AAV9-Mediated Intra-Striatal Delivery of GNAO1 Reduces Hyperlocomotion in Gnao1 Heterozygous R209H Mutant Mice.

Mutations in the GNAO1 gene, which encodes the abundant brain G-protein Gαo, result in neurologic disorders characterized by developmental delay, epilepsy, and movement abnormalities. There are over 50 mutant alleles associated with GNAO1 disorders; the R209H mutation results in dystonia, choreoathetosis, and developmental delay without seizures. Mice heterozygous for the human mutant allele (Gnao1 +/R209H) exhibit hyperactivity in open field tests but no seizures. We developed self-complimentary adeno-associated virus vectors (scAAV9) expressing two splice variants of human GNAO1 Gαo isoforms 1 (GoA, GNAO1.1) and 2 (GoB, GNAO1.2). Bilateral intra-striatal injections of either scAAV9-GNAO1.1 or scAAV9-GNAO1.2 significantly reversed mutation-associated hyperactivity in open field tests. GNAO1 overexpression did not increase seizure susceptibility, a potential side-effect of GNAO1 vector treatment. This represents the first report of successful preclinical gene therapy for GNAO1 encephalopathy applied in vivo Further studies are needed to uncover the molecular mechanism that results in behavior improvements after scAAV9-mediated Gαo expression and to refine the vector design. Significance Statement GNAO1 mutations cause a spectrum of developmental, epilepsy, and movement disorders. Here, we show that intra-striatal delivery of scAAV9-GNAO1 to express the wild-type Gαo protein reduces the hyperactivity of the Gnao1 +/R209H mouse model, which carries one of the most common movement disorder-associated mutations. This is the first report of a gene therapy for GNAO1 encephalopathy applied in vivo on a patient-allele model.

Alpha-synuclein inclusion responsive microglia are resistant to CSF1R inhibition.

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 determined that microglia proximal to PFF-induced nigral α-syn inclusions increase in soma size, upregulate major-histocompatibility complex-II (MHC-II) expression, and increase expression of a suite of inflammation-associated transcripts. This microglial response is observed months prior to degeneration, suggesting that microglia reacting to α-syn inclusion may contribute to neurodegeneration and could represent a potential target for novel therapeutics. The goal of this study was to determine whether colony stimulating factor-1 receptor (CSF1R)-mediated microglial depletion impacts the magnitude of α-syn aggregation, nigrostriatal degeneration, or the response of microglial in the context of 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, 600 mg/kg), a CSF1R inhibitor, to deplete microglia for a period of either 2 or 6 months. RESULTS: CSF1R inhibition resulted in significant depletion (~ 43%) of ionized calcium-binding adapter molecule 1 immunoreactive (Iba-1ir) microglia within the SNpc. However, CSF1R inhibition did not impact the increase in microglial number, soma size, number of MHC-II immunoreactive microglia or microglial expression of Cd74, Cxcl10, Rt-1a2, Grn, Csf1r, Tyrobp, and Fcer1g associated with phosphorylated α-syn (pSyn) nigral inclusions. Further, accumulation of pSyn and degeneration of nigral neurons was not impacted by CSF1R inhibition. Paradoxically, long term CSF1R inhibition resulted in increased soma size of remaining Iba-1ir microglia in both control and PFF rats, as well as expression of MHC-II in extranigral regions. CONCLUSIONS: Collectively, our results suggest that CSF1R inhibition does not impact the microglial response to nigral pSyn inclusions and that CSF1R inhibition is not a viable disease-modifying strategy for PD.

Deficits in basal and evoked striatal dopamine release following alpha-synuclein preformed fibril injection: An in vivo microdialysis study.

