Abstract:The last decade has witnessed the identification of several families affected by hereditary non-syndromic hearing loss (NSHL) caused by mutations in the SMPX gene and the loss of function has been suggested as the underlying mechanism. In the attempt to confirm this hypothesis we generated an Smpx-deficient zebrafish model, pointing out its crucial role in proper inner ear development. Indeed, a marked decrease in the number of kinocilia together with structural alterations of the stereocilia and the kinociliu… Show more
“…The Smpx localization in the primary cilium strongly supports the observation that the Smpx-positive cells are the actual mechanosensory HCs responsible for the conversion of mechanical stimuli into electric signal 27 . This data is in line to what we previously reported in the inner ear, where Smpx was localized exclusively in the HCs of the sensory patches 11 , 17 . Figure 2 Smpx protein localization in the neuromast mechanosensory hair cells.…”
Section: Resultssupporting
confidence: 93%
“…As we previously demonstrated, the zebrafish Smpx protein inside the inner ear HC was mainly localized at the level of the cuticular plate (CP), an actin-rich structure that is necessary for the correct development of both stereocilia and kinocilia, and to carry out mechanotransduction 17 . Employing a zebrafish model of Smpx deficiency, we showed that the protein exerted a primary function in the proper structural organization of the inner ear HCs sensory bundle as in that both stereocilia and kinocilia displayed evident morphological alterations in terms of number and length 11 , as later also confirmed in a knock-out mouse model 18 . Moreover, we demonstrated that inner ear HCs of Smpx-deficient embryos lack of mechanotransduction activity, providing the first possible explanation for the reported cases of SMPX -linked hearing loss in human patients 4 – 14 .…”
Section: Introductionmentioning
confidence: 65%
“…The role of Smpx in proper neuromast development was analyzed, at first, by gene knockdown through the microinjection of a previously validated ATG-targeting smpx -specific morpholino 11 , then by CRISPR/Cas9-mediated F0 gene knockout. Being Smpx expressed in the mechanosensory HCs of the neuromasts, we employed the FM 4–64 vital dye to stain such cells in vivo 28 , 29 .…”
The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating auditory function. In the last decade, several mutations in SMPX have been associated with X-chromosomal progressive non syndromic hearing loss in humans and, in line with this, Smpx-deficient animal models, namely zebrafish and mouse, showed significant impairment of inner ear HCs development, maintenance, and functioning. In this work, we uncovered smpx expression in the neuromast mechanosensory HCs of both Anterior and Posterior Lateral Line (ALL and PLL, respectively) of zebrafish larvae and focused our attention on the PLL. Smpx was subcellularly localized throughout the cytoplasm of the HCs, as well as in their primary cilium. Loss-of-function experiments, via both morpholino-mediated gene knockdown and CRISPR/Cas9 F0 gene knockout, revealed that the lack of Smpx led to fewer properly differentiated and functional neuromasts, as well as to a smaller PLL primordium (PLLp), the latter also Smpx-positive. In addition, the kinocilia of Smpx-deficient neuromast HCs appeared structurally and numerically altered. Such phenotypes were associated with a significant reduction in the mechanotransduction activity of the neuromast HCs, in line with their positivity for Smpx. In summary, this work highlights the importance of Smpx in lateral line development and, specifically, in proper HCs differentiation and/or maintenance, and in the mechanotransduction process carried out by the neuromast HCs. Because lateral line HCs are both functionally and structurally analogous to the cochlear HCs, the neuromasts might represent an invaluable—and easily accessible—tool to dissect the role of Smpx in HCs development/functioning and shed light on the underlying mechanisms involved in hearing loss.
