Lysosomal dysfunction in Parkinson disease

Mutations in ATP13A2 (PARK9) cause an autosomal recessive form of early-onset parkinsonism with pyramidal degeneration and dementia called Kufor-Rakeb Syndrome (KRS). The ATP13A2 gene encodes a transmembrane lysosomal P5-type ATPase (ATP13A2) whose physiological function in mammalian cells, and hence its potential role in Parkinson disease (PD), remains elusive. In this context, we have recently shown that KRS-linked mutations in ATP13A2 leads to several lysosomal alterations in ATP13A2 KRS patient-derived fibroblasts, including impaired lysosomal acidification, decreased proteolytic processing of lysosomal enzymes, reduced degradation of lysosomal substrates and diminished lysosomal-mediated clearance of autophagosomes (AP). Similar alterations are observed in stable ATP13A2-knockdown dopaminergic cell lines, which are associated with cell death. Restoration of ATP13A2 levels in ATP13A2-mutant/depleted cells is able to restore lysosomal function and attenuate cell death. Relevant to PD, we have determined that ATP13A2 levels are decreased in dopaminergic nigral neurons from sporadic PD patients. Interestingly in these patients, the main signal of ATP13A2 is detected in the Lewy bodies. Our results unravel an instrumental role of ATP13A2 in lysosomal function and in cell viability. Altogether, our results validate ATP13A2 as a likely therapeutic target against PD degeneration.     

Lysosomal dysfunction in neurodegeneration: The role of ATP13A2/PARK9

Neuronal homeostasis and survival critically depend on an efficient autophagy-lysosomal degradation pathway, especially since neurons cannot reduce the concentration of misfolded proteins and damaged organelles by cell division. While increasing evidence implicates lysosomal dysfunction in the pathogenesis of neurodegenerative disorders, the molecular underpinnings of the role of lysosomes in neurodegeneration remain largely unknown. To this end, studies of neurodegenerative disorders caused by mutations in lysosomal proteins offer an opportunity to elucidate such mechanisms and potentially identify specific therapeutic targets. One of these disorders is Kufor-Rakeb syndrome, caused by mutations in the lysosomal protein ATP13A2/PARK9 and characterized by early-onset Parkinsonism, pyramidal degeneration and dementia. We found that loss of ATP13A2 function results in impaired lysosomal function and, consequently, accumulation of SNCA/α-synuclein and neurotoxicity. Our results suggest that targeting of ATP13A2 to lysosomes to enhance lysosomal function may result in neuroprotection in Kufor-Rakeb syndrome. From a broader perspective, these findings, together with other recent studies of lysosomal dysfunction in neurodegeneration, suggest that strategies to upregulate lysosomal function in neurons represent a promising therapeutic approach for neurodegenerative disorders.     

Nanoparticles restore lysosomal acidification defects: Implications for Parkinson and other lysosomal-related diseases

Lysosomal impairment causes lysosomal storage disorders (LSD) and is involved in pathogenesis of neurodegenerative diseases, notably Parkinson disease (PD). Strategies enhancing or restoring lysosomal-mediated degradation thus appear as tantalizing disease-modifying therapeutics. Here we demonstrate that poly(DL-lactide-co-glycolide) (PLGA) acidic nanoparticles (aNP) restore impaired lysosomal function in a series of toxin and genetic cellular models of PD, i.e. ATP13A2-mutant or depleted cells or glucocerebrosidase (GBA)-mutant cells, as well as in a genetic model of lysosomal-related myopathy. We show that PLGA-aNP are transported to the lysosome within 24 h, lower lysosomal pH and rescue chloroquine (CQ)-induced toxicity. Re-acidification of defective lysosomes following PLGA-aNP treatment restores lysosomal function in different pathological contexts. Finally, our results show that PLGA-aNP may be detected after intracerebral injection in neurons and attenuate PD-related neurodegeneration in vivo by mechanisms involving a rescue of compromised lysosomes.     

Lysosomal dysfunction in neurodegeneration

Neuronal homeostasis and survival critically depend on an efficient autophagy-lysosomal degradation pathway, especially since neurons cannot reduce the concentration of misfolded proteins and damaged organelles by cell division. While increasing evidence implicates lysosomal dysfunction in the pathogenesis of neurodegenerative disorders, the molecular underpinnings of the role of lysosomes in neurodegeneration remain largely unknown. To this end, studies of neurodegenerative disorders caused by mutations in lysosomal proteins offer an opportunity to elucidate such mechanisms and potentially identify specific therapeutic targets. One of these disorders is Kufor-Rakeb syndrome, caused by mutations in the lysosomal protein ATP13A2/PARK9 and characterized by early-onset Parkinsonism, pyramidal degeneration and dementia. We found that loss of ATP13A2 function results in impaired lysosomal function and, consequently, accumulation of SNCA/α-synuclein and neurotoxicity. Our results suggest that targeting of ATP13A2 to lysosomes to enhance lysosomal function may result in neuroprotection in Kufor-Rakeb syndrome. From a broader perspective, these findings, together with other recent studies of lysosomal dysfunction in neurodegeneration, suggest that strategies to upregulate lysosomal function in neurons represent a promising therapeutic approach for neurodegenerative disorders.     

Genetic perspective on the role of the autophagy-lysosome pathway in Parkinson disease

Parkinson disease (PD), once considered as a prototype of a sporadic disease, is now known to be considerably affected by various genetic factors, which interact with environmental factors and the normal process of aging, leading to PD. Large studies determined that the hereditary component of PD is at least 27%, and in some populations, single genetic factors are responsible for more than 33% of PD patients. Interestingly, many of these genetic factors, such as LRRK2, GBA, SMPD1, SNCA, PARK2, PINK1, PARK7, SCARB2, and others, are involved in the autophagy-lysosome pathway (ALP). Some of these genes encode lysosomal enzymes, whereas others correspond to proteins that are involved in transport to the lysosome, mitophagy, or other autophagic-related functions. Is it possible that all these factors converge into a single pathway that causes PD? In this review, we will discuss these genetic findings and the role of the ALP in the pathogenesis of PD and will try to answer this question. We will suggest a novel hypothesis for the pathogenic mechanism of PD that involves the lysosome and the different autophagy pathways.     

Hormesis: Protecting neurons against cellular stress in Parkinson disease

Protein folding stress is a salient feature of the most frequent neurodegenerative diseases. Although the accumulation of abnormally folded proteins is a well-characterized event underlying the pathology, the way cells respond to this phenomenon is not well understood. Signs of endoplasmic reticulum (ER) stress are a common marker of neurodegeneration in many diseases, which may represent two contrasting processes: cell protection events due to activation of adaptive programs, or a chronic stress state that culminates in apoptosis to eliminate irreversibly injured cells. Autophagy has been proposed as a protective mechanism to overcome neurodegeneration that is also modulated by ER stress. In this issue of autophagy Bertrand Mollereau's group provides novel evidence indicating that engagement of nonharmful levels of ER stress protects against experimental Parkinson disease. At the mechanistic level, a homeostatic crosstalk between ER stress signaling and the autophagy pathway was proposed to mediate the therapeutic effects. This study, together with recent findings, supports the involvement of a "hormesis mechanism" to handle degeneration through preconditioning mediated by a dynamic balance between ER stress and autophagy. The implications for aging and future therapeutic development are discussed.     

Cryo-EM reveals mechanistic insights into lipid-facilitated polyamine export by human ATP13A2

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LAMP2A as a therapeutic target in Parkinson disease

Abnormal aggregation of SNCA/α-synuclein plays a crucial role in Parkinson disease (PD) pathogenesis. SNCA levels determine its toxicity, and its accumulation, even to a small extent, may be a risk factor for neurodegeneration. One of the main pathways for SNCA degradation is chaperone-mediated autophagy (CMA), a selective form of autophagy, while aberrant SNCA may act as a CMA inhibitor. In the current punctum we summarize our recent data showing that induction of CMA, via overexpression of the protein controlling its rate-limiting step, the lysosomal receptor LAMP2A, effectively decreases SNCA levels and ameliorates SNCA-induced neurodegeneration, both in neuronal cell culture systems and in the rat brain. Such findings suggest that modulation of LAMP2A and, consequently, CMA, represents a viable therapeutic target for PD and other synucleinopathies where SNCA accumulation and aggregation plays a fundamental role.     

BAX channel activity mediates lysosomal disruption linked to Parkinson disease

Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP). However, the molecular mechanisms underlying PD-linked LMP and subsequent lysosomal defects remain virtually unknown, thereby precluding their potential therapeutic targeting. Here we show that the pro-apoptotic protein BAX (BCL2-associated X protein), which permeabilizes mitochondrial membranes in PD models and is activated in PD patients, translocates and internalizes into lysosomal membranes early following treatment with the parkinsonian neurotoxin MPTP, both in vitro and in vivo, within a time-frame correlating with LMP, lysosomal disruption, and autophagosome accumulation and preceding mitochondrial permeabilization and dopaminergic neurodegeneration. Supporting a direct permeabilizing effect of BAX on lysosomal membranes, recombinant BAX is able to induce LMP in purified mouse brain lysosomes and the latter can be prevented by pharmacological blockade of BAX channel activity. Furthermore, pharmacological BAX channel inhibition is able to prevent LMP, restore lysosomal levels, reverse AP accumulation, and attenuate mitochondrial permeabilization and overall nigrostriatal degeneration caused by MPTP, both in vitro and in vivo. Overall, our results reveal that PD-linked lysosomal impairment relies on BAX-induced LMP, and point to small molecules able to block BAX channel activity as potentially beneficial to attenuate both lysosomal defects and neurodegeneration occurring in PD.     

