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

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

Increased hippocampal volume in exercising mice: comparison of control conditions with in vivo voxel based morphometry

Introduction Both human and animal studies have shown that physical exercise (primarily aerobic exercise) may have facilitating effects on brain plasticity and cognition. In rodents, improvements of various forms of learning and memory induced by wheel-running have been associated with numerous neuroplastic changes such as increased hippocampal neurogenesis. A few studies, using magnetic resonance imaging (MRI), consistently reported hippocampal volumetric increase relative to non-exercising mice. However, the control group is commonly limited either to a locked wheel or no wheel. Methods In the present study, we intended to test whether 6 weeks of voluntary wheel-running exercise during adulthood induced a detectable volumetric change in mice brain in comparison to non-exercised control mice housed either with a locked wheel or without such wheel. 54 C57Bl6 males were randomly assigned to one of the three groups and individually housed for 6 weeks before imaging session. MRI (Agilent 9.4T) acquisition consisted in 3D T2 volume sequence (voxel size: 0.21 mm isotropic) using a dedicated surface coil receiver. We used Dartel soware for the preprocessing of the data, and the Voxel Based Morphometry was done with SPM mouse toolbox (F test, threshold p < .001 uncor). A small volume correction was applied to limit the analysis to the hippocampus. Results/Discussion VBM analysis shows significant clusters with increased grey matter volume in the hippocampus (cluster sizes 1531 and 3460, p < .001) when we compare the wheel vs locked wheel groups. Regarding the wheel vs no wheel comparison, significant clusters were observed in the hippocampus (cluster sizes 955 and 238, p < .001). Interestingly, no dierences were found when we compare the two control groups (locked wheel vs no wheel). Conclusions In this study, we replicate previous studies depicting an increased hippocampal volume under physical exercise in mice using VBM. Moreover, we certified here that attempting to study the impact of physical exercise on brain volume, control groups with a locked wheel or no wheel are equivalent

[18F]UCB-H BINDING QUANTIFICATION IN RAT BRAIN: FROM MODELLING TO SUV

Introduction Image quantification in Positron Emission Tomography (PET) is usually achieved through the invasive and sometimes infeasible arterial blood sampling [1, 2]. Alternative methods have been proposed, but a validation of their results is necessary [3, 4]. In the scope of improving the use of [18F]UCB-H, a specific biomarker for the Synaptic Vesicle protein 2A (SV2A) [5, 6, 7, 8], we have compared the distribution volume (VT) obtained through full kinetic modelling using a Population Based Input Function (PBIF) [9], and the Standardized Uptake Value (SUV). Methods Twelve Sprague Dawley male rats were pre-treated with vehicle (saline), 1 or 10 mg/kg of SV2A ligand (Keppra®, IP). Thirty minutes later, [18F]UCB-H was injected (IV) and a 90 min microPET dynamic acquisition was started followed by a T2 structural MRI. Primary image analysis was focused in examining tracer measurement stability through 10 min time windows. Subsequently, we calculated the correlation between VT (90 minutes) and SUV values over consecutive 20-minute time frames searching for the optimal frame to perform a static acquisition [10]. Finally, we did a supplementary test-retest static acquisition, from 60 to 80 minutes, in order to test group differences in SUV. Results/Discussion Evaluation of ten minutes time windows showed more stability in VT than in SUV measures, for all the groups. This change in signal seems to decrease in late time frames. We found also a strong correlation (R2>0.6) between dynamic VT and twenty minutes frame SUV, especially between 20 min and 60 min. From this, we can infer that an optimal frame to perform a static acquisition with [18F]UCB-H would be between 50 and 80 minutes. Using a static acquisition from 60 to 80 minutes, the SUV highlighted statistically significant differences between the group injected with vehicle and the other groups (p<0.01), but not between groups pre-treated with 1mg/kg and 10mg/kg of Keppra®. Conclusions Our work shows that a strong correlation between the SUV and the VT parameter based on a PBIF does exist. This opens the way to a possible simplification for SV2A in vivo imaging with [18F]UCB-H. Despite the fact that SUV is affected by many factors [11] and that it can overestimate results relative to VT [10], it is able to detect important differences in SV2A expression. Based on these results, SUV could become an interesting and easy to obtain parameter to study group differences in the context of several diseases. References 1. Acton PD et al. Radiologic Clinics of North America. 2004; 42(6):1055. 2. Kinahan PE & Fletcher JW. Seminars in Ultrasound, CT and MRI 2010; 31(6): 496. 3. Heurling K et al. Brain Res. 2017; 1670:220. 4. Tomasi G et al. Molecular Imaging and Biology. 2012; 14(2):131. 5. Bretin F et al. EJNMMI res. 2013; 3(1):35. 6. Warnock GI et al. J Nucl Med. 2014; 55(8):1336. 7. Bretin F et al. Molecular Imaging and Biology. 2015; 17(4):557. 8. Salmon E et al. Alzheimer's & Dementia. 2017; 13(7):781. 9. Becker G et al. Molecular Pharmaceutics. 2017; 14(8):2719. 10. Lockhart SN et al. PLoS One. 2016; 11(6):e0158460. 11. Boellaard R. J Nucl Med. 2009; 50(Suppl 1):11S-20S. Acknowledgement This work was funded by University of Liège, F.R.S.-FNRS, Walloon Region and UCB Pharma. Alain Plenevaux is research director from F.R.S.-FNRS.SV2A Projec

