Cocaine regulates ezrin–radixin–moesin proteins and RhoA signaling in the nucleus accumbens

The ezrin–radixin–moesin (ERM) proteins are a family of widely distributed membrane-associated proteins and have been implicated not only in cell-shape determination but also in signaling pathway. The nucleus accumbens (NAcc) is an important neuronal substrate mediating the effects of drugs of abuse. However, it has not been determined yet how ERM proteins are regulated in this site by drugs of abuse. Here we show in rat that the phosphorylation levels of ERM protein are dose- and time-dependently decreased in the NAcc by a single injection of cocaine (15 or 30 mg/kg i.p.). Further, we show that the amount of active RhoA, a small GTPase protein, is significantly reduced in the NAcc by cocaine, while the phosphorylation levels of ERM protein are also decreased by bilateral microinjections in this site of the Rho kinase inhibitors. Together, these results suggest that cocaine reduces phosphorylated ERM levels in the NAcc by making downregulation of RhoA–Rho kinase signaling, which may importantly contribute to initiate synaptic changes in this site leading to drug addiction.     

LyGDI functions in cancer metastasis by anchoring Rho proteins to the cell membrane

Rho family GTPases play an important role in a number of processes related to metastasis, and RhoGDP dissociation, inhibitors (RhoGDIs) regulate Rho family proteins. We cloned genomic DNA from colon carcinoma SW480 cells capable of transforming nonmetastatic ras-transformed 1-1ras1000 cells into metastatic cells. This DNA contained a truncated human ras homolog gene family GDP dissociation inhibitor beta (ARHGDIB) gene, resulting in a C-terminal truncated form of LyGDI (Delta C-LyGDI, 166-201 deletion), a member of the RhoGDIs. The stable expression of Delta C-LyGDI induced pulmonary metastasis in 1-1ras1000 cells, whereas expression of full-length LyGDI did not induce metastasis. Delta C-LyGDI was preferentially localized in the membrane, detected in a NP-40-insoluble fraction, and co-purified with radixin, moesin, Rac1, Cdc42, and RhoA. In Delta C-LyGDI transfectant, an activation state of Rac1 was elevated and Delta C-LyGDI was associated with Rac1-GTP. In keeping with the observed localization of Rac1 to the cell membrane and the elevated level of Rac1-GTP, Delta C-LyGDI transfectants were found to be more invasive than mock transfectant. These results suggest that LyGDI functions in the cell membrane to afford spatial regulation of Rho family GTPase signaling through ezrin radixin moesin (ERM) proteins during metastasis.     

Evaluation of dual nasal delivery of infectious hematopoietic necrosis virus and enteric red mouth vaccines in rainbow trout (Oncorhynchus mykiss)

Farmed fish are susceptible to different infectious disease agents including viruses and bacteria. Thus, multivalent vaccines or vaccination programs against two or more pathogens are valuable tools in aquaculture. Recently, nasal vaccines have been shown to be very effective in rainbow trout. The current study investigates, for the first time, the use of the nasal route in dual vaccination trials against two important aquatic diseases, infectious hematopoietic necrosis virus (IHN) and enteric red mouth (ERM) disease. Rainbow trout received live attenuated IHN virus (IHNV) vaccine and the ERM bacterin using four different vaccine delivery methods and were challenged with virulent IHNV or Yersinia ruckeri 7 (100 deg day) and 28 (400 deg day) days post-vaccination. The highest survival rates against IHNV at day 7 were obtained by nasal vaccination either when IHNV and ERM were delivered separately into each nare or when they were premixed and delivered to both nasal rosettes (group D). Protection at 28 days against IHNV was similar in all four vaccinated groups. Early protection against ERM was highest in fish that received each vaccine in separate nares (group B), whereas protection at 28 days was highest in the i.p. vaccinated group (group E), followed by the nasally vaccinated group (group B). Survival results were supported by histological observations of the left and right olfactory organ which showed strong immune responses one day (14 deg days) after vaccination in group B vaccinated fish. These data indicate that dual vaccination against two different pathogens via the nasal route is a very effective vaccination strategy for use in aquaculture, particularly when each nare is used separately during delivery. Further long-term studies should evaluate the contribution of adaptive immunity to the protection levels observed.     

Inhibition of RAS Activation Due to a Homozygous Ezrin Variant in Patients with Profound Intellectual Disability

Gain‐of‐function alterations in several components and modulators of the Ras‐MAPK pathway lead to dysregulation of the pathway and cause a broad spectrum of autosomal dominant developmental disorders, collectively known as RASopathies. These findings demonstrate the importance of tight multilevel Ras regulation to safeguard signaling output and prevent aberrant activity. We have recently identified ezrin as a novel regulatory element required for Ras activation. Homozygosity mapping and exome sequencing have now revealed the first presumably disease‐causing variant in the coding gene EZR in two siblings with a profound intellectual disability. Localization and membrane targeting of the altered ezrin protein appeared normal but molecular modeling suggested protein interaction surfaces to be disturbed. Functional analysis revealed that the altered ezrin protein is no longer able to bind Ras and facilitate its activation. Furthermore, expression of the altered ezrin protein in different cell lines resulted in abnormal cellular processes, including reduced proliferation and neuritogenesis, thus revealing a possible mechanism for its phenotype in humans. To our knowledge, this is the first report of an autosomal recessively inherited loss‐of‐function mutation causing reduced Ras activity and thus extends and complements the pathogenicity spectrum of known Ras‐MAPK pathway disturbances.     

Juxtanodin in retinal pigment epithelial cells: Expression and biological activities in regulating cell morphology and actin cytoskeleton organization

Juxtanodin (JN, also known as ermin) was initially identified as an actin cytoskeleton‐related oligodendroglial protein in the rat central nervous system. It was subsequently also found in the rat olfactory neuroepithelium, especially at the apical junctional belt of the sustentacular cells. We further examined JN expression and functional roles in the retina using fluorescence histochemistry, confocal microscopy, immuno‐electron microscopy, molecular biology, and cell culture. Prominent JN expression was found in the photoreceptor‐supporting retinal pigment epithelium (RPE), especially in a zone corresponding to the apices of RPE cells, at the roots of the RPE microvilli, and at the base of RPE cells next to the Bruch's membrane. Partial co‐localization of JN immunoreactivity with F‐actin (labeled with phalloidin) was observed at the apices and bases of RPE cells. No JN was detected in other cell types of the retina. In cultured human RPE cell line ARPE‐19, expression of extrinsic JN up‐regulated formation of actin cytoskeleton stress fibers, caused redistribution of more F‐actin fibers to the cell periphery, and promoted spreading/enlargement of transfected cells. These findings suggest possible roles of JN in RPE molecular transport, phagocytosis and formation of outer blood‐retinal barrier, or possible involvement of JN expression perturbations in pathogenesis of such retinal disorders as proliferative vitreoretinopathy and age‐related macular degeneration.