The cochlear matrisome: Importance in hearing and deafness.

The extracellular matrix (ECM) consists in a complex meshwork of collagens, glycoproteins, and proteoglycans, which serves a scaffolding function and provides viscoelastic properties to the tissues. ECM acts as a biomechanical support, and actively participates in cell signaling to induce tissular changes in response to environmental forces and soluble cues. Given the remarkable complexity of the inner ear architecture, its exquisite structure-function relationship, and the importance of vibration-induced stimulation of its sensory cells, ECM is instrumental to hearing. Many factors of the matrisome are involved in cochlea development, function and maintenance, as evidenced by the variety of ECM proteins associated with hereditary deafness. This review describes the structural and functional ECM components in the auditory organ and how they are modulated over time and following injury.

Sox9 Inhibits Cochlear Hair Cell Fate by Upregulating Hey1 and HeyL Antagonists of Atoh1.

It is widely accepted that cell fate determination in the cochlea is tightly controlled by different transcription factors (TFs) that remain to be fully defined. Here, we show that Sox9, initially expressed in the entire sensory epithelium of the cochlea, progressively disappears from differentiating hair cells (HCs) and is finally restricted to supporting cells (SCs). By performing ex vivo electroporation of E13.5-E14.5 cochleae, we demonstrate that maintenance of Sox9 expression in the progenitors committed to HC fate blocks their differentiation, even if co-expressed with Atoh1, a transcription factor necessary and sufficient to form HC. Sox9 inhibits Atoh1 transcriptional activity by upregulating Hey1 and HeyL antagonists, and genetic ablation of these genes induces extra HCs along the cochlea. Although Sox9 suppression from sensory progenitors ex vivo leads to a modest increase in the number of HCs, it is not sufficient in vivo to induce supernumerary HC production in an inducible Sox9 knockout model. Taken together, these data show that Sox9 is downregulated from nascent HCs to allow the unfolding of their differentiation program. This may be critical for future strategies to promote fully mature HC formation in regeneration approaches.

Generation of a Well-Characterized Homozygous Chromodomain-Helicase-DNA-Binding Protein 4G1003D Mutant hESC Line Using CRISPR/eCas9 (ULIEGEe001-A-1).

The chromatin remodeler Chromodomain-helicase-DNA-binding protein 4 (CHD4) is crucial for the development of multiple organ systems. Functional mutations of CHD4 have recently been described in a developmental disorder, namely Siffrim-Hitz-Weiss syndrome (SIHIWES). Herein, we have generated a homozygous CHD4G1003D hESC line (WAe025-A-1) using CRISPR/eCas9-based gene editing in the WA-25 hESC line. The edited hESC line maintains normal karyotype, pluripotency, and ability to differentiate into three germ layers. This cell line will be a valuable resource for studying the functional role of CHD4 during the development and disease modeling of SIHIWES in vitro.

Re-Evaluating the Relevance of the Oxygen-Glucose Deprivation Model in Ischemic Stroke: The Example of Cdk Inhibition.

Previous research has shown that cyclin-dependent kinases (Cdks) that play physiological roles in cell cycle regulation become activated in post-mitotic neurons after ischemic stroke, resulting in apoptotic neuronal death. In this article, we report our results using the widely used oxygen-glucose deprivation (OGD) in vitro model of ischemic stroke on primary mouse cortical neurons to investigate whether Cdk7, as part of the Cdk-activating kinase (CAK) complex that activates cell cycle Cdks, might be a regulator of ischemic neuronal death and may potentially constitute a therapeutic target for neuroprotection. We found no evidence of neuroprotection with either pharmacological or genetic invalidation of Cdk7. Despite the well-established idea that apoptosis contributes to cell death in the ischemic penumbra, we also found no evidence of apoptosis in the OGD model. This could explain the absence of neuroprotection following Cdk7 invalidation in this model. Neurons exposed to OGD seem predisposed to die in an NMDA receptor-dependent manner that could not be prevented further downstream. Given the direct exposure of neurons to anoxia or severe hypoxia, it is questionable how relevant OGD is for modeling the ischemic penumbra. Due to remaining uncertainties about cell death after OGD, caution is warranted when using this in vitro model to identify new stroke therapies.

ATP-Dependent Chromatin Remodellers in Inner Ear Development.

During transcription, DNA replication and repair, chromatin structure is constantly modified to reveal specific genetic regions and allow access to DNA-interacting enzymes. ATP-dependent chromatin remodelling complexes use the energy of ATP hydrolysis to modify chromatin architecture by repositioning and rearranging nucleosomes. These complexes are defined by a conserved SNF2-like, catalytic ATPase subunit and are divided into four families: CHD, SWI/SNF, ISWI and INO80. ATP-dependent chromatin remodellers are crucial in regulating development and stem cell biology in numerous organs, including the inner ear. In addition, mutations in genes coding for proteins that are part of chromatin remodellers have been implicated in numerous cases of neurosensory deafness. In this review, we describe the composition, structure and functional activity of these complexes and discuss how they contribute to hearing and neurosensory deafness.

