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Abstract Parkinson’s disease (PD) is characterized by neuroinflammation, progressive loss of dopaminergic neurons, and accumulation of α-synuclein (α-Syn) into insoluble aggregates called Lewy pathology. The Line 61 α-Syn mouse is an established preclinical model of PD; Thy-1 is used to promote human α-Syn expression, and features of sporadic PD develop at 9–18 months of age. To accelerate the PD phenotypes, we injected sonicated human α-Syn preformed fibrils (PFFs) into the striatum, which produced phospho-Syn (p-α-Syn) inclusions in the substantia nigra pars compacta and significantly increased MHC Class II-positive immune cells. Additionally, there was enhanced infiltration and activation of innate and adaptive immune cells in the midbrain. We then used this new model, Line 61-PFF, to investigate the effect of inhibiting the JAK/STAT signaling pathway, which is critical for regulation of innate and adaptive immune responses. After administration of the JAK1/2 inhibitor AZD1480, immunofluorescence staining showed a significant decrease in p-α-Syn inclusions and MHC Class II expression. Flow cytometry showed reduced infiltration of CD4+ T-cells, CD8+ T-cells, CD19+ B-cells, dendritic cells, macrophages, and endogenous microglia into the midbrain. Importantly, single-cell RNA-Sequencing analysis of CD45+ cells from the midbrain identified 9 microglia clusters, 5 monocyte/macrophage (MM) clusters, and 5 T-cell (T) clusters, in which potentially pathogenic MM4 and T3 clusters were associated with neuroinflammatory responses in Line 61-PFF mice. AZD1480 treatment reduced cell numbers and cluster-specific expression of the antigen-presentation genes H2-Eb1, H2-Aa, H2-Ab1, and Cd74 in the MM4 cluster and proinflammatory genes such as Tnf, Il1b, C1qa, and C1qc in the T3 cluster. Together, these results indicate that inhibiting the JAK/STAT pathway suppresses the activation and infiltration of innate and adaptive cells, reducing neuroinflammation in the Line 61-PFF mouse model.

Abstract Background LRRK2-targeting therapeutics that inhibit LRRK2 kinase activity have advanced to clinical trials in idiopathic Parkinson’s disease (iPD). LRRK2 phosphorylates Rab10 on endolysosomes in phagocytic cells to promote some types of immunological responses. The identification of factors that regulate LRRK2-mediated Rab10 phosphorylation in iPD, and whether phosphorylated-Rab10 levels change in different disease states, or with disease progression, may provide insights into the role of Rab10 phosphorylation in iPD and help guide therapeutic strategies targeting this pathway. Methods Capitalizing on past work demonstrating LRRK2 and phosphorylated-Rab10 interact on vesicles that can shed into biofluids, we developed and validated a high-throughput single-molecule array assay to measure extracellular pT73-Rab10. Ratios of pT73-Rab10 to total Rab10 measured in biobanked serum samples were compared between informative groups of transgenic mice, rats, and a deeply phenotyped cohort of iPD cases and controls. Multivariable and weighted correlation network analyses were used to identify genetic, transcriptomic, clinical, and demographic variables that predict the extracellular pT73-Rab10 to total Rab10 ratio. Results pT73-Rab10 is absent in serum from Lrrk2 knockout mice but elevated by LRRK2 and VPS35 mutations, as well as SNCA expression. Bone-marrow transplantation experiments in mice show that serum pT73-Rab10 levels derive primarily from circulating immune cells. The extracellular ratio of pT73-Rab10 to total Rab10 is dynamic, increasing with inflammation and rapidly decreasing with LRRK2 kinase inhibition. The ratio of pT73-Rab10 to total Rab10 is elevated in iPD patients with greater motor dysfunction, irrespective of disease duration, age, sex, or the usage of PD-related or anti-inflammatory medications. pT73-Rab10 to total Rab10 ratios are associated with neutrophil degranulation, antigenic responses, and suppressed platelet activation. Conclusions The extracellular serum ratio of pT73-Rab10 to total Rab10 is a novel pharmacodynamic biomarker for LRRK2-linked innate immune activation associated with disease severity in iPD. We propose that those iPD patients with higher serum pT73-Rab10 levels may benefit from LRRK2-targeting therapeutics that mitigate associated deleterious immunological responses.

Disease-associated microglia (DAM) are a subset of microglia that appear at various stages of central nervous system neurodegenerative diseases. DAM were identified using single-cell RNA sequencing within Alzheimer’s Disease (AD) where they were characterized by their unique localization near amyloid-β plaques and their phagocytic and lipid-metabolizing features. Unfortunately, activation and etiology of DAM are only understood within the context of AD where Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), a receptor for amyloid-β, appears to be the key regulator in microglial transition to a DAM state. Despite this reliance on TREM2 in AD, DAM appear across other neurodegenerative diseases in which TREM2 may not be a critical player. This begs the question of if DAM are truly the same across all neurodegenerative diseases or if there exists a heterogeneity to DAM across neurodegenerative pathologies. Investigation into this critical gap in the field regarding DAM etiology and activation, as well as DAM function, could be delineated utilizing models of Parkinson’s disease (PD) to complement studies in models of AD. Though highly underexplored regarding DAM, PD with its pattern of protein aggregation-associated pathology like AD could serve as the spatiotemporal comparison against AD findings to ascertain the nature of DAM. The experimental vehicle that could guide the future of such investigation is the multi-omics model. With a compound approach focusing on exploring triggers for DAM at the chromatin or mRNA level and related protein output, it becomes possible to strongly characterize and firmly answer the question of what is a DAM.

