Sporadic Parkinson's Disease Brains Research Articles

Glucocerebrosidase deficiency promotes release of α-synuclein fibrils from cultured neurons

Mutations in the GBA gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase), are the most important genetic risk factor for Parkinson disease (PD). GCase activity is also decreased in sporadic PD brains and with normal ageing. Loss of GCase activity impairs the autophagy lysosomal pathway resulting in increased α-synuclein (α-syn) levels. Furthermore, elevated α-syn results in decreased GCase activity. Although the role of α-syn in PD remains unclear, evidence indicates that aggregated α-syn fibrils are a pathogenic species in PD, passing between neurons and inducing endogenous native α-syn to aggregate; spreading pathology through the brain. We have investigated if preformed α-syn fibrils (PFFs) impair GCase activity in mouse cortical neurons and differentiated dopaminergic cells, and whether GCase deficiency in these models increased the transfer of α-syn pathology to naïve cells. Neurons treated with PFFs induced endogenous α-syn to become insoluble and phosphorylated at Ser129 to a greater extent than monomeric α-syn-treatment. PFFs, but not monomeric α-syn, inhibited lysosomal GCase activity in these cells and induced the unfolded protein response. Neurons in which GCase was inhibited by conduritol β-epoxide did not increase the amount of insoluble monomeric α-syn or its phosphorylation status. Instead the release of α-syn fibrils from GCase deficient cells was significantly increased. Co-culture studies showed that the transfer of α-syn pathology to naïve cells was greater from GCase deficient cells. This study suggests that GCase deficiency increases the spread of α-syn pathology and likely contributes to the earlier age of onset and increased cognitive decline associated with GBA-PD.

Synj1 haploinsufficiency causes dopamine neuron vulnerability and alpha-synuclein accumulation in mice.

Synaptojanin1 (synj1) is a phosphoinositide phosphatase with dual SAC1 and 5'-phosphatase enzymatic activities in regulating phospholipid signaling. The brain-enriched isoform has been shown to participate in synaptic vesicle (SV) recycling. More recently, recessive human mutations were identified in the two phosphatase domains of SYNJ1, including R258Q, R459P and R839C, which are linked to rare forms of early-onset Parkinsonism. We now demonstrate that Synj1 heterozygous deletion (Synj1+/-), which is associated with an impaired 5'-phosphatase activity, also leads to Parkinson's disease (PD)-like pathologies in mice. We report that male Synj1+/- mice display age-dependent motor function abnormalities as well as alpha-synuclein accumulation, impaired autophagy and dopaminergic terminal degeneration. Synj1+/- mice contain elevated 5'-phosphatase substrate, PI(4,5)P2, particularly in the midbrain neurons. Moreover, pharmacological elevation of membrane PI(4,5)P2 in cultured neurons impairs SV endocytosis, specifically in midbrain neurons, and further exacerbates SV trafficking defects in Synj1+/- midbrain neurons. We demonstrate down-regulation of SYNJ1 transcript in a subset of sporadic PD brains, implicating a potential role of Synj1 deficiency in the decline of dopaminergic function during aging.

The role of glucocerebrosidase in Parkinson disease pathogenesis.

GBA encodes the lysosomal enzyme glucocerebrosidase (GCase), an enzyme involved in sphingolipid metabolism. Mutations in the GBA gene are numerically the most important risk factor for developing Parkinson disease (PD) accounting for at least 5% of all PD cases. Furthermore, loss of GCase activity is found in sporadic PD brains. Lysosomal dysfunction is thought to play a principal role in PD pathogenesis and in particular its effect on the metabolism of α-synuclein. A hallmark of PD is the presence of intraneuronal protein inclusions called Lewy bodies, which are composed mainly of α-synuclein. Cellular and animal models of GCase deficiency result in lysosomal dysfunction, and in particular the autophagy lysosome pathway, resulting in the accumulation of α-synuclein. Some forms of mutant GCase unfold in the endoplasmic reticulum activating the unfolded protein response, which might also contribute to PD pathogenesis. It has also been suggested that accumulation of GCase substrates glucosylceramide/glucosylsphingosine may contribute to GBA-PD pathogenesis. Mitochondrial dysfunction and neuroinflammation are associated with GCase deficiency and have also been implicated in the aetiology of PD. This review discusses these points and highlights potential treatments that might be effective in treating GCase deficiency in PD.

No evidence for substrate accumulation in Parkinson brains with GBA mutations.

