Decreased expression of synaptic genes in the vestibular ganglion of rodents following subchronic ototoxic stress.

The vestibular ganglion contains primary sensory neurons that are postsynaptic to the transducing hair cells (HC) and project to the central nervous system. Understanding the response of these neurons to HC stress or loss is of great interest as their survival and functional competence will determine the functional outcome of any intervention aiming at repair or regeneration of the HCs. We have shown that subchronic exposure to the ototoxicant 3,3'-iminodipropionitrile (IDPN) in rats and mice causes a reversible detachment and synaptic uncoupling between the HCs and the ganglion neurons. Here, we used this paradigm to study the global changes in gene expression in vestibular ganglia using RNA-seq. Comparative gene ontology and pathway analyses of the data from both model species indicated a robust downregulation of terms related to synapses, including presynaptic and postsynaptic functions. Manual analyses of the most significantly downregulated transcripts identified genes with expressions related to neuronal activity, modulators of neuronal excitability, and transcription factors and receptors that promote neurite growth and differentiation. For choice selected genes, the mRNA expression results were replicated by qRT-PCR, validated spatially by RNA-scope, or were demonstrated to be associated with decreased expression of the corresponding protein. We conjectured that decreased synaptic input or trophic support on the ganglion neurons from the HC was triggering these expression changes. To support this hypothesis, we demonstrated decreased expression of BDNF mRNA in the vestibular epithelium after subchronic ototoxicity and also downregulated expression of similarly identified genes (e.g Etv5, Camk1g, Slc17a6, Nptx2, Spp1) after HC ablation with another ototoxic compound, allylnitrile. We conclude that vestibular ganglion neurons respond to decreased input from HCs by decreasing the strength of all their synaptic contacts, both as postsynaptic and presynaptic players.

Tumor suppressor p53 regulates heat shock factor 1 protein degradation in Huntington's disease.

p53 and HSF1 are two major transcription factors involved in cell proliferation and apoptosis, whose dysregulation contributes to cancer and neurodegeneration. Contrary to most cancers, p53 is increased in Huntington's disease (HD) and other neurodegenerative diseases, while HSF1 is decreased. p53 and HSF1 reciprocal regulation has been shown in different contexts, but their connection in neurodegeneration remains understudied. Using cellular and animal models of HD, we show that mutant HTT stabilized p53 by abrogating the interaction between p53 and E3 ligase MDM2. Stabilized p53 promotes protein kinase CK2 alpha prime and E3 ligase FBXW7 transcription, both of which are responsible for HSF1 degradation. Consequently, p53 deletion in striatal neurons of zQ175 HD mice restores HSF1 abundance and decrease HTT aggregation and striatal pathology. Our work shows the mechanism connecting p53 stabilization with HSF1 degradation and pathophysiology in HD and sheds light on the broader molecular differences and commonalities between cancer and neurodegeneration.

Assessment of cochlear toxicity in response to chronic 3,3'-iminodipropionitrile in mice reveals early and reversible functional loss that precedes overt histopathology.

The peripheral auditory and vestibular systems rely on sensorineural structures that are vulnerable to ototoxic agents that cause hearing loss and/or equilibrium deficits. Although attention has focused on hair cell loss as the primary pathology underlying ototoxicity, evidence from the peripheral vestibular system indicates that hair cell loss during chronic exposure is preceded by synaptic uncoupling from the neurons and is potentially reversible. To determine if synaptic pathology also occurs in the peripheral auditory system, we examined the extent, time course, and reversibility of functional and morphological alterations in cochleae from mice exposed to 3,3'-iminodipropionitrile (IDPN) in drinking water for 2, 4 or 6 weeks. Functionally, IDPN exposure caused progressive high- to low-frequency hearing loss assessed by measurement of auditory brainstem response wave I absolute thresholds and amplitudes. The extent of hearing loss scaled with the magnitude of vestibular dysfunction assessed behaviorally. Morphologically, IDPN exposure caused progressive loss of outer hair cells (OHCs) and synapses between the inner hair cells (IHCs) and primary auditory neurons. In contrast, IHCs were spared from ototoxic damage. Importantly, hearing loss consistent with cochlear synaptopathy preceded loss of OHCs and synapses and, moreover, recovered if IDPN exposure was stopped before morphological pathology occurred. Our observations suggest that synaptic uncoupling, perhaps as an early phase of cochlear synaptopathy, also occurs in the peripheral auditory system in response to IDPN exposure. These findings identify novel mechanisms that contribute to the earliest stages of hearing loss in response to ototoxic agents and possibly other forms of acquired hearing loss.

Quantitative Assessment of Anti-Gravity Reflexes to Evaluate Vestibular Dysfunction in Rats.

The tail-lift reflex and the air-righting reflex are anti-gravity reflexes in rats that depend on vestibular function. To obtain objective and quantitative measures of performance, we recorded these reflexes with slow-motion video in two experiments. In the first experiment, vestibular dysfunction was elicited by acute exposure to 0 (control), 400, 600, or 1000 mg/kg of 3,3'-iminodipropionitrile (IDPN), which causes dose-dependent hair cell degeneration. In the second, rats were exposed to sub-chronic IDPN in the drinking water for 0 (control), 4, or 8 weeks; this causes reversible or irreversible loss of vestibular function depending on exposure time. In the tail-lift test, we obtained the minimum angle defined during the lift and descent maneuver by the nose, the back of the neck, and the base of the tail. In the air-righting test, we obtained the time to right the head. We also obtained vestibular dysfunction ratings (VDRs) using a previously validated behavioral test battery. Each measure, VDR, tail-lift angle, and air-righting time demonstrated dose-dependent loss of vestibular function after acute IDPN and time-dependent loss of vestibular function after sub-chronic IDPN. All measures showed high correlations between each other, and maximal correlation coefficients were found between VDRs and tail-lift angles. In scanning electron microscopy evaluation of the vestibular sensory epithelia, the utricle and the saccule showed diverse pathological outcomes, suggesting that they have a different role in these reflexes. We conclude that these anti-gravity reflexes provide useful objective and quantitative measures of vestibular function in rats that are open to further development.

Calyx junction dismantlement and synaptic uncoupling precede hair cell extrusion in the vestibular sensory epithelium during sub-chronic 3,3'-iminodipropionitrile ototoxicity in the mouse.

The cellular and molecular events that precede hair cell (HC) loss in the vestibular epithelium during chronic ototoxic exposure have not been widely studied. To select a study model, we compared the effects of sub-chronic exposure to different concentrations of 3,3'-iminodipropionitrile (IDPN) in the drinking water of two strains of mice and of both sexes. In subsequent experiments, male 129S1/SvImJ mice were exposed to 30 mM IDPN for 5 or 8 weeks; animals were euthanized at the end of the exposure or after a washout period of 13 weeks. In behavioral tests, IDPN mice showed progressive vestibular dysfunction followed by recovery during washout. In severely affected animals, light and electron microscopy observations of the vestibular epithelia revealed HC extrusion towards the endolymphatic cavity. Comparison of functional and ultrastructural data indicated that animals with fully reversible dysfunction did not have significant HC loss or stereociliary damage, but reversible dismantlement of the calyceal junctions that characterize the contact between type I HCs (HCI) and their calyx afferents. Immunofluorescent analysis revealed the loss of calyx junction proteins, Caspr1 and Tenascin-C, during exposure and their recovery during washout. Synaptic uncoupling was also recorded, with loss of pre-synaptic Ribeye and post-synaptic GluA2 puncta, and differential reversibility among the three different kinds of synaptic contacts existing in the epithelium. qRT-PCR analyses demonstrated that some of these changes are at least in part explained by gene expression modifications. We concluded that calyx junction dismantlement and synaptic uncoupling are early events in the mouse vestibular sensory epithelium during sub-chronic IDPN ototoxicity.

Physiological assesment of vestibular function and toxicity in humans and animals.

Physiological methods that can be similarly recorded in humans and animals have a major role in sensory toxicology, as they provide a bridge between human sensory perception data and the molecular and cellular data obtained in animal studies. Vestibular toxicity research lags well behind other sensory systems in many aspects, including the availability of methods for functional assessment in animals that could be robustly translated to human significance. Here we review the methods available for the assessment of vestibular function in both humans and laboratory animals, with an emphasis on their similarity or divergence, to highlight their potential utility for the predictive assessment of vestibular toxicity.