Loss of Lipid Carrier ApoE Exacerbates Brain Glial and Inflammatory Responses after Lysosomal GBA1 Inhibition.

Tightly regulated and highly adaptive lipid metabolic and transport pathways are critical to maintaining brain cellular lipid homeostasis and responding to lipid and inflammatory stress to preserve brain function and health. Deficits in the lipid handling genes APOE and GBA1 are the most significant genetic risk factors for Lewy body dementia and related dementia syndromes. Parkinson's disease patients who carry both APOE4 and GBA1 variants have accelerated cognitive decline compared to single variant carriers. To investigate functional interactions between brain ApoE and GBA1, in vivo GBA1 inhibition was tested in WT versus ApoE-deficient mice. The experiments demonstrated glycolipid stress caused by GBA1 inhibition in WT mice induced ApoE expression in several brain regions associated with movement and dementia disorders. The absence of ApoE in ApoE-KO mice amplified complement C1q elevations, reactive microgliosis and astrocytosis after glycolipid stress. Mechanistically, GBA1 inhibition triggered increases in cell surface and intracellular lipid transporters ABCA1 and NPC1, respectively. Interestingly, the absence of NPC1 in mice also triggered elevations of brain ApoE levels. These new data show that brain ApoE, GBA1 and NPC1 functions are interconnected in vivo, and that the removal or reduction of ApoE would likely be detrimental to brain function. These results provide important insights into brain ApoE adaptive responses to increased lipid loads.

Viral-like TLR3 induction of cytokine networks and α-synuclein are reduced by complement C3 blockade in mouse brain.

Inflammatory processes and mechanisms are of central importance in neurodegenerative diseases. In the brain, α-synucleinopathies such as Parkinson's disease (PD) and Lewy body dementia (LBD) show immune cytokine network activation and increased toll like receptor 3 (TLR3) levels for viral double-stranded RNA (dsRNA). Brain inflammatory reactions caused by TLR3 activation are also relevant to understand pathogenic cascades by viral SARS-CoV-2 infection causing post- COVID-19 brain-related syndromes. In the current study, following regional brain TLR3 activation induced by dsRNA in mice, an acute complement C3 response was seen at 2 days. A C3 splice-switching antisense oligonucleotide (ASO) that promotes the splicing of a non-productive C3 mRNA, prevented downstream cytokines, such as IL-6, and α-synuclein changes. This report is the first demonstration that α-synuclein increases occur downstream of complement C3 activation. Relevant to brain dysfunction, post-COVID-19 syndromes and pathological changes leading to PD and LBD, viral dsRNA TLR3 activation in the presence of C3 complement blockade further revealed significant interactions between complement systems, inflammatory cytokine networks and α-synuclein changes.

Crucial neuroprotective roles of the metabolite BH4 in dopaminergic neurons.

Dopa-responsive dystonia (DRD) and Parkinson's disease (PD) are movement disorders caused by the dysfunction of nigrostriatal dopaminergic neurons. Identifying druggable pathways and biomarkers for guiding therapies is crucial due to the debilitating nature of these disorders. Recent genetic studies have identified variants of GTP cyclohydrolase-1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis, as causative for these movement disorders. Here, we show that genetic and pharmacological inhibition of BH4 synthesis in mice and human midbrain-like organoids accurately recapitulates motor, behavioral and biochemical characteristics of these human diseases, with severity of the phenotype correlating with extent of BH4 deficiency. We also show that BH4 deficiency increases sensitivities to several PD-related stressors in mice and PD human cells, resulting in worse behavioral and physiological outcomes. Conversely, genetic and pharmacological augmentation of BH4 protects mice from genetically- and chemically induced PD-related stressors. Importantly, increasing BH4 levels also protects primary cells from PD-affected individuals and human midbrain-like organoids (hMLOs) from these stressors. Mechanistically, BH4 not only serves as an essential cofactor for dopamine synthesis, but also independently regulates tyrosine hydroxylase levels, protects against ferroptosis, scavenges mitochondrial ROS, maintains neuronal excitability and promotes mitochondrial ATP production, thereby enhancing mitochondrial fitness and cellular respiration in multiple preclinical PD animal models, human dopaminergic midbrain-like organoids and primary cells from PD-affected individuals. Our findings pinpoint the BH4 pathway as a key metabolic program at the intersection of multiple protective mechanisms for the health and function of midbrain dopaminergic neurons, identifying it as a potential therapeutic target for PD.

