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Pain related channels are differentially expressed in neuronal and non-neuronal cells of glabrous skin of fabry knockout male mice.

Lakomá J, Rimondini R, Donadio V, Liguori R, Caprini M - PLoS ONE (2014)

Bottom Line: Additionally, KO mice showed a decreased and scattered pattern of neuronal terminations consistent with the reduction in neuronal terminations in skin biopsies of patients with small fiber neuropathies.At the molecular level KO animals showed an increase in the expression of TRPV1 and Nav1.8, and a decrease in the expression of TRPM8.Notably, these alterations are observed in young animals.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Human and General Physiology, Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Bologna, Italy.

ABSTRACT
Fabry disease (FD) is one of the X-linked lysosomal storage disorders caused by deficient functioning of the alpha-galactosidase A (α-GalA) enzyme. The α-GalA deficiency leads to multi-systemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide in the endothelium and vascular smooth muscles. A hallmark symptom of FD patients is peripheral pain that appears in the early stage of the disease. Pain in FD patients is a peripheral small-fiber idiopathic neuropathy, with intra-epidermal fiber density and integrity being used for diagnosing FD in humans. However, the molecular correlates underlying pain sensation in FD remain elusive. Here, we have employed the α-GalA gene KO mouse as a model of FD in rodents to investigate molecular changes in their peripheral nervous system that may account for their algesic symptoms. The α-GalA mice display neuropathic pain as evidenced by thermal hyperalgesia and mechanical allodynia, with histological analyses showing alterations in cutaneous innervation. Additionally, KO mice showed a decreased and scattered pattern of neuronal terminations consistent with the reduction in neuronal terminations in skin biopsies of patients with small fiber neuropathies. At the molecular level KO animals showed an increase in the expression of TRPV1 and Nav1.8, and a decrease in the expression of TRPM8. Notably, these alterations are observed in young animals. Taken together, our findings imply that the α-GalA KO mouse is a good model in which to study the peripheral small fiber neuropathy exhibited by FD patients, and provides molecular evidence for a hyperexcitability of small nociceptors in FD.

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The expression of pain receptors in α-GalA KO males frontal paws.A) Immunohistochemistry of 12 µm frozen coronal sections of α-GalA KO males (n = 3) showed different expression at the protein level of the specific pain (Nav1.8), heat (TRPV1) and cold (TRPM8, Immunological Science) receptors in comparison to their WT controls (n = 3). B) The western blot analysis of Nav1.8 expression (∼220 kDa) in the whole protein extract from skin of frontal paws confirmed 1.5 fold increased protein expression of Nav1.8 in case of α-GalA KO males (n = 5) when compared to WT males (n = 5), p = 0.0246. The western blot analysis of TRPV1 expression (∼92 kDa) revealed 1.4 fold increased protein expression in case of α-GalA KO males (n = 3) when compared to WT (n = 3), p = 0.0344. The analysis of TRPM8 (Santa Cruz) expression (∼127 kDa) in the whole protein extract from skin of frontal paws showed the almost half expression decrease in protein expression of TRPM8 in case of α-GalA KO (n = 3) males when compared to WT (n = 3), p = 0.0518. Original images of presented blots are part of supplementary material (Figure S4). Scale bar represents 100 µm.
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pone-0108641-g004: The expression of pain receptors in α-GalA KO males frontal paws.A) Immunohistochemistry of 12 µm frozen coronal sections of α-GalA KO males (n = 3) showed different expression at the protein level of the specific pain (Nav1.8), heat (TRPV1) and cold (TRPM8, Immunological Science) receptors in comparison to their WT controls (n = 3). B) The western blot analysis of Nav1.8 expression (∼220 kDa) in the whole protein extract from skin of frontal paws confirmed 1.5 fold increased protein expression of Nav1.8 in case of α-GalA KO males (n = 5) when compared to WT males (n = 5), p = 0.0246. The western blot analysis of TRPV1 expression (∼92 kDa) revealed 1.4 fold increased protein expression in case of α-GalA KO males (n = 3) when compared to WT (n = 3), p = 0.0344. The analysis of TRPM8 (Santa Cruz) expression (∼127 kDa) in the whole protein extract from skin of frontal paws showed the almost half expression decrease in protein expression of TRPM8 in case of α-GalA KO (n = 3) males when compared to WT (n = 3), p = 0.0518. Original images of presented blots are part of supplementary material (Figure S4). Scale bar represents 100 µm.

Mentions: About 50% of C-fiber and 10% of Aδ fibers in naive rats express Nav1.8 channels [24]. This percentage is increased in conditions of pain [25], [26]. More Nav1.8 channels are expressed on C-fibers and Aδ fibers in rat digital nerves after neuropathic injury [27]. In our immunological studies we have observed increased expression of Nav1.8 in epidermis of frontal paw glabrous skin in case of α-GalA(−/0) mice in comparison with control (Figure 4A, n = 3 animals for WT, n = 3 animals for KO). The western blot analysis of Nav1.8 expression in the whole protein extract from skin of frontal paws confirmed that the ratio of Nav1.8 protein to β-actin was higher in KO males respectively to their control. Specifically, the higher protein expression of Nav1.8 in case of α-Gal A KO males is about 1.5 fold increase compared to WT (Figure 4Bp = 0.0246, n = 5 animals for WT, n = 5 animals for KO). Interestingly, the expression of the voltage-gated sodium channel Nav1.7 which plays important roles in inflammatory and neuropathic pain remained unaffected in control and FD mice (Figure S2, n = 2 animals for WT, n = 2 animals for KO, p = 0.1495).


