Anne Luebke - US grants

DESCRIPTION: The goal of the proposed study is to examine the expression of the three known alpha-bungarotoxin (alpha-BTX) sensitive subunits, alpha 7, alpha 8, and alpha 9, of the nicotinic acetylcholine receptor. The degenerate polymerize chain reaction (PCR) cloning technique will be used to clone probes for three nicotinic acetylcholine receptor subunits in the guinea pig cochlea. DNA sequencing will be performed on these cloned fragments to determine their nucleotide sequence, thus, establishing their identity as one of the known receptor subunits or perhaps a new auditory-specific form of an alpha-BTX sensitive nicotinic ACh receptor. The second specific aim is to determine the cellular expression pattern of these nicotinic ACh receptor subunits in the guinea pig cochlea. This will be accomplished by in situ hybridization techniques using a colorimetric detection protocol, rather than radioisotopic detection, to provide cellular resolution. Additionally, co-localization studies are proposed using riboprobes labeled with different fluorophores in conjunction with confocal microscopy.

DESCRIPTION: The objective of this proposal is to study the molecular mechanisms by which neurotransmitters released by olivocochlear efferents influence outer hair cells (OHCs) of the cochlea. One theory about the contributions that cochlear efferents make to hearing is that they protect the cochlea from high frequency acoustic over-stimulation, whereas another theory is that they enhance sound recognition in the presence of background noise. The primary neurotransmitters of the efferents projecting on high frequency OHCs are acetylcholine (ACh) and calcitonin gene-related peptide (CGRP). The proposed experiments will test the hypothesis that CGRP exerts its effects by initiating a cascade of events resulting in desensitization of the cholinergic receptors present on OHCs. The first specific aim is to clone full-length cDNAs for ACh and CGRP receptor subtypes from the guinea pig cochlea using as probes partial cDNAs encoding the guinea pig a9 ACh and CGRP receptors. In situ hybridization techniques will be used to establish which cells in the guinea pig organ of Corti express these receptors. The second specific aim will use immunohistochemical and Western blotting techniques to verify the presence and distribution of proteins for the cochlear ACh a9 and CGRP efferent receptors. The third specific aim employs electrophysiological techniques to investigate whether the cloned CGRP and Ach receptors are able to interact functionally, such that CGRP preincubation causes second messenger effects, which leads to Ach receptor desensitization. Information gained from these studies may contribute to future drug therapies that target the cochlear efferents system, and thus, help decrease the occurrence of, and improve the treatment for patients with high-frequency hearing loss.

DESCRIPTION (provided by applicant): The proposed study will determine the efficacy of in vivo gene delivery to the adult mouse cochlea using a modified 2nd generation adenovirus [El-, E2b-, E3-] and test cochlear function, as assessed by distortion product to acoustic emissions (DPOAEs) and auditory brainstem responses (ABRs). The magnitude of DPOAEs are reliable indicators of functioning outer hair cells, while ABR threshold measures are an indicator of functioning inner hair cells. Our initial findings support the ability of adenovirus to deliver transgenes to guinea pig cochlear hair cells in vivo, but it has not yet been determined if such gene-transter techniques can be used to change endogenous levels of genes present in hair cells, and if gene-transfer can be used to affect functional changes in cochlear physiology. The first Specific Aim is to deliver a modified 2nd generation adenovirus to the adult mouse cochlea. We will also determine if there is any toxicity associated with introducing and expressing a foreign transgene by comparing serum levels of transaminase and liver pathology from animals that have had equal titers of modified 2nd generation adenovirus containing a foreign transgene (LacZ) to that of modified 2nd generation adenovirus infection without a foreign transgene, Throughout the experiment, DPOAEs and ABRs will be monitored to determine if there is any loss of cochlear function due to viral infection and possible immune clearance of hair cells. The second Specific Aim will determine if protein expression in the cochlea can be modulated using virally-mediated gene transfer using the KCN04 channel as a model. We will overexpress functional KCN04 channels and dominant-negative mutant (G285S) KCN04 channels. We will then use gene transfer to determine if over-expression of normal and dominant-negative mutant KCN04 channels can be used to change the ability of adult hair cells to transduce auditory signals. In both Aims we will use molecular biological cloning, virus production, immunohistochemical, Western blotting, DPOAE and ABR measurements, and aseptic surgical techniques. KCNQ4 mutations are found in the human autosomal dominant non-syndromic deafness disorder, DFNA2, and families with this mutation exhibit a significant hearing loss. Once we can mimic human disease using gene transfer, future studies can employ gene transfer techniques to rescue mutant phenotypes. Information gained from these studies will aid development of theraupeutic strategies targeted at genes involved in deafness.

