Glaucoma is a progressive optic neuropathy characterized by axonal degeneration and retinal ganglion cells loss. Several factors have been postulated to play a role in glaucoma, elevated intraocular pressure (IOP) being the best well-known causative factor. The mechanisms leading to ocular hypertension and glaucoma are still not fully understood. An increasing number of evidence indicates a role of autophagy in the pathophysiological process of ocular hypertension and glaucoma. However, while all of the studies agree that autophagy is induced in RGCs in response to injury, autophagy was found to either protect or promote cell death depending on the experimental model used. In order to gain more insight into both, the role of autophagy in the pathogenesis of glaucoma and the effect of chronic IOP elevation in the autophagy pathway, we have investigated here for the first time autophagy in the iridocorneal angle region, retinal ganglion cell bodies, and ON axons in the spontaneous ocular hypertensive DBA/2J mouse glaucoma model and in the transgenic DBA/2J::GFP-LC3 mice, generated in our laboratory. Our results indicate decreased autophagic flux in the outflow pathway cells in the DBA/2J mice, characterized by increased levels of LC3-II and p62 together with a decrease in the lysosomal marker LAMP1, evaluated by western blot and immunofluorescence. Elevated presence of autophagic vacuoles in the DBA/2J and, in particular, in the DBA/2J::GFP-LC3 mice was also observed. Expression of the GFP-LC3 transgene was associated to higher cumulative IOP in the DBA/2J background. In addition to higher elevation in IOP, DBA/2J::GFP-LC3 were characterized by further RGCs and exacerbated axonal degeneration compared to DBA/2J. This was accompanied by the notable high presence of autophagic figures within degenerating axons. These results strongly suggest overactivation of autophagy as a potential cellular mechanism leading to ON degeneration in the chronic hypertensive DBA/2J mice.

Activation of autophagy is one of the responses elicited by high intraocular pressure (IOP) and mechanical stretch in trabecular meshwork (TM) cells. However, the mechanosensor and the molecular mechanisms by which autophagy is induced by mechanical stretch in these or other cell types is largely unknown. Here, we have investigated the mechanosensor and downstream signaling pathway that regulate cyclic mechanical stretch (CMS)-induced autophagy in TM cells. We report that primary cilia act as a mechanosensor for CMS-induced autophagy and identified a cross-regulatory talk between AKT1 and noncanonical SMAD2/3 signaling as critical components of primary cilia-mediated activation of autophagy by mechanical stretch. Furthermore, we demonstrated the physiological significance of our findings in ex vivo perfused eyes. Removal of primary cilia disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge, strongly supporting a role of primary cilia-mediated autophagy in regulating IOP homeostasis.

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.

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFβ/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.

Improving speed and image quality of Magnetic Resonance Imaging (MRI) using deep learning reconstruction is an active area of research. The fastMRI dataset contains large volumes of raw MRI data, which has enabled significant advances in this field. While the impact of the fastMRI dataset is unquestioned, the dataset currently lacks clinical expert pathology annotations, critical to addressing clinically relevant reconstruction frameworks and exploring important questions regarding rendering of specific pathology using such novel approaches. This work introduces fastMRI+, which consists of 16154 subspecialist expert bounding box annotations and 13 study-level labels for 22 different pathology categories on the fastMRI knee dataset, and 7570 subspecialist expert bounding box annotations and 643 study-level labels for 30 different pathology categories for the fastMRI brain dataset. The fastMRI+ dataset is open access and aims to support further research and advancement of medical imaging in MRI reconstruction and beyond.