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-β 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-β 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-β peptide, ubiquitinated proteins and other autophagic substrates within autolysosomes/lysosomes and reduced intraneuronal amyloid-β peptide. The amelioration of lysosomal function in TgCRND8 markedly decreased extracellular amyloid deposition and total brain amyloid-β 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.
autophagy; lysosome; cystatin B; cathepsin; Alzheimer’s disease
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 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 upon fusion with bi-directionally 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, APP, and neurofilament protein hyperphosphorylation. Restoration of lysosomal proteolysis reversed the affected movements of proteolytic Rab7 vesicles, which in turn, largely 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.
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.
A recently proposed therapeutic approach for lysosomal storage disorders (LSDs) relies upon the ability of transcription factor EB (TFEB) to stimulate autophagy and induce lysosomal exocytosis leading to cellular clearance. This approach is particularly attractive in glycogen storage disease type II [a severe metabolic myopathy, Pompe disease (PD)] as the currently available therapy, replacement of the missing enzyme acid alpha-glucosidase, fails to reverse skeletal muscle pathology. PD, a paradigm for LSDs, is characterized by both lysosomal abnormality and dysfunctional autophagy. Here, we show that TFEB is a viable therapeutic target in PD: overexpression of TFEB in a new muscle cell culture system and in mouse models of the disease reduced glycogen load and lysosomal size, improved autophagosome processing, and alleviated excessive accumulation of autophagic vacuoles. Unexpectedly, the exocytosed vesicles were labelled with lysosomal and autophagosomal membrane markers, suggesting that TFEB induces exocytosis of autophagolysosomes. Furthermore, the effects of TFEB were almost abrogated in the setting of genetically suppressed autophagy, supporting the role of autophagy in TFEB-mediated cellular clearance.
acid alpha-glucosidase; autophagy; lysosomal storage; Pompe disease; TFEB
The role of autophagy, a catabolic lysosome-dependent pathway, has recently been recognized in a variety of disorders, including Pompe disease, the genetic deficiency of the glycogen-degrading lysosomal enzyme acid-alpha glucosidase. Accumulation of lysosomal glycogen, presumably transported from the cytoplasm by the autophagic pathway, occurs in multiple tissues, but pathology is most severe in skeletal and cardiac muscle. Skeletal muscle pathology also involves massive autophagic buildup in the core of myofibers. To determine if glycogen reaches the lysosome via autophagy and to ascertain whether autophagic buildup in Pompe disease is a consequence of induction of autophagy and/or reduced turnover due to defective fusion with lysosomes, we generated muscle-specific autophagy-deficient Pompe mice. We have demonstrated that autophagy is not required for glycogen transport to lysosomes in skeletal muscle. We have also found that Pompe disease involves induction of autophagy but manifests as a functional deficiency of autophagy because of impaired autophagosomal–lysosomal fusion. As a result, autophagic substrates, including potentially toxic aggregate-prone ubiquitinated proteins, accumulate in Pompe myofibers and may cause profound muscle damage.
Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ1–42. Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APPSwInd and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβx-42 sandwich ELISA measures in APPSwInd mice of 10–11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ1–38 occurs as Aβ1–42 levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ1–42 accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders.
Abnormal presence of autophagic vacuoles is evident in brains of patients with Parkinson's disease (PD), in contrast to the rare detection of autophagosomes in a normal brain. However, the actual cause and pathological significance of these observations remain unknown. Here, we demonstrate a role for mitochondrial metabolism in the regulation of the autophagy-lysosomal pathway in ex vivo and in vitro models of PD. We show that transferring mitochondria from PD patients into cells previously depleted of mitochondrial DNA is sufficient to reproduce the alterations in the autophagic system observed in PD patient brains. Although the initial steps of this pathway are not compromised, there is an increased accumulation of autophagosomes associated with a defective autophagic activity. We prove that this functional decline was originated from a deficient mobilization of autophagosomes from their site of formation toward lysosomes due to disruption in microtubule-dependent trafficking. This contributed directly to a decreased proteolytic flux of α-synuclein and other autophagic substrates. Our results lend strong support for a direct impact of mitochondria in autophagy as defective autophagic clearance ability secondary to impaired microtubule trafficking is driven by dysfunctional mitochondria. We uncover mitochondria and mitochondria-dependent intracellular traffic as main players in the regulation of autophagy in PD.
