Alcohol represents the third leading cause of preventable death in the United States. Yet, despite its prevalent role in impeding human health, there is much to understand about how it elicits its effects on the body and how the body and brain change when an individual becomes physiologically dependent upon alcohol. The work presented herein represents an effort to elucidate the acute and chronic effects of alcohol on the nervous system. We investigate two specific protein pathways and their role in alcohol's effects on the body. The first begins with brain-derived neurotrophic factor (BDNF), which acts on TrkB, and ends with KCC2. We demonstrate that BDNF expression is increased in the VTA during withdrawal from chronic but not acute alcohol exposure and that this increase persists for at least seven days. Concomitantly, we demonstrate that the activation of GABAA channels on produces less inhibition of VTA GABA neurons in mice treated with chronic intermittent ethanol exposure than in alcohol naïve mice. This effect likewise persisted for at least seven days. We illustrate that BDNF has no apparent direct effect on VTA GABA neuron firing rate. The second pathway begins with the T cell marker CD5 and ends with the anti-inflammatory cytokine, IL-10. We demonstrate that in a genetic CD5 knockout (CD5 KO) mouse model both alcohol consumption as well as the sedative properties of alcohol are reduced. Since CD+ B cells secrete more IL-10 than CD5- B cells, we also demonstrate the effects of IL-10 on VTA neurons. We show that IL-10 has direct effects on VTA dopamine (DA) neurons by increasing their firing activity. We relatedly illustrate that IL-10 produces an increase in DA release in the nucleus accumbens (NAc). However, contrary to our hypotheses, we show that IL-10 produces conditioned place aversion rather than conditioned place preference in a place conditioning paradigm, suggesting that IL-10 might mediate pain-induced secretions of DA. Collectively, these results suggest two potential therapeutic targets to reduce alcohol consumption that need further validation. They also suggest a novel mechanism for the sedative effects of alcohol at moderate and high doses.

Alcohol represents the third leading cause of preventable death in the United States. Yet, despite its prevalent role in impeding human health, there is much to understand about how it elicits its effects on the body and how the body and brain change when an individual becomes physiologically dependent upon alcohol. The work presented herein represents an effort to elucidate the acute and chronic effects of alcohol on the nervous system. We investigate two specific protein pathways and their role in alcohol’s effects on the body. The first begins with brain-derived neurotrophic factor (BDNF), which acts on TrkB, and ends with KCC2. We demonstrate that BDNF expression is increased in the VTA during withdrawal from chronic but not acute alcohol exposure and that this increase persists for at least seven days. Concomitantly, we demonstrate that the activation of GABAA channels on produces less inhibition of VTA GABA neurons in mice treated with chronic intermittent ethanol exposure than in alcohol naïve mice. This effect likewise persisted for at least seven days. We illustrate that BDNF has no apparent direct effect on VTA GABA neuron firing rate. The second pathway begins with the T cell marker CD5 and ends with the anti-inflammatory cytokine, IL-10. We demonstrate that in a genetic CD5 knockout (CD5 KO) mouse model both alcohol consumption as well as the sedative properties of alcohol are reduced. Since CD+ B cells secrete more IL-10 than CD5- B cells, we also demonstrate the effects of IL-10 on VTA neurons. We show that IL-10 has direct effects on VTA dopamine (DA) neurons by increasing their firing activity. We relatedly illustrate that IL-10 produces an increase in DA release in the nucleus accumbens (NAc). However, contrary to our hypotheses, we show that IL-10 produces conditioned place aversion rather than conditioned place preference in a place conditioning paradigm, suggesting that IL-10 might mediate pain-induced secretions of DA. Collectively, these results suggest two potential therapeutic targets to reduce alcohol consumption that need further validation. They also suggest a novel mechanism for the sedative effects of alcohol at moderate and high doses.

