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Background: Parkinson’s disease (PD) is a neurodegenerative disorder that is characterized by the presence of proteinaceous alpha-synuclein (α-syn) inclusions (Lewy bodies), markers of neuroinflammation and the progressive loss of nigrostriatal dopamine (DA) neurons. These pathological features can be recapitulated in vivo using the α-syn preformed fibril (PFF) model of synucleinopathy. We have previously described the time course of microglial major-histocompatibility complex-II (MHC-II) expression and alterations in microglia morphology in the PFF model in rats. Specifically, the peaks of α-syn inclusion formation, MHC-II expression, and reactive morphology in the substantia nigra pars compacta (SNpc) all occur two months post PFF injection, months before neurodegeneration occurs. These results suggest that activated microglia may contribute to neurodegeneration and could represent a potential target for novel therapeutics. The goal of this study was to determine whether microglial depletion could impact the magnitude of α-syn aggregation, nigrostriatal degeneration, or related microglial activation during the α-syn PFF model. Methods: Male Fischer 344 rats were injected intrastriatally with either α-syn PFFs or saline. Rats were continuously administered Pexidartinib (PLX3397B, 600mg/kg), a colony stimulating factor-1 receptor (CSF1R) inhibitor, to deplete microglia for a period of either two or six months. Results: PLX3397B administration resulted in significant depletion (45-53%) of ionized calcium-binding adapter molecule 1 immunoreactive (Iba-1ir) microglia within the SNpc. Microglial depletion did not impact accumulation of phosphorylated α-syn (pSyn) within SNpc neurons and did not alter pSyn associated microglial reactivity or expression of MHC-II. Further, microglial depletion did not impact SNpc neuron degeneration. Paradoxically, long term microglial depletion resulted in increased soma size of remaining microglia in both control and PFF rats, as well as expression of MHC-II in extranigral regions. Conclusions: Collectively, our results suggest that microglial depletion is not a viable disease-modifying strategy for PD and that partial microglial depletion can induce a heightened proinflammatory state in remaining microglia.

Background Alpha-synuclein (α-syn) preformed fibril (PFF)-induced pathology can be used to study the features and progression of synucleinopathies, such as Parkinson's disease. Intrastriatal injection of mouse α-syn PFFs produce accumulation of α-syn pathology in both mice and rats. Previous studies in mice have revealed that greater sequence homology between the α-syn amino acid sequence used to produce PFFs with that of the endogenous host α-syn increases α-syn pathology in vivo. New methods Based on the prediction that greater sequence homology will result in more α-syn pathology, PFFs generated from recombinant rat α-syn (rPFFs) were used instead of PFFs produced from recombinant mouse α-syn (mPFFs), which are normally used in the model. Rats received unilateral intrastriatal injections of either rPFFs or mPFFs and accumulation of α-syn phosphorylated at serine 129 (pSyn) was examined at 1-month post-surgery. Results Rats injected with mPFFs exhibited abundant accumulation of α-syn inclusions in the substantia nigra and cortical regions, whereas in rats injected with rPFFs had significantly fewer SNpc neurons containing pSyn inclusions (≈60% fewer) and little, if any, pSyn inclusions were observed in the cortex. Conclusions Our results suggest that additional factors beyond the degree of sequence homology between host α-syn and injected recombinant α-syn impact efficiency of seeding and subsequent inclusion formation. More practically, these findings caution against the use of rPFFs in the rat preformed fibril model.

Subthalamic nucleus deep brain stimulation (STN DBS) protects dopaminergic neurons of the substantia nigra pars compacta (SNpc) against 6-OHDA and MPTP. We evaluated STN DBS in a parkinsonian model that displays α-synuclein pathology using unilateral, intranigral injections of recombinant adeno-associated virus pseudotype 2/5 to overexpress wildtype human α-synuclein (rAAV2/5 α-syn). A low titer of rAAV2/5 α-syn results in progressive forelimb asymmetry, loss of striatal dopaminergic terminal density and modest loss of SNpc dopamine neurons after eight weeks, corresponding to robust human-Snca expression and no effect on rat-Snca, Th, Bdnf or Trk2. α-syn overexpression increased phosphorylation of ribosomal protein S6 (p-rpS6) in SNpc neurons, a readout of trkB activation. Rats received intranigral injections of rAAV2/5 α-syn and three weeks later received four weeks of STN DBS or electrode implantation that remained inactive. STN DBS did not protect against α-syn-mediated deficits in forelimb akinesia, striatal denervation or loss of SNpc neuron, nor did STN DBS elevate p-rpS6 levels further. ON stimulation, forelimb asymmetry was exacerbated, indicating α-syn overexpression-mediated neurotransmission deficits. These results demonstrate that STN DBS does not protect the nigrostriatal system against α-syn overexpression-mediated toxicity. Whether STN DBS can be protective in other models of synucleinopathy is unknown.