Scaling brain neurogenesis across evolution.

A genetic change could explain increased cortical neurogenesis in modern humans.

Cyclin-dependent kinase 7 contributes to myelin maintenance in the adult central nervous system and promotes myelin gene expression.

Mechanisms regulating oligodendrocyte differentiation, developmental myelination and myelin maintenance in adulthood are complex and still not completely described. Their understanding is crucial for the development of new protective or therapeutic strategies in demyelinating pathologies such as multiple sclerosis. In this perspective, we have investigated the role of Cyclin-dependent kinase 7 (Cdk7), a kinase involved in cell-cycle progression and transcription regulation, in the oligodendroglial lineage. We generated a conditional knock-out mouse model in which Cdk7 is invalidated in post-mitotic oligodendrocytes. At the end of developmental myelination, the number and diameter of myelinated axons, as well as the myelin structure, thickness and protein composition, were normal. However, in young adult and in aged mice, there was a higher number of small caliber myelinated axons associated with a decreased mean axonal diameter, myelin sheaths of large caliber axons were thinner, and the level of some major myelin-associated proteins was reduced. These defects were accompanied by the appearance of an abnormal clasping phenotype. We also used an in vitro oligodendroglial model and showed that Cdk7 pharmacological inhibition led to an altered myelination-associated morphological modification combined with a decreased expression of myelin-specific genes. Altogether, we identified novel functions for Cdk7 in CNS myelination.

The ubiquitin-proteasome system in normal hearing and deafness.

Post-translational modifications of proteins are essential for the proper development and function of many tissues and organs, including the inner ear. Ubiquitination is a highly selective post-translational modification that involves the covalent conjugation of ubiquitin to a substrate protein. The most common outcome of protein ubiquitination is degradation by the ubiquitin-proteasome system (UPS), preventing the accumulation of misfolded, damaged, and excess proteins. In addition to proteasomal degradation, ubiquitination regulates other cellular processes, such as transcription, translation, endocytosis, receptor activity, and subcellular localization. All of these processes are essential for cochlear development and maintenance, as several studies link impairment of UPS with altered cochlear development and hearing loss. In this review, we provide insight into the well-oiled machinery of UPS with a focus on its confirmed role in normal hearing and deafness and potential therapeutic strategies to prevent and treat UPS-associated hearing loss.

Dispensability of Tubulin Acetylation for 15-protofilament Microtubule Formation in the Mammalian Cochlea.

The development of hearing in mammals requires the formation and maturation of a highly organized and specialized epithelium known as the organ of Corti. This epithelium contains two types of cells, the sensory cells, which are the true receptors of auditory information, and the surrounding supporting cells, which are composed of a highly developed cytoskeleton essential to the architecture of the mature organ of Corti. The supporting cells are the only mammalian cells reported to contain the unusual 15-protofilament microtubules. In this paper, we show that 15-protofilament microtubules appear between the second and fourth day after birth in the pillar cells of the organ of Corti in mice. We also show that contrary to what has been described in the nematode worm Caenorhabiditis. elegans, microtubule acetylation is not essential for the formation of 15-protofilament microtubules in mice but is required for fine-tuning of their diameter.Key words: Acetylation, cytoskeleton, microtubule, inner ear, supporting cells.

Core cell cycle machinery is crucially involved in both life and death of post-mitotic neurons.

A persistent dogma in neuroscience supported the idea that terminally differentiated neurons permanently withdraw from the cell cycle. However, since the late 1990s, several studies have shown that cell cycle proteins are expressed in post-mitotic neurons under physiological conditions, indicating that the cell cycle machinery is not restricted to proliferating cells. Moreover, many studies have highlighted a clear link between cell cycle-related proteins and neurological disorders, particularly relating to apoptosis-induced neuronal death. Indeed, cell cycle-related proteins can be upregulated or overactivated in post-mitotic neurons in case of acute or degenerative central nervous system disease. Given the considerable lack of effective treatments for age-related neurological disorders, new therapeutic approaches targeting the cell cycle machinery might thus be considered. This review aims at summarizing current knowledge about the role of the cell cycle machinery in post-mitotic neurons in healthy and pathological conditions.

Auditory function and dysfunction: estrogen makes a difference.

