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In myelinating Schwann cells, communication between myelin layers is mediated by gap junction channels (GJC) formed by docked connexin 32 hemichannels (HCs). Mutations in Cx32 cause the X-linked Charcot–Marie–Tooth disease (CMT1X), a degenerative neuropathy with no cure. A molecular link between Cx32 dysfunction and CMT1X pathogenesis is still missing. Here, we describe the high resolution cryo-EM structures of the Cx32 GJC and HC, along with two CMT1X-linked mutants, W3S and R22G. While the structures of wild-type and mutant GJCs are virtually identical, the HCs show a major difference: in the W3S and R22G mutant HCs, the N-terminal helix partially occludes the pore, consistent with an impaired HC activity. Our results suggest that HC dysfunction may be involved in the pathogenesis of CMT1X.One-Sentence SummaryConnexin 32 channel structures reveal a gating helix defect in CMT1X disease-associated mutant hemichannels

The epidermis forms an essential barrier against a variety of insults. The overall goal of this study was to shed light not only on the effects of accidental epidermal injury, but also on the mechanisms that support laser skin resurfacing with intra-epidermal focal laser-induced photodamage, a widespread medical practice used to treat a range of skin conditions. To this end, we selectively photodamaged a single keratinocyte with intense, focused and pulsed laser radiation, triggering Ca2+ waves in the epidermis of live anesthetized mice with ubiquitous expression of a genetically encoded Ca2+ indicator. Waves expanded radially and rapidly, reaching up to eight orders of bystander cells that remained activated for tens of minutes, without displaying oscillations of the cytosolic free Ca2+ concentration (${[ {{\rm{C}}{{\rm{a}}^{2 + }}} ]_c}$). By combining in vivo pharmacological dissection with mathematical modeling, we demonstrate that Ca2+ wave propagation depended primarily on the release of ATP, a prime damage-associated molecular patterns (DAMPs), from the hit cell. Increments of the ${[ {{\rm{C}}{{\rm{a}}^{2 + }}} ]_c}$ in bystander cells were chiefly due to Ca2+ release from the endoplasmic reticulum (ER), downstream of ATP binding to P2Y purinoceptors. ATP-dependent ATP release though connexin hemichannels (HCs) affected wave propagation at larger distances, where the extracellular ATP concentration was reduced by the combined effect of passive diffusion and hydrolysis due to the action of ectonucleotidases, whereas pannexin channels had no role. Bifurcation analysis suggests basal keratinocytes have too few P2Y receptors (P2YRs) and/or phospholipase C (PLC) to transduce elevated extracellular ATP levels into inositol trisphosphate (IP3) production rates sufficiently large to sustain ${[ {{\rm{C}}{{\rm{a}}^{2 + }}} ]_c}$ oscillations.

Perfluoroalkyl substances (PFASs) are synthetic chemicals widely used in industrial and consumer products. The environmental spreading of PFASs raises concerns for their impact on human health. In particular, the bioaccumulation in humans due to environmental exposure has been reported also in total brain samples and PFAS exposure has been associated with neurodevelopmental disorders. In this study we aimed to investigate the specific PFAS bioaccumulation in different brain areas. Our data reported major accumulation in the brainstem region, which is richly populated by dopaminergic neurons (DNs), in brain autopsy samples from people resident in a PFAS-polluted area of Italy. Since DNs are the main source of dopamine (DA) in the mammalian central nervous system (CNS), we evaluated the possible functional consequences of perfluoro-octanoic acid (PFOA) exposure in a human model of DNs obtained by differentiation of human induced pluripotent stem cells (hiPSCs). Particularly, we analyzed the specific effect of the exposure to PFOA for 24 h, at the concentration of 10 ng/ml, at 3 different steps of dopaminergic differentiation: the neuronal commitment phase (DP1), the neuronal precursor phase (DP2) and the mature dopaminergic differentiation phase (DP3). Interestingly, compared to untreated cells, exposure to PFOA was associated with a reduced expression of Tyrosine Hydroxylase (TH) and Neurofilament Heavy (NFH), both markers of dopaminergic maturation at DP2 phase. In addition, cells at DP3 phase exposed to PFOA showed a severe reduction in the expression of the Dopamine Transporter (DAT), functionally involved in pre-synaptic dopamine reuptake. In this proof-of-concept study we show a significant impact of PFOA exposure, mainly on the most sensitive stage of neural dopaminergic differentiation, prompting the way for further investigations more directly relevant to risk assessment of these chemicals.

