Mitochondria are dynamic organelles that constantly undergo fusion and fission to maintain their normal functionality. Impairment of mitochondrial dynamics is implicated in various neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS) is an adult-onset neuromuscular degenerative disorder characterized by motor neuron death and muscle atrophy. ALS onset and progression clearly involve motor neuron degeneration but accumulating evidence suggests primary muscle pathology may also be involved. Here, we examined mitochondrial dynamics in live skeletal muscle of an ALS mouse model (G93A) harboring a superoxide dismutase mutation (SOD1G93A). Using confocal microscopy combined with overexpression of mitochondria-targeted photoactivatable fluorescent proteins, we discovered abnormal mitochondrial dynamics in skeletal muscle of young G93A mice before disease onset. We further demonstrated that similar abnormalities in mitochondrial dynamics were induced by overexpression of mutant SOD1G93A in skeletal muscle of normal mice, indicating the SOD1 mutation drives ALS-like muscle pathology in the absence of motor neuron degeneration. Mutant SOD1G93A forms aggregates inside muscle mitochondria and leads to fragmentation of the mitochondrial network as well as mitochondrial depolarization. Partial depolarization of mitochondrial membrane potential in normal muscle by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused abnormalities in mitochondrial dynamics similar to that in the SOD1G93A model muscle. A specific mitochondrial fission inhibitor (Mdivi-1) reversed the SOD1G93A action on mitochondrial dynamics, indicating SOD1G93A likely promotes mitochondrial fission process. Our results suggest that accumulation of mutant SOD1G93A inside mitochondria, depolarization of mitochondrial membrane potential and abnormal mitochondrial dynamics are causally linked and cause intrinsic muscle pathology, which occurs early in the course of ALS and may actively promote ALS progression.

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The generation of hair cells (HCs) from the differentiation of proliferating supporting cells (SCs) appears to be an ideal approach for replacing lost HCs in the cochlea and is promising for restoring hearing after damage to the organ of Corti. We show here that extensive proliferation of SCs followed by mitotic HC generation is achieved through a genetic reprogramming process involving the activation of ␤-catenin to upregulate Wnt signaling, the deletion of Notch1 to downregulate Notch signaling, and the overexpression of Atoh1 in Sox2 ϩ SCs in neonatal mouse cochleae. We used RNA sequencing to compare the transcripts of the cochleae from control mice and from mice with ␤-catenin activation, Notch1 deletion, and ␤-catenin activation combined with Notch1 deletion in Sox2 ϩ SCs. We identified the genes involved in the proliferation and transdifferentiation process that are either controlled by individual signaling pathways or by the combination of Wnt and Notch signaling. Moreover, the proliferation of SCs induced by Notch1 deletion disappears after deleting ␤-catenin in Notch1 knock-out Sox2 ϩ cells, which further demonstrates that Notch signaling is an upstream and negative regulator of Wnt signaling.

BackgroundInner ear supporting cells (SCs) in the neonatal mouse cochlea are a potential source for hair cell (HC) regeneration, but several studies have shown that the regeneration ability of SCs decreases dramatically as mice age and that lost HCs cannot be regenerated in adult mice. To better understand how SCs might be better used to regenerate HCs, it is important to understand how the gene expression profile changes in SCs at different ages.MethodsHere, we used Sox2GFP/+ mice to isolate the Sox2+ SCs at postnatal day (P)3, P7, P14, and P30 via flow cytometry. Next, we used RNA-seq to determine the transcriptome expression profiles of P3, P7, P14, and P30 SCs. To further analyze the relationships between these age-related and differentially expressed genes in Sox2+ SCs, we performed gene ontology (GO) analysis.ResultsConsistent with previous reports, we also found that the proliferation and HC regeneration ability of isolated Sox2+ SCs significantly decreased as mice aged. We identified numerous genes that are enriched and differentially expressed in Sox2+ SCs at four different postnatal ages, including cell cycle genes, signaling pathway genes, and transcription factors that might be involved in regulating the proliferation and HC differentiation ability of SCs. We thus present a set of genes that might regulate the proliferation and HC regeneration ability of SCs, and these might serve as potential new therapeutic targets for HC regeneration.ConclusionsIn our research, we found several genes that might play an important role in regulating the proliferation and HC regeneration ability of SCs. These datasets are expected to serve as a resource to provide potential new therapeutic targets for regulating the ability of SCs to regenerate HCs in postnatal mammals.

One of the most unfortunate side effects of aminoglycoside (AG) antibiotics such as neomycin is that they target sensory hair cells (HCs) and can cause permanent hearing impairment. We have observed HC loss and microglia-like cell (MLC) activation in the inner ear (cochlea) following neomycin administration. We focused on CX3CL1, a membrane-bound glycoprotein expressed on neurons and endothelial cells, as a way to understand how the MLCs are activated and the role these cells play in HC loss. CX3CL1 is the exclusive ligand for CX3CR1, which is a chemokine receptor expressed on the surface of macrophages and MLCs. In vitro experiments showed that the expression levels of CX3CL1 and CX3CR1 increased in the cochlea upon neomycin treatment, and CX3CL1 was expressed on HCs, while CX3CR1 was expressed on MLCs. When cultured with 1 μg/mL exogenous CX3CL1, MLCs were activated by CX3CL1, and the cytokine level was increased in the cochleae leading to apoptosis in the HCs. In CX3CR1 knockout mice, a significantly greater number of cochlear HCs survived than in wild-type mice when the cochlear explants were cultured with neomycin in vitro. Furthermore, inhibiting the activation of MLCs with minocycline reduced the neomycin-induced HC loss and improved the hearing function in neomycin-treated mice in vivo. Our results demonstrate that CX3CL1-induced MLC activation plays an important role in the induction of HC death and provide evidence for CX3CL1 and CX3CR1 as promising new therapeutic targets for the prevention of hearing loss.

Cochlear supporting cells (SCs) have been shown to be a promising resource for hair cell (HC) regeneration in the neonatal mouse cochlea. Previous studies have reported that Lgr5+ SCs can regenerate HCs both in vitro and in vivo and thus are considered to be inner ear progenitor cells. Lgr5+ progenitors are able to regenerate more HCs than Lgr5- SCs, and it is important to understand the mechanism behind the proliferation and HC regeneration of these progenitors. Here, we isolated Lgr5+ progenitors and Lgr5- SCs from Lgr5-EGFP-CreERT2/Sox2-CreERT2/Rosa26-tdTomato mice via flow cytometry. As expected, we found that Lgr5+ progenitors had significantly higher proliferation and HC regeneration ability than Lgr5- SCs. Next, we performed RNA-Seq to determine the gene expression profiles of Lgr5+ progenitors and Lgr5- SCs. We analyzed the genes that were enriched and differentially expressed in Lgr5+ progenitors and Lgr5- SCs, and we found 8 cell cycle genes, 9 transcription factors, and 24 cell signaling pathway genes that were uniquely expressed in one population but not the other. Last, we made a protein–protein interaction network to further analyze the role of these differentially expressed genes. In conclusion, we present a set of genes that might regulate the proliferation and HC regeneration ability of Lgr5+ progenitors, and these might serve as potential new therapeutic targets for HC regeneration.

Accumulation of abnormal protein inclusions is implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Autophagy, an intracellular process targeting misfolded proteins and damaged organelles for lysosomal degradation, plays crucial roles in survival and diseased conditions. Efforts were made to understand the role of autophagy in motor neuron degeneration and to target autophagy in motor neuron for ALS treatment. However, results were quite contradictory. Possible autophagy defects in other cell types may also complicate the results. Here, we examined autophagy activity in skeletal muscle of an ALS mouse model G93A. Through overexpression of a fluorescent protein LC3‐RFP, we found a basal increase in autophagosome formation in G93A muscle during disease progression when the mice were on a regular diet. As expected, an autophagy induction procedure (starvation plus colchicine) enhanced autophagy flux in skeletal muscle of normal mice. However, in response to the same autophagy induction procedure, G93A muscle showed significant reduction in the autophagy flux. Immunoblot analysis revealed that increased cleaved caspase‐3 associated with apoptosis was linked to the cleavage of several key proteins involved in autophagy, including Beclin‐1, which is an essential molecule connecting autophagy and apoptosis pathways. Taking together, we provide the evidence that the cytoprotective autophagy pathway is suppressed in G93A skeletal muscle and this suppression may link to the enhanced apoptosis during ALS progression. The abnormal autophagy activity in skeletal muscle likely contributes muscle degeneration and disease progression in ALS.

Hair cell (HC) loss is the major cause of permanent sensorineural hearing loss in mammals. Unlike lower vertebrates, mammalian cochlear HCs cannot regenerate spontaneously after damage, although the vestibular system does maintain limited HC regeneration capacity. Thus HC regeneration from the damaged sensory epithelium has been one of the main areas of research in the field of hearing restoration. Hedgehog signaling plays important roles during the embryonic development of the inner ear, and it is involved in progenitor cell proliferation and differentiation as well as the cell fate decision. In this study, we show that recombinant Sonic Hedgehog (Shh) protein effectively promotes sphere formation, proliferation, and differentiation of Lgr5+ progenitor cells isolated from the neonatal mouse cochlea. To further explore this, we determined the effect of Hedgehog signaling on cell proliferation and HC regeneration in cultured cochlear explant from transgenic R26-SmoM2 mice that constitutively activate Hedgehog signaling in the supporting cells of the cochlea. Without neomycin treatment, up-regulation of Hedgehog signaling did not significantly promote cell proliferation or new HC formation. However, after injury to the sensory epithelium by neomycin treatment, the over-activation of Hedgehog signaling led to significant supporting cell proliferation and HC regeneration in the cochlear epithelium explants. RNA sequencing and real-time PCR were used to compare the transcripts of the cochleae from control mice and R26-SmoM2 mice, and multiple genes involved in the proliferation and differentiation processes were identified. This study has important implications for the treatment of sensorineural hearing loss by manipulating the Hedgehog signaling pathway.