Conditional deletion of Pip5k1c in sensory ganglia and effects on nociception and inflammatory sensitization

Phosphatidylinositol 4-phosphate 5-kinase type 1 gamma (Pip5k1c) generates phosphatidylinositol 4,5-bisphosphate, also known as PI(4,5)P2 or PIP2. Many pronociceptive signaling pathways and receptor tyrosine kinases signal via PIP2 hydrolysis. Previously, we found that pain signaling and pain sensitization were reduced in Pip5k1c+/− global heterozygous knockout mice. Here, we sought to evaluate the extent to which dorsal root ganglia selective deletion of Pip5k1c affected nociception in mice. Initially, we crossed sensory neuron-selective Advillin-Cre mice with a conditional Pip5k1c knockout (cKO) allele (Pip5k1cfl/fl). However, these mice displayed an early onset proprioceptive deficit. To bypass this early onset phenotype, we used two different tamoxifen-inducible Cre lines (Brn3a-Cre-ERT2 and Advillin-Cre-ERT2) to conditionally delete Pip5k1c in adults. Tamoxifen induced high efficiency deletion of PIP5K1C in dorsal root ganglia and slightly reduced PIP5K1C in spinal cord and brain in Brn3a-Cre-ERT2 × Pip5k1cfl/fl (Brn3a cKO) mice while PIP5K1C was selectively deleted in dorsal root ganglia with no changes in spinal cord or brain in Advillin-Cre-ERT2 × Pip5k1cfl/fl (Advil cKO) mice. Acute thermosensation and mechanosensation were not altered in either line relative to wild-type mice. However, thermal hypersensitivity and mechanical allodynia recovered more rapidly in Brn3a cKO mice, but not Advil cKO mice, following hind paw inflammation. These data collectively suggest that PIP5K1C regulates nociceptive sensitization in more regions of the nervous system than dorsal root ganglia alone

Lipid kinases as therapeutic targets for chronic pain

Existing analgesics are not efficacious in treating all patients with chronic pain and have harmful side effects when used long-term. A deeper understanding of pain signaling and sensitization could lead to the development of more efficacious analgesics. Nociceptor sensitization occurs under conditions of inflammation and nerve injury where diverse chemicals are released and signal through receptors to reduce the activation threshold of ion channels, leading to an overall increase in neuronal excitability [98; 28]. Drugs that inhibit specific receptors have so far been unsuccessful in alleviating pain, possibly because they do not simultaneously target the diverse receptors that contribute to nociceptor sensitization. Hence, focus has shifted towards targeting downstream convergence points of nociceptive signaling [98]. Lipid mediators, including phosphatidylinositol 4,5-bisphosphate (PIP2), are attractive targets as these molecules are required for signaling downstream of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). Furthermore, PIP2 regulates the activity of various ion channels [80]. Thus, PIP2 sits at a critical convergence point for multiple receptors, ion channels and signaling pathways that promote and maintain chronic pain. Decreasing the amount of PIP2 in neurons was recently shown to attenuate pronociceptive signaling and could provide a novel approach for treating pain. Here, we review the lipid kinases that are known to regulate pain signaling and sensitization and speculate on which additional lipid kinases might regulate signaling in nociceptive neurons

The C-type natriuretic peptide induces thermal hyperalgesia through a noncanonical Gβγ-dependent modulation of TRPV1 channel

Natriuretic peptides (NPs) control natriuresis and normalize changes in blood pressure. Recent studies suggest that NPs are also involved in the regulation of pain sensitivity, although the underlying mechanisms remain largely unknown. Many biological effects of NPs are mediated by guanylate cyclase (GC)-coupled NP receptors, NPR-A and NPR-B, whereas the third NP receptor, NPR-C, lacks the GC kinase domain and acts as the NP clearance receptor. In addition, NPR-C can couple to specific Gα(i)-βγ-mediated intracellular signaling cascades in numerous cell types. We found that NPR-C is co-expressed in TRPV1-expressing mouse DRG neurons. NPR-C can be co-immunoprecipitated with Gα(i), and CNP treatment induced translocation of PKCε to the plasma membrane of these neurons, which was inhibited by pertussis toxin pre-treatment. Application of CNP potentiated capsaicin- and proton-activated TRPV1 currents in cultured mouse DRG neurons, and increased neuronal firing frequency, an effect that was absent in DRG neurons from TRPV1(−/−) mice. CNP-induced sensitization of TRPV1 activity was attenuated by pre-treatment of DRG neurons with the specific inhibitors of Gβγ, PLCβ or PKC, but not of PKA, and was abolished by mutations at two PKC phosphorylation sites in TRPV1. Further, CNP injection into mouse hind paw led to the development of thermal hyperalgesia that was attenuated by administration of specific inhibitors of Gβγ or TRPV1, and was also absent in TRPV1(−/−) mice. Thus, our work identifies the Gβγ-PLCβ-PKC-dependent potentiation of TRPV1 as a novel signaling cascade recruited by CNP in mouse DRG neurons that can lead to enhanced nociceptor excitability and thermal hypersensitivity