BACKGROUNDThe mammalian skin, the body’s largest single organ, is a highly organized tissue that forms an essential barrier against dehydration, pathogens, light and mechanical injury. Damage triggers perturbations of the cytosolic free Ca2+ concentration ([Ca2+]c) that spread from cell to cell (known as intercellular Ca2+ waves) in different epithelia, including epidermis. Ca2+ waves are considered a fundamental mechanism for coordinating multicellular responses, however the mechanisms underlying their propagation in the damaged epidermis are incompletely understood.AIM OF THE PROJECTTo dissect the molecular components contributing to Ca2+ wave propagation in murine model of epidermal photodamage.METHODSTo trigger Ca2+ waves, we used intense and focused pulsed laser radiation and targeted a single keratinocyte of the epidermal basal layer in the earlobe skin of live anesthetized mice. To track photodamage-evoked Ca2+ waves, we performed intravital multiphoton microscopy in transgenic mice with ubiquitous expression of the sensitive and selective Ca2+ biosensor GCaMP6s. To dissect the molecular components contributing to Ca2+ wave propagation, we performed in vivo pharmacological interference experiments by intradermal microinjection of different drugs.EXPERIMENTAL RESULTSThe major effects of drugs that interfere with degradation of extracellular ATP or P2 purinoceptors suggest that Ca2+ waves in the photodamaged epidermis are primarily due to release of ATP from the target cell, whose plasma membrane integrity was compromised by laser irradiation. The limited effect of the Connexin 43 (Cx43) selective inhibitor TAT-Gap19 suggests ATP-dependent ATP release though connexin hemichannels (HCs) plays a minor role, affecting Ca2+ wave propagation only at larger distances, where the concentration of ATP released from the photodamaged cell was reduced by the combined effect of passive diffusion and hydrolysis due to the action of ectonucleotidases. The ineffectiveness of probenecid suggests pannexin channels have no role. As GCaMP6s signals in bystander keratinocytes were augmented by exposure to the Ca2+ chelator EGTA in the extracellular medium, the corresponding transient increments of the [Ca2+]c should be ascribed primarily to Ca2+ release from the ER, downstream of ATP binding to P2Y purinoceptors, with Ca2+ entry through plasma membrane channels playing a comparatively negligible role. The effect of thapsigargin (a well-known inhibitor of SERCA pumps) and carbenoxolone (a recently recognized inhibitor of Ca2+ release through IP3 receptors) support this conclusion.CONCLUSIONSThe one presented here is an experimental model for accidental skin injury that may also shed light on the widespread medical practice of laser skin resurfacing, used to treat a range of pathologies from photodamage and acne scars to hidradenitis suppurativa and posttraumatic scarring from basal cell carcinoma excision. The results of our experiments support the notion that Ca2+ waves reflect chiefly the sequential activation of bystander keratinocytes by the ATP released through the compromised plasma membrane of the cell hit by laser radiation. We attributed the observed increments of the [Ca2+]c chiefly to signal transduction through purinergic P2Y receptors. Several studies have highlighted fundamental roles of P2Y receptors during inflammatory and infectious diseases, and the initial phase of wound healing involves acute inflammation. In addition, hyaluronan is a major component of the extracellular matrix and its synthesis is rapidly upregulated after tissue wounding via P2Y receptor activation. It is tempting to speculate that response coordination after injury in the epidermis occurs via propagation of the ATP-dependent intercellular Ca2+ waves described in this work.

In mammals, the sense of hearing arises through a complex sequence of morphogenetic events that drive the sculpting of the auditory sensory epithelium into its terminally functional three-dimensional shape. While the majority of the underlying mechanisms remain unknown, it has become increasingly clear that Ca2+ signaling is at center stage and plays numerous fundamental roles both in the sensory hair cells and in the matrix of non-sensory, epithelial and supporting cells, which embed them and are tightly interconnected by a dense network of gap junctions formed by connexin 26 (Cx26) and connexin 30 (Cx30) protein subunits. In this review, we discuss the intricate interplay between Ca2+ signaling, connexin expression and function, apoptosis and autophagy in the crucial steps that lead to hearing acquisition.

Macrophages are highly plastic and dynamic cells that exert much of their function through phagocytosis. Phagocytosis depends on a coordinated, finely tuned, and compartmentalized regulation of calcium concentrations. We examined the role of mitochondrial calcium uptake and mitochondrial calcium uniporter (MCU) in macrophage polarization and function. In primary cultures of human monocyte-derived macrophages, calcium uptake in mitochondria was instrumental for alternative (M2) macrophage polarization. Mitochondrial calcium uniporter inhibition with KB-R7943 or MCU knockdown, which prevented mitochondrial calcium uptake, reduced M2 polarization, while not affecting classical (M1) polarization. Challenging macrophages with E. coli fragments induced spikes of mitochondrial calcium concentrations, which were prevented by MCU inhibition or silencing. In addition, mitochondria remodelled in M2 macrophages during phagocytosis, especially close to sites of E. coli internalization. Remarkably, inhibition or knockdown of MCU significantly reduced the phagocytic capacity of M2 macrophages. KB-R7943, which also inhibits the membrane sodium/calcium exchanger and Complex I, reduced mitochondria energization and cellular ATP levels, but such effects were not observed with MCU silencing. Therefore, phagocytosis inhibition by MCU knockdown depended on the impaired mitochondrial calcium buffering rather than changes in mitochondrial and cellular energy status. These data uncover a new role for MCU in alternative macrophage polarization and phagocytic activity.

