TopBP1 assembles nuclear condensates to switch on ATR signaling.

ATR checkpoint signaling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micrometer-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse, and co-localize with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slow down replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR activation domain disrupt TopBP1 condensation and consequently ATR/Chk1 signaling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signaling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.

Gangliosides interact with synaptotagmin to form the high-affinity receptor complex for botulinum neurotoxin B.

Botulinum neurotoxin type B (BoNT/B) recognizes nerve terminals by binding to 2 receptor components: a polysialoganglioside, predominantly GT1b, and synaptotagmin 1/2. It is widely thought that BoNT/B initially binds to GT1b then diffuses in the plane of the membrane to interact with synaptotagmin. We have addressed the hypothesis that a GT1b-synaptotagmin cis complex forms the BoNT/B receptor. We identified a consensus glycosphingolipid-binding motif in the extracellular juxtamembrane domain of synaptotagmins 1/2 and confirmed by Langmuir monolayer, surface plasmon resonance, and circular dichroism that GT1b interacts with synaptotagmin peptides containing this sequence, inducing α-helical structure. Molecular modeling and tryptophan fluorescence spectroscopy were consistent with the intertwining of GT1b and synaptotagmin, involving cis interactions between the oligosaccharide and ceramide moieties of GT1b and the juxtamembrane and transmembrane domains of synaptotagmin, respectively. Furthermore, a point mutation on synaptotagmin, located outside of the BoNT/B-binding segment, inhibited GT1b binding and blocked GT1b-induced potentiation of BoNT/B binding to synaptotagmin-expressing cells. Our findings are consistent with a model in which a preassembled GT1b-synaptotagmin complex constitutes the high-affinity BoNT/B receptor.

Spatial organization and functions of Chk1 activation by TopBP1 biomolecular condensates.

Assembly of TopBP1 biomolecular condensates triggers activation of the ataxia telangiectasia-mutated and Rad3-related (ATR)/Chk1 signaling pathway, which coordinates cell responses to impaired DNA replication. Here, we used optogenetics and reverse genetics to investigate the role of sequence-specific motifs in the formation and functions of TopBP1 condensates. We propose that BACH1/FANCJ is involved in the partitioning of BRCA1 within TopBP1 compartments. We show that Chk1 is activated at the interface of TopBP1 condensates and provide evidence that these structures arise at sites of DNA damage and in primary human fibroblasts. Chk1 phosphorylation depends on the integrity of a conserved arginine motif within TopBP1's ATR activation domain (AAD). Its mutation uncouples Chk1 activation from TopBP1 condensation, revealing that optogenetically induced Chk1 phosphorylation triggers cell cycle checkpoints and slows down replication forks in the absence of DNA damage. Together with previous work, these data suggest that the intrinsically disordered AAD encodes distinct molecular steps in the ATR/Chk1 pathway.

Spatially Addressable Multiplex Biodetection by Calibrated Micro/Nanostructured Surfaces.

A challenge of any biosensing technology is the detection of very low concentrations of analytes. The fluorescence interference contrast (FLIC) technique improves the fluorescence-based sensitivity by selectively amplifying, or suppressing, the emission of a fluorophore-labeled biomolecule immobilized on a transparent layer placed on top of a mirror basal surface. The standing wave of the reflected emission light means that the height of the transparent layer operates as a surface-embedded optical filter for the fluorescence signal. FLIC extreme sensitivity to wavelength is also its main problem: small, e.g., 10 nm range, variations of the vertical position of the fluorophore can translate in unwanted suppression of the detection signal. Herein, we introduce the concept of quasi-circular lenticular microstructured domes operating as continuous-mode optical filters, generating fluorescent concentric rings, with diameters determined by the wavelengths of the fluorescence light, in turn modulated by FLIC. The critical component of the lenticular structures was the shallow sloping side wall, which allowed the simultaneous separation of fluorescent patterns for virtually any fluorophore wavelength. Purposefully designed microstructures with either stepwise or continuous-slope dome geometries were fabricated to modulate the intensity and the lateral position of a fluorescence signal. The simulation of FLIC effects induced by the lenticular microstructures was confirmed by the measurement of the fluorescence profile for three fluorescent dyes, as well as high-resolution fluorescence scanning using stimulated emission depletion (STED) microscopy. The high sensitivity of the spatially addressable FLIC technology was further validated on a diagnostically important target, i.e., the receptor-binding domain (RBD) of the SARS-Cov2 via the detection of RBD:anti-S1-antibody.