Acid ceramidase involved in pathogenic cascade leading to accumulation of α-synuclein in iPSC model of GBA1-associated Parkinson's disease.

Bi-allelic mutations in GBA1, the gene that encodes ß-glucocerebrosidase (GCase), cause Gaucher disease (GD), whereas mono-allelic mutations do not cause overt pathology. Yet mono- or bi-allelic GBA1 mutations are the highest known risk factor for Parkinson's disease (PD). GCase deficiency results in the accumulation of glucosylceramide (GluCer) and its deacylated metabolite glucosylsphingosine (GluSph). Brains from patients with neuronopathic GD have high levels of GluSph, and elevation of this lipid in GBA1-associated PD has been reported. To uncover the mechanisms involved in GBA1-associated PD, we used human induced pluripotent stem cell-derived dopaminergic (DA) neurons from patients harboring heterozygote mutations in GBA1 (GBA1/PD-DA neurons). We found that compared with gene-edited isogenic controls, GBA1/PD-DA neurons exhibit mammalian target of rapamycin complex 1 (mTORC1) hyperactivity, a block in autophagy, an increase in the levels of phosphorylated α-synuclein (129) and α-synuclein aggregation. These alterations were prevented by incubation with mTOR inhibitors. Inhibition of acid ceramidase, the lysosomal enzyme that deacylates GluCer to GluSph, prevented mTOR hyperactivity, restored autophagic flux and lowered α-synuclein levels, suggesting that GluSph was responsible for these alterations. Incubation of gene-edited wild type (WT) controls with exogenous GluSph recapitulated the mTOR/α-synuclein abnormalities of GBA1/PD neurons, and these phenotypic alterations were prevented when GluSph treatment was in the presence of mTOR inhibitors. We conclude that GluSph causes an aberrant activation of mTORC1, suppressing normal lysosomal functions, including the clearance of pathogenic α-synuclein species. Our results implicate acid ceramidase in the pathogenesis of GBA1-associated PD, suggesting that this enzyme is a potential therapeutic target for treating synucleinopathies caused by GCase deficiency.

Glycosphingolipid metabolism and its role in ageing and Parkinson's disease.

It is well established that lysosomal glucocerebrosidase gene (GBA) variants are a risk factor for Parkinson's disease (PD), with increasing evidence suggesting a loss of function mechanism. One question raised by this genetic association is whether variants of genes involved in other aspects of sphingolipid metabolism are also associated with PD. Recent studies in sporadic PD have identified variants in multiple genes linked to diseases of glycosphingolipid (GSL) metabolism to be associated with PD. GSL biosynthesis is a complex pathway involving the coordinated action of multiple enzymes in the Golgi apparatus. GSL catabolism takes place in the lysosome and is dependent on the action of multiple acid hydrolases specific for certain substrates and glycan linkages. The finding that variants in multiple GSL catabolic genes are over-represented in PD in a heterozygous state highlights the importance of GSLs in the healthy brain and how lipid imbalances and lysosomal dysfunction are associated with normal ageing and neurodegenerative diseases. In this article we will explore the link between lysosomal storage disorders and PD, the GSL changes seen in both normal ageing, lysosomal storage disorders (LSDs) and PD and the mechanisms by which these changes can affect neurodegeneration.

Fibroblasts from idiopathic Parkinson's disease exhibit deficiency of lysosomal glucocerebrosidase activity associated with reduced levels of the trafficking receptor LIMP2.

