Nicotinamide Pathway-Dependent Sirt1 Activation Restores Calcium Homeostasis to Achieve Neuroprotection in Spinocerebellar Ataxia Type 7

Abstract

Sirtuin 1 (Sirt1) is a NAD⁺-dependent deacetylase capable of countering age-related neurodegeneration, but the basis of Sirt1 neuroprotection remains elusive. Spinocerebellar ataxia type 7 (SCA7) is an inherited CAG-polyglutamine repeat disorder. Transcriptome analysis of SCA7 mice revealed downregulation of calcium flux genes accompanied by abnormal calcium-dependent cerebellar membrane excitability. Transcription-factor binding-site analysis of downregulated genes yielded Sirt1 target sites, and we observed reduced Sirt1 activity in the SCA7 mouse cerebellum with NAD⁺ depletion. SCA7 patients displayed increased poly(ADP-ribose) in cerebellar neurons, supporting poly(ADP-ribose) polymerase-1 upregulation. We crossed Sirt1-overexpressing mice with SCA7 mice and noted rescue of neurodegeneration and calcium flux defects. NAD⁺ repletion via nicotinamide riboside ameliorated disease phenotypes in SCA7 mice and patient stem cell-derived neurons. Sirt1 thus achieves neuroprotection by promoting calcium regulation, and NAD⁺ dysregulation underlies Sirt1 dysfunction in SCA7, indicating that cerebellar ataxias exhibit altered calcium homeostasis because of metabolic dysregulation, suggesting shared therapy targets.

Keywords: NAD; Purkinje neuron; calcium; cerebellum; neurodegeneration; neuronal excitability; neuroprotection; potassium channel; sirtuin; spinocerebellar ataxia.

Figure 1.. SCA7 transgenic mice exhibit decreased expression of calcium regulatory genes.

(A) Hierarchical clustering of enriched pathways from DAVID analysis of cerebellar transcriptome data from SCA7 mice and controls (WT) at presymptomatic and symptomatic ages reveals genes involved in calcium signaling and phosphatidyl-inositol signaling. See also Figure S1A and Table S1. (B) Here we see a diagram of the cell membrane, membrane-based cell signaling pathways, and the ER, indicating proteins involved in regulating calcium homeostasis. Genes that encode these proteins or regulators are shown in italic font. Genes, which when mutated, produce a phenotype of ataxia are shown in bold. A red triangle indicates gene mutations that yield ataxia in humans, while a red square indicates genes that yield ataxia in mice. Note that all listed genes (in italics) were found to be down-regulated in cerebellum of SCA7 transgenic mice. (C) We performed qRT-PCR analysis on RNA’s isolated from cerebellar tissue from 30 week-old fxSCA7 92Q and WT control mice. WT: n=7, fxSCA7 92Q: n=8; three technical replicates; two-tailed t-test, *P <0.05, ** P <0.01. Error bars = s.e.m. (D) Representative immunostaining of cerebellar sections from 25 week-old fxSCA7 92Q and WT control mice for calbindin (green) and Ca_v3.1 (red). Scale bar = 100 μm (E) Representative higher magnification images of cerebellar sections from 25 week-old fxSCA7 92Q and WT control mice immunostained for calbindin (green) and Ca_v3.1 (red). Note mislocalization of Purkinje cell soma (arrow). Scale bar = 60 μm. See also Figure S1 and Table S1.

Figure 2.. Altered calcium regulatory gene expression is accompanied by cerebellar degeneration and abnormal Purkinje cell electrophysiology.

