Structural Alterations Reduce Nociception in Mice Carrying the L799P-Gain-of-Function Variant in NaV1.9 Sodium Channel
PNS 2022 Annual Meeting eLibrary. Schmidt A. 04/14/22; 356096; 1243
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Structural Alterations Reduce Nociception in Mice Carrying the L799P-Gain-of-Function Variant in NaV1.9 Sodium Channel
Poster No: 1243
Presenter: Annika Schmidt
Institution: Institute of Human Genetics
Introduction: Pain protects the body from injuries while impaired nociception leads to severe mutilations and tissue damage. SCN11A encodes the voltage-gated sodium ion channel NaV1.9 primarily expressed in nociceptors. The gain-of-function variant Scn11a-L799P (orthologous to p.L811P in humans) leads to overactivity of this channel and disturbed nociceptive transmission. So far, the underlying pathomechanisms of this channelopathy have not been conclusively clarified, i.e., whether the loss of pain perception is due to neurodegeneration or to altered transmission of the corresponding neurons.
Methods: Investigations were performed on heterozygous knock-in mice carrying the Scn11a-L799P variant and compared with wild type specimen. Electron micrographs of peripheral nerves (sciatic and sural) were analyzed regarding axon size distribution and cluster size of unmyelinated nerve fibers. Frozen sections of hindpaw glabrous skin were stained free floating using protein gene product 9.5 (PGP 9.5) antibodies to determine intraepidermal nerve fibre density (IENFD). Expression of collagen type IV alpha 3 chain (Col4a3) was analyzed by quantitative real time PCR (qPCR) as well as immunofluorescence stainings on frozen sections, as this collagen was strongly upregulated in the transcriptomes from dorsal root ganglia (DRGs) of knock-in mice.
Results: Highly elevated Col4a3 expression levels in heterozygous knock-in animals were verified by qPCR as well as immunofluorescence stainings of DRG tissue sections. This matches findings from transcriptome analyses in DRGs. Analyses of axon number and axonal size distribution in peripheral nerves indicated distinct changes within small unmyelinated axons. Preliminary evaluation of IENFD did not show marked differences.
Conclusions: Increased expression of Col4a3 indicates an expanded extracellular matrix production. However, this collagen subtype has previously not been allocated to nervous tissue but points towards compensatory structural alterations in heterozygous animals. A different axon size distribution in peripheral nerves implies additional compensation mechanisms. Further analyses of DRGs by single cell sequencing will provide additional insights in this pain-related channelopathy.
References: Yes
References 1: Leipold, E., et al., A de novo gain-of-function mutation in SCN11A causes loss of pain perception. Nat Genet, 2013. 45(11): p. 1399-404.
References 2: Ebbinghaus M, et al., Gain-of-function mutation in SCN11A causes itch and affects neurogenic inflammation and muscle function in Scn11a+/L799P mice. PLoS One, 2020. 15(8): e0237101.
References 3:
References 4:
Grant Support: This project was funded by grants of the Deutsche Forschungsgemeinschaft (DFG) to Prof. Ingo Kurth.
Keywords: NaV1.9, Scn11a, channelopathy, electron microscopy, Col4a3
Poster No: 1243
Presenter: Annika Schmidt
Institution: Institute of Human Genetics
Introduction: Pain protects the body from injuries while impaired nociception leads to severe mutilations and tissue damage. SCN11A encodes the voltage-gated sodium ion channel NaV1.9 primarily expressed in nociceptors. The gain-of-function variant Scn11a-L799P (orthologous to p.L811P in humans) leads to overactivity of this channel and disturbed nociceptive transmission. So far, the underlying pathomechanisms of this channelopathy have not been conclusively clarified, i.e., whether the loss of pain perception is due to neurodegeneration or to altered transmission of the corresponding neurons.
Methods: Investigations were performed on heterozygous knock-in mice carrying the Scn11a-L799P variant and compared with wild type specimen. Electron micrographs of peripheral nerves (sciatic and sural) were analyzed regarding axon size distribution and cluster size of unmyelinated nerve fibers. Frozen sections of hindpaw glabrous skin were stained free floating using protein gene product 9.5 (PGP 9.5) antibodies to determine intraepidermal nerve fibre density (IENFD). Expression of collagen type IV alpha 3 chain (Col4a3) was analyzed by quantitative real time PCR (qPCR) as well as immunofluorescence stainings on frozen sections, as this collagen was strongly upregulated in the transcriptomes from dorsal root ganglia (DRGs) of knock-in mice.
Results: Highly elevated Col4a3 expression levels in heterozygous knock-in animals were verified by qPCR as well as immunofluorescence stainings of DRG tissue sections. This matches findings from transcriptome analyses in DRGs. Analyses of axon number and axonal size distribution in peripheral nerves indicated distinct changes within small unmyelinated axons. Preliminary evaluation of IENFD did not show marked differences.
Conclusions: Increased expression of Col4a3 indicates an expanded extracellular matrix production. However, this collagen subtype has previously not been allocated to nervous tissue but points towards compensatory structural alterations in heterozygous animals. A different axon size distribution in peripheral nerves implies additional compensation mechanisms. Further analyses of DRGs by single cell sequencing will provide additional insights in this pain-related channelopathy.
References: Yes
References 1: Leipold, E., et al., A de novo gain-of-function mutation in SCN11A causes loss of pain perception. Nat Genet, 2013. 45(11): p. 1399-404.
References 2: Ebbinghaus M, et al., Gain-of-function mutation in SCN11A causes itch and affects neurogenic inflammation and muscle function in Scn11a+/L799P mice. PLoS One, 2020. 15(8): e0237101.
References 3:
References 4:
Grant Support: This project was funded by grants of the Deutsche Forschungsgemeinschaft (DFG) to Prof. Ingo Kurth.
