Nogo-A mRNA Research Articles

Comparison and effect of moxibustion and acupuncture on Nogo/NgR signaling pathway in rats with cerebral ischemia/reperfusion injury.

In China, acupuncture and moxibustion have been used effectively to treat various diseases for thousands of years. However, the evidence for a difference in the efficacies of moxibustion and acupuncture in cerebral infarction treatment is scarce. We aimed to compare the effects of acupuncture and moxibustion treatment on the Nogo/NgR signaling pathway in rats with cerebral ischemia/reperfusion (I/R) injury. Eighty male SD rats were randomly divided into five groups, based on treatment received: sham surgery (sham group), middle cerebral artery occlusion (MCAO, MCAO group), MCAO and NEP(1-40) inhibitor injection (MCAO+block group), MCAO and moxibustion (MCAO+moxi group), and MCAO and minimal acupuncture (MCAO+MA group). Neurological status was evaluated before treatment, and cerebral infarction volume (IV) and neurological function; Nogo-A, NgR, p75NTR, and LINGO-1 expressions; and NgR and LINGO-1 co-expression were assessed after treatment. After treatment, barring Nogo-A mRNA and protein expression in the MCAO+block group, the Longa score and IV significantly decreased; Nogo-A, NgR, p75NTR, and LINGO-1 mRNA and protein expressions as well as NgR and LINGO-1 co-expression significantly decreased in cerebral tissues; whereas the BWT score increased (P<0.01) in the MCAO+moxi group, compared with the MCAO group. Except for NgR and LINGO-1 protein expressions, there were no significant differences in the abovementioned parameters between rats that underwent acupuncture and moxibustion. Acupuncture and moxibustion have similar effects on Nogo/NgR signaling pathway inhibition after cerebral infarction.

Effect of moxibustion on Nogo/neurotrophin receptor signaling pathway in rats with cerebral ischemia/reperfusion injury

To observe the effect of moxibustion treatment on the expression of Nogo-A, Nogo receptor (NgR), neurotrophin receptor p75 (p75NTR) and leucine rich repeat and Ig domain containing 1 (Lingo-1) in brain tissue of rats with cerebral ischemia/reperfusion injury (CI/RI), so as to analyze its mechanism underlying improvement of CI/RI. Male SD rats were randomly divided into sham operation group (16 rats), model group (17 rats), NEP1-40 (extracellular peptide residues 1-40, a blocker targeting NgR) group (model+blocker, 17 rats) and moxibustion group (model+moxibustion, 17 rats). The CI/RI model was established by occlusion of the left middle cerebral artery (MCAO). Moxibustion was applied to "Baihui"(GV20), right "Quchi"(LI11) and "Zusanli"(ST36) for 20 min, once a day for 14 days, with 2 days' rest after the top 7 days' intervention. For rats of the NEP1-40 group, 30 μL PBS containing 18 μg NEP 1-40 was injected into the epidural inferior vena (L5-S1) via a polyvinyl chloride conduit. The neurological deficit state in each group was evaluated by Longa's 5-point scale and Feeney's 7-point scale of beam walking test (BWT). The cerebral infarct volume was assessed by 2,3,5-triphenyltetrazole chloride staining. The brain tissue between the central anterior and posterior sulcus was taken for observing the expression of NgR and Lingo-1 by fluorescence double-label method, and for determining the expression levels of Nogo-A, NgR, p75NTR and Lingo-1 mRNAs and proteins by real-time quantitative PCR and Western blot, respectively. After modeling, the Longa's score, infarct volu-me percent, expression levels of Nogo-A, NgR, Lingo-1 and p75NTR mRNAs and proteins were significantly increased (P<0.01) and BWT score was obviously decreased (P<0.01) in the model group relevant to the sham operation group. In comparison with the model group, the increase of Longa's score, infarct volume percentage, expression levels of Nogo-A, NgR, Lingo-1 and p75NTR mRNAs and proteins and decrease of BWT score in NEP1-40 and moxibustion groups were reversed (P<0.01) except Nogo-A protein in the NEP1-40 group. The effect of moxibustion was significantly superior to that of blocker NEP1-40 in redu-cing the infarct volume percentage, and down-regulating the expression of Nogo-A mRNA and protein, p75NTR mRNA and protein, NgR and Lingo-1 proteins (P<0.01, P<0.05). Moxibustion, similar to blocker NEP1-40 of NgR, can improve neurological dysfunction in CI/RI rats, which may be related to its functions in reducing cerebral infarction and down-regulating the activity of Nogo/neurotrophin receptor signaling pathway.

