Implication de la protéine SV2A dans l'épilepsie: études précliniques

Abstract

This PhD thesis deals with epilepsy and, in particular, with the role the Synaptic Vesicle 2A (SV2A) protein plays on the development and severity of this disease. Epilepsy is a group of neurodegenerative diseases whose most notable feature is the presence of spontaneous and recurrent brain seizures. Despite the extensive efforts made to treat epilepsy, 65 million people worldwide suffer from this disease. Moreover, with the available treatments, the seizures cannot be efficiently controlled in 30% of these patients. Amongst the antiepileptic drugs currently in use, levetiracetam is the one with the fewest side effects, easy to use, and a high efficacy controlling the seizures. These drugs typically target the SV2A protein, a transmembrane protein implicated in the synaptic transmission, and indispensable for life. In the existing literature on this topic, the homozygous mutation of SV2A has been associated with intractable epilepsy, growth retardation, and premature death both in mice and humans. However, to date, few studies have analysed the influence and the expression of this protein in the course of the epileptic process. Thus, the aim of this thesis is to further the understanding of the relationship between the SV2A protein and the epileptic disease, considering different perspectives and using diverse techniques. In the first part of this manuscript (Chapters 3 and 4), we study the variations in SV2A levels owing to the progression of temporal lobe epilepsy, making use of the kainic acid (KASE) rat model. In particular, Chapter 3 delves into the characteristics and the specificity of [18F]UCB-H, a radiotracer with an affinity for SV2A of 7.8 M (pIC50). On the one hand, we demonstrate the importance of high enantiomeric purity ((R)-enantiomer) and high affinity for the target (pIC50 > 6.8) to obtain PET images of good quality. On the other hand, we perform a competition assay between [18F]UCB-H and different SV2 ligands, demonstrating that this radiotracer displays more affinity for SV2A than for SV2B or SV2C, which are paralogs sharing around 60% of their sequences. In addition, in Chapter 3 we detail the methodology developed and validated to quantify the [18F]UCB-H uptake in the rat brain. The conclusion of this part is that it is possible to perform 60-minute dynamic scans (Vt) and 20-minute static scans (SUV20-40) with [18F]UCB-H. This methodology is applied in Chapter 4, where we analyse variations in SV2A levels with [18F]UCB-H in the KASE rat model, a well-known model of temporal lobe epilepsy (TLE). The results show a progressive increase in SV2A levels through the rat brain development. This increase is affected by the progression of epilepsy, as demonstrated by the significant group differences (Sham vs KASE) in all the phases of the disease. Moreover, these differences evolve progressively and differently in all brain regions. In addition, in Chapter 4 we also explore the relationship between variations in SV2A levels and other neuropathological correlates present in the epileptic process: the brain hypometabolism (quantified with [18F]FDG) and the severity of epilepsy (evaluated with electroencephalography). Our results propose that variations in SV2A levels could be: (1) positively correlated to a previous brain hypometabolism, and (2) positively correlated to the number of electrographic seizures observed in epileptic animals during the chronic phase of TLE.In the second part of this manuscript (Chapters 5 and 6) we assess the cognitive and behavioural impact of the decrease in SV2A expression in the brain, specifically in the hippocampus. Therefore, Chapter 5 is dedicated to the validation of a conditional SV2A knockout mice model (cKO) with [18F]UCB-H in vitro autoradiography. This model was developed with the Cre/loxP technique to study the influence of the specific decrease of SV2A expression in CA3 glutamatergic neurons in epilepsy. Then, in Chapter 6, we evaluate the cognitive and the behavioural aspects of this model with multiple tests (elevated plus maze, actometers, contextual fear conditioning test, and Barnes maze). The final goal of this evaluation is to shed light on the underlying molecular processes of the clinical features observed in the epileptic disease. Our results show a relationship between the decrease in SV2A expression at the hippocampus and the existence of anxiety-like features and spatial memory problems.Combined together, our findings represent a step forwards in the understanding, not only of the relationship between the SV2A protein and epilepsy, but also of the epileptic disease itself. Thus, with this work we expect firstly to further the existing knowledge about the underlying processes of epilepsy, and secondly to guide future researches, raising new key questions which may open the door to improve both the detection and treatment of epilepsy