Altered interaction between striatonigral dopaminergic (DA) inputs and local acetylcholine (ACh) in striatum has long been hypothesized to play a central role in the pathophysiology of dystonia and dyskinesia. Indeed, previous research using several genetic mouse models of human isolated dystonia identified a shared endophenotype with paradoxical excitation of striatal cholinergic interneuron (ChIs) activity in response to activation of dopamine D2 receptors (D2R). These mouse models lack a dystonic motor phenotype, which leaves a critical gap in comprehending the role of DA and ACh transmission in the manifestations of dystonia. To tackle this question, we used a combination of ex vivo slice physiology and in vivo monitoring of striatal ACh dynamics in the inducible, phenotypically penetrant, transgenic mouse model of paroxysmal non-kinesiogenic dyskinesia (PNKD), an animal with both dystonic and dyskinetic features. We found that, similarly to genetic models of isolated dystonia, the PNKD mouse displays D2R-induced paradoxical excitation of ChI firing in ex vivo striatal brain slices. In vivo, caffeine triggers dystonic symptoms while reversing the D2R-mediated excitation of ChIs and desynchronizing ACh release in PNKD mice. In WT littermate controls, caffeine stimulates spontaneous locomotion through a similar but reversed mechanism involving an excitatory switch of the D2R control of ChI activity, associated with enhanced synchronization of ACh release. These observations suggest that the “paradoxical excitation” of cholinergic interneurons described in isolated dystonia models could represent a compensatory or protective mechanism that prevents manifestation of movement abnormalities and that phenotypic dystonia is possible only when this is absent. These findings also suggest that D2Rs may play an important role in synchronizing the ChI network leading to rhythmic ACh release during heightened movement states. Dysfunction of this interaction and corresponding desynchrony of ACh release may contribute to aberrant movements.

Abstract Parkinson’s disease is the fastest-growing neurologic disease with seemingly no means of prevention. Intrinsic risk factors (age, sex, and genetics) are inescapable, but environmental factors are not. We identified repeated blows to the head in sports/combat as a potential new risk factor. 23% of PD cases in females were attributable to pesticide/herbicide exposure, and 30% of PD in males were attributable to pesticides/herbicides, military-related chemical exposures, and repeated blows to the head, and therefore could have potentially been prevented.

Abstract Introduction Foslevodopa/foscarbidopa, a soluble formulation of levodopa/carbidopa (LD/CD) prodrugs for the treatment of Parkinson’s disease (PD), is administered as a 24-hour/day continuous subcutaneous infusion (CSCI) with a single infusion site. The efficacy and safety of foslevodopa/foscarbidopa versus oral immediate-release LD/CD was previously demonstrated in patients with PD in a 12-week, randomized, double-blind, phase 3 trial (NCT04380142). We report the results of a separate 52-week, open-label, phase 3 registrational trial (NCT03781167) that evaluated the safety/tolerability and efficacy of 24-hour/day foslevodopa/foscarbidopa CSCI in patients with advanced PD. Methods Male and female patients with levodopa-responsive PD and ≥ 2.5 hours of “Off” time/day received 24-hour/day foslevodopa/foscarbidopa CSCI at individually optimized therapeutic doses (approximately 700–4250 mg of LD per 24 hours) for 52 weeks. The primary endpoint was safety/tolerability. Secondary endpoints included changes from baseline in normalized “Off” and “On” time, percentage of patients reporting morning akinesia, Movement Disorder Society Unified Parkinson’s Disease Rating Scale (MDS-UPDRS), Parkinson’s Disease Sleep Scale–2 (PDSS-2), 39-item Parkinson’s Disease Questionnaire (PDQ-39), and EuroQol 5-dimension questionnaire (EQ-5D-5L). Results Of 244 enrolled patients, 107 discontinued, and 137 completed treatment. Infusion site events were the most common adverse events (AEs). AEs were mostly nonserious (25.8% of patients reported serious AEs) and mild/moderate in severity. At week 52, “On” time without troublesome dyskinesia and “Off” time were improved from baseline (mean [standard deviation (SD)] change in normalized “On” time without troublesome dyskinesia, 3.8 [3.3] hours; normalized “Off” time, −3.5 [3.1] hours). The percentage of patients experiencing morning akinesia dropped from 77.7% at baseline to 27.8% at week 52. Sleep quality (PDSS-2) and quality of life (PDQ-39 and EQ-5D-5L) also improved. Conclusion Foslevodopa/foscarbidopa has the potential to provide a safe and efficacious, individualized, 24-hour/day, nonsurgical alternative for patients with PD. Trial Registration Number ClinicalTrials.gov identifier NCT03781167.

Dystonia refers to a heterogeneous group of movement disorders characterized by involuntary, sustained muscle contractions leading to repetitive twisting movements and abnormal postures. Dystonia has a broad clinical spectrum and can affect different body regions, causing significant disability and reduced quality of life. Despite significant progress in understanding the disorder, many challenges in dystonia research remain. This mini-review aims to highlight the major challenges facing basic and translational research in this field, including 1) heterogeneity of the disorder, 2) limited understanding of its pathophysiology, 3) complications of using animal models, 4) lack of a framework linking genes, biochemistry, circuits, and clinical phenomenology, and 5) limited research funding. Identifying and discussing these challenges can help prioritize research efforts and resources, highlight the need for further investigation and funding, and inspire action towards addressing these challenges.

