A role for the hearing gene Cib2 in somatic sensation

Hearing and touch are two of our senses that rely on mechanotransduction to convert mechanical stimulus into electrochemical activity. Several studies have demonstrated that touch and hearing share common genetic determinants; mutations in genes causing deafness are also associated with reduced tactile sensitivity. Furthermore, transcriptome analysis from several studies has revealed that the same genes are expressed in both systems. However, the role of these genes has yet to be determined. In this work, I demonstrated that the calcium and integrin binding protein 2 (CIB2), a key component of mechanotransduction in the auditory system, is also implicated in somatosensory mechanosensation. CIB2 is an auxiliary subunit of the mechanotransduction channel in the auditory system that modulates channel function and determines correct channel positioning. Mutations in Cib2 cause hearing loss in humans and mice, as well as the cessation of mechanotransduction in the auditory system. By applying immunohistochemistry, I demonstrated the expression of CIB2 in a subset of DRG neurons, mainly nociceptors and, to a lesser extent, mechanoreceptors. CIB2 was also present in the terminal endings of sensory neurons that innervate skin end organs. In particular, CIB2 was found in terminal afferents innervating Meissner’s corpuscles and hair follicles, both associated with rapidly adapting mechanoreceptors. The impact of CIB2 on the physiological properties of different types of mechanoreceptors and nociceptors was investigated using an ex-vivo skin nerve preparation from the saphenous and tibial nerve. I showed that genetic ablation of Cib2 had a profound impact on rapidly adapting mechanoreceptors (RAMs). In a velocity and vibration experiment, RAMs lacking Cib2 exhibited dramatically higher frequency sensitivity. Thus, CIB2 influences RAM adaption rate and functions as a brake on their velocity sensitivity. CIB2 exhibits structural similarities with KChIP proteins, which are like CIB2, calcium sensors. Additionally, KChIP proteins have been shown to modulate Kv4 channel surface expression, assembly, and kinetics, leading us to the hypothesis that CIB2 influences RAM adaptation by interacting with and regulating a potassium channel. KCNQ4 inhibition or genetic deletion in RAMs had previously been shown to enhance velocity and vibration responses, similarly to observations in Cib2 knockout mice. I found that inhibiting KCNQ4 did not affect RAM responses in Wnt1-Cre; Cib2fl/ fl mice, showing that in mice lacking Cib2, KCNQ4 does not function effectively in RAMs. We were able to study the influence of CIB2 on KCNQ4 kinetics by employing a whole-cell patch clamp and transiently overexpressing CIB2 and KCNQ4 in the CHO cell line. We discovered that the deactivation time for KCNQ4 was slower after CIB2 overexpression. Furthermore, KCNQ4 mRNA was found in 60% of Cib2-positive neurons. Thus, we conclude that CIB2 interacts with and modulates KCNQ4. The absence of CIB2 in C-fibers, clearly altered their physiological properties. C-fires lacking Cib2 showed signs of sensitization as they displayed a prominent increase in after-charge responses. However, genetic deletion of Cib2 did not affect another type of nociceptor, the Aδ nociceptors. Finally, we assessed the behavioral impact of peripheral Cib2 ablation on light touch and mechanical pain. By using multiple behavioral assays, I found that mice lacking Cib2 exhibit a profound loss of touch sensation. Therefore, I performed an electrical search experiment using an ex-vivo skin nerve preparation to search for mechanically insensitive fibers and found that half of the Aβ-fibers were mechanically silent. These findings suggest that CIB2 is interacting with a mechanosensitive channel, the nature of which is yet to be investigated. This study allowed us to conclude that CIB2 interacts with and modulates ion channels (potassium and mechanosensitive channels) in the somatosensory system and therefore regulates sensory mechanotransduction. We hypothesize that CIB2, as in the auditory system, is an auxiliary subunit of channels in the somatosensory system.Hören und Tasten sind zwei unserer Sinne, die auf Mechanotransduktion beruhen, um mechanische Reize in elektrochemische Aktivität umzuwandeln. Mehrere Studien haben gezeigt, dass Tastsinn und Gehör gemeinsame genetische Determinanten haben; Mutationen in Genen, die Taubheit