Rao Fu

C-jun phosphorylation contributes to down regulation of neuronal nitric oxide synthase protein and motoneurons death in injured spinal cords following root-avulsion of the brachial plexus

Previous studies have shown that c-jun and neuronal nitric oxide synthase (nNOS) are both induced in injured motoneurons, but their roles in motoneuron death remain unclear. We hypothesized that nNOS might be the downstream effector of c-jun N-terminal kinase (JNK)/c-jun in avulsion-induced motoneuron death. Here, we found that brachial root-avulsion induced a temporary increase in JNK activity and three- and four-fold increases in phospho-c-jun and c-jun, respectively; however, brachial root-avulsion caused a decrease in nNOS protein expression from 4 h to 14 days post-injury. At 14 days post-injury, almost all nNOS-positive motoneurons were co-localized with phospho-c-jun-positive motoneurons in ipsilateral ventral horns. The JNK inhibitor SP600125, applied immediately post-injury, resulted in an upregulation of nNOS protein both in injured spinal cords and motoneurons and caused a slight alleviation of motoneuron death by inhibiting c-jun phosphorylation at 14 days post-injury. Our results demonstrated that the JNK/c-jun signal transduction pathway is involved in root-avulsion. The inhibition of c-jun phosphorylation prevents nNOS levels from dropping below baseline levels in the spinal cord and partially alleviates motoneuron death following root-avulsion. Therefore, inhibiting c-jun phosphorylation or up-regulating the nNOS protein in injured spinal cords at the early stage might be used in the future as the molecular-target strategies to prevent the motoneurons degeneration in root-avulsion.

Intrathecal application of short interfering RNA knocks down c-jun expression and augments spinal motoneuron death after root avulsion in adult rats

The immediate-early gene, c-jun, is expressed in spinal motoneurons after spinal root avulsion. The expression of c-jun was suggested to be necessary for motoneuron survival and regeneration after avulsion. In the present study, a small interfering RNA (siRNA) was delivered intrathecally to the injured spinal segments immediately after root avulsion in rats to knock down expression of the c-jun gene in injured spinal motoneurons so as to explore the role of c-jun in the motoneurons in vivo. Our results showed that the siRNA not only inhibited the expression of both c-jun mRNA and protein but also augmented the death of injured motoneurons at day 14 post-injury. These findings indicated that induction of c-jun gene expression plays a pivotal role in the survival of injured motoneurons. Meanwhile, these results suggest that siRNAs applied intrathecally can effectively mediate the expression of the c-jun gene in injured motoneurons.

Lithium enhances survival and regrowth of spinal motoneurons after ventral root avulsion

BackgroundDuring the clinical treatment of the brachial plexus root avulsion (BPRA), reimplantation surgery can not completely repair the motor function of the hand because the axonal growth velocity of the spinal motoneurons (MNs) is too slow to re-innervate the intrinsic hand muscles before muscle atrophy. Here, we investigated whether lithium can enhance the regenerative capacity of the spinal MNs in a rat model of BPRA.ResultsThe avulsion and immediate reimplantation of the C7 and C8 ventral roots were performed and followed with daily intraperitoneal administration of a therapeutic concentrationof LiCl. After a 20 week long-term rehabilitation, the motor function recovery of the injured forepaw was studied by a grasping test. The survival and regeneration of MNs were checked by choline acetyltransferase (ChAT) immunofluorescence and by Fluoro-Gold (FG) retrograde labeling through the median and ulnar nerves of the ventral horn MNs. The number and diameter of the nerve fibers in the median nerve were assessed by toluidine blue staining. Our results showed that lithium plus reimplantation therapy resulted in a significantly higher grasping strength of the digits of the injured forepaw. Lithium plus reimplantation allowed 45.1% ± 8.11% of ChAT-positive MNs to survive the injury and increased the number and diameter of nerve fibers in the median nerve. The number of FG-labeled regenerative MNs was significantly elevated in all of the reimplantation animals. Our present data proved that lithium can enhance the regenerative capacity of spinal MNs.ConclusionsThese results suggest that immediate administration of lithium could be used to assist reimplantation surgery in repairing BPRA injuries in clinical treatment.

