PKA mediates modality-specific modulation of the mechanically gated ion channel PIEZO2.

PKA is a downstream effector of many inflammatory mediators that induce pain hypersensitivity by increasing the mechanosensitivity of nociceptive sensory afferent. Here, we examine the molecular mechanism underlying PKA-dependent modulation of the mechanically activated ion channel PIEZO2, which confers mechanosensitivity to many nociceptors. Using phosphorylation site prediction algorithms, we identified multiple putative and highly conserved PKA phosphorylation sites located on intracellular intrinsically disordered regions of PIEZO2. Site-directed mutagenesis and patch-clamp recordings showed that substitution of one or multiple putative PKA sites within a single intracellular domain does not alter PKA-induced PIEZO2 sensitization, whereas mutation of a combination of nine putative sites located on four different intracellular regions completely abolishes PKA-dependent PIEZO2 modulation, though it remains unclear whether all or just some of these nine sites are required. By demonstrating that PIEZO1 is not modulated by PKA, our data also reveal a previously unrecognized functional difference between PIEZO1 and PIEZO2. Moreover, by demonstrating that PKA only modulates PIEZO2 currents evoked by focal mechanical indentation of the cell, but not currents evoked by pressure-induced membrane stretch, we provide evidence suggesting that PIEZO2 is a polymodal mechanosensor that engages different protein domains for detecting different types of mechanical stimuli.

Structural basis of activation of the tumor suppressor protein neurofibromin.

Mutations in the NF1 gene cause the familial genetic disease neurofibromatosis type I, as well as predisposition to cancer. The NF1 gene product, neurofibromin, is a GTPase-activating protein and acts as a tumor suppressor by negatively regulating the small GTPase, Ras. However, structural insights into neurofibromin activation remain incompletely defined. Here, we provide cryoelectron microscopy (cryo-EM) structures that reveal an extended neurofibromin homodimer in two functional states: an auto-inhibited state with occluded Ras-binding site and an asymmetric open state with an exposed Ras-binding site. Mechanistically, the transition to the active conformation is stimulated by nucleotide binding, which releases a lock that tethers the catalytic domain to an extended helical repeat scaffold in the occluded state. Structure-guided mutational analysis supports functional relevance of allosteric control. Disease-causing mutations are mapped and primarily impact neurofibromin stability. Our findings suggest a role for nucleotides in neurofibromin regulation and may lead to therapeutic modulation of Ras signaling.

Quiescence, Stemness and Adipogenic Differentiation Capacity in Human DLK1-/CD34+/CD24+ Adipose Stem/Progenitor Cells.

We explore the status of quiescence, stemness and adipogenic differentiation capacity in adipose stem/progenitor cells (ASCs) ex vivo, immediately after isolation from human subcutaneous white adipose tissue, by sorting the stromal vascular fraction into cell-surface DLK1+/CD34-, DLK1+/CD34dim and DLK1-/CD34+ cells. We demonstrate that DLK1-/CD34+ cells, the only population exhibiting proliferative and adipogenic capacity, express ex vivo the bonafide quiescence markers p21Cip1, p27Kip1 and p57Kip2 but neither proliferation markers nor the senescence marker p16Ink4a. The pluripotency markers NANOG, SOX2 and OCT4 are barely detectable in ex vivo ASCs while the somatic stemness factors, c-MYC and KLF4 and the early adipogenic factor C/EBPß are highly expressed. Further sorting of ASCs into DLK1-/CD34+/CD24- and DLK1-/CD34+/CD24+ fractions shows that KLF4 and c-MYC are higher expressed in DLK1-/CD34+/CD24+ cells correlating with higher colony formation capacity and considerably lower adipogenic activity. Proliferation capacity is similar in both populations. Next, we show that ASCs routinely isolated by plastic-adherence are DLK1-/CD34+/CD24+. Intriguingly, CD24 knock-down in these cells reduces proliferation and adipogenesis. In conclusion, DLK1-/CD34+ ASCs in human sWAT exist in a quiescent state, express high levels of somatic stemness factors and the early adipogenic transcription factor C/EBPß but senescence and pluripotency markers are barely detectable. Moreover, our data indicate that CD24 is necessary for adequate ASC proliferation and adipogenesis and that stemness is higher and adipogenic capacity lower in DLK1-/CD34+/CD24+ relative to DLK1-/CD34+/CD24- subpopulations.

Control of mechanical pain hypersensitivity in mice through ligand-targeted photoablation of TrkB-positive sensory neurons.

