Altered Capicua expression drives regional Purkinje neuron vulnerability through ion channel gene dysregulation in spinocerebellar ataxia type 1

Selective neuronal vulnerability in neurodegenerative disease is poorly understood. Using the ATXN1[82Q] model of spinocerebellar ataxia type 1 (SCA1), we explored the hypothesis that regional differences in Purkinje neuron degeneration could provide novel insights into selective vulnerability. ATXN1[82Q] Purkinje neurons from the anterior cerebellum were found to degenerate earlier than those from the nodular zone, and this early degeneration was associated with selective dysregulation of ion channel transcripts and altered Purkinje neuron spiking. Efforts to understand the basis for selective dysregulation of channel transcripts revealed modestly increased expression of the ATXN1 co-repressor Capicua (Cic) in anterior cerebellar Purkinje neurons. Importantly, disrupting the association between ATXN1 and Cic rescued the levels of these ion channel transcripts, and lentiviral overexpression of Cic in the nodular zone accelerated both aberrant Purkinje neuron spiking and neurodegeneration. These findings reinforce the central role for Cic in SCA1 cerebellar pathophysiology and suggest that only modest reductions in Cic are needed to have profound therapeutic impact in SCA1

Dendritic degeneration in SCA1 is associated with reduced expression of K⁺ channels found in Purkinje neuron dendrites.

<p><b>(A)</b> Measurement of the capacitance of the distal dendritic arbor (C₂) reveals dendritic degeneration starting at five weeks of age in ATXN1[82Q] Purkinje neurons. <b>(B)</b> Measurement of the capacitance of the soma and proximal dendrite (C₁) reveals no change in ATXN1[82Q] Purkinje neurons. <b>(C and D)</b> Evaluation of published RNA sequencing data [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198040#pone.0198040.ref024" target="_blank">24</a>] for all voltage-gated ion channel genes reveals altered expression of several subfamilies of ion channel genes in ATXN1[82Q] mice at five weeks of age <b>(C)</b> and twelve weeks of age <b>(D)</b>. Only ion channel genes where a statistically significant change in expression at these ages is shown here. The entire list of all known 145 ion channel genes with official IUPHAR classification [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198040#pone.0198040.ref025" target="_blank">25</a>] whose expression was assessed is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198040#pone.0198040.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198040#pone.0198040.s002" target="_blank">S2</a> Tables. K⁺ channel genes make up a significant proportion of dysregulated genes at both ages. Those genes where expression is dysregulated at both five and twelve weeks of age are underlined. In <b>(A-B)</b> N = 3–6 mice were utilized per group. Where error bars are present, data are mean ± SEM. * = <i>P<0</i>.<i>05</i>. Statistical significance derived by two-way ANOVA <b>(A</b>, <b>B)</b>.</p

Normalizing intrinsic dendritic excitability limits SCA1 Purkinje neuron dendritic degeneration.

<p><b>(A)</b> Representative images at the cerebellar primary fissure in wild-type mice, ATXN1[82Q] mice exposed to sham surgery and vehicle, ATXN1[82Q] mice exposed to sham surgery and baclofen, and ATXN1[82Q] mice exposed to BK-AAV and baclofen. Scale bar, 50 μm. <b>(B)</b> Molecular layer thinning is reduced at fourteen weeks in ATXN1[82Q] mice injected with BK-AAV and treated with baclofen. Data are mean ± SEM. * = statistically-significant difference from wild-type and ATXN1[82Q] BK-AAV + Baclofen groups; # = statistically-significant difference from all other groups. Statistical significance derived by one-way ANOVA with Holm-Sidak multiple comparison test.</p

Increased Purkinje neuron dendritic membrane excitability is present at five weeks.