Activation of phospholipase-Cγ and protein kinase C signal pathways helps the survival of spinal motoneurons injured by root avulsion

J. Neurochem. (2012) 121, 362–372.

Efficient labeling in vitro with non-ionic gadolinium magnetic resonance imaging contrast agent and fluorescent transfection agent in bone marrow stromal cells of neonatal rats

Although studies have been undertaken on gadolinium labeling-based molecular imaging in magnetic resonance imaging (MRI), the use of non-ionic gadolinium in the tracking of stem cells remains uncommon. To investigate the efficiency in tracking of stem cells with non-ionic gadolinium as an MRI contrast agent, a rhodamine-conjugated fluorescent reagent was used to label bone marrow stromal cells (BMSCs) of neonatal rats in vitro, and MRI scanning was undertaken. The fluorescent-conjugated cell uptake reagents were able to deliver gadodiamide into BMSCs, and cell uptake was verified using flow cytometry. In addition, the labeled stem cells with paramagnetic contrast medium remained detectable by an MRI monitor for a minimum of 28 days. The present study suggested that this method can be applied efficiently and safely for the labeling and tracking of bone marrow stromal cells in neonatal rats.

Involvement of phospholipase C-γ in the pro-survival role of glial cell line-derived neurotrophic factor in developing motoneurons in rat spinal cords

The glial cell line-derived neurotrophic factor (GDNF) has been proven to be the most powerful neurotrophic factor in neuronal development. However, it remains uncertain as to which intracellular signaling pathway interacting with GDNF is invovlved in motoneuron (MN) development. In this study, we investigated whether phosphoinositide phospholipase C-γ (PLC-γ) is involved in GDNF-promoted MN development. The primary spinal MNs from 12- to 14-day-old embryos of Sprague-Dawley rats were cultured and survival was sustained by GDNF. A specific inhibitor of PLC-γ, 1-[6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl]-1H-pyrrole-2,5-dione (U73122), was used to block the pro-survival effect of GDNF. Our results showed that MN-like cells appeared at 72 h after initial implantation and were sustained for a period of up to seven days under GDNF treatment. These cultured MNs expressed neuron-specific enolase, SMI-32, 75-kDa low-affinity neurotrophic receptor and choline acetyltransferase. The survival rate of the cultured MNs at 24 h was significantly lower in the GDNF + U73122-treated group (31.87±2.17%), compared either with that of the GDNF- (81.38±1.13%) or GDNF + DMSO (79.39±1.22%)-treated groups. The present data suggest that PLC-γ may be one of the intracellular signals that play a role in the survival-promoting effects of GDNF in developing spinal MNs.

Suppression of c-jun influences nNOS expression in differentiated PC12 cells.

In various animal models of central neuronal diseases, both c-jun and nNOS genes are expressed inside injured neurons; however, the mechanism of these two genes in neuronal diseases remains uncertain. Our previous studies have shown that c-jun expression always occurs prior to expression of nNOS in motoneuron injuries. We aimed to determine whether there is a correlation between c-jun and nNOS, and whether the crosstalk between these two genes regulated the pathological progression of injury-induced neuronal degeneration. In the present study, we used the neuron-like differentiated PC12 cells, which express c-jun and nNOS, to examine whether c-jun is the upstream molecule modulating nNOS expression. The c-jun small interfering RNAs (c-jun siRNA) were transfected into PC12 cells and cells were treated for 72 h in vitro. Western blotting and immunofluorescence were used to check the protein levels and the expression of c-jun and nNOS in differentiated PC12 cells. The results from the immunofluorescence experiments showed that the c-jun and nNOS proteins were co-expressed in the differentiated PC12 cells. The results from the western blotting experiments revealed that the protein levels of c-jun were significantly decreased by c-jun siRNA. Moreover, the nNOS protein levels were also downregulated in differentiated PC12 cells following c-jun siRNA treatment. The present study found that siRNA used against c-jun not only knocked down c-jun, but also downregulated the nNOS protein expression in differentiated PC12 cells. These results indicate that there is a functional relationship between c-jun and nNOS in differentiated PC12 cells.

BCR-ABL tyrosine kinase inhibitor pharmacophore model derived from a series of phenylaminopyrimidine-based (PAP) derivatives

To reveal novel insights into the inhibition of BCR-ABL tyrosine kinase, pharmacophore mapping studies were performed for a series of phenylaminopyrimidine-based (PAP) derivatives, including imatinib (Gleevec). A seven-point pharmacophore model with one hydrophobic group (H), two hydrogen bond donors (D) and four aromatic rings (R) was developed using phase (pharmacophore alignment & scoring engine). The pharmacophore hypothesis yielded a statistically significant 3D-QSAR model, with a correlation coefficient of 0.886 and a survival score of 4.97 for training set molecules. The model showed excellent predictive power, with a correlation coefficient of Q(2)=0.768 for an external test set of ten molecules. The results obtained from our studies provide a valuable tool for designing new lead molecules with potent activity.