Alzheimer's Disease (AD) is a neurodegenerative disorder that affects around 50 million people worldwide and causes cognitive decline, brain atrophy and death. Despite extensive basic and clinical studies and drug development efforts, currently no effective treatments are available for AD. The amyloid β (Aβ) peptide is neurotoxic and is tightly associated with AD pathology, but the molecular mechanism of its action remains unclear. There are various forms of Aβ in the brain, ranging from the full length Aβ1-42 to shorter peptides, such as a strongly toxic Aβ25-35 fragment. The Amyloid Cascade Hypothesis (ACH) postulated that extracellular Aβ deposits cause the disease. More recently, the soluble Aβ oligomers came into the focus of research as they proved to be the major neurotoxic entities. One of the mechanisms by which Aβ peptides, including Aβ25-35, kill neurons is membrane perforation and disruption of cellular homeostasis. Although direct membrane interaction and pore formation by Aβ has been documented, the detailed structural aspects of membrane pores remain elusive. Here, we quantitatively describe the structure of Aβ25-35 in aqueous buffer and in lipid environment, its binding to membranes, pore formation, and the details of membrane pores. We have shown that membrane binding of Aβ25-35 is electrostatically driven. Aβ25-35 forms β-barrel like structures ranging from hexamers to octamers and then assemble into supra-molecular structures forming calcium-conducting pores in the membrane with radius of 6 Å to 7 Å. The structural features of Aβ25-35 pores depend on the content of cholesterol in the membranes. Moreover, the aggregation and structural changes of a series of Aβ fragments have been analyzed to identify the segment(s) of highest propensity for fibrillogenesis that might serve as initiators of Aβ aggregation and conversion into toxic species. Finally, the structures of the full-length Aβ1-42 and a hypertoxic version pEAβ 3-42, in lipid environment have been analyzed by solid state nuclear magnetic resonance. Collectively, these studies will elucidate the structural details of membrane pores formed by Aβ peptides as targets for new anti-AD therapies.

<p>Lysosomal storage diseases (LSDs) are devastating neurological disorders caused by mutations in lysosomal hydrolases that result in accumulations of hydrolase substrates. Tay-Sachs disease (TSD) is an LSD that specifically results in the accumulation of GM2 gangliosides causing the activation of inflammatory signaling pathways, and leading to microglial activation and apoptotic cell death. The detailed mechanisms through which cell death occurs have not been completely elucidated, however, excitotoxicity is thought to play a major role. Here, we investigated the role of hypoxia-inducible factor-1α (HIF- 1α) and its effector microRNA, miR-210, and the impact they have on the expression of important molecules involved in excitotoxicity, namely neuronal pentraxin 1 (NPTX1) and potassium channel KCNK2 (KCNK2). We discovered that TSD neuroglia are inefficient at stabilizing HIF-1α in hypoxic conditions. Furthermore, miR-210 expression is significantly higher in TSD neuroglia compared to normal neuroglia at baseline and during hypoxia. In addition, TSD neuroglia expressed <em>NPTX1</em>, <em>NPTX2 </em>and <em>KCNK2 </em>at higher levels, and neuronal pentraxin receptor at lower levels than normal neuroglia, implicating excitotoxicity in disease pathogenesis. We also confirmed that miR-210 binds to the 3’ UTR of <em>NPTX1 </em>to repress its expression in TSD neuroglia. The presence of reverse hypoxia response elements in the promoter of KCNK2 and the repression of <em>KCNK2 </em>expression by HIF-1α stabilization suggest that KCNK2 is directly regulated by HIF-1α. Moreover, the glucosylceramide synthase inhibitor, NBDNJ, which is used to reduce ganglioside synthesis, caused expression of <em>NPTX1 </em>to decrease but <em>KCNK2 </em>expression to increase, indicating this drug can modify multiple parameters of disease. This study identifies major gene expression changes between normal and TSD neuroglia that affect the excitability and therefore the viability of TSD cells. This information provides new insight into the mechanisms of neurodegeneration experienced by TSD neuroglia.</p> / Master of Science (MSc)

