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Glucocerebrosidase deficiency promotes release of ?-synuclein fibrils from cultured neurons.

ABSTRACT: 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 ?-synuclein (?-syn) levels. Furthermore, elevated ?-syn results in decreased GCase activity. Although the role of ?-syn in PD remains unclear, evidence indicates that aggregated ?-syn fibrils are a pathogenic species in PD, passing between neurons and inducing endogenous native ?-syn to aggregate; spreading pathology through the brain. We have investigated if preformed ?-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 ?-syn pathology to naïve cells. Neurons treated with PFFs induced endogenous ?-syn to become insoluble and phosphorylated at Ser129 to a greater extent than monomeric ?-syn-treatment. PFFs, but not monomeric ?-syn, inhibited lysosomal GCase activity in these cells and induced the unfolded protein response. Neurons in which GCase was inhibited by conduritol ?-epoxide did not increase the amount of insoluble monomeric ?-syn or its phosphorylation status. Instead the release of ?-syn fibrils from GCase deficient cells was significantly increased. Co-culture studies showed that the transfer of ?-syn pathology to naïve cells was greater from GCase deficient cells. This study suggests that GCase deficiency increases the spread of ?-syn pathology and likely contributes to the earlier age of onset and increased cognitive decline associated with GBA-PD.

SUBMITTER: Gegg ME

PROVIDER: S-EPMC7322566 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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

Gegg Matthew E ME Verona Guglielmo G Schapira Anthony H V AHV

Human molecular genetics 20200601 10

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 aggrega ...[more]

PMID: 32391886

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Project description:Synucleinopathies are characterized by the accumulation of insoluble ?-synuclein (?Syn). To test whether ?Syn aggregates modulate synaptic activity, we used a recently developed model in primary neurons for inducing ?Syn pathology. We demonstrated that preformed fibrils (PFFs) generated with recombinant human ?Syn compromised synaptic activity in a time- and dose-dependent manner and that the magnitude of these deficits correlated with the formation of ?Syn pathology in cultured excitatory hippocampal neurons from both sexes of mice. Remarkably, acute passive infusion of ?Syn PFFs from whole-cell patch-clamp pipette decreased mEPSC frequency within 10 min followed by induction of ?Syn pathology within 1 d. Moreover, by direct addition of ?Syn PFFs into culture medium, the formation of misfolded ?Syn inclusions dramatically compromised the colocalization of synaptic markers and altered dynamic changes of dendritic spines, but the viability of neurons was not affected up to 7 d post-treatment with ?Syn PFFs. Our data indicate that intraneuronal ?Syn fibrils impaired the initiation of synaptogenesis and their physiological functions, thereby suggesting that targeting synaptic dysfunction in synucleinopathies may provide a promising therapeutic direction.SIGNIFICANCE STATEMENT Under pathological conditions, the presynaptic protein ?-synuclein (?Syn) aggregates to form intraneuronal inclusions. To understand how and to what extent ?Syn aggregates modulate synaptic activity before neuron loss, we demonstrate that ?Syn preformed fibrils (PFFs) reduced synaptic activity in a dose- and time-dependent manner. The magnitude of these deficits correlated with the deposition of ?Syn pathology, which dramatically compromised the colocalization of synaptic markers and altered the dendritic spine dynamics. The present work further highlights the impact of ?Syn PFFs on synaptogenesis and physiological function, which may be applicable to other types of synucleinopathies.

Project description:Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinson's disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. We find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. These results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

Project description:The distinct neuropathological features of the different α-Synucleinopathies, as well as the diversity of the α-Synuclein (α-Syn) intracellular inclusion bodies observed in post mortem brain sections, are thought to reflect the strain diversity characterizing invasive α-Syn amyloids. However, this "one strain, one disease" view is still hypothetical, and to date, a possible disease-specific contribution of non-amyloid factors has not been ruled out. In Multiple System Atrophy (MSA), the buildup of α-Syn inclusions in oligodendrocytes seems to result from the terminal storage of α-Syn amyloid aggregates first pre-assembled in neurons. This assembly occurs at the level of neuronal cytoplasmic inclusions, and even earlier, within neuronal intranuclear inclusions (NIIs). Intriguingly, α-Syn NIIs are never observed in α-Synucleinopathies other than MSA, suggesting that these inclusions originate (i) from the unique molecular properties of the α-Syn fibril strains encountered in this disease, or alternatively, (ii) from other factors specifically dysregulated in MSA and driving the intranuclear fibrillization of α-Syn. We report the isolation and structural characterization of a synthetic human α-Syn fibril strain uniquely capable of seeding α-Syn fibrillization inside the nuclear compartment. In primary mouse cortical neurons, this strain provokes the buildup of NIIs with a remarkable morphology reminiscent of cat's eye marbles (see video abstract). These α-Syn inclusions form giant patterns made of one, two, or three lentiform beams that span the whole intranuclear volume, pushing apart the chromatin. The input fibrils are no longer detectable inside the NIIs, where they become dominated by the aggregation of endogenous α-Syn. In addition to its phosphorylation at S129, α-Syn forming the NIIs acquires an epitope antibody reactivity profile that indicates its organization into fibrils, and is associated with the classical markers of α-Syn pathology p62 and ubiquitin. NIIs are also observed in vivo after intracerebral injection of the fibril strain in mice. Our data thus show that the ability to seed NIIs is a strain property that is integrally encoded in the fibril supramolecular architecture. Upstream alterations of cellular mechanisms are not required. In contrast to the lentiform TDP-43 NIIs, which are observed in certain frontotemporal dementias and which are conditional upon GRN or VCP mutations, our data support the hypothesis that the presence of α-Syn NIIs in MSA is instead purely amyloid-strain-dependent.

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Glucocerebrosidase deficiency promotes release of ?-synuclein fibrils from cultured neurons.

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

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