The contamination of seafood with methylmercury (MeHg) is a global health issue, as MeHg is a well known neurotoxin. Since dietary nutrients may interact with MeHg toxicity, and oxidative stress is one of the primary mechanisms underlying MeHg neurotoxicity, we characterized dietary antioxidant-MeHg interactions. Firstly, we used an ethnobotanical study to confirm the antioxidant activity of Northern Labrador Tea, Rhododendron tomentosum ssp. subarcticum (Tea), for the Canadian Inuit, a population with elevated MeHg exposure. Secondly, we determined the ability of Tea to ameliorate MeHg-induced toxicity in a rat perinatal exposure study. MeHg exposure (2 mg/KgBW/d) was associated with perturbed development and behaviour, elevated brain N-methyl-D-aspartate receptors, and serum lipid peroxidation. Surprisingly, Tea co-exposure (100 mg/KgBW/d) modulated MeHg’s effects on brain NMDA-R levels and lipid peroxidation, but also increased mercury serum concentrations. Thirdly, using a toxicogenomics approach we determined that MeHg exposure caused the down-regulation of Nr4a2 and its protein product Nurr1. These novel MeHg targets are implicated in developmental learning functions and were corrected with MeHg + Tea co-exposure. Lastly, we conducted a risk assessment survey and cross-sectional dietary epidemiology study in Costa Rica to further investigate dietary nutrient-MeHg interactions. Costa Rica is a Central American country with multiple sources of Hg and a high per capital fish consumption. Here, 5 of the 14 populations we studied exceeded the recommended MeHg provisional tolerable daily intake (pTDI) of 0.2 µg/KgBW/d. In Heredia the pTDI was exceeded by 34% of woman participants, primarily associated with canned tuna consumption. Interestingly, we detected that Hg body burden was significantly reduced by the consumption of antioxidant-rich dietary items. Considering our collective results, we hypothesized that MeHg toxicokinetics may be altered by dietary nutrients at the site of intestinal absorption from the disruption of gut flora, or at the site of cellular demethylation in tissues from the improvement of cellular redox state. The interaction of dietary nutrients on MeHg outcomes has a large impact on risk assessment and may provide a public health approach for managing the risk associated with MeHg exposure without reducing local fish consumption.

The contamination of seafood with methylmercury (MeHg) is a global health issue, as MeHg is a well known neurotoxin. Since dietary nutrients may interact with MeHg toxicity, and oxidative stress is one of the primary mechanisms underlying MeHg neurotoxicity, we characterized dietary antioxidant-MeHg interactions. Firstly, we used an ethnobotanical study to confirm the antioxidant activity of Northern Labrador Tea, Rhododendron tomentosum ssp. subarcticum (Tea), for the Canadian Inuit, a population with elevated MeHg exposure. Secondly, we determined the ability of Tea to ameliorate MeHg-induced toxicity in a rat perinatal exposure study. MeHg exposure (2 mg/KgBW/d) was associated with perturbed development and behaviour, elevated brain N-methyl-D-aspartate receptors, and serum lipid peroxidation. Surprisingly, Tea co-exposure (100 mg/KgBW/d) modulated MeHg’s effects on brain NMDA-R levels and lipid peroxidation, but also increased mercury serum concentrations. Thirdly, using a toxicogenomics approach we determined that MeHg exposure caused the down-regulation of Nr4a2 and its protein product Nurr1. These novel MeHg targets are implicated in developmental learning functions and were corrected with MeHg + Tea co-exposure. Lastly, we conducted a risk assessment survey and cross-sectional dietary epidemiology study in Costa Rica to further investigate dietary nutrient-MeHg interactions. Costa Rica is a Central American country with multiple sources of Hg and a high per capital fish consumption. Here, 5 of the 14 populations we studied exceeded the recommended MeHg provisional tolerable daily intake (pTDI) of 0.2 µg/KgBW/d. In Heredia the pTDI was exceeded by 34% of woman participants, primarily associated with canned tuna consumption. Interestingly, we detected that Hg body burden was significantly reduced by the consumption of antioxidant-rich dietary items. Considering our collective results, we hypothesized that MeHg toxicokinetics may be altered by dietary nutrients at the site of intestinal absorption from the disruption of gut flora, or at the site of cellular demethylation in tissues from the improvement of cellular redox state. The interaction of dietary nutrients on MeHg outcomes has a large impact on risk assessment and may provide a public health approach for managing the risk associated with MeHg exposure without reducing local fish consumption.