Axons use a conserved program to actively drive their own destruction after injury. Axon degeneration is present in many neurological disorders and an axon death program could be a major pharmaceutical target to preserve neuronal function. This intrinsic signaling cascade activates pro-degenerative dSarm/Sarm1, rapidly depletes axonal stores of NAD+, and terminates in cytoskeletal breakdown. Conversely, loss of dSarm/Sarm1, maintenance of NAD+ levels or its biosynthetic enzyme Nmnat, result in long-term morphological perseveration of severed axons. Exactly how dSarm/Sarm1 and loss of NAD+ execute axon death remains poorly defined. We sought to uncover novel regulators of axon death and maintenance by performing a deficiency screen and a forward genetic mutagenesis screen in axotomized Drosophila wing sensory neurons. We identified a BTB domain protein enriched in neurons, we named Axundead (Axed), which is specifically required for axon death. Severed axons harboring loss of function mutations in axed, similar to dSarm mutants, remain preserved for 50 days post axotomy. Spontaneous neurodegeneration induced by activated dSarm or dNmnat depletion are both suppressed in axed mutants, but not in dSarm mutant alleles. Additionally, severed axed mutant axons also expressing activated dSarm or lacking Nmnat are preserved. These results indicate that dSarm acts upstream of dNmnat loss, and both events precede essential Axed function and axon destruction. Thus, the axon death pathway converges on Axed function.

Progesterone regulates female reproduction in many ways, yet it is still unclear how signals are conveyed through nuclear and extranuclear receptors. The traditional notion was that progesterone binds classical progesterone receptors to alter gene transcription. This view has been challenged by the discovery of additional progesterone signaling molecules important for progesterone actions in non-neural cells. In granulosa cells, the progesterone receptor membrane component 1 (Pgrmc1) mediates progesterone effects by forming a receptor complex with binding partner, Serpine mRNA binding protein 1, but it is unknown whether these molecules function similarly in the brain. To begin to address these issues, I investigated the neural role of Pgrmc1 in female mouse brain, rat brain and in neural cells. By examining the neuroanatomical localization, hormonal regulation, and colocalization of Pgrmc1 within key neurons in the neural control of ovulation, Pgrmc1 emerged as a candidate signaling molecule likely to mediate progesterone functions. Furthermore, Pgrmc1 levels regulate the expression of several diverse genes and signaling pathways in neural cells. Taken together, these results demonstrate that Pgrmc1 function is likely to impact diverse neural functions.

An essential process to the development of the neural network of the nervous system is axon guidance. The noncanonical Wnt/Planar Cell Polarity pathway has been identified as an integral component in controlling the projection of axons during axon guidance. Prickle, ROR1 and ROR2 are PCP related proteins that do not have clearly defined roles in the process. This study aims to use zebrafish CoPA neurons as a model to study the roles of Prickle, ROR1, and ROR2 in axon guidance. Using in situ hybridization, morpholino knockdown, and CRISPR/Cas9 loss of function experiments were able to identify ror1, ror2 and prickle as potential required components in CoPA neuron axon guidance. Elucidating the role of these protein in axon guidance not only will increase our knowledge of the PCP pathway but it will also increase our understanding of the development of the nervous system.

Cannabis (Marijuana) has multiple effects on the human body, such as analgesia, euphoria and memory impairment. Delta-9 tetrahydrocannabinol (D9-THC), the active ingredient in cannabis, binds to cannabinoid receptors, seven-transmembrane G protein-coupled receptors (GPCRs) that mediate a variety of physiological functions. GPCRs were believed to function only in homomeric forms, however, recent findings show that different GPCRs can also form heteromeric complexes that may expand their signaling properties. In this study, we focused on Cannabinoid CB1 receptor (CB1R) heteromers with the mu-opioid receptor (MOR) and the Dopamine type 2 receptor (D2R), respectively. We utilized a variety of techniques, such as the calcium mobilization assay, a luciferase complementation assay and an electrophysiology assay to study the pharmacology of the CB1R-MOR and CB1R-D2R heteromers. Our data demonstrate that co-expression of CB1R enhances the Gi signaling through MOR and inhibits the beta-arrestin recruitment to MOR. We also show that co-application of CB1R ligands can further accentuate the MOR signaling modulation. Co-expression of a CB1R transmembrane domain 5 (TM5), but not a TM1, mini-gene abrogated the signaling change suggesting that it is likely due to heteromerization of MOR and CB1R. Utilizing this herteromeric signaling could provide a novel therapeutic approach that may yield potent analgesic effects with reduced side effects. We have also found that CB1R switched its signaling specificity from Gi to Gs upon its heteromerizaiton with D2R. In conclusion, our data show that CB1R expands its signaling repertory and modulates the partner receptor signaling upon heteromerization.