Xuesaitong exerts long-term neuroprotection for stroke recovery by inhibiting the ROCKII pathway, in vitro and in vivo

Ethnopharmacological relevanceXuesaitong (XST) is a traditional Chinese medicine injection with neuroprotective properties and has been extensively used to treat stroke for many years. The main component of XST is Panax notoginseng saponins (PNS), which is the main extract of the Chinese herbal medicine Panax notoginseng. Aim of the studyIn this study, we investigated whether XST provided long-term neuroprotection by inhibiting neurite outgrowth inhibitor-A (Nogo-A) and the ROCKII pathway in experimental rats after middle cerebral artery occlusion (MCAO) and in SH-SY5Y cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R). Materials and methodsRats with permanent MCAO were administered XST, Y27632, XST plus Y27632, and nimodipine for 14 and 28 days. Successful MCAO onset was confirmed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Neurological deficit score (NDS) was used to assess neurological impairment. Hematoxylin-eosin (HE) staining and immunohistochemical (IHC) analysis of synaptophysin (SYN) and postsynaptic density protein-95 (PSD-95) were performed to evaluate cerebral ischemic injury and the neuroprotective capability of XST. Nogo-A levels and the ROCKII pathway were detected by IHC analysis, western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR) to explore the protective mechanism of XST. OGD/R model was established in SH-SY5Y cells. Cell counting kit 8 (CCK8) was applied to detect the optimum OGD time and XST concentration. The expression levels Nogo-A and ROCKII pathway were determined using western blotting. ResultsOur results showed that XST reduced neurological dysfunction and pathological damage, promoted weight gain and synaptic regeneration, reduced Nogo-A mRNA and protein levels, and inhibited the ROCKII pathway in MCAO rats. CCK8 assay displayed that the optimal OGD time and optimal XST concentration were 7 h and 20 μg/mL respectively in SH-SY5Y cells. XST could evidently inhibit OGD/R-induced Nogo-A protein expression and ROCKII pathway activation in SH-SY5Y cells. ConclusionsThe present study suggested that XST exerted long-term neuroprotective effects that assisted in stroke recovery, possibly through inhibition of the ROCKII pathway.

Anti-apoptotic efficacy of Qingnao Yizhi formula in hypoxia/reoxygenation primary cortical neurons through the promotion of synaptic plasticity by modulating the NogoA-Nogo receptor/Rho-Rho kinase signaling pathway.

To evaluate the anti-apoptotic efficacy of Qingnao Yizhi formula (，QNYZ) in cultured cerebral cortical neuronal cells (CNCs) and the regulation of the NogoA-Nogo receptor (NgR)/Rho-Rho kinase (ROCK) signaling pathway. Primary cultured CNCs were randomly divided into the following groups: normal control group (N-C), hypoxia-reoxygenation group (H/R), high-dose QNYZ group (Q-H), low-dose QNYZ group (Q-L) butylphthalide (NBP) group, and Y-27632 (a selective ROCK transduction pathway inhibiter) group. Except those in the N-C group, CNCs were placed in hypoxic conditions for 24 h and then in reoxygenation conditions for 24 h. Cell media was changed every 48 h, and various assays were performed on the 7th day. Cell viability was evaluated by measuring mitochondrial dehydrogenase activity, using a CCK-8 assay, in triplicate. Synapsin (SYN) protein concentrations were evaluated by enzyme-linked immunosorbent assay. NogoA and RhoA protein expression were evaluated through Western blotting. The gene expression of NogoA, NgR, RhoA, and ROCK was evaluated by reverse transcription-polymerase chain reaction. Cell apoptosis was measured using a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay. Compared with the N-C group, the cell viability of the H/R group decreased significantly (P < 0.05). The cell viability values for the Q-H and Q-L groups increased compared with that for the H/R group, and the difference was significant for the Q-H group (P < 0.05). The NogoA and RhoA protein levels and the NogoA, NgR, RhoA, and ROCK mRNA expression levels increased in the H/R group, compared with the N-C group, and decreased significantly in the Q-H and Q-L groups (P < 0.05) and in the Y-27632 group (P < 0.05) compared with the H/R group. The SYN levels in the Q-H, Q-L, and NBP groups significantly increased compared with that in the H/R group (P < 0.05). Compared with the H/R group, the numbers of apoptotic cells in the Q-H, Q-L, and NBP groups significantly decreased (P < 0.05). The presented study demonstrated that QNYZ exerted anti-apoptotic effects on H/R-induced CNCs, possibly through the modulation of the NogoA-NgR/Rho-ROCK signaling pathway and the promotion of synaptic plasticity in H/R CNCs.

