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.

Progressive disorders of airway protection, namely cough (dystussia) and swallowing (dysphagia) dysfunction, are highly prevalent in Parkinson’s disease (PD), impacting quality of life and contributing to the development of aspiration pneumonia – a leading cause of death in this population. To date, dysphagia rehabilitation has remained the primary (and often only) treatment target of choice by clinicians managing dysphagia in patients with PD and other neurodegenerative disease. This is a major concern since the progressive nature of PD makes it somewhat unreasonable to expect that treatments can fully rehabilitate swallowing dysfunction and eliminate chronic aspiration. Instead, rehabilitating cough dysfunction can serve as an adjunctive approach to promote pulmonary health. Considering that impairments in motor control and organization are primary features of PD, skill training may have a necessary role in cough rehabilitation. Despite a growing body of research supporting the feasibility and effectiveness of cough skill training, a significant gap remains in our understanding of optimal skill training parameters that maximize treatment outcomes through motor learning. This document addresses this gap in the literature in a series of three research studies. Chapter 1 will begin by reviewing the current body of literature related to normal and disordered mechanisms of airway protection dysfunction in PD, skill training as an efficacious approach to rehabilitate cough dysfunction, and motor learning considerations to maximize treatment outcomes. Chapter 2 will characterize motor performance and learning during a voluntary cough skill training paradigm, and evaluate the contributions of physiologic (i.e., lung volume) and treatment-specific (i.e., biofeedback) factors to treatment response in PD. Chapter 3 will then characterize trajectories of motor performance during multiple sessions of sensorimotor cough skill training and explore the role of task-specific predictors (i.e., variability, motor learning) on motor performance. Chapter 4 will examine the effects of cough skill training with variable practice on motor performance and motor learning and characterize contributions of laryngeal and respiratory subsystems to cough strength. This document will then conclude (Chapter 5) by synthesizing results from these studies and discussing clinical implications, limitations, and potential directions for future research.

Focused ultrasound (FUS) can modulate activity in the central nervous system of animals, however the mechanism of action is not yet fully understood. FUS is a promising technique for clinical use in treating both physiological and psychological pathology of the nervous system. FUS can noninvasively penetrate the skull deep into the brain and modulate brain targets with millimeter-scale resolution. FUS is less invasive than deep brain stimulation (DBS) and can target deeper structures with greater resolution than transcranial magnetic stimulation (TMS). Functional ultrasound imaging (fUSI) is an emerging modality for monitoring stimulus-evoked brain activity. However, the thick skull of large animals poses a significant obstacle for the noninvasive translation of the technique to nonhuman primates and humans. In this dissertation, FUS is performed in mice and nonhuman primates and an fUSI technique is developed for transcranially imaging FUS-evoked responses in both species. The first aim of this dissertation established a procedure for performing high-resolution FUS in mice in vivo. FUS-evoked motor responses were evaluated using four-limb electromyography (EMG). A detailed quantitative analysis of several EMG characteristics demonstrated that observed motor responses exhibited brain target-specific differences. FUS in the brain was also shown to modulate cardiorespiratory activity. However, simulations conceded that intracranial reverberations may activate brain structures outside acoustic foci, suggesting that direct detection of brain activity is preferable to responses like EMG and cardiorespiratory activity. The second aim of this dissertation developed an fUSI system for monitoring FUS-evoked responses in mice in vivo. fUSI was validated using electrical peripheral nerve stimulation to elicit somatosensory-evoked responses, a well-characterized approach in established techniques like functional magnetic resonance imaging (fMRI). fUSI was later integrated into an ultrasoundbased optogenetic stimulation procedure. Lastly, a dual FUS-fUSI transducer system for performing neuromodulation and functional activity monitoring was developed and successfully demonstrated in mice in vivo. The final aim of this dissertation was to adapt the FUS-fUSI procedure developed in mice for use in nonhuman primates. Two approaches were developed and tested in vivo. The first approach employed a low-frequency ultrasound array for both neuromodulation and activity monitoring. The second approach implemented a dual FUS-fUSI transducer system similar to that used in mice. Preliminary evidence indicated that the adapted dual transducer system can successfully perform fully noninvasive neuromodulation and functional activity monitoring transcranially in nonhuman primates in vivo. The findings presented in this dissertation provide a framework for performing fully noninvasive ultrasound-mediated neuromodulation and functional activity monitoring in non human primates and describes a road map for further translating the technique for clinical use in human subjects. A fully noninvasive FUS-fUSI technique can provide an invaluable tool for clinicians to treat diseases of the nervous system not indicated for invasive procedures, opening the door to a wide range of therapeutic applications.

