Advancing Age and the rs6265 BDNF SNP are Permissive to Graft-induced Dyskinesias in Parkinsonian Rats.

The rs6265 single nucleotide polymorphism (SNP) in the gene for brain-derived neurotrophic factor is a common variant that alters therapeutic outcomes for individuals with Parkinson's disease (PD). We previously investigated the effects of this SNP on the experimental therapeutic approach of neural grafting, demonstrating that young adult parkinsonian rats carrying the variant Met allele exhibited enhanced graft function compared to wild-type rats, and also exclusively developed aberrant graft-induced dyskinesias (GID). Aging is the primary risk factor for PD and reduces graft efficacy. Here we investigated whether aging interacts with this SNP to further alter cell transplantation outcomes. We hypothesized that aging would dampen enhancement of graft function associated with this genetic variant and exacerbate GID in all grafted subjects. Unexpectedly, beneficial graft function was maintained in aged rs6265 subjects. However, aging was permissive to GID induction, regardless of genotype, with the greatest incidence and severity found in rs6265 expressing animals.

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.

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.

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.

A comparison of machine learning approaches for the quantification of microglial cells in the brain of mice, rats and non-human primates.

Microglial cells are brain-specific macrophages that swiftly react to disruptive events in the brain. Microglial activation leads to specific modifications, including proliferation, morphological changes, migration to the site of insult, and changes in gene expression profiles. A change in inflammatory status has been linked to many neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. For this reason, the investigation and quantification of microglial cells is essential for better understanding their role in disease progression as well as for evaluating the cytocompatibility of novel therapeutic approaches for such conditions. In the following study we implemented a machine learning-based approach for the fast and automatized quantification of microglial cells; this tool was compared with manual quantification (ground truth), and with alternative free-ware such as the threshold-based ImageJ and the machine learning-based Ilastik. We first trained the algorithms on brain tissue obtained from rats and non-human primate immunohistochemically labelled for microglia. Subsequently we validated the accuracy of the trained algorithms in a preclinical rodent model of Parkinson's disease and demonstrated the robustness of the algorithms on tissue obtained from mice, as well as from images provided by three collaborating laboratories. Our results indicate that machine learning algorithms can detect and quantify microglial cells in all the three mammalian species in a precise manner, equipotent to the one observed following manual counting. Using this tool, we were able to detect and quantify small changes between the hemispheres, suggesting the power and reliability of the algorithm. Such a tool will be very useful for investigation of microglial response in disease development, as well as in the investigation of compatible novel therapeutics targeting the brain. As all network weights and labelled training data are made available, together with our step-by-step user guide, we anticipate that many laboratories will implement machine learning-based quantification of microglial cells in their research.

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.

The Role of Striatal Cav1.3 Calcium Channels in Therapeutics for Parkinson's Disease.

Parkinson's disease (PD) is a relentlessly progressive neurodegenerative disorder with typical motor symptoms that include rigidity, tremor, and akinesia/bradykinesia, in addition to a host of non-motor symptoms. Motor symptoms are caused by progressive and selective degeneration of dopamine (DA) neurons in the SN pars compacta (SNpc) and the accompanying loss of striatal DA innervation from these neurons. With the exception of monogenic forms of PD, the etiology of idiopathic PD remains unknown. While there are a number of symptomatic treatment options available to individuals with PD, these therapies do not work uniformly well in all patients, and eventually most are plagued with waning efficacy and significant side-effect liability with disease progression. The incidence of PD increases with aging, and as such the expected burden of this disease will continue to escalate as our aging population increases (Dorsey et al. Neurology 68:384-386, 2007). The daunting personal and socioeconomic burden has pressed scientists and clinicians to find improved symptomatic treatment options devoid side-effect liability and meaningful disease-modifying therapies. Federal and private sources have supported clinical investigations over the past two-plus decades; however, no trial has yet been successful in finding an effective therapy to slow progression of PD, and there is currently just one FDA approved drug to treat the antiparkinsonian side-effect known as levodopa-induced dyskinesia (LID) that impacts approximately 90% of all individuals with PD. In this review, we present biological rationale and experimental evidence on the potential therapeutic role of the L-type voltage-gated Cav1.3 calcium (Ca2+) channels in two distinct brain regions, with two distinct mechanisms of action, in impacting the lives of individuals with PD. Our primary emphasis will be on the role of Cav1.3 channels in the striatum and the compelling evidence of their involvement in LID side-effect liability. We also briefly discuss the role of these same Ca2+ channels in the SNpc and the longstanding interest in Cav1.3 in this brain region in halting or delaying progression of PD.