Antenatal taurine supplementation improves neural axon development in fetal rats with intrauterine growth restriction by inhibiting the activity of Rho-ROCK signaling pathway

Objective To investigate the effects of prenatal taurine supplementation on the Rho-ROCK signaling pathway activity and synaptophysin (Syp) expression in brain tissues of rats with intrauterine growth restriction. Methods Eighteen pregnant Sprague-Dawley rats were randomly divided into control group, fetal growth restriction (FGR) group and taurine group, with six rats in each group. Low-protein diet was given in FGR and taurine groups to establish an FGR model. Taurine 300 mg/(kg·d) was supplemented from gestational day 12 until delivery in taurine group. The mRNA expression levels of neurite growth inhibitor-A(Nogo-A), neurite growth inhibitor receptor (NgR), Rho-A and ROCKⅡin fetal rat brain were detected using reverse transcriptase polymerase chain reaction (n=24), which are the key signaling molecules of the Rho-ROCK signal pathway. The protein expression levels of Nogo-A and NgR were detected by Western blot (n=12). The mean optical density in Nogo-A, NgR and Syp was determined by immunohistochemistry (n=18). One-way analysis of variance and LSD-t test were used for statistical analysis. Results (1) Expression of mRNA: the expression levels of Nogo-A, NgR, Rho-A and ROCKⅡ mRNA in fetal rat brain were 4.09±1.34, 3.01±0.77, 39.89±7.71 and 7.82±1.83, respectively in FGR group, and were significantly higher than in control group (1.00±0.13, 1.00±0.10, 1.02±0.30 and 1.00±0.10) (t=4.735, 5.204, 7.682 and 10.675, all P 0.05). (2) Expression of protein by Western blot: the expressions of Nogo-A and NgR protein in fetal rat brain were 1.51±0.09 and 0.31±0.05 in FGR group, 0.82±0.06 and 0.06±0.01 in taurine group, and 1.04±0.10 and 0.09±0.12 in control group. The expression was significantly higher in FGR group than in control group (t=9.644 and 5.285, both P 0.05). (3) Positive expression of protein: the positive expressions of Nogo-A and NgR protein in fetal rat brain were 0.28±0.06 and 0.11±0.02 in FGR group, 0.10±0.02 and 0.04±0.01 in taurine group, and 0.07±0.01 and 0.04±0.01 in control group. The expression was significantly higher in FGR group than in control group (t=9.778 and 7.645, both P 0.05). The positive expression of Syp protein in fetal rat brain was 0.08±0.01 in FGR group, and was significantly lower than in control group (0.16±0.04, t=4.600, P 0.05). Conclusions Prenatal taurine supplementation can improve neural axon development via down-regulating the expressions of the key molecules of Rho-ROCK signal pathway in fetal rat brain tissue. Key words: Fetal growth retardation; Taurine; Rho-associated kinases; Synaptophysin; Signal transduction; Axons; Rats, Sprague-Dawley

Neuroprotective effects of electroacupuncture on early- and late-stage spinal cord injury.