verursachen, werden auch mit einer verringerten Tastsensibilität in Verbindung gebracht. Darüber hinaus haben Transkriptomanalysen in mehreren Studien ergeben, dass in beiden Systemen die gleichen Gene exprimiert werden. Die Rolle dieser Gene ist jedoch noch nicht geklärt. In dieser Arbeit habe ich gezeigt, dass das Calcium- und Integrin-bindende Protein 2 (CIB2), eine Schlüsselkomponente der Mechanotransduktion im auditorischen System, auch an der somatosensorischen Mechanosensation beteiligt ist. CIB2 ist eine Hilfsuntereinheit des Mechanotransduktionskanals im auditorischen System, die die Kanalfunktion moduliert und die korrekte Kanalpositionierung bestimmt. Mutationen in Cib2 führen bei Menschen und Mäusen zu Hörverlust und zur Unterbrechung der Mechanotransduktion im auditorischen System. Mit Hilfe der Immunhistochemie konnte ich die Expression von CIB2 in einer Untergruppe von DRG-Neuronen nachweisen, hauptsächlich in Nozizeptoren und in geringerem Maße in Mechanorezeptoren. CIB2 war auch in den Endigungen der sensorischen Neuronen vorhanden, die die Endorgane der Haut innervieren. Insbesondere wurde CIB2 in terminalen Afferenzen gefunden, die Meissner-Körperchen und Haarfollikel innervieren, die beide mit schnell adaptierenden Mechanorezeptoren assoziiert sind. Die Auswirkungen von CIB2 auf die physiologischen Eigenschaften verschiedener Arten von Mechanorezeptoren und Nozizeptoren wurden anhand eines Ex-vivo-Hautnervenpräparats aus dem Nervus saphenus und dem Nervus tibialis untersucht. Ich konnte zeigen, dass die genetische Ablation von Cib2 eine tiefgreifende Auswirkung auf die sich schnell anpassenden Mechanorezeptoren (RAMs) hat. In einem Geschwindigkeits- und Vibrationsexperiment zeigten RAMs, denen Cib2 fehlte, eine dramatisch höhere Frequenzempfindlichkeit. Somit beeinflusst CIB2 die Anpassungsgeschwindigkeit der RAMs und wirkt als Bremse für ihre Geschwindigkeitsempfindlichkeit. CIB2 weist strukturelle Ähnlichkeiten mit KChIP-Proteinen auf, die wie CIB2 Calcium-Sensoren sind. Darüber hinaus wurde gezeigt, dass KChIP-Proteine die Oberflächenexpression, den Zusammenbau und die Kinetik von Kv4-Kanälen modulieren, was uns zu der Hypothese führt, dass CIB2 die RAM-Anpassung durch Interaktion mit einem Kaliumkanal und dessen Regulierung beeinflusst. Die Hemmung oder genetische Deletion von KCNQ4 in RAMs hatte zuvor gezeigt, dass sich die Reaktionen auf Geschwindigkeit und Vibration verstärken, ähnlich wie bei Cib2-Knockout-Mäusen beobachtet wurde. Ich fand heraus, dass die Hemmung von KCNQ4 die RAM-Reaktionen in Wnt1-Cre; Cib2fl/ fl-Mäusen nicht beeinflusste, was zeigt, dass KCNQ4 in Mäusen, denen Cib2 fehlt, nicht effektiv in RAMs funktioniert. Wir konnten den Einfluss von CIB2 auf die Kinetik von KCNQ4 untersuchen, indem wir eine Ganzzell-Patch-Clamp-Anlage einsetzten und CIB2 und KCNQ4 in CHO-Zellen vorübergehend überexprimierten. Wir entdeckten, dass die Deaktivierungszeit für KCNQ4 bei CIB2-Überexpression langsamer war. Außerdem wurde KCNQ4 mRNA in 60% der Cib2-positiven Neuronen gefunden. Daraus schließen wir, dass CIB2 mit KCNQ4 interagiert und es moduliert. Das Fehlen von CIB2 in C-Fasern veränderte eindeutig deren physiologische Eigenschaften. C-Fasern, denen Cib2 fehlte, zeigten Anzeichen einer Sensibilisierung, da sie eine deutliche Zunahme der Nachladungsreaktionen zeigten. Die genetische Deletion von Cib2 hatte jedoch keine Auswirkungen auf einen anderen Nozizeptortyp, die Aδ-Nozizeptoren. Schließlich untersuchten wir die verhaltensbezogenen Auswirkungen der peripheren Cib2-Ablation auf leichte Berührungen und mechanische Schmerzen. Mit Hilfe mehrerer Verhaltenstests stellte ich fest, dass Mäuse, denen Cib2 fehlt, einen tiefgreifenden Verlust des Tastsinns aufweisen. Daher führte ich ein elektrisches Suchexperiment mit einem ex-vivo Hautnervenpräparat durch, um nach mechanisch unempfindlichen Fasern zu suchen, und stellte fest, dass die Hälfte der Aβ-Fasern mechanisch stumm waren. Diese Ergebnisse deuten darauf hin, dass CIB2 mit einem mechanosensitiven Kanal interagiert, dessen Natur noch untersucht werden muss. Diese Studie erlaubt uns die Schlussfolgerung, dass CIB2 mit Ionenkanälen (Kalium- und mechanosensitiven Kanälen) im somatosensorischen System interagiert und diese moduliert und somit die sensorische Mechanotransduktion reguliert. Wir stellen die Hypothese auf, dass CIB2, wie im auditorischen System, eine Hilfsuntereinheit von Kanälen im somatosensorischen System ist