Significance The selective phosphorylation of spatially distinct PKA targets is key for the pleiotropy of the cAMP cascade. This characteristic of the pathway is currently attributed to the ability of phosphodiesterases or adenylate cyclases to create subcellular sites (microdomains) where the concentration of cAMP is distinct from that of the surrounding areas. The role of phosphatases in this process has not been tested. Here we show that limited access of phosphatases to the PKA targets present at the outer mitochondrial membrane generates distinct microdomains of PKA phosphorylated proteins despite there being no differences in the local cAMP levels. These results describe an alternative mechanism capable of generating functional cAMP/PKA-dependent microdomains and may be extrapolated to the compartmentalization of other kinase-dependent events., Evidence supporting the heterogeneity in cAMP and PKA signaling is rapidly accumulating and has been largely attributed to the localization or activity of adenylate cyclases, phosphodiesterases, and A-kinase–anchoring proteins in different cellular subcompartments. However, little attention has been paid to the possibility that, despite homogeneous cAMP levels, a major heterogeneity in cAMP/PKA signaling could be generated by the spatial distribution of the final terminators of this cascade, i.e., the phosphatases. Using FRET-based sensors to monitor cAMP and PKA-dependent phosphorylation in the cytosol and outer mitochondrial membrane (OMM) of primary rat cardiomyocytes, we demonstrate that comparable cAMP increases in these two compartments evoke higher levels of PKA-dependent phosphorylation in the OMM. This difference is most evident for small, physiological increases of cAMP levels and with both OMM-located probes and endogenous OMM proteins. We demonstrate that this disparity depends on differences in the rates of phosphatase-dependent dephosphorylation of PKA targets in the two compartments. Furthermore, we show that the activity of soluble phosphatases attenuates PKA-driven activation of the cAMP response element-binding protein while concurrently enhancing PKA-dependent mitochondrial elongation. We conclude that phosphatases can sculpt functionally distinct cAMP/PKA domains even in the absence of gradients or microdomains of this messenger. We present a model that accounts for these unexpected results in which the degree of PKA-dependent phosphorylation is dictated by both the subcellular distribution of the phosphatases and the different accessibility of membrane-bound and soluble phosphorylated substrates to the cytosolic enzymes.

Aims: Diabetes is associated with an excess release of neutrophil extracellular traps (NETs) and an enhanced NETosis, a neutrophil cell death programme instrumental to anti-microbial defences, but also involved in tissue damage. We herein investigated whether the antidiabetic drug metformin protects against NETosis. Methods: We measured NET components in the plasma of patients with pre-diabetes who were randomized to receive metformin or placebo for 2 months. To control for the effect on glucose, we also measured NET components in the plasma of patients with type 2 diabetes before and after treatment with insulin or dapagliflozin. In vitro, we used static and dynamic imaging with advanced live confocal two-photon microscopy to evaluate the effects of metformin on cellular events during NETosis. We examined putative molecular mechanisms by monitoring chromatin decondensation and DNA release in vitro. Results: Metformin, as compared to placebo, significantly reduced the concentrations of NET components elastase, proteinase-3, histones and double strand DNA, whereas glucose control with insulin or dapagliflozin exerted no significant effect. In vitro, metformin prevented pathologic changes in nuclear dynamics and DNA release, resulting in a blunted NETosis in response to phorbol myristate acetate and calcium influx. Metformin prevented membrane translocation of PKC-?II and activation of NADPH oxidase in neutrophils, both of which diminished the NETosis response. Conclusions: Metformin treatment reduced the concentrations of NET components independently from glucose control. This effect was reproducible in vitro and was related to the inhibitory effect exerted by metformin on the PKC-NADPH oxidase pathway. © 2018, Springer-Verlag Italia S.r.l., part of Springer Nature.

Hair cells of the inner ear detect sound stimuli, inertial or gravitational forces by deflection of their apical stereocilia. A small number of stereociliary cation-selective mechanotransduction (MET) channels admit K+ and Ca2+ ions into the cytoplasm promoting hair cell membrane depolarization and, consequently, neurotransmitter release at the cell basolateral pole. Ca2+ influx into the stereocilia compartment is counteracted by the unusual w/a splicing variant of plasma-membrane calcium-pump isoform 2 (PMCA2) which, unlike other PMCA2 variants, increases only marginally its activity in response to a rapid variation of the cytoplasmic free Ca2+ concentration ([Ca2+)(c)). Missense mutations of PMCA2w/a cause deafness and loss of balance in humans. Mouse models in which the pump is genetically ablated or mutated show hearing and balance impairment, which correlates with defects in homeostatic regulation of stereociliary [Ca2+](c), decreased sensitivity of mechanotransduction channels to hair bundle displacement and progressive degeneration of the organ of Corti. These results highlight a critical role played by the PMCA2w/a pump in the control of hair cell function and survival, and provide mechanistic insight into the etiology of deafness and vestibular disorders.

Electrolyte-gated organic field-effect transistors (EGOFETs) have been fabricated using 6,13-Bis (triisopropyl-silylethynyl) pentacene (TIPS-pentacene) as an organic semiconductor active material. Despite the organic semiconductor being deposited in air by drop casting, our EGOFETs showed performance comparable with state-of-the-art technologies, and devices kept their functionalities while gated with saline solution at 37 °C for several days. In addition, the biocompatibility of the material was successfully investigated, promoting the use of TIPS-pentacene -based EGOFETs for biosensing applications.