Regulation of intracellular manganese homeostasis by Kufor-Rakeb syndrome-associated ATP13A2 protein

Mutations in the ATP13A2 gene are associated with Kufor-Rakeb syndrome (KRS) and are found also in patients with various other types of parkinsonism. ATP13A2 encodes a predicted lysosomal P5-type ATPase that plays important roles in regulating cation homeostasis. Disturbance of cation homeostasis in brains is indicated in Parkinson disease pathogenesis. In this study, we explored the biological function of ATP13A2 as well as the pathogenic mechanism of KRS pathogenic ATP13A2 mutants. The results revealed that wild-type ATP13A2, but not the KRS pathogenic ATP13A2 mutants, protected cells from Mn(2+)-induced cell death in mammalian cell lines and primary rat neuronal cultures. In addition, wild-type ATP13A2 reduced intracellular manganese concentrations and prevented cytochrome c release from mitochondria compared with the pathogenic mutants. Furthermore, endogenous ATP13A2 was up-regulated upon Mn(2+) treatment. Our results suggest that ATP13A2 plays important roles in protecting cells against manganese cytotoxicity via regulating intracellular manganese homeostasis. The study provides a potential mechanism of KRS and parkinsonism pathogenesis.     

Genetic animal models for evaluating the role of autophagy in etiopathogenesis of Parkinson disease

Parkinson disease (PD) is the most common neurodegenerative movement disorder and is characterized pathologically by the formation of ubiquitin and SNCA/α-synuclein-containing inclusions (Lewy bodies), dystrophic midbrain dopaminergic (DAergic) terminals, and degeneration of midbrain DAergic neurons. The vast majority of PD occurs sporadically, while approximately 5% of all PD cases are inherited. Genetic mutations of a few genes have been identified as causes of familiar PD, i.e., mutations in SNCA, PARK2/parkin, UCHL1, PARK7/DJ1, PINK1 and LRRK2, leading to DAergic cell death, but variable pathological changes. The evidence supports the hypothesis that several pathogenic mechanisms are likely involved at initial stages of the disease, and eventually they merge to cause parkinsonism. The current challenge facing PD research is to unravel the components in these pathways that contribute to the pathogenesis of PD. Accumulating evidence has implicated dysfunctional autophagy, a regulated lysosomal pathway with a capacity for clearing protein aggregates and cellular organelles, as one of the pathogenic systems contributing to the development of idiopathic PD.     

Genetic animal models for evaluating the role of autophagy in etiopathogenesis of Parkinson disease

Parkinson disease (PD) is the most common neurodegenerative movement disorder and is characterized pathologically by the formation of ubiquitin and SNCA/α-synuclein-containing inclusions (Lewy bodies), dystrophic midbrain dopaminergic (DAergic) terminals, and degeneration of midbrain DAergic neurons. The vast majority of PD occurs sporadically, while approximately 5% of all PD cases are inherited. Genetic mutations of a few genes have been identified as causes of familiar PD, i.e., mutations in SNCA, PARK2/parkin, UCHL1, PARK7/DJ1, PINK1 and LRRK2, leading to DAergic cell death, but variable pathological changes. The evidence supports the hypothesis that several pathogenic mechanisms are likely involved at initial stages of the disease, and eventually they merge to cause parkinsonism. The current challenge facing PD research is to unravel the components in these pathways that contribute to the pathogenesis of PD. Accumulating evidence has implicated dysfunctional autophagy, a regulated lysosomal pathway with a capacity for clearing protein aggregates and cellular organelles, as one of the pathogenic systems contributing to the development of idiopathic PD.     

Structural mechanisms for gating and ion selectivity of the human polyamine transporter ATP13A2

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Functional impairment in RHOT1/Miro1 degradation and mitophagy is a shared feature in familial and sporadic Parkinson disease

Mitophagy is a conserved and highly regulated process of selective degradation crucial in maintaining normal cellular physiology. Genetic defects and cellular aberrations affecting mitophagy have been associated with the development of Parkinson disease. In their recently published article (highlighted in a punctum in this issue of the journal) Hsieh et al. present a putative mitophagy marker, which serves as a mechanistic link between sporadic and familial Parkinson disease.     

