Novel treatments for optic neuropathies and glaucoma

Abstract

The present disclosure provides novel approaches and methods for treating various eye diseases or conditions including optic neuropathies. In particular, the disclosed methods and treatments are directed to those eye diseases or conditions involving mitochondrial dysfunction. More particularly, the methods and treatments provided can restore mitochondrial homeostasis.

Description

NOVEL TREATMENTS FOR OPTIC NEUROPATHIES AND GLAUCOMA

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the prior filed U.S. provisional application 63 / 579,380, filed August 29, 2023, the entire contents of which are incorporated herein by reference.

[0003] REFERENCE TO GOVERNMENT GRANTS

[0004] This invention was made with government support under EY028223 awarded by National Institutes of Health. The Government has certain rights in the invention.

[0005] FIELD OF THE INVENTION

[0006] The present disclosure provides novel approaches and methods for treating various eye diseases or conditions including optic neuropathies. In particular, the disclosed methods and treatments are directed to those eye diseases or conditions involving mitochondrial dysfunctions. More particularly, the methods and treatments provided can restore mitochondrial homeostasis.

[0007] BACKGROUNDOptic neuropathies are a group of neurodegenerative diseases that result in the dysfunction and death of retinal ganglion cells (RGCs), the projection neurons of the retina that deliver visual signals to the brain. Glaucoma accounts for the majority of optic neuropathies, being a leading cause of blindness worldwide and a leading cause of blindness in individuals of African descent. See Leske, “Open-angle glaucoma-an epidemiologic overview,” (2007) Ophthalmic Epidemiol. 14(4): pp. 166-172; Quigley et al., “The number of people with glaucoma worldwide in 2010 and 2020,” (2006) Br J Ophthalmol. 90(3): pp. 262-267. Nearly 18 million Americans are suffering from glaucoma, with an economic burden of treating and preventing the disease reaching $5.8 billion annually and increasing to $17.3 billion by 20501. RGCs die in the glaucoma, causing irreversible vision loss. RGC sensitivity to intraocular pressure (IOP) has been the hallmark feature of glaucoma. However, even with IOP management, an estimated 40-50% of patients go on to losing vision2. Furthermore, over 50% of primary' open-angle glaucoma (POAG) patients with optic nerve damage show normal eye pressure, regardless of treatment, but tend to have increased IOP at follow-ups3. Glaucoma is largely axogenic which probably explains why significant vision loss occurs even with minor RGC body loss in non-human primate glaucoma model4. This interval provides an obvious window of opportunity for neuroprotective therapeutic intervention, especially for patients who do not respond to hypotensive regimens or other treatments, including for secondary glaucoma patients. Currently, our understanding on RGC intrinsic mechanisms that can safely be stimulated in combination with IOP management for novel neuroprotective therapies is limited.SUBSTITUTE SHEET (RULE 26)

[0008] The early pathogenesis of glaucoma progression is marked by metabolic disruption with depleted adenosine-5’ -triphosphate (ATP) supply in RGCs5'8. As such, this has been shown that elevated IOP lowers ATP level in the RGC axons at optic nerve5. IOP elevation first impairs the anterograde axonal transport and at later stages affects retrograde transport (brain to retina) with eventual cell body death6, 10, RGC sensitivity to metabolic disruption is due in part to the high ATP consumption need for unmyelinated intraretinal and laminar axons where transport is slow with a lack of saltatory conduction requiring densely packed mitochondria11'13. Depleted ATP supply first affects anterograde transport to onset distal axonopathy due to less efficient ATP hydrolysis by the anterograde motor kinesins than the retrograde motor dynein6. Mutations in the mitochondrial quality control proteins such as Mitofusin 1 / 2 (MFN1 / 2) and mitophagy protein ()PTN'-M‘ (-13% prevalence) are routinely found among normal tension glaucoma patients. Disrupted mitochondrial homeostasis is the hallmark for these diseases. See Wolf et al., “Evaluation of nine candidate genes in patients with normal tension glaucoma: a case control study,” (2009) BMC Med Genet. 10: p. 91; Rezaie et al., “Adult-onset primary open-angle glaucoma caused by mutations in optineurin,” (2002) Science 295(5557): pp. 1077-1079; Danese et al., “Pathological mitophagy disrupts mitochondrial homeostasis in Leber's hereditary optic neuropathy,” (2022) Cell Rep. 40(3): p. 111124; Sladen et al., “CRISPR-Cas9 correction of OPA1 c, 1334G>A: p.R445H restores mitochondrial homeostasis in dominant optic atrophy patient- derived iPSCs.” (2021 ) Mol Ther Nucleic Acids. 26: pp. 432-443; Geyman et al., “Noninvasive Detection of Mitochondrial Dysfunction in Ocular Hypertension and Primary Open-angle Glaucoma,” (2018) J Glaucoma. 27(7): pp. 592-599. In addition to glaucoma, mitochondrial dysfunctions have been the hallmark for other optic neuropathies such as, Leber’s hereditary optic neuropathy (LHON) and dominant optic neuropathy (DOA). Mutations in the mitochondrial electron transport chain protein ND1 are causal for LHON and mutations in inner mitochondrial membrane protein OPA1 required for mitochondrial fusion for dominant optic neuropathy (DOA).

[0009] The inventors have previously shown that glaucomatous human stem cell differentiated RGC’s (hRGCs) with the mitophagy protein Optineurin (OPTN) mutation (E50K). possess less mitochondrial mass compared to the wild-type (WT) cells but with higher mitochondrial ATP production load leading to mitochondrial damage and neurodegeneration. See Surma et al., “Enhanced mitochondrial biogenesis promotes neuroprotection in human pluripotent stem cell derived retinal ganglion cells,” (2023) Communications Biology. 6(1): p. 218. Dysfunctional mitochondria are the primary source for toxic reactive oxygen species (ROS), which at elevated level leads to cell death. See Kim et al., “The Role of Oxidative Stress in Neurodegenerative Diseases,” (2015) Exp Neurobiol. 24(4): pp. 325-340. Furthermore, people with high intraocularpressure (IOP), both with glaucoma symptoms and those yet to suffer any symptoms, showed prominent signs of increased ROS in the retinal nerve fiber layer (NFL). See Geyman et al. 2018. Their unique structure and need for continuous firing make RGCs particularly vulnerable to mitochondrial dysfunctions. A long portion of RGC axons spanning from the cell body through the optic nen e head (ONH) to the posterior end of the lamina cribrosa remain unmyelinated where action potential propagates slowly, needing more ATP and thus more mitochondria. See Quigley, “Understanding Glaucomatous Optic Neuropathy: The Synergy Between Clinical Observation and Investigation,” (2016) Annu Rev Vis Sci. 2: pp. 235-254. Among the different regions of RGCs, maximum mitochondrial density and activity have been reported at the ONH of the human eye. See Zhu J. Li P. Zhou YG, Ye J. Altered Energy Metabolism During Early Optic Nerve Crush Injury: Implications of Warburg-Like Aerobic Glycolysis in Facilitating Retinal Ganglion Cell Survival,” (2020) Neurosci Bull. 36(7): pp. 761-777.

[0010] Currently, there is no neuroprotection therapy available for LHON or DOA and for primary open angle glaucoma (POAG) patients. Thus, this is critical to identity’ a therapeutic strategy that would restore mitochondrial homeostasis in RGCs leading to restoration of metabolic homeostasis for neuroprotection therapy for above optic neuropathies. RGC neuroprotection therapy can be applied in combination with IOP lowering treatment to obtain long-term robust glaucoma therapy.