Myocardial infarction (MI), often referred to as a heart attack, is a serious health issue in the United States. There is a well-documented link between MI and major depressive disorder (MDD), with a high incidence of MDD occurring after an MI. Overlapping pathologies have been observed within the hippocampus of the brain in animal models of MI and depression. These observations suggest that pathobiological cross-talk between the heart and brain could have a role in the etiology of MDD that occurs after an MI. Spinal cord stimulation (SCS) has previously been shown to have both cardioprotective and neuroprotective effects post-MI, and hence may protect individuals from developing depression post-MI. In this study, we examined the potential biochemical mechanisms that might underlie the neuroprotective actions of SCS following MI. Brain tissues were obtained from three groups of canines: sham-operated animals, animals subjected to experimental myocardial infarction/mitral regurgitation (MI/MR), and animals subjected to MI/MR that were simultaneously administered SCS. The whole hippocampus and hippocampal dentate gyrus were dissected from frozen brains. Quantitative endpoint-PCR and RT-qPCR techniques were employed to measure select biochemical mediators of neuroprotection, i.e. adenosine A2A receptor, ghrelin, and ghrelin receptor expression in hippocampal samples. SCS induced a significant decrease in A2A receptor expression and a dramatic increase in ghrelin expression in MI/MR canines as compared to the MI/MR group without SCS. These findings suggest that adenosine receptors and ghrelin may play a biochemical role in SCS-induced neuroprotection of the hippocampus. Understanding the neuroprotective actions of SCS has the potential to aid the development of new treatments or preventative measures for depression following a heart attack.

<p><strong>Background</strong><strong></strong></p> <p>IgE antibodies have high antigen specificity and are the hallmark biomarkers of allergy. IgE binds to high-affinity IgE receptors, known as FcεRI, which are expressed especially on mast cells and basophils. In allergic individuals, antigen binding to IgE that is associated with FcεRI leads to crosslinking of adjacent receptors and subsequently to cell activation, degranulation and/or secretion of bioactive molecules. These molecules together cause minor local tissue reactions such as oedema or itch, but also can cause major systemic reactions such as hypotension, cardiac and respiratory distress or even laryngeal swelling and death. The role of the nervous system in these reactions is usually thought of as secondary. However, in recent years there have been a number of studies suggesting the expression of FcεRI on neurons, opening the possibility that nerves are directly involved in antigen-specific responses and making a previously unrecognized contribution to allergic disease. <strong></strong></p> <p>Based on these previous observations regarding neuronal FcεRI, the current study employed both <em>in vivo</em> and <em>in vitro</em> approaches with the following objectives:<strong></strong> <ol> <li>To confirm the presence of FcεRI on peripheral nerves and demonstrate that they are functionally active under different conditions of IgE sensitization.</li> <li>To examine the pathways involved in neuronal activation by IgE bound to FcεRI and compare and contrast these to those already established for mast cells and basophils.</li> </ol></p> <p><strong>Methods and Results</strong></p> <p>A potential role of neuronal FcεRI in the IgE-dependent allergen avoidance behaviour of sensitized mice presented with antigen in sucrose solution was assessed based on published evidence for the involvement of peripheral nerves in this response.</p> <p>Chimeric mice with a wild-type nervous system but lacking FcεRI on hematopoietic cells including mast cells and basophils, failed to exhibit aversive behaviour, whereas mice with FcεRI-bearing hematopoietic cells demonstrated the normal aversive response confirming that FcεRI expression on mast cells is necessary for development of allergen avoidance. While immunohistochemical staining could detect IgE bound to mast cells in tissue samples, no IgE was detected on nerves where the nerves were identified by using a pan-neuronal marker, PGP 9.5, in the intestine of either normal or passively sensitized C57BL/6 and BALB/c mice. Similarly, traditional FACS analysis clearly identified FcεRI on cultured mast cells, but these methods provided no evidence for expression of FcεRI or IgE binding on superior cervical ganglion (SCG) and dorsal root ganglion (DRG) neurons in culture.</p> <p>To determine evidence for functional FcεRI on neurons <em>in vitro</em>, intracellular calcium increase was assessed as a measure of cell activation following sensitization and antigen challenge. Using both microscopy and FACS analysis, calcium fluorophore (Fluo-3, AM) increase could be detected in SCG or DRG that were activated with the calcium ionophore A23187 but not following antigen challenge.</p> <p><strong>Summary: </strong>The current study found no evidence for the presence of the FcεRI on neurons <em>in situ</em> or their sensitization by IgE actively or passively using several different approaches both in tissues and cultured SCG or DRG neurons. Possible explanations for the resultant discrepancy with previously published works are discussed.</p> / Master of Science (MSc)

