Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity

Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary1. Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da2,3. Using genome-wide linkage analyses, we discovered an association between nerve-injury–induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7. Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain

Identification of Genes for Spontaneous Neuropathic Pain in Mice: Whole Genome and Candidate Gene Approaches

Chronic neuropathic pain (NP) affects many people worldwide; causing suffering that is difficult to treat, incurable and not preventable. There is growing hope that pain genetics may identify novel treatment targets. In this dissertation we report on candidate NP genes using a mouse NP model produced by hindpaw denervation. Previous research showed that inbred A/J (A-mice) but not C57BL6/J (B-mice) express highly variable levels of self-mutilation of the denervated hindpaw (`autotomy', a behaviour related to NP). This suggested that genetic and environmental factors interact (GXE) in controlling this variance. Using this NP model in recombinant inbred mice, a region on chromosome-15 (`Pain1') was identified as harbouring autotomy gene(s). Here we report that Csf2rb1, a gene encoding the colony stimulating factor-2Beta1 common receptor of GM-CSF (granulocyte-macrophage colony stimulating factor), and interleukins 3 and 5, is a candidate autotomy gene in Pain1. Up-regulation in Csf2rb1 expression levels in the lumbar spinal cord correlated autotomy levels in denervated A and B mice vs. their na誰ve or sham groups. Csf2rb1-expressing cells were labelled immunohistologically in several CNS structures known to process pain inputs, including spinal dorsal horn, central canal, and select spinal white matter regions, hippocampal dentate gyrus, ventricle linings and periventricular and arcuate hypothalamus nuclei. CSF2RB1 protein levels were increased in spinal cord and brain of denervated A mice expressing autotomy vs. non-autotomizing A and B mice, and vs. their control groups (na誰ve and sham A and B mice). Based on cyto-morphology and co-localization with Vimentin, but not GFAP (astrocytes), OX42 (microglia), NeuN (neurons), MAP2 (neurons), and NG2 (oligodendrocytes) markers, Csf2rb1-expressing cells were identified as ependymal cells/radial glia/tanycytes. Previous studies showed that C3H/HeN mice express significantly more pain behaviour than C3H/HeJ mice in several models. This contrast has been attributed to a mutation in Tlr4 encoding Toll-like receptor-4 in C3H/HeJ mice. We show here that denervated C3H/HeN mice express higher autotomy levels than C3H/HeJ mice. Spinal Csf2rb1 expression levels increased significantly post-denervation in C3H/HeN but not C3H/HeJ mice. Thus, we propose that spontaneous NP behaviour in mice is associated with up-regulated Csf2rb1 and CSF2RB1 levels in the CNS and associated with TLR4 signalling.Ph.D

Susceptibility to chronic pain following nerve injury is genetically affected by CACNG2

Chronic neuropathic pain is affected by specifics of the precipitating neural pathology, psychosocial factors, and by genetic predisposition. Little is known about the identity of predisposing genes. Using an integrative approach, we discovered that CACNG2 significantly affects susceptibility to chronic pain following nerve injury. CACNG2 encodes for stargazin, a protein intimately involved in the trafficking of glutamatergic AMPA receptors. The protein might also be a Ca2+ channel subunit. CACNG2 has previously been implicated in epilepsy. Initially, using two fine-mapping strategies in a mouse model (recombinant progeny testing [RPT] and recombinant inbred segregation test [RIST]), we mapped a pain-related quantitative trait locus (QTL) (Pain1) into a 4.2-Mb interval on chromosome 15. This interval includes 155 genes. Subsequently, bioinformatics and whole-genome microarray expression analysis were used to narrow the list of candidates and ultimately to pinpoint Cacng2 as a likely candidate. Analysis of stargazer mice, a Cacng2 hypomorphic mutant, provided electrophysiological and behavioral evidence for the gene's functional role in pain processing. Finally, we showed that human CACNG2 polymorphisms are associated with chronic pain in a cohort of cancer patients who underwent breast surgery. Our findings provide novel information on the genetic basis of neuropathic pain and new insights into pain physiology that may ultimately enable better treatments

Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity

Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary(1). Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da(2,3). Using genome-wide linkage analyses, we discovered an association between nerve-injury–induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7. Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain