Abstract
Purpose::
Genetic and biochemical evidence suggests that innate immunity plays a pivotal role in retinal degenerative disorders. Our previous work in the mouse model for X-linked juvenile retinoschisis (RS1h-/Y) has identified microglia activation preceding photoreceptor apoptosis and retinal degeneration. In this study, our aim was to further characterize underlying early signaling events, transcriptional markers, and effector mechanisms in microglia activation.
Methods::
Early postnatal retinae and ex vivo microglia cells from retinoschisin-deficient and wild type mice were subjected to DNA-microarray and realtime qRT-PCR analysis. Cells were functionally characterized and lineage markers and molecular activation patterns were examined. RAW264.7 and BV-2 cells were used as model system to study TLR4-dependent activation of microglia-specific genes and to characterize TLR4-responsive Egr1 promoter elements.
Results::
After isolation, purification and culture, phase contrast micrographs and latex bead phagocytosis assays demonstrate a highly activated state of retinoschisin-deficient microglia cells compared to controls. Starting from the induced transcripts in retinal samples of RS1h-/Y mice, a transcriptional network of TLR4-activated genes composed of Casp11, Spp1, Clec7a, Fcer1g, as well as a group of highly expressed constitutive markers (Tyrobp, CCl6, Cd68, and S100A6) could be specifically allocated to the microglia population in the diseased retina. Furthermore, LPS-stimulation of RAW264.7 and BV-2 cells mimicks endogenous microglia activation, up-regulates the TLR4-dependent gene cluster and targets a specific region in the proximal Egr1 promoter.
Conclusions::
TLR4 signaling and up-regulation of Egr1 are early events during microglia activation in Rs1h-deficiency leading to transcriptional induction of pro-inflammatory and activation-induced apoptosis pathways. Targeting of these pathways may point to novel therapeutic options in neurodegenerative retinal disease.
Keywords: microglia • retinal degenerations: cell biology • immunomodulation/immunoregulation