Animal models that accurately recapitulate the accumulation of alpha-synuclein (α-syn) inclusions, progressive neurodegeneration of the nigrostriatal system and motor deficits can be useful tools for Parkinson's disease (PD) research. The preformed fibril (PFF) synucleinopathy model in rodents generally displays these PD-relevant features, however, the magnitude and predictability of these events is far from established. We therefore sought to optimize the magnitude of α-syn accumulation and nigrostriatal degeneration, and to understand the time course of both. Rats were injected unilaterally with different quantities of α-syn PFFs (8 or 16 μg of total protein) into striatal sites selected to concentrate α-syn inclusion formation in the substantia nigra pars compacta (SNpc). Rats displayed an α-syn PFF quantity-dependent increase in the magnitude of ipsilateral SNpc inclusion formation at 2 months and bilateral loss of nigral dopamine neurons at 6 months. Unilateral 16 μg PFF injection also resulted in modest sensorimotor deficits in forelimb adjusting steps associated with degeneration at 6 months. Bilateral injection of 16 μg α-syn PFFs resulted in symmetric bilateral degeneration equivalent to the ipsilateral nigral degeneration observed following unilateral 16 μg PFF injection (~50% loss). Bilateral PFF injections additionally resulted in alterations in several gait analysis parameters. These α-syn PFF parameters can be applied to generate a reproducible synucleinopathy model in rats with which to study pathogenic mechanisms and vet potential disease-modifying therapies.

Use of the in vivo alpha-synuclein preformed fibril (α-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson's disease synucleinopathy and nigrostriatal degeneration. The standardization of α-syn PFF generation and in vivo application is critical in order to ensure consistent, robust α-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric α-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of α-syn PFFs into rats or mice. Starting with monomeric α-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed α-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated α-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.

Background Converging evidence suggests a role for microglia-mediated neuroinflammation in Parkinson’s disease (PD). Animal models of PD can serve as a platform to investigate the role of neuroinflammation in degeneration in PD. However, due to features of the previously available PD models, interpretations of the role of neuroinflammation as a contributor to or a consequence of neurodegeneration have remained elusive. In the present study, we investigated the temporal relationship of neuroinflammation in a model of synucleinopathy following intrastriatal injection of pre-formed alpha-synuclein fibrils (α-syn PFFS). Methods Male Fischer 344 rats (N = 114) received unilateral intrastriatal injections of α-syn PFFs, PBS, or rat serum albumin with cohorts euthanized at monthly intervals up to 6 months. Quantification of dopamine neurons, total neurons, phosphorylated α-syn (pS129) aggregates, major histocompatibility complex-II (MHC-II) antigen-presenting microglia, and ionized calcium-binding adaptor molecule-1 (Iba-1) immunoreactive microglial soma size was performed in the substantia nigra. In addition, the cortex and striatum were also examined for the presence of pS129 aggregates and MHC-II antigen-presenting microglia to compare the temporal patterns of pSyn accumulation and reactive microgliosis. Results Intrastriatal injection of α-syn PFFs to rats resulted in widespread accumulation of phosphorylated α-syn inclusions in several areas that innervate the striatum followed by significant loss (~ 35%) of substantia nigra pars compacta dopamine neurons within 5–6 months. The peak magnitudes of α-syn inclusion formation, MHC-II expression, and reactive microglial morphology were all observed in the SN 2 months following injection and 3 months prior to nigral dopamine neuron loss. Surprisingly, MHC-II immunoreactivity in α-syn PFF injected rats was relatively limited during the later interval of degeneration. Moreover, we observed a significant correlation between substantia nigra pSyn inclusion load and number of microglia expressing MHC-II. In addition, we observed a similar relationship between α-syn inclusion load and number of microglia expressing MHC-II in cortical regions, but not in the striatum. Conclusions Our results demonstrate that increases in microglia displaying a reactive morphology and MHC-II expression occur in the substantia nigra in close association with peak numbers of pSyn inclusions, months prior to nigral dopamine neuron degeneration, and suggest that reactive microglia may contribute to vulnerability of SNc neurons to degeneration. The rat α-syn PFF model provides an opportunity to examine the innate immune response to accumulation of pathological α-syn in the context of normal levels of endogenous α-syn and provides insight into the earliest neuroinflammatory events in PD. Electronic supplementary material The online version of this article (10.1186/s12974-018-1171-z) contains supplementary material, which is available to authorized users.

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.

Gene therapy methods are increasingly used to model Parkinson's disease (PD) in animals in an effort to test experimental therapeutics within a more relevant context to disease pathophysiology and neuropathology. We have detailed several criteria that are critical or advantageous to accurately modeling PD in a murine model or in a nonhuman primate. Using these criteria, we then evaluate approaches made to model PD using viral vectors to date, including both adeno-associated viruses and lentiviruses. Lastly, we comment on the consideration of aging as a critical factor for modeling PD.

Gene therapy methods are increasingly used to model Parkinson's disease (PD) in animals in an effort to test experimental therapeutics within a more relevant context to disease pathophysiology and neuropathology. We have detailed several criteria that are critical or advantageous to accurately modeling PD in a murine model or in a nonhuman primate. Using these criteria, we then evaluate approaches made to model PD using viral vectors to date, including both adeno-associated viruses and lentiviruses. Lastly, we comment on the consideration of aging as a critical factor for modeling PD.