RNA microarray analysis in prenatal mouse cochlea reveals novel IGF-I target genes: implication of MEF2 and FOXM1 transcription factors

Background: Insulin-like growth factor-I (IGF-I) provides pivotal cell survival and differentiation signals during inner ear development throughout evolution. Homozygous mutations of human IGF1 cause syndromic sensorineural deafness, decreased intrauterine and postnatal growth rates, and mental retardation. In the mouse, deficits in IGF-I result in profound hearing loss associated with reduced survival, differentiation and maturation of auditory neurons. Nevertheless, little is known about the molecular basis of IGF-I activity in hearing and deafness. Methodology/Principal Findings: A combination of quantitative RT-PCR, subcellular fractionation and Western blotting, along with in situ hybridization studies show IGF-I and its high affinity receptor to be strongly expressed in the embryonic and postnatal mouse cochlea. The expression of both proteins decreases after birth and in the cochlea of E18.5 embryonic Igf1(-/-) null mice, the balance of the main IGF related signalling pathways is altered, with lower activation of Akt and ERK1/2 and stronger activation of p38 kinase. By comparing the Igf1(-/-) and Igf1(+/+) transcriptomes in E18.5 mouse cochleae using RNA microchips and validating their results, we demonstrate the up-regulation of the FoxM1 transcription factor and the misexpression of the neural progenitor transcription factors Six6 and Mash1 associated with the loss of IGF-I. Parallel, in silico promoter analysis of the genes modulated in conjunction with the loss of IGF-I revealed the possible involvement of MEF2 in cochlear development. E18.5 Igf1(+/+) mouse auditory ganglion neurons showed intense MEF2A and MEF2D nuclear staining and MEF2A was also evident in the organ of Corti. At P15, MEF2A and MEF2D expression were shown in neurons and sensory cells. In the absence of IGF-I, nuclear levels of MEF2 were diminished, indicating less transcriptional MEF2 activity. By contrast, there was an increase in the nuclear accumulation of FoxM1 and a corresponding decrease in the nuclear cyclin-dependent kinase inhibitor p27(Kip1). Conclusions/Significance: We have defined the spatiotemporal expression of elements involved in IGF signalling during inner ear development and reveal novel regulatory mechanisms that are modulated by IGF-I in promoting sensory cell and neural survival and differentiation. These data will help us to understand the molecular bases of human sensorineural deafness associated to deficits in IGF-I

AKT Signaling Mediates IGF-I Survival Actions on Otic Neural Progenitors

Background: Otic neurons and sensory cells derive from common progenitors whose transition into mature cells requires the coordination of cell survival, proliferation and differentiation programmes. Neurotrophic support and survival of post-mitotic otic neurons have been intensively studied, but the bases underlying the regulation of programmed cell death in immature proliferative otic neuroblasts remains poorly understood. The protein kinase AKT acts as a node, playing a critical role in controlling cell survival and cell cycle progression. AKT is activated by trophic factors, including insulin-like growth factor I (IGF-I), through the generation of the lipidic second messenger phosphatidylinositol 3-phosphate by phosphatidylinositol 3-kinase (PI3K). Here we have investigated the role of IGF-dependent activation of the PI3K-AKT pathway in maintenance of otic neuroblasts. Methodology/Principal Findings: By using a combination of organotypic cultures of chicken (Gallus gallus) otic vesicles and acoustic-vestibular ganglia, Western blotting, immunohistochemistry and in situ hybridization, we show that IGF-I-activation of AKT protects neural progenitors from programmed cell death. IGF-I maintains otic neuroblasts in an undifferentiated and proliferative state, which is characterised by the upregulation of the forkhead box M1 (FoxM1) transcription factor. By contrast, our results indicate that post-mitotic p27Kip-positive neurons become IGF-I independent as they extend their neuronal processes. Neurons gradually reduce their expression of the Igf1r, while they increase that of the neurotrophin receptor, TrkC. Conclusions/Significance: Proliferative otic neuroblasts are dependent on the activation of the PI3K-AKT pathway by IGF-I for survival during the otic neuronal progenitor phase of early inner ear development

RAF Kinase Activity Regulates Neuroepithelial Cell Proliferation and Neuronal Progenitor Cell Differentiation during Early Inner Ear Development

Background: Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood. Methodology/Principal Findings: By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation. Conclusions/Significance: We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG

Cabbage and fermented vegetables : From death rate heterogeneity in countries to candidates for mitigation strategies of severe COVID-19

