Small-molecule inhibition of Lats kinases may promote Yap-dependent proliferation in postmitotic mammalian tissues

AbstractHippo signaling is an evolutionarily conserved pathway that restricts growth and regeneration predominantly by suppressing the activity of the transcriptional coactivator Yap. Using a high-throughput phenotypic screen, we identified a potent and non-toxic activator of Yap. In vitro kinase assays show that the compound acts as an ATP-competitive inhibitor of Lats kinases—the core enzymes in Hippo signaling. The substance prevents Yap phosphorylation and induces proliferation of supporting cells in the murine inner ear, murine cardiomyocytes, and human Müller glia in retinal organoids. RNA sequencing indicates that the inhibitor reversibly activates the expression of transcriptional Yap targets: upon withdrawal, a subset of supporting-cell progeny exits the cell cycle and upregulates genes characteristic of sensory hair cells. Our results suggest that the pharmacological inhibition of Lats kinases may promote initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear.

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Small-molecule inhibition of Lats kinases promotes Yap-dependent proliferation in postmitotic mammalian tissues

10.1101/2020.02.11.944157 ◽

2020 ◽

Author(s):

Nathaniel Kastan ◽

Ksenia Gnedeva ◽

Theresa Alisch ◽

Aleksandra A. Petelski ◽

David J. Huggins ◽

...

Keyword(s):

Inner Ear ◽

Hair Cells ◽

Competitive Inhibitor ◽

Reversible Inhibitor ◽

Hippo Signaling ◽

Supporting Cells ◽

Small Molecule Inhibition ◽

Phenotypic Screen ◽

Evolutionarily Conserved ◽

Mammalian Tissues

SummaryHippo signaling is an evolutionarily conserved pathway that restricts organ growth during development and suppresses regeneration in mature organs1–3. Using a high-throughput phenotypic screen, we have identified a potent, non-toxic, and reversible inhibitor of Hippo signaling. An ATP-competitive inhibitor of Lats kinases, the compound causes Yap-dependent proliferation of murine supporting cells in the inner ear, murine cardiomyocytes, and human Müller glia in retinal organoids. RNA sequencing indicates that the substance fosters both the G1-S and G2-M checkpoint transitions and yields supporting cells capable of transdifferentiation. Upon withdrawal of the compound, a subset of supporting cells move their nuclei into the hair-cell layer and express genes characteristic of hair cells. Viral transfection of Atoh1 induces the expression of hair cellspecific proteins in progeny. The compound promotes the initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear.

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Generation of inner ear hair cells by direct lineage conversion of primary somatic cells

eLife ◽

10.7554/elife.55249 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 3

Author(s):

Louise Menendez ◽

Talon Trecek ◽

Suhasni Gopalakrishnan ◽

Litao Tao ◽

Alexander L Markowitz ◽

...

Keyword(s):

Hair Cell ◽

Inner Ear ◽

Hair Cells ◽

Cell Loss ◽

Electrophysiological Properties ◽

Sensory Hair Cells ◽

Channel Expression ◽

Direct Lineage Conversion ◽

Tail Tip

The mechanoreceptive sensory hair cells in the inner ear are selectively vulnerable to numerous genetic and environmental insults. In mammals, hair cells lack regenerative capacity, and their death leads to permanent hearing loss and vestibular dysfunction. Their paucity and inaccessibility has limited the search for otoprotective and regenerative strategies. Growing hair cells in vitro would provide a route to overcome this experimental bottleneck. We report a combination of four transcription factors (Six1, Atoh1, Pou4f3, and Gfi1) that can convert mouse embryonic fibroblasts, adult tail-tip fibroblasts and postnatal supporting cells into induced hair cell-like cells (iHCs). iHCs exhibit hair cell-like morphology, transcriptomic and epigenetic profiles, electrophysiological properties, mechanosensory channel expression, and vulnerability to ototoxin in a high-content phenotypic screening system. Thus, direct reprogramming provides a platform to identify causes and treatments for hair cell loss, and may help identify future gene therapy approaches for restoring hearing.

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A Novel Small Molecule Neurotrophin-3 Analogue Promotes Inner Ear Neurite Outgrowth and Synaptogenesis In vitro

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.666706 ◽

2021 ◽

Vol 15 ◽

Author(s):

Judith S. Kempfle ◽

Marlon V. Duro ◽

Andrea Zhang ◽

Carolina D. Amador ◽

Richard Kuang ◽

...

Keyword(s):

Inner Ear ◽

Neurite Outgrowth ◽

Small Molecule ◽

Hair Cells ◽

Spiral Ganglion ◽

Sensory Hair ◽

Neurotrophin 3 ◽

Sensory Hair Cells ◽

Ganglion Neurons

Sensorineural hearing loss is irreversible and is associated with the loss of spiral ganglion neurons (SGNs) and sensory hair cells within the inner ear. Improving spiral ganglion neuron (SGN) survival, neurite outgrowth, and synaptogenesis could lead to significant gains for hearing-impaired patients. There has therefore been intense interest in the use of neurotrophic factors in the inner ear to promote both survival of SGNs and re-wiring of sensory hair cells by surviving SGNs. Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) represent the primary neurotrophins in the inner ear during development and throughout adulthood, and have demonstrated potential for SGN survival and neurite outgrowth. We have pioneered a hybrid molecule approach to maximize SGN stimulation in vivo, in which small molecule analogues of neurotrophins are linked to bisphosphonates, which in turn bind to cochlear bone. We have previously shown that a small molecule BDNF analogue coupled to risedronate binds to bone matrix and promotes SGN neurite outgrowth and synaptogenesis in vitro. Because NT-3 has been shown in a variety of contexts to have a greater regenerative capacity in the cochlea than BDNF, we sought to develop a similar approach for NT-3. 1Aa is a small molecule analogue of NT-3 that has been shown to activate cells through TrkC, the NT-3 receptor, although its activity on SGNs has not previously been described. Herein we describe the design and synthesis of 1Aa and a covalent conjugate of 1Aa with risedronate, Ris-1Aa. We demonstrate that both 1Aa and Ris-1Aa stimulate neurite outgrowth in SGN cultures at a significantly higher level compared to controls. Ris-1Aa maintained its neurotrophic activity when bound to hydroxyapatite, the primary mineral component of bone. Both 1Aa and Ris-1Aa promote significant synaptic regeneration in cochlear explant cultures, and both 1Aa and Ris-1Aa appear to act at least partly through TrkC. Our results provide the first evidence that a small molecule analogue of NT-3 can stimulate SGNs and promote regeneration of synapses between SGNs and inner hair cells. Our findings support the promise of hydroxyapatite-targeting bisphosphonate conjugation as a novel strategy to deliver neurotrophic agents to SGNs encased within cochlear bone.

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SoxC transcription factors are essential for the development of the inner ear

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1517371112 ◽

2015 ◽

Vol 112 (45) ◽

pp. 14066-14071 ◽

Cited By ~ 18

Author(s):

Ksenia Gnedeva ◽

A. J. Hudspeth

Keyword(s):

Transcription Factors ◽

Hair Cell ◽

Inner Ear ◽

Hair Cells ◽

Supporting Cell ◽

Sensory Epithelia ◽

Enhanced Expression ◽

Mechanosensory Receptors

Hair cells, the mechanosensory receptors of the inner ear, underlie the senses of hearing and balance. Adult mammals cannot adequately replenish lost hair cells, whose loss often results in deafness or balance disorders. To determine the molecular basis of this deficiency, we investigated the development of a murine vestibular organ, the utricle. Here we show that two members of the SoxC family of transcription factors, Sox4 and Sox11, are down-regulated after the epoch of hair cell development. Conditional ablation of SoxC genes in vivo results in stunted sensory organs of the inner ear and loss of hair cells. Enhanced expression of SoxC genes in vitro conversely restores supporting cell proliferation and the production of new hair cells in adult sensory epithelia. These results imply that SoxC genes govern hair cell production and thus advance these genes as targets for the restoration of hearing and balance.

