To Study The Function And Signal Pathway Of The Nystagmus-associated Protein FRMD7during Neuronal Development

Idiopathic congenital nystagmus （ICN） is an oculomotor disorder that arises from a primary defect in the brain regions involved in ocular motor control. It also termed as congenital motor nystagmus and exhibits various patterns of inheritance, although X-linked inheritance with incomplete penetrance and variable expressivity is probably the most common pattern. ICN is characterized by involuntary horizontal oscillations of both eyes, and its onset occurs within6months of birth. Patients with ICN experience a significant decrease in their quality of life over their lifetime, and there is no effective treatment available.Mutations of the FERM domain containing protein7（FRMD7; NM₁94277） gene are known to be one of the main causes of X-linked idiopathic congenital nystagmus in2006. To date, more than40mutations of FRMD7have been reported from various ethnic backgrounds. In2007, mutation screening in the FRMD7gene identified two novel missense mutations （c.781C>G and c.886G>C）（HM070040and HM070041） and one reported nonsense mutation （c.1003C>T）. This study widens the mutation spectrum of the FRMD7gene.The human FRMD7gene comprises12exons, encodes a714-residue polypeptide, and shares a four-point-one, ezrin, radixin, moesin （FERM） domain at its N-terminus. The FERM family of proteins regulates the adhesion and morphogenesis of cells by modulating changes in the cytoskeleton, where the FRMD7protein is thought to be involved in signal transduction between the plasma membrane and cytoskeleton. FRMD7downregulation has been shown to alter the development of neurites by influencing the dynamics of F-actin during retinoic acid （RA）-induced differentiation in mouse neuroblastoma （Neuro-2a） cells. However, the precise mechanism by which FRMD7influences F-actin, and its involvement in ICN pathogenesis, remains to be elucidated.Furthermore, FRMD7shares close amino acid sequence homology with two other FERM domain containing proteins:FARP1（FERM, RhoGEF and pleckstrin domain protein1; chondrocyte-derived ezrin-like protein; NM₀05766） and FARP2（FERM, RhoGEF and pleckstrin domain protein2; NM₀14808）. FARP1and FARP2play important roles in neuronal development through activating Rho GTPase signaling. Rho GTPases are key regulators of the actin cytoskeleton in eukaryotic cells and mediate morphological changes during neuronal development and plasticity, such as the growth of neurites, axon guidance and dendrite elaboration. RhoA, Cdc42and Racl form the archetypal trio of Rho GTPases whose function as signaling switches resides in their ability to cycle between active GTP-bound states and inactive GDP-bound states. The Rho GDP dissociation inhibitor （GDI） forms a complex with the GDP-bound inactive form of Rho GTPases and inhibits their activation. This last complex is not activated by the GDP/GTP exchange factor for Rho family members, suggesting the presence of another factor necessary for this activation.Therefore, in the present study, we investigated the function and signal conduction pathway of the FRMD7during neuronal development, the results include as follows:1.The immunohistochemistry experiments show at16-17wpc, FRMD7was observed to be highly expressed in the pons, medulla oblongata, and midbrain （brainstem）, which is the important region associated with ocular motor control. FRMD7was also observed in the cerebellar region and diencephalons at16-17wpc. During the later stages of fetal development （i.e.,21and25wpc）, overall expression of FRMD7was found to significantly decrease in the brainstem, and from the cortex, limited expression was manifested in the cortex plate.2. Immunoflurence show full-length FRMD7was found to be uniformly distributed in the cytoplasm. In contrast, when various regions of FRMD7were overexpressed, only the C-terminal regions of FRMD7（ΔFERM and ΔFERM+FA） resulted in a distribution pattern that was similar to that of full-length FRMD7. However, for these modified FRMD7proteins, the expression levels were lower, and some aggregates were observed in the cytoplasm. For FRMD7constructs containing only the N-terminal FERM domain （aa1-279） of FRMD7, or a combination of the FERM domain and the FA domain （adjacent to the FERM domain; aa294-336）, a predominantly nuclear distribution was observed.3. Both wild-type FRMD7and c.781C>G and c.886G>C FRMD7mutants exhibited a diffuse distribution in the cytoplasm, and also in actin-rich regions. However, another FRMD7mutant, c.1003C>T, exhibited changes in its subcellular distribution pattern and did not co-localize with F-actin.4. Overexpression of FRMD7showed a remarkable ability to increase the length of neurites compared with the control cells under the same serum-depleted RA-induced conditions. We observed a significant increase in neurite length after FRMD7transfection （control=68.99±2.59, FRMD7=94.40±2.62, P<0.01）. However, the number of cells with neurites was not significantly different （control38.63±3.65, FRMD741.87±3.46）.5. A time-course RT-PCR experiment was performed to study with eleven neuron-specific genes and their mRNA expression profiles after FRMD7transfection. The mRNA expression of Mtap2, neurofilament genes （NF-L and NF-M） and NeuN were highly upregulated48hours after transfection compared to control cells （P<0.05）. Other genes showed no significant alteration （P>0.05）.6. In vivo Co-IP experiments showed mFRMD7interact with RhoGDIa; in vitro GST pull down showed RhoGDIa directly interacted with full-length FRMD7, but not Nr-ferm （aa:1-279; the N-terminal FERM domain of the full-length FRMD7） or Cr-fragment （aa:280-703; truncated FERM domain of FRMD7）.7. Rho GTPases pull down assay revealed that extracts of Neuro-2a cells transfected with Racl and FRMD7contained increased amounts of GTP-Racl compared with the control cells. Conversely, little or no activation of RhoA or Cdc42was detected.8. Endogenous Racl was co-immunoprecipitated with Myc-tagged RhoGDIa （Myc-RhoGDIa） from the lysate of Neuro-2a cells transiently expressing Myc-RhoGDIa alone, however, in the lysate of cells transiently expressing both Myc-RhoGDIα and FLAG-tagged FRMD7, less endogenous Racl was co-immunoprecipitated with Myc-RhoGDIa （p<0.05）, but FLAG-tagged FRMD7was co-immunoprecipitated with Myc-RhoGDIα.9. Mouse embryonic fibroblast cell line （NIH3T3） transformation assay showed GFP-FRMD7-transfected cells displayed lamellipodia and ruffles, indicative of Racl. Stress fiber formation was significantly suppressed in the cells overexpressing FRMD7compared with the control cells expressing GFP.10. Racl activation experiments showed that the human wild-type FRMD7protein had the ability to activate Racl signaling, however two missense mutant FRMD7（c.781C>G and c.886G>C） proteins previously reported by us generated less GTP-Racl compared with the wild-type （P<0.05）. Furthermore, little activation of Racl was detected in another truncated mutant-type group, c.1003C>T,（P<0.01）.11. In vivo Co-IP experiments showed human wild-type FRMD7interact with RhoGDIa; however three mutation-type FRMD7proteins have weaken this ability to interact with RhoGDIa and released less Racl from Racl-RhoGDIα complex compared to the wild-type.Collectively, our results indicate that FRMD7may play an important role in the brainstem in the early stages of development of the human fetal brain, and provides clues for the mechanism of mutation FRMD7, which may be involved in influencing F-actin dynamics. Furthermore, FRMD7promotes the extension of neurites and may be involved in regulating the movement of cytoskeletal proteins, which influences not only F-actin but also neurofilament and microtubule dynamics. Finally, FRMD7interacts with one of the Rho GTPase regulators, RhoGDIa, and activates the Rho subfamily member Racl, which regulates reorganization of actin filaments and controls neuronal outgrowth. We also demonstrate that human mutant FRMD7influences Racl signaling activation by weakening the ability to interact with RhoGDIα that leading to less release Racl from Racl-RhoGDIα complex, which can lead to abnormal neuronal outgrowth and cause the X-linked ICN.