Granulocyte-colony stimulating factor gene therapy as a novel therapeutics for stroke in a mouse model.

BACKGROUND: Global ischemia is the resulting effect of a cardiopulmonary arrest (CPA). Presently there is no effective treatment to address neurological deficits in patients who survived a CPA. Granulocyte-colony stimulating factor is a growth factor (G-CSF) with a plethora of beneficial effects, including neuroprotection. Clinical application of human G-CSF (hG-CSF) is limited due to its plasma half-life of 4 h. Therefore, novel approaches need to be investigated that would (1) enable prolonged manifestation of hG-CSF and (2) demonstrate G-CSF efficacy from studying the underlying protective mechanisms of hG-CSF. In our previous work, we used the self-complementary adeno-associated virus (stereotype2: scAAV2) as a vector to transfect the hG-CSF gene into the global ischemic brain of a mouse. As an extension of that work, we now seek to elucidate the protective mechanisms of hG-CSF gene therapy against endoplasmic reticulum induced stress, mitochondrial dynamics and autophagy in global ischemia. METHOD: A single drop of either AAV-CMV-hG-CSF or AAV-CMV-GFP was dropped into the conjunctival sac of the Swiss Webster mouse's left eye, 30-60 min after bilateral common artery occlusion (BCAO). The efficacy of the expressed hG-CSF gene product was analyzed by monitoring the expression levels of endoplasmic reticulum stress (ER), mitochondrial dynamics and autophagic proteins over 4- and 7-days post-BCAO in vulnerable brain regions including the striatum, overlying cortex (frontal brain regions) and the hippocampus (middle brain regions). Statistical analysis was performed using mostly One-Way Analysis of variance (ANOVA), except for behavioral analysis, which used Repeated Measures Two-Way ANOVA, post hoc analysis was performed using the Tukey test. RESULTS: Several biomarkers that facilitated cellular death, including CHOP and GRP78 (ER stress) DRP1 (mitochondrial dynamics) and Beclin 1, p62 and LC3-ll (autophagy) were significantly downregulated by hG-CSF gene transfer. hG-CSF gene therapy also significantly upregulated antiapoptotic Bcl2 while downregulating pro-apoptotic Bax. The beneficial effects of hG-CSF gene therapy resulted in an overall improvement in functional behavior. CONCLUSION: Taken together, this study has substantiated the approach of sustaining the protein expression of hG-CSF by eye drop administration of the hG-CSF gene. In addition, the study has validated the efficacy of using hG-CSF gene therapy against endoplasmic reticulum induced stress, mitochondrial dynamics and autophagy in global ischemia.

Granulocyte-colony stimulating factor protects against endoplasmic reticulum stress in an experimental model of stroke.

Granulocyte-colony stimulating factor (G-CSF) is an endogenous growth factor that exhibits a diverse range of neuroprotective mechanisms against a variety of neurological disorders including ischemic stroke. We investigated the anti-apoptotic mechanisms of G-CSF against endoplasmic reticulum (ER) stress induced apoptosis. Sprague-Dawley rats were subjected to transient occlusion of the middle cerebral artery (MCAO) for 90 min. Rats were injected with G-CSF (n = 15; 50 µg/kg body weight s.c.) for 4 days, starting 24 h post-MCAO and brains were harvested after 4 days reperfusion (n = 16). Key proteins in ER stress apoptosis were analyzed by immunoblotting. G-CSF reduced infarct volume to 53% and improved neurological deficits. G-CSF treatment significantly (P < .05) attenuated the expression of proteins involved in ER stress apoptosis pathway; ATF4, ATF6, p-p38MAPK, pJNK and CHOP. G-CSF treatment also re-established ER homeostasis evident by the reduction of the intraluminal ER stress sensor, GRP78 as well as reducing the overall cellular stress level protein, HSP27. G-CSF also up-regulated anti-apoptotic proteins pAKT and Bcl-2 while down-regulated the pro-apoptotic protein Bax. G-CSF exerts neuroprotection from cerebral ischemia through the preservation of the ER, resulting in the attenuation of pro-apoptotic proteins and the potentiation of anti-apoptotic proteins.