Autophagy in the Aging and Experimental Ocular Hypertensive Mouse Model

Abstract

Purpose To investigate autophagy in the outflow pathway and ganglion cell layer in the aging and ocular hypertensive mouse. Methods Both 4-month-old and 18-month-old C57BL/6J and GFP-LC3 mice were subjected to unilateral injection of hypertonic saline into a limbal vein, causing sclerosis of the outflow pathway and subsequent elevation of intraocular pressure (IOP). IOP was measured on a weekly basis using a rebound tonometer. Protein expression levels of LC3B, Lamp1, and p62 were evaluated by western blot and/or immunofluorescence. Retinal ganglion cell (RGC) count was performed in whole retinal flat mounts using an anti-Brn3a antibody. Optic nerves were fixed with 4% paraformaldehyde and resin-embedded for axon counts and electron microscopy. Results In contrast to 18-month-old mice, which developed sustained elevated IOP with a single injection, 4-month-old mice were refractory to high elevations of IOP. Interestingly, both the percentage of animals that developed elevated IOP and the mean ∆IOP were significantly higher in the transgenic mice compared to C57BL/6J. Immunofluorescence and western blot analysis showed dysregulated autophagy in the iridocorneal and retina tissues from 18-month-old mice compared to 4-month-old ones. Moreover, the LC3-II/LC3-I ratio correlated with IOP. As expected, injected hypertensive eyes displayed axonal degeneration and RGC death. RGC and axon loss were significantly exacerbated with aging, especially when combined with GFP-LC3 expression. Autophagic structures were observed in the degenerating axons. Conclusions Our results indicate dysregulation of autophagy in the trabecular meshwork and retinal tissues with aging and suggest that such dysregulation of autophagy contributes to neurodegeneration in glaucoma.

Primary cilia and the reciprocal activation of AKT and SMAD2/3 regulate stretch-induced autophagy in trabecular meshwork cells

“…These results very strongly indicate the role of PC-mediated autophagy in regulating IOP homeostasis. From a pathological point of view, previous work conducted in cultured TM cells isolated from glaucomatous eyes (64) and in glaucoma mouse models have revealed dysregulated autophagy in the TM of aging and hypertensive eyes (65)(66)(67)(68)(69)(70)(71)(72). Future studies, specifically directed at evaluating PC in open-angle glaucoma models or human samples, will be conducted to establish a direct connection between dysregulated autophagy in glaucoma with defective ciliogenesis or cilia function and to investigate the AKT-SMAD2/3 cross talk.…”

Autophagy in healthy aging and disease

“…Accumulating evidence in aged mice, as well as in rodent models recapitulating characteristic features of human diseases, has shown that compromised autophagy is among the most common factors contributing to the collapse of tissue homeostasis. In particular, ageassociated dysregulation of autophagy (demonstrated by the accumulation of autophagosomes), possibly due to impaired lysosomal fusion and/or degradation, is associated with cellular dysfunction and/or death, which contribute to neurodegeneration, as well as cardiac and skeletal muscle aging [49][50][51][52][53] . In hematopoietic stem cells (HSCs), autophagy has been shown to delay aging via activation of downstream sirtuin-3 (SIRT3), a key mitochondrial protein capable of rejuvenating blood and protecting against oxidative stress in mice and human HSC-enriched cells 54 .…”

Dexamethasone Downregulates Autophagy through Accelerated Turn-Over of the Ulk-1 Complex in a Trabecular Meshwork Cells Strain: Insights on Steroid-Induced Glaucoma Pathogenesis

“…Furthermore, TMCs were reported to modulate autophagy in response to several stimuli ranging from oxidative stress to mechanical stretch [ 58 , 59 , 60 ]. Main evidence for this is: (i) a decrease in autophagy markers, such as LC3B-II, in TMCs isolated from glaucomatous subjects along with a reduced lysosome basification [ 18 , 59 ]; (ii) a dysregulation of autophagy upon chronic oxidative stress exposure in cultivated TMCs [ 59 ]; (iii) the role of autophagy in TGF-β-mediated TM stiffness [ 61 ]; (iv) autophagy is a central pathway in retina neurodegeneration during ageing [ 62 ]; (v) a defective autophagy flux in the DBA/J2 mouse, a glaucoma murine model characterized by a spontaneous development of IOP [ 63 ]; (vi) the protective effect of rapamycin, an autophagy inducer, against RGCs apoptosis and IOP elevation in a rat model of glaucoma [ 64 ]. Very recently, a dysregulation of autophagy, based on a reduced content of LC3B-II and Beclin-1, has been reported in TMCs of DBA/J2 mouse treated with GCs and, further, a protective role of rapamycin, again, in TMCs homeostasis and TM remodeling, was uncovered [ 65 , 66 ].…”