Decrease in SV2A expression in the hippocampus involves changes in cognition and anxiety-like features.

The Synaptic Vesicle Protein 2A (SV2A) is a transmembrane protein whose link with the epilepsy has been reported in multiples articles. However, the behavioral consequences of the decrease in its expression remain still unclear. The purpose of our research is to better understand the role of this protein through the evaluation of cKO (Grik4 +/-, SV2A lox/lox) mice of both sexes, which present a specific decrease in the hippocampus. After a first evaluation of the SV2A levels in the hippocampus with the in vitro [18F]UCB-H autoradiography, differences in brain metabolism were assessed with [18F]FDG in mPET and ex vivo autoradiography. Finally, the phenotype of cKO mice was analyzed with a behavioral test battery. Our results showed a strong reduction of SV2A expression in the whole hippocampus of cKO mice, with regard to the WT mice, not accompanied by statistically significant differences in brain metabolism between groups, either in vivo or ex vivo. No statistically significant differences were found in spontaneous locomotor activity or fear-linked memory. However, cKO males showed significant more anxiety than WT (less percent of entries in open arms) and females presented spatial memory deficits measured in the Barnes Maze (less time spend in quadrant during the test). These results could explain the comorbidity between anxiety, memory impairment and epilepsy present both in animal models and in humans, suggesting an important role of SV2A in the symptomatology of other neurodegenerative diseases, such as the Alzheimer disease, or in anxiety-related pathologies

Behavioural phenotyping of SV2A lox/lox mice: Motor and anxiety-like features

Background: Epilepsy is one of the most common neurological disorders (Alexopoulos, 2004). Current anti-epileptic drugs, such as Levetiracetam (Keppra®) or Brivaracetam, mainly target the trans-membrane Synaptic Vesicle Protein 2A (Hamann et al., 2008). Studies on homozygous SV2A KO mice phenotype, prove the mice to suffer severe seizures and die within 3 weeks (Crowder et al., 1999), establishing a link between this protein and the epilepsy. In 2009, the availability of heterozygous SV2A (+/-) mice as research tool enabled shedding light on the role of protein SV2A, revealing no motor differences but anxiety-like features in these mice compared with the WT (Lamberty et al., 2009), and a pro-epileptic phenotype (Crowder et al., 1999; Kaminski et al., 2008). Recently, a floxed SV2A mouse model has been produced with the Cre/loxP recombination system, this model allows invalidating the protein in CA3 hippocampal region, not followed by epileptic seizures (Menten-Dedoyart et al., 2016). Objectives: Perform a first behavioural phenotyping of SV2A lox/lox mice. Methodology: Two experiments were conducted in parallel to evaluate the effect of 3 different genotypes in the phenotype: WT (Grik4-/-, SV2A lox/lox), HZ (Grik4 +/-, SV2A lox/+) and cKO (Grik4 +/-, SV2A lox/lox) in male (n = 42) and female (n = 33) separately . Mice were housed individually along the experiment, with standard food and water ad libitum. After an acclimatization period of 2 weeks, anxiety-like features as well as exploration abilities were evaluated in an elevated plus-maze (EPM) single session of 5 minutes). 3 days later, spontaneous locomotor activity and habituation to the environment were measured during 1 hour, 3 consecutive days, in the activity chambers (ACT). Results: One-way ANOVA in EPM data presented no significant differences between groups, either in males or in females. A significant difference was found, between time spent in close arms vs open arms (p0.05). Statistical significant differences were found between the 3 days (p<0.01; η2p = 0.716 males; η2p = 0.663 females). Conclusion: Results indicate that a decrease in the hippocampal expresion of SV2A protein does not lead to major behavioral changes. Regarding locomotor activity, the results found in heterozygous SV2A (+/-) mice are in line with (Lamberty et al., 2009), however, our mice did not present anxiety-like features, being necessary a global decrease in brain SV2A levels and not only a partial loss in a restricted region of the brain. Further analyses increasing the number of mice per group, will allow us to intensify our power value from 50-60% (females-males) up to 80%, with large effect size and a signification of p<0.05. An additional test to evaluate the spatial memory may help us better understand the effect a specific reduction in SV2A hippocampal expression has on the phenotype of mice.ARC 13/17 - 07 (SYNAP - SV2A

