Jianxin Bao - US grants

Age-related hearing loss (Presbycusis) is a major health concern for the elderly population. Loss ofspiral ganglion neurons (SGNs) is consistently associated with Presbycusis. The sequence of causative molecular events for age-related loss of SGNs is unknown. This proposal focuses on possible roles of Neuregulin-1 may have on this aging process. My recent work revealed: (a) the cytoplasmic domain of Neuregulin-I (NRG-CD) is highly expressed in SGNs; (b) NRG-CD can translocate into the nucleus and regulate gene expression including that of apoptotic genes; and (c) during aging there is a correlation between nuclear translocation of NRG-CD and up-regulation of BAK, a pro-apoptotic gene. The general hypothesis is that NRG-CD plays an essential role in age-related loss of SGNs. I will first determine the temporal profile of NRG-CD nuclear translocation and subsequent gene expression profile during age-related SGN loss. I will then assess how direct manipulation of nuclear NRG-CD in vivo influences age-related loss of SGNs. Furthermore, I will decipher gene expression profiles directly regulated by NRG-CD nuclear translocation during age-related loss of SGNs. The ultimate goal of this research is to develop methods for prevention and treatment of age-related loss of SGNs based on identifying molecular candidates involved in this aging process. The career development of the candidate during this award will be provided by the mentor: Loma Role (for neuroscience and physiological expertise), and two outstanding co-mentors: Ronald Liem (for aging and molecular expertise), and Robin Davis (auditory neuroscience expertise). In addition, collaborative relationships with three experts on transgenic and microarray methods have also been arranged with Drs. Richard Axel, Rene Hen, and Anthony Ferrante. From the conduct of this research under their supervision, the candidate will expand his capabilities with advanced methods of transgenic and DNA microarray technologies, acquire new skills in molecular and genetic analysis of aging. The goal of this award is to elucidate the role of NRG-CD in age-related loss of SGNs as well as to provide the candidate with the continuity of support necessary to develop into an independent investigator.

DESCRIPTION (provided by applicant): One major health concern for the elderly population is age-related hearing loss (presbycusis), which is consistently associated with age-related loss of hair cells and spiral ganglion neurons (SGNs). The sequence of causative molecular events for age-related loss of SGNs is unknown. Certain nicotinic acetylcholine receptors (nAChRs) are highly expressed in SGNs. Recently, nAChRs were found to contribute to age-related neuronal degeneration in the central nervous system, and it is unknown whether nACHRs also contribute to age-related loss of neurons in the peripheral nervous system. Preliminary works have revealed: (a) alterations of the expression level of alpha 4, alpha 5, and beta 2 nAChR subunits in SGNs during aging; and (b) an accelerated age-related hearing loss and correspondent loss of SGNs in mice lacking the beta 2 subunit, but not in mice lacking the alpha 5 subunit. This application focuses on possible roles nAChRs may have on age-related loss of SGNs. The general hypothesis is that certain nAChR subunits play an essential role in age-related loss of SGNs. The two specific Aims are: (a) to determine the temporal profile of nAChRs expression during age-related SGN loss, and (b) to assess how direct manipulation of nAChRs in vivo influences age-related loss of SGNs. This application introduces a powerful new approach, the conditional tissue-specific transgenic method, to directly test possible involvements of nAChRs in the age-related loss of SGNS. The ultimate goal of this research is to develop methods for prevention and treatment of age-related loss of SGNs, based on identifying molecular candidates involved in this aging process.

DESCRIPTION (provided by applicant): Age related functional decline of nervous system is consistently observed, the sequence of causative molecular events for age related functional decline is unknown. This proposal focuses on possible roles of transmembrane Neuregulin-1 (Nrg-1) may have on this aging process. Our recent work revealed: (a) the cytoplasmic domain of Neuregulin-1 (Nrg-ICD) can translocate into the nucleus and regulate gene expression including that of PSD-95 and apoptotic genes;(b) Nrg-ICD regulates PSD-95 expression by binding to a transcription factor, Eos, and (c) during aging there is a correlation between nuclear translocation of Nrg-ICD and up-regulation of PSD-95 and BAK, a pro-apoptotic gene. The general hypothesis is that NRG-1 plays an essential role in age related neuronal changes. To test this hypothesis, we have made a conditional tissue-specific transgenic mouse model, which conditionally expresses Nrg-1 in several specific regions including hippocampus, spiral ganglion neurons, and outer-hair cells. By taking advantage of this mouse model, we will examine three related issues: (1) whether conditional overexpression of Nrg-1 in adult mice can alter age related hearing loss;(2) whether a conditional overexpression of Nrg-1 in adult mice can alter age related neuronal changes in hippocampus;(3) whether caloric restriction, the most effective way to delay the aging process, affects the role of Nrg-1 signaling pathways on age related neuronal changes. The ultimate goal of our research is to develop methods for prevention and treatment of age related functional decline of nervous system based on identifying molecular candidates involved in the aging process