Parkinson's disease (PD) is characterized by the accumulation of misfolded alpha-synuclein (α-syn) protein, forming intraneuronal Lewy body (LB) inclusions. The α-syn preformed fibril (PFF) model of PD recapitulates α-syn aggregation, progressive nigrostriatal degeneration and motor dysfunction; however, little is known about the time course of PFF-induced alterations in basal and evoked dopamine (DA). In vivo microdialysis is well suited for identifying small changes in neurotransmitter levels over extended periods. In the present study, adult male Fischer 344 rats received unilateral, intrastriatal injections of either α-syn PFFs or phosphate-buffered saline (PBS). At 4 or 8 months post-injection (p.i.), animals underwent in vivo microdialysis to evaluate basal extracellular striatal DA and metabolite levels, local KCl-evoked striatal DA release and the effects of systemic levodopa (l-DOPA). Post-mortem analysis demonstrated equivalent PFF-induced reductions in tyrosine hydroxylase (TH) immunoreactive nigral neurons (~50%) and striatal TH (~20%) at both time points. Compared with reduction in striatal TH, reduction in striatal dopamine transporter (DAT) was more pronounced and progressed between the 4- and 8-month p.i. intervals (36% âž” 46%). Significant PFF-induced deficits in basal and evoked striatal DA, as well as deficits in motor performance, were not observed until 8 months p.i. Responses to l-DOPA did not differ regardless of PBS or PFF treatment. These results suggest that basal and evoked striatal DA are maintained for several months following PFF injection, with loss of both associated with motor dysfunction. Our studies provide insight into the time course and magnitude of PFF-induced extracellular dopaminergic deficits in the striatum.

Transcriptomic profiling of early synucleinopathy in rats induced with preformed fibrils.

Examination of early phases of synucleinopathy when inclusions are present, but long before neurodegeneration occurs, is critical to both understanding disease progression and the development of disease modifying therapies. The rat alpha-synuclein (α-syn) preformed fibril (PFF) model induces synchronized synucleinopathy that recapitulates the pathological features of Parkinson's disease (PD) and can be used to study synucleinopathy progression. In this model, phosphorylated α-syn (pSyn) inclusion-containing neurons and reactive microglia (major histocompatibility complex-II immunoreactive) peak in the substantia nigra pars compacta (SNpc) months before appreciable neurodegeneration. However, it remains unclear which specific genes are driving these phenotypic changes. To identify transcriptional changes associated with early synucleinopathy, we used laser capture microdissection of the SNpc paired with RNA sequencing (RNASeq). Precision collection of the SNpc allowed for the assessment of differential transcript expression in the nigral dopamine neurons and proximal glia. Transcripts upregulated in early synucleinopathy were mainly associated with an immune response, whereas transcripts downregulated were associated with neurotransmission and the dopamine pathway. A subset of 29 transcripts associated with neurotransmission/vesicular release and the dopamine pathway were verified in a separate cohort of males and females to confirm reproducibility. Within this subset, fluorescent in situ hybridization (FISH) was used to localize decreases in the Syt1 and Slc6a3 transcripts to pSyn inclusion-containing neurons. Identification of transcriptional changes in early synucleinopathy provides insight into the molecular mechanisms driving neurodegeneration.

Neuroinflammatory gene expression profiles of reactive glia in the substantia nigra suggest a multidimensional immune response to alpha synuclein inclusions.

Parkinson's disease (PD) pathology is characterized by alpha-synuclein (α-syn) aggregates, degeneration of dopamine neurons in the substantia nigra pars compacta (SNpc), and neuroinflammation. The presence of reactive glia correlates with deposition of pathological α-syn in early-stage PD. Thus, understanding the neuroinflammatory response of microglia and astrocytes to synucleinopathy may identify therapeutic targets. Here we characterized the neuroinflammatory gene expression profile of reactive microglia and astrocytes in the SNpc during early synucleinopathy in the rat α-syn pre-formed fibril (PFF) model. Rats received intrastriatal injection of α-syn PFFs and expression of immune genes was quantified with droplet digital PCR (ddPCR), after which fluorescent in situ hybridization (FISH) was used to localize gene expression to microglia or astrocytes in the SNpc. Genes previously associated with reactive microglia (Cd74, C1qa, Stat1, Axl, Casp1, Il18, Lyz2) and reactive astrocytes (C3, Gbp2, Serping1) were significantly upregulated in the SN of PFF injected rats. Localization of gene expression to SNpc microglia near α-syn aggregates identified a unique α-syn aggregate microglial gene expression profile characterized by upregulation of Cd74, Cxcl10, Rt-1a2, Grn, Csf1r, Tyrobp, C3, C1qa, Serping1 and Fcer1g. Importantly, significant microglial upregulation of Cd74 and C3 were only observed following injection of α-syn PFFs, not α-syn monomer, confirming specificity to α-syn aggregation. Serping1 expression also localized to astrocytes in the SNpc. Interestingly, C3 expression in the SNpc localized to microglia at 2- and 4-months post-PFF, but to astrocytes at 6-months post-PFF. We also observed expression of Rt1-a2 and Cxcl10 in SNpc dopamine neurons. Cumulatively our results identify a dynamic, yet reproducible gene expression profile of reactive microglia and astrocytes associated with early synucleinopathy in the rat SNpc.