In mammals, the sense of hearing arises through a complex sequence of morphogenetic events that drive the sculpting of the auditory sensory epithelium into its terminally functional three-dimensional shape. While the majority of the underlying mechanisms remain unknown, it has become increasingly clear that Ca

Two-photon excitation (2PE) microscopy [1] has proven to be an excellent technique for in vivo fluorescence imaging of deep structures and tissues due to low absorption and scattering of infrared light. The combination of two laser beams introduces the possibility of further imaging capabilities, such as simultaneous excitation of three fluorophores (multiphoton-multicolor [2]) and sub-diffraction resolution of cellular structures (STimulated Emission Depletion, STED [3,4,5,6,7]). Here we present a cost-effective solution that combines both techniques by use of a homemade optical bench equipped with a pulsed laser split in two beams by an Optical Parametric Oscillator (OPO). Preliminary experiments were successfully performed with the multiphoton-multicolor technique, setting proper conditions to perform experiments for the study of neutrophil mobilization in skull bone marrow of diabetic mice. In the STED configuration, we successfully tested the fluorescence depletion effect and the optical generation of a doughnut-shaped point-spread-function by the depletion laser, both conditions required to obtain super-resolution. A preliminary super-resolution performance was obtained by visualizing the outer mitochondrial membrane stained with Alexa 647 dye.

Mutations of the GJB1 gene encoding connexin 32 (Cx32) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX1), a demyelinating peripheral neuropathy for which there is no cure. A growing body of evidence indicates that ATP release through Cx32 hemichannels in Schwann cells could be critical for nerve myelination, but it is unknown if CMTX1 mutations alter the cytosolic Ca2+-dependent gating mechanism that controls Cx32 hemichannel opening and ATP release. The current study uncovered that loss of the C-terminus in Cx32 (R220X mutation), which causes a severe CMTX1 phenotype, inhibits hemichannel opening during a canonical IP3-mediated increase in cytosolic Ca2+ in HeLa cells. Interestingly, the gating function of R220X hemichannels was completely restored by both the intracellular and extracellular application of a peptide that mimics the Cx32 cytoplasmic loop. All-atom molecular dynamics simulations suggest that loss of the C-terminus in the mutant hemichannel triggers abnormal fluctuations of the cytoplasmic loop which are prevented by binding to the mimetic peptide. Experiments that stimulated R220X hemichannel opening by cell depolarization displayed reduced voltage sensitivity with respect to wild-type hemichannels which was explained by loss of subconductance states at the single channel level. Finally, experiments of intercellular diffusion mediated by wild-type or R220X gap junction channels revealed similar unitary permeabilities to ions, signalling molecules (cAMP) or larger solutes (Lucifer yellow). Taken together, our findings support the hypothesis that paracrine signalling alteration due to Cx32 hemichannel dysfunction underlies CMTX1 pathogenesis and suggest a candidate molecule for novel studies investigating a therapeutic approach.

cAMP/PKA signalling is compartmentalised with tight spatial and temporal control of signal propagation underpinning specificity of response. The cAMP-degrading enzymes, phosphodiesterases (PDEs), localise to specific subcellular domains within which they control local cAMP levels and are key regulators of signal compartmentalisation. Several components of the cAMP/PKA cascade are located to different mitochondrial sub-compartments, suggesting the presence of multiple cAMP/PKA signalling domains within the organelle. The function and regulation of these domains remain largely unknown. Here, we describe a novel cAMP/PKA signalling domain localised at mitochondrial membranes and regulated by PDE2A2. Using pharmacological and genetic approaches combined with real-time FRET imaging and high resolution microscopy, we demonstrate that in rat cardiac myocytes and other cell types mitochondrial PDE2A2 regulates local cAMP levels and PKA-dependent phosphorylation of Drp1. We further demonstrate that inhibition of PDE2A, by enhancing the hormone-dependent cAMP response locally, affects mitochondria dynamics and protects from apoptotic cell death. DOI: http://dx.doi.org/10.7554/eLife.21374.001

Confocal imaging of fluorescent probes offers a powerful, non-invasive tool which enables data collection from vast population of cells at high spatial and temporal resolution. Spinning disk confocal microscopy parallelizes the imaging process permitting the study of dynamic events in populations of living cells on the millisecond time scale. Several spinning disk microscopy solutions are commercially available, however these are often poorly configurable and relatively expensive. This chapter describes a procedure to assemble a cost-effective homemade spinning disk system for fluorescence microscopy, which is highly flexible and easily configurable. We finally illustrate a reliable protocol to obtain high-quality Ca(2+) and voltage imaging data from cochlear preparations.

Confocal imaging of fluorescent probes offers a powerful, non-invasive tool which enables data collection from vast population of cells at high spatial and temporal resolution. Spinning disk confocal microscopy parallelizes the imaging process permitting the study of dynamic events in populations of living cells on the millisecond time scale. Several spinning disk microscopy solutions are commercially available, however these are often poorly configurable and relatively expensive. This chapter describes a procedure to assemble a cost-effective homemade spinning disk system for fluorescence microscopy, which is highly flexible and easily configurable. We finally illustrate a reliable protocol to obtain high-quality Ca(2+) and voltage imaging data from cochlear preparations.

