Charcot Marie Tooth disease (CMT) represents the most common inheritable peripheral group of motor and sensory disorders; affecting 1 in 2500 people worldwide. Individuals with CMT experience slow progressing weakness of the muscle, atrophy, mild loss of motor coordination and in some cases loss of sensory function in the hands and feet which could ultimately affect mobility. Dynein is an essential molecular motor that functions to transport cargos in all cells. A point mutation in the dynein heavy chain was discovered to cause CMT disease in humans, specifically CMT type 2O. We generated a knock-in mouse model bearing the same mutation(H304R) in the dynein heavy chain to study the disease. We utilized behavioral assays to determine whether our mutant mice had a phenotype linked to CMT disease. The mutant mice had motor coordination defects and reduced muscle strength compared to normal mice. To better understand the disease pathway, we obtained homozygous mutants from a heterozygous cross, and the homozygotes show even more severe deficits compared to heterozygotes. They also developed an abnormal gait which separates them from heterozygous mice. In view of the locomotor deficits observed in mutants, we examined the neuromuscular junction (NMJ) for possible impairments. We identified defects in innervation at the later stages of the study and abnormal NMJ architecture in the muscle as well. The dysmorphology of the NMJ was again worse in the homozygous mutants with reduced complexity and denervation at all the timepoints assessed. Our homozygous dynein mutants can live up to two years and therefore make the design of longitudinal studies possible. Altogether, this mouse model provides dynein researchers an opportunity to work towards establishing the link between dynein mutations, dynein dysfunction and the onset and progression of disease.

Huntington Disease (HD) is an autosomal dominant neurodegenerative disorder caused by a trinucleotide repeat expansion mutation in the huntingtin (HTT) gene. HD is characterized by neurological symptoms, including motor, cognitive, and psychiatric decline. A non-neurological symptom, metabolic dysfunction, is also common in HD and can cause weight change, a feature recapitulated in HD mice. There is a need for a better understanding of the weight changes associated with HD, because patients with a higher body mass index show slower disease progression. Our lab has shown that metabolic dysfunction in HD mice is the result of a disrupted circadian feeding rhythm but when HTT expression is suppressed in the brain, this is normalized. Additionally, when a normal circadian feeding rhythm is enforced, HTT protein in the brain is reduced. Together, this suggests that HTT is involved in gut-brain feedback. The gut brain axis (GBA) is the bidirectional communication pathway between the gut and central nervous system. One major component of the GBA is the gut microbiome, which has been implicated in neurological disorders. The microbiome interacts with the brain by producing short chain fatty acids, neurotransmitters, and metabolites. A plasma metabolomics study of HD mutation carriers revealed a reduction of Indole-3-propionic acid (IPA) in HD. IPA is a metabolite exclusively produced by bacteria and is a known antioxidant and anti-inflammatory. Inflammation and oxidative stress are features of HD and a reduction in circulating IPA may contribute to these aspects of the disease. This research was conducted to investigate the relationship between the relative abundance of IPA producing bacteria in the gut and the amount of plasma IPA in HD model mice. This will provide a basis for further investigation into the connection between the microbiome, bacterial metabolites, and the potential to target gut dysbiosis as a therapeutic outlet for HD.

Epileptogenesis is the complex process of the brain developing epileptic acitivity. Due to the role of glutamate and the hippocampus in synaptic plasticity a dysregulation in glutamate neurotransmission and hippocampal dysfunction are implicated in the process of epileptogenesis. However, the exact causal factors that promote epileptogenesis are unknown. We study presynaptic proteins that regulate glutamate neurotransmission and their role in epileptogenesis. The presynaptic protein, tomosyn, is believed to be a negative regulator of glutamate neurotransmission; however, no one has studied the effects of this protein on glutamate transmission in vivo. Furthermore, evidence suggests that mice lacking tomosyn have a kindling phenotype. Thus, in vivo glutamate recordings in mice lacking tomosyn have the potential to elucidate the exact role of tomosyn in glutamate neurotransmission and its potential relationship to epileptogenesis. Here we used biosensors to measure glutamate in the dentate gyrus (DG), CA3, and CA1 of the hippocampus in tomosyn wild-type (Tom+/+), heterozygous (Tom+/-), and knock out (Tom-/-) mice. We found that, in the DG, that glutamate release increases as tomosyn expression decreases across genotype. This suggests that tomosyn dysregulation in the DG leads to an increase in glutamate release, which may explain why these mice have an epileptogenic phenotype.