Lysosomal dysfunction is a central pathway associated with Parkinson's disease (PD) pathogenesis. Haploinsufficiency of the lysosomal hydrolase GBA (encoding glucocerebrosidase (GCase)) is one of the largest genetic risk factors for developing PD. Deficiencies in the activity of the GCase enzyme have been observed in human tissues from both genetic (harboring mutations in the GBA gene) and idiopathic forms of the disease. To understand the mechanisms behind the deficits of lysosomal GCase enzyme activity in idiopathic PD, this study utilized a large cohort of fibroblast cells from control subjects and PD patients with and without mutations in the GBA gene (N370S mutation) (control, n = 15; idiopathic PD, n = 31; PD with GBA N370S mutation, n = 6). The current data demonstrates that idiopathic PD fibroblasts devoid of any mutations in the GBA gene also exhibit reduction in lysosomal GCase activity, similar to those with the GBA N370S mutation. This reduced GCase enzyme activity in idiopathic PD cells was accompanied by decreased expression of the GBA trafficking receptor, LIMP2, and increased ER retention of the GBA protein in these cells. Importantly, in idiopathic PD fibroblasts LIMP2 protein levels correlated significantly with GCase activity, which was not the case in control subjects or in genetic PD GBA N370S cells. In conclusion, idiopathic PD fibroblasts have decreased GCase activity primarily driven by altered LIMP2-mediated transport of GBA to lysosome and the reduced GCase activity exhibited by  the genetic GBA N370S derived PD fibroblasts occurs through a different mechanism.

Cell type-specific lipid storage changes in Parkinson's disease patient brains are recapitulated by experimental glycolipid disturbance.

Neurons are dependent on proper trafficking of lipids to neighboring glia for lipid exchange and disposal of potentially lipotoxic metabolites, producing distinct lipid distribution profiles among various cell types of the central nervous system. Little is known of the cellular distribution of neutral lipids in the substantia nigra (SN) of Parkinson's disease (PD) patients and its relationship to inflammatory signaling. This study aimed to determine human PD SN neutral lipid content and distribution in dopaminergic neurons, astrocytes, and microglia relative to age-matched healthy subject controls. The results show that while total neutral lipid content was unchanged relative to age-matched controls, the levels of whole SN triglycerides were correlated with inflammation-attenuating glycoprotein non-metastatic melanoma protein B (GPNMB) signaling in human PD SN. Histological localization of neutral lipids using a fluorescent probe (BODIPY) revealed that dopaminergic neurons and midbrain microglia significantly accumulated intracellular lipids in PD SN, while adjacent astrocytes had a reduced lipid load overall. This pattern was recapitulated by experimental in vivo inhibition of glucocerebrosidase activity in mice. Agents or therapies that restore lipid homeostasis among neurons, astrocytes, and microglia could potentially correct PD pathogenesis and disease progression.

Upregulating ß-hexosaminidase activity in rodents prevents α-synuclein lipid associations and protects dopaminergic neurons from α-synuclein-mediated neurotoxicity.

Sandhoff disease (SD) is a lysosomal storage disease, caused by loss of ß-hexosaminidase (HEX) activity resulting in the accumulation of ganglioside GM2. There are shared features between SD and Parkinson's disease (PD). α-synuclein (aSYN) inclusions, the diagnostic hallmark sign of PD, are frequently found in the brain in SD patients and HEX knockout mice, and HEX activity is reduced in the substantia nigra in PD. In this study, we biochemically demonstrate that HEX deficiency in mice causes formation of high-molecular weight (HMW) aSYN and ubiquitin in the brain. As expected from HEX enzymatic function requirements, overexpression in vivo of HEXA and B combined, but not either of the subunits expressed alone, increased HEX activity as evidenced by histochemical assays. Biochemically, such HEX gene expression resulted in increased conversion of GM2 to its breakdown product GM3. In a neurodegenerative model of overexpression of aSYN in rats, increasing HEX activity by AAV6 gene transfer in the substantia nigra reduced aSYN embedding in lipid compartments and rescued dopaminergic neurons from degeneration. Overall, these data are consistent with a paradigm shift where lipid abnormalities are central to or preceding protein changes typically associated with PD.

