BackgroundOtosclerosis is a common cause of adult-onset progressive hearing loss, affecting 0.3%–0.4% of the population. It results from dysregulation of bone homeostasis in the otic capsule, most commonly leading to fixation of the stapes bone, impairing sound conduction through the middle ear. Otosclerosis has a well-known genetic predisposition including familial cases with apparent autosomal dominant mode of inheritance. While linkage analysis and genome-wide association studies suggested an association with several genomic loci and with genes encoding structural proteins involved in bone formation or metabolism, the molecular genetic pathophysiology of human otosclerosis is yet mostly unknown.MethodsWhole-exome sequencing, linkage analysis, generation of CRISPR mutant mice, hearing tests and micro-CT.ResultsThrough genetic studies of kindred with seven individuals affected by apparent autosomal dominant otosclerosis, we identified a disease-causing variant inSMARCA4, encoding a key component of the PBAF chromatin remodelling complex. We generated CRISPR-Cas9 transgenic mice carrying the human mutation in the mouseSMARCA4orthologue. MutantSmarca4+/E1548Kmice exhibited marked hearing impairment demonstrated through acoustic startle response and auditory brainstem response tests. Isolated ossicles of the auditory bullae of mutant mice exhibited a highly irregular structure of the incus bone, and their in situ micro-CT studies demonstrated the anomalous structure of the incus bone, causing disruption in the ossicular chain.ConclusionWe demonstrate that otosclerosis can be caused by a variant inSMARCA4, with a similar phenotype of hearing impairment and abnormal bone formation in the auditory bullae in transgenic mice carrying the human mutation in the mouseSMARCA4orthologue.

Optical imaging with mechanical contrast is critical for material and biological discovery since it allows contactless light-radiation force-excitation within the sample, as opposed to traditional mechanical imaging. Whilst optical microscopy based on stimulated Brillouin scattering (SBS) enables mechanical imaging of materials and living biological systems with high spectrospatial resolution, its temporal resolution remains limited. Here, we develop Brillouin gain microscopy (BGM) with a 200-fold higher temporal resolution by detecting the Brillouin gain at a mechanically contrasting frequency in the sample with high sensitivity. Using BGM, we demonstrate mechanical imaging of materials, living organisms and cells at high spectro-spatiotemporal resolution.

Background Mitochondrial DNA (mtDNA) is present at high copy numbers in animal cells, and though characterized by a single haplotype in each individual due to maternal germline inheritance, deleterious mutations and intact mtDNA molecules frequently co-exist (heteroplasmy). A number of factors, such as replicative segregation, mitochondrial bottlenecks, and selection, may modulate the exitance of heteroplasmic mutations. Since such mutations may have pathological consequences, they likely survive and are inherited due to functional complementation via the intracellular mitochondrial network. Here, we hypothesized that compromised mitochondrial fusion would hamper such complementation, thereby affecting heteroplasmy inheritance. Results We assessed heteroplasmy levels in three Caenorhabditis elegans strains carrying different heteroplasmic mtDNA deletions (ΔmtDNA) in the background of mutant mitofusin ( fzo-1 ). Animals displayed severe embryonic lethality and developmental delay. Strikingly, observed phenotypes were relieved during subsequent generations in association with complete loss of ΔmtDNA molecules. Moreover, deletion loss rates were negatively correlated with the size of mtDNA deletions, suggesting that mitochondrial fusion is essential and sensitive to the nature of the heteroplasmic mtDNA mutations. Introducing the ΔmtDNA into a fzo-1 ; pdr-1; +/ΔmtDNA (PARKIN ortholog) double mutant resulted in a skewed Mendelian progeny distribution, in contrast to the normal distribution in the fzo-1; +/ΔmtDNA mutant, and severely reduced brood size. Notably, the ΔmtDNA was lost across generations in association with improved phenotypes. Conclusions Taken together, our findings show that when mitochondrial fusion is compromised, deleterious heteroplasmic mutations cannot evade natural selection while inherited through generations. Moreover, our findings underline the importance of cross-talk between mitochondrial fusion and mitophagy in modulating the inheritance of mtDNA heteroplasmy.

The quality control machinery regulates the cellular proteome to ensure proper protein homeostasis (proteostasis). In Caenorhabditis elegans, quality control networks are downregulated cell-nonautonomously by the gonadal longevity pathway or metabolic signaling at the onset of reproduction. However, how signals are mediated between the gonad and the somatic tissues is not known. Gonadotropin-releasing hormone (GnRH)-like signaling functions in the interplay between development and reproduction and have conserved roles in regulating reproduction, metabolism, and stress. We, therefore, asked whether GnRH-like signaling is involved in proteostasis collapse at the onset of reproduction. Here, we examine whether C. elegans orthologues of GnRH receptors modulate heat shock survival. We find that gnrr-2 is required for proteostasis remodeling in different somatic tissues during the transition to adulthood. We show that gnrr-2 likely functions in neurons downstream of the gonad in the gonadal-longevity pathway and modulate the somatic regulation of transcription factors HSF-1, DAF-16, and PQM-1. In parallel, gnrr-2 modulates egg-laying rates, vitellogenin production, and thus reproductive capacity. Taken together, our data suggest that gnrr-2 plays a GnRH-associated role, mediating the cross-talk between the reproduction system and the soma in the decision to commit to reproduction.

Deleterious and intact mitochondrial DNA (mtDNA) mutations frequently co-exist in cells (heteroplasmy). Such mutations likely survive and are inherited due to complementation via the intra-cellular mitochondrial network. Hence, we hypothesized that compromised mitochondrial fusion would hamper such complementation, thereby affecting heteroplasmy inheritance. To test this hypothesis, we assessed heteroplasmic patterns in three Charcot-Marie-Tooth disease type 2A pedigrees, which carry a mutated mitofusin 2 (MFN2). We found reduced prevalence of a potentially functional mtDNA heteroplasmic mutation in these patients, as compared to healthy maternal relatives, while neutral heteroplasmic mutations fluctuated randomly. Secondly, we found that MFN2 dysfunction in a Caenorhabditis elegans model carrying a large heteroplasmic mtDNA deletion (ΔmtDNA) led to a severe developmental delay and embryonic lethality. Strikingly, these phenotypes were relieved during subsequent generations in association with complete ΔmtDNA removal. Such ΔmtDNA loss occurred during both gametogenesis and embryogenesis. Therefore, mitochondrial fusion is essential for inheritance of mtDNA heteroplasmy.

Protein homeostasis is remodeled early in Caenorhabditis elegans adulthood, resulting in a sharp decline in folding capacity and reduced ability to cope with chronic and acute stress. Endocrine signals from the reproductive system can ameliorate this proteostatic collapse and reshape the quality control network. Given that environmental conditions, such as food availability, impact reproductive success, we asked whether conditions of dietary restriction (DR) can also reverse the decline in quality control function at the transition to adulthood, and if so, whether gonadal signaling and dietary signaling remodel the quality control network in a similar or different manner. For this, we employed the eat-2 genetic model and bacterial deprivation protocol. We found that animals under DR maintained heat shock response activation and high protein folding capacity during adulthood. However, while gonadal signaling required DAF-16, DR-associated rescue of quality control functions required the antagonistic transcription factor, PQM-1. Bioinformatic analyses supported a role for DAF-16 in acute stress responses and a role for PQM-1 in cellular maintenance and chronic stress. Comparing the stress activation and folding capacities of dietary- and gonadal-signaling mutant animals confirmed this prediction and demonstrated that each differentially impacts cellular quality control capabilities. These data suggest that the functional mode of cellular quality control networks can be differentially remodeled, affecting an organism's ability to respond to acute and chronic stresses during adulthood.