<p>Allosteric modulators are a newly emerging concept in the field of drug discovery which have shown remarkable success in their ability to alter G-protein coupled receptor (GPCR) activity in a precise and subtle manner. A GPCR of particular interest for allosteric targeting is the dopamine D2 receptor. This receptor has repeatedly been implicated in the etiology of complex neurological and neuropsychiatric disorders including Parkinson’s disease and schizophrenia. Previous studies from our lab have effectively developed allosteric modulators targeting the D2 receptor based on the pharmacophore of the endogenous tripeptide L-prolyl-L-leucyl-glycinamide (PLG). PLG and its potent peptidomimetics, particularly 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) (PCT/CA2011/000968), have shown robust preclinical efficacy in treating models of Parkinson’s disease, depression, tardive dyskinesia and schizophrenia. These ligands modulate agonist binding to the D2 receptor in a biphasic manner, although further information on their mechanisms of action are currently unknown. Therefore, the overarching objective of this thesis was to enhance our knowledge on the mechanisms of action of the promising D2 allosteric ligands PLG and PAOPA. Results of the studies presented here show PAOPA to cause significant upregulation of D2 regulatory proteins and downstream signaling kinases, as well as cause an increase in D2 internalization. Additionally, the PLG allosteric binding site was narrowed down to be localized between transmembrane domains 5 and 6 on the D2 receptor. The collection of work presented here enhance our understanding of the mechanisms of action of the potentially therapeutic D2 allosteric ligands PLG and PAOPA, progressing them closer to helping clinically affected populations. The findings of these studies prove globally significant as they highlight the diverse cellular pathways which could be affected by allosteric modulators, and bring to light the importance of studying these candidate ligands for eventual improvements in the treatment of human health.</p> / Doctor of Philosophy (Medical Science)

Epilepsy has been previously attributed to either increased excitation or decreased inhibition. With this closed frame of mind, modern medicine has been unable to develop a permanent treatment against the mechanisms of epilepsy. In order to treat patients with intractable seizures, especially those caused by developmental malformations, it is essential to understand the entirety of mechanisms that could possibly play a role in the abnormal cortical function. One such developmental malformation is known as polymicrogyria. Epileptogenesis occurs in an area laterally adjacent to this malformation known as the paramicrogyral region (PMR). Past studies have narrowed down the potential cause of this increased network excitation to a certain type of inhibitory interneuron, the somatostatin (SS) interneuron. Additionally, previous studies have shown an increase in the mGlu5 receptor on this interneurons in the PMR region only and not in control tissue, meaning that targeting these receptors as treatment will not affect normal functioning tissue. These results lead to our hypothesis: blockade of the mGluRs will decrease the 2 activity of SS interneurons and thereby prevent the generation of epileptiform activity and increased SS output in malformed cortex. Utilizing the freeze-lesion model for microgyria in transgenic mice expressing Channelrhodopsin optogenetic channels in SS interneurons, we assessed the contribution of these SS interneurons in four different animal groups: control or PMR treated with either Gabapentin, a current AED (antiepileptic drug), or MTEP, an mGlu5 receptor antagonist. We tested the effects of these two drugs on SS interneuron output to determine whether they decrease the over activation in the PMR that has been previously studied. The following study revealed no correlation between Gabapentin-treated animals and a decrease in epileptiform activity. Additionally, no significant difference was seen between the MTEP-treated groups in the protocols that were measured.

