Phosphatidylinositol phosphates (PtdInsPs) are ubiquitous membrane phospholipids that play diverse roles in cell growth and differentiation. To clarify the regulation mechanism acting on neurofilament light chain (NF-L) self assembly, we examined the effects of various PtdInsPs on this process. We found that PtdInsPs, including PI(4,5)P2, directly bind to the positively charged Arg54 of murine NF-L, and this binding promotes NF-L self assembly in vitro. Mutant NF-L (R53A/R54A) proteins lacking binding affinity to PtdInsPs did not have the same effect, but the mutant NF-L proteins showed greater self assembly than the wild-type in the absence of any PtdInsP. These results collectively suggest that Arg54 plays a pivotal role in NF-L self assembly by binding with PtdInsPs.
Animals ; Fluorescent Antibody Technique ; Mice ; Mutation/genetics ; Neurofilament Proteins/genetics/*metabolism ; Phosphatidylinositol Phosphates/*metabolism ; Phospholipase C gamma/metabolism ; *Protein Multimerization

OBJECTIVETo observe the cellular expression of (R127W) HSPB1 and its influence on neurofilament light chain (NFL) self-assembly and co-localization with NFL.

METHODSEukaryotic expression vectors pEGFPN1-(wt) HSPB1 and pEGFPN1- (R127W) HSPB1 were constructed. Hela cells were transiently transfected with pEGFPN1-(wt) HSPB1 or pEGFPN1- (R127W) HSPB1 and observed under a confocal microscope. Hela cells were also transiently co-transfected with Pcl-NFL and pEGFPN1-(wt)HSPB1, or pCL-NFL and pEGFPN1-(R127W)HSPB1. The self-assembly of NFL was observed and the co-localization study of HSPB1/ (R127W)HSPB1 with NFL was carried out in these two cell models by immunofluorescence technique.

RESULTSThe aggregates formed by EGFP-(R127W)HSPB1 predominantly located around the nucleus, and EGFP-(wt)HSPB1 showed diffusion pattern in Hela cells. When co expressed with EGFP-(wt)HSPB1, NFL formed homogeneous structure in cytosol. When co-expressed with EGFP-(R127W)HSPB1, however, NFL had amorphous staining pattern predominantly consisting of NFL aggregates, and NFL co-localized with (R127W)HSPB1 in these aggregates.

CONCLUSIONThe R127W mutant of HSPB1 may have reduced capacity to serve as a chaperone to prevent aggregate formation, and fail to correctly organize the neurofilament network. Dysfunction of the axon cytoskeleton and axon transport may be the primary mechanism of R127W mutation of HSPB1 in the pathogenesis of Charcot-Marie-Tooth disease.

Base Sequence ; Charcot-Marie-Tooth Disease ; genetics ; metabolism ; Gene Expression Regulation ; Genetic Vectors ; genetics ; HSP27 Heat-Shock Proteins ; genetics ; metabolism ; HeLa Cells ; Humans ; Intracellular Space ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Neurofilament Proteins ; metabolism ; Protein Binding ; genetics ; Protein Transport ; Transfection

OBJECTIVETo study the possible mechanism of the intracellular aggregate formation of small heat shock protein HSPB8 (HSPB8)(K141N) mutation resulting in axonal Charcot-Marie-Tooth disease type 2L(CMT2L).

METHODSThe cell models which transiently expressed pEGFPN1-HSPB8 and pEGFPN1-(K141N)HSPB8 were established. The immunofluorescent co-location study of EGFP-(K141N)HSPB8 and HSPB1, EGFP-(K141N)HSPB8 and neurofilament light chain (NEFL) was carried out in the SHSY5Y cell models. The aggregate formation of EGFP-(K141N)HSPB8 in cell models was investigated and the possible mechanism of cellular aggregate formation was analyzed by t test and analysis of variance between group(ANOVA).

RESULTSEGFP-(K141N)HSPB8 formed large aggregate which predominantly located around the nucleus in cell models. EGFP-(K141N)HSPB8 co-localized perfectly with HSPB1 and NEFL in the SHSY5Y cell models. The aggregate formation was different in different cell types, there were fewer aggregates formed in an sHSPs deficient milieu than in HEK293T cells.

CONCLUSION(K141N)HSPB8 formed aggregates predominantly locate around the nucleus in cells. (K141N)HSPB8 co-localizes perfectly with HSPB1 and NEFL. The aggregate formation may be due to (K141N)HSPB8 conformational change leading to self aggregation and its abnormal interaction with other sHSPs such as HSPB1.