[0011] Unfortunately, despite the strong association of mitochondrial dysfunctions with glaucoma and mitochondrial optic neuropathies (MONs) and the clinical desirability of neuroprotective approaches, the development of clinically effective neuroprotective strategies to support RGC survival remains an ongoing challenge. See Danesh-Meyer, “Neuroprotection in glaucoma: recent and future directions,” (2011) Curr Opin Ophthalmol. 22(2): pp. 78-86; Levin et al., “History of neuroprotection and rationale as a therapy for glaucoma,” (2008) Am J Manag Care. 14(1 Suppl): SI 1-14; Krupin et al., “Low-Pressure Glaucoma Study G. A randomized trial of brimonidine versus timolol in preserving visual function: results from the Low-Pressure Glaucoma Treatment Study.” (2011) Am J Ophthalmol. 151(4): pp. 671-681. Years of studies have led to important scientific advances including the findings of reduced level of pyruvate and nicotinamide adenine dinucleotide (NAD) in the retinas of animal models of glaucoma. See Harder et al., “Disturbed glucose and pyruvate metabolism in glaucoma with neuroprotection by pyruvate or rapamycin,” (2020) Proc Natl Acad Sci USA 117(52): pp. 33619-33627; Williams et al., “Vitamin B(3) modulates mitochondrial vulnerability and prevents glaucoma in aged mice.” (2017) Science 355(6326): pp. 756-760; Williams et al., “Glaucoma as a Metabolic Optic Neuropathy: Making the Case for Nicotinamide Treatment in Glaucoma,” (2017) J Glaucoma26(12): pp. 1161-1168. These chemicals are required for mitochondrial oxidative phosphorylation (OXPHOS), hence their decline is assumed to be causal for glaucoma which led to the ongoing clinical trial (NCT05405868) for promoting mitochondrial OXPHOS by supplementing nicotinamide. However, conflicting evidence has also been reported that optic nerve crush (ONC) injun’ significantly increased mitochondrial OXPHOS in the mouse retina and optic nerve. See Zhu et al. 2020. Promising studies show constitutive activation of mitochondrial fusion14, or by over-expression of mitochondrial anterograde transporter Armcxl15> 16can regenerate RGC axons post optic nerve crush (ONC). In a separate study, supplementation of oxidative phosphorylation (OXPHOS) precursor metabolites such as pyruvates17, and nicotinamide18showed RGC protection in elevated 1OP retina. While these mitochondria targeted approaches are attractive, there are challenges to be overcome. For example, constitutive transport of mitochondria to distal axons can deplete supply to the proximal unmyelinated RGC axons that are particularly ATP-demanding. Similarly, daily supplementation with pyruvate or constitutive mito-fusion can both activate OXPHOS and in long run that can lead to mitochondrial damage and release of toxic cytochrome c and buildup of toxic oxidative byproducts above the basal level leading to neurodegeneration19'21. To protect RGCs in glaucoma, our long-term goal is to restore metabolic imbalance by improving mitochondrial health and regaining ATP homeostasis without constitutive changes.

[0012] The inventors recently have discovered that in glaucomatous OPTNE50KhRGCs mitochondrial metabolism is in fact higher than the WT hRGCs. Rather, mutant cells have less mitochondrial mass to meet the metabolic demand causing mitochondrial bum out leading to mitochondrial swelling, disrupted homeostasis and neurodegeneration. However, activating mitochondrial biogenesis through targeting TBK1 mediated activation of biogenesis master regulator PGC-la / I) restored organelle homeostasis ultimately leading to neuroprotection of E50K hRGCs against a mitochondrial stressor. See Surma et al. 2023. Mitochondrial biogenesis relies on synthesis of 13 proteins from mitochondrial DNA (mtDNA) and -1000 proteins from the nuclear DNA. Peroxisome proliferator-activated receptor gamma co-activator (PGCla) is a transcriptional co-activator that drives most of these nuclear genes, including mitochondrial transcription factor Tfam, which is required for mtDNA transcription. Hence, PGCla is considered as the mitochondrial biogenesis master regulator. See Ventura-Clapier et al., “Transcriptional control of mitochondrial biogenesis: the central role of PGC-la,” (2008) Cardiovascular Research 79(2): pp. 208-217. Over-expression of PGC-la in differentiated neurons from patient-derived induced pluripotent stem cells (iPSCs) with LHON disease reversed the pathological conditions. See Danese et al. 2022.

[0013] Thus, there remains a need to identify mechanisms for restoring mitochondrial homeostasis in RGCs that hopefully can provide more effective neuroprotective agents. The inventors have found that in optic neuropathy, RGCs need to produce more ATPs from fewer healthy mitochondria to combat the stress causing more mitochondrial damage and oxidative stress leading to neurodegeneration. However, a transient activation of biogenesis of more healthy mitochondria and / or restoring health of existing mitochondrial population can reduce ATP production load per mitochondrion which can restore the organelle homeostasis leading to RGC protection.

[0014] Thus, there remains a need to produce novel approaches to the prevention and treatment of glaucoma and related retinal diseases. The present disclosure addresses these needs.

[0015] SUMMARY OF THE INVENTION

[0016] The inventors have discovered that under optic neuropathy RGCs need to produce more ATPs from a smaller number of functional mitochondria leading to mitochondrial damage, disrupted metabolic homeostasis, and neurodegeneration. The inventors they have discovered a novel pharmacological agent that restores mitochondrial homeostasis leading to the restoration of metabolic homeostasis with strong RGC protection effect in multiple glaucoma models. See FIG. 1.

[0017] Progressive loss of retinal ganglion cells (RGCs) is the hallmark for optic neuropathies including glaucoma, with no available FDA approved therapy for neuroprotection. Thus, there is a critical need to find the molecular and pharmacological target(s) in human RGCs for developing a neuroprotective therapy. Disrupted mitochondrial homeostasis is the primary suspect for optic neuropathies such as Leber’s hereditary optic neuropathy (LHON), dominant optic atrophy (DO A) and for some forms of glaucoma.

[0018] The rationale for the current disclosure is that despite strong clinical desires we are extremely limited on ways to restore mitochondrial homeostasis which can lead to ganglion cell protection for optic neuropathy. Optic nerve injury is the hallmark for glaucoma and disrupted energy homeostasis has been reported in the animal model of optic nerve crush (ONC) injury’. Using a robust human stem cell differentiated RGC (hRGC) model and small molecule screening, here, inventors discovered novel compounds that potentially restore mitochondrial health leading to enhanced survival of hRGCs in an inherited form of glaucomatous Optineurin mutation (OPTNE50K). Furthermore, one of these compounds provided robust RGC protection, axon regeneration and vision maintenance in mice long after ONC injury with no systemic toxicity. The discovery that restoration of mitochondrial homeostasis could lead to RGC protection is innovativewhich led the inventors to identify a novel compound and its target signaling pathway without relying on a priori notions about which pathway and molecules are involved in RGC survival.

[0019] Based on an innovative screening technique, the inventors have discovered a novel compound WAY-100635 maleate (WAY), a serotonin receptor 5-HT1A antagonist that results in mitochondrial biogenesis activation, suppression of apoptosis in human stem cell derived glaucomatous RGCs (hRGCs). Furthermore, WAY provides robust neuroprotection, axon regeneration and vision maintenance in vivo. The strong cell biology approaches for investigating RGC protection mechanisms using hRGCs and its application for developing in vivo neuroprotection is novel. Inhibition of 5-HT1A receptor by WAY has been shown to alleviate learning and memory impairments in primates and is also indicative of beneficial effects for other neurodegenerative conditions. See Harder et al., “The 5-HT1A antagonist, WAY-100635, ameliorates the cognitive impairment induced by fornix transection in the marmoset,” (1996) Psychopharmacology (Berl) 127(3): pp. 245-254; Harder et al.. “The 5-HT1A antagonist, WAY- 100635, alleviates cognitive impairments induced by dizocilpine (MK-801) in monkeys,” (2000) Neuropharmacology 39(4): pp. 547-552.

[0020] In addition to WAY, the inventors have identified 4-Imidazoleacetic acid hydrochloride (I4AA) and L-glutamic acid as activators of mito-biogenesis and ouabain as a potential activator of mitophagy that led to the suppression of apoptosis in glaucomatous hRGCs further establishes the innovative aspects of the current disclosure.

[0021] The inventors further disclose and have identified BX795 as an activator of mito- biogenesis and RTA-408 (omaveloxolone) as an activator of the oxidative stress response master regulator Nrf2 that led to robust reduction of cellular apoptosis in glaucomatous hRGCs. Thus, I4AA, L-glutamic acid, ouabain, BX795 and RTA-408, also show suppression of cell death in hRGCs. Thus, based on these conceptual and technical innovative approaches the inventors have discovered the above compounds that present novel uses for optic neuropathy therapy development.

[0022] Thus, in embodiments, the cunent invention provides methods for treating various eye diseases or conditions including optic neuropathies. In particular, the disclosed methods and treatments are directed to those eye diseases or conditions involving mitochondrial dysfunction. More particularly, the methods and treatments provided can restore mitochondrial homeostasis. Even more particularly, the eye diseases or conditions contemplated to be treated include glaucoma and optic neuropathies, where the optic neuropathies can include: ischemic optic neuropathy, anterior ischemic optic neuropathy, posterior ischemic optic neuropathy, radiation optic neuropathy, optic neuritis, compressive optic neuropathy, infiltrative optic neuropathy, traumaticoptic neuropathy, mitochondrial optic neuropathy, nutritional optic neuropathy, toxic optic neuropathy, and hereditary or inherited optic neuropathy.