Stroke is a leading cause of death and disability worldwide. Although rapid recognition and prompt treatment have dropped mortality rates, most stroke survivors are left with permanent disability. Approximately 87% of all strokes result from the thromboembolic occlusion of the cerebrovasculature (ischemic strokes). Potential stroke therapeutics have included anti-inflammatory drugs, as well as many other targets with the goal of mitigating the acute and chronic inflammatory responses typically seen in an ischemic stroke. While these approaches have had great success in preclinical studies, their clinical translation has been less successful. Master inflammatory cytokines, such as IL-1, are of particular interest. IL-1’s isoforms, IL-1α and IL-1β, were long thought to have similar function. While IL-1β has been extensively studied in stroke, the role of IL-1α during post stroke inflammation has been overlooked. Because IL-1 inhibitors have been unsuccessful in clinical application, we reasoned that IL-1α may provide previously unknown benefits to the brain after injury. We hypothesized that IL-1α could be protective or even accelerate reparative processes in the brain such as producing new blood vessels (angiogenesis) or neurons (neurogenesis). To test that IL-1α is protective after stroke, we tested IL-1α’s protective effects on primary cortical neurons in in vitro models of stroke. We showed that IL-1α was directly protective on primary cortical neurons in a dose-dependent fashion. We then performed mouse middle cerebral artery occlusion stroke studies to determine the safety of giving IL-1α in vivo. These studies showed that administering IL-1α acutely was neuroprotective. However, intravenous (IV) administration of IL-1α resulted in transient, hemodynamic changes following drug delivery. To minimize these systemic effects, we administered IL-1α intra-arterially (IA) directly into the stroke affected brain tissue, allowing us to significantly lower the concentration of administered IL-1α. In comparison to IV, IA IL-1α showed greater histological protection from ischemic injury as well as improved functional recovery following stroke, all without systemic side effects. To test that IL-1α could aid in neurorepair following stroke, we tested IL-1α’s ability to help damaged blood vessels repair in vitro. We found that IL-1α significantly increased brain endothelial cell activation, proliferation, migration, and capillary formation. We tested IL-1α’s proangiogenic properties in vivo by administering IL-1α three days following stroke. Delayed administration allowed us to separate IL-1α’s acute neuroprotective effects from potential subacute angiogenic effects. We found that mice receiving IL-1α performed significantly better on behavioral tests and also showed greater vascularization within the penumbra two weeks following stroke. We also found that IL-1α treated animals showed more endothelial activation than vehicle treated animals. Finally, our studies showed that IL-1α treated animals showed increased early-phase neurogenesis with evidence of increased proliferation at the subventricular zone suggesting that IL-1α’s beneficial effects are even more far-reaching than previously thought. In conclusion, our experiments suggest that the inflammatory cytokine IL-1α is neuroprotective and neuroreparative in experimental ischemic stroke and worthy of further study as a novel stroke therapy.

Schizophrenia is primarily treated with the use of antipsychotic medications. However, antipsychotics used have severe, dose-dependent side effects in schizophrenia patients. Therefore, there is a need for new adjunctive drugs that lower the effective dose of first line schizophrenia drugs and improve patient symptoms. Neuroinflammation is associated with microglial activation in schizophrenia, and increased tumor necrosis factor-alpha (TNF) has shown to be associated with Metabolic Syndrome in schizophrenia patients. A newly developed anti-neuroinflammatory, PD2024, reduces TNF-alpha action in vitro and in vivo, and has been shown to be well-tolerated in rat and dog studies with no adverse effects. The purpose of this research is to evaluate the effect of PD2024 in two well-defined schizophrenia models in rats. The neonatal quinpirole model has been established through administration of the dopamine D2-like agonist quinpirole (NQ) or saline control (NS) postnatally from days 1-21. NQ treatment results in increases of dopamine D2 receptor sensitivity throughout the animal’s lifetime without changing receptor number, mimicking a hallmark of schizophrenia. The polyinosinic:polycytidylic acid (Poly I:C) model is based on mimicking an increase immune response during early brain development, which has been shown to increase the prevalence of schizophrenia. Poly I:C (2 mg/kg) was administered during the neonatal period at postnatal days (P)5-7 to produce this effect. Both models were given PD2024 at 10mg/kg orally through the diet from P30-67. Prepulse inhibition (PPI) was used to test sensorimotor gating deficits in the rats. PPI has past research showing its use as a quantitative phenotype for evaluating schizophrenia-associated behavioral and neurobiological deficits. In our PPI test, rats are exposed to three different, randomly ordered noise trials. The trials included a pulse trial with a 120-decibel startle pulse, a prepulse trial with an auditory click at 73, 76, or 82-decibels, and a no stimulus trial without any additional noise. The rats were given 25 randomized trials, comprised of 5 pulse, 15 prepulse (5 each of 73, 76, and 82dB) and 5 no stimulus trials. Background noise was 70dB, and the rats were tested during adolescence (days 45-46) and adulthood (60-65). In NQ adolescent rats, PPI was significantly improved in the PD2024-treated compared to NQ controls. NQ-PD2024 and NS rats were statistically equivalent throughout the trials. These results were reflected in the NQ adult model as well. The Poly I:C adolescents treated with PD2024 also demonstrated improved PPI performance compared to Poly I:C controls. This improvement was also shown in the adult Poly I:C rats. Overall, the PPI deficits in both models improved between 15 to 30% in adolescence and adulthood. These results indicate that PD2024 is effective in treating schizophrenia-associated behaviors.