The pathogenic mechanisms underlying acute pancreatitis are not clear. Two key pathologic acinar cell responses of this disease are vacuole accumulation and trypsinogen activation. We show here that both result from defective autophagy, by comparing the autophagic responses in rodent models of acute pancreatitis to physiologic autophagy triggered by fasting. Pancreatitis-induced vacuoles in acinar cells were greater in number and much larger than those induced with fasting. Degradation of long-lived proteins, a measure of autophagic efficiency, was markedly inhibited in in vitro pancreatitis, while it was stimulated by acinar cell starvation. Further, processing of the lysosomal proteases cathepsin L (CatL) and CatB into their fully active, mature forms was reduced in pancreatitis, as were their activities in the lysosome-enriched subcellular fraction. These findings indicate that autophagy is retarded in pancreatitis due to deficient lysosomal degradation caused by impaired cathepsin processing. Trypsinogen activation occurred in pancreatitis but not with fasting and was prevented by inhibiting autophagy. A marker of trypsinogen activation partially localized to autophagic vacuoles, and pharmacologic inhibition of CatL increased the amount of active trypsin in acinar cells. The results suggest that retarded autophagy is associated with an imbalance between CatL, which degrades trypsinogen and trypsin, and CatB, which converts trypsinogen into trypsin, resulting in intra-acinar accumulation of active trypsin in pancreatitis. Thus, deficient lysosomal degradation may be a dominant mechanism for increased intra-acinar trypsin in pancreatitis.
Macroautophagy, a major pathway for organelle and protein turnover, has been implicated in the neurodegeneration of Alzheimer's disease (AD). The basis for the profuse accumulation of autophagic vacuoles (AVs) in affected neurons of the AD brain, however, is unknown. In this study, we show that constitutive macroautophagy in primary cortical neurons is highly efficient, as newly formed autophagosomes are rapidly cleared by fusion with lysosomes, accounting for their scarcity in the healthy brain. Even after macroautophagy is strongly induced by suppressing mTOR kinase activity with rapamycin or nutrient deprivation, active cathepsin-positive autolysosomes rather than LC3-II-positive autophagosomes predominate, implying efficient autophagosome clearance in healthy neurons. By contrast, selectively impeding late steps in macroautophagy by inhibiting cathepsin-mediated proteolysis within autophagosomes with cysteine- and aspartyl-protease inhibitors caused a marked accumulation of electron-dense double membrane-limited AVs, containing cathepsin D and incompletely degraded LC3-II in perikarya and neurites. Similar structures accumulated in large numbers when fusion of autophagosomes with lysosomes was slowed by disrupting their transport on microtubules with vinblastine. Finally, we find that the autophagic vacuoles accumulating after protease inhibition or prolonged vinblastine treatment strongly resembled AVs that collect in dystrophic neurites in the AD brain and in an AD mouse model. We conclude that macroautophagy is constitutively active and highly efficient in healthy neurons, and that the autophagic pathology observed in AD most likely arises from impaired clearance of AVs rather than strong autophagy induction alone. Therapeutic modulation of autophagy in AD may, therefore, require targeting late steps in the autophagic pathway.
Aging [Ageing]; Alzheimer; Autophagy; Culture; Lysosome; Neuron; Neuron Death; neuronal death; Neuropathology; neuroprotection; Neurotoxicity; Storage
Abnormally swollen regions of axons and dendrites (neurites) filled mainly with autophagy-related organelles represent the highly characteristic and widespread form of “neuritic dystrophy” in Alzheimer disease (AD), which implies dysfunction of autophagy and axonal transport. In this punctum, we discuss our recent findings that autophagic/lysosomal degradation is critical to proper axonal transport of autophagic vacuoles (AVs) and lysosomes. We showed that lysosomal protease inhibition induces defective axonal transport of specific cargoes, causing these cargoes to accumulate in axonal swellings that biochemically and morphologically resemble the dystrophic neurites in AD. Our findings suggest that a cargo-specific failure of axonal transport promotes neuritic dystrophy in AD, which involves a mechanism distinct from the global axonal transport deficits seen in some other neurodegenerative diseases.