[unreadable] DESCRIPTION (provided by applicant): Age related functional decline of nervous system is consistently observed, the sequence of causative molecular events for age related functional decline is unknown. This proposal focuses on possible roles of transmembrane Neuregulin-1 (Nrg-1) may have on this aging process. Our recent work revealed: (a) the cytoplasmic domain of Neuregulin-1 (Nrg-ICD) can translocate into the nucleus and regulate gene expression including that of PSD-95 and apoptotic genes; (b) Nrg-ICD regulates PSD-95 expression by binding to a transcription factor, Eos, and (c) during aging there is a correlation between nuclear translocation of Nrg-ICD and up-regulation of PSD-95 and BAK, a pro-apoptotic gene. The general hypothesis is that NRG-1 plays an essential role in age related neuronal changes. To test this hypothesis, we have made a conditional tissue-specific transgenic mouse model, which conditionally expresses Nrg-1 in several specific regions including hippocampus, spiral ganglion neurons, and outer-hair cells. By taking advantage of this mouse model, we will examine three related issues: (1) whether conditional overexpression of Nrg-1 in adult mice can alter age related hearing loss; (2) whether a conditional overexpression of Nrg-1 in adult mice can alter age related neuronal changes in hippocampus; (3) whether caloric restriction, the most effective way to delay the aging process, affects the role of Nrg-1 signaling pathways on age related neuronal changes. The ultimate goal of our research is to develop methods for prevention and treatment of age related functional decline of nervous system based on identifying molecular candidates involved in the aging process [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Noise is the most common occupational and environmental hazard. Noise-induced hearing loss (NIHL) is the second most common form of sensorineural hearing deficit, after age-related hearing loss (presbycusis). Although promising approaches have been identified for reducing NIHL that are mainly based on the free radical pathway, currently there are no effective medications to prevent NIHL. Development of an efficacious treatment has been hampered by the complex array of cellular and molecular pathways involved in NIHL. In this proposal, we have turned this difficulty into an advantage by asking whether NIHL can be effectively prevented by a combination therapy targeting multiple signaling pathways. The immediate goal of this work is to explore this therapeutic direction for NIHL. The long-term goal is to develop an effective drug combination that dramatically prevents permanent noise-induced hearing loss. We have recently found that antiepileptic drugs blocking T-type calcium channels have both prophylactic and therapeutic effects for NIHL. NIHL can also be prevented by an up-regulation of glucocorticoid signaling pathways. In the R21 phase of this application, we will develop a combination therapy with anticonvulsants and synthetic glucocorticoids in one mouse NIHL condition, which has dramatic changes for both temporary and permanent threshold shifts. In the R33 phase, we will expand to two other NIHL mouse models that are similar to human conditions caused by a nearby explosion or manufacturing noise. We will also determine if the similar combination therapy can ameliorate permanent hearing loss in chinchillas. The innovative aspects of our approach are two-fold: (1) we will focus on FDA-approved drugs, which have already been extensively studied in animals with abundant pharmacological information; (2) the strategy of our combination therapy is to intervene in multiple signaling pathways instead of focusing only on the well-studied free radical pathway. This pharmacological approach may also reveal novel molecular targets critical to the development of NIHL. We are aware of the high-risk nature of this project because we do not know whether intervention in multiple signaling pathways will have a synergistic effect against NIHL. However, synergistic effects have been observed in drug combination treatments for acquired immunodeficiency syndrome and cancers. Our study can potentially have a high impact not only in the field of NIHL but also in other areas of communication science. For example, if successful, a similar approach can be explored for the treatment of presbycusis and tinnitus. In summary, this proposal will develop a paradigm-shifting approach to test novel drug combinations against NIHL.

