Shared and organ-specific gene-expression programs during the development of the cochlea and the superior olivary complex.

The peripheral and central auditory subsystems together form a complex sensory network that allows an organism to hear. The genetic programs of the two subsystems must therefore be tightly coordinated during development. Yet, their interactions and common expression pathways have never been systematically explored. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression and are essential for normal development of the auditory system. We performed mRNA and small-RNA sequencing of organs from both auditory subsystems at three critical developmental timepoints (E16, P0, P16) to obtain a comprehensive and unbiased insight of their expression profiles. Our analysis reveals common and organ-specific expression patterns for differentially regulated mRNAs and miRNAs, which could be clustered with a particular selection of functions such as inner ear development, Wnt signalling, K+ transport, and axon guidance, based on gene ontology. Bioinformatics detected enrichment of predicted targets of specific miRNAs in the clusters and predicted regulatory interactions by monitoring opposite trends of expression of miRNAs and their targets. This approach identified six miRNAs as strong regulatory candidates for both subsystems. Among them was miR-96, an established critical factor for proper development in both subsystems, demonstrating the strength of our approach. We suggest that other miRNAs identified by this analysis are also common effectors of proper hearing acquirement. This first combined comprehensive analysis of the developmental program of the peripheral and central auditory systems provides important data and bioinformatics insights into the shared genetic program of the two sensory subsystems and their regulation by miRNAs.

Loss of miR-183/96 Alters Synaptic Strength via Presynaptic and Postsynaptic Mechanisms at a Central Synapse.

A point mutation in miR-96 causes non-syndromic progressive peripheral hearing loss and alters structure and physiology of the central auditory system. To gain further insight into the functions of microRNAs (miRNAs) within the central auditory system, we investigated constitutive Mir-183/96dko mice of both sexes. In this mouse model, the genomically clustered miR-183 and miR-96 are constitutively deleted. It shows significantly and specifically reduced volumes of auditory hindbrain nuclei, because of decreases in cell number and soma size. Electrophysiological analysis of the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) demonstrated strongly altered synaptic transmission in young-adult mice. We observed an increase in quantal content and readily releasable vesicle pool size in the presynapse while the overall morphology of the calyx was unchanged. Detailed analysis of the active zones (AZs) revealed differences in its molecular composition and synaptic vesicle (SV) distribution. Postsynaptically, altered clustering and increased synaptic abundancy of the AMPA receptor subunit GluA1 was observed resulting in an increase in quantal amplitude. Together, these presynaptic and postsynaptic alterations led to a 2-fold increase of the evoked excitatory postsynaptic currents in MNTB neurons. None of these changes were observed in deaf Cldn14ko mice, confirming an on-site role of miR-183 and miR-96 in the auditory hindbrain. Our data suggest that the Mir-183/96 cluster plays a key role for proper synaptic transmission at the calyx of Held and for the development of the auditory hindbrain.SIGNIFICANCE STATEMENT The calyx of Held is the outstanding model system to study basic synaptic physiology. Yet, genetic factors driving its morphologic and functional maturation are largely unknown. Here, we identify the Mir-183/96 cluster as an important factor to regulate its synaptic strength. Presynaptically, Mir-183/96dko calyces show an increase in release-ready synaptic vesicles (SVs), quantal content and abundance of the proteins Bassoon and Piccolo. Postsynaptically, the quantal size as well as number and size of GluA1 puncta were increased. The two microRNAs (miRNAs) are thus attractive candidates for regulation of synaptic maturation and long-term adaptations to sound levels. Moreover, the different phenotypic outcomes of different types of mutations in the Mir-183 cluster corroborate the requirement of mutation-tailored therapies in patients with hearing loss.

Expression pattern of cochlear microRNAs in the mammalian auditory hindbrain.

The auditory system comprises the auditory periphery, engaged in sound transduction and the central auditory system, implicated in auditory information processing and perception. Recently, evidence mounted that the mammalian peripheral and central auditory systems share a number of genes critical for proper development and function. This bears implication for auditory rehabilitation and evolution of the auditory system. To analyze to which extent microRNAs (miRNAs) belong to genes shared between both systems, we characterize the expression pattern of 12 cochlea-abundant miRNAs in the central auditory system. Quantitative real-time PCR (qRT-PCR) demonstrated expression of all 12 genes in the cochlea, the auditory hindbrain and the non-auditory prefrontal cortex (PFC) at embryonic stage (E)16 and postnatal stages (P)0 and P30. Eleven of them showed differences in expression between tissues and nine between the developmental time points. Hierarchical cluster analysis revealed that the temporal expression pattern in the auditory hindbrain was more similar to the PFC than to the cochlea. Spatiotemporal expression analysis by RNA in situ hybridization demonstrated widespread expression throughout the cochlear nucleus complex (CNC) and the superior olivary complex (SOC) during postnatal development. Altogether, our data indicate that miRNAs represent a relevant class of genetic factors functioning across the auditory system. Given the importance of gene regulatory network (GRN) components for development, physiology and evolution, the 12 miRNAs provide promising entry points to gain insights into their molecular underpinnings in the auditory system.

miR-96 is required for normal development of the auditory hindbrain.

The peripheral deafness gene Mir96 is expressed in both the cochlea and central auditory circuits. To investigate whether it plays a role in the auditory system beyond the cochlea, we characterized homozygous Dmdo/Dmdo mice with a point mutation in miR-96. Anatomical analysis demonstrated a significant decrease in volume of auditory nuclei in Dmdo/Dmdo mice. This decrease resulted from decreased cell size. Non-auditory structures in the brainstem of Dmdo/Dmdo mice or auditory nuclei of the congenital deaf Cldn14-/- mice revealed no such differences. Electrophysiological analysis in the medial nucleus of the trapezoid body (MNTB) showed that principal neurons fired preferentially multiple action potentials upon depolarization, in contrast to the single firing pattern prevalent in controls and Cldn14-/- mice. Immunohistochemistry identified significantly reduced expression of two predicted targets of the mutated miR-96, Kv1.6 and BK channel proteins, possibly contributing to the electrophysiological phenotype. Microscopic analysis of the Dmdo/Dmdo calyx of Held revealed a largely absent compartmentalized morphology, as judged by SV2-labeling. Furthermore, MNTB neurons from Dmdo/Dmdo mice displayed larger synaptic short-term depression, slower AMPA-receptor decay kinetics and a larger NMDA-receptor component, reflecting a less matured stage. Again, these synaptic differences were not present between controls and Cldn14-/- mice. Thus, deafness genes differentially affect the auditory brainstem. Furthermore, our study identifies miR-96 as an essential gene regulatory network element of the auditory system which is required for functional maturation in the peripheral and central auditory system alike.