Here, the authors perform large-scale high-resolution Parkinson’s disease metagenomics analyses, revealing widespread dysbiosis characterized by overabundance of pathogens, immunogens, toxicants, and curli, reduction in neuroprotective and antiinflammatory molecules, and dysregulated neuroactive signaling.

Abstract Parkinson’s disease (PD) is a multi-stage neurodegenerative disorder with largely unknown etiology. Recent findings have identified PD-associated autoimmune features including roles for T cells. To further characterize the role of T cells in PD, we performed RNA sequencing on PBMC and peripheral CD4 and CD8 memory T cell subsets derived from PD patients and age-matched healthy controls. When the groups were stratified by their T cell responsiveness to alpha-synuclein (α-syn) as a proxy for an ongoing inflammatory autoimmune response, the study revealed a broad differential gene expression profile in memory T cell subsets and a specific PD associated gene signature. We identified significant enrichment of transcriptomic signatures previously associated with PD, including for oxidative stress, phosphorylation, autophagy of mitochondria, cholesterol metabolism and inflammation, and the chemokine signaling proteins CX3CR1, CCR5, and CCR1. In addition, we identified genes in these peripheral cells that have previously been shown to be involved in PD pathogenesis and expressed in neurons, such as LRRK2, LAMP3, and aquaporin. Together, these findings suggest that features of circulating T cells with α-syn-specific responses in PD patients provide insights into the interactive processes that occur during PD pathogenesis and suggest potential intervention targets.

Abstract Background Leucine rich repeat kinase 2 (LRRK2) and SNCA are genetically linked to late-onset Parkinson’s disease (PD). Aggregated α-synuclein pathologically defines PD. Recent studies identified elevated LRRK2 expression in pro-inflammatory CD16+ monocytes in idiopathic PD, as well as increased phosphorylation of the LRRK2 kinase substrate Rab10 in monocytes in some LRRK2 mutation carriers. Brain-engrafting pro-inflammatory monocytes have been implicated in dopaminergic neurodegeneration in PD models. Here we examine how α-synuclein and LRRK2 interact in monocytes and subsequent neuroinflammatory responses. Methods Human and mouse monocytes were differentiated to distinct transcriptional states resembling macrophages, dendritic cells, or microglia, and exposed to well-characterized human or mouse α-synuclein fibrils. LRRK2 expression and LRRK2-dependent Rab10 phosphorylation were measured with monoclonal antibodies, and myeloid cell responses to α-synuclein fibrils in R1441C-Lrrk2 knock-in mice or G2019S-Lrrk2 BAC mice were evaluated by flow cytometry. Chemotaxis assays were performed with monocyte-derived macrophages stimulated with α-synuclein fibrils and microglia in Boyden chambers. Results α-synuclein fibrils robustly stimulate LRRK2 and Rab10 phosphorylation in human and mouse macrophages and dendritic-like cells. In these cells, α-synuclein fibrils stimulate LRRK2 through JAK-STAT activation and intrinsic LRRK2 kinase activity in a feed-forward pathway that upregulates phosphorylated Rab10. In contrast, LRRK2 expression and Rab10 phosphorylation are both suppressed in microglia-like cells that are otherwise highly responsive to α-synuclein fibrils. Corroborating these results, LRRK2 expression in the brain parenchyma occurs in pro-inflammatory monocytes infiltrating from the periphery, distinct from brain-resident microglia. Mice expressing pathogenic LRRK2 mutations G2019S or R1441C have increased numbers of infiltrating pro-inflammatory monocytes in acute response to α-synuclein fibrils. In primary cultured macrophages, LRRK2 kinase inhibition dampens α-synuclein fibril and microglia-stimulated chemotaxis. Conclusions Pathologic α-synuclein activates LRRK2 expression and kinase activity in monocytes and induces their recruitment to the brain. These results predict that LRRK2 kinase inhibition may attenuate damaging pro-inflammatory monocyte responses in the brain.

Introduction: Internal tremor (IT) occurs in > 30 % of people with Parkinson’s Disease (PwPD), but remains largely uninvestigated. Our objective was to describe demographic characteristics and associated symptoms in PwPD who reported IT. Methods: This was a matched case-control survey study. Data were from PwPD in the Fox Insight study who answered the Patient Report of Problems (PD-PROP) assessment, a series of open-ended questions that asks people to report in their own words their most bothersome PD-related problems. Cases were those who reported IT ≥ 1 times compared with PwPD controls who did not report IT and were matched 1:3 by age and disease duration. Results: 243 PwPD reported IT as a bothersome problem. Mean (SD) age of cases was 64.9 (9.4) years and disease duration was 3.8 (4.0) years. The proportion of women was greater among cases compared to controls (74 % vs 47 %, p

Abstract The causes of complex diseases remain an enigma despite decades of epidemiologic research on environmental risks and genome-wide studies that have uncovered tens or hundreds of susceptibility loci for each disease. We hypothesize that the microbiome is the missing link. Genetic studies have shown that overexpression of alpha-synuclein, a key pathological protein in Parkinson’s disease (PD), can cause familial PD and variants at alpha-synuclein locus confer risk of idiopathic PD. Recently, dysbiosis of gut microbiome in PD was identified: altered abundances of three microbial clusters were found, one of which was composed of opportunistic pathogens. Using two large datasets, we found evidence that the overabundance of opportunistic pathogens in PD gut is influenced by the host genotype at the alpha-synuclein locus, and that the variants responsible modulate alpha-synuclein expression. Results put forth testable hypotheses on the role of gut microbiome in the pathogenesis of PD, the incomplete penetrance of PD susceptibility genes, and potential triggers of pathology in the gut.