DESCRIPTION (provided by applicant): Age-related hearing loss (presbycusis) is the most common neurodegenerative disease, afflicting nearly half of the population over 75 years of age. According to the National Council on Aging, presbycusis leads to decreased quality of life, can increase emotional distress such as sadness and depression, and leads to withdrawal from family, friends, and community. Current treatment options, which only a fraction of older adults with presbycusis receive, are primarily limited to hearing aids, which often fail to restore optima auditory function. We recently discovered that trimethadione and ethosuximide, two antiepileptic drugs that block T-type calcium channels, can effectively ameliorate noise-induced and age-related hearing loss in mice. Unfortunately, both drugs cause undesirable side effects at dosages used for epilepsy prophylaxis. Here, we propose two specific aims to translate our findings for potential use in humans. The studies in specific aim 1 will allow us to determine the pharmacodynamic properties of ethosuximide and related compounds against presbycusis. These experiments will provide structure-activity relationship information to facilitate chemical optimization of these compounds against presbycusis. The data will also address whether ethosuximide and related compounds can attenuate presbycusis at dosages lower than those prescribed when the drugs are used as anticonvulsants. In specific aim 2, we will develop combination therapies targeting both calcium and free radical pathways. Combination therapies will include one inhibitor of T-type calcium channels (either ethosuximide or zonisamide) plus one or two antioxidants (vitamin C or E). If a synergistic effect is discovered, the dosage of each compound can be reduced. We will also test whether the same combinations can prevent accelerated age-related hearing loss in mice exposed to noise at a young age. After treatment, quantitative histological analysis of cochleae will be used to identify cellular targets. A batteryof behavioral tests will be used to evaluate potential neurological side effects. We expect this project to result in new drugs or drug combinations that effectively mitigate presbycusis. Importantly, because we focus on the discovery of new uses for drugs already approved by the U.S. Food and Drug Administration, our work has the potential for rapid translation into clinical practice. Overall, this project represents an extraordinary opportunity that brings a multidisciplinary team together for the purpose of developing an effective drug-based intervention for presbycusis.

? DESCRIPTION (provided by applicant): Noise is the most common occupational and environmental hazard, thus it is not surprising that noise-induced hearing loss (NIHL) is the second most common form of sensorineural hearing deficit, second only to age-related hearing loss. Although therapeutics that target the free radical pathway have shown promise for reducing NIHL, there are no medications approved by the U.S. Food and Drug Administration for NIHL. Development of an efficacious treatment has been hampered by the complex array of cellular and molecular pathways involved in NIHL. Therefore, a new approach is necessary to combat these multiple signaling pathways in order for therapeutics to be successful at preventing and treating NIHL. Recent preclinical studies have demonstrated that calcium channel blockers and anti-inflammatory drugs can prevent NIHL. Tetrandrine (TET), a bis-benzylisoquinoline alkaloid originally purified from a Chinese medicinal herb, shows strong promise as a NIHL therapeutic due to its antioxidant and anti-inflammatory properties, as well as its ability to block calcium channels. Moreover, the use of TET clinically has shown a long-term safety profile. Based on our preliminary findings that TET could protect against NIHL, in this Phase I STTR application, we will determine the median effective dose (ED50) for this drug to prevent (Aim 1) and treat (Aim 2) NIHL in two different animal models. The innovative aspects in our approach for using TET are: (1) TET already has well-characterized pharmacological properties and a long history of clinical applications; and (2) TET is known to intervene in multiple signaling pathways involved in NIHL. We will use the data from these studies to complete an Investigational New Drug (IND)-enabling data package for our SBIR phase II clinical studies of this agent. The goal of this program is to bring TET to market for use in prevention and treatment of NIHL.

PROJECT SUMMARY In spite of recent new drug developments, platinum-based drugs such as cisplatin are still widely used to treat solid organ malignancies. Besides its limited efficacy, serious side effects have been associated with the use of cisplatin, such as bilateral and irreversible hearing loss. In the cochlea, cisplatin can trigger the production of reactive oxygen species, and activate several other signaling pathways. A variety of agents, mainly based on their antioxidant properties, have been tested against cisplatin-induced ototoxicity. However, many of them show limited efficacies, and also interfere with the therapeutic effect of cisplatin. Extensive in vitro studies have indicated that flunarizine (Sibelium), a drug that blocks T-type calcium channels, can protect cochlear cells against cisplatin-induced cytotoxicity. This drug also has strong anti-tumor activities that act synergistically on several important aspects of cancer treatment. Our preliminary studies have found that flunarizine can synergistically induce cell death with cisplatin in one lung cancer cell line, and it can also protect noise-induced hearing loss. Based on these findings, we propose to develop a new cancer drug combination by testing whether flunarizine can synergistically induce cancer cell death with cisplatin, and at the same time, prevent cisplatin-induced ototoxicity. Because lung cancer is the leading cause of cancer-related death globally, and cisplatin is widely used to treat this disease, here, we will first determine the cytotoxic effects of flunarizine and cisplatin in two lung cancer cell lines: A549 and H1975, and determine whether flunarizine and cisplatin have synergistic effects in these cancer cells when used in combination (Aim 1). We will then determine whether flunarizine can prevent cisplatin- induced ototoxicity in vivo (Aim 2). In short, based on previous studies and our preliminary data, our project goal is to repurpose flunarizine to combine with cisplatin against solid tumors with a focus on lung cancer. This project will generate data important to complete an Investigational New Drug (IND)-enabling data package for future clinical studies. The ultimate goal is to develop an effective cancer drug combination with fewer side effects.

