Evidence for Paracrine Protective Role of Exogenous αA-Crystallin in Retinal Ganglion Cells

Expression and secretion of neurotrophic factors have long been known as a key mechanism of neuroglial interaction in the central nervous system. In addition, several other intrinsic neuroprotective pathways have been described, including those involving small heat shock proteins such as α-crystallins. While initially considered as a purely intracellular mechanism, both αA-crystallins and αB-crystallins have been recently reported to be secreted by glial cells. While an anti-apoptotic effect of such secreted αA-crystallin has been suggested, its regulation and protective potential remain unclear. We recently identified residue threonine 148 (T148) and its phosphorylation as a critical regulator of αA-crystallin intrinsic neuroprotective function. In the current study, we explored how mutation of this residue affected αA-crystallin chaperone function, secretion, and paracrine protective function using primary glial and neuronal cells. After demonstrating the paracrine protective effect of αA-crystallins secreted by primary Müller glial cells (MGCs), we purified and characterized recombinant αA-crystallin proteins mutated on the T148 regulatory residue. Characterization of the biochemical properties of these mutants revealed an increased chaperone activity of the phosphomimetic T148D mutant. Consistent with this observation, we also show that exogeneous supplementation of the phosphomimetic T148D mutant protein protected primary retinal neurons from metabolic stress despite similar cellular uptake. In contrast, the nonphosphorylatable mutant was completely ineffective. Altogether, our study demonstrates the paracrine role of αA-crystallin in the central nervous system as well as the therapeutic potential of functionally enhanced αA-crystallin recombinant proteins to prevent metabolic-stress induced neurodegeneration.