Abstract Increasing evidence supports the role of brain and systemic inflammation in the etiology of Parkinson disease (PD). We used gene expression profiling to examine the activation state of peripheral blood monocytes in 18 patients with early, untreated PD and 16 healthy control (HC) subjects. Monocytes were isolated by negative selection, and gene expression studied by RNA-seq and gene set enrichment analysis. A computational model that incorporated case/control status, sex, and the interaction between case/control status and sex was utilized. We found that there was a striking effect of sex on monocyte gene expression. There was inflammatory activation of monocytes in females with PD, with enrichment of gene sets associated with interferon gamma stimulation. In males, the activation patterns were more heterogeneous. These data point to the importance of systemic monocyte activation in PD, and the importance of studies which examine the differential effects of sex on pathophysiology of the disease.

Abstract The Trial of Parkinson’s And Zoledronic acid (TOPAZ, https://clinicaltrials.gov/ct2/show/NCT03924414 ) is a unique collaboration between experts in movement disorders and osteoporosis to test the efficacy of zoledronic acid, an FDA-approved parenteral treatment for osteoporosis, for fracture prevention in people with neurodegenerative parkinsonism. Aiming to enroll 3,500 participants age 65 years or older, TOPAZ is one of the largest randomized, placebo-controlled clinical trials ever attempted in parkinsonism. The feasibility of TOPAZ is enhanced by its design as a U.S.- wide home-based trial without geographical limits. Participants receive information from multiple sources, including specialty practices, support groups and websites. Conducting TOPAZ in participants’ homes takes advantage of online consent technology, the capacity to confirm diagnosis using telemedicine and the availability of research nursing to provide screening and parenteral therapy in homes. Home-based clinical research may provide an efficient, convenient, less expensive method that opens participation in clinical trials to almost anyone with parkinsonism.

Abstract The self-antigen α-synuclein (α-syn) was recently shown to be associated with Parkinson’s disease (PD). Here we mapped the T cell receptor (TCR) repertoire of α-syn-specific T cells from six PD patients. The self-antigen α-syn-specific repertoire was compared to the repertoire of T cells specific for pertussis (PT), as a representative foreign antigen that most individuals are exposed to, revealing that the repertoire for α-syn was as diverse as the repertoire for PT. The diversity of PT-specific clonotypes was similar between individuals with PD diagnosis and age-matched healthy controls. We found that the TCR repertoire was specific to each PD patient, and no shared TCRs among patients were defined, likely due to differences in HLA expression that select for different subsets of epitope-specific TCR rearrangements. This study provides the first characterization of α-syn-specific TCR clonotypes in individuals with PD. Antigen-specific TCRs can serve as immunotherapeutics and diagnostics, and means to track longitudinal changes in specific T cells, and disease progression.

This review provides an overview of the synaptic dysfunctions of neuronal circuits and underlying neurochemical alterations observed in the hyperkinetic movement disorders, dystonia and dyskinesia. These disorders exhibit similar changes in expression of synaptic plasticity and neuromodulation. This includes alterations in physical attributes of synapses, synaptic protein expression, and neurotransmitter systems, such as glutamate and gamma-aminobutyric acid (GABA), and neuromodulators, such as dopamine, acetylcholine, serotonin, adenosine, and endocannabinoids. A full understanding of the mechanisms and consequences of disruptions in synaptic function and plasticity will lend insight into the development of these disorders and new ways to combat maladaptive changes.

α-Synuclein-specific T cell reactivity is preferentially associated with Parkinson’s disease (PD) patients, but the temporal relation with diagnosis was previously unknown. This study reveals that α-syn-reactive T cells are highest before and shortly after diagnosis of motor PD, and then decrease.

There is a growing interest in using 18F-DPA-714 PET to study neuroinflammation and microglial activation through imaging the 18-kDa translocator protein (TSPO). Although quantification of 18F-DPA-714 binding can be achieved through kinetic modeling analysis with an arterial input function (AIF) measured with blood sampling procedures, the invasiveness of such procedures has been an obstacle for wide application. To address these challenges, we developed an image-derived input function (IDIF) that noninvasively estimates the arterial input function from the images acquired for 18F-DPA-714 quantification. Methods: The method entails three fully automatic steps to extract the IDIF, including a segmentation of voxels with highest likelihood of being the arterial blood over the carotid artery, a model-based matrix factorization to extract the arterial blood signal, and a scaling optimization procedure to scale the extracted arterial blood signal into the activity concentration unit. Two cohorts of human subjects were used to evaluate the extracted IDIF. In the first cohort of five subjects, arterial blood sampling was performed, and the calculated IDIF was validated against the measured AIF through the comparison of distribution volumes from AIF (VT,AIF) and IDIF (VT,IDIF). In the second cohort, PET studies from twenty-eight healthy controls without arterial blood sampling were used to compare VT,IDIF with VT,REF measured using a reference region-based analysis to evaluate whether it can distinguish high-affinity (HAB) and mixed-affinity (MAB) binders. Results: In the arterial blood-sampling cohort, VT derived from IDIF was found to be an accurate surrogate of the VT from AIF. The bias of VT, IDIF was −5.8 ± 7.8% when compared to VT,AIF, and the linear mixed effect model showed a high correlation between VT,AIF and VT, IDIF (p < 0.001). In the nonblood-sampling cohort, VT, IDIF showed a significance difference between the HAB and MAB healthy controls. VT, IDIF and standard uptake values (SUV) showed superior results in distinguishing HAB from MAB subjects than VT,REF. Conclusions: A novel IDIF method for 18F-DPA-714 PET quantification was developed and evaluated in this study. This IDIF provides a noninvasive alternative measurement of VT to quantify the TSPO binding of 18F-DPA-714 in the human brain through dynamic PET scans.

