HDAC6 inhibition as a mechanism to prevent neurodegeneration in the mSOD1G93A mouse model of ALS

Abstract

The loss of upper and lower motor neurons, and their axons is central to the loss of motor function and death in amyotrophic lateral sclerosis (ALS). Due to the diverse range of genetic and environmental factors that contribute to the pathogenesis of ALS, there have been difficulties in developing effective therapies for ALS. One emerging dichotomy is that protection of the neuronal cell soma does not prevent axonal vulnerability and degeneration, suggesting the need for targeted therapeutics to prevent axon degeneration. Post-translational modifications of protein acetylation can alter the function, stability and half-life of individual proteins, and can be enzymatically modified by histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs), which add, or remove acetyl groups, respectively. Maintenance of post-translational microtubule acetylation has been suggested as a mechanism to stabilize axons, prevent axonal loss and neurodegeneration in ALS. This study used an orally dosed potent HDAC6 inhibitor, ACY-738, prevent deacetylation and stabilize microtubules in the mSOD1^G93A mouse model of ALS. Co-treatment with riluzole was performed to determine any effects or drug interactions and potentially enhance preclinical research translation. This study shows ACY-738 treatment increased acetylation of microtubules in the spinal cord of mSOD1^G93A mice, reduced lower motor neuron degeneration in female mice, ameliorated reduction in peripheral nerve axon puncta size, but did not prevent overt motor function decline. The current study also shows peripheral nerve axon puncta size to be partially restored after treatment with riluzole and highlights the importance of co-treatment to measure the potential effects of therapeutics in ALS.

Keywords: ACY-738; ALS; Axon degeneration; Neurofilament; Riluzole; SOD1.

Fig. 1

ACY-738 inhibits HDAC6 activity in the spinal cord of mSOD1^G93A mice. Schematic illustration of clinicopathological features of the mSOD1^G93A mouse model and experimental timeline for behavioural testing and endpoint (a). Indirect ELISA of the ratio of acetylated α-tubulin to α-tubulin show acute treatment of ACY-738 for 3 days in C57BL/6 mice led to increased cortical acetylated α-tubulin (b). Treatment administered by intraperitoneal (IP) injection (20 mg/kg bodyweight), oral dosing via porridge (625 mg/kg chow) or formulated into pellets (625 mg/kg chow) did not significantly alter acetylated α-tubulin levels (b). Bar graph of ratios for relative indirect ELISA absorbance show treatment with ACY-738, and co-dosing ACY-738+riluzole increased acetylated α-tubulin in 20-week-old mSOD1^G93A mice compared to riluzole only, and untreated control groups (c). Data presented represent mean ± 95 % confidence intervals. N = 5–8 per treatment group for mSOD1^G93A mice, and n = 2 for WT controls.

Fig. 2

HDAC6i does not prevent weight loss and muscle deterioration in mSOD1^G93A mice. Body weight is unaltered by chronic treatment with ACY-738 (salmon), ACY-738 + riluzole (red), riluzole (blue) in mSOD1^G93A mice compared to untreated mSOD1^G93A (light blue) controls, however, are significantly lower than C57BL/6 controls (black) at endpoint (a). Treatment with ACY-738 did not rescue disease-associated weight loss in male (b - left), or female (b – right) mSOD1^G93A mice at 20 weeks of age. Gastrocnemius weight is significantly decreased in pooled male and female mSOD1^G93A mice compared to C57BL/6 control group, regardless of treatment with ACY-738 or riluzole (c). Data presented represent mean ± 95 % CI. N = 5 for C57BL/6 mice and n = 11–19 per mSOD1^G93A treatment group.

