Autophagic lysosome reformation dysfunction in glucocerebrosidase deficient cells: relevance to Parkinson disease.
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ABSTRACT:
SUBMITTER: Magalhaes J
PROVIDER: S-EPMC5179940 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Lysosomes are the primary degradative compartment within cells and there have been significant advances over the past decade toward understanding how lysosome homeostasis is maintained. Lysosome repopulation ensures sustained autophagy function, a fundamental process that protects against disease. During macroautophagy/autophagy, cellular debris is sequestered into phagophores that mature into autophagosomes, which then fuse with lysosomes to generate autolysosomes in which contents are degraded. Autophagy cannot proceed without the sufficient generation of lysosomes, and this can be achieved via their de novo biogenesis. Alternatively, during autophagic lysosome reformation (ALR), lysosomes are generated via the recycling of autolysosome membranes. During this process, autolysosomes undergo significant membrane remodeling and scission to generate membrane fragments, that mature into functional lysosomes. By utilizing membranes already formed during autophagy, this facilitates an efficient pathway for re-deriving lysosomes, particularly under conditions of prolonged autophagic flux. ALR dysfunction is emerging as an important disease mechanism including for neurodegenerative disorders such as hereditary spastic paraplegia and Parkinson disease, neuropathies including Charcot-Marie-Tooth disease, lysosome storage disorders, muscular dystrophy, metabolic syndrome, and inflammatory and liver disorders. Here, we provide a comprehensive review of ALR, including an overview of its dynamic spatiotemporal regulation by MTOR and phosphoinositides, and the role ALR dysfunction plays in many diseases.
| S-EPMC10240999 | biostudies-literature
Project description:Autophagy allows cells to adapt to changes in their environment by coordinating the degradation and recycling of cellular components and organelles to maintain homeostasis. Lysosomes are organelles critical for terminating autophagy via their fusion with mature autophagosomes to generate autolysosomes that degrade autophagic materials; therefore, maintenance of the lysosomal population is essential for autophagy-dependent cellular clearance. Here, we have demonstrated that the two most common autosomal recessive hereditary spastic paraplegia gene products, the SPG15 protein spastizin and the SPG11 protein spatacsin, are pivotal for autophagic lysosome reformation (ALR), a pathway that generates new lysosomes. Lysosomal targeting of spastizin required an intact FYVE domain, which binds phosphatidylinositol 3-phosphate. Loss of spastizin or spatacsin resulted in depletion of free lysosomes, which are competent to fuse with autophagosomes, and an accumulation of autolysosomes, reflecting a failure in ALR. Moreover, spastizin and spatacsin were essential components for the initiation of lysosomal tubulation. Together, these results link dysfunction of the autophagy/lysosomal biogenesis machinery to neurodegeneration.
| S-EPMC4348974 | biostudies-literature
Project description:Autophagy is a conserved cellular process to degrade and recycle cytoplasmic components. During autophagy, lysosomes fuse with an autophagosome to form an autolysosome. Sequestered components are degraded by lysosomal hydrolases and presumably released into the cytosol by lysosomal efflux permeases. Following starvation-induced autophagy, lysosome homeostasis is restored by autophagic lysosome reformation (ALR) requiring activation of the "target of rapamycin" (TOR) kinase. Spinster (Spin) encodes a putative lysosomal efflux permease with the hallmarks of a sugar transporter. Drosophila spin mutants accumulate lysosomal carbohydrates and enlarged lysosomes. Here we show that defects in spin lead to the accumulation of enlarged autolysosomes. We find that spin is essential for mTOR reactivation and lysosome reformation following prolonged starvation. Further, we demonstrate that the sugar transporter activity of Spin is essential for ALR.
| S-EPMC3093520 | biostudies-literature
Project description:Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders characterized by leg spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that spg11 and zfyve26 KO mice developed motor impairments within the same course of time. This correlated with enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agreement with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at autolysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15.Abbreviations: ALR: autophagic lysosome reformation; AP-5: adaptor protein complex 5; BFP: blue fluorescent protein; dKO: double knockout; EBSS: Earle's balanced salt solution; FBA: foot base angle; GFP: green fluorescent protein; HSP: hereditary spastic paraplegia; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; SQSTM1/p62: sequestosome 1; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; SPG: spastic paraplegia gene; TGN: trans-Golgi network; WT: wild type.