[0023] In any embodiment, the methods and treatments disclosed herein include the administration of a therapeutically effective amount of a compound or pharmaceutical composition that includes one or more of WAY- 100635 maleate or other pharmaceutically acceptable salt. L- Glutamic acid hydrochloride, Ouabain, 4-Imidazoleacetic acid hydrochloride (I4AA), BX-795 hydrochloride and RTA-408 (Omaveloxolone).

[0024] These and other embodiments and features of the disclosure can become more apparent through reference to the following description, the accompanying figures, and the claims. Furthermore, it is to be understood that the features of the vanous embodiments described herein are not mutually exclusive and can exist in various combinations and permutations.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 depicts that under optic neuropathy RGCs need to produce more ATPs from a smaller number of functional mitochondria to combat the stress leading to mitochondrial damage, metabolic disruption and neurodegeneration. We have discovered a novel pharmacological compound that transiently activates of mitochondrial biogenesis and / or other neuroprotective mechanisms that can restore mitochondrial health ultimately leading to the restoration of metabolic homeostasis for RGC protection therapy.FIG. 2A. B shows that the small molecule screen identifies compound for increasing mitochondrial biogenesis. (FIG. 2A) Small molecule screen on H7-hRGCWT(differentiated from H7-hESC reporter line) identified drugs showing >40% change (outside of blue box) to mitochondria mass by MTDR labeling, with WAY-100635 maleate (WAY) indicated by the arrow. (FIG. 2B) Manual validation of WAY treatment shows increased MTDR labelled mitochondrial mass in H7- hRGCWT. Control (CTR) represents untreated cells as WAY is water soluble. Unpaired student’s t-test, n=3. Error bars are SEM. Unpaired student’s t-test, ****, p < 0.0001, n=3.

[0027] FIG. 3A - I depicts that WAY transiently activates cyclic AMP (cAMP) level and promotes hRGC survival. (FIG. 3A) Illustration for mechanism of action by antagonist WAY or agonist DPAT through 5-HT1A GPCR. (FIG. 3B) Representative max-projections and central z- plane of confocal IF images of H7-hRGCWTagainst cAMP (anti-cAMP antibody, R&D systems) and DAPI after indicated timepoints of 5 pM WAY treatments. Insets show perinuclear cAMP that increases with WAY treatment. (FIG. 3C) Presents quantification of cAMP intensity per cell area from sum projections, normalized to control, n = 15-18 cells per condition. (FIG. 3D) Representative max projections of H7-hRGCWTagainst cAMP and DAPI after Ih pretreatment with WAY and then 6h WAY with indicated 8-OH-DPAT concentrations, n = 5 images containingan average of 80-150 cells per image. (FIG. 3E) Quantification of cAMP intensity per area from sum projections, normalized to control shows WAY pretreatment increases cAMP level but addition of DP AT suppresses it. DMSO treatment used for control as DP AT solubilizes in DMSO. (FIG. 3F-I) Caspase-3 / 7 activity for apoptosis was measured using ApoToxGlo-Triplex assay kit in hRGCs normalized to the DMSO control, n=3. Error bars are SEM. One-way ANOVA with Dunnett’s, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0028] FIG. 4A-F shows that WAY promotes neuroprotection in mice against ONC injury. (FIG. 4A) Schematic illustration of unilateral ONC in mouse. (FIG. 4B) Representative tiled confocal image (10X / 0.3NA) of mouse retinal flat mount with immunohistochemistry (IHC) against RGC specific RBPMS protein. Dotted circle and white boxes on the circles represent central and peripheral retinal regions for image acquisitions and quantifications. (FIG. 4C) Representative high-resolution confocal images (40X / 1.3NA) of RGCs from indicated retinas from peripheral white boxes (FIG. 4B). C57BL / 6J (C57) mice were treated daily with WAY (5 mg / kg) by intraperitoneal injection (IP) 5 days before ONC until tissue collection on indicated "‘day post crush (dpc)”. (FIG. 4D) Quantification of RGC numbers / mm2from central and peripheral retina shows significant protection by WAY on 6 and 14 dpc. (FIG. 4E) Regenerated or protected RGC axons are detected by confocal IHC images of optic nerve cryosections against growth associated protein 43 (GAP43) following published methods by Larry Benowitz lab22, 23. GAP43 antibody is shared by Larry Benowitz. (FIG. 4F) Quantification of RGC axon numbers at 0.5 mm from crush site on serial cryosections. Error bars are SEM. (FIG. 4D) Two-way ANOVA with Tukey’s, ****, p < 0.0001, ***, p < 0.001, n = 24-48 images from 3-6 mice for each group. (FIG. 4F) Student’s t- test, **, p < 0.007, n = 3 mice per group.

[0029] FIG. 5A-F shows that WAY maintains visual acuity (VA) in mice after ONC injury. (FIG. 5A) Schematic illustration of the WAY treatment, ONC and subsequent VA measurements. (FIG. 5B) VA measured by scoring head movements at increasing spatial frequency but at fixed speed and contrast. (FIG. 5C, E) Quantification of VA in response to both clockwise and counterclockwise rotation of the sinusoidal gratings. One-way ANOVA with Tukey’s correction. (FIG. 6D, F) VA in response to the individual clockwise or counterclockwise movement of the gratings. Error bars are SEM. Two-way ANOVA with Tukey’s correction, **p < 0.01, ***p < 0.001, ****p < 0.0001. n=4 mice / group

[0030] FIG. 6A-E shows that WAY maintains contrast sensitivity (CS) in mice after ONC injury. CS measurements done on the same mice by OKR as illustrated in FIG. 5A. (FIG. 6A) CS measured by scoring head movements at decreasing contrast but at fixed speed and spatial frequency. (FIG. 6B, D) CS presented here as Michelson contrast (max-min / max+min); max, minrepresent maximum or minimum contrast that the mouse responded (Prusky et al. 2006). Data shown combined response to both clockwise and counterclockwise rotations. One-way ANOVA with Tukey’s correction. (FIG. 7C, E) CS data in response to the individual clockwise or counterclockwise movement of the gratings. Error bars are SEM. Two-way ANOVA with Tukey 's correction, **p < 0.01. ***p < 0.001, ****p < 0.0001. n=4 mice / group.

[0031] FIG. 7A, B shows that WAY maintains visual depth perception after ONC. (FIG. 7A) Animals were tested in a visual cliff apparatus with approximately 2 feet tall cliff (deep) side with similar surface pattern under the glass for optical illusion. (FIG. 7B) Latency time to step down from the platform. Each data point is average from 5 measurements. One-way ANOVA with Dunnett’s, **, p < 0.005. n=3 mice / group.

[0032] FIG. 8A-D shows WAY prevents RGC and visual acuity loss in glaucoma mouse. (FIG. 8A) Intraocular pressure (IOP) is induced by unilateral (left eye, inset) injection of 2 pL of 1 pm, and 2 pL of 6 pm beads (Polyscience, Inc.) (MB) or PBS (sham) into the anterior chamber of C57 mouse eye. IOP measured by rebound tonometer shows elevation and then reduction. The amplitude and time course of IOP changes with elevated IOP for about a month and then decline to the basal level agrees with published unilateral single bead injection data in C57 mouse24. WAY (5 mg / kg) was treated by IP daily 6 days prior to MB until the day post injection (dpi) of measurements, shows transient faster elevation and reduction of IOP. (FIG. 8B) Confocal IHC image of RBPMS positive RGCs, (FIG. 8C) RGC number counted 57 days after MB injection as in FIG. 4D. (FIG. 8D) Visual acuity measured by OptoMotry (Cerebral Mechanics) where mouse exposed to 4 adjoining LCD monitors with moving sinusoidal gratings (12deg / sec clockwise or counterclockwise) with randomly varying frequency but 100% contrast. Spatial frequency in cycles per degree (c / deg) measured for different condition with mouse identity masked. (FIG. 8C) n = 24 images from 3 mice / group. (FIG. 8D) 3 mice / group. Two-way ANOVA with Tukey’s, ****, p < 0.0001. ***, p < 0.001.

[0033] FIG. 9A-D shows robust 5-HT1A expression in hRGCs and in mouse RGCs. (FIG. 9A) Confocal images show 5-HT1A membrane localization in H7-hRGCs and (FIG. 9C) shows on ganglion cell layer of mouse retinal cryosection. (FIG. 9B) Western blots detect 5-HT1 A protein expression in hRGCs, and (FIG. 9D) detects 5-HT1A expression in RGCs isolated from mouse retina.

