The autophagic machinery is necessary for removal of cell corpses from the developing retinal neuroepithelium

Abstract

Autophagy is a homoeostatic process necessary for the clearance of damaged or superfluous proteins and organelles. The recycling of intracellular constituents also provides energy during periods of metabolic stress, thereby contributing to cell viability. In addition, disruption of autophagic machinery interferes with embryonic development in several species, although the underlying cellular processes affected remain unclear. Here, we investigate the role of autophagy during the early stages of chick retina development, when the retinal neuroepithelium proliferates and starts to generate the first neurons, the retinal ganglion cells. These two developmental processes are accompanied by programmed cell death. Upon treatment with the autophagic inhibitor 3-methyladenine, retinas accumulated numerous TdT-mediated dUTP nick-end labelling-positive cells that correlated with a lack of the 'eat-me' signal phosphatidylserine (PS). In consequence, neighbouring cells did not engulf apoptotic bodies and they persisted as individual cell corpses, a phenotype that was also observed after blockade of phagocytosis with phospho-L-Serine. Supplying the retinas with methylpyruvate, a cell-permeable substrate for ATP production, restored ATP levels and the presentation of PS at the cell surface. Hence, engulfment and lysosomal degradation of apoptotic bodies were also re-established. Together, these data point to a novel role for the autophagic machinery during the development of the central nervous system.

Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice

“…32 Autophagy may also be involved in additional cytoprotective functions by removing axonal debris or remodeling cytoskeleton, 33 and as a pro-survival response after growth factor deprivation that results from RGC disconnection from their targets and through provision of ATP to the retina. 34 Altogether these data show for the first time the cytoprotective role of autophagy in cell soma after axonal insult and may provide the basis for new regeneration therapies, in which prolongation of neuronal soma survival is crucial for axonal maintenance and regeneration. It is important to remark the great degree of neuroprotection achieved in such a drastic model.…”

“…Ultrastructural analysis was assessed as previously described, 34 and observed using a Zeiss EM 902 transmission electron microscope (Zeiss, Oberkochem, Germany), at 90 kV, on ultra-thin sections (50 nm) stained with uranyl acetate and lead citrate.…”

“…Retina isolation and immunohistofluorescence was performed as previously described 34 with Brn-3a (Millipore, Billerica, MA, USA), cleaved caspase 3 (Cell Signaling, Boston, MA, USA) and alexa-conjugated secondary antibodies (Invitrogen). GCL was analyzed on a confocal microscope (TCS SP2; Leica Microsystems, Barcelona, Spain).…”

“…RNA isolation and RT were performed for individual retinas as previously described. 34 Q-PCR was performed with 100 ng cDNA with a TaqMan Universal PCR Master mix using probes from the Universal ProbeLibrary Set in a 7900 HT-Fast Real-Time PCR System (Roche Applied Biosystems, Pleasanton, CA, USA). Each value was adjusted by using 18S RNA levels as a reference.…”

The disruption of the rod-derived cone viability gene leads to photoreceptor dysfunction and susceptibility to oxidative stress

“…TUNEL staining, although a classic marker for apoptosis may not be reliable in some forms of RD, as the highly efficient autophagocytic mechanisms inherent to retinal function may achieve a rapid clearance of cell debris with each wave of cell loss. 28 Apart from the kinetics of rod death, the nuclear fragmentation of the dying cells may never be detectable where autophagy arises in close parallel with apoptosis, as has been described to occur in the rd1 mouse and in models of light damage. 29 The ultrastructural images here support the possibility that vacuolation and the infiltration of autophagosomes is occurring in the cytoplasm of Nxnl1À/À ONL cells and may explain the absence of TUNEL-positive cells.…”