Xiaojuan Zhu

PtdIns (3,4,5) P3 Recruitment of Myo10 Is Essential for Axon Development

Myosin X (Myo10) with pleckstrin homology (PH) domains is a motor protein acting in filopodium initiation and extension. However, its potential role has not been fully understood, especially in neuronal development. In the present study the preferential accumulation of Myo10 in axon tips has been revealed in primary culture of hippocampal neurons with the aid of immunofluorescence from anti-Myo10 antibody in combination with anti-Tuj1 antibody as specific marker. Knocking down Myo10 gene transcription impaired outgrowth of axon with loss of Tau-1-positive phenotype. Interestingly, inhibition of actin polymerization by cytochalasin D rescued the defect of axon outgrowth. Furthermore, ectopic expression of Myo10 with enhanced green fluorescence protein (EGFP) labeled Myo10 mutants induced multiple axon-like neurites in a motor-independent way. Mechanism studies demonstrated that the recruitment of Myo10 through its PH domain to phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5) P3) was essential for axon formation. In addition, in vivo studies confirmed that Myo10 was required for neuronal morphological transition during radial neuronal migration in the developmental neocortex.

Both Myosin-10 Isoforms Are Required for Radial Neuronal Migration in the Developing Cerebral Cortex

During embryonic development of the mammalian cerebral cortex, postmitotic cortical neurons migrate radially from the ventricular zone to the cortical plate. Proper migration involves the correct orientation of migrating neurons and the transition from a multipolar to a mature bipolar morphology. Herein, we report that the 2 isoforms of Myosin-10 (Myo10) play distinct roles in the regulation of radial migration in the mouse cortex. We show that the full-length Myo10 (fMyo10) isoform is located in deeper layers of the cortex and is involved in establishing proper migration orientation. We also demonstrate that fMyo10-dependent orientation of radial migration is mediated at least in part by the netrin-1 receptor deleted in colorectal cancer. Moreover, we show that the headless Myo10 (hMyo10) isoform is required for the transition from multipolar to bipolar morphologies in the intermediate zone. Our study reveals divergent functions for the 2 Myo10 isoforms in controlling both the direction of migration and neuronal morphogenesis during radial cortical neuronal migration.

Down-regulation of neogenin accelerated glioma progression through promoter Methylation and its overexpression in SHG-44 Induced Apoptosis.

Background Dependence receptors have been proved to act as tumor suppressors in tumorigenesis. Neogenin, a DCC homologue, well known for its fundamental role in axon guidance and cellular differentiation, is also a dependence receptor functioning to control apoptosis. However, loss of neogenin has been reported in several kinds of cancers, but its role in glioma remains to be further investigated. Methodology/Principal Findings Western blot analysis showed that neogenin level was lower in glioma tissues than in their matching surrounding non-neoplastic tissues (n = 13, p<0.01). By immunohistochemical analysis of 69 primary and 16 paired initial and recurrent glioma sections, we found that the loss of neogenin did not only correlate negatively with glioma malignancy (n = 69, p<0.01), but also glioma recurrence (n = 16, p<0.05). Kaplan-Meier plot and Cox proportional hazards modelling showed that over-expressive neogenin could prolong the tumor latency (n = 69, p<0.001, 1187.6±162.6 days versus 687.4±254.2 days) and restrain high-grade glioma development (n = 69, p<0.01, HR: 0.264, 95% CI: 0.102 to 0.687). By Methylation specific polymerase chain reaction (MSP), we reported that neogenin promoter was methylated in 31.0% (9/29) gliomas, but absent in 3 kinds of glioma cell lines. Interestingly, the prevalence of methylation in high-grade gliomas was higher than low-grade gliomas and non-neoplastic brain tissues (n = 33, p<0.05) and overall methylation rate increased as glioma malignancy advanced. Furthermore, when cells were over-expressed by neogenin, the apoptotic rate in SHG-44 was increased to 39.7% compared with 8.1% in the blank control (p<0.01) and 9.3% in the negative control (p<0.01). Conclusions/Significance These observations recapitulated the proposed role of neogenin as a tumor suppressor in gliomas and we suggest its down-regulation owing to promoter methylation is a selective advantage for glioma genesis, progression and recurrence. Furthermore, the induction of apoptosis in SHG-44 cells after overexpression of neogenin, indicated that neogenin could be a novel target for glioma therapy.

