Sensory neurons from Nf1 haploinsufficient mice exhibit increased excitability.
ABSTRACT Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by tumor formation. People with NF1 also can experience more intense painful responses to stimuli, such as minor trauma, than normal. NF1 results from a heterozygous mutation of the NF1 gene, leading to decreased levels of neurofibromin, the protein product of the NF1 gene. Neurofibromin is a guanosine triphosphatase activating protein (GAP) for Ras and accelerates the conversion of active Ras-GTP to inactive Ras-GDP; therefore mutation of the NF1 gene frequently results in an increase in activity of the Ras transduction cascade. Using patch-clamp electrophysiological techniques, we examined the excitability of capsaicin-sensitive sensory neurons isolated from the dorsal root ganglia of adult mice with a heterozygous mutation of the Nf1 gene (Nf1+/-), analogous to the human mutation, in comparison to wildtype sensory neurons. Sensory neurons from adult Nf1+/- mice generated a more than twofold higher number of action potentials in response to a ramp of depolarizing current as wild-type neurons. Consistent with the greater number of action potentials, Nf1+/- neurons had lower firing thresholds, lower rheobase currents, and shorter firing latencies than wild-type neurons. Interestingly, nerve growth factor augmented the excitability of wild-type neurons in a concentration-related manner but did not further alter the excitability of the Nf1+/- sensory neurons. These data clearly suggest that GAPs, such as neurofibromin, can play a key role in the excitability of nociceptive sensory neurons. This increased excitability may explain the painful conditions experienced by people with NF1.
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ABSTRACT: Major aspects of neuronal function are regulated by Ca(2+) including neurotransmitter release, excitability, developmental plasticity, and gene expression. We reported previously that sensory neurons isolated from a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/-) exhibited both greater excitability and evoked release of neuropeptides compared to wildtype mice. Furthermore, augmented voltage-dependent sodium currents but not potassium currents contribute to the enhanced excitability. To determine the mechanisms giving rise to the enhanced release of substance P and calcitonin gene-related peptide in the Nf1+/- sensory neurons, the potential differences in the total voltage-dependent calcium current (ICa) as well as the contributions of individual Ca(2+) channel subtypes were assessed. Whole-cell patch-clamp recordings from small diameter capsaicin-sensitive sensory neurons demonstrated that the average peak ICa densities were not different between the two genotypes. However, by using selective blockers of channel subtypes, the current density of N-type (Cav2.2) ICa was significantly larger in Nf1+/- neurons compared to wildtype neurons. In contrast, there were no significant differences in L-, P/Q- and R-type currents between the two genotypes. Quantitative real-time PCR measurements made from the isolated but intact dorsal root ganglia indicated that N-type (Cav2.2) and P/Q-type (Cav2.1) Ca(2+) channels exhibited the highest mRNA expression levels although there were no significant differences in the levels of mRNA expression between the genotypes. These results suggest that the augmented N-type (Cav2.2) ICa observed in the Nf1+/- sensory neurons does not result from genomic differences but may reflect post-translational or some other non-genomic modifications. Thus, our results demonstrate that sensory neurons from Nf1+/- mice, exhibit increased N-type ICa and likely account for the increased release of substance P and calcitonin gene-related peptide that occurs in Nf1+/- sensory neurons.Neuroscience 04/2014; · 3.12 Impact Factor
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ABSTRACT: This study examined whether mice with a deficiency of neurofibromin, a Ras GTPase activating protein, exhibit a nociceptive phenotype and probed a possible contribution by calcitonin gene-related peptide. In the absence of inflammation, Nf1+/- mice (B6.129S6 Nf1 /J) and wild type littermates responded comparably to heat or mechanical stimuli, except for a subtle enhanced mechanical sensitivity in female Nf1+/- mice. Nociceptive phenotype was also examined after inflammation induced by capsaicin and formalin, which release endogenous calcitonin gene-related peptide. Intraplantar injection of capsaicin evoked comparable heat hyperalgesia and mechanical hypersensitivity in Nf1+/- and wild type mice of both genders. Formalin injection caused a similar duration of licking in male Nf1+/- and wild type mice. Female Nf1+/- mice licked less than wild type mice, but displayed other nociceptive behaviors. In contrast, intraplantar injection of CGRP caused greater heat hyperalgesia in Nf1+/- mice of both genders compared to wild type mice. Male Nf1+/- mice also exhibited greater mechanical hypersensitivity; however, female Nf1+/- mice exhibited less mechanical hypersensitivity than their wild type littermates. Transcripts for calcitonin gene-related peptide were similar in the dorsal root ganglia of both genotypes and genders. Transcripts for receptor activity-modifying protein-1, which is rate-limiting for the calcitonin gene-related peptide receptor, in the spinal cord were comparable for both genotypes and genders. The increased responsiveness to intraplantar calcitonin gene-related peptide suggests that the peripheral actions of calcitonin gene-related peptide are enhanced as a result of the neurofibromin deficit. The analgesic efficacy of calcitonin gene-related peptide receptor antagonists may therefore merit investigation in neurofibromatosis patients.PLoS ONE 01/2014; 9(9):e106767. · 3.53 Impact Factor
