Here, we show that loss of
Slc4a11, which leads to excessive glutamine dependent mitoROS, interferes with TFEB nuclear translocation, and lysosomal biogenesis and function, resulting in aberrant autophagy. Quenching of mitoROS by MitoQ rescues each of these parameters both in vitro and in vivo, resulting in a partial reversal of corneal edema in the mouse model of CHED. This is consistent with a previous study that showed partial alleviation of corneal edema in the CHED mouse when mitoROS was reduced by bypassing glutamine catabolism with dimethyl-α-ketoglutarate eye drops.
6 Taken together, these results confirm that excessive mitochondrial derived ROS drives the disease phenotype in CHED.
Corneal endothelial cell dysfunction, as evidenced by corneal edema, is apparent at eye opening in the CHED mouse. Whereas cell morphology is altered, cell density is not significantly different from WT at 10 weeks of age.
15 Significant reduction in cell density is noted in KO at 40 weeks of age.
15 Therefore, we set out to determine if enhanced autophagy slows cell death in these early stages. However, we found that mitochondrial ROS impedes lysosomal function and autophagy in
Slc4a11 KO corneal endothelial cells. In the KO cells, we observed modest upregulation of several autophagy associated proteins, however, autophagy flux was aberrant. In order for efficient degradation of substrates in the autophagosomes, normal activity of the lysosomal system is crucial. Therefore, we analyzed whether lysosomes are functional in
Slc4a11 KO cells. Lysosomal mass was decreased and lysosomal pH was increased in these cells. Most importantly, nuclear translocation of the master regulator of lysosomal function and biogenesis, TFEB was decreased. All these data indicate that autophagy, which in many cells enhance survival, is compromised in
Slc4a11 KO cells.
Inter-organelle communication has been recognized to be an essential component for normal cellular physiology.
33 Mitochondria-lysosome cross-talk is well studied in this regard. Loss of mitochondrial function in many cases impairs lysosomal activities.
34,35 This sometimes leads to decreased p-mTOR/mTOR ratio that reduces TFEB phosphorylation and increases nuclear translocation, which enhances lysosomal biogenesis.
33,36 This mechanism of action is evident in several cell types during acute mitochondrial stress but not during chronic stress.
36 However, in the
Slc4a11 KO cells, a reduction in TFEB levels and activities, rather than an increase, was observed. These data suggest that chronic mitochondrial stress in
Slc4a11 KO cells may prevent TFEB translocation, which disrupts lysosome functions. Paradoxically, acute application of MitoQ in WT cells, while not producing a detectable change in MitoROS level (see
Supplementary Fig. S4), decreased TFEB nuclear localization, lysosomal content and acidification. This suggests that some small amount of mitochondrial ROS, which we could not detect, may play a role in normal lysosomal biogenesis and function
36 or there is an off target effect of MitoQ.
Phosphorylation by mTOR prevents TFEB nuclear translocation, and thereby inhibits its transcriptional activities.
28,37 In the present study, nuclear levels of TFEB were decreased even with low pmTOR/mTOR ratio in
Slc4a11 KO cells, suggesting alternate mechanisms of TFEB regulation in these cells. Protein kinase B regulates TFEB through mTOR independent mechanisms,
38 and protein kinase C mediated inactivation of Glycogen Synthase Kinase β (GSK3β) is known to improve TFEB nuclear translocation.
39 In certain cell types during chronic stress conditions, the status of mTOR activation is not sufficient to enhance TFEB activities,
33,35,36 and it is stipulated that, in such situations, cells may conserve the limited functionality of damaged organelles rather than spend energy to recycle materials.
35 Whether chronic oxidative stress in
Slc4a11 KO cells supersedes mTOR regulation of TFEB will require additional studies.
Loss of lysosomal proteolytic activities compromise protein degradation pathways in many neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis.
40–42 In addition, impediments in autophagy due to lysosome dysfunctions are present in several ocular diseases, including glaucoma,
43 retinitis pigmentosa,
44 age related macular degeneration,
45 and granular corneal dystrophy.
46 To the best of our knowledge, the present study is the first to identify and characterize autophagy impairment and lysosomal malfunctions in CHED. Several studies have identified mitochondrial dysfunction, or oxidative stress as the trigger behind cell death in FECD, CHED, keratoconus, and granular corneal dystrophy.
4,5,47–50 Here, we further characterize the cross-talk between mitochondria and lysosomes in the corneal endothelial cells, and reveal that an important physiological process (autophagy) is disabled by increased mitochondrial ROS.
CHED is a disease with no cure. The prevalent form of treatment involves endothelial keratoplasty, an invasive procedure with significant risk of graft rejection. Our study shows that the use of a mitochondrial ROS quencher, MitoQ, can decrease endothelial cell loss and corneal edema in a mouse model of CHED. In the
Slc4a11 KO animal model, corneal edema precedes endothelial cell loss, indicating that cell function and not cell loss is the cause of the initial edema. However, with increasing age a continued increase in corneal edema is observed as well as a significant endothelial cell loss. At some point, cell loss will contribute to edema. MitoQ treatment reversed both corneal edema and endothelial cell loss in
Slc4a11 KO animals. We associate the edema reversal to rescue of cell function. Moreover, there is rescue of cell loss because both edema and cell loss are ameliorated by the decreased mitoROS levels. Animal studies involving type 2 diabetes,
32 kidney disease,
51,52 and sensorineural hearing loss
53 have shown promising results using MitoQ as a therapeutic agent. MitoQ was initially synthesized as a lipophilic molecule capable of specifically accumulating in the mitochondria.
54 It was used toward rescuing mitochondrial dysfunction in Parkinson's disease,
55 although clinical trials were ultimately unsuccessful, possibly because damage was already too extensive.
55 However, no major side effects were seen when given the maximum oral dose of MitoQ (80 mg/day) for a year, indicating that it was well tolerated.
55 Because repurposing of old drugs can circumvent many years of additional research that may go into the development of a new drug, MitoQ stands as a promising candidate toward the treatment of disease, such as CHED.