Previous studies have shown that the neurite growth inhibitor Nogo-A can cause secondary neural damage by activating RhoA. In the present study, we hypothesized that electroacupuncture promotes neurological functional recovery after spinal cord injury by inhibiting RhoA expression. We established a rat model of acute spinal cord injury using a modification of Allen's method. The rats were given electroacupuncture treatment at Dazhui (Du14), Mingmen (Du4), Sanyinjiao (SP6), Huantiao (GB30), Zusanli (ST36) and Kunlun (BL60) acupoints with a sparse-dense wave at a frequency of 4 Hz for 30 minutes, once a day, for a total of 7 days. Seven days after injury, the Basso, Beattie and Bresnahan (BBB) locomotor scale and inclined plane test scores were significantly increased, the number of apoptotic cells in the spinal cord tissue was significantly reduced, and RhoA and Nogo-A mRNA and protein expression levels were decreased in rats given electroacupuncture compared with rats not given electroacupuncture. Four weeks after injury, pathological tissue damage in the spinal cord at the site of injury was alleviated, the numbers of glial fibrillary acidic protein- and neurofilament 200-positive fibers were increased, the latencies of somatosensory-evoked and motor-evoked potentials were shortened, and their amplitudes were increased in rats given electroacupuncture. These findings suggest that electroacupuncture treatment reduces neuronal apoptosis and decreases RhoA and Nogo-A mRNA and protein expression at the site of spinal cord injury, thereby promoting tissue repair and neurological functional recovery.

Nogo-A expression dynamically varies after spinal cord injury.

The mechanism involved in neural regeneration after spinal cord injury is unclear. The myelin-derived protein Nogo-A, which is specific to the central nervous system, has been identified to negatively affect the cytoskeleton and growth program of axotomized neurons. Studies have shown that Nogo-A exerts immediate and chronic inhibitory effects on neurite outgrowth. In vivo, inhibitors of Nogo-A have been shown to lead to a marked enhancement of regenerative axon extension. We established a spinal cord injury model in rats using a free-falling weight drop device to subsequently investigate Nogo-A expression. Nogo-A mRNA and protein expression and immunoreactivity were detected in spinal cord tissue using real-time quantitative PCR, immunohistochemistry and western blot analysis. At 24 hours after spinal cord injury, Nogo-A protein and mRNA expression was low in the injured group compared with control and sham-operated groups. The levels then continued to drop further and were at their lowest at 3 days, rapidly rose to a peak after 7 days, and then gradually declined again after 14 days. These changes were observed at both the mRNA and protein level. The transient decrease observed early after injury followed by high levels for a few days indicates Nogo-A expression is time dependent. This may contribute to the lack of regeneration in the central nervous system after spinal cord injury. The dynamic variation of Nogo-A should be taken into account in the treatment of spinal cord injury.

Nogo-A and its functions beyond axonal inhibition: the controversial role of Nogo-A in Parkinson's disease.

Nogo-A belongs to the reticulon family (RTN4) and is generally assumed to be one of the most potent myelin associated neurite outgrowth inhibitors in the central nervous system (CNS). Together with other inhibitors such as the myelin associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), several semaphorins and ephrins as well as chondriotin sulphate proteoglycans, Nogo-A contributes to a nonpermissive environment in the brain and spinal cord. Based on their seminal observation that neurons grown in close proximity to CNS derived oligodendrocytes showed a robust decrease in neurite outgrowth and number of processes, Schwab and Caroni (1988) postulated for nonpermissive substrate properties present in the CNS myelin. In further studies, they identified a high molecular weight component from the CNS myelin called NI-250 which was later renamed Nogo-A. In the course it could be demonstrated that Nogo-A is expressed on the surface of oligodendrocytes and is responsible for the inhibition of axonal sprouting. Since the nonpermissive environment plays a very important role in brain injuries and neurodegenerative diseases, Nogo-A and its receptors were extensively studied.

Age-dependent decline of nogo-a protein in the mouse cerebrum.

Nogo-A, a myelin-associated neurite growth inhibitory protein, is implicated in synaptic plasticity. It binds to its receptor namely the Nogo-66 receptor1 (NgR1) and regulates filamentous (F) actin dynamics via small GTPases of the Rho family, RhoA kinase (ROCK), LimK and cofilin. These proteins are associated with the structural plasticity, one of the components of synaptic plasticity, which is known to decline with normal aging. So, the level of Nogo-A and its receptor NgR1 are likely to vary during normal brain aging. However, it is not clearly understood how the levels of Nogo-A and its receptor NgR1 change in the cerebrum during aging. Several studies show an age- and gender-dependent decline in synaptic plasticity. Therefore, the present study was planned to analyze the relative changes in the mRNA and protein levels of Nogo-A and NgR1 in both male and female mice cerebrum during normal aging. Western blot analysis has shown decrease in Nogo-A protein level during aging in both male and female mice cerebrum. This was further confirmed by immunofluorescence analysis. RT-PCR analysis of Nogo-A mRNA showed no significant difference in the above-mentioned groups. This was also supported by in situ hybridization. NgR1 protein and its mRNA expression levels showed no significant alteration with aging in the cerebrum of both male and female mice. Taken together, we speculate that the downregulation of Nogo-A protein might have a role in the altered synaptic plasticity during aging.