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ABSTRACT: Neurofibromatosis type I (Nf1) is a GTPase activating protein (GAP) that inactivates the oncoprotein Ras and plays important roles in nervous system development and learning. Alternative exon 23a falls within the Nf1 GAP domain and is tightly regulated in favor of skipping in neurons; however, its biological function is not fully understood.Here, we generated mouse embryonic stem (ES) cells with constitutive endogenous Nf1 exon 23a inclusion, termed Nf1 23aIN/23aIN cells, by mutating the splicing signals surrounding the exon to better match consensus sequences. We also made Nf1 23aΔ/23aΔ cells lacking the exon. Active Ras levels are high in WT and Nf1 23aIN/23aIN ES cells, where Nf1 exon 23a inclusion is high, and low in Nf1 23aΔ/23aΔ cells. Upon neuronal differentiation, active Ras levels are high in Nf1 23aIN/23aIN cells, where exon inclusion remains high, but Ras activation is low in the other two genotypes, where the exon is skipped. Signaling downstream of Ras is significantly elevated in Nf1 23aIN/23aIN neurons. These results suggest that exon 23a suppresses the Ras-GAP activity of Nf1. Therefore, regulation of Nf1 exon 23a inclusion serves as a mechanism for providing appropriate levels of Ras signaling, and may be important in modulating Ras-related neuronal functions.Molecular and Cellular Biology 04/2014; · 5.04 Impact Factor
Sensory Neurons from Nf1 Haploinsufficient Mice Exhibit Increased Excitability
Yue Wang1, G. D. Nicol1, D. Wade Clapp2,3 and Cynthia M. Hingtgen4,1
1: Department of Pharmacology and Toxicology
2: Department of Pediatrics
3: Department of Microbiology and Immunology
4: Department of Neurology
All at Indiana University School of Medicine, Indianapolis, IN
Running head: Nf1+/- Sensory Neurons Exhibit Increased Excitability
Cynthia M. Hingtgen, MD, PhD
Stark Neurosciences Research Institute
Indiana University School of Medicine
450 West Walnut Street, R2-466
Indianapolis, IN 46202
Articles in PresS. J Neurophysiol (August 10, 2005). doi:10.1152/jn.00489.2005
Copyright © 2005 by the American Physiological Society.
Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by tumor
formation. People with NF1 also can experience more intense painful responses to
stimuli, such as minor trauma, than normal. NF1 results from a heterozygous mutation
of the NF1 gene, leading to decreased levels of neurofibromin, the protein product of the
NF1 gene. Neurofibromin is a guanosine triphosphatase activating protein (GAP) for
Ras and accelerates the conversion of active Ras-GTP to inactive Ras-GDP; therefore,
mutation of the NF1 gene frequently results in an increase in activity of the Ras
transduction cascade. Using patch-clamp electrophysiological techniques, we examined
the excitability of capsaicin-sensitive sensory neurons isolated from the dorsal root
ganglia of adult mice with a heterozygous mutation of the Nf1 gene (Nf1+/-), analogous
to the human mutation, in comparison to wildtype sensory neurons. Sensory neurons
from adult Nf1+/- mice generated a more than two-fold higher number of action
potentials in response to a ramp of depolarizing current as wildtype neurons.
Consistent with the greater number of action potentials, Nf1+/- neurons had lower firing
thresholds, lower rheobase currents, and shorter firing latencies than wildtype neurons.
Interestingly, nerve growth factor augmented the excitability of wildtype neurons in a
concentration-related manner, but did not further alter the excitability of the Nf1+/-
sensory neurons. These data clearly suggest that GAPs, such as neurofibromin, can
play a key role in the excitability of nociceptive sensory neurons. This increased
excitability may explain the painful conditions experienced by people with NF1.
Keywords: dorsal root ganglia, neurofibromin, nerve growth factor, nociceptors, Ras
Neurofibromatosis type 1 (NF1) is a common autosomal dominant disease with
an incidence of 1 in 3,500 people (Lakkis and Tennekoon 2000). It is characterized by
formation of neurofibromas (complex tumors composed of axonal processes, Schwann
cells, fibroblasts and mast cells), as well as malignant tumors such as
neurofibrosarcomas, malignant astrocytomas and myeloid leukemias. In addition to
tumor formation, some people with NF1 also experience a more intense painful
response to stimuli, such as minor injuries, than normal (Riccardi and Eichner 1992;
Creange et al. 1999; Wolkenstein et al. 2001). Although the mechanism by which the
NF1 mutation causes these symptoms has not been elucidated, it is likely that the
abnormal painful states involve the increased sensitivity of small diameter nociceptive
sensory neurons; cells that are known to mediate the transmission of pain.
