Activation of the melanocortin-1 receptor attenuates neuronal apoptosis after traumatic brain injury by upregulating Merlin expression.

Traumatic brain injury (TBI) is a common disease worldwide with high mortality and disability rates. Besides the primary mechanical injury, the secondary injury associated with TBI can also induce numerous pathological changes, such as brain edema, nerve apoptosis, and neuroinflammation, which further aggravates neurological dysfunction and even causes the death due to the primary injury. Among them, neuronal apoptosis is a key link in the injury. Melanocortin-1 receptor (MC1R) is a G protein coupled receptor, belonging to the melanocortin receptor family. Studies have shown that activation of MC1R inhibits oxidative stress and apoptosis, and confers neuroprotective effects against various neurological diseases. Merlin is a protein product of the NF2 gene, which is widely expressed in the central nervous system (CNS) of mice, rats, and humans. Studies have indicated that Merlin is associated with MC1R. In this study, we explored the anti-apoptotic effects and potential mechanisms of MC1R. A rat model of TBI was established through controlled cortical impact. The MC1R-specific agonist Nle4-D-Phe7-α-Melanocyte (NDP-MSH) and the inhibitor MSG-606 were employed to explore the effects of MC1R and Merlin following TBI and investigated the associated mechanisms. The results showed that the expression levels of MC1R and Merlin were upregulated after TBI, and activation of MC1R promoted Merlin expression. Further, we found that MC1R activation significantly improved neurological dysfunction and reduced brain edema and neuronal apoptosis induced by TBI in rats. Mechanistically, its neuroprotective function and anti-apoptotic were partly associated with MC1R activation. In conclusion, we demonstrated that MC1R activation after TBI may inhibit apoptosis and confer neuroprotection by upregulating the expression of Merlin.

PLPP/CIN-mediated NF2 S10 dephosphorylation distinctly regulates kainate-induced seizure susceptibility and neuronal death through PAK1-NF-κB-COX-2-PTGES2 signaling pathway.

BACKGROUND: Pyridoxal-5'-phosphate phosphatase/chronophin (PLPP/CIN) selectively dephosphorylates serine (S) 10 site on neurofibromin 2 (NF2, also known as merlin (moesin-ezrin-radixin-like protein) or schwannomin). p21-activated kinase 1 (PAK1) is a serine/threonine protein kinase, which is involved in synaptic activity and plasticity in neurons. NF2 and PAK1 reciprocally regulate each other in a positive feedback manner. Thus, the aim of the present study is to investigate the effects of PLPP/CIN-mediated NF2 S10 dephosphorylation on PAK1-related signaling pathways under physiological and neuroinflammatory conditions, which are largely unknown. METHODS: After kainate (KA) injection in wild-type, PLPP/CIN-/- and PLPP/CINTg mice, seizure susceptibility, PAK1 S204 autophosphorylation, nuclear factor-κB (NF-κB) p65 S276 phosphorylation, cyclooxygenase-2 (COX-2) upregulation, prostaglandin E synthase 2 (PTGES2) induction and neuronal damage were measured. The effects of 1,1'-dithiodi-2-naphthtol (IPA-3, a selective inhibitor of PAK1) pretreatment on these responses to KA were also validated. RESULTS: PLPP/CIN overexpression increased PAK1 S204 autophosphorylation concomitant with the enhanced NF2 S10 dephosphorylation in hippocampal neurons under physiological condition. Following KA treatment, PLPP/CIN overexpression delayed the seizure on-set and accelerated PAK1 S204 phosphorylation, NF-κB p65 S276 phosphorylation, COX-2 upregulation and PTGES2 induction, which were ameliorated by PLPP/CIN deletion or IPA-3. Furthermore, IPA-3 pretreatment shortened the latency of seizure on-set without affecting seizure severity (intensity) and ameliorated CA3 neuronal death induced by KA. CONCLUSIONS: These findings indicate that PLPP/CIN may regulate seizure susceptibility (the latency of seizure on-set) and CA3 neuronal death in response to KA through NF2-PAK1-NF-κB-COX-2-PTGES2 signaling pathway.

Strain-Dependent Restriction of Human Cytomegalovirus by Zinc Finger Antiviral Proteins.

Cellular antiviral factors that recognize viral nucleic acid can inhibit virus replication. These include the zinc finger antiviral protein (ZAP), which recognizes high CpG dinucleotide content in viral RNA. Here, we investigated the ability of ZAP to inhibit the replication of human cytomegalovirus (HCMV). Depletion of ZAP or its cofactor KHNYN increased the titer of the high-passage HCMV strain AD169 but had little effect on the titer of the low-passage strain Merlin. We found no obvious difference in expression of several viral proteins between AD169 and Merlin in ZAP knockdown cells, but observed a larger increase in infectious virus in AD169 compared to Merlin in the absence of ZAP, suggesting that ZAP inhibited events late in AD169 replication. In addition, there was no clear difference in the CpG abundance of AD169 and Merlin RNAs, indicating that genomic content of the two virus strains was unlikely to be responsible for differences in their sensitivity to ZAP. Instead, we observed less ZAP expression in Merlin-infected cells late in replication compared to AD169-infected cells, which may be related to different abilities of the two virus strains to regulate interferon signaling. Therefore, there are strain-dependent differences in the sensitivity of HCMV to ZAP, and the ability of low-passage HCMV strain Merlin to evade inhibition by ZAP is likely related to its ability to regulate interferon signaling, not the CpG content of RNAs produced from its genome. IMPORTANCE Determining the function of cellular antiviral factors can inform our understanding of virus replication. The zinc finger antiviral protein (ZAP) can inhibit the replication of diverse viruses. Here, we examined ZAP interaction with the DNA virus human cytomegalovirus (HCMV). We found HCMV strain-dependent differences in the ability of ZAP to influence HCMV replication, which may be related to the interaction of HCMV strains with the type I interferon system. These observations affect our current understanding of how ZAP restricts HCMV and how HCMV interacts with the type I interferon system.

Locally released small (non-protein) ninhydrin-reacting molecules underlie developmental differences of cultured medullary versus spinal dorsal horn neurons.

Neurons located in the trigeminal subnucleus caudalis (Vc) play crucial roles in pain and sensorimotor functions in the orofacial region. Because of many anatomical and functional similarities with the spinal dorsal horn (SDH), Vc has been termed the medullary dorsal horn--analogous to the SDH. Here, we report that when compared with embryonic SDH neurons in culture, neurons isolated from the Vc region showed significantly slower growth, lower glutamate receptor activity, and more cells undergoing cell death. SDH neuron development was inhibited in co-cultures of SDH and Vc tissues while Vc neuron development was promoted by co-culture with SDH tissues. Furthermore, we identified that small (non-protein) ninhydrin-reacting molecules purified from either embryonic or post-natal Vc-conditioned medium inhibited neuronal growth whereas ninhydrin-reacting molecules from SDH-conditioned medium promoted neuronal growth. These findings suggest the involvement of locally released factors in the region-specific regulation of neuronal development in Vc and SDH, central nervous system regions playing critical roles in pain, and point to novel avenues for investigating central nervous system regionalization and for designing therapeutic approaches to manage neurodegenerative diseases and pain.

Point mutation in the NF2 gene of HEI-193 human schwannoma cells results in the expression of a merlin isoform with attenuated growth suppressive activity.

Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by the formation of bilateral schwannomas of the eighth cranial nerve. Although the protein product of the NF2 gene (merlin) is a classical tumor suppressor, the mechanism by which merlin suppresses cell proliferation is not fully understood. The availability of isolated tumor cells would facilitate a better understanding of the molecular function of merlin, but primary schwannoma cells obtained from patients grow slowly and do not yield adequate numbers for biochemical analysis. In this study, we have examined the NF2 mutation in HEI-193 cells, an immortalized cell line derived from the schwannoma of an NF2 patient. Previous work showed that the NF2 mutation in HEI-193 cells causes a splicing defect in the NF2 transcript. We have confirmed this result and further identified the resultant protein product as an isoform of merlin previously designated as isoform 3. The level of isoform 3 proteins in HEI-193 cells is comparable to the levels of merlin isoforms 1 and 2 in normal human Schwann cells and several other immortalized cell lines. In contrast to many mutant forms of merlin, isoform 3 is as resistant to proteasomal degradation as isoforms 1 and 2 and can interact with each of these isoforms in vivo. Cell proliferation assays showed that, in NF2(-/-) mouse embryonic fibroblasts, exogenously expressed merlin isoform 3 does exhibit growth suppressive activity although it is significantly lower than that of identically expressed merlin isoform 1. These results indicate that, although HEI-193 cells have undetectable levels of merlin isoforms 1 and 2, they are, in fact, not a merlin-null model because they express the moderately active growth suppressive merlin isoform 3.

A clue to the therapy of neurofibromatosis type 2: NF2/merlin is a PAK1 inhibitor.

