Neuromuscular synapse formation is driven by two nerve-derived molecules, agrin and neuregulin. Agrin is believed to signal through a Muscle Specific Kinase (MuSK)/Lrp4 receptor complex to cluster existing acetylcholine receptors (AChRs) in the postsynaptic membrane via a rapsyn-mediated mechanism, while neuregulin signals via ErbB kinase receptors to induce synaptic gene transcription. Recent studies suggest that neuregulin-ErbB signalling may also cross-signal to the agrin-MuSK pathway to modulate agrin’s ability to cluster AChRs. This thesis aimed to further elucidate this idea. Results of this thesis present two novel findings. First, there is a direct interaction between two tyrosine kinase signalling pathways at the neuromuscular synapse and second, neuregulin plays an important role in modulating, modifying and refining AChRs at developing synapses. Here I show that neuregulin can modulate two distinct processes. In the presence of agrin, neuregulin was able to potentiate both agrin-induced AChR clustering and agrin-induced AChR cluster dispersal, and this modulation by neuregulin occurred independently of any transcriptional mechanism. In vitro, I observed a marked effect by neuregulin on the number and size of AChR clusters that were induced by agrin. Treatment of myotubes for 4hrs with agrin and neuregulin led to a significant potentiation in agrin-induced AChR clustering compared to agrin treatment alone. Neuregulin on its own had no measurable effect on AChR clustering. When incubation times were much longer (12hrs), neuregulin promoted a further significant decrease in AChR cluster number compared to agrin treatment alone. Thus at 12hrs, rather than inhibit AChR clustering, as has been previously suggested, neuregulin promoted the dispersal of AChRs from pre-existing agrin-induced clusters. Follow-up in vivo studies into the potentiating ability of neuregulin in agrin-induced AChR clustering showed that the injection of exogenous neuregulin into developing mouse sternomastoid musculature led to an increase in the size of AChRs. Collectively, these data suggest interactions between the signalling pathways initiated by agrin and neuregulin. Subsequent investigation into the second messengers downstream of agrin-MuSK and neuregulin-ErbB signalling revealed that cyclin-dependent kinase 5 (Cdk5) and Shp2 played a role in neuregulin’s modulation of AChR cluster formation and dispersal. It appears that neuregulin enhances the phosphorylation status of MuSK by inhibiting the Shp2-dependent negative feedback loop on MuSK phosphorylation, thereby leading to an increase in AChR cluster numbers. By contrast, the way in which neuregulin disperses agrin-induced AChR clusters seems to occur partially, via a Cdk5 signalling-dependent mechanism. While it is accepted that neuregulin acts in a transcriptional manner during neuromuscular synapse formation, real-time PCR and immunoblot results suggest that transcriptional regulation was not involved in neuregulin’s modulation of agrin-induced AChR clustering.

Inflammation can be up-regulated by microglia and macrophages through the release of pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α). Excess production of TNF-α can lead to a variety of diseases and even tissue necrosis. Recently, the expression of alpha seven acetylcholine receptors (α7AChR) by microglia have been shown to decrease the amount of TNF-α released. This anti-inflammatory pathway has been studied extensively where researchers are able to reduce TNF-α concentration through α7AChR expression and increases in the concentration of its ligand. I have shown that Neuregulin is able to increase the expression of α7AChR in microglia and macrophages. Using three immortalized cell lines, BV-2, EOC-20 and RAW 264.7, and primary microglial cells harvest from mice I investigated the role that neuregulin plays in the anti-inflammatory process. Neuregulin signals through the ErbB receptors, a family of tyrosine kinase receptors, to facilitate the effects on ACh expression. My results show that ErbB4 is expressed in BV-2, EOC-20 and RAW 264.7 cell lines while ErbB2-4 receptors are expressed in primary microglia. As well, I was able to show that ErbB4 became phosphorylated upon binding to NRG in immortalized cell lines. Using an Enzyme Linked Immunsorbent Assay to analyze TNF- α concentration in microglia and macrophages, I was able to demonstrate that increased levels of α7AChRs did not result in a reduction in TNF-α concentration. These results showed that NRG is able to increase α7AChRs in microglia and macrophages after the phosphorylation of the ErbB4 receptors. As well, this increase in α7AChR does not relate to a reduction in TNF-α, thus under these experimental conditions does not have an effect on the anti-inflammatory pathway.