DESCRIPTION (provided by applicant): Difficulty in filtering relevant auditory information in background noise is one of the key features of autism spectrum disorders (ASD) and such filtering difficulties can significantly impair a person's social communication abilities. Parents and teachers often report that children with autism have particular difficulty attending to and understanding speech in noisy environments. These observations are supported by studies using standardized questionnaires to measure sensory functioning. While people with ASD have difficulties filtering speech information in background noise, there may be variability in filtering abilities, and some of this variability maybe be related to processing of: i) the auditory target (speech or non-speech signals), ii) auditory background noise (masker type), and iii) the ability of the auditory system to spatially separate the target from the masker (spatial release from masking). The physiological basis for the ability to filter relevant auditory information is believed to be due to regulation of cochlear activity by the CNS via descending auditory pathways-the olivocochlear efferent system. The medial olivocochlear (MOC) efferent system mediates active micromechanical contractile properties of cochlear outer hair cells (OHCs) which then modulate inner hair cell afferent firing rates. Moreover, the contractile activity of the OHCs can be evaluated in human subjects, because their contractions generate acoustic signals (otoacoustic emissions;OAEs), which can be recorded in the external ear canal, making it possible to directly measure auditory filtering processes at the cochlear periphery. It has previously been shown that adolescents and children with ASD have reduced MOC efferent feedback strengths using transient OAEs (TrOAEs). In Aim 1, we will investigate auditory filtering and spatial release from masking ability in adolescents with high functioning autism and typically developing controls, using both speech and musical tones as targets, and both speech-shaped and synthesized babble noise as maskers. We will use psychophysics and adaptive tracking to measure target thresholds in the different conditions to evaluate whether ASD is associated with impaired performance on speech-specific listening tasks. In Aim 2, we will measure cochlear MOC efferent feedback strength using two different otoacoustic emission-based tests: distortion-product OAEs (DPOAEs) and transient OAEs (TrOAEs) with binaural broadband stimulation to maximally activate MOC efferent feedback. We will test the hypothesis that MOC efferent feedback strength and R/L ear symmetry is impaired in ASD. Information gained from these studies will allow us to determine if a non-invasive measure of efferent feedback strength can serve as a physiological indicator of auditory filtering capabilities in this disorder. PUBLIC HEALTH RELEVANCE: This research will advance our understanding of how individuals with autism can hear speech sounds in the presence of background noise. This ability to filter relevant auditory information is critical for speech perception and the social use of language more generally. Using miniature speaker-microphone earplugs, we will also measure acoustic signals (otoacoustic emissions) generated by sensory cells in the inner ear as these emissions are suppressed in the presence of background noise. We will determine if individuals with autism have reduced noise-induced suppression of these emissions, and if such autism-specific differences in emissions are related to auditory filtering capabilities in this population.