<p><b>(A)</b> Representative image of dendritic patch clamp recording configuration with patch pipette (red) recording from a Purkinje neuron dendrite (yellow arrows). <b>(B)</b> Representative dendritic whole-cell patch clamp recordings from a five week old wild-type Purkinje neuron (soma-to-patch distance: 53.9 μm) and five week old ATXN1[82Q] Purkinje neuron (soma-to-patch distance: 58.2 μm). Traces have been aligned at the threshold of the spike. <b>(C)</b> Scatter-plots with single-exponent decay best-fit lines for back-propagating action potential (bAP) amplitude measurements in wild-type and ATXN1[82Q] Purkinje neurons at five weeks of age show less attenuation of back-propagating action potentials in ATXN1[82Q] mice. Each data point represents a recording from a separate cell. Best-fit line is an exponential decay model with two parameters, of the form <i>y</i> = <i>Ae</i>^−<i>bx</i>, consistent with a previous study of action potential backpropagation in Purkinje neurons [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198040#pone.0198040.ref037" target="_blank">37</a>] and showing R² values of 0.9717 (wild-type) and 0.9873 (ATXN1[82Q]). <b>(D)</b> Scatter-plots with single-exponent growth best-fit lines for bAP half-width measurements in wild-type and ATXN1[82Q] Purkinje neurons at five weeks of age show wider bAPs in ATXN1[82Q] mice. Each data point represents a recording from a separate cell. Best-fit line is an exponential growth model with two parameters, of the form <i>y</i> = <i>Ae</i>^<i>bx</i>. <b>(E)</b> Representative action potential from a somatic whole-cell recording in wild-type and ATXN1[82Q] Purkinje neurons at five weeks of age. The peak-to-trough amplitude of action potentials (summarized in <b>(F)</b>) and the action potential half-width (summarized in <b>(G)</b>) recorded at the soma do not differ significantly between wild-type and ATXN1[82Q] Purkinje neurons at five weeks of age. In <b>(C</b>, <b>D</b>, <b>F</b>, and <b>G)</b> N = 5–10 mice were utilized per genotype. Where error bars are present, data are mean ± SEM. * = <i>P<0</i>.<i>05</i>; NS = Not significant. Statistical significance derived by extra sum of squares F test to determine whether one should reject the null hypothesis that data are best described by a single curve rather than two curves separated by genotype <b>(C</b>, <b>D)</b> or unpaired two-tailed Student’s T-Test <b>(F</b>, <b>G)</b>.</p

Increased dendritic membrane excitability persists in fifteen week atrophic SCA1 Purkinje neurons.

<p><b>(A)</b> Representative dendritic whole-cell patch clamp recordings from a fifteen week old wild-type Purkinje neuron (soma-to-patch distance: 45.0 μm) and fifteen week old ATXN1[82Q] Purkinje neuron (soma-to-patch distance: 48.6 μm). Traces have been aligned at the threshold of the spike. <b>(B)</b> Scatter-plots with single-exponent decay best-fit lines for back-propagating action potential (bAP) amplitude measurements in wild-type and ATXN1[82Q] Purkinje neurons at fifteen weeks of age show less attenuation of bAPs in ATXN1[82Q] mice. Each data point represents a recording from a separate cell. Best-fit line is an exponential decay model with two parameters, of the form <i>y</i> = <i>Ae</i>^−<i>bx</i>, consistent with a previous study of action potential backpropagation in Purkinje neurons [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198040#pone.0198040.ref037" target="_blank">37</a>] and showing R² values of 0.9834 (wild-type) and 0.9346 (ATXN1[82Q]). <b>(C)</b> Scatter-plots with single-exponent growth best-fit lines for bAP half-width measurements in wild-type and ATXN1[82Q] Purkinje neurons at fifteen weeks of age show wider bAPs in ATXN1[82Q] mice. Each data point represents a recording from a separate cell. Best-fit line is an exponential growth model with two parameters, of the form <i>y</i> = <i>Ae</i>^<i>bx</i>. <b>(D)</b> Representative action potential from a somatic whole-cell recording in wild-type and ATXN1[82Q] Purkinje neurons at fifteen weeks of age. The peak-to-trough amplitude of action potentials (summarized in <b>(E)</b>) and the action potential half-width (summarized in <b>(F)</b>) recorded at the soma do not differ significantly between wild-type and ATXN1[82Q] Purkinje neurons at fifteen weeks of age. In <b>(B</b>, <b>C</b>, <b>E</b>, and <b>F)</b> N = 5–10 mice were utilized per genotype. Where error bars are present, data are mean ± SEM. * = <i>P<0</i>.<i>05</i>; NS = Not significant. Statistical significance derived by extra sum of squares F test to determine whether one should reject the null hypothesis that data are best described by a single curve rather than two curves separated by genotype <b>(B</b>, <b>C)</b> or unpaired two-tailed Student’s T-Test <b>(E</b>, <b>F)</b>.</p

Rescuing intrinsic dendritic excitability improves SCA1 mouse motor performance.

