Chimera RNA interference knockdown of γ-synuclein in human cortical astrocytes results in mitotic catastrophe

Elevated levels of gamma-synuclein (gamma-syn) expression have been noted in the progression of glioblastomas, and also in the cerebrospinal fluid of patients diagnosed with neurodegenerative diseases. gamma-Syn can be either internalized from the extracellular milieu or expressed endogenously by human cortical astrocytes. Internalized gamma-syn results in increased cellular proliferation, brain derived neurotrophic factor release and astroprotection. However, the function of endogenous gamma-syn in primary astrocytes, and the relationship to these two opposing disease states are unknown. gamma-Syn is expressed by astrocytes in the human cortex, and to gain a better understanding of the role of endogenous gamma-syn, primary human cortical astrocytes were treated with chimera RNA interference (RNAi) targeting gamma-syn after release from cell synchronization. Quantitative polymerase chain reaction analysis demonstrated an increase in endogenous gamma-syn expression 48 hours after release from cell synchronization, while RNAi reduced gamma-syn expression to control levels. Immunocytochemistry of Ki67 and 5-bromodeoxyuridine showed chimera RNAi gamma-syn knockdown reduced cellular proliferation at 24 and 48 hours after release from cell synchronization. To further investigate the consequence of gamma-syn knockdown on the astrocytic cell cycle, phosphorylated histone H3 pSer10 (pHH3) and phosphorylated cyclin dependent kinase-2 pTyr15 (pCDK2) levels were observed via western blot analysis. The results revealed an elevated expression of pHH3, but not pCDK2, indicating gamma-syn knockdown leads to disruption of the cell cycle and chromosomal compaction after 48 hours. Subsequently, flow cytometry with propidium iodide determined that increases in apoptosis coincided with gamma-syn knockdown. Therefore, gamma-syn exerts its effect to allow normal astrocytic progression through the cell cycle, as evidenced by decreased proliferation marker expression, increased pHH3, and mitotic catastrophe after knockdown. In this study, we demonstrated that the knockdown of gamma-syn within primary human cortical astrocytes using chimera RNAi leads to cell cycle disruption and apoptosis, indicating an essential role for gamma-syn in regulating normal cell division in astrocytes. Therefore, disruption to gamma-syn function would influence astrocytic proliferation, and could be an important contributor to neurological diseases.