Alzheimer disease; autophagy; axonal transport; dystrophic neurites; lysosomes; proteolysis
The mechanism of widespread neuronal death occurring in Alzheimer's disease (AD) remains enigmatic even after extensive investigation during the last two decades. Amyloid beta 42 peptide (Aβ1–42) is believed to play a causative role in the development of AD. Here we expressed human Aβ1–42 and amyloid beta 40 (Aβ1–40) in Drosophila neurons. Aβ1–42 but not Aβ1–40 causes an extensive accumulation of autophagic vesicles that become increasingly dysfunctional with age. Aβ1–42-induced impairment of the degradative function, as well as the structural integrity, of post-lysosomal autophagic vesicles triggers a neurodegenerative cascade that can be enhanced by autophagy activation or partially rescued by autophagy inhibition. Compromise and leakage from post-lysosomal vesicles result in cytosolic acidification, additional damage to membranes and organelles, and erosive destruction of cytoplasm leading to eventual neuron death. Neuronal autophagy initially appears to play a pro-survival role that changes in an age-dependent way to a pro-death role in the context of Aβ1–42 expression. Our in vivo observations provide a mechanistic understanding for the differential neurotoxicity of Aβ1–42 and Aβ1–40, and reveal an Aβ1–42-induced death execution pathway mediated by an age-dependent autophagic-lysosomal injury.
The autophagy pathway is the major degradation pathway of the cell for long-lived proteins and organelles. Dysfunction of autophagy has been linked to several neurodegenerative disorders that are associated with an accumulation of misfolded protein aggregates. Alzheimer’s disease, the most common neurodegenerative disorder, is characterized by 2 aggregate forms, tau tangles and amyloid-β plaques. Autophagy has been linked to Alzheimer’s disease pathogenesis through its merger with the endosomal-lysosomal system, which has been shown to play a role in the formation of the latter amyloid-β plaques. However, the precise role of autophagy in Alzheimer’s disease pathogenesis is still under contention. One hypothesis is that aberrant autophagy induction results in an accumulation of autophagic vacuoles containing amyloid-β and the components necessary for its generation, whereas other evidence points to impaired autophagic clearance or even an overall reduction in autophagic activity playing a role in Alzheimer’s disease pathogenesis. In this review, we discuss the current evidence linking autophagy to Alzheimer’s disease as well as the uncertainty over the exact role and level of autophagic regulation in the pathogenic mechanism of Alzheimer’s disease.
Alzheimer’s disease; amyloidogenesis; autophagy; dysfunction; endosomal-lysosomal pathway
In Pompe disease, a lysosomal glycogen storage disorder, cardiac and skeletal muscle abnormalities are responsible for premature death and severe weakness. Swollen glycogen-filled lysosomes, the expected pathology, are accompanied in skeletal muscle by a secondary pathology – massive accumulation of autophagic debris – that appears to contribute greatly to the weakness. We have tried to reproduce these defects in murine, Pompe myotubes derived from either primary myoblasts or myoblasts with extended proliferative capacity. The cells accumulated large lysosomes filled with glycogen, but, to our disappointment, did not have autophagic buildup even though basal autophagy was intact. When we suppressed autophagy by knocking down Atg7, we found that glycogen uptake by lysosomes was not affected, suggesting that macroautophagy is not the major pathway for glycogen delivery to lysosomes. But two apparently incidental observations – a peculiar distribution of both microinjected dextran and of small acidic structures adjacent to the interior membrane of large alkalinized glycogen-containing lysosomes – raised the possibility that glycogen traffics to the lysosomes by microautophagy or/and by the engulfment of small lysosomes by large ones. The cultured myotubes, therefore, appear to be a useful model for studying the mechanisms involved in glycogen accumulation in Pompe disease and to test substrate deprivation approaches.
lysosomal storage; glycogen; Pompe disease; myotubes; Atg7
The role of autophagy, a catabolic lysosome-dependent pathway, has recently been recognized in a variety of disorders, including Pompe disease, which results from a deficiency of the glycogen-degrading lysosomal hydrolase acid-alpha glucosidase (GAA). Skeletal and cardiac muscle are most severely affected by the progressive expansion of glycogen-filled lysosomes. In both humans and an animal model of the disease (GAA KO), skeletal muscle pathology also involves massive accumulation of autophagic vesicles and autophagic buildup in the core of myofibers, suggesting an induction of autophagy. Only when we suppressed autophagy in the skeletal muscle of the GAA KO mice did we realize that the excess of autophagy manifests as a functional deficiency. This failure of productive autophagy is responsible for the accumulation of potentially toxic aggregate-prone ubiquitinated proteins, which likely cause profound muscle damage in Pompe mice. Also, by generating muscle-specific autophagy-deficient wild-type mice, we were able to analyze the role of autophagy in healthy skeletal muscle.