New Frontiers in Neurodegeneration and Regeneration Associated with Brain-Derived Neurotrophic Factor and the rs6265 Single Nucleotide Polymorphism.

Brain-derived neurotrophic factor is an extensively studied neurotrophin implicated in the pathology of multiple neurodegenerative and psychiatric disorders including, but not limited to, Parkinson's disease, Alzheimer's disease, Huntington's disease, traumatic brain injury, major de-pressive disorder, and schizophrenia. Here we provide a brief summary of current knowledge on the role of BDNF and the common human single nucleotide polymorphism, rs6265, in driving the pathogenesis and rehabilitation in these disorders, as well as the status of BDNF-targeted therapies. A common trend has emerged correlating low BDNF levels, either detected within the central nervous system or peripherally, to disease states, suggesting that BDNF replacement therapies may hold clinical promise. In addition, we introduce evidence for a distinct role of the BDNF pro-peptide as a biologically active ligand and the need for continuing studies on its neurological function outside of that as a molecular chaperone. Finally, we highlight the latest research describing the role of rs6265 expression in mechanisms of neurodegeneration as well as paradoxical advances in the understanding of this genetic variant in neuroregeneration. All of this is discussed in the context of personalized medicine, acknowledging there is no "one size fits all" therapy for neurodegenerative or psychiatric disorders and that continued study of the multiple BDNF isoforms and genetic variants represents an avenue for discovery ripe with therapeutic potential.

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.

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease.

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson's disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor. It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10-15 years of treatment. Understanding the mechanisms of this malady and developing both novel and effective anti-dyskinesia treatments requires consistent and accurate modeling for pre-clinical testing of therapeutic interventions. A detailed method for reliable induction and comprehensive rating of LIDs following 6-OHDA-induced nigral lesioning in a rat model of PD is presented here. Dependable LID assessment in rats provides a powerful tool that can be readily utilized across laboratories to test emerging therapies focused on reducing or eliminating this common treatment-induced burden for individuals with PD.

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.

Striatal Afferent BDNF Is Disrupted by Synucleinopathy and Partially Restored by STN DBS.

Preclinical studies show a link between subthalamic nucleus (STN) deep brain stimulation (DBS) and neuroprotection of nigrostriatal dopamine (DA) neurons, potentially through brain-derived neurotrophic factor (BDNF) signaling. However, the question of whether DBS of the STN can be disease-modifying in Parkinson's disease (PD) remains unanswered. In particular, the impact of STN DBS on α-synuclein (α-syn) aggregation, inclusion-associated neuroinflammation, and BDNF levels has yet to be examined in the context of synucleinopathy. To address this, we examined the effects of STN DBS on BDNF using the α-syn preformed fibril (PFF) model in male rats. While PFF injection resulted in accumulation of phosphorylated α-syn (pSyn) inclusions in the substantia nigra pars compacta (SNpc) and cortical areas, STN DBS did not impact PFF-induced accumulation of pSyn inclusions in the SNpc. In addition, nigral pSyn inclusions were associated with increased microgliosis and astrogliosis; however, the magnitude of these processes was not altered by STN DBS. Total BDNF protein was not impacted by pSyn inclusions, but the normally positive association of nigrostriatal and corticostriatal BDNF was reversed in rats with PFF-induced nigrostriatal and corticostriatal inclusions. Despite this, rats receiving both STN DBS and PFF injection showed increased BDNF protein in the striatum, which partially restored the normal corticostriatal relationship. Our results suggest that pathologic α-syn inclusions disrupt anterograde BDNF transport within nigrostriatal and corticostriatal circuitry. Further, STN DBS has the potential to exert protective effects by modifying the long-term neurodegenerative consequences of synucleinopathy.SIGNIFICANCE STATEMENT An increase in brain-derived neurotrophic factor (BDNF) has been linked to the neuroprotection elicited by subthalamic nucleus (STN) deep brain stimulation (DBS) in neurotoxicant models of Parkinson's disease (PD). However, whether STN DBS can similarly increase BDNF in nigrostriatal and corticostriatal circuitry in the presence of α-synuclein (α-syn) inclusions has not been examined. We examined the impact of STN DBS on rats in which accumulation of α-syn inclusions is induced by injection of α-syn preformed fibrils (PFFs). STN DBS significantly increased striatal BDNF protein in rats seeded with α-syn inclusions and partially restored the normal corticostriatal BDNF relationship. These findings suggest that STN DBS can drive BDNF in the parkinsonian brain and retains the potential for neuroprotection in PD.

