Co-induction of the heat shock response ameliorates disease progression in a mouse model of human spinal and bulbar muscular atrophy: implications for therapy

Abstract

Spinal and bulbar muscular atrophy, also known as Kennedy's disease, is an adult-onset hereditary neurodegenerative disorder caused by an expansion of the polyglutamine repeat in the first exon in the androgen receptor gene. Pathologically, the disease is defined by selective loss of spinal and bulbar motor neurons causing bulbar, facial and limb weakness. Although the precise disease pathophysiology is largely unknown, it appears to be related to abnormal accumulation of the pathogenic androgen receptor protein within the nucleus, leading to disruption of cellular processes. Using a mouse model of spinal and bulbar muscular atrophy that exhibits many of the characteristic features of the human disease, in vivo physiological assessment of muscle function revealed that mice with the pathogenic expansion of the androgen receptor develop a motor deficit characterized by a reduction in muscle force, abnormal muscle contractile characteristics, loss of functional motor units and motor neuron degeneration. We have previously shown that treatment with arimoclomol, a co-inducer of the heat shock stress response, delays disease progression in the mutant superoxide dismutase 1 mouse model of amyotrophic lateral sclerosis, a fatal motor neuron disease. We therefore evaluated the therapeutic potential of arimoclomol in mice with spinal and bulbar muscular atrophy. Arimoclomol was administered orally, in drinking water, from symptom onset and the effects established at 18 months of age, a late stage of disease. Arimoclomol significantly improved hindlimb muscle force and contractile characteristics, rescued motor units and, importantly, improved motor neuron survival and upregulated the expression of the vascular endothelial growth factor which possess neurotrophic activity. These results provide evidence that upregulation of the heat shock response by treatment with arimoclomol may have therapeutic potential in the treatment of spinal and bulbar muscular atrophy and may also be a possible approach for the treatment of other neurodegenerative diseases.

Figure 1

Examination of expression levels of heat shock proteins in mice with spinal and bulbar muscular atrophy. The expression levels of Hsp70 and Hsp90 were analysed in spinal cord of (A) presymptomatic (3 month), (B) symptomatic (12 month) and (C) late stage (18 month) wild-type (WT) and AR100 mice. Densitometric analysis of bands was performed using values normalized to actin. Data are displayed as mean ± SEM and are representative of three independent experiments. Statistical analysis was performed using a two sample t-test (n ≥ 3, *P < 0.05). AU = arbitrary units.

Figure 2

Arimoclomol induces heat shock proteins in spinal and bulbar muscular atrophy mice. To investigate the effects of arimoclomol, an intraperitoneal injected dose of 120 mg/kg/body weight was given daily to 12-month-old AR100 mice after the onset of symptoms for a period of 16 days. Arimoclomol significantly upregulated the expression of Hsp70 in (A) spinal cord and (B) hindlimb tibialis anterior (TA) muscles in comparison with vehicle-treated (WT) mice. Furthermore, no increase was observed in (C) cortex or liver of AR100 mice. Data are displayed as mean ± SEM and are representative of three independent experiments. Statistical analysis was performed using either a two sample t-test or one-way ANOVA followed by the Student–Newman–Keuls and Tukey’s Honestly Significantly Different post hoc tests (n ≥ 3, *P < 0.05). Arim = arimoclomol; V = vehicle; AU = arbitrary units.

Figure 3

Arimoclomol upregulates vascular endothelial growth factor (Vegf) in spinal cord and muscles of mice with spinal and bulbar muscular atrophy. The expression of several genes was investigated using quantitative PCR after treatment with arimoclomol, with an intraperitoneal injected dose of 120 mg/kg/body weight given daily to 12-month-old AR100 mice after the onset of symptoms for a period of 16 days. There was a significant increase in levels of vascular endothelial growth factor both in (A) spinal cord and (B) hindlimb tibialis anterior muscles after administration of arimoclomol compared with vehicle-treated AR100 mice. Data are displayed as mean ± SEM and are representative of three independent experiments. Statistical analysis was performed using a two sample t-test (n ≥ 3, *P < 0.05).