A label-free biosensor assay for botulinum neurotoxin B in food and human serum.

Botulinum neurotoxins (BoNTs) are among the most toxic substances known. Surveillance and diagnostics require methods for rapid detection of BoNTs in complex media such as foodstuffs and human serum. We have developed in vitro assays to specifically detect the protease activity of botulinum neurotoxin B (BoNT/B) on a time scale of minutes. Cleavage of the BoNT/B substrate VAMP2, a membrane SNARE protein associated with synaptic vesicles, was monitored using real-time surface plasmon resonance to measure vesicle capture by specific antibodies coupled to microchips. The assay is functional in low-ionic-strength buffers and stable over a wide range of pH values (5.5-9.0). Endoproteolytic cleavage of VAMP2 was detected in 10 min with 2 pM native BoNT/B holotoxin. Contamination of liquid food products such as carrot juice, apple juice, and milk with low picomolar amounts of BoNT/B was revealed within 3h. BoNT/B activity was detected in sera from patients with type B botulism but not in healthy controls or patients with other neurological diseases. This robust, sensitive, and rapid protein chip assay is appropriate for monitoring BoNT/B in food products and diagnostic tests for type B botulism and could replace the current in vivo mouse bioassay.

Neuroglial and synaptic rearrangements associated with photic entrainment of the circadian clock in the suprachiasmatic nucleus.

Rhythmic biological functions in mammals are orchestrated by a circadian timekeeper in the suprachiasmatic nucleus of the hypothalamus (SCN) which precisely adjusts clock outputs to solar time through the process of photic synchronization. Entrainment to the 24-h light-dark cycle is known to act on the molecular loops which trigger circadian oscillations but is also thought to involve day-night adjustments in the intercellular phasing of the multiple component SCN oscillators. This view is supported by data showing that the SCN undergoes important rearrangements of its neuroglial architecture throughout the 24-h cycle. The present paper highlights our data showing in rat that the two main sources of SCN efferents, composed of neurons synthesizing either vasopressin (AVP) or vasoactive intestinal peptide (VIP), are differentially involved in day-night SCN neuroglial plasticity. We found that the synaptic inputs received by the VIP neurons, which are major integrators of photic signals in the retinorecipient SCN subregion, increased during the day while those received by the AVP neurons remained unchanged at day and night. Glutamatergic axons, known to convey photic information from the retina, together with nonglutamatergic axons, contribute to the synaptic remodellings on VIP neurons. Experimental data providing strong indication that these plastic events may subserve synchronization of the clock to the light-dark cycle and that the daily fluctuations of plasma glucocorticoid hormones may act as temporal endocrine signals that may modulate SCN neuroglial plasticity through the rhythmic release of serotonin are also reviewed.

Daily changes in synaptic innervation of VIP neurons in the rat suprachiasmatic nucleus: contribution of glutamatergic afferents.