Protection of NEP1-40 on retinal cells following retinal ischemia reperfusion injury

Background The retina ischemia reperfusion injury (RIRI) can lead to apoptosis,which is associated with many genes.It is very significant to explore the protection of drugs on RIRI-induced apoptosis.Objective This study was to explore the relationship between the expression of Nogo-A and the cell apoptosis as well as the therapeutic effect of NEP1-40 in RIRI retina.Methods The device of raising intraocular pressure (IOP)was used to elevate the IOP for 60 minutes and then restore the IOP to normal for the establishment of RIRI models.Seventy-eight SD rats were randomized to the normal group,RIRI group and NEP1-40 group.PBS of 5 ml/(kg · d)was injected intraperitoneally in the rats of the RIRI group,and NEP1-40 of 8 mg/(L · kg) was used in the same way in the NEP1-40 group.The rats were sacrificed in overdose anesthesia at 6 hours,12 hours and 1 day,2,3,7 days after RIRI to prepare the retinal specimens.The ultrastructure of rat retinas was examined under the transmission electron microscope.Cell apoptosis was assessed by the TUNEL method,and the apoptotic index (AI) was calculated.The expressions of Nogo-A protein and mRNA in rat retinas were detected by immunohistochemistry and semiquantitative reverse transcription PCR (RT-PCR).Results The ultrastructure of retinal cells were normal in the normal group.Retinal cell organelle dissolution,mitochondria swelling,cavitation,chromatin edge heterochromatin and apoptotic body were seen in the rats of the RIRI group from 12 hours through 7 days after injection.However,only the slight loose of outer membranous disk,inner and outer nuclear layer and retinal ganglion cells,the nucleus gap broadening,shortness of some mitochondrial cristae in the NEP1-40 group.TUNEL-positive cells appeared 6 hours after RIRI and reached peak in 1 day,and gradually declined after that in the RIRI group.A similar pattern was seen in the rats of the NEP1-40 group with a more mild manifestation.Significant differences were seen in the AI values among the different groups at various time points (Fgroup =100.850,P =0.000 ; Ftime =34.309,P =0.000),and the AI values were significantly higher in the RIRI group and NEP1-40 group compared with normal group;while the AI values in the NEP1-40 group was lower than those of the RIRI group (all at P＜0.05).The expressions of Nogo-A protein and mRNA showed a coincident pattern with apoptosis procedure,with a gradually elevated level from 6 hours through 7 days after RIRI and a peak in 2 days,and those in the NEP1-40 group were weaker in comparison with the RIRI group in various time points.Significant differences were detected in the expression of Nogo-A protein and the expression of Nogo-A mRNA among different groups and various time points(protein:Fgroup =164.139,P =0.000;Ftime =21.772,P =0.000.mRNA:Fgroup =93.889,P =0.000 ; Ftime =6.349,P =0.000).Conclusions Nogo-A probably plays an important role in RIRI.NEP1-40 can protect retinal cells from apoptosis following RIRI through down-regulating the expression of Nogo-A. Key words: NEP1-40; Retina; Ischemia reperfusion injury; Nogo-A; Apoptosis

Synthetic microRNA-mediated downregulation of Nogo-A in transgenic rats reveals its role as regulator of synaptic plasticity and cognitive function

