Nicole Elaine Chambers

Loss-of-function mutations in GNAL have been linked to an adult-onset, isolated dystonia that is largely indistinguishable from idiopathic dystonia. GNAL encodes Gα _olf , a heterotrimeric G-protein α subunit with a defined molecular function to increase the production of the second messenger cAMP. Gα _olf is abundant in the striatum, and is the only stimulatory G-protein in many cell types of the striatum. Due to the defined molecular signaling pathway and expression pattern of Gα _olf , the clear genetic link to dystonia makes GNAL an exciting target to understand the pathological mechanisms of not only this genetic dystonia, but also the larger idiopathic disease. To better understand GNAL -linked dystonia, we generated a novel genetic mouse model that allows us to conditionally knock out Gnal in a site and time-specific manner. In the current study we used genetic or AAV based approaches to express Cre to knockout striatal Gnal in our novel Gnal fl/fl model. We then performed motor behavioral testing and ex vivo whole-cell patch clamp electrophysiology of striatal spiny projection neurons to interrogate how loss of Gnal leads to dystonia. Mice with conditional striatal knockout of Gnal show hindlimb clasping, other dystonia-like postures, less motor coordination, slowness, and torticollis as compared to age-matched controls. Furthermore, striatal spiny projection neurons show increased excitability in Gnal knockout animals. These exciting data are the first to report uninduced, overt dystonia in a mouse model of GNAL- linked dystonia, and directly correlate these with changes in spiny projection neuron electrophysiological properties. Our results show that adult loss of Gnal in the striatum leads to the development of dystonia, through homeostatic, paradoxical increases in spiny projection neuron excitability, and suggest that therapeutic strategies aimed at decreasing this hyperexcitable phenotype may provide symptomatic relief for patients with disease. One Sentence Summary: When Gnal is knocked out in the striatum of mice we observe overt behavioral symptoms and hyperexcitability in striatal spiny projection neurons.

Standard treatment for Parkinson’s disease (PD) is dopamine replacement therapy with L-DOPA. However, chronic treatment often results in abnormal involuntary movements called L-DOPA-induced dyskinesia (LID). Prior evidence indicates that heightened striatal cholinergic tone may contribute to LID. Restoring cholinergic inhibition by targeting the inhibitory M ₄ muscarinic acetylcholine (ACh) receptor (M ₄ ) reduces LID in preclinical models. Although intrinsic striatal sources of ACh have been considered for their role in LID, extrinsic sources of ACh such as the pedunculopontine nucleus (PPN) have not been well investigated for their role in LID. Therefore, the current study employed hemiparkinsonian Long-Evans rats with a PPN-targeted cannula ipsilateral to 6-OHDA lesion. Following chronic treatment with L-DOPA, we examined the effect of local unilateral PPN infusion of M ₄ PAM VU0467154 on LID, motor performance, and c-fos expression within the PPN. It was expected that PPN infusion of VU0467154 would reduce LID, reduce L-DOPA’s motor benefit, and globally reduce c-fos expression in the PPN. Contrary to our expectations, PPN infusion of M ₄ PAM did not significantly affect LID severity. Furthermore, the group receiving M ₄ PAM showed slightly elevated motor improvement compared to L-DOPA, and decreased c-fos expression specifically in PPN cholinergic neurons. These results suggest that local PPN ACh dynamics differ from those of the striatum. Specifically, our results suggest that PPN cholinergic neurons may be a promising therapeutic target for augmenting L-DOPA-mediated motor benefit without increasing LID.

The heterotrimeric G-protein α subunit, Gα _olf , acts to transduce extracellular signals through G-protein coupled receptors (GPCRs) and stimulates adenylyl cyclase mediated production of the second messenger cyclic adenosine monophosphate. Numerous mutations in the GNAL gene, which encodes Gα _olf , have been identified as causative for an adult-onset dystonia. These mutations disrupt GPCR signaling cascades in in vitro assays through several mechanisms, and this disrupted signaling is hypothesized to lead to dystonic motor symptoms in patients. However, the cells and circuits that mutations in GNAL corrupt are not well understood. Published patterns of Gα _olf expression outside the context of the striatum are sparse, conflicting, often lack cell type specificity, and may be confounded by expression of the close GNAL homolog of GNAS . Here, we use RNAScope in-situ hybridization to quantitatively characterize Gnal mRNA expression in brain tissue from wildtype C57BL/6J adult mice. We observed widespread expression of Gnal puncta throughout the brain, suggesting Gα _olf is expressed in more brain structures and neuron types than previously accounted for. We quantify transcripts at a single cell level, and use neuron type specific markers to further classify and understand patterns of GNAL expression. Our data suggests that brain regions classically associated with motor control, initiation, and regulation show the highest expression of GNAL , with Purkinje Cells of the cerebellum showing the highest expression of any neuron type examined. Subsequent conditional Gnal knockout in Purkinje cells led to markedly decreased intracellular cAMP levels and downstream cAMP-dependent enzyme activation. Our work provides a detailed characterization of Gnal expression throughout the brain and the biochemical consequences of loss of Gα _olf signaling in vivo in neurons that highly express Gnal .

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% ➔ 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.

Barbeau's seesaw hypothesis of dopamine-acetylcholine balance has predominated movement disorders literature for years. Both the simplicity of the explanation and the matching efficacy of anticholinergic treatment in movement disorders seem to support this hypothesis. However, evidence from translational and clinical studies in movement disorders indicates that many features of this simple balance are lost, broken, or absent from movement disorders models or in imaging studies of patients with these disorders. This review reappraises the dopamine-acetylcholine balance hypothesis in light of recent evidence and describes how the Gα_i/o coupled muscarinic M₄ receptor acts in opposition to dopamine signaling in the basal ganglia. We highlight how M₄ signaling can ameliorate or exacerbate movement disorders symptoms and physiological correlates of these symptoms in specific disease states. Furthermore, we propose future directions for investigation of this mechanisms to fully understand the potential efficacy of M₄ targeting therapeutics in movement disorders. Overall, initial evidence suggest that M₄ is a promising pharmaceutical target to ameliorate motor symptoms of hypo- and hyper-dopaminergic disorders.

Pedunculopontine nucleus (PPN) cholinergic neurons are implicated in freezing of gait in Parkinson's disease (PD) and motor stereotypy in normal animals, but the causal role of these neurons on specific gait parameters and treatment‐induced dyskinesia remains speculative. Therefore, we examined whether selective cholinergic lesion of the rostral PPN affects PD motor and gait deficits, L‐DOPA‐induced dyskinesia and motor improvement, and DA‐agonist‐induced dyskinesia. Sprague–Dawley rats were assigned to one unilaterally lesioned group: Sham lesion, PPN cholinergic lesion with diphtheria urotensin II fusion toxin, medial forebrain bundle dopamine lesion with 6‐hydroxydopamine, or dual acetylcholine and dopamine lesion. We used gait analysis and forepaw adjusting steps to examine PD gait and motor deficits. Forepaw adjusting steps were also used to assess motor improvement with L‐DOPA treatment. The abnormal involuntary movements scale measured L‐DOPA and dopamine D1‐ and D2‐receptor agonist‐induced dyskinesia. Lesions, verified via tyrosine hydroxylase and choline acetyltransferase immunohistochemistry reduced an average of 95% of nigral dopamine neurons and 80% of PPN cholinergic neurons, respectively. Rats receiving acetylcholine and dual lesion demonstrated enhanced freezing, and acetylcholine lesioned rats exhibited increased print area and stand index. Dopamine and dual lesion produced similar forepaw adjusting steps task on and off L‐DOPA. Relative to DA lesioned rats, dual lesioned rats displayed reduced L‐DOPA and DA agonist‐induced dyskinesia at specific time points. Our results indicate that PPN cholinergic neurons affect gait parameters related to postural stability. Therefore, therapeutically targeting PPN cholinergic neurons could reduce intractable postural instability in PD without affecting motor benefits or side effects of L‐DOPA treatment.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by hypokinetic motor features; however, patients also display non-motor symptoms like sleep disorders. The standard treatment for PD is dopamine replacement with L-DOPA; however, symptoms including gait deficits and sleep disorders are unresponsive to L-DOPA. Notably, these symptoms have been linked to aberrant activity in the pedunculopontine nucleus (PPN). Of late, clinical trials involving PPN deep brain stimulation (DBS) have been employed to alleviate gait deficits. Although preclinical evidence implicating PPN cholinergic neurons in gait dysfunction was initially promising, DBS trials fell short of expected outcomes. One reason for the failure of DBS may be that the PPN is a heterogenous nucleus that consists of GABAergic, cholinergic, and glutamatergic neurons that project to a diverse array of brain structures. Second, DBS trials may have been unsuccessful because PPN neurons are susceptible to mitochondrial dysfunction, Lewy body pathology, and degeneration in PD. Therefore, pharmaceutical or gene-therapy strategies targeting specific PPN neuronal populations or projections could better alleviate intractable PD symptoms. Unfortunately, how PPN neuronal populations and their respective projections influence PD motor and non-motor symptoms remains enigmatic. Herein, we discuss normal cellular and neuroanatomical features of the PPN, the differential susceptibility of PPN neurons to PD-related insults, and we give an overview of literature suggesting a role for PPN neurons in motor and sleep deficits in PD. Finally, we identify future approaches directed towards the PPN for the treatment of PD motor and sleep symptoms.

In the majority of Parkinson’s disease (PD) patients, long-term dopamine (DA) replacement therapy leads to dyskinesia characterized by abnormal involuntary movements (AIMs). There are various mechanisms of dyskinesia, such as the sensitization of striatal DA D1 receptors (D₁R) and upregulation of DA D3 receptors (D₃R). These receptors interact physically and functionally in D₁R-bearing medium spiny neurons to synergistically drive dyskinesia. However, the cross-receptor-mediated effects due to D₁R-D₃R cooperativity are still poorly understood. In pursuit of this, we examined whether or not pharmacological D₁R or D₃R stimulation sensitizes the dyskinetic response to the appositional agonist, a process known as cross-sensitization. First, we established D₁R-D₃R behavioral synergy in a cohort of 6-OHDA-lesioned female adult Sprague-Dawley rats. Then, in a new cohort, we tested for cross-sensitization in a between-subject design. Five groups received a sub-chronic regimen of either saline, the D₁R agonist SKF38393 (1.0 mg/kg), or the D₃R agonist PD128907 (0.3 mg/kg). For the final injection, each group received an acute injection of the other agonist. AIMs were monitored following each injection. Sub-chronic administration of both SKF38393 and PD128907 induced the development of dyskinesia. More importantly, cross-agonism tests revealed reciprocal cross-sensitization; chronic treatment with either SKF38393 or PD128907 induced sensitization to a single administration of the other agonist. This reciprocity was not marked by changes to either D₁R or D₃R striatal mRNA expression. The current study provides key behavioral data demonstrating the role of D3R in dyskinesia and provides behavioral evidence of D1R and D3R functional interactions.