4-aminopyridine reverses ataxia and cerebellar firing deficiency in a mouse model of spinocerebellar ataxia type 6

Abstract

Spinocerebellar ataxia type 6 (SCA6) is a devastating midlife-onset autosomal dominant motor control disease with no known treatment. Using a hyper-expanded polyglutamine (84Q) knock-in mouse, we found that cerebellar Purkinje cell firing precision was degraded in heterozygous (SCA6(84Q/+)) mice at 19 months when motor deficits are observed. Similar alterations in firing precision and motor control were observed at disease onset at 7 months in homozygous (SCA6(84Q/84Q)) mice, as well as a reduction in firing rate. We further found that chronic administration of the FDA-approved drug 4-aminopyridine (4-AP), which targets potassium channels, alleviated motor coordination deficits and restored cerebellar Purkinje cell firing precision to wildtype (WT) levels in SCA6(84Q/84Q) mice both in acute slices and in vivo. These results provide a novel therapeutic approach for treating ataxic symptoms associated with SCA6.

Figure 1. Firing precision deficits in heterozygous SCA6^84Q/+ mice.

(A) 19-month-old heterozygous WT and SCA6^84Q/+ mice. (B) Sample traces from a Purkinje cell from WT (black, top) and SCA6^84Q/+ mice (blue, bottom) illustrate the loss of Purkinje cell firing precision at 19 months. (C) Purkinje cell firing precision, as measured by the coefficient of variation (CV) of inter-spike intervals of Purkinje cell action potentials, is reduced in 19-month-old heterozygous SCA6^84Q/+ mice (WT: CV = 0.08 ± 0.005; SCA6^84Q/+: CV = 0. 11 ± 0.010; P = 0.0047). (D) However, no changes in firing frequency was observed in 19-month-old SCA6^84Q/+ mice (WT: frequency = 65.8 ± 4.9 Hz, SCA6^84Q/84Q: frequency = 53.6 ± 4.23 Hz, P = 0.071; N = 11 WT and N = 19 SCA6^84Q/+ for (C,D)). (E) Motor coordination deficit was observed in SCA6^84Q/+ mice at 19 months (WT mice spent 78.7 ± 10.3 s on Rotarod before falling on day 4, N = 7, grey/black markers; SCA6^84Q/84Q mice spend 26.3 ± 4.2 s on Rotarod on day 4, N = 7, light/dark blue markers; P = 0.0016). Comparisons made with Student’s t test; ***P < 0.005.

Figure 2. Firing precision and rate deficits in homozygous SCA6^84Q/84Q mice.

(A) 7-month-old homozygous WT and SCA6^84Q/84Q mice. (B) Sample traces show action potentials from a single Purkinje cell from a WT (black, top) and an SCA6^84Q/84Q (orange, bottom) mouse at 7 months. (C) Changes in firing precision demonstrated by an elevated CV of inter-spike intervals for Purkinje cell action potential firing in SCA6^84Q/84Q mice (WT: CV = 0.09 ± 0.005; SCA6^84Q/84Q: CV = 0.14 ± 0.01; P < 0.0001). (D) Histogram showing firing rates of individual Purkinje cells from SCA6^84Q/84Q mice (orange) and WT Purkinje cells (black); note the absence of Purkinje cells firing at frequencies >100 Hz in SCA6^84Q/84Q mice. Average WT Purkinje cell firing rate = 71.7 ± 6.1 Hz; average SCA6^84Q/84Q Purkinje cell firing rate = 56.4 ± 3.9 Hz; P = 0.041; N = 28 for WT, N = 26 for SCA6^84Q/84Q for (C,D). Comparisons made with Student’s t tests; ***P < 0.005.

Figure 3. Acute 4-AP rescues firing precision deficits in SCA6^84Q/84Q mice.

(A) Acute application of 5 μM 4-AP in cerebellar slices from SCA6^84Q/84Q and WT mice. (B) Representative traces from SCA6^84Q/84Q mouse Purkinje cell before (orange, top) and after (red, bottom) 4-AP wash-in. (C) Purkinje cell spiking becomes more regular with the application of 4-AP (CV was reduced after 4-AP application, CV_before = 0.15 ± 0.02; CV_after = 0.10 ± 0.009; paired Student’s t test, P = 0.0027; N = 11), while (D) not altering firing rate (frequency_before = 60.7 ± 5.6 Hz; frequency_after = 61.8 ± 5.6 Hz; P = 0.15). ***P < 0.005.

Figure 4. Chronic orally administered 4-AP improves motor coordination in SCA6^84Q/84Q mice.