Utilization of airway protective rehabilitation among individuals with neurodegenerative disease is astoundingly low. Yet, due to progressive decline in airway protective function and resulting health consequences such as aspiration pneumonia, the need for rehabilitation is clear. Moreover, a growing literature supports the benefit of airway protective rehabilitation in neurodegenerative populations. Therefore, it is a healthcare priority to increase treatment utilization in order to improve health and quality of life for individuals with neurodegenerative disease. Improving treatment accessibility and relevance are two approaches that have the potential to improve utilization. Despite the need to increase treatment accessibility and the growing evidence base to support the use of telehealth to increase access, a significant gap remains in our understanding of the feasibility and acceptability of telehealth to manage dysphagia in neurodegenerative disease. Moreover, little is known about patient perspectives which are critical to refine person-centered models of care that are relevant to patient’s needs. To address this important clinical research gap, this dissertation includes a series of three research studies aimed at improving accessible and relevant rehabilitation for airway protective dysfunction in neurodegenerative disease. Chapter 1 will provide an overview of the current literature as it relates to airway protective dysfunction in neurodegenerative disease, existing rehabilitation approaches, telehealth to manage dysphagia, and the need for personalized care. Chapter 2 will examine the feasibility of rehabilitating airway protection via telehealth in individuals with neurodegenerative movement disorders. Chapter 3 will then explore speech language pathologists’ (SLPs) perspectives and experiences using telehealth to manage dysphagia. Chapter 4 will characterize patient perspectives on airway protective dysfunction and treatment experience following cough skill training (CST). I will conclude (Chapter 5) by synthesizing the findings from chapters 2-4 and suggesting directions for future research.

Human microglia play a pivotal role in neurological diseases, but few targeted therapies that directly modulate microglial state or function exist due to an incomplete understanding of microglial heterogeneity. This thesis aims to advance our understanding of microglial heterogeneity by using single-cell RNA sequencing to profile live human microglia from autopsies or surgical resections across diverse neurological diseases and using computational tools to infer chemical and genetic regulators of specific microglial substates. Chapter 1 provides an overview of microglial ontogeny, function, and known heterogeneity, especially in disease contexts. It also describes the steadily increasing disease burden seen in neurological disease as well as the lack of efficacious treatments and future directions for microglia-targeted therapies. Chapter 2 focuses on microglial heterogeneity in an understudied disease, ALS, describing population structure shifts seen in ALS across cortex and spinal cord. Chapter 3 instead focuses on exploring underlying cross-disease microglial population structure, identifying subsets with metabolic and functional properties, as well as subsets enriched in susceptibility genes for neurodegenerative disease. We then demonstrate applications of this type of data by using our resource to annotate other datasets. Chapter 4 leverages this data in another way, by identifying and validating candidates for chemically and genetically inducing subtype-specific states in vitro. Notably, we show that Camptothecin downregulates the transcriptional signature of disease-enriched subsets and upregulates a signature previously shown to be depleted in Alzheimer’s. Finally, I review our findings and discuss future directions for the field.

