Motor neuron degeneration correlates with respiratory dysfunction in SCA1

Abstract

Spinocerebellar ataxia type 1 (SCA1) is characterized by adult-onset cerebellar degeneration with attendant loss of motor coordination. Bulbar function is eventually impaired and patients typically die from an inability to clear the airway. We investigated whether motor neuron degeneration is at the root of bulbar dysfunction by studying SCA1 knock-in (Atxn1^154Q/+ ) mice. Spinal cord and brainstem motor neurons were assessed in Atxn1^154Q/+ mice at 1, 3 and 6 months of age. Specifically, we assessed breathing physiology, diaphragm histology and electromyography, and motor neuron histology and immunohistochemistry. Atxn1^154Q/+ mice show progressive neuromuscular respiratory abnormalities, neurogenic changes in the diaphragm, and motor neuron degeneration in the spinal cord and brainstem. Motor neuron degeneration is accompanied by reactive astrocytosis and accumulation of Atxn1 aggregates in the motor neuron nuclei. This observation correlates with previous findings in SCA1 patient tissue. Atxn1^154Q/+ mice develop bulbar dysfunction because of motor neuron degeneration. These findings confirm the Atxn1^154Q/+ line as a SCA1 model with face and construct validity for this understudied disease feature. Furthermore, this model is suitable for studying the pathogenic mechanism driving motor neuron degeneration in SCA1 and possibly other degenerative motor neuron diseases. From a clinical standpoint, the data indicate that pulmonary function testing and employment of non-invasive ventilator support could be beneficial in SCA1 patients. The physiological tests used in this study might serve as valuable biomarkers for future therapeutic interventions and clinical trials.This article has an associated First Person interview with the first author of the paper.

Keywords: Bulbar dysfunction; Motor neuron degeneration; SCA1; Spinocerebellar ataxia type 1.

Fig. 1.

Atxn1^154Q/+ mice show progressive neuromuscular respiratory dysfunction with neurogenic changes in the diaphragm. (A) Age-matched wild-type and Atxn1^154Q/+ mice (gray and black lines, respectively) were assessed by whole-body unrestrained plethysmography at 1, 3 and 6 months of age. Traces show, from left to right, average tidal volume, respiratory rate, minute ventilation and inter-breath interval irregularity, ±s.d. Statistical significance (*) between groups at 6 months was demonstrated using a two-way ANOVA (genotype×age), followed by a Tukey-Kramer post-hoc analysis (*P<0.05, n=15 mice per group). (B) Diaphragm muscles from Atxn1^154Q/+ and age-matched wild-type control mice at 1, 3 and 6 months of age were stained with Hematoxylin and Eosin. Diaphragms of 6 month old Atxn1^154Q/+ mice show a number of small angular fibers (arrows), consistent with a chronic neurogenic underlying process. (C) Diaphragms from Atxn1^154Q/+ mice and age-matched wild-type control mice at 1, 3 and 6 months of age were subjected to esterase enzyme histochemistry. Atxn1^154Q/+ mice at 6 months showed increased esterase activity (darker fibers), which indicates denervation. Scale bar: 50 μm. (D) Representative needle electromyography traces of diaphragms obtained from Atxn1^154Q/+ mice (lower trace) and age-matched wild-type control mice (upper trace) at 6 months of age. (E) Quantification of motor unit action potential (MUAP) amplitude and frequency illustrated as mean value in bar graphs±s.d. Statistical significance (*) between groups was demonstrated using a two-tailed Student's t-test (*P<0.05, n=4 mice per group).

Fig. 2.

Motor neuron degeneration in the spinal cord and hypoglossal nucleus. Representative motor neurons from (A) the anterior horn of the cervical spinal cord and (B) the hypoglossal nucleus of the brainstem from Atxn1^154Q/+ mice and age-matched wild-type control mice at 1, 3 and 6 months of age, stained with Hematoxylin and Eosin. Arrowheads point to typical healthy motor neurons, which are pyramidal in shape. By contrast, motor neurons in the Atxn1^154Q/+ mice (arrows) become rounded and small by 6 months of age. Scale bar: 50 μm.

Fig. 3.

Reactive astrocytosis in motor neuron areas of aged Atxn1^154Q/+ mice. Diaminobenzidine (DAB) immunohistochemistry was performed with an antibody against glial fibrillary astrocyte protein in paraffin-embedded tissue from Atxn1^154Q/+ mice and age-matched wild-type control mice at 1, 3 and 6 months of age. Arrowheads highlight astrocytes in areas of healthy motor neurons. By 6 months of age, the Atxn1^154Q/+ astrocytes show a significant increase in density as well as notable reactive hypertrophy (arrows) in (A) the anterior horn of the cervical spinal cord and (B) the hypoglossal nucleus of the brainstem. Astrocyte density was determined by counting the number of astrocytes per region of interest (ROI, defined as an area of 46,225 μm²). Bars illustrate the mean number of astrocytes per ROI±standard deviation. Statistical significance between all groups and Atxn1^154Q/+ mice at 6 months was demonstrated using a two-way ANOVA (genotype×age), followed by a Tukey-Kramer post-hoc analysis (*P<0.05, n=3 mice per group). Scale bar: 50 μm.

Fig. 4.

Age-dependent formation of Atxn1 nuclear inclusions in motor neurons of Atxn1^154Q/+ mice. Representative motor neurons from (A) the anterior horn of the cervical spinal cord and (B) the hypoglossal nucleus of the brainstem. DAB immunohistochemistry was performed with an antibody against Atxn1 in paraffin-embedded tissue from Atxn1^154Q/+ mice and age-matched wild-type control mice at 1, 3 and 6 months of age. Motor neurons in the Atxn1^154Q/+ mice develop sparse, small nuclear aggregates at 3 months of age, which increase in size and frequency by 6 months of age (arrows). Scale bar: 10 μm. (C) Quantification of the number of motor neurons with detectable nuclear aggregates of Atxn1 protein in Atxn1^154Q/2 mice at 40× magnification. A total of 100-200 motor neurons were counted per animal and averages calculated for three mice per group. Bars illustrate mean±s.d. for each group. Statistical significance between 3 and 6 months for spinal cord (#) and brainstem (*) was demonstrated using a two-tailed Student's t-test (P<0.05).