Deletion of Socs3 in LysM+ cells and Cx3cr1 resulted in age-dependent development of retinal microgliopathy

Abstract

Background We generated a mouse model of primary microglial dysfunction by deleting two negative immune regulatory genes, Cx3cr1 and Socs3 (in LysM+ cells). This study aimed to understand how primary microglial dysfunction impacts retinal neurons during aging. Methods The LysMCre-Socs3fl/flCx3cr1gfp/gfp double knockout (DKO), LysMCre-Socs3fl/fl, Cx3cr1gfp/gfp and Socs3fl/fl mice were maintained up to 12 months. Eyes were collected and processed for immunohistochemistry of IBA-1, cone arrestin, secretagogin, PKCα and GABA. Brain microglia from DKO and WT mice were stimulated with LPS + IFN-γ or IL-4. The expression of TNF-α, IL-1β, IL-6, iNOS, IL-12p40, IL-23p19, CCL2, CCL5, CXCL2, IL-10, CD206 and Arg1 were examined by qRT-PCR and protein production was measured by Luminex assay. Retinal explants from C57BL/6 J mice were co-cultured with microglia from DKO or WT mice for 24 h, after which the number of cone arrestin+ cells in retinal flatmount were quantified. Results In 3–5 month old mice, the number of microglia in retinal ganglion cell layer (GCL) and inner plexiform layer (IPL) were comparable in all strains of mice. The DKO mice had a significantly higher number of microglia in the outer plexiform layer (OPL) but significantly lower numbers of cone arrestin+, secretagogin+ and GABA+ cells compared to Socs3fl/fl and single KO mice. During aging, 57% of the DKO mice died before 12 months old. The 10–12 months old DKO mice had significantly higher numbers of microglia in GCL/IPL and OPL than age-matched Socs3fl/fl and single KO mice. The aged DKO mice developed retinal pigment epithelial (RPE) dysmorphology accompanied by subretinal microglial accumulation. The number of photoreceptors, bipolar cells (Secretagogin+ or PKCα+) and GABA+ amacrine cells was significantly lower in aged DKO mice compared to age-matched Socs3fl/fl and single KO mice. Microglia from DKO mice showed significantly higher levels of phagocytic activity and produced higher levels of TNF-α, IL-6, CCL2, CCL5, CXCL2 and CXCL10 compared to microglia from Socs3fl/fl mice. Co-culture of retinal explants with LPS + IFN-γ or IL-4 pre-treated DKO microglia significantly reduced cone photoreceptor survival. Conclusions The LysMCre-Socs3fl/flCx3cr1gfp/gfp DKO mice displayed primary microglial dysfunction and developed age-related retinal microgliopathy characterized by aggragated microglial activation and multiple retinal neuronal and RPE degeneration. Trial registration Not applicable. The article does not contain any results from human participants.

Minocycline Inhibits Microglial Activation and Improves Visual Function in a Chronic Model of Age-Related Retinal Degeneration

“…Neither WT nor DKO mice showed significant weight loss ( Figure 1 ) or behaviour change during the study. We previously reported ~50% of natural death in DKO mice by 12 months of age [ 24 ]. In this study, the vehicle-treated group lost two mice (out of 16) and the minocycline-treated group lost one mouse (out of 12) at 7 months old.…”

“…The LysMCre-Socs3 fl/fl Cx3cr1 gfp/gfp DKO mouse line was generated by crossing bred the LysMCre-Socs3 fl/fl mice with the Cx3cr1 gfp/gfp mice (all in C57BL/6J background) as described previously by us [ 24 ]. The Socs3 fl/fl mice were used as wild-type (WT) controls in all experiments.…”

“…Fundus images were obtained after anesthesia and pupil dilation of the mice described previously [ 24 ]. Images were captured with a Nikon D90 camera via an endoscope or the Micron IV system (Phoenix Research Labs, Pleasanton, CA, USA).…”

“…We previously reported a mouse model of age-related retinal microgliopathy, whereby, two immune checkpoint genes Cx3cr1 and Socs3 were deleted in microglial cells (i.e., the LysMCre-Socs3 fl/fl Cx3cr1 gfp/gfp double knockout (DKO) mice) leading to uncontrolled microglial activation, retinal neuron and RPE degeneration when mice reach 10~12 months of age [ 24 ]. The DKO mice, therefore, are an ideal model to test the long-term therapeutic potential of minocycline.…”

More than meets the eye: The role of microglia in healthy and diseased retina

“…Interestingly, the impairment of Cx3CL1-Cx3CR1 signaling in mice, particularly important for microglia activity ( 110 ), was associated with the extension of microglia branches inside the ONL and morphological and functional alterations in photoreceptors during early postnatal development, particularly in cones ( 111 ). Similarly, specific depletion of microglia at adult stages was shown to affect photoreceptor synapses and visual performance ( 112 , 113 ). Of note, an additional role for microglia in axon guidance of ganglion cells through nerve growth factor (NGF) signaling was suggested ( 114 ), thus corroborating a multi-faceted spectrum of activities for microglia also in the context of the retina.…”