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Bone marrow mesenchymal stem cells are progenitors in vitro for inner ear hair cells

Molecular and Cellular Neuroscience ◽

10.1016/j.mcn.2006.10.003 ◽

2007 ◽

Vol 34 (1) ◽

pp. 59-68 ◽

Cited By ~ 76

Author(s):

Sang-Jun Jeon ◽

Kazuo Oshima ◽

Stefan Heller ◽

Albert S.B. Edge

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Inner Ear ◽

Hair Cells ◽

Marrow Mesenchymal Stem Cells

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In Silico Electrophysiology of Inner-Ear Mechanotransduction Channel TMC1 Models

10.1101/2021.09.17.460860 ◽

2021 ◽

Author(s):

Sanket Walujkar ◽

Jeffrey M Lotthammer ◽

Collin R Nisler ◽

Joseph C Sudar ◽

Angela Ballesteros ◽

...

Keyword(s):

Inner Ear ◽

Conformational Changes ◽

Hair Cells ◽

Head Movements ◽

Mechanical Stimuli ◽

Ion Conduction ◽

Sensory Hair Cells ◽

Activation Mechanisms ◽

Dynamics Simulations ◽

Molecular Components

Inner-ear sensory hair cells convert mechanical stimuli from sound and head movements into electrical signals during mechanotransduction. Identification of all molecular components of the inner-ear mechanotransduction apparatus is ongoing; however, there is strong evidence that TMC1 and TMC2 are pore-forming subunits of the complex. We present molecular dynamics simulations that probe ion conduction of TMC1 models built based on two different structures of related TMEM16 proteins. Unlike most channels, the TMC1 models do not show a central pore. Instead, simulations of these models in a membrane environment at various voltages reveal a peripheral permeation pathway that is exposed to lipids and that shows cation permeation at rates comparable to those measured in hair cells. Furthermore, our analyses suggest that TMC1 gating mechanisms involve protein conformational changes and tension-induced lipid-mediated pore widening. These results provide insights into ion conduction and activation mechanisms of hair-cell mechanotransduction channels essential for hearing and balance.

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Electrospun regenerated silk fibroin is a promising biomaterial for the maintenance of inner ear progenitors in vitro

Journal of Biomaterials Applications ◽

10.1177/08853282211051501 ◽

2021 ◽

pp. 088532822110515

Author(s):

Guangfei Li ◽

Yanbo Yin ◽

Yaopeng Zhang ◽

Jingfang Wu ◽

Shan Sun

Keyword(s):

Inner Ear ◽

Silk Fibroin ◽

Hair Cells ◽

Engineering Research ◽

Cell Mortality ◽

Regenerated Silk Fibroin ◽

Significant Difference ◽

Student’S T ◽

New Strategies

Objective We sought to determine the biocompatibility of electrospun regenerated silk fibroin (RSF) mats with inner ear progenitors, especially their effect on the differentiation of inner ear progenitors into hair cells. Methods Neonatal mouse cochleae (n = 20) were collected and digested and allowed to form spheres over several days. Cells digested from the spheres were then seeded onto aligned or random RSF mats, with laminin-coated coverslips serving as controls. The inner ear progenitor cell mortality was examined by TUNEL labeling, and the adhesion of cells to the RSF mats or coverslip was determined by scanning electron microscopy. Finally, the number of hair cells that differentiated from inner ear progenitors was determined by Myosin7a expression. Unpaired Student’s t-tests and one-way ANOVA followed by a Dunnett’s multiple comparisons test were used in this study ( p < 0.05). Results After 5 days of culture, the inner ear progenitors had good adhesion to both the aligned and random RSF mats and there was no significant difference in TUNEL+ cells between the mats compared to the coverslip ( p > 0.05). After 7 days of in vitro differentiation culture, the percentage of differentiated hair cells on the control, aligned, and random RSF mats was 2.5 ± 0.5%, 2.7 ± 0.4%, and 2.4 ± 0.2%, respectively, and there was no significant difference between Myosin7a+ cells on either RSF mat compared to controls ( p > 0.05). Conclusion The aligned and random RSF mats had excellent biocompatibility with inner ear progenitors and helped the inner ear progenitors maintain their stemness. Our results thus indicate that RSF mats represent a useful scaffold for the development of new strategies for inner ear tissue engineering research.

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