Initialement découvert au cerveau, le Brain-derived neutrophic factor (BDNF) est un facteur de croissance dont les mécanismes de relâche et la signalisation ont été bien étudiés dans le système nerveux central. Il est aussi retrouvé en concentrations importantes dans la circulation où il est emmagasiné dans les plaquettes avec des niveaux pouvant atteindre 100 à 1000 fois ceux des neurones. Malgré l’abondance du BDNF dans les plaquettes, sa fonction dans la physiologie plaquettaire n’a jamais été étudiée. Le but de ce projet était donc d’investiguer le rôle du BDNF dans la fonction plaquettaire et les mécanismes de signalisation impliqués dans la réponse plaquettaire au BDNF. Lorsque les plaquettes sont isolées et re-suspendues dans un tampon physiologique dépourvu de protéines plasmatiques, le BDNF induit une agrégation plaquettaire complète et biphasique qui dépend des voies secondaires de l’agrégation. La neurotrophine NT4 ainsi qu’un anticorps activateur du récepteur TrkB ont tous les deux induit une agrégation plaquettaire similaire à celle du BDNF suggérant un récepteur commun, le TrkB. Par immunobuvardage, cytométrie en flux et microscopie électronique, nous avons pu confirmer que les plaquettes expriment une forme tronquée du récepteur TrkB, au niveau intracellulaire et à leur surface. Les tests fonctionnels nous ont mené à impliquer les voies de rhoGTPase Rac1, la protéine kinase C (PKC) et la voie phosphoinositide 3-kinase (PI3K)/Akt dans l’agrégation plaquettaire induite par le BDNF. Une fois activées par le BDNF, les plaquettes relâchent plusieurs cytokines proinflammatoires et proangiogéniques qui peuvent jouer un rôle important dans le maintien et la réparation de l’intégrité vasculaire. Parmi les agents relâchés, on retrouve des facteurs de croissances comme le PDGF et le VEGF, mais aussi des chimiokines comme l’IL8 et ENA-78. Finalement, lorsque les expériences d’agrégation ont été répétées en plasma riche en plaquettes, l’effet pro-agrégant du BDNF était perdu, possiblement via une liaison de BDNF avec la protéine plasmatique α2-macroglobuline (α2M). Cette liaison à α2M, suggérée par des expériences de co-immunoprécipitation, réduit la biodisponibilité du BDNF et pourrait aider à contenir la réponse plaquettaire au BDNF aux sites de lésions vasculaires. / The Brain-Derived Neutrophic Factor (BDNF) is a growth factor that was initially discovered in the brain. BDNF has both an autocrine and a paracrine role in neurons and its release and signaling mechanisms have been extensively studied in the central nervous system. Surprisingly, large quantities of BDNF have been reported in circulation, where it is essentially stored in platelets with concentrations reaching 100-1000-fold those of neurons. Despite this abundance, the function of BDNF in platelet biology has not been explored. Thus, this project sought to investigate the effect of BDNF on platelet function and the mechanisms underlying platelet responses to BDNF. In washed platelets, BDNF induced complete biphasic platelet aggregation that in part relied on amplification from secondary mediators. The low-affinity agonist neurotrophin-4 and an activating antibody raised against the canonical BDNF receptor TrkB induced similar platelet responses, implicating TrkB. Platelets express, both at their surface and in their intracellular compartment, a truncated form of TrkB lacking a tyrosine kinase domain. The BDNF-induced aggregation of washed platelets was prevented by inhibitors of the Rac1, PKC, and PI3K/Akt. Platelets exposed to BDNF secreted pro-angiogenic and pro-inflammatory cytokines, which may play a role in maintaining vascular homeostasis. Finally, in platelet-rich plasma, exogenous BDNF failed to induce aggregation and BDNF immunoprecipitates contained α2-macroglobulin immunoreactivity. Hence, platelets are rich in BDNF, which induce platelet aggregation via TrkB activation. The restriction of BDNF bioavailablility by plasma protein binding may serve to target BDNF-mediated platelet activation to sites of vascular injury.

Basal forebrain cholinergic neurons (BFCNs) are critical for learning and memory. Profound and early BFCN degeneration is a hallmark of aging and Alzheimer’s disease (AD). BFCNs depend for their survival on the retrograde axonal transport of neurotrophins, proteins critical for neuronal function. Neurotrophins like brain derived neurotrophic factor (BDNF) and pro-nerve growth factor (proNGF) are retrogradely transported to BFCNs from their synaptic targets. In AD, neurotrophin levels are increased within BFCN target areas and reduced in the basal forebrain, implicating dysfunctional neurotrophin transport in AD pathogenesis. However, neurotrophin transport within this highly susceptible neuronal population is currently poorly understood. We began by establishing protocols for the accurate quantification of axonal transport in BFCNs using microfluidic culture. We then determined the effect of age on neurotrophin transport. BFCNs were left in culture for up to 3 weeks to model aging in vitro. BFCNs initially displayed robust neurotrophin transport, which diminished with in vitro age. We observed that the levels of proNGF receptor tropomyosin-related kinase-A (TrkA) were reduced in aged neurons. Additionally, neurotrophin transport in BFCNs derived from 3xTg-AD mice, an AD model, was also impaired. Next, we sought to determine a mechanism for these transport deficits. First, we determined that proNGF transport was solely contingent upon the levels of TrkA. We then found that elevation of oxidative stress, an established AD contributor, significantly reduced both TrkA levels and proNGF retrograde transport. TrkA levels are partially regulated by protein tyrosine phosphatase-1B (PTP1B), an enzyme whose activity is reduced by oxidation. PTP1B antagonism significantly reduced TrkA levels and proNGF retrograde transport in BFCNs. Treatment of BFCNs with PTP1B-activating antioxidants rescued TrkA levels, proNGF transport, and proNGF-mediated axonal degeneration. Our results suggest that oxidative stress contributes to BFCN degeneration in aging and AD by impairing retrograde neurotrophin transport via oxidative PTP1B-mediated TrkA loss. / Thesis / Doctor of Philosophy (PhD) / During aging and Alzheimer’s disease (AD), the connections between neurons, a type of brain cell, break down, causing memory loss. This breakdown begins in a brain area called the basal forebrain. Basal forebrain neurons rely upon the transport of nutrients along their connections with other neurons, called axons, for proper function. This transport process becomes impaired in AD. Our goal was to understand why this happens. First, we determined that axonal transport was impaired with age and in basal forebrain neurons of mice genetically predisposed to develop AD. We recreated these impairments by increasing the levels of harmful molecules called reactive oxidative species (ROS). ROS levels increase with age and become abnormally high during AD. We found that increased ROS impair axonal transport and contribute to the breakdown of basal forebrain neurons. Our work suggests that reducing ROS will help prevent the breakdown of basal forebrain neurons in AD.

