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Nedd4-2 haploinsufficiency causes hyperactivity and increased sensitivity to inflammatory stimuli

View Article: PubMed Central - PubMed

ABSTRACT

Nedd4-2 (NEDD4L in humans) is a ubiquitin protein ligase best known for its role in regulating ion channel internalization and turnover. Nedd4-2 deletion in mice causes perinatal lethality associated with increased epithelial sodium channel (ENaC) expression in lung and kidney. Abundant data suggest that Nedd4-2 plays a role in neuronal functions and may be linked to epilepsy and dyslexia in humans. We used a mouse model of Nedd4-2 haploinsufficiency to investigate whether an alteration in Nedd4-2 levels of expression affects general nervous system functions. We found that Nedd4-2 heterozygous mice are hyperactive, have increased basal synaptic transmission and have enhanced sensitivity to inflammatory pain. Thus, Nedd4-2 heterozygous mice provide a new genetic model to study inflammatory pain. These data also suggest that in human, SNPs affecting NEDD4L levels may be involved in the development of neuropsychological deficits and peripheral neuropathies and may help unveil the genetic basis of comorbidities.

No MeSH data available.


Nedd4-2 heterozygous mice have increased sensitivity to chemical stimuli.(A) In the two-temperature choice test there is no difference between mutants and controls in response to variable temperature. Side A of the chamber was kept at the constant temperature of 32 °C whereas side B of the chamber had variable temperature ranging from 4 to 50 °C as described in Methods. (B) Nedd4-2 heterozygous mice do not have an altered threshold in the Hargreaves test compared to controls. Graph showing the average time (in seconds) by which the mice responded to the thermal stimuli. (C) Nedd4-2 +/− mice have increased sensitivity in the early Phase II formalin test (two-way ANOVA, P < 0.05). Time course of the nocifensive response to formalin injection. n = 9–11 for each genotype in each test. *Indicates p < 0.05. (D) Quantification of total nocifensive responses from (C). (E,F) Nedd4-2 loss increases TrkA expression levels in DRG. Western blot analysis of TrkA and Nedd4-2 protein in E13.5 mouse DRGs (E) and quantification of relative TrkA protein levels (F). TrkA band intensity was normalized relative to the intensity of the β-actin band used as control for loading control. Data are expressed as percentage of the mean TrkA levels in WT DRG. N = 3 sets of pooled DRG from different embryos per genotype *p = 0.0147, Error bars are S.E.M. (G–K) Western blot analysis of TrkA (G), Nav1.7 (H) and Nav1.8 (I) protein levels in lysates from +/+ and +/− adult mouse DRG. β-actin was used as a control for loading. (J,K) Quantification of TrkA (J) and Nav1.7 (K) band intensity as shown respectively in (G,H) (n = 3 sets of pooled DRG from different mice).
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f5: Nedd4-2 heterozygous mice have increased sensitivity to chemical stimuli.(A) In the two-temperature choice test there is no difference between mutants and controls in response to variable temperature. Side A of the chamber was kept at the constant temperature of 32 °C whereas side B of the chamber had variable temperature ranging from 4 to 50 °C as described in Methods. (B) Nedd4-2 heterozygous mice do not have an altered threshold in the Hargreaves test compared to controls. Graph showing the average time (in seconds) by which the mice responded to the thermal stimuli. (C) Nedd4-2 +/− mice have increased sensitivity in the early Phase II formalin test (two-way ANOVA, P < 0.05). Time course of the nocifensive response to formalin injection. n = 9–11 for each genotype in each test. *Indicates p < 0.05. (D) Quantification of total nocifensive responses from (C). (E,F) Nedd4-2 loss increases TrkA expression levels in DRG. Western blot analysis of TrkA and Nedd4-2 protein in E13.5 mouse DRGs (E) and quantification of relative TrkA protein levels (F). TrkA band intensity was normalized relative to the intensity of the β-actin band used as control for loading control. Data are expressed as percentage of the mean TrkA levels in WT DRG. N = 3 sets of pooled DRG from different embryos per genotype *p = 0.0147, Error bars are S.E.M. (G–K) Western blot analysis of TrkA (G), Nav1.7 (H) and Nav1.8 (I) protein levels in lysates from +/+ and +/− adult mouse DRG. β-actin was used as a control for loading. (J,K) Quantification of TrkA (J) and Nav1.7 (K) band intensity as shown respectively in (G,H) (n = 3 sets of pooled DRG from different mice).