“…The Smpx localization in the primary cilium strongly supports the observation that the Smpx-positive cells are the actual mechanosensory HCs responsible for the conversion of mechanical stimuli into electric signal 27 . This data is in line to what we previously reported in the inner ear, where Smpx was localized exclusively in the HCs of the sensory patches 11 , 17 . Figure 2 Smpx protein localization in the neuromast mechanosensory hair cells.…”
Section: Resultssupporting
confidence: 93%
“…As we previously demonstrated, the zebrafish Smpx protein inside the inner ear HC was mainly localized at the level of the cuticular plate (CP), an actin-rich structure that is necessary for the correct development of both stereocilia and kinocilia, and to carry out mechanotransduction 17 . Employing a zebrafish model of Smpx deficiency, we showed that the protein exerted a primary function in the proper structural organization of the inner ear HCs sensory bundle as in that both stereocilia and kinocilia displayed evident morphological alterations in terms of number and length 11 , as later also confirmed in a knock-out mouse model 18 . Moreover, we demonstrated that inner ear HCs of Smpx-deficient embryos lack of mechanotransduction activity, providing the first possible explanation for the reported cases of SMPX -linked hearing loss in human patients 4 – 14 .…”
Section: Introductionmentioning
confidence: 65%
“…The role of Smpx in proper neuromast development was analyzed, at first, by gene knockdown through the microinjection of a previously validated ATG-targeting smpx -specific morpholino 11 , then by CRISPR/Cas9-mediated F0 gene knockout. Being Smpx expressed in the mechanosensory HCs of the neuromasts, we employed the FM 4–64 vital dye to stain such cells in vivo 28 , 29 .…”
The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating auditory function. In the last decade, several mutations in SMPX have been associated with X-chromosomal progressive non syndromic hearing loss in humans and, in line with this, Smpx-deficient animal models, namely zebrafish and mouse, showed significant impairment of inner ear HCs development, maintenance, and functioning. In this work, we uncovered smpx expression in the neuromast mechanosensory HCs of both Anterior and Posterior Lateral Line (ALL and PLL, respectively) of zebrafish larvae and focused our attention on the PLL. Smpx was subcellularly localized throughout the cytoplasm of the HCs, as well as in their primary cilium. Loss-of-function experiments, via both morpholino-mediated gene knockdown and CRISPR/Cas9 F0 gene knockout, revealed that the lack of Smpx led to fewer properly differentiated and functional neuromasts, as well as to a smaller PLL primordium (PLLp), the latter also Smpx-positive. In addition, the kinocilia of Smpx-deficient neuromast HCs appeared structurally and numerically altered. Such phenotypes were associated with a significant reduction in the mechanotransduction activity of the neuromast HCs, in line with their positivity for Smpx. In summary, this work highlights the importance of Smpx in lateral line development and, specifically, in proper HCs differentiation and/or maintenance, and in the mechanotransduction process carried out by the neuromast HCs. Because lateral line HCs are both functionally and structurally analogous to the cochlear HCs, the neuromasts might represent an invaluable—and easily accessible—tool to dissect the role of Smpx in HCs development/functioning and shed light on the underlying mechanisms involved in hearing loss.
“…Atp2b2 encodes plasma membrane Ca 2+ ‐ATPase type 2 (PMCA2) that is important for maintaining calcium homeostasis in stereocilia (Street et al, 1998). Other RBM24 targets Grxcr1 , Grxcr2 , Ptprq , and Smpx have also been shown to play important roles in stereocilia development and/or maintenance (Ghilardi et al, 2021; Goodyear et al, 2003; Liu et al, 2018; Odeh et al, 2010; Tu et al, 2021). Therefore, it seems that RBM24 acts downstream of ATOH1 and mainly regulates hair bundle development and function.…”
As the sensory receptor cells in vertebrate inner ear and lateral lines, hair cells are characterized by the hair bundle that consists of one tubulin‐based kinocilium and dozens of actin‐based stereocilia on the apical surface of each hair cell. Hair cell development is tightly regulated, and deficits in this process usually lead to hearing loss and/or balance dysfunctions. RNA‐binding motif protein 24 (RBM24) is an RNA‐binding protein that is specifically expressed in the hair cells in the inner ear. Previously, we showed that RBM24 affects hair cell development in zebrafish by regulating messenger RNA (mRNA) stability. In the present work, we further investigate the role of RBM24 in hearing and balance using conditional knockout mice. Our results show that Rbm24 knockout results in severe hearing and balance deficits. Hair cell development is significantly affected in Rbm24 knockout cochlea, as the hair bundles are poorly developed and eventually degenerated. Hair bundle disorganization is also observed in Rbm24 knockout vestibular hair cells, although to a lesser extent. Consistently, significant hair cell loss is observed in the cochlea but not vestibule. RNAseq analysis identified several genes whose mRNA stability or pre‐mRNA alternative splicing is affected by Rbm24 knockout. Among them are Cdh23, Pcdh15, and Myo7a, which have been shown to play important roles in stereocilia development as well as mechano‐electrical transduction. Taken together, our present work suggests that RBM24 is required for mouse hair cell development through regulating pre‐mRNA alternative splicing as well as mRNA stability.
“…Thus, smpx , gsdmeb , and otofa are possible target mRNAs of Rbm24a for hair cell morphogenesis and function. Consistently, zebrafish smpx morphants show structural alterations of stereocilia and kinocilia in hair cells as well as impaired hair cell activity [ 49 ].…”
Section: Rbp-mediated Post-transcriptional Regulation In Inner Ear Ha...mentioning
RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level. They play major roles in the tissue- and stage-specific expression of protein isoforms as well as in the maintenance of protein homeostasis. The inner ear is a bi-functional organ, with the cochlea and the vestibular system required for hearing and for maintaining balance, respectively. It is relatively well documented that transcription factors and signaling pathways are critically involved in the formation of inner ear structures and in the development of hair cells. Accumulating evidence highlights emerging functions of RBPs in the post-transcriptional regulation of inner ear development and hair cell function. Importantly, mutations of splicing factors of the RBP family and defective alternative splicing, which result in inappropriate expression of protein isoforms, lead to deafness in both animal models and humans. Because RBPs are critical regulators of cell proliferation and differentiation, they present the potential to promote hair cell regeneration following noise- or ototoxin-induced damage through mitotic and non-mitotic mechanisms. Therefore, deciphering RBP-regulated events during inner ear development and hair cell regeneration can help define therapeutic strategies for treatment of hearing loss. In this review, we outline our evolving understanding of the implications of RBPs in hair cell formation and hearing disease with the aim of promoting future research in this field.
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