Pain related channels are differentially expressed in neuronal and non-neuronal cells of glabrous skin of fabry knockout male mice.

Lakomá J, Rimondini R, Donadio V, Liguori R, Caprini M - PLoS ONE (2014)

The expression of pain receptors in α-GalA KO males frontal paws.A) Immunohistochemistry of 12 µm frozen coronal sections of α-GalA KO males (n = 3) showed different expression at the protein level of the specific pain (Nav1.8), heat (TRPV1) and cold (TRPM8, Immunological Science) receptors in comparison to their WT controls (n = 3). B) The western blot analysis of Nav1.8 expression (∼220 kDa) in the whole protein extract from skin of frontal paws confirmed 1.5 fold increased protein expression of Nav1.8 in case of α-GalA KO males (n = 5) when compared to WT males (n = 5), p = 0.0246. The western blot analysis of TRPV1 expression (∼92 kDa) revealed 1.4 fold increased protein expression in case of α-GalA KO males (n = 3) when compared to WT (n = 3), p = 0.0344. The analysis of TRPM8 (Santa Cruz) expression (∼127 kDa) in the whole protein extract from skin of frontal paws showed the almost half expression decrease in protein expression of TRPM8 in case of α-GalA KO (n = 3) males when compared to WT (n = 3), p = 0.0518. Original images of presented blots are part of supplementary material (Figure S4). Scale bar represents 100 µm.
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pone-0108641-g004: The expression of pain receptors in α-GalA KO males frontal paws.A) Immunohistochemistry of 12 µm frozen coronal sections of α-GalA KO males (n = 3) showed different expression at the protein level of the specific pain (Nav1.8), heat (TRPV1) and cold (TRPM8, Immunological Science) receptors in comparison to their WT controls (n = 3). B) The western blot analysis of Nav1.8 expression (∼220 kDa) in the whole protein extract from skin of frontal paws confirmed 1.5 fold increased protein expression of Nav1.8 in case of α-GalA KO males (n = 5) when compared to WT males (n = 5), p = 0.0246. The western blot analysis of TRPV1 expression (∼92 kDa) revealed 1.4 fold increased protein expression in case of α-GalA KO males (n = 3) when compared to WT (n = 3), p = 0.0344. The analysis of TRPM8 (Santa Cruz) expression (∼127 kDa) in the whole protein extract from skin of frontal paws showed the almost half expression decrease in protein expression of TRPM8 in case of α-GalA KO (n = 3) males when compared to WT (n = 3), p = 0.0518. Original images of presented blots are part of supplementary material (Figure S4). Scale bar represents 100 µm.
Mentions: About 50% of C-fiber and 10% of Aδ fibers in naive rats express Nav1.8 channels [24]. This percentage is increased in conditions of pain [25], [26]. More Nav1.8 channels are expressed on C-fibers and Aδ fibers in rat digital nerves after neuropathic injury [27]. In our immunological studies we have observed increased expression of Nav1.8 in epidermis of frontal paw glabrous skin in case of α-GalA(−/0) mice in comparison with control (Figure 4A, n = 3 animals for WT, n = 3 animals for KO). The western blot analysis of Nav1.8 expression in the whole protein extract from skin of frontal paws confirmed that the ratio of Nav1.8 protein to β-actin was higher in KO males respectively to their control. Specifically, the higher protein expression of Nav1.8 in case of α-Gal A KO males is about 1.5 fold increase compared to WT (Figure 4Bp = 0.0246, n = 5 animals for WT, n = 5 animals for KO). Interestingly, the expression of the voltage-gated sodium channel Nav1.7 which plays important roles in inflammatory and neuropathic pain remained unaffected in control and FD mice (Figure S2, n = 2 animals for WT, n = 2 animals for KO, p = 0.1495).

Bottom Line: Additionally, KO mice showed a decreased and scattered pattern of neuronal terminations consistent with the reduction in neuronal terminations in skin biopsies of patients with small fiber neuropathies.At the molecular level KO animals showed an increase in the expression of TRPV1 and Nav1.8, and a decrease in the expression of TRPM8.Notably, these alterations are observed in young animals.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Human and General Physiology, Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Bologna, Italy.

ABSTRACT
Fabry disease (FD) is one of the X-linked lysosomal storage disorders caused by deficient functioning of the alpha-galactosidase A (α-GalA) enzyme. The α-GalA deficiency leads to multi-systemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide in the endothelium and vascular smooth muscles. A hallmark symptom of FD patients is peripheral pain that appears in the early stage of the disease. Pain in FD patients is a peripheral small-fiber idiopathic neuropathy, with intra-epidermal fiber density and integrity being used for diagnosing FD in humans. However, the molecular correlates underlying pain sensation in FD remain elusive. Here, we have employed the α-GalA gene KO mouse as a model of FD in rodents to investigate molecular changes in their peripheral nervous system that may account for their algesic symptoms. The α-GalA mice display neuropathic pain as evidenced by thermal hyperalgesia and mechanical allodynia, with histological analyses showing alterations in cutaneous innervation. Additionally, KO mice showed a decreased and scattered pattern of neuronal terminations consistent with the reduction in neuronal terminations in skin biopsies of patients with small fiber neuropathies. At the molecular level KO animals showed an increase in the expression of TRPV1 and Nav1.8, and a decrease in the expression of TRPM8. Notably, these alterations are observed in young animals. Taken together, our findings imply that the α-GalA KO mouse is a good model in which to study the peripheral small fiber neuropathy exhibited by FD patients, and provides molecular evidence for a hyperexcitability of small nociceptors in FD.

Show MeSH
Related in: MedlinePlus