Calcium- and Integrin-Binding Protein 2 (CIB2) in physiology and disease: bright and dark sides

Calcium- and integrin-binding protein 2 (CIB2) is a small EF-hand protein capable of binding Mg2+ and Ca2+ ions. While its biological function remains largely unclear, an increasing number of studies have shown that CIB2 is an essential component of the mechano-transduction machinery that operates in cochlear hair cells. Mutations in the gene encoding CIB2 have been associated with non-syndromic deafness. In addition to playing an important role in the physiology of hearing, CIB2 has been implicated in a multitude of very different processes, ranging from integrin signaling in platelets and skeletal muscle to autophagy, suggesting extensive functional plasticity. In this review, we summarize the current understanding of biochemical and biophysical properties of CIB2 and the biological roles that have been proposed for the protein in a variety of processes. We also highlight the many molecular aspects that remain unclarified and deserve further investigation

A Novel C-Terminal CIB2 (Calcium and Integrin Binding Protein 2) Mutation Associated with Non-Syndromic Hearing Loss in a Hispanic Family

Hearing loss is a complex disorder caused by both genetic and environmental factors. Previously, mutations in CIB2 have been identified as a common cause of genetic hearing loss in Pakistani and Turkish populations. Here we report a novel (c.556C\u3eT; p.(Arg186Trp)) transition mutation in the CIB2 gene identified through whole exome sequencing (WES) in a Caribbean Hispanic family with non-syndromic hearing loss. CIB2 belongs to the family of calcium-and integrin-binding (CIB) proteins. The carboxy-termini of CIB proteins are associated with calcium binding and intracellular signaling. The p.(Arg186Trp) mutation is localized within predicted type II PDZ binding ligand at the carboxy terminus. Our ex vivo studies revealed that the mutation did not alter the interactions of CIB2 with Whirlin, nor its targeting to the tips of hair cell stereocilia. However, we found that the mutation disrupts inhibition of ATP-induced Ca2+ responses by CIB2 in a heterologous expression system. Our findings support p.(Arg186Trp) mutation as a cause for hearing loss in this Hispanic family. In addition, it further highlights the necessity of the calcium binding property of CIB2 for normal hearing

Oligomeric state, hydrodynamic properties and target recognition of human Calcium and Integrin Binding protein 2 (CIB2)