The BDNF Val66Met polymorphism (rs6265) enhances dopamine neuron graft efficacy and side-effect liability in rs6265 knock-in rats.

Prevalent in approximately 20% of the worldwide human population, the rs6265 (also called 'Val66Met') single nucleotide polymorphism (SNP) in the gene for brain-derived neurotrophic factor (BDNF) is a common genetic variant that can alter therapeutic responses in individuals with Parkinson's disease (PD). Possession of the variant Met allele results in decreased activity-dependent release of BDNF. Given the resurgent worldwide interest in neural transplantation for PD and the biological relevance of BDNF, the current studies examined the effects of the rs6265 SNP on therapeutic efficacy and side-effect development following primary dopamine (DA) neuron transplantation. Considering the significant reduction in BDNF release associated with rs6265, we hypothesized that rs6265-mediated dysfunctional BDNF signaling contributes to the limited clinical benefit observed in a subpopulation of PD patients despite robust survival of grafted DA neurons, and further, that this mutation contributes to the development of aberrant graft-induced dyskinesias (GID). To this end, we generated a CRISPR knock-in rat model of the rs6265 BDNF SNP to examine for the first time the influence of a common genetic polymorphism on graft survival, functional efficacy, and side-effect liability, comparing these parameters between wild-type (Val/Val) rats and those homozygous for the variant Met allele (Met/Met). Counter to our hypothesis, the current research indicates that Met/Met rats show enhanced graft-associated therapeutic efficacy and a paradoxical enhancement of graft-derived neurite outgrowth compared to wild-type rats. However, consistent with our hypothesis, we demonstrate that the rs6265 genotype in the host rat is strongly linked to development of GID, and that this behavioral phenotype is significantly correlated with neurochemical signatures of atypical glutamatergic neurotransmission by grafted DA neurons.

Preclinical evidence in support of repurposing sub-anesthetic ketamine as a treatment for L-DOPA-induced dyskinesia.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Pharmacotherapy with L-DOPA remains the gold-standard therapy for PD, but is often limited by the development of the common side effect of L-DOPA-induced dyskinesia (LID), which can become debilitating. The only effective treatment for disabling dyskinesia is surgical therapy (neuromodulation or lesioning), therefore effective pharmacological treatment of LID is a critical unmet need. Here, we show that sub-anesthetic doses of ketamine attenuate the development of LID in a rodent model, while also having acute anti-parkinsonian activity. The long-term anti-dyskinetic effect is mediated by brain-derived neurotrophic factor-release in the striatum, followed by activation of ERK1/2 and mTOR pathway signaling. This ultimately leads to morphological changes in dendritic spines on striatal medium spiny neurons that correlate with the behavioral effects, specifically a reduction in the density of mushroom spines, a dendritic spine phenotype that shows a high correlation with LID. These molecular and cellular changes match those occurring in hippocampus and cortex after effective sub-anesthetic ketamine treatment in preclinical models of depression, and point to common mechanisms underlying the therapeutic efficacy of ketamine for these two disorders. These preclinical mechanistic studies complement current ongoing clinical testing of sub-anesthetic ketamine for the treatment of LID by our group, and provide further evidence in support of repurposing ketamine to treat individuals with PD. Given its clinically proven therapeutic benefit for both treatment-resistant depression and several pain states, very common co-morbidities in PD, sub-anesthetic ketamine could provide multiple therapeutic benefits for PD in the future.

Striatal Nurr1, but not FosB expression links a levodopa-induced dyskinesia phenotype to genotype in Fisher 344 vs. Lewis hemiparkinsonian rats.