Sensory hair cells of the inner ear detect sound stimuli, inertial or gravitational forces. These mechanical inputs cause deflection of the cell stereociliary bundle and activate a small number of cation-selective mechano-transduction (MET) channels that admit K+ and Ca2+ ions into the cytoplasm. Stereociliary Ca2+ levels are homeostatically regulated by an unusual splicing isoform (w/a) of plasma membrane calcium-pump isoform 2 (PMCA2w/a), ablation or missense mutations of which cause deafness and loss of balance in humans and mice. At variance with other PMCA2 isoforms, PMCA2w/a expressed in CHO transfectants increases only marginally its activity in response to a rapid increase of the cytoplasmic free Ca2+ concentration ([Ca2+]c). In this expression system, deafness-related mutations of PMCA2w/a decrease the pump ability to extrude Ca2+ both at steady state and in response to a [Ca2+]c rise. Consistent with these findings, mouse strains in which the pump is genetically ablated or mutated show hearing impairment correlated with defects in homeostatic regulation of stereociliary Ca2+, decreased sensitivity of the MET channels to hair bundle displacement, and morphological abnormalities in the organ of Corti. These results highlight a critical role played by PMCA2w/a in the control of hair cell function and survival and provide mechanistic insight into the etiology of deafness and vestibular disorders.

The mechanotransduction process in hair cells in the inner ear is associated with the influx of calcium from the endolymph. Calcium is exported back to the endolymph via the splice variant w/a of the PMCA2 of the stereocilia membrane. To further investigate the role of the pump, we have identified and characterized a novel ENU-induced mouse mutation, Tommy, in the PMCA2 gene. The mutation causes a non-conservative E629K change in the second intracellular loop of the pump that harbors the active site. Tommy mice show profound hearing impairment from P18, with significant differences in hearing thresholds between wild type and heterozygotes. Expression of mutant PMCA2 in CHO cells shows calcium extrusion impairment; specifically, the long term, non-stimulated calcium extrusion activity of the pump is inhibited. Calcium extrusion was investigated directly in neonatal organotypic cultures of the utricle sensory epithelium in Tommy mice. Confocal imaging combined with flash photolysis of caged calcium showed impairment of calcium export in both Tommy heterozygotes and homozygotes. Immunofluorescence studies of the organ of Corti in homozygous Tommy mice showed a progressive base to apex degeneration of hair cells after P40. Our results on the Tommy mutation along with previously observed interactions between cadherin-23 and PMCA2 mutations in mouse and humans underline the importance of maintaining the appropriate calcium concentrations in the endolymph to control the rigidity of cadherin and ensure the function of interstereocilia links, including tip links, of the stereocilia bundle.

Mutations in the GJB2 and GJB6 genes, respectively, coding for connexin26 (Cx26) and connexin30 (Cx30) proteins, are the most common cause for prelingual non-syndromic deafness in humans. In the inner ear, Cx26 and Cx30 are expressed in different non-sensory cell types, where they largely co-localize and may form heteromeric gap junction channels. Here, we describe the generation and characterization of a mouse model for human bilateral middle/high-frequency hearing loss based on the substitution of an evolutionarily conserved threonine by a methionine residue at position 5 near the N-terminus of Cx30 (Cx30T5M). The mutation was inserted in the mouse genome by homologous recombination in mouse embryonic stem cells. Expression of the mutated Cx30T5M protein in these transgenic mice is under the control of the endogenous Cx30 promoter and was analysed via activation of the lacZ reporter gene. When probed by auditory brainstem recordings, Cx30(T5M/T5M) mice exhibited a mild, but significant increase in their hearing thresholds of about 15 dB at all frequencies. Immunolabelling with antibodies to Cx26 or Cx30 suggested normal location of these proteins in the adult inner ear, but western blot analysis showed significantly down-regulated the expression levels of Cx26 and Cx30. In the developing cochlea, electrical coupling, probed by dual patch-clamp recordings, was normal. However, transfer of the fluorescent tracer calcein between cochlear non-sensory cells was reduced, as was intercellular Ca(2+) signalling due to spontaneous ATP release from connexin hemichannels. Our findings link hearing loss to decreased biochemical coupling due to the point-mutated Cx30 in mice.

Although it is widely accepted that mitochondria in living cells can efficiently uptake Ca(2+) during stimulation because of their vicinity to microdomains of high [Ca(2+)], the direct proof of Ca(2+) hot spots' existence is still lacking. Thanks to a GFP-based Ca(2+) probe localized on the cytosolic surface of the outer mitochondrial membrane, we demonstrate that, upon Ca(2+) mobilization, the [Ca(2+)] in small regions of the mitochondrial surface reaches levels 5- to 10-fold higher than in the bulk cytosol. We also show that the [Ca(2+)] to which mitochondria are exposed during capacitative Ca(2+) influx is similar between near plasma membrane mitochondria and organelles deeply located in the cytoplasm, whereas it is 2- to 3-fold higher in subplasma membrane mitochondria upon activation of voltage-gated Ca(2+) channels. These results demonstrate that mitochondria are exposed to Ca(2+) hot spots close to the ER but are excluded from the regions where capacitative Ca(2+) influx occurs.

The inner ear contains delicate sensory receptors that have adapted to detect the minutest mechanical disturbances. Ca2+ ions are implicated in all steps of the transduction process, as well as in its regulation by an impressive ensemble of finely tuned feedback control mechanisms. Recent studies have unveiled some of the key players, but things do not sound quite right yet.