No description available.

BDNF/TrkB são descritos em diversas neoplasias onde iniciam sinais mitogênicos, facilitam o crescimento tumoral, previnem apoptose e regulam angiogênese e metástase. Outros fatores de crescimento também são importantes para tumorigênese, como GRP/GRPR e EGF/EGFR. O objetivo geral deste trabalho foi investigar o papel de BDNF/TrkB em câncer colorretal avaliando possíveis interações com GRPR e EGFR. Verificamos que BDNF e seu receptor, TrkB, estão presentes em amostras de pacientes com câncer colorretal esporádico, e os níveis de BDNF encontram-se mais elevados no tecido neoplásico que no tecido adjacente ao tumor. O tratamento com RC- 3095, um antagonista de GRPR, na linhagem celular de câncer colorretal humana, HT-29, causa diminuição nos níveis de NGF secretados pelas células e aumento de BDNF em relação ao controle não tratado. RC-3095 inibe a proliferação e viabilidade celular das linhagens HT-29 (EGFR positiva) e SW-620 (EGFR negativa), embora apenas em HT-29 ocorra um aumento significativo na expressão de mRNA de BDNF. Por isso, um anticorpo monoclonal anti-EGFR, cetuximabe, foi combinado a RC-3095, nas células HT-29, sendo capaz de prevenir tal aumento, sugerindo que este efeito seja mediado por EGFR. Os tratamentos com um inibidor de Trks, K252a (1000 nM) ou com cetuximabe (10 nM) também inibem a proliferação celular. Entretanto, a combinação de BDNF a cetuximabe previne este efeito, enquanto que a combinação de doses não efetivas de K252a (10 nM) à cetuximabe (1 nM) inibe a proliferação celular de HT- 29. Além disso, cetuximabe também causa aumento na expressão de mRNA de TrkB e BDNF, após 600 minutos de tratamento. Nossos resultados sugerem que a inibição da proliferação celular in vitro ou do crescimento tumoral in vivo devem acontecer através do bloqueio combinado entre GRPR e TrkB em células de câncer colorretal EGFR positivas, e que BDNF também esteja envolvido em mecanismos de resistência a fármacos. Por isso, o bloqueio de BDNF / TrkB pode emergir como potencial alvo antitumoral. / BDNF / TrkB are described in various cancers where they participate in tumor growth, apoptosis, angiogenesis and metastasis. Furthermore, other growth factors are also important to tumorigenesis as GRP/GRPR and EGF/EGFR. Therefore, the aim of this study was to investigate the role of BDNF/TrkB in colorectal cancer evaluating the interactions with GRPR and EGFR. We found that BDNF and its receptor, TrkB, are present in samples from patients diagnosed with sporadic colorectal cancer, and BDNF levels were higher in tumor tissue compared to adjacent tumor tissue. Treatment with RC-3095, GRPR antagonist, in human colorectal cancer cell line, HT-29 caused a decrease in NGF levels secreted by cells, and generated increase of BDNF when compared to untreated control. RC-3095 inhibited the proliferation and cell viability in HT-29 (EGFR positive) and SW-620 (EGFR negative), but only HT-29 cells showed a significant increase in BDNF mRNA expression. Therefore, a monoclonal anti-EGFR antibody, cetuximab was combined with RC-3095 in HT-29 cells, and was able to prevent such an increase, suggesting that this effect is mediated by EGFR. The treatment with a Trk inhibitor, K252a (1000 nM) or cetuximab (10 nM), inhibited cell proliferation. However, the combination of BDNF with cetuximab prevented this effect, whereas the combination of ineffective doses of K252a (10 nM) with cetuximab (1 nM) still inhibited cell proliferation of HT-29. Furthermore, cetuximab also caused an increase in BDNF and TrkB mRNA expression, 600 minutes after treatment. In summary, our results suggest that inhibition of cell proliferation in vitro or tumor growth in vivo must occur between the combination of GRPR and TrkB in EGFR positive colorectal cancer cells, and that BDNF is also involved in drug resistance mechanisms. Therefore, blockage of BDNF / TrkB may emerge as potential antitumor target.

