Cone snails are carnivorous marine mollusks, utilizing their neuropeptide-rich venom for prey capture. The venom of Conus brunneus, a wide-spread Eastern Pacific vermivore, has not been extensively studied. In the current work, peptides from the dissected venom were characterized and tested using preliminary bioassays. Six peptides (A-F) were isolated and tested. Three peptide identities were determined by comparison with previously reported data: bru9a (A), bru3a (F), and an a-conotoxin (E). Preliminary screening in a stroke-related model of induced glutamate excitotoxicity in primary neuronal cells and PC12 cell cultures indicated potential neuroprotective activity of peptide fractions A, D, and F. Further testing is necessary to determine and verify structure, activity, target, and mechanism of action of the promising peptides from C. brunneus, which may prove effective neuropharmacological agents to treat stroke. / by Rebecca A. Crouch. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

by Sau Cheung Tiu. / Thesis (M.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 348-388). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Patients with giant axonal neuropathy (GAN) exhibit loss of motor and sensory function and typically live for less than 30 years. GAN is caused by autosomal recessive mutations leading to low levels of gigaxonin, a ubiquitously-expressed cytoplasmic protein whose cellular roles are poorly understood. GAN pathology is characterized by aggregates of intermediate filaments (IFs) in multiple tissues. Disorganization of the neuronal intermediate filament (nIF) network is a feature of several neurodegenerative disorders, including amyotrophic lateral sclerosis, Parkinson's disease and axonal Charcot-Marie-Tooth disease. In GAN such changes are often striking: peripheral nerve biopsies show enlarged axons with accumulations of neurofilaments; so called "giant axons." Interestingly, IFs also accumulate in other cell types in patients. These include desmin in muscle fibers, GFAP (glial fibrillary acidic protein) in astrocytes, and vimentin in multiple cell types including primary cultures of biopsied fibroblasts. These findings suggest that gigaxonin may be a master regulator of IFs, and understanding its function(s) could shed light on GAN as well as the numerous other diseases in which IFs accumulate. However, an interaction between gigaxonin and IFs has not been detected and how IF accumulation is triggered in the absence of functional gigaxonin has not been determined. To address these questions I undertook a proteomic screen to identify the normal binding partners of gigaxonin. Prominent among them were several classes of IFs, including the neurofilament subunits whose accumulation leads to the axonal swellings for which GAN is named. Strikingly, human motor neurons (MNs) differentiated from GAN iPSCs recapitulate this key phenotype. Accumulation of nIFs can be rescued by reintroduction of gigaxonin, by viral delivery or genetic correction. GAN iPS-MNs do not display survival vulnerability in the presence of trophic factors, but do display increased cell death in the presence of oxidative stress. Preliminary experiments suggest that in iPS-MNs nIFs are degraded by contributions from both the proteasome and lysosome. Gigaxonin interacts with the autophagy protein p62 which has been implicated in the clearance of ubiquitin aggregates by the lysosome, and this interaction is greatly enhanced in conditions of oxidative stress. My data provide the first direct link between gigaxonin loss and IF aggregation, and suggest that gigaxonin may be a substrate adaptor for the degradation of IFs by autophagy, pointing to future approaches for reversing the phenotype in human patients.

This thesis describes the development of an systems biology method to study transcriptional programs that are activated during early and late phases of cell-fusion mediated reprogramming, as well as an implementation of systems-level analysis using reverse-engineered regulatory networks to study CNS disorders like Alcohol Addiction, and neurodegenerative disorders like Alzheimer's Disease (AD), and Parkinson's Disease (PD). The results will show an unprecedented view into the mechanisms underlying complex processes and diseases, and will demonstrate the predictive power of these methodologies that extended far beyond their original contexts.