Advancements in neural interfacing technologies, such as microelectrode arrays, have significantly contributed to understanding brain function and treating neurological disorders. Decoding the intricacies and functioning of neural circuits is key to further unlocking its potential. Two key approaches, electrical neural recording and optical imaging, have been the basis of stimulating and monitoring neural circuits. Despite the remarkable progress, several key issues such as reliable stimulation of neurons, closed-loop stimulation and monitoring, and undesired background fluorescence during widefield optical imaging remain challenging. After giving a brief background on electrode and microLED arrays, the dissertation delves into the design, microfabrication, and characterization of microelectrode arrays for neural electrical stimulation, recordings, and microLED arrays as a light source for improving optical microscopy. We first discuss a dense conformal electrode array for high spatial resolution stimulation in electrosensory systems. The performance metrics of the integrated system are thoroughly examined through meticulous characterization and optimization processes. Special emphasis is placed on evaluating biocompatibility, electrical properties, and spatial resolution to ensure robust and reliable neural stimulation capability. Next, we discuss a microelectrode device that combines simultaneous electrical recording and 2-photon imaging. We use an Indium Tin Oxide (ITO) material to fabricate a transparent electrode array with a design capable of single neuron recordings. The design, microfabrication, and electrooptical characterization are presented to demonstrate the device’s capability. A system integrating the array with a GRIN lens is also presented to record and image deeper into the brain tissue. Combining both the electrical and optical recordings of neuron ensembles and finding correlations can shed further light on the functioning of neural circuits. To address the problem of unwanted background fluorescence during neural cell imaging, two microLED arrays as light sources are presented. With a microstripe array, we implement optical sectioning structured illumination microscopy (OS-SIM), and with the 2D microLED array, we implemented targeted illumination to reject background fluorescence and improve contrast. We examine the capability of the microLED as a light source with luminance-current-voltage, directivity, and transient measurements. Both implementations highlight the novel non-display application of microLED to address challenges in neural imaging. This research represents a significant contribution to the burgeoning field of neural engineering, offering novel methodologies and technologies that promise to revolutionize our approach to understanding brain functions.

Indiana University-Purdue University Indianapolis (IUPUI) / The main, underlying cause of neurodegenerative disease is the progressive loss of neuronal structure or function, whereby central and/or peripheral nervous system circuitry is severely and irreversibly damaged, resulting in the manifestation of clinical symptoms and signs. Neurodegenerative research has revealed many similarities among these diseases: although their clinical presentation and outcomes may differ, many parallels in their pathological mechanisms can be found. Unraveling these relationships and similarities could provide the potential for the discovery of therapeutic advances such that a treatment for one neurologic disease may also be effective for several other neurodegenerative disorders. There is growing awareness that due to the complexity of pathophysiological processes in human disease, specifically targeting or inactivating a single degenerative process or a discrete cellular molecular pathway may be ineffective in the treatment of these multifaceted disorders. Rather, potential therapeutics with a multi-target approach may be required to successfully and effectively control disease progression. Recent advances in neurodegenerative research involve the creation of animal disease models that closely mimic their human counterparts. The use of both toxin- exposure and genetic animal models in combination may give insight into the underlying pathologic mechanisms of neurodegenerative disorders (target identification) leading to the development and screening of prospective treatments and determination of their neuroprotective mechanism (target validation). Taken together, ideal candidates for the treatment of neurodegenerative disease would need to exert their neuroprotective effect on multiple pathological pathways. Previous studies from this laboratory and collaborators have shown that the naturally-occurring compound, caffeic acid phenethyl ester (CAPE), is efficacious for the treatment against neurodegeneration. Because of its versatile abilities, CAPE was chosen for this study as this compound may be able to target the pathogenic pathways shared by two different animal models of neurodegeneration and may exhibit neuroprotection. In addition, adipose-derived stem cell conditioned media (ASC-CM), a biologically-derived reagent containing a multitude of neuroprotective and neurotrophic factors, was selected as ASC-CM has been previously shown to be neuroprotective by using both animal and cell culture models of neurodegeneration.