Piperlongumine restores the balance of autophagy and apoptosis by increasing BCL2 phosphorylation in rotenone-induced Parkinson disease models

Parkinson disease (PD) is the second most common neurodegenerative disorder after Alzheimer disease and is caused by genetics, environmental factors and aging, with few treatments currently available. Apoptosis and macroautophagy/autophagy play critical roles in PD pathogenesis; as such, modulating their balance is a potential treatment strategy. BCL2 (B cell leukemia/lymphoma 2) is a key molecule regulating this balance. Piperlongumine (PLG) is an alkaloid extracted from Piper longum L. that has antiinflammatory and anticancer effects. The present study investigated the protective effects of PLG in rotenone-induced PD cell and mouse models. We found that PLG administration (2 and 4 mg/kg) for 4 wk attenuated motor deficits in mice and prevented the loss of dopaminergic neurons in the substantia nigra induced by oral administration of rotenone (10 mg/kg) for 6 wk. PLG improved cell viability and enhanced mitochondrial function in primary neurons and SK-N-SH cells. These protective effects were exerted via inhibition of apoptosis and induction of autophagy through enhancement of BCL2 phosphorylation at Ser70. These results demonstrate that PLG exerts therapeutic effects in a rotenone-induced PD models by restoring the balance between apoptosis and autophagy.

Abbreviations: 6-OHDA, 6-hydroxydopamine; ACTB, actin, beta; BafA1, bafilomycin A₁; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BCL2, B cell leukemia/lymphoma2; BECN1, Beclin 1, autophagy related; CoQ10, coenzyme Q₁₀; COX4I1/COX IV, cytochrome c oxidase subunit 4I1; CsA, cyclosporine A; ED50, 50% effective dose; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; HPLC, high-performance liquid chromatography; JC-1, tetraethylbenz-imidazolylcarbocyanine iodide; LC3, microtubule-associated protein 1 light chain3; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LDH, lactate dehydrogenase; l-dopa, 3, 4-dihydroxyphenyl-l-alanine; MAPK8/JNK1, mitogen-activated protein kinase 8; MMP, mitochondrial membrane potential; mPTP, mitochondrial permeability transition pore; mRFP, monomeric red fluorescent protein; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NFE2L2/NRF2, nuclear factor, erythroid derived 2, like 2; PD, Parkinson disease; PLG, piperlongumine; pNA, p-nitroanilide; PI, propidium iodide; PtdIns3K, phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol-3-phosphate; PTX, paclitaxel; Rap, rapamycin; SQSTM1/p62, sequestosome 1; TH, tyrosine hydroxylase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WIPI2, WD repeat domain, phosphoinositide interacting 2; ZFYVE1/DFCP1, zinc finger, FYVE domain containing 1.     

Cd, Mn, Ni and Se toxicity to Saccharomyces cerevisiae lacking YPK9p the orthologue of human ATP13A2

The Saccharomyces cerevisiae gene YPK9 encodes a putative integral membrane protein which is 58% similar and 38% identical in amino acid sequence to the human lysosomal P_5B ATPase ATP13A2. Mutations in ATP13A2 have been found in patients with Kufor-Rakeb syndrome, a form of juvenile Parkinsonism. We report that Ypk9p localizes to the yeast vacuole and that deletion of YPK9 confers sensitivity for growth for cadmium, manganese, nickel or selenium. These results suggest that Ypk9p may play a role in sequestration of divalent heavy metal ions. Further studies on the function of Ypk9p/ATP13A2 may help to define the molecular basis of Kufor-Rakeb syndrome and provide a potential link to environmental factors such as heavy metals contributing to some forms of Parkinsonism.     

Cholesterol and multilamellar bodies: Lysosomal dysfunction in GBA-Parkinson disease

Lipid and cholesterol metabolism might play a role in the pathogenesis of Parkinson disease (PD). However, the association between cholesterol and PD is not clearly established. Cholesterol accumulation is closely related to the expression of multilamellar bodies (MLBs). Also, cholesterol controls autophagosome transport. Thus, impaired cholesterol and autophagosome trafficking might lead to robust autophagic vacuole accumulation. Our recent work provides the first evidence that the presence of the N370S GBA mutation produces an accumulation of cholesterol, which alters autophagy-lysosome function with the appearance of MLBs, rendering the cell more vulnerable and sensitive to apoptosis.     

Glucocerebrosidase deficits in sporadic Parkinson disease

Parkinson disease (PD) is a progressive neurodegenerative movement disorder characterized pathologically by abnormal SNCA/α-synuclein protein inclusions in neurons. Impaired lysosomal autophagic degradation of cellular proteins is implicated in PD pathogenesis and progression. Heterozygous GBA mutations, encoding lysosomal GBA/glucocerebrosidase (glucosidase, β, acid), are the greatest genetic risk factor for PD, and reduced GBA and SNCA accumulation are related in PD models. Here we review our recent human brain tissue study demonstrating that GBA deficits in sporadic PD are related to the early accumulation of SNCA, and dysregulation of chaperone-mediated autophagy (CMA) pathways and lipid metabolism.