Inflammation can be up-regulated by microglia and macrophages through the release of pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α). Excess production of TNF-α can lead to a variety of diseases and even tissue necrosis. Recently, the expression of alpha seven acetylcholine receptors (α7AChR) by microglia have been shown to decrease the amount of TNF-α released. This anti-inflammatory pathway has been studied extensively where researchers are able to reduce TNF-α concentration through α7AChR expression and increases in the concentration of its ligand. I have shown that Neuregulin is able to increase the expression of α7AChR in microglia and macrophages. Using three immortalized cell lines, BV-2, EOC-20 and RAW 264.7, and primary microglial cells harvest from mice I investigated the role that neuregulin plays in the anti-inflammatory process. Neuregulin signals through the ErbB receptors, a family of tyrosine kinase receptors, to facilitate the effects on ACh expression. My results show that ErbB4 is expressed in BV-2, EOC-20 and RAW 264.7 cell lines while ErbB2-4 receptors are expressed in primary microglia. As well, I was able to show that ErbB4 became phosphorylated upon binding to NRG in immortalized cell lines. Using an Enzyme Linked Immunsorbent Assay to analyze TNF- α concentration in microglia and macrophages, I was able to demonstrate that increased levels of α7AChRs did not result in a reduction in TNF-α concentration. These results showed that NRG is able to increase α7AChRs in microglia and macrophages after the phosphorylation of the ErbB4 receptors. As well, this increase in α7AChR does not relate to a reduction in TNF-α, thus under these experimental conditions does not have an effect on the anti-inflammatory pathway.

Despite being first identified over a century ago, Alzheimer’s disease (AD) is a complex neurological disorder that still has not been properly characterized. Most cases are sporadic in nature, with an unidentifiable cause, but early-onset familial Alzheimer’s disease (FAD) is induced by genetic mutations in certain key genes. FAD mutations in the full length amyloid precursor protein (flAPP) increases production of the amyloid beta (Aβ) peptide responsible for plaque formation commonly associated with the disease, leading to neuronal death. A mutation in the PS1 gene (mPS1) results in increased APP cleavage into Aβ1-42, also leading to early AD formation. Although discoveries of FAD mutations have enabled concentrated studies into AD pathogenesis, its cause is still unknown. In this thesis, experimental projects were designed to study how signaling pathways associated with markers of AD, including APP and PS1 gene mutations, could result in neuronal dysfunction associated with disease pathology, and how these pathways could be manipulated for use as potential therapeutic targets. Cortical neurons isolated from FAD mPS1 mice (expressing the Met146Val mPS1 protein) were analyzed to establish neuronal viability in response to Aβ1-42 insult compared to healthy neurons. mPS1 neurons were no more susceptible to cell death compared to wild-type neurons, because of an increased activation of the transcription factor nuclear factor kappa B (NF-κB) protein brought about by elevated endoplasmic reticulum (ER) calcium release due to the PS1 mutation. However, NF-κB inhibition in the mPS1 neurons caused increased pro-apoptotic protein CHOP expression leading to significantly higher cell death versus controls when neurons were exposed to Aβ1-42. Following this study, the role of the neurotrophic protein neuregulin on cytoplasmic calcium levels of hippocampal neurons was examined, with the intent of assessing the contributioin of that signaling pathway to AD neuropathology in AD transgenic mice. Neuregulin has been shown to modify glutamatergic channels at neuronal synapses, but how this could affect cytoplasmic calcium levels in neurons was uncertain. Long term treatment (24 hours), but not short-term (1 hr), with neuregulin increased glutamatergic-induced intracellular calcium levels in hippocampal neurons, through a PI3K-mediated mechanism. This study demonstrated that inhibition of the NRG/ErbB axis could be a possible therapeutic target to reduce excitotoxic levels of calcium leading to neuronal death in AD, or enhance synaptic plasticity and memory in AD-initiated areas of deficit. Finally, interactions between the neurotrophic insulin pathway and amyloid peptides were studied using an amyloid precursor protein (APP) overexpressing mouse model, the TgCRND8 strain. Despite insulin depletion induced by streptozotocin injection, young diabetic TgCRND8 mice displayed no impairment in insulin signaling compared to controls, likely due to activation of the insulin signaling pathway by sAPPα. This indicates a possible biological role for sAPPα that prevents diabetic-induced insulin signaling impairment. Thus, the data from these three projects elucidated different components of AD pathogenesis and possibly targets of future AD treatment.

Despite being first identified over a century ago, Alzheimer’s disease (AD) is a complex neurological disorder that still has not been properly characterized. Most cases are sporadic in nature, with an unidentifiable cause, but early-onset familial Alzheimer’s disease (FAD) is induced by genetic mutations in certain key genes. FAD mutations in the full length amyloid precursor protein (flAPP) increases production of the amyloid beta (Aβ) peptide responsible for plaque formation commonly associated with the disease, leading to neuronal death. A mutation in the PS1 gene (mPS1) results in increased APP cleavage into Aβ1-42, also leading to early AD formation. Although discoveries of FAD mutations have enabled concentrated studies into AD pathogenesis, its cause is still unknown. In this thesis, experimental projects were designed to study how signaling pathways associated with markers of AD, including APP and PS1 gene mutations, could result in neuronal dysfunction associated with disease pathology, and how these pathways could be manipulated for use as potential therapeutic targets. Cortical neurons isolated from FAD mPS1 mice (expressing the Met146Val mPS1 protein) were analyzed to establish neuronal viability in response to Aβ1-42 insult compared to healthy neurons. mPS1 neurons were no more susceptible to cell death compared to wild-type neurons, because of an increased activation of the transcription factor nuclear factor kappa B (NF-κB) protein brought about by elevated endoplasmic reticulum (ER) calcium release due to the PS1 mutation. However, NF-κB inhibition in the mPS1 neurons caused increased pro-apoptotic protein CHOP expression leading to significantly higher cell death versus controls when neurons were exposed to Aβ1-42. Following this study, the role of the neurotrophic protein neuregulin on cytoplasmic calcium levels of hippocampal neurons was examined, with the intent of assessing the contributioin of that signaling pathway to AD neuropathology in AD transgenic mice. Neuregulin has been shown to modify glutamatergic channels at neuronal synapses, but how this could affect cytoplasmic calcium levels in neurons was uncertain. Long term treatment (24 hours), but not short-term (1 hr), with neuregulin increased glutamatergic-induced intracellular calcium levels in hippocampal neurons, through a PI3K-mediated mechanism. This study demonstrated that inhibition of the NRG/ErbB axis could be a possible therapeutic target to reduce excitotoxic levels of calcium leading to neuronal death in AD, or enhance synaptic plasticity and memory in AD-initiated areas of deficit. Finally, interactions between the neurotrophic insulin pathway and amyloid peptides were studied using an amyloid precursor protein (APP) overexpressing mouse model, the TgCRND8 strain. Despite insulin depletion induced by streptozotocin injection, young diabetic TgCRND8 mice displayed no impairment in insulin signaling compared to controls, likely due to activation of the insulin signaling pathway by sAPPα. This indicates a possible biological role for sAPPα that prevents diabetic-induced insulin signaling impairment. Thus, the data from these three projects elucidated different components of AD pathogenesis and possibly targets of future AD treatment.

No description available.

