Distribution and Activity of Mitochondrial Proteins in Vascular and Avascular Retinas: Implications for Retinal Metabolism

Abstract

PURPOSE. Understanding the energetics of retinal neurons and glia is crucial for developing therapies for diseases that feature deficits in nutrient or oxygen availability. Herein, we performed a detailed characterization of the distribution and activity of mitochondrial proteins in the vascularized retinas of rat and marmoset, and the avascular retinas of rabbit and guinea pig. Further, we delineated expression of ubiquitous mitochondrial creatine kinase (uMtCK). METHODS. Expression of eight mitochondrial proteins was investigated using Western blotting, single-and double-labeling immunohistochemistry. Activities of cytochrome c oxidase, succinate dehydrgogenase, and isocitrate dehydrogenase were determined by enzyme histochemistry using unfixed tissue sections. RESULTS. In vascularized retinas, immunoreactivities were characterized by strong, punctate labeling in the plexiform layers, photoreceptor inner segments, somas of various cell types, notably retinal ganglion cells (RGCs), and the basolateral surface of the retinal pigment epithelium. In avascular retinas, immunoreactivities featured intense labeling of inner segments, together with weak, but unambiguous, staining of both plexiform layers. RGCs were relatively enriched. In Müller cells of avascular retinas, mitochondria were restricted to scleral-end processes. For each species, enzyme activity assays yielded similar results to the protein distributions. Labeling for uMtCK in vascular and avascular retinas was fundamentally similar, being restricted to neuronal populations, most notably inner segments and RGCs. Of all of the mitochondrial proteins, uMtCK displayed the strongest labeling in avascular retinas. uMtCK was not detectable in Müller cells in any species. CONCLUSIONS. The current findings advance our understanding of the metabolic similarities and differences between vascular and avascular retinas.

Retinal energy metabolism in health and glaucoma

“…We have recently shown that in vascularized retinas (marmoset and rat) the IPL displays intense punctate immunoreactivity for mitochondrial proteins. (Chidlow et al, 2019) Conversely, in the avascular retina (rabbit and guinea pig), intense labeling of photoreceptor inner segments was observed, but the inner and OPL display only weakly punctate staining, indicating that avascular retinas are generating ATP via nonaerobic glycolytic mechanisms. Enzyme histochemistry assays for three mitochondrial enzymes were also consistent with this pattern.…”

“…Enzyme histochemistry assays for three mitochondrial enzymes were also consistent with this pattern. (Chidlow et al, 2019)…”

“…(Perge et al, 2009) These calculations were performed on guinea pig optic nerve and conceivably the avascular guinea pig retina may not extrapolate to vascularised retinas; however, our own data suggest that the RGC somata and axons share a similar mitochondrial profile in both vascular and avascular retinas. (Chidlow et al, 2019) Furthermore, using transmission electron microscopy, Wang et al demonstrate that the unmyelinated portion of the RGC axons in primates (including humans) display varicosities rich in mitochondria that likely serve local high-energy demands of unmyelinated fibers. (Wang et al, 2003) In the optic nerve the unmyelinated prelaminar and laminar regions are rich in both cytochrome c oxidase and succinate dehydrogenase; however, myelination of fibres as they exited the lamina cribrosa is associated with an abrupt reduction in enzyme activity.…”

Energy Metabolism in the Inner Retina in Health and Glaucoma

“…It is worth noting that the axon volume is estimated to comprise 80% of the total RGC volume based on published data [127,153,169], and the lack of saltatory conduction in the unmyelinated intraocular portion of the RGC axons both place a particular bioenergetic burden on this cellular component. While the cell soma is the principal site for energy production and protein synthesis, there is evidence that the axons supplement some of these functions as they share a similar mitochondrial profile with the somata [170].…”

Investigations Into Bioenergetic Neuroprotection of Cone Photoreceptors: Relevance to Retinitis Pigmentosa

“…Functions ascribed to the creatine kinase/phosphocreatine kinase system include spatiotemporal buffering of ATP and improving the efficiency of oxidative phosphorylation. The creatine transporter, which is responsible for creatine uptake into cells, and creatine kinase, which catalyzes the reversible conversion of creatine into high energy phosphocreatine, are both expressed in photoreceptors (Acosta et al, 2005; Linton et al, 2010; Rueda et al, 2016; Chidlow et al, 2019); thus, supplementation with creatine is a neuroprotective strategy worthy of investigation.…”