Silencing of Glucocerebrosidase Gene in Drosophila Enhances the Aggregation of Parkinson's Disease Associated ?-Synuclein Mutant A53T and Affects Locomotor Activity.
ABSTRACT: Background: Mutations in glucocerebrosidase (GBA), a lysosomal enzyme are the most common genetic risk factor for developing Parkinson's disease (PD). We studied how reduced GCase activity affects ?-synuclein (?-syn) and its mutants (A30P and A53T) aggregation, neurodegeneration, sleep and locomotor behavior in a fly model of PD. Methods: We developed drosophila with GBA gene knockdown (RNAi) (with reduced GCase activity) that simultaneously expresses either wildtype (WT) or mutants such as A30P or A53T ?-syn. Western blot and confocal microscopy were performed to study the ?-syn aggregation and neurodegeneration in these flies. We also studied the sleep and locomotor activity of those flies using Drosophila activity monitor (DAM) system. Results: Western blot analysis showed that GBA RNAi A53T ?-syn flies (30 days old) had an increased level of Triton insoluble synuclein (that corresponds to ?-syn aggregates) compared to corresponding A53T flies without GBA RNAi (control), while mRNA expression of ?-syn remained unchanged. Confocal imaging of whole brain staining of 30 days old drosophila showed a statistically significant decrease in neuron numbers in PPL1 cluster in flies expressing ?-syn WT, A30P and A53T in the presence GBA RNAi compared to corresponding control. Staining with conformation specific antibody for ?-syn aggregates showed an increased number of neurons staining for ?-syn aggregates in A53T fly brain with GBA RNAi compared to control A53T flies, thus confirming our protein analysis finding that under decreased GBA enzyme activity, mutant A53T aggregates more than the control A53T without GBA silencing. Sleep analysis revealed decreased total activity in GBA silenced flies expressing mutant A53T compared to both A53T control flies and GBA RNAi flies without synuclein expression. Conclusion: In A53T flies with reduced GCase activity, there is increased ?-syn aggregation and dopamine (DA) neuronal loss. This study demonstrates that reduced GCase activity both in the context of heterozygous GBA1 mutation associated with PD and in old age, contribute to increased aggregation of mutant ?-syn A53T and exacerbates the phenotype in a fly model of PD.
Project description:?-Synuclein (?-syn) is a synaptic protein in which four mutations (A53T, A30P, E46K and gene triplication) have been found to cause an autosomal dominant form of Parkinson's disease (PD). It is also the major component of intraneuronal protein aggregates, designated as Lewy bodies (LBs), a prominent pathological hallmark of PD. How ?-syn contributes to LB formation and PD is still not well-understood. It has been proposed that aggregation of ?-syn contributes to the formation of LBs, which then leads to neurodegeneration in PD. However, studies have also suggested that aggregates formation is a protective mechanism against more toxic ?-syn oligomers. In this study, we have generated ?-syn mutants that have increased propensity to form aggregates by attaching a CL1 peptide to the C-terminal of ?-syn. Data from our cellular study suggest an inverse correlation between cell viability and the amount of ?-syn aggregates formed in the cells. In addition, our animal model of PD indicates that attachment of CL1 to ?-syn enhanced its toxicity to dopaminergic neurons in an age-dependent manner and induced the formation of Lewy body-like ?-syn aggregates in the substantia nigra. These results provide new insights into how ?-syn-induced toxicity is related to its aggregation.
Project description:Mutations in alpha-synuclein (alpha-Syn) cause Parkinson's disease (PD) in a small number of pedigrees with familial PD. Moreover, alpha-Syn accumulates as a major component of Lewy bodies and Lewy neurites, intraneuronal inclusions that are neuropathological hallmarks of PD. To better understand the pathogenic relationship between alterations in the biology of alpha-Syn and PD-associated neurodegeneration, we generated multiple lines of transgenic mice expressing high levels of either wild-type or familial PD-linked Ala-30 --> Pro (A30P) or Ala-53 --> Thr (A53T) human alpha-Syns. The mice expressing the A53T human alpha-Syn, but not wild-type or the A30P variants, develop adult-onset neurodegenerative disease with a progressive motoric dysfunction leading to death. Pathologically, affected mice exhibit neuronal abnormalities (in perikarya and neurites) including pathological accumulations of alpha-Syn and ubiquitin. Consistent with abnormal neuronal accumulation of alpha-Syn, brain regions with pathology exhibit increases in detergent-insoluble alpha-Syn and alpha-Syn aggregates. Our results demonstrate that the A53T mutant alpha-Syn causes significantly greater in vivo neurotoxicity as compared with other alpha-Syn variants. Further, alpha-Syn-dependent neurodegeneration is associated with abnormal accumulation of detergent-insoluble alpha-Syn.
