<p>This thesis aims to investigate how one fundamental component of the inner-ear (cochlear) response to all sounds, the temporal fine structure (TFS), is used by the auditory system in everyday hearing. Although it is well known that neurons in the cochlea encode the TFS through exquisite phase locking, how this initial/peripheral temporal code contributes to everyday hearing and how its degradation contributes to perceptual deficits are foundational questions in auditory neuroscience and clinical audiology that remain unresolved despite extensive prior research. This is largely because the conventional approach to studying the role of TFS involves performing perceptual experiments with acoustic manipulations of stimuli (such as sub-band vocoding), rather than direct physiological or behavioral measurements of TFS coding, and hence is intrinsically limited. The present thesis addresses these gaps in three parts: 1) developing assays that can quantify TFS coding at the individual level 2) comparing individual differences in TFS coding to differences in speech-in-noise perception across a range of real-world listening conditions, and 3) developing deep neural network (DNN) models of speech separation/enhancement to complement the individual-difference approach. By comparing behavioral and electroencephalogram (EEG)-based measures, Part 1 of this work identified a robust test battery that measures TFS processing in individual humans. Using this battery, Part 2 subdivided a large sample of listeners (N=200) into groups with “good” and “poor” TFS sensitivity. A comparison of speech-in-noise scores under a range of listening conditions between the groups revealed that good TFS coding reduces the negative impact of reverberation on speech intelligibility, and leads to reduced reaction times suggesting lessened listening effort. These results raise the possibility that cochlear implant (CI) sound coding strategies could be improved by attempting to provide usable TFS information, and that these individualized TFS assays can also help predict listening outcomes in reverberant, real-world listening environments. Finally, the DNN models (Part 3) introduced significant improvements in speech quality and intelligibility, as evidenced by all acoustic evaluation metrics and test results from CI listeners (N=8). These models can be incorporated as “front-end” noise-reduction algorithms in hearing assistive devices, as well as complement other approaches by serving as a research tool to help generate and rapidly sub-select the most viable hypotheses about the role of TFS coding in complex listening scenarios.</p>

<p>Tauopathies are neurodegenerative diseases defined by the accumulation of pathological tau protein in neurons and glia. Alzheimer’s disease (AD), the most common tauopathy, is characterized by the presence of neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein aggregates in neurons. Emerging evidence suggests that the NFT burden correlates with neuron death and cognitive decline, contributing to disease progression. Besides AD, a similar deposition of tau inclusions is found to be associated with neurodegeneration in the brains of patients with other tauopathies including progressive supranuclear palsy, corticobasal degeneration, and Pick’s disease. These diseases display clinical, biochemical, and neuropathological heterogeneity. Little is known about how tau aggregation can lead to varied phenotypes in tauopathies, and there is no disease-modifying treatment. Thus, it is necessary to understand the role of diverse tau proteoforms in tauopathies for the development of new therapeutics to treat tauopathies, including AD.</p> <p>In the studies summarized in Chapter 2, we investigated how the molecular diversity of tau proteoforms could impact antibody-based assays of a phospho-tau variant serving as an AD biomarker. A tau variant phosphorylated on threonine 181 (pT181-tau) has been widely investigated as a potential AD biomarker in cerebrospinal fluid (CSF) and blood. pT181-tau is present in NFTs of AD brains, and CSF levels of pT181-tau correlate with overall NFT burden. Various immuno-based analytical methods, including Western blotting and ELISA, have been used to quantify pT181-tau in human biofluids. The reliability of these methods depends on the affinity and binding specificity of the antibodies used to measure pT181-tau levels. Although both of these properties could in principle be affected by phosphorylation within or near the antibody’s cognate antigen, such effects have not been extensively studied. Here, we developed a bio-layer interferometry (BLI)-based analytical assay to assess the degree to which the affinity of pT181-tau antibodies is altered by the phosphorylation of serine or threonine residues near the target epitope. Our results revealed that phosphorylation near T181 negatively affected the binding of pT181-tau antibodies to their cognate antigen to varying degrees. In particular, two of three antibodies tested showed a complete loss of affinity for the pT181 target when S184 or S185 was phosphorylated.</p> <p>In the studies outlined in Chapter 3, we examined the relative abilities of different tau proteoforms to induce seeded tau aggregation and to themselves undergo seeded aggregation in cultured cells. Accumulating evidence suggests that tau aggregates, including NFTs, spread in a stereotypical pattern across neuroanatomically connected brain regions. This spreading phenomenon is thought to occur via a prion-like mechanism involving the release of tau aggregates from a diseased neuron into the extracellular space, aggregate uptake by neighboring healthy neurons, and the formation of new aggregates in the cytosol of the recipient cells via a seeding process. Although research over the past decade has revealed key molecular events involved in the cell-to-cell transmission of tau aggregates, the impact of the protein’s domain structure and phosphorylation profile on the efficiency of prion-like propagation remains poorly defined. Here, we compared three tau variants – K18, 0N4R, and 2N4R – in terms of their aggregation and seeding efficiencies in recombinant protein solutions and in cell culture models. Our results revealed that K18 had the highest fibrillization rate and yield among the three tau variants. Recombinant preformed fibrils (PFFs) derived from all three variants had similar seeding efficiencies. Additionally, we investigated the relationship between tau phosphorylation and aggregation. We found that hyperphosphorylated tau did not undergo self-assembly in the absence of heparin, whereas it formed fibrils at low yield in the presence of the cofactor. Moreover, hyperphosphorylated tau PFFs produced under these conditions induced seeded tau aggregation in cell culture.</p> <p>Taken together, these results point to critical roles of tau proteoforms as both AD biomarkers and drivers of disease progression. Our results indicate that the presence of different combinations of phosphorylated residues near a target phospho-tau antigen can affect the accuracy of antibody-based biomarker assays. In addition, the domain structure and phosphorylation profiles of tau proteoforms associated with AD and other tauopathies likely have a profound influence on the evolution of tau pathology in these disorders. Our findings highlight the importance of accounting for the molecular diversity of tau proteoforms in tauopathies and provide valuable insights into molecular determinants influencing tau aggregation and propagation in the brains of patients.</p>