Quinpirole inhibits levodopa-induced dyskinesias at structural and behavioral levels: Efficacy negated by co-administration of isradipine.

Dopamine depletion associated with parkinsonism induces plastic changes in striatal medium spiny neurons (MSN) that are maladaptive and associated with the emergence of the negative side-effect of standard treatment: the abnormal involuntary movements termed levodopa-induced dyskinesia (LID). Prevention of MSN dendritic spine loss is hypothesized to diminish liability for LID in Parkinson's disease. Blockade of striatal CaV1.3 calcium channels can prevent spine loss and significantly diminish LID in parkinsonian rats. While pharmacological antagonism with FDA approved CaV1 L-type channel antagonist dihydropyridine (DHP) drugs (e.g, isradipine) are potentially antidyskinetic, pharmacologic limitations of current drugs may result in suboptimal efficacy. To provide optimal CaV1.3 antagonism, we investigated the ability of a dual pharmacological approach to more potently antagonize these channels. Specifically, quinpirole, a D2/D3-type dopamine receptor (D2/3R) agonist, has been demonstrated to significantly reduce calcium current activity at CaV1.3 channels in MSNs of rats by a mechanism distinct from DHPs. We hypothesized that dual inhibition of striatal CaV1.3 channels using the DHP drug isradipine combined with the D2/D3 dopamine receptor agonist quinpirole prior to, and in conjunction with, levodopa would be more effective at preventing structural modifications of dendritic spines and providing more stable LID prevention. For these proof-of-principle studies, rats with unilateral nigrostriatal lesions received daily administration of vehicle, isradipine, quinpirole, or isradipine + quinpirole prior to, and concurrent with, levodopa. Development of LID and morphological analysis of dendritic spines were assessed. Contrary to our hypothesis, quinpirole monotherapy was the most effective at reducing dyskinesia severity and preventing abnormal mushroom spine formation on MSNs, a structural phenomenon previously associated with LID. Notably, the antidyskinetic efficacy of quinpirole monotherapy was lost in the presence of isradipine co-treatment. These findings suggest that D2/D3 dopamine receptor agonists when given in combination with levodopa and initiated in early-stage Parkinson's disease may provide long-term protection against LID. The negative interaction of isradipine with quinpirole suggests a potential cautionary note for co-administration of these drugs in a clinical setting.

Developmental exposure to the Parkinson's disease-associated organochlorine pesticide dieldrin alters dopamine neurotransmission in α-synuclein pre-formed fibril (PFF)-injected mice.

Parkinson's disease (PD) is the fastest-growing neurological disease worldwide, with increases outpacing aging and occurring most rapidly in recently industrialized areas, suggesting a role of environmental factors. Epidemiological, post-mortem, and mechanistic studies suggest that persistent organic pollutants, including the organochlorine pesticide dieldrin, increase PD risk. In mice, developmental dieldrin exposure causes male-specific exacerbation of neuronal susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and synucleinopathy. Specifically, in the α-synuclein (α-syn) pre-formed fibril (PFF) model, exposure leads to increased deficits in striatal dopamine (DA) turnover and motor deficits on the challenging beam. Here, we hypothesized that alterations in DA handling contribute to the observed changes and assessed vesicular monoamine transporter 2 (VMAT2) function and DA release in this dieldrin/PFF 2-hit model. Female C57BL/6 mice were exposed to 0.3 mg/kg dieldrin or vehicle every 3 days by feeding, starting at 8 weeks of age and continuing throughout breeding, gestation, and lactation. Male offspring from independent litters underwent unilateral, intrastriatal injections of α-syn PFFs at 12 weeks of age, and vesicular 3H-DA uptake assays and fast-scan cyclic voltammetry were performed 4 months post-PFF injection. Dieldrin-induced an increase in DA release in striatal slices in PFF-injected animals, but no change in VMAT2 activity. These results suggest that developmental dieldrin exposure increases a compensatory response to synucleinopathy-triggered striatal DA loss. These findings are consistent with silent neurotoxicity, where developmental exposure to dieldrin primes the nigrostriatal striatal system to have an exacerbated response to synucleinopathy in the absence of observable changes in typical markers of nigrostriatal dysfunction and degeneration.