[0034] FIG. 10A-D depicts that WAY transiently activates mitochondrial biogenesis in hRGCs. (FIG. 10A) Representative confocal images (63X / 1.4NA) of H7-hRGCWTwith Tom20, TdTomato, and DAPI after 5 pM WAY treatment at indicated timepoints. (FIG. 10B) Quantification of Tom20 intensity per cell area from sum projections of confocal z-stacks,normalized to control, n = 19-30 cells per condition. (FIG. 10C) Representative max projections of confocal IF images of H7-hRGCWTagainst PGCl after indicated timepoints of 5 pM WAY treatments. (FIG. 10D) Quantification of PGCla nuclear to cytoplasm intensity' ratio, from sum projections, normalized to control, n = 22-33 cells per condition. Error bars are SEM, One-way ANOVA with Dunnetf s, * p < 0.05. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0035] FIG. 1 1 A-F shows that WAY transiently activates mitochondrial biogenesis with neuroprotection effect to OPTNE50K. H7-hRGCs with OPTNE50Kmutation are treated with 5 pM WAY for indicated timepoints. (FIG. 11 A) Images show Mitochondria by confocal IF against Tom20. (FIG. 1 IB) Quantification of Tom20 intensity per cell area from sum projections of confocal z-stacks, normalized to control, n = 23-29 cells per condition. (FIG. 11C) Representative live cell confocal images of JC1 labelled mitochondria in H7-hRGCE50K. (FIG. 1 ID) Quantification of the red-to-green fluorescence intensity ratios from the sum projections of confocal z-stacks, normalized to the corresponding control values, n = 17-24 cells. (FIG. HE) Mean firing rate averaged from 16 sensors / well over multiple wells, n= 4-5. (FIG. 1 IF) Shown are action potential firing spikes from a single MEA sensor monitored over 3 minutes for respective conditions. (FIG. 1 IB) One-way ANOVA with Dunnett’s. (FIG. 11D) Unpaired student’s t-test. (FIG. HE) Two- way ANOVA with Tukey's. * p < 0.05, ** p < 0.01, **** p < 0.0001.

[0036] FIG. 12A, B shows WAY restores OXPHOS and glycolysis balance in OPTNE50KhRGCs. hRGCs are treated with 5 pM WAY for 24 hr follow ed by seahorse analysis. (FIG. 12A) Seahorse Mito Stress test measures mitochondrial ATP production from change in oxygen consumption rate (OCR) under sequential inhibition of ETC proteins. (FIG. 12B) Glycoly tic rate assay measures OCR, and extra cellular acidification rate (ECAR) under sequential inhibition of mitochondrial ETC proteins and glycolysis to calculate mitoOCR and glycolytic proton efflux rate (gly coPER) and their ratio. Measurements are normalized to cell area, n = 4 - 6. Two-way ANOVA with Tukey’s correction. * p < 0.05, ** p < 0.01. All error bars are SEM.

[0037] FIG. 13A-C depicts CRISPR mediated gene KO in hRGCs using lentivirus (LV). (FIG. 13A) Confocal image (63x / 1.4NA) of WT H7-hRGCs transduced with the LV containing viral vector with EFla-GFP cassette or no GFP vector (Control LV). (FIG. 13B) Cas9 mRNA level in H7-hRGCs 4 days after LV-EFla-Cas9 transduction after digestion of DNA. Error bars are SEM. One-way ANOVA with Dunnett’s, *, p < 0.05, ****, p < 0.0001, n=3. (FIG. 13C) TBK1 KO in iPSC-hRGCs with OPTNE50Kmutation by LV-U6-gRNA (TBK1) and the LV-EFla-Cas9. 8 days post transduction.

[0038] FIG. 14 shows that LC-MS chromatograms detect WAY in mice retina. Plasma and retina isolated from mice 4h after IP injection. The blue filled peak is the analyte of interest. WAYretention time is 5.5 minutes. Representative chromatogram for treated retinal tissue is from 4 independent experiments.

[0039] FIG. 15 A, B shows that WAY does not show systemic toxicity in mice. H&E-stained histological images (40x / 0.6NA) of (FIG. 14A) kidney tissue and (FIG. 15B) liver tissue sections, from naive or WAY treated (5 mg / kg body weight) C57BL / 6J mice by IP injection once daily for 30 days.

[0040] FIG. 16 shows that the small molecule screen identifies compound for activator of mitobiogenesis and mitophagy. Drugs showing >40% change (outside of box) in H7-hRGCWT. Error bars are SEM. Arrows indicate compounds that show reduced apoptosis in H7-hRGCs in the secondary screen for potential RGC neuroprotection therapy.

[0041] FIG. 17A, B shows that L-Glutamic acid (L-Glut) promotes mitochondrial mass and lowers apoptosis in hRGCs. (FIG. 17A) Manual validation of L-Glut treatment at 5 pM for 24h shows increase in mitochondrial mass compared to the untreated control (CTR) in H7-hRGCx lthat was identified from high throughput screen in FIG. 16. L-Glut is water soluble hence we used untreated cells as control. Error bars are SEM. Unpaired student’s t-test. (FIG. 17B) Apoptosis measurements by Apo-Tox Gio Triplex assay in presence of L-Glut (5 pM) at the indicated time points for hRGCs differentiated from H7 WT, H7-OPTNE50K, patient derived iPSCs with OPTNE50Kmutation and iPSCs with OPTNE50Kmutation corrected to WT cells. Error bars are SEM. One-Way ANOVA with Dunnett’s.* p<0.05. ** p<0.01, ***p<0.001, **** p<0.000L n=3

[0042] FIG. 18 A, B shows that MAA promotes mitochondrial mass and lowers apoptosis in hRGCs. (FIG. 18A) Manual validation of MAA treatment at 5 pM for 24h shows increase in mitochondrial mass compared to the untreated control (CTR) in H7-hRGCWTthat was identified from high throughput screen in FIG. 16. MAA is water soluble hence we used untreated cells as control. (FIG. 18B) Apoptosis measurements by Apo-Tox Gio Triplex assay in presence of MAA (5 pM) after 24h treatments to the indicated hRGCs. Error bars are SEM. Unpaired student’s t-test, * p<0.05. ** p<0.01, ***p<0.00I, n=3.

[0043] FIG. 19 A, B shows that Ouabain promotes mitochondrial degradation and lowers apoptosis in hRGCs. (FIG. 19A) Manual validation of ouabain treatment at 5 pM for 24h shows reduced mitochondrial mass in H7-hRGCWTcompared to the DMSO control (CTR) that was identified from the high throughput screen in figure 15. (FIG. 19B) Apoptosis measurements in the WT and OPTNE50KhRGCs by Apo-Tox Gio Triplex assay in presence of ouabain (5 pM) for 24h. Error bars are SEM. Unpaired Student’s t-test. * p<0.05, **** pO.OOOL n=3.

[0044] FIG. 20A-C shows that Nrf2 activation in OPTNE50KhRGCs is defective. (FIG. 20 A) Western blot images of Nrf2 and p-Nrf2 in presence of 10 pM CCCP; (FIG. 20B, C) Protein bandintensity normalized with-respect-to corresponding GAPDH and successively to the DMSO control to identify the changes over time. Error bars are SEM. n=3.

[0045] FIG. 21 A-D shows that the Nrf2 activator RTA-408 reduces ROS level in the WT and glaucoma causing OPTNE50Kmutant hRGCs. (FIG. 20 A) Max projection confocal z-stacks of hRGCs treated with lOOnM RTA-408 or DMSO for 24h. ROS level detected in the live cells by DCFDA which fluoresces green upon oxidation by ROS. (FIG. 20B-D) Quantification of ROS intensity from sum projections of confocal z-stacks, normalized to cell area. Error bars are SEM. Student’s t-test, *, p-value < 0.09, **, <0.01. n=13-16.

[0046] FIG. 22 shows that Nrf2 activation by RTA-408 reduces apoptosis in WT and glaucoma causing OPTNE50KhRGCs. Caspase-3 / 7 activity was measured in hRGCs after 24h DMSO or 100 nM RTA-408 using ApoToxGlo-Triplex assay kit. Error bars are SEM. Student’s t-test, **, p-value < 0.01, n=3.

[0047] DETAILED DESCRIPTION

[0048] The inventors have discovered that under optic neuropathy RGCs need to produce more ATPs from a smaller number of functional mitochondria leading to mitochondrial damage, oxidative stress and neurodegeneration. Transient activation of mitochondrial biogenesis restores mitochondrial homeostasis, leading to RGC protection. See FIG. 1.

[0049] Accordingly, the present disclosure provides novel approaches and methods for treating various eye diseases or conditions including optic neuropathies. In particular, the disclosed methods and treatments are directed to those eye diseases or conditions involving mitochondrial dysfunctions. More particularly, the methods and treatments provided can restore mitochondrial homeostasis.

