Shanting Zhao

Src controls neuronal migration by regulating the activity of FAK and cofilin.

Migration of postmitotic neurons in the developing cortex along radial glial fiber is essential for the formation of cortical layers. Several neurological diseases are caused by defects in neuronal migration, underlining the importance of this process for brain function. Multiple molecules are involved in this process. However, the precise mechanisms are largely unknown. In the present study, we examined the expression of Src in the developing cortex and investigated the role of Src in neuronal migration and its cellular and molecular mechanisms. Our results showed that Src was strongly expressed in the cerebral cortex during corticogenesis and mainly targeted to the leading processes of migrating neurons. Overexpression of wildtype Src (Src-WT) and its mutants, constitutively active Src (Src-CA) and dominant negative Src (Src-DN) in the mouse brain by in utero electroporation perturbed neuronal migration through affecting the adhesion properties and cytoskeletal dynamics of migrating neurons. Overexpression of Src-WT and Src-CA induced aggregation and branching of migrating neurons, whereas overexpression of Src-DN led to abnormal elongation of the leading processes of migrating neurons. Furthermore, we showed that Src activates the focal adhesion kinase (FAK) and cofilin by regulating their phosphorylation levels. We conclude that Src controls neuronal migration by regulating adhesion properties and F-actin dynamics of migrating neurons.

Exposure to swainsonine impairs adult neurogenesis and spatial learning and memory

Swainsonine (SW) is an indolizidine triol plant alkaloid isolated from the species Astragalus, colloquially termed locoweed. Ingestion induces severe neurological symptoms of livestock and wildlife, including ataxia, trembling, exaggerated fright reactions. Toxicity to the central and peripheral nervous system is caused by inhibition of lysosomal a-mannosidase (AMA) and accumulation of intracellular oligosaccharide. However, the effects of SW on adult neurogenesis and cognition have remained unclear. Therefore, the present study was conducted to examine the effects of SW on adult neurogenesis and learning as well as memory performance in adult mice. SW (10μg/mL in drinking water) was administered orally to mice for 4 weeks. Our results showed that SW reduced proliferation and survival of neural progenitor cells (NPCs) in culture, and in the hippocampus of adult mice. In addition, exposure to SW led to down-regulation of doublecortin (DCX) and synaptophysin (SYP) in the hippocampus. However, caspase 3 and glial fibrillary acidic protein (GFAP) levels were significantly increased in SW-treated mice. Finally, SW-treated mice exhibited deficits in hippocampus-dependent spatial learning and memory. Our findings suggest that SW affects adult neurogenesis and cognitive function.

Spag6 Negatively Regulates Neuronal Migration During Mouse Brain Development

Sperm-associated antigen 6 (Spag6) is a Chlamydomonas reinhardtii PF16 homologous gene detected in the human testis and is crucial for sperm motility. Neuronal migration is a dynamic process requiring coordinated cytoskeletal remodeling, and Spag6 is co-localized with microtubules in Chinese hamster ovary cells and COS-1 cells. However, the role of Spag6 in neuronal migration remains unclear. Here, we demonstrated that Spag6 was continuously expressed in the developing cerebral cortex. Using in utero electroporation (IUE), we found that overexpression of Spag6 delayed the rate of neuronal migration, rather than affecting the ultimate fate of cortical neurons. Furthermore, overexpression of Spag6 caused a significant decrease in neurite number and length of cortical neurons. Our results indicated that Spag6 controlled neuronal migration as well as neurite branching and elongation.

The aspartic acid of Fyn at 390 is critical for neuronal migration during corticogenesis

The mammalian cerebral cortex develops through the coordinated migration of postmitotic neurons. Fyn, a member of the Src tyrosine kinase family (SFKs), is involved in the neuronal migration and the absence of Fyn leads to abnormal migration. However, the molecular mechanism whereby Fyn acts on migrating neurons has remained unclear. Here, we employed two Fyn mutants (Fyn259T and FynD390A) to investigate the function of Fyn kinase domain in neuronal migration. Using in utero electroporation, we co-transfected the migrating neurons in embryonic cortex with these mutants combined with plasmid expressing GFP. Interestingly, although both of them impaired neuronal migration, FynD390A, rather than Fyn259T, induced remarkable morphology change. Our work provides in vivo and in vitro evidence that the aspartic acid of Fyn at 390 is indispensable for the radial migration, and it is required for precise cooperation with focal adhesion kinase.

The function of sperm-associated antigen 6 in neuronal proliferation and differentiation

Sperm-associated antigen 6 (SPAG6) is initially found in human testis and is essential for sperm motility and male fertility. Later studies indicate that it also express in the chick Central Nervous System and human embryonic stem cells. However, the function of Spag6 in cortical development is still largely unclear. Using in utero electroporation, we showed that overexpression of Spag6 induced the transfected cells excluded from the proliferation zone of the mouse cortex. Ki67 Co-labeling and BrdU incorporation experiment suggested that overexpression of Spag6 inhibited proliferation of neural progenitor cells. Furthermore, we demonstrated that Spag6-overexpressing cells preferred to differentiated into neurons, which could be labeled by Brn2, rather than GFAP positive astrocytes. Taken together, our data indicate that Spag6 plays an essential role in the process of neuronal proliferation and differentiation.