The Lipid Kinase PIP5K1C Regulates Pain Signaling and Sensitization

SummaryNumerous pain-producing (pronociceptive) receptors signal via phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. However, it is currently unknown which lipid kinases generate PIP2 in nociceptive dorsal root ganglia (DRG) neurons and if these kinases regulate pronociceptive receptor signaling. Here, we found that phosphatidylinositol 4-phosphate 5 kinase type 1C (PIP5K1C) is expressed at higher levels than any other PIP5K and, based on experiments with Pip5k1c+/− mice, generates at least half of all PIP2 in DRG neurons. Additionally, Pip5k1c haploinsufficiency reduces pronociceptive receptor signaling and TRPV1 sensitization in DRG neurons as well as thermal and mechanical hypersensitivity in mouse models of chronic pain. We identified a small molecule inhibitor of PIP5K1C (UNC3230) in a high-throughput screen. UNC3230 lowered PIP2 levels in DRG neurons and attenuated hypersensitivity when administered intrathecally or into the hindpaw. Our studies reveal that PIP5K1C regulates PIP2-dependent nociceptive signaling and suggest that PIP5K1C is a therapeutic target for chronic pain

Chemokine co-receptor CCR5/CXCR4-dependent modulation of Kv2.1 channel confers acute neuroprotection to HIV-1 glycoprotein gp120 exposure.

Infection with human immunodeficiency virus-1 (HIV-1) within the brain has long been known to be associated with neurodegeneration and neurocognitive disorder (referred as HAND), a condition characterized in its early stages by declining cognitive function and behavioral disturbances. Mechanistically, the HIV-1 coat glycoprotein 120 (gp120) has been suggested to be a critical factor inducing apoptotic cell death in neurons via the activation of p38 mitogen-activated protein kinase (MAPK), upon chronic exposure to the virus. Here we show that acute exposure of neurons to HIV-1 gp120 elicits a homeostatic response, which provides protection against non-apoptotic cell death, involving the major somatodendritic voltage-gated K⁺ (Kv) channel Kv2.1 as the key mediator. The Kv2.1 channel has recently been shown to provide homeostatic control of neuronal excitability under conditions of seizures, ischemia and neuromodulation/neuroinflammation. Following acute exposure to gp120, cultured rat hippocampal neurons show rapid dephosphorylation of the Kv2.1 protein, which ultimately leads to changes in specific sub-cellular localization and voltage-dependent channel activation properties of Kv2.1. Such modifications in Kv2.1 are dependent on the activation of the chemokine co-receptors CCR5 and CXCR4, and subsequent activation of the protein phosphatase calcineurin. This leads to the overall suppression of neuronal excitability and provides neurons with a homeostatic protective mechanism. Specific blockade of calcineurin and Kv2.1 channel activity led to significant enhancement of non-apoptotic neuronal death upon acute gp120 treatment. These observations shed new light on the intrinsic homeostatic mechanisms of neuronal resilience during the acute stages of neuro-HIV infections

Acute gp120 treatment provides neuroprotection via calcineurin-dependent dephosphorylation of Kv2.1.

