AAV-mediated transfer of RhoA shRNA and CNTF promotes retinal ganglion cell survival and axon regeneration.

The aim of the present study was to determine whether adeno-associated viral vector (AAV) mediated transfer of ciliary neurotrophic factor (CNTF) and RhoA shRNA has additive effects on promoting the survival and axon regeneration of retinal ganglion cells (RGCs) after optic nerve crush (ONC). Silencing effects of AAV-RhoA shRNA were confirmed by examining neurite outgrowth in PC12 cells, and by quantifying RhoA expression levels with western blotting. Young adult Fischer rats received an intravitreal injection of (i) saline, (ii) AAV green fluorescent protein (GFP), (iii) AAV-CNTF, (iv) AAV-RhoA shRNA, or (v) a combination of both AAV-CNTF and AAV-RhoA shRNA. Two weeks later, the ON was completely crushed. Three weeks after ONC, RGC survival was estimated by counting ßIII-tubulin-positive neurons in retinal whole mounts. Axon regeneration was evaluated by counting GAP-43-positive axons in the crushed ON. It was found that AAV-RhoA shRNA decreased RhoA expression levels and promoted neurite outgrowth in vitro. In the ONC model, AAV-RhoA shRNA by itself had only weak beneficial effects on RGC axon regeneration. However, when combined with AAV-CNTF, AAV-RhoA shRNA significantly improved the therapeutic effect of AAV-CNTF on axon regeneration by nearly two fold, even though there was no significant change in RGC viability. In sum, this combination of vectors increases the regenerative response and can lead to more successful therapeutic outcomes following neurotrauma.

Nogo-p4 Suppresses TrkA Signaling Induced by Low Concentrations of Nerve Growth Factor Through NgR1 in Differentiated PC12 Cells.

BACKGROUND: Regeneration of injured axons in adult mammalian central nervous system (CNS) is not spontaneous. Nogo is a major inhibitory molecule contributing to axon regeneration failure. The molecular mechanisms of Nogo inhibition of axon regeneration are not completely understood. To further investigate the underlying mechanisms, we studied the effects of Nogo-p4, a 25-amino acid core inhibitory fragment of Nogo, on nerve growth factor (NGF)-induced TrkA signaling. METHODS: NGF-differentiated PC12 cells were used as cell models. The effects of Nogo-p4 on two key components of TrkA signaling, phosphorylated Erk1/2 and Akt, were analyzed by western blot. Co-immunoprecipitation experiments were performed to detect the formation of NgR1/p75 complexes. Neurite growth was quantified by measuring the neurite length. RESULTS: Nogo-p4 did not significantly affect TrkA signaling induced by 100 ng/ml NGF, but signaling was suppressed when an NGF concentration of 5 ng/ml was used. Further investigation demonstrated that Nogo-p4 affected TrkA signaling in an NGF concentration-dependent manner. Nogo-p4 suppression of TrkA signaling was strong at low (1 and 5 ng/ml), moderate at intermediate (25 ng/ml), but absent at high (50 and 100 ng/ml) NGF concentrations. NEP1-40 attenuated, and NgR1 overexpression enhanced, Nogo-p4 suppression of TrkA signaling induced by low concentrations of NGF. High but not low concentrations of NGF reduced the formation of NgR1/p75 complexes triggered by Nogo-p4. Nogo-p4 strongly inhibited neurite growth induced by low rather than high concentrations of NGF. CONCLUSION: Nogo-p4 binding with NgR1 suppresses TrkA signaling induced by low concentrations of NGF in differentiated PC12 cells. Suppression of NGF-induced TrkA signaling may be another mechanism by which Nogo inhibits neurite growth.

p300 expression is induced by oxygen deficiency and protects neuron cells from damage.

Low oxygen level or oxygen deficiency (hypoxia) is a major factor causing neuronal damage in many diseases. Inducing cell adaptation to hypoxia is an effective method for neuroprotection that can be achieved by either inhibiting the death effectors or enhancing the survival factors. Transcription coactivator p300 is necessary for hypoxia-induced transcriptional activation and plays an important role in neuron survival. However, the alteration of p300 expression under hypoxia condition and its role in hypoxia-induced neuronal damage remain unclear. In this study, the distribution of p300 in rat brain and the alteration of its expression in rat hippocampus during hypobaric hypoxia exposure were detected. In addition, the role of p300 in neuronal-like PC12 cell damage induced by oxygen deficiency (3% oxygen) was evaluated. Our results showed that p300 protein was mainly expressed in the cells expressed beta-tubulin III in the cerebral cortex, hippocampus, cerebellum cortex, medulla oblongata and hypothalamus. Less or no positive signal of p300 expression was observed in beta-tubulin III negative cells. This indicated that p300 was predominantly expressed in neurons of rat brain. Furthermore, p300 expression was up-regulated in rat hippocampus during hypoxia exposure and in neuronal-like PC12 cells under 3% oxygen condition. Interestingly, neuronal-like PC12 cell damage induced by oxygen deficiency (3% oxygen) was increased by suppression of p300 expression with short hairpin RNA (shRNA). These data indicate that p300 is an important molecule for neuroprotection under hypoxia.

Marked effect of RhoA-specific shRNA-producing plasmids on neurite growth in PC12 cells.

In order to promote neurite outgrowth, we constructed a plasmid producing RhoA-specific small hairpin RNA (shRNA) to block RhoA expression and tested its actions in PC12 cells. Our results show that the shRNA-mediated RNA interference (RNAi) successfully suppressed RhoA expression. As a consequence of RhoA knockdown, F-actin levels were significantly reduced and processes were markedly induced. These processes express two neurite markers, neurofilament and betaIII tubulin. This study demonstrates that plasmid-producing shRNA specific for RhoA can act as an effective tool to induce neurite outgrowth and further confirms the neurite growth-inhibitory role of RhoA. This shRNA may thus be a useful tool in promoting neurite outgrowth and could be applicable in neural repair after CNS injury.

Chemotactic effect of ciliary neurotrophic factor on macrophages in retinal ganglion cell survival and axonal regeneration.

PURPOSE: To examine whether ciliary neurotrophic factor (CNTF) has a chemotactic effect on macrophages and whether macrophages are involved in CNTF-induced retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve (ON) injury. METHODS: Adult Fischer 344 rats received an autologous peripheral nerve graft onto transected ON for injured axons to grow. CNTF was applied intravitreally. When needed, clodronate liposomes were applied intravitreally or intravenously to deplete macrophages in the eye. A chemotaxis microchamber system was used to examine whether CNTF has a chemotactic effect on macrophages in vitro, whereas immunohistochemistry was used to identify the location of macrophages/microglia in the retina. The effects of CNTF on RGC neurite outgrowth and macrophage/microglia proliferation were tested in retinal explants. RESULTS: Intravitreal CNTF significantly enhanced RGC survival and axonal regeneration as well as the number of macrophages in the eye. Removal of macrophages significantly reduced CNTF-induced RGC survival and axon regeneration. A chemotaxis assay showed a clear chemotactic effect of CNTF on blood-derived but not peritoneal macrophages. Immunohistochemistry revealed that local microglia was located in a region from the nerve fiber layer (NFL) to the inner nuclear layer, whereas blood-derived macrophages were in the NFL. In vitro experiments revealed that CNTF did not enhance neurite outgrowth or macrophage/microglia proliferation in retinal explants. CONCLUSIONS: CNTF is a chemoattractant but not a proliferation enhancer for blood-derived macrophages, and blood-borne macrophages recruited into the eye by CNTF participate in RGC protection. This finding thus adds an important category to the existing understanding of the biological actions of CNTF.

PI3K/akt, JAK/STAT and MEK/ERK pathway inhibition protects retinal ganglion cells via different mechanisms after optic nerve injury.

Recently we unexpectedly found that PI3K/akt, JAK/STAT and MEK/ERK pathway inhibitors enhanced retinal ganglion cell (RGC) survival after optic nerve (ON) axotomy in adult rat, a phenomenon contradictory to conventional belief that these pathways are pro-survival. In this study we showed that: (i) the RGC protection was pathway inhibition-dependent; (ii) inhibition of PI3K/akt and JAK/STAT, but not MEK/ERK, activated macrophages in the eye, (iii) macrophage removal from the eye using clodronate liposomes significantly impeded PI3K/akt and JAK/STAT inhibition-induced RGC survival and axon regeneration whereas it only slightly affected MEK/ERK inhibition-dependent protection; (iv) in the absence of recruited macrophages in the eye, inhibition of PI3K/akt or JAK/STAT did not influence RGC survival; and (v) strong PI3K/akt, JAK/STAT and MEK/ERK pathway activities were located in RGCs but not macrophages after ON injury. In retinal explants, in which supply of blood-derived macrophages is absent, MEK/ERK inhibition promoted RGC survival whereas PI3K/akt or JAK/STAT inhibition had no effect on RGC viability. However, MEK/ERK inhibition exerted opposite effects on the viability of purified adult RGCs at different concentrations in vitro, suggesting that this pathway may be bifunctional depending on the level of pathway activity. Our data thus demonstrate that inhibition of the PI3K/akt or JAK/STAT pathway activated macrophages to facilitate RGC protection after ON injury whereas the two pathways per se did not modulate RGC viability under the injury conditions (in the absence of the pathway activators). In contrast, the MEK/ERK pathway inhibition protected RGCs via macrophage-independent mechanism(s).