Cortical injuries, such as those caused by stroke and other insults, are the leading cause of death and disability worldwide. While thrombolytics can be used to restore blood flow immediately following the onset of symptoms of an ischemic stroke, there are currently no neurorestorative therapeutics that can enhance long-term recovery of function following injury. Neuregulins are a family of growth factors involved with the survival and function of neurons and glia. Glial Growth Factor 2 (GGF2) is an isoform of neuregulin-1 that has demonstrated significant effects in the recovery of function in rodent models of stroke. Histological analyses suggest GGF2 promotes recovery by enhancing endogenous mechanisms to reduce inflammation and promote plasticity. To further explore the efficacy of GGF2, we used our rhesus monkey model of cortical injury and fine motor impairment to compare the rate and pattern of recovery in monkeys treated with GGF2. Twenty-four young adult male monkeys (ages 4-10 years old) were pre-trained on our task of fine motor function of the hand before undergoing surgery to produce a cortical lesion limited to the hand representation of the primary motor cortex on one side. Intravenous (IV) administration of GGF2 (0.5 mg/kg) began 24 hours after surgery and continued daily for 7 days. This was followed by 21 days of sub-cutaneous administration of GGF2 at two different dose levels (0.1 mg/kg or 0.3 mg/kg). Post-operative testing began two weeks after the lesion and continued for 12 weeks. All trials were video recorded and latency to retrieve a reward was quantitatively measured to assess the trajectory of post-operative response latency and grasp pattern compared to pre-operative levels. The results showed no significant differences between the groups in the recovery of fine motor function. Moreover, all vehicle control monkeys returned to their pre-operative levels of latency and grasp pattern despite no significant differences in lesion volume from the experimental groups. In addition to measures of behavioral recovery, we processed the brain tissue with immunohistochemistry to investigate the role of GGF2 treatment in reducing the pro-inflammatory response of microglia and enhancing axonal sprouting and synaptogenesis following injury. All groups had a greater density of Iba1+ microglia in the perilesional grey matter and sublesional white matter, but there were no significant differences in the numerical density or phenotypes of microglia between the groups. We also found no significant differences in axonal sprouting between the groups. However, GGF2 treatment did enhance expression of synaptophysin in the contralesional hemisphere of monkeys that received subcutaneous doses of GGF2 following the initial 7 days of intravenous GGF2 treatment. This suggests that high dose GGF2 treatment may enhance plasticity of compensatory circuits involving the intact hemisphere and that this effect is dose dependent. In addition, we followed up these analyses using a subset of monkeys from the larger GGF2 study to optimize and validate a method that labels newly synthesized myelin. This is accomplished by in vivo administration of a choline analog, propargylcholine (P-Cho) that labels newly synthesized myelin and can be visualized post-mortem. Our results demonstrate effective and stable incorporation of P-Cho with post injection survival of 1 to 6 weeks. Using this method to quantify new myelin after cortical injury to the primary motor cortex, showed significantly greater P-Cho labeling and co-localization with myelin basic protein (MBP) in the white matter underlying the ipsilesional hemisphere when compared with the contralesional hemisphere. This validates P-Cho for assessing myelin plasticity in a nonhuman primate brain and how it might be used to assess therapeutics aimed at inducing remyelination and enhancing myelin synthesis. Finally, this dissertation also includes the comparison of sex differences in recovery of motor function after cortical injury. In a cohort of aged male and female monkeys, postmortem analysis showed no differences in lesion volume between the males and females. However, behaviorally, the females returned to their pre-operative latency and grasp patterns significantly faster and more completely than the males. These findings demonstrate the need for additional studies to further investigate the role of estrogens and other sex hormones that may differentially affect recovery outcomes in the primate brain. Collectively, the results presented in this dissertation highlight the complexity of evaluating treatments and mechanisms underlying recovery of function by enhancing neuroplasticity. Specifically, we were unable to effectively evaluate GGF2 as a treatment due to the behavioral recovery of all control monkeys. Follow up studies should investigate treatment with GGF2 in aging monkeys and compare the results with our findings. Additionally, it is necessary to further explore the recovery of fine motor function in young monkeys. Finally, our study showing sex differences in recovery of function provides evidence that sex hormones may play a significant role in providing neuroprotection in the aging brain following cortical injury. Future studies should measure post-operative estrogen levels and evaluate supplementation as a potential treatment option.

Introduction: Bipolar disorder is associated with cortical abnormalities, including deficits in intracortical myelination. Intracortical myelin follows an inverted-U trajectory over the lifetime, but this trajectory is blunted in individuals with bipolar disorder. Little is understood about which genetic factors contribute to these deficits. Neuregulin-1, a cell-signalling protein, has been shown to contribute to cortical abnormalities and increase susceptibility to related disorders. Assessing the prevalence of neuregulin-1 polymorphisms, notably rs6994992, in bipolar disorder may elucidate the genetic contributors of intracortical myelin deficits and increase our understanding of factors causing susceptibility to bipolar disorder. Methods: 67 participants with bipolar disorder type I and 75 healthy control participants were included. T1-weighted MRI images were collected and processed to create R1 cortical maps, a proxy measure of intracortical myelin. Participant blood samples were genotyped at the rs6994992 locus. Linear models were used to test whether intracortical myelin can be predicted by age, bipolar diagnosis and NRG1 genotype. Results: Intracortical myelin is significantly predicted by age, diagnosis and genotype together in the motor cortex (left: R2 = 0.09, p < 0.01, right: R2 = 0.06, p < 0.05), the right premotor cortex (R2 = 0.095, p < 0.001), and the right inferior frontal cortex (R2 = 0.098, p < 0.001). Age is a significant individual predictor of intracortical myelin in the right dorsal anterior cingulate cortex, the bilateral motor cortex, the right premotor cortex, and the right inferior frontal cortex. Conclusions and Future Directions: Our study suggests that the right premotor, bilateral primary motor, and right inferior frontal cortices are regions of interest for understanding how intracortical myelin changes throughout the lifetime, especially in bipolar disorder. Future work should examine the impact of polygenic risk scores of bipolar disorder on intracortical myelin. / Thesis / Master of Science (MSc) / Bipolar disorder is associated with neurobiological changes, including cortical abnormalities, contributing to a greater disorder burden. Cortical myelination changes throughout the lifetime and larger deficits are found in individuals with bipolar disorder. However, the role of genetics in these intracortical myelin deficits is largely unknown. This thesis investigates how intracortical myelin content in various regions of the cortex is impacted by age, bipolar disorder diagnosis, and neuregulin gene variants. The goal of this research is to contribute to a better understanding of how genetics and age impact intracortical myelin in bipolar disorder to better understand the neurobiological changes of the disorder.

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No description available.