Arrhythmogenic Ca2+ signalling may be influenced by alterations in intracellular pH (pHi), as found in myocardial ischaemia. Here we characterise the effects of altering pHi on Ca2+ waves in rat isolated ventricular myocytes.Ca2+ waves were induced by raising [Ca2+]o, and imaged confocally using fluo-3. Parallel measurements of pHi were made using cSNARF-1. pHi changes were induced by superfusion of acetate (20-80mM) or trimethylamine (20mM).Reducing pHi acutely decreased (to 16±6% of control) then increased (to 360±62% by 60s) wave frequency. Inhibiting the sarcolemmal Na+/H+ exchanger (NHE) with 30μM 5-(N,N-dimethyl)amiloride suppressed this secondary increase. A linear relationship was found between pHi and wave velocity: intracellular acidosis increased velocity up to 140±3%. Acidosis also slowed wave decay and increased diastolic Ca2+, while alkalosis decreased diastolic Ca2+.A stable longitudinal pHi gradient (pHi 6.6-7.3) was generated in single myocytes by microperfusing half of the cell with normal Tyrode and half with 80mM acetate, using a double-barrelled micropipette. With NHE inhibited, a decrease in wave frequency was seen in the acidic microdomain and an increase in the more alkaline microdomain. Conversely, wave velocity was increased in acidic and decreased in alkaline microdomains. Thus, wave properties map onto a spatial gradient of pHi.Wave velocity may be influenced by multiple pH-dependent components. Computational modelling of Ca2+ waves predicts that, at steady-state, sarco/endoplasmic reticulum Ca2+ ATPase inhibition decreases wave velocity, due to a fall in sarcoplasmic reticulum Ca2+ content (Swietach et al, 2010). However, decreasing the kon of Ca2+ buffers predicted an increase in wave velocity, suggesting the effect of pHi changes on velocity is multifactorial.We conclude that pHi heterogeneity locally regulates Ca2+ waves, suggesting that it may contribute to the heterogeneity of Ca2+ signalling observed in myocardial ischaemia.

Phosphatidylinositol phosphate kinase type 1γ (PIPKIγ) is a key enzyme in the generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] and is expressed at high levels in the nervous system. Homozygous knockout mice lacking this enzyme die postnatally within 24 h, whereas PIPKIγ +/− siblings breed normally and have no reported phenotype. Here we show that adult PIPKIγ +/− mice have dramatically elevated hearing thresholds for high-frequency sounds. During the first postnatal week we observed a reduction of ATP-dependent Ca 2+ signaling activity in cochlear nonsensory cells. Because Ca 2+ signaling under these conditions depends on inositol-1,4,5-trisphosphate generation from phospholipase C (PLC)-dependent hydrolysis of PI(4,5)P 2 , we conclude that ( i ) PIPKIγ is primarily responsible for the synthesis of the receptor-regulated PLC-sensitive PI(4,5)P 2 pool in the cell syncytia that supports auditory hair cells; ( ii ) spatially graded impairment of this signaling pathway in cochlear nonsensory cells causes a selective alteration in the acquisition of hearing in PIPKIγ +/− mice. This mouse model also suggests that PIPKIγ may determine the level of gap junction contribution to cochlear development.

Objective: Connexins are transmembrane proteins that form intercellular junctional channels in vertebrates and are known or suspected to be involved in a wide variety of biological processes including cardiac development and function, hearing, haematopoesis, regeneration, lens transparency, fertility, immune system function and protection from oxidative stress. Connexin mutations can cause developmental and physiological defects, and link to various diseases. In particular, defective permeation of cAMP or inositol-1,4,5-trisphosphate (InsP3) through connexin channels is associated with peripheral neuropathies and deafness, respectively. Here we present a method to estimate the permeability of single-gap junction channels to second messengers. Study design: Using HeLa cells that overexpressed wild-type human connexin 26 (HCx26wt) as a model system, we combined measurements of junctional conductance and fluorescence resonance energy transfer (FRET) emission ratio of biosensors selective for cAMP and...

Connexins are transmembrane proteins that form intercellular junctional channels in vertebrates and are known or suspected to be involved in a wide variety of biological processes including cardiac development and function, hearing, hematopoesis, regeneration, lens transparency, fertility, immune system function and protection from oxidative stress. Connexin mutations can cause developmental and physiological defects, and link to various diseases. In particular, defective permeation of cAMP or inositol-1,4,5-trisphosphate (InsP3) through connexin channels is associated with peripheral neuropathies and deafness, respectively.

