Understanding Exposure-Receptor Occupancy Relationships for Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators across a Range of Preclinical and Clinical Studies.

The metabotropic glutamate receptor 5 (mGlu5) is a recognized central nervous system therapeutic target for which several negative allosteric modulator (NAM) drug candidates have or are continuing to be investigated for various disease indications in clinical development. Direct measurement of target receptor occupancy (RO) is extremely useful to help design and interpret efficacy and safety in nonclinical and clinical studies. In the mGlu5 field, this has been successfully achieved by monitoring displacement of radiolabeled ligands, specifically binding to the mGlu5 receptor, in the presence of an mGlu5 NAM using in vivo and ex vivo binding in rodents and positron emission tomography imaging in cynomolgus monkeys and humans. The aim of this study was to measure the RO of the mGlu5 NAM HTL0014242 in rodents and cynomolgus monkeys and to compare its plasma and brain exposure-RO relationships with those of clinically tested mGlu5 NAMs dipraglurant, mavoglurant, and basimglurant. Potential sources of variability that may contribute to these relationships were explored. Distinct plasma exposure-response relationships were found for each mGlu5 NAM, with >100-fold difference in plasma exposure for a given level of RO. However, a unified exposure-response relationship was observed when both unbound brain concentration and mGlu5 affinity were considered. This relationship showed <10-fold overall difference, was fitted with a Hill slope that was not significantly different from 1, and appeared consistent with a simple Emax model. This is the first time this type of comparison has been conducted, demonstrating a unified brain exposure-RO relationship across several species and mGlu5 NAMs with diverse properties. SIGNIFICANCE STATEMENT: Despite the long history of mGlu5 as a therapeutic target and progression of multiple compounds to the clinic, no formal comparison of exposure-receptor occupancy relationships has been conducted. The data from this study indicate for the first time that a consistent, unified relationship can be observed between exposure and mGlu5 receptor occupancy when unbound brain concentration and receptor affinity are taken into account across a range of species for a diverse set of mGlu5 negative allosteric modulators, including a new drug candidate, HTL0014242.

AMPA receptors mediate the pro-cognitive effects of electrical and optogenetic stimulation of the medial prefrontal cortex in antidepressant non-responsive Wistar-Kyoto rats.

BACKGROUND: The chronic mild stress (CMS) procedure is a widely used animal model of depression, and its application in Wistar-Kyoto (WKY) rats has been validated as a model of antidepressant-refractory depression. While not responding to chronic treatment with antidepressant drugs, WKY rats do respond to acute deep brain stimulation (DBS) of the medial prefrontal cortex (mPFC). In antidepressant-responsive strains there is evidence suggesting a role for AMPA subtype of glutamate receptor in the action mechanism of both antidepressants and DBS. METHODS: Animals were subjected to CMS for 6 to 8 weeks; sucrose intake was monitored weekly and novel object recognition (NOR) test was conducted following recovery from CMS. Wistars were treated chronically with venlafaxine (VEN), while WKY were treated acutely with either DBS, optogenetic stimulation (OGS) of virally-transduced (AAV5-hSyn-ChR2-EYFP) mPFC or ventral hippocampus, or acute intra-mPFC injection of the AMPA receptor positive allosteric modulator CX-516. The AMPA receptor antagonist NBQX was administered, at identical sites in mPFC, immediately following the exposure trial in the NOR. RESULTS: Sucrose intake and NOR were suppressed by CMS, and restored by VEN in Wistars and by DBS, OGS, or CX-516 in WKY. However, OGS of the ventral hippocampal afferents to mPFC was ineffective. A low dose of NBQX selectively blocked the procognitive effect of VEN, DBS and OGS. CONCLUSIONS: These results suggest that activation of AMPA receptors in the mPFC represents a common pathway for the antidepressant effects of both conventional (VEN) and novel (DBS, OGS) antidepressant modalities, in both antidepressant responsive (Wistar) and antidepressant-resistant (WKY) rats.

Moving Towards Therapy in SCA1: Insights from Molecular Mechanisms, Identification of Novel Targets, and Planning for Human Trials.

