Tools for analysis and conditional deletion of subsets of sensory neurons [version 1; peer review: 4 approved]

Background: Somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons. // Methods: A floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Nav1.8Cre and then crossed to CGRPCreER (Calca), ThCreERT2, Tmem45bCre, Tmem233Cre, Ntng1Cre and TrkBCreER (Ntrk2) lines. Pain behavioural assays included Hargreaves’, hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests. // Results: Motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Nav1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model. // Conclusions: Ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders

GPR80/99, proposed to be the P2Y15 receptor activated by adenosine and AMP, is not a P2Y receptor

The orphan receptor GPR80 (also called GPR99) was recently reported to be the P2Y15 receptor activated by AMP and adenosine and coupled to increases in cyclic AMP accumulation and intracellular Ca2+ mobilization (Inbe et al. J Biol Chem 2004; 279: 19790–9[12]). However, the cell line (HEK293) used to carry out those studies endogenously expresses A2A and A2B adenosine receptors as well as multiple P2Y receptors, which complicates the analysis of a potential P2Y receptor. To determine unambiguously whether GPR80 is a P2Y receptor subtype, HA-tagged GPR80 was either stably expressed in CHO cells or transiently expressed in COS-7 and HEK293 cells, and cell surface expression was verified by radioimmunoassay (RIA). COS-7 cells overexpressing GPR80 showed a consistent twofold increase in basal inositol phosphate accumulation. However, neither adenosine nor AMP was capable of promoting accumulation of either cyclic AMP or inositol phosphates in any of the three GPR80-expressing cells. A recent paper (He et al. Nature 2004; 429: 188–93 [15]) reported that GPR80 is a Gq-coupled receptor activated by the citric acid cycle intermediate, α-ketoglutarate. Consistent with this report, α-ketoglutarate promoted inositol phosphate accumulation in CHO and HEK293 cells expressing GPR80, and pretreatment of GPR80-expressing COS-7 cells with glutamate dehydrogenase, which converts α-ketoglutarate to glutamate, decreased basal levels of inositol phosphates. Taken together, these data demonstrate that GPR80 is not activated by adenosine, AMP or other nucleotides, but instead is activated by α-ketoglutarate. Therefore, GPR80 is not a new member of the P2Y receptor family

Long-term (trophic) purinergic signalling: purinoceptors control cell proliferation, differentiation and death

The purinergic signalling system, which uses purines and pyrimidines as chemical transmitters, and purinoceptors as effectors, is deeply rooted in evolution and development and is a pivotal factor in cell communication. The ATP and its derivatives function as a 'danger signal' in the most primitive forms of life. Purinoceptors are extraordinarily widely distributed in all cell types and tissues and they are involved in the regulation of an even more extraordinary number of biological processes. In addition to fast purinergic signalling in neurotransmission, neuromodulation and secretion, there is long-term (trophic) purinergic signalling involving cell proliferation, differentiation, motility and death in the development and regeneration of most systems of the body. In this article, we focus on the latter in the immune/defence system, in stratified epithelia in visceral organs and skin, embryological development, bone formation and resorption, as well as in cancer. Cell Death and Disease (2010) 1, e9; doi:10.1038/cddis.2009.11; published online 14 January 201

Multiple P2Y receptors couple to calcium-dependent, chloride channels in smooth muscle cells of the rat pulmonary artery