BDNF/TrkB são descritos em diversas neoplasias onde iniciam sinais mitogênicos, facilitam o crescimento tumoral, previnem apoptose e regulam angiogênese e metástase. Outros fatores de crescimento também são importantes para tumorigênese, como GRP/GRPR e EGF/EGFR. O objetivo geral deste trabalho foi investigar o papel de BDNF/TrkB em câncer colorretal avaliando possíveis interações com GRPR e EGFR. Verificamos que BDNF e seu receptor, TrkB, estão presentes em amostras de pacientes com câncer colorretal esporádico, e os níveis de BDNF encontram-se mais elevados no tecido neoplásico que no tecido adjacente ao tumor. O tratamento com RC- 3095, um antagonista de GRPR, na linhagem celular de câncer colorretal humana, HT-29, causa diminuição nos níveis de NGF secretados pelas células e aumento de BDNF em relação ao controle não tratado. RC-3095 inibe a proliferação e viabilidade celular das linhagens HT-29 (EGFR positiva) e SW-620 (EGFR negativa), embora apenas em HT-29 ocorra um aumento significativo na expressão de mRNA de BDNF. Por isso, um anticorpo monoclonal anti-EGFR, cetuximabe, foi combinado a RC-3095, nas células HT-29, sendo capaz de prevenir tal aumento, sugerindo que este efeito seja mediado por EGFR. Os tratamentos com um inibidor de Trks, K252a (1000 nM) ou com cetuximabe (10 nM) também inibem a proliferação celular. Entretanto, a combinação de BDNF a cetuximabe previne este efeito, enquanto que a combinação de doses não efetivas de K252a (10 nM) à cetuximabe (1 nM) inibe a proliferação celular de HT- 29. Além disso, cetuximabe também causa aumento na expressão de mRNA de TrkB e BDNF, após 600 minutos de tratamento. Nossos resultados sugerem que a inibição da proliferação celular in vitro ou do crescimento tumoral in vivo devem acontecer através do bloqueio combinado entre GRPR e TrkB em células de câncer colorretal EGFR positivas, e que BDNF também esteja envolvido em mecanismos de resistência a fármacos. Por isso, o bloqueio de BDNF / TrkB pode emergir como potencial alvo antitumoral. / BDNF / TrkB are described in various cancers where they participate in tumor growth, apoptosis, angiogenesis and metastasis. Furthermore, other growth factors are also important to tumorigenesis as GRP/GRPR and EGF/EGFR. Therefore, the aim of this study was to investigate the role of BDNF/TrkB in colorectal cancer evaluating the interactions with GRPR and EGFR. We found that BDNF and its receptor, TrkB, are present in samples from patients diagnosed with sporadic colorectal cancer, and BDNF levels were higher in tumor tissue compared to adjacent tumor tissue. Treatment with RC-3095, GRPR antagonist, in human colorectal cancer cell line, HT-29 caused a decrease in NGF levels secreted by cells, and generated increase of BDNF when compared to untreated control. RC-3095 inhibited the proliferation and cell viability in HT-29 (EGFR positive) and SW-620 (EGFR negative), but only HT-29 cells showed a significant increase in BDNF mRNA expression. Therefore, a monoclonal anti-EGFR antibody, cetuximab was combined with RC-3095 in HT-29 cells, and was able to prevent such an increase, suggesting that this effect is mediated by EGFR. The treatment with a Trk inhibitor, K252a (1000 nM) or cetuximab (10 nM), inhibited cell proliferation. However, the combination of BDNF with cetuximab prevented this effect, whereas the combination of ineffective doses of K252a (10 nM) with cetuximab (1 nM) still inhibited cell proliferation of HT-29. Furthermore, cetuximab also caused an increase in BDNF and TrkB mRNA expression, 600 minutes after treatment. In summary, our results suggest that inhibition of cell proliferation in vitro or tumor growth in vivo must occur between the combination of GRPR and TrkB in EGFR positive colorectal cancer cells, and that BDNF is also involved in drug resistance mechanisms. Therefore, blockage of BDNF / TrkB may emerge as potential antitumor target.