The spinocerebellar ataxias (SCAs) are a group of neurodegenerative disorders inherited in an autosomal dominant fashion. The SCAs result in progressive gait imbalance, incoordination of the limbs, speech changes, and oculomotor dysfunction, among other symptoms. Over the past few decades, significant strides have been made in understanding the pathogenic mechanisms underlying these diseases. Although multiple efforts using a combination of genetics and pharmacology with small molecules have been made towards developing new therapeutics, no FDA approved treatment currently exists. In this review, we focus on SCA1, a common SCA subtype, in which some of the greatest advances have been made in understanding disease biology, and consequently potential therapeutic targets. Understanding of the underlying basic biology and targets of therapy in SCA1 is likely to give insight into treatment strategies in other SCAs. The diversity of the biology in the SCAs, and insight from SCA1 suggests, however, that both shared treatment strategies and specific approaches tailored to treat distinct genetic causes of SCA are likely needed for this group of devastating neurological disorders.

TAK-137, an AMPA-R potentiator with little agonistic effect, has a wide therapeutic window.

Activation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPA-R) is a promising strategy to treat psychiatric and neurological diseases if issues of bell-shaped response and narrow safety margin against seizure can be overcome. Here, we show that structural interference at Ser743 in AMPA-R is a key to lower the agonistic effect of AMPA-R potentiators containing dihydropyridothiadiazine 2,2-dioxides skeleton. With this structural insight, TAK-137, 9-(4-phenoxyphenyl)-3,4-dihydropyrido[2,1-c][1,2,4]thiadiazine 2,2-dioxide, was discovered as a novel AMPA-R potentiator with a lower agonistic effect than an AMPA-R potentiator LY451646 ((R)-N-(2-(4'-cyanobiphenyl-4-yl)propyl)propane-2-sulfonamide) in rat primary neurons. TAK-137 induced brain-derived neurotrophic factor in neurons in rodents and potently improved cognition in both rats and monkeys. Compared to LY451646, TAK-137 had a wider safety margin against seizure in rats. TAK-137 enhanced neural progenitor proliferation over a broader range of doses in rodents. Thus, TAK-137 is a promising AMPA-R potentiator with potent procognitive effects and lower risks of bell-shaped response and seizure. These data may open the door for the development of AMPA-R potentiators as therapeutic drugs for psychiatric and neurological diseases.

Rat Cerebrospinal Fluid Treatment Method through Cisterna Cerebellomedullaris Injection.

Drugs that lack the ability to cross the blood-brain barrier (BBB) need to be placed directly into the central nervous system. Our laboratory studies the involvement of the glutamatergic system in the aggressiveness of glioma, and some ligands of glutamate receptors cannot permeate the BBB. Here, glioma-implanted rats were treated by a technique that delivers ligands directly into the cerebrospinal fluid by puncture into the cisterna cerebellomedullaris. Rats were anesthetized and fixed in a rodent stereotactic device. The head was gently tilted downwards at an angle that allowed exposure of the cisterna. Injection into the cisterna was done freehand using a gingival needle coupled to a microsyringe. The efficiency of intracisternal injection was demonstrated using a methylene blue solution. This type of injection is adaptable for any rodent model using small volumes of a variety of other drugs, and is an interesting method for neuroscience studies.

Glutamatergic Modulators in Depression.

LEARNING OBJECTIVE: After participating in this activity, learners should be better able to evaluate the evidence supporting the antidepressant effects of glutamatergic modulators.Both preclinical and clinical studies have implicated glutamatergic system dysfunction in the pathophysiology of mood disorders such as bipolar depression and major depressive disorder. In particular, rapid reductions in depressive symptoms have been noted in response to subanesthetic doses of the glutamatergic modulator ketamine in subjects with major depressive disorder or bipolar depression. These results have prompted the repurposing or development of other glutamatergic modulators, both as monotherapy or adjunctive to other therapies. Here, we highlight the evidence supporting the antidepressant effects of various glutamatergic modulators, including (1) broad glutamatergic modulators (ketamine, esketamine, dextromethorphan, dextromethorphan-quinidine [Nuedexta], AVP-786, nitrous oxide [N2O], AZD6765), (2) subunit (NR2B)-specific N-methyl-D-aspartate (NMDA) receptor antagonists (CP-101,606/traxoprodil, MK-0657 [CERC-301]), (3) glycine-site partial agonists (D-cycloserine, GLYX-13, sarcosine, AV-101), and (4) metabotropic glutamate receptor modulators (AZD2066, RO4917523/basimglurant, JNJ40411813/ADX71149, R04995819 [RG1578]).

Fasoracetam in adolescents with ADHD and glutamatergic gene network variants disrupting mGluR neurotransmitter signaling.