Calcium- and Integrin-Binding protein 2 (CIB2) is a small and ubiquitously expressed protein with largely unknown biological function but ascertained role in hearing physiology and disease. Recent studies found that CIB2 binds Ca2+ with moderate affinity and dimerizes under conditions mimicking the physiological ones. Here we provided new lines of evidence on CIB2 oligomeric state and the mechanism of interaction with the alpha 7B integrin target. Based on a combination of native mass spectrometry, chemical cross-linking/mass spectrometry, analytical gel filtration, dynamic light scattering and molecular dynamics simulations we conclude that CIB2 is monomeric under all tested conditions and presents uncommon hydrodynamic properties, most likely due to the high content of hydrophobic solvent accessible surface. Surface plasmon resonance shows that the interaction with alpha 7B occurs with relatively low affinity and is limited to the cytosolic region proximal to the membrane, being kinetically favored in the presence of physiological Mg2+ and in the absence of Ca2+. Although CIB2 binds to an alpha 7B peptide in a 1:1 stoichiometry, the formation of the complex might induce binding of another CIB2 molecule

Calcium sensor proteins in hearing and sight. Biochemical investigation of diseases-associated variants.

Calcium is a cation which plays a pivotal role as second messenger, thus its concentration in cells needs to be finely regulated. Many systems work for that purpose, including Ca2+ sensor proteins, which undergo conformational changes upon Ca2+ coordination via EF-hands. Ca2+ sensors can be ubiquitous or tissue specific. Examples in this sense are represented by Guanylate Cyclase Activating Protein 1 (GCAP1) and Calcium- and Integrin-Binding Protein 2 (CIB2), involved in sight and hearing respectively. Missense point mutations in GCAP1 and CIB2 were found to be associated with genetic diseases characterized by retinal dystrophies and/or deafness. During my PhD, I focused my attention on the characterization of two point mutations namely p.Glu111Val (E111V) in GCAP1, leading to Cone/Rod dystrophy in an Italian family, and p.Glu64Asp (E64D) in CIB2, linked to Usher syndrome type 1J (USH1J), a rare disease characterized by the copresence of blindness and deafness. In particular, I spent the first part of the PhD investigating the role of CIB2 which is still under debate, finding that it is per se uncapable to work as a Ca2+ sensor under physiological conditions and that the conservative mutation linked to USH1J perturbs an allosteric communication between pseudo-EF1 and EF3, thus blocking the protein in an unfunctional conformation. Then, I characterized E111V GCAP1, finding that it is incapable of regulating its molecular target (Guanylate Cyclase), leading to a constitutive active enzyme and thus a progressively high concentrations of Ca2+ and cGMP in cells, which may explain the pathological phenotype. Looking for a potential therapeutic approach for Cone-Rod dystrophies, we found that the well-established Ca2+-relay model, explaining the gradual activation of Guanylate Cyclase by multiple GCAP molecules following gradual changes in intracellular Ca2+ concentrations, seems to be species-specific, since it apparently does not work in the same way in humans as in mouse and bovine photoreceptors. Finally, we identified a general method for the characterization of the interaction between a ubiquitous Ca2+ sensor protein (calmodulin) and inorganic CaF2 nanoparticles, suggesting their suitability as devices for nanomedicine applications

CIB2 Interacts with TMC1 and TMC2 and is Essential for Mechanotransduction in Auditory Hair Cells

Inner ear hair cells detect sound through deflection of stereocilia, the microvilli-like projections that are arranged in rows of graded heights. Calcium and integrin-binding protein 2 is essential for hearing and localizes to stereocilia, but its exact function is unknown. Here, we have characterized two mutant mouse lines, one lacking calcium and integrin-binding protein 2 and one carrying a human deafness-related Cib2 mutation, and show that both are deaf and exhibit no mechanotransduction in auditory hair cells, despite the presence of tip links that gate the mechanotransducer channels. In addition, mechanotransducing shorter row stereocilia overgrow in hair cell bundles of both Cib2 mutants. Furthermore, we report that calcium and integrin-binding protein 2 binds to the components of the hair cell mechanotransduction complex, TMC1 and TMC2, and these interactions are disrupted by deafness-causing Cib2 mutations. We conclude that calcium and integrin-binding protein 2 is required for normal operation of the mechanotransducer channels and is involved in limiting the growth of transducing stereocilia

Genetic linkage analysis of DFNB40 and DFNB48 loci in families with autosomal recessive non-syndromic hearing loss (ARNSHL) from western provinces of Iran