It is estimated that one in 59 children in the US are affected by autism spectrum disorder (ASD). ASD is distinguished by social and communication deficits that can be displayed throughout a wide range of severity. This resulting spectrum of behaviors observed in ASD suggests that a complex etiology is involved. Previous studies have shown a genetic susceptibility to autism including paternal age, twin and sibling concordance. Genetic sequencing of those affected as well as first order relatives have identified alterations in genes associated with neuronal synaptic communication. However, very little information is available regarding the pathophysiology of synapses in ASD. Neuronal communication between anterior cingulate cortical neurons via synapses with other brain regions is vital in the execution of social behaviors in individuals. The aim of this study was to evaluate the protein expression of the synaptic marker spinophilin and post-synaptic density protein-95 (PSD-95) in postmortem ASD gray matter brain tissue from the anterior cingulate and frontal cortex to compare to typically developing (TD) control brain tissue. Postmortem brain tissue of ASD and TD subjects was acquired from nationally funded brain repositories previously matched by brain area, age and gender. Immunoblotting for spinophilin and PSD-95 was performed using anterior cingulate and frontal cortical gray matter brain tissue from matched ASD and TD brain tissue. Spinophilin and PSD-95 protein amounts for all donors were normalized using GAPDH. Frontal cortical tissue demonstrated no significant differences in spinophilin protein expression between TD and ASD groups (N=6). Anterior cingulate tissue demonstrated no significant differences in spinophilin protein expression between TD and ASD groups (N=5). PSD-95 protein expression levels did not result in any significant differences between ASD donors and their control pairs for either brain tissue region. Although no changes were detected in the frontal cortex or anterior cingulate cortex, more brain areas and subjects must be evaluated to determine if spinophilin or PSD-95 can be reliable markers for synaptic alterations in ASD. These data are critical in determining synaptic pathology in ASD which may lead to future treatments.

In this two-part study, I investigated whether the cyclic-adenosine monophosphate response element-binding (CREB) protein has the potential to be clinically modulated as a therapeutic target for the treatment of neurodegenerative diseases. Part one consisted of a systematic review that was conducted on select articles gathered through a stepwise method to explore (1) the relationship between diseased, neurodegenerative brains and levels of active, phosphorylated CREB (pCREB), (2) increased activation of CREB as a treatment for neurodegenerative symptoms, and (3) a potential therapeutic drug for neurodegenerative diseases that can target CREB signaling. The results of the systematic review showed evidence that suggested excitotoxic concentrations of N-methyl-D-aspartate (NMDA) results in decreased pCREB levels, while decreased pCREB levels were associated with impaired cognition and behavior, increased cell death, as well as decreased CRE-gene transcription and long-term potentiation (LTP). Part two consisted of a systematic review and meta-analysis on clinical trials that used the phosphodiesterase type IV inhibitor, roflumilast, on healthy and schizophrenic patients. It was found that 100 µM roflumilast was able to improve verbal learning in healthy and schizophrenic subjects (ES = 64). Initial evidence indicates that future research on neurodegenerative diseases should further investigate CREB’s potential to be clinically modulated and research investigating PDE4 inhibitor drug therapy for the treatment of neurodegeneration should be expanded upon further in subsequent studies.

Investigations of human biology and disease have been hindered by the use of animal models. The information obtained from such studies often results in clinically irrelevant results and drug trial failures. Additionally, several governing bodies have been formulating legislation to move away from animal models and toward more ethical and efficient testing platforms for drug discovery and cosmetic research. As an answer to these issues, "body-on-a-chip" systems have been a rapidly developing field which easily recapitulates in vivo functionality, providing a more relevant, repeatable, and ethical testing platform to better predict biology. These systems can be used as human-based testing platforms to evaluate human physiology, disease progression, and drug responsiveness for specific cell types and multi-organ systems. Diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) have significant research challenges, specifically with translating research findings into treatment plans. The complexity of the neuromuscular reflex arc, the biological system affected by these diseases, is difficult to study with traditional molecular techniques, namely because the many components of this disease system interact with each other using complex pathways. This work pushes the existing platform to a more complete human model of neuromuscular disease with the incorporation of gamma motoneurons, development of the first human induced pluripotent cell (iPSC) derived intrafusal fibers, and proposals to incorporate nociceptive neurons all on a functionally interrogative platform. The incorporation of these components will allow for a more complete, clinically relevant model to study neuromuscular disorders and for preclinical dug discovery.