Proteases play an indispensable role in medicine, serving both as drug targets and as therapeuticagents. Protease inhibitors are a key component of our antiviral arsenal, and are widely used to combat human immunodeficiency virus (HIV), hepatitis C virus (HCV), and, most recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nirmatrelvir, an inhibitor of the 3-chymotrypsin like (3CL) protease essential for SARS-CoV-2 replication, was granted emergency use authorization in late 2021, formulated with ritonavir and sold under the brand name Paxlovid. Since then, Paxlovid use has become widespread, raising the possibility that nirmatrelvir resistance could emerge in circulating SARS-CoV-2. It is therefore important to understand how the 3CL protease might mutate to lose nirmatrelvir sensitivity so that circulating variants can be monitored for these mutations and future generations of inhibitors can be designed to prevent cross-resistance. The most widely used therapeutic proteases are the botulinum neurotoxins (BoNTs),which are effective in the treatment of a wide array of movement, pain, and autonomic disorders. These toxins exert their therapeutic effect by cleaving members of the SNARE protein family inside neuronal cytosol, preventing neurotransmitter release. Much work has been dedicated to engineering members of the BoNT family to extend their therapeutic utility, including altering receptor tropism, extending half-life, and modifying protease specificity. While significant progress has been made in each of these areas, the extent to which these proteases can be reprogrammed to target the proteins that cause human disease remains unexplored. In this dissertation, we employ diverse methods to study and direct protease evolutionand to facilitate protein engineering more broadly. To investigate the emergence of nirmatrelvir resistance, we passage SARS-CoV-2 in increasing concentrations of the drug and sequence the 3CL protease gene over time in resistant lineages. We then validate the observed 3CL protease mutations by incorporating them into recombinant SARS-CoV-2 and testing the nirmatrelvir sensitivity of the resulting viruses. We find that the development of nirmatrelvir resistance typically begins with the acquisition of a precursor mutation such as T21I, P252L, or T304I, which confers a low level of resistance and enables strong resistance conferring mutations, such as E166V, to emerge without imposing a significant fitness cost. To explore the programmability of the BoNT proteases, we engineer type E (BoNT/E) to cleave proteins involved in neurodegenerative disease. To accomplish this, we employ targeted mutation based on a structural model as well as the continuous evolution platform known as OrthoRep, selecting for variants that cleave the desired substrates with a circuit that links protease activity to the growth of a Saccharomyces cerevisiae (yeast) cellular chassis. We first use this approach to target ATXN3, the protein whose aggregation causes type 3 spinocerebellar ataxia (SCA3). We then profile the substrate specificities of the BoNT/E variants that emerged during our ATXN3 engineering, identifying patterns that can inform the selection of new targets. Based on one of these patterns, we generate a new BoNT/E variant capable of cleaving TDP-43, a protein implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Finally, to facilitate future protein engineering campaigns, we develop a machine learning pipeline that uses deep mutational scanning (DMS) data to model a protein’s fitness landscape and predict optimized variants with user-defined constraints. We name this pipeline OptiProt, and we demonstrate its utility by applying it to the human chaperone DNAJB6, which rescues cellular toxicity in a yeast model of ALS. In this context, we probe OptiProt’s engineering capabilities with a range of challenges. We first predict hyper-functional variants with up to 50 mutations. We then restore activity to variants harboring one of two known loss-offunction mutations. Finally, to demonstrate the OptiProt’s amenability to complex engineering constraints, we restore activity to a variant harboring a deleterious mutation while simultaneously mutating a set of highly conserved residues. Altogether, the work presented in this dissertation highlights the value of laboratory protease evolution in two clinically relevant applications and provides a tool that can facilitate future work in this field.

The functional compartmentalization underlying neuronal polarity makes tightly regulated intracellular transport between the cell body, axons, and dendrites essential for proper development and homeostatic maintenance. Disruptions to neuronal trafficking are a major cause of neurodegenerative disease. Pathogenic variants in the microtubule motor protein KIF1A cause KIF1A Associated Neurological Disorder (KAND), a spectrum of rare neurodegenerative conditions. KAND is clinically and genetically heterogeneous, with a broad phenotypic spectrum and over a hundred pathogenic variants identified. KAND is poorly understood at both the clinical and molecular level, and there is currently no treatment. This work characterizes the natural history of KAND and describes a novel heuristic severity score. This severity score is then used to show how the location of pathogenic missense variants within the KIF1A motor domain correlates with disease severity, providing evidence the clinical phenotypic heterogeneity in KAND reflects and parallels the molecular phenotypes. Insights from the neuropathology of deceased KAND patients is used to focus a histopathologic assessment of the C3-Kif1aLgdg mouse model. C3-Kif1aLgdg/Lgdg mice have a cerebellar axonal torpedo phenotype, paralleling some of the pathological changes seen in the patients. Phenotypically, the C3-Kif1aLgdg mice were found to recapitulate some of the symptoms seen in patients including progressive spasticity and gait abnormalities associated with hind limb paralysis. To model the disease at a cellular level, iPSCs were derived from affected individuals and successfully used to generate neural stem cells and neurons. These patient-derived neurons were found to have increased markers of protein aggregates, a cellular phenotype that can be used to test potential treatments. Taken together, these studies provide foundational knowledge for future therapeutic development.

