ASD risk genes including Mef2c are differentially expressed during mouse

Dysfunction of the peripheral auditory nerve (AN) contributes to dynamic changes throughout the central auditory system, resulting in abnormal auditory processing, including hypersensitivity. Altered sound sensitivity 38 is frequently observed in autism spectrum disorder (ASD), suggesting that AN deficits and changes in auditory 39 information processing may contribute to ASD-associated symptoms, including social communication deficits 40 and hyperacusis. The MEF2C transcription factor is associated with risk for several neurodevelopmental 41 disorders, and mutations or deletions of MEF2C produce a haploinsufficiency syndrome characterized by ASD, 42 language and cognitive deficits. A mouse model of this syndromic ASD (i.e., Mef2c +/- or Mef2c -Het) 43 recapitulates many of the MEF2C Haploinsufficiency syndrome-linked behaviors including communication 44 deficits. We show here that Mef2c -Het mice exhibit functional impairment of the peripheral AN and a modest 45 reduction in hearing sensitivity. We find that MEF2C is expressed during development in multiple AN and 46 cochlear cell types, and in Mef2c -Het mice, we observe multiple cellular and molecular alterations associated 47 with the AN, including abnormal myelination, neuronal degeneration, neuronal mitochondria dysfunction, and 48 increased macrophage activation and cochlear inflammation. These results reveal the importance of MEF2C 49 function in inner ear development and function and the engagement of immune cells and other non-neuronal 50 cells, which suggests that microglia/macrophages and other non-neuronal cells might contribute, directly or 51 indirectly, to AN dysfunction and ASD-related phenotypes. Finally, our study establishes a comprehensive 52 approach for characterizing AN function at the physiological, cellular, and molecular levels in mice, which can 53 be applied to animal models with a wide range of human auditory processing impairments. This is the first report of peripheral auditory nerve (AN) impairment in a mouse model of human MEF2C haploinsufficiency syndrome that has well-characterized ASD related behaviors including communication deficits, hyperactivity, repetitive behavior, and social deficits. We identify multiple underlying cellular, sub- cellular, and molecular abnormalities that may contribute to peripheral AN impairment. Our findings also highlight the important roles of immune cells (e.g., cochlear macrophages) and other non-neuronal elements (e.g., glial cells and cells in the stria vascularis) in auditory impairment in ASD. The methodological significance 61 of the study is the establishment of a comprehensive approach for evaluating peripheral AN function and impact of peripheral AN deficits with minimal hearing loss. repetitive This mouse model of syndromic autism provides a means to directly investigate the relationship between peripheral AN dysfunction and ASD behaviors. Here we report that this mouse model of human MCHS has functional impairments of the AN at the physiological, cellular, and molecular levels. Further, our findings highlight the importance of immune cells and non-neuronal elements as regulatory factors in auditory impairment in ASD. Microdissections were performed to isolate the AN from the rest of the cochlear structures, taking care to preserve peripheral fibers. Samples were created by pooling ANs collected from either one mouse (aging study) or two mice (Mef2c deficiency study). Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen Inc, Germantown, MD) per the manufacturer's instructions. Quality of each total RNA preparation was assessed by 4200 TapeStation (Agilent Technologies, Santa Clara, CA). Low-quality samples showing degradation or contamination were excluded. mice AN identified 258 genes that were differentially expressed (fold change >1.5 and p adj <0.10). Enrichment analysis of these genes showed that they were highly representative of neurogenic function, with the top 15 significant biological processes including categories relating to glia cells, myelination, and other neural developmental processes. To clarify these findings, all significant biological processes were refined by eliminating redundant categories. This refinement revealed that there was a cluster of highly similar processes that were centrally linked to sensory perception of sound. This cluster included several processes relating to glial cell function and myelination and also processes relating to the development of tissues derived from mesenchymal cell lineages. ASD models exhibited auditory impairment but limited regarding possible in peripheral auditory organ or pathological alterations of AN degeneration and dysfunction, which is critical for understanding the link between AN function and ASD characteristics, such as sensory hypersensitivity and social communication deficits. Here we report that a model of human MEF2C hypofunction with well-defined ASD-like (Harrington a., has a modest reduction in hearing sensitivity combined with AN functional deficits. This study is significant in that it: 1) links a monogenic ASD animal model with a clear impairment in peripheral AN structure and function, and 2) reveals a critical new role for Mef2c peripheral AN Based on these observations, we hypothesize that Mef2c hypofunction contributes to aging-like degeneration via a negative impact on mitochondrial function and cellular metabolism. postmortem brain tissues from individuals with autism has revealed expression changes in microglial-specific genes and genes related to inflammatory response Recent studies revealed that MEF2C expression in microglia/macrophages is also important for brain development and maintenance of cognitive function in adults Here our study revealed that MEF2C is expressed in neurons as well as in microglia/macrophages in the AN of the peripheral auditory system. In the peripheral auditory system, we found that Mef2c -Het mice demonstrated an increase in macrophage activation in the AN and the stria vascularis, an increase in mRNA expression of pro-inflammatory genes, and morphological disruption of microvasculature. There are at least three factors that may lead to increased IBA1 expressing cells in the cochlea: 1) as a direct result of Mef2c deficiency (since MEF2C is highly expressed in IBA1 + cochlear macrophage); 2) as a result of dysfunction of the spiral ganglion neuron, and 3) a consequence of the degeneration of glial cells in the AN. Our data showing increased inflammatory cells in the strial vascularis also suggest the direct impact of Mef2c deficiency in cochlear macrophages in multiple cochlear locations. Though the current study did not establish whether increased macrophage activation was a direct result of Mef2c hypofunction in those cells, SGN aging-like degeneration, or both, our data clearly reveal that the AN functional decline is accompanied by increased macrophage activation and a shift in the cochlea toward pro-inflammatory status.