Reduction of inflammation and mitochondrial degeneration in mutant SOD1 mice through inhibition of voltage-gated potassium channel Kv1.3

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no effective therapy, causing progressive loss of motor neurons in the spinal cord, brainstem, and motor cortex. Regardless of its genetic or sporadic origin, there is currently no cure for ALS or therapy that can reverse or control its progression. In the present study, taking advantage of a human superoxide dismutase-1 mutant (hSOD1-G93A) mouse that recapitulates key pathological features of human ALS, we investigated the possible role of voltage-gated potassium channel Kv1.3 in disease progression. We found that chronic administration of the brain-penetrant Kv1.3 inhibitor, PAP-1 (40 mg/Kg), in early symptomatic mice (i) improves motor deficits and prolongs survival of diseased mice (ii) reduces astrocyte reactivity, microglial Kv1.3 expression, and serum pro-inflammatory soluble factors (iii) improves structural mitochondrial deficits in motor neuron mitochondria (iv) restores mitochondrial respiratory dysfunction. Taken together, these findings underscore the potential significance of Kv1.3 activity as a contributing factor to the metabolic disturbances observed in ALS. Consequently, targeting Kv1.3 presents a promising avenue for modulating disease progression, shedding new light on potential therapeutic strategies for ALS.

Keywords: ALS; Kv1.3 channels; inflammation; mitochondria; mutant SOD1.

FIGURE 1

Disease onset evaluation. Histograms representing the performance of WT (n = 20, white) and hSOD1G93A (n = 31, black) animals at 9 and 10 weeks of age on the rotarod (A), hanging wire test (score, B), and latency (C), strength test (normalized for animal weight) (D). Data are the mean ± standard error (SE); *p < 0.05, **p < 0.01, Unpaired Student’s t-test. Three independent experiments.

FIGURE 2

Effect of PAP-1 on motor activity: Body weight, growth rate (A,B) and Behavioral tests (C–F) of hSOD1G93A mice treated with PAP-1 (40 mg/kg) (green squares, n = 18) and with vehicle (black circles, n = 21); panel (C) Rotarod test, panel (D) Hanging Wire Test score panel (E) Hanging Wire test latency, panel (F) Grip strength test normalized with weight (g). Data are expressed as Mean ± SE; *p < 0.05, **p < 0.01, by Two-way ordinary ANOVA.

FIGURE 3

Analysis of survival (days) of hSOD1G93A mice treated with PAP-1 (40 mg/kg) (green line, n = 11) and vehicle (black line, n = 9). Kaplan-Meier, LogRank Test, p = 0.043.

FIGURE 4

(A) Mean area of GFAP + cells (expressed as % of spinal cord area) in hSOD1G93A mice (17–18 weeks old) treated with vehicle (oil) or PAP-1. Each circle represents one mouse (n = 9 mice per condition, **p < 0.001 two-tailed Student’s t-test). Right: representative immunofluorescence image of GFAP+ cells in spinal cord sections. Scale bar: 50 μm. (B) The mean area of Iba1+ cells (expressed as % of spinal cord area) in hSOD1G93A mice (17–18 weeks old) treated with vehicle (oil) or PAP-1. Each circle represents one mouse (n = 10–12 per treatment). Right: representative immunofluorescence image of Iba1+ cells in the ventral horn of spinal cord sections. Scale bar: 50 μm. (C) Kv1.3+ immunoreactivity (expressed as % of spinal cord area) in hSOD1G93A mice (18 weeks old) treated with oil or PAP-1. Each circle represents one mouse (n = 7 mice per treatment). Right: representative immunofluorescence image of Kv1.3+ staining in spinal cord sections. Scale bar: 50 μm. (D) Colocalization of Iba1 and Kv1.3 covered area in the spinal cord in hSOD1G93A mice (17–18 weeks old) treated with vehicle (oil) or PAP-1. (n = 6–7, **p < 0.01 two-tailed Student’s t-test). Right: representative immunofluorescence image showing the microglial expression of Kv1.3 as indicated by white arrows. Scale bar: 50 μm. Dashed square: 40x magnification of co-localization signals (E) Quantification of Smi32+ MNs in the ventral horns of the spinal cord in hSOD1G93A mice treated with vehicle (oil) or PAP-1 (n = 8 per treatment). Scale bar: 50 μm. Right: representative immunofluorescence image of Smi32+ – MNs in spinal cord sections. Scale bar: 50 μm. All data are expressed as mean area ± SE.

FIGURE 5

(A) Heatmap for the inflammatory factor levels measured in the serum of hSOD1G93A mice treated with PAP-1 or vehicle, n = 4 from 14 pooled samples per condition, two independent experiments; (B): representative immune-array dot blots of hSOD1G93A mice sera.

FIGURE 6

(A) Representative single plane confocal images of KV1.3 (red), TOMM20 (green), merge channels, and Hoechst staining (blue) for nuclei visualization in lumbar spinal cord region of hSOD1-G93A mice at early symptomatic stage (10 weeks old). 60x objective (scale:10 um) yellow stars indicate the co-localizing signals; (B) Representative images of the mitochondrial network in alpha motor neurons of healthy WT littermates, hSOD1G93A mice treated with vehicle or PAP-1 at late symptomatic stage (17–18 weeks old). (C) 3D confocal reconstruction of the mitochondrial network in single motor neurons labeled with SMI-32 (pink), and with anti-TOMM20 antibody (green). (D) Quantification of the cytosolic volume occupied by the mitochondrial network in WT, hSOD1G93A mice treated with vehicle or with the potassium channel inhibitor PAP-1. Data are expressed as mean ± SD n = 11–12 cells, 3 animals per group, by One way ANOVA. (E) OCR experiments: quantification of the ATP-linked OCR in NSC-34 and NSC-34 cells expressing hSOD1-G93A (G93A) treated with PAP-1 for 72 h. Values are expressed as mean ± SD of three independent experiments conducted in quadruplicate on each cell line, n = 12 ****p < 0.0001 by One way ANOVA. *p < 0.05, **p < 0.01.