Over 50 million people globally suffer from neurodegenerative disorders—a number that is steadily increasing as the general population ages. Yet, effective treatments for neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) remain limited, primarily due to the presence of a natural protective biological barrier lining cerebral blood vessels called the blood-brain barrier (BBB). The blood- brain barrier prevents passage of nearly 98% of small molecules from blood vessels to brain tissue, while most therapies designed for neurodegenerative disorders, such as gene therapies, are considered large-molecule drugs, making development of efficacious pharmacological treatments extremely challenging. Present strategies to bypass the BBB for drug delivery broadly fall into two categories: non-invasive but non-targeted methods, or targeted but invasive surgical procedures such as direct intracranial injection. Currently, the only method poised clinically to provide both non-invasive and targeted drug delivery to the brain is focused ultrasound (FUS). When combined with intravenously administered ultrasound contrast agents called microbubbles which oscillate within blood vessels in response to FUS pressure waves, FUS can safely and reversibly open the blood-brain barrier (BBB) in a highly targeted manner. This enhances drug delivery to brain regions affected by neurodegenerative disorders through a physical mechanism known as acoustic cavitation. A majority of FUS research to date has centered around development and clinical translation of stereotactic FUS guided by magnetic resonance imaging (MRI) for treatment monitoring, commonly referred to as MRgFUS. However, MRgFUS exhibits cost, accessibility, and portability barriers to implementation in medical centers globally. Alternatively, our group has developed cost-effective and accessible ultrasound-guided FUS (USgFUS) configurations, which have the potential to enable BBB opening and drug delivery treatment outside of an MRI with treatment guidance facilitated by neuro-navigation technology and cavitation monitoring. While most USgFUS systems developed prior to this dissertation achieve therapeutic opening of the BBB and cavitation monitoring with separate ultrasound transducers, this thesis focuses primarily on development and optimization of a single-transducer technique for both therapy and monitoring called theranostic ultrasound (ThUS). In Aim 1, we show that a repurposed diagnostic ultrasound array reprogrammed with focused imaging pulses can produce therapeutically relevant ultrasound energy through primate skulls, and can induce multi-site modulatory drug and gene delivery depending on the ThUS parameters applied. In Aims 2 and 3, we apply ThUS-mediated drug and gene delivery for pre-clinical neuroscience and therapeutic applications in PD, respectively. In Aim 2, we demonstrated non-invasive delivery of specialized genes and nanoparticles which together enable remote stimulation and recording of neuronal activity, a synergistic process which could enable remote brain-to-brain communication. In Aim 3, we leveraged ThUS-mediated gene therapy to restore degenerated neurons in a PD mouse model, achieving nearly 85% restoration of diseased dopaminergic neurons non-invasively. Finally, in Aim 4, we translated ThUS-mediated BBB opening to non-human primates (NHP) to determine initial feasibility of targeted gene expression facilitated by a low frequency, custom ThUS array. We demonstrated that both conventional USgFUS and ThUS configurations can safely induce targeted gene expression in brain regions implicated in PD in rhesus macaques, motivating translation of USgFUS for gene therapy in the clinic. The aims in this dissertation collectively underscore the growing number of pre-clinical applications which could benefit from ThUS technology, while propelling USgFUS methodologies as a whole to the brink of clinical translation for unprecedented access to efficacious non-invasive gene therapy for neurodegenerative disorders in the future.