The glutamatergic neurotransmitter system may play an important role in attention-deficit hyperactivity disorder (ADHD). This 5-week, open-label, single-blind, placebo-controlled study reports the safety, pharmacokinetics and responsiveness of the metabotropic glutamate receptor (mGluR) activator fasoracetam (NFC-1), in 30 adolescents, age 12-17 years with ADHD, harboring mutations in mGluR network genes. Mutation status was double-blinded. A single-dose pharmacokinetic profiling from 50-800 mg was followed by a single-blind placebo at week 1 and subsequent symptom-driven dose advancement up to 400 mg BID for 4 weeks. NFC-1 treatment resulted in significant improvement. Mean Clinical Global Impressions-Improvement (CGI-I) and Severity (CGI-S) scores were, respectively, 3.79 at baseline vs. 2.33 at week 5 (P < 0.001) and 4.83 at baseline vs. 3.86 at week 5 (P < 0.001). Parental Vanderbilt scores showed significant improvement for subjects with mGluR Tier 1 variants (P < 0.035). There were no differences in the incidence of adverse events between placebo week and weeks on active drug. The trial is registered at https://clinicaltrials.gov/ct2/show/study/NCT02286817 .

AMPAkines and morphine provide complementary analgesia.

Glutamate signaling in the central nervous system is known to play a key role in pain regulation. AMPAkines can enhance glutamate signaling through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. previous studies have shown that AMPAkines are effective analgesic agents, and their site of action is likely in the brain. It is not known, however, if AMPAkines can provide complementary analgesia in combination with opioids, the most commonly used analgesics. Here, we show that the co-administration of an AMPAkine with morphine can provide additional analgesia, both in naïve rats and in rats that experience postoperative pain. Furthermore, we show that this AMPAkine can be administered directly into the prefrontal cortex to provide analgesia, and that prefrontal AMPAkine infusion, similar to systemic administration, can provide added pain relief to complement morphine analgesia.

Ketamine and Beyond: Investigations into the Potential of Glutamatergic Agents to Treat Depression.

Clinical and preclinical studies suggest that dysfunction of the glutamatergic system is implicated in mood disorders such as major depressive disorder and bipolar depression. In clinical studies of individuals with major depressive disorder and bipolar depression, rapid reductions in depressive symptoms have been observed in response to subanesthetic-dose ketamine, an agent whose mechanism of action involves the modulation of glutamatergic signaling. The findings from these studies have prompted the repurposing and/or development of other glutamatergic modulators for antidepressant efficacy, both as monotherapy or as an adjunct to conventional monoaminergic antidepressants. This review highlights the evidence supporting the antidepressant effects of subanesthetic-dose ketamine as well as other glutamatergic modulators, such as D-cycloserine, riluzole, CP-101,606, CERC-301 (previously known as MK-0657), basimglurant, JNJ-40411813, dextromethorphan, nitrous oxide, GLYX-13, and esketamine.

Neuropeptide cycloprolylglycine is an endogenous positive modulator of AMPA receptors.

We have previously shown that neuropeptide cycloprolylglycine (CPG) increases the content of brain-derived neurotrophic factor (BDNF) in the culture of neuronal cells under normal conditions and in pathology. This is the first study to show that CPG at a physiological concentration of 10-6 M significantly enhances the transmembrane AMPA currents in rat cerebellar Purkinje cells. Thus, CPG is a positive endogenous modulator of AMPA receptors. It was assumed that the neuropsychotropic effects of CPG are implemented as a result of BDNF accumulation after the activation of AMPA receptors by this neuropeptide.

Suppression of the swallowing reflex by stimulation of the red nucleus.

We study whether the red nucleus is involved in control of swallowing. The swallowing reflex was induced in anesthetized rats by repetitive electrical stimulation of the superior laryngeal nerve. The electromyographic activities of the mylohyoid and thyrohyoid muscles were recorded in order to identify the swallowing reflex. Repetitive electrical stimulation applied to the red nucleus reduced the number of swallows. The onset latency of the first swallow was increased during repetitive electrical stimulation applied to the magnocellular part of the red nucleus. Microinjection of monosodium glutamate into the red nucleus also reduced the number of swallows. The onset latency of the first swallow was increased after microinjection of monosodium glutamate into the magnocellular part of the red nucleus. These results imply that the red nucleus is involved in the control of swallowing.

Glutamatergic agents for schizophrenia: current evidence and perspectives.