Background: Sensorineural hearing loss (SNHL) is the most common sensory disorder and 1 in every 500-1000 newborns is affected. Non-syndromic SNHL accounts for 70% of hereditary hearing loss and 80% of SNHL cases have an autosomal recessive mode of inheritance (ARNSHL). The Purpose of the recent study is genetic linkage analysis to determine the prevalence of DFNB40 and DFNB48 loci in studying families with ARNSHL from the western provinces of Iran. Methods: In this study, 60 families from 3 provinces of Iran involving Hamedan, Kohgiluyeh and Boyer-Ahmad and Chaharmahal and Bakhtiari with autosomal recessive non syndromic hearing loss were examined. The selected families in this study were consanguineous and had at least two patients. They also were negative for GJB2 mutations. Linkage analysis was performed by using 6 markers short tandem repeat (STR) for the DFNB40 locus and 7 markers STR for the DFNB48 locus. Findings: After examining different families, it was revealed that none of them showed linkage to the DFNB40 and DFNB48 loci. Conclusion: The recent study suggests that DFNB40 and DFNB48 loci might not play an important role in causing hearing loss in the mentioned provinces. However, further studies are necessary to determine more precisely the role of these loci in the Iranian population. © 2016, Isfahan University of Medical Sciences(IUMS). All rights reserved

CMB Lensing Power Spectrum Biases from Galaxies and Clusters using High-angular Resolution Temperature Maps

The lensing power spectrum from cosmic microwave background (CMB) temperature maps will be measured with unprecedented precision with upcoming experiments, including upgrades to ACT and SPT. Achieving significant improvements in cosmological parameter constraints, such as percent level errors on sigma_8 and an uncertainty on the total neutrino mass of approximately 50 meV, requires percent level measurements of the CMB lensing power. This necessitates tight control of systematic biases. We study several types of biases to the temperature-based lensing reconstruction signal from foreground sources such as radio and infrared galaxies and the thermal Sunyaev-Zel'dovich effect from galaxy clusters. These foregrounds bias the CMB lensing signal due to their non-Gaussian nature. Using simulations as well as some analytical models we find that these sources can substantially impact the measured signal if left untreated. However, these biases can be brought to the percent level if one masks galaxies with fluxes at 150 GHz above 1 mJy and galaxy clusters with masses above M_vir = 10^14 M_sun. To achieve such percent level bias, we find that only modes up to a maximum multipole of l_max ~ 2500 should be included in the lensing reconstruction. We also discuss ways to minimize additional bias induced by such aggressive foreground masking by, for example, exploring a two-step masking and in-painting algorithm.Comment: 14 pages, 14 figures, to be submitted to Ap

Integrated multiomics approach identifies calcium and integrin-binding protein-2 as a novel gene for pulse wave velocity

Background: Carotid-femoral pulse wave velocity (PWV) is an important measure of arterial stiffness, which is an independent predictor of cardiovascular morbidity and mortality. In this study, we used an integrated genetic, epigenetic and transcriptomics approach to uncover novel molecular mechanisms contributing to PWV. Methods and results: We measured PWV in 1505 healthy twins of European descendent. A genomewide association analysis was performed using standardized residual of the inverse of PWV. We identified one single-nucleotide polymorphism (rs7164338) in the calcium and integrin-binding protein-2 (CIB2) gene on chromosome 15q25.1 associated with PWV [beta = -0.359, standard error (SE) = 0.07, P = 4.8 x 10(-8)]. The same variant was also associated with increased CIB2 expression in leucocytes (beta = 0.034, SE = 0.008, P = 4.95 x 10(-5)) and skin (beta = 0.072, SE = 0.01, P = 2.35 x 10(-9)) and with hypomethylation of the gene promoter (beta = -.899, SE = 0.098, P = 3.63 x 10(-20)). Conclusion: Our data indicate that reduced methylation of the CIB2 promoter in individuals carrying rs7164338 may lead to increased CIB2 expression. Given that CIB2 is thought to regulate intracellular calcium levels, an increase in protein levels may prevent the accumulation of serum calcium and phosphate, ultimately slowing down the process of vascular calcification. This study shows the power of integrating multiple omics to discover novel cardiovascular mechanisms

Novel HIV-1 Knockdown Targets Identified by an Enriched Kinases/Phosphatases shRNA Library Using a Long-Term Iterative Screen in Jurkat T-Cells

HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA) library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies