Bcl-2 Reduces The Expression Of BMP4 After Cerebral Ischemia By β-catenin To Promote Neuronal Regeneration In Striatum

Ischemic stroke ranks as the third major cause of clinical mortality and the leading cause of handicap in adults.The severity of an acute ischemic stroke depends mainly on the degree and duration of local cerebral blood flow（ICBF） reduction.Prompt reperfusion improves neurological deficits,spontaneous electrical activity,energy metabolism,cerebral protein synthesis（CPS）,and tissue repair,among which neurogenesis may have important functional and therapeutic implications.However,it has been reported that the majority of newborn neurons underwent apoptosis soon after their birth.Therefore,attention was given to the study of how to promote neurogenesis and survival of newborn neurons.Bcl-2,an anti-apoptosis protein,plays an important role in both central nervous system development and neuropathological processes.In addition,several lines of evidence suggest that besides its anti-apoptotic activity,Bcl-2 plays an active role in neuronal differentiation and axon outgrowth,and the mechanism mediated in this process is independent of apoptosis.To investigate the concrete molecular mechanism that Bcl-2 overexpression promotes ischemia-induced neurogenesis in rat brain,we performed occlusion of the middle cerebral artery（MCAO） combined with Bcl-2 expressing plasmid andβ-catenin siRNA intraventricular injection.Double immunohistochemical staining was used to detect newborn neurons of different phenotype and the effects of Bcl-2 overproduction and endogenousβ-catenin on neurogenesis were analyzed in the adult rat brain after ischemia.Western blot was used to probe the expression of BMP4, Noggin,β-catenin,and BMP7 in the ischemic striatum.The results were summarized as follows:1.Expression of BMP4,Noggin,andβ-catenin in the striatum after cerebral ischemia.To evaluate the effect of BMP4,Noggin,andβ-catenin on endogenous neurogenesis after MCAO,we first analyzedβ-catenin protein expression in the ischemic striatum.Immunohistochemistry and Western blot were performed.We found thatβ-catenin immunoreactivity preferentially localized to the cell membrane and immunostaining with anti-β-catenin antibodies revealed intense labeling of ringlike cell structures in the normal striatum.However,β-catenin-positive cells and protein were markedly decreased in the ischemic striatum at 3 d after MCAO.Both BMP4 and Noggin positive cells and protein were significantly increased in the ischemic striatum at 3,7,and 14 d after MCAO,and arrived at the peak at 3 d following stroke.These results demonstrated that BMP4,Noggin,andβ-catenin might play vital roles in ischemic-induced striatal neurogenesis.2.Bcl-2 overproduction down-regulated the expression of BMP4 and rescuedβ-catenin protein in the ischemic striatum.To observe the effect of Bcl-2 overproduction on the expression of BMP4,Noggin, andβ-catenin in the ischemic striatum,we injected Bcl-2 expressing or control plasmid into the lateral ventricle after ischemia（i.c.v.）.Bcl-2 overproduction significantly down-regulated the BMP4 positive cells quantity at 3,7,and 14 days after MCAO.But Bcl-2 overproduction could not up-regulated the Noggin positive cells quantity at 3,7,and 14 days after MCAO.On the contrary,Bcl-2 plasmid i.c.v. administration significantly decreased Noggin positive cell number at 3 days after MCAO.Moreover,Bcl-2 overproduction could significantly up-regulate theβ-catenin positive cells quantity at 3 days after MCAO.Western blot also obtained similar results.These results suggested that Bcl-2 overproduction might decrease the expression of BMP4 thoughβ-catenin.3.Bcl-2 overexpression had no effect on the expression of BMP7 in the ischemic striatum.To exclude