BDNF/TrkB são descritos em diversas neoplasias onde iniciam sinais mitogênicos, facilitam o crescimento tumoral, previnem apoptose e regulam angiogênese e metástase. Outros fatores de crescimento também são importantes para tumorigênese, como GRP/GRPR e EGF/EGFR. O objetivo geral deste trabalho foi investigar o papel de BDNF/TrkB em câncer colorretal avaliando possíveis interações com GRPR e EGFR. Verificamos que BDNF e seu receptor, TrkB, estão presentes em amostras de pacientes com câncer colorretal esporádico, e os níveis de BDNF encontram-se mais elevados no tecido neoplásico que no tecido adjacente ao tumor. O tratamento com RC- 3095, um antagonista de GRPR, na linhagem celular de câncer colorretal humana, HT-29, causa diminuição nos níveis de NGF secretados pelas células e aumento de BDNF em relação ao controle não tratado. RC-3095 inibe a proliferação e viabilidade celular das linhagens HT-29 (EGFR positiva) e SW-620 (EGFR negativa), embora apenas em HT-29 ocorra um aumento significativo na expressão de mRNA de BDNF. Por isso, um anticorpo monoclonal anti-EGFR, cetuximabe, foi combinado a RC-3095, nas células HT-29, sendo capaz de prevenir tal aumento, sugerindo que este efeito seja mediado por EGFR. Os tratamentos com um inibidor de Trks, K252a (1000 nM) ou com cetuximabe (10 nM) também inibem a proliferação celular. Entretanto, a combinação de BDNF a cetuximabe previne este efeito, enquanto que a combinação de doses não efetivas de K252a (10 nM) à cetuximabe (1 nM) inibe a proliferação celular de HT- 29. Além disso, cetuximabe também causa aumento na expressão de mRNA de TrkB e BDNF, após 600 minutos de tratamento. Nossos resultados sugerem que a inibição da proliferação celular in vitro ou do crescimento tumoral in vivo devem acontecer através do bloqueio combinado entre GRPR e TrkB em células de câncer colorretal EGFR positivas, e que BDNF também esteja envolvido em mecanismos de resistência a fármacos. Por isso, o bloqueio de BDNF / TrkB pode emergir como potencial alvo antitumoral. / BDNF / TrkB are described in various cancers where they participate in tumor growth, apoptosis, angiogenesis and metastasis. Furthermore, other growth factors are also important to tumorigenesis as GRP/GRPR and EGF/EGFR. Therefore, the aim of this study was to investigate the role of BDNF/TrkB in colorectal cancer evaluating the interactions with GRPR and EGFR. We found that BDNF and its receptor, TrkB, are present in samples from patients diagnosed with sporadic colorectal cancer, and BDNF levels were higher in tumor tissue compared to adjacent tumor tissue. Treatment with RC-3095, GRPR antagonist, in human colorectal cancer cell line, HT-29 caused a decrease in NGF levels secreted by cells, and generated increase of BDNF when compared to untreated control. RC-3095 inhibited the proliferation and cell viability in HT-29 (EGFR positive) and SW-620 (EGFR negative), but only HT-29 cells showed a significant increase in BDNF mRNA expression. Therefore, a monoclonal anti-EGFR antibody, cetuximab was combined with RC-3095 in HT-29 cells, and was able to prevent such an increase, suggesting that this effect is mediated by EGFR. The treatment with a Trk inhibitor, K252a (1000 nM) or cetuximab (10 nM), inhibited cell proliferation. However, the combination of BDNF with cetuximab prevented this effect, whereas the combination of ineffective doses of K252a (10 nM) with cetuximab (1 nM) still inhibited cell proliferation of HT-29. Furthermore, cetuximab also caused an increase in BDNF and TrkB mRNA expression, 600 minutes after treatment. In summary, our results suggest that inhibition of cell proliferation in vitro or tumor growth in vivo must occur between the combination of GRPR and TrkB in EGFR positive colorectal cancer cells, and that BDNF is also involved in drug resistance mechanisms. Therefore, blockage of BDNF / TrkB may emerge as potential antitumor target.