We have generated a transgenic rat model using RNAi and used it to study the role of the membrane protein Nogo-A in synaptic plasticity and cognition. The membrane protein Nogo-A is expressed in CNS oligodendrocytes and subpopulations of neurons, and it is known to suppress neurite growth and regeneration. The constitutively expressed polymerase II-driven transgene was composed of a microRNA-targeting Nogo-A placed into an intron preceding the coding sequence for EGFP, thus quantitatively labeling cells according to intracellular microRNA expression. The transgenic microRNA in vivo efficiently reduced the concentration of Nogo-A mRNA and protein preferentially in neurons. The resulting significant increase in long-term potentiation in both hippocampus and motor cortex indicates a repressor function of Nogo-A in synaptic plasticity. The transgenic rats exhibited prominent schizophrenia-like behavioral phenotypes, such as perseveration, disrupted prepulse inhibition, and strong withdrawal from social interactions. This fast and efficient microRNA-mediated knockdown provides a way to silence gene expression in vivo in transgenic rats and shows a role of Nogo-A in regulating higher cognitive brain functions.

Time Course and Spatial Profile of Nogo-A Expression in Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice

Inhibition of the myelin-associated neurite outgrowth inhibitor Nogo-A has been found to be beneficial in experimental autoimmune encephalomyelitis (EAE), but there are little data on its expression dynamics during the disease course. We analyzed Nogo-A mRNA and protein during the course of EAE in 27 C57BL/6 mice and in 8 controls. Histopathologic and molecular analyses were performed on Day 0 (naive), preclinical (Day 10), acute (Days 18-22) and chronic (Day 50) time points. In situ hybridization and real-time polymerase chain reaction analyses revealed reduced Nogo-A mRNA expression at preclinical (p < 0.0001) and acute phases (p < 0.0001), followed by upregulation during the chronic phase (p < 0.0001). Nogo-A mRNA was expressed in neurons and oligodendrocytes. By immunohistochemistry and Western blot, there was increased Nogo-A protein expression (p < 0.001) in the chronic phase. Moreover, spatial differences were observed within EAE lesions. The pattern of Nogo-A protein expression inversely correlated with axonal regeneration growth-associated protein 43-positive axons (60% of which were Nogo-A contact-free during the acute phase) and axonal injury (β-amyloid precursor protein-positive axons). Cortical Nogo-66 receptor protein and mRNA levels increased during the chronic phase. The results indicate that Nogo-A and Nogo receptor are actively regulated in EAE lesions; this may indicate a specific time window for localized axonal regeneration in the acute phase of EAE.

Expression of Nogo-A, NgR mRNA and protein in the retina of rats with chronic elevated intraocular pressure

To investigate the expression of Nogo-A and its receptor NgR mRNA and protein in the retina of rats with chronic elevated intraocular pressure (IOP). Experimental study. Rat chronic ocular hypertension (OHT) was induced by obstructing episcleral veins and temporal limber veins. The retinal tissues were collected at day 3, 7, 14 and 28 after the IOP elevated in rats. Each group includes 16 rats and one group served as normal control. Expression of Nogo-A and NgR mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR), while the protein levels of Nogo-A and NgR were expressed by the western blot in rat retinal tissues in different groups. Expression of Nogo-A, NgR mRNA and protein were analysed by one way analysis of variance (ANOV) and multiple comparison ANOV. IOP in rat OHT group was significantly increased after day 3 lasting for 28 day. Compared with control group, the level of Nogo-A mRNA and protein in rat chronic OHT groups were significantly (mRNA: F = 7.464, protein: F = 5.677; P < 0.01) increased at day 7, 14, and 28 (mRNA: 0.661 ± 0.065 vs 0.831 ± 0.055, 0.813 ± 0.063, 0.844 ± 0.077, protein: 1.284 ± 0.043 vs 1.359 ± 0.033, 1.381 ± 0.063, 1.361 ± 0.044), respectively. There is a tendency of increase of Nogo-A mRNA expression at day 3 but did not reach statistical significance. However, the level of NgR in the retina in rats with the chronic OHT was not significantly (mRNA:F = 0.598, protein:F = 0.460; P > 0.01) changed compared with normal group. The increased expression of Nogo-A in retina of rat OHT indicates that Nogo-A may play a primary role in obstructing regeneration of optic nerve, which is mediated by other receptors and elements rather than NgR.