Amyloid plaques, a neuropathological hallmark of Alzheimer’s disease (AD), are largely composed of amyloid beta (Aβ) peptide, derived from cleavage of amyloid precursor protein (APP) by β- and γ-secretase. The endosome is increasingly recognized as an important crossroads for APP and the secretases, with major implications for APP processing and amyloidogenesis. Amongst various posttranslational modifications affecting APP, ubiquitination of cytodomain lysines may represent a key signal controlling endosomal sorting. Here, we show that substitution of APP COOH-terminal lysines with arginines disrupts APP ubiquitination, though the pool of ubiquitinated APP is small or transient. Nonetheless, this small deficiency in ubiquitination can have a significant impact on APP, such that the number of lysines mutated trends toward an increase in APP metabolism. An APP mutant lacking all COOH-terminal lysines undergoes the most pronounced increase in processing, leading to accumulation of both secreted and intracellular Aβ40, without change in Aβ42. This phenotype is abolished by artificial ubiquitination of APP using rapalog-mediated proximity inducers. Lack of APP COOH-terminal lysines does not affect APP endocytosis, but leads to a redistribution of APP from endosomal intraluminal vesicles (ILVs) to the endosomal limiting membrane, with subsequent decrease in APP COOH-terminal fragment (CTF) content of secreted exosomes, but minimal effects on APP lysosomal degradation. Both the secreted and intracellular increase in Aβ40 is abolished by depletion of presenilin 2 (PSEN2), recently shown to be enriched on the endosomal limiting membrane compared to presenilin 1 (PSEN1). In a separate set of studies, we found that a familial AD mutant, L723P, which occurs immediately next to a string of three lysines in the juxtamembrane region, behaves more similarly to other FAD-causing mutations. APP L723P exhibits a selective increase in Aβ42, and a delay in degradation, but no change in exosomal content, despite some missorting to the endosomal limiting membrane. Our findings demonstrate that ubiquitin can act as a signal for endosomal sorting at five lysines in the APP cytodomain, disruption of which prevents sequestration of APP in ILVs and results in the processing of a larger pool of APP-CTF by PSEN2 on the endosomal membrane.

Amyotrophic lateral sclerosis (ALS) is an aggressive neurodegenerative disease in which motor neurons selectively degenerate, leading to paralysis and death. Rare causal mutations in FUS and TARDBP implicated RNA binding proteins and RNA metabolism in ALS disease mechanisms. The absence of faithful animal models has impeded precise understanding of the impact of ALS mutations on all functions of ALS-associated proteins. In my graduate studies, I used a novel, animal model of FUS-ALS to explore gain of function disease mechanisms and observed specific, aberrant interactions between mutant FUS and other RNA binding proteins including hnRNP U. Genetic experiments indicate loss of hnRNP U is toxic to motor neurons, suggesting mutant FUS toxicity may result from hnRNP U sequestration and loss of function. In a parallel series of experiments, I also generated novel knock-in mouse models of ALS expressing pathogenic TARDBP mutations to address the flaws of existing model systems and to study the functional consequences of disease-related mutations. We demonstrate that the ALS mutant alleles TDP-43M337V and TDP-43G298S are fully functional and are insufficient to cause age-dependent motor neuron pathology, indicating that physiological levels of mutant TDP-43 are alone insufficient to initiate disease. This model enables future exploration of the interaction between genetic and environmental factors that lead to TDP-43 toxicity in ALS and related disorders. Collectively, our findings suggest a gain of function mechanism of toxicity in which mutations and aging, with other factors, alter the local concentration of RNA binding proteins, leading motor neurons to degenerate.

Purpose: The purpose of this study was to explore the efficacy of an interdisciplinary intervention on interdisciplinary teamwork and patient functional outcomes in an acute inpatient rehabilitation unit at a mid-sized regional hospital. Design: Pilot mixed-methods pre-post intervention study. Methods: Interdisciplinary teamwork and patient functional outcomes were measured before and after a teamwork intervention. Interdisciplinary teamwork was measured with the Healthcare Team Vitality Instrument (HTVI) and a qualitative staff questionnaire developed by a content expert. Patient functional outcomes were measured by aggregated Functional Independence Measure (FIM®) scores. Findings: Post-intervention FIM® gain scores increased significantly (p = .008). Staff questionnaire revealed improvement in interdisciplinary teamwork, with the major themes of teamwork and appreciation/respect. Post-intervention HTVI showed no significant change (p=.528). Conclusions: Initial results of this intervention are promising; additional research is needed to study the effectiveness of this intervention in a variety of acute rehabilitation settings. Clinical Relevance: Rehabilitation leaders can implement low-cost teamwork interventions to improve interdisciplinary teamwork and patient outcomes.