Anxiety-like features and spatial memory problems as a consequence of hippocampal SV2A expression.

The Synaptic Vesicle Protein 2A (SV2A) is a transmembrane protein whose presence is reduced both in animal models and in patients with chronic epilepsy. Besides its implication in the epileptic process, the behavioural consequences of the changes in its expression remain unclear. The purpose of our research is to better understand the possible role(s) of this protein through the phenotype of cKO (Grik4 Cre+/-, SV2A lox/lox) mice, male and female, which present a specific decrease of SV2A expression levels in the hippocampal glutamatergic neurons but without any epileptic seizures. In this study, we compare the cKO mice with cHZ (Grik4 Cre+/-, SV2A lox/+) and WT (Grik4 Cre+/+, SV2A lox/lox) mice through a battery of tests, used to evaluate different features: the anxiety-related features (Elevated Plus Maze), the locomotor activity (Activity Chambers), the contextual fear-related memory (Contextual Fear Conditioning), and the spatial memory (Barnes Maze). Our results showed statistically significant differences in the habituation to a new environment, an increase in the anxiety levels and spatial memory deficit in the cHZ and cKO groups, compared to the WT group. No statistically significant differences due to the genotype appeared in the spontaneous locomotor activity or the fear-linked memory. However, sexual differences were observed in this last feature. These results highlight not only an important role of the SV2A protein in the cognitive and anxiety problems typically encountered in epileptic patients, but also a possible role in the symptomatology of other neurodegenerative diseases, such as the Alzheimer’s disease

Fully Automated Synthesis and Evaluation of [ 18 F]BPAM121: Potential of an AMPA Receptor Positive Allosteric Modulator as PET Radiotracer

Alzheimer's disease (AD) remains a significant burden on society. In the search for new AD drugs, modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are of particular interest, as loss of synaptic AMPARs has been linked to AD learning and memory deficits. Previously reported fluorine-containing BPAM121, an AMPA positive allosteric modulator (pam) with high activity, low toxicity, and slow metabolism, was considered to be a perfect 18 F-labeled candidate for positron emission tomography (PET) AD diagnostic investigations. For the preclinical use of this compound, an automated synthesis avoiding human radiation exposure was developed. The detailed production of [ 18 F]BPAM121 in relatively high quantity using a commercial FASTlab synthesizer from GE Healthcare coupled with a full set of quality controls is presented, along with procedures for the synthesis of the tosylated precursor and the fluorinated reference. To evaluate the clinical usefulness of [ 18 F]BPAM121 as a potential AD diagnostic, some in vivo studies in mice were then realized, alongside blocking and competition studies. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinhei

Exercise against cocaine sensitization in mice: a [18F]fallypride micro-PET study.

Wheel-running exercise in laboratory rodents (animal model useful to study the neurobiology of aerobic exercise) decreases behavioural markers of vulnerability to addictive properties of various drugs of abuse including cocaine. However, neurobiological mechanisms underpinning this protective effect are far from fully characterized. Here, 28-day-old female C57BL/6J mice were housed with (n=48) or without (n=48) a running wheel for 6 weeks before being tested for acute locomotor responsiveness and initiation of locomotor sensitization to intraperitoneal injections of 8 mg/kg cocaine. The long-term expression of sensitization took place 3 weeks after the last session. On the day after, all mice underwent a micro-PET imaging session with [18F]fallypride radiotracer (dopamine 2/3 receptors antagonist). Exercised mice were less sensitive to acute and sensitized cocaine hyperlocomotor effects, such attenuation being particularly well-marked for long-term expression of sensitization (η²p = 0.262). Chronic administration of cocaine was associated with a clear-cut increase of [18F]fallypride binding potential in mouse striatum (η²p = 0.170) while wheel-running exercise was associated with a moderate decrease in dopamine 2/3 receptors density in striatum (η²p = 0.075), a mechanism that might contribute to protective properties of exercise against drugs of abuse vulnerability