<p><b>(A)</b> Representative traces from Purkinje neurons at five weeks of age where dendritic calcium spikes were evoked with somatic current injection (injected current amount indicated on the trace). Recordings are from wild-type mice treated with GFP-AAV (left), ATXN1[82Q] mice treated with GFP-AAV (middle), and ATXN1[82Q] mice treated with BK-AAV and recorded in the presence of 2 μm baclofen (right). <b>(B)</b> At five weeks of age, Purkinje neurons from ATXN1[82Q] mice which had been injected with BK-AAV and recorded in the presence of 2 μm baclofen and 1 μM TTX show a normalization of their dendritic calcium spike threshold. Data are mean ± SEM. * = <i>P<0</i>.<i>05</i>. Statistical significance derived by one-way ANOVA with Holm-Sidak multiple comparison test with. N = 4–6 mice were utilized per group. <b>(C)</b> Input resistance is consistent across all conditions tested when measured in normal aCSF with 1 μm TTX. Data are mean ± SEM. * = <i>P<0</i>.<i>05</i>. Statistical significance derived by one-way ANOVA with Holm-Sidak multiple comparison test with. N = 4–6 mice were utilized per group. <b>(D)</b> Rotarod task performance was improved in ATXN1[82Q] mice injected with BK-AAV and treated with baclofen, when compared to ATXN1[82Q] mice that received sham surgery and were treated with vehicle. Wild-type mice that were treated with vehicle were used as a control. Data are mean ± SEM. * = statistically-significant difference from Wild-Type + Vehicle; #* = statistically-significant difference ATXN1[82Q] + Sham + Vehicle. Statistical significance derived by repeated-measures two-way ANOVA with Holm-Sidak multiple comparison test. <b>(E)</b> Rotarod task performance was not improved in ATXN1[82Q] mice treated with baclofen alone, when compared to ATXN1[82Q] mice that were treated with vehicle. Untreated wild-type mice were used as a control. Data are mean ± SEM. * = statistically-significant difference from ATXN1[82Q] + Vehicle and ATXN1[82Q] + Baclofen; NS = not significant.</p

A Chlorzoxazone‐Baclofen Combination Improves Cerebellar Impairment in Spinocerebellar Ataxia Type 1

BackgroundA combination of central muscle relaxants, chlorzoxazone and baclofen (chlorzoxazone‐baclofen), has been proposed for treatment of cerebellar symptoms in human spinocerebellar ataxia. However, central muscle relaxants can worsen balance. The optimal dose for target engagement without toxicity remains unknown. Using the genetically precise Atxn1154Q/2Q model of spinocerebellar ataxia type 1, we aimed to determine the role of cerebellar dysfunction in motor impairment. We also aimed to identify appropriate concentrations of chlorzoxazone‐baclofen needed for target engagement without toxicity to plan for human clinical trials.MethodsWe use patch clamp electrophysiology in acute cerebellar slices and immunostaining to identify the specific ion channels targeted by chlorzoxazone‐baclofen. Behavioral assays for coordination and grip strength are used to determine specificity of chlorzoxazone‐baclofen for improving cerebellar dysfunction without off‐target effects in Atxn1154Q/2Q mice.ResultsWe identify irregular Purkinje neuron firing in association with reduced expression of ion channels Kcnma1 and Cacna1g in Atxn1154Q/2Q mice. Using in vitro electrophysiology in brain slices, we identified concentrations of chlorzoxazone‐baclofen that improve Purkinje neuron spike regularity without reducing firing frequency. At a disease stage in Atxn1154Q/2Q mice when motor impairment is due to cerebellar dysfunction, orally administered chlorzoxazone‐baclofen improves motor performance without affecting muscle strength.ConclusionWe identify a tight relationship between baclofen‐chlorzoxazone concentrations needed to engage target and levels above which cerebellar function will be compromised. We propose to use this information for a novel clinical trial design, using sequential dose escalation within each subject, to identify dose levels that are likely to improve ataxia symptoms while minimizing toxicity. © 2020 International Parkinson and Movement Disorder SocietyPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/167017/1/mds28355_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167017/2/mds28355.pd

Targeting potassium channels to treat cerebellar ataxia.

ObjectivePurkinje neuron dysfunction is associated with cerebellar ataxia. In a mouse model of spinocerebellar ataxia type 1 (SCA1), reduced potassium channel function contributes to altered membrane excitability resulting in impaired Purkinje neuron spiking. We sought to determine the relationship between altered membrane excitability and motor dysfunction in SCA1 mice.MethodsPatch-clamp recordings in acute cerebellar slices and motor phenotype testing were used to identify pharmacologic agents which improve Purkinje neuron physiology and motor performance in SCA1 mice. Additionally, we retrospectively reviewed records of patients with SCA1 and other autosomal-dominant SCAs with prominent Purkinje neuron involvement to determine whether currently approved potassium channel activators were tolerated.ResultsActivating calcium-activated and subthreshold-activated potassium channels improved Purkinje neuron spiking impairment in SCA1 mice (P &lt; 0.05). Additionally, dendritic hyperexcitability was improved by activating subthreshold-activated potassium channels but not calcium-activated potassium channels (P &lt; 0.01). Improving spiking and dendritic hyperexcitability through a combination of chlorzoxazone and baclofen produced sustained improvements in motor dysfunction in SCA1 mice (P &lt; 0.01). Retrospective review of SCA patient records suggests that co-treatment with chlorzoxazone and baclofen is tolerated.InterpretationTargeting both altered spiking and dendritic membrane excitability is associated with sustained improvements in motor performance in SCA1 mice, while targeting altered spiking alone produces only short-term improvements in motor dysfunction. Potassium channel activators currently in clinical use are well tolerated and may provide benefit in SCA patients. Future clinical trials with potassium channel activators are warranted in cerebellar ataxia