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Peer reviewe

Exploring with [18F]UCB-H the in vivo cariations in SV2A expression through the kainic acid rat model of temporal lobe epilepsy

Purpose The main purpose of this study was to understand how the positron emission tomography (PET) measure of the synaptic vesicle 2A (SV2A) protein varies in vivo during the development of temporal lobe epilepsy (TLE) in the kainic acid rat model. Procedures Twenty Sprague Dawley male rats were administered with multiple systemic doses of saline (control group, n = 5) or kainic acid (5 mg/kg/injection, epileptic group, n = 15). Both groups were scanned at the four phases of TLE (early, latent, transition, and chronic phase) with the [F-18]UCB-H PET radiotracer and T2-structural magnetic resonance imaging. At the end of the scans (3 months post-status epilepticus), rats were monitored for 7 days with electroencephalography for the detection of spontaneous electrographic seizures. Finally, the immunofluorescence staining for SV2A expression was performed. Results Control rats presented a significant increase in [F-18]UCB-H binding at the last two scans, compared with the first ones (p < 0.001). This increase existed but was lower in epileptic animals, producing significant group differences in all the phases of the disease (p < 0.028). Furthermore, the quantification of the SV2A expression in vivo with the [F-18]UCB-H radiotracer or ex vivo with immunofluorescence led to equivalent results, with a positive correlation between both. Conclusions Even if further studies in humans are required, the ability to detect a progressive decrease in SV2A expression during the development of temporal lobe epilepsy supports the use of [F-18]UCB-H as a useful tool to differentiate, in vivo, between healthy and epileptic animals along with the development of the epileptic disease

Associative memory and its cerebral correlates in Alzheimer’s disease: Evidence for distinct deficits of relational and conjunctive memory

This study investigated the impact of Alzheimer's disease (AD) on conjunctive and relational binding in episodic memory. Mild AD patients and controls had to remember item-color associations by imagining color either as a contextual association (relational memory) or as a feature of the item to be encoded (conjunctive memory). Patients' performance in each condition was correlated with cerebral metabolism measured by FDG-PET. The results showed that AD patients had an impaired capacity to remember item-color associations, with deficits in both relational and conjunctive memory. However, performance in the two kinds of associative memory varied independently across patients. Partial least square analyses revealed that poor conjunctive memory was related to hypometabolism in an anterior temporal-posterior fusiform brain network, whereas relational memory correlated with metabolism in regions of the default mode network. These findings support the hypothesis of distinct neural systems specialized in different types of associative memory and point to heterogeneous profiles of memory alteration in Alzheimer's disease as a function of damage to the respective neural networks

EPILEPSY AND THE SV2A PROTEIN: new insights about the disease.

Around two million of people worldwide are affected by neurodegenerative diseases, such as Alzheimer, Parkinson or Epilepsy. Despite the social and the economic impact of these diseases, their causes still remain unclear. In the case of the epilepsy, for example, around 25% of the patients suffer drug-resistant epilepsy, for which there is no medicament able to mitigate the epileptic crises or the associated symptomatology, such as cognitive problems and mood disorders. In 1974, UCB Pharma synthetized a new antiepileptic drug with a high therapeutic index: the Levetiracetam. The target of this medicament is the Synaptic Vesicle Protein 2A (SV2A) whose specific role in the pathology is still unknown. The main goal of my thesis is to better understand the relationship between this protein and the epilepsy. On the one hand, the production and phenotyping of conditional knockout mice for the SV2A protein allowed us to discover a possible implication of this protein in the spatial memory and anxiety process, an important part of the epileptic symptomatology. On the other hand, the synthesis of the radiotracer [18F]UCB-H, with a high affinity for the SV2A protein, enabled the in vivo evaluation (with the mPET technique) of a rat model of the temporal lobe epilepsy through the disease process. Results showed a strong correlation between the severity of the epilepsy (EEG technique) and the SV2A levels in different brain regions, highlighting the importance of this protein in the development of the disease. In summary, although further studies in humans are necessary, this protein emerges as an important key in clinical diagnosis and medical research, being implicated in all the aspects of the epileptic disease

