Limits...
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.


Related in: MedlinePlus

Nedd4-2 is widely expressed in the adult mouse brain.(A–F) β-Galactosidase staining of heterozygous mouse brain sections showing widespread expression of NEDD4-2 in the adult brain. High expression in entorhinal cortex cortical layers II and III, striatum, DG and CA regions of hippocampus and cerebellum. FrA- Frontal association cortex, OC-orbital cortex, AO- anterior olfactory nucleus, LV-lateral ventricle, CTX-cortex, HIP-hippocampus, STR-striatum, TH-thalamus, HT-hypothalamus, 3V- third ventricle, EC- entorhinal cortex, IC- internal capsule, AMY- amygdala, SC-superior colliculus, PAG- periaqueductal gray, GN- geniculate nucleus, RN-reticular nucleus, SN-substantia nigra, InfC- inferior colliculus, CBM- cerebellum, TgN- tegmental nucleus, PBN- parabrachial nucleus, 4V- 4th ventricle, VN- vestibular nucleus. (G) β-Gal staining of an adult Nedd4-2 heterozygous mouse spinal cord section at the lumbar level showing widespread expression of NEDD4-2 in the gray matter. (H,I) Staining of a section from an adult heterozygous mouse lumbar DRG shows Nedd4-2 expression in virtually all DRG neurons (I); (H) shows an unstained adjacent DRG section.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5015076&req=5

f2: Nedd4-2 is widely expressed in the adult mouse brain.(A–F) β-Galactosidase staining of heterozygous mouse brain sections showing widespread expression of NEDD4-2 in the adult brain. High expression in entorhinal cortex cortical layers II and III, striatum, DG and CA regions of hippocampus and cerebellum. FrA- Frontal association cortex, OC-orbital cortex, AO- anterior olfactory nucleus, LV-lateral ventricle, CTX-cortex, HIP-hippocampus, STR-striatum, TH-thalamus, HT-hypothalamus, 3V- third ventricle, EC- entorhinal cortex, IC- internal capsule, AMY- amygdala, SC-superior colliculus, PAG- periaqueductal gray, GN- geniculate nucleus, RN-reticular nucleus, SN-substantia nigra, InfC- inferior colliculus, CBM- cerebellum, TgN- tegmental nucleus, PBN- parabrachial nucleus, 4V- 4th ventricle, VN- vestibular nucleus. (G) β-Gal staining of an adult Nedd4-2 heterozygous mouse spinal cord section at the lumbar level showing widespread expression of NEDD4-2 in the gray matter. (H,I) Staining of a section from an adult heterozygous mouse lumbar DRG shows Nedd4-2 expression in virtually all DRG neurons (I); (H) shows an unstained adjacent DRG section.

Mentions: Nedd4-2 has a role in the regulation of ion channels levels in lung and kidney346. However, limited information is available on its function in the adult nervous system215. To address this, we targeted the Nedd4-2 gene in mouse by employing an embryonic stem cell line with a gene trap cassette inserted in the intron between exon 10 and 11 (Fig. 1). The cassette used for gene inactivation contains a strong splice acceptor from the Engrail2 gene and the β-geo cassette (fusion between the neo and the β-galactosidase gene) such that, after exon 10 the splicing will occur to the Engrail2 exon and will stop at the β-geo fusion gene preventing splicing to Nedd4-2 exon 11 (Fig. 1A). Importantly, the promoterless β-galactosidase gene driven by the endogenous Nedd4-2 gene allows for the study of Nedd4-2 expression (Fig. 2). Mating of heterozygous mice yielded progeny with WT, Nedd4-2 +/− and Nedd4-2 −/− animals present at the expected ratio. While at birth Nedd4-2 −/− pups were indistinguishable from WT littermates, most of them died early post-natally. This is in agreement with two previous studies reporting perinatal lethality due to lung inflammation and respiratory distress34. Western blot analysis of DRG protein extracts from Nedd4-2 −/− mice showed a complete loss of Nedd4-2 protein suggesting effective and complete inactivation of the gene (Fig. 1C). Importantly, DRG from heterozygous mice showed a reduction in Nedd4-2 protein level of at least 50% suggesting a lack of compensatory mechanisms to keep Nedd4-2 levels constant after loss of one allele. This result is in agreement with the findings of Boase et al.3 reporting a similar 50% reduction of Nedd4-2 protein level in the lung from heterozygous animals.