This research delves into idiopathic autism spectrum disorder (ASD), investigating the role of TrkB signaling pathways and BDNF regulation in the cortex. Additionally, it explores offering insights into maternal influences on mouse models. / Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by challenges in social interactions and repetitive behaviors. Prevalence of ASD is estimated to be 1 in 54 globally and is rising recently in many countries including Canada. ASD affects individuals differently, making diagnosis challenging. At present, no molecular diagnosis of ASD is available. Further, available medications only manage some symptoms of the disease and have adverse side effects in children. Therefore, there is a need for accurate molecular diagnostic tools to aid in molecular detection and treatment of ASD. To this end, a better understanding of the underlying molecular mechanisms that link ASD etiology to ASD-related behavior is crucial. While genetic factors contribute to syndromic ASD, most cases of ASD are idiopathic with unknown causes, influenced by a combination of epigenetic and environmental factors. TrkB and its downstream signaling pathways, such as Akt and Erk, are hyper-activated in syndromic ASD and hypo-activated in idiopathic cases. Therefore, drugs like rapamycin that inhibit the mTOR pathway downstream of TrkB are beneficial for syndromic ASD but not idiopathic cases. Additionally, insulin-like growth factor 1 (IGF-1), which mitigates ASD-related synaptic disruptions via Akt and Erk signaling, shows unchanged mRNA and protein levels along with its receptor in the idiopathic ASD fusiform gyrus. In ASD with either genetic or epigenetic/environmental causes, disruptions in synaptic connectivity are observed. Synaptic function is regulated by signaling pathways involving brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), as well as their downstream signaling cascades such as MAPK and Akt. The existing literature suggests that there is an association between BDNF and TrkB signaling pathways and ASD. However, a serious gap in knowledge about the precise molecular role of TrkB in ASD pathology is that our current understanding is correlational in nature and based on observational studies that lack causal experiments. This underscores the importance of further research to understand the causative role of TrkB and its related molecular events in idiopathic ASD. The present work aims to provide a deeper understanding about the causative role of molecular mechanisms underlying TrkB signaling in ASD. ASD mouse models exhibit behaviors and molecular features resembling those observed in human ASD. Therefore, these mouse models are helpful tools for studying ASD. However, understudied physiological confounding factors, such as maternal age and parity, can introduce biases and add to data variability, thus negatively impacting the reproducibility and translational value of ASD mouse models. To achieve a reliable mouse model of ASD, we conducted our first study that examines the impact of maternal age and parity on pregnancy complications, neurodevelopment, and social behavior in mice. Results demonstrate that older maternal age and prior motherhood interact to ensure a normal, steady developmental rate and provide protective effects against anxiety, social impairment, and olfactory deficits. Given the current lack of clarity regarding the causative impact of TrkB on ASD pathology, our subsequent investigation sought to establish a causal relationship between TrkB signaling and ASD. We used the TrkB agonist, LM22A-4 treatment in a validated ASD mouse model. Our results demonstrate that treatment with LM22A-4 effectively rescues the core symptoms associated with ASD (social impairment and repetitive behavior). These findings indicate that impaired TrkB signaling is responsible for ASD-like behavior of valproic acid (VPA)-exposed mice. However, unlike TrkB-related molecular events occurring in the fusiform gyrus of idiopathic ASD, TrkB isoform protein levels, BDNF species, Akt, and Erk total protein levels and activation remained unchanged in VPA-exposed cortices compared to healthy control mice. Since our VPA mouse model does not replicate human idiopathic ASD, our study cannot draw a conclusion on how disruptions in these signaling pathways may contribute to the development and manifestation of ASD symptoms. Cortex is responsible for various aspects of social behavior that are impaired in ASD. However, regulatory mechanisms that are involved in ASD upstream of cortical TrkB and BDNF are not well known. BDNF expression is highly cell-and tissue-specific and is regulated by different sets of transcription factors in specific tissues. While NURR1, the BDNF regulator in midbrain neurons, is associated with ASD pathology, its specific role in regulation of cortical BDNF is not yet well-established. Our third study aimed to understand the role of NURR1 in regulating BDNF specifically in the cortex. We showed that in resting and depolarized neurons, when NURR1 is knocked down, BDNF mRNA levels remained unchanged, suggesting that NURR1 does not regulate BDNF in cortical neurons and highlighting the tissue-specificity of BDNF regulation. In summary, we address the understudied effects of maternal factors on mouse models, which enhances the reliability of ASD research. Further, our studies significantly enhance the understanding of ASD by elucidating the role of TrkB and its downstream signaling pathways in the behavioral aspects of the disorder. We also contribute to the knowledge of BDNF regulation in the cortex, a brain tissue with crucial roles in various aspects of social behavior. In a forward-looking approach, the results of our studies provide valuable insights into mouse modeling of idiopathic ASD and the potential role of TrkB in ASD behavioral symptoms. / Thesis / Candidate in Philosophy / Autism spectrum disorder (ASD) is a condition that is accompanied by challenges in social interaction and repetitive behaviors. ASD is a complicated condition because we do not fully understand all the details of how it works in the body. Studying ASD is important as it is the most challenging condition in children and it is becoming more common, especially in the last two decades. While scientists are developing molecular tools to improve ASD diagnosis and understand its biology, these tools are not widely used in clinics for ASD diagnosis yet. Also, the approved medications available can only