Kelvin Y. Kwan - US grants

DESCRIPTION (provided by applicant): Auditory neuropathy caused by the loss of spiral ganglion neurons (SGN) loss results in hearing loss. Standard remediation, such as the use of hearing aids and cochlear implants are ineffective after the loss of SGNs. Many daily ototoxic insults such as loud sounds result in acute loss of synaptic contacts, degeneration of the auditory nerve fibers and loss of SGN over an extended period of time. Given an ever-increasing aging population that is exposed to loud sounds from portable music players, degeneration of SGNs will become a major health concern. Stem cell replacement therapies hold great promise to repopulate lost SGNs and restore hearing function. In order to properly use stem cells to treat hearing loss, identifying genes that can recapitulate SGN development in stem cells will accelerate efforts for replacement therapies. Using an immortalized otic progenitor (iMOP) cell line, we identified novel candidate transcription factors and chromatin remodeling proteins that are important in SGN development and regeneration. One of the candidates is Chd4, a chromatin remodeling protein that is part of the nucleosome remodeling and deacetylase (NuRD) complex. We propose that Chd4 is essential for neuronal specification, axon guidance and synaptogenesis during SGN development. We will test the role of Chd4 in specifying neuronal fate and neurite extension using iMOP-derived neurons and an inner ear Chd4 knockout animal. Using an iMOP-derived neuron and deafferented cochlear explant co-culture system, we will study how Chd4 promotes axon guidance to the hair cell targets. Finally, we will generate a tamoxifen inducible Chd4 knockout animal to determine defects in synaptogenesis during SGN maturation.

PROJECT SUMMARY Hearing loss during early childhood significantly affects learning and acquisition of social skills, while hearing loss in adults can often result in social isolation and inability to perform many routine social functions. A leading cause of sensorineural hearing loss is the loss of sensory hair cells of the inner ear. A lifetime exposure to aminoglycoside and loud sounds will result in an estimated 15% of adult Americans (~36 million) having some form of hearing loss. A promising approach to mitigate hearing loss and deafness is a cell replacement therapy by transdifferentiating supporting cells into hair cells. Unfortunately, current approaches for transdifferentiation rely on viral delivery may be unsafe and impractical for clinical translation. Therefore, there is a critical need to develop alternative platforms to regulate gene expression and induce transdifferentiation in an efficient, non-viral manner suitable for hearing restoration. To this end, our long-term goal is to develop NanoScript, an innovative, tunable nanoparticle-based artificial transcription factor platform capable of effectively regulating gene expression in a non-viral manner. Using NanoScript, we will transdifferentiate supporting cells into functional hair cells. NanoScript consists of a nanoparticle functionalized with specific small molecules and peptides that are designed to mimic the individual domains of natural transcription factor (TF) proteins. TFs are endogenous, multi-domain proteins that orchestrate many cellular functions including differentiation. Since NanoScript is a functional replica of TF proteins, it can replace virally-delivered TFs for regenerative medicine-based applications. The overall objective of this proposal is to design three NanoScripts that mimic three TFs essential for hair cell differentiation (Gfi1, Pou4f3, and Atoh1; GPA). We will test whether GPA-NanoScript binds to the same DNA sequence and activate gene expression in vitro. Next we will determine if addition of epigenetic modulators to GPA-NanoScript will bind to the same targets as the TF proteins, locally alter the chromatin structure and enhance gene expression. Finally, we will use cochlear explants to determine whether GPA-NanoScript promotes transdifferentiation of supporting cells into hair cells. Generation of nascent hair cells using an ex vivo model will serve as a springboard to test NanoScript technology for regenerative medicine. It will also establish NanoScript as an effective and non-viral tool for researchers to generate functional cells via direct reprogramming.