[0050] Definitions

[0051] Various quantities, such as amounts, sizes, dimensions, proportions, and the like, are presented in a range format throughout this disclosure. It should be understood that the description of a quantity in range format is merely for convenience and brevity7and should not be construed as an inflexible limitation on the scope of any embodiment. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as all individual numerical values within that range unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3. from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example. 1. 1, 2, 2.3. 4.62, 5. and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within thedisclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

[0052] The terminology used herein is to describe particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a.” “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It can be further understood that the terms “includes”, “comprises”, “including” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. Additionally, it should be appreciated that items included in a list in the form of “at least one of A, B, and C” can mean (A); (B); (C): (A and B); (B and C); (A and C); or (A, B, and C). Similarly, items listed in the form of “at least one of A, B. or C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C).

[0053] Unless expressly stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0054] In any of the embodiments disclosed herein, the terms “treating” or “to treat” includes restraining, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder.

[0055] In any of the embodiments disclosed herein, the term “patient” refers to a human.

[0056] Optic neuropathy

[0057] The inventors contemplate that the disclosed methods and treatments can be used for the treatment of any of the known optic neuropathies. The skilled artisan can recognize that optic neuropathy is damage to the optic nerve, which provides visual signals to the brain from the eye, from any cause. Damage and death of these nerve cells, or neurons, leads to characteristic features of optic neuropathy. The main symptom is loss of vision, with colors appearing subtly washed out in the affected eye. A pale disc is characteristic of long-standing optic neuropathy. In many cases, only one eye is affected, and patients may not be aw are of the loss of color vision until the doctor asks them to cover the healthy eye. Optic neuropathy is often called optic atrophy, to describe the loss of some or most of the fibers of the optic nerve. Optic neuropathies capable of being treating by the current invention include:

[0058] Ischemic optic neuropathy in which there is insufficient blood flow (ischemia) to the optic nerve. The anterior optic nerve is supplied by the short posterior ciliary artery and choroidal circulation, while the retrobulbar optic nen e is supplied intraorbitally by apial plexus, which arises from the ophthalmic artery, internal carotid artery, anterior cerebral artery, and anterior communicating arteries. Ischemic optic neuropathies are classified based on the location of the damage and the cause of reduced blood flow, if known.

[0059] Anterior ischemic optic neuropathy (AION) includes diseases that affect the optic nerve head and cause swelling of the optic disc. These diseases often cause sudden rapid visual loss in one eye. Inflammatory diseases of the blood vessels, like giant-cell arteritis, polyarteritis nodosa, Churg-Strauss syndrome, granulomatosis with polyangiitis, and rheumatoid arthritis can cause arteritic AIONs (AAION). The vast majority of AIONs are nonarteritic AIONs (NAION). The most common acute optic neuropathy in patients over 50 years of age, NAION has an annual incidence of 2.3-10.2 / 100,000. NAION presents as apainless loss of vision, often when awakening, that occurs over hours to days. Most patients lose the lower half of their visual field (an inferior altitudinal loss), though superior altitudinal loss is also common. The pathophysiology of NAION is unknown, but it is related to poor circulation in the optic nerve head. NAION is often associated with diabetes mellitus, elevated intraocular pressure (acute glaucoma, eye surgery), high cholesterol, hypercoagulable states, a drop in blood pressure (bleeding, cardiac arrest, perioperative esp. cardiac and spine procedures), and sleep apnea. Rarely, amiodarone, interferonalpha, and erectile dysfunction drugs have been associated with this disease.

[0060] Posterior ischemic optic neuropathy is a syndrome of sudden visual loss with optic neuropathy without initial disc swelling with subsequent development of optic atrophy. This can occur in patients who are predisposed to AAION and NAION as described above as well as those who had cardiac and spine surgery or serious episodes of hypotension.

[0061] Radiation optic neuropathy (RON) is also thought to be due to ischemia of the optic nen e that occurs 3 months to 8 or more years after radiation therapy to the brain and orbit. It occurs most often around 1.5 years after treatment and results in irreversible and severe vision loss, which may also be associated with damage to the retina (radiation retinopathy). This is thought to be due to damage to dividing glial and vascular endothelial cells. RON can present with transient visual loss followed by acute painless visual loss in one or both eyes several weeks later. The risk of RON is significantly increased with radiation doses over 50 Gy.

[0062] Optic neuritis is inflammation of the optic nerve, which is associated with swelling and destruction of the myelin sheath covering the optic nerve. Young adults, usually females, are most commonly affected. Symptoms of optic neuritis in the affected eye include pain on eye movement,sudden loss of vision, and decrease in color vision (especially reds). Optic neuritis, when combined with the presence of multiple demyelinating white matter brain lesions on MRI, is suspicious for multiple sclerosis. Several causes and clinical courses are possible for the optic neuritis which are classified thusly: i) Single isolated optic neuritis; ii) relapsing isolated optic neuritis; iii) chronic relapsing inflammatory optic neuropathy; iv) neuromyelitis optica spectrum disorder; v) multiple sclerosis associated optic neuritis; and various unclassified optic neuritis. Medical examination of the optic nerve with an ophthalmoscope may reveal a swollen optic nerve, but the nerve may also appear normal. Presence of an afferent pupillary defect decreased color vision, and visual field loss (often central) are suggestive of optic neuritis. Recovery of visual function is expected within 10 weeks. However, attacks may lead to permanent axonal loss and thinning of the retinal nerve fiber layer.

[0063] Compressive optic neuropathy includes tumors, infections, and inflammatory processes which can cause lesions within the orbit and, less commonly, the optic canal. These lesions may compress the optic nerve, resulting optic disc swelling and progressive visual loss. Implicated orbital disorders include optic gliomas, meningiomas, hemangiomas, lymphangiomas, dermoid cysts, carcinoma, lymphoma, multiple myeloma, inflammatory orbital pseudotumor, and thyroid ophthalmopathy. Patients often have bulging out of the eye (proptosis) with mild color deficits and almost normal vision with disc swelling.

[0064] Infiltrative optic neuropathy are disorders caused by infiltrative processes. The optic nerve can be infiltrated by a variety of manners, including tumors, inflammation, and infections. Tumors that can infiltrate the optic nerve can be primary7(optic gliomas, capillary7hemangiomas, and cavernous hemangiomas) or secondary7(metastatic carcinoma, nasopharyngeal carcinoma, lymphoma, and leukemia). The most common inflammatory disorder that infiltrates the optic nerve is sarcoidosis. Opportunistic fungi, viruses, and bacteria may also infiltrate the optic nerve. The optic nerve may be elevated if the infiltration occurs in the proximal portion of the nerve. The appearance of the nerve on examination depends on the portion of the nerve that is affected.

[0065] Traumatic optic neuropathy includes disorders caused when the optic nerve is damaged when exposed to direct or indirect injury. Direct optic nerve injuries are caused by trauma to the head or orbit that crosses normal tissue planes and disrupts the anatomy and function of the optic nerve; e.g., a bullet or forceps that physically injures the optic nerve. Indirect injuries, like blunt trauma to the forehead during a motor vehicle accident, transmit force to the optic nerve without transgressing tissue planes. This type of force causes the optic nerve to absorb excess energy at the time of impact. The most common site of injury of the optic nerve is the intracanalicular portion of the nerve. Deceleration injuries from motor vehicle or bicycle accidents account for 17 to 63percent of cases. Falls are also a common cause, and optic neuropathy most commonly occurs when there is a loss of consciousness associated with multi-system trauma and serious brain injury. In less than three percent of patients, an orbital hemorrhage after an injection behind the eye (retrobulbar block) can cause injury to the optic nen e, but this is readily manageable if it does not involve direct optic nene injury’ and is caught early. The role of high-dose steroids and orbital decompression in treating these patients is controversial and, if administered, must be done very soon after injury with minimal effects. In patients with an orbital fracture, vomiting or nose blowing can force air into the orbit and possibly compromise the integrity of the optic nerve.

[0066] Mitochondrial optic neuropathies are described in detail herein. Mitochondria play a central role in maintaining the life cycle of retinal ganglion cells because of their high energy dependence. Mitochondria are made within the central somata of the retinal ganglion cell, transported down axons, and distributed where they are needed. Genetic mutations in mitochondrial DNA, vitamin depletion, alcohol and tobacco abuse, and use of certain drugs can cause derangements in efficient transport of mitochondria, which can cause a primary or secondary optic neuropathy.

