Lingzhen Song

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.

Protective effects of lithium against lead-induced toxicities in multiple systems of adult mouse

Occupational and environmental exposures to lead (Pb), one of the toxic metal pollutants, is of global concern. The present study aims to investigate the protective effects of lithium (Li) against Pb-induced damage in vivo and in vitro. For this purpose, 3-month-old mice received Li (250 mg per kg body weight, i.p.) and 2 hours later water containing Pb (20 mg per kg body weight, i.p.) for 2-weeks. Treatment of mice with Pb induced remarkable morphological damage in multiple organs, such as swelling and necrosis in the liver, kidney and spleen. Immunohistochemistry demonstrated that the number of newly generated cells and immature neurons in the hippocampus was significantly decreased in mice exposed to Pb when compared with those that received saline for control or Li. Furthermore, in mice exposed to Pb a higher percentage of newly generated cells differentiated into glial cells and fewer into neurons, and less newborn cells survived compared to those in controls and Li-treated mice. In mice exposed to Pb cognitive tests were impaired. Interestingly, pre-administration of Li markedly decreased Pb-induced pathological and neurological lesions in vivo and in vitro. Specifically, the reduction of hippocampal neurogenesis resulting from Pb exposure was prevented by administration of Li. In addition, we found that pretreatment with Li effectively prevented cognitive impairment in mice exposed to Pb. Furthermore, Li pretreatment significantly improved Pb-induced depletion in p-GSK-3β (Ser9) and microRNA-34c levels in the hippocampus. Collectively our findings point to the capacity of Li to attenuate Pb-induced damage.

Morphological changes of radial glial cells during mouse embryonic development.

During brain development, the radial glial cell acts as a scaffold to support radial migration of postmitotic neurons. However, the morphological changes of radial glial cells during embryo development are poorly understood. We used in utero electroporation and immunohistochemistry to study the dynamics of radial glial cells accompanied by cortical development in mice from embryonic day 14 to postnatal day 0. We found that different segments of radial glial cells changed by the growth of different layers of cortex, such as marginal zone, cortical plate, intermediate zone and ventricular zone. Moreover, the length, angle and number of branches of the radial glial cell changed significantly at the late stage of neurogenesis. All these changes were consistent with the distinct phases of locomotion. Thus, we speculated that morphological changes of the radial glial cell were associated with the neuronal migration and dendritic development.

Subcellular localization and function of mouse radial spoke protein 3 in mammalian cells and central nervous system

Radial spoke protein 3 (RSP3) was first identified in Chlamydomonas as a component of the radial spoke. The mammalian homologue of the Chlamydomonas RSP3 gene is mainly expressed in testis and developing central nervous system (CNS). However, the subcellular localization and function of mammalian RSP3 in the developing brain and mammalian cells remain poorly understood. Here we show that the mouse RSP3 accumulates at the perinuclear region of Chinese hamster ovary (CHO) and 293T cells. Detailed analysis shows that the mouse RSP3 is not co-localized with the endoplasmic reticulum or Golgi apparatus markers in CHO cells. Using in utero electroporation, we found that over-expression of mammalian RSP3 increases the percentage of neurons reaching the upper cortical plate. In vivo analysis shows that the mouse RSP3 mainly accumulates in the proximal cytoplasmic dilation of the leading process of the migrating cortical neurons. Furthermore, we find that the mammalian RSP3 concentrates in the ependymal cilia as a component of the cilia. Thus, our data provide the first evidence for the subcellular localization and function of mammalian RSP3 in mammalian cells and developing CNS.