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

Abstract

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.

Small Heat Shock Proteins in Retinal Diseases

“…It has been shown that phosphorylation of HspB4 at serine 148 (mice) and threonine 148 (human) is dramatically reduced in diabetic retinopathy, which could increase stress and apoptosis in retinal cells ( Ruebsam et al, 2018 ). HspB4 is highly expressed in Müller glia and protects retinal neurons via a paracrine mechanism ( Nath et al, 2022 ). Further, it has been shown that phosphorylation of threonine 148 in HspB4 enhances its cytoprotective function, which could reduce inflammation in the diabetic retina ( Nath et al, 2021 ).…”

Phosphorylation of αB-Crystallin Involves Interleukin-1β-Mediated Intracellular Retention in Retinal Müller Cells: A New Mechanism Underlying Fibrovascular Membrane Formation

“… 47 , 48 Furthermore, retinal Müller cell-derived αA-crystallin was shown to have neuroprotective functions by anti-apoptotic effects in a paracrine manner. 49 Therefore, it is estimated that extracellular αB-crystallin from retinal Müller cells acts on adjacent cells to serve neuroprotective effects by paracrine, and IL-1β-mediated secretion reduction of αB-crystallin damages in the neural retina under diabetic conditions. As impairment of the neural function, such as the reduction of oscillatory potential amplitudes in electroretinograms, can be confirmed in patients with early diabetic retinopathy, 50 the reduction of extracellular αB-crystallin by IL-1β may have the potential to deteriorate the pathogenesis of diabetic retinopathy.…”

Retinal Proteomic Analysis in a Mouse Model of Endotoxin-Induced Uveitis Using Data-Independent Acquisition-Based Mass Spectrometry

“…Crystallin is a major protein of the lens and can be further categorized into α, β, and γ subtypes. It is also expressed in the retina [ 45 , 46 ]. Crystallin proteins are reported to be dramatically elevated in experimental autoimmune uveitis (EAU)-induced retinal mitochondrial protein, EAU-induced aqueous and vitreous protein, and EIU-induced vitreous protein profiles [ 15 , 47 , 48 ], and are suggested as potential biomarkers and therapeutic targets for uveitis [ 14 ].…”