Quantification of [18F]UCB-H Binding in the Rat Brain: From Kinetic Modelling to Standardised Uptake Value

Purpose: [18F]UCB-H is a specific positron emission tomography (PET) biomarker for the Synaptic Vesicle protein 2A (SV2A), the binding site of the antiepileptic drug levetiracetam. With a view to optimising acquisition time and simplifying data analysis with this radiotracer, we compared two parameters: the distribution volume (Vt) obtained from Logan graphical analysis using a Population-Based Input Function, and the Standardised Uptake Value (SUV). Procedures: Twelve Sprague Dawley male rats, pre-treated with three different doses of levetiracetam were employed to develop the methodology. Three additional kainic acid (KA) treated rats (temporal lobe epilepsy model) were also used to test the procedure. Image analyses focused on: (i) length of the dynamic acquisition (90 versus 60 min); (ii) correlations between Vt and SUV over 20-min consecutive time-frames; (iii) and (iv) evaluation of differences between groups using the Vt and the SUV; and (v) preliminary evaluation of the methodology in the KA epilepsy model. Results: A large correlation between the Vt issued from 60 to 90-min acquisitions was observed. Further analyses highlighted a large correlation (r 9 0.8) between the Vt and the SUV. Equivalent differences between groups were detected for both parameters, especially in the 20–40 and 40– 60-min time-frames. The same results were also obtained with the epilepsy model. Conclusions: Our results enable the acquisition setting to be changed from a 90-min dynamic to a 20-min static PET acquisition. According to a better image quality, the 20–40-min time-frame appears optimal. Due to its equivalence to the Vt, the SUV parameter can be considered in order to quantify [18F]UCB-H uptake in the rat brain. This work, therefore, establishes a starting point for the simplification of SV2A in vivo quantification with [18F]UCB-H, and represents a step forward to the clinical application of this PET radiotracer.SV2A projec

EPILEPSY AND THE SV2A PROTEIN: new insights about the disease.

Around two million of people worldwide are affected by neurodegenerative diseases, such as Alzheimer, Parkinson or Epilepsy. Despite the social and the economic impact of these diseases, their causes still remain unclear. In the case of the epilepsy, for example, around 25% of the patients suffer drug-resistant epilepsy, for which there is no medicament able to mitigate the epileptic crises or the associated symptomatology, such as cognitive problems and mood disorders. In 1974, UCB Pharma synthetized a new antiepileptic drug with a high therapeutic index: the Levetiracetam. The target of this medicament is the Synaptic Vesicle Protein 2A (SV2A) whose specific role in the pathology is still unknown. The main goal of my thesis is to better understand the relationship between this protein and the epilepsy. On the one hand, the production and phenotyping of conditional knockout mice for the SV2A protein allowed us to discover a possible implication of this protein in the spatial memory and anxiety process, an important part of the epileptic symptomatology. On the other hand, the synthesis of the radiotracer [18F]UCB-H, with a high affinity for the SV2A protein, enabled the in vivo evaluation (with the mPET technique) of a rat model of the temporal lobe epilepsy through the disease process. Results showed a strong correlation between the severity of the epilepsy (EEG technique) and the SV2A levels in different brain regions, highlighting the importance of this protein in the development of the disease. In summary, although further studies in humans are necessary, this protein emerges as an important key in clinical diagnosis and medical research, being implicated in all the aspects of the epileptic disease.SV2A projec

THE SV2A PROTEIN: IMAGING SYNAPTIC DENSITY DURING THE PROGRESSION OF THE TEMPORAL LOBE EPILEPSY IN THE KASE RAT MODEL

Introduction The temporal lobe epilepsy (TLE) is the most common epileptic disorder. New antiepileptic drugs target the Synaptic Vesicle protein 2A (SV2A) (1). Nevertheless, the prevailing literature addressing the relation between this protein and the epilepsy is limited (2, 3). This study provides insights on the role of the SV2A protein during the four stages of TLE (4, 5), throughout its in vivo study with the [18F]UCB-H radiotracer (6). Methods Twenty-four male Sprague-Dawley were subjected to multiple injections (7) of i) Saline (Sham), or ii) 5mg/kg of Kainic Acid (KA). The rats not reacting to KA (NKA) were also scanned. In each TLE stages, a [18F]UCB-H dynamic scan was performed, followed by a T2-structural MRI. EEG recordings were performed to determine the number of crises. Data processing was done with PMOD 3.6. Results were expressed as SUV and statistically analyzed with the SPSS and the SPM. Results During the acute phase, statistically significant differences were found between Sham and KA in striatum, cerebellum, and medulla. In the latent phase, these SUV differences were detected between the NKA and KA in the same regions along with hippocampus and thalamus. When the spontaneous crises started, these group differences became statistically significant in all the regions but the cortex. During the chronic phase, all the regions showed statistically significant differences between groups. Furthermore, the voxel-wise analysis highlighted statistically significant differences in voxels at the level of amygdala and hippocampus. Conclusions These results show that [18F]UCB-H is able to detect early modifications in SV2A expression (3 days after the TLE model creation), in particular in regions implicated in the epileptic process. This radiotracer can potentially be used as a suitable biomarker for the early detection of the epileptic disease, being able to distinguish between stages in this neurodegenerative disease.SV2A projec

Evaluating the specificity of [18F] UCB-H for the isoform SV2A, compared with isoforms SV2B and SV2C

Background: SV2A is the most studied isoform of the Synaptic Vesicle 2 proteins, which are involved in the synaptic vesicle trafficking, being important in normal and pathological process, like the epilepsy (1, 2). [18F]UCB-H was developed like a tool to study the role of this isoform with neuroimaging techniques (3, 4). The objective of this study was to evaluate its specificity to this isoform comparing with the others, through a competition assay in rats with ex-vivo autoradiography and mPET imaging. Methods: Forty male Sprague-Dawley were used in ex-vivo autoradiography experiments (N=20) and in microPET imaging (N=20). Animals were pre-treated 30 minutes before the injection of [18F]UCB-H with a dose IP either of vehicle, Keppra (SV2A ligand), UCB068 (SV2B ligand) or UCB054 (SV2C ligand). Ex-vivo autoradiography was carried out 5 minutes after radiotracer injection while mPET images were acquiring with a dynamic scanner of 1 hour. Data were expressed in Standard Uptake Value and then, the area under the curve was calculated for the total process. Results: In ex-vivo autoradiography, ANOVA of two-ways showed statistical significant differences in brain uptake of [18F]UCB-H among the groups pretreated with Keppra or the ligand for SV2B and the control group. Regarding mPET data, statistical significant differences were found between the group injected with keppra and the rest of groups. Conclusion: Even if a considerable affinity between the ligands UCB068 and UCB054, and the receptor for the isoform SV2A exists, it is only detected during the first 5 minutes (ex-vivo technique), being certainly due to a nonspecific binding. This binding is not strong enough to show a direct competition with the radiotracer during a mPET acquisition. These results allow us to conclude that [18F]UCB-H is a suitable radiotracer for the imaging of the isoform SV2A in vivo, allowing us the clinical study about the molecular base of a disease with a high population impact, like the epilepsy.ARC 13/17 - 07 (SYNAP - SV2A

