MicroRNA-23a inhibits melanoma cell proliferation, migration, and invasion in mice through a negative feedback regulation of sdcbp and the MAPK/ERK signaling pathway.

Melanoma is the main cause of death associated with skin cancer. Surgical resection and adjuvant therapy are currently effective treatments, but the recurrence rate is very high. The understanding of microRNA (miR) dynamics after surgical resection of melanoma is essential to accurately explain the changes in the recurrence of melanoma. In this study, we hypothesized that microRNA-23a (miR-23a) affects the cell proliferation, migration, and invasion of melanoma with a mechanism related to SDCBP and the MAPK/ERK signaling pathway. To validate this, we performed a series of experiments in cells of melanoma modeled. Initially, positive expression of SDCBP and morphology of normal and melanoma tissues and cells were observed. Expression of miR-23a, SDCBP, and MAPK/ERK signaling pathway-related genes was identified in melanoma tissues. Melanoma cells transfected with mimic or inhibitor of miR-23a or si-SDCBP were detected to validate effect of miR-23a on SDCBP and the MAPK/ERK signaling pathway. MTT assay, scratch test, transwell assay, and flow cytometry were performed to evaluate cell viability, invasion, metastasis, and apoptosis in vitro, respectively. Tumorigenicity assay in nude mice was conducted to test the tumorigenesis of the transfected cells in vivo. High positive expression of SDCBP and abnormal morphology were observed in melanoma tissues and cells. Reduced expression of miR-23a and increased expression of SDCBP and MAPK/ERK signaling pathway-related genes were identified in the melanoma tissues of melanoma mice. Overexpressed miR-23a dampened SDCBP and the MAPK/ERK signaling pathway. The melanoma cells with overexpressed miR-23a presented ascended cell apoptosis and descended cell proliferation, migration, invasion as well as tumor size. Taken together, our study demonstrated that miR-23a could inhibit the development of melanoma in mice through a negative feedback regulation of SDCBP and the MAPK/ERK signaling pathway. © 2018 IUBMB Life, 71(5):587-600, 2019.

Insulin-like growth factor binding protein 4 inhibits proliferation of bone marrow mesenchymal stem cells and enhances growth of neurospheres derived from the stem cells.

Insulin-like growth factor binding protein 4 (IGFBP-4) was reported to trigger cellular senescence and reduce cell growth of bone marrow mesenchymal stem cells (BMSCs), but its contribution to neurogenic differentiation of BMSCs remains unknown. In the present study, BMSCs were isolated from the femur and tibia of young rats to investigate effects of IGFBP-4 on BMSC proliferation and growth of neurospheres derived from BMSCs. Bone marrow mesenchymal stem cell proliferation was assessed using CCK-8 after treatment with IGFBP-4 or blockers of IGF-IR and ß-catenin. Phosphorylation levels of Akt, Erk, and p38 in BMSCs were analysed by Western blotting. Bone marrow mesenchymal stem cells were induced into neural lineages in NeuroCult medium; the number and the size of BMSC-derived neurospheres were counted after treatment with IGFBP-4 or the blockers. It was shown that addition of IGFBP-4 inhibited BMSC proliferation and immunodepletion of IGFBP-4 increased the proliferation. The blockade of IGF-IR with AG1024 increased BMSC proliferation and reversed IGFBP-4-induced proliferation inhibition; however, blocking of ß-catenin with FH535 did not. p-Erk was significantly decreased in IGFBP-4-treated BMSCs. IGFBP-4 promoted the growth of neurospheres derived from BMSCs, as manifested by the increases in the number and the size of the derived neurospheres. Both AG1024 and FH535 inhibited the formation of NeuroCult-induced neurospheres, but FH535 significantly inhibited the growth of neurospheres in NeuroCult medium with EGF, bFGF, and IGFBP-4. The data suggested that IGFBP-4 inhibits BMSC proliferation through IGF-IR pathway and promotes growth of BMSC-derived neurospheres via stabilizing ß-catenin.