Neuronal Nogo‐A regulates glutamate receptor subunit expression in hippocampal neurons

Nogo-A and its cognate receptor NogoR1 (NgR1) are both expressed in neurons. To explore the function of these proteins in neurons of the CNS, we carried out a series of studies using postnatal hippocampal neurons in culture. Interfering with the binding of Nogo-A to NgR1 either by adding truncated soluble fragment of NgR1 (NgSR) or by reducing NgR1 protein with a specific siRNA, resulted in a marked reduction in Nogo-A expression. Inhibition of Rho-ROCK or MEK-MAPK signaling resulted in a similar reduction in neuronal Nogo-A mRNA and protein. Reducing Nogo-A protein levels by siRNA resulted in an increase in the post-synaptic scaffolding protein PSD95, as well as increases in GluA1/GluA2 AMPA receptor and GluN1/GluN2A/GluN2B NMDA glutamate receptor subunits. siRNA treatment to reduce Nogo-A resulted in phosphorylation of mTOR; addition of rapamycin to block mTOR signaling prevented the up-regulation in glutamate receptor subunits. siRNA reduction of NgR1 resulted in increased expression of the same glutamate receptor subunits. Taken together the results suggest that transcription and translation of Nogo-A in hippocampal neurons is regulated by a signaling through NgR1, and that interactions between neuronal Nogo-A and NgR1 regulate glutamatergic transmission by altering NMDA and AMPA receptor levels through an rapamycin-sensitive mTOR-dependent translation mechanism.

Induction of Nogo‐A in Cardiomyopathy

The Nogo isoforms A, B, and C are part of a superfamily of proteins termed the reticulons, from the same gene (Nogo/RTN4), share a c‐terminal domain, and are thought to have preferential ER localization. Nogo‐A has been traditionally described as a myelin associated inhibitor of axonal sprouting, with a molecular weight of ~160 kilodaltons (kDa) limited tissue distribution, and multiple transmembrane domains. There is a vast amount of data examining the function of the Nogo reticulon protein family, particularly with respect to neuronal plasticity however, very little is known about the Nogo proteins in the heart. In failing human heart samples we saw a 3 fold increase in Nogo‐A, vs nonfailing by western immunoanalysis (n=5). RT‐PCR of isolated human cardiac fibroblasts showed a 3 fold increase in nogo‐A mRNA with 2 hours of cyclic stretch. Western analysis of PKC epsilon overexpressing heart extracts showed increased levels of Nogo‐A, corresponding to the development of the dilated phenotype. Isolated cardiomyocyte experiment demonstrate an increase in Nogo‐A with mechanical stretch but not with hypertrophic agonist treatment. We propose an increase Nogo‐A in the heart is an adaptive mechanoresponse.

Soluble Nogo Receptor Down-regulates Expression of Neuronal Nogo-A to Enhance Axonal Regeneration

Nogo-A, a member of the reticulon family, is present in neurons and oligodendrocytes. Nogo-A in central nervous system (CNS) myelin prevents axonal regeneration through interaction with Nogo receptor 1, but the function of Nogo-A in neurons is less known. We found that after axonal injury, Nogo-A is increased in dorsal root ganglion (DRG) neurons unable to regenerate following a dorsal root injury or a sciatic nerve ligation-cut injury and that exposure in vitro to CNS myelin dramatically enhanced neuronal Nogo-A mRNA and protein through activation of RhoA while inhibiting neurite growth. Knocking down neuronal Nogo-A by small interfering RNA results in a marked increase of neurite outgrowth. We constructed a nonreplicating herpes simplex virus vector (QHNgSR) to express a truncated soluble fragment of Nogo receptor 1 (NgSR). NgSR released from QHNgSR prevented myelin inhibition of neurite extension by hippocampal and DRG neurons in vitro. NgSR prevents RhoA activation by myelin and decreases neuronal Nogo-A. Subcutaneous inoculation of QHNgSR to transduce DRG neurons resulted in improved regeneration of myelinated fibers in both the dorsal root and the spinal dorsal root entry zone, with concomitant improvement in sensory behavior. The results indicate that neuronal Nogo-A is an important intermediate in neurite growth dynamics and its expression is regulated by signals related to axonal injury and regeneration, that CNS myelin appears to activate signaling events that mimic axonal injury, and that NgSR released from QHNgSR may be used to improve recovery after injury.

