ALP dynamics. In the Q175 and/or TRGL/Q175 mice, mHTT was detected in autophagic vacuoles and also exhibited high level colocalization with autophagy receptors p62/SQSTM1 and ubiquitin in the IBs. Compared to the robust lysosomal pathology in late-stage human HD striatum, ALP alterations in Q175 models are also late-onset but milder that included a lowered phospho-p70S6K level, lysosome depletion and autolysosome elevation including more poorly acidified autolysosomes and larger-sized lipofuscin granules, reflecting impaired autophagic flux. Administration of a mTOR inhibitor to 6-mo-old TRGL/Q175 normalized lysosome number, ameliorated aggresome pathology while reducing mHTT-, p62- and ubiquitin-immunoreactivities, suggesting beneficial potential of autophagy modulation at early stages of disease progression.

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.

Lysosomal dysfunction in Down syndrome and Alzheimer mouse models is caused by v-ATPase inhibition by Tyr682-phosphorylated APP ßCTF.

Lysosome dysfunction arises early and propels Alzheimer's disease (AD). Herein, we show that amyloid precursor protein (APP), linked to early-onset AD in Down syndrome (DS), acts directly via its ß-C-terminal fragment (ßCTF) to disrupt lysosomal vacuolar (H+)-adenosine triphosphatase (v-ATPase) and acidification. In human DS fibroblasts, the phosphorylated 682YENPTY internalization motif of APP-ßCTF binds selectively within a pocket of the v-ATPase V0a1 subunit cytoplasmic domain and competitively inhibits association of the V1 subcomplex of v-ATPase, thereby reducing its activity. Lowering APP-ßCTF Tyr682 phosphorylation restores v-ATPase and lysosome function in DS fibroblasts and in vivo in brains of DS model mice. Notably, lowering APP-ßCTF Tyr682 phosphorylation below normal constitutive levels boosts v-ATPase assembly and activity, suggesting that v-ATPase may also be modulated tonically by phospho-APP-ßCTF. Elevated APP-ßCTF Tyr682 phosphorylation in two mouse AD models similarly disrupts v-ATPase function. These findings offer previously unknown insight into the pathogenic mechanism underlying faulty lysosomes in all forms of AD.

A Comprehensive Enumeration of the Human Proteostasis Network. 2. Components of the Autophagy-Lysosome Pathway.

The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Establishing and maintaining proteostasis is the province of the proteostasis network, approximately 2,700 components that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental entity in biology that is essential for cellular health and has direct relevance to many diseases of protein conformation. However, it is not well defined or annotated, which hinders its functional characterization in health and disease. In this series of manuscripts, we aim to operationally define the human proteostasis network by providing a comprehensive, annotated list of its components. We provided in a previous manuscript a list of chaperones and folding enzymes as well as the components that make up the machineries for protein synthesis, protein trafficking into and out of organelles, and organelle-specific degradation pathways. Here, we provide a curated list of 838 unique high-confidence components of the autophagy-lysosome pathway, one of the two major protein degradation systems in human cells.

Posttranscriptional regulation of neurofilament proteins and tau in health and disease.

Neurofilament and tau proteins are neuron-specific cytoskeletal proteins that are enriched in axons, regulated by many of the same protein kinases, interact physically, and are the principal constituents of neurofibrillary lesions in major adult-onset dementias. Both proteins share functions related to the modulation of stability and functions of the microtubule network in axons, axonal transport and scaffolding of organelles, long-term synaptic potentiation, and learning and memory. Expression of these proteins is regulated not only at the transcriptional level but also through posttranscriptional control of pre-mRNA splicing, mRNA stability, transport, localization, local translation and degradation. Current evidence suggests that posttranscriptional determinants of their levels are usually regulated by RNA-binding proteins and microRNAs primarily through 3'-untranslated regions of neurofilament and tau mRNAs. Dysregulations of neurofilament and tau expression caused by mutations or pathologies of RNA-binding proteins such as TDP43, FUS and microRNAs are increasingly recognized in association with varied neurological disorders. In this review, we summarize the current understanding of posttranscriptional control of neurofilament and tau by examining the posttranscriptional regulation of neurofilament and tau by RNA-binding proteins and microRNAs implicated in health and diseases.

Autophagy is a novel pathway for neurofilament protein degradation in vivo.

