Neuronal PC12 cells induced by nerve growth factor (NGF) have been considered to be postmitotic and lack the ability to divide. However, in this study, we not only detected DNA synthesis but also observed cell division in some morphologically differentiated neuronal PC12 cells bearing long neurites. More interestingly, in addition to the division of perikaryon, the neurites located on the division site of the cell membrane also divided into two parts and were allocated to the two daughter cells. These results demonstrate that the morphologically differentiated neuronal PC12 cells still retain the ability to divide. This is the first report that neuronal PC12 cells as well as their neurites can divide.
Animals ; Cell Differentiation ; drug effects ; Cell Division ; drug effects ; DNA Replication ; drug effects ; physiology ; Nerve Growth Factor ; pharmacology ; Neurites ; drug effects ; Neurons ; cytology ; PC12 Cells ; Rats

OBJECTIVETo study the role of adaptor protein Dok6 in neurite outgrowth in PC12 cells.

METHODSSeries of fusion clones were constructed by fusing different domains of Dok6 into mutant TrkC/Y516F. These constructs were transiently transfected into PC12 cells separately and the expression levels of fusion proteins were detected by Western blot. Neurite outgrowth in these PC12 cells was tested after stimulation of NT-3.

RESULTSEach fusion clone was stably expressed in PC12 cells. The fusion clones that fused both TrkC/Y516F-Dok6 (PTB+C) and TrkC/Y516F-Dok6C rescued the loss of neurite outgrowth in PC12 cells resulting from the mutation in tyrosine 516, while fusion clones that fused with single TrkC/Y516F-Dok6PTB did not show such effect.

CONCLUSIONDok6 can promote neurite outgrowth induced by NT-3 stimulation through its C-terminal in TrkC-positive PC12 cells.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Neurites ; drug effects ; physiology ; Neurotrophin 3 ; pharmacology ; PC12 Cells ; Rats ; Receptor, trkC ; metabolism ; Transfection

We previously reported that nidogen is an extracellular matrix protein regulating Schwann cell proliferation and migration. Since Schwann cells play a critical role in peripheral nerve regeneration, nidogen may play a role in it via regulation of Schwann cells. Here, we demonstrate direct evidence that nidogen induces elongation of regenerative axon growth of adult sensory neurons, and that the effect is Schwann cell dependent. Continuous infusion of recombinant ectodomain of tumor endothelial marker 7, which specifically blocks nidogen function in Schwann cells, suppressed regenerative neurite growth in a sciatic nerve axotomy model. Taken together, it is likely that nidogen is required for proper regeneration of peripheral nerves after injury.
Animals ; Axotomy ; Cell Movement ; Cell Proliferation ; Male ; Membrane Glycoproteins/*physiology ; Membrane Proteins/pharmacology ; *Nerve Regeneration ; Nerve Tissue Proteins/pharmacology ; Neurites/drug effects/*physiology/ultrastructure ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/pharmacology ; Schwann Cells/cytology/*physiology ; Sensory Receptor Cells/*physiology

Netrin is a neuronal guidance molecule implicated in the development of spinal commissural neurons and cortical neurons. The attractive function of netrin requires the receptor, Deleted in Colorectal Cancer (DCC), while the receptor Unc5h is involved in the repulsive action of netrin during embryonic development. Although the expression of netrin and its receptor has been demonstrated in the adult nervous system, the function of netrin in adult neurons has not yet been elucidated. Here, we show that netrin treatment inhibited neurite outgrowth of adult dorsal root ganglion (DRG) neurons in explant and dissociated cultures. In addition, unc5h1-3 mRNAs, but not the dcc mRNA, are abundantly expressed in the adult DRG. An in situ hybridization study demonstrated that unc5h mRNAs were expressed in DRG neurons. This finding indicates that netrin/Unc5h signaling may play a role in the neurite outgrowth of adult DRG neurons and that netrin may be involved in the regulation of peripheral nerve regeneration.
Animals ; Axons/*drug effects/physiology ; Cells, Cultured ; Ganglia, Spinal/cytology/drug effects/metabolism ; Gene Expression/drug effects ; In Situ Hybridization ; Male ; Nerve Growth Factors/*pharmacology ; Nerve Regeneration/drug effects ; Neurites/drug effects/physiology ; Neurons/*drug effects/metabolism/physiology ; RNA, Messenger/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface/genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Tissue Culture Techniques ; Tumor Suppressor Proteins/*pharmacology

OBJECTIVESHeparin-binding neurite-promoting factor (HBNF) is a heparin-binding protein primarily found in the brain, which can stimulate neurite outgrowth in vitro. We expressed recombinant human heparin-binding neurite-promoting factor (hrHBNF) using a yeast system, and observed its activity in stimulating neurite outgrowth in vitro.

METHODScDNA encoding mature human HBNF was amplified from total RNA isolated from an 18-week aborted human fetal brain by RT-PCR method. After amplification, the HBNF cDNA gene was cloned into pPIC9K, a shuttle expression vector for yeast system. The positive clone of expression vector bearing HBNF cDNA gene was obtained by screening. Verified recombinant vector was then used to transform Pichia strain GS115 by electroporation. His(+) transformants were selected on minimal dextrose medium (MD) plates which were histidine free. His(+) yeast recombinants with multi-copy inserts were screened in vivo by their resistance to G418. PCR analysis was used to confirm the integration of the HBNF cDNA gene into the Pichia genome. Secreted expression of hrHBNF protein in culture medium was obtained when the positive clone containing the HBNF cDNA gene was induced by methanol. The hrHBNF product purified by gel chromatography was added to cultured rat pheochromocytoma (PC12) cells to observe its ability to stimulate neurite outgrowth.