Parkinson's disease (PD) is defined by motor symptoms such as tremor at rest, bradykinesia, postural instability, and stiffness. In addition to the classical motor defects that define PD, up to 80% of patients experience cognitive changes and psychiatric disturbances, referred to as PD dementia (PDD). Pathologically, PD is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and intracellular inclusions, called Lewy bodies and Lewy neurites, composed mostly of α-synuclein. Much of PD research has focused on the role of α-synuclein aggregates in degeneration of SNpc dopamine neurons because of the impact of loss of striatal dopamine on the classical motor phenotypes. However, abundant Lewy pathology is also found in other brain regions including the cortex and limbic brain regions such as the amygdala, which may contribute to non-motor phenotypes. Little is known about the consequences of α-synuclein inclusions in these brain regions, or in neuronal subtypes other than dopamine neurons. This project expands knowledge on how α-synuclein inclusions disrupt behavior, specifically non-motor symptoms of synucleinopathies. We show that bilateral injections of fibrils into the striatum results in robust bilateral α-synuclein inclusion formation in the cortex and amygdala. Inclusions in the amygdala and prefrontal cortex primarily localize to excitatory neurons, but unbiased stereology shows no significant loss of neurons in the amygdala or cortex. Fibril injected mice show defects in a social dominance behavioral task and fear conditioning, tasks that are associated with prefrontal cortex and amygdala function. Together, these observations suggest that seeded α-synuclein inclusion formation impairs behaviors associated with cortical and amygdala function, without causing cell loss, in brain areas that may play important roles in the complex cognitive features of PDD.

Dystonia and levodopa-induced dyskinesia (LID) are both hyperkinetic movement disorders. Dystonia arises most often spontaneously, although it may be seen after stroke, injury, or as a result of genetic causes. LID is associated with Parkinson's disease (PD), emerging as a consequence of chronic therapy with levodopa, and may be either dystonic or choreiform. LID and dystonia share important phenomenological properties and mechanisms. Both LID and dystonia are generated by an integrated circuit involving the cortex, basal ganglia, thalamus and cerebellum. They also share dysregulation of striatal cholinergic signaling and abnormalities of striatal synaptic plasticity. The long duration nature of both LID and dystonia suggests that there may be underlying epigenetic dysregulation as a proximate cause. While both may improve after interventions such as deep brain stimulation (DBS), neither currently has a satisfactory medical therapy, and many people are disabled by the symptoms of dystonia and LID. Further study of the fundamental mechanisms connecting these two disorders may lead to novel approaches to treatment or prevention.

Abstract Background Parkinson’s disease (PD) is characterized by intracellular alpha-synuclein (α-syn) inclusions, progressive death of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and activation of the innate and adaptive immune systems. Disruption of immune signaling between the central nervous system (CNS) and periphery, such as through targeting the chemokine receptor type 2 (CCR2) or the major histocompatibility complex II (MHCII), is neuroprotective in rodent models of PD, suggesting a key role for innate and adaptive immunity in disease progression. The purpose of this study was to investigate whether genetic knockout or RNA silencing of the class II transactivator (CIITA), a transcriptional co-activator required for MHCII induction, is effective in reducing the neuroinflammation and neurodegeneration observed in an α-syn mouse model of PD. Methods In vitro, we utilized microglia cultures from WT or CIITA −/− mice treated with α-syn fibrils to investigate inflammatory iNOS expression and antigen processing via immunocytochemistry (ICC). In vivo, an adeno-associated virus (AAV) was used to overexpress α-syn in WT and CIITA −/− mice as a model for PD. Concurrently with AAV-mediated overexpression of α-syn, WT mice received CIITA-targeted shRNAs packaged in lentiviral constructs. Immunohistochemistry and flow cytometry were used to assess inflammation and peripheral cell infiltration at 4 weeks post transduction, and unbiased stereology was used 6 months post transduction to assess neurodegeneration. Results Using ICC and DQ-ovalbumin, we show that CIITA −/− microglial cultures failed to upregulate iNOS and MHCII expression, and had decreased antigen processing in response to α-syn fibrils when compared to WT microglia. In vivo, global knock-out of CIITA as well as local knockdown using lentiviral shRNAs targeting CIITA attenuated MHCII expression, peripheral immune cell infiltration, and α-syn-induced neurodegeneration. Conclusion Our data provide evidence that CIITA is required for α-syn-induced MHCII induction and subsequent infiltration of peripheral immune cells in an α-syn mouse model of PD. Additionally, we demonstrate that CIITA in the CNS drives neuroinflammation and neurodegeneration. These data provide further support that the disruption or modulation of antigen processing and presentation via CIITA is a promising target for therapeutic development in preclinical animal models of PD.

