Evidence for Paracrine Protective Role of Exogenous alpha A-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 alpha-crystallins. While initially considered as a purely intracellular mechanism, both alpha A-crystallins and alpha B-crystallins have been recently reported to be secreted by glial cells. While an anti-apoptotic effect of such secreted alpha A-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 alpha A-crystallin intrinsic neuroprotective function. In the current study, we explored how mutation of this residue affected alpha A-crystallin chaperone function, secretion, and paracrine protective function using primary glial and neuronal cells. After demonstrating the paracrine protective effect of alpha A-crystallins secreted by primary Muller glial cells (MGCs), we purified and characterized recombinant alpha A-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 alpha A-crystallin in the central nervous system as well as the therapeutic potential of functionally enhanced alpha A-crystallin recombinant proteins to prevent metabolic-stress induced neurodegeneration.