Alzheimer’s disease (AD) is a devastating neurodegenerative condition in which a patient’s cognitive functioning, memory, and physical health progressively deteriorate. In order to treat physiological deterioration in AD, a neuroprotective recombinant human- erythropoietin (EPO) fusion protein was used. In addition to its ability to target amyloid beta (Aβ) aggregation, EPO has been shown to reduce inflammation, oxidative stress and synaptic loss. Recombinant human-erythropoietin (EPO) was combined with a chimeric transferrin receptor (TfR) monoclonal antibody (cTfRMAb) to form a fusion protein (cTfRMAb-EPO) that is able to cross the blood-brain barrier (BBB) by binding to the TfR expressed on the luminal side of the BBB. Thirty eight male APPswePSEN1dE9 (APP/PS1) mice were separated into four treatment groups (wildtype (WT) treated with saline, APP/PS1 treated with saline (TG), APP/PS1 treated with cTfRMAb-EPO (cTfRMAb-EPO), and APP/PS1 treated with rHu-EPO alone (rhu-EPO)) and were subcutaneously injected with their respective treatments twice a week for six weeks. Recognition memory and locomotive behavior were tested through the novel object recognition (NOR) task and open field (OF) test when the mice were 8 months old and again at 11 months old (after 8 weeks of treatment) to determine treatment effects. Both behavioral tests demonstrated a clear age effect in mice between 8- and 11-months old. In the NOR task, no significant differences in recognition memory were observed in TG, cTfRMAb-EPO, or rHu-EPO groups. Lastly, the OF test demonstrated no significant behavioral differences among treatment groups.

Les protéines de la famille TET catalysent l'oxydation des 5-méthylcytosines (5mC) en 5-hydroxyméthylcytosines (5hmC) et jouent ainsi un rôle dans la régulation épigénétique de la transcription et dans le processus de déméthylation de l'ADN. Les interactions entre la méthylation de l'ADN et les autres marques épigénétiques sont encore mal connues.Des mutations inactivatrices du gène TET2 ont été décrites dans les hémopathies myéloïdes et lymphoïdes et l'inactivation conditionnelle (cKO) de ce gène évaluée chez la souris a permis d'identifier de multiples anomalies de l'hématopoïèse, ainsi que le développement tardif d'hémopathies myéloïdes. Cette latence importante suggère la nécessité d'évènements oncogéniques coopératifs pour la transformation hématopoïétique. Chez l'Homme, les mutations de TET2 sont observées en association avec de nombreuses autres mutations, et en particulier avec des mutations du gène DNMT3A impliqué dans la méthylation de novo des cytosines de l'ADN, et avec des mutations du gène EZH2 responsable de methylation de la lysine 27 de l'histone H3. Nous avons testé fonctionnellement ces associations en utilisant des modèles murins.L'utilisation d'un modèle de transplantation de moelle osseuse nous a permis d'identifier une coopération de l'inactivation de Tet2 et du mutant DNMT3AR882H dans la transformation des lignées myéloïdes et lymphoïde T, correspondant aux hémopathies humaines porteuses de ces mutations. Dans la transformation lymphoïde, nos données indiquent que la dérégulation de la méthylation entraine une surexpression du gène NOTCH1 et de l'activité de la voie de signalisation correspondante.L'analyse de souris invalidées de manière conditionnelle pour Tet2 et Ezh2 a montré que les souris correspondantes meurent d'aplasie médullaire, dont l'origine est imputée à la disparition de cellules souches hématopoïétiques capables de reconstituer l'hématopoïèse à long terme (LT-HSC). Ezh2 et Tet2 ont donc des rôles primordiaux dans le maintien de l'autorenouvellement des cellules souches hématopoïétiques, dont les mécanismes moléculaires, génétiques et épigénétiques restent à définir. / TET family proteins catalyzing the conversion of 5-methylcytosines (5mC) into 5-hydroxymethylcytosines (5hmC) are crucial for epigenetic regulation of transcription and for DNA demethylation. Interactions between DNA methylation and other epigenetic marks are not fully understood.TET2 inactivating mutations have been identified in both myeloid and lymphoid malignancies. The conditional inactivation (cKO) of this gene in mice highlights pleiotropic hematopoietic abnormalities as well as myeloid transformation at late stages. This latency suggest cooperativity between Tet2 and other oncogenic events during transformation. Human TET2 mutations are frequently found associated with other mutations, and more particularly with mutations in DNMT3A, involved in de novo methylation of cytosines and with mutations in EZH2, responsible for lysine 27 of histone H3 methylation. We decided to functionally assess these mutation associations in mice.Bone marrow transplantation of Tet2 inactivated and DNMT3AR882H mutated cells allowed us to identify myeloid and T-cell transformations, corresponding to human hematological disorders harboring these mutations. Our results on T-cell transformations clearly demonstrate that the deregulation of methylation leads to NOTCH1 overexpression and activation of the corresponding signaling pathway.Analyses of Tet2 and Ezh2 inactivated mice show that these Ezh2 Tet2 mice succumb to bone marrow exhaustion, attributed to long term hematopoietic stem cells (LT-HSC) disappearance. Ezh2 and Tet2 show major roles in LT-HSC maintenance whose molecular, genetic and epigenetic mechanism remains to be investigated.

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.