Mechanical allodynia is a major symptom of neuropathic pain whereby innocuous touch evokes severe pain. Here we identify a population of peripheral sensory neurons expressing TrkB that are both necessary and sufficient for producing pain from light touch after nerve injury in mice. Mice in which TrkB-Cre-expressing neurons are ablated are less sensitive to the lightest touch under basal conditions, and fail to develop mechanical allodynia in a model of neuropathic pain. Moreover, selective optogenetic activation of these neurons after nerve injury evokes marked nociceptive behavior. Using a phototherapeutic approach based upon BDNF, the ligand for TrkB, we perform molecule-guided laser ablation of these neurons and achieve long-term retraction of TrkB-positive neurons from the skin and pronounced reversal of mechanical allodynia across multiple types of neuropathic pain. Thus we identify the peripheral neurons which transmit pain from light touch and uncover a novel pharmacological strategy for its treatment.

Myotubularin related protein-2 and its phospholipid substrate PIP2 control Piezo2-mediated mechanotransduction in peripheral sensory neurons.

Piezo2 ion channels are critical determinants of the sense of light touch in vertebrates. Yet, their regulation is only incompletely understood. We recently identified myotubularin related protein-2 (Mtmr2), a phosphoinositide (PI) phosphatase, in the native Piezo2 interactome of murine dorsal root ganglia (DRG). Here, we demonstrate that Mtmr2 attenuates Piezo2-mediated rapidly adapting mechanically activated (RA-MA) currents. Interestingly, heterologous Piezo1 and other known MA current subtypes in DRG appeared largely unaffected by Mtmr2. Experiments with catalytically inactive Mtmr2, pharmacological blockers of PI(3,5)P2 synthesis, and osmotic stress suggest that Mtmr2-dependent Piezo2 inhibition involves depletion of PI(3,5)P2. Further, we identified a PI(3,5)P2 binding region in Piezo2, but not Piezo1, that confers sensitivity to Mtmr2 as indicated by functional analysis of a domain-swapped Piezo2 mutant. Altogether, our results propose local PI(3,5)P2 modulation via Mtmr2 in the vicinity of Piezo2 as a novel mechanism to dynamically control Piezo2-dependent mechanotransduction in peripheral sensory neurons.

LAMTOR/Ragulator is a negative regulator of Arl8b- and BORC-dependent late endosomal positioning.

Signaling from lysosomes controls cellular clearance and energy metabolism. Lysosomal malfunction has been implicated in several pathologies, including neurodegeneration, cancer, infection, immunodeficiency, and obesity. Interestingly, many functions are dependent on the organelle position. Lysosomal motility requires the integration of extracellular and intracellular signals that converge on a competition between motor proteins that ultimately control lysosomal movement on microtubules. Here, we identify a novel upstream control mechanism of Arl8b-dependent lysosomal movement toward the periphery of the cell. We show that the C-terminal domain of lyspersin, a subunit of BLOC-1-related complex (BORC), is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. In addition, we establish lyspersin as the linker between BORC and late endosomal/lysosomal adaptor and mitogen activated protein kinase and mechanistic target of rapamycin activator (LAMTOR) complexes and show that epidermal growth factor stimulation decreases LAMTOR/BORC association, thereby promoting BORC- and Arl8b-dependent lysosomal centrifugal transport.

Nerve growth factor and nociception: from experimental embryology to new analgesic therapy.

Nerve growth factor (NGF) is central to the development and functional regulation of sensory neurons that signal the first events that lead to pain. These sensory neurons, called nociceptors, require NGF in the early embryo to survive and also for their functional maturation. The long road from the discovery of NGF and its roles during development to the realization that NGF plays a major role in the pathophysiology of inflammatory pain will be reviewed. In particular, we will discuss the various signaling events initiated by NGF that lead to long-lasting thermal and mechanical hyperalgesia in animals and in man. It has been realized relatively recently that humanized function blocking antibodies directed against NGF show remarkably analgesic potency in human clinical trials for painful conditions as varied as osteoarthritis, lower back pain, and interstitial cystitis. Thus, anti-NGF medication has the potential to make a major impact on day-to-day chronic pain treatment in the near future. It is therefore all the more important to understand the precise pathways and mechanisms that are controlled by NGF to both initiate and sustain mechanical and thermal hyperalgesia. Recent work suggests that NGF-dependent regulation of the mechanosensory properties of sensory neurons that signal mechanical pain may open new mechanistic avenues to refine and exploit relevant molecular targets for novel analgesics.

Adipogenic differentiation is impaired in replicative senescent human subcutaneous adipose-derived stromal/progenitor cells.

We demonstrate that adipose-derived stromal/progenitor cells isolated from abdominal subcutaneous fat pads of adult donors successively enter replicative senescence after long-term cultivation. This is characterized by enlarged cell size, flattened morphology, and upregulated senescence-associated ß-galactosidase activity. Moreover, the senescence- associated cyclin-dependent kinase inhibitors p16(Ink4A) and p21(Cip1) were induced correlating with activation of the G1/S cell cycle inhibitor retinoblastoma protein and terminal proliferation arrest. The number of cells in the adipose-derived stromal/progenitor cell population with high adipogenic capacity declined inversely with the increase of senescent cells. Adipogenic hormone cocktail induced expression of the adipogenic key regulators peroxisome proliferator-activated receptor-γ2 and CCAAT/enhancer-binding protein α was significantly reduced in senescent adipose-derived stromal/ progenitor cells. Furthermore, the expression of the adipogenic differentiation genes fatty acid binding protein-4, adiponectin, and leptin and the formation of fat droplets were impaired. We conclude cellular senescence contributes to dysfunctions in adipose-derived stromal/progenitor cell replication, adipogenesis, triglyceride storage, and adipokine secretion.

The transcription factor c-Maf in sensory neuron development.

The proto-oncogene c-Maf has been shown to be an important transcriptional regulator in the differentiation of a number of cellular contexts, like the eye and hematopoietic system. Here we discuss the recent progress made in understanding c-Maf function in the nervous system.

The transcription factor c-Maf controls touch receptor development and function.

The sense of touch relies on detection of mechanical stimuli by specialized mechanosensory neurons. The scarcity of molecular data has made it difficult to analyze development of mechanoreceptors and to define the basis of their diversity and function. We show that the transcription factor c-Maf/c-MAF is crucial for mechanosensory function in mice and humans. The development and function of several rapidly adapting mechanoreceptor types are disrupted in c-Maf mutant mice. In particular, Pacinian corpuscles, a type of mechanoreceptor specialized to detect high-frequency vibrations, are severely atrophied. In line with this, sensitivity to high-frequency vibration is reduced in humans carrying a dominant mutation in the c-MAF gene. Thus, our work identifies a key transcription factor specifying development and function of mechanoreceptors and their end organs.

The human papillomavirus type 16 E7 oncoprotein targets Myc-interacting zinc-finger protein-1.

We demonstrate that HPV-16 E7 forms a complex with Miz-1. UV-induced expression of the CDK-inhibitor p21(Cip1) and subsequent cell cycle arrest depends upon endogenous Miz-1 in HPV-negative C33A cervical cancer cells containing mutated p53. Transient expression of E7 in C33A inhibits UV-induced expression of p21(Cip1) and overcomes Miz-1-induced G1-phase arrest. The C-terminal E7Δ79LEDLL83-mutant with reduced Miz-1-binding capacity was impaired in its capability to repress p21(Cip1) expression; whereas the pRB-binding-deficient E7C24G-mutant inhibited p21(Cip1) expression similar to wild-type E7. Using ChIP, we demonstrate that endogenous E7 is bound to the endogenous p21(Cip1) core-promoter in CaSki cells and RNAi-mediated knock down of Miz-1 abrogates E7-binding to the p21(Cip1) promoter. Co-expression of E7 with Miz-1 inhibited Miz-1-induced p21(Cip1) expression from the minimal-promoter via Miz-1 DNA-binding sites. Co-expression of E7Δ79LEDLL83 did not inhibit Miz-1-induced p21(Cip1) expression. E7C24G retained E7-wild-type capability to inhibit Miz-1-dependent transactivation. These findings suggest that HPV-16 E7 can repress Miz-1-induced p21(Cip1) gene expression.

Subcellular localization of the human papillomavirus 16 E7 oncoprotein in CaSki cells and its detection in cervical adenocarcinoma and adenocarcinoma in situ.

E7 is the major oncoprotein of high-risk human papillomaviruses (HPV) which causes cervical cancer. To date E7 oncoproteins have not been investigated in cervical adenocarcinoma. In this study we generated a rabbit monoclonal anti-HPV-16 E7 antibody, RabMab42-3, which recognizes a conformational epitope in the E7 carboxy-terminal zinc-finger resulting in a strong increase in the sensitivity for the detection of cell-associated HPV-16 E7 protein relative to conventional polyclonal anti-HPV-16 E7 antibodies. Using RabMab42-3, we show that the subcellular localization of endogenous HPV-16 E7 oncoprotein varies during the cell cycle in cervical cancer cells. Moreover, we demonstrate for the first time that the HPV-16 E7 oncoprotein is abundantly expressed in cervical adenocarcinoma in situ and adenocarcinoma, suggesting an important role of HPV-16 E7 for the development of these tumors. Our findings suggest that the HPV-16 E7 oncoprotein could be a useful marker for the detection of cervical adenocarcinoma and their precursors.

