A Protein Aggregation Inhibitor, Leuco-Methylthioninium Bis(Hydromethanesulfonate), Decreases α-Synuclein Inclusions in a Transgenic Mouse Model of Synucleinopathy.

Karima Schwab,Silke Frahm,Franz Theuring,Charles R Harrington,Valeria Melis,John M D Storey,Mandy Magbagbeolu,Thomas C Baddeley,Gernot Riedel,Elizabeth A Goatman,Janet E Rickard,Michael G Leith,Claude M Wischik,Morag Douglas,David Horsley

doi:10.3389/fnmol.2017.00447

Abstract

α-Synuclein (α-Syn) aggregation is a pathological feature of synucleinopathies, neurodegenerative disorders that include Parkinson’s disease (PD). We have tested whether N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diaminium bis(hydromethanesulfonate) (leuco-methylthioninium bis(hydromethanesulfonate); LMTM), a tau aggregation inhibitor, affects α-Syn aggregation in vitro and in vivo. Both cellular and transgenic models in which the expression of full-length human α-Syn (h-α-Syn) fused with a signal sequence peptide to promote α-Syn aggregation were used. Aggregated α-Syn was observed following differentiation of N1E-115 neuroblastoma cells transfected with h-α-Syn. The appearance of aggregated α-Syn was inhibited by LMTM, with an EC50 of 1.1 μM, with minimal effect on h-α-Syn mRNA levels being observed. Two independent lines of mice (L58 and L62) transgenic for the same fusion protein accumulated neuronal h-α-Syn that, with aging, developed into fibrillary inclusions characterized by both resistance to proteinase K (PK)-cleavage and their ability to bind thiazin red. There was a significant decrease in α-Syn-positive neurons in multiple brain regions following oral treatment of male and female mice with LMTM administered daily for 6 weeks at 5 and 15 mg MT/kg. The early aggregates of α-Syn and the late-stage fibrillar inclusions were both susceptible to inhibition by LMTM, a treatment that also resulted in the rescue of movement and anxiety-related traits in these mice. The results suggest that LMTM may provide a potential disease modification therapy in PD and other synucleinopathies through the inhibition of α-Syn aggregation.

Highlights

Protein conformational disorders are a group of diseases which evolve due to protein misfolding leading to the formation of aggregated structures (Carrell and Gooptu, 1998; Uversky and Fink, 2004; Lashuel et al, 2013)
The level of immunoreactivity was minimal in the DH60.21 cell line, derived from N1E-115 mouse neuroblastoma cells and constitutively expressing full-length human α-Syn fused with an N-terminal signal sequence peptide (SSFsyn), in the absence of differentiation (Figure 1A, lane 5)
We report the development of two novel model systems for testing the efficacy of synuclein aggregation inhibitors as a potential treatment for diseases characterized by pathological aggregation of synuclein, Parkinson’s disease (PD) and dementia with Lewy bodies

Summary

Introduction

Protein conformational disorders are a group of diseases which evolve due to protein misfolding leading to the formation of aggregated structures (Carrell and Gooptu, 1998; Uversky and Fink, 2004; Lashuel et al, 2013). PD and dementia with Lewy bodies are the most common disorders associated with synuclein pathology and are characterized by intra-neuronal deposits of α-Syn fibrils most prominently found in midbrain regions and cerebral cortex (Uversky, 2003). This pathology spreads progressively from medulla oblongata through midbrain to neocortex (Braak et al, 2003). There is intra-neuronal expression of h-α-Syn in both neocortex and archicortex in these mouse lines, and both are associated with a behavioral hypoactivity state

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