Microglial depletion does not impact alpha-synuclein aggregation or nigrostriatal degeneration in the rat preformed fibril model.

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.

Downregulation of striatal CaV1.3 inhibits the escalation of levodopa-induced dyskinesia in male and female parkinsonian rats of advanced age.

In the past 25 years, the prevalence of Parkinson's disease (PD) has nearly doubled. Age remains the primary risk factor for PD and as the global aging population increases this trend is predicted to continue. Even when treated with levodopa, the gold standard dopamine (DA) replacement therapy, individuals with PD frequently develop therapeutic side effects. Levodopa-induced dyskinesia (LID), a common side effect of long-term levodopa use, represents a significant unmet clinical need in the treatment of PD. Previously, in young adult (3-month-old) male parkinsonian rats, we demonstrated that the silencing of CaV1.3 (Cacan1d) L-type voltage-gated calcium channels via striatal delivery of rAAV-CaV1.3-shRNA provides uniform protection against the induction of LID, and significant reduction of established severe LID. With the goal of more closely replicating a clinical demographic, the current study examined the effects of CaV1.3-targeted gene therapy on LID escalation in male and female parkinsonian rats of advanced age (18-month-old at study completion). We tested the hypothesis that silencing aberrant CaV1.3 channel activity in the parkinsonian striatum would prevent moderate to severe dyskinesia with levodopa dose escalation. To test this hypothesis, 15-month-old male and female F344 rats were rendered unilaterally parkinsonian and primed with low-dose (3-4 mg/kg) levodopa. Following the establishment of stable, mild dyskinesias, rats received an intrastriatal injection of either the Cacna1d-specific rAAV-CaV1.3-shRNA vector (CAV-shRNA), or the scramble control rAAV-SCR-shRNA vector (SCR-shRNA). Daily (M-Fr) low-dose levodopa was maintained for 4 weeks during the vector transduction and gene silencing window followed by escalation to 6 mg/kg, then to 12 mg/kg levodopa. SCR-shRNA-shRNA rats showed stable LID expression with low-dose levodopa and the predicted escalation of LID severity with increased levodopa doses. Conversely, complex behavioral responses were observed in aged rats receiving CAV-shRNA, with approximately half of the male and female subjects-therapeutic 'Responders'-demonstrating protection against LID escalation, while the remaining half-therapeutic 'Non-Responders'-showed LID escalation similar to SCR-shRNA rats. Post-mortem histological analyses revealed individual variability in the detection of Cacna1d regulation in the DA-depleted striatum of aged rats. However, taken together, male and female therapeutic 'Responder' rats receiving CAV-shRNA had significantly less striatal Cacna1d in their vector-injected striatum relative to contralateral striatum than those with SCR-shRNA. The current data suggest that mRNA-level silencing of striatal CaV1.3 channels maintains potency in a clinically relevant in vivo scenario by preventing dose-dependent dyskinesia escalation in rats of advanced age. As compared to the uniform response previously reported in young male rats, there was notable variability between individual aged rats, particularly females, in the current study. Future investigations are needed to derive the sex-specific and age-related mechanisms which underlie variable responses to gene therapy and to elucidate factors which determine the therapeutic efficacy of treatment for PD.

BDNF rs10501087, rs1491850 and rs11030094 polymorphisms associated with delayed progression in early-stage Parkinson's disease.