Axon guidance pathways served as common targets for human speech/language evolution and related disorders.

Human and several nonhuman species share the rare ability of modifying acoustic and/or syntactic features of sounds produced, i.e. vocal learning, which is the important neurobiological and behavioral substrate of human speech/language. This convergent trait was suggested to be associated with significant genomic convergence and best manifested at the ROBO-SLIT axon guidance pathway. Here we verified the significance of such genomic convergence and assessed its functional relevance to human speech/language using human genetic variation data. In normal human populations, we found the affected amino acid sites were well fixed and accompanied with significantly more associated protein-coding SNPs in the same genes than the rest genes. Diseased individuals with speech/language disorders have significant more low frequency protein coding SNPs but they preferentially occurred outside the affected genes. Such patients' SNPs were enriched in several functional categories including two axon guidance pathways (mediated by netrin and semaphorin) that interact with ROBO-SLITs. Four of the six patients have homozygous missense SNPs on PRAME gene family, one youngest gene family in human lineage, which possibly acts upon retinoic acid receptor signaling, similarly as FOXP2, to modulate axon guidance. Taken together, we suggest the axon guidance pathways (e.g. ROBO-SLIT, PRAME gene family) served as common targets for human speech/language evolution and related disorders.

Recombinant insulin-like growth factor binding protein-4 inhibits proliferation and promotes differentiation of neural progenitor cells.

Insulin-like growth factor (IGF) is involved in regulating many processes during neural development, and IGF binding protein-4 (IGFBP4) functions as a modulator of IGF actions or in an IGF-independent manner (e.g., via inhibiting Wnt/ß-catenin signaling). In the present study, neural progenitor cells (NPCs) were isolated from the forebrain of newborn mice to investigate effects of IGFBP4 on the proliferation and differentiation of NPCs. The proliferation of NPCs was evaluated using Cell Counting Kit-8 (CCK-8) after treatment with or without IGFBP4 as well as blockers of IGF-IR and ß-catenin. Phosphorylation levels of Akt, Erk1, 2 and p38 were analyzed by Western blotting. The differentiation of NPCs was evaluated using immunofluorescence and Western blotting. It was shown that exogenous IGFBP4 significantly inhibited the proliferation of NPCs and it did not induce a more pronounced inhibition of cell proliferation after blockade of IGF-IR but it did after antagonism of ß-catenin. Akt phosphorylation was significantly decreased and phosphorylation levels of Erk1, 2 and p38 were not significantly changed in IGFBP4-treated NPCs. Excessive IGFBP4 significantly promoted NPCs to differentiate into astrocytes and neurons. These data suggested that exogenous IGFBP4 inhibits proliferation and promotes differentiation of neural progenitor cells mainly through IGF-IR signaling pathway.

Long-term protective effects of AAV9-mesencephalic astrocyte-derived neurotrophic factor gene transfer in parkinsonian rats.

Intrastriatal injection of mesencephalic astrocyte-derived neurotrophic factor (MANF) protein has been shown to provide neuroprotective and neurorestorative effects in a 6-hydroxydopamine (6-OHDA) - lesioned rat model of Parkinson's disease. Here, we used an adeno-associated virus serotype 9 (AAV9) vector to deliver the human MANF (hMANF) gene into the rat striatum 10days after a 6-OHDA lesion to examine long-term effects of hMANF on nigral dopaminergic neurons and mechanisms underlying MANF neuroprotection. Intrastriatal injection of AAV9-hMANF vectors led to a robust and widespread expression of the hMANF gene in the injected striatum up to 24weeks. Increased levels of hMANF protein were also detected in the ipsilateral substantia nigra. The hMANF gene transfer promoted the survival of nigral dopaminergic neurons, regeneration of striatal dopaminergic fibers and an upregulation of striatal dopamine levels, resulting in a long-term improvement of rotational behavior up to 16weeks after viral injections. By using SH-SY5Y cells, we found that intra- and extracellular application of MANF protected cells against 6-OHDA-induced toxicity via inhibiting the endoplasmic reticulum stress and activating the PI3K/Akt/mTOR pathway. Our results suggest that AAV9-mediated hMANF gene delivery into the striatum exerts long-term neuroprotective and neuroregenerative effects on the nigrostriatal dopaminergic system in parkinsonian rats, and provide insights into mechanisms responsible for MANF neuroprotection.