BACKGROUND: Neurofibromatosis type 2 is a group of tumors caused by loss-of-function mutations of a tumor suppressor gene encoding NF2/merlin. Development of chemotherapeutics for this disease, which often threatens the life of young children, has been hampered by a limited information on the signaling function of NF2. NF2 can inhibit Ras-induced malignant transformation. However, the primary (signaling) target of NF2 in the oncogenic pathway has not been previously identified. RESULTS: Here, using a series of NF2 constructs, we show that NF2 inhibits directly the Rac/CDC42-dependent Ser/Thr kinase PAK1, which is essential for both Ras transformation and neurofibromatosis type 1 (NF1), through two separate domains. A mutant of NF2, that lacks the PAK1-inhibiting domain of 78 amino acids (NF78C, residues 447-524), fails to suppress Ras transformation. Furthermore, PAK1-specific inhibitors CEP-1347 and WR-PAK18 selectively inhibit the growth of NF2-deficient cancer cells, but not NF2-positive cells. CONCLUSIONS: These results suggest that PAK1 is essential for the malignant growth of NF2-deficient cells, and that PAK1-blocking drugs could be potentially useful forthe treatment of neurofibromatosis types 2, in addition to Ras-induced cancers and neurofibromatosis type 1.

Merlin neutralizes the inhibitory effect of Mdm2 on p53.

The stability of p53 tumor suppressor is regulated by Mdm2 via the ubiquitination and proteasome-mediated proteolysis pathway. The c-Abl and PTEN tumor suppressors are known to stabilize p53 by blocking the Mdm2-mediated p53 degradation. This study investigated the correlation between p53 and merlin, a neurofibromatosis 2 (NF2)-related tumor suppressor, in association with the Mdm2 function. The results showed that merlin increased the p53 stability by inhibiting the Mdm2-mediated degradation of p53, which accompanied the increase in the p53-dependent transcriptional activity. The stabilization of p53 by merlin appeared to be accomplished through Mdm2 degradation, and the N-terminal region of merlin was responsible for this novel activity. This study also showed that overexpression of merlin-induced apoptosis of cells depending preferentially on p53 in response to the serum starvation or a chemotherapeutic agent. These results suggest that merlin could be a positive regulator of p53 in terms of tumor suppressor activity, and provide the promising therapeutic means for treating tumors with non-functional merlin or Mdm2 overexpression.

Merlin, the product of the Nf2 tumor suppressor gene, is an inhibitor of the p21-activated kinase, Pak1.

The Nf2 tumor suppressor gene codes for merlin, a protein whose function has been elusive. We describe a novel interaction between merlin and p21-activated kinase 1 (Pak1), which is dynamic and facilitated upon increased cellular confluence. Merlin inhibits the activation of Pak1, as the loss of merlin expression results in the inappropriate activation of Pak1 under conditions associated with low basal activity. Conversely, the overexpression of merlin in cells that display a high basal activity of Pak1 resulted in the inhibition of Pak1 activation. This inhibitory function of merlin is mediated through its binding to the Pak1 PBD and by inhibiting Pak1 recruitment to focal adhesions. This link provides a possible mechanism for the effect of loss of merlin expression in tumorigenesis.

Merlin suppresses the SRE-dependent transcription by inhibiting the activation of Ras-ERK pathway.

The neurofibromatosis type 2 (NF2) gene encodes an intracellular membrane-associated protein called merlin or schwannomin, which is known to be a tumor suppressor. Numerous studies have suggested that merlin is involved in the regulation of cell growth and proliferation. Previously, merlin/schwannomin was reported to block Ras-induced cell proliferation and anchorage-independent cell growth. Also, the N-terminus of merlin was found to suppress cell proliferation, although it appears to be less effective than full-length merlin. However, the inhibitory mechanism of merlin is unknown. In this report, merlin is shown to be effective at suppressing serum/Ras-induced and Elk-mediated SRE dependent transactivation, and serum-induced ERK phosphorylation in NIH3T3 cells. In addition, merlin inhibited serum-induced Elk phosphorylation, a downstream effector of ERKs. Also, the N-terminal deficient merlin mutant could not block serum-induced and Elk-mediated SRE dependent transactivation, although the C-terminal deficient merlin mutant could. These results suggest that merlin inhibits SRE dependent transactivation by repressing serum-induced ERK phosphorylation and its downstream effector, Elk phosphorylation. Also, the N-terminus of merlin may be important for its inhibitory effect. Our results show that merlin acts as a negative regulator of the SRE signaling pathway via the Ras-ERKs pathway.