Parkinson's disease (PD) is heterogenous in its presentation, progression and response to therapies. Genetic polymorphisms may account for some of this variability. Several single nucleotide polymorphisms (SNPs) in the brain-derived neurotrophic factor gene BDNF have been associated with differing clinical outcomes from different dopaminergic replacement strategies, and one of these, the rs6265 SNP, has been associated with a milder clinical phenotype in the unmedicated, early-stage of PD. We examined if other BDNF SNPs with potential pharmacogenetic effects also are associated with different rates of disease progression. The Deprenyl And Tocopherol Antioxidative Therapy Of Parkinsonism (DATATOP) study was analyzed retrospectively. DNA samples (n = 217) were genotyped for the BDNF rs908867, rs11030094, rs10501087, rs1157659, and rs1491850 SNPs, and the primary endpoint was time to initiate symptomatic pharmacotherapy. Genotypes were compared using the Cox proportional hazard ratio (HR) with baseline age, sex, site, time since PD diagnosis and rs6265 genotype as covariates. The primary endpoint was associated with a delay with three SNPs: rs10501087 [HR (95% Confidence Interval) = 28.3 (3.6-223.1, p = 0.002) and 7.6 (1.9-29.8, p = 0.004) for T/T and T/C subjects, respectively, vs. C/C subjects], rs1491850 [HR = 3.3 (1.3-8.4, p = 0.04) and 2.8 (1.3-6.4, p = 0.03) for T/T and T/C subjects, respectively, vs. C/C subjects] and rs11030094 [HR = 2.5 (1.1-5.6, p = 0.03) and 2.0 (1.3-6.4, p = 0.03) for A/A and A/G subjects, respectively, vs. G/G subjects]. From the primary endpoint, specific rs10501087, rs1491850, and rs11030094 SNP genotypes are associated with a slower rate of PD progression in the unmedicated state. A prospective clinical trial examining many BDNF SNPs is warranted.

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling.

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. Large-scale testing in a surveillance or screening capacity for asymptomatic carriers of COVID-19 provides both data on viral spread and the follow-up ability to rapidly test individuals during suspected outbreaks. The COVID-19 early detection program at Michigan State University has been utilizing large-scale testing in a surveillance or screening capacity since fall of 2020. The methods adapted here take advantage of the reliability, large sample volume, and self-collection benefits of saliva, paired with a cost-effective, reagent conserving two-dimensional pooling scheme. The process was designed to be adaptable to supply shortages, with many components of the kits and the assay easily substituted. The processes outlined for collecting and processing SARS-CoV-2 samples can be adapted to test for future viral pathogens reliably expressed in saliva. By providing this blueprint for universities or other organizations, preparedness plans for future viral outbreaks can be developed.

Leveraging the preformed fibril model to distinguish between alpha-synuclein inclusion- and nigrostriatal degeneration-associated immunogenicity.

Neuroinflammation has become a well-accepted pathologic hallmark of Parkinson's disease (PD). However, it remains unclear whether inflammation, triggered by α-syn aggregation and/or degeneration, contributes to the progression of the disease. Studies examining neuroinflammation in PD are unable to distinguish between Lewy body-associated inflammation and degeneration-associated inflammation, as both pathologies are present simultaneously. Intrastriatal and intranigral injections of alpha-synuclein (α-syn) preformed fibrils (PFFs) results in two distinct pathologic phases: Phase 1: The accumulation and peak formation of α-syn inclusions in nigrostriatal system and, Phase 2: Protracted dopaminergic neuron degeneration. In this review we summarize the current understanding of neuroinflammation in the α-syn PFF model, leveraging the distinct Phase 1 aggregation phase and Phase 2 degeneration phase to guide our interpretations. Studies consistently demonstrate an association between pathologic α-syn aggregation in the substantia nigra (SN) and activation of the innate immune system. Further, major histocompatibility complex-II (MHC-II) antigen presentation is proportionate to inclusion load. The α-syn aggregation phase is also associated with peripheral and adaptive immune cell infiltration to the SN. These findings suggest that α-syn like aggregates are immunogenic and thus have the potential to contribute to the degenerative process. Studies examining neuroinflammation during the neurodegenerative phase reveal elevated innate, adaptive, and peripheral immune cell markers, however limitations of single time point experimental design hinder interpretations as to whether this neuroinflammation preceded, or was triggered by, nigral degeneration. Longitudinal studies across both the aggregation and degeneration phases of the model suggest that microglial activation (MHC-II) is greater in magnitude during the aggregation phase that precedes degeneration. Overall, the consistency between neuroinflammatory markers in the parkinsonian brain and in the α-syn PFF model, combined with the distinct aggregation and degenerative phases, establishes the utility of this model platform to yield insights into pathologic events that contribute to neuroinflammation and disease progression in PD.

Beta2-adrenoreceptor agonist clenbuterol produces transient decreases in alpha-synuclein mRNA but no long-term reduction in protein.

