| Mammalian neocortex contains six layers and is formed by the directional migration of post-mitotic neurons from the ventricular zone toward the pial surface.When neurons reach their destinations in the cortical plate,apical dendrites extended up to the pial surface,and their axons grow down to the ventricle to establish synaptic connections.Neuronal migration,which is critical for brain function,is a multifaceted process and dependent on a wide variety of cellular functions that modulate cell shape,polarity and motility.Several neurological diseases are caused by defects in neuronal migration,including epilepsy,dysgnosia and mental retardation.Normal central nervous system development relies upon the presence of LIMK1,deletion of which has been implicated in the development of Williams syndrome,a complex human developmental disorder characterized by mental retardation and profound deficits in visuospatial cognition.The LIM kinase family is composed of LIMK1 and LIMK2.LIMK1 contains two LIM domains,an S/P-rich domain and a kinase domain,and it plays a central role in numerous cellular processes,including cell proliferation,the establishment of cell morphology,cell motility and structural remodeling.LIMK1 is restricted to neuronal tissues and accumulates at high levels in mature synapses.In the nervous system,it has been shown that LIMK1 is important for neurite outgrowth,spine morphology and synaptic plasticity.LIMK1 knockout in mice or genetic mutation in humans is associated with spine abnormalities and cognitive impairments.Moreover,the overexpression of wildtype LIMK1 in cultured hippocampal pyramidal neurons induces growth cone formation and axon outgrowth at early time points.LIMK1 is activated downstream of Rho and Rac1,and evidences indicate that ROCK activates LIMK1 in vitro and in vivo by phosphorylation at Thr-508.In addition,as a serine/threonine kinase,LIMK1 influences the architecture of the actin cytoskeleton by phosphorylating and inactivating its substrate,Cofilin family proteins.However,it remains largely unknown whether LIMK1 functions in neuronal migration,and if so,how it contributes to those deficits.In present study,overexpression of LIMK1 and its mutants,constitutively active LIMK1(LIMK1-CA)and dominant-negative LIMK1(LIMK1-DN)were constructed.Using in utero electroporation,combined with immunochemistry were used to study the effect of LIMK1 on neuronal migration.To further study the mechanism of LIMK1's influence on neuronal migration,we constructed Cofilin-S3 A to rescue the migration deficiency,and clarify the relationship between LIMK1 and Cofilin on the process of neuronal migration.Here,we provide some main findings:1.We successfully cloned LIMK1 gene and constructed the LIMK1 overexpression plasmid.Using in utero electroporation,we overexpressed LIMK1-WT in the developing mouse cortical neurons at E15.5 and found overexpression of LIMK1-WT significantly inhibited neuronal migration,induced neurites and promoted the branching of leading process.2.Using point mutation,we constructed LIMK1 dominant negative plasmid,LIMK1-DN.We transfected the plasmid into the mouse cortical neurons and found LIMK1-DN not only blocked neuronal migration,but also induced the quantity of leading processes.3.To elucidate the phosphorylation site of LIMK1 in cortical development,we constructed continuously active form of LIMK1,LIMK1-CA.the result showed LIMK1-CA significantly impaired neuronal migration,and influenced neuronal morphology,the length of LIMK1-CA changed obviously.4.To clarify the mechanism of migration deficiency,we used point mutant to overexpress the nonphosphorylated substrate of limk1,Cofilin-S3 A.Co-transfected LIMK1-CA and Cofilin-S3 A efficiently rescued migration deficiency.Under high-power magnification,we surprisely observed that the length of leading process was resumed.Taken together,we hypothesized that there is a balance between activation and inactivation of LIMK1 in vivo.Once the balance was broken,neuronal migration would be blocked. |
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