Presenilin mutations are the main cause of familial Alzheimer's disease (FAD). Presenilins also play a key role in Ca 2+ homeostasis, and their FAD-linked mutants affect cellular Ca 2+ handling in several ways. We previously have demonstrated that FAD-linked presenilin 2 (PS2) mutants decrease the Ca 2+ content of the endoplasmic reticulum (ER) by inhibiting sarcoendoplasmic reticulum Ca 2+ -ATPase (SERCA) activity and increasing ER Ca 2+ leak. Here we focus on the effect of presenilins on mitochondrial Ca 2+ dynamics. By using genetically encoded Ca 2+ indicators specifically targeted to mitochondria (aequorin- and GFP-based probes) in SH-SY5Y cells and primary neuronal cultures, we show that overexpression or down-regulation of PS2, but not of presenilin 1 (PS1), modulates the Ca 2+ shuttling between ER and mitochondria, with its FAD mutants strongly favoring Ca 2+ transfer between the two organelles. This effect is not caused by a direct PS2 action on mitochondrial Ca 2+ -uptake machinery but rather by an increased physical interaction between ER and mitochondria that augments the frequency of Ca 2+ hot spots generated at the cytoplasmic surface of the outer mitochondrial membrane upon stimulation. This PS2 function adds further complexity to the multifaceted nature of presenilins and to their physiological role within the cell. We also discuss the importance of this additional effect of FAD-linked PS2 mutants for the understanding of FAD pathogenesis.

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26(Sox10Cre) mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10-Cre line. Cx26(Sox10Cre) mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26(Sox10Cre) mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans.

Ca2+ acts as a fundamental signal transduction element in the inner ear, delivering information about sound acceleration and gravity through a small number of mechano-transduction channels in the hair cell stereocilia as far as to the ribbon synapse, where it drives neurotransmission. The genetic approach is proving fundamental in unravelling the molecular basis of these important biological functions. In particular, ablation or missense mutations of the PMCA2 Ca2+-pump of stereocilia cause deafness and loss of balance. To investigate the physiological significance of these genetic defects, we used a combination of confocal fluorescence microscopy and cytosolic Ca2+ photoliberation. The study of Ca2+-extrusion in hair cells from neonatal mice inner ear permitted us to show that Ca2+ extrusion was compromised by various degrees in PMCA2 knockout mice as well as in the mutant deafwaddler and Oblivion mice. We suggest that the consequent reduced endolymphatic Ca2+ concentration can trouble the finely tuned control mechanisms of signal transduction, eventually resulting in hair cell death.

Connexin 26 (Cx26) and connexin 30 (Cx30) form hemichannels that release ATP from the endolymphatic surface of cochlear supporting and epithelial cells and also form gap junction (GJ) channels that allow the concomitant intercellular diffusion of Ca2+ mobilizing second messengers. Released ATP in turn activates G-protein coupled P2Y2 and P2Y4 receptors, PLC-dependent generation of IP3, release of Ca2+ from intracellular stores, instigating the regenerative propagation of intercellular Ca2+ signals (ICS). The range of ICS propagation is sensitive to the concentration of extracellular divalent cations and activity of ectonucleotidases. Here, the expression patterns of Cx26 and Cx30 were characterized in postnatal cochlear tissues obtained from mice aged between P5 and P6. The expression gradient along the longitudinal axis of the cochlea, decreasing from the basal to the apical cochlear turn (CT), was more pronounced in outer sulcus (OS) cells than in inner sulcus (IS) cells. GJ-mediated dye coupling was maximal in OS cells of the basal CT, inhibited by the nonselective connexin channel blocker carbenoxolone (CBX) and absent in hair cells. Photostimulating OS cells with caged inositol (3,4,5) tri-phosphate (IP3) resulted in transfer of ICS in the lateral direction, from OS cells to IS cells across the hair cell region (HCR) of medial and basal CTs. ICS transfer in the opposite (medial) direction, from IS cells photostimulated with caged IP3 to OS cells, occurred mostly in the basal CT. In addition, OS cells displayed impressive rhythmic activity with oscillations of cytosolic free Ca2+ concentration ([Ca2+]i) coordinated by the propagation of Ca2+ wavefronts sweeping repeatedly through the same tissue area along the coiling axis of the cochlea. Oscillations evoked by uncaging IP3 or by applying ATP differed greatly, by as much as one order of magnitude, in frequency and waveform rise time. ICS evoked by direct application of ATP propagated along convoluted cellular paths in the OS, which often branched and changed dynamically over time. Potential implications of these findings are discussed in the context of developmental regulation and cochlear pathophysiology. Electronic supplementary material The online version of this article (doi:10.1007/s11302-010-9192-9) contains supplementary material, which is available to authorized users.

