Parkinson’s disease is a common neurological disorder with a complex etiology. The disease is characterized by a progressive loss of dopaminergic cells in the substantia nigra, which leads to motor function and sometimes cognitive function disabilities. One of the pathological hallmarks in Parkinson’s disease is the cytoplasmic inclusions called Lewy bodies found in the dopamine neurons. The aggregated protein α-synuclein is a main component of Lewy bodies. In view of severe symptoms and the upcoming of problematic side effects that are developed by the current most commonly used treatment in Parkinson’s disease, new treatment strategies need to be elucidated. One such strategy is replacing the lost dopamine neurons with new dopamine-rich tissue. To improve survival of the implanted neurons, neurotrophic factors have been used. Glial cell line-derived neurotrophic factor (GDNF), which was discovered in 1993, improves survival of ventral mesencephalic dopamine neurons and enhances dopamine nerve fiber formation according to several studies. Thus, GDNF can be used to improve dopamine-rich graft outgrowth into the host brain as well as inducing sprouting from endogenous remaining nerve fibers. This study was performed on Gdnf gene-deleted mice to investigate the role of GDNF on the nigrostriatal dopamine system. The transplantation technique was used to create a nigrostriatal microcircuit from ventral mesencephalon (VM) and the lateral ganglionic eminence (LGE) from different Gdnf gene-deleted mice. The tissue was grafted into the lateral ventricle of wildtype mice. The results revealed that reduced concentrations of GDNF, as a consequence from the Gdnf gene deletion, had effects on survival of dopamine neurons and the dopamine innervation of the nigrostriatal microcircuit. All transplants had survived at 3 months independently of Gdnf genotype, however, the grafts derived from Gdnf gene-deleted tissue had died at 6 months. Transplants with partial Gdnf gene deletion survived up to 12 months after transplantation. Moreover, the dopaminergic innervation of striatal co-grafts was impaired in Gdnf gene-deleted tissue. These results highlight the role of GDNF for long-term maintenance of the nigrostriatal dopamine system. To further investigate the role of GDNF expression on survival and organization of the nigrostriatal dopamine system, VM and LGE as single or combined to double co-grafts created from mismatches in Gdnf genotypes were transplanted into the lateral ventricle of wildtype mice. Survival of the single grafts was monitored over one year using a 9.4T MR scanner. The size of single LGE transplants was significantly reduced by the lack of GDNF already at 2 weeks postgrafting while the size of single VM was maintained over time, independently of GDNF expression. The double grafts were evaluated at 2 months, and the results revealed that lack of GDNF in LGE reduced the dopamine cell survival, while no loss of dopamine neurons was found in VM single grafts. The dopaminergic innervation of LGE was affected by absence of GDNF, which also caused a disorganization of the striatal portion of the co-grafts. Small, cytoplasmic inclusions were frequently found in the dopamine neurons in grafts lacking GDNF expression. These inclusions were not possible to classify as Lewy bodies by immunohistochemistry and the presence of phospho-α-synuclein and ubiquitin; however, mitochondrial dysfunction could not be excluded. To further study the death of the dopamine neurons by the deprivation of GDNF, the attention was turned to how Lewy bodies are developed. With respect to the high levels of α-synuclein that was found in the striatum, this area was selected as a target to inject the small molecule – FN075, which stimulates α-synuclein aggregation, to further investigate the role of α-synuclein in the formation of cytoplasmic inclusions. The results revealed that cytoplasmic inclusions, similar to those found in the grafts, was present at 1 month after the injection, while impairment in sensorimotor function was exhibited, the number of dopamine neurons was not changed at 6 months after the injection. Injecting the templator to the substantia nigra, however, significantly reduced the number of TH-positive neurons at 3 months after injection. In conclusion, these studies elucidate the role of GDNF for maintenance and survival of the nigrostriatal dopamine system and mechanisms of dopamine cell death using small molecules that template the α-synuclein aggregation.