BackgroundTo establish whether Parkinson's disease (PD) brains previously described to have decreased glucocerebrosidase activity exhibit accumulation of the lysosomal enzyme's substrate, glucosylceramide, or other changes in lipid composition.MethodsLipidomic analyses and cholesterol measurements were performed on the putamen (n = 5‐7) and cerebellum (n = 7‐14) of controls, Parkinson's disease brains with heterozygote GBA1 mutations (PD+GBA), or sporadic PD.ResultsTotal glucosylceramide levels were unchanged in both PD+GBA and sporadic PD brains when compared with controls. No changes in glucosylsphingosine (deacetylated glucosylceramide), sphingomyelin, gangliosides (GM2, GM3), or total cholesterol were observed in either putamen or cerebellum.ConclusionsThis study did not demonstrate glucocerebrosidase substrate accumulation in PD brains with heterozygote GBA1 mutations in areas of the brain with low α‐synuclein pathology. © 2015 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Lysosomal impairment in Parkinson's disease

Impairment of autophagy-lysosomal pathways (ALPs) is increasingly regarded as a major pathogenic event in neurodegenerative diseases, including Parkinson's disease (PD). ALP alterations are observed in sporadic PD brains and in toxic and genetic rodent models of PD-related neurodegeneration. In addition, PD-linked mutations and post-translational modifications of α-synuclein impair its own lysosomal-mediated degradation, thereby contributing to its accumulation and aggregation. Furthermore, other PD-related genes, such as leucine-rich repeat kinase-2 (LRRK2), parkin, and phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), have been mechanistically linked to alterations in ALPs. Conversely, mutations in lysosomal-related genes, such as glucocerebrosidase (GBA) and lysosomal type 5 P-type ATPase (ATP13A2), have been linked to PD. New data offer mechanistic molecular evidence for such a connection, unraveling a causal link between lysosomal impairment, α-synuclein accumulation, and neurotoxicity. First, PD-related GBA deficiency/mutations initiate a positive feedback loop in which reduced lysosomal function leads to α-synuclein accumulation, which, in turn, further decreases lysosomal GBA activity by impairing the trafficking of GBA from the endoplasmic reticulum-Golgi to lysosomes, leading to neurodegeneration. Second, PD-related mutations/deficiency in the ATP13A2 gene lead to a general lysosomal impairment characterized by lysosomal membrane instability, impaired lysosomal acidification, decreased processing of lysosomal enzymes, reduced degradation of lysosomal substrates, and diminished clearance of autophagosomes, collectively contributing to α-synuclein accumulation and cell death. According to these new findings, primary lysosomal defects could potentially account for Lewy body formation and neurodegeneration in PD, laying the groundwork for the prospective development of new neuroprotective/disease-modifying therapeutic strategies aimed at restoring lysosomal levels and function.

MicroRNA-205 regulates the expression of Parkinson's disease-related leucine-rich repeat kinase 2 protein

Recent genome-wide association studies indicate that a simple alteration of Leucine-rich repeat kinase 2 (LRRK2) gene expression may contribute to the etiology of sporadic Parkinson's disease (PD). However, the expression and regulation of LRRK2 protein in the sporadic PD brains remain to be determined. Here, we found that the expression of LRRK2 protein was enhanced in the sporadic PD patients using the frontal cortex tissue from a set of 16 PD patients and 7 control samples. In contrast, no significant difference was detected in the level of LRRK2 mRNA expression between the control and PD cases, suggesting a potential post-transcriptional modification of the LRRK2 protein expression in the sporadic PD brains. Indeed, it was identified that microRNA-205 (miR-205) suppressed the expression of LRRK2 protein through a conserved-binding site at the 3'-untranslated region (UTR) of LRRK2 gene. Interestingly, miR-205 expression was significantly downregulated in the brains of patients with sporadic PD, showing the enhanced LRRK2 protein levels. Also, in vitro studies in the cell lines and primary neuron cultures further established the role of miR-205 in modulating the expression of LRRK2 protein. In addition, introduction of miR-205 prevented the neurite outgrowth defects in the neurons expressing a PD-related LRRK2 R1441G mutant. Together, these findings suggest that downregulation of miR-205 may contribute to the potential pathogenic elevation of LRRK2 protein in the brains of patients with sporadic PD, while overexpression of miR-205 may provide an applicable therapeutic strategy to suppress the abnormal upregulation of LRRK2 protein in PD.

Glucocerebrosidase deficiency in substantia nigra of parkinson disease brains

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, α-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 α-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. ANN NEUROL 2012;72:455–463.

TRAF6 promotes atypical ubiquitination of mutant DJ-1 and alpha-synuclein and is localized to Lewy bodies in sporadic Parkinson's disease brains

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the Substantia Nigra and the formation of ubiquitin- and alpha-synuclein (aSYN)-positive cytoplasmic inclusions called Lewy bodies (LBs). Although most PD cases are sporadic, families with genetic mutations have been found. Mutations in PARK7/DJ-1 have been associated with autosomal recessive early-onset PD, while missense mutations or duplications of aSYN (PARK1, PARK4) have been linked to dominant forms of the disease. In this study, we identify the E3 ubiquitin ligase tumor necrosis factor-receptor associated factor 6 (TRAF6) as a common player in genetic and sporadic cases. TRAF6 binds misfolded mutant DJ-1 and aSYN. Both proteins are substrates of TRAF6 ligase activity in vivo. Interestingly, rather than conventional K63 assembly, TRAF6 promotes atypical ubiquitin linkage formation to both PD targets that share K6-, K27- and K29- mediated ubiquitination. Importantly, TRAF6 stimulates the accumulation of insoluble and polyubiquitinated mutant DJ-1 into cytoplasmic aggregates. In human post-mortem brains of PD patients, TRAF6 protein colocalizes with aSYN in LBs. These results reveal a novel role for TRAF6 and for atypical ubiquitination in PD pathogenesis.

A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

The aim of this study was to develop a new model of sporadic Parkinson disease (PD) based on silencing of the SKP1A gene, a component of the ubiquitin-proteasome/E3 ligase complex, Skp1, Cullin 1, F-box protein, which was found to be highly decreased in the substantia nigra of sporadic PD patients. Initially, an embryonic mouse substantia nigra-derived cell line (SN4741 cells) was infected with short hairpin RNA lentiviruses encoding the murine transcript of the SKP1A gene or with scrambled vector. SKP1A silencing resulted in increased susceptibility to neuronal damages induced by the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium ion and serum starvation, in parallel with a decline in the expression of the dopaminergic markers, dopamine transporter and vesicular monoamine transporter-2. SKP1A-deficient cells presented a delay in completion of the cell cycle and the inability to arrest at the G(0)/G(1) phase when induced to differentiate. Instead, the cells progressed through S phase, developing rounded aggregates with characteristics of aggresomes including immunoreactivity for gamma-tubulin, alpha-synuclein, ubiquitin, tyrosine hydroxylase, Hsc-70 (70-kDa heat shock cognate protein), and proteasome subunit, and culminating in a lethal phenotype. Conversely, stably enforced expression of wild type SKP1A duplicated the survival index of naïve SN4741 cells under proteasomal inhibition injury, suggesting a new structural role of SKP1 in dopaminergic neuronal function, besides its E3 ligase activity. These results link, for the first time, SKP1 to dopamine neuronal function and survival, suggesting an essential role in sporadic PD. In summary, this new model has reproduced to a significant extent the molecular alterations described in sporadic PD at the cellular level, implicating Skp1 as a potential modifier in sporadic PD neurodegeneration.

Parkin is associated with actin filaments in neuronal and nonneural cells

Inactivating mutations of the gene encoding parkin are responsible for autosomal recessive juvenile parkinsonism (AR-JP). However, little information is known about the function and distribution of parkin. We generated antibodies to two different peptides of parkin. By Western blot analysis and immunohistochemistry, we found that parkin is a 50-kd protein that is expressed in neuronal processes and cytoplasm of selected neurons in the basal ganglia, midbrain, cerebellum, and cerebral cortex. Unlike ubiquitin and α-synuclein, parkin labeling was not found in Lewy bodies of four sporadic Parkinson disease brains. Parkin was colocalized with actin filaments but not with microtubules in COS1 kidney cells and nerve growth factor–induced PC12 neurons. These results point to the importance of the cytoskeleton and associated proteins in neurodegeneration. Ann Neurol 2000;48:737–744

Parkin is associated with actin filaments in neuronal and nonneural cells

Inactivating mutations of the gene encoding parkin are responsible for autosomal recessive juvenile parkinsonism (AR-JP). However, little information is known about the function and distribution of parkin. We generated antibodies to two different peptides of parkin. By Western blot analysis and immunohistochemistry, we found that parkin is a 50-kd protein that is expressed in neuronal processes and cytoplasm of selected neurons in the basal ganglia, midbrain, cerebellum, and cerebral cortex. Unlike ubiquitin and alpha-synuclein, parkin labeling was not found in Lewy bodies of four sporadic Parkinson disease brains. Parkin was colocalized with actin filaments but not with microtubules in COS1 kidney cells and nerve growth factor-induced PC12 neurons. These results point to the importance of the cytoskeleton and associated proteins in neurodegeneration.