Fusion of Ssm6a with a protein scaffold retains selectivity on NaV 1.7 and improves its therapeutic potential against chronic pain.

Voltage-gated sodium channel NaV 1.7 serves as an attractive target for chronic pain treatment. Several venom peptides were found to selectively inhibit NaV 1.7 but with intrinsic problems. Among them, Ssm6a, a recently discovered centipede venom peptide, shows the greatest selectivity against NaV 1.7, but dissociates from the target too fast and loses bioactivity in synthetic forms. As a disulfide-rich venom peptide, it is difficult to optimize Ssm6a by artificial mutagenesis and produce the peptide with common industrial manufacturing methods. Here, we developed a novel protein scaffold fusion strategy to address these concerns. Instead of directly mutating Ssm6a, we genetically fused Ssm6a with a protein scaffold engineered from human muscle fatty acid-binding protein. The resultant fusion protein, SP-TOX, maintained the selectivity and potency of Ssm6a upon NaV 1.7 but dissociated from target at least 10 times more slowly. SP-TOX dramatically reduced inflammatory pain in a rat model through DRG-targeted delivery. Importantly, SP-TOX can be expressed cytosolically in Escherichia coli and purified in a cost-effective way. In summary, our study provided the first example of cytosolically expressed fusion protein with high potency and selectivity on NaV 1.7. Our protein scaffold fusion approach may have its broad application in optimizing disulfide-rich venom peptides for therapeutic usage.

Conversion of adult human peripheral blood mononuclear cells into induced neural stem cell by using episomal vectors.

Human neural stem cells (NSCs) hold great promise for research and therapy in neural diseases. Many studies have shown direct induction of NSCs from human fibroblasts, which require an invasive skin biopsy and a prolonged period of expansion in cell culture prior to use. Peripheral blood (PB) is routinely used in medical diagnoses, and represents a noninvasive and easily accessible source of cells. Here we show direct derivation of NSCs from adult human PB mononuclear cells (PB-MNCs) by employing episomal vectors for transgene delivery. These induced NSCs (iNSCs) can expand more than 60 passages, can exhibit NSC morphology, gene expression, differentiation potential, and self-renewing capability and can give rise to multiple functional neural subtypes and glial cells in vitro. Furthermore, the iNSCs carry a specific regional identity and have electrophysiological activity upon differentiation. Our findings provide an easily accessible approach for generating human iNSCs which will facilitate disease modeling, drug screening, and possibly regenerative medicine.

Malignant transformation of bone marrow stromal cells induced by the brain glioma niche in rats.

Normal human embryonic stem cells (hESCs) can develop neoplastic cancer stem cell (CSC) properties after coculture with transformed hESCs in vitro. In the present study, the influence of the tumor microenvironment on malignant transformation of bone marrow stromal cells (BMSCs) was studied after allografting a mixture of enhanced green fluorescent protein (EGFP)-labeled BMSCs and C6 glioma cells into the rat brain to understand the influence of the cellular environment, especially the tumor environment, on the transformation of grafted BMSCs in the rat brain. We performed intracerebral transplantation in the rat brain using EGFP-labeled BMSCs coinjected with C6 tumor cells. After transplantation, the EGFP-labeled cells were isolated from the tumor using fluorescence-activated cell sorting, and the characteristics of the recovered cells were investigated. Glioma-specific biomarkers of the sorted cells and the biological characteristics of the tumors were analyzed. The BMSCs isolated from the cografts were transformed into glioma CSCs, as indicated by the marked expression of the glioma marker GFAP in glioma cells, and of Nestin and CD133 in neural stem cells and CSCs, as well as rapid cell growth, decreased level of the tumor suppressor gene p53, increased level of the oncogene murine double minute gene 2 (MDM2), and recapitulation of glioma tissues in the brain. These data suggest that BMSCs can be transformed into CSCs, which can be further directed toward glioma formation under certain conditions, supporting the notion that the tumor microenvironment is involved in transforming normal BMSCs into glial CSCs.

Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1(Ser137) involvement in spindle formation and REC8 cleavage.

Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1(Ser137) and pPlk1(Thr210)) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1(Ser137) with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1(Thr210) was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1(Ser137) was colocalized with microtubule organizing center (MTOC) proteins, γ-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1(Thr210) was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1(Ser137) accumulation at MTOCs and between chromosome arms, consequently disturbed γ-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1(Ser137) and pPlk1(Thr210), as well as the subcellular distribution of pPlk1(Thr210), were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1(Ser137) is the action executor, in mouse oocytes during meiotic division.

The influence of GAP-43 on orientation of cell division through G proteins.

Recent studies have shown that GAP-43 is highly expressed in horizontally dividing neural progenitor cells, and G protein complex are required for proper mitotic-spindle orientation of those progenitors in the mammalian developing cortex. In order to verify the hypothesis that GAP-43 may influence the orientation of cell division through interacting with G proteins during neurogenesis, the GAP-43 RNA from adult C57 mouse was cloned into the pEGFP-N1 vector, which was then transfected into Madin-Darby Canine Kidney (MDCK) cells cultured in a three-dimensional (3D) cell culture system. The interaction of GAP-43 with Gαi was detected by co-immunoprecipitation (co-IP), while cystogenesis of 3D morphogenesis of MDCK cells and expression of GAP-43 and Gαi were determined by immunofluorescence and Western blotting. The results showed are as follows: After being transfected by pEGFP-N1-GAP-43, GAP-43 was localized on the cell membrane and co-localized with Gαi, and this dramatically induced a defective cystogenesis in 3D morphogenesis of MDCK cells. The functional interaction between GAP-43 and Gαi proteins was proven by the co-IP assay. It can be considered from the results that the GAP-43 is involved in the orientation of cell division by interacting with Gαi and this should be an important mechanism for neurogenesis in the mammalian brain.

Comparison of intracerebral transplantation effects of different stem cells on rodent stroke models.

In the present study, induced pluripotent stem cells (iPSCs), induced neural stem cells (iNSCs), mesenchymal stem cells (MSCs) and an immortalized cell line (RMNE6), representing different characteristics of stem cells, were transplanted into normal and/or injured brain areas of rodent stroke models, and their effects were compared to select suitable stem cells for cell replacement stroke therapy. The rat and mice ischaemic models were constructed using the middle cerebral artery occlusion technique. Both electrocoagulation of the artery and the intraluminal filament technique were used. The behaviour changes and fates of grafted stem cells were determined mainly by behaviour testing and immunocytochemistry. Following iPSC transplantation into the corpora striata of normal mice, a tumour developed in the brain. The iNSCs survived well and migrated towards the injured area without differentiation. Although there was no tumourigenesis in the brain of normal or ischaemic mice after the iNSCs were transplanted in the cortices, the behaviour in ischaemic mice was not improved. Upon transplanting MSC and RMNE6 cells into ischaemic rat brains, results similar to iNSCs in mice were seen. However, transplantation of RMNE6 caused a brain tumour. Thus, tumourigenesis and indeterminate improvement of behaviour are challenging problems encountered in stem cell therapy for stroke, and the intrinsic characteristics of stem cells should be remodelled before transplantation.

The experimental therapy on brain ischemia by improvement of local angiogenesis with tissue engineering in the mouse.

