Therapeutic Role of Neuregulin 1 Type III in SOD1-Linked Amyotrophic Lateral Sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating motoneuron (Mn) disease without effective cure currently available. Death of MNs in ALS is preceded by failure of neuromuscular junctions and axonal retraction. Neuregulin 1 (NRG1) is a neurotrophic factor highly expressed in MNs and neuromuscular junctions that support axonal and neuromuscular development and maintenance. NRG1 and its ErbB receptors are involved in ALS. Reduced NRG1 expression has been found in ALS patients and in the ALS SOD1^G93A mouse model; however, the expression of the isoforms of NRG1 and its receptors is still controversial. Due to the reduced levels of NRG1 type III (NRG1-III) in the spinal cord of ALS patients, we used gene therapy based on intrathecal administration of adeno-associated virus to overexpress NRG1-III in SOD1^G93A mice. The mice were evaluated from 9 to 16 weeks of age by electrophysiology and rotarod tests. At 16 weeks, samples were harvested for histological and molecular analyses. Our results indicate that overexpression of NRG1-III is able to preserve neuromuscular function of the hindlimbs, improve locomotor performance, increase the number of surviving MNs, and reduce glial reactivity in the treated female SOD1^G93A mice. Furthermore, the NRG1-III/ErbB4 axis appears to regulate MN excitability by modulating the chloride transporter KCC2 and reduces the expression of the MN vulnerability marker MMP-9. However, NRG1-III did not have a significant effect on male mice, indicating relevant sex differences. These findings indicate that increasing NRG1-III at the spinal cord is a promising approach for promoting MN protection and functional improvement in ALS.

Keywords: Amyotrophic lateral sclerosis; ErbB receptor; motoneuron disease; motor system; mouse; neuregulin.

Fig. 1

Neuregulin 1 type III expression in ALS patients and SOD1^G93A mice. (a) Microphotographs of pan-NRG1 and NRG1 type III labeling by DAB staining in the spinal cord ventral horn of healthy and ALS patients. Higher magnification images show that pan-NRG1 labeling may colocalize with Iba1 in ALS patients (scale bar = 100 μm). (b) Quantitative PCR of NRG1 isoform mRNA reveals a downregulation of type III whereas type I is increased in the spinal cord of SOD1^G93A mice at 16 weeks of age. Data are shown as mean ± SEM. Student t, *p < 0.05 versus WT. (c) mRNA expression shows that intrathecal administration of AAVrh10-NRG1-III induced overexpression of NRG1-III in the spinal cord of both WT and SOD1^G93A mice. Data are shown as mean ± SEM. One-way ANOVA and Tukey’s post hoc test, *p < 0.05 versus WT mock and ^#p < 0.05 versus SOD mock. (d) Viral genome analysis in lumbar spinal cord corroborated efficient intrathecal AAV injection in the treated mice. Data are shown as mean ± SEM. One-way ANOVA and Tukey’s post hoc test, *p < 0.05 versus WT mock and ^#p < 0.05 versus SOD mock. (e) Microphotographs of NRG1-III labeling in the ventral horn of the spinal cord confirms the downregulation of this isoform in the SOD1^G93A mice that is recovered upon viral-mediated overexpression (scale bar = 100 μm). Data are shown as mean ± SEM

Fig. 2

NRG1-III overexpression slows the disease progression of SOD1^G93A female mice. Electrophysiological tests show that AAV-NRG1-III injection produced significant preservation of the CMAP amplitude of (a) plantar, (b) tibialis anterior, and (c) gastrocnemius hindlimb muscles in the SOD1^G93A mice. Two-way ANOVA followed by Tukey’s post hoc test, ^#p < 0.05 versus SOD mock mice; *p < 0.05 versus WT mock. (d) AAV-NRG1-III gene therapy increased the amplitude of MEPs in SOD1^G93A mice, indicating improved connectivity between upper and lower MNs (^#p < 0.05 vs SOD mock mice). Student t test, *p < 0.05 versus SOD mock. (e) Electrophysiological estimation of motor unit number (MUNE) and mean amplitude of single motor unit action potential (SMUA) of the tibialis anterior muscle shows preservation of large motor units in AAV-NRG1-III treated compared with mock SOD1^G93A mice (*p < 0.05 vs SOD mock mice). Student t test, *p < 0.05 versus SOD mock. The frequency distribution of the TA motor units demonstrates a shift to the right in the treated group. (f) NRG1-III overexpression produced improvement in the rotarod performance of treated SOD1^G93A mice during the follow-up time. Two-way ANOVA followed by Tukey’s post hoc test, ^#p < 0.05 versus SOD mock mice. (g) The onset of locomotion dysfunction was delayed but without significant differences. (h) Overexpression of NRG1-III slightly improved the survival of the treated mice without reaching statistical significance (n = 9 mice per group, Mantel-Cox test)