Understanding the specificity of homeobox genes has been hampered by the lack of verified direct transcriptional targets. The Dlx family of homeobox genes is expressed in the ganglionic eminences of the developing forebrain. Dlx1/Dlx2 double knockout (DKO) mice die at birth. Phenotypic analyses demonstrate abnormal development of the basal telencephalon, including defects in neuronal differentiation in the basal ganglia, reduced expression of GABA in the basal telencephalon, and loss of migration of GABAergic inhibitory interneurons to the neocortex. The mechanisms underlying DLX protein regulation of differentiation and migration of GABAergic interneurons are poorly defined. We have successfully applied chromatin immunoprecipitation to identify potential direct transcriptional targets of DLX homeoproteins from embryonic tissues in vivo. Reporter gene assays demonstrated the transcriptional significance of the binding of DLX proteins to different downstream regulatory elements, which were confirmed in vitro by electrophoretic mobility shift assay and site-directed mutagenesis. The functional significance of DLX mediated transcriptional regulation of these targets was further elaborated through several series of loss-of-function assays including gene expression in Dlx1/2 knockout embryonic forebrain tissues, as well as siRNA or Lentiviral mediated shRNA knockdown experiments with primary forebrain cultures. Quantitative analysis of the regulatory effect of Dlx genes on various forebrain markers of differentiation and migration was performed using in situ hybridization, high-performance liquid chromatography coupled with cell counting. Neuronal migration was assessed by forebrain explants and diI labelling of migratory cells from ganglionic eminence to neocortex. We have demonstrated that DLX1 and DLX2 can transcriptionally activate (Gad1, Gad2) or repress (Nrp2) different downstream targets. In the Dlx1/2 DKO, reduction of GABA expression and failure of GABAergic interneurons to migrate to the neocortex is partly due to loss or aberrant expression of these DLX downstream targets. In the triple Dlx1/2; Nrp2KO, partial restoration of tangential migration of GABAergic interneurons from basal ganglia to the neocortex was successfully established signifying the importance of DLX regulation of Semaphorin-Neuropilin signalling during forebrain development.

Understanding the specificity of homeobox genes has been hampered by the lack of verified direct transcriptional targets. The Dlx family of homeobox genes is expressed in the ganglionic eminences of the developing forebrain. Dlx1/Dlx2 double knockout (DKO) mice die at birth. Phenotypic analyses demonstrate abnormal development of the basal telencephalon, including defects in neuronal differentiation in the basal ganglia, reduced expression of GABA in the basal telencephalon, and loss of migration of GABAergic inhibitory interneurons to the neocortex. The mechanisms underlying DLX protein regulation of differentiation and migration of GABAergic interneurons are poorly defined. We have successfully applied chromatin immunoprecipitation to identify potential direct transcriptional targets of DLX homeoproteins from embryonic tissues in vivo. Reporter gene assays demonstrated the transcriptional significance of the binding of DLX proteins to different downstream regulatory elements, which were confirmed in vitro by electrophoretic mobility shift assay and site-directed mutagenesis. The functional significance of DLX mediated transcriptional regulation of these targets was further elaborated through several series of loss-of-function assays including gene expression in Dlx1/2 knockout embryonic forebrain tissues, as well as siRNA or Lentiviral mediated shRNA knockdown experiments with primary forebrain cultures. Quantitative analysis of the regulatory effect of Dlx genes on various forebrain markers of differentiation and migration was performed using in situ hybridization, high-performance liquid chromatography coupled with cell counting. Neuronal migration was assessed by forebrain explants and diI labelling of migratory cells from ganglionic eminence to neocortex. We have demonstrated that DLX1 and DLX2 can transcriptionally activate (Gad1, Gad2) or repress (Nrp2) different downstream targets. In the Dlx1/2 DKO, reduction of GABA expression and failure of GABAergic interneurons to migrate to the neocortex is partly due to loss or aberrant expression of these DLX downstream targets. In the triple Dlx1/2; Nrp2KO, partial restoration of tangential migration of GABAergic interneurons from basal ganglia to the neocortex was successfully established signifying the importance of DLX regulation of Semaphorin-Neuropilin signalling during forebrain development.

No description available.