<p><b><i>A</i></b>, Representative images of MAP2-positive cultured rat hippocampal neurons (green) labeled with ethidium homodimer (EthD-1), which is an indicator of dead or dying cells. Under control and gp120 treatment (10 nM, 30 min) conditions (top row), the majority of MAP2-positive cells were EthD-1 negative. However, when gp120 was applied in the presence of the calcineurin inhibitor FK506 (10 µM) or Kv2.1-blocking toxin ScTx-1 (100 nM; bottom row), MAP2-positive cells with EthD-1-positive nuclei became more prevalent. <b><i>B</i></b>, Quantification of (EthD-1)-positive neurons (MAP2-positive) following different drug treatment conditions as detailed in the bottom of the graph. Acute gp120 (10 nM, 30 min), only upon co-application with FK506 (10 µM) or ScTx-1 (100 nM) led to a significant increase in the extent of neuronal death. No change in the extent of neuronal death was observed upon co-application of gp120 either with CXCR4 inhibitor WZ811 (WZ811) or CCR5 inhibitor Maraviroc (100 nM) or p38 MAPK inhibitor SB203580 (10 µM). <b><i>C</i></b>, Quantification of cleaved-caspase-3 (c-C3)-positive neurons (MAP2-positive) as a measure of cells undergoing apoptotic death, following different drug treatment conditions as detailed in the bottom of the graph. None of the treatment conditions led to a significant increase in the number of c-C3-positive neurons. Treatment of neurons with the inhibitors alone did not alter the percentage of EthD-1-positive (<b><i>B</i></b>) and c-C3-positive neurons (<b><i>C</i></b>). For both the panels data are presented as mean ± SEM. n = 1006 (control/untreated), 909 (gp120 alone), 684 (WZ811+gp120), 665 (Maraviroc+gp120), 655 (SB230580+gp120), 886 (FK506+gp120), 839 (ScTx-1+gp120), 960 (WZ811 alone), 669 (Maraviroc alone), 1023 (SB203580 alone), 1134 (FK506 alone), and 681 (ScTx-1 alone) treatment groups, obtained from ≥4 batches of cultured rat hippocampal neurons. **p<0.01 and ***p<0.001 indicate significantly different in comparison to control/untreated conditions; ^# p<0.05 and ^{# # #} p<0.001 indicate significantly different in comparison to gp120-treatment conditions, one way ANOVA with post-hoc Bonferroni’s correction).</p

Anabolic Factors and Myokines Improve Differentiation of Human Embryonic Stem Cell Derived Skeletal Muscle Cells

International audienceSkeletal muscle weakness is linked to many adverse health outcomes. Current research to identify new drugs has often been inconclusive due to lack of adequate cellular models. We previously developed a scalable monolayer system to differentiate human embryonic stem cells (hESCs) into mature skeletal muscle cells (SkMCs) within 26 days without cell sorting or genetic manipulation. Here, building on our previous work, we show that differentiation and fusion of myotubes can be further enhanced using the anabolic factors testosterone (T) and follistatin (F) in combination with a cocktail of myokines (C). Importantly, combined TFC treatment significantly enhanced both the hESC-SkMC fusion index and the expression levels of various skeletal muscle markers, including the motor protein myosin heavy chain (MyHC). Transcriptomic and proteomic analysis revealed oxidative phosphorylation as the most up-regulated pathway, and a significantly higher level of ATP and increased mitochondrial mass were also observed in TFC-treated hESC-SkMCs, suggesting enhanced energy metabolism is coupled with improved muscle differentiation. This cellular model will be a powerful tool for studying in vitro myogenesis and for drug discovery pertaining to further enhancing muscle development or treating muscle diseases

Acute HIV-1 gp120 induces calcium (Ca²⁺) flux and dephosphorylation of Kv2.1 in hippocampal neurons.

<p><b><i>A</i></b>, Representative traces of ratiometric Ca²⁺ imaging in cultured rat hippocampal neurons using Fura-2. In the left panel, HH buffer (wash) was perfused over the coverslip, prior to a pulse of 50 mM KCl (K50) to demonstrate depolarization-induced Ca²⁺ influx in each neuron. Following exposure to 1 nM gp120 (center panel), neurons demonstrate transient or sustained changes in the fluorescence ratio (340: 380 nm), an indicator of increased intracellular Ca²⁺. This effect was partially washed out upon washing with HH buffer. With 10 nM gp120 (right panel), the magnitude of Ca²⁺ influx is increased, such that several neurons show sustained elevations in [Ca²⁺]_i that persist for minutes after gp120 perfusion has ended. <b><i>B</i></b>, Immunoblot analysis of Kv2.1 phosphorylation levels upon acute gp120 treatment (1 and 10 nM, 30 min) of cultured rat hippocampal neurons, showing increased electrophoretic mobility of Kv2.1 immunoreactive bands in a dose-dependent manner. This apparent decrease in molecular weight can be attributed solely to channel dephosphorylation, since alkaline phosphatase (AP) treatment of samples (shown in <b><i>C</i></b>) decreases the molecular weight of the channel protein to the same, minimal level as predicted from the deduced amino acid sequence of Kv2.1. Numbers on the left in panels <b><i>B</i></b> & <b><i>C</i></b> denote the approximate molecular weight. <b><i>D</i></b>, Quantification of the extent of Kv2.1 phosphorylation upon gp120 exposure as shown in panels <b><i>B</i></b> & <b><i>C</i></b>). Densitometric analysis of Kv2.1 immunoreactive band intensities at molecular weight range of ~90 to ~150 kDa (see methods for details), showing phospho-Kv2.1 with peak intensity at ~130 kDa and dephospho-Kv2.1 with peak intensity at ~100 kDa. Data are presented as mean (n = 4-5 for each group).</p