In NF1 there is a mutation of one allele of the NF1 gene (NF1+/-). This results in
reduced expression of the protein product of the NF1 gene, neurofibromin, in many cells,
including neurons (Bollag and McCormick 1991; Largaespada et al. 1996; Zhang et al.
1998; Cichowski and Jacks 2001). Neurofibromin is a guanosine triphosphatase
activating protein (GAP) that accelerates the conversion of the active form of the small G
protein, Ras (Ras-GTP), to its inactive form (Ras-GDP; Martin et al. 1990; Wallace et al.
1990; Li et al. 1990). In many cell types, mutation of the NF1 gene or its mouse
correlate (Nf1), frequently results in increased basal and cytokine-stimulated Ras-GTP
and enhanced activity of the downstream effectors of the Ras transduction cascade. For
example, investigators have shown that the level of Ras-GTP is elevated in human NF1
neurogenic tumors (Guha et al. 1996), in mast cells from mice with a heterozygous
mutation of the Nf1 gene (Nf1+/-; Ingram et al. 2001), and in Schwann cells from
embryonic mice with a homozygous mutation of the Nf1 gene (Nf1-/-; Sherman et al.
2000). In addition, the sensory neurons from embryonic Nf1-/- mice demonstrate
increased Ras activity (Klesse and Parada 1998; Vogel et al. 2000).
Among the many growth factors that activate the Ras transduction cascade,
nerve growth factor (NGF) has been explored extensively for its role in pain signaling.
NGF plays a critical role in the development and maintenance of sensory neurons,
however, a growing body of evidence has demonstrated that NGF is an important
mediator of the enhanced pain sensation (hyperalgesia) that occurs with inflammation.
The content of NGF is elevated in inflamed skin (Weskamp and Otten 1987) and
peripheral tissue (Aloe et al. 1992a,b). Mendell and coworkers demonstrated that NGF
produces both thermal and mechanical hyperalgesia (Lewin and Mendell 1993; Lewin et
al. 1993). In addition, the hyperalgesia associated with inflammation is diminished by
an anti-NGF antibody (Woolf et al. 1994). By using a skin-nerve preparation, Rueff and
Mendell (1996) demonstrated that NGF can increase the firing frequency of isolated
saphenous nerve in response to heat stimulation. NGF also enhanced the excitability of
isolated sensory neurons in culture by increasing a TTX-resistant sodium current and by
suppressing a delayed-rectifier potassium current (Zhang et al. 2002). Although it is
clear that NGF can sensitize sensory neurons to noxious stimuli, the intracellular
cascades by which NGF exerts its effects remain poorly understood. The stimulation of
either the TrkA or p75 receptor by NGF can lead to the activation of Ras transduction
cascade (Blochl et al. 2004; Corbett and Alber 2001; Huang and Reichardt 2003; Susen
et al. 1999). In addition, recent studies have suggested that NGF can activate
downstream effectors of the Ras transduction cascade to affect changes in adult
sensory neurons (Ganju et al. 1998; Bron et al. 2003; Zhuang et al. 2004). For example,
Bron and colleagues have shown that NGF-induced increases in phosphorylated
extracellular signal-regulated kinase (pERK) and phosphorylated Akt (pAkt), two
downstream effectors of Ras activation, are associated with increases in the expression
of the heat- and capsaicin-activated receptor, TRPV1, in DRG neurons and that
constitutively active Ras mimics the action of NGF to increase TRPV1 expression in
isolated sensory neurons (Bron et al., 2003). Based on the hypothesis that NGF-
induced alteration in peripheral pain signaling may, in part, be related to activation of the
Ras transduction cascade, the enhanced painful sensations experienced by people with
NF1 could result from altered control of the Ras cascade because of decreased
To test the hypothesis that the NF1 mutation results in increased sensory neuron
excitability, we used a mouse model of NF1. These mice have a heterozygous mutation
of the Nf1 gene (Nf1+/-), similar to that seen in the human disorder (Jacks et al, 1994).
In this report, we demonstrate that capsaicin-sensitive sensory neurons from Nf1+/- mice
exhibit enhanced excitability. Treatment of wildtype neurons with NGF mimics the
increased excitability of Nf1+/- neurons. These results suggest that decreased GAP
levels correlate with enhanced neuronal excitability, and are consistent with the idea that
GAP-regulated signaling pathways are important in the modulation of sensory neuron
Materials and Methods:
Mice heterozygous for the Nf1 mutation on a background of C57BL/6J were
originally developed by Dr. Tyler Jacks (Jacks et al. 1994). All animals were housed and
bred in the Indiana University Laboratory Animal Research Center and used in
accordance with National Institute of Health Guide for Care and Use of Laboratory
Animals (NIH Publications No. 80-23) revised 1996.
Horse serum, F-12 medium, L-glutamine, and penicillin/streptomycin were
purchased from Invitrogen (Carlsbad, CA). Nerve growth factor (NGF) was purchased