Estrogen is the major female hormone involved in reproductive functions, but it also exerts a variety of additional roles in non-reproductive organs. In this review, we highlight the preclinical and clinical studies that have pointed out sex differences and estrogenic influence on audition. We also describe the experimental evidences supporting a protective role of estrogen towards acquired forms of hearing loss. Although a high level of endogenous estrogen is associated with a better hearing function, hormonal treatments at menopause have provided contradictory outcomes. The various factors that are likely to explain these discrepancies include the treatment regimen as well as the hormonal status and responsiveness of the patients. The complexity of estrogen signaling is being untangled and many downstream effectors of its genomic and non-genomic actions have been identified in other systems. Based on these advances and on the common physio-pathological events that underlie age-related, drug or noise-induced hearing loss, we discuss potential mechanisms for their protective actions in the cochlea.

Proteostasis is essential during cochlear development for neuron survival and hair cell polarity.

Protein homeostasis is essential to cell function, and a compromised ability to reduce the load of misfolded and aggregated proteins is linked to numerous age-related diseases, including hearing loss. Here, we show that altered proteostasis consequent to Elongator complex deficiency also impacts the proper development of the cochlea and results in deafness. In the absence of the catalytic subunit Elp3, differentiating spiral ganglion neurons display large aggresome-like structures and undergo apoptosis before birth. The cochlear mechanosensory cells are able to survive proteostasis disruption but suffer defects in polarity and stereociliary bundle morphogenesis. We demonstrate that protein aggregates accumulate at the apical surface of hair cells, where they cause a local slowdown of microtubular trafficking, altering the distribution of intrinsic polarity proteins and affecting kinocilium position and length. Alleviation of protein misfolding using the chemical chaperone 4-phenylbutyric acid during embryonic development ameliorates hair cell polarity in Elp3-deficient animals. Our study highlights the importance of developmental proteostasis in the cochlea and unveils an unexpected link between proteome integrity and polarized organization of cellular components.

EphA4-ADAM10 Interplay Patterns the Cochlear Sensory Epithelium through Local Disruption of Adherens Junctions.

The cochlear sensory epithelium contains a functionally important triangular fluid-filled space between adjacent pillar cells referred to as the tunnel of Corti. However, the molecular mechanisms leading to local cell-cell separation during development remain elusive. Here we show that EphA4 associates with ADAM10 to promote the destruction of E-cadherin-based adhesions between adjacent pillar cells. These cells fail to separate from each other, and E-cadherin abnormally persists at the pillar cell junction in EphA4 forward-signaling-deficient mice, as well as in the presence of ADAM10 inhibitor. Using immunolabeling and an in situ proximity ligation assay, we found that EphA4 forms a complex with E-cadherin and its sheddase ADAM10, which could be activated by ephrin-B2 across the pillar cell junction to trigger the cleavage of E-cadherin. Altogether, our findings provide a new molecular insight into the regulation of adherens junctions, which might be extended to a variety of physiological or pathological processes.

Pluripotent stem cell-derived cochlear cells: a challenge in constant progress.

Hearing loss is a common affection mainly resulting from irreversible loss of the sensory hair cells of the cochlea; therefore, developing therapies to replace missing hair cells is essential. Understanding the mechanisms that drive their formation will not only help to unravel the molecular basis of deafness, but also give a roadmap for recapitulating hair cells development from cultured pluripotent stem cells. In this review, we provide an overview of the molecular mechanisms involved in hair cell production from both human and mouse embryonic stem cells. We then provide insights how this knowledge has been applied to differentiate induced pluripotent stem cells into otic progenitors and hair cells. Finally, we discuss the current limitations for properly obtaining functional hair cell in a Petri dish, as well as the difficulties that have to be overcome prior to consider stem cell therapy as a potential treatment for hearing loss.

ATAT1-enriched vesicles promote microtubule acetylation via axonal transport.

Microtubules are polymerized dimers of α- and ß-tubulin that underlie a broad range of cellular activities. Acetylation of α-tubulin by the acetyltransferase ATAT1 modulates microtubule dynamics and functions in neurons. However, it remains unclear how this enzyme acetylates microtubules over long distances in axons. Here, we show that loss of ATAT1 impairs axonal transport in neurons in vivo, and cell-free motility assays confirm a requirement of α-tubulin acetylation for proper bidirectional vesicular transport. Moreover, we demonstrate that the main cellular pool of ATAT1 is transported at the cytosolic side of neuronal vesicles that are moving along axons. Together, our data suggest that axonal transport of ATAT1-enriched vesicles is the predominant driver of α-tubulin acetylation in axons.

Proliferation of hippocampal progenitors relies on p27-dependent regulation of Cdk6 kinase activity.

Neural stem cells give rise to granule dentate neurons throughout life in the hippocampus. Upon activation, these stem cells generate fast proliferating progenitors that complete several rounds of divisions before differentiating into neurons. Although the mechanisms regulating the activation of stem cells have been intensively studied, little attention has been given so far to the intrinsic machinery allowing the expansion of the progenitor pool. The cell cycle protein Cdk6 positively regulates the proliferation of hippocampal progenitors, but the mechanism involved remains elusive. Whereas Cdk6 functions primarily as a cell cycle kinase, it can also act as transcriptional regulator in cancer cells and hematopoietic stem cells. Using mouse genetics, we show here that the function of Cdk6 in hippocampal neurogenesis relies specifically on its kinase activity. The present study also reveals a specific regulatory mechanism for Cdk6 in hippocampal progenitors. In contrast to the classical model of the cell cycle, we observe that the Cip/Kip family member p27, rather than the Ink4 family, negatively regulates Cdk6 in the adult hippocampus. Altogether, our data uncover a unique, cell type-specific regulatory mechanism controlling the expansion of hippocampal progenitors, where Cdk6 kinase activity is modulated by p27.

Genetic and pharmacological inhibition of Cdk1 provides neuroprotection towards ischemic neuronal death.

Cell cycle proteins are mainly expressed by dividing cells. However, it is well established that these molecules play additional non-canonical activities in several cell death contexts. Increasing evidence shows expression of cell cycle regulating proteins in post-mitotic cells, including mature neurons, following neuronal insult. Several cyclin-dependent kinases (Cdks) have already been shown to mediate ischemic neuronal death but Cdk1, a major cell cycle G2/M regulator, has not been investigated in this context. We therefore examined the role of Cdk1 in neuronal cell death following cerebral ischemia, using both in vitro and in vivo genetic and pharmacological approaches. Exposure of primary cortical neurons cultures to 4 h of oxygen-glucose deprivation (OGD) resulted in neuronal cell death and induced Cdk1 expression. Neurons from Cdk1-cKO mice showed partial resistance to OGD-induced neuronal cell death. Addition of R-roscovitine to the culture medium conferred neuroprotection against OGD-induced neuronal death. Transient 1-h occlusion of the cerebral artery (MCAO) also leads to Cdk1 expression and activation. Cdk1-cKO mice displayed partial resistance to transient 1-h MCAO. Moreover, systemic delivery of R-roscovitine was neuroprotective following transient 1-h MCAO. This study demonstrates that promising neuroprotective therapies can be considered through inhibition of the cell cycle machinery and particularly through pharmacological inhibition of Cdk1.

Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis.

Interneurons navigate along multiple tangential paths to settle into appropriate cortical layers. They undergo a saltatory migration paced by intermittent nuclear jumps whose regulation relies on interplay between extracellular cues and genetic-encoded information. It remains unclear how cycles of pause and movement are coordinated at the molecular level. Post-translational modification of proteins contributes to cell migration regulation. The present study uncovers that carboxypeptidase 1, which promotes post-translational protein deglutamylation, controls the pausing of migrating cortical interneurons. Moreover, we demonstrate that pausing during migration attenuates movement simultaneity at the population level, thereby controlling the flow of interneurons invading the cortex. Interfering with the regulation of pausing not only affects the size of the cortical interneuron cohort but also impairs the generation of age-matched projection neurons of the upper layers.

The miR-183/ItgA3 axis is a key regulator of prosensory area during early inner ear development.

MicroRNAs are important regulators of gene expression and are involved in cellular processes such as proliferation or differentiation, particularly during development of numerous organs including the inner ear. However, it remains unknown if miRNAs are required during the earliest stages of otocyst and cochlear duct development. Here, we report that a conditional loss of Dicer expression in the otocyst impairs the early development of the inner ear as a result of the accumulation of DNA damage that trigger p53-mediated apoptosis. Moreover, cochlear progenitors in the prosensory domain do not exit the cell cycle. Our unbiased approach identified ItgA3 as a target of miR-183, which are both enriched in the otic vesicle. We observed that the repression of integrin alpha 3 by miR-183 controls cell proliferation in the developing cochlea. Collectively, our results reveal that Dicer and miRNAs play essential roles in the regulation of early inner ear development.

Loss of Elp3 Impairs the Acetylation and Distribution of Connexin-43 in the Developing Cerebral Cortex.

The Elongator complex is required for proper development of the cerebral cortex. Interfering with its activity in vivo delays the migration of postmitotic projection neurons, at least through a defective α-tubulin acetylation. However, this complex is already expressed by cortical progenitors where it may regulate the early steps of migration by targeting additional proteins. Here we report that connexin-43 (Cx43), which is strongly expressed by cortical progenitors and whose depletion impairs projection neuron migration, requires Elongator expression for its proper acetylation. Indeed, we show that Cx43 acetylation is reduced in the cortex of Elp3cKO embryos, as well as in a neuroblastoma cell line depleted of Elp1 expression, suggesting that Cx43 acetylation requires Elongator in different cellular contexts. Moreover, we show that histones deacetylase 6 (HDAC6) is a deacetylase of Cx43. Finally, we report that acetylation of Cx43 regulates its membrane distribution in apical progenitors of the cerebral cortex.