Cell Line ; Cell Line, Tumor ; Cell Nucleus ; metabolism ; Charcot-Marie-Tooth Disease ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; HSP27 Heat-Shock Proteins ; HeLa Cells ; Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Kidney ; cytology ; metabolism ; Microscopy, Confocal ; Neoplasm Proteins ; genetics ; metabolism ; Neuroblastoma ; genetics ; metabolism ; pathology ; Neurofilament Proteins ; genetics ; metabolism ; Point Mutation ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

OBJECTIVE: To investigate the effect of tetramethylpyrazine on spinal cord repair through hypothesized pathways including regulation of caspase-3, neurofilament protein NF-L, NF-H, and NF-M expression. METHODS: Allenos weight drop method was used to establish acute spinal cord injury (SCI) rat model at T10 section and was conducted in 80 adult Sprague Dawley rats that were subsequently divided into pseudo-therapy group (sodium chloride group, n=40) and tetramethylpyrazine group (treatment group, n=40). Eight normal rats were included into the pseudo-surgery normal group (n=1 for each time point). The function of spinal cord was evaluated with animal behavioral scores by measuring modified Rivilin loxotic plate degree and counting BBB score at 1, 3, 7, 14, and 21 d postoperatively. The injured spinal cord tissue samples were harvested at 1 h, 3 h, 6 h, 1 d, 3 d, 7 d, 14 d, and 21 d postoperatively (n=5 for each time point) for the preparation of continuous histological sections that were analyzed the expression of caspase-3,NF-L,NF-H, and NF-M by immunohistochemistry method. RESULTS: At postoperative Day 7, 14, and 21, animal behavioral scores revealed higher modified Rivlin loxotic plate degree and BBB scores in the treatment group when compared with those of the control group (P<0.05). The cells in the injured spinal cord tissue expressed significantly less caspase-3 and significantly more NF (NF-L, NF-H and NF-M) in the treatment group than those in the control group at Day 3, 7, and 14 (P<0.05). There was positive correlation between modified Rivlin loxotic plate degree and NF expression, BBB score and NF expression; and negative correlations between BBB score and caspase-3 expression, caspase-3 expression and NF expression. CONCLUSION Tetramethylpyrazine improves spinal cord healing through regulation of caspase-3 and neurofilament protein expression.
Animals ; Caspase 3 ; biosynthesis ; genetics ; Female ; Male ; Nerve Regeneration ; drug effects ; Neurofilament Proteins ; biosynthesis ; genetics ; Pyrazines ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism ; physiopathology

OBJECTIVETo investigate the isolation and expansion of mesenchymal stem cells (MSCs) from human umbilical cord Wharton's jelly and their biological identities, and explore the possibility of inducing human umbilical cord-derived MSCs to differentiate into neurocyte-like cells.

METHODSThe growth and proliferative abilities of human umbilical cord-derived MSCs were observed, and their immunophenotypes were determined by flow cytometry. Salvia miltiorrhiza and beta-sulfhydryl alcohol were adopted to induce the cells to differentiate. The differentiated and undifferentiated cells were identified with immunocytochemistry. The pleiotrophin and nestin genes were measured by RT-PCR.

RESULTSA population of human umbilical cord-derived MSCs were isolated from human umbilical Wharton's jelly; they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling, and they were expanded as undifferentiated cells in culture for more than 10 passages, indicating their proliferative capacity. The human umbilical cord-derived MSCs were positive for CD(29), CD(44), CD(59), CD(105), but negative or weakly expressed the markers of hematopoietic cells such as CD(14), CD(33), CD(34), CD(28), CD(45) and CD(117). The important GVHD correlation markers were negative or weakly expressed, including CD(80) (B7-1), CD(86) (B7-2), CD(40) and CD(40L). Salvia miltiorrhiza beta-sulfhydryl alcohol could induce the MSCs to express nestin, a marker of neuronal precursor stem cells at early stage of differentiation. Later, they exhibited neural phenotypes, expressing beta-tubulin III and neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that the MSCs could express pleiotrophin either before or after the induction of salvia miltiorrhiza, furthermore, after the induction the expression was markedly enhanced and the nestin gene was also expressed.

CONCLUSIONThe human MSCs could be isolated from human umbilical cord Wharton's jelly, and it was easy to propagate these MSCs. The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier, which may suggest potential clinical significance. The MSCs are capable of differentiating into neurocyte-like cells and they may represent an alternative stem cell source for CNS cells transplantation.