Le gène SYNGAP1 code pour la protéine Synaptic Ras GTPase-Activating protein 1 et est essentiel pour le développement normal de la fonction synaptique et de la cognition. Les mutations dans le gène SYNGAP1 qui provoquent la perte d'une seule copie du gène (haplo-insuffisance) sont associées à un handicap intellectuel, comorbide avec un trouble du spectre autistique et l'épilepsie. Les individus présentant des mutations SYNGAP1 montrent un large éventail de caractéristiques phénotypiques telles que l'encéphalopathie épileptique, des déficits moteurs, des déficits sensoriels et d'autres anomalies comportementales et cognitives. De manière intéressante, les modèles de souris transgéniques Syngap1 haplo-insuffisantes reproduisent les déficits comportementaux et cognitifs observés chez les individus SYNGAP1. Plusieurs études se sont concentrées sur le rôle de Syngap1 dans les synapses glutamatergiques, révélant qu'il est un régulateur négatif de Ras, impliqué dans le trafic des récepteurs AMPA au niveau de la membrane postsynaptique des neurones excitateurs. Syngap1 est fortement impliqué dans la maturation des épines dendritiques et dans la régulation de la plasticité synaptique et de l'homéostasie neuronale. Toutefois, le rôle de Syngap1 dans les neurones GABAergiques est moins bien exploré. Les interneurones GABAergiques forment une population hétérogène, les sous-types dominants étant les interneurones exprimant la Parvalbumine (PV) et la Somatostatine (SST) dérivés de l'éminence ganglionnaire médiane (MGE). Des études récentes ont révélé le rôle multifacette de Syngap1 dans les interneurones GABAergiques, notamment son implication dans la migration des interneurones, le branchement axonal des cellules PV et la régulation des synapses inhibitrices sur les somas postsynaptiques. Cependant, si et comment Syngap1 affecte les types cellulaires spécifiques d’interneurones dérivés de MGE tels que les interneurones PV et/ou SST n’est pas connu, et cela est exploré dans ma thèse. De plus, nous avons exploré si et comment l'haplo-insuffisance de Syngap1 induite pré- ou post-natalement spécifiquement dans les sous-types d'interneurones PV et SST contribue aux modalités comportementales, cognitives et sensorielles chez les souris adultes. Des stratégies génétiques ont été utilisées pour induire l'haplo-insuffisance de Syngap1, 1. pré-natalement dans les cellules PV et SST en utilisant la lignée de souris Nkx2.1_Cre, 2. pré-natalement dans les cellules SST en utilisant la ligne de souris SST_Cre et 3. post-natalement dans les cellules PV en utilisant la lignée de souris PV_Cre. Nous avons constaté que la réduction de Syngap1 pré-natalement dans les cellules PV et SST (en utilisant le promoteur Nkx2.1 pour cibler les interneurones dérivés de MGE) influence le traitement sensoriel auditif, en augmentant notamment les oscillations gamma de base, en affectant l'entraînement auditif et en échouant à s'habituer aux sons répétitifs. De plus, ces souris présentent des déficits de comportement social et une flexibilité cognitive altérée dans le comportement d'extinction de la peur. De telles altérations du traitement sensoriel, ainsi que des déficits comportementaux et cognitifs, n'ont pas été observés observés lorsque Syngap1 a été supprimé dans les cellules SST prénatales (en utilisant le promoteur SST). La suppression postnatale de Syngap1 dans les cellules PV montre quant à elle une habituation auditive accrue. Cependant, ces souris transgéniques ne présentent aucun déficit de comportement social ou d'extinction de la peur. Ces résultats suggèrent que les cellules PV pré- et/ou péri-natales sont particulièrement vulnérables à l'haplo-insuffisance de Syngap1 pendant une fenêtre temporelle sensible précoce lors du développement cérébral chez la souris. Alors que des modèles de souris conditionnelles spécifiques aident à comprendre la fonction biologique fondamentale de Syngap1, ils n'englobent pas la complexité du trouble génétique causé par SYNGAP1-ID. Nous avons donc étendu notre étude pour comprendre si les cellules PV sont altérées dans un modèle murin d'haplo-insuffisance germinale de Syngap1. En raison de leur innervation unique du soma et des dendrites proximales de leurs cibles postsynaptiques, les cellules PV influencent fortement l'activité du réseau et sont impliquées dans des fonctions cognitives supérieures telles que l'attention sélective, la mémoire de travail et la flexibilité cognitive, en particulier dans le cortex préfrontal (PFC). Nous avons étudié la connectivité synaptique des cellules PV et avons constaté qu'elles reçoivent des entrées excitatrices réduites dans les cortex préfrontal et auditif adultes. En parallèle, nous avons montré une connectivité réduite des cellules PV sur les cellules excitatrices avec moins de recrutement dans le PFC des souris adultes. Les souris transgéniques germinales présentent également des déficits de flexibilité cognitive (comme dans le comportement d'extinction de la peur) et dans l'apprentissage de la peur contextuelle. Ces résultats suggèrent un déséquilibre global entre l'excitation et l'inhibition dû à des altérations dans la connectivité des cellules PV. Nos études explorent donc le rôle de Syngap1 dans des lignées transgéniques haplo-insuffisantes