Abstract
Purpose:
Diabetic retinopathy is a neurodegenerative disease characterized by diminished retinal AKT activity and neuroinflammation. AKT is crucial to neurotrophic signal transduction. We hypothesized that AKT activity is a key determinant of the response of retinal neurons to a pro-inflammatory stimuli, and examined how manipulation of AKT affected the response of R28 cells to treatment with TNF-alpha.
Methods:
R28 cells were cultured on laminin in the presence of a cAMP analogue to induce neuronal differentiation. Serum deprivation (SD) and pharmacological inhibitors were used to negate AKT activity. IGF-1 treatment was used to acutely activate AKT. AKT activation and AKT substrate phosphorylation was evaluated by Western blotting with antibodies to phosphorylated and total proteins. Apoptotic death was evaluated by caspase-3/7 activity, DNA fragmentation ELISA and lactate dehydrogenase (LDH) release. Messenger RNA levels were measured by qRT-PCR. Expression of FOXO1 and BIM, a FOXO1-target gene, was knocked down using RNA silencing.
Results:
AKT activation coincided with phosphorylation of several protein substrates of AKT kinase, with FOXO1 being most prominent. SD resulted in dephosphorylation of FOXO1 at the threonine 24 activation site, leading to nuclear localization of the protein and increased expression of BIM. IGF-1 reversed these effects. SD and inhibition of AKT activity caused a significant increase TNF alpha-induced cell death response, coinciding with increased BIM expression. BIM was the only member of the pro-apoptotic BH3-only family of genes that was responsive to AKT deactivation and TNF-alpha treatment. Knockdown of FOXO1 and BIM significantly diminished R28 cell death in response to SD and TNF alpha-treatment.
Conclusions:
In the absence of AKT activity R28 retinal neuronal cells exhibited a greater apoptotic response to an inflammatory stimuli, modeled by TNF-alpha treatment that coincided with increased expression of BIM. Activation of FOXO1 and BIM expression are partially responsible for R28 cell death in response to TNF-alpha treatment in the absence of AKT activity.
Keywords: 426 apoptosis/cell death •
499 diabetic retinopathy •
557 inflammation