Experimental studies of mitochondrial and lysosomal function in in vitro and in vivo models relevant to Parkinson's disease genetic risk.

Several studies have identified the involvement of mitochondrial and lysosomal dysfunction in Parkinson's disease (PD) pathology. In this review we discuss recent work that has identified deficits in mitophagy, mitochondrial network formation, increased sensitivity to mitochondrial stressors and alterations in proteins regulating mitochondrial fission and fusion associated with patient-derived fibroblasts harboring mutations in LRRK2 gene and from sporadic PD patient cells. We further focus on alterations of lysosomal enzymes, in particular glucocerebrosidase activity, and resultant lipid dyshomeostasis in PD and aging, in human tissue and in vivo rodent models. Future studies aimed at understanding the convergence of mitochondrial and lysosomal pathways will be of essence for the identification of unique cellular defects in PD and for the development of new treatments.

Splice-Switching Antisense Oligonucleotides Reduce LRRK2 Kinase Activity in Human LRRK2 Transgenic Mice.

Parkinson's disease (PD) is a progressive neurological disorder estimated to affect 7-10 million people worldwide. There is no treatment available that cures or slows the progression of PD. Elevated leucine-rich repeat kinase 2 (LRRK2) activity has been associated with genetic and sporadic forms of PD and, thus, reducing LRRK2 function is a promising therapeutic strategy. We have previously reported that an antisense oligonucleotide (ASO) that blocks splicing of LRRK2 exon 41, which encodes part of the kinase domain, reverses aberrant endoplasmic reticulum (ER) calcium levels and mitophagy defects in PD patient-derived cell lines harboring the LRRK2 G2019S mutation. In this study, we show that treating transgenic mice expressing human wild-type or G2019S LRRK2 with a single intracerebroventricular injection of ASO induces exon 41 skipping and results in a decrease in phosphorylation of the LRRK2 kinase substrate RAB10. Exon 41 skipping also reverses LRRK2 kinase-dependent changes in LC3B II/I ratios, a marker for the autophagic process. These results demonstrate the potential of LRRK2 exon 41 skipping as a possible therapeutic strategy to modulate pathogenic LRRK2 kinase activity associated with PD development.

Advantages and Recent Developments of Autologous Cell Therapy for Parkinson's Disease Patients.

Parkinson's Disease (PD) is a progressive degenerative disease characterized by tremor, bradykinesia, rigidity and postural instability. There are approximately 7-10 million PD patients worldwide. Currently, there are no biomarkers available or pharmaceuticals that can halt the dopaminergic neuron degeneration. At the time of diagnosis about 60% of the midbrain dopamine (mDA) neurons have already degenerated, resulting in a depletion of roughly 70% of striatal dopamine (DA) levels and synapses. Symptomatic treatment (e.g., with L-dopa) can initially restore DA levels and motor function, but with time often lead to side-effects like dyskinesia. Deep-brain-stimulation can alleviate these side-effects and some of the motor symptoms but requires repeat procedures and adds limitations for the patients. Restoration of dopaminergic synapses using neuronal cell replacement therapy has shown benefit in clinical studies using cells from fetal ventral midbrain. This approach, if done correctly, increases DA levels and restores synapses, allowing biofeedback regulation between the grafted cells and the host brain. Drawbacks are that it is not scalable for a large patient population and the patients require immunosuppression. Stem cells differentiated in vitro to mDA neurons or progenitors have shown promise in animal studies and is a scalable approach that allows for cryopreservation of transplantable cells and rigorous quality control prior to transplantation. However, all allogeneic grafts require immunosuppression. HLA-donor-matching, reduces, but does not completely eliminate, the need for immunosuppression, and is currently investigated in a clinical trial for PD in Japan. Since immune compatibility is very important in all areas of transplantation, these approaches may ultimately be of less benefit to the patients than an autologous approach. By using the patient's own somatic cells, reprogrammed to induced pluripotent stem cells (iPSCs) and differentiated to mDA neurons immunosuppression is not required, and may also present with several biological and functional advantages in the patients, as described in this article. The proof-of-principle of autologous iPSC mDA restoration of function has been shown in parkinsonian non-human primates (NHPs), and this can now be investigated in clinical trials in addition to the allogeneic and HLA-matched approaches. In this review, we focus on the autologous approach of cell therapy for PD.