Antigens, CD ; immunology ; Carrier Proteins ; genetics ; Cell Differentiation ; physiology ; Cells, Cultured ; Cytokines ; genetics ; Female ; Flow Cytometry ; Glial Fibrillary Acidic Protein ; metabolism ; Humans ; Immunohistochemistry ; Infant, Newborn ; Intermediate Filament Proteins ; genetics ; Male ; Mesenchymal Stromal Cells ; immunology ; metabolism ; physiology ; Nerve Tissue Proteins ; genetics ; Nestin ; Neurofilament Proteins ; metabolism ; Neurons ; metabolism ; physiology ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction ; Tubulin ; metabolism ; Umbilical Cord ; cytology

When we treated rat bone marrow stromal cells (rBMSCs) with neuronal differentiation induction media, typical unfolded protein response (UPR) was observed. BIP/GRP78 protein expression was time-dependently increased, and three branches of UPR were all activated. ATF6 increased the transcription of XBP1 which was successfully spliced by IRE1. PERK was phosphorylated and it was followed by eIF2alpha phosphorylation. Transcription of two downstream targets of eIF2alpha, ATF4 and CHOP/GADD153, were transiently up-regulated with the peak level at 24 h. Immunocytochemical study showed clear coexpression of BIP and ATF4 with NeuN and Map2, respectively. UPR was also observed during the neuronal differentiation of mouse embryonic stem (mES) cells. Finally, chemical endoplasmic reticulum (ER) stress inducers, thapsigargin, tunicamycin, and brefeldin A, dose-dependently increased both mRNA and protein expressions of NF-L, and, its expression was specific to BIP-positive rBMSCs. Our results showing the induction of UPR during neuronal differentiations of rBMSCs and mES cells as well as NF-L expression by ER stress inducers strongly suggest the potential role of UPR in neuronal differentiation.
Activating Transcription Factor 4/genetics/metabolism ; Animals ; Apoptosis/drug effects ; Bone Marrow Cells/*cytology ; Cell Differentiation ; Culture Media/pharmacology ; Embryonic Stem Cells/*cytology ; Endoplasmic Reticulum/genetics/metabolism ; Gene Expression/drug effects ; Heat-Shock Proteins/*genetics/metabolism ; Mice ; Microtubule-Associated Proteins/genetics/metabolism ; Molecular Chaperones/*genetics/metabolism ; Nerve Tissue Proteins/genetics/*metabolism ; Neurofilament Proteins/genetics/metabolism ; Neurons/*cytology ; Nuclear Proteins/genetics/metabolism ; Protein Folding ; Rats ; Stromal Cells

OBJECTIVETo study the molecular mechanism underlying multidrug resistance (MDR) and identify unknown genes that might be involved in drug resistance development in K562/A02 cells.

METHODSK562/A02 was induced by gradually increasing the ADM concentration in culture medium of K562 cells, the differential expression of associated genes between K562 and K562/A02 was determined with cDNA microarray. Overexpression of neurofilament protein NF-H gene in K562/A02 cells was confirmed with RT-PCR and immunocytochemistry. Anti-sense oligodeoxynucleotides were transfected into K562/A02 cells by lipofectamine in order to further analyze the role of NF-H in drug resistance.

RESULTSComparing with the expression profiles, we found upregulation of 5 transcripts and downregulation of 7 transcripts in response to MDR of K562/A02 cells. The overexpression of NF-H, one of the 5 upregulated genes, was confirmed. After being treated with antisense oligodeoxynucleotides of NF-H and mdr1, the cellular adriamycin concentration increased significantly, but antisense NF-H alone did not have significant effect on drug resistance phenotype.

CONCLUSIONThe development of MDR in K562/A02 cells is multifactorial. NF-H may be involved in the drug resistance of K562/A02, which may provide a new marker of diagnosis and a new target of therapy.

Doxorubicin ; metabolism ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Genes, MDR ; Humans ; K562 Cells ; Neurofilament Proteins ; biosynthesis ; genetics ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Oligonucleotide Array Sequence Analysis ; RNA, Messenger ; biosynthesis ; genetics ; Up-Regulation

The retinal activity for vision requires a precise synaptic connectivity. Shank proteins at postsynaptic sites of excitatory synapses play roles in signal transmission into the postsynaptic neuron. However, the correlation of Shank 2 expression with neuronal differentiation in the developing retina remains to be elucidated regardless of previous evidences of Shank 2 expression in retina. Herein, we demonstrated that with progression of development, Shank 2 is initially detected in the inner plexiform layer at P2, and then intensively detected in inner plexiform layer, outer plexiform layer, and ganglion cell layer at P14, which was closely colocalized to the neurofilament expression. Shank 2 was, however, not colocalized with glial fibrillary acidic protein. Shank 2 expression was increased in the differentiated retinoblastoma cells, which was mediated by ERK 1/2 activation. Moreover, Shank 2 expression was colocalized with neurofilament at the dendritic region of cells. In conclusion, our data suggests that Shank 2 is expressed in the neurons of the developing retina and could play a critical role in the neuronal differentiation of the developing retina.
Adaptor Proteins, Signal Transducing/genetics/*metabolism ; Animals ; Astrocytes/cytology/metabolism ; Cell Differentiation/*physiology ; Enzyme Activation ; Extracellular Signal-Regulated MAP Kinases/metabolism ; *Gene Expression Regulation, Developmental ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins/genetics/*metabolism ; Neurofilament Proteins/metabolism ; Neurons/cytology/*physiology ; *Retina/cytology/growth & development/physiology ; Retinoblastoma/metabolism/pathology

OBJECTIVEIn order to explore the effects of lead on the growth and development of cultured hippocampal neural cells and on the expression of Oct-2, the II subtype POU domain protein.

METHODSExperiment cell model was established using primary culture of hippocampal neural cells from SD rat embryos. Target cells were exposed to lead acetate in the different concentrations, i.e. 10(-1), 10(0), 10(1), 10(2), 10(3) micromol/L, while the control group was given the same quantity of the culture medium. The immunohistochemistry method was utilized to detect the expressions of Neurofilament (NF) and Glial Fibrillary Acidic Protein (GFAP), the markers for neuron and astrocyte, respectively, and the expression of Oct-2 as well.

RESULTSThe results showed that 10 micromol/L lead acetate treatment caused diminishing of neuronal cell body and the decreases of both axon lengths and inter-cellular connections. In addition, 1 micromol/L lead acetate significantly increased the number of GFAP-positive cells compared with the control group (P < 0.05). By image analysis system, 1 micromol/L lead acetate treatment was found to induce a statistically significant increase of the positive area rate concerning Oct-2 expression in hippocampal neurons and astrocytes, while both positive area rate and integral density of light of Oct-2 expression were found to increase markedly in the groups treated by 10 micromol/L lead acetate (P < 0.01).

CONCLUSIONSLead acetate treatment may contribute to the inhibitions of both growth and differentiation of hippocampus neurons, and to the stimulation of glial cell hyperplasia simultaneously. In addition, the CNS impairments caused by lead is partly correlated with the enhancement of Oct-2 expression.

Animals ; Astrocytes ; metabolism ; Cell Division ; drug effects ; Cells, Cultured ; DNA-Binding Proteins ; biosynthesis ; genetics ; Dose-Response Relationship, Drug ; Embryo, Mammalian ; Female ; Glial Fibrillary Acidic Protein ; biosynthesis ; genetics ; Hippocampus ; cytology ; metabolism ; Lead ; toxicity ; Neurofilament Proteins ; biosynthesis ; genetics ; Neurons ; cytology ; metabolism ; Octamer Transcription Factor-2 ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Transcription Factors ; biosynthesis ; genetics

OBJECTIVETo observe the effect of Draconis Sanguis-containing serum on the expressions of NGF, BDNF, CNTF, LNG-FR, TrkA, GDNF, GAP-43 and NF-H in Schwann cells, and investigate the possible mechanism of Draconis Sanguis to promote peripheral nerve regeneration.

METHODSD rats were randomly divided into 2 groups: the Draconis Sanguis group (orally administered with Draconis Sanguis-containing balm solution) and the blank group (equivoluminal balm) to prepare Draconis Sanguis-containing serum and blank control serum. Schwann cells were extracted from double sciatic nerves of three-day-old SD rats, divided into 2 groups: the Draconis Sanguis group and the blank control group, and respectively cultured with 10% Draconis Sanguis-containing serum or blank control serum. The mRNA expressions of NGF, BDNF, CNTF and other genes in Schwann cells were measured by RT-PCR analysis 48 hours later.

RESULTMost of the Schwann cells were bipolar spindle and arranged shoulder to shoulder or end to end under the microscope and identified to be positive with the immunocytochemical method. To compare with the blank group, mRNA expressions of NGF, LNGFR, GDNF and GAP-43 significantly increased (P < 0.01). Whereas that of BDNF decreased significantly (P < 0.05), and so did that of TrkA, CNTF (P < 0.01), with no remarkable difference in NF-H-mRNA.

CONCLUSIONTraditional Chinese medicine Draconis Sanguis may show effect in nerve regeneration by up-regulating mRNA expressions of NGF, LNGFR, GDNF and GAP-43 and down-regulating mRNA expressions of TrkA, BDNF and CNTF.

Animals ; Arecaceae ; chemistry ; Brain-Derived Neurotrophic Factor ; genetics ; metabolism ; Cells, Cultured ; Ciliary Neurotrophic Factor ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; GAP-43 Protein ; genetics ; metabolism ; Gene Expression ; drug effects ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; metabolism ; Male ; Nerve Growth Factor ; genetics ; metabolism ; Nerve Regeneration ; drug effects ; Neurofilament Proteins ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, trkA ; genetics ; metabolism ; Schwann Cells ; drug effects ; physiology ; Serum ; chemistry