Epigenetic Regulation of Dpp6 Expression by Dnmt3b and Its Novel Role in the Inhibition of RA Induced Neuronal Differentiation of P19 Cells

DNA methylation is an important mechanism of gene silencing in mammals catalyzed by a group of DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b which are required for the establishment of genomic methylation patterns during development and differentiation. In this report, we studied the role of DNA methyltransferases during retinoic acid induced neuronal differentiation of P19 cells. We observed an increase in the mRNA and protein level of Dnmt3b, whereas the expression of Dnmt1 and Dnmt3a was decreased after RA treatment of P19 cells which indicated that Dnmt3b is more important during neuronal differentiation of P19 cells. Dnmt3b enriched chromatin library from RA treated P19 cells identified dipeptidyl peptidase 6 (Dpp6) gene as a novel target of Dnmt3b. Further, quantitative ChIP analysis showed that the amount of Dnmt3b recruited on Dpp6 promoter was equal in both RA treated as well as untreated p19 cells. Bisulfite genomic sequencing, COBRA, and methylation specific PCR analysis revealed that Dpp6 promoter was heavily methylated in both RA treated and untreated P19 cells. Dnmt3b was responsible for transcriptional silencing of Dpp6 gene as depletion of Dnmt3b resulted in increased mRNA and protein expression of Dpp6. Consequently, the average methylation of Dpp6 gene promoter was reduced to half in Dnmt3b knockdown cells. In the absence of Dnmt3b, Dnmt3a was associated with Dpp6 gene promoter and regulated its expression and methylation in P19 cells. RA induced neuronal differentiation was inhibited upon ectopic expression of Dpp6 in P19 cells. Taken together, the present study described epigenetic silencing of Dpp6 expression by DNA methylation and established that its ectopic expression can act as negative signal during RA induced neuronal differentiation of P19 cells.

RING finger protein 10 regulates retinoic acid-induced neuronal differentiation and the cell cycle exit of P19 embryonic carcinoma cells.

Rnf10 is a member of the RING finger protein family. Recently, a number of RING finger proteins were reported to be involved in neuronal differentiation, development, and proliferation. In this study, we observed that the mRNA levels and protein expression of Rnf10 increase significantly upon the retinoic acid‐induced neuronal differentiation of P19 cells. Knockdown of Rnf10 by RNA interference significantly impaired neuronal differentiation of P19 cells by attenuating the expression of neuronal markers. Cell cycle profiling revealed that Rnf10‐depleted cells were unable to establish cell cycle arrest after RA treatment. In agreement with flow cytometry analysis, increased cell proliferation was observed after RA induction in Rnf10 knockdown cells as determined by a BrdU incorporation assay. Moreover, like Rnf10, the mRNA levels and protein expression of p21 and p27 also increased upon RA induction. Rnf10 knockdown only resulted in a reduction of p21 expression, while p27 and p57 expression remained unchanged, indicating that Rnf10 may regulate cell cycle exit through the p21 pathway. Ectopic p21 expression partially rescued the effect of Rnf10 depletion on the neuronal differentiation of P19 cells. Collectively, these results showed that increase in Rnf10 expression upon RA induction is necessary for the positive regulation of cyclin kinase inhibitor p21 expression, which leads to cell cycle arrest and is critical for neuronal differentiation. J. Cell. Biochem. 114: 2007–2015, 2013.

Doxycycline can stimulate cytoprotection in neural stem cells with oxygen-glucose deprivation-reoxygenation injury: A potential approach to enhance effectiveness of cell transplantation therapy

A substantial loss of transplanted neural stem cells is a major limitation to cell transplantation therapy of stroke. In this study, we provided in vitro evidence that doxycycline preconditioning of neural stem cells have resulted in decreased cell death and increased cell viability after oxygen-glucose deprivation-reoxygenation conditions that best mimics cerebral ischemia-reperfusion injury. Resistance to oxidative stress is one of the mechanisms of doxycycline-induced cytoprotection in neural stem cells as it significantly reduced the superoxide anion production. Moreover, doxycycline preconditioning also induced the expression of Nrf2 which is a basic transcription factor for a series of antioxidative and cytoprotective genes. Collectively, we suggested that doxycycline preconditioning of neural stem cells is a potential strategy to improve effectiveness of cell transplantation therapy.

Headless Myo10 is a regulator of microtubule stability during neuronal development.