The SH2 domain is crucial for function of Fyn in neuronal migration and cortical lamination

Neurons in the developing brain form the cortical plate (CP) in an inside-out manner, in which the late-born neurons are located more superficially than the early-born neurons. Fyn, a member of the Src family kinases, plays an important role in neuronal migration by binding to many substrates. However, the role of the Src-homology 2 (SH2) domain in function of Fyn in neuronal migration remains poorly understood. Here, we demonstrate that the SH2 domain is essential for the action of Fyn in neuronal migration and cortical lamination. A point mutation in the Fyn SH2 domain (FynR176A) impaired neuronal migration and their final location in the cerebral cortex, by inducing neuronal aggregation and branching. Thus, we provide the first evidence of the Fyn SH2 domain contributing to neuronal migration and neuronal morphogenesis. [BMB Reports 2015; 48(2): 97-102]

The mouse radial spoke protein 3 is a nucleocytoplasmic shuttling protein that promotes neurogenesis

Radial spoke protein 3 (RSP3) was first identified in Chlamydomonas as a component of radial spoke, which is important for flagellar motility. The mammalian homolog of the Chlamydomonas RSP3 protein is found to be a mammalian protein kinase A-anchoring protein that binds ERK1/2. Here we show that mouse RSP3 is a nucleocytoplasmic shuttling protein. The full-length RSP3–EGFP fusion protein is mainly located in the cytoplasm of Chinese hamster ovary cells. However, by using deletion mutants of RSP3, we identified two nuclear localization signals and a nuclear export signal in RSP3. Moreover, using in utero electroporation, we found that overexpression of RSP3 in the developing cerebral cortex promotes neurogenesis. The layer II/III of the neocortex was much thicker in the RSP3-transfected region than that of the untransfected region in the neocortex. We also show that RSP3 is specifically located in the primary cilia of the radial glial cells, where it acts as a signaling mediator that regulates neurogenesis. Thus, our results suggest that RSP3 is a nucleocytoplasmic shuttling protein and plays an essential role in neurogenesis.

Neuronal maturation and laminar formation in the chicken optic tectum are accompanied by the transition of phosphorylated cofilin from cytoplasm to nucleus.

Laminar formation in the chicken optic tectum requires processes that coordinate proliferation, migration and differentiation of neurons, in which the dynamics of actin filaments are crucial. Cofilin plays pivotal roles in regulating actin arrangement via its phosphorylation on Ser3. Given poor studies on the profile of phosphorylated cofilin (p-cofilin) in the developing tectum, we investigated its expression pattern. As determined by immunofluorescence histochemistry and western blotting, p-cofilin could be detected in most tectal layers except for the neural epithelium. In addition, we found p-cofilin was expressed both in the cytoplasm and the nucleus. During development, the expression of the cytoplasmic p-cofilin was decreasing and the nuclear p-cofilin was gradually increasing, but the total level of p-cofilin was down regulated. Double-labeling experiments revealed that the nuclear p-cofilin could be labeled in mature neurons but undetected in immature neurons. Furthermore, the number of cells co-stained with nuclear p-cofilin and NeuN was up-regulated during lamination and 60% cells were detected to be mature neurons that can express nuclear p-cofilin just at the first appearance of completed laminae. Our results demonstrate that the maturation of neurons is accompanied by this cytoplasm-to-nucleus transition of p-cofilin, and the nuclear p-cofilin can work effectively as a marker in the laminar formation of the chicken optic tectum.

Aberrant expression of LIMK1 impairs neuronal migration during neocortex development.

Neuronal migration is essential for the formation of cortical layers, and proper neuronal migration requires the coordination of cytoskeletal regulation. LIMK1 is a serine/threonine protein kinase that mediates actin dynamics by regulating actin depolymerization factor/cofilin. However, the role of LIMK1 in neuronal migration and its potential mechanism remains elusive. Here, we found that using the in utero electroporation to overexpress LIMK1 and its mutants, constitutively active LIMK1 (LIMK1-CA) and dominant-negative LIMK1 (LIMK1-DN), impaired neuronal migration in the embryonic mouse brain. In addition, the aberrant expression of LIMK1-WT and LIMK1-CA induced abnormal branching and increased the length of the leading process, while LIMK1-DN-transfected neurons gave rise to two leading processes. Furthermore, the co-transfection of LIMK1-CA and cofilin-S3A partially rescued the migration deficiency and fully rescued the morphological changes in migrating neurons induced by LIMK1-CA. Our results indicated that LIMK1 negatively regulated neuronal migration by affecting the neuronal cytoskeleton and that its effects were partly mediated by cofilin phosphorylation.

N-cadherin regulates beta-catenin signal and its misexpression perturbs commissural axon projection in the developing chicken spinal cord

N-cadherin is a calcium-sensitive cell adhesion molecule that plays an important role in the formation of the neural circuit and the development of the nervous system. In the present study, we investigated the function of N-cadherin in cell–cell connection in vitro with HEK293T cells, and in commissural axon projections in the developing chicken spinal cord using in ovo electroporation. Cell–cell connections increased with N-cadherin overexpression in HEK293T cells, while cell contacts disappeared after co-transfection with an N-cadherin-shRNA plasmid. The knockdown of N-cadherin caused the accumulation of β-catenin in the nucleus, supporting the notion that N-cadherin regulates β-catenin signaling in vitro. Furthermore, N-cadherin misexpression perturbed commissural axon projections in the spinal cord. The overexpression of N-cadherin reduced the number of axons that projected alongside the contralateral margin of the floor plate, and formed intermediate longitudinal commissural axons. In contrast, the knockdown of N-cadherin perturbed commissural axon projections significantly, affecting the projections alongside the contralateral margin of the floor plate, but did not affect intermediate longitudinal commissural axons. Taken together, these findings suggest that N-cadherin regulates commissural axon projections in the developing chicken spinal cord.