Adipokine profile and insulin sensitivity in formerly obese women subjected to bariatric surgery or diet-induced long-term caloric restriction.

To better understand the contribution of the fat mass to the effects of long-term caloric restriction in humans, we compared adipokine profile and insulin sensitivity in long-term calorically restricted formerly obese women (CRW) subjected to different interventions, bariatric surgery, or reducing diet, with age- and BMI-matched obese (OW) and normal-weight women (NW) eating ad libitum. Our key findings are that despite a considerably stronger weight loss induced by bariatric surgery, both long-term caloric restriction interventions improved insulin sensitivity to the same degree and led to significantly lower retinol-binding protein-4 and interleukin-6 serum levels than in OW, suggesting that lowering of these two adipokines contributes to the improved insulin sensitivity. Moreover, serum leptin was considerably lower in CRW than in OW as well as in NW, suggesting that CRW develop hypoleptinemia.

Pyruvate kinase isoenzyme M2 is a glycolytic sensor differentially regulating cell proliferation, cell size and apoptotic cell death dependent on glucose supply.

The glycolytic key regulator pyruvate kinase M2 (M2-PK or PKM2) can switch between a highly active tetrameric and an inactive dimeric form. The transition between the two conformations regulates the glycolytic flux in tumor cells. We developed specific M2-PK-binding peptide aptamers which inhibit M2-PK, but not the 96% homologous M1-PK isoenzyme. In this study we demonstrate that, at normal blood glucose concentrations, peptide aptamer-mediated inhibition of M2-PK induces a significant decrease of the population doubling (PDL rate) and cell proliferation rate as well as an increase in cell size, whereas under glucose restriction an increase in PDL and cell proliferation rates but a decrease in cell size was observed. Moreover, M2-PK inhibition rescues cells from glucose starvation-induced apoptotic cell death by increasing the metabolic activity. These findings suggest that M2-PK is a metabolic sensor which regulates cell proliferation, cell growth and apoptotic cell death in a glucose supply-dependent manner.

Peripheral sensitisation of nociceptors via G-protein-dependent potentiation of mechanotransduction currents.

Mechanical stimuli impinging on the skin are converted into electrical signals by mechanically gated ion channels located at the peripheral nerve endings of dorsal root ganglion (DRG) neurons. Under inflammatory conditions sensory neurons are commonly sensitised to mechanical stimuli; a putative mechanism that may contribute to such sensitisation of sensory neurons is enhanced responsiveness of mechanotransduction ion channels. Here we show that the algogens UTP and ATP potentiate mechanosensitive RA currents in peptidergic nociceptive DRG neurons and reduce thresholds for mechanically induced action potential firing in these neurones. Pharmacological characterisation suggests that this effect is mediated by the Gq-coupled P2Y(2) nucleotide receptor. Moreover, using the in vitro skin nerve technique, we show that UTP also increases action potential firing rates in response to mechanical stimuli in a subpopulation of skin C-fibre nociceptors. Together our findings suggest that UTP sensitises a subpopulation of cutaneous C-fibre nociceptors via a previously undescribed G-protein-dependent potentiation of mechanically activated RA-type currents.

Presynaptic inhibition via a phospholipase C- and phosphatidylinositol bisphosphate-dependent regulation of neuronal Ca2+ channels.

Presynaptic inhibition of transmitter release is commonly mediated by a direct interaction between G protein betagamma subunits and voltage-activated Ca2+ channels. To search for an alternative pathway, the mechanisms by which presynaptic bradykinin receptors mediate an inhibition of noradrenaline release from rat superior cervical ganglion neurons were investigated. The peptide reduced noradrenaline release triggered by K+-depolarization but not that evoked by ATP, with Ca2+ channels being blocked by Cd2+. Bradykinin also reduced Ca2+ current amplitudes measured at neuronal somata, and this effect was pertussis toxin-insensitive, voltage-independent, and developed slowly within 1 min. The inhibition of Ca2+ currents was abolished by a phospholipase C inhibitor, but it was not altered by a phospholipase A2 inhibitor, by the depletion of intracellular Ca2+ stores, or by the inactivation of protein kinase C or Rho proteins. In whole-cell recordings, the reduction of Ca2+ currents was irreversible but became reversible when 4 mM ATP or 0.2 mM dioctanoyl phosphatidylinositol-4,5-bisphosphate was included in the pipette solution. In contrast, the effect of bradykinin was entirely reversible in perforated-patch recordings but became irreversible when the resynthesis of phosphatidylinositol-4,5-bisphosphate was blocked. Thus, the inhibition of Ca2+ currents by bradykinin involved a consumption of phosphatidylinositol-4,5-bisphosphate by phospholipase C but no downstream effectors of this enzyme. The reduction of noradrenaline release by bradykinin was also abolished by the inhibition of phospholipase C or of the resynthesis of phosphatidylinositol-4,5-bisphosphate. These results show that the presynaptic inhibition was mediated by a closure of voltage-gated Ca2+ channels through depletion of membrane phosphatidylinositol bisphosphates via phospholipase C.