ABSTRACT Epigenetic regulation of gene expression occurs via heritable changes in DNA and associated histone proteins. Such modifications, which include methylation, acetylation, and nucleosome repositioning, have a major and poorly understood role in development and disease. Recent studies have begun to explore epigenetics of hearing and balance disorders which negatively impact quality of life and impose a significant socioeconomic burden on millions of Americans. In both children and adults with hearing or balance disorders, development of the cochlear epithelium, vestibular epithelia and associated neurons are often disrupted. During inner ear development, neurosensory progenitors from the otic vesicle give rise to sensory hair cells and vestibulocochlear neurons. Mutations and epigenetic changes in genes that perturb otic development often cause improper hair cell and neuron formation, resulting in hearing loss. The chromodomain helicase DNA binding protein 7 (CHD7) is an ATP dependent epigenetic chromatin remodeler implicated in inner ear development. Mutations in CHD7 cause CHARGE syndrome (ocular Coloboma, Heart defects, Atresia of the choanae, Retardation of growth and development, Genital hypoplasia and pubertal delay, and Ear abnormalities). Patients with CHD7 loss are often diagnosed with mixed conductive and sensorineural hearing loss; however, the pathogenic mechanisms that cause sensorineural hearing loss are not known. In the inner ear, CHD7 is dynamically expressed in neurosensory progenitors, mesenchyme, sensory epithelium, and other otic cell types. However, it is unclear which otic cell type(s) and what type of cis-regulatory element(s) are perturbed in the presence of pathogenic CHD7 mutations. In addition, CHD7 has been shown to reposition nucleosomes in vitro, yet the chromatin remodeling activity of CHD7 in vivo during otic development has not been determined. Enrichment of CHD7 at different cis-regulatory elements is cell type dependent, and our preliminary studies have identified CHD7 binding to the promoter of long noncoding RNA transcripts preceding neuronal differentiation in immortalized multipotent otic progenitors (iMOPs). We hypothesize that CHD7 forms a chromatin remodeling complex in otic neurosensory progenitors and binds to cis-regulatory elements to regulate transcription. We will test our hypothesis using a combination of mouse genetics, single-cell sequencing approaches, and super-resolution microscopy. Results from these studies will help identify mechanisms underlying sensorineural hearing loss, enhance understanding of epigenetic regulation of inner ear neurosensory cell development, and contribute knowledge to help design regenerative or restorative therapies for the inner ear.

ABSTRACT Sporadic Alzheimer?s disease (AD) is an age-dependent neurodegenerative disorder characterized by progressive cognitive dysfunction and memory loss. Pathogenic ?-amyloid (A?) plaques are a hallmark of AD, starting first in the hippocampus before spreading to other parts of the brain. Initial research has primarily focused on how A? aggregates are formed. Despite progress made in understanding progression of AD pathology, fundamental questions remain about what molecular determinants contribute to the onset of AD. Age-dependent genomic changes likely play a role in AD but the molecular players involved have yet to be defined. We show that Chd4, a nucleosome repositioning enzyme, dramatically decreases expression with age in pyramidal neurons of the hippocampus. We propose that Chd4 deletion mimics the epigenomic landscape of old neurons to accelerate the onset of AD pathology. The goal of the proposal is to address the function of Chd4 in the context of AD. We will use tools in single-cell transcriptomics and super-resolution microscopy to determine the molecular changes that occur in pyramidal neurons (Aim1) and the composition of synaptic proteins as well as the integrity of neuronal circuits in the hippocampus (Aim2) after deleting Chd4. We will also determine if A? plaques and memory loss are observed in animals lacking Chd4 (Aim3).