Sv2a protein levels in the kainic acid epilepsy rat model during the acute phase

Introduction The Kaïnic Acid model (KA) is one of the most validated models of temporal lobe epilepsy (TLE) (Lévesque et al.,2016). Its administration induces status epilepticus (SE), characterized by an extensive neuronal damage in limbic structures (Sperk et al.,1983). Post-mortem studies, such as the epilepsy model presented in (Wang et al., 2014), show a reduction of SV2A protein levels during the chronic phase, however, no data have been reported during the acute phase (0-48h after KA injection).. The present pilot study is undertaken to evaluate in vivo, with the specific radiotracer [18F]UCB-H (Bretin et al., 2015; Warnock et al., 2014), the SV2A expression 24h after a SE produced by KA administration. Methods Two Sprague-Dawley rats were scanned at two different times: baseline, and 24h after three systemic injections of 5mg/kg KA. The scanning process consisted of a first scan with microPET (Focus 120), during 1 hour, using [18F]UCB-H (41 ± 5 MBq IV tail vein). This is followed by MRI (9.4T Agilent, anatomical T2). A coregistration was performed with PMOD 3.6 software. Data were expressed as SUV and AUC were calculated for the different brain regions. Results [18F]UCB-H microPET images exhibited a small reduction (around 10%) in SV2A brain levels after KA injections compared to the baseline, marked in thalamus, hippocampus and amygdale. MRI images obtained 24h after KA injections are in accordance with previous histological studies, revealing inflammatory edema, tissue necrosis and increased ventricle volume (Sperk et al.,1983). Conclusions These preliminary results obtained in KA treated rats show that [18F]UCB-H is able to detect alterations in SV2A levels in relevant regions for epilepsy. This radiotracer emerges as a valuable tool to follow in vivo SV2A through longitudinal studies. KA model in rats deserves as a tool for the study of epilepsy, exhibiting the same features than the human disease. References [1] Lévesque et al., J Neurosci Methods, 2016 [2] Sperk et al., Neuroscience, 1983 [3] Wang et al., J Mol Neurosci., 2014 [4] Bretin et al., Molecular Imaging and Biology, 2015 [5] Warnock et al., J Nucl Med., 2014 [5] Van Nieuwenhuyse et al., Brain Research, 2015 [6] Hellier et al., Epilepsy Res., 1998The SV2A protein projec

IN VIVO STUDY OF THE SV2A PROTEIN IN AN EPILEPTIC RAT MODEL

Introduction Epilepsy is one of the commonest neurological disorders, affecting more than 60 million people worldwide [1]. New and effective antiepileptic drugs mainly target the SV2A protein [2] but its actual role is still largely unknown. [18F]UCB-H was developed as a tool to study in vivo the brain expression of this isoform [3, 4]. Due to the fact that only post-mortem studies were reported so far [5] the present pilot study was undertaken in order to evaluate for the first time in vivo in rats the SV2A expression in the validated Kaïnic Acid (KA) epilepsy model [6]. Methods Three male Sprague-Dawley were used, one injected with saline (Sham) and two with multiple KA systemic injections (5mg/kg x 3) [9]. SV2A brain levels were estimated at day 75, when spontaneous seizures started to appear. Animals were anesthetized (2.5 to 3 % isoflurane), and scanned for 1 hour with [18F]UCB-H (41 ± 5 MBq IV tail vein) in a Focus 120 microPET system and with MRI (9.4T Agilent, anatomical T2). Coregistration was done with PMOD 3.6 software. Data were expressed in SUV and areas under the curve were calculated for the different regions. Results [18F]UCB-H microPET images showed an important reduction (20-30%) for SV2A after KA injections mainly localized in amygdala, hippocampus, lateral parietal association cortex and cingulate cortex. The rest of the brain was globally unchanged. MRI revealed atrophy and inflammation in amygdala and hippocampus. Conclusions These preliminary results in KA treated rats presenting spontaneous seizures showed that [18F]UCB-H microPET was able to detect important reductions for the SV2A proteins in relevant regions for epilepsy [5]. Accordingly to this, we can infer that the KA model in rats deserves for further development and validation as a tool for the study of epilepsy. [18F]UCB-H appears as a valuable tool to follow in vivo SV2A proteins through longitudinal protocols and in turn to better understand its actual role in epilepsy. References/acknowledgements 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. [1] Alexopoulos, Epileptology, 2004 [2] Hamann et al., Eur J Pharmacol, 2008 [3] Bretin et al., Molecular Imaging and Biology, 2015 [4] Warnock et al., J Nucl Med., 2014 [5] Wang et al., J Mol Neurosci., 2014 [6] Hellier et al., Epilepsy Res., 1998ARC 13/17 - 07 (SYNAP - SV2A