Autoinhibition of transmitter release from PC12 cells and sympathetic neurons through a P2Y receptor-mediated inhibition of voltage-gated Ca2+ channels.

Although feedback inhibition of noradrenaline release by coreleased nucleotides is a well known phenomenon, it remained unclear which P2 receptor subtypes and associated signalling cascades may be involved. In the rat pheochromocytoma cell line PC12, 2-methylthio-ADP reduced noradrenaline release triggered by K+ depolarization more potently than ADP and ATP, whereas UDP or UTP failed to do so. The inhibition by ADP was abolished by pertussis toxin and antagonized by reactive blue 2, 2-methylthio-AMP, and AR-C69931MX, but not by suramin. AR-C69931MX acted as a competitive antagonist with an apparent affinity of 2 nm, but did not alter noradrenaline release, when PC12 cells were continuously superfused. However, when the superfusion was halted during K+ depolarization, release was significantly reduced and this inhibition was attenuated by AR-C69931MX, thus revealing ongoing autoinhibition. Rises in cellular cyclic AMP did not alter depolarization-evoked release nor its reduction by ADP, even though the nucleotide did inhibit cyclic AMP accumulation. ADP and the direct Ca2+ channel blocker Cd2+ inhibited voltage-activated Ca2+ currents, but not ATP-induced currents, and both agents reduced K+-evoked, but not ATP-evoked, release. Hence, if voltage-gated Ca2+ channels do not contribute to stimulation-evoked release, ADP fails to exert its inhibitory action. In primary cultures of rat sympathetic neurons, ADP also reduced Ca2+ currents and K+-evoked noradrenaline release, and AR-C69931MX acted again as competitive antagonist with an apparent affinity of 3 nm. These results show that P2Y12 receptors mediate an autoinhibition of transmitter release from PC12 cells and sympathetic neurons through an inhibition of voltage-gated Ca2+ channels.

Attenuation of the P2Y receptor-mediated control of neuronal Ca2+ channels in PC12 cells by antithrombotic drugs.

1. In PC12 cells, adenine nucleotides inhibit voltage-activated Ca(2+) currents and adenylyl cyclase activity, and the latter effect was reported to involve P2Y(12) receptors. To investigate whether these two effects are mediated by one P2Y receptor subtype, we used the antithrombotic agents 2-methylthio-AMP (2-MeSAMP) and N(6)-(2-methyl-thioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (AR-C69931MX). 2. ADP reduced A(2A) receptor-dependent cyclic AMP synthesis with half maximal effects at 0.1-0.17 micro M. In the presence of 30 micro M 2-MeSAMP or 100 nM AR-C69931MX, concentration response curves were shifted to the right by factors of 39 and 30, indicative of pA(2) values of 6.1 and 8.5, respectively. 3. The inhibition of Ca(2+) currents by ADP was attenuated by 10-1000 nM AR-C69931MX and by 3-300 micro M 2-MeSAMP. ADP reinhibited Ca(2+) currents after removal of 2-MeSAMP within less than 15 s, but required 2 min to do so after removal of AR-C69931MX. 4. ADP inhibited Ca(2+) currents with half maximal effects at 5-20 micro M. AR-C69931MX (10-100 nM) displaced concentration response curves to the right, and the resulting Schild plot showed a slope of 1.09 and an estimated pK(B) value of 8.7. Similarly, 10-100 micro M 2-MeSAMP also caused rightward shifts resulting in a Schild plot with a slope of 0.95 and an estimated pK(B) of 5.4. 5. The inhibition of Ca(2+) currents by 2-methylthio-ADP and ADPbetaS was also antagonized by AR-C69931MX, which (at 30 nM) caused a rightward shift of the concentration response curve for ADPbetaS by a factor of 3.8, indicative of a pA(2) value of 8.1. 6. These results show that antithrombotic drugs antagonize the inhibition of neuronal Ca(2+) channels by adenine nucleotides, which suggests that this effect is mediated by P2Y(12) receptors.