<p><strong>Purpose:</strong> Microglia are the innate immune cells of the central nervous system. Activated microglia release nitric oxide, glutamate, and superoxide radicals, which are harmful to retinal ganglion cells (RGCs). They may also benefit surviving cells by removing toxic cellular debris or by secretion of neurotrophic factors. The paradoxical role of microglia remains controversial because the nature and time-course of the injury that determines whether microglia acquire a neuroprotective or pro-inflammatory phenotype is unknown. HAPI cells are an immortalized microglial cell line, whose phenotype can be manipulated <em>in vitro</em>. It is my HYPOTHESIS that pharmacological manipulation of microglia to acquire either a pro-inflammatory or pro-survival phenotype will exacerbate neuronal cell death or enhance neuronal survival after injury, respectively.</p> <p><strong>Method:</strong> Lipopolysaccharides (LPS) were used to hyper-stimulate the HAPI cells and minocycline to maintain the HAPI cells in a quiescent state. Prior to the experiments, the HAPI cells were labelled with Wheat Germ Agglutinin conjugated to Texas Red. The HAPI cells were cultured and exposed to minocycline (10 µg/mL for 1 hour) or LPS (1 µg/mL for 24 hours). Sprague-Dawley rats then recieved intraocular (30,000 cells) or tail vein (5 million cells) injections of either the minocycline treated HAPI cells or the LPS treated HAPI cells and an optic nerve crush. Retinas were examined at 4-14 days later and the number of surviving RGCs will be determined by Brn3a labelling of RGCs. BM88 antibody labelling was done to determine the severity of the injury and to determine molecular changes after neuroinflammation.</p> <p><strong>Results: </strong>Injection of untreated HAPI cells resulted in the greater loss of RGCs early after ONC when injected into the vitreous and later after ONC when injected into the tail vein. LPS activated HAPI cells injected into the vitreous resulted in greater RGC loss with and without injury. When injected into the tail vein with ONC there was no loss of RGCs 4 days after ONC but later there was greater loss of RGCs. Minocycline treated HAPI cells injected into the vitreous resulted in greater RGC survival than when untreated HAPI cells were injected. However, when injected into the tail vein with ONC there was greater loss of RGCs. There was also BM88 down regulation after injury and this was more pronounced after HAPI cell injection.</p> <p><strong>Conclusion:</strong> Neuroprotection or cytotoxicity of microglia depends on the type of activation, time course of the injury, and if the microglia act on the axon or cell body of the retinal ganglion cell.</p> / Doctor of Philosophy (PhD)

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the progressive death of dopaminergic neurons present in the substantia nigra. The clinical presentation of PD includes tremors, slowed movement (bradykinesia), muscle and limb rigidity, and difficulty with walking and balancing. While many environmental factors can affect the onset and progression of the disease, genetic mutations have a large influence. Of the identified PD-linked genetic mutations, mutations in the leucine-rich repeat kinase 2 (LRRK2) are one of the most common genetic causes of PD. Located in endosomes, LRRK2 has been shown to play a role in the sorting and endocytosis of synaptic vesicles, a process that is largely mediated by the retromer complex. Mutations in Vps35, a core component of the retromer cargo-recognition complex, have also been identified as a significant cause of late-onset autosomal dominant familial PD. While the exact molecular mechanisms by which LRRK2 and Vps35 mutations induce PD remain largely unknown, their influence on several cellular processes, including vesicular trafficking and breakdown, and endosomal sorting and recycling, strongly implicate the retromer and autophagy in PD pathology. Recent findings that transgenic expression of Vps35 is able to rescue the PD-related phenotypes caused by LRRK2 mutant forms provide further insight into the interplay of these genes in the context of PD and point to these -genes as potential therapeutic targets. This review outlines the current studies involving these genetic mutations and their interactions with various cellular processes and pathways so as to gain a better understanding of the molecular mechanisms underlying PD pathology for the ultimate purpose of developing safe and effective treatments for PD.