PROJECT SUMMARY The most common occupational and environmental hazard in this country is noise. It's not surprising, then, that noise-induced hearing loss (NIHL) is the second most common form of sensorineural hearing deficit, behind only age-related hearing loss (presbycusis). Although therapeutics that target the free radical pathway have shown promise for reducing NIHL, there are no effective nutraceuticals or approved medications for NIHL. Development of an efficacious treatment has been hampered by the complex array of cellular and molecular pathways involved in NIHL. Our preliminary studies have shown that the Chinese herb Stephania tetrandra and its main extract, tetrandrine (TET), can effectively protect against NIHL in mice. Clinical use of this herb has exhibited an excellent long-term safety profile. Moreover, TET possesses strong antioxidant and anti-inflammatory properties, and can also block calcium channels, thus targeting three main signaling pathways that underlie NIHL. Consistent with the funding priorities at National Center for Complementary and Integrative Health, in this Phase I application, we will determine the median effective dose (ED50) for this herb extract to prevent (Aim 1) and treat (Aim 2) NIHL in rats using a long noise exposure model (8 hours per day) similar to most industrial working environments. The innovative aspects of our approach are: (1) developing a nutraceutical against NIHL, thus breaking a major access barrier for customers, who are more likely to visit audiologists for hearing treatment; (2) testing in a rat model with cumulative noise exposure similar to that encountered by factory workers; (3) selecting an herb with well-characterized pharmacological properties and a long safety history of clinical applications; and (4) targeting multiple signaling pathways that underlie NIHL. If the outcome of this study is successful, we will then obtain institutional review board (IRB) approval to conduct SBIR Phase II studies in humans of this nutraceutical against NIHL. The goal of our program is to bring this nutraceutical to market for use in the prevention and treatment of NIHL. If this approach is successful, similar nutraceuticals will be evaluated for potential prevention of presbycusis.

Abstract Noise is the number one occupational and environmental hazard, and noise- induced hearing loss (NIHL) is one common form of sensorineural hearing deficit, only second to age-related hearing loss. Currently no drug preventing NIHL is approved by the United States Food and Drug Administration (FDA). One major challenge for the introduction of such interventions is the need to identify appropriate human populations available for evaluating the effectiveness of new otoprotective agents. In this STTR Phase I project, we propose to determine the extent of hearing loss in unoperated ears of patients after drilling during skull-based surgery. Based on our preliminary studies, we have reduced the variability of electrocochleography for detecting hearing loss. By working with our collaborators at Washington University School of Medicine in St. Louis, in two out three patients, we have detected temporal hearing threshold shifts in unoperated ears after the skull-based surgery. A recent seminal discovery in animals revealed that, even without permanent threshold shift, a loss of synapses between inner hair cells and spiral ganglion neurons in the inner ear is the primary pathology for NIHL. This form loss of synapses is called cochlear synaptopathy, or hidden hearing loss. So far, no longitudinal studies have been reported to confirm the existence of hidden hearing loss in people although two human studies implicated possibility of cochlear synaptopathy by comparing groups with different risks for noise exposure. Here, we will continue to recruit qualified patients scheduled for skull-based surgery. Functional assays, such as audiometric testing, electrocochleography, distortion product otoacoustic emissions, and the Words-in-Noise test with multi-talker babble will be repeated on their unoperated ears before the surgery, one day and one-month post- surgery. By the project?s end, we expect to quantify the extent of both temporary and permanent threshold shift in patients; and confirm the existence of hidden hearing loss in a certain percentage of these volunteers. The main innovative aspect of this study is to identify a new population with unavoidable noise exposure under a laboratory-like setting using improved functional assays. If successful, In the STTR or SBIR Phase II project, we will carry our FDA-approved clinical trial (NCT02049073) based on this population. Thus, this project will not only identify a new population for future clinical studies of new otoprotective agents, but also the first epidemiological study of hidden hearing loss in this population. Our long-term goal is to develop an effective medical intervention for NIHL.