Fig. 3

Effect of NRG1-III overexpression on MN preservation and glial reactivity in SOD1^G93A female mice. (a) Representative images of the ventral horn of L4 spinal cord sections stained with cresyl violet of wild-type and SOD1^G93A mice, treated with NRG1-III or with mock vector (scale bar = 100 μm). (b) Histological analysis showed higher number of MNs in the ventral horn of the treated mice compared with that of mock mice. One-way ANOVA followed by Tukey’s post hoc test, *p < 0.05 versus SOD mock mice. (c) Representative confocal images of astrocytes labeled against GFAP, and microglia labeled against Iba-1, in the spinal cord ventral horn of SOD1^G93A mice (scale bar = 100 μm). (d) AAV-NRG1-III therapy reduced the astrocyte and microglial reactivity in the spinal cord. Student t, *p < 0.05. Data are shown as mean ± SEM

Fig. 4

NRG1-III overexpression does not produce beneficial effects on male SOD1^G93A mice. Electrophysiological tests showed that there were no differences in the CMAP amplitude of plantar (a) and gastrocnemius (b) muscles in the male SOD1^G93A mice receiving either AAV-NRG1-III or mock vector. Two-way ANOVA followed by Tukey’s post hoc test, *p < 0.05 versus SOD mock. (c) Treatment with NRG1-III did not improve the rotarod performance of the male SOD1^G93A mice. (d) Representative images of the ventral horn at L4 spinal cord of wild-type and SOD1^G93A mice, treated with NRG1-III or with mock vector (scale bar = 100 μm). (e) Histological analysis showed a similar number of MNs in the spinal cord ventral horn of the treated SOD1^G93A mice compared with that of the mock mice. One-way ANOVA followed by Tukey’s post hoc test, *p < 0.05 versus SOD mock. Data are shown as mean ± SEM

Fig. 5

Effect of NRG1-III overexpression on NRG1/ErbB4 signaling and MN excitability markers in SOD1^G93A mice. (a) Treatment with NRG1-III tended to increase the ErbB4 receptor expression to normal levels in the SOD1^G93A mice. One-way ANOVA followed by Tukey’s post hoc test, *p < 0.05 versus SOD mock mice. (b) Confocal images showed ErbB4 translocation to the nucleus of MNs in the SOD1^G93A mice (scale bar = 20 μm). The integrated density of ErbB4 immunolabeling was significantly increased in the nucleus in SOD1^G93A mice. Student t, *p < 0.05. (c) Microphotographs of spinal cord samples labeled for ErbB4 showed also presence of ErbB4 in the nucleus of MNs in ALS patients but not in healthy subjects (scale bar = 20 μm). (d) Representative images of spinal cord ventral horn MNs immunolabeled for ChAT (green) and MMP-9 (red). NRG1-III overexpression increased the number of MMP-9-negative MNs (scale bar = 100 μm). Student t, *p < 0.05. (e) Representative images of KCC2 (red) labeling in the membrane of MNs labeled with FluoroNissl (green) (scale bar = 50 μm). Higher magnification images (bottom) show that KCC2 staining was decreased specially around the MN soma (scale bar = 25 μm). NRG1-III treatment rescued the KCC2 downregulation observed in the SOD1^G93A mice. One-way ANOVA followed by Tukey’s post hoc test, *p < 0.05 versus SOD mock mice. (f) NRG1-III increases Akt phosphorylation (both Ser473 and Thr308) and diminishes Erk2 activation in SOD1^G93A-treated mice, as demonstrated by Western blot. At least 3 different Western blots were used for quantification; relative phosphorylation compared to total protein was normalized by GAPDH and represented by fold-change compared to WT animals. One-way ANOVA followed by Tukey’s post hoc test, *p < 0.05 versus SOD mock. Data are shown as mean ± SEM