How macroautophagy/autophagy influences neurofilament (NF) proteins in neurons, a frequent target in neurodegenerative diseases and injury, is not known. NFs in axons have exceptionally long half-lives in vivo enabling formation of large stable supporting networks, but they can be rapidly degraded during Wallerian degeneration initiated by a limited calpain cleavage. Here, we identify autophagy as a previously unrecognized pathway for NF subunit protein degradation that modulates constitutive and inducible NF turnover in vivo. Levels of NEFL/NF-L, NEFM/NF-M, and NEFH/NF-H subunits rise substantially in neuroblastoma (N2a) cells after blocking autophagy either with the phosphatidylinositol 3-kinase (PtdIns3K) inhibitor 3-methyladenine (3-MA), by depleting ATG5 expression with shRNA, or by using both treatments. In contrast, activating autophagy with rapamycin significantly lowers NF levels in N2a cells. In the mouse brain, NF subunit levels increase in vivo after intracerebroventricular infusion of 3-MA. Furthermore, using tomographic confocal microscopy, immunoelectron microscopy, and biochemical fractionation, we demonstrate the presence of NF proteins intra-lumenally within autophagosomes (APs), autolysosomes (ALs), and lysosomes (LYs). Our findings establish a prominent role for autophagy in NF proteolysis. Autophagy may regulate axon cytoskeleton size and responses of the NF cytoskeleton to injury and disease.

Preclinical and randomized clinical evaluation of the p38α kinase inhibitor neflamapimod for basal forebrain cholinergic degeneration.

The endosome-associated GTPase Rab5 is a central player in the molecular mechanisms leading to degeneration of basal forebrain cholinergic neurons (BFCN), a long-standing target for drug development. As p38α is a Rab5 activator, we hypothesized that inhibition of this kinase holds potential as an approach to treat diseases associated with BFCN loss. Herein, we report that neflamapimod (oral small molecule p38α inhibitor) reduces Rab5 activity, reverses endosomal pathology, and restores the numbers and morphology of BFCNs in a mouse model that develops BFCN degeneration. We also report on the results of an exploratory (hypothesis-generating) phase 2a randomized double-blind 16-week placebo-controlled clinical trial (Clinical trial registration: NCT04001517/EudraCT #2019-001566-15) of neflamapimod in mild-to-moderate dementia with Lewy bodies (DLB), a disease in which BFCN degeneration is an important driver of disease expression. A total of 91 participants, all receiving background cholinesterase inhibitor therapy, were randomized 1:1 between neflamapimod 40 mg or matching placebo capsules (taken orally twice-daily if weight <80 kg or thrice-daily if weight >80 kg). Neflamapimod does not show an effect in the clinical study on the primary endpoint, a cognitive-test battery. On two secondary endpoints, a measure of functional mobility and a dementia rating-scale, improvements were seen that are consistent with an effect on BFCN function. Neflamapimod treatment is well-tolerated with no study drug associated treatment discontinuations. The combined preclinical and clinical observations inform on the validity of the Rab5-based pathogenic model of cholinergic degeneration and provide a foundation for confirmatory (hypothesis-testing) clinical evaluation of neflamapimod in DLB.

The three-dimensional landscape of cortical chromatin accessibility in Alzheimer's disease.

To characterize the dysregulation of chromatin accessibility in Alzheimer's disease (AD), we generated 636 ATAC-seq libraries from neuronal and nonneuronal nuclei isolated from the superior temporal gyrus and entorhinal cortex of 153 AD cases and 56 controls. By analyzing a total of ~20 billion read pairs, we expanded the repertoire of known open chromatin regions (OCRs) in the human brain and identified cell-type-specific enhancer-promoter interactions. We show that interindividual variability in OCRs can be leveraged to identify cis-regulatory domains (CRDs) that capture the three-dimensional structure of the genome (3D genome). We identified AD-associated effects on chromatin accessibility, the 3D genome and transcription factor (TF) regulatory networks. For one of the most AD-perturbed TFs, USF2, we validated its regulatory effect on lysosomal genes. Overall, we applied a systematic approach to understanding the role of the 3D genome in AD. We provide all data as an online resource for widespread community-based analysis.

Autolysosomal acidification failure as a primary driver of Alzheimer disease pathogenesis.

Genetic evidence has increasingly linked lysosome dysfunction to an impaired autophagy-lysosomal pathway (ALP) flux in Alzheimer disease (AD) although the relationship of these abnormalities to other pathologies is unclear. In our recent investigation on the origin of impaired autophagic flux in AD, we established the critical early role of defective lysosomes in five mouse AD models. To assess in vivo alterations of autophagy and ALP vesicle acidification, we expressed eGFP-mRFP-LC3 specifically in neurons. We discovered that autophagy dysfunction in these models arises from exceptionally early failure of autolysosome/lysosome acidification, which then drives downstream AD pathogenesis. Extreme autophagic stress in compromised but still intact neurons causes autophagic vacuoles (AVs) containing toxic APP metabolites, Aß/ß-CTFs, to pack into huge blebs and protrude from the perikaryon membrane. Most notably, AVs also coalesce with ER tubules and yield fibrillar ß-amyloid within these tubules. Collectively, amyloid immunoreactivity within these intact neurons assumes the appearance of amyloid-plaques, and indeed, their eventual death transforms them into extracellular plaque lesions. Quantitative analysis confirms that neurons undergoing this transformation are the principal source of ß-amyloid-plaques in APP-AD models. These findings prompt reconsideration of the conventionally accepted sequence of events in plaque formation and may help explain the inefficacy of Aß/amyloid vaccine therapies.

