Reducing C-terminal truncation mitigates synucleinopathy and neurodegeneration in a transgenic model of multiple system atrophy

Abstract

Multiple system atrophy (MSA) is a sporadic orphan neurodegenerative disorder. No treatment is currently available to slow down the aggressive neurodegenerative process, and patients die within a few years after disease onset. The cytopathological hallmark of MSA is the accumulation of alpha-synuclein (α-syn) aggregates in affected oligodendrocytes. Several studies point to α-syn oligomerization and aggregation as a mediator of neurotoxicity in synucleinopathies including MSA. C-terminal truncation by the inflammatory protease caspase-1 has recently been implicated in the mechanisms that promote aggregation of α-syn in vitro and in neuronal cell models of α-syn toxicity. We present here an in vivo proof of concept of the ability of the caspase-1 inhibitor prodrug VX-765 to mitigate α-syn pathology and to mediate neuroprotection in proteolipid protein α-syn (PLP-SYN) mice, a transgenic mouse model of MSA. PLP-SYN and age-matched wild-type mice were treated for a period of 11 wk with VX-765 or placebo. VX-765 prevented motor deficits in PLP-SYN mice compared with placebo controls. More importantly, VX-765 was able to limit the progressive toxicity of α-syn aggregation by reducing its load in the striatum of PLP-SYN mice. Not only did VX-765 reduce truncated α-syn, but it also decreased its monomeric and oligomeric forms. Finally, VX-765 showed neuroprotective effects by preserving tyrosine hydroxylase-positive neurons in the substantia nigra of PLP-SYN mice. In conclusion, our results suggest that VX-765, a drug that was well tolerated in a 6 wk-long phase II trial in patients with epilepsy, is a promising candidate to achieve disease modification in synucleinopathies by limiting α-syn accumulation.

Keywords: alpha-synuclein; caspase-1; multiple system atrophy; truncation.

Fig. 1.

VX-765 treatment reversed α-syn–induced pathology in PLP-SYN mice by decreasing α-syn load in the striatum and rescuing motor performance. (A) Placebo-treated PLP-SYN mice produced more errors per step compared with VX-765–treated PLP-SYN mice in the challenging beam test. (B–F) Representative immunoblot levels of oligomeric (D), monomeric (E), and truncated (F) α-syn in placebo- (B) and VX-765– (C) treated PLP-SYN mice. (G) Analysis of human α-syn mRNA levels in VX-765–treated PLP-SYN mice compared with placebo-treated mice. (H–M) Immunohistochemical analysis of α-syn load (H–J) and insolubility (K–M) in the striatum of placebo- (I and K) and VX-765– (J and L) treated PLP-SYN mice. In all panels, n = 8 per experimental group—except for mRNA α-syn (G), n = 5 for placebo-treated and n = 6 for VX-765–treated PLP-SYN mice. Error bars indicate SE. *P < 0.05, **P < 0.01, ***P < 0.001. FL, full length; Hmw, high molecular weight; PK, proteinase-K; Trc, truncated.

Fig. 2.

(A–E) Representative immunoblot levels of oligomeric (C), monomeric (D), and truncated (E) α-syn in the cortex showing no significant difference between placebo- (A) and VX-765– (B) treated PLP-SYN mice. (F–K) α-syn immunohistochemistry assessing the load (F–H) and insolubility (I–K) of α-syn in placebo- (G and I) and VX-765– (H and J) treated PLP-SYN mice. In all panels, n = 8 per experimental group. Error bars indicate SE. FL, full length; Hmv, high molecular weight; PK, proteinase-K; Trc, truncated.

Fig. S1.

Individual correlation plots of truncated α-syn and full-length α-syn levels in the striatum of PLP-SYN mice receiving placebo (black circles) or VX-765 (red circles). Circle size reflects the load of high-molecular weight α-syn as measured by Western blot.

Fig. 3.

Representative immunoblot levels of pro–IL-1β (A–C and E–G) and IL-1β (A, B, D–F, and H) in the striatum (A–D) and cortex (E–H) showing pro–IL-1β in placebo- (A) and VX-765– (B) treated PLP-SYN mice in the striatum and placebo- (E) and VX-765– (F) treated PLP-SYN mice in the cortex. (C and E) Pro–IL-1β levels were not different between groups in striatum and cortex. (D) VX-765–treated PLP-SYN mice showed lower IL-1β levels in the striatum than placebo-treated PLP-SYN mice, whereas (H) no differences between groups was observed in the cortex. In all panels, n = 8 per experimental group. Error bars indicate SE. **P < 0.01.

Fig. 4.

PLP-SYN mice treated with VX-765 showed no loss of TH-positive neurons in the SN. (A and B) Representative nigral sections from placebo- (A) and VX-765– (B) treated PLP-SYN mice. (C) Statistical analysis of TH+ stereological counting showing a loss of nigral TH+ neurons in placebo-treated PLP-SYN mice compared with VX-765–treated PLP-SYN mice, as well as placebo- and VX-765–treated WT mice. In all panels, n = 8 per experimental group. Error bars indicate SE. *P < 0.05, placebo PLP-SYN vs. VX-765 PLP-SYN; ^#P < 0.05, placebo PLP-SYN vs. VX-765 WT; ^$P < 0.05, placebo PLP-SYN vs. placebo WT.

Fig. S2.

Statistical analysis of Nissl counts showing a significant effect of VX-765 treatment compared with placebo. In all panels, n = 8 per experimental group. Error bars indicate SE. *P < 0.05, treatment vs. placebo.

Fig. S3.

Analysis of VX-765 brain levels in VX-765–treated WT (n = 4) and PLP-SYN mice (n = 6).