<p>Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear.</p> <p><br></p> <p>Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative <em>in vitro</em> model is critical to elucidate the signaling pathways orchestrating CSF production in the CP.</p> <p><br></p> <p>This research aims to characterize an <em>in vitro</em> culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.</p>

<p>  </p> <p>Cilia are cell appendages that sense our environment and are critical in cell-to-cell communication. Dysfunction of cilia can result in several disease states including obesity. While cilia in the brain are known to be important for feeding behavior, it is unclear how they regulate energy homeostasis. Classically, cilia coordinate signaling through surface receptors called G-protein coupled receptors (GPCRs). For example, cilia mediated GPCR signaling is critical for both our senses of vision and smell. How cilia regulate the signaling of GPCRs in other areas of the body including the brain is only now emerging. To answer cell biology questions around cilia mediated GPCR signaling in neurons, we developed a system for primary neuronal cultures. We discovered that the cilia mediated hedgehog pathway influences the ability of neurons to respond to GPCR ligands. For the first time, this result highlights the role of the hedgehog pathway in neurons. We continue to explore how cilia integrate the hedgehog pathway and GPCR signaling in the central nervous system, and the potential connections to energy homeostasis. We discovered that hedgehog pathway activity in feeding centers of the brain changes based upon feeding conditions like fasting. We also learned that activating the hedgehog pathway in these brain regions is sufficient to cause obesity in mice. These novel results highlight an unrecognized role for the hedgehog pathway in the regulation of feeding behavior. Overall, this work provides a better understanding of ciliopathy associated obesity and may reveal more common mechanisms of obesity in the general population. In addition, this work implicates the hedgehog pathway in regulating behaviors and new modes of cell-cell communication within the central nervous system.</p>

<p>Sudden unexpected death in epilepsy (SUDEP) is a fatal complication of epilepsy that kills 1̃2 of every 10,000 epileptic patients every year. SUDEP has proven difficult to study because it frequently occurs unobserved and cannot be predicted. What limited clinical data exists suggests that SUDEP occurs as a cardiorespiratory collapse immediately following a seizure. In this work, I explore how a group of autonomic reflexes termed collectively as “oxygen‐conserving reflexes (OCRs)” lead to sudden death when activated during seizures. I also demonstrate multiple physiological parallels between the OCR‐mediated deaths that I report and the clinical data on cases of human SUDEP. Additionally, I explore the neural pathway underlying OCRs, identify the carotid body as a potential target for intervention, and demonstrate the efficacy of electroceutical intervention in reducing the mortality risk of OCR activation during seizures. This work seeks to both offer a neural explanation for SUDEP as well as present a promising target and means for potential intervention.</p>