Autism Spectrum Disorder (ASD) is a behaviorally diagnosed disorder of early onset characterized by impairments in social communication and restricted and repetitive behaviors. While accurate diagnosis can reliably be made by 24 months and signs of the disorder are present even earlier, the majority of children with ASD are not diagnosed until after age 4. Some of the earliest signs of ASD involve auditory processing deficits, including absent or atypical response to speech (such as one's name being called). Testing early auditory processing is difficult in very young, minimally verbal children, but the integrity of outer hair cell function can be evaluated reliably using otoacoustic emissions (OAEs). Sound causes contractions of the outer hair cells and generates acoustic signals (OAEs), which can be recorded in the external ear canal. Measuring these signals is noninvasive and reliable, and is a routine approach to testing auditory functioning in children as young as infancy. We have recently shown that high functioning children and adolescents with ASD (with normal hearing thresholds) have reduced OAEs using multiple tests in the 1 kHz frequency region critical for speech, yet the other frequencies tested between 0.5-8 kHz were unaffected. Moreover, we found that lower OAEs in this 1 kHz region were significantly related to greater autism severity in the ASD group. OAEs can also be used to measure the strength of descending pathways from the midbrain to the cochlea. This olivocochlear [OC] efferent system has cell bodies found in the superior olivary complex, which has previously been found to be either absent, greatly reduced, or disorganized in autopsy and MRI studies of adults with ASD. This OC efferent feedback system can be activated with sound and because the medial OC (MOC) system projects to outer hair cells, it is possible to measure efferent feedback processes at the cochlear periphery using OAEs. It has previously been shown that children with ASD have reduced MOC efferent feedback strength and greater right/left (R/L) asymmetries in their responses. In Aim 1, we will investigate baseline cochlear function using two different OAE measures, in young children (4-6 yrs) with ASD, including minimally verbal and verbal subsamples, and age-matched typical controls. We predict that children with ASD will have reduced OAEs in the 1 kHz speech frequency region, with minimally verbal children having more severely reduced OAEs than those with better early language skills. In Aim 2, we will measure MOC efferent feedback strength, again using two different OAE-based tests with binaural stimulation, and test the hypothesis that MOC efferent feedback strength and R/L ear symmetry are impaired in ASD in the 1 kHz speech frequency region, and that these will be more pronounced in minimally verbal children. Information gained from these studies will allow us to determine if non-invasive measures of cochlear function and MOC efferent feedback may serve as objective early indicators of auditory processing differences in ASD.!

Migraine is a severe and chronic neurological disorder that affects ~18% of people worldwide, majority female (3:1). Migraine is characterized by recurrent attacks of debilitating headaches and nausea, with heightened sensory sensitivities, such as light (photophobia) and sound (phonophobia). Recently it has become recognized that ~42% of migraine patients also suffer from balance problems and dizziness, termed vestibular migraine (VM). VM is a major cause of vertigo in dizziness clinics, and is estimated to affect 1% of the overall population. Clinically, triptans (serotonin 5-HT 1B/1D receptor agonists) can reduce photophobia and headaches in migraine, but often do not relieve other VM symptoms. Thus, there is an urgent need for validated preclinical models for VM. The neuropeptide calcitonin gene-related peptide (CGRP) is recognized as a key player in migraine based on the efficacy of CGRP blockers in clinical trials against headache, and injection of CGRP triggers migraine. We have developed CGRP-sensitized transgenic mouse models for migraine that have heightened responses to light (photophobia) when CGRP is delivered either centrally or systemically. We have shown that CGRP blockers can block CGRP-induced photosensitivity in these mice, yet CGRP blockers delivered systemically cannot effectively penetrate the CNS, and these blockers are not effective against centrally-induced photophobia, suggesting that photosensitivity in migraine involves both systemic and central components. CGRP is also widely distributed throughout the vestibular CNS, and CGRP-containing efferent fibers project to all inner ear endorgans. CGRP is also signaling in the vestibular and auditory systems, as we discovered in a different mouse model, that the loss of CGRP reduces the vestibulo-ocular reflex (VOR) gain, reduces otolith- evoked dynamics reduces, reduces sound-evoked activity in the cochlear nerve, and impairs rotarod (balance) performance. Yet, it is not yet known if CGRP is acting centrally or peripherally in VM; which would influence treatment routes and efficacy. We propose to measure mouse homologues of clinical VM symptoms namely; phonophobia, imbalance, nausea, and motion-sickness susceptibility; and will investigate these measures in three mouse lines: i) wildtype (WT) littermate controls and CGRP-sensitized mouse models with ii) neuronally- sensitized CGRP receptors (nestinRAMP1) or iii) globally-sensitized CGRP receptors (global RAMP1). Our hypothesis is that elevated CGRP-sensitivity will contribute to increased sensitivities found in VM; and that these VM symptoms will be ameliorated by CGRP blockers. Information gained from these studies will provide a direct assessment of whether CGRP-sensitized mouse models that have been shown to mirror the photophobia of migraine, can also mirror deficits observed in VM. Such a preclinical model of VM could be used for future translational studies to develop and test new therapeutics. !