Mentions: The hyperactivity of Nedd4-2 heterozygous mice and the higher response at low stimulus intensity in the basal synaptic transmission suggest that haploinsufficiency of this E3 ligase could have other consequences on nervous system function. Since Nedd4-2 deficiency in a conditional mouse model has been linked to neuropathic pain we investigated whether its partial systemic loss, which could mimic a human condition caused by the presence of loss of function SNPs, affects pain sensitivity10. In the two-temperature choice test, in which mice can choose to spend time on a plate at either 32 °C or a plate at different temperatures, we found that WT and Nedd4-2 heterozygous mice spent the same amount of time at either cold or hot temperatures (Fig. 5A) suggesting a similar sensitivity to thermal stimulation. This result was further confirmed in the Hargreaves test, that showed no difference in the latency threshold for flinching or paws withdrawal to a radiant heat stimulus in the two groups of mice (Fig. 5B) suggesting no changes in sensitivity to noxious thermal stimuli. However, when we measured spontaneous pain behavior in the formalin test we found that Nedd4−2 +/−mice were more sensitive than controls. In Phase I, corresponding to acute nociception immediately after the injection, there was no significant difference between genotypes. However, in the early Phase II, during the central sensitization and inflammation period mutant mice displayed a significant increase in licking and lifting/favoring of the injected paw as compared with their WT littermates suggesting a central consequence of increased peripheral activity due to loss of one Nedd4-2 allele (Fig. 5C,D). Taken together, these results indicate that Nedd4-2 heterozygous mice are normal in response to thermal stimuli but are hypersensitive to inflammatory stimulus. Next, we investigated the level of expression of some putative Nedd4-2 substrates that have been linked to this phenotype. Analysis of the sodium channel Nav1.710 and Nav1.8 and the tyrosine kinase receptor TrkA1718 in adult heterozygous DRG showed no change in their level of the expression (Fig. 5G–K). The lack of change in Nav1.7 was not surprising because its level was unchanged even in mice with a more drastic reduction of Nedd4-2 in DRG. However, Nav1.8 lack of up-regulation was more surprising because of the drastic increase observed in the Nedd4-2 conditional mutant mouse model10. These data suggest that most likely a combination of subtle changes in other sodium channels shown to be substrate of Nedd4-29 may be involved in this phenotype. However, TrkA is the only Trk receptor targeted by this E3 ligase19. Indeed, analysis of DRG of Nedd4-2 −/− embryos showed a significant increase in the level of TrkA compared to WT controls confirming that TrkA is a direct Nedd4-2 substrate in vivo and there is no significant redundancy by other Nedd4 ligases (Fig. 5E,F). This result suggests that partial loss of Nedd4-2 in the adult may impact, although not significantly, the level of TrkA which could result in the increase in the pain sensitivity phenotype20.


