Abstract
We aimed to investigate fractalkine (CX3CL1) protein expression in wild type (wt) retina and its alterations during retinal degeneration in mouse model (rd10) of retinitis pigmentosa. Forms of retinal protein CX3CL1, total protein and mRNA levels of CX3CL1 were analyzed at postnatal days (P) 5, 10, 14, 22, 30, 45, and 60 by Western blotting and real-time PCR. Cellular sources of CX3CL1 were investigated by in situ hybridization histochemistry (ISH) and using transgenic (CX3CL1cherry) mice. The immunoblots revealed that in both, wt and rd10 retinas, a membrane integrated ∼100 kDa CX3CL1 form and a cleaved ∼85 kDa CX3CL1 form were present at P5. At P10, accumulation of another presumably intra-neuronal ∼95 kDa form and a decrease in the ∼85-kDa form were observed. From P14, a ∼95 kDa form became principal in wt retina, while in rd10 retinas a soluble ∼85 kDa form increased at P45 and P60. In comparison, retinas of rd10 mice had significantly lower levels of total CX3CL1 protein (from P10 onwards) and lower CX3CL1 mRNA levels (from P14), even before the onset of primary rod degeneration. ISH and mCherry reporter fluorescence showed neurons in the inner retina layers as principal sites of CX3CL1 synthesis both in wt and rd10 retinas. In conclusion, our results demonstrate that CX3CL1 has a distinctive course of expression and functional regulation in rd10 retina starting at P10. The biological activity of CX3CL1 is regulated by conversion of a membrane integrated to a soluble form during neurogenesis and in response to pathologic changes in the adult retinal milieu. Viable mature neurons in the inner retina likely exhibit a dynamic intracellular storage depot of CX3CL1.
Highlights
Retinitis pigmentosa (RP) constitutes a large, heterogeneous group of inherited retinal neurodegenerative conditions
The analyses revealed CX3CL1 mRNA expression in neurons of inner- and outermost-subtiers of the inner nuclear layer and in the ganglion cell layer in both wt and rd10 retinas (Fig. 3A-D), while no expression was detected in the distal retina or in cells associated with the retinal vasculature
The,100 kDa CX3CL1 form found in our study, has been suggested previously to represent the mature full-length form and the only CX3CL1 form present at the cell surface, while the,85kDa has been considered as a soluble CX3CL1 form
Summary
Retinitis pigmentosa (RP) constitutes a large, heterogeneous group of inherited retinal neurodegenerative conditions. Retinal remodeling is a universal pathologic process subsequent to retinal degenerative disease that results in deafferentiation of the neural retina from photoreceptor input. Downstream neuronal elements respond to loss of input with a phased revision of retinal structure and function found at the molecular, synaptic, cell, and tissue levels and involving all cell classes in the retina, including neurons and glia [3,4]. Neurons, express ‘‘on and off‘‘ signals to maintain their milieu through the release of factors that control innate immune cell function, including microglia [5,6,7,8]. Microglia are resident cells of the nervous tissue involved in regulatory processes critical for development, maintenance of the neural environment, injury and repair and, as such, are sensors of events occurring within their immediate environment [9]. It was demonstrated that all retinal microglial cells express a receptor for CX3CL1, a chemokine receptor 1 (CX3CR1) [12]
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