[0067] Nutritional optic neuropathies may be present in a patient with obvious evidence of under-nutrition (weight loss and wasting). Months of depletion are usually necessary to deplete body stores of most nutrients. Undernourished patients often have many vitamin and nutrient deficiencies and have low serum protein levels. However, the optic neuropathy associated with pernicious anemia and vitamin B12 deficiency can even be seen in well-nourished individuals. Gastric bypass surgery' may also cause a vitamin B12 deficiency from poor absorption. Patients who have nutritional optic neuropathy may notice that colors are not as vivid or bright as before and that the color red is washed out. This normally occurs in both eyes at the same time and is not associated with any eye pain. They might initially notice a blur or fog, followed by a drop in vision. While vision loss may be rapid, progression to blindness is unusual. These patients tend to have blind spots in the center of their vision with preserved peripheral vision. In most cases, the pupils continue to respond normally to light.

[0068] Toxic optic neuropathies. The most recognized cause of toxic optic neuropathy is methanol poisoning. This can be a life-threatening event that normally accidentally occurs when the person mistook or substituted, methanol for ethyl alcohol. The patient initially has nausea and vomiting, followed by respiratory distress, headache, and visual loss 18-48 hours after consumption. Without treatment, patients can go blind, and their pupils can dilate and stop reacting to light. Ethylene glycol, a component of automobile antifreeze, is a poison that is toxic to the whole body including the optic nen e. Consumption can be fatal, or recovery' can occur withpermanent neurologic and ophthalmologic deficits. While the visual loss is not very common, increased intracranial pressure can cause bilateral optic disc swelling from cerebral edema. A clue to the cause of intoxication is the presence of oxalate crystals in the urine. Like methanol intoxication, treatment is ethanol consumption. Ethambutol, a drug commonly used to treat tuberculosis, is notorious for causing toxic optic neuropathy. Patients with vision loss from ethambutol toxicity lose vision in both eyes equally. This initially presents with problems with colors (dyschromatopsia) and can leave central visual deficits. If vision loss occurs while using ethambutol, it would be best to discontinue this medication under a doctor's supervision. Vision can improve slowly after discontinuing ethambutol but rarely returns to baseline. Amiodarone is an anti arrhythmic medication commonly used for abnormal heart rhythms (atrial or ventricular tachyarrhythmias). Most patients on this medication get comeal epithelial deposits, but this medication has also been controversially associated with NAION. Patients on amiodarone with new visual symptoms should be evaluated by an ophthalmologist. Tobacco exposure, most commonly through pipe and cigar smoking, can cause optic neuropathy. Middle-aged or elderly men are often affected and present with painless, slowly progressive, color distortion and visual loss in both eyes. The mechanism is unclear, but this has been reported to be more common in individuals who are already suffering from malnutrition.

[0069] Hereditary or inherited optic neuropathies ty pically manifest as a symmetric bilateral central visual loss. Optic nerve damage in most inherited optic neuropathies is permanent and progressive. Leber's hereditary optic neuropathy (LHON) is the most frequently occurring mitochondrial disease, and this inherited form of acute or subacute vision loss predominantly affects young males. LHON usually presents with rapid vision loss in one eye followed by involvement of the second eye (usually within months). Visual acuity often remains stable and poor (around or below 20 / 200) with a residual central visual field defect. Patients with the 14484 / ND6 mutation are most likely to have visual recovery. Dominant optic atrophy is an autosomal dominant disease caused by a defect in the nuclear gene OPAL A slowly progressive optic neuropathy, dominant optic atrophy, usually presents in the first decade of life and is bilaterally symmetrical. Examination of these patients shows loss of visual acuity, temporal pallor of the optic discs, centrocecal scotomas with peripheral sparing, and subtle impairments in color vision. Behr's syndrome is a rare autosomal recessive disorder characterized by early -onset optic atrophy, ataxia, and spasticity. Berk-Tabatznik syndrome is a condition that shows symptoms of short stature, congenital optic atrophy and brachytelephalangy. This condition is extremely rare.

[0070] Small molecules discovered for novel use for optic neuropathy treatment

[0071] Disclosed herein are several small molecules useful in various aspects of the current invention for RGC protection. Any embodiments disclosed herein can include the administration to a patient in need, a therapeutically effective amount of one or more of: WAY-100635 maleate or other pharmaceutically acceptable salt, L-Glutamic acid hydrochloride, Ouabain, 4- Imidazoleacetic acid hydrochloride (I4AA), BX-795 hydrochloride and RTA-408 (Omaveloxolone).

[0072] Method of Treatment

[0073] A method of treatment described herein comprises administering a pharmaceutical composition containing WAY- 100635 maleate for treating optic neurodegenerative diseases, such as glaucoma and inherited optic neuropathies. The primary goal of this method is to achieve retinal ganglion cell (RGC) neuroprotection, axonal regeneration, and the maintenance of visual functions through various biological mechanisms.

[0074] Administration Regimen

[0075] In some embodiments, the pharmaceutical composition is administered intraperitoneally to a patient diagnosed with an optic neurodegenerative disease. The administration may include daily doses, ranging from about 0. 1 mg / kg to about 45 mg / kg body weight, determined according to therapeutic requirements, patient's medical condition, and tolerance. Alternative routes of administration, including intravenous, subcutaneous, or oral, may also be considered based on formulation stability and patient compliance.

[0076] Mechanistic Pathways

[0077] Restoration of Mitochondrial Homeostasis: The methods disclosed herein comprise the administration of WAY-100635 maleate, which acts as a serotonin receptor 5-HT1A antagonist. Without being limited by theory, the inhibition of this receptor may lead to the transient activation of mitochondrial biogenesis in retinal ganglion cells. Consequently, there is a production of healthy mitochondria, which may be essential for sustaining cellular energy demands and overall cell survival.

[0078] Suppression of Apoptosis: The administration of a pharmaceutical composition comprising WAY-100635 maleate may lead to the suppression of apoptosis in RGCs. Without being limited by theory. WAY- 100635 maleate may inhibit the progression of programmed cell death, preserving the cellular integrity of RGCs, which are crucial for transmitting visual information from the retina to the brain.

[0079] Neuroprotective Signaling Pathways: In some embodiments, the administration of a pharmaceutical composition comprising WAY-100635 maleate may modulate neuroprotective signaling pathways that may enhance the survival and function of RGCs. This includes pathwayspotentially involving proteins such as PGC-lot, SIRT1, and CaMKII / p38 MAPK. which promote cellular survival and neuro-regeneration. A pharmaceutical composition comprising WAY- 100635 maleate may assist in axonal regeneration following optic nerve injury. In some embodiments, a method of administering WAY-100635 maleate to a patient having optic nerve injun’ promotes the regeneration of damaged axons, facilitating the re-establishment of neural pathways necessary for vision restoration. Furthermore, the method may include visual inspection, vision test, and MRI scans of patient’s brain to detect if visual signal is being properly processed in transmitted the brain.

[0080] Dosage and Administration

[0081] A therapeutically effective dose of WAY-100635 maleate may range from approximately 0. 1 mg / kg to 45 mg / kg body weight. The determination of a therapeutically effective dose is based on clinical evaluations, pharmacokinetics, and patient-specific factors.

[0082] Administration Routes: In some embodiments, the pharmaceutical composition is administered by intraperitoneal injection. Alternatively, or in addition to, the composition may be administered by other routes such as intravenous, subcutaneous, and oral administration are considered herein.

[0083] Safety and Efficacy

[0084] Preclinical studies utilizing mouse models with optic nerve crush injuries have demonstrated that a composition comprising WAY- 100635 maleate significantly protects RGCs. assists in axonal regeneration, and improves measurements of visual functions, such as visual acuitj’ and contrast sensitivity measured by optokinetic response (OKR). Behavioral vision tests, including visual cliff tests, further confirm these observations. No systemic toxicity was noted in preliminary studies with prolonged administration of WAY- 100635 maleate at therapeutic doses. More details on these preclinical studies is provided in the EXAMPLES SECTION herein.

[0085] Subjects or patients who may benefit from this treatment, include but are not limited to, encompass individuals diagnosed with optic neurodegenerative diseases, such as glaucoma, hereditary optic neuropathies, and other conditions characterized by the progressive degeneration of retinal ganglion cells (RGCs). Specific patient populations include those: diagnosed with glaucoma, including primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and angle-closure glaucoma, where increased intraocular pressure or other factors lead to RGC death and vision loss, exhibiting inherited optic neuropathies, such as Leber hereditary optic neuropathy (LHON) and dominant optic atrophy (DO A), which result in genetic mutations causing mitochondrial dysfunction and subsequent RGC deterioration, those suffering from optic nen e injuries, including traumatic optic neuropathy, such as where mechanical damage to theoptic nerve results in axonal loss and impaired vision, those patients diagnosed with optic neuritis or anterior ischemic optic neuropathy (AION), where inflammation or ischemia leads to acute RGC damage and potential permanent vision loss, those patients experiencing toxic or nutritional optic neuropathies, where exposure to certain toxins or nutritional deficiencies cause mitochondrial damage and RGC apoptosis, or those patients presenting with secondary optic neuropathies due to systemic conditions like diabetes, multiple sclerosis, and other diseases where associated complications lead to optic nerve degeneration and visual impairment.

[0086] These subjects are anticipated to gain therapeutic benefits from the administration of WAY- 100635 maleate and / or alternative compounds targeting mitochondrial dysfunction, leading to enhanced mitochondrial biogenesis, reduction of apoptosis, and promotion of neuroprotective and regenerative pathways in retinal ganglion cells. The resultant therapeutic outcomes include preservation of vision, improved visual acuity, and potentially the regeneration of damaged optic nerve fibers.

[0087] Routes of Administration

[0088] The pharmaceutical composition containing WAY-100635 maleate can be administered to individuals diagnosed with optic neurodegenerative diseases utilizing various administration routes. The following describes potential routes of administration, each with distinct advantages depending on the formulation stability, patient compliance, and targeted delivery:

[0089] Intraperitoneal Administration: Intraperitoneal injections involve delivering the pharmaceutical composition directly into the peritoneal cavity’, allowing for efficient absorption and systemic distribution. This route is commonly used in preclinical studies and provides consistent therapeutic dosing.

[0090] Intravenous Administration: Intravenous (IV) administration entails injecting the pharmaceutical composition directly into the bloodstream. This route ensures rapid onset of action and precise control over the administration dosage, making it suitable for acute therapeutic intervention.

[0091] Subcutaneous Administration: Subcutaneous injections involve delivering the pharmaceutical composition into the subcutaneous tissue, which houses a network of small blood vessels for gradual absorption. This route is beneficial for sustained release formulations and enhanced patient compliance due to ease of administration.

[0092] Oral Administration: Oral administration involves ingestion of the pharmaceutical composition in the form of tablets, capsules, or liquid solutions. This route is non-invasive and highly convenient for patients, although it requires consideration of formulation stability andbioavailability. Various excipients and delivery systems may be employed to enhance solubility and absorption of WAY-100635 maleate.

[0093] Intravitreal Administration: Intravitreal injections administer the pharmaceutical composition directly into the vitreous humor of the eye, potentially bypassing systemic circulation and delivering higher local concentrations to the retinal ganglion cells. This route is particularly effective for localized treatment of ocular conditions.

[0094] Topical Administration: Topical ocular administration involves applying the pharmaceutical composition, such as eye drops or ophthalmic gels, directly to the ocular surface. This route may be used to target anterior segment conditions and offers non-invasive delivery, with considerations for periocular penetration.

[0095] Each route of administration may be optimized based on therapeutic objectives, pharmacokinetics, and patient-specific factors. Additionally, formulation strategies, like encapsulation in nanoparticles or use of sustained release systems, may be employed to improve stability, efficacy, and patient compliance across different administration routes.Dosage and Dosage Schedules

[0096] The administration of the therapeutic composition comprising WAY-100635 maleate may be executed in a manner optimized to achieve retinal ganglion cell (RGC) neuroprotection, axonal regeneration, and the maintenance of visual function. The following provides exemplary therapeutic effective amounts; however, dosage ranges, frequency, and potential adjustments pursuant to individual patient responses and therapeutic requirements are considered by those having ordinary' skill in the art when w riting prescriptions and treatment plans.

[0097] The administration regimen for the pharmaceutical composition may comprise dosage ranges from about 0.1 mg / kg to about 45 mg / kg body weight. Specific dosage ranges within this spectrum include, but are not limited to about 0. 1 mg / kg to about 40 mg / kg, about 0.1 mg / kg to about 35 mg / kg, about 0.1 mg / kg to about 30 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 0. 1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 15 mg / kg, about 0. 1 mg / kg to about 10 mg / kg, about 0. 1 mg / kg to about 5 mg / kg. about 0. 1 mg / kg to about 1 mg / kg, or about 0. 1 mg / kg to about 0.5 mg / kg. Additional dosage ranges include about 0.5 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 35 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 0.5 mg / kg to about 25 mg / kg, about 0.5 mg / kg to about 20 mg / kg, about 0.5 mg / kg to about 15 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 5 mg / kg. or about 0.5 mg / kg to about 1 mg / kg. Additional dosage ranges include about 1 mg / kg to about 45 mg / kg, about 1 mg / kg to about 40 mg / kg, about 1 mg / kg to about 35 mg / kg, about 1 mg / kg to about 30 mg / kg, about 1 mg / kg to about 25 mg / kg, about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 15 mg / kg,about 1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 5 mg / kg. Additional dose ranges include about 5 mg / kg to about 45 mg / kg, about 5 mg / kg to about 40 mg / kg, about 5 mg / kg to about 35 mg / kg, about 5 mg / kg to about 30 mg / kg, about 5 mg / kg to about 25 mg / kg, about 5 mg / kg to about 20 mg / kg, about 5 mg / kg to about 15 mg / kg, or about 5 mg / kg to about 10 mg / kg. Further dosage ranges include about 10 mg / kg to about 45 mg / kg, about 10 mg / kg to about 40 mg / kg, about 10 mg / kg to about 35 mg / kg, about 10 mg / kg to about 30 mg / kg, about 10 mg / kg to about 25 mg / kg, about 10 mg / kg to about 20 mg / kg, or about 10 mg / kg to about 15 mg / kg. Further dosage ranges include about 15 mg / kg to about 45 mg / kg, about 15 mg / kg to about 40 mg / kg, about 15 mg / kg to about 35 mg / kg, about 15 mg / kg to about 30 mg / kg, about 15 mg / kg to about 25 mg / kg, about 15 mg / kg to about 20 mg / kg. about 20 mg / kg to about 45 mg / kg, about 20 mg / kg to about 40 mg / kg, about 20 mg / kg to about 35 mg / kg, about 20 mg / kg to about 30 mg / kg, about 20 mg / kg to about 25 mg / kg, about 25 mg / kg to about 45 mg / kg, about 25 mg / kg to about 40 mg / kg, about 25 mg / kg to about 35 mg / kg, about 25 mg / kg to about 30 mg / kg, about 30 mg / kg to about 45 mg / kg, about 30 mg / kg to about 40 mg / kg. about 30 mg / kg to about 35 mg / kg, about 35 mg / kg to about 45 mg / kg, about 35 mg / kg to about 40 mg / kg, or about 40 mg / kg to about 45 mg / kg.

[0098] The dosage range administer in the methods disclosed herein may be modified to accommodate various factors, including but not limited to:

[0099] Severity of the Optic Neurodegen erative Disease: Patients exhibiting more advanced stages of optic neurodegeneration may require higher doses within the specified range to achieve the desired therapeutic effects.

[0100] Patient-Specific Factors: Variables such as age. weight, metabolic rate, and existing comorbidities may influence the dosage needed to reach therapeutic efficacy.

[0101] Clinical Response and Tolerance: Adjustments to the dosage may be made based on the patient's response to the treatment and tolerance to the therapeutic agent. Continuous monitoring and evaluations by healthcare professionals may be employed to tailor the dosage to individual needs.

[0102] Dosage Schedules

[0103] The administration of WAY-100635 maleate may follow a structured schedule to maximize therapeutic outcomes while minimizing potential adverse effects. For example, a method may comprise administered a pharmaceutical composition comprising WAY-100635 maleate as an initial dose, a maintenance dose, and / or long-term dosing.

[0104] Initial Dosing Phase: Patients may begin treatment with a dose in the lower end of the effective range (0.1 mg / kg to 5 mg / kg per day). After initial administration, patients may beassessed for therapeutic response and tolerance. Adjustments to the dosing amount may be made based on observed efficacy and any adverse reactions.

[0105] Maintenance Dosing: Depending on a patient response, the dosage may be gradually increased to maintenance levels within the specified range (0.5 mg / kg to 45 mg / kg per day). Maintenance dosing aims to stabilize mitochondrial biogenesis, reduce apoptosis, and promote neuroprotective pathways in RGCs.

[0106] Long-Term Treatment: For sustained therapeutic outcomes, long-term administration of WAY- 100635 maleate may be administered, with periodic evaluations to ensure continued efficacy and safety. Long-term dosages may be maintained within the effective range, subject to adjustments based on clinical evaluations and patient-specific considerations.

[0107] Alternative Dosage Schedules: Alternative routes, such as intravenous, subcutaneous, or oral administration, may adopt different schedules based on the pharmacokinetics associated with each route. Periodic breaks from administration may be considered in certain cases to assess the natural progression of the disease and the long-term efficacy of the treatment.

[0108] Special Considerations

[0109] Pediatric and Geriatric Patients: Pediatric and geriatric populations may require carefully tailored dosages due to differences in metabolism, drug absorption, and overall health status. Lower initial doses and gradual titration may be recommended in these populations to minimize potential adverse effects.