(A) In acute cerebellar slices from 25 week-old fxSCA7 92Q and WT mice (n = 3 /group), we measured total Purkinje cell capacitance, which is a function of membrane surface area, separately in the anterior lobe and posterior nodular zone of the cerebellum. WT cells: n=8-12, fxSCA7 92Q cells: n =10-12; two-tailed t-test, *P <0.05. (B) Measurement of Purkinje cell capacitance in fxSCA7 92Q and WT mice (n = 3 /group) at 40 weeks of age. WT cells: n=6-9, fxSCA7 92Q cells: n =6-9; two-tailed t-test, ***P < 0.001, **P < 0.01. (C) To quantify Purkinje cell neuron loss, we measured linear Purkinje cell density in the anterior and posterior cerebellum of 40 week-old fxSCA7 92Q and WT mice (n = 5 / group). Two-tailed t-test, *P <0.05. (D) Summary of acute cerebellar slice recordings from 14 week-old fxSCA7 92Q and WT control mice (n = 5-6 / group). The coefficient of variation (CV) of the interspike interval (ISI) of Purkinje cell firing is similar in SCA7 and control mice. WT cells: n = 22-38, fxSCA7 92Q cells: n = 19-25. (E) Summary of acute cerebellar slice recordings from 25 week-old fxSCA7 92Q and WT control mice (n = 4-5 / group). The ISI CV is significantly higher in the posterior cerebellum of SCA7 mice at this age. WT cells: n = 29-32, fxSCA7 92Q cells: n = 19-24; Mann-Whitney U-test, **P <0.01. (F) Summary of acute cerebellar slice recordings from 40 week-old fxSCA7 92Q and WT control mice (n = 5-7 / group). The ISI CV is significantly higher in both the anterior and the posterior cerebellum of SCA7 mice at this age. WT cells: n = 29-34, fxSCA7 92Q cells: n = 41-49; Mann-Whitney U-test, *P <0.05. (G) Representative traces of Purkinje cell firing from the posterior nodular zone of the cerebellum of 25 week-old fxSCA7 92Q and WT mice using extracellular recordings from acute cerebellar slices, illustrating irregular spiking in fxSCA7 92Q Purkinje cells (red carats). (H) In acute cerebellar slices from 25 week-old fxSCA7 92Q mice (n = 3) and WT mice (n = 2), we performed whole-cell patch-clamp recordings from Purkinje neurons from the posterior cerebellum in the presence of tetrodotoxin and determined the threshold to dendritic calcium spikes to increasing depolarizing current steps. WT cells: n=9, fxSCA7 92Q cells: n=11; two-tailed t-test, **P <0.01. (I) Representative traces of individual spikes from Purkinje cells of fxSCA7 92Q and WT mice at 25 weeks of age, illustrating greater AHP decay in fxSCA7 92Q Purkinje cells from the nodular zone of the cerebellum. (J) In spikes observed upon whole-cell patch-clamp of Purkinje cells in the nodular zone of midline cerebellar slices from 25 week-old fxSCA7 92Q and WT mice (n = 5 / group), AHP decay was more rapid in fxSCA7 92Q mice. The AHP amplitude was measured at defined points in the ISI: maximal AHP, mean ISI*0.5, mean ISI*0.65, and mean ISI*0.85. WT cells: n=30, fxSCA7 92Q cells: n=23; two-way repeated-measures ANOVA with Holm-Sidak post-test, ***P <0.001. (K) Representative extracellular recordings from acute cerebellar slices at 26 weeks, showing Purkinje cell spiking from neurons in the nodular zone of the cerebellum from mice following cerebellar delivery of an adeno-associated virus (AAV) encoding either BK or GFP. Irregular spiking is indicated with red carats. (L) Quantification of data in (K) for 26-week old WT mice treated with GFP-AAV (n = 3), fxSCA7 92Q mice treated with GFP-AAV (n = 3), and fxSCA7 92Q mice treated with BK-AAV (n = 4). WT + GFP-AAV cells: n=22, fxSCA7 92Q + GFP-AAV cells: n=22, fxSCA7 92Q + BK-AAV cells: n=21; one-way ANOVA with Holm-Sidak post-test, **P <0.01. (M) We cultured primary granule cell neurons from cerebella from P7 fxSCA7 92Q and WT mice, and performed live cell imaging with a calcium-sensitive dye after membrane depolarization with 40 mM KCl. The variance of the resulting calcium amplitude curve was calculated to determine calcium handling. WT: n=6, fxSCA7 92Q: n=7; three technical replicates; two-tailed t-test, ****P <0.0001. Error bars = s.e.m. See also Figure S2 and S3.

Figure 3.. SCA7 transcriptome abnormalities stem from Sirt1 dysfunction secondary to NAD⁺ depletion.