The daily temporal organization of rhythmic functions in mammals, which requires synchronization of the circadian clock to the 24-h light-dark cycle, is believed to involve adjustments of the mutual phasing of the cellular oscillators that comprise the time-keeper within the suprachiasmatic nucleus of the hypothalamus (SCN). Following from a previous study showing that the SCN undergoes day/night rearrangements of its neuronal-glial network that may be crucial for intercellular phasing, we investigated the contribution of glutamatergic synapses, known to play major roles in SCN functioning, to such rhythmic plastic events. Neither expression levels of the vesicular glutamate transporters nor numbers of glutamatergic terminals showed nycthemeral variations in the SCN. However, using quantitative imaging after combined immunolabelling, the density of synapses on neurons expressing vasoactive intestinal peptide, known as targets of the retinal input, increased during the day and both glutamatergic and non-glutamatergic synapses contributed to the increase (+36%). This was not the case for synapses made on vasopressin-containing neurons, the other major source of SCN efferents in the non-retinorecipient region. Together with electron microscope observations showing no differences in the morphometric features of glutamatergic terminals during the day and night, these data show that the light synchronization process in the SCN involves a selective remodelling of synapses at sites of photic integration. They provide a further illustration of how the adult brain may rapidly and reversibly adapt its synaptic architecture to functional needs.

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA.

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA's family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That's the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest. This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

Paraspeckles as rhythmic nuclear mRNA anchorages responsible for circadian gene expression.

Circadian clocks regulate rhythmic gene expression levels by means of mRNA oscillations that are mainly driven by post-transcriptional regulation. We identified a new post-transcriptional mechanism, which involves nuclear bodies called paraspeckles. Major components of paraspeckles including the long noncoding RNA Neat1, which is the structural component, and its major protein partners, as well as the number of paraspeckles, follow a circadian pattern in pituitary cells. Paraspeckles are known to retain within the nucleus RNAs containing inverted repeats of Alu sequences. We showed that a reporter gene in which these RNA duplex elements were inserted in the 3'-UTR region displayed a circadian expression. Moreover, circadian endogenous mRNA associated with paraspeckles lost their circadian pattern when paraspeckles were disrupted. This work not only highlights a new paraspeckle-based post-transcriptional mechanism involved in circadian gene expression but also provides the list of all mRNA associated with paraspeckles in the nucleus of pituitary cells.

Circadian RNA expression elicited by 3'-UTR IRAlu-paraspeckle associated elements.

Paraspeckles are nuclear bodies form around the long non-coding RNA, Neat1, and RNA-binding proteins. While their role is not fully understood, they are believed to control gene expression at a post-transcriptional level by means of the nuclear retention of mRNA containing in their 3'-UTR inverted repeats of Alu sequences (IRAlu). In this study, we found that, in pituitary cells, all components of paraspeckles including four major proteins and Neat1 displayed a circadian expression pattern. Furthermore the insertion of IRAlu at the 3'-UTR of the EGFP cDNA led to a rhythmic circadian nuclear retention of the egfp mRNA that was lost when paraspeckles were disrupted whereas insertion of a single antisense Alu had only a weak effect. Using real-time video-microscopy, these IRAlu were further shown to drive a circadian expression of EGFP protein. This study shows that paraspeckles, thanks to their circadian expression, control circadian gene expression at a post-transcriptional level.

In vitro impact of pegvisomant on growth hormone-secreting pituitary adenoma cells.

Pegvisomant (PEG), an antagonist of growth hormone (GH)-receptor (GHR), normalizes insulin-like growth factor 1 (IGF1) oversecretion in most acromegalic patients unresponsive to somatostatin analogs (SSAs) and/or uncontrolled by transsphenoidal surgery. The residual GH-secreting tumor is therefore exposed to the action of circulating PEG. However, the biological effect of PEG at the pituitary level remains unknown. To assess the impact of PEG in vitro on the hormonal secretion (GH and prolactin (PRL)), proliferation and cellular viability of eight human GH-secreting tumors in primary cultures and of the rat somatolactotroph cell line GH4C1. We found that the mRNA expression levels of GHR were characterized in 31 human GH-secreting adenomas (0.086 copy/copy ß-Gus) and the GHR was identified by immunocytochemistry staining. In 5/8 adenomas, a dose-dependent inhibition of GH secretion was observed under PEG with a maximum of 38.2±17% at 1µg/mL (P<0.0001 vs control). A dose-dependent inhibition of PRL secretion occurred in three mixed GH/PRL adenomas under PEG with a maximum of 52.8±11.5% at 10µg/mL (P<0.0001 vs control). No impact on proliferation of either human primary tumors or GH4C1 cell line was observed. We conclude that PEG inhibits the secretion of GH and PRL in primary cultures of human GH(/PRL)-secreting pituitary adenomas without effect on cell viability or cell proliferation.