Project description:Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson's disease (PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant (GBA-/-) medaka that are completely deficient in glucocerebrosidase (GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (?-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of ?-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of ?-syn accumulation caused by GCase deficiency, and the minimal contribution of ?-syn to the pathogenesis of neuronopathic GD.
Project description: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.
Project description:The aggregation of ?-synuclein (?-Syn) is linked to Parkinson's disease. The mechanism of early aggregation steps and the effect of pathogenic single-point mutations remain elusive. We report here a single-molecule fluorescence study of ?-Syn dimerization and the effect of mutations. Specific interactions between tethered fluorophore-free ?-Syn monomers on a substrate and fluorophore-labeled monomers diffusing freely in solution were observed using total internal reflection fluorescence microscopy. The results showed that wild-type (WT) ?-Syn dimers adopt two types of dimers. The lifetimes of type 1 and type 2 dimers were determined to be 197 ± 3 ms and 3334 ± 145 ms, respectively. All three of the mutations used, A30P, E46K, and A53T, increased the lifetime of type 1 dimer and enhanced the relative population of type 2 dimer, with type 1 dimer constituting the major fraction. The kinetic stability of type 1 dimers (expressed in terms of lifetime) followed the order A30P (693 ± 14 ms) > E46K (292 ± 5 ms) > A53T (226 ± 6 ms) > WT (197 ± 3 ms). Type 2 dimers, which are more stable, had lifetimes in the range of several seconds. The strongest effect, observed for the A30P mutant, resulted in a lifetime 3.5 times higher than observed for the WT type 1 dimer. This mutation also doubled the relative fraction of type 2 dimer. These data show that single-point mutations promote dimerization, and they suggest that the structural heterogeneity of ?-Syn dimers could lead to different aggregation pathways.
Project description:The etiology of Parkinson's disease (PD) converges on a common pathogenic pathway of mitochondrial defects in which ?-Synuclein (?Syn) is thought to play a role. However, the mechanisms by which ?Syn and its disease-associated allelic variants cause mitochondrial dysfunction remain unknown. Here, we analyzed mitochondrial axonal transport and morphology in human-derived neurons overexpressing wild-type (WT) ?Syn or the mutated variants A30P or A53T, which are known to have differential lipid affinities. A53T ?Syn was enriched in mitochondrial fractions, inducing significant mitochondrial transport defects and fragmentation, while milder defects were elicited by WT and A30P. We found that ?Syn-mediated mitochondrial fragmentation was linked to expression levels in WT and A53T variants. Targeted delivery of WT and A53T ?Syn to the outer mitochondrial membrane further increased fragmentation, whereas A30P did not. Genomic editing to disrupt the N-terminal domain of ?Syn, which is important for membrane association, resulted in mitochondrial elongation without changes in fusion-fission protein levels, suggesting that ?Syn plays a direct physiological role in mitochondrial size maintenance. Thus, we demonstrate that the association of ?Syn with the mitochondria, which is modulated by protein mutation and dosage, influences mitochondrial transport and morphology, highlighting its relevance in a common pathway impaired in PD.
Project description:Many neuropathological and experimental studies suggest that the degeneration of dopaminergic terminals and axons precedes the demise of dopaminergic neurons in the substantia nigra, which finally results in the clinical symptoms of Parkinson disease (PD). The mechanisms underlying this early axonal degeneration are, however, still poorly understood. Here, we examined the effects of overexpression of human wildtype alpha-synuclein (?Syn-WT), a protein associated with PD, and its mutant variants ?Syn-A30P and -A53T on neurite morphology and functional parameters in rat primary midbrain neurons (PMN). Moreover, axonal degeneration after overexpression of ?Syn-WT and -A30P was analyzed by live imaging in the rat optic nerve in vivo. We found that overexpression of ?Syn-WT and of its mutants A30P and A53T impaired neurite outgrowth of PMN and affected neurite branching assessed by Sholl analysis in a variant-dependent manner. Surprisingly, the number of primary neurites per neuron was increased in neurons transfected with ?Syn. Axonal vesicle transport was examined by live imaging of PMN co-transfected with EGFP-labeled synaptophysin. Overexpression of all ?Syn variants significantly decreased the number of motile vesicles and decelerated vesicle transport compared with control. Macroautophagic flux in PMN was enhanced by ?Syn-WT and -A53T but not by ?Syn-A30P. Correspondingly, colocalization of ?Syn and the autophagy marker LC3 was reduced for ?Syn-A30P compared with the other ?Syn variants. The number of mitochondria colocalizing with LC3 as a marker for mitophagy did not differ among the groups. In the rat optic nerve, both ?Syn-WT and -A30P accelerated kinetics of acute axonal degeneration following crush lesion as analyzed by in vivo live imaging. We conclude that ?Syn overexpression impairs neurite outgrowth and augments axonal degeneration, whereas axonal vesicle transport and autophagy are severely altered.