Correction to: Reduced sphingolipid hydrolase activities, substrate accumulation and ganglioside decline in Parkinson's disease.

The original article [1] contains an error in the y-axes of Fig. 8's sub-figures whereby 'CSF' is mistakenly mentioned instead of 'serum'.

Reduced sphingolipid hydrolase activities, substrate accumulation and ganglioside decline in Parkinson's disease.

BACKGROUND: Haploinsufficiency in the Gaucher disease GBA gene, which encodes the lysosomal glucocerebrosidase GBA, and ageing represent major risk factors for developing Parkinson's disease (PD). Recently, more than fifty other lysosomal storage disorder gene variants have been identified in PD, implicating lysosomal dysfunction more broadly as a key risk factor for PD. Despite the evidence of multiple lysosomal genetic risks, it remains unclear how sphingolipid hydrolase activities, other than GBA, are altered with ageing or in PD. Moreover, it is not fully known if levels of glycosphingolipid substrates for these enzymes change in vulnerable brain regions of PD. Finally, little is known about the levels of complex gangliosides in substantia nigra which may play a significant role in ageing and PD. METHODS: To study sphingolipid hydrolase activities and glycosphingolipid expression in ageing and in PD, two independent cohorts of human substantia nigra tissues were obtained. Fluorescent 4-methylumbelliferone assays were used to determine multiple enzyme activities. The lysosomal GBA and non-lysosomal GBA2 activities were distinguished using the inhibitor NB-DGJ. Sensitive and quantitative normal-phase HPLC was performed to study glycosphingolipid levels. In addition, glycosphingolipid levels in cerebrospinal fluid and serum were analysed as possible biomarkers for PD. RESULTS: The present study demonstrates, in two independent cohorts of human post-mortem substantia nigra, that sporadic PD is associated with deficiencies in multiple lysosomal hydrolases (e.g. α-galactosidase and ß-hexosaminidase), in addition to reduced GBA and GBA2 activities and concomitant glycosphingolipid substrate accumulation. Furthermore, the data show significant reductions in levels of complex gangliosides (e.g. GM1a) in substantia nigra, CSF and serum in ageing, PD, and REM sleep behaviour disorder, which is a strong predictor of PD. CONCLUSIONS: These findings conclusively demonstrate reductions in GBA activity in the parkinsonian midbrain, and for the first time, reductions in the activity of several other sphingolipid hydrolases. Furthermore, significant reductions were seen in complex gangliosides in PD and ageing. The diminished activities of these lysosomal hydrolases, the glycosphingolipid substrate accumulation, and the reduced levels of complex gangliosides are likely major contributors to the primary development of the pathology seen in PD and related disorders with age.

Novel Results and Concepts Emerging From Lipid Cell Biology Relevant to Degenerative Brain Aging and Disease.

While very rare familial forms of proteinopathy can cause Parkinson's disease (PD), Lewy body dementia (LBD) and age-related dementias, recent in-depth studies of lipid disturbances in the majority of the common forms of these diseases instead suggest a primary pathogenesis in lipid pathways. This review synthesizes a perspective from new data that point to an interdependence of lipids and proteinopathy. This article describes disturbed relationships in lipid homeostasis that causes neuropathology to develop over time and with age, which includes altered mechanisms of glia-neuron exchange of lipids and inflammatory signals.

Lipid and immune abnormalities causing age-dependent neurodegeneration and Parkinson's disease.

