The CaV1.2 L-type calcium channel regulates bone homeostasis in the middle and inner ear.

Bone remodeling of the auditory ossicles and the otic capsule is highly restricted and tightly controlled by the osteoprotegerin (OPG)/receptor activator of nuclear factor kappa-Β ligand (RANKL)/receptor activator of nuclear factor kappa-Β (RANK) system. In these bony structures, a pathological decrease in OPG expression stimulates osteoclast differentiation and excessive resorption followed by accrual of sclerotic bone, ultimately resulting in the development of otosclerosis, a leading cause of deafness in adults. Understanding the signaling pathways involved in maintaining OPG expression in the ear would shed light on the pathophysiology of otosclerosis and other ear bone-related diseases. We and others previously demonstrated that Ca2+ signaling through the L-type CaV1.2 Ca2+ channel positively regulates OPG expression and secretion in long bone osteoblasts and their precursor cells in vitro and in vivo. Whether CaV1.2 regulates OPG expression in ear bones has not been investigated. We drove expression of a gain-of-function CaV1.2 mutant channel (CaV1.2TS) using Col2a1-Cre, which we found to target osteochondral/osteoblast progenitors in the auditory ossicles and the otic capsule. Col2a1-Cre;CaV1.2TS mice displayed osteopetrosis of these bones shown by µCT 3D reconstruction, histological analysis, and lack of bone sculpting, findings similar to phenotypes seen in mice with an osteoclast defect. Consistent with those observations, we found that Col2a1-Cre;CaV1.2TS mutant mice showed reduced osteoclasts in the otic capsule, upregulated mRNA expression of Opg and Opg/Rankl ratio, and increased mRNA expression of osteoblast differentiation marker genes in the otic capsule, suggesting both an anti-catabolic and anabolic effect of CaV1.2TS mutant channel contributed to the observed morphological changes of the ear bones. Further, we found that Col2a1-Cre;CaV1.2TS mice experienced hearing loss and displayed defects of body balance in behavior tests, confirming that the CaV1.2-dependent Ca2+ influx affects bone structure in the ear and consequent hearing and vestibular functions. Together, these data support our hypothesis that Ca2+ influx through CaV1.2TS promotes OPG expression from osteoblasts, thereby affecting bone modeling/remodeling in the auditory ossicles and the otic capsule. These data provide insight into potential pathological mechanisms underlying perturbed OPG expression and otosclerosis.

Thermal Excitation of the Mechanotransduction Apparatus of Hair Cells.

Although a hair bundle is normally deflected by mechanical stimuli, we found that irradiation of a hair cell from the bullfrog's sacculus with ultraviolet light causes rapid motion of the hair bundle toward its tall edge. This movement is associated with opening of mechanotransduction channels and disappears when tip links are disrupted. We localized the absorptive element responsible for the motion to the region directly below the hair bundle and measured an action spectrum similar to the absorption spectra of mitochondrial constituents. Temperature measurements revealed heating around the site of absorption; direct heating of the hair bundle confirmed that the response to light is mediated through heat. Although mechanical offsets of the hair bundle revealed that heat softens gating springs, it also acts directly to open transduction channels. This study identifies an unconventional method of hair-cell stimulation and clarifies the previously unexplained sensitivity of auditory organs to thermal stimulation.

Direct mechanical stimulation of tip links in hair cells through DNA tethers.

Mechanoelectrical transduction by hair cells commences with hair-bundle deflection, which is postulated to tense filamentous tip links connected to transduction channels. Because direct mechanical stimulation of tip links has not been experimentally possible, this hypothesis has not been tested. We have engineered DNA tethers that link superparamagnetic beads to tip links and exert mechanical forces on the links when exposed to a magnetic-field gradient. By pulling directly on tip links of the bullfrog's sacculus we have evoked transduction currents from hair cells, confirming the hypothesis that tension in the tip links opens transduction channels. This demonstration of direct mechanical access to tip links additionally lays a foundation for experiments probing the mechanics of individual channels.

NOVA2-mediated RNA regulation is required for axonal pathfinding during development.

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

Control of a hair bundle's mechanosensory function by its mechanical load.

Hair cells, the sensory receptors of the internal ear, subserve different functions in various receptor organs: they detect oscillatory stimuli in the auditory system, but transduce constant and step stimuli in the vestibular and lateral-line systems. We show that a hair cell's function can be controlled experimentally by adjusting its mechanical load. By making bundles from a single organ operate as any of four distinct types of signal detector, we demonstrate that altering only a few key parameters can fundamentally change a sensory cell's role. The motions of a single hair bundle can resemble those of a bundle from the amphibian vestibular system, the reptilian auditory system, or the mammalian auditory system, demonstrating an essential similarity of bundles across species and receptor organs.