Abstract
Purpose: :
Recent studies suggest, the soluble form of fractalkine (FKN, a membrane-anchored chemokine) reduces the release of microglial pro-inflammatory cytokines and increases neutrophin production. Based on this evidence, we expected FKN cleavage to be affected in the retinal degenerations. In our studies we aim to compare in vivo levels and localization of FKN in a mouse model (rd10) of autosomal recessive retinitis pigmentosa and control C57BL/6J (wt) mouse retinas.
Methods: :
Cryostat sections from fixed eyes sampled of age-matched rd10 and wt mice were used for immunohistochemistry (IHC). Western blot (WB) analyses were performed using corresponding neuroretina lysates. Rabbit polyclonal antibody to recombinant chemotactic domain of human FKN was used in both IHC and WB assay. Mouse anti-GAPDH antibody was used as a loading control.
Results: :
FKN protein was detected in retinas of both rd10 and wt mice. At postnatal days 22, 30 and 45, from IHC staining of retinal sections, we observed expression of FKN in a subset of neurons, Mueller cells and in superficial blood vessels. Wt mice, however had additional weak and diffuse but specific FKN immunoreactivity. WB analysis of wt retinal lysates revealed numerous species of FKN in the lysates: (1) ~ 120 kDa and 100 kDa full-length membrane-anchored fragments ; (2) ~ 60 kDa and 50 kDa intracellular precursor forms, and (3) a low molecular weight fragment at ~ 35 kDa that corresponds to the cleaved soluble form. While the bands at 100 and 35 kDa were the most intense in wt, WB analysis of rd10 retinal lysates revealed a reduction in the level of the 120 kDa form, absence of the 100 kDa band and a faint 35 kDa fragment. No changes in the level of intracellular precursor forms (60 and 50 kDa) were detected indicating that protein was obtained from intact rd10 mouse retinal tissue. There were no sex-related differences.
Conclusions: :
Our observations are consistent with the findings showing both the stromal and parenchymal origin of fractalkine in the retina. The diffuse fractalkine signal in control mouse retinas may originate from immunoreactivity to the soluble form of the protein. The protein expression data indicate that fractalkine protein homeostasis is altered in the retina of rd10 mice by diminishing of the soluble FKN levels. We are currently generating a mouse model that will allow us to extend our knowledge on microglia-neuron interaction through FKN signalling axis.
Keywords: retinal degenerations: hereditary • cytokines/chemokines • microglia