(A) Here we see a diagram of the cell membrane, membrane-based cell signaling pathways, and the ER, indicating proteins involved in regulating calcium homeostasis and flux. Genes that encode these proteins or regulators are shown in italic font. Red stars demarcate the 11 calcium regulatory genes that contain PPAR response elements (PPREs) in their promoters. (B) We prepared cerebellar protein lysates from 30 week-old fxSCA7-92Q mice and WT controls, and then performed immunoprecipitation of PGC-1α. Immunoblotting of PGC-1α IP’s with antibodies directed against either anti-acetyl-lysine or anti-PGC-1α was performed, revealing a marked increase in acetylated PGC-1α in SCA7 cerebellum. We quantified these results by densitometry analysis of the acetyl-lysine and PGC-1α bands in the graph below. WT: n=4, fxSCA7 92Q: n=4; two-tailed t-test, *P <0.05. (C) PARP1 immunoblot analysis of cerebellar protein lysates from 8.5 week-old SCA7 266Q mice and WT controls, quantified by densitometry analysis of PARP1 and β-actin (loading control) bands in the graph below. WT: n=4, SCA7 266Q: n=5; two-tailed t-test, *P <0.05. (D) Mass spectrometry measurement of NAD⁺ levels in the cerebellum and cortex of 8.5 week-old SCA7-266Q mice and WT controls. WT: n=5, SCA7 266Q: n=5; two-tailed t-test, *P <0.05. (E) γH2Ax immunostaining of cortical neurons cultured from SCA7 210Q k.i. mice and WT littermates, at baseline and after 1 hr of H₂0₂. LEFT: Representative image set. γH2Ax (red), DAPI (blue). RIGHT: Quantification of γH2Ax+ signal / soma area; n = 3 mice/genotype; n = 3 cultures / mouse; n = 50 neurons / culture; ***P <.001, *P <.05, two-tailed t-test. Scale bar = 20 μm. Error bars = s.e.m.

Figure 4.. Sirt1 transgenic over-expression ameliorates SCA7 disease phenotypes.

(A) We performed a neurological screening battery on cohorts of mice (n = 8 - 12 / group), of indicated genotypes at indicated ages. One-way ANOVA with Tukey’s post-hoc test, *P<0.05. (B) Sirt1 uOE – SCA7 266Q mice display increased calbindin immunoreactivity and decreased gliosis. Green = calbindin; Red = Glial Fibrillary Acidic Protein; Blue = DAPI. Scale bar = 200 μm (C) Quantification of Purkinje cell calbindin immunoreactivity, based upon (B). n = 3 mice / group, 66 neurons / genotype; one-way ANOVA with Tukey post-hoc test, *P <0.05. (D) Quantification of Purkinje cell soma area, based upon (B). n = 3 mice / group, 36 - 82 neurons / genotype; one-way ANOVA with Tukey post-hoc test, *P <0.05. (E) Kaplan-Meier plot shows that Sirt1 over-expression significantly extends the survival compared to SCA7-266Q mice. SCA7: n = 21, Sirt1-SCA7: n = 32; log-rank test, P <0.01. (F) We performed qRT-PCR analysis on RNA’s isolated from cerebellar tissue from 36 week-old fxSCA7 92Q, Sirt1 uOE - fxSCA7 92Q, and WT control mice. n = 5 mice / group; three technical replicates; two-tailed t-test, *P <0.05, ** P <0.01. For comparisons not achieving significance, there was a strong trend (P < 0.08). (G) Validation of SCA7 transcriptome analysis. LEFT: Nanostring analysis of the 100 genes found to display significant expression alterations comparing cerebellar RNAs from fxSCA7 92Q mice (n = 5) and WT littermate controls (n = 6); two-tailed t-test, P < 0.05. MIDDLE: Nanostring analysis of the 96 expression-altered genes in the Sirt1 uOE-fxSCA7 92Q mice (n = 5) and fxSCA7 92Q mice (n = 5). Fisher’s exact test, P < 0.00001. RIGHT: Inspection of the promoters of the 58 rescued genes for the presence or absence of the PPRE. Fisher’s exact test, P = 0.0003. Error bars = s.e.m.

Figure 5.. Purkinje neuron membrane excitability is improved upon Sirt1 over-expression.