Levodopa (L-DOPA) remains the most effective pharmacological treatment for Parkinson Disease (PD) but its use is limited by the development of debilitating drug-related side effects, particularly L-DOPA induced dyskinesia (LID). LID is a consequence of long-term L-DOPA use, and in model systems is characterized by a “priming effect”, whereby initial administrations of L-DOPA trigger a sensitized biochemical and transcriptional response upon subsequent dopaminergic stimulation. Preliminary studies into the mechanisms underlying this cellular memory have indicated an important role for epigenetic change but many of the downstream mechanisms remain unknown. The family of bromodomain and extraterminal (BET) proteins, which bind acetylated histones, play a critical effector role in the regulation of transcription. BET proteins have been implicated in several forms of neural plasticity, but their potential relevance to LID remains unexplored. Using the 6-OHDA rodent model of LID, we show that dyskinesia development induces alterations in BET protein expression along with enhanced occupation of sites at the promoter and enhancer regions of genes dysregulated during dyskinesia development. When BET function was blocked using the pharmacologic inhibitor JQ1, LID was prevented. In addition, we found that JQ1 treatment blocked the transcriptional upregulation of several immediate-early genes known to participate in the pathogenesis of dyskinesia. Together, these results demonstrate an essential role for BET protein activity as an epigenetic “reader” of the altered histone acetylation required for LID development and suggest that modulation of BET protein function is a potential therapeutic avenue for the prevention or reversal of LID in PD.

Immune modulation: translating the benefits The immune system modulating drug sargramostim shows promising results in a small clinical trial with Parkinson’s disease (PD) patients. Previous studies have shown that sargramostim increases the number of regulatory T cells, attenuates immune responses, and confers neuroprotection in animal models of neurodegenerative disease. To determine whether these findings translate to humans, Howard E. Gendelman at the University of Nebraska Medical Center, USA, and colleagues examined the effects of sargramostim in 20 patients with PD. Despite the high number of mild to moderate reported adverse events, the drug was generally well tolerated and led to an increase in regulatory T cell number and activity. Moreover, preliminary assessments after 6 and 8 weeks of treatment suggested an overall improvement in the motor skills of patients that received the drug compared with those that received a placebo.

DYT1 dystonia is an early-onset, hyperkinetic movement disorder caused by a deletion in the gene TOR1A, which encodes the protein torsinA. Several lines of evidence show that in animal models of DTY1 dystonia, there is impaired basal dopamine (DA) release and enhanced acetylcholine tone. Clinically, anticholinergic drugs are the most effective pharmacological treatment for DYT1 dystonia, but the currently used agents are non-selective muscarinic antagonists and associated with side effects. We used a DYT1 ∆GAG knock-in mouse model (DYT1 KI) to investigate whether nicotine and/or a non-desensitizing nicotinic agonist, AZD1446, would increase DA output in DYT1 dystonia. Using in vivo microdialysis, we found that DYT1 KI mice showed significantly increased DA output and greater sensitivity to nicotine compared to wild type (WT) littermate controls. In contrast, neither systemic injection (0.25–0.75 mg/kg) or intrastriatal infusion (30 μM–1 mM) of AZD1446 had a significant effect on DA efflux in WT or DYT1 KI mice. In vitro, we found that AZD1446 had no effect on the membrane properties of striatal spiny projection neurons (SPNs) and did not alter the spontaneous firing of ChI interneurons in either WT or DYT1 KI mice. We did observe that the firing frequency of dopaminergic neurons was significantly increased by AZD1446 (10 μM), an effect blocked by dihydro-beta-erythroidine (DHβE 3 μM), but the effect was similar in WT and DYT1 KI mice. Our results support the view that DYT1 models are associated with abnormal striatal cholinergic transmission, and that the DYT1 KI animals have enhanced sensitivity to nicotine. We found little effect of AZD1446 in this model, suggesting that other approaches to nicotinic modulation should be explored.

Gene-based therapies for neurological diseases continue to develop briskly. As disease mechanisms are elucidated, flexible gene delivery platforms incorporating transcriptional regulatory elements, therapeutic genes and targeted delivery are required for the safety and efficacy of these approaches. Adenovirus serotype 5 (Ad5)-based vectors can carry large genetic payloads to provide this flexibility, but do not transduce neuronal cells efficiently. To address this, we have developed a tropism-modified Ad5 vector with neuron-selective targeting properties for evaluation in models of Parkinson disease therapy. A panel of tropism-modified Ad5 vectors was screened for enhanced gene delivery in a neuroblastoma cell line model system. We used these observations to design and construct an unbiased Ad vector platform, consisting of an unmodified Ad5 and a tropism-modified Ad5 vector containing the fiber knob domain from canine Ad serotype 2 (Ad5-CGW-CK2). Delivery to the substantia nigra or striatum showed that this vector produced a neuronally-restricted pattern of gene expression. Many of the transduced neurons were from regions with afferent projections to the injection site, implicating that the vector binds the presynaptic terminal resulting in presynaptic transduction. We show that Ad5-CGW-CK2 can selectively transduce neurons in the brain and hypothesize that this modular platform is potentially adaptable to clinical use.