| S-EPMC8632344 | biostudies-literature
Project description:The regulation of autophagy-dependent lysosome homeostasis in vivo is unclear. We showed that the inositol polyphosphate 5-phosphatase INPP5K regulates autophagic lysosome reformation (ALR), a lysosome recycling pathway, in muscle. INPP5K hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] to phosphatidylinositol 4-phosphate [PI(4)P], and INPP5K mutations cause muscular dystrophy by unknown mechanisms. We report that loss of INPP5K in muscle caused severe disease, autophagy inhibition, and lysosome depletion. Reduced PI(4,5)P2 turnover on autolysosomes in Inpp5k-/- muscle suppressed autophagy and lysosome repopulation via ALR inhibition. Defective ALR in Inpp5k-/- myoblasts was characterized by enlarged autolysosomes and the persistence of hyperextended reformation tubules, structures that participate in membrane recycling to form lysosomes. Reduced disengagement of the PI(4,5)P2 effector clathrin was observed on reformation tubules, which we propose interfered with ALR completion. Inhibition of PI(4,5)P2 synthesis or expression of WT INPP5K but not INPP5K disease mutants in INPP5K-depleted myoblasts restored lysosomal homeostasis. Therefore, bidirectional interconversion of PI(4)P/PI(4,5)P2 on autolysosomes was integral to lysosome replenishment and autophagy function in muscle. Activation of TFEB-dependent de novo lysosome biogenesis did not compensate for loss of ALR in Inpp5k-/- muscle, revealing a dependence on this lysosome recycling pathway. Therefore, in muscle, ALR is indispensable for lysosome homeostasis during autophagy and when defective is associated with muscular dystrophy.
| S-EPMC7773396 | biostudies-literature
Project description:Gaucher disease is caused by mutations in the glucocerebrosidase gene, which encodes the lysosomal hydrolase glucosylceramidase. Patients with Gaucher disease and heterozygous glucocerebrosidase mutation carriers are at increased risk of developing Parkinson's disease. Indeed, glucocerebrosidase mutations are the most frequent risk factor for Parkinson's disease in the general population. Therefore there is an urgent need to understand the mechanisms by which glucocerebrosidase mutations predispose to neurodegeneration to facilitate development of novel treatments. To study this we generated fibroblast lines from skin biopsies of five patients with Gaucher disease and six heterozygous glucocerebrosidase mutation carriers with and without Parkinson's disease. Glucosylceramidase protein and enzyme activity levels were assayed. Oxidative stress was assayed by single cell imaging of dihydroethidium. Glucosylceramidase enzyme activity was significantly reduced in fibroblasts from patients with Gaucher disease (median 5% of controls, P = 0.0001) and heterozygous mutation carriers with (median 59% of controls, P = 0.001) and without (56% of controls, P = 0.001) Parkinson's disease compared with controls. Glucosylceramidase protein levels, assessed by western blot, were significantly reduced in fibroblasts from Gaucher disease (median glucosylceramidase levels 42% of control, P < 0.001) and heterozygous mutation carriers with (median 59% of control, P < 0.001) and without (median 68% of control, P < 0.001) Parkinson's disease. Single cell imaging of dihydroethidium demonstrated increased production of cytosolic reactive oxygen species in fibroblasts from patients with Gaucher disease (dihydroethidium oxidation rate increased by a median of 62% compared to controls, P < 0.001) and heterozygous mutation carriers with (dihydroethidium oxidation rate increased by a median of 68% compared with controls, P < 0.001) and without (dihydroethidium oxidation rate increased by a median of 70% compared with controls, P < 0.001) Parkinson's disease. We hypothesized that treatment with the molecular chaperone ambroxol hydrochloride would improve these biochemical abnormalities. Treatment with ambroxol hydrochloride increased glucosylceramidase activity in fibroblasts from healthy controls, Gaucher disease and heterozygous glucocerebrosidase mutation carriers with and without Parkinson's disease. This was associated with a significant reduction in dihydroethidium oxidation rate of ∼50% (P < 0.05) in fibroblasts from controls, Gaucher disease and heterozygous mutation carriers with and without Parkinson's disease. In conclusion, glucocerebrosidase mutations are associated with reductions in glucosylceramidase activity and evidence of oxidative stress. Ambroxol treatment significantly increases glucosylceramidase activity and reduces markers of oxidative stress in cells bearing glucocerebrosidase mutations. We propose that ambroxol hydrochloride should be further investigated as a potential treatment for Parkinson's disease.
| S-EPMC3999713 | biostudies-literature
Project description:Parkinson disease (PD) is the second most common neurodegenerative disorder after Alzheimer disease, whereas Gaucher disease (GD) is the most frequent lysosomal storage disorder caused by homozygous mutations in the glucocerebrosidase (GBA1) gene. Increased risk of developing PD has been observed in both GD patients and carriers. It has been estimated that GBA1 mutations confer a 20- to 30-fold increased risk for the development of PD, and that at least 7-10% of PD patients have a GBA1 mutation. To date, mutations in the GBA1 gene constitute numerically the most important risk factor for PD. The type of PD associated with GBA1 mutations (PD-GBA1) is almost identical to idiopathic PD, except for a slightly younger age of onset and a tendency to more cognitive impairment. Importantly, the pathology of PD-GBA1 is identical to idiopathic PD, with nigral dopamine cell loss, Lewy bodies, and neurites containing alpha-synuclein. The mechanism by which GBA1 mutations increase the risk for PD is still unknown. However, given that clinical manifestation and pathological findings in PD-GBA1 patients are almost identical to those in idiopathic PD individuals, it is likely that, as in idiopathic PD, alpha-synuclein accumulation, mitochondrial dysfunction, autophagic impairment, oxidative and endoplasmic reticulum stress may contribute to the development and progression of PD-GBA1. Here, we review the GBA1 gene, its role in GD, and its link with PD. The impact of glucocerebrosidase 1 (GBA1) mutations on functioning of endoplasmic reticulum (ER), lysosomes, and mitochondria. GBA1 mutations resulting in production of misfolded glucocerebrosidase (GCase) significantly affect the ER functioning. Misfolded GCase trapped in the ER leads to both an increase in the ubiquitin-proteasome system (UPS) and the ER stress. The presence of ER stress triggers the unfolded protein response (UPR) and/or endoplasmic reticulum-associated degradation (ERAD). The prolonged activation of UPR and ERAD subsequently leads to increased apoptosis. The presence of misfolded GCase in the lysosomes together with a reduction in wild-type GCase levels lead to a retardation of alpha-synuclein degradation via chaperone-mediated autophagy (CMA), which subsequently results in alpha-synuclein accumulation and aggregation. Impaired lysosomal functioning also causes a decrease in the clearance of autophagosomes, and so their accumulation. GBA1 mutations perturb normal mitochondria functioning by increasing generation of free radical species (ROS) and decreasing adenosine triphosphate (ATP) production, oxygen consumption, and membrane potential. GBA1 mutations also lead to accumulation of dysfunctional and fragmented mitochondria. This article is part of a special issue on Parkinson disease.
| S-EPMC5111601 | biostudies-literature
Project description:Mutations in both copies (homozygous or compound heterozygous) of the gene encoding the lysosomal enzyme glucocerebrosidase, which cleaves the glycolipid glucocerebroside into glucose and ceramide cause Gaucher disease. However, multiple independent studies have also reported an association between GBA mutations and Parkinsonism with an increased frequency of heterozygous GBA mutations in various cohorts of patients with parkinsonism and other Lewy body disorders. Furthermore, GBA mutation carriers exhibit diverse parkinsonian phenotypes and present a diffuse pattern of Lewy body distribution in the cerebral cortex. This review provides an overview of the genetic basis for this association in various diseases with dysfunction of the central nervous system in which affected individuals developed Parkinsonian symptoms. The emerging clinical, pathological, and genetic studies in neuronal synucleinopathies suggest a common underlying mechanism in the etiology of these neurodegenerative disorders.
| S-EPMC3338066 | biostudies-literature
Project description:ObjectiveMutations in the glucocerebrosidase gene (GBA) represent a significant risk factor for developing Parkinson disease (PD). We investigated the enzymatic activity of glucocerebrosidase (GCase) in PD brains carrying heterozygote GBA mutations (PD+GBA) and sporadic PD brains.MethodsGCase activity was measured using a fluorescent assay in cerebellum, frontal cortex, putamen, amygdala, and substantia nigra of PD+GBA (n = 9-14) and sporadic PD brains (n = 12-14). Protein expression of GCase and other lysosomal proteins was determined by western blotting. The relation between GCase, ?-synuclein, and mitochondria function was also investigated in vitro.ResultsA significant decrease in GCase activity was observed in all PD+GBA brain areas except the frontal cortex. The greatest deficiency was in the substantia nigra (58% decrease; p < 0.01). GCase activity was also significantly decreased in the substantia nigra (33% decrease; p < 0.05) and cerebellum (24% decrease; p < 0.05) of sporadic PD brains. GCase protein expression was lower in PD+GBA and PD brains, whereas increased C/EBP homologous protein and binding immunoglobulin protein levels indicated that the unfolded protein response was activated. Elevated ?-synuclein levels or PTEN-induced putative kinase 1 deficiency in cultured cells had a significant effect on GCase protein levels.InterpretationGCase deficiency in PD brains with GBA mutations is a combination of decreased catalytic activity and reduced protein levels. This is most pronounced in the substantia nigra. Biochemical changes involved in PD pathogenesis affect wild-type GCase protein expression in vitro, and these could be contributing factors to the GCase deficiency observed in sporadic PD brains.
| S-EPMC3638323 | biostudies-literature
Project description:Heterozygous glucocerebrosidase (GBA) mutations are the leading genetic risk factor for Parkinson disease, yet imaging correlates, particularly transcranial sonography, have not been extensively described.To determine whether GBA mutation heterozygotes with Parkinson disease demonstrate hyperechogenicity of the substantia nigra, transcranial sonography was performed in Ashkenazi Jewish Parkinson disease subjects, tested for the eight most common Gaucher disease mutations and the LRRK2 G2019S mutation, and in controls. [(18)F]-fluorodeoxyglucose or [(18)F]-fluorodopa positron emission tomography is also reported from a subset of Parkinson disease subjects with heterozygous GBA mutations.Parkinson disease subjects with heterozygous GBA mutations (n = 23) had a greater median maximal area of substantia nigral echogenicity compared to controls (n = 34, aSNmax = 0.30 vs. 0.18, p = 0.007). There was no difference in median maximal area of nigral echogenicity between Parkinson disease groups defined by GBA and LRRK2 genotype: GBA heterozygotes; GBA homozygotes/compound heterozygotes (n = 4, aSNmax = 0.27); subjects without LRRK2 or GBA mutations (n = 32, aSNmax = 0.27); LRRK2 heterozygotes/homozygotes without GBA mutations (n = 27, aSNmax = 0.28); and GBA heterozygotes/LRRK2 heterozygotes (n = 4, aSNmax = 0.32, overall p = 0.63). In secondary analyses among Parkinson disease subjects with GBA mutations, maximal area of nigral echogenicity did not differ based on GBA mutation severity or mutation number. [(18)F]-fluorodeoxyglucose (n = 3) and [(18)F]-fluorodopa (n = 2) positron emission tomography in Parkinson disease subjects with heterozygous GBA mutations was consistent with findings in idiopathic Parkinson disease.Both transcranial sonography and positron emission tomography are abnormal in GBA mutation associated Parkinson disease, similar to other Parkinson disease subjects.
| S-EPMC3567285 | biostudies-literature