CNC

Overview

Project Summary

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD), is the most common autosomal dominant ataxia worldwide. It is caused by the expansion of CAG trinucleotide repeat in ATXN3/MJD1 gene, being translated into an expanded polyglutamine (polyQ) stretch within ataxin-3 protein, which gains toxic properties and triggers degeneration, leading to neuronal loss and patients’ death. Unfortunately, SCA3 still LACKS AN EFFICIENT TREATMENT allowing disease modification. Direct reprogramming of endogenous glial cells into functional neurons have shown to promote significant recovery in several neurodegenerative disorders, but it has not been explored so far to treat ataxias. The aim of this proposal is to explore the efficiency of direct in vivo reprogramming of astrocytes into cerebellar neurons in SCA3. This would bring great impact to the clinics as patients would profit from a treatment developed from their own endogenous cells. For that purpose, induced differentiation of astrocytes into cerebellar neurons will be performed by using two transcription factors (TFs), Achaete-scute homolog 1 (Ascl1) and Neurogenin2 (Neuro2), already established. Adeno-associated virus of serotype 5 (AAV5), which infects preferentially astrocytes, will simultaneously overexpress GFAP and one of the TFs (or both) under a system that will allow to track the astrocyte induced-neurons by triggering the expression of mCherry (a red fluorescent probe). A transgenic SCA3 mouse model, allowing both for neuropathological and phenotypic assessments, will be used. Post-symptomatic transgenic mice will be injected in the vermis with AAV5 expressing constructs: 1) overexpressing Ascl1; 2) overexpressing Neurog2; 3) overexpressing Ascl1 and Neurog2; 4) expressing mCherry only - control. First, efficiency of the reprogramming constructs at two different dosages will be assessed and the best strategy selected. Then, transgenic mice will be treated with the best construct and evaluated by rotarod (motor performance evaluation) before and every 3 weeks after injection, until they reach 2-3 months post-injection. Wild type littermates will also be used as controls to evaluate toxicity of these constructs and compare the efficiency of reprogramming in normal and SCA3 mice. Cerebellar neuropathologic assessments will be performed to determine: the number and fate of reprogrammed neurons; number of ataxin-3 aggregates; cerebellar volume and Purkinje cell number, known to be severely reduced in this SCA3 mouse model. We expect to show that this combined approach can increase the percentage of healthy neurons in the cerebellum of SCA3 mice, becoming a promising and valuable therapy for this disease, and for other cerebellar ataxias.

Main Goals

1 - Selection of the most efficient strategy to form in vivo induced neurons reprogrammed from astrocytes in SCA3 mice;

2 - Assessing the potential of direct reprogramming of astrocytes into cerebellar neurons to correct motor and neuropathological impairments in SCA3 mice;

3 - Fostering science dissemination through ART, SCIENCE AND EDUCATION.