ß2-adrenoreceptor (ß2AR) agonists have been associated with a decreased risk of developing Parkinson's disease (PD) and are hypothesized to decrease expression of both alpha-synuclein mRNA (Snca) and protein (α-syn). Effects of ß2AR agonist clenbuterol on the levels of Snca mRNA and α-syn protein were evaluated in vivo (rats and mice) and in rat primary cortical neurons by two independent laboratories. A modest decrease in Snca mRNA in the substantia nigra was observed after a single acute dose of clenbuterol in rats, however, this decrease was not maintained after multiple doses. In contrast, α-syn protein levels remained unchanged in both single and multiple dosing paradigms. Furthermore, clenbuterol did not decrease Snca in cultured rat primary cortical neurons, or decrease Snca or α-syn in mice. Additionally, compared to the single-dose paradigm, repeat dosing resulted in substantially lower levels of clenbuterol in plasma and brain tissue in rodents. Based on our observations of a transient decrease in Snca and no effect on α-syn protein in this preclinical study, these data support the conclusion that clenbuterol is not likely a viable disease-modifying strategy for PD.

Dopaminergic Positron Emission Tomography Imaging in the Alpha-Synuclein Preformed Fibril Model Reveals Similarities to Early Parkinson's Disease.

BACKGROUND: Positron emission tomography (PET) imaging in early Parkinson's disease (PD) subjects reveals that increased dopamine (DA) turnover and reduced dopamine transporter (DAT) density precede decreases in DA synthesis and storage. The rat α-synuclein preformed fibril (α-syn PFF) model provides a platform to investigate DA dynamics during multiple stages of α-syn inclusion-triggered nigrostriatal degeneration. OBJECTIVES: We investigated multiple aspects of in vivo dopaminergic deficits longitudinally and similarities to human PD using translational PET imaging readouts. METHODS: Longitudinal imaging was performed every 2 months in PFF and control rats for 7 months. [18 F]-Fluoro-3,4-dihydroxyphenyl-L-alanine (FDOPA) imaging was performed to investigate DA synthesis and storage (Kocc ) and DA turnover, estimated by its inverse, the effective distribution volume ratio (EDVR). 11 C-Methylphenidate (MP) was used to estimate DAT density (BPND ). RESULTS: Early DA turnover increases and DAT binding decreases were observed in the ipsilateral striatum of PFF rats, progressing longitudinally. EDVR decreased 26%, 38%, and 47%, and BPND decreased 36%, 50%, and 65% at the 2-, 4-, and 6-month time points, respectively, compared to ipsilateral control striatum. In contrast, deficits in DA synthesis and storage were not observed in the ipsilateral striatum of PFF rats compared to control injections and were relatively preserved up to 6 months (Kocc decreased 20% at 6 months). CONCLUSIONS: The relative preservation of DA synthesis and storage compared to robust progressive deficits in DAT density and increases in DA turnover in the rat α-syn PFF model display remarkable face validity to dopaminergic alterations in human PD. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Behavioral phenotyping of a rat model of the BDNF Val66Met polymorphism reveals selective impairment of fear memory.

The common brain-derived neurotrophic factor (BDNF) Val66Met polymorphism is associated with reduced activity-dependent BDNF release and increased risk for anxiety disorders and PTSD. Here we behaviorally phenotyped a novel Val66Met rat model with an equivalent valine to methionine substitution in the rat Bdnf gene (Val68Met). In a three-day fear conditioning protocol of fear learning and extinction, adult rats with the Met/Met genotype demonstrated impaired fear memory compared to Val/Met rats and Val/Val controls, with no genotype differences in fear learning or extinction. This deficit in fear memory occurred irrespective of the sex of the animals and was not seen in adolescence (4 weeks of age). There were no changes in open-field locomotor activity or anxiety measured in the elevated plus maze (EPM) nor in other types of memory measured using the novel-object recognition test or Y-maze. BDNF exon VI expression in the dorsal hippocampus was higher and BDNF protein level in the ventral hippocampus was lower in female Val/Met rats than female Val/Val rats, with no other genotype differences, including in total BDNF, BDNF long, or BDNF IV mRNA. These data suggest a specific role for the BDNF Met/Met genotype in fear memory in rats. Further studies are required to investigate gene-environment interactions in this novel animal model.

BDNF rs6265 Genotype Influences Outcomes of Pharmacotherapy and Subthalamic Nucleus Deep Brain Stimulation in Early-Stage Parkinson's Disease.