Pompe disease; lysosome; muscle-specific autophagy deficiency; protein inclusions
Alterations in the metabolism of amyloid precursor protein (APP) are believed to
play a central role in Alzheimer disease pathogenesis. Burgeoning data indicate
that APP is proteolytically processed in endosomal-autophagic-lysosomal
compartments. In this study, we used both in vivo and
in vitro paradigms to determine whether alterations in
macroautophagy affect APP metabolism. Three mouse models of glycosphingolipid
storage diseases, namely Niemann-Pick type C1, GM1 gangliosidosis, and Sandhoff
disease, had mTOR-independent increases in the autophagic vacuole
(AV)-associated protein, LC3-II, indicative of impaired lysosomal flux. APP
C-terminal fragments (APP-CTFs) were also increased in brains of the three mouse
models; however, discrepancies between LC3-II and APP-CTFs were seen between
primary (GM1 gangliosidosis and Sandhoff disease) and secondary (Niemann-Pick
type C1) lysosomal storage models. APP-CTFs were proportionately higher than
LC3-II in cerebellar regions of GM1 gangliosidosis and Sandhoff disease,
although LC3-II increased before APP-CTFs in brains of NPC1 mice. Endogenous
murine Aβ40 from RIPA-soluble extracts was increased in brains of all
three mice. The in vivo relationship between AV and APP-CTF
accumulation was also seen in cultured neurons treated with agents that impair
primary (chloroquine and leupeptin + pepstatin) and secondary (U18666A
and vinblastine) lysosomal flux. However, Aβ secretion was unaffected by
agents that induced autophagy (rapamycin) or impaired AV clearance, and
LC3-II-positive AVs predominantly co-localized with degradative LAMP-1-positive
lysosomes. These data suggest that neuronal macroautophagy does not directly
regulate APP metabolism but highlights the important anti-amyloidogenic role of
lysosomal proteolysis in post-secretase APP-CTF catabolism.
Alzheimer Disease; Amyloid; Autophagy; Endocytosis; Lysosomal Storage Disease; Lysosomes; Neurobiology; Neuron; Secretases; Storage Diseases
Alterations in macroautophagy (hereafter referred to as “autophagy”) are a common feature of lysosomal storage disorders, and have been hypothesized to play a major role in the pathogenesis of these diseases. We have recently reported multiple defects in autophagy contributing to the lysosomal storage disorder Niemann-Pick type C (NPC). These include increased formation of autophagosomes, slowed turnover of autophagosomes secondary to impaired lysosomal proteolysis, and delivery of stored lipids to the lysosome via autophagy. The study summarized here describes novel methods for the interrogation of individual stages of the autophagic pathway, and suggests mechanisms by which lipid storage may result in broader lysosomal dysfunction.
Niemann-Pick; autophagy; lysosome; lipid storage; cholesterol; cathepsin; neurodegeneration
Autophagy mediates lysosomal degradation of cytosolic components. Recent work has associated autophagic dysfunction with pathologies, including cancer and cardiovascular disease. To date, the identification of clinically-applicable drugs that modulate autophagy has been hampered by the lack of standardized assays capable of precisely reporting autophagic activity.
We developed and implemented a high-content, flow-cytometry-based screening approach for rapid, precise, and quantitative measurements of pharmaceutical control over autophagy. Our assay allowed for time-resolved individual measurements of autolysosome formation and degradation, and endolysosomal activities under both basal and activated autophagy conditions. As proof of concept, we analyzed conventional autophagy regulators, including cardioprotective compounds aminoimidazole carboxamide ribonucleotide (AICAR), rapamycin, and resveratrol, and revealed striking conditional dependencies of rapamycin and autophagy inhibitor 3-methyladenine (3-MA). To identify novel autophagy modulators with translational potential, we screened the Prestwick Chemical Library of 1,120 US Food and Drug Administration (FDA)-approved compounds for impact on autolysosome formation. In all, 38 compounds were identified as potential activators, and 36 as potential inhibitors of autophagy. Notably, amongst the autophagy enhancers were cardiac glycosides, from which we selected digoxin, strophanthidin, and digoxigenin for validation by standard biochemical and imaging techniques. We report the induction of autophagic flux by these cardiac glycosides, and the concentrations allowing for specific enhancement of autophagic activities without impact on endolysosomal activities.