This article describes pathogenic concepts and factors, in particular glycolipid abnormalities, that create cell dysfunction and synaptic loss in neurodegenerative diseases. By phenocopying lysosomal storage disorders, such as Gaucher disease and related disorders, age- and dose-dependent changes in glycolipid cell metabolism can lead to Parkinson's disease and related dementias. Recent results show that perturbation of sphingolipid metabolism can precede or is a part of abnormal protein handling in both genetic and idiopathic Parkinson's disease and Lewy body dementia. In aging and genetic predisposition with lipid disturbance, α-synuclein's normal vesicular and synaptic role may be detrimentally shifted toward accommodating and binding such lipids. Specific neuronal glycolipid, protein, and vesicular interactions create potential pathophysiology that is amplified by astroglial and microglial immune mechanisms resulting in neurodegeneration. This perspective provides a new logic for therapeutic interventions that do not focus on protein aggregation, but rather provides a guide to the complex biology and the common sequence of events that lead to age-dependent neurodegenerative disorders.

Mitochondrial clearance and maturation of autophagosomes are compromised in LRRK2 G2019S familial Parkinson's disease patient fibroblasts.

This study utilized human fibroblasts as a preclinical discovery and diagnostic platform for identification of cell biological signatures specific for the LRRK2 G2019S mutation producing Parkinson's disease (PD). Using live cell imaging with a pH-sensitive Rosella biosensor probe reflecting lysosomal breakdown of mitochondria, mitophagy rates were found to be decreased in fibroblasts carrying the LRRK2 G2019S mutation compared to cells isolated from healthy subject (HS) controls. The mutant LRRK2 increased kinase activity was reduced by pharmacological inhibition and targeted antisense oligonucleotide treatment, which normalized mitophagy rates in the G2019S cells and also increased mitophagy levels in HS cells. Detailed mechanistic analysis showed a reduction of mature autophagosomes in LRRK2 G2019S fibroblasts, which was rescued by LRRK2 specific kinase inhibition. These findings demonstrate an important role for LRRK2 protein in regulation of mitochondrial clearance by the lysosomes, which is hampered in PD with the G2019S mutation. The current results are relevant for cell phenotypic diagnostic approaches and potentially for stratification of PD patients for targeted therapy.

Neurite Collapse and Altered ER Ca2+ Control in Human Parkinson Disease Patient iPSC-Derived Neurons with LRRK2 G2019S Mutation.

The Parkinson disease (PD) genetic LRRK2 gain-of-function mutations may relate to the ER pathological changes seen in PD patients at postmortem. Human induced pluripotent stem cell (iPSC)-derived neurons with the PD pathogenic LRRK2 G2019S mutation exhibited neurite collapse when challenged with the ER Ca2+ influx sarco/ER Ca2+-ATPase inhibitor thapsigargin (THP). Baseline ER Ca2+ levels measured with the ER Ca2+ indicator CEPIA-ER were lower in LRRK2 G2019S human neurons, including in differentiated midbrain dopamine neurons in vitro. After THP challenge, PD patient-derived neurons displayed increased Ca2+ influx and decreased intracellular Ca2+ buffering upon membrane depolarization. These effects were reversed following LRRK2 mutation correction by antisense oligonucleotides and gene editing. Gene expression analysis in LRRK2 G2019S neurons identified modified levels of key store-operated Ca2+ entry regulators, with no alterations in ER Ca2+ efflux. These results demonstrate PD gene mutation LRRK2 G2019S ER calcium-dependent pathogenic effects in human neurons.

Lipid-dependent deposition of alpha-synuclein and Tau on neuronal Secretogranin II-positive vesicular membranes with age.

