Dataset Information

Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents.

ABSTRACT: Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting approximately one-percent of the population over the age of sixty. Although many animal models have been developed to study this disease, each model presents its own advantages and caveats. A unique model has arisen to study the role of alpha-synuclein (aSyn) in the pathogenesis of PD. This model involves the conversion of recombinant monomeric aSyn protein to a fibrillar form-the aSyn pre-formed fibril (aSyn PFF)-which is then injected into the brain or introduced to the media in culture. Although many groups have successfully adopted and replicated the aSyn PFF model, issues with generating consistent pathology have been reported by investigators. To improve the replicability of this model and diminish these issues, The Michael J. Fox Foundation for Parkinson's Research (MJFF) has enlisted the help of field leaders who performed key experiments to establish the aSyn PFF model to provide the research community with guidelines and practical tips for improving the robustness and success of this model. Specifically, we identify key pitfalls and suggestions for avoiding these mistakes as they relate to generating the aSyn PFFs from monomeric protein, validating the formation of pathogenic aSyn PFFs, and using the aSyn PFFs in vivo or in vitro to model PD. With this additional information, adoption and use of the aSyn PFF model should present fewer challenges, resulting in a robust and widely available model of PD.

SUBMITTER: Polinski NK

PROVIDER: S-EPMC6004926 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents.

Polinski Nicole K NK Volpicelli-Daley Laura A LA Sortwell Caryl E CE Luk Kelvin C KC Cremades Nunilo N Gottler Lindsey M LM Froula Jessica J Duffy Megan F MF Lee Virginia M Y VMY Martinez Terina N TN Dave Kuldip D KD

Journal of Parkinson's disease 20180101 2

Parkinson's disease (PD) is the second most common neurodegenerative disease, affecting approximately one-percent of the population over the age of sixty. Although many animal models have been developed to study this disease, each model presents its own advantages and caveats. A unique model has arisen to study the role of alpha-synuclein (aSyn) in the pathogenesis of PD. This model involves the conversion of recombinant monomeric aSyn protein to a fibrillar form-the aSyn pre-formed fibril (aSyn ...[more]

PMID: 29400668

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Project description:Previous studies demonstrate that intrastriatal injections of fibrillar alpha-synuclein (α-syn) into mice induce Parkinson's disease (PD)-like Lewy body (LB) pathology formed by aggregated α-syn in anatomically interconnected regions and significant nigrostriatal degeneration. The aim of the current study was to evaluate whether exogenous mouse α-syn pre-formed fibrils (PFF) injected into the striatum of rats would result in accumulation of LB-like intracellular inclusions and nigrostriatal degeneration. Sprague-Dawley rats received unilateral intrastriatal injections of either non-fibrillized recombinant α-syn or PFF mouse α-syn in 1- or 2- sites and were euthanized at 30, 60 or 180 days post-injection (pi). Both non-fibrillized recombinant α-syn and PFF α-syn injections resulted in phosphorylated α-syn intraneuronal accumulations (i.e., diffuse Lewy neurite (LN)- and LB-like inclusions) with significantly greater accumulations following PFF injection. LB-like inclusions were observed in several areas that innervate the striatum, most prominently the frontal and insular cortices, the amygdala, and the substantia nigra pars compacta (SNpc). α-Syn accumulations co-localized with ubiquitin, p62, and were thioflavin-S-positive and proteinase-k resistant, suggesting that PFF-induced pathology exhibits properties similar to human LBs. Although α-syn inclusions within the SNpc remained ipsilateral to striatal injection, we observed bilateral reductions in nigral dopamine neurons at the 180-day time-point in both the 1- and 2-site PFF injection paradigms. PFF injected rats exhibited bilateral reductions in striatal dopaminergic innervation at 60 and 180 days and bilateral decreases in homovanillic acid; however, dopamine reduction was observed only in the striatum ipsilateral to PFF injection. Although the level of dopamine asymmetry in PFF injected rats at 180 days was insufficient to elicit motor deficits in amphetamine-induced rotations or forelimb use in the cylinder task, significant disruption of ultrasonic vocalizations was observed. Taken together, our findings demonstrate that α-syn PFF are sufficient to seed the pathological conversion and propagation of endogenous α-syn to induce a progressive, neurodegenerative model of α-synucleinopathy in rats.

Project description:The endocytic pathway is both an essential route of molecular uptake in cells and a potential entry point for pathology-inducing cargo. The cell-to-cell spread of cytotoxic aggregates, such as those of α-synuclein (α-syn) in Parkinson's Disease (PD), exemplifies this duality. Here we used a human iPSC-derived induced neuronal model (iNs) prone to death mediated by aggregation in late endosomes and lysosomes of endogenous α-syn, seeded by internalized pre-formed fibrils of α-syn (PFFs). This PFF-mediated death was not observed with parental iPSCs or other non-neuronal cells. Using live-cell optical microscopy to visualize the read out of biosensors reporting endo-lysosome wounding, we discovered that up to about 10% of late endosomes and lysosomes in iNs exhibited spontaneous constitutive perforations, regardless of the presence of internalized PFFs. This wounding, absent in parental iPSCs and non-neuronal cells, corresponded to partial damage by nanopores in the limiting membranes of a subset of endolysosomes directly observed by volumetric focused ion beam scanning electron microscopy (FIB-SEM) in iNs and in CA1 pyramidal neurons from mouse brain, and not found in iPSCs or in other non-neuronal cells in culture or in mouse liver and skin. We suggest that the compromised limiting membranes in iNs and neurons in general are the primary conduit for cytosolic α-syn to access PFFs entrapped within endo-lysosomal lumens, initiating PFF-mediated α-syn aggregation. Significantly, eradicating the intrinsic endolysosomal perforations in iNs by inhibiting the endosomal Phosphatidylinositol-3-Phosphate/Phosphatidylinositol 5-Kinase (PIKfyve kinase) using Apilimod or Vacuolin-1 markedly reduced PFF-induced α-syn aggregation, despite PFFs continuing to enter the endolysosomal compartment. Crucially, this intervention also diminished iN death associated with PFF incubation. Our results reveal the surprising presence of intrinsically perforated endo-lysosomes in neurons, underscoring their crucial early involvement in the genesis of toxic α-syn aggregates induced by internalized PFFs. This discovery offers a basis for employing PIKfyve kinase inhibition as a potential therapeutic strategy to counteract synucleinopathies.

Dataset Information

Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents.

Publications

Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents.

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