DYT1 dystonia, a common and severe primary dystonia, is caused by a 3-bp deletion in TOR1A which encodes torsinA, a protein found in the endoplasmic reticulum. Several cellular functions are altered by the mutant protein, but at a systems level the link between these and the symptoms of the disease is unclear. The most effective known therapy for DYT1 dystonia is the use of anticholinergic drugs. Previous studies have revealed that in mice, transgenic expression of human mutant torsinA under a non-selective promoter leads to abnormal function of striatal cholinergic neurons. To investigate what pathological role torsinA plays in cholinergic neurons, we created a mouse model in which the Dyt1 gene, the mouse homolog of TOR1A, is selectively deleted in cholinergic neurons (ChKO animals). These animals do not have overt dystonia, but do have subtle motor abnormalities. There is no change in the number or size of striatal cholinergic cells or striatal acetylcholine content, uptake, synthesis, or release in ChKO mice. There are, however, striking functional abnormalities of striatal cholinergic cells, with paradoxical excitation in response to D2 receptor activation and loss of muscarinic M2/M4 receptor inhibitory function. These effects are specific for cholinergic interneurons, as recordings from nigral dopaminergic neurons revealed normal responses. Amphetamine stimulated dopamine release was also unaltered. These results demonstrate a cell-autonomous effect of Dyt1 deletion on striatal cholinergic function. Therapies directed at modifying the function of cholinergic neurons may prove useful in the treatment of the human disorder.

Dystonia is a clinical syndrome with sustained muscle contraction, twisting, and abnormal postures. A number of different genetic forms have been defined, but most cases are sporadic in nature and of uncertain cause. Relatively few cases of dystonia have been studied pathologically. In primary dystonias, where dystonia is the main symptom, most reports describe little or no detectable neuropathology, although changes in brainstem neurons have been described in some cases. Secondary dystonias are associated with degenerative or destructive diseases of the nervous system; the pathology may be located in the basal ganglia, but in some cases the primary pathological changes are found in the cerebellum or cerebellar outflow pathways, suggesting that both regions may be involved in the pathogenesis of dystonic symptoms. Overall the number of well-documented pathological cases available for study is few, and there is an urgent need for additional postmortem studies. This article is part of a Special Issue entitled “Advances in dystonia”.

DYT1 dystonia is an inherited disease linked to mutation in the TOR1A gene encoding for the protein torsinA. Although the mechanism by which this genetic alteration leads to dystonia is unclear, multiple lines of clinical evidence suggest a link between dystonia and a reduced dopamine D2 receptor (D2R) availability. Based on this evidence, herein we carried out a comprehensive analysis of electrophysiological, behavioral and signaling correlates of D2R transmission in transgenic mice with the DYT1 dystonia mutation. Electrophysiological recordings from nigral dopaminergic neurons showed a normal responsiveness to D2-autoreceptor function. Conversely, postsynaptic D2R function in hMT mice was impaired, as suggested by the inability of a D2R agonist to re-establish normal corticostriatal synaptic plasticity and supported by the reduced sensitivity to haloperidol-induced catalepsy. Although an in situ hybridization analysis showed normal D1R and D2R mRNA expression levels in the striata of hMT mice, we found a significant decrease of D2R protein, coupled to a reduced ability of D2Rs to activate their cognate Go/i proteins.Of relevance, we found that pharmacological blockade of adenosine A2A receptors (A2ARs) fully restored the impairment of synaptic plasticity observed in hMT mice.Together, our findings demonstrate an important link between torsinA mutation and D2R dysfunction and suggest that A2AR antagonism is able to counteract the deficit in D2R-mediated transmission observed in mutant mice, opening new perspectives for the treatment of this movement disorder.

VPS41 is a protein identified as a potential therapeutic target for Parkinson's disease (PD) as a result of a high-throughput RNAi screen in Caenorhabditis elegans. VPS41 has a plausible mechanistic link to the pathogenesis of PD, as in yeast it is known to participate in trafficking of proteins to the lysosomal system and several recent lines of evidence have pointed to the importance of lysosomal system dysfunction in the neurotoxicity of alpha-synuclein (α-syn). We found that expression of the human form of VPS41 (hVPS41) prevents dopamine (DA) neuron loss induced by α-syn overexpression and 6-hydroxydopamine (6-OHDA) neurotoxicity in C. elegans. In SH-SY5Y neuroblastoma cell lines stably transfected with hVPS41, we determined that presence of this protein conferred protection against the neurotoxins 6-OHDA and rotenone. Overexpression of hVPS41 did not alter the mitochondrial membrane depolarization induced by these neurotoxins. hVPS41 did, however, block downstream events in the apoptotic cascade including activation of caspase-9 and caspase-3, and PARP cleavage. We also observed that hVPS41 reduced the accumulation of insoluble high-molecular weight forms of α-syn in SH-SY5Y cells after treatment with rotenone. These data show that hVPS41 is protective against both α-syn and neurotoxic-mediated injury in invertebrate and cellular models of PD. These protective functions may be related to enhanced clearance of misfolded or aggregated protein, including α-syn. Our studies indicate that hVPS41 may be a useful target for developing therapeutic strategies for human PD.