Progressive hearing loss is common in the human population, but we have few clues to the molecular basis. Mouse mutants with progressive hearing loss offer valuable insights, and ENU (N-ethyl-N-nitrosourea) mutagenesis is a useful way of generating models. We have characterised a new ENU-induced mouse mutant, Oblivion (allele symbol Obl), showing semi-dominant inheritance of hearing impairment. Obl/+ mutants showed increasing hearing impairment from post-natal day (P)20 to P90, and loss of auditory function was followed by a corresponding base to apex progression of hair cell degeneration. Obl/Obl mutants were small, showed severe vestibular dysfunction by 2 weeks of age, and were completely deaf from birth; sensory hair cells were completely degenerate in the basal turn of the cochlea, although hair cells appeared normal in the apex. We mapped the mutation to Chromosome 6. Mutation analysis of Atp2b2 showed a missense mutation (2630C→T) in exon 15, causing a serine to phenylalanine substitution (S877F) in transmembrane domain 6 of the PMCA2 pump, the resident Ca2+ pump of hair cell stereocilia. Transmembrane domain mutations in these pumps generally are believed to be incompatible with normal targeting of the protein to the plasma membrane. However, analyses of hair cells in cultured utricular maculae of Obl/Obl mice and of the mutant Obl pump in model cells showed that the protein was correctly targeted to the plasma membrane. Biochemical and biophysical characterisation showed that the pump had lost a significant portion of its non-stimulated Ca2+ exporting ability. These findings can explain the progressive loss of auditory function, and indicate the limits in our ability to predict mechanism from sequence alone., Author Summary Progressive hearing loss is very common in the human population, but we know little about the causes. Environmental and genetic factors each may contribute. Knowledge of the genetic variants involved in hearing loss and understanding of the molecular and cellular mechanism of their action will aid the development of better treatments. One of the few genes known to be involved, in both mouse and humans, is Atp2b2, which encodes a calcium pump. We have discovered a new mutation in this gene leading to hearing loss in the mouse mutant oblivion. The mutation leads to a serine to phenylalanine substitution in a transmembrane domain. Mutations affecting such transmembrane domains are usually expected to interfere with the normal process of inserting the protein in the membrane and transporting it to its final destination on the plasma membrane of the cell. Surprisingly, the pump is produced and is targeted to the plasma membrane, in both cultured cells expressing the mutant gene and sensory hair cells from the oblivion mutant inner ear. However, we show it has impaired calcium pumping ability, which can account for the progressive hearing loss as well as the progressive degeneration of the sensory hair cells that we observe in the mutants.

Autosomal dominant optic atrophy (ADOA), the commonest cause of inherited optic atrophy, is caused by mutations in the ubiquitously expressed gene optic atrophy 1 (OPA1), involved in fusion and biogenesis of the inner membrane of mitochondria. Bioenergetic failure, mitochondrial network abnormalities and increased apoptosis have all been proposed as possible causal factors. However, their relative contribution to pathogenesis as well as the prominent susceptibility of the retinal ganglion cell (RGC) in this disease remains uncertain. Here we identify a novel deletion of OPA1 gene in the GTPase domain in three patients affected by ADOA. Muscle biopsy of the patients showed neurogenic atrophy and abnormal morphology and distribution of mitochondria. Confocal microscopy revealed increased mitochondrial fragmentation in fibroblasts as well as in myotubes, where mitochondria were also unevenly distributed, with clustered organelles alternating with areas where mitochondria were sparse. These abnormalities were not associated with altered bioenergetics or increased susceptibility to pro-apoptotic stimuli. Therefore, changes in mitochondrial shape and distribution can be independent of other reported effects of OPA1 mutations, and therefore may be the primary cause of the disease. The arrangement of mitochondria in RGCs, which degenerate in ADOA, may be exquisitely sensitive to disturbance, and this may lead to bioenergetic crisis and/or induction of apoptosis. Our results highlight the importance of mitochondrial dynamics in the disease per se, and point to the loss of the fine positioning of mitochondria in the axons of RGCs as a possible explanation for their predominant degeneration in ADOA.

Signal transduction by auditory and vestibular hair cells involves an impressive ensemble of finely tuned control mechanisms, strictly dependent on the local intracellular Ca(2+) concentration ([Ca(2+)](i)). The study of Ca(2+) dynamics in hair cells typically combines Ca(2+)-sensitive fluorescent indicators (dyes), patch clamp and optical microscopy to produce images of the patterns of fluorescence of a Ca(2+) indicator following various stimulation protocols. Here we describe a novel method that combines electrophysiological recordings, fluorescence imaging and numerical simulations to effectively deconvolve Ca(2+) signals within cytoplasmic microdomains that would otherwise remain inaccessible to direct observation. The method relies on the comparison of experimental data with virtual signals derived from a Monte Carlo reaction-diffusion model based on a realistic reconstruction of the relevant cell boundaries in three dimensions. The model comprises Ca(2+) entry at individual presynaptic active zones followed by diffusion, buffering, extrusion and release of Ca(2+). Our results indicate that changes of the hair cell [Ca(2+)](i) during synaptic transmission are primarily controlled by the Ca(2+) endogenous buffers both at short (

Gap junction channels assembled from connexin protein subunits mediate intercellular transfer of ions and metabolites. Impaired channel function is implicated in several hereditary human diseases. In particular, defective permeation of cAMP or inositol-1,4,5-trisphosphate (InsP(3)) through connexin channels is associated with peripheral neuropathies and deafness, respectively. Here we present a method to estimate the permeability of single gap junction channels to second messengers. Using HeLa cells that overexpressed wild-type human connexin 26 (HCx26wt) as a model system, we combined measurements of junctional conductance and fluorescence resonance energy transfer (FRET) emission ratio of biosensors selective for cAMP and InsP(3). The unitary permeabilities to cAMP (47 x 10(-3) +/- 15 x 10(-3) microm(3)/s) and InsP(3) (60 x 10(-3) +/- 12 x 10(-3) microm(3)/s) were similar, but substantially larger than the unitary permeability to lucifer yellow (LY; 7 +/- 3 x 10(-3) microm(3)/s), an exogenous tracer. This method permits quantification of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signaling pathways.