Suboptimal outcomes in schizophrenia are a consequence of lacking insight into the etiology, biomarkers and treatment-relevant subgroups, the therapeutic restriction to dopaminergic-modulating antipsychotics that fail to significantly improve negative and cognitive symptoms, non-adherence, and, in the case of treatment-resistance, the underutilization of clozapine. Evidence suggests additional, extra-dopaminergic abnormalities in amino acid neurotransmission, particularly the glutamatergic system. Antidopaminergic antipsychotics modulate this system on several levels, as do mood stabilizers, including lamotrigine, topiramate and pregabaline. Recently, agonists at metabotropic glutamate receptors and glycine uptake inhibitors failed in large placebo-controlled trials for schizophrenia. Problems to overcome for successfully leveraging glutamatergic agents for schizophrenia are patient selection, focus on positive symptoms and late disease stages, and dose-response relationships. Because glutamate guides processes of brain development and maturation, clinical research should focus on the at-risk mental state or first-episode psychosis, address cognition and negative symptoms and use monotherapy designs in parallel to augmentation strategies.

Effects of muscle pain induced by glutamate injections during sustained clenching on the contraction pattern of masticatory muscles.

AIMS: To evaluate the contraction pattern of masticatory muscles during sustained clenching tasks with or without experimental pain induced by glutamate injection into the masseter muscle. It was hypothesized that acute muscle pain could induce compensatory changes in the electromyographic (EMG) activity of the masticatory muscles. METHODS: Fifteen volunteers (seven males, mean age ± SD = 29.7 ± 1.1 years; eight females, mean age ± SD = 23.5 ± 1.2 years) were recruited in a crossover experimental study. All subjects participated in two randomized 20-minute experimental sessions. Each subject was asked to clench at 25% of the maximum voluntary contraction (MVC). After 10 minutes, isotonic saline or glutamate was injected in random order into the right masseter. EMG activity (root mean square [RMS] and mean power frequency [MPF]) was assessed in the masseter and anterior temporalis muscles on both sides. Pain and fatigue were assessed by 0-10 numeric rating scales (NRS) every minute. Differences between conditions (isotonic saline vs glutamate) for all the outcome parameters were analyzed by using a mixed effect model. RESULTS: The EMG activity of the masticatory muscles and pain and fatigue scores were not dependent on isotonic saline/glutamate injection (all P > .05). The RMS in the temporalis and masseter muscles increased with time (right masseter P = 0.001, left masseter P = .004, right temporalis P = .22, left temporalis P = .006), whereas the MPF decreased (right masseter P = .0001, left masseter P < .0001, right temporalis P = .51, left temporalis P = .0005). Scores for fatigue and pain increased during the experimental sessions (all P < .05). CONCLUSION: Intramuscular injection of glutamate caused more pain than isotonic saline but did not affect the contraction pattern of the masticatory muscles during a sustained clenching task. This finding strongly suggests the adaptive capacity of the stomatognathic system in the presence of acute nociceptive inputs.

Differential behavioral profile induced by the injection of dipotassium chlorazepate within brain areas that project to the nucleus accumbens septi.

BACKGROUND: The effect of the agonism on γ-aminobutyric acid (GABA) receptors was studied within medial prefrontal cortex (mPFC), amygdala (AMY) and ventral hipocampus (VH) in the plus-maze test in male rats bilaterally cannulated. These structures send glutamatergic projections to the nucleus accumbens septi (NAS), in which interaction and integration between these afferent pathways has been described. In a previous study of our group, blockade of glutamatergic transmission within NAS induced an anxiolytic like effect. METHODS: Three rat groups received either saline or dipotassium chlorazepate (1 or 2 µg/1 µl solution) 15 min before testing. Time spent in the open arms (TSOA), time per entry (TPE), extreme arrivals (EA), open and closed arms entries (OAE, CAE) and relationship between open- and closed-arms quotient (OCAQ) were recorded. RESULTS: In the AMY injected group TSOA, OAE and EA were increased by the higher doses of dipotassium chlorazepate (p < 0.01). In the mPFC, TPE was decreased by both doses (p < 0.05). Injection within ventral hippocampus (VH) decreased TSOA, OAE and OCAQ with lower doses (p < 0.05). When the three studied saline groups were compared, TSOA, OAE, EA and OCAQ were enhanced in the VH group when compared to mPFC and AMY (p < 0.001). Insertion of inner canula (p < 0.001, p < 0.01, p < 0.01) and saline injection showed an increasing significant difference (p < 0.001 in all cases) with the action of guide cannula alone within VH in TSOA, OAE and EA. CONCLUSION: We conclude that the injection of dipotassium chlorazepate has a differential effect depending of the brain area, leading to facilitatory and inhibitory effects on anxiety processing.

Metabotropic glutamate receptors as therapeutic targets for schizophrenia.

Treatment options for schizophrenia that address all symptom categories (positive, negative, and cognitive) are lacking in current therapies for this disorder. Compounds targeting the metabotropic glutamate (mGlu) receptors hold promise as a more comprehensive therapeutic alternative to typical and atypical antipsychotics and may avoid the occurrence of extrapyramidal side effects that accompany these treatments. Activation of the group II mGlu receptors (mGlu(2) and mGlu(3)) and the group I mGlu(5) are hypothesized to normalize the disruption of thalamocortical glutamatergic circuitry that results in abnormal glutamaterigic signaling in the prefrontal cortex (PFC). Agonists of mGlu(2) and mGlu(3) have demonstrated efficacy for the positive symptom group in both animal models and clinical trials with mGlu(2) being the subtype most likely responsible for the therapeutic effect. Limitations in the chemical space tolerated by the orthosteric site of the mGlu receptors has led to the pursuit of compounds that potentiate the receptor's response to glutamate by acting at less highly conserved allosteric sites. Several series of selective positive allosteric modulators (PAMs) for mGlu(2) and mGlu(5) have demonstrated efficacy in animal models used for the evaluation of antipsychotic agents. In addition, evidence from animal studies indicates that mGlu(5) PAMs hold promise for the treatment of cognitive deficits that occur in schizophrenia. Hopefully, further optimization of allosteric modulators of mGlu receptors will yield clinical candidates that will allow full evaluation of the potential efficacy of these compounds in the treatment of multiple symptom domains in schizophrenia patients in the near future.

Metabotropic glutamate receptors and interacting proteins: evolving drug targets.

The correct targeting, localization, regulation and signaling of metabotropic glutamate receptors (mGluRs) represent major mechanisms underlying the complex function of neuronal networks. These tasks are accomplished by the formation of synaptic signal complexes that integrate functionally related proteins such as neurotransmitter receptors, enzymes and scaffold proteins. By these means, proteins interacting with mGluRs are important regulators of glutamatergic neurotransmission. Most described mGluR interaction partners bind to the intracellular C-termini of the receptors. These domains are extensively spliced and phosphorylated, resulting in a high variability of binding surfaces offered to interacting proteins. Malfunction of mGluRs and associated proteins are linked to neurodegenerative and neuropsychiatric disorders including addiction, depression, epilepsy, schizophrenia, Alzheimer's, Huntington's and Parkinson's disease. MGluR associated signal complexes are dynamic structures that assemble and disassemble in response to the neuronal fate. This, in principle, allows therapeutic intervention, defining mGluRs and interacting proteins as promising drug targets. In the last years, several studies elucidated the geometry of mGluRs in contact with regulatory proteins, providing a solid fundament for the development of new therapeutic strategies. Here, I will give an overview of human disorders directly associated with mGluR malfunction, provide an up-to-date summary of mGluR interacting proteins and highlight recently described structures of mGluR domains in contact with binding partners.

Effects of the mGlu2/3 agonist LY379268 and the mGlu5 antagonist MTEP on ethanol seeking and reinforcement are differentially altered in rats with a history of ethanol dependence.

BACKGROUND: Growing evidence supports a role of metabotropic glutamate receptors (mGluRs) in ethanol reinforcement, ethanol seeking, and ethanol withdrawal. To extend the understanding of the role of mGluRs in the addiction-relevant effects of ethanol as well as of the treatment target potential of these receptors for alcohol abuse, the effects of a selective mGlu2/3 agonist (LY379268) and a selective mGlu5 antagonist (MTEP) were tested on two processes central to alcohol addiction: ethanol reinforcement and stress-induced reinstatement of ethanol seeking in rats with a history of ethanol dependence. METHODS: Following operant ethanol self-administration training, male Wistar rats were made dependent by intragastric ethanol intubations. Ethanol dependence was confirmed by the presence of somatic withdrawal signs. Following 2 weeks of withdrawal, stable ethanol self-administration was reestablished, and the effects of LY379268 (0-3 mg/kg subcutaneous) and MTEP (0-3 mg/kg, intraperitoneal) on ethanol self-administration were determined in both nondependent and postdependent rats. A second set of rats underwent extinction training and then was tested for the effects of LY379268 or MTEP on reinstatement of ethanol seeking induced by footshock stress. RESULTS: LY379268 and MTEP dose-dependently reduced both ethanol self-administration and reinstatement of ethanol seeking induced by footshock stress. Additionally, LY379268 was more effective than MTEP in inhibiting both behaviors in postdependent than in nondependent animals. CONCLUSIONS: These findings suggest that neuroadaptation associated with chronic ethanol exposure or withdrawal alters the sensitivity of mGlu2/3 receptors, with implications for the understanding of the neural basis of alcohol dependence and the treatment target potential of these receptors.

Adjunctive alpha2-adrenoceptor blockade enhances the antipsychotic-like effect of risperidone and facilitates cortical dopaminergic and glutamatergic, NMDA receptor-mediated transmission.

Compared to both first- and second-generation antipsychotic drugs (APDs), clozapine shows superior efficacy in treatment-resistant schizophrenia. In contrast to most APDs clozapine possesses high affinity for alpha2-adrenoceptors, and clinical and preclinical studies provide evidence that the alpha2-adrenoceptor antagonist idazoxan enhances the antipsychotic efficacy of typical D2 receptor antagonists as well as olanzapine. Risperidone has lower affinity for alpha2-adrenoceptors than clozapine but higher than most other APDs. Here we examined, in rats, the effects of adding idazoxan to risperidone on antipsychotic effect using the conditioned avoidance response (CAR) test, extrapyramidal side-effect (EPS) liability using the catalepsy test, brain dopamine efflux using in-vivo microdialysis in freely moving animals, cortical N-methyl-D-aspartate (NMDA) receptor-mediated transmission using intracellular electrophysiological recording in vitro, and ex-vivo autoradiography to assess the in-vivo alpha2A- and alpha2C-adrenoceptor occupancies by risperidone. The dose of risperidone needed for antipsychotic effect in the CAR test was approximately 0.4 mg/kg, which produced 11% and 17% in-vivo receptor occupancy at alpha2A- and alpha2C-adrenoceptors, respectively. Addition of idazoxan (1.5 mg/kg) to a low dose of risperidone (0.25 mg/kg) enhanced the suppression of CAR, but did not enhance catalepsy. Both cortical dopamine release and NMDA receptor-mediated responses were enhanced. These data propose that the therapeutic effect of risperidone in schizophrenia can be enhanced and its EPS liability reduced by adjunctive treatment with an alpha2-adrenoceptor antagonist, and generally support the notion that the potent alpha2-adrenoceptor antagonistic action of clozapine may be highly important for its unique efficacy in schizophrenia.

Deranged calcium signaling and neurodegeneration in spinocerebellar ataxia type 2.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited, neurodegenerative disease caused by an expansion of polyglutamine tracts in the cytosolic protein ataxin-2 (Atx2). Cerebellar Purkinje cells (PCs) are predominantly affected in SCA2. The cause of PC degeneration in SCA2 is unknown. Here we demonstrate that mutant Atx2-58Q, but not wild-type (WT) Atx2-22Q, specifically associates with the cytosolic C-terminal region of type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1), an intracellular calcium (Ca(2+)) release channel. Association with Atx2-58Q increased the sensitivity of InsP(3)R1 to activation by InsP(3) in planar lipid bilayer reconstitution experiments. To validate physiological significance of these findings, we performed a series of experiments with an SCA2-58Q transgenic mouse model that expresses human full-length Atx2-58Q protein under the control of a PC-specific promoter. In Ca(2+) imaging experiments, we demonstrated that stimulation with 3,5-dihydroxyphenylglycine (DHPG) resulted in higher Ca(2+) responses in 58Q PC cultures than in WT PC cultures. DHPG-induced Ca(2+) responses in 58Q PC cultures were blocked by the addition of ryanodine, an inhibitor of the ryanodine receptor (RyanR). We further demonstrated that application of glutamate induced more pronounced cell death in 58Q PC cultures than in WT PC cultures. Glutamate-induced cell death of 58Q PC cultures was attenuated by dantrolene, a clinically relevant RyanR inhibitor and Ca(2+) stabilizer. In whole animal experiments, we demonstrated that long-term feeding of SCA1-58Q mice with dantrolene alleviated age-dependent motor deficits (quantified in beam-walk and rotarod assays) and reduced PC loss observed in untreated SCA2-58Q mice by 12 months of age (quantified by stereology). Results of our studies indicate that disturbed neuronal Ca(2+) signaling may play an important role in SCA2 pathology and also suggest that the RyanR constitutes a potential therapeutic target for treatment of SCA2 patients.