the influence of Bcl-2 overexpression on the other member of BMP, such as BMP7,we observed the expression of BMP7 in the ischemic striatum. Immunohistochemistry and Western blot were performed.Our findings indicated that BMP7 immunoreactivity preferentially localized to neuron-like cells.BMP7 positive cells and protein were significantly increased in the ischemic striatum at 3,7,and 14 d after MCAO,and arrived at the crest at 3 d following stroke,whereas Bcl-2 overexpression had no effect on the expression of BMP7 in the ischemic striatum at 3, 7,and 14 days after MCAO.4.β-catenin siRNA counteracted the down-regualation effect of Bcl-2 overexpression on BMP4 in the ischemic striatum.To investigate the interactional relationship of BMP4 andβ-catenin,we injected Bcl-2 expressing plasmid and/orβ-catenin siRNA into the lateral ventricle after ischemia.β-catenin siRNA markedly counteracted the down-regualation effect of Bcl-2 overexpression on BMP4 in the ischemic striatum at 3 d after MCAO,and decreased the expression ofβ-catenin at the same time.These results revealed that Bcl-2 overexpression decreased the expression of BMP4 thoughβ-catenin in the ischemic striatum.5.Bcl-2 overexpression protected brain against ischemic injury and increased neurogenesis in ischemic brain.To determine the effect of Bcl-2 on neurogenesis in ischemic rat brain,we injected Bcl-2 expressing plasmid and/orβ-catenin siRNA into the lateral ventricle after ischemia.Bcl-2 overexpression reduced infarct volume in ischemic brain,suggesting its neuroprotection.Using such an experimental model,we further studied the effects of Bcl-2 overproduction on neurogenesis in the adult rat brain after ischemia.Our results revealed that Bcl-2 increased ipsilateral SVZ expansion,enhanced migration of SVZ progenitors and neurogenesis in ischemic striatum.Bcl-2 overexpression enhanced migration of SVZ progenitors to ischemic striatum and neurogenesis in striatum.The results showed that Bcl-2 increased the numbers of neuronal progenitors（BrdU⁺-DCX⁺） at 1 week of reperfusion;increased newborn immature neurons（BrdU⁺-Tuj-1⁺） and newborn mature neurons（BrdU⁺-MAP-2⁺） in the ipsilateral striatum at 3～28 days;and increased newborn GABAergic (BrdU⁺-GAD₆₇⁺) and cholinergic（BrdU⁺-ChAT⁺） neurons in the ipsilateral striatum.6.β-catenin siRNA counteracted the neuroprotection effect of Bcl-2 overexpression and inhibited neurogenesis in the ischemic striatum.To measure the effect ofβ-catenin siRNA on neurogenesis in ischemic rat brain,we injected Bcl-2 expressing plasmid and/orβ-catenin siRNA into the lateral ventricle after ischemia.Our results showed thatβ-catenin siRNA i.c.v,administration not only significantly enlarged the infarct volume and reduced SVZ expansion at 3 d after MCAO but also decreased the BrdU⁺-Tuj-1⁺,BrdU⁺-DCX⁺,and BrdU⁺-MAP-2⁺ cells in the ipsilateral ischemic striatum at 3,7,14,and 28 days after MCAO.Interestly,the BrdU⁺-GFAP⁺ cells were significantly increased at the same time point.Moreover,β-catenin siRNA i.c.v,administration markedly suppressed newborn GABAergic (BrdU⁺-GAD₆₇⁺) and cholinergic（BrdU⁺-ChAT⁺） neurons in the ipsilateral striatum. These findings suggested thatβ-catenin siRNA counteracted the neuroprotection effect of Bcl-2 overexpression and inhibited neurogenesis in the ischemic striatum.Conclusion:1.Bcl-2 overexpression enhances ischemia-induced striatal neurogenesis and accelerates differentiation and maturity of newborn neurons in adult rat brain.2.Bcl-2 overexpression promotes neurogenesis following stroke in adult rat brain by downregulating expression of the BMP4 viaβ-catenin.3.Endogenousβ-catenin plays an important role in the proliferation,migration,and differentiation of neural progenitor cells in the ischemic striatum.