[0110] Concurrent Medications: Patients receiving concurrent medications may be monitored for potential drug interactions that may influence the pharmacokinetics or dynamics of WAY- 100635 maleate. Dosage adjustments may be made based on comprehensive evaluations of all medications taken by the patient.

[0111] Adjustment Based on Disease Progression: Dosages may be subject to change in response to the progression of optic neurodegenerative diseases, with more aggressive dosing schedules considered for rapidly advancing cases.

[0112] Through adherence prescribed dosage and dosing schedules, the therapeutic application of WAY-100635 maleate aims to maximize benefit while minimizing potential risks to patients with optic neurodegenerative diseases.

[0113] The administration of WAY-100635 maleate in combination with another compound, such as L-Glutamic acid hydrochloride, may provide a synergistic therapeutic effect for the treatment of optic neurodegenerative diseases. This combination approach may enhance the overall efficacy of the treatment by targeting multiple mechanistic pathways involved in neuroprotection and RGC survival.

[0114] The pharmaceutical composition comprising WAY- 100635 maleate may be administered with other compositions or may comprise additional compositions such as L- Glutamic acid hydrochloride, 4-Imidazoleacetic acid hydrochloride, BX-795 hydrochloride, RTA-408, or Ouabain. In some embodiments, the pharmaceutical composition comprises WAY- 100635 and L-Glutamic acid hydrochloride may be administered according to a regimen tailored to a patient's condition and therapeutic requirements. The combination may be delivered via intraperitoneal injections, with dosages adjusted based on pharmacokinetic profiles and patient tolerance. As an example, a pharmaceutical composition may comprise WAY- 100635 maleate in an amount of about 0.5 mg / kg to about 5 mg / kg body weight and L-Glutamic acid hydrochloride in an amount of about 10 mg / kg to about 50 mg / kg. The pharmaceutical composition comprising WAY-100635 maleate in an amount of about 5 mg / kg to about 30 mg / kg body and L-Glutamic acid hydrochloride in an amount of about 50 mg / kg to about 100 mg / kg may be administered a patient in need thereof.

[0115] Methods

[0116] ESC maintenance

[0117] Human embryonic stem cell line (H7-hESCs) from WiCell, Madison. WI, was used as a reporter line. The H7-hESCs were modified by CRISPR to introduce a multicistronic BRN3B- P2A-tdTomato-P2A-Thyl.2 construct into the endogenous BRN3B locus, specifically expressed in retinal ganglion cells (RGCs), serving as an isogenic control. See Sluch et al. 2017. As previously described, another set of H7-hESC reporter cells with a CRISPR-induced mutation (H7- E50K) in the OPTN gene was also generated. See VanderWall et al. 2020. Additionally, patient- derived induced pluripotent stem cells (iPSCs) earn ing the E50K mutation (iPSC-E50K), as well as iPSCs with the OPTNE’0Kmutation corrected to wild type (iPSC-E50Kcorr) was generated by CRISPR. See Ohlemacher et al., “Stepwise Differentiation of Retinal Ganglion Cells from Human Pluripotent Stem Cells Enables Analysis of Glaucomatous Neurodegeneration." (2016) Stem Cells 34(6): pp. 1553-1562. All the cell lines were cultured in mTeSRl media (mT) at 37°C in a 5% CO2 incubator on matrigel (MG)-coated plates. Routine clump passaging using Gentle Cell Dissociation Reagent (GD) was performed when the cells reached 70-80% confluency. The media was aspirated, GD was added to the cells and incubated at 37°C in a 5% CO2 incubator for 5 minutes. After the aspiration of GD. mT media was used to break up the colonies into small clumps through repeated pipetting and the clumps were then seeded onto new MG plates.

[0118] hRGC differentiation and immunopurification

[0119] The stem cell colonies were split into individual cells using accutase for 10 minutes. The cells were treated with double the volume of mT containing 5 pM blebbistatin (blebb) to halt the accutase activity. Following this, the cells were centrifuged at 150 xg for 6 minutes and then resuspended in mT supplemented with blebb (5 pM). Cells were counted using a hemacytometer and subsequently, 100,000 cells were seeded in each well of a 24-well plate coated with matrigel (MG). The next day, the media was replaced with mT without blebb. After 24 hours, iNS media was added and differentiation with small molecules was carried out according to previous protocols. See Das et al. 2020. The differentiation of human retinal ganglion cells (hRGCs) w as monitored by observing the expression of tdTomato and the cells were purified between days 45- 55. using THY1.2 microbeads and the magnetic-activated cell sorting system (MACS) from Miltenyi Biotec, following the previously described method. Id.; Sluch et al. 2017. Next, the purified hRGCs were suspended in iNS media, counted using a hemocytometer and seeded onto MG-coated plates, coverslips, or MatTek dishes for subsequent experiments.

[0120] Small Molecule Screening

[0121] Cold matrigel (30pl) was added to each w ell of a 384 well plate using Thermo Scientific Multidrop 384 and incubated at 37°C 5% CO2 overnight. Matrigel solution was removed from wells by pouring out onto paper towels and then 50 pl of cell suspension in iNS (10,000 hRGCs) was added to each well and incubated 48h. Media was removed using an automatic vacuum and 50 pl of iNS with 10 nM MTDR was added to each well. After 15 min incubation, drugs were added to wells robotically, using Tecan EVO 100 with PIN tool, from the standard drug plate (0.5 pl of ImM stock, for 5 pM final concentration) and incubated another 24h. Media was aspirated from wells using an automatic vacuum and 15 pl accutase was added to each well and incubated for 20 minutes. 50 pl iNS was added to each well, mixed by pipette up / down and transferred (Tecan EVO150MCA with disposable tips) to 384 well U-bottom plate. The plate w as then placed into the flow cytometer (Thermo Fisher Attune NxT flow cytometer, with autosampler) and single-cell suspensions were analyzed for the MTDR signal intensity from the drug-treated RGCs compared to the DMSO controls.

[0122] Flow cytometry

[0123] Purified hRGCs were seeded at 30.000 cells per well of a 96-well MG-coated plate and maintained for 3 days. For measuring mitochondrial mass, hRGCs w ere labelled with mitochondria specific MTDR dye. To measure mitochondrial biogenesis, hRGCs were labeled with MTDR first and treated with WAY at different time points. After treatments, hRGCs were dissociated to singlecell suspension using accutase and analyzed using Attune NxT flow cytometer (Thermo Fisher).

[0124] Western blot

[0125] The purified hRGCs were seeded at a density of 500,000 cells per well in 24-well plates coated with MG and were cultured for 3 days. Following this, the cells were subjected to WAY treatment at specified time points. As a control, distilled water (dFhO) was utilized since the WAY was dissolved in dFFO. Subsequently, the cells were lysed and collected in 100 pl of M-PER extraction buffer containing 5mM EDTA and protease inhibitors. The protein content was determined using the DC Protein Assay Kit II (Bio-Rad) with bovine serum albumin (BSA) as a standard and quantified using a microplate reader. For preparing the loading samples, heat denaturation was carried out at 95 °C for 5 minutes with Laemmli sample buffer (IX). Approximately 10-20 pg of protein per sample were loaded onto Bio-Rad Mini-PROTEAN TGX precast gels using Tris-Glycine-SDS buffer (Bio-Rad) as the running buffer. The gel electrophoresis was conducted at 100 V until the dye front reached the bottom of the gel. A transfer sandwich was created using a PVDF membrane, activated with methanol and placed in Tris- Glycine transfer buffer (Bio-Rad) containing 20% methanol. The transfer was carried out for 2 hours at 30 V and 4°C. For protein visualization, the membranes were blocked in 5% skim milk in TBST (TBS buffer with 0.5% Tween20) for 2 h at room temperature and incubated overnight at 4°C in 1: 1000 dilution of primary antibodies for Tom20 (Santa Cruz), 5HT1A (Abeam), or GAPDH (CST). Membranes were then washed three times for 5 minutes each in TBST, followed by 2h of incubation in 5% milk in TBST containing anti-rabbit HRP-linked secondary antibody (CST) at 1 : 10,000 dilution. The membranes were washed three times with TBST and placed in Clarity Max Western ECL (Bio-Rad) substrate for 5 min. The membranes were imaged in a BioRad ChemiDoc Gel Imager and the raw- integrated density7for each band was measured and normalized with respect to the corresponding GAPDH or actin loading control using Image J. In addition, the treatment conditions were further normalized to their respective controls within each experiment. The quantification of protein bands was performed based on the expected size of the proteins, as indicated in product datasheets and published literature.

[0126] