Converging evidence from genetic, pathological and experimental studies have increasingly suggested an important role for autophagy impairment in Parkinson's Disease (PD). Genetic studies have identified mutations in genes encoding for components of the autophagy-lysosomal pathway (ALP), including glucosidase beta acid 1 (GBA1), that are associated with increased risk for developing PD. Observations in PD brain tissue suggest an aberrant regulation of autophagy associated with the aggregation of α-synuclein (α-syn). As autophagy is one of the main systems involved in the proteolytic degradation of α-syn, pharmacological enhancement of autophagy may be an attractive strategy to combat α-syn aggregation in PD. Here, we review the potential of autophagy enhancement as disease-modifying therapy in PD based on preclinical evidence. In particular, we provide an overview of the molecular regulation of autophagy and targets for pharmacological modulation within the ALP. In experimental models, beneficial effects on multiple pathological processes involved in PD, including α-syn aggregation, cell death, oxidative stress and mitochondrial dysfunction, have been demonstrated using the autophagy enhancers rapamycin and lithium. However, selectivity of these agents is limited, while upstream ALP signaling proteins are involved in many other pathways than autophagy. Broad stimulation of autophagy may therefore cause a wide spectrum of dose-dependent side-effects, suggesting that its clinical applicability is limited. However, recently developed agents selectively targeting core ALP components, including Transcription Factor EB (TFEB), lysosomes, GCase as well as chaperone-mediated autophagy regulators, exert more specific effects on molecular pathogenetic processes causing PD. To conclude, the targeted manipulation of downstream ALP components, rather than broad autophagy stimulation, may be an attractive strategy for the development of novel pharmacological therapies in PD. Further characterization of dysfunctional autophagy in different stages and molecular subtypes of PD in combination with the clinical translation of downstream autophagy regulation offers exciting new avenues for future drug development.
Mutations in the GBA gene, encoding the lysosomal hydrolase glucocerebrosidase (GCase), are the most common known genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). The present study aims to gain more insight into changes in lysosomal activity in different brain regions of sporadic PD and DLB patients, screened for GBA variants. Enzymatic activities of GCase, β-hexosaminidase, and cathepsin D were measured in the frontal cortex, putamen, and substantia nigra (SN) of a cohort of patients with advanced PD and DLB as well as age-matched non-demented controls (n = 15/group) using fluorometric assays. Decreased activity of GCase (− 21%) and of cathepsin D (− 15%) was found in the SN and frontal cortex of patients with PD and DLB compared to controls, respectively. Population stratification was applied based on GBA genotype, showing substantially lower GCase activity (~ − 40%) in GBA variant carriers in all regions. GCase activity was further significantly decreased in the SN of PD and DLB patients without GBA variants in comparison to controls without GBA variants. Our results show decreased GCase activity in brains of PD and DLB patients with and without GBA variants, most pronounced in the SN. The results of our study confirm findings from previous studies, suggesting a role for GCase in GBA-associated as well as sporadic PD and DLB.
Growing evidence suggests a crucial role of neuroinflammation in the pathophysiology of Parkinson’s disease (PD). Neuroinflammation is linked to the accumulation and aggregation of a-synuclein (αSyn), the primary pathological hallmark of PD. Toll-like receptors 4 (TLR4) can have implications in the development and progression of the pathology. In this study, we analyzed the expression of TLR4 in the substantia nigra (SN) and medial temporal gyrus (GTM) of well-characterized PD patients and age-matched controls. We also assessed the co-localization of TLR4 with pSer129 αSyn. Using qPCR, we observed an upregulation of TLR4 expression in the SN and GTM in PD patients compared to controls, which was accompanied by a reduction in αSyn expression likely due to the depletion of dopaminergic (DA) cells. Additionally, using immunofluorescence and confocal microscopy, we observed TLR4-positive staining and co-localization with pSer129-αSyn in Lewy bodies of DA neurons in the SN, as well as in pyramidal neurons in the GTM of PD donors. Furthermore, we observed a co-localization of TLR4 and Iba-1 in glial cells of both SN and GTM. Our findings provide evidence for the increased expression of TLR4 in the PD brain and suggest that the interaction between TLR4 and pSer129-αSyn could play a role in mediating the neuroinflammatory response in PD.
b-galactosidase (b-gal), a lysosomal enzyme involved the removal of b-linked terminal galactosyl residues of many glycoconjugates, is deficient the neurodegenerative lysosomal disorder GM1-gangliosidosis (GM1). GM1-/-mice closely mimic the most fundamental aspects of the neuropathological and neurochemical abnormalities of the human disorder. Bone marrow progenitor cells have been used as a source of corrective protein because of their ability to repopulate the recipients and to supply functional enzyme to different cells by in trans correction. Hematopoietic progenitors transduced with a murine stem cell virus (MSCV)-based bicistronic retroviral vector over-expressing b-gal and the green fluorescent protein (GFP) marker were used for transplantation into sublethally irradiated GM1-/-mice. Transduction efficiency of total BM cells with the MSCV-b-gal ranged from 25–89% prior to the transplantation. b-gal expressing BM-derived cells were detected histologically and enzymatically many tissues including spleen, liver, lungs, intestine and kidney after one, three and six months post transplantation (BMT). GFP-expressing cells of the erythroid, myeloid or lymphoid lineage were identified by FACS analysis of peripheral blood samples, collected at different time points after transplantation. Secondary transplantations demonstrated consistent long-term in vivo b-gal expression all tissues including brain. In addition, the thin layer chromatography of brain lipids showed a reduction the GM1-ganglioside content brainstem and cerebellum after three and six months post BMT. In line with the biochemical and histological findings, different behavioral tests including open field, rotorod and walking pattern, indicated a clear improvement of the neurological function transplanted GM1-/-compared to untreated littermates. Taken together these results have encouraged the use of ex-vivo gene therapy for the treatment of GM1.