Project description:The presynaptic protein ?-synuclein (?-syn), particularly in its amyloid form, is widely recognized for its involvement in Parkinson disease (PD). Recent genetic studies reveal that mutations in the gene GBA are the most widespread genetic risk factor for parkinsonism identified to date. GBA encodes for glucocerebrosidase (GCase), the enzyme deficient in the lysosomal storage disorder, Gaucher disease (GD). In this work, we investigated the possibility of a physical linkage between ?-syn and GCase, examining both wild type and the GD-related N370S mutant enzyme. Using fluorescence and nuclear magnetic resonance spectroscopy, we determined that ?-syn and GCase interact selectively under lysosomal solution conditions (pH 5.5) and mapped the interaction site to the ?-syn C-terminal residues, 118-137. This ?-syn-GCase complex does not form at pH 7.4 and is stabilized by electrostatics, with dissociation constants ranging from 1.2 to 22 ?m in the presence of 25 to 100 mm NaCl. Intriguingly, the N370S mutant form of GCase has a reduced affinity for ?-syn, as does the inhibitor conduritol-?-epoxide-bound enzyme. Immunoprecipitation and immunofluorescence studies verified this interaction in human tissue and neuronal cell culture, respectively. Although our data do not preclude protein-protein interactions in other cellular milieux, we suggest that the ?-syn-GCase association is favored in the lysosome, and that this noncovalent interaction provides the groundwork to explore molecular mechanisms linking PD with mutant GBA alleles.
Project description:Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body-like structures that contain ?-synuclein (?-Syn). We reveal that Munc18-1 binds ?-Syn, and its EIEE mutants coaggregate ?-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of ?-Syn(WT) and that of the Parkinson's disease-causing ?-Syn(A30P) mutant, an effect rescued by Munc18-1(WT) expression, indicative of chaperone activity. Coexpression of the ?-Syn(A30P) mutant with Munc18-1 reduced the number of ?-Syn(A30P) aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for ?-Syn leading to aggregation-induced neurodegeneration.
Project description:Parkinson's disease (PD) pathology is characterized by the formation of intra-neuronal inclusions called Lewy bodies, which are comprised of alpha-synuclein (?-syn). Duplication, triplication or genetic mutations in ?-syn (A53T, A30P and E46K) are linked to autosomal dominant PD; thus implicating its role in the pathogenesis of PD. In both PD patients and mouse models, there is increasing evidence that neuronal dysfunction occurs before the accumulation of protein aggregates (i.e., ?-syn) and neurodegeneration. Characterization of the timing and nature of symptomatic dysfunction is important for understanding the impact of ?-syn on disease progression. Furthermore, this knowledge is essential for identifying pathways and molecular targets for therapeutic intervention. To this end, we examined various functional and morphological endpoints in the transgenic mouse model expressing the human A53T ?-syn variant directed by the mouse prion promoter at specific ages relating to disease progression (2, 6 and 12 months of age). Our findings indicate A53T mice develop fine, sensorimotor, and synaptic deficits before the onset of age-related gross motor and cognitive dysfunction. Results from open field and rotarod tests show A53T mice develop age-dependent changes in locomotor activity and reduced anxiety-like behavior. Additionally, digigait analysis shows these mice develop an abnormal gait by 12 months of age. A53T mice also exhibit spatial memory deficits at 6 and 12 months, as demonstrated by Y-maze performance. In contrast to gross motor and cognitive changes, A53T mice display significant impairments in fine- and sensorimotor tasks such as grooming, nest building and acoustic startle as early as 1-2 months of age. These mice also show significant abnormalities in basal synaptic transmission, paired-pulse facilitation and long-term depression (LTD). Combined, these data indicate the A53T model exhibits early- and late-onset behavioral and synaptic impairments similar to PD patients and may provide useful endpoints for assessing novel therapeutic interventions for PD.