An optical biosensor assay for rapid dual detection of Botulinum neurotoxins A and E.

The enzymatic activity of the pathogenic botulinum neurotoxins type A and E (BoNT/A and E) leads to potentially lethal paralytic symptoms in humans and their prompt detection is of crucial importance. A chip assay based on Surface Plasmon Resonance monitoring of the cleavage products is a simple method that we have previously established to detect BoNT/A activity. We have now developed a similar format assay to measure BoNT/E activity. A monoclonal antibody specifically recognizing SNAP25 cleaved by BoNT/E was generated and used to measure the appearance of the neo-epitope following injection of BoNT/E over SNAP-25 immobilized on a chip. This assay detects BoNT/E activity at 1 LD50/ml within minutes and linear dose-responses curves were obtained using a multiplexed biosensor. A threshold of 0.01 LD50/ml was achieved after 5 h of cleavage. This assay is 10-fold more sensitive than the in vivo assay for direct detection of BoNT/E in serum samples. The SNAP25 chip assay is able to discriminate in an automated manner the presence of BoNT/E, BoNT/A or a combination of both toxins.

One-way calcium spill-over during signal transduction in Paramecium cells: from the cell cortex into cilia, but not in the reverse direction.

We asked to what extent Ca(2+) signals in two different domains of Paramecium cells remain separated during different stimulations. Wild-type (7S) and pawn cells (strain d4-500r, without ciliary voltage-dependent Ca(2+)-channels) were stimulated for trichocyst exocytosis within 80 ms by quenched-flow preparation and analysed by energy-dispersive X-ray microanalysis (EDX), paralleled by fast confocal fluorochrome analysis. We also analysed depolarisation-dependent calcium signalling during ciliary beat rerversal, also by EDX, after 80-ms stimulation in the quenched-flow mode. EDX and fluorochrome analysis enable to register total and free intracellular calcium concentrations, [Ca] and [Ca(2+)], respectively. After exocytosis stimulation we find by both methods that the calcium signal sweeps into the basis of cilia, not only in 7S but also in pawn cells which then also perform ciliary reversal. After depolarisation we see an increase of [Ca] along cilia selectively in 7S, but not in pawn cells. Opposite to exocytosis stimulation, during depolarisation no calcium spill-over into the nearby cytosol and no exocytosis occurs. In sum, we conclude that cilia must contain a very potent Ca(2+) buffering system and that ciliary reversal induction, much more than exocytosis stimulation, involves strict microdomain regulation of Ca(2+) signals.

Weightlessness affects cytoskeleton of rat utricular hair cells during maturation in vitro.

The aim of this study was to investigate whether an altered gravitational environment affected the phenotype of vestibular hair cells during maturation. We developed, using an automated incubator, a 3D culture of utricles from newborn rats. These cultures were subjected to weightlessness for 1 or 3 days, and then compared with control cultures developed in natural and induced 1G gravity. Immunocytochemistry for alpha-tubulin and calretinin revealed disorganisation of the microtubules and a loss of hair cell shape in cells subjected to weightlessness during maturation. We conclude that the lack of gravitational strain affected cytoskeletal dynamics, resulting in loss of the specific morphological phenotype of the cells.

Ghrelin receptor (GHS-R1a) and its constitutive activity in somatotroph adenomas: a new co-targeting therapy using GHS-R1a inverse agonists and somatostatin analogs.