Large differences in COVID-19 death rates exist between countries and between regions of the same country. Some very low death rate countries such as Eastern Asia, Central Europe, or the Balkans have a common feature of eating large quantities of fermented foods. Although biases exist when examining ecological studies, fermented vegetables or cabbage have been associated with low death rates in European countries. SARS-CoV-2 binds to its receptor, the angiotensin-converting enzyme 2 (ACE2). As a result of SARS-CoV-2 binding, ACE2 downregulation enhances the angiotensin II receptor type 1 (AT(1)R) axis associated with oxidative stress. This leads to insulin resistance as well as lung and endothelial damage, two severe outcomes of COVID-19. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the most potent antioxidant in humans and can block in particular the AT(1)R axis. Cabbage contains precursors of sulforaphane, the most active natural activator of Nrf2. Fermented vegetables contain many lactobacilli, which are also potent Nrf2 activators. Three examples are: kimchi in Korea, westernized foods, and the slum paradox. It is proposed that fermented cabbage is a proof-of-concept of dietary manipulations that may enhance Nrf2-associated antioxidant effects, helpful in mitigating COVID-19 severity.Peer reviewe

Nrf2-interacting nutrients and COVID-19 : time for research to develop adaptation strategies

There are large between- and within-country variations in COVID-19 death rates. Some very low death rate settings such as Eastern Asia, Central Europe, the Balkans and Africa have a common feature of eating large quantities of fermented foods whose intake is associated with the activation of the Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) anti-oxidant transcription factor. There are many Nrf2-interacting nutrients (berberine, curcumin, epigallocatechin gallate, genistein, quercetin, resveratrol, sulforaphane) that all act similarly to reduce insulin resistance, endothelial damage, lung injury and cytokine storm. They also act on the same mechanisms (mTOR: Mammalian target of rapamycin, PPAR gamma:Peroxisome proliferator-activated receptor, NF kappa B: Nuclear factor kappa B, ERK: Extracellular signal-regulated kinases and eIF2 alpha:Elongation initiation factor 2 alpha). They may as a result be important in mitigating the severity of COVID-19, acting through the endoplasmic reticulum stress or ACE-Angiotensin-II-AT(1)R axis (AT(1)R) pathway. Many Nrf2-interacting nutrients are also interacting with TRPA1 and/or TRPV1. Interestingly, geographical areas with very low COVID-19 mortality are those with the lowest prevalence of obesity (Sub-Saharan Africa and Asia). It is tempting to propose that Nrf2-interacting foods and nutrients can re-balance insulin resistance and have a significant effect on COVID-19 severity. It is therefore possible that the intake of these foods may restore an optimal natural balance for the Nrf2 pathway and may be of interest in the mitigation of COVID-19 severity

Primary antibodies.

1<p>Antibody type: RbP, rabbit polyclonal; MouM, mouse monoclonal; GtP, goat polyclonal.</p>2<p>Technique: IHF, Immunohistofluorescence. WB, Western Blotting.</p>3<p>Monoclonal antibody developed by Thomas Jessell and Jane Dodd and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD, which is maintained by the Department of Biological Sciences, University of Iowa, Iowa City, IA 52242.</p

IGF-I protects otic progenitors from programmed cell death.

<p>(<b>A</b>) Spatiotemporal pattern of TUNEL-positive cells in otic vesicles. Otic vesicles were isolated from HH18 chicken embryos and cultured in serum-free medium either without additives (0S; a–e and a′–e′) or supplemented with IGF-I (10 nM; g–k and g′–k′). Cell death was visualized by TUNEL staining (green, a′–k′). Light microscopy images are also shown for comparison (a–k). Arrows and arrowheads show the accumulation of TUNEL-positive cells in the otic epithelium and in the AVG, respectively (j′, k′). Panels j″ and k″ show the boxed areas of the ventral part of the AVG in j′ and k′ respectively, where TUNEL-positive cells accumulate. Representative images of six otic vesicles per condition and from at least three independent experiments are shown, and they were obtained from compiled confocal microscopy projections of otic vesicles. Orientation: A, anterior; D, dorsal. Scale bars, 150 µm. (<b>B</b>) TUNEL-positive cells were counted in the presence and absence of IGF-I (close and open circles, respectively). Data are shown as mean±SEM. Statistical significance between the control and IGF-I conditions at each time-point was estimated by ANOVA and using the Bonferroni and Tukey's post-hoc tests for multiple comparisons: *P<0.05, **P< 0.01 and ***P<0.001 (0S versus IGF-I at each time-point).</p

The PI3K-AKT pathway mediates IGF-I survival of otic neuroblasts.