Numerous genes, and alterations in their expression, have been identified as risk factors for developing levodopa-induced dyskinesia (LID). However, our understanding of the complexities of molecular changes remains insufficient for development of clinical treatment. In the current study we used gene array, in situ hybridization, immunohistochemistry, and microdialysis to provide a unique compare and contrast assessment of the relationship of four candidate genes to LID, employing three genetically distinct rat strains (Sprague-Dawley (SD), Fischer-344 (F344) and Lewis-RT.1) showing differences in dyskinesia susceptibility and 'first-ever LID' versus 'chronic LID' expression in subjects displaying equal dyskinesia severity. In these studies, rat strains were easily distinguishable for their LID propensity with: 1) a majority of SD rats expressing LID (LID+) and a subset being resistant (LID-); 2) all F344 rats readily developing (LID+); and 3) all Lewis rats being LID-resistant (LID-). Following chronic levodopa, LID+ SD rats showed significant increases in candidate gene expression: Nr4a2/(Nurr1) > > Trh > Inhba = Fosb. However, SD rats with long-standing striatal dopamine (DA) depletion treated with first-ever versus chronic high-dose levodopa revealed that despite identical levels of LID severity: 1) Fosb and Nurr1 transcripts but not protein were elevated with acute LID expression; 2) FOSB/ΔFOSB and NURR1 proteins were elevated only with chronic LID; and 3) Trh transcript and protein were elevated only with chronic LID. Strikingly, despite similar levodopa-induced striatal DA release in both LID-expressing F344 and LID-resistant Lewis rats, Fosb, Trh, Inhba transcripts were significantly elevated in both strains; however, Nurr1 mRNA was significantly increased only in LID+ F344 rats. These findings suggest a need to reevaluate currently accepted genotype-to-phenotype relationships in the expression of LID, specifically that of Fosb, a transcription factor generally assumed to play a causal role, and Nurr1, a transcription factor that has received significant attention in PD research linked to its critical role in the survival and function of midbrain DA neurons but who's striatal expression, generally below levels of detection, has remained largely unexplored as a regulator of LID. Finally these studies introduce a novel 'model' (inbred F344 vs inbred Lewis) that may provide a powerful tool for investigating the role for 'dyskinesia-resistance' genes downstream of 'dyskinesia-susceptibility' genes in modulating LID expression, a concept that has received considerably less attention and offers a new ways of thinking about antidyskinetic therapies.

Striatal Nurr1 Facilitates the Dyskinetic State and Exacerbates Levodopa-Induced Dyskinesia in a Rat Model of Parkinson's Disease.

The transcription factor Nurr1 has been identified to be ectopically induced in the striatum of rodents expressing l-DOPA-induced dyskinesia (LID). In the present study, we sought to characterize Nurr1 as a causative factor in LID expression. We used rAAV2/5 to overexpress Nurr1 or GFP in the parkinsonian striatum of LID-resistant Lewis or LID-prone Fischer-344 (F344) male rats. In a second cohort, rats received the Nurr1 agonist amodiaquine (AQ) together with l-DOPA or ropinirole. All rats received a chronic DA agonist and were evaluated for LID severity. Finally, we performed single-unit recordings and dendritic spine analyses on striatal medium spiny neurons (MSNs) in drug-naïve rAAV-injected male parkinsonian rats. rAAV-GFP injected LID-resistant hemi-parkinsonian Lewis rats displayed mild LID and no induction of striatal Nurr1 despite receiving a high dose of l-DOPA. However, Lewis rats overexpressing Nurr1 developed severe LID. Nurr1 agonism with AQ exacerbated LID in F344 rats. We additionally determined that in l-DOPA-naïve rats striatal rAAV-Nurr1 overexpression (1) increased cortically-evoked firing in a subpopulation of identified striatonigral MSNs, and (2) altered spine density and thin-spine morphology on striatal MSNs; both phenomena mimicking changes seen in dyskinetic rats. Finally, we provide postmortem evidence of Nurr1 expression in striatal neurons of l-DOPA-treated PD patients. Our data demonstrate that ectopic induction of striatal Nurr1 is capable of inducing LID behavior and associated neuropathology, even in resistant subjects. These data support a direct role of Nurr1 in aberrant neuronal plasticity and LID induction, providing a potential novel target for therapeutic development.SIGNIFICANCE STATEMENT The transcription factor Nurr1 is ectopically induced in striatal neurons of rats exhibiting levodopa-induced dyskinesia [LID; a side-effect to dopamine replacement strategies in Parkinson's disease (PD)]. Here we asked whether Nurr1 is causing LID. Indeed, rAAV-mediated expression of Nurr1 in striatal neurons was sufficient to overcome LID-resistance, and Nurr1 agonism exacerbated LID severity in dyskinetic rats. Moreover, we found that expression of Nurr1 in l-DOPA naïve hemi-parkinsonian rats resulted in the formation of morphologic and electrophysiological signatures of maladaptive neuronal plasticity; a phenomenon associated with LID. Finally, we determined that ectopic Nurr1 expression can be found in the putamen of l-DOPA-treated PD patients. These data suggest that striatal Nurr1 is an important mediator of the formation of LID.

Cell therapy for Parkinson's disease: Why it doesn't work every time.