Afin de répondre correctement aux nombreux changements se produisant à chaque instant dans leur environnement, les cellules utilisent une panoplie de messagers moléculaires dont la synchronisation est essentielle à la signalisation cellulaire appropriée. La superfamille des récepteurs nucléaires compte quarante-huit membres impliqués dans ces processus de signalisation et influence ainsi plusieurs fonctions physiologiques. Les récepteurs nucléaires de la sous-famille NR4A composée de Nur77/NR4A1, Nurr1/NR4A2 et Nor1/NR4A3 sont des facteurs critiques du développement et de la maintenance du système nerveux. Nor1/NR4A3 en particulier est essentiel aux processus de guidage axonal et de survie neuronale au niveau de l’hippocampe. Les NR4A se démarquent des autres récepteurs nucléaires puisqu’ils sont considérés comme des récepteurs orphelins constitutivement actifs, ce qui veut dire qu’ils ne nécessitent pas d’interaction avec un ligand afin d’être activés. Il devient ainsi important d’identifier de nouveaux mécanismes de régulation pour cette sous-famille de récepteur afin d’améliorer notre compréhension et potentiellement contrôler leurs activités dans un contexte neuronal. L’activité des récepteurs nucléaires peut être régulée de plusieurs façons, indépendamment de leur association avec un ligand endogène. Les modifications post-traductionnelles représentent un aspect crucial de la signalisation cellulaire permettant de réguler la fonction des protéines cibles de manière spécifique au contexte. La SUMOylation et la phosphorylation sont des exemples de modifications post-traductionnelles avec le potentiel de réguler l’activité transcriptionnelle, la stabilité et l’expression des gènes cibles des récepteurs nucléaires. Dans cette thèse, l’impact de la SUMOylation retrouvée sur un motif consensus ainsi que sur un motif non-consensus de SUMOylation phosphorylé du récepteur Nor1 est étudié. Dans la première étude, un motif de SUMOylation non-consensus nouvellement découvert sur Nor1 est décrit. Ce nouveau motif nommé pSuM a été identifié pour la première fois sur le récepteur nucléaire des estrogènes ERβ et sur le récepteur farnésoÏde FXR. Nous avons identifié un motif pSuM situé à la lysine 137 de Nor1 qui sert de cible fonctionnelle de SUMO2. Le pSuM se démarque du motif de SUMOylation consensus puisqu’il nécessite une phosphorylation afin d’être SUMOylé. Dans le cas de Nor1, nos résultats démontrent que la sérine 139 est phosphorylée par la voie des MAPK. La SUMOylation sur ce site mène à une réduction de l’activité transcriptionnelle et du recrutement à la chromatine de Nor1 ainsi que de l’expression des gènes sensibles à Nor1. Une particularité intéressante du pSuM de Nor1 est qu’il possède également une extension phosphorylable par la kinase CK2 qui est essentielle au processus de SUMOylation. Cette extension a également un effet sur la stabilité et la compétence transcriptionnelle de Nor1. En utilisant des lignées SH-SY5Y exprimant de manière stable différents mutants SUMO de Nor1, il est démontré que la SUMOylation du pSuM diminue la prolifération et la survie cellulaire en réponse au stress oxydant. Dans la seconde étude, la SUMOylation de Nor1 sur un motif de SUMOylation canonique situé sur la Lysine 89 est caractérisée. Il est démontré que ce site de SUMOylation est ciblé principalement par SUMO1 et qu’il est important afin de maintenir une compétence transcriptionnelle et une stabilité optimale du récepteur. Cette SUMOylation régule également la prolifération et la survie en réponse à un traitement au nocodazole des lignées stables ainsi que la stabilité des microtubules. En conclusion, ces études identifient de nouveaux mécanismes de SUMOylation et phosphorylation utilisés dans la régulation de l'activité du récepteur nucléaire Nor1. Elles permettent également d’approfondir nos connaissances des rôles joués par Nor1 dans la neuroprotection en réponse au stress oxydant ainsi que dans la régulation de la stabilité du réseau de microtubules, ce qui apporte une nouvelle fonction de Nor1. Puisque Nor1 et les autres NR4A sont fortement impliqués dans la formation