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.

A gene toolbox for monitoring autophagy transcription.

Autophagy is a highly dynamic and multi-step process, regulated by many functional protein units. Here, we have built up a comprehensive and up-to-date annotated gene list for the autophagy pathway, by combining previously published gene lists and the most recent publications in the field. We identified 604 genes and created main categories: MTOR and upstream pathways, autophagy core, autophagy transcription factors, mitophagy, docking and fusion, lysosome and lysosome-related genes. We then classified such genes in sub-groups, based on their functions or on their sub-cellular localization. Moreover, we have curated two shorter sub-lists to predict the extent of autophagy activation and/or lysosomal biogenesis; we next validated the "induction list" by Real-time PCR in cell lines during fasting or MTOR inhibition, identifying ATG14, ATG7, NBR1, ULK1, ULK2, and WDR45, as minimal transcriptional targets. We also demonstrated that our list of autophagy genes can be particularly useful during an effective RNA-sequencing analysis. Thus, we propose our lists as a useful toolbox for performing an informative and functionally-prognostic gene scan of autophagy steps.

Neurofilament Proteins as Biomarkers to Monitor Neurological Diseases and the Efficacy of Therapies.

Biomarkers of neurodegeneration and neuronal injury have the potential to improve diagnostic accuracy, disease monitoring, prognosis, and measure treatment efficacy. Neurofilament proteins (NfPs) are well suited as biomarkers in these contexts because they are major neuron-specific components that maintain structural integrity and are sensitive to neurodegeneration and neuronal injury across a wide range of neurologic diseases. Low levels of NfPs are constantly released from neurons into the extracellular space and ultimately reach the cerebrospinal fluid (CSF) and blood under physiological conditions throughout normal brain development, maturation, and aging. NfP levels in CSF and blood rise above normal in response to neuronal injury and neurodegeneration independently of cause. NfPs in CSF measured by lumbar puncture are about 40-fold more concentrated than in blood in healthy individuals. New ultra-sensitive methods now allow minimally invasive measurement of these low levels of NfPs in serum or plasma to track disease onset and progression in neurological disorders or nervous system injury and assess responses to therapeutic interventions. Any of the five Nf subunits - neurofilament light chain (NfL), neurofilament medium chain (NfM), neurofilament heavy chain (NfH), alpha-internexin (INA) and peripherin (PRPH) may be altered in a given neuropathological condition. In familial and sporadic Alzheimer's disease (AD), plasma NfL levels may rise as early as 22 years before clinical onset in familial AD and 10 years before sporadic AD. The major determinants of elevated levels of NfPs and degradation fragments in CSF and blood are the magnitude of damaged or degenerating axons of fiber tracks, the affected axon caliber sizes and the rate of release of NfP and fragments at different stages of a given neurological disease or condition directly or indirectly affecting central nervous system (CNS) and/or peripheral nervous system (PNS). NfPs are rapidly emerging as transformative blood biomarkers in neurology providing novel insights into a wide range of neurological diseases and advancing clinical trials. Here we summarize the current understanding of intracellular NfP physiology, pathophysiology and extracellular kinetics of NfPs in biofluids and review the value and limitations of NfPs and degradation fragments as biomarkers of neurodegeneration and neuronal injury.

Assessing Rab5 Activation in Health and Disease.

The endocytic pathway is a system of dynamically communicating vesicles, known as early endosomes, that internalize, sort, and traffic nutrients, trophic factors, and signaling molecules to sites throughout the cell. In all eukaryotic cells, early endosome functions are regulated by Rab5 activity, dependent upon its binding to GTP, whereas Rab5 bound to GDP represents the biologically inactive form. An increasing number of neurodegenerative diseases are associated with endocytic dysfunction and, in the case of Alzheimer's disease (AD) and Down syndrome (DS), an early appearing highly characteristic reflection of endocytic pathway dysfunction is an abnormal enlargement of Rab5 positive endosomes. In AD and DS, endosome enlargement accompanying accelerated endocytosis and fusion, upregulated transcription of endocytosis-related genes, and aberrant signaling by endosomes are caused by pathological Rab5 overactivation. In this chapter, we describe a battery of methods that have been used to assess Rab5 activation in models of AD/DS and are applicable to other cell and animal disease models. These methods include (1) fluorescence recovery after photobleaching (FRAP) assay; (2) quantitative measurement of endosome size by light, fluorescence and electron microscopy; (3) detection of GTP-Rab5 by in situ immunocytochemistry in vitro and ex vivo; (4) immunoprecipitation and GTP-agarose pull-down assay; (5) biochemical detection of Rab5 in endosome-enriched subcellular fractions obtained by OptiPrep™ density gradient centrifugation of mouse brain.