Our systematic analysis of autophagic and endolysosomal activities outperformed conventional autophagy assays and highlights the complexity of drug influence on autophagy. We demonstrate conditional dependencies of established regulators. Moreover, we identified new autophagy regulators and characterized cardiac glycosides as novel potent inducers of autophagic flux.
Mutations in the glucocerebrosidase (gba) gene cause Gaucher disease (GD), the most common lysosomal storage disorder, and increase susceptibility to Parkinson’s disease (PD). While the clinical and pathological features of idiopathic PD and PD related to gba (PD-GBA) mutations are very similar, cellular mechanisms underlying neurodegeneration in each are unclear. Using a mouse model of neuronopathic GD, we show that autophagic machinery and proteasomal machinery are defective in neurons and astrocytes lacking gba. Markers of neurodegeneration—p62/SQSTM1, ubiquitinated proteins, and insoluble α-synuclein—accumulate. Mitochondria were dysfunctional and fragmented, with impaired respiration, reduced respiratory chain complex activities, and a decreased potential maintained by reversal of the ATP synthase. Thus a primary lysosomal defect causes accumulation of dysfunctional mitochondria as a result of impaired autophagy and dysfunctional proteasomal pathways. These data provide conclusive evidence for mitochondrial dysfunction in GD and provide insight into the pathogenesis of PD and PD-GBA.
•Autophagic and proteasomal pathways are suppressed in gba knockout mice•α-Synuclein accumulates and forms deposits in gba knockout mouse brainstem•Neurons and astrocytes from gba knockout mice harbor dysfunctional mitochondria•Mitochondria do not recruit Parkin and accumulate in gba knockout neurons
The accumulation of misfolded proteins in insoluble aggregates within the neuronal cytoplasm is one of the common pathological hallmarks of most adult-onset human neurodegenerative diseases. The clearance of these misfolded proteins may represent a promising therapeutic strategy in these diseases. The two main routes for intracellular protein degradation are the ubiquitin–proteasome and the autophagy–lysosome pathways. In this review, we will focus on the autophagic pathway, by providing some examples of how impairment at different steps in this degradation pathway is related to different neurodegenerative diseases. We will also consider that upregulating autophagy may be useful in the treatment of some of these diseases. Finally, we discuss how antioxidants, which have been considered to be beneficial in neurodegenerative diseases, can block autophagy, thus potentially compromising their therapeutic potential.
►Autophagy compromise occurs in different neurodegenerative diseases. ►Upregulating autophagy may be useful in the treatment of some neurodegenerative diseases. ►Many different reactive oxygen species scavengers impair autophagy
Autophagy; Neurodegeneration; Huntington's disease
Autophagy is an evolutionarily conserved catabolic pathway of lysosome-dependent turnover of damaged proteins and organelles. When nutrients are in short supply, bulk removal of cytoplasmic components by autophagy replenishes depleted energy stores, a process critical for maintaining cellular homeostasis. However, prolonged activation of autophagic pathways can result in cell death. Longstanding evidence has linked the stimulation of lysosomal pathways to pathologic cardiac remodeling and a number of cardiac diseases, including heart failure and ischemia. Only recently, however, has work begun to parse cyto-protective autophagy from autophagy that contributes to disease pathogenesis. Current thinking suggests that the effects of autophagy exist on a continuum, with the eliciting triggers, the duration and amplitude of autophagic flux, and possibly the targeted intra cellular cargo as critical determinants of the end result. Deciphering how autophagy participates in basal homeostasis of the heart, in aging, and in disease pathogenesis may uncover novel insights with clinical relevance in the treatment of heart disease.
Although traditionally regarded as a cellular adaptive process triggered by nutrient deprivation, autophagy in neurons appears to provide an important neuroprotective mechanism. Neurons in the brain are protected from starvation, and neuronal autophagy serves a critical role in the turnover of abnormal proteins and damaged organelles. As post-mitotic, highly polarized cells with active protein trafficking, neurons rely heavily on an efficient autophagic pathway. Using human embryonic stem cell-derived neurons engineered to mimic the cholesterol lysosomal storage disease Niemann Pick type C1 (NPC1), we have shown that excessive activation and impaired progression of the autophagic pathway conspire to cause abnormal mitochondrial clearance. Defective mitophagy is exceptionally severe in human NPC1 neurons, as compared with patient fibroblasts, and may explain the selective neuronal failure observed in NPC1 and related neurodegenerative disorders.
