Construction of a Global Pain Systems Network Highlights Phospholipid Signaling as a Regulator of Heat Nociception

The ability to perceive noxious stimuli is critical for an animal's survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species. © 2012 Neely et al

Construction of a Global Pain Systems Network Highlights Phospholipid Signaling as a Regulator of Heat Nociception

The ability to perceive noxious stimuli is critical for an animal's survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species

A Genome-wide Drosophila Screen for Heat Nociception Identifies α2δ3 as an Evolutionarily Conserved Pain Gene

Worldwide, acute and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knock-down in Drosophila, we report a global screen for an innate behavior and identify hundreds of novel genes implicated in heat nociception, including the α2δ-family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain evoked signals from the thalamus to higher order pain centers. Intriguingly, in α2δ3 mutant mice thermal pain and tactile stimulation triggered strong cross-activation or synesthesia of brain regions involved in vision, olfaction, and hearing

The GIY-YIG Type Endonuclease Ankyrin Repeat and LEM Domain-Containing Protein 1 (ANKLE1) Is Dispensable for Mouse Hematopoiesis

<div><p>Ankyrin repeat and LEM-domain containing protein 1 (ANKLE1) is a GIY-YIG endonuclease with unknown functions, mainly expressed in mouse hematopoietic tissues. To test its potential role in hematopoiesis we generated <i>Ankle1</i>-deficient mice. <i>Ankle1</i>^Δ/Δ mice are viable without any detectable phenotype in hematopoiesis. Neither hematopoietic progenitor cells, myeloid and lymphoid progenitors, nor B and T cell development in bone marrow, spleen and thymus, are affected in <i>Ankle1</i>^Δ/Δ<i>-</i>mice. Similarly embryonic stress erythropoiesis in liver and adult erythropoiesis in bone marrow and spleen appear normal. To test whether ANKLE1, like the only other known GIY-YIG endonuclease in mammals, SLX1, may contribute to Holliday junction resolution during DNA repair, <i>Ankle1</i>-deficient cells were exposed to various DNA-damage inducing agents. However, lack of <i>Ankle1</i> did not affect cell viability and, unlike depletion of <i>Slx1</i>, <i>Ankle1</i>-deficiency did not increase sister chromatid exchange in Bloom helicase-depleted cells. Altogether, we show that lack of <i>Ankle1</i> does neither affect mouse hematopoiesis nor DNA damage repair in mouse embryonic fibroblasts, indicating a redundant or non-essential function of ANKLE1 in mouse.</p></div

Flow cytometric analysis of erythroid differentiation in <i>Ankle1</i>^Δ/Δ mice.

<p>Cells were labeled with antibodies against Ter119 and CD71 (A) Representative dot plots of fetal liver cells from <i>Ankle1</i>^<i>+/+</i> and <i>Ankle1</i>^Δ/Δ embryos (E 13.5), regions 1–5 (R1–R5) represent five erythroid maturation stages, bar graphs show the percentage of labeled cells in each differentiation stage, n _litter = 3; Representative dot plots of (B) Bone marrow and (C) Splenic cells from an 9–10 months old <i>Ankle1</i>^<i>+/+</i> and an <i>Ankle1</i>^Δ/Δ mouse, regions 1–4 (R1–R4) represent four erythroid maturation stages, bar graphs show the percentage of labeled cells in each differentiation stage, n = 5; error bars: standard deviation, students t test was used for determination of statistical significant differences (p≤0.05).</p

Involvement of ANKLE1 in Holliday junction resolution.

<p>(A) mRNA expression profile of <i>Ankle1</i> and known proteins involved in Holliday junction resolution after double strand break repair in somatic cells, representative end point PCR, <i>Actin</i> and <i>Gapdh</i> serve as loading control; (B) Western blot and qPCR analysis of iMEFs 48 h after transfection with indicated siRNAs; Scatter plot of sister chromatid exchange (SCE) frequencies in (C) Wild-type iMEFs and (D) Wild-type vs. <i>Ankle1</i>^Δ/Δ iMEFs following siRNA treatment, each dot represents one metaphase spread, horizontal bars represent the mean, error bars: standard error of the mean, p values were determined using the students t test, n.s. not significant, * significant (p≤0.05), ** very significant (p≤0.01), *** extremely significant (p≤0.001).</p

Flow cytometric analysis of lymphoid differentiation in 8 weeks old <i>Ankle1</i>^Δ/Δ mice.

<p>(A) Representative dot plots of B cell progenitors (B220⁺ IgM^-) and B cells (B220⁺ IgM⁺) in white blood cells (WBC) from bone marrow of an <i>Ankle1</i>^<i>+/+</i> and an <i>Ankle1</i>^Δ/Δ mouse and relative quantification of the B220⁺ IgM^- and B220⁺ IgM⁺ cells in a bar graph; (B) Representative dot plots of consecutive developmental stages of T cell progenitors in thymus of an <i>Ankle1</i>^<i>+/+</i> and an <i>Ankle1</i>^Δ/Δ mouse and its relative quantification; Relative quantification of (C) B220⁺ and (D) CD4⁺ and CD8⁺ WBCs in spleen; error bars: standard deviation, paired students t test was used for determination of statistical significant differences (p≤0.05), n = 5.</p

Flow cytometric analysis of early hematopoietic development in 9–10 months old <i>Ankle1</i>^Δ/Δ mice.

<p>(A) Representative dot plot of lineage negative bone marrow cells (BMCs) of an <i>Ankle1</i>^<i>+/+</i> and an <i>Ankle1</i>^Δ/Δ mouse and relative quantification of the KLS cells (c-Kit+ and Sca-1+, boxed) depicted in a scatter plot; (B) Representative dot plot of c-Kit+ Lin- IL7Rα- Sca-1- BMCs of an <i>Ankle1</i>^<i>+/+</i> and an <i>Ankle1</i>^Δ/Δ mouse and relative quantification of the boxed, CD16/32^low CD34⁺ common myeloid progenitors (CMPs) in a scatter plot; (C) Representative dot plot of IL7Rα⁺ Lin^- BMC of an <i>Ankle1</i>^<i>+/+</i> and an <i>Ankle1</i>^Δ/Δ mouse and relative quantification of the c-Kit⁺ Sca-1⁺, common lymphoid progenitors (CLPs) depicted in a scatter plot; scatter plots indicate the mean (n = 5), error bars: standard deviation, paired students t test was used for determination of statistical significant differences (p≤0.05).</p