Final Report Summary - PAINEURONS (Functional significance of nociceptive primary sensory neurons diversity)
The main objectives of my ERC grant were towfold: expanding our understating of the molecular mechanisms that underlie primary sensory neurons diversity and contribute to understanding the functional significance of this neuronal diversity.
Primary sensory neurons can be distinguished by many morphological, functional, anatomical and few molecular characteristics. Along with extensive morphological and electrophysiological characterizations, the last two decades, provided compelling evidence describing the molecular identities of these primary sensory neuronal subtypes. Deciphering how these neuronal subpopulations of nociceptive neurons are molecularly specified and functionally diversified will greatly expand our understanding of the pain biology.
Unlike anatomical, morphological or electrophysiological properties, molecular differences can be genetically manipulated providing a powerful tool to get new insights into the functional relevance of nociceptive neurons diversity. We designed a strategy that allowed us to expand the molecular characterization of the nociceptive system by identifying new factors expressed in specific subsets of DRG neurons (Legha et al., 2010). This work allowed expanding the repertoire of genes selectively expressed in discrete subpopulations of nociceptive neurons that is currently used as a resource for the sensory biology community. Out of our several characterized genes, two were selected for further studies: a new EF hand and PHD-containing protein (now named GINIP) and a CC-chemokine-like protein TAFA4.
For each of these newly identified genes, we applied a strategy that allowed us to:
a- Precisely map the subpopulations of neurons expressing each of these candidate genes
b- Launch a yeast two hybrid essay to identify protein partners that could illuminate the functions of GINIP and TAFA4 novel proteins.
c- Generate sophisticated mouse models in which each candidate gene will be inactivated by knocking-in a series of cassettes. A two loxP-flanked first cassette expressing the fluorescent protein Venus and a second cassette that will drive the expression an inducible “killer molecule” hDTR (human diphtheria toxin receptor) in a tissue specific manner.
1- Summary related to GINIP work: The above mentioned strategy was very successful for the ginip gene. Detailed on the expression pattern, identification of GINIP partners and the functional role of GINIP in sensory physiology can be found in (Gaillard et al., Neuron. 2014, October 1;84(1):123-36). Ranked as highly interesting achievement by the faculty of 1000
- We found that GINIP marks two functionally distinct subpopulations of nonpeptidergic C-fibers: the cutaneous free nerve ending MRGPRD-expressing known to transmit high threshold mechanical stimuli (HTMRs) and the C- low threshold mechanorecptors (C-LTMRs) that exclusively innervate the hair follicles.
- Using yeast Two Hybrid system in combination with biochemistry, we showed that GINIP interacts specifically with the active form of Gαi proteins.
- GINIP plays an important role in modulating injury-induced mechanical pain, through tight modulation of synaptic activity in spinal cord laminae II interneurons.
We also showed that
- GINIP is dispensable for development and maturation of GINIP+ neurons
- GINIP is dispensable for temperature sensation
- GINIP is dispensable for acute mechanical pain
The versatility of GINIP mouse model was used in a second story aimed at deciphering the functional specialization of GINIP-expressing neurons. As this mouse model allows selective ablation of GINIP-expressing neurons in a living animal, we succeeded to show that GINIP-expressing neurons were:
- Dispensable for temperature sensation. That these neurons were required for sensing and transducing formalin-evoked pain, and participate to some extent to sensing and transducing acute and injury-induced mechanical pain. This work will be ready for submission in the next few months.
The versatility of GINIP mouse model was used in a third story in which we combined the genetic labeling of GINIP-expressing, FACS sorting of well-defined subpopulations of cutaneous sensory neurons and RNA deep sequencing. This work describes the identification of the transcriptional signatures that confer the functional specialization of the free nerve endings C-HTMRs and hair follicles-innervating C-LTMRs. This work also demonstrated that C-LTMRs share common molecular signatures with the Aβ and A| LTMRs (Please see future plans). We also provide two expression libraries describing the expression profiles of over 100 genes in DRG neurons and functionally validated the specific expression of a T-type channel in C-LTMRs but not in C-HTMRs. Detailed information can be found in (Reynders et al., Cell Reports 2015) and the technical issues are described in (Reynders and Moqrich, Genomics data 2015).