CONTEXT: The ghrelin receptor GHS-R1a is highly expressed in human somatotroph adenomas and exhibits unusually high basal signaling activity. In humans, the suppression of this constitutive activity by mutation induces a short stature. OBJECTIVE: Using a GHS-R1a inverse agonist, modified substance P (MSP), we explored the role of GHS-R1a constitutive activity in GH hypersecretion from somatotroph adenomas and as a putative therapeutic target. DESIGN: The effects of MSP were assessed on GH secretion from 19 human somatotroph tumors in vitro. Moreover, these effects were compared with those of octreotide (somatostatin receptor subtype 2 [sst2] agonist) and with the combination of both drugs. Expression and localization of GHS-R1a and sst2 were studied. RESULTS: For all tumors, MSP inhibited GH secretion in a dose-dependent manner from 13 to 64%. Moreover, MSP enhanced octreotide-induced GH inhibition. For five tumors, the effects of combined MSP plus octreotide treatment were significantly higher than the sum of effects of each drug alone. MSP increased the membrane localization of GHS-R1a and of microdomains colocalizing sst2-GHS-R1a, highlighting the cooperation between the two drugs. CONCLUSIONS: The GHS-R1a inverse agonist could open new therapeutic options for acromegalic patients, particularly patients partially sensitive to octreotide whose GH secretion is not completely controlled by the sst2 agonist.

Pasireotide and octreotide antiproliferative effects and sst2 trafficking in human pancreatic neuroendocrine tumor cultures.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) raise difficult therapeutic problems despite the emergence of targeted therapies. Somatostatin analogs (SSA) remain pivotal therapeutic drugs. However, the tachyphylaxis and the limited antitumoral effects observed with the classical somatostatin 2 (sst2) agonists (octreotide and lanreotide) led to the development of new SSA, such as the pan sst receptor agonist pasireotide. Our aim was to compare the effects of pasireotide and octreotide on cell survival, chromogranin A (CgA) secretion, and sst2 phosphorylation/trafficking in pancreatic NET (pNET) primary cells from 15 tumors. We established and characterized the primary cultures of human pancreatic tumors (pNETs) as powerful preclinical models for understanding the biological effects of SSA. At clinically relevant concentrations (1-10 nM), pasireotide was at least as efficient as octreotide in inhibiting CgA secretion and cell viability through caspase-dependent apoptosis during short treatments, irrespective of the expression levels of the different sst receptors or the WHO grade of the parental tumor. Interestingly, unlike octreotide, which induces a rapid and persistent partial internalization of sst2 associated with its phosphorylation on Ser341/343, pasireotide did not phosphorylate sst2 and induced a rapid and transient internalization of the receptor followed by a persistent recycling at the cell surface. These results provide the first evidence, to our knowledge, of striking differences in the dynamics of sst2 trafficking in pNET cells treated with the two SSAs, but with similar efficiency in the control of CgA secretion and cell viability.

A substrate sensor chip to assay the enzymatic activity of Botulinum neurotoxin A.

Botulinum neurotoxin A (BoNT/A) induces muscle paralysis by enzymatically cleaving the presynaptic SNARE protein SNAP-25, which results in lasting inhibition of acetylcholine release at the neuromuscular junction. A rapid and sensitive in vitro assay for BoNT/A is required to replace the mouse lethality assay (LD50) in current use. We have developed a fully automated sensor to assay the endoprotease activity of BoNT/A. We produced monoclonal antibodies (mAbs) that recognize SNAP-25 neo-epitopes specifically generated by BoNT/A action. Recombinant SNAP-25 was coupled to the sensor surface of a surface plasmon resonance (SPR) system and samples containing BoNT/A were injected over the substrate sensor. Online substrate cleavage was monitored by measuring binding of mAb10F12 to a SNAP-25 neo-epitope. The SNAP-25-chip assay was toxin serotype-specific and detected 55 fM BoNT/A (1 LD50/ml) in 5 min and 0.4 fM (0.01 LD50/ml) in 5h. Time-course and dose-response curves were linear, yielding a limit of quantification of 0.03 LD50/ml. This label-free method is 100 times more sensitive than the mouse assay, potentially providing rapid read-out of small amounts of toxin for environmental surveillance and the quality control of pharmaceutical preparations.

