RESEARCH STATEMENT                                                                            HOLLY A. HOLMAN, Ph.D.

I have pioneered novel techniques for live cell calcium signaling in mammalian vestibular and auditory systems. My research focuses on signaling within the vestibular and auditory sensory epithelia during development, aging, and vestibular dysfunction. Recent reports from my lab has shown that signaling among glial-like supporting cells is critical for development and function of auditory and vestibular peripheral organs. Whether supporting cell signaling affects sensory hair cell and neuronal signaling, or vice-versa, and whether this signaling changes following ototoxicity my lab seeks to better understand. We know that hair cells are susceptible to apoptosis following platinum-based chemotherapy drugs and aminoglycoside antibiotics. Understanding the mechanisms and the role supporting cells play in ototoxicity can provide targets for reducing or even preventing hearing loss and vestibular dysfunction.

My lab has developed several new methods for studying glial-like supporting cell signaling in mammalian auditory and vestibular systems. To date, my lab has demonstrated glial-like supporting cells in the cochlea, utricle, semicircular canal cristae with robust spontaneous Ca²⁺ transients during neonatal development. These supporting cells also respond to ATP, acetylcholine (ACh), and muscarine (Musc) with evoked Ca²⁺ transients that are orders of magnitude larger than their neighboring sensory hair cells. These evoked responses are present during neonatal development and are maintained in a rhythmic and robust response in geriatric mice (two years). In the utricle, we demonstrated spontaneous Ca²⁺ transients in primarily type I hair cells, which diminish with the onset of ACh and Musc evoked Ca²⁺ transients during the first postnatal week. The molecular mechanisms responsible for this switch from spontaneous to evoked cholinergic Ca²⁺ responses and how this affects vestibular function is one of the main projects I am developing.

The overall goal of my lab is to demonstrate the relationship between signaling in glial-like supporting cells, their neighboring hair cells and synaptic contacts, at different stages of development, in old age, and following ototoxicity.

Major Research Projects

o   Develop optical methods for recording glial-like supporting cell-hair cell-neuronal signaling in semi-intact preparations and in vivo.

o   Demonstrate sensory hair cell and non-sensory supporting cell lineages specifically in the mammalian vestibular system.

o   Determine the function of calcium microenvironments (i.e. cupula, otoconia and tectorial membrane) in hair bundles and their mechanotransduction.

o   Examine human temporal bone immunohistochemistry evolutionarily conserved GAD signaling molecules.

o   Design and develop a multimetric vestibular system functional testing method.

Understanding whether supporting cell signaling mechanisms in the mouse auditory and vestibular systems are evolutionarily conserved in humans and other species is a critical component to my research. To address this, I established a collaboration with the NIDCD National Temporal Bone Laboratory at UCLA Health. This project received NIH funding in 2018, and preliminary data suggests GAD signaling molecules are also present in human sensory epithelia, although much more work must be conducted.

Supporting Cell-Hair Cell-Neuronal Signaling

Hair cells are the primary sensory receptors crucial in the transduction of mechanical stimuli into electrical signals. Hair cells are surrounded by supporting cells that have the capacity to regenerate hair cells under certain conditions in the vestibular organs. My lab has identified a niche of supporting cells in the crista ampullaris with progenitor-like cell characteristics (Holman et al., 2020). Spontaneous and ATP evoked Ca²⁺ transients in these and other vestibular supporting cells and hair cells.  We have identified different supporting cell types in the utricle and semicircular canal crista based on their unique spontaneous and evoked Ca²⁺ responses (Holman et al., 2019, Holman et al, 2020, Rabbitt and Holman, 2021). ATP co-exists with acetylcholine (ACh) in certain presynaptic nerve terminals and evokes Ca²⁺ transients in cochlear hair cells through muscarinic receptor activation. We recently demonstrated that ACh evokes Ca²⁺ transients in vestibular supporting cells that are orders of magnitude larger than reported in the cochlea (Rabbitt and Holman, 2021). My lab is continuing to study this signaling to better understand the mechanisms responsible for these robust ACh and ATP evoked Ca²⁺ transients in the vestibular supporting cells.

Mechanotransduction and Calcium Signaling

Using a transgenic mouse we have demonstrated intracellular calcium [Ca²⁺]i transients in the long stereocilia of semicircular canal cristae. My lab is developing optical methods to image [Ca²⁺]i through the intact membranous labyrinth and ampullae during physiological stimulation, while maintaining ion homeostasis. We are examining hair bundle elongation and puncta at putative mechanotransduction (MET) channels. We quantify the genetically encoded calcium (GCaMP) signal using the dual reporter tdTomato, which is constitutively expressed in these cells and hair bundles.

Vestibular Sensory and Non-sensory Cell Lineage Tracing

Independent studies show that the rate-limiting enzyme glutamate decarboxylase 2 (Gad2) is expressed in the vestibular neural epithelium. Using the Gad2-Cre transgenic mouse, my lab has demonstrated Gad2 dependent GCaMP5G-tdTomato expressing glial-like supporting cells and hair cells during neonatal development in the mouse (postnatal day 0, the earliest age examined to date; Holman et al., 2020). We are collaborating and developing methods to perform genetic lineage tracing to track supporting cell and hair cell migration, proliferation, and differentiation in vivo. Using this transgenic mouse method will allow indefinite labeling of cells with tdTomato induction of Gad2-Cre (Figure 1 A-B).

Multimetric Functional Vestibular System Testing Method

My lab has designed a multimetric vestibular function testing platform that includes recording the vestibular ocular reflex (VOR). The first phase of testing measures rate and kinematics of mice traversing a balance beam. Two-dimensional and three-dimensional marker less pose estimation analysis is performed based on transfer learning with deep neural networks in FluoRender (Yong Wan, Scientific Computing and Imaging Institute, Univ of Utah). In the second phase, calcium imaging of vestibular peripheral organs using a miniscope is added during during vestibular stimuli (Nick Frost, Dept. of Neurology, Univ of Utah). In the third phase, eye-tracking (Behrad Noudoost, Moran Eye Center, Univ of Utah), and foot strike measurements are recorded (Robert Hitchcock and Tomasz Petelenz  Dept. of Biomedical Engineering, Univ of Utah). The goal of this interdisciplinary project is to determine the role of glial-like supporting cells and their signaling in maintaining vestibular function. A commercial rotarod is also used as a control for vestibular function testing in collaboration with David Krizaj (Moran Eye Center, Univ of Utah).

Human Temporal Bone Collaboration

My lab has established a human translational collaboration to examine the anatomy and pathology of the GAD pathway in temporal bone specimens. This project is an extension of my training from the Massachusetts Eye and Ear (NIDCD National Temporal Bone, Hearing and Balance Pathology Resource Registry (2018)). With NIH funding, my laboratory is leading studies on the role(s) of GADs in glial-like supporting cells and the human sensory epithelium (National Institute on Deafness and Other Communication Disorders (NIDCD) National Temporal Bone Laboratory, UCLA Health).

REFERENCES

Holman HA, Poppi LA, Frerck M, Rabbitt RD. Spontaneous and Acetylcholine Evoked Calcium Transients in the Developing Mouse Utricle. Front Cell Neurosci. 2019 May 7;13:186. doi: 10.3389/fncel.2019.00186. PMID: 31133810; PMCID: PMC6514437.

Holman HA, Wan Y, Rabbitt RD. Developmental GAD2 Expression Reveals Progenitor-like Cells with Calcium Waves in Mammalian Crista Ampullaris. iScience. 2020 Aug 21;23(8):101407. doi: 10.1016/j.isci.2020.101407.

Rabbitt RD, Holman HA. ATP and ACh Evoked Calcium Transients in the Neonatal Mouse Cochlear and Vestibular Sensory Epithelia. Front Neurosci. 2021 Sep 8;15:710076. doi: 10.3389/fnins.2021.710076.