2- Summary related to TAFA4 work: Delfini M.C* Mantilleri A* et al., Cell Reports, 2013 Oct
31;5(2):378-88. Ranked as a significant achievement by the faculty of 1000.
The second candidate gene that we studied for the last four years encodes a chemokine-like protein named TAFA4. TAFA4 belong to a new family composed of 5 members. Using the same strategy we applied for GINIP we found that TAFA4 is specifically expressed in a particular subpopulation of DRG neurons called C-Low threshold mechanoreceptors or C-LTMRs. Having no good antibody for TAFA4 we used a series of double in situ hybridization or single in situ hybridization followed by immunolabeling. We found that TAFA4 is expressed in a small population of Ret+ nonpeptidergic neurons. We also found that TAFA4 is co-expressed with the tyrosine hydroxylase and the VGLUT3; two molecular markers of C-LTMRs. Attempts to apply our versatile construct to tafa4 locus were unsuccessful. As this project involved the work of a PhD student (Annabelle Mantilleri), we decided to generate a simple knock-in mouse model expressing the fluorescent marker Venus from tafa4 locus. TAFA4venus mice were extremely valuable as they allowed us to decipher the molecular and electrophysiological properties of TAFA4-expressing neurons, to identify TAFA4 as a strong modulator of injury-induced and to obtain a patent on TAFA4 as a powerful analgesic drug. n°EP14723045.2 filed the 06/05/2014 and entitled “Tafa4 compounds and uses thereof for treating pain”. This patent has been given PCT international extension PCT/EP2014/059247 filed the 06/05/2014.
Thanks to the work by Reynders et al., Cell Reports, 2015), we succeeded to identify few genes whose expression was restricted to C-LTMRs. Such genes are valuable tools to design new strategies aimed at addressing important biological questions related to C-LTMRs’ biology, including their in vivo functional specialization and their circuitry. Indeed, the recent work from our team consolidated the idea of the dual functions of C-LTMRs in sensing gentle touch under normal conditions and modulating pain sensitivity under pathology (Delfini et al., 2013). However, the cellular and functional organization of spinal networks enabling C-LTMRs to mediate both pleasant and noxious touch remains totally unexplored. To solve this issue, we decided to generate new versatile mice models by taking advantage of our newly identified C-LTMRs’ enriched genes. bhlh9a (a transcription factor) and gm7271 (a non-coding RNA) genes have been chosen for this purpose. The locus of each of these genes will be genetically manipulated (using homologous recombination in embryonic stem cells) to allow the expression of a series of molecules, including a fluorescent transynaptic marker; the wheat germ agglutinin WGA, the light activated cationic channel Channelrhodopsin 2 (ChRh2) and the human diphtheria toxin receptor. These reporter molecules will be expressed specifically in C-LTMRs. In addition to addressing these important biological questions, each mouse model will be used to study the functional role of bhlh9a and gm7271 in C-LTMRs development and function.
Also thanks to the work by (Reynders et al., Cell Reports, 2015) we identified a Myosin protein highly enriched in a subset of primary sensory neurons that sense gentle touch. Mice lacking this myosin protein developed a long lasting and irreversible inflammatory, neuropathic and postoperative pain chronic pain. Most importantly, mice lacking one copy of this gene developed the same hypersensitivity phenotype as the homozygous mice, suggesting that this myosin protein plays a critical role in the modulation of the molecular and cellular events that underlie the transition from acute to chronic pain under pathological conditions. Furthermore, this finding also suggests that a loss-of-function mutation in this gene might represent the genetic risk factor that predisposes a subpopulation of human patients to develop Chronic Post-Surgical Pain (CPSP).