help with managing some of the behavioral symptoms like self-harming behavior. Despite the pressing need to find a solution, our recent advancements have not yet brought us closer to a cure for ASD, mainly because of the complexity of the disorder. Therefore, identifying the specific ASD-related mechanisms at the molecular level that contribute to ASD-related behaviors is crucial for gaining a deeper understanding of the disease. In ASD, there are problems with how brain cells communicate with each other. This communication is controlled by certain molecules in the brain, such as brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), along with other molecules. There is evidence suggesting a link between these molecules and ASD, but we have not fully understood their precise roles because most of the current knowledge is based on observations and correlations, rather than on establishing cause-and-effect relationships. To bridge this gap, our research focused on understanding TrkB's role in ASD. We required reliable mouse models. Since we aimed to induce ASD-like behaviors in mice using an ASD-causing chemical, it was crucial to ensure they were healthy beforehand. We needed to confirm that any social deficits or repetitive behaviors were not due to other factors, such as adverse infancy experiences or impaired interactions between mother and infant. We discovered that sexually mature dams aged between 3 to 6 months, with a history of previous pregnancies and motherhood, give birth to healthier litters. These litters can serve as a more dependable source for our animal behavioral studies. Many cases of ASD in humans are caused by non-genetic factors such as environmental influences like pesticides, air pollution, and the use of certain drugs during pregnancy. In cases of human ASD triggered by non-genetic factors, there is an increase in proBDNF, the precursor of BDNF. However, this proBDNF does not efficiently convert to BDNF. With insufficient BDNF and TrkB receptors, molecules like Akt (protein kinase B, also PKB) and Erk (Extracellular Signal-Regulated Kinase), which are crucial for neuron communication, are also less active downstream. This imbalance disrupts neuron connections, leading to ASD behaviors. In our research, the ASD-causing chemical which we used is valproic acid. It is originally an anti-seizure medication. When pregnant women took valproic acid, the chance of their child having ASD increased. Scientists used this information to inject pregnant mice with valproic acid, and as a result, all the offspring showed ASD-like behaviors. We anticipated that by isolating the brains of these offspring and measuring protein levels of BDNF, TrkB, Akt, and Erk, we would observe a similar pattern to that seen in humans with non-genetic ASD cases. We focused on studying the cortex, a region of the brain responsible for regulating social behaviors in both mice and humans. Since ASD is associated with challenges in social behaviors, we isolated the cortex from mouse brains to analyze protein levels. A chemical known as LM22A-4 with a structure resembling BDNF can bind to TrkB and activate it. We expected that the offspring of pregnant dams injected with valproic acid, which led to reduced TrkB axis activation in their brains, would show improvement in ASD behavior. This anticipation stems from the understanding that LM22A-4 activates the TrkB axis, thus compensating for its reduction, which is thought to be causing ASD-like behaviors. The offspring of mothers injected with valproic acid exhibited ASD-like behaviors, unlike the control mice. Control mice were offspring of pregnant dams injected with a solution containing only the substances used to dissolve valproic acid, typically water and salt (saline). Mice prenatally exposed to valproic acid (VPA) exhibited ASD-like behaviors, but treatment with LM22A-4 helped alleviate these behaviors, promoting more typical behavior patterns. LM22A-4, by activating TrkB receptors, helped to protect the brain from harm caused by exposure to valproic acid before birth. This could mean that valproic acid-induced changes in TrkB-related molecular mechanisms are involved in social behavior difficulties and increased repetitive behaviors seen in autism. Nevertheless, the levels of TrkB, BDNF, proBDNF, Akt, and Erk in the cortex of offspring from mothers injected with valproic acid were like those in the offspring from mothers injected with the saline solution. Therefore, the BDNF and TrkB signaling pathways remained unchanged in the cortex of our valproic acid model in this study, and they differ from those observed in human idiopathic ASD. We also speculated that a protein, called NURR1 acting upstream of BDNF and TrkB might be involved in the process. NURR1 acts as a regulatory protein that binds to the BDNF, increasing the production of copies from the BDNF. We also used a small RNA that targets a specific region in the Nurr1 and inhibits its protein production We anticipated a reduction in Nurr1 levels. As NURR1 acts as an upregulator of BDNF, lower levels of Nurr1 would result in decreased BDNF production. Activating NURR1 resulted in increased BDNF mRNA levels. However, when NURR1 was reduced, BDNF mRNA levels remained unaffected. This led us to conclude that if NURR1 levels decrease, other proteins may step in to maintain BDNF mRNA levels. Therefore, in the cortex, unlike in some other brain regions, the presence of NURR1 is not essential for regulating Bdnf. In summary, before inducing ASD-like behavior in mice using valproic acid, it is crucial to ensure the health of the mice. We used sexually mature mothers with prior pregnancy experience to provide a healthy baseline. We showed valproic acid induced ASD-like behaviors in mice offspring. We also observed that LM22A-4 treatment alleviated ASD-like behaviors of offspring. In our study, we demonstrated that the levels of BDNF, TrkB, Erk, and Akt proteins in the cortex of mice exposed to valproic acid were not affected. For this reason, our mouse model does not resemble human non-genetic ASD. Finally, NURR1's role in BDNF regulation varies by brain region. Lowering NURR1 did not affect BDNF mRNA levels, suggesting compensatory mechanisms. Our findings suggest new directions for further research to better understand the roles of TrkB and BDNF in non-genetic ASD. Overall, this study provides valuable knowledge that can contribute to advancing our understanding of idiopathic ASD-related molecular mechanisms.

Objectifs: Hepatocyte Growth Factor (HGF) améliore la régénération axonale et la survie des motoneurones lors du développement embryonnaire. Son rôle dans la régénération des nerfs périphériques lésés chez l’adulte n’a pas encore été étudié. Notre objectif est de déterminer l’expression de HGF dans la moelle épinière murine suite à une axotomie, avec ou sans stimulation électrique, directe ou transcutanée. Méthodes: Soixante souris C57BL/6 adultes ont été divisées en 5 groupes : Contrôle (n=12), Placebo (n=12), Axotomie (n=12, lacération et réparation immédiate du nerf sciatique), Directe (n=12, lacération, réparation immédiate et stimulation électrique directe proximale du nerf sciatique, 1h, 20 Hz) et Transcutanée (n=12, lacération, réparation immédiate et stimulation électrique transcutanée proximale du nerf sciatique, 1h, 20 Hz). Les moelles épinières ont été recueillies 1, 3, 7 et 14 jours suivant l’intervention. L’expression de HGF a été évaluée par technique d’hybridation in situ. Résultats: Nos résultats démontrent une augmentation de l’expression de HGF dans les moelles épinières murines suite à l’axotomie. Cette augmentation est plus rapide suite à la stimulation électrique, autant directe que transcutanée. L’expression de HGF devient localisée aux zones motrices de la moelle épinière murine dans les groupes Axotomie, Directe et Transcutanée. Conclusions: HGF, facteur neurotrophique impliqué de le développement et la survie des motoneurones, a une expression altérée suite à la lacération du nerf sciatique. Ceci suggère fortement qu’il participe aussi à la régénération des nerfs moteurs. De plus, l’expression plus rapide de HGF suite à la stimulation électrique suggère son implication dans l’augmentation de la régénération nerveuse. / Purpose: Hepatocyte Growth Factor (HGF) plays a role in promoting axonal growth and survival of motoneurons during embryonic development. This factor might also be important in directing the regeneration of adult motoneurons following laceration. We aim to identify the expression patterns of HGF following axotomy, with or without direct or transcutaneous electrical nerve stimulation in a mouse model. Methods: Sixty adult C57BL/6 mice were divided into 5 groups: Control (n=12), Sham (n=12), Axotomy (n=12, sciatic nerve laceration and immediate repair), Direct (n=12, sciatic nerve laceration, immediate repair and application of direct electrical stimulation on the proximal nerve end, 1h, 20 Hz) and Transcutaneous (n=12, nerve laceration and immediate repair followed by proximal transcutaneous electrical stimulation, 1h, 20 Hz). Spinal cords were harvested at 1, 3, 7 and 14 days post-surgery. The expression patterns of HGF were measured using in situ hybridization. Results: Our results showed an upregulation of HGF expression in mouse spinal cords following sciatic nerve axotomy. This occurred more quickly following electrical stimulation in both Direct and Transcutaneous groups. The expression pattern of HGF became localized to the motor neuron pools in the Axotomy, Direct and Transcutaneous groups. Conclusions: HGF, a growth factor involved in directing the outgrowth of motor axons in development, has an altered expression pattern following sciatic nerve laceration, suggesting it may also play a role in directing motoneuron regeneration. Furthermore, rapid change in the expression pattern of HGF following electrical stimulation suggests it may also be involved in the upregulation of nerve regeneration following electrical stimulation.