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ANR funded project

JCJC : Sciences de la vie, de la santé et des écosystèmes : Physiologie, métabolisme, physiopathologie, santé publique (JCJC SVSE 1) 2010
Projet Mechataxia

Physiopathological mechanisms of episodic (EA2) and related progressive ataxia

One third of inherited mutations in human diseases lead to a disruption of the reading frame, producing truncated proteins that have deleterious cellular effects. Two main general cellular mechanisms appear to be linked in this type of diseases: the RNA and protein quality controls. Indeed, transcripts with a premature stop codon are generally subject to the non-sense mRNA decay (NMD), whereas truncated proteins are often subject to protein quality control and subsequent proteasomal degradation.
The goal of this project is to study the pathogenic mechanisms of the episodic ataxia type 2 (EA2), a prototype of dominant inherited diseases due to non-sense mutations in a Voltage Dependant Calcium Channel, CaV2.1. The genetic bases of these diseases provide unique opportunities to study the underlying mechanisms, from the molecular to organism level.
CaV2.1 channels are mainly expressed in presynaptic nerve terminals where their opening is linked to the rapid release of synaptic vesicles. Mutations in the CaV2.1 gene (CACNA1A) cause several autosomal dominant movement disorders including familial hemiplegic migraine (FHM-1), spinocerebellar ataxia type 6 (SCA6) and episodic ataxia type 2 (EA2). EA2 is characterized by variable symptoms including periodic attacks, gait ataxia, vertigo and progressive ataxia with cerebellar atrophy and Purkinje neuron loss. Functional expression studies have established that non- or hypo-conducting channels cause the disorder, which is largely associated with expression of truncated forms of CaV2.1. We and other groups have shown that EA2 mutants exert a dominant-negative effect in vitro. Truncated EA2 mutants are misfolded, intracellularly retained and subjected to endoplasmic reticulum associated degradation (ERAD). This misfolded mutants bind to nascent wild-type Cav subunits and induce their subsequent proteasomal degradation, thereby abolishing channel activity.
However, the origin of the dominance and the molecular events leading to neuronal degeneration are still matter of debate. The goal of this project is to decipher each molecular event implicated in the disease and characterise their interrelationships. In particular, we will investigate 1) implication of the NMD in EA2 as a potential protective cellular mechanism, 2) the key actors of the ERAD process, such as the E3 ligase(s), implicated in EA2, and 3) different strategies to bypass the NMD.
Altogether the findings will help to understand the impact of the EA2 mutation on neuronal dysfunction and degeneration in vivo.
To this aim, novel methodology and tools are available. An EA2 knock-in mouse has been generated with a non-sense mutation as no adequate EA2 mouse models are actually available. Alternatively, we are using a lentiviral-based vector strategy to express channel mutants directly in primary neuron as well as in mouse brain. We will use pharmacology and RNA silencing strategies to inhibit the NMD in heterozygote EA2 KI mice, in order to study different degrees of the disease severity.
We expect to implicate the NMD in EA2 both in vitro and in vivo in our EA2-KI mouse model. Moreover, we attempt to identify the E3 ligase(s) implicated in the misfolded channel degradation and to investigate their impact in vivo. Finally, directly targeting the cerebellum with EA2 mutant lentiviral vectors and manipulating the NMD in EA2 KI mice will allow us to identify the nature of the cellular stress leading to neuronal degeneration. Therefore, accomplishment of this project should define new targets for EA2 and potentially for other similar inherited diseases.



ANR grant: 273 170 euros
Beginning and duration: - 36 mois


ANR Programme: JCJC : Sciences de la vie, de la santé et des écosystèmes : Physiologie, métabolisme, physiopathologie, santé publique (JCJC SVSE 1) 2010

Project ID: ANR-10-JCJC-1102

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The project coordinator is the author of this abstract and is therefore responsible for the content of the summary. The ANR disclaims all responsibility in connection with its content.