[Hypoxia promotes apoptosis of germ cells in rat testes].

OBJECTIVE: To explore the effects of hypobaric hypoxia on the apoptosis of germ cells in male rats. METHODS: Adult male Wistar rats were randomly divided into four groups: a control group raised at sea level; a 5 d, a 15 d and a 30 d hypoxic group raised in a hypobaric chamber simulating 5000 m altitude for 5 days, 15 days and 30 days respectively. Flow cytometry and TUNEL were used to evaluate the apoptosis of germ cells in the testis. Bax and Bcl-2 in the testis were measured by Western blot. RESULTS: Seminiferous tubules with apoptotic germ cells were significantly more in the hypoxic groups than in the control (P < 0.01). Most apoptotic germ cells were spermatogonia and spermatocytes. Compared with the control group, apoptotic germ cells detected by PI flow cytometry were significantly increased in the hypoxic 15 d and 30 d groups (P < 0.05); Bax was significantly higher (P < 0.05), and so was the ratio of Bax to Bcl-2 in the hypoxic 30 d group (P < 0.01). CONCLUSION: Hypoxia promotes apoptosis of testicular germ cells in male rats. Chronic hypoxia increases Bax expression in the rat testis.

[Isolation and identification of differentially expressed proteins in hippocampus of mice during hypobaric hypoxic delayed preconditioning].

AIM: To explore the differentially expressed proteins between hypobaric hypoxic delayed preconditioning (HHDP) and normal mouse hippocampus. METHODS: After the animal model of HHDP was constructed, hippocampal proteins were obtained by a series of abstraction with lysis solution containing high concentration urea. As soon as isoelectric focusing and SDS-PAGE was performed. The resolved proteins in the 2-DE gels were visualized by Coomassie blue R-250. The gels were scanned, and the images were processed with PDQuest software. Differential proteins were exactly excised from the gels, destained and digested with trypsin. The peptides were isolated and sent for MALDI-TOF-MS testing. Database searching was performed using peptide masses obtained from MALDI-TOF-MS. RESULTS: Averages of 481 +/- 38 and 477 +/- 21 protein spots were detected in control gels and preconditioning gels, respectively. 169 +/- 6 protein spots were matched between these two types of gels. Among the matched spots, while the quantities of 21 +/- 12 spots in control gels increased by above 2 times than that in preconditioning one, the quantities of 33 +/- 10 spots in preconditioning gels increased by the same times than that in control one. The correlation coefficient between these two patterns were 0.7748 +/- 0.0267. 12 spots in preconditioning gels significantly increased compared with the control (P < 0.05, n = 4). Among 12 spots excised from the gels, perfect peptide mass fingerprinting spectrums of 8 spots were acquired. The results showed that one protein was fructose biphosphate aldolase A. Three proteins matched nothing might be new proteins. The other four proteins just matched the partial sequences of the proteins of database were no coincidence to it's isoelectric point and molecular weight. So they might be homological proteins. CONCLUSION: Many proteins, for example fructose biphosphate aldolase A, has been differentially expressed in hippocampus of mice during HHDP. This may be one of the molecule mechanisms of HHDP.

[Effect of hypoxia on expression of iNOS mRNA in cultured rat astrocytes].

AIM: To explore the effects of hypoxia on expression of inducible nitric oxide synthase (iNOS) mRNA in cultured rat astrocytes. METHODS: Cultured rat astrocytes were randomly divided into 4 groups: glutamate group (G), hypoxic group (H), hypoxia + glutamate group (H + G) and the control (C). Cells of control group were exposed to normoxic (95% air, 5% CO2) condition, and cells of G and H + G were incubated with 100 micromol/L L-glutamate, cells of H and H + G exposed to hypoxic conditions (5% CO2, 95% N2) at 37 degrees C. Each group had five timepoints which included 0 h, 3 h, 6 h, 12 h, 24 h, respectively. Expression of mRNAs of iNOS were detected with reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Expression of iNOS mRNA was not detectable in G and C, while it increased dramatically and continuously from 6 h to 24 h in H and G + H. Expression of iNOS mRNA was significantly higher in H than both in G and C at 6 h, 12 h and 24 h, and expression of iNOS mRNA was the highest of all groups in G + H. CONCLUSION: Hypoxia upregulates the expression of iNOS mRNA in cultured astrocytes. Glutamate does not induce the expression of iNOS mRNA but enhance the effect of hypoxia, which is maybe one of the adaptive mechanisms of hypoxia-induced cerebral dilation.