Ras and Rap1 govern spatiotemporal dynamic of activated ERK in pituitary living cells.

The Ras/Raf/MEK/ERK is a conserved signalling pathway involved in the control of fundamental cellular processes. Despite extensive research, how this pathway can process a myriad of diverse extracellular inputs into substrate specificity to determine biological outcomes is not fully understood. It has been established that the ERK1/2 pathway is an integrative point in the control of the pituitary function exerted by various extracellular signals. In addition we previously established that the GTPases Ras and Rap1 play a key role in the regulation of ERK1/2-dependent prolactin transcription by EGF or the cAMP-dependent neuropeptide VIP. In this report, using the FRET-based biosensor of ERK activity (EKAR) in the pituitary GH4C1 cell line, we show that both EGF and VIP tightly control the spatiotemporal dynamic of activated ERK with different magnitude and duration. Importantly, we provide the first evidence of a differential control of cytoplasmic and nuclear pools of activated ERK by the GTPases Ras and Rap1. Ras is required for nuclear magnitude and duration of EGF-dependent ERK activation, whereas it is required for both VIP-activated cytoplasmic and nuclear ERK pools. Rap1 is exclusively involved in VIP-activated ERK nuclear pool. Moreover, consistent with the control of the nuclear pool of activated ERK by the GTPases, we observe the same differential role of Ras and Rap1 on ERK nuclear translocation triggered by EGF or VIP. Together these findings identify Ras and Rap1 as determinant partners in shaping nuclear and cytoplasmic ERK kinetics in response to EGF and VIP, which in turn should control pituitary secretion.

High-magnification observation of seminiferous tubules through the tunica albuginea by two-photon laser scanning microscopy.

Testicular sperm extraction is widely used in the treatment of male infertility in cases of non-obstructive azoospermia. Identifying spermatogenetic foci within the testes is critical for testicular sperm extraction. Two-photon laser scanning microscopy (TPLSM) is an autofluorescence-based microscopy technique that allows observation at a cellular level in the depth of fresh living tissues and does not require any histological processing (fixation or staining). The wavelengths previously used have shown no phototoxicity on sperm. We used TPLSM to detect spermatogenetic foci in fresh mouse testicular parenchyma without disrupting the tunica albuginea. Fresh surgically retrieved testes were observed using TPLSM within 1 h after extraction. Contralateral testes for each animal were observed using standard histology. Using TPLSM we were able to observe and measure the diameter of seminiferous tubules through the tunica albuginea, similar to the histological control. Structures within epithelial tubules were also observed, although their nature has yet to be identified. TPLSM is a real-time microscopy technique that could detect spermatogenetic foci.

Receptors for leptin in the otic labyrinth and the cochlear-vestibular nerve of guinea pig are modified in hormone-induced anorexia.

Metabolic syndromic inner ear pathology is a recognized condition in clinical practice but the possible causes remain controversial. We have previously reported that chronically-implanted estrogen implants in guinea pig results in hyperprolactinemia and hearing loss together with otic bone dysmorphology. The animals also present with anorexia. The hormone leptin has major roles in the regulation of satiety as well as bone metabolism and so we hypothesized that leptin might contribute to pathology of the otic labyrinth. We employed immunohistochemistry to investigate leptin receptor (ObR) expression. In control animals, ObR immunolabeling was not detected in the bone of the otic capsule but immunolabeling was observed in the cochlear-vestibular nerve. The labeling was associated with the astrocytic glial dome area, which marks the transition between central and peripheral parts of the nerve. In estrogen-treated animals, positive-ObR immunolabeling was observed in osteoblasts in new bone of the otic capsule and the ObR labeling was reduced in the cochlear-vestibular nerve compared to controls. The data provide evidence that leptin may target the labyrinth - affecting the bone and the nerve - and so could contribute to ongoing protection of the inner ear. Leptin disturbance might contribute to metabolic syndromes involving the audiovestibular system.