Neural restoration has proven to be difficult after brain stroke, especially in its chronic stage. This is mainly due to the generation of an unpropitious niche in the injured area, including loss of vascular support but production of numerous inhibitors against neuronal regeneration. Reconstruction of a proper niche for promoting local angiogenesis, therefore, should be a key approach for neural restoration after stroke. In the present study, a new biomaterial composite that could be implanted in the injured area of the brain was created for experimental therapy of brain ischemia in the mouse. This composite was made using a hyaluronic acid (HA)-based biodegradable hydrogel scaffold, mixed with poly(lactic-co-glycolic acid) (PLGA) microspheres containing vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1), two factors that stimulate angiogenesis. In addition, the antibody of Nogo receptor (NgR-Ab), which can bind to multiple inhibitory myelin proteins and promote neural regeneration, was covalently attached to the hydrogel, making the hydrogel more bioactive and suitable for neural survival. This composite (HA-PLGA) was implanted into the mouse model with middle cerebral artery occlusion (MCAO) to explore a new approach for restoration of brain function after ischemia. A good survival and proliferation of human umbilical artery endothelial cells (HUAECs) and neural stem cells (NSCs) were seen on the HA hydrogel with PLGA microspheres in vitro. This new material was shown to have good compatibility with the brain tissue and inhibition to gliosis and inflammation after its implantation in the normal or ischemic brain of mice. Particularly, good angiogenesis was found around the implanted HA-PLGA hydrogel, and the mouse models clearly showed a behavioral improvement. The results in this present study indicate, therefore, that the HA-PLGA hydrogel is a promising material, which is able to induce angiogenesis in the ischemic region by releasing VEGF and Ang1, thus creating a suitable niche for neural restoration in later stages of stroke. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

Configuration of fibrous and adipose tissues in the cavernous sinus.

OBJECTIVE: Three-dimensional anatomical appreciation of the matrix of the cavernous sinus is one of the crucial necessities for a better understanding of tissue patterning and various disorders in the sinus. The purpose of this study was to reveal configuration of fibrous and adipose components in the cavernous sinus and their relationship with the cranial nerves and vessels in the sinus and meningeal sinus wall. MATERIALS AND METHODS: Nineteen cadavers (8 females and 11 males; age range, 54-89 years; mean age, 75 years) were prepared as transverse (6 sets), coronal (3 sets) and sagittal (10 sets) plastinated sections that were examined at both macroscopic and microscopic levels. RESULTS: Two types of the web-like fibrous networks were identified and localized in the cavernous sinus. A dural trabecular network constituted a skeleton-frame in the sinus and contributed to the sleeves of intracavernous cranial nerves III, IV, V1, V2 and VI. A fine trabecular network, or adipose tissue, was the matrix of the sinus and was mainly distributed along the medial side of the intracavernous cranial nerves, forming a dumbbell-shaped adipose zone in the sinus. CONCLUSIONS: This study revealed the nature, fine architecture and localization of the fine and dural trabecular networks in the cavernous sinus and their relationship with intracavernous cranial nerves and vessels. The results may be valuable for better understanding of tissue patterning in the cranial base and better evaluation of intracavernous disorders, e.g. the growth direction and extent of intracavernous tumors.

Chlorotoxin-conjugated graphene oxide for targeted delivery of an anticancer drug.

Current chemotherapy for glioma is rarely satisfactory due to low therapeutic efficiency and systemic side effects. We have developed a glioma-targeted drug delivery system based on graphene oxide. Targeted peptide chlorotoxin-conjugated graphene oxide (CTX-GO) sheets were successfully synthesized and characterized. Doxorubicin was loaded onto CTX-GO (CTX-GO/DOX) with high efficiency (570 mg doxorubicin per gram CTX-GO) via noncovalent interactions. Doxorubicin release was pH-dependent and showed sustained-release properties. Cytotoxicity experiments demonstrated that CTX-GO/DOX mediated the highest rate of death of glioma cells compared with free doxorubicin or graphene oxide loaded with doxorubicin only. Further, conjugation with chlorotoxin enhanced accumulation of doxorubicin within glioma cells. These findings indicate that CTX-GO is a promising platform for drug delivery and provide a rationale for developing a glioma-specific drug delivery system.

