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

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

Expression of tripartite motif-containing protein 28 in primary breast carcinoma predicts metastasis and is involved in the stemness, chemoresistance, and tumor growth

The prediction of who develops metastasis has been the most difficult aspect in the management of breast cancer patients. The lymph node metastasis has been the most useful predictor of prognosis and patient management. However, a good proportion of patients with lymph node positivity remain disease free for 5 years or more, while about a third of those who were lymph node negative develop distant metastasis within the same period. This warrants a robust biomarker(s), preferably gene expression based. In order to elucidate gene-based biomarkers for prognosis of breast cancers, gene expression profiling of primary tumors and follow-up for over 5 years has been performed. The analysis revealed a network of genes centered around the tripartite motif-containing protein 28 as an important indicator of disease progression. Short hairpin RNA-mediated knockdown of tripartite motif-containing protein 28 in breast cancer cells revealed a decreased expression of epithelial-to-mesenchymal transition markers and increased expression of epithelial markers, decreased migration and invasion, and increased chemosensitivity to doxorubicin, 5-fluorouracil, and methotrexate. Furthermore, knockdown of tripartite motif-containing protein 28 resulted in the decrease of stemness as revealed by sphere formation assay as well as decreased expression of CD44 and Bmi1. Moreover, tripartite motif-containing protein 28 knockdown significantly reduced the tumor size and lung metastasis in orthotopic tumor xenograft assay in immunocompromised mice. The tumor size was further reduced when these mice were treated with doxorubicin. These data provide evidence for tripartite motif-containing protein 28 as a biomarker and a potential therapeutic target for breast cancer

PI3Kγ acts in DRG neurons as a negative regulator of thermal and TRPV1 responses.

<p>(A) Representative temperature response ramps and Arrhenius plots for heat-activated currents measured in single DRG neurons isolated from wild type (WT) and <i>PI3Kγ</i> mutant (KO) mice. For temperature response ramps, red lines depict temperature ramps and black lines depict inward current. (B) Q10 as a measure of the rate of inward current changes in response to temperature. n = 37 for isolated WT; n = 9 for <i>PI3Kγ</i> KO DRG neurons. (C,D) Capsaicin sensitivity of DRG neurons isolated from WT and <i>PI3Kγ</i> KO mice. (C) Representative capsaicin responses from a single DRG neuron. (D) Dose-response curves to different concentrations of capsaicin. The capsaicin EC50 is indicated. Numbers indicate numbers of single neurons tested with the indicated capsaicin doses at the respective data points. Electrophysiology data was generated by single neuron patch clamping. Data are presented as mean +/− sem. ** p<0.01, *** p<0.001 (Mann-Whitney u-test).</p

PI5Kα signaling controls thermal and capsaicin nociception <i>in vivo</i>.

<p>(A) Thermal pain thresholds of wild type (WT) and <i>PIP5Kα</i>^−/− (KO) littermates in response to radiant heat (Hargreaves; n = 6 for WT; n = 6 for KO mice). (B) <i>PIP5Kα</i> KO mice also show enhanced thermal sensitivity in the hot plate assay (n = 12 for WT; n = 12 for KO mice) and (C) an exaggerated capsaicin-evoked behavioral response (n = 12 for WT; n = 9 for KO mice). (D) PIP5Kα KO mice exhibited normal mechanical pain (force threshold latency) as assessed by the von Frey test (n = 9 for WT; n = 9 for KO mice). All data are presented as mean +/− sem. *<i>p</i><0.05; **<i>p</i><0.01 (t-test).</p

A global network map of thermal nociception.

<p>The systems network includes data from significantly enriched <i>Drosophila</i> KEGG pathways and GO processes, mouse and human KEGG pathways and C2 gene sets. Pathways, processes and gene sets that share a role in a biological process were pooled into functional classes while the underlying genes that constitute them are depicted with a connection to their respective functional class. Functional classes (gold), genes representing direct hits with a thermal nociception phenotype (red), their first degree binding partners (green), and developmental lethal genes (blue) represent the nodes in the network. Only select KEGG pathways, biological processes and C2 gene sets were used to build systems map. For the entire list of individual pathways, gene sets, and processes see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003071#pgen.1003071.s010" target="_blank">Tables S5</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003071#pgen.1003071.s011" target="_blank">S6</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003071#pgen.1003071.s012" target="_blank">S7</a>.</p

Multi-gene testing in neurological disorders showed an improved diagnostic yield: data from over 1000 Indian patients

Background Neurological disorders are clinically heterogeneous group of disorders and are major causes of disability and death. Several of these disorders are caused due to genetic aberration. A precise and confirmatory diagnosis in the patients in a timely manner is essential for appropriate therapeutic and management strategies. Due to the complexity of the clinical presentations across various neurological disorders, arriving at an accurate diagnosis remains a challenge. Methods We sequenced 1012 unrelated patients from India with suspected neurological disorders, using TruSight One panel. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. Results We were able to detect mutations in 197 genes in 405 (40%) cases and 178 mutations were novel. The highest diagnostic rate was observed among patients with muscular dystrophy (64%) followed by leukodystrophy and ataxia (43%, each). In our cohort, 26% of the patients who received definitive diagnosis were primarily referred with complex neurological phenotypes with no suggestive diagnosis. In terms of mutations types, 62.8% were truncating and in addition, 13.4% were structural variants, which are also likely to cause loss of function. Conclusion In our study, we observed an improved performance of multi-gene panel testing, with an overall diagnostic yield of 40%. Furthermore, we show that NGS (next-generation sequencing)-based testing is comprehensive and can detect all types of variants including structural variants. It can be considered as a single-platform genetic test for neurological disorders that can provide a swift and definitive diagnosis in a cost-effective manner