In conclusion, thr ERC grant not only allowed us to extend our understanding of primary sensory neurons’ diversity and function but also opened new avenues of investigation aimed at understanding the circuitry of C-LTMRs and to provide mechanistic understanding of chronic pain establishment and perpetuation.
Primary sensory neurons can be distinguished by many morphological, functional, anatomical and few molecular characteristics. Along with extensive morphological and electrophysiological characterizations, the last two decades, provided compelling evidence describing the molecular identities of these primary sensory neuronal subtypes. Deciphering how these neuronal subpopulations of nociceptive neurons are molecularly specified and functionally diversified will greatly expand our understanding of the pain biology.
Unlike anatomical, morphological or electrophysiological properties, molecular differences can be genetically manipulated providing a powerful tool to get new insights into the functional relevance of nociceptive neurons diversity. We designed a strategy that allowed us to expand the molecular characterization of the nociceptive system by identifying new factors expressed in specific subsets of DRG neurons (Legha et al., 2010). This work allowed expanding the repertoire of genes selectively expressed in discrete subpopulations of nociceptive neurons that is currently used as a resource for the sensory biology community. Out of our several characterized genes, two were selected for further studies: a new EF hand and PHD-containing protein (now named GINIP) and a CC-chemokine-like protein TAFA4.
For each of these newly identified genes, we applied a strategy that allowed us to:
a- Precisely map the subpopulations of neurons expressing each of these candidate genes
b- Launch a yeast two hybrid essay to identify protein partners that could illuminate the functions of GINIP and TAFA4 novel proteins.
c- Generate sophisticated mouse models in which each candidate gene will be inactivated by knocking-in a series of cassettes. A two loxP-flanked first cassette expressing the fluorescent protein Venus and a second cassette that will drive the expression an inducible “killer molecule” hDTR (human diphtheria toxin receptor) in a tissue specific manner.
1- Summary related to GINIP work: The above mentioned strategy was very successful for the ginip gene. Detailed on the expression pattern, identification of GINIP partners and the functional role of GINIP in sensory physiology can be found in (Gaillard et al., Neuron. 2014, October 1;84(1):123-36). Ranked as highly interesting achievement by the faculty of 1000
- We found that GINIP marks two functionally distinct subpopulations of nonpeptidergic C-fibers: the cutaneous free nerve ending MRGPRD-expressing known to transmit high threshold mechanical stimuli (HTMRs) and the C- low threshold mechanorecptors (C-LTMRs) that exclusively innervate the hair follicles.
- Using yeast Two Hybrid system in combination with biochemistry, we showed that GINIP interacts specifically with the active form of Gαi proteins.
- GINIP plays an important role in modulating injury-induced mechanical pain, through tight modulation of synaptic activity in spinal cord laminae II interneurons.
We also showed that
- GINIP is dispensable for development and maturation of GINIP+ neurons
- GINIP is dispensable for temperature sensation
- GINIP is dispensable for acute mechanical pain
The versatility of GINIP mouse model was used in a second story aimed at deciphering the functional specialization of GINIP-expressing neurons. As this mouse model allows selective ablation of GINIP-expressing neurons in a living animal, we succeeded to show that GINIP-expressing neurons were:
- Dispensable for temperature sensation. That these neurons were required for sensing and transducing formalin-evoked pain, and participate to some extent to sensing and transducing acute and injury-induced mechanical pain. This work will be ready for submission in the next few months.
The versatility of GINIP mouse model was used in a third story in which we combined the genetic labeling of GINIP-expressing, FACS sorting of well-defined subpopulations of cutaneous sensory neurons and RNA deep sequencing. This work describes the identification of the transcriptional signatures that confer the functional specialization of the free nerve endings C-HTMRs and hair follicles-innervating C-LTMRs. This work also demonstrated that C-LTMRs share common molecular signatures with the Aβ and A| LTMRs (Please see future plans). We also provide two expression libraries describing the expression profiles of over 100 genes in DRG neurons and functionally validated the specific expression of a T-type channel in C-LTMRs but not in C-HTMRs. Detailed information can be found in (Reynders et al., Cell Reports 2015) and the technical issues are described in (Reynders and Moqrich, Genomics data 2015).