et maintenance du système nerveux et que les modifications post-traductionnelles peuvent réguler ces fonctions, la découverte et la caractérisation de nouveaux mécanismes de régulation de ces récepteurs ont le potentiel de nous fournir des nouvelles connaissances utiles dans le cadre des maladies neurodégénératives et autres conditions pathologiques. / To answer the many changes happening every instant in its surroundings, cells require a fine-tuned array of molecular messengers to carry on proper signal transduction and homeostasis. The superfamily of nuclear receptors contains forty-eight members implicated in a wide variety of cellular and physiological functions. The nuclear receptors of the NR4A subfamily containing Nur77/NR4A1, Nurr1/NR4A2 and Nor1/NR4A3 are heavily implicated in the development and maintenance of the nervous system. In particular, Nor1/NR4A3 has been shown to be essential for axonal guidance and neuronal survival in the hippocampus. This subfamily also operates differently from other nuclear receptors as they are considered constitutively active orphan nuclear receptors without known endogenous ligand. Therefore, there is an increasing need to identify critical mechanisms that regulate NR4A nuclear receptors and to better understand the control of their activities in a neuronal context. Nuclear receptor activity can be regulated in various ways independently of their interaction with an endogenous ligand. One is through post-translational modifications which allow the regulation of protein function depending on the cellular context. SUMOylation and phosphorylation are post-translational modifications with the potential to regulate nuclear receptor activity, stability and target gene expression. In this thesis, the impact of a canonical SUMOylation site and a phosphorylation dependant SUMOylation motif on the orphan nuclear receptor Nor1 are studied. In the first study, a newly identified non-canonical SUMOylation motif on Nor1 was described. This new motif named pSuM was first identified on the nuclear estrogen receptor ERβ and farnesoid X receptor FXR. We report that this pSuM is located at Lys-137 on Nor1 and is a target of SUMO2. The pSuM differs from traditional SUMOylation motif since it requires to be phosphorylated for SUMOylation to occur. For Nor1, our evidence showed that the obligate phosphorylation of the pSuM on Ser-139 occurred through the MAPK pathway. SUMOylation of Nor1 pSuM reduced Nor1 transcriptional competence, responsive gene expression and chromatin binding. Interestingly, the pSuM of Nor1 also possesses an extension phosphorylated by the CK2 kinase, which is essential to achieve the SUMOylation process. This extension also affected Nor1 protein stability and transcriptional activity. Using stable SH-SY5Y cell lines expressing different SUMO mutants of Nor1, we also showed that Nor1 pSuM SUMOylation reduced cell proliferation and survival to oxidative stress. In the second study, the SUMOylation of Nor1 on a canonical SUMOylation site found at Lys-89 was characterized. This SUMOylation site was found to be targeted mainly by SUMO1 and to be important in maintaining optimal transcriptional competency and stability of the receptor. This SUMOylation also regulated proliferation and survival to a nocodazole treatment of stable cell lines, as well as microtubule network stability. In conclusion, these studies provide novel mechanisms in the regulation of Nor1 activity by SUMOylation and phosphorylation. They also helped to expand our knowledge on the role played by Nor1 in neuroprotection in response to oxidative stress, as well as in the regulation of microtubule stability, which identified a new function of Nor1. Since Nor1 and other NR4A receptors are implicated in the formation and maintenance of the nervous system, the identification of post-translational modifications as a regulatory mechanism uncovers novel opportunities in our understanding of these receptors and provide new insights for neurodegenerative diseases and other neuropathological conditions.

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.