(A) Experimental set-up. (B) Rotarod performance was reduced in SCA6^84Q/84Q mice (orange) compared to litter-matched WT mice (black; P < 0.0001). Chronic 4-AP administration improved the motor coordination in SCA6^84Q/84Q mice (SCA6^84Q/84Q + 4-AP, red; different from SCA6^84Q/84Q with vehicle, orange; P < 0.0001), although rescue was incomplete to WT levels (P < 0.0001). Oral administration of 4-AP caused no change in WT mice performance (grey; P = 0.99; ANOVA followed by post-hoc Tukey test for all comparisons; N = 11 for WT with vehicle; N = 11 for WT + 4-AP; N = 9 for SCA6^84Q/84Q mice with vehicle; N = 11 for SCA6^84Q/84Q + 4-AP). (C) Chronic 4-AP administration improved motor coordination after 1 week (red bars: Pre-drug versus 1 week 4-AP, P < 0.0001), continued to improve after 2 weeks, (1 versus 2 weeks 4-AP, P = 0.0024) and showed further improvement after 1 month of 4-AP (2 weeks versus 1 month 4-AP, P = 0.02; N = 11 SCA6^84Q/84Q mice); motor performance remained elevated for as long as 3 months (1 versus 3 months not different, P = 0.45; ANOVA followed by post-hoc Tukey test). Motor performance improved in SCA6^84Q/84Q mice without drug after 1 week because of motor learning (Pre-trial versus 1 week no drug, P < 0.0001), and then plateaued (1 versus 2 weeks, P = 0.20; 2 weeks versus 1 month, P = 0.27; 1 versus 3 months, P = 0.84). (D) Improved performance requires the continual administration of 4-AP, as withdrawal of 4-AP resulted in declining Rotarod performance levels (1 week − 4-AP after 1 week + 4-AP, versus 1 week + 4-AP, P < 0.0001; significantly different; N = 5 SCA6^84Q/84Q mice; paired Student’s t test, Bonferroni corrected, α = 0.025). Dashed orange line shows 2 week SCA6^84Q/84Q mice – 4-AP. (E) There was a strong correlation between motor performance and drug intake in SCA6^84Q/84Q mice, while (F), the performance of WT mice was not correlated to drug intake (±4-AP). *P < 0.05, ***P < 0.005.

Figure 5. Chronic 4-AP restores in vitro Purkinje cell firing precision in SCA6^84Q/84Q mice.

(A) After chronic 4-AP or vehicle treatment for 3 months, Purkinje cell action potentials were recorded in the absence of 4-AP in the extracellular solution in acute slices. (B) Sample recordings from age-matched WT (top, black traces), vehicle-treated SCA6^84Q/84Q (middle, orange traces), and chronic 4-AP-treated SCA6^84Q/84Q mice (bottom, red traces) showing that the firing precision is recovered in 4-AP-treated mice. (C) Spike precision, as measured by the CV of inter-spike intervals from Purkinje cell spike trains from WT, vehicle-treated SCA6^84Q/84Q, and 4-AP-treated SCA6^84Q/84Q mice, shows a significant reduction after 4-AP treatment to levels that are indistinguishable from WT (WT: CV = 0.09 ± 0.01; SCA6^84Q/84Q with vehicle: CV = 0.13 ± 0.01; SCA6^84Q/84Q + 4-AP: CV = 0.10 ± 0.01). (D) Firing frequency is however unaffected by 4-AP treatment in SCA6^84Q/84Q mice, and both show significant reduction from WT levels (WT: frequency = 76.6 ± 5.5 Hz; SCA6^84Q/84Q with vehicle: frequency = 58.8 ± 3.7 Hz; SCA6^84Q/84Q + 4-AP: frequency = 59.7 ± 3.9 Hz). N = 20 for WT, N = 18 for untreated SCA6^84Q/84Q, and N = 20 for SCA6^84Q/84Q + 4-AP for (C,D). (E,F) For individual animals, 4-AP treatment produced a significant increase in the time spent on the Rotarod (X-axis for both graphs, P < 0.0005). (E) For individual mice, 4-AP treatment significantly reduced the average CV of recorded Purkinje cells (red) from untreated (orange) SCA6^84Q/84Q mice (P = 0.011 for CV), while (F), there was no significant difference on an animal-by-animal basis for Purkinje cell firing frequency (P = 0.75 for frequency). For (E,F), open circles show data for individual mice, while filled circle show averages. N = 5–8 Purkinje cells/animal for N = 3 vehicle-treated and N = 3 4-AP-treated SCA6^84Q/84Q mice; Comparisons made with one-way ANOVA followed by post-hoc Tukey test for (C,D), and Student’s t test for (E,F); *P < 0.05; **P < 0.01; ***P < 0.005.

Figure 6. Chronic 4-AP restores in vivo Purkinje cell firing precision in SCA6^84Q/84Q mice.

(A) Sample recordings from age-matched WT (top, black trace), vehicle-treated SCA6^84Q/84Q (middle, orange trace), and chronic 4-AP-treated SCA6^84Q/84Q mice (bottom, red trace) showing the firing precision lost in SCA6^84Q/84Q mice is recovered in 4-AP-treated mice. (B) Firing frequency is significantly reduced in SCA6^84Q/84Q mice but is unaffected by 4-AP treatment (WT: frequency = 27.2 ± 2.4 Hz; SCA6^84Q/84Q with vehicle: frequency = 19.0 ± 2.5 Hz; SCA6^84Q/84Q + 4-AP: frequency = 26.3 ± 2.3 Hz). (C) CV does not accurately measure regularity in in vivo Purkinje cell spike trains, and no significant changes are observed across genotype and condition with CV (WT: CV = 0.64 ± 0.10; SCA6^84Q/84Q with vehicle: CV = 0.77 ± 0.06; SCA6^84Q/84Q + 4-AP: CV = 0.69 ± 0.15). (D) However, CV2, a better measure of spike regularity in in vivo Purkinje cell recordings, reveals that Purkinje cells fire with significantly less firing precision in anaesthetized SCA6^84Q/84Q mice and that firing precision is recovered upon chronic 4-AP treatment (WT: CV2 = 0.51 ± 0.04; SCA6^84Q/84Q with vehicle: CV2 = 0.63 ± 0.04; SCA6^84Q/84Q + 4-AP: CV2 = 0.43 ± 0.04). N = 16 cells for WT; N = 17 cells for SCA6^84Q/84Q mice and N = 11 for SCA6^84Q/84Q + 4-AP mice. Comparisons made with one-way ANOVA followed by post-hoc Student’s t test for (B–D); *P < 0.05; ***P < 0.005.