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a resting tremor combined with varying degrees of rigidity and bradykinesia. Introduced in the 1950s, thalamotomy is used as a surgical procedure to improve brain function in patients and serves as an effective treatment method for the PD tremor where connections within the thalamus are cut. In 1987, deep brain stimulation (DBS), chronic electrical stimulation of deep neural structures using electrodes, was introduced as a clinical treatment for medically refractory tremor in patients with PD. Though thalamotomy has historically been the primary treatment method for PD, an increasing number of patients have chosen to undergo DBS as it has become increasingly touted as an alternative to ablative therapies. The proposed study examines the advantages and disadvantages of both treatment methods to improve cardinal features in highly-skilled, career-oriented PD patients who actively use motor functions in their work. As an alternative to a simple finger-tapping test used for normal PD patients, a more complex strength-dexterity (S-D) test would be performed on 50 skilled patients to evaluate and compare the effectiveness of tremor suppression between both surgeries. The goal of this experiment is to determine which treatment produces the most short-term benefits for the patient to continue with his or her career with minimal future management required. The results of this study will help determine the preferred treatment method when taking into consideration other external factors such as cost, continual management, and preference for short-term vs. long-term results.

Axonal pathology is a key contributor to long-term disability in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS), but the mechanisms that underlie axonal insults remain unclear. While most axonal pathologies characterized in MS are a direct consequence of myelin loss, we propose that axonal pathologies also occur independent of demyelination. In support of this idea, we recently reported that mice that develop experimental autoimmune encephalomyelitis (EAE), a model commonly used to mimic the pathogenesis of MS, exhibit a structural and functional disruption of the axon initial segment (AIS), a subdomain of the axon that acts as the trigger-zone for action potential generation. Importantly, this disruption is independent of myelin loss. Although the mechanism responsible for AIS disruption remains unclear, we observed an attenuation of the AIS insult following treatment with a known scavenger of oxygen free radicals. To further investigate the role of oxidative stress in modulating AIS stability, we employed an in vitro model in which neurons were exposed to a spontaneous reactive oxygen and nitrogen species generator. Through this approach, we demonstrated that oxidative stress is capable of AIS modulation acting through induction of cytosolic calcium (Ca2+) influx from both extracellular and intracellular sources, resulting in calpain protease activation. Furthermore, because rises in intracellular Ca2+ are central to these and other mechanisms of AIS disruption, we next investigated the cisternal organelle (CO), an AIS-localized Ca2+-regulating structure. Although this organelle could prove to be central to AIS modulation, very little is known about the mechanisms regulating its stability. Through this line of investigation, we provide the first evidence of pathological alteration to the CO in a disease state. This disruption precedes loss of AIS protein clustering and axo-axonic GABAergic input in both EAE and MS postmortem tissue. Overall, these studies reveal a primary axonal insult, independent of myelin loss, in a disease classically characterized as a white-matter pathology. Instead, this insult is most likely driven by oxidative stress through local Ca2+ dysregulation at the AIS, providing novel therapeutic targets for MS.

SARM1 is an injury-induced nicotinamide adenine dinucleotide nucleosidase (NADase) that was previously shown to promote axonal degeneration in response to traumatic, toxic, and excitotoxic stressors. This raises the question of whether a SARM1-dependent program of axonal degeneration is central to a common pathway contributing to disease burden in neurological disorders. The degree to and mechanism by which SARM1 inactivation decreases the pathophysiology of such disorders is of interest to establish the rationale to pursue SARM1 as a therapeutic target. In this study, we compare the course and pathology of experimental autoimmune encephalomyelitis (EAE) in Sarm1-knockout (KO) mice and wild-type littermates to test the contribution of SARM1-dependent axonal degeneration specifically in the context of chronic, immune-mediated central nervous system (CNS) inflammation. The question of whether SARM1 loss in Sarm1-KO mice would inhibit, promote, or have a negligible impact on EAE-induced axonal degeneration and more broadly CNS inflammation was explored using a variety of analyses: quantification of clinical score in a chronic EAE model, CNS immune infiltrate profile, axon initial segment morphology in layer V cortical neurons, axonal transport disruption and transection in the lumbar spinal cord. Additionally, we have proposed a method for detecting SARM1 activation in situusing a novel SARM1-mCitrine bimolecular fluorescence complementation (BiFC) technique. Successful implementation of such a molecular tool would allow for a detailed, mechanistic approach to enhance our understanding of upstream intracellular signals that trigger SARM1 activation.