Ho3+ doped NaGdF4 nanoparticles as MRI/optical probes for brain glioma imaging.

Lanthanide-ion doped NaGdF4 nanoparticles (NPs) have been exploited as a new generation of MRI/optical probes. However, it remains difficult for these NPs to image tiny brain gliomas due to low specificity and limited accumulation. To circumvent this obstacle, chlorotoxin (CTX) was conjugated onto Ho3+ doped NaGdF4 (CTX-NaGdF4:Ho3+) to render a glioma-specific targeted MRI/optical probe. Both confocal laser scanning microscopy and flow cytometry demonstrated the targeting ability of CTX-NaGdF4:Ho3+ NPs towards glioma cells in vitro. Furthermore, in vivo MRI and fluorescence imaging of the tiny brain gliomas in mice confirmed that the CTX-NaGdF4:Ho3+ NPs could lead to a significant contrast enhancement effect and a clearer boundary between glioma and normal tissue. In addition, the CTX-NaGdF4:Ho3+ NPs exhibited a low cytotoxicity and no detectable tissue damages. Therefore, the CTX-NaGdF4:Ho3+ NPs could potentially serve as an MRI/optical probe for the detection of tiny brain gliomas.

Subthalamic hGAD65 gene therapy and striatum TH gene transfer in a Parkinson's disease rat model.

The aim of the present study is to detect a combination method to utilize gene therapy for the treatment of Parkinson's disease (PD). Here, a PD rat model is used for the in vivo gene therapy of a recombinant adeno-associated virus (AAV2) containing a human glutamic acid decarboxylase 65 (rAAV2-hGAD65) gene delivered to the subthalamic nucleus (STN). This is combined with the ex vivo gene delivery of tyrosine hydroxylase (TH) by fibroblasts injected into the striatum. After the treatment, the rotation behavior was improved with the greatest efficacy in the combination group. The results of immunohistochemistry showed that hGAD65 gene delivery by AAV2 successfully led to phenotypic changes of neurons in STN. And the levels of glutamic acid and GABA in the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNr) were obviously lower than the control groups. However, hGAD65 gene transfer did not effectively protect surviving dopaminergic neurons in the SNc and VTA. This study suggests that subthalamic hGAD65 gene therapy and combined with TH gene therapy can alleviate symptoms of the PD model rats, independent of the protection the DA neurons from death.

Emerging restorative treatments for Parkinson's disease: manipulation and inducement of dopaminergic neurons from adult stem cells.

Parkinson's disease (PD) is a common neurodegenerative disease, characterized by a selective loss of midbrain Dopaminergic (DA) neurons. To address this problem, various types of stem cells that have potential to differentiate into DA neurons are being investigated as cellular therapies for PD, including cells derived from embryonic or adult donor tissue, and embryonic stem cells. These cell sources, however, have raised certain questions with regard to ethical and rejection issues. Recent progress in adult stems has further proved that the cells derived from adult tissue could be expanded and differentiated into DA precursor cells in vitro, and cell therapy with adult stem cells could produce a clear improvement for PD models. Using adult stem cells for clinic application may not only overcome the ethical problem inherent in using human fetal tissue or embryonic stem cells, but also open the possibility for autologous transplantation. The patient-specific adult stem cell is therefore a potential and prospective candidate for PD treatment.

Proteomic analysis of cPKCßII-interacting proteins involved in HPC-induced neuroprotection against cerebral ischemia of mice.