Stabilized microtubules are required for neuronal morphogenesis and migration. However, the underlying mechanism is not fully understood. In this study, we demonstrate that myosin X (Myo10), which is composed of full‐length myosin X (fMyo10) and headless myosin X (hMyo10), is important for axon development. fMyo10 is involved in axon elongation, whereas hMyo10 is critical for Tau‐1 positive axon formation through stabilizing microtubules. Furthermore, in vivo studies reveal that hMyo10‐mediated microtubule stability has a profound effect on both neuronal migration and dendritic arborization in the mammalian cerebral cortex. Taken together, our findings suggest that hMyo10 is involved in neuronal development both in vitro and in vivo by regulating microtubule stability.

Myosin X regulates neuronal radial migration through interacting with N-cadherin.

Proper brain function depends on correct neuronal migration during development, which is known to be regulated by cytoskeletal dynamics and cell-cell adhesion. Myosin X (Myo10), an uncharacteristic member of the myosin family, is an important regulator of cytoskeleton that modulates cell motilities in many different cellular contexts. We previously reported that Myo10 was required for neuronal migration in the developing cerebral cortex, but the underlying mechanism was still largely unknown. Here, we found that knockdown of Myo10 expression disturbed the adherence of migrating neurons to radial glial fibers through abolishing surface Neuronal cadherin (N-cadherin) expression, thereby impaired neuronal migration in the developmental cortex. Next, we found Myo10 interacted with N-cadherin cellular domain through its FERM domain. Furthermore, we found knockdown of Myo10 disrupted N-cadherin subcellular distribution and led to localization of N-cadherin into Golgi apparatus and endosomal sorting vesicle. Taking together, these results reveal a novel mechanism of Myo10 interacting with N-cadherin and regulating its cell-surface expression, which is required for neuronal adhesion and migration.

RA-Induced Transcriptional Silencing of Checkpoint Kinase-2 through Promoter Methylation by Dnmt3b Is Required for Neuronal Differentiation of P19 Cells

In a previous study, we identified several novel targets of Dnmt3b using a chromatin library from retinoic acid (RA)-treated P19 cells. The present study describes the regulation of expression and function of checkpoint kinase (Chk2), which was one of the target genes of Dnmt3b. Chromatin immunoprecipitation followed by quantitative PCR analysis showed that recruitment of Dnmt3b on Chk2 promoter is induced following RA treatment of P19 cells. Both bisulfite genomic sequence and COBRA analyses showed that the methylation level of Chk2 promoter is progressively increased during RA-induced neuronal differentiation of P19 cells. Concomitantly, both mRNA and protein expression of Chk2 are reduced as determined by real-time PCR and Western blot analysis, respectively. Suppression of Dnmt3b expression by lentiviral-mediated shRNA resulted in increased expression and reduced methylation of Chk2, which clearly showed that Dnmt3b is responsible for transcriptional silencing of Chk2 gene in RA-treated P19 cells. Neuronal differentiation of P19 cells was inhibited upon enforced Chk2 expression in P19 cells, which showed that the decrease in endogenous expression of Chk2 is essential for normal differentiation. Ectopic Chk2 expression also negatively regulated cell cycle arrest and apoptosis following RA treatment, which could also contribute to impaired neuronal differentiation. Together, this study described the regulation of Chk2 expression through promoter methylation and also presented a novel role of Chk2 during neuronal differentiation, which is independent of its previously known function in DNA damage response.

Gβ2 Regulates the Multipolar-Bipolar Transition of Newborn Neurons in the Developing Neocortex

Proper neuronal migration is critical for the formation of the six-layered neocortex in the mammalian brain. However, the precise control of neuronal migration is not well understood. Heterotrimeric guanine nucleotide binding proteins (G proteins), composed of Gα and Gβγ, transduce signals from G protein-coupled receptors to downstream effectors and play crucial roles in brain development. However, the functions of individual subunits of G proteins in prenatal brain development remain unclear. Here, we report that Gβ2 is expressed in the embryonic neocortex, with abundant expression in the intermediate zone, and is significantly upregulated in differentiated neurons. Perturbation of Gβ2 expression impairs the morphogenetic transformation of migrating neurons from multipolar to bipolar and subsequently delays neuronal migration. Moreover, Gβ2 acts as a scaffold protein to organize the MP1-MEK1-ERK1/2 complex and mediates the phosphorylation of ERK1/2. Importantly, expression of a constitutively active variant of MEK1 rescues the migration defects that are caused by the loss of Gβ2. In conclusion, our findings reveal that Gβ2 regulates proper neuronal migration during neocortex development by activating the ERK1/2 signaling pathway.