conditionnelles et germinales en se concentrant sur des types cellulaires GABAergiques distincts (cellules PV et/ou SST), et montrent que les déficits des cellules PV, pendant une fenêtre de développement précoce, sont un facteur prédominant contribuant à la physiopathologie sous-jacente des mutations de Syngap1. Une meilleure compréhension du rôle de Syngap1 dans différents types cellulaires et stades de développement aidera à concevoir des stratégies d'intervention thérapeutique optimales. / SYNGAP1 gene encodes for the Synaptic Ras GTPase-Activating protein 1, and is critical for the normal development of synaptic function and cognition. Mutations in SYNGAP1 gene that cause loss of single copy of the gene (haploinsufficiency) are associated with intellectual disability, comorbid with autism spectrum disorder and epilepsy. Individuals with SYNGAP1 mutations show a broad spectrum of phenotypic features such as epileptic encephalopathy, motor deficits, sensory deficits and other behavioral and cognitive abnormalities. Interestingly, transgenic Syngap1 haploinsufficient mouse models phenocopy the behavioral and cognitive deficits as in SYNGAP1 individuals. Several studies have focused on the role of Syngap1 in glutamatergic synapses and revealed it to be a negative regulator of Ras, involved in the trafficking of AMPA receptors at the postsynaptic membrane of excitatory neurons. Syngap1 is strongly implicated in dendritic spine maturation and in regulating synaptic plasticity and neuronal homeostasis. The role of Syngap1 in GABAergic neurons however is less well explored. GABAergic interneurons form a heterogenous population, the dominant subtypes being the Parvalbumin (PV) and Somatostatin (SST) expressing interneurons derived from Medial Ganglionic Eminence (MGE). Recent studies have divulged the multi-faceted role of Syngap1 in GABAergic interneurons such as its involvement with interneuron migration, PV cell axonal branching, and regulation of inhibitory synapses onto postsynaptic somata. However, whether and how Syngap1 affects specific MGE-derived interneuron cell types such as PV and/or SST interneurons is unknown and explored in my thesis. Further, we explored whether and how Syngap1 haploinsufficiency induced either pre- or postnatally specifically in PV and SST interneuron subtypes, contributes to behavioral, cognitive and sensory related modalities in adult mice. Genetic strategies were used to induce Syngap1 haploinsufficiency, 1. prenatally in PV and SST cells using the Nkx2.1_Cre driver line, 2. prenatally in SST cells using SST_Cre driver line and 3. postnatally in PV cells using the PV_Cre driver line. We found that reduction of Syngap1 prenatally in both PV and SST cells (using the Nkx2.1 promoter to target MGE-derived interneurons) influences auditory sensory processing, in particular increasing the baseline gamma oscillations, affecting auditory entrainment and failing to habituate to repetitive sounds. In addition, these mice show deficits in social behavior and impaired cognitive flexibility in fear extinction behavior. Such sensory processing alterations, as well as behavioral and cognitive deficits were not observed when Syngap1 was deleted in prenatal SST cells (using the SST promoter). Postnatal deletion of Syngap1 in PV cells in turn showed increased auditory habituation, however these transgenic mice show no deficits in either social or fear extinction behavior. These results suggest that pre- and/or perinatal PV cells are particularly vulnerable to Syngap1 haploinsufficiency at an early sensitive time window during mouse brain development. While specific conditional mouse models help in understanding the fundamental biological function of the Syngap1, they do not encompass the complexity of the genetic disorder caused in SYNGAP1-ID. We therefore extended our study to understand whether PV cells are altered in a mouse model of germline Syngap1 haploinsufficiency. Due to their unique innervation of the soma and proximal dendrites of their postsynaptic targets, PV cells strongly influence network activity and are involved in higher cognitive functions such as selective attention, working memory and cognitive flexibility particularly in the prefrontal cortex (PFC). We investigated PV cell synaptic connectivity and found that they receive reduced excitatory inputs in the adult prefrontal and auditory cortex. In parallel, we showed reduced PV connectivity onto excitatory cells with less recruitment in the PFC of adult mice. The germline transgenic mice also showed deficits in cognitive flexibility (such as in fear extinction behavior) and cued contextual fear conditioning. These results suggest an overall E/I imbalance due to alterations in PV cell connectivity. Our studies therefore explore the role of Syngap1 in both conditional and germline haploinsufficient transgenic lines focusing on distinct GABAergic cell types (PV and/or SST cells), and shows that PV cell deficits, during an early developmental window, is a predominant contributing factor to the pathophysiology underlying Syngap1 mutations. A better understanding of the role of Syngap1 in different cell types and developmental stages will help in designing optimal therapeutic intervention strategies.

No description available.