<p>(<b>A</b>) Expression of inner ear <i>Akt1</i> (white bars) and <i>Akt3</i> (black bars) mRNA analyzed by qRT-PCR at different stages, using eukaryotic 18s rRNA as the endogenous control gene. Gene expression was calculated as 2 ^−ΔΔCt and normalized to the levels at HH17. The results are expressed as the means±SEM from four independent experiments performed in triplicate. (<b>B</b>) Distribution of activated AKT (red, pAKT, Ser 473) in the developing inner ear. At HH17, pAKT is strongly expressed at the edges of the otic pore and in the mitotic epithelial cells (a, arrows and arrowheads respectively). At stage HH18, both epithelial and migrating Islet-1-positive otic neuroblasts (green) accumulate cytoplasmic pAKT (b, arrow and arrowhead, respectively). At stage HH19, the otic epithelium and ganglionic neuroblasts strongly express pAKT (c and c′, arrow and arrowheads, respectively). Panel c′ corresponds to the boxed area in c. Orientation: A, anterior; D, dorsal. (<b>C</b>) Schematic representation of the two main signaling cascades triggered by IGF-I: PI3K-AKT and RAF-MEK-ERK. LY294002 (LY) inhibits PI3K activation, whilst AKTi VIII (AKTi) inhibits AKT phosphorylation. The arrows denote the facilitating action whereas crosses indicate inhibitory influence. (<b>D</b>) Levels of activated AKT (pAKT), AKT and FoxM1 with different treatments. Both pAKT and FoxM1 levels are increased in presence of IGF-I. Quiescent HH18 otic vesicles (15 per condition) were incubated for 30 min (pAKT Ser473 and Thr 308) or 20 h (FoxM1) in the presence of IGF-I (10 nM), LY (25 µM), AKTi, (50 µM), or combinations of IGF-I and the inhibitors. Representative blots are shown from four independent experiments. The data are shown as the mean±SEM and the statistical significance between the different conditions was estimated by ANOVA: **P<0.01, ***P<0.005, versus IGF-I. (<b>E</b>) Effects of AKT-inhibition on Islet-1-positive neuroblast population. Otic vesicles were isolated from HH18 chicken embryos and cultured for 20 h in 0S culture conditions (a), in the presence of IGF-I (10 nM, b), LY (25 µM, c), or a combination of both (d). Immunostaining for TuJ-1 (red) and Islet-1 (green) were performed. Islet-1-positive population increases in the presence of IGF-I compared to 0S (a and b, dashed areas). In the presence of LY, there are fewer Islet-1-positive neuroblasts and generalized TuJ-1 expression in the AVG (c, arrow). In the presence of LY and IGF-I, the Islet-1-positive population partially recovers (d, dashed areas). Representative images of five otic vesicles per condition and from at least three independent experiments are shown, and they were obtained from compiled confocal microscopy projections of otic vesicles. Orientation: A, anterior; D, dorsal. Scale bar, 150 µm.</p

IGF-I promotes BrdU incorporation in the otic vesicle.

<p>(<b>A</b>) Spatiotemporal pattern of BrdU incorporation in cultured otic vesicles. Otic vesicles were isolated from HH18 chicken embryos and cultured in serum-free medium without additives (0S; a–e and a′–e′) or supplemented with IGF-I (10 nM; g–k and g′–k′). Immunostaining for incorporated BrdU was performed (green), and light microscopy images are shown for comparison (a–k). The arrowhead points to the accumulation of BrdU-positive cells in the neurogenic region in the otic epithelium (c′), whilst the arrows indicate the areas of low BrdU incorporation in the AVG (c′, j′, k′). Panels j″ and k″ correspond to the boxed areas of the ventral part of the AVG in j′ and k′, respectively, where BrdU incorporation is very low. Representative images of six otic vesicles per condition and from at least three independent experiments are shown, and they were obtained from compiled confocal microscopy projections of otic vesicles. Orientation, A, anterior; D, dorsal. Scale bars, 150 µm. (<b>B</b>) Quantification (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030790#s2" target="_blank">Materials and Methods</a>) of the areas (µm²) of both otic vesicle (circles) and AVG (triangles) in the presence or absence of IGF-I (closed and open figures, respectively). Data are shown as mean±SEM. Statistical significance between the control and IGF-I conditions at each time-point studied was estimated by ANOVA, followed by Bonferroni and Tukey's multiple comparisons: *P<0.05, ***P<0.001 (0S versus IGF-I at each time-point).</p