Photoreceptors are highly specialized cells required for phototransduction within the retina and thus crucial for visual perception. Inherited retinal diseases are mainly caused by mutations in photoreceptor-specific genes, the majority of which is regulated by the key transcription factor cone rod homeobox (CRX). Using genome-wide chromatin immunoprecipitation data (CRX ChIP-seq), we have identified a novel sterile alpha motif (SAM) domain containing protein, SAMD7, as a CRX target. SAMD7 is expressed in the mouse retina and pineal gland and localizes to the cytoplasm and nucleus of photoreceptor cells. SAMD7 expression is regulated by CRX, which binds to two specific regions in the promoter and first intron (enhancer) of the SAMD7 gene. Consequently, CRX knock-down leads to a significant decrease of SAMD7 enhancer activity and protein levels in the retina. Functionally, SAMD7 acts as a transcriptional repressor of CRX-mediated photoreceptor gene expression, indicating that mutations in or dysregulation of SAMD7 could lead to disturbed photoreceptor homeostasis and ultimately retinal degeneration.
Microglial cells are the resident macrophages of the central nervous system (CNS), including the retina, and play pivotal roles in innate immune responses and regulation of homeostasis in the healthy and degenerating CNS. Reactive microgliosis is a common hallmark of neurodegenerative diseases and chronic pro-inflammatory microglial reactivity contributes to disease progression. We have previously identified activated microglia/macrophage whey acidic protein (AMWAP) as a biomarker for microglial reactivity and counter-regulator of pro-inflammatory response. AMWAP is actively secreted from lipopolysaccharide (LPS)-activated microglia and recombinant AMWAP is taken up by microglial cells in a paracrine fashion and effectively reduces TLR2- and TLR4-mediated pro-inflammatory gene expression. AMWAP exerts its anti-inflammatory function through blockade of NFκB activation, as it inhibits proteolysis of the NFκB pathway mediators IRAK-1 and IκBα without preventing IκBα phosphorylation and ubiquitination or affecting overall 20S proteasome activity. Functionally, AMWAP reduces pro-inflammatory microglial nitric oxide (NO) secretion and neurotoxicity on photoreceptor cells in vitro. Further, AMWAP promotes filopodia formation of microglia and increases the phagocytic recognition and uptake of apoptotic photoreceptor debris, common features of homeostatic regulatory microglia. We therefore hypothesize that anti-inflammatory whey acidic proteins (WAPs) could have therapeutic potential in neurodegenerative diseases of the brain and retina.
The translocator protein (18 kDa) (TSPO) is a mitochondrial protein expressed in reactive glial cells and a biomarker for gliosis in the brain but has not been investigated in a retinal context so far. Various TSPO ligands have been shown to reduce neuroinflammation in neurodegenerative mouse models. We could show strong upregulation of TSPO transcript and protein levels in reactive microglial cells in vitro, microglia of the retinoschisin-deficient retinal degeneration mouse model as well as TSPO expression in microglia of the human retina. TSPO mRNA expression is high in the developing mouse retina and declines to low levels in the adult tissue. The synthetic TSPO ligand XBD173 effectively suppresses pro-inflammatory microglial gene expression, migration, proliferation, NO secretion and neurotoxicity on photoreceptors in vitro. Further, XBD173 treatment promotes filopodia formation and increases the phagocytic recognition and uptake of latex beads and apoptotic photoreceptor debris by murine and human microglial cells in vitro. Finally, XBD173 effectively reduces the number of amoeboid alerted microglia in organotypic murine retinal explant cultures stimulated with LPS. In conclusion, we have identified TSPO as a novel marker for microglial reactivity in the retina and a potential therapeutic target to reduce chronic neuroinflammation during retinal degeneration.
In summary, our studies on the novel photoreceptor gene regulator SAMD7 and the microglial reactivity markers AMWAP and TSPO provide insights into potential disease mechanisms of retinal degeneration and suggest future strategies of identifying and therapeutically modulating pro-inflammatory microglial reactivity in degenerative diseases of the CNS and retina