Keywords: NaV1.9, Scn11a, channelopathy, electron microscopy, Col4a3
Structural Alterations Reduce Nociception in Mice Carrying the L799P-Gain-of-Function Variant in NaV1.9 Sodium Channel
Poster No: 1243
Presenter: Annika Schmidt
Institution: Institute of Human Genetics
Introduction: Pain protects the body from injuries while impaired nociception leads to severe mutilations and tissue damage. SCN11A encodes the voltage-gated sodium ion channel NaV1.9 primarily expressed in nociceptors. The gain-of-function variant Scn11a-L799P (orthologous to p.L811P in humans) leads to overactivity of this channel and disturbed nociceptive transmission. So far, the underlying pathomechanisms of this channelopathy have not been conclusively clarified, i.e., whether the loss of pain perception is due to neurodegeneration or to altered transmission of the corresponding neurons.
Methods: Investigations were performed on heterozygous knock-in mice carrying the Scn11a-L799P variant and compared with wild type specimen. Electron micrographs of peripheral nerves (sciatic and sural) were analyzed regarding axon size distribution and cluster size of unmyelinated nerve fibers. Frozen sections of hindpaw glabrous skin were stained free floating using protein gene product 9.5 (PGP 9.5) antibodies to determine intraepidermal nerve fibre density (IENFD). Expression of collagen type IV alpha 3 chain (Col4a3) was analyzed by quantitative real time PCR (qPCR) as well as immunofluorescence stainings on frozen sections, as this collagen was strongly upregulated in the transcriptomes from dorsal root ganglia (DRGs) of knock-in mice.
Results: Highly elevated Col4a3 expression levels in heterozygous knock-in animals were verified by qPCR as well as immunofluorescence stainings of DRG tissue sections. This matches findings from transcriptome analyses in DRGs. Analyses of axon number and axonal size distribution in peripheral nerves indicated distinct changes within small unmyelinated axons. Preliminary evaluation of IENFD did not show marked differences.
Conclusions: Increased expression of Col4a3 indicates an expanded extracellular matrix production. However, this collagen subtype has previously not been allocated to nervous tissue but points towards compensatory structural alterations in heterozygous animals. A different axon size distribution in peripheral nerves implies additional compensation mechanisms. Further analyses of DRGs by single cell sequencing will provide additional insights in this pain-related channelopathy.
References: Yes
References 1: Leipold, E., et al., A de novo gain-of-function mutation in SCN11A causes loss of pain perception. Nat Genet, 2013. 45(11): p. 1399-404.
References 2: Ebbinghaus M, et al., Gain-of-function mutation in SCN11A causes itch and affects neurogenic inflammation and muscle function in Scn11a+/L799P mice. PLoS One, 2020. 15(8): e0237101.
References 3:
References 4:
Grant Support: This project was funded by grants of the Deutsche Forschungsgemeinschaft (DFG) to Prof. Ingo Kurth.
Keywords: NaV1.9, Scn11a, channelopathy, electron microscopy, Col4a3
Poster No: 1243
Presenter: Annika Schmidt
Institution: Institute of Human Genetics
Introduction: Pain protects the body from injuries while impaired nociception leads to severe mutilations and tissue damage. SCN11A encodes the voltage-gated sodium ion channel NaV1.9 primarily expressed in nociceptors. The gain-of-function variant Scn11a-L799P (orthologous to p.L811P in humans) leads to overactivity of this channel and disturbed nociceptive transmission. So far, the underlying pathomechanisms of this channelopathy have not been conclusively clarified, i.e., whether the loss of pain perception is due to neurodegeneration or to altered transmission of the corresponding neurons.
Methods: Investigations were performed on heterozygous knock-in mice carrying the Scn11a-L799P variant and compared with wild type specimen. Electron micrographs of peripheral nerves (sciatic and sural) were analyzed regarding axon size distribution and cluster size of unmyelinated nerve fibers. Frozen sections of hindpaw glabrous skin were stained free floating using protein gene product 9.5 (PGP 9.5) antibodies to determine intraepidermal nerve fibre density (IENFD). Expression of collagen type IV alpha 3 chain (Col4a3) was analyzed by quantitative real time PCR (qPCR) as well as immunofluorescence stainings on frozen sections, as this collagen was strongly upregulated in the transcriptomes from dorsal root ganglia (DRGs) of knock-in mice.
Results: Highly elevated Col4a3 expression levels in heterozygous knock-in animals were verified by qPCR as well as immunofluorescence stainings of DRG tissue sections. This matches findings from transcriptome analyses in DRGs. Analyses of axon number and axonal size distribution in peripheral nerves indicated distinct changes within small unmyelinated axons. Preliminary evaluation of IENFD did not show marked differences.
Conclusions: Increased expression of Col4a3 indicates an expanded extracellular matrix production. However, this collagen subtype has previously not been allocated to nervous tissue but points towards compensatory structural alterations in heterozygous animals. A different axon size distribution in peripheral nerves implies additional compensation mechanisms. Further analyses of DRGs by single cell sequencing will provide additional insights in this pain-related channelopathy.
References: Yes
References 1: Leipold, E., et al., A de novo gain-of-function mutation in SCN11A causes loss of pain perception. Nat Genet, 2013. 45(11): p. 1399-404.
References 2: Ebbinghaus M, et al., Gain-of-function mutation in SCN11A causes itch and affects neurogenic inflammation and muscle function in Scn11a+/L799P mice. PLoS One, 2020. 15(8): e0237101.
References 3:
References 4:
Grant Support: This project was funded by grants of the Deutsche Forschungsgemeinschaft (DFG) to Prof. Ingo Kurth.
Keywords: NaV1.9, Scn11a, channelopathy, electron microscopy, Col4a3
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