BACKGROUND: Uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP) act via P2Y receptors to evoke contraction of rat pulmonary arteries, whilst adenosine 5'-triphosphate (ATP) acts via P2X and P2Y receptors. Pharmacological characterisation of these receptors in intact arteries is complicated by release and extracellular metabolism of nucleotides, so the aim of this study was to characterise the P2Y receptors under conditions that minimise these problems. METHODS: The perforated-patch clamp technique was used to record the Ca(2+)-dependent, Cl(- )current (I(Cl,Ca)) activated by P2Y receptor agonists in acutely dissociated smooth muscle cells of rat small (SPA) and large (LPA) intrapulmonary arteries, held at -50 mV. Contractions to ATP were measured in isolated muscle rings. Data were compared by Student's t test or one way ANOVA. RESULTS: ATP, UTP and UDP (10(-4)M) evoked oscillating, inward currents (peak = 13–727 pA) in 71–93% of cells. The first current was usually the largest and in the SPA the response to ATP was significantly greater than those to UTP or UDP (P < 0.05). Subsequent currents tended to decrease in amplitude, with a variable time-course, to a level that was significantly smaller for ATP (P < 0.05), UTP (P < 0.001) and UDP (P < 0.05) in the SPA. The frequency of oscillations was similar for each agonist (mean≈6–11.min(-1)) and changed little during agonist application. The non-selective P2 receptor antagonist suramin (10(-4)M) abolished currents evoked by ATP in SPA (n = 4) and LPA (n = 4), but pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (10(-4)M), also a non-selective P2 antagonist, had no effect (n = 4, 5 respectively). Currents elicited by UTP (n = 37) or UDP (n = 14) were unaffected by either antagonist. Contractions of SPA evoked by ATP were partially inhibited by PPADS (n = 4) and abolished by suramin (n = 5). Both antagonists abolished the contractions in LPA. CONCLUSION: At least two P2Y subtypes couple to I(Cl,Ca )in smooth muscle cells of rat SPA and LPA, with no apparent regional variation in their distribution. The suramin-sensitive, PPADS-resistant site activated by ATP most resembles the P2Y(11 )receptor. However, the suramin- and PPADS-insensitive receptor activated by UTP and UDP does not correspond to any of the known P2Y subtypes. These receptors likely play a significant role in nucleotide-induced vasoconstriction

Identification of Colorectal Cancer Related Genes with mRMR and Shortest Path in Protein-Protein Interaction Network

One of the most important and challenging problems in biomedicine and genomics is how to identify the disease genes. In this study, we developed a computational method to identify colorectal cancer-related genes based on (i) the gene expression profiles, and (ii) the shortest path analysis of functional protein association networks. The former has been used to select differentially expressed genes as disease genes for quite a long time, while the latter has been widely used to study the mechanism of diseases. With the existing protein-protein interaction data from STRING (Search Tool for the Retrieval of Interacting Genes), a weighted functional protein association network was constructed. By means of the mRMR (Maximum Relevance Minimum Redundancy) approach, six genes were identified that can distinguish the colorectal tumors and normal adjacent colonic tissues from their gene expression profiles. Meanwhile, according to the shortest path approach, we further found an additional 35 genes, of which some have been reported to be relevant to colorectal cancer and some are very likely to be relevant to it. Interestingly, the genes we identified from both the gene expression profiles and the functional protein association network have more cancer genes than the genes identified from the gene expression profiles alone. Besides, these genes also had greater functional similarity with the reported colorectal cancer genes than the genes identified from the gene expression profiles alone. All these indicate that our method as presented in this paper is quite promising. The method may become a useful tool, or at least plays a complementary role to the existing method, for identifying colorectal cancer genes. It has not escaped our notice that the method can be applied to identify the genes of other diseases as well

Novel gene HCS1 is specifically expressed in the giant interneurones of the terrestrial snail

THE terrestrial snail Helix lucorum is a promising model for molecular neurobiology since its central nervous system (CNS) is simple and contains many morphologically and functionally identified large neurones. Among these, the giant interneurones located in pleural and parietal ganglia are especially interesting because they trigger the withdrawal behaviour of the snail and participate in aversive conditioning. Here we describe the identification and characterization of a gene named HCS1 which is preferentially expressed in these interneurones. It encodes a putative protein 100 amino acids long containing an N-terminal hydrophobic leader peptide. No sequences with significant homology to HCS1 were found in the protein (Swiss-Prot) and nucleotide (EMBLbank) data libraries. We suppose that the product of this gene is a secreted protein, presumably a neuropeptide or a growth factor