RESULTSIn the recombinant expression vector, the insert was sequenced to show exactly the sequence encoding human HBNF according to Genbank data. The HBNF cDNA gene was cloned downstream to the alpha-factor, and its open reading frame was in frame with the alpha-factor signal sequence in pPIC9K. SDS-PAGE showed that the molecular weight of the induced expression product was about 18 kDa, consistent with that of human HBNF reported in the literature. The protein product did promote neurite outgrowth in cultured rat pheochromocytoma (PC12) cells.

CONCLUSIONRecombinant human heparin-binding neurite-promoting factor can be expressed with a yeast system, and its product possesses the biological activity to promote neurite outgrowth.

Animals ; Base Sequence ; Carrier Proteins ; biosynthesis ; genetics ; pharmacology ; Cytokines ; biosynthesis ; genetics ; pharmacology ; DNA, Complementary ; chemistry ; Genetic Vectors ; Humans ; Molecular Sequence Data ; Neurites ; drug effects ; physiology ; PC12 Cells ; Pichia ; genetics ; Rats ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the effects of androgen on the expression of phosphacan and NG2 proteoglycan (NG2) and neurite regeneration in neonatal rats with hypoxic-ischemic brain damage (HIBD) and the potential mechanism underlying the protective effect of androgen against HIBD.

METHODSOne hundred and twenty neonatal Sprague-Dawley rats were randomly divided into three groups: sham-operated, HIBD and androgen treatment. HIBD was induced by the ligation of left common carotid artery and hypoxia exposure. The androgen treatment group rats were injected with testosterone propionate (25 mg/kg) immediately after HIBD. Phosphacan and NG2 expression in the cortex and the hippocampus was detected with the immunohistochemical method 24 and 72 hrs and 7 and 10 days after hypoxia-ischemia (HI). The ultrastructure and neurite regeneration of neurons in the cortex and the hippocampus were observed under a transmission electron microscope.

RESULTSThe neurite regeneration was obvious in the sham-operated group, but seldom in the HIBD group. The androgen treatment group showed increased neurite regeneration compared with the HIBD group. There were fewer phosphacan and NG2 positive cells in the cortex and the hippocampus in the sham-operated group. Phosphacan and NG2 expression in the cortex and the hippocampus was observed at 24 hrs, increased at 72 hrs, and peaked at 7 days after HI in the HIBD group and remained at a higher expression 10 days after HI than in the sham-operated group. The levels of phosphacan and NG2 expression in the cortex and the hippocampus in the androgen treatment group were significantly reduced compared with those in the HIBD group 24 and 72 hrs and 7 and 10 days after HI (P<0.01).

CONCLUSIONSPhosphacan and NG2 may be important inhibitory factors for neurite regeneration following HIBD in neonatal rats. The neuroprotection of androgen against neonatal HIBD is produced possibly through an inhibition of phosphacan and NG2 expression.

Animals ; Animals, Newborn ; Antigens ; analysis ; Brain Chemistry ; drug effects ; Female ; Hypoxia-Ischemia, Brain ; physiopathology ; Immunohistochemistry ; Male ; Microscopy, Electron, Transmission ; Nerve Regeneration ; drug effects ; Neurites ; physiology ; ultrastructure ; Proteoglycans ; analysis ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor-Like Protein Tyrosine Phosphatases, Class 5 ; analysis ; Testosterone Propionate ; pharmacology

Down syndrome cell adhesion molecule (DSCAM) acts as a netrin-1 receptor and mediates attractive response of axons to netrin-1 in neural development. However, the signaling mechanisms of netrin-DSCAM remain unclear. Here we report that AMP-activated protein kinase (AMPK) interacts with DSCAM through its γ subunit, but does not interact with DCC (deleted in colorectal cancer), another major receptor for netrin-1. Netrin-treatment of cultured cortical neurons leads to increased phosphorylation of AMPK. Both AMPK mutant with dominant-negative effect and AMPK inhibitor can significantly suppress netrin-1 induced neurite outgrowth. Together, these findings demonstrate that AMPK interacts with DSCAM and plays an important role in netrin-1 induced neurite outgrowth. Our study uncovers a previously unknown component, AMPK, in netrin-DSCAM signaling pathway.
AMP-Activated Protein Kinases ; antagonists & inhibitors ; genetics ; metabolism ; Animals ; Cell Adhesion Molecules ; genetics ; metabolism ; Cells, Cultured ; HEK293 Cells ; Humans ; Mice ; Nerve Growth Factors ; pharmacology ; Netrin-1 ; Neurites ; physiology ; Neurons ; cytology ; drug effects ; metabolism ; Phosphorylation ; Protein Binding ; Protein Kinase Inhibitors ; pharmacology ; RNA Interference ; RNA, Small Interfering ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Signal Transduction ; drug effects ; Transfection ; Tumor Suppressor Proteins ; pharmacology