This project will determine whether formal musical training is associated with enhanced neural processing and perception of sounds, including speech in noisy backgrounds. Music forms an important part of the lives of millions of people around the world, and it is one of the few universals shared by all known human cultures. Yet its utility and potential evolutionary advantages remain a mystery. This project will test the hypothesis that early musical exposure has benefits that extend beyond music to critical aspects of human communication, such as speech perception in noise. In addition, the investigators will test whether early musical training is associated with less severe effects of aging on the ability to understand speech in noisy backgrounds. Degraded ability to understand speech in noise is a common complaint among older listeners and hearing loss has been shown to be associated with social isolation and more rapid cognitive and health declines. If formal musical training is shown to affect improved perception and speech communication in later life, the outcomes could have a potentially major impact on quality of life,

Earlier studies have suggested relationships between early musical training and improved auditory neural processing and perception, but the studies' impact has been limited by small sample numbers and inconsistent methods between different studies. This project will test a large number of participants (N=360) with uniform recruitment criteria and testing protocols across six different sites. Measures will include the neural frequency following response (FFR) to speech sounds, behavioral frequency selectivity, speech perception in noise, speech perception against a background of competing talkers, pitch discrimination, and auditory masking. The participants will also complete other assessments, including a personality inventory questionnaire, a profile of musical perception skills, a spatial reasoning test to assess general cognitive ability, as well as a background questionnaire to determine socio-economic status, education, and musical background. Participants will be selected to span a wide range of ages and musical experience. The neural data and the speech perception measures will be related to factors of musical training, such as the number of years of musical training and the age at which musical training began. Scientific rigor will be assured by preregistering the study and the analyses and by making the data and analysis code publicly available via a dedicated website.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The aims of this research proposal are to provide a definitive answer to the question of whether formal musical training is associated with enhanced perception of sound. Musicians have been reported to possess enhanced auditory processing of sounds both in quiet and in noise ? a result of processes that may begin as early in the auditory pathway as the cochlea itself. These enhancements may underlie improved abilities to understand speech-in-noise, and because of their profound implications, these studies have generated tremendous interest. However, many of the conclusions were based on studies that had i) relatively few participants, ii) mostly adult participants and iii) no assessment of audiometric or cochlear function in the extended high-frequency (EHF) ranges. These proposed experiments are aimed to remedy these deficiencies by recruiting 150 youth musicians, and assessing both audiometric and cochlear EHF regions. Interestingly, even in people with clinically normal hearing thresholds there are differences in hearing in noise abilities. In fact, while there is no clear relationship between normal standard thresholds (0.25-8kHz) and hearing in noise abilities, there is a proposed relationship between extended high-frequency (EHF) hearing thresholds (8-20 kHz) and improved hearing in noise abilities. In addition to audiometric EHF thresholds, cochlear outer hair cell health can also be assessed non-invasively using distortion-product otoacoustic emissions (DPOAEs) both in the standard and EHF frequency ranges. As mentioned early, most previous studies have sorted their adult participants into ?musicians? or ?non- musicians? based on years of musical training. Our laboratory has recently shown (in both children and adults) that musical aptitude and musical training significantly predict hearing-in-noise abilities. Our hypothesis is that this `musician advantage' in auditory processing is related to both EHF cochlear function and musical aptitude. For both aims, we will recruit ~150 youth (11-18 years) who are students at The Hochstein School as well as other area youth musicians. The Hochstein School enrolls ~ 715 students/year in this age range in their classes and ensembles, providing a natural recruitment platform to assess these behavioral and physiological measures. The specific aims are to investigate whether the `musician advantage' in auditory processing is related to i) EHF audiometric and cochlear function and ii) musical aptitude. Information gained from these studies in youth musicians will provide a rigorous assessment of whether the `musician advantage' in auditory processing exists for all types of sound, and if this advantage is related to musical aptitude, musical training, and EHF cochlear health. We will also be examining what extent music and speech hearing-in-noise abilities overlap, and will be establishing norms and outcome measures that can be used to assess future music-based interventions.