human stem cells; cholesterol; neurodegeneration; lysosomal storage disease; mitophagy
Overall autophagy was measured in isolated hepatocytes as the sequestration and lysosomal hydrolysis of electroinjected [14C]lactose, using HPLC to separate the degradation product [14C]glucose from undegraded lactose. In addition, the sequestration step was measured separately as the transfer from cytosol to sedimentable cell structures of electroinjected [3H]raffinose or endogenous lactate dehydrogenase (LDH; in the presence of leupeptin to inhibit lysosomal proteolysis). Inhibitor effects at postsequestrational steps could be detected as the accumulation of autophaged lactose (which otherwise is degraded intralysosomally), or of LDH in the absence of leupeptin. Asparagine, previously shown to inhibit autophagic but not endocytic protein breakdown, strongly suppressed the autophagic hydrolysis of electroinjected lactose. Vinblastine, which inhibits both types of degradation, likewise suppressed lactose hydrolysis. Asparagine had little or no effect on sequestration, but caused an accumulation of autophaged LDH and lactose, indicating inhibition at a postsequestrational step. Neither asparagine nor vinblastine affected the degradation of intralysosomal lactose preaccumulated in the presence of the reversible lysosome inhibitor propylamine. However, if lactose was preaccumulated in the presence of asparagine, both asparagine and vinblastine suppressed its subsequent degradation. The data thus indicate that autophagic-lysosomal delivery, i.e., the transfer of autophaged material from prelysosomal vacuoles to lysosomes, is inhibited selectively by asparagine and non-selectively by vinblastine.
Saposin C deficiency, a rare variant form of Gaucher disease, is due to mutations in the prosaposin gene (PSAP) affecting saposin C expression and/or function. We previously reported that saposin C mutations affecting one cysteine residue result in autophagy dysfunction. We further demonstrated that the accumulation of autophagosomes, observed in saposin C-deficient fibroblasts, is due to an impairment of autolysosome degradation, partially caused by the reduced amount and enzymatic activity of CTSB (cathepsin B) and CTSD (cathepsin D). The restoration of both proteases in pathological fibroblasts results in almost completely recovery of autophagic flux and lysosome homeostasis.
Gaucher disease; saposin C deficiency; glucosylceramidase deficiency; lysosomal storage disorders; glucosylceramide; autophagy; cathepsins; overexpression; autophagic lysosome reformation
Lysosomes are the final destination of the autophagic pathway. It is in the acidic milieu of the lysosomes that autophagic cargo is metabolized and recycled. One would expect that diseases with primary lysosomal defects would be among the first systems in which autophagy would be studied. In reality, this is not the case. Lysosomal storage diseases, a group of more than 60 diverse inherited disorders, have only recently become a focus of autophagic research. Studies of these clinically severe conditions promise not only to clarify pathogenic mechanisms, but also to expand our knowledge of autophagy itself. In this chapter, we will describe the lysosomal storage diseases in which autophagy has been explored, and present the approaches used to evaluate this essential cellular pathway.
Pompe disease is caused by an inherited deficiency of acid α-glucosidase (GAA), a lysosomal enzyme that catalyzes the breakdown of glycogen to glucose. In the absence of GAA, enlarged, glycogen-laden lysosomes accumulate in multiple tissues, although the major clinical manifestations are seen in cardiac and skeletal muscle. For many years, it was believed that the rupture of glycogen-filled lysosomes was the major cause of the profound muscle damage observed in patients with Pompe disease. Here, we present evidence that a failure of productive autophagy in muscle tissue contributes strongly to disease pathology in both patients with Pompe disease and GAA-knockout mice. In the GAA-knockout mouse model, progressive accumulation of autophagic vesicles is restricted to Type II-rich muscle fibers. Not only does this build-up of autophagosomes disrupt the contractile apparatus in the muscle fibers, it also interferes with enzyme replacement therapy by acting as a sink for the recombinant enzyme and preventing its efficient delivery to the lysosomes. Our data indicate that a re-examination of the presumed pathological mechanism in Pompe disease is necessary, and suggest that successful treatment of patients with Pompe disease will require consideration of the dramatic failure of autophagy that occurs in this disease.
autophagy; enzyme replacement therapy; glycogen; Pompe disease; skeletal muscle