OPTN E50K mutation is the most common mutation for NTG [2, 3], causing severe symptoms in patients . Its pathological mechanism is still obscure, and systematic analysis is rare. In the present study, CRISPR/Cas9 was used to produce OPTN E50K mice. Proteomic analysis was performed to elucidate proteomic and biochemical pathway alterations during NTG development with respect to mitochondrial function, autophagy, and protein synthesis. Comparative analysis between NTG and AD also provided evidences for shared retinal pathological manifestations, suggesting common pathogenesis between those disorders.
CNS neurons are highly energy-demanding cells, dependent on mitochondrial OXPHOS . Thus, mitochondrial dysfunction is a major cause of aging and neurodegenerative disease, including glaucoma . In old E50K mice, 180 DEPs are identified as mitochondrial matrix and membrane components. Complex I, the largest multi-subunit complex, coupling NADH-ubiquinone electron transfer with the generation of a proton gradient for ATP synthesis, was the most severely affected in old E50K mice [29, 30]. Damage to that complex is associated with ROS generation , triggering oxidative stress and gradual cell death, thereby partly explaining the previous finding of E50K mutation selectively inducing ROS-mediated RGC death . Another factor behind this mechanism is complex IV disruptions, of which 16 DEPs were found in old OPTN E50K mice. RGCs, unlike other neurons, are directly exposed to light, thus cytochrome oxidase (COX) in complex IV is important to help absorb light and avoid retinal damage , which was lost with complex IV dysfunction. A key molecule in energy homeostasis is the AMPK complex, which senses low cellular ATP levels to trigger autophagy and inhibit protein synthesis in response to energetic stress and mitochondrial insults [33, 34]. However, this capacity is attenuated under aging and pathological conditions, where severe mitochondrial damage disturbs AMPK signaling, causing apoptosis . The finding of these associations is in agreement with our IPA results in old OPTN E50K mice indicating significant inhibition in OXPHOS and AMPK signaling.
Autophagy is a fundamental cellular process mediating cellular component degradation, and defects in this process plays an important role in neurodegenerative disorder pathogenesis [35, 36]. mTOR is its central regulator, found in our IPA analysis to target various downstream effectors, such as PI3K/AKT signaling, mitochondrial protein synthesis, and mitochondrial-lysosomal homeostasis. Recently, mitochondria and lysosomes are considered mutually functional in maintaining homeostasis [37, 38], where mitochondrial dysfunction leads to lysosomal impairment and autophagy by-product buildup, whereas lysosomal defects trigger functional and morphological mitochondrial defects. This is supported by our IPA analysis showing that mTOR signaling-associated canonical pathways of mitochondria function and phagosome maturation were both disrupted. This connection between mTOR, mitochondria, and lysosomes is facilitated by V-ATPase, which ensures low lysosomal pH to maintain lysosomal hydrolase activity and autophagic vesicle fusion [39, 40]. Loss of V-ATPase subunits triggers accumulation of non-functional lysosomes and inhibition of autophagy, as shown in a previous study where downregulation of the Atp6v0c subunit is observed to promote accumulation of autophagosomes with high basal LC3 II levels, thereby adversely affecting autophagy-lysosome pathway (ALP) function . This was consistent with our findings showing Atp6v0c down-regulation, coupled with higher LC3 II levels, indicating autophagy dysfunction, in old OPTN E50K mice. Furthermore, our findings indicating SNARE family inhibition and RAB family downregulation are in accordance with reduced autophagy activity being present in E50K mutants, as both protein families are associated with membrane fusion. We thus speculate that E50K mutant autophagy blockage stems from the impairment of multiple cellular pathways.
Our results showed the OPTN E50K mutation inducing several age-related proteomic features, even at younger ages. Similar DEP distribution was found between proteins in young OPTN E50K and old WT retinas, particularly those associated with EIF2, eIF4, p70S6K, and mTOR signaling, as well as those found in the ribosome, all of which are involved in protein synthesis and energy metabolism, suggesting a common energy stress response occurring in both retinal types. This association, particularly with ribosomal proteins, is supported by recent studies highlighting that ribosomal dysfunction may cause neuronal synaptic functional and plasticity impairment [42, 43]. The similarities for DEPs are further supported by our GSEA analysis finding 16 biological processes, associated with transcription, translation and protein folding in endoplasmic reticulum, being affected in both young OPTN E50K and old WT retinas, with the same trends. Interestingly, 8 processes for young OPTN E50K mice were discovered to also be affected with old mutants, but with opposite trends. We postulate that aging-driven dysfunctions in transcriptional and protein homeostasis play an important role in old-age glaucoma, which is aggravated by the E50K mutation. This is consistent with clinical findings showing that NTG patients with E50K mutation had early and severe visual dysfunction.
The retina responds similarly to the brain under pathological conditions, owing to it being an extension of the CNS [44, 45]. Being the only place allowing direct visualization of neurons and blood vessels, early non-invasive detection and monitoring of cerebral changes can take place, resulting in greater interest in identifying ocular indicators for neurodegenerative diseases, such as AD [44, 46–48]. Various studies have proven AD ocular involvement, including RGC loss, retinal thinning, astrogliosis, inflammatory changes, and Aβ plaques [47, 48]. Some of these processes, particularly RGC loss, GCL thinning, and Aβ presence, are also present in glaucoma [49, 50]. A recent study also observed OPTN within AD neurofibrillary tangles and dystrophic neurites , supporting our usage of the OPTN E50K mouse model to investigate underlying common pathologies between NTG and AD. We first examined Aβ deposits within NTG retinas, and observed its age-related elevation and disease-specific enhancement in OPTN E50K mice, even at early stages. This aggregation was also observed at 2–3 months in AD mice model retinas, prior to their brain appearance . Based on this observation, it is likely that the OPTN E50K mutation may be connected to Aβ accumulation for both NTG and AD through its impairment of autophagy, which would otherwise remove Aβ peptides [52–54]. Another important pathologic process for both diseases is neuroinflammation triggered by astrocytes and microglia in response to protein aggregates and degenerated neurons [49, 55]. Its occurrence was supported by our findings where astrocytes and microglia were significantly activated in old OPTN E50K mice. Both AD and NTG share other pathological biochemical alterations, such as mitochondria, mRNA splicing and crystallin protein dysfunction [27, 56]. In particular, alternative splicing dysregulation is a common characteristic for several nervous system age-related illnesses, and glaucoma is no exception, as our GSEA analysis showed that the spliceosome was significantly up-regulated in old OPTN E50K mice, involving 45 DEPs [57–59]. As for crystallin proteins, involved in eye development and protein folding, their levels were decreased in both NTG and AD mice [26, 27, 60–63]. Indeed, a reduction in abundance was observed in previous studies for 7 such proteins in association with high IOP [26, 64], as well as here for young OPTN E50K mice. In particular, 2 crystallin proteins within those 7, Crybb3 and Cryab, were also found to have significantly decreased in AD mice retina , further supporting findings highlighting biomolecular similarities between glaucoma and AD. We also found this crystallin decrease to be associated with neurofibril formation and glial cell inflammation, consistent with previous studies [61–63]. Overall, the shared pathological alterations between NTG and AD provide promising targets for glaucoma treatment, as well as possible markers to diagnose CNS disease in patients.