| Stroke is the second fatal disease in the world and the leading cause of death inChina. Moreover, stroke remains to be the most important cause of adult disability.The damage of synapses and the loss of neurons are the crucial reasons ofneurological dysfunction after stroke. Up to now, there is no safety and effectivetreatment for stroke, except for reperfusion therapy (t-PA, vascular recanalization).And new strategies including anti-excitatory toxicity, anti-oxidant, anti-apoptosis andanti-inflammion treatments were all failed in clinical translation because ofineffectiveness or side effects. Thus, development of novel treatment strategies forstroke, especially in suppression apoptosis, prompting neurogenesis and rebuildingneural circuits, is in high priority.The scientific discovery of neurogenesis in adult mammalian is believed to be amilestone in improving neurological recovery after stroke. And as a member of bHLH family, Ngn2plays an important role in neurogenesis of adult mammalian.Many studies have demonstrated that Ngn2is not only the initiation factor ofneurogenesis, but also the regulator of neuronal differentiation, subtype specification,axons projection, dendrites formation, maturation and integration in various regionsof brain. Moreover, latest researches have found that Ngn2is sufficient to stimulateembryonic stem cells, astrocytes, and oligodendrocytes precursor cells differentiationinto glutamate neurons. Meanwhile, Ngn2promotes the translated neural precursorcells to survive, differentiate, migrate and mature. These results indicate that Ngn2may be an attractive candidate for the treatment of stroke, especially in rehabilitationtreatment. However, as a macromolecular peptide, Ngn2can not cross the bloodbrain barrier (BBB), that limits the use of Ngn2in the treatment of central nervoussystem diseases. In the past, we have fused Ngn2, protein transduction domain (PTD)of TAT protein and LBD (laminin binding domain from the first135amino acids ofagrin) to TAT-LBD-Ngn2protein, which may cross the cell membrane and BBB, toaggregate at lesion regions. Moreover, the TAT-LBD-Ngn2fusion protein provides afoundation for the further study on the neuroprotective effect on ischemic stroke.Cultured hippocampal neurons exposed to oxygen-glucose deprivation(OGD),mice global cerebral ischemia model, neurological behavior, immunohistochemistry,and western blot techniques were used in this study to investigate the short-term andlong term neuroprotective effects of TAT-LBD-Ngn2, to confirm the effect ofTAT-LBD-Ngn2on neurogenesis, and to clarify the underlying mechanisms. Thepresent study is aimed to represent a promising therapeutic target and candidate drugfor treatment of ischemic stroke, and provide novel evidence and insights on theneuroprotective effect of Ngn2.Experiment1The protective effect of TAT-LBD-Ngn2againstOGD damage in cultured neuronsPurpose: To investigate the neuroprotective effects of TAT-LBD-Ngn2onprimary neurons exposed to OGD, and to preliminarily clarify the mechanisms in vitro.Methods: The primary cultured hippocampal neurons were respectivelycultured in the presence of TAT-Ngn2, TAT-LBD and TAT-LBD-Ngn2, at aconcentration of125μg/L. Six hours later, the transduction ability and neurite growthwere investigated by immunofluoresence analysis. Then the neurons were dividedinto5groups: Control group, OGD group, OGD+TAT-LBD-Ngn2(62.5μg/L) group,OGD+TAT-LBD-Ngn2(125μg/L) group and OGD+TAT-LBD-Ngn2(250μg/L)group. At24h after OGD, the cell viability was assessed by MTT assay, and neuralinjury was evaluated by LDH release. To determine whether TAT-LBD-Ngn2canreduce neural apoptosis induced by OGD, TUNEL staining was performed. At thesame time, Elisa and western blot were conducted to investigate the expression ofapoptosis-related proteins, such as Caspase-3, Bcl-2and Bax.Results: We found that both TAT-LBD-Ngn2and TAT-Ngn2could cross the cellmembrane into hippocampal neurons, and promote neurite growth (P<0.05) in vitro.At24h after reperfusion, the cell viability significantly increased (P<0.01), and theLDH release decreased (P<0.01) in TAT-LBD-Ngn2group, which showed adose-dependent effect. TAT-LBD-Ngn2also statistically reduced the number ofTUNEL labeling-positive neurons (P<0.05). Meanwhile, the expression ofpro-apoptotic protein(Caspase-3and Bax) decrease (P<0.01), and the expression ofanti-apoptotic protein Bcl-2increased(P<0.01) in TAT-LBD-Ngn2groups. Theanti-apoptotic effect of TAT-LBD-Ngn2was also strengthened with the increasedosage.Conclusion: TAT-LBD-Ngn2could cross the cell membrane, and promoteneurite growth. In addition, TAT-LBD-Ngn2could reduce neurons apoptosis andalleviate ischemia reperfusion injury by suppressing the intrinsic and extrinsicapoptotic pathways.