Nedd4-2 haploinsufficiency causes hyperactivity and increased sensitivity to inflammatory stimuli
Nedd4-2 is widely expressed in the adult mouse brain.(A–F) β-Galactosidase staining of heterozygous mouse brain sections showing widespread expression of NEDD4-2 in the adult brain. High expression in entorhinal cortex cortical layers II and III, striatum, DG and CA regions of hippocampus and cerebellum. FrA- Frontal association cortex, OC-orbital cortex, AO- anterior olfactory nucleus, LV-lateral ventricle, CTX-cortex, HIP-hippocampus, STR-striatum, TH-thalamus, HT-hypothalamus, 3V- third ventricle, EC- entorhinal cortex, IC- internal capsule, AMY- amygdala, SC-superior colliculus, PAG- periaqueductal gray, GN- geniculate nucleus, RN-reticular nucleus, SN-substantia nigra, InfC- inferior colliculus, CBM- cerebellum, TgN- tegmental nucleus, PBN- parabrachial nucleus, 4V- 4th ventricle, VN- vestibular nucleus. (G) β-Gal staining of an adult Nedd4-2 heterozygous mouse spinal cord section at the lumbar level showing widespread expression of NEDD4-2 in the gray matter. (H,I) Staining of a section from an adult heterozygous mouse lumbar DRG shows Nedd4-2 expression in virtually all DRG neurons (I); (H) shows an unstained adjacent DRG section.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015076&req=5

f2: Nedd4-2 is widely expressed in the adult mouse brain.(A–F) β-Galactosidase staining of heterozygous mouse brain sections showing widespread expression of NEDD4-2 in the adult brain. High expression in entorhinal cortex cortical layers II and III, striatum, DG and CA regions of hippocampus and cerebellum. FrA- Frontal association cortex, OC-orbital cortex, AO- anterior olfactory nucleus, LV-lateral ventricle, CTX-cortex, HIP-hippocampus, STR-striatum, TH-thalamus, HT-hypothalamus, 3V- third ventricle, EC- entorhinal cortex, IC- internal capsule, AMY- amygdala, SC-superior colliculus, PAG- periaqueductal gray, GN- geniculate nucleus, RN-reticular nucleus, SN-substantia nigra, InfC- inferior colliculus, CBM- cerebellum, TgN- tegmental nucleus, PBN- parabrachial nucleus, 4V- 4th ventricle, VN- vestibular nucleus. (G) β-Gal staining of an adult Nedd4-2 heterozygous mouse spinal cord section at the lumbar level showing widespread expression of NEDD4-2 in the gray matter. (H,I) Staining of a section from an adult heterozygous mouse lumbar DRG shows Nedd4-2 expression in virtually all DRG neurons (I); (H) shows an unstained adjacent DRG section.
Mentions: Nedd4-2 has a role in the regulation of ion channels levels in lung and kidney346. However, limited information is available on its function in the adult nervous system215. To address this, we targeted the Nedd4-2 gene in mouse by employing an embryonic stem cell line with a gene trap cassette inserted in the intron between exon 10 and 11 (Fig. 1). The cassette used for gene inactivation contains a strong splice acceptor from the Engrail2 gene and the β-geo cassette (fusion between the neo and the β-galactosidase gene) such that, after exon 10 the splicing will occur to the Engrail2 exon and will stop at the β-geo fusion gene preventing splicing to Nedd4-2 exon 11 (Fig. 1A). Importantly, the promoterless β-galactosidase gene driven by the endogenous Nedd4-2 gene allows for the study of Nedd4-2 expression (Fig. 2). Mating of heterozygous mice yielded progeny with WT, Nedd4-2 +/− and Nedd4-2 −/− animals present at the expected ratio. While at birth Nedd4-2 −/− pups were indistinguishable from WT littermates, most of them died early post-natally. This is in agreement with two previous studies reporting perinatal lethality due to lung inflammation and respiratory distress34. Western blot analysis of DRG protein extracts from Nedd4-2 −/− mice showed a complete loss of Nedd4-2 protein suggesting effective and complete inactivation of the gene (Fig. 1C). Importantly, DRG from heterozygous mice showed a reduction in Nedd4-2 protein level of at least 50% suggesting a lack of compensatory mechanisms to keep Nedd4-2 levels constant after loss of one allele. This result is in agreement with the findings of Boase et al.3 reporting a similar 50% reduction of Nedd4-2 protein level in the lung from heterozygous animals.

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.


Related in: MedlinePlus