2- Summary related to TAFA4 work: Delfini M.C* Mantilleri A* et al., Cell Reports, 2013 Oct
31;5(2):378-88. Ranked as a significant achievement by the faculty of 1000.
The second candidate gene that we studied for the last four years encodes a chemokine-like protein named TAFA4. TAFA4 belong to a new family composed of 5 members. Using the same strategy we applied for GINIP we found that TAFA4 is specifically expressed in a particular subpopulation of DRG neurons called C-Low threshold mechanoreceptors or C-LTMRs. Having no good antibody for TAFA4 we used a series of double in situ hybridization or single in situ hybridization followed by immunolabeling. We found that TAFA4 is expressed in a small population of Ret+ nonpeptidergic neurons. We also found that TAFA4 is co-expressed with the tyrosine hydroxylase and the VGLUT3; two molecular markers of C-LTMRs. Attempts to apply our versatile construct to tafa4 locus were unsuccessful. As this project involved the work of a PhD student (Annabelle Mantilleri), we decided to generate a simple knock-in mouse model expressing the fluorescent marker Venus from tafa4 locus. TAFA4venus mice were extremely valuable as they allowed us to decipher the molecular and electrophysiological properties of TAFA4-expressing neurons, to identify TAFA4 as a strong modulator of injury-induced and to obtain a patent on TAFA4 as a powerful analgesic drug. n°EP14723045.2 filed the 06/05/2014 and entitled “Tafa4 compounds and uses thereof for treating pain”. This patent has been given PCT international extension PCT/EP2014/059247 filed the 06/05/2014.
Thanks to the work by Reynders et al., Cell Reports, 2015), we succeeded to identify few genes whose expression was restricted to C-LTMRs. Such genes are valuable tools to design new strategies aimed at addressing important biological questions related to C-LTMRs’ biology, including their in vivo functional specialization and their circuitry. Indeed, the recent work from our team consolidated the idea of the dual functions of C-LTMRs in sensing gentle touch under normal conditions and modulating pain sensitivity under pathology (Delfini et al., 2013). However, the cellular and functional organization of spinal networks enabling C-LTMRs to mediate both pleasant and noxious touch remains totally unexplored. To solve this issue, we decided to generate new versatile mice models by taking advantage of our newly identified C-LTMRs’ enriched genes. bhlh9a (a transcription factor) and gm7271 (a non-coding RNA) genes have been chosen for this purpose. The locus of each of these genes will be genetically manipulated (using homologous recombination in embryonic stem cells) to allow the expression of a series of molecules, including a fluorescent transynaptic marker; the wheat germ agglutinin WGA, the light activated cationic channel Channelrhodopsin 2 (ChRh2) and the human diphtheria toxin receptor. These reporter molecules will be expressed specifically in C-LTMRs. In addition to addressing these important biological questions, each mouse model will be used to study the functional role of bhlh9a and gm7271 in C-LTMRs development and function.
Also thanks to the work by (Reynders et al., Cell Reports, 2015) we identified a Myosin protein highly enriched in a subset of primary sensory neurons that sense gentle touch. Mice lacking this myosin protein developed a long lasting and irreversible inflammatory, neuropathic and postoperative pain chronic pain. Most importantly, mice lacking one copy of this gene developed the same hypersensitivity phenotype as the homozygous mice, suggesting that this myosin protein plays a critical role in the modulation of the molecular and cellular events that underlie the transition from acute to chronic pain under pathological conditions. Furthermore, this finding also suggests that a loss-of-function mutation in this gene might represent the genetic risk factor that predisposes a subpopulation of human patients to develop Chronic Post-Surgical Pain (CPSP).
In conclusion, thr ERC grant not only allowed us to extend our understanding of primary sensory neurons’ diversity and function but also opened new avenues of investigation aimed at understanding the circuitry of C-LTMRs and to provide mechanistic understanding of chronic pain establishment and perpetuation.