PROJECT SUMMARY Hearing loss during early childhood significantly affects learning and acquisition of social skills, while hearing loss in adults can often result in social isolation and the inability to perform many routine social functions. A leading cause of sensorineural hearing loss is the loss of sensory hair cells of the inner ear. A lifetime exposure to aminoglycoside and loud sounds will result in an estimated 15% of adult Americans (~36 million) having some form of hearing loss. A promising approach to mitigate hearing loss and deafness is a cell replacement therapy by transdifferentiating supporting cells into hair cells. Unfortunately, current approaches for transdifferentiation rely on viral delivery may be unsafe and impractical for clinical translation. Therefore, there is a critical need to develop alternative platforms for regulating gene expression and inducing transdifferentiation in an efficient, non- viral manner that is suitable for restoration of hearing. To this end, our long-term goal is to develop NanoScript, an innovative, tunable nanoparticle-based artificial transcription factor platform capable of effectively regulating gene expression in a non-viral manner. Using NanoScript, we will transdifferentiate supporting cells into functional hair cells. NanoScript consists of a nanoparticle functionalized with specific small molecules and peptides that are designed to mimic the individual domains of natural transcription factor (TF) proteins. TFs are endogenous, multi-domain proteins that orchestrate many cellular functions, including differentiation. Since NanoScript is a functional replica of TF proteins, it can replace virally-delivered TFs for regenerative medicine-based applications. The overall objective of this proposal is to design three NanoScripts that mimic three TFs essential for hair cell differentiation (Gfi1, Pou4f3, and Atoh1; GPA). We will test whether GPA-NanoScript binds to the same DNA sequence and activate gene expression in vitro. Next, we will determine if the addition of epigenetic modulators to GPA-NanoScript will bind to the same targets as the TF proteins, locally alter the chromatin structure and enhance gene expression. Finally, we will use cochlear explants to determine whether GPA-NanoScript promotes transdifferentiation of supporting cells into hair cells by single-cell transcriptome analysis. Generation of nascent hair cells using an ex vivo model will serve as a springboard to test NanoScript technology for regenerative medicine. It will also establish NanoScript as an effective and non-viral tool for researchers to generate functional cells via direct reprogramming.

PROJECT SUMMARY Hearing loss during early childhood significantly affects learning and acquisition of social skills, while hearing loss in adults can often result in social isolation and the inability to perform many routine social functions. A leading cause of sensorineural hearing loss is the loss of sensory hair cells of the inner ear. A lifetime exposure to aminoglycoside and loud sounds will result in an estimated 15% of adult Americans (~36 million) having some form of hearing loss. A promising approach to mitigate hearing loss and deafness is a cell replacement therapy by transdifferentiating supporting cells into hair cells. Unfortunately, current approaches for transdifferentiation rely on viral delivery may be unsafe and impractical for clinical translation. Therefore, there is a critical need to develop alternative platforms for regulating gene expression and inducing transdifferentiation in an efficient, non- viral manner that is suitable for restoration of hearing. To this end, our long-term goal is to develop NanoScript, an innovative, tunable nanoparticle-based artificial transcription factor platform capable of effectively regulating gene expression in a non-viral manner. Using NanoScript, we will transdifferentiate supporting cells into functional hair cells. NanoScript consists of a nanoparticle functionalized with specific small molecules and peptides that are designed to mimic the individual domains of natural transcription factor (TF) proteins. TFs are endogenous, multi-domain proteins that orchestrate many cellular functions, including differentiation. Since NanoScript is a functional replica of TF proteins, it can replace virally-delivered TFs for regenerative medicine-based applications. The overall objective of this proposal is to design three NanoScripts that mimic three TFs essential for hair cell differentiation (Gfi1, Pou4f3, and Atoh1; GPA). We will test whether GPA-NanoScript binds to the same DNA sequence and activate gene expression in vitro. Next, we will determine if the addition of epigenetic modulators to GPA-NanoScript will bind to the same targets as the TF proteins, locally alter the chromatin structure and enhance gene expression. Finally, we will use cochlear explants to determine whether GPA-NanoScript promotes transdifferentiation of supporting cells into hair cells by single-cell transcriptome analysis. Generation of nascent hair cells using an ex vivo model will serve as a springboard to test NanoScript technology for regenerative medicine. It will also establish NanoScript as an effective and non-viral tool for researchers to generate functional cells via direct reprogramming.