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Published on: July 31, 2014
Granulocyte-colony stimulating factor gene therapy as a novel therapeutics for stroke in a mouse model
Janet M Menzie-Suderam1,2, Jigar Modi1,3, Hongyaun Xu1
1Department of Biomedical Sciences, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL, 33431, USA.
Insights
Gene therapy using human granulocyte-colony stimulating factor (hG-CSF) delivered via eye drops shows promise for treating neurological deficits after global ischemia. This approach effectively reduces cellular stress and improves outcomes by targeting endoplasmic reticulum stress, mitochondrial dynamics, and autophagy.
Area of Science:
- Neuroscience
- Gene Therapy
- Cellular Biology
Background:
- Global ischemia, often resulting from cardiopulmonary arrest, leads to neurological deficits with no effective treatments.
- Human granulocyte-colony stimulating factor (hG-CSF) offers neuroprotection but has a short plasma half-life, limiting clinical use.
- Novel strategies are needed for prolonged hG-CSF expression and to understand its protective mechanisms.
Purpose of the Study:
- To investigate the protective mechanisms of hG-CSF gene therapy against endoplasmic reticulum stress, mitochondrial dynamics, and autophagy in global ischemia.
- To evaluate the efficacy of sustained hG-CSF expression achieved through gene therapy delivered via eye drops.
Main Methods:
- AAV-CMV-hG-CSF or AAV-CMV-GFP was administered via eye drops to mice after bilateral common carotid artery occlusion (BCAO).
- Expression levels of proteins involved in ER stress, mitochondrial dynamics, and autophagy were monitored in vulnerable brain regions over 4 and 7 days post-BCAO.
- Statistical analysis included One-Way ANOVA and Repeated Measures Two-Way ANOVA with Tukey's post hoc test.
Main Results:
- hG-CSF gene therapy significantly downregulated biomarkers of cellular death, including ER stress markers (CHOP, GRP78), mitochondrial dynamics marker (DRP1), and autophagy markers (Beclin 1, p62, LC3-II).
- The therapy upregulated the anti-apoptotic protein Bcl2 and downregulated the pro-apoptotic protein Bax.
- Functional behavioral tests indicated an overall improvement following hG-CSF gene therapy.
Conclusions:
- Sustained protein expression of hG-CSF is achievable through eye drop administration of the hG-CSF gene.
- hG-CSF gene therapy effectively mitigates endoplasmic reticulum stress, modulates mitochondrial dynamics, and influences autophagy in the context of global ischemia.
- This study validates hG-CSF gene therapy as a promising approach for neuroprotection after global ischemia.
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.