This report demonstrates insoluble alpha-synuclein (aSYN)+ aggregates in human sporadic Parkinson's disease (PD) midbrain that are linearly correlated with loss of glucocerebrosidase (GCase) activity. To identify early protein-lipid interactions that coincide with loss of lipid homeostasis, an aging study was carried out in mice with age-dependent reductions in GCase function. The analysis identified aberrant lipid-association by aSYN and hyperphosphorylated Tau (pTau) in a specific subset of neurotransmitter-containing, Secretogranin II (SgII)+ large, dense-core vesicles (LDCVs) responsible for neurotransmission of dopamine and other monoamines. The lipid vesicle-accumulation was concurrent with loss of PSD-95 suggesting synaptic destabilization. aSYN overexpression in the absence of lipid deregulation did not recapitulate the abnormal association with SgII+ vesicles. These results show lipid-dependent changes occur with age in neuronal vesicular membrane compartments that accumulate lipid-stabilized aSYN and pTau.

The glycoprotein GPNMB is selectively elevated in the substantia nigra of Parkinson's disease patients and increases after lysosomal stress.

GPNMB is a glycoprotein observed upon tissue damage and inflammation and is associated with astrocytes, microglia, and macrophages. Gene variations in GPNMB are linked with Parkinson's disease (PD) risk, and changes in protein levels of GPNMB have been found in lysosomal storage disorders, including Gaucher's disease with glucocerebrosidase (GCase) deficiency. In the current study, GPNMB increases were seen in the substantia nigra (SN) of PD patients compared to age-matched controls. Such PD patients have a decrease in GCase activity and corresponding elevation of glycosphingolipids in the SN (Rocha et al., 2015a). Interestingly, transgenic mice modelling synucleinopathy did not show GPNMB elevations or altered GCase activity levels compared to wild-type mice. However, upon CBE-induced GCase lysosomal dysfunction with elevated glycosphingolipids in wild-type mice, there were similar changes in GPNMB levels in the brain as seen in PD patient brains. These results indicate that GPNMB levels do not depend on alpha-synuclein load per se but relate directly to the lipidopathy changes induced by CBE-mediated GCase inhibition. The experimental modelling of elevating glycolipids resulted in GPNMB elevations with glial activation in several brain regions in mice. This is the first demonstration of region-specific elevations of GPNMB protein in Parkinson's disease. The presence of GPNMB in PD patient substantia nigra, the induction of GPNMB after experimental glycosphingolipid increases, but not with pure alpha-synucleinopathy, point towards the potential for primary lipid-induced degeneration in PD.

Glycosphingolipid levels and glucocerebrosidase activity are altered in normal aging of the mouse brain.

Aging is the predominant risk factor for both genetic and sporadic Parkinson's disease (PD). The majority of PD cases are nonfamilial, and the connection between aging and PD-associated genes is not well understood. Haploinsufficiency of the GBA gene, leading to a reduction in glucocerebrosidase (GCase) activity, is one of the most common genetic risk factors for PD. Furthermore, GCase activity is also reduced in brain regions of sporadic PD patients, with a corresponding accumulation of its glycosphingolipid (GSL) substrates. Recent findings in PD patients and aging control cases, and in human PD patient induced pluripotent stem cell neurons, have shown an age-dependent reduction in GCase activity and an elevation of some GSLs. We therefore asked whether aging-induced changes to both lysosomal and nonlysosomal GCase activity and GSL homeostasis in the brain could also be reflected in other nonhuman mammalian systems. Increases in brain polyubiquitin and the lysosomal-associated membrane protein, LAMP2A, were found in 24-month-old wild-type mice compared to 1.5-month-old mice. A lipidomic analysis was performed on brains of wild-type mice of different strains between 1.5 and 24 months of age. Aging created GSL changes that are reminiscent of sporadic PD. Levels of glucosylceramide, glucosylsphingosine, lactosylceramide, and GM1a were elevated in the brain of aged mice, and levels of complex gangliosides, GD1a, GD1b, and GT1b, were reduced with age. Parallel biochemical analyses revealed a change in lipid metabolism probably mediated by lysosomal hydrolases, with reduced GCase and increased neuraminidase activity. Based on these data, we hypothesize that perturbation of GSL metabolism in the aging brain may precede or may be part of abnormal protein handling and may accelerate PD pathophysiological processes in vulnerable neurons in PD and other age-related neurodegenerative disorders.