Apoptosis is a mechanism of programmed cell death involved in the homeostasis of nervous tissues and it’s dysregulation has been associated with the pathology of neurodegenerative diseases, stroke, and neurotrauma. Therefore, therapeutic strategies to prevent neuronal cell apoptosis is critical in Neurologic diseases (Siren, Fratelli et al.
2001; Pregi, Vittori et al. 2006) Recent experimental studies raised the possibility of using hMSCs in neurological disease therapy. There is increasing evidence that hMSC promote fuctional recovery in animal models. In this regard, hMSCs are considered to have neuroprotective role.(Bang, Lee et al. 2005) This study investigates that hMSCs have protective effects against apoptosic stress by expression of anti-apoptotic protein Bcl-2.
Staurosporine is able to induce a prominent neuritogenesis in human neuroblastoma cells. Because of this neurotrophin-like effect, staurosporine has been proposed as a potential prototype for future neurothrphic drugs, that could stimulate the process of regeneration and neurite outgrowth in damaged neurons.(Rasouly and Lazarovici 1994) However staurosproine can also be toxic for cells of neural origine at the concentrations and neurite outgrowth occurs early and cell death phenomenon takes place at later times.(Boix, Llecha et al. 1997) SH-SY5Ys have been used extensively in the study of neuronal cell death. Staurosporine-induced cell death in SH-SY5Ys is accompanied by endogenous Bax translocation and concomitant translocation of cytochome c from the
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mitochondrial to the cytosolic fractions.(McGinnis, Gnegy et al. 1999; Lopez and Ferrer 2000) Our results indicate that staurosporine not only triggers the apoptotic phenomenon upon the SH-SY5Ys but also induce cell death in concentration-, and time-dependent manner. We decided that 0.25uM of staurosporine treatment for 24 hours was optimal to observe the effect of hBM-MSCs against the STS-induced apoptotic cell death. Also I obtained the results that treatment with both fresh culture media including 10% FBS and culture media of the hMSCs failed to rescue the apoptotic stress of SH-SY5Ys. Isele N et al reported that the protective effects of hMSCs conditioned medium was further accelerated when hMSCs were incubated with Neurobasal medium of neurons previously exposed to apoptotic stress indication a potential cross talk between stressed neurons and hMSCs. But heating of the conditioned medium abolish the protective effect, suggesting that heat labile factors, i.e. protein, released by hMSCs mediated the observed anti-apoptotic effect in the neurons(Isele, Lee et al. 2006). And we found that the presence of hMSCs reduced cell death, which suggested that hMSCs exert protective roles on cell death. This indicates the release of protective factors which exert their effects across species barriers, which is typical for growth factors and cytokines. In addition, these results offer the perspective to use hMSCs in forcal cerebral ischemia. Li et al. reported that hMSCs treatment of stroke in rats increased astrocytic proliferation and activation in the subventricular zone, and an increased expression of an axonal marker(GAP-43) was observed among reactive astrocytes in the scar boundary zone and SVZ. (Li, Chen et al. 2005) This indicates that the beneficial beneficial functional recovery that hMSCs provide after stroke may be derived from the
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protection of astrocytes from cell death post-ischemia. The increased survival of astorcytes in the injured brain might subsequently increase neuronal cell survival, enhance neuroregeneration, and promote plasticity.(Gao, Li et al. 2005)
Another important aspect is that the neuroprotective effect of hMSCs required coculture with SH-SY5Ys induced cell death. When cultured media of hMSCs was supplied simultaneously with the apoptotic challenge, the neuroprotective effect was not detectable, whereas coculture of apoptosis induced SH-SY5Ys with hMSCs significantly attenuated neuronal apoptosis. This supports the idea that hMSCs mediated neuroprotection depends on expression of survival factors.
To further understand the mechanism underlying hMSCs effects on apoptosis, we examined Bcl-2 family protein. Bax translocates from the cytosol to the mitochondria in neuronal cells undergoing apoptosis. Bax has been previously found in various cell types.(Jurgensmeier, Xie et al. 1998) Bax translocation is accompanied by release of cytochrome c from the mitochondria in both CFNs and SH-SY5Ys. Cytochrom c released from the mitochondria is a cofactor for caspase-3 activity.(McGinnis, Gnegy et al. 1999) Apoptotic signals induced Bax translocation in SH-SY5Ys suggest that Bax translocation is a universal component of apoptosis in neuronal cells. Bax expression is known to be critical for neuronal apoptosis. Anti-apoptotic protein such as Bcl-2 inhibit the mitochondrial permeability transition and cytochrom c release, although effects downstream of cychrome c release have been documented.(Kluck, Bossy-Wetzel et al.
1997; Rosse, Olivier et al. 1998) Cytochrome c plays an active role in the apoptotic cascade by facilitating activation of downstream caspases through processing of
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procaspase-9.(Li, Nijhawan et al. 1997) While it has been suggested that the prima(Susin, Zamzami et al. 1996; Kluck, Bossy-Wetzel et al. 1997)ry site for Bcl-2 regulation of apoptosis is by preventing cytochrome release.(Susin, Zamzami et al.
1996; Kluck, Bossy-Wetzel et al. 1997) (Murphy, Ranganathan et al. 2000)In monitoring Bax expression during coculturing period, Bax expression was increased in time dependent manner. It was peaked at 5days. While Bcl-2 expression was gradually increased. These our results suggest that the mechanism by which Bcl-2 blocks drug-induced apoptosis is by preventing Bax overexpression. These finding suggests that the hMSCs neuroprotective effects on apoptosis induced cells might partially be derived from upregulation of anti-apoptotic protein and downregulation of pro-apoptotic protein.
In summary, out findings strongly suggest that hMSCs secrete protective factors that prevent neuronal apoptosis that prevent neuronal apoptosis through stimulation of expression of endogenous anti-apoptotic protein. These findings suggest that the therapeutic benefit hMSCs provide to injured brain after ischemic infarct may be inpart due to hMSCs stimulation of neuroprotection.
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