`Experiment2The neuroprotective effects of TAT-LBD-Ngn2oncerebral ischemic injury in vivo Purpose: To investigate the neuroprotective effect of TAT-LBD-Ngn2onglobal cerebral ischemia injury, and to preliminarily clarify the anti-apoptoticmechanisms in vivo.Methods: BCCAO was used as a model of global cerebral ischemia (GCI).Male C57BL/6mice were randomly divided into two groups: control group (PBS200μL, qd ip, n=30) and TAT-LBD-Ngn2(TAT-LBD-Ngn2250μg/kg, qd ip, n=30).At24h after injection of medicines, fluorescent staining and western blot wereperformed to investigate whether TAT-LBD-Ngn2could cross BBB and cellmembrane. The total motor score (TMS)(9possible points) was calculated to detectmotor deficits at1,2,3d after BCCAO. In addition, TUNEL staining was conductedto determine the neuron apoptosis at3d after BCCAO. At the same time, theexpression of neurotrophic factors including BDNF and NGF were investigated byimmunohistochemistry.Results: The model of GCI was successfully established. We found thatTAT-LBD-Ngn2could cross the BBB and transduce into hippocampal and corticalneurons. Moreover, Ngn2distributed much more in hippocampus and cortex inTAT-LBD-Ngn2group(P<0.01), due to the laminin binding domains which inducedNgn2aggregation in the cerebral lesion regions. TAT-LBD-Ngn2treatmentsignificantly improved TMS scores (P<0.05) after GCI injury. Meanwhile,TAT-LBD-Ngn2could attenuate the number of TUNEL labeling-positive cells(P<0.05) in the hippocampal CA1subregion, and improve the expression of BDNFrather than NGF in cortex.Conclusion: TAT-LBD-Ngn2could cross BBB and aggregated at lesion regions.Moreover, TAT-LBD-Ngn2could alleviate neuron I/R injury and improveneurological function by increasing BDNF expression and suppressing neuronapoptosis.Experiment3The effect of TAT-LBD-Ngn2on neurogenesis aftercerebral ischemia Purpose: To investigate the long-term neuroprotective effect ofTAT-LBD-Ngn2, especially the recovery of neurological function. Moreover, topreliminary discuss the effect of neurogenesis in the neuroprotection..Methods: BCCAO was used as a model of GCI. Male C57BL/6mice wererandomly divided into two groups: control group (PBS200μL, qd ip, n=30) andTAT-LBD-Ngn2(TAT-LBD-Ngn2250μg/kg, qd ip, n=30). At14,28d after BCCAO,Morris water maze was conducted to determine the spatial learning and memoryfunction. Fluorescent staining was performed to investigate the number andmorphology of neurons in hippocampal CA1subregion, the new born neurons werelabeled by BrdU and DCX in DG, and functional synaptic connections were labeledby SYN.Results: The escape latency got shortened (P<0.05), and the number ofplatform-site crossover and time spent in target quandrant increased (P<0.05) inTAT-LBD-Ngn2group at14and28d after BCCAO, indicating a better spatial learningand memory functions. In addition, TAT-LBD-Ngn2, at a concentration of250μg/kg,could significantly increase the number of neuron in hippocampal CA1subregion(P<0.05), the numbers of BrdU and DCX labeling-positive neuron in DG (P<0.05).Meanwhile, the expression of SYN increased in TAT-LBD-Ngn2group compared withControl group in cortex and hippocampus.Conclusion: TAT-LBD-Ngn2could promote the survival of neurons inhippocampal CA1subregion. TAT-LBD-Ngn2also improves spatial learning andmemory functions after I/R injury by promoting neurogenesis and rebuilding thesynaptic structure.Summary: According to these results, TAT-LBD-Ngn2could cross blood brainbarrier and cell membrane, and gather into the neurons in the lesion regions.Moreover, TAT-LBD-Ngn2could increase the expression of BDNF, suppress theneuron apoptosis to alleviate early stage ischemic cerebral injury. TAT-LBD-Ngn2also enhances neurogenesis, and rebuilds synaptic structure to promote neurologicalrecovery after ischemic cerebral injury. All these results indicate that TAT-LBD-Ngn2can be a candidate drug for clinicalstroke treatment, which could alleviate cerebral injury in short-term, and promoteneurological recovery in long-term. The present study not only develops a newdirection of macromolecular peptides treatment for stroke, but also provides a newdrug target and research basis in drugs development of stroke. |
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