Hypoxic preconditioning (HPC) initiates intracellular signaling pathway to provide protection against subsequent cerebral ischemic injuries, and its mechanism may provide molecular targets for therapy in stroke. According to our study of conventional protein kinase C ßII (cPKCßII) activation in HPC, the role of cPKCßII in HPC-induced neuroprotection and its interacting proteins were determined in this study. The autohypoxia-induced HPC and middle cerebral artery occlusion (MCAO)-induced cerebral ischemia mouse models were prepared as reported. We found that HPC reduced 6 h MCAO-induced neurological deficits, infarct volume, edema ratio and cell apoptosis in peri-infarct region (penumbra), but cPKCßII inhibitors Go6983 and LY333531 blocked HPC-induced neuroprotection. Proteomic analysis revealed that the expression of four proteins in cytosol and eight proteins in particulate fraction changed significantly among 49 identified cPKCßII-interacting proteins in cortex of HPC mice. In addition, HPC could inhibit the decrease of phosphorylated collapsin response mediator protein-2 (CRMP-2) level and increase of CRMP-2 breakdown product. TAT-CRMP-2 peptide, which prevents the cleavage of endogenous CRMP-2, could inhibit CRMP-2 dephosphorylation and proteolysis as well as the infarct volume of 6 h MCAO mice. This study is the first to report multiple cPKCßII-interacting proteins in HPC mouse brain and the role of cPKCßII-CRMP-2 in HPC-induced neuroprotection against early stages of ischemic injuries in mice.

[Bone marrow stem cell-derived astrocytes are involved in glia limitans formation in rats after brain injury].

OBJECTIVE: To investigate the involvement of bone marrow stem cell-derived astrocytes (BMDSCs) in the formation of glia limitans after brain injury. METHODS: In a female SD rat model of brain injury, green fluorescence protein (GFP)-labeled BMDSCs from male SD rats were transplanted via the caudal vein 24 h after the injury. The rats were sacrificed at 2, 4 and 8 weeks after the transplantation, and immunohistochemistry for glial fibrillary acidic protein (GFAP) was performed to observe the astrocytes. The fluorescence emitted by GFP was observed to identify the presence of the bone marrow-derived stem cells, and the GFAP(+)/GFP(+) cells in the glia limitnas were detected under fluorescence microscopy. RESULTS The GFAP(+)/GFP(+) cells were found in the glia limitans between the brain lesion and normal brain tissue. CONCLUSION: Bone marrow stem cell-derived astrocytes is involved in glia limitans formation after brain injury, which can be of significance in brain injury recovery and implantation of engineered materials.

Role of Nurr1 and Ret in inducing rat embryonic neural precursors to dopaminergic neurons.

BACKGROUND: Nurr1, a member of the nuclear receptor superfamily of transcription factors, is highly expressed in midbrain dopaminergic (DA) neurons. Ret is a member of the receptor tyrosine kinase (RTK) superfamily and a critical signal transducing subunit of receptors for glial cell line-derived neurotrophic factor (GDNF). Both Nurr1 and Ret play important roles in the development of DA neurons. PURPOSE: To investigate possible correlation between Nurr1 and Ret on inducing expression of tyrosine hydroxylase (TH) in neural precursor cells. METHODS: Neural precursors isolated from rat embryonic mesencephalon (E13.5d) were transfected with vectors containing Nurr1 or Ret and treated with 9-cis-retinoic acid (RA) and/or GDNF for 3 days. RT-PCR and immunocytochemistry was used to test the expression of Nurr1 and Ret and the TH positive cells. RESULTS: The number of TH positive cells was increased from 1.53 +/- 0.12 to 3.83 +/- 0.56% after the cells were transfected with Nurr1. Increased endogenous Nurr1 by RA lead to 1.8 times (2.86 +/- 0.32% versus 1.53 +/- 0.12% in the controls) increase in TH positive cells. A double inducing effect by both endogenous and exogenous Nurr1 on the expression of TH was observed by 3.3 times increase in the positive cells (from 5.03 +/- 0.76 to 1.53 +/- 0.12% in control). Ret expression was induced by overexpression of Nurr1. Overexpressed Ret had no inducing effect on the expression of Nurr1 and the number of TH positive cells. The gene of dopamine transporter (DAT) was clearly induced in the cells transfected with Ret. CONCLUSION: Nurr1, required for the expression of Ret, had inducing effect on TH positive cells, and Ret may promote maturation of DA neuron by up-regulating DAT expression through its ligand. As a cooperator, Ret seems to work together with Nurr1 in the development of DA neurons.