The purpose of this COVID-19 research supplement is to critically evaluate if a calcitonin gene-related peptide (CGRP) receptor antagonist can mitigate both neuroinflammatory and hyper-immune responses to SARS-CoV-2 infection. In December 2019, the coronavirus disease (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) was identified. There are now over ~5.5 million confirmed cases worldwide (1.6 million US), and 345,000 deaths (~100,000 US). COVID-19 causes a respiratory illness like the flu with symptoms such as fever, cough, loss of smell, fatigue, sputum production, shortness of breath, sore throat, headache, chills, and nausea or vomiting. Approximately 80% of people have mild disease and recover. However, in those remaining 20%, COVID-19 is severe and there is evidence that progression to the most serious type of COVID-19 illness is related to a hyper-immune response (ie, cytokine storm). Currently, there are no effective vaccines or treatments available for COVID-19. In this supplement we will test the ability of CGRP-receptor antagonists to inhibit the neuroimmune consequences of SARS-CoV-2 infection, using temperature and nausea as an indicator of SARS-CoV2 infection, as we are doing in our parent grant to assess migraine nausea pain. A humanized mouse model has been developed for studying SARS-CoV2, where mice express the human angiotensin-converting enzyme 2 (hACE2), enabling us to model Covid-19 in the mouse. The FDA has recently approved Biohaven Pharmaceuticals to proceed to a phase 2 clinical trial of its CGRP-receptor antagonist (vazegepant; currently in phase 3 trials for migraine) to treat patients with severe COVID-19, suggesting that the neuroinflammatory reaction that is initiated by CGRP in response to SARS- CoV2 could be a therapeutic target for treating severe Covid-19, and that the non-invasive readouts of neuroinflammation that we are developing could be used to rapidly identify at risk patients. Our hypothesis is that mild to severe COVID-19 symptoms will occur in the transgenic hACE2 mouse that has been infected with SARS-CoV-2, and that a CGRP receptor antagonist will mitigate these symptoms. The specific aims are to test the following hypotheses that transgenic mice and non-carrier littermates infected with SARS-CoV-2 will exhibit: aim 1) mild severe symptoms based on viral load, and if these symptoms are less severe when treated with a CGRP-receptor antagonist; and aim 2) reduced fever and nausea-like pain when treated with a CGRP- receptor antagonist. Information gained from these studies will provide a direct assessment of whether a CGRP-receptor antagonist can mitigate both mild and severe symptoms associated with SARS-CoV-2 infection. This proposal also impacts the development of robust preclinical in vivo assays of COVID-19 symptoms, paving the way to develop and test future therapeutics for COVID-19.

Currently, 5.5 million Americans (~10%) over age 65 have Alzheimer's disease (AD), and this number will more than double by 2050. AD is definitively diagnosed from post-mortem analyses of neurodegenera- tion with the presence of ?-amyloid (A?) containing plaques and tau-containing neurofibrillary tangles. As A? accumulation in the brain can precede cognitive impairment by decades, treatments aimed at facilitating clearance of A? are most effective at early stages, when the disease is difficult to diagnose. The inability to detect AD early enough to apply meaningful interventions is a major barrier to AD's successful manage- ment. AD patients have a higher prevalence of vestibular impairment relative to healthy age-matched controls, with postural sway serving as an excellent predictor of the overall cognitive assessment score in AD. Gait metrics discriminated better between patients with mild cognitive impairments and healthy con- trols, more so than verbal-fluency tests. Additionally, patients with AD have difficulty suppressing incongru- ent brain signals when maintaining balance compared to age-matched controls. In addition to vestibular imbalances, AD patients have difficulties understanding speech in background noise, a deficit known as auditory processing disorder (APD). The mechanisms of APD are unclear, yet circuitry in auditory-specific regions of the brainstem are known to play a role in speech processing and noise masking. Moreover, better peripheral frequency resolution thresholds correlate with improved hearing in noise abilities. Preclinical models have been extremely useful to test hypotheses about AD pathophysiology and to assess putative interventions. However, it is unknown if current AD mouse models exhibit alterations in postural sway, gait, or APD, as observed in early pre-dementia AD patients. It is also not well established if increased A? deposition occurs in the brainstems of AD mouse models, similar to post-mortem histological evidence of early A? deposition in brainstems of patients with little evidence of cognitive impairment. We propose to measure mouse homologues of these early AD symptoms in the 5xFAD AD mouse model crossed to mouse lines without age-related hearing loss. In Aim 1, we will measure sway and gait distur- bances. In Aim 2, we will use auditory brainstem responses (ABRs) measured in simultaneous notched noise to measure peripheral frequency resolution thresholds and auditory filter shapes. Lastly, we will determine if these deficits correlate with plaque deposition, demyelination, or inflammation in the vestibular and auditory brainstem and CNS. The impact of this proposal will be in developing robust preclinical assays of early AD, paving the way to develop and test future therapeutics for pre-dementia AD.