Putative neuropeptides and an EF-hand motif region are encoded by a novel gene expressed in the four giant interneurons of the terrestrial snail

Nine giant interneurons located in the pleural and parietal ganglia of the terrestrial snail Helix lucorum L. were reported to be a key element in the network controlling withdrawal behaviour of the animal. Using a combination of complementary DNA subtraction cloning and differential screening approaches we have isolated a novel gene named HCS2 which is expressed predominantly in a subset of these interneurons. The predicted amino acid sequence of the HCS2 protein contains at the N-terminus a hydrophobic leader sequence and four putative neuropeptides, and at the C-terminus a perfect match to the consensus motif of the EF-hand family of the Ca2+-binding proteins. All four predicted neuropeptides bear a C-terminal signature sequence Tyr-Pro-Arg-X (where X is Ile, Leu, Val or Pro), and three of them are likely to be amidated. Physiological action of three synthetic peptides corresponding to the predicted mature HCS2 peptides mimics fairly well the described action of parietal interneurons on follower motoneurons controlling pneumostome closure. In situ hybridization experiments demonstrated that the HCS2 gene is selectively expressed in the four parietal giant interneurons, as well as in several small unidentified neurons. The onset of the HCS2 transcription during embryogenesis coincides temporally with the time-point when the first withdrawal responses of the embryo to tactile stimulation appear.We propose that the HCS2 gene encodes a hybrid precursor protein whose processed products act as neuromodulators or neurotransmitters mediating the withdrawal reactions of the snail, and in addition may participate in the calcium regulatory pathways or calcium homeostasis in command neurons

Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors

The efficacy of fast synaptic inhibition is critically dependent on the accumulation of GABAA receptors at inhibitory synapses, a process that remains poorly understood. Here, we examined the dynamics of cell surface GABAA receptors using receptor subunits modified with N-terminal extracellular ecliptic pHluorin reporters. In hippocampal neurons, GABAA receptors incorporating pHluorin-tagged subunits were found to be clustered at synaptic sites and also expressed as diffuse extrasynaptic staining. By combining FRAP (fluorescence recovery after photobleaching) measurements with live imaging of FM4-64-labeled active presynaptic terminals, it was evident that clustered synaptic receptors exhibit significantly lower rates of mobility at the cell surface compared with their extrasynaptic counterparts. To examine the basis of this confinement, we used RNAi to inhibit the expression of gephyrin, a protein shown to regulate the accumulation of GABAA receptors at synaptic sites. However, whether gephyrin acts to control the actual formation of receptor clusters, their stability, or is simply a global regulator of receptor cell surface number remains unknown. Inhibiting gephyrin expression did not modify the total number of GABAA receptors expressed on the neuronal cell surface but significantly decreased the number of receptor clusters. Live imaging revealed that clusters that formed in the absence of gephyrin were significantly more mobile compared with those in control neurons. Together, our results demonstrate that synaptic GABAA receptors have lower levels of lateral mobility compared with their extrasynaptic counterparts, and suggest a specific role for gephyrin in reducing the diffusion of GABAA receptors, facilitating their accumulation at inhibitory synapses

Differential splicing creates a diversity of transcripts from a neurospecific developmentally regulated gene encoding a protein with new zinc-finger motifs

We have cloned a novel neurospecific gene, named neuro-d4, by differential screening a rat cerebral cortex cDNA library. Northern blot hybridization showed that neuro-d4 expression is restricted to neuronal tissues both in newborn and adult animals. The level of neuro-d4 mRNA in the rat central nervous system is high during the later stages of embryonic development and gradually decreases during the postnatal period. In situ hybridization suggests that the gene transcripts are localized in neuronal cell bodies. Nucleotide sequences of overlapped cDNA clones and all 12 exons in genomic clone were determined. The deduced protein has consensus sequences for a nuclear localization signal, a Krüppel-type zinc-finger and a new type of cysteine/histidine-rich motif resembling zinc-fingers. Several differential splicing variants were found, each of which influences the structure of the encoded protein