Figure 4

Deterioration of muscle force in hindlimb muscles of mice with spinal and bulbar muscular atrophy. In vivo assessment of hindlimb muscle force revealed a reduction in (A) maximal twitch and (B) tetanic tension in tibialis anterior (TA) muscles of 18-month-old AR100 mice (n = 11) compared with AR20 (n = 11) and wild-type (WT) mice (n = 8). Analysis of twitch contraction and relaxation rates of hindlimb muscles showed significant slowing of (C) tibialis anterior muscle time to peak contraction and (D) tibialis anterior muscle half relaxation time, indicative of muscle deterioration. In extensor digitorum longus (EDL) muscles, a similar loss of (E) twitch and (F) tetanic tension was also observed in AR100 mice, as well as a slowing of (G) the time to peak contraction and (H) half relaxation time. Values are expressed as mean ± SEM. Statistical analysis was performed using the Kruskal–Wallis test, *P < 0.05.

Figure 5

Development of a progressive neuromuscular phenotype in mice with spinal and bulbar muscular atrophy, manifested by a loss of motor units and the development of abnormal muscle fatigue characteristics. (A) The fatigue characteristics of extensor digitorum longus (EDL) muscles in 18-month-old AR100 mice (n = 11) compared with AR20 (n = 11) and wild-type (WT) mice (n = 8) were examined. As can be seen in the fatigue traces, in AR100 mice, the extensor digitorum longus muscles become resistant to fatigue and can maintain force when repeatedly stimulated. From such traces, a fatigue index was calculated for each muscle, where a fatigue index of 1.0 indicates that a muscle is completely fatigue resistant. The bar chart shows that there a significant decrease in the fatigue index of AR100 extensor digitorum longus muscles. (B) The number of functional motor units innervating extensor digitorum longus was established. Typical motor unit traces from extensor digitorum longus muscles of 18-month-old wild-type, AR20 and AR100 mice are shown. Motor unit numbers were significantly reduced in extensor digitorum longus muscle of AR100, but not AR20 mice. Analysis of muscles was also performed in presymptomatic 3-month-old mice, which showed no pathological alteration in (C) maximal twitch and (D) tetanic tension, or (E) the time to peak contraction and (F) half relaxation time in tibialis anterior muscle (TA). Values are expressed as mean ± SEM. Statistical analysis was performed using the Kruskal–Wallis test, *P < 0.05.

Figure 6

Pathological changes in muscle and spinal cord of mice with spinal and bulbar muscular atrophy. (A) The mean weight of tibialis anterior muscles (TA), extensor digitorum longus (EDL) and soleus muscles of wild-type, AR20 and AR100 mice shows a significant decrease in the weight of fast twitch muscles in 18-month-old AR100 mice, but not in slow twitch muscles such as soleus. (B) The presence of degenerating motor neurons can be observed in the ventral horn of 18-month-old AR100 mice (arrow indicates degenerating neuron). (C) Surviving motor neurons in the ventral horn sciatic motor pool were counted and the results shown in the bar chart, with values expressed as a percentage of wild-type surviving motor neurons. By 18 months of age, a significant number of motor neurons have died in AR100 mice compared with wild-type (WT), whereas no motor neuron death occurs in AR20 mice. For each genotype, n ≥ 7, with one-way ANOVA and post hoc analysis performed to test for significance, with *P < 0.05 and ***P < 0.001; error bars represent SEM.

Figure 7

Treatment with arimoclomol delays disease progression in mice with spinal and bulbar muscular atrophy. (A) Body weight was recorded monthly from 4–18 months of age in arimoclomol and vehicle-treated AR100 mice. Body weight begins to decline from 13 months in vehicle-treated AR100 mice, but is maintained in mice treated with arimoclomol. Arimoclomol increased the maximal (B) twitch tension and (C) maximal tetanic force in tibialis anterior (TA) muscles of AR100 mice (n = 10) compared with vehicle-treated mice (n = 8) at 18 months of age. (D) Arimoclomol prevents the change in the fatigue characteristics and (E) increases motor unit survival in extensor digitorum longus muscles of AR100 mice. (F) There is a significant increase in the weight of tibialis anterior and extensor digitorum longus muscles (EDL) in arimoclomol-treated AR100 mice, but no change in the soleus muscle, which is unaffected in AR100 mice at this stage. (G) These improvements in muscle function in arimoclomol-treated AR100 mice are reflected in a significant increase in motor neuron survival. Values are expressed as mean ± SEM. Statistical analysis was performed using the Mann–Whitney U test, *P < 0.05, **P < 0.01 and ***P < 0.001.