Nedd4-2 haploinsufficiency causes hyperactivity and increased sensitivity to inflammatory stimuli
Nedd4-2 heterozygous mice have increased sensitivity to chemical stimuli.(A) In the two-temperature choice test there is no difference between mutants and controls in response to variable temperature. Side A of the chamber was kept at the constant temperature of 32 °C whereas side B of the chamber had variable temperature ranging from 4 to 50 °C as described in Methods. (B) Nedd4-2 heterozygous mice do not have an altered threshold in the Hargreaves test compared to controls. Graph showing the average time (in seconds) by which the mice responded to the thermal stimuli. (C) Nedd4-2 +/− mice have increased sensitivity in the early Phase II formalin test (two-way ANOVA, P < 0.05). Time course of the nocifensive response to formalin injection. n = 9–11 for each genotype in each test. *Indicates p < 0.05. (D) Quantification of total nocifensive responses from (C). (E,F) Nedd4-2 loss increases TrkA expression levels in DRG. Western blot analysis of TrkA and Nedd4-2 protein in E13.5 mouse DRGs (E) and quantification of relative TrkA protein levels (F). TrkA band intensity was normalized relative to the intensity of the β-actin band used as control for loading control. Data are expressed as percentage of the mean TrkA levels in WT DRG. N = 3 sets of pooled DRG from different embryos per genotype *p = 0.0147, Error bars are S.E.M. (G–K) Western blot analysis of TrkA (G), Nav1.7 (H) and Nav1.8 (I) protein levels in lysates from +/+ and +/− adult mouse DRG. β-actin was used as a control for loading. (J,K) Quantification of TrkA (J) and Nav1.7 (K) band intensity as shown respectively in (G,H) (n = 3 sets of pooled DRG from different mice).
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f5: Nedd4-2 heterozygous mice have increased sensitivity to chemical stimuli.(A) In the two-temperature choice test there is no difference between mutants and controls in response to variable temperature. Side A of the chamber was kept at the constant temperature of 32 °C whereas side B of the chamber had variable temperature ranging from 4 to 50 °C as described in Methods. (B) Nedd4-2 heterozygous mice do not have an altered threshold in the Hargreaves test compared to controls. Graph showing the average time (in seconds) by which the mice responded to the thermal stimuli. (C) Nedd4-2 +/− mice have increased sensitivity in the early Phase II formalin test (two-way ANOVA, P < 0.05). Time course of the nocifensive response to formalin injection. n = 9–11 for each genotype in each test. *Indicates p < 0.05. (D) Quantification of total nocifensive responses from (C). (E,F) Nedd4-2 loss increases TrkA expression levels in DRG. Western blot analysis of TrkA and Nedd4-2 protein in E13.5 mouse DRGs (E) and quantification of relative TrkA protein levels (F). TrkA band intensity was normalized relative to the intensity of the β-actin band used as control for loading control. Data are expressed as percentage of the mean TrkA levels in WT DRG. N = 3 sets of pooled DRG from different embryos per genotype *p = 0.0147, Error bars are S.E.M. (G–K) Western blot analysis of TrkA (G), Nav1.7 (H) and Nav1.8 (I) protein levels in lysates from +/+ and +/− adult mouse DRG. β-actin was used as a control for loading. (J,K) Quantification of TrkA (J) and Nav1.7 (K) band intensity as shown respectively in (G,H) (n = 3 sets of pooled DRG from different mice).
Mentions: The hyperactivity of Nedd4-2 heterozygous mice and the higher response at low stimulus intensity in the basal synaptic transmission suggest that haploinsufficiency of this E3 ligase could have other consequences on nervous system function. Since Nedd4-2 deficiency in a conditional mouse model has been linked to neuropathic pain we investigated whether its partial systemic loss, which could mimic a human condition caused by the presence of loss of function SNPs, affects pain sensitivity10. In the two-temperature choice test, in which mice can choose to spend time on a plate at either 32 °C or a plate at different temperatures, we found that WT and Nedd4-2 heterozygous mice spent the same amount of time at either cold or hot temperatures (Fig. 5A) suggesting a similar sensitivity to thermal stimulation. This result was further confirmed in the Hargreaves test, that showed no difference in the latency threshold for flinching or paws withdrawal to a radiant heat stimulus in the two groups of mice (Fig. 5B) suggesting no changes in sensitivity to noxious thermal stimuli. However, when we measured spontaneous pain behavior in the formalin test we found that Nedd4−2 +/−mice were more sensitive than controls. In Phase I, corresponding to acute nociception immediately after the injection, there was no significant difference between genotypes. However, in the early Phase II, during the central sensitization and inflammation period mutant mice displayed a significant increase in licking and lifting/favoring of the injected paw as compared with their WT littermates suggesting a central consequence of increased peripheral activity due to loss of one Nedd4-2 allele (Fig. 5C,D). Taken together, these results indicate that Nedd4-2 heterozygous mice are normal in response to thermal stimuli but are hypersensitive to inflammatory stimulus. Next, we investigated the level of expression of some putative Nedd4-2 substrates that have been linked to this phenotype. Analysis of the sodium channel Nav1.710 and Nav1.8 and the tyrosine kinase receptor TrkA1718 in adult heterozygous DRG showed no change in their level of the expression (Fig. 5G–K). The lack of change in Nav1.7 was not surprising because its level was unchanged even in mice with a more drastic reduction of Nedd4-2 in DRG. However, Nav1.8 lack of up-regulation was more surprising because of the drastic increase observed in the Nedd4-2 conditional mutant mouse model10. These data suggest that most likely a combination of subtle changes in other sodium channels shown to be substrate of Nedd4-29 may be involved in this phenotype. However, TrkA is the only Trk receptor targeted by this E3 ligase19. Indeed, analysis of DRG of Nedd4-2 −/− embryos showed a significant increase in the level of TrkA compared to WT controls confirming that TrkA is a direct Nedd4-2 substrate in vivo and there is no significant redundancy by other Nedd4 ligases (Fig. 5E,F). This result suggests that partial loss of Nedd4-2 in the adult may impact, although not significantly, the level of TrkA which could result in the increase in the pain sensitivity phenotype20.

View Article: PubMed Central - PubMed

ABSTRACT

Nedd4-2 (NEDD4L in humans) is a ubiquitin protein ligase best known for its role in regulating ion channel internalization and turnover. Nedd4-2 deletion in mice causes perinatal lethality associated with increased epithelial sodium channel (ENaC) expression in lung and kidney. Abundant data suggest that Nedd4-2 plays a role in neuronal functions and may be linked to epilepsy and dyslexia in humans. We used a mouse model of Nedd4-2 haploinsufficiency to investigate whether an alteration in Nedd4-2 levels of expression affects general nervous system functions. We found that Nedd4-2 heterozygous mice are hyperactive, have increased basal synaptic transmission and have enhanced sensitivity to inflammatory pain. Thus, Nedd4-2 heterozygous mice provide a new genetic model to study inflammatory pain. These data also suggest that in human, SNPs affecting NEDD4L levels may be involved in the development of neuropsychological deficits and peripheral neuropathies and may help unveil the genetic basis of comorbidities.

No MeSH data available.