Fig. 3

Motor function decline is unaltered by treatment with ACY-738 in mSOD1^G93A mice. Mixed effects models of all-paw grip-strength (a-c) for female (left) and male (right) mSOD1^G93A mice (yellow), compared to C57BL/6 (blue) control littermates, showed a significant reduction in all-paw grip-strength in mSOD1^G93A mice (a). Treatment with ACY-738 (b) or riluzole (c) (red) led to sex-specific alterations in all-paw grip strength in mSOD1^G93A animals. Wire-hang performance was significantly reduced in mSOD1^G93A (yellow) mice compared to C57BL/6 control littermates (blue) (d). Wire-hang was unaltered after treatment with ACY-738 (e), or riluzole (f) (red) in mSOD1^G93A mice. Kaplan-Meier time-to-event analysis demonstrates a significant effect of ACY-738 treatment on muscle tremor onset, compared to untreated mSOD1^G93A controls (g – left, ACY-738 = red), whereas riluzole treatment does not alter tremor onset in mSOD1^G93A mice (g – right, riluzole = red). Data presented represent conditional mean ± 95 % CI for predicted values. N = 11–19 per treatment group, and n = 5 for C57BL/6 controls.

Fig. 4

Motor neurons are rescued by ACY-738 treatment, and riluzole treatment improves axon fibres in mSOD1^G93A mice. Representative images of cresyl violet labelled lumbar spinal cord from ACY-738 and riluzole treated (left), vehicle treated mSOD1^G93A mice (middle), and C57BL/6 vehicle controls (right) (a). Motor neurons are significantly reduced in the lumbar spinal cord of mSOD1^G93A mice compared to C57BL/6 controls (b). Lower motor neurons are rescued by treatment with riluzole or ACY-738 individually, but not co-treatment in female mSOD1^G93A mice (c). Representative images of sciatic nerve from ACY-738 and riluzole treated (left), vehicle-treated mSOD1^G93A mice (middle), and C57BL/6 vehicle controls (right) (d). The average size of SMI312 positive axons in the sciatic nerve are significantly reduced in mSOD1^G93A mice compared to C57BL/6 control littermates (e). Riluzole restores the average size of SMI312 positive axons in the sciatic nerve of mSOD1^G93A mice (f). The average size of ChAT positive axons in the sciatic nerve are significantly reduced in mSOD1^G93A mice compared to C57BL/6 control littermates (g). Combined treatment of riluzole and ACY-738 restores the average size of ChAT positive axons in the sciatic nerve of mSOD1^G93A mice (h). Data presented as conditional means and 95 % confidence intervals. n = 6–9 per treatment group, and n = 4 for C57BL/6 controls.

Fig. 5

Astrocytes are altered after treatment with riluzole and ACY-738 in mSOD1^G93A mice. Representative images of Iba1+ve microglia in the lumbar spinal cord of mSOD1^G93A mice treated with ACY-738 (left), untreated (middle), and C57BL/6 wildtype (right) mice (a). Scatter plot showing mSOD1^G93A mice (right) have significantly increased microglia in the lumbar spinal cord compared to C57BL/6 WT (left) control littermates (b). Scatter plot showing mSOD1^G93A microglia in the lumbar spinal cord are unaltered after treatment with ACY-738, riluzole, or both (c). Representative images of GFAP positive astrocytes in the lumbar spinal cord of mSOD1^G93A mice treated with ACY-738 (left), untreated (middle), and C57BL/6 wildtype (right) mice (d). mSOD1^G93A mice have significantly increased GFAP astrocyte reactivity in the lumbar spinal cord compared to C57BL/6 WT controls (e). Female mSOD1^G93A mice treated with ACY-738 alone have significantly enriched GFAP reactivity compared to untreated littermates (f). Data presented as conditional means and 95 % confidence intervals. N = 5–9 per treatment group, and n = 5 for C57BL/6 controls.

Fig. 6

Serum NFL is significantly increased with disease progression but is unaltered by treatment with ACY-738 or riluzole. Plots showing predicted values of NFL in mSOD1^G93A mice treated with riluzole (a), or ACY-738 (b), at 14, and 20 weeks of age respectively. There was a significant increase in the level of serum NFL (log pg/uL) between 14 and 20 weeks of age. Data presented as conditional means and 95 % confidence intervals.