Abstract Transcription factor EB is a master regulator of genes involved in the maintenance of autophagic and lysosomal homeostasis, processes which have been implicated in the pathogenesis of GBA -related and sporadic Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). TFEB activation at the lysosomal level results in its translocation from the cytosol to the nucleus. Here, we aimed at investigating whether TFEB subcellular localization is altered in post-mortem human brain of aged individuals with either prodromal PD/DLB (incidental Lewy body disease, iLBD, N=3), GBA -related PD/DLB (N=9) or sPD/DLB (N=9), compared to control subjects (N=12). We scanned nigral dopaminergic neurons using high-resolution confocal and stimulated emission depletion (STED) microscopy and semi-quantitatively scored the observed TFEB subcellular localization patterns. In line with previous studies, we observed reduced nuclear TFEB immunoreactivity in PD/DLB patients compared to controls, both sporadic and GBA -related cases, as well as in iLBD cases. Nuclear depletion of TFEB was more pronounced in neurons with Ser129-phosphorylated (pSer129) aSyn cytopathology and in cases carrying pathogenic GBA variants. Interestingly, we further observed previously unidentified TFEB-immunopositive somatic clusters in human brain dopaminergic neurons and in human embryonic stem cell (hESC)-derived neurons, which localized at the Golgi apparatus. The TFEB clustering was more frequently observed and more severe in iLBD, sPD/DLB and GBA -PD/DLB compared to controls, particularly in pSer129 aSyn-positive neurons but also in neurons without apparent cytopathology. Notably, increased frequency of cytoplasmic TFEB clusters in aSyn-negative cells correlated with reduced total GBA enzymatic activity and higher Braak LB stage. In the studied patient population, altered TFEB distribution was accompanied by a reduction in overall mRNA expression levels of selected CLEAR genes, indicating a possible early dysfunction of lysosomal regulation. Overall, these findings suggest the early cytoplasmic TFEB retention and accumulation at the Golgi prior pSer129 aSyn accumulation in incidental, GBA -related and sporadic PD/DLB and indicate TFEB as potential as early therapeutic target for synucleinopathies
Abstract Transcription factor EB (TFEB) is a master regulator of genes involved in the maintenance of autophagic and lysosomal homeostasis, processes which have been implicated in the pathogenesis of GBA -related and sporadic Parkinson’s disease (PD), and dementia with Lewy bodies (DLB). TFEB activation results in its translocation from the cytosol to the nucleus. Here, we investigated TFEB subcellular localization and its relation to intracellular alpha-synuclein (aSyn) accumulation in post-mortem human brain of individuals with either incidental Lewy body disease (iLBD), GBA -related PD/DLB ( GBA -PD/DLB) or sporadic PD/DLB (sPD/DLB), compared to control subjects. We analyzed nigral dopaminergic neurons using high-resolution confocal and stimulated emission depletion (STED) microscopy and semi-quantitatively scored the TFEB subcellular localization patterns. We observed reduced nuclear TFEB immunoreactivity in PD/DLB patients compared to controls, both in sporadic and GBA -related cases, as well as in iLBD cases. Nuclear depletion of TFEB was more pronounced in neurons with Ser129-phosphorylated (pSer129) aSyn accumulation in all groups. Importantly, we observed previously-unidentified TFEB-immunopositive perinuclear clusters in human dopaminergic neurons, which localized at the Golgi apparatus. These TFEB clusters were more frequently observed and more severe in iLBD, sPD/DLB and GBA -PD/DLB compared to controls, particularly in pSer129 aSyn-positive neurons, but also in neurons lacking detectable aSyn accumulation. In aSyn-negative cells, cytoplasmic TFEB clusters were more frequently observed in GBA -PD/DLB and iLBD patients, and correlated with reduced GBA enzymatic activity as well as increased Braak LB stage. Altered TFEB distribution was accompanied by a reduction in overall mRNA expression levels of selected TFEB-regulated genes, indicating a possible early dysfunction of lysosomal regulation. Overall, we observed cytoplasmic TFEB retention and accumulation at the Golgi in cells without apparent pSer129 aSyn accumulation in iLBD and PD/DLB patients. This suggests potential TFEB impairment at the early stages of cellular disease and underscores TFEB as a promising therapeutic target for synucleinopathies.