INTRODUCTION: The efficacy of pharmacotherapy and deep brain stimulation of the subthalamic nucleus in treating Parkinson's disease motor symptoms is highly variable and may be influenced by patient genotype. The relatively common (prevalence about one in three) and protein-altering rs6265 single nucleotide polymorphism (C > T) in the gene BDNF has been associated with different clinical outcomes with levodopa. OBJECTIVE: We sought to replicate this reported association in early-stage Parkinson's disease subjects and to examine whether a difference in clinical outcomes was present with subthalamic nucleus deep brain stimulation. MATERIALS AND METHODS: Fifteen deep brain stimulation and 13 medical therapy subjects were followed for 24 months as part of the Vanderbilt DBS in Early Stage PD clinical trial (NCT00282152, FDA IDE #G050016). Primary outcome measures were the Unified Parkinson's Disease Rating Scale (UPDRS) and Parkinson's Disease Questionnaire-39. RESULTS: Outcomes with drug therapy in subjects carrying the rs6265 T allele were significantly worse following 12 months of treatment compared to C/C subjects (UPDRS: +20 points, p = 0.019; PDQ-39: +16 points, p = 0.018). In contrast, rs6265 genotype had no effect on overall motor response to subthalamic nucleus deep brain stimulation at any time point; further, rs6265 C/C subjects treated with stimulation were associated with worse UPDRS part II scores at 24 months compared to medical therapy. CONCLUSIONS: Genotyping for the rs6265 polymorphism may be useful for predicting long-term response to drug therapy and counseling Parkinson's disease patients regarding whether to consider earlier subthalamic nucleus deep brain stimulation. Validation in a larger cohort of early-stage Parkinson's disease subjects is warranted.

Preformed fibrils generated from mouse alpha-synuclein produce more inclusion pathology in rats than fibrils generated from rat alpha-synuclein.

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.

Synucleinopathy-associated pathogenesis in Parkinson's disease and the potential for brain-derived neurotrophic factor.

The lack of disease-modifying treatments for Parkinson's disease (PD) is in part due to an incomplete understanding of the disease's etiology. Alpha-synuclein (α-syn) has become a point of focus in PD due to its connection to both familial and idiopathic cases-specifically its localization to Lewy bodies (LBs), a pathological hallmark of PD. Within this review, we will present a comprehensive overview of the data linking synuclein-associated Lewy pathology with intracellular dysfunction. We first present the alterations in neuronal proteins and transcriptome associated with LBs in postmortem human PD tissue. We next compare these findings to those associated with LB-like inclusions initiated by in vitro exposure to α-syn preformed fibrils (PFFs) and highlight the profound and relatively unique reduction of brain-derived neurotrophic factor (BDNF) in this model. Finally, we discuss the multitude of ways in which BDNF offers the potential to exert disease-modifying effects on the basal ganglia. What remains unknown is the potential for BDNF to mitigate inclusion-associated dysfunction within the context of synucleinopathy. Collectively, this review reiterates the merit of using the PFF model as a tool to understand the physiological changes associated with LBs, while highlighting the neuroprotective potential of harnessing endogenous BDNF.

α-Synuclein antisense oligonucleotides as a disease-modifying therapy for Parkinson's disease.

Parkinson's disease (PD) is a prevalent neurodegenerative disease with no approved disease-modifying therapies. Multiplications, mutations, and single nucleotide polymorphisms in the SNCA gene, encoding α-synuclein (aSyn) protein, either cause or increase risk for PD. Intracellular accumulations of aSyn are pathological hallmarks of PD. Taken together, reduction of aSyn production may provide a disease-modifying therapy for PD. We show that antisense oligonucleotides (ASOs) reduce production of aSyn in rodent preformed fibril (PFF) models of PD. Reduced aSyn production leads to prevention and removal of established aSyn pathology and prevents dopaminergic cell dysfunction. In addition, we address the translational potential of the approach through characterization of human SNCA-targeting ASOs that efficiently suppress the human SNCA transcript in vivo. We demonstrate broad activity and distribution of the human SNCA ASOs throughout the nonhuman primate brain and a corresponding decrease in aSyn cerebral spinal fluid (CSF) levels. Taken together, these data suggest that, by inhibiting production of aSyn, it may be possible to reverse established pathology; thus, these data support the development of SNCA ASOs as a potential disease-modifying therapy for PD and related synucleinopathies.

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.