PROJECT SUMMARY Despite recent advances in cancer therapies, platinum-based drugs are still widely used for the treatment of solid tumors. A primary example is cisplatin, which is frequently used to treat lung, bladder, testicular, and cervical cancers. However, limitations of this therapy include the development of drug resistance in cancer cells as well as severe side effects, such as nephrotoxicity and neurotoxicity. One major type of peripheral sensory neurotoxicity is ototoxicity, or drug-induced damage to the inner ear, and cisplatin is the most ototoxic compound that can cause bilateral and irreversible hearing loss. While DNA damage by cisplatin is the major cytotoxic mechanism in cancer cells, cisplatin-induced ototoxicity appears to result from toxic levels of reactive oxygen species and other signaling dysregulation within various cellular compartments. A variety of agents have been tested against cisplatin-induced ototoxicity, mainly based on their antioxidant properties. However, many of them show limited efficacies, and also interfere with the therapeutic effect of cisplatin. In our preliminary studies, we have found that tetrandrine (TET), a compound isolated from the herb Stephanai tetrandra, can protect against noise-induced hearing loss. Previous studies have also revealed that TET can act synergistically with other cancer treatments in several important aspects: increasing tumor cell cytotoxicity, enhancing radiosensitization, reducing multidrug resistance, and inhibiting angiogenesis. At the molecular level, TET can block calcium channels, downregulate multiple drug-resistant proteins, and modulate signaling pathways important for the inflammatory response. Based on these findings, we propose a proof-of-concept project to test whether TET can synergistically work with cisplatin to induce cancer cell death and, at the same time, prevent cisplatin- induced ototoxicity. Because lung cancer is the leading cause of cancer-related death globally, and because cisplatin is widely used to treat this disease, we will be focusing on lung cancer cell lines for this study. We will first study the cytotoxic effects of TET and cisplatin in three lung cancer cell lines and one cochlear cell line, and determine whether TET and cisplatin have synergistic effects when used in combination against these cancer cells while TET has no ototoxic effects on the cochlear cell line (Aim 1). We will then determine whether the TET can prevent cisplatin-induced ototoxicity in vivo while preserving or enhancing anti-tumor efficacy (Aim 2). Our project goal is to develop a new product to combine with cisplatin against lung cancer. This project will generate data important for developing new approaches to reduce cisplatin-induced ototoxicity.

Subjective tinnitus is the perception of a phantom sound, which negatively impacts the quality of life for millions of people worldwide. Despite the great demand for remedy, there are no FDA-approved drugs to prevent or treat tinnitus. Possible causes of tinnitus are complicated. The current view is that hyperactivity in the central auditory nervous system contributes to the majority of tinnitus cases. This abnormal electrical activity, including an increase in delta-band activity, may be the direct result of an increase in T-type calcium channel activity. Other studies suggest that inflammatory responses within the brain may be involved in the development and persistence of tinnitus; therefore, drug candidates targeting both inflammatory and calcium signaling pathways may act synergistically to prevent and treat tinnitus. Tetrandrine (TET), an approved drug used in China, exhibits both anti-inflammatory and calcium channel-blocking properties. Using a new tinnitus detection method in mice, we have shown that both salicylate-induced and noise-induced tinnitus can be effectively treated by TET in a dose-dependent manner. Our hypothesis is that TET or its chemical analogs can be developed as drugs to prevent and treat tinnitus. In our proposed experiments, we have two parallel goals: (1) obtain investigational new drug (IND)-enabling toxicity and pharmacokinetics data for TET (Aim 1) and (2) optimize second-generation products with structure-activity relationship studies of TET and its chemical analogs (Aim 2). Successful accomplishment of Aim 1 will enable TET to advance into clinical development. In addition, the studies of Aim 2 will enable us to identify additional candidates in case TET fails at clinical stages. By targeting multiple cellular signaling pathways that impinge upon tinnitus, our study will open new areas for the treatment and prevention of tinnitus. The extensive body of data publicly available for TET and its analogs will help us significantly reduce development time and costs.