(A) Representative extracellular recordings from acute cerebellar slices of Purkinje cells from ~30 week-old fxSCA7 92Q, Sirt1 uOE-fxSCA7 92Q, and WT mice, illustrating irregular spiking (red carats), which is improved upon Sirt1 over-expression. (B) Quantification of data from (A) for fxSCA7 92Q mice (n = 11), Sirt1 uOE-fxSCA7 92Q mice (n = 9), and WT mice (n = 8), indicating the CV of the ISI of Purkinje cell firing from the nodular zone of the cerebellum. WT cells: n=43, fxSCA7 92Q cells: n=64, Sirt1 uOE-fxSCA7 92Q cells: n=61; one-way ANOVA with Holm-Sidak post-test, *P <0.05, n.s. = not significant. (C) Distribution of ISI CV of Purkinje cell firing from fxSCA7 92Q, Sirt1 uOE-fxSCA7 92Q, and WT mice, showing a shift of the distribution towards WT in Sirt1 uOE-fxSCA7 92Q mice. (D) Overlaid whole-cell patch-clamp traces of individual spikes from Purkinje cells in the nodular zone of the cerebellum of fxSCA7 92Q, Sirt1 uOE-fxSCA7 92Q, and WT mice at ~30 weeks of age, illustrating greater AHP decay in fxSCA7 92Q Purkinje cells, which is normalized in Sirt1 uOE-fxSCA7 92Q Purkinje cells. (E) Quantification of data from (D) for fxSCA7 92Q mice (n = 11), Sirt1 uOE-fxSCA7 92Q mice (n = 9), and WT mice (n = 8), measuring the decay of Purkinje cell spike-AHP. The AHP amplitude was measured at defined points in the Inter Spike Interval (ISI): maximal AHP, mean ISI*0.5, mean ISI*0.65, and mean ISI*0.85. WT cells: n=32, fxSCA7 92Q cells: n=50, Sirt1 uOE-fxSCA7 92Q cells: n=53; two-way ANOVA with Holm-Sidak post-test, *P <0.05: fxSCA7 92Q compared to WT; ^#P <0.01: fxSCA7 92Q compared to Sirt1 uOE-fxSCA7 92Q. (F) In acute cerebellar slices from 35 week-old fxSCA7 92Q mice (n = 5), Sirt1 uOE-fxSCA7 92Q mice (n = 6), and WT mice (n = 7), we measured total Purkinje cell capacitance, which is a function of membrane surface area, in the nodular zone of the cerebellum. WT cells: n=17, fxSCA7 92Q cells: n=19, Sirt1 uOE-fxSCA7 92Q cells: n=15; one-way ANOVA with Hold-Sidak post-test, *P <0.05, n.s. = not significant. Error bars = s.e.m. See also Figure S6.

Figure 6.. Nicotinamide riboside and Sirt1 rescue disease phenotypes in SCA7 mice and patient neurons.

(A) We performed a neurological screening battery on cohorts of SCA7 266Q knock-in mice (n = 11 – 17 / group), maintained on a diet supplemented with nicotinamide riboside (NR) or vehicle, at the indicated ages. Two-tailed t-test, *P<0.05. (B) Kaplan-Meier plot of SCA7-266Q mice. Vehicle-supplemented diet: n = 11, NR-supplemented diet: n = 17; log-rank test, P <0.05. (C) We cultured NPCs from SCA7 patients and related, unaffected controls (n = 2 individuals / genotype), treated and/or transfected the cultures as indicated, and quantified cell death. Three technical replicates; two-way ANOVA with post-hoc Tukey test, *P <0.05, **P <0.01. (D) We cultured NPCs from a SCA7 patient and related, unaffected control in the presence of 1 mM NR or vehicle, performed live cell imaging with a calcium-sensitive dye after 100 mM KCl depolarization, and quantified NPCs that exhibited a sustained increase in calcium concentration. Two unique clones / individual; two technical replicates; two-tailed t-test, ****P <0.0001. (E) We cultured NPCs from a SCA7 patient and related, unaffected control in the presence of 1 mM NR or vehicle, performed live cell imaging with a calcium-sensitive dye after 100 mM KCl depolarization, and calculated the variance of the resulting calcium amplitude curve. Two unique clones / individual; two technical replicates; two-tailed t-test, ****P <0.0001. Error bars = s.e.m.

Figure 7.. Human SCA7 patients display increased poly(ADP-ribose) in cerebellar neuronal nuclei.

(A) Representative images of control and SCA7 patient post-mortem cerebellar sections immunostained for poly(ADP-ribose), followed by 3,3-diaminobenzidine detection (brown) and counterstained with hematoxylin (blue). Poly(ADP-ribose)-positive nuclei stain brown, while poly(ADP-ribose)-negative nuclei stain blue, and Purkinje cells are indicated with a red arrow. Note greater fraction of cerebellar neuronal nuclei stain brown in SCA7 patient. Scale bar = 50 μM (B) We determined the percentage of poly(ADP-ribose)-positive neuronal nuclei in cerebellar sections from four SCA7 patients and three unaffected control individuals by counting at least 300 cerebellar neuronal nuclei / individual (n ≥ 100 neuronal nuclei / section, 3 section images / individual) as either poly(ADP-ribose)-positive (brown) or poly(ADP-ribose)-negative (blue). Two-tailed t-test, *P <0.05. (C) We quantified poly(ADP-ribose) immunoreactivity in cerebellar sections from four SCA7 patients and three unaffected control individuals by measuring 3,3-diaminobenzidine (DAB) pixel intensity in at least 300 cerebellar neuronal nuclei / individual (n ≥ 100 neuronal nuclei / section, 3 section images / individual). Two-tailed t-test, ****P <0.0001. Error bars = s.e.m.