DYT1 dystonia is caused by a deletion in a glutamic acid residue in the C-terminus of the protein torsinA, whose function is still largely unknown. Alterations in GABAergic signaling have been involved in the pathogenesis of dystonia. We recorded GABA- and glutamate-mediated synaptic currents from a striatal slice preparation obtained from a mouse model of DYT1 dystonia. In medium spiny neurons (MSNs) from mice expressing human mutant torsinA (hMT), we observed a significantly higher frequency, but not amplitude, of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature currents (mIPSCs), whereas glutamate-dependent spontaneous excitatory synaptic currents (sEPSCs) were normal. No alterations were found in mice overexpressing normal human torsinA (hWT). To identify the possible sources of the increased GABAergic tone, we recorded GABAergic Fast-Spiking (FS) interneurons that exert a feed-forward inhibition on MSNs. However, both sEPSC and sIPSC recorded from hMT FS interneurons were comparable to hWT and non-transgenic (NT) mice. In physiological conditions, dopamine (DA) D2 receptor act presynaptically to reduce striatal GABA release. Of note, application of the D2-like receptor agonist quinpirole failed to reduce the frequency of sIPSCs in MSNs from hMT as compared to hWT and NT mice. Likewise, the inhibitory effect of quinpirole was lost on evoked IPSCs both in MSNs and FS interneurons from hMT mice. Our findings demonstrate a disinhibition of striatal GABAergic synaptic activity, that can be at least partially attributed to a D2 DA receptor dysfunction.

Alpha-synuclein has been implicated in Parkinson disease, yet the mechanism by which alpha-synuclein causes cell injury is not understood. Using a transgenic mouse model, we evaluated the effect of alpha-synuclein overexpression on gene expression in the substantia nigra. Nigral mRNA from wild type and alpha-synuclein transgenic mice was analyzed using Affymetrix gene arrays. At 3 months, before pathological changes are apparent, we observed modest alterations in gene expression. However, nearly 200 genes were altered in expression at 9 months, when degenerative changes are more apparent. Functional genomic analysis revealed that the genes altered at 9 months were predominantly involved in gene transcription. As in human Parkinson disease, gene expression changes in the transgenic model were also modulated by gender. These data demonstrate that alterations of gene expression are widespread in this animal model, and suggest that transcriptional dysregulation may be a disease mechanism that can be targeted therapeutically.

To identify gene expression patterns in human dopamine (DA) neurons in the substantia nigra pars compacta (SNc) of male and female control and Parkinson disease (PD) patients, we harvested DA neurons from frozen SNc from 16 subjects (4 male PDs, 4 female PDs, 4 male and 4 female controls) using Laser Capture microdissection and microarrays. We assessed for enrichment of functional categories with a hypergeometric distribution. The data were validated with QPCR.We observed that gender has a pervasive effect on gene expression in DA neurons. Genes upregulated in females relative to males are mainly involved in signal transduction and neuronal maturation, while in males some of the upregulated genes (alpha-synuclein and PINK1) were previously implicated in the pathogenesis of PD. In females with PD we found alterations in genes with protein kinase activity, genes involved in proteolysis and WNT signaling pathway, while in males with PD there were alterations in protein-binding proteins and copper-binding proteins.Our data reveal broad gender-based differences in gene expression in human dopaminergic neurons of SNc that may underlie the predisposition of males to PD. Moreover, we show that gender influences the response to PD, suggesting that the nature of the disease and the response to treatment may be gender-dependent.

An immunohistochemical technique was developed to visualize nitric oxide synthase (NOS)-immunopositive neurons in fresh-frozen tissue sections of rat brain for laser capture microdissection (LCM) and mRNA analysis. The effect of tissue fixation and the choice of fluorophore were investigated. Here we describe a rapid immunofluorescence protocol that allows the processing of fresh-frozen tissue sections within eight minutes and subsequent mRNA extraction and real-time PCR from pools of 20 NOS-immunopositive LCM neurons. The cellular complement of a subset of ionotropic glutamate receptors, specifically N-methyl-D-aspartate receptor subunit mRNAs, was examined because these receptor complexes are thought to mediate the effects of fast and slow glutamate excitotoxicity. Real-time PCR data revealed that striatal NOS interneurons express the mRNAs encoding NR1, NR2A, NR2B, and NR2D but not NR2C. These LCM mRNA data are consistent with previous in situ hybridization studies and demonstrate the utility of rapid immuno-LCM with real-time quantitative PCR for the study of mRNA abundance in discrete populations of neurons within the mammalian brain.

We have used a quantitativein situhybridization method with human ribonucleotide probes to examine the regional and cellular distribution ofN-methyl-d-aspartate receptor (NMDAR) subunit mRNAs in the human cerebellum. Purkinje cells showed very dense labeling for NMDAR1 mRNA, dense labeling for NMDAR2A mRNA, and moderate labeling for NMDAR2D mRNA, whereas labeling for NMDAR2C mRNA was low. Granule cells showed high hybridization signals for the NMDAR1 and NMDAR2C mRNAs and moderate signals for the NMDAR2A and NMDAR2D mRNAs. In addition intense labeling with the NMDAR2B probe was observed in medium-sized neurons with chromophilic cell bodies in the upper part of the granule cell layer, most likely representing Golgi cells. Neurons in the molecular layer, i.e., basket cells and stellate cells, showed moderate hybridization signals for NMDAR1 and NMDAR2D and low signal for NMDAR2C. Each type of cerebellar neuron analyzed displayed a distinct NMDAR2 subunit profile, suggesting that they are likely to have NMDA receptors with distinct properties.