The clinical experience with cell replacement therapy for advanced PD has yielded notable successes and failures. A recent autopsy case report of an individual that received implants of fetal dopamine neurons 16 years previously, but at no time experienced clinical benefit despite the best documented survival of grafted neurons and most extensive reinnervation of the striatum, raises sobering issues. With good reason, a great deal of effort in cell replacement science continues to focus on optimizing the cell source and implantation procedure. Here, we describe our preclinical studies in aged rats indicating that despite survival of large numbers of transplanted dopamine neurons and dense reinnervation of the striatum, synaptic connections between graft and host are markedly decreased and behavioral recovery is impaired. This leads us to the hypothesis that the variability in therapeutic response to dopamine neuron grafts may be less about the viability of transplanted neurons and more about the integrity of the aged, dopamine-depleted striatum and its capacity for repair. Replacement of dopamine innervation only can be fully effective if the correct target is present. © 2019 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Genetic silencing of striatal CaV1.3 prevents and ameliorates levodopa dyskinesia.

BACKGROUND: Levodopa-induced dyskinesias are an often debilitating side effect of levodopa therapy in Parkinson's disease. Although up to 90% of individuals with PD develop this side effect, uniformly effective and well-tolerated antidyskinetic treatment remains a significant unmet need. The pathognomonic loss of striatal dopamine in PD results in dysregulation and disinhibition of striatal CaV1.3 calcium channels, leading to synaptopathology that appears to be involved in levodopa-induced dyskinesias. Although there are clinically available drugs that can inhibit CaV1.3 channels, they are not adequately potent and have only partial and transient impact on levodopa-induced dyskinesias. METHODS: To provide unequivocal target validation, free of pharmacological limitations, we developed a CaV1.3 shRNA to provide high-potency, target-selective, mRNA-level silencing of striatal CaV1.3 channels and examined its ability to impact levodopa-induced dyskinesias in severely parkinsonian rats. RESULTS: We demonstrate that vector-mediated silencing of striatal CaV1.3 expression in severely parkinsonian rats prior to the introduction of levodopa can uniformly and completely prevent induction of levodopa-induced dyskinesias, and this antidyskinetic benefit persists long term and with high-dose levodopa. In addition, this approach is capable of ameliorating preexisting severe levodopa-induced dyskinesias. Importantly, motoric responses to low-dose levodopa remained intact in the presence of striatal CaV1.3 silencing, indicating preservation of levodopa benefit without dyskinesia liability. DISCUSSION: The current data provide some of the most profound antidyskinetic benefit reported to date and suggest that genetic silencing of striatal CaV1.3 channels has the potential to transform treatment of individuals with PD by allowing maintenance of motor benefit of levodopa in the absence of the debilitating levodopa-induced dyskinesia side effect. © 2019 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Regulation of dopamine neurotransmission from serotonergic neurons by ectopic expression of the dopamine D2 autoreceptor blocks levodopa-induced dyskinesia.

Levodopa-induced dyskinesias (LID) are a prevalent side effect of chronic treatment with levodopa (L-DOPA) for the motor symptoms of Parkinson's disease (PD). It has long been hypothesized that serotonergic neurons of the dorsal raphe nucleus (DRN) are capable of L-DOPA uptake and dysregulated release of dopamine (DA), and that this "false neurotransmission" phenomenon is a main contributor to LID development. Indeed, many preclinical studies have demonstrated LID management with serotonin receptor agonist treatment, but unfortunately, promising preclinical data has not been translated in large-scale clinical trials. Importantly, while there is an abundance of convincing clinical and preclinical evidence supporting a role of maladaptive serotonergic neurotransmission in LID expression, there is no direct evidence that dysregulated DA release from serotonergic neurons impacts LID formation. In this study, we ectopically expressed the DA autoreceptor D2Rs (or GFP) in the DRN of 6-hydroxydopamine (6-OHDA) lesioned rats. No negative impact on the therapeutic efficacy of L-DOPA was seen with rAAV-D2Rs therapy. However, D2Rs treated animals, when subjected to a LID-inducing dose regimen of L-DOPA, remained completely resistant to LID, even at high doses. Moreover, the same subjects remained resistant to LID formation when treated with direct DA receptor agonists, suggesting D2Rs activity in the DRN blocked dyskinesogenic L-DOPA priming of striatal neurons. In vivo microdialysis confirmed that DA efflux in the striatum was reduced with rAAV-D2Rs treatment, providing explicit evidence that abnormal DA release from DRN neurons can affect LID. This is the first direct evidence of dopaminergic neurotransmission in DRN neurons and its modulation with rAAV-D2Rs gene therapy confirms the serotonin hypothesis in LID, demonstrating that regulation of serotonergic neurons achieved with a gene therapy approach offers a novel and potent antidyskinetic therapy.

Subthalamic Nucleus Deep Brain Stimulation Does Not Modify the Functional Deficits or Axonopathy Induced by Nigrostriatal α-Synuclein Overexpression.

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.