Regional‐specific regulation of BDNF and trkB correlates with nigral dopaminergic cell sprouting following unilateral nigrostriatal axotomy

Axotomy is a powerful stimulus of axon growth and plastic changes. We investigated the potential role of BDNF/trkB signaling in the sprouting of dopaminergic nigral axons in response to axotomy of the medial forebrain bundle. Tyrosine hydroxylase immunohistochemistry revealed the existence of sprouting mechanisms in the axotomized substantia nigra (SN). Time-course changes of trkB mRNA expression demonstrated a robust increase in an area projecting from the rostral tip of the SN to the glial scar, which coincided with evidence of nigral dopaminergic sprouting. In addition, we found an early loss of this messenger in areas related to the knife cut, which recovered by 7 days postlesion. TrkB down-regulation appeared to be associated to the lesion-induced local damage, as it was restricted to an area showing Fluoro-Jade B- and TUNEL positive cells. In trkB-depleted areas, an inverse correlation between mRNA expressions of BDNF and trkB was apparent. Specific induction of BDNF mRNA was mostly seen in border of areas devoid of trkB mRNA. In contrast, in the areas exhibiting trkB mRNA expression, no BDNF mRNA was detected. We suggest that trkB levels could be a determinant element in regulating BDNF expression. Finally, the search for molecules involved in either promoting or inhibiting axonal growth, demonstrated up-regulation of GAP-43 and Nogo-A mRNA at sites close to the knife transections as early as 1 day postlesion. However, overall, Nogo-A induction was more robust than that seen for GAP-43.

Differential expression patterns of messenger RNAs encoding nogo receptors and their ligands in the rat central nervous system

Nogo receptors (NgR1, -2, and -3) and their ligands, i.e., myelin-derived neurite outgrowth inhibitor (Nogo)-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), have been considered to play pivotal roles in controlling axonal regeneration and neuronal plasticity. We show here that NgR1-3 mRNAs were differentially expressed exclusively in neurons situated in the telencephalon, diencephalons, and cerebellum, whereas we could not detect any NgR1-3 mRNA expression in the mesencephalon, pons, medulla oblongata, and spinal cord. On the other hand, Nogo-A mRNA was abundantly expressed in both neurons and oligodendrocytes throughout the central nervous system (CNS). MAG and OMgp mRNAs were also abundantly expressed in oligodendrocytes throughout the CNS. Interestingly, we did not detect NgR1-3 mRNAs in monoaminergic neurons in the substantia nigra, ventral tegmental area, locus caeruleus, and raphe nuclei, which are known to have high regenerative capacity. In addition, although neurons in the reticular thalamus and cerebellar nuclei are also known to show high capacity for regeneration, NgR1-3 mRNAs were not detected there. These data indicate that NgR1-3, Nogo-A, MAG, and OMgp mRNAs are differentially expressed in the rat CNS and suggest that the level of NgR1-3 expression in a neuron might determine its regenerative capacity.

Knocking-down of Nogo-A gene expression in PC12 cell line by plasmid-based RNAi

To study the inhibitory effect of Nogo-A shRNA on cell line PC12, the Nogo-A shRNA (short hairpin RNA, or shRNA) was designed and synthesized. The annealed shRNA template was inserted into plasmid pGenesil-1 containing enhanced green fluorescent protein (EGFP) gene by gene cloning technique to generate eukaryotic expression vector. The recombinant plasmid was transfected into PC12 cells by lipofecamine2000 and the mRNA and protein expression level of Nogo-A gene was detected by RT-PCR and Western blotting 48 h after the transfection. Gene sequencing showed that that the Nogo-A shRNA eukaryotic expression vector was successfully constructed. No significant change was found in the Nogo-A mRNA and protein expression level in empty vector-transfected group as compared with controls (P>0.05), while the expression level in shRNA-transfected group decreased significantly (P<0.05). It is concluded that the pGenesil-1/Nogo-AshRNA recombinant plasmid can effectively suppress the expression of Nogo-A gene in PC12 cells.

Expression of Nogo-A mRNA and Nogo-A protein in brain tissue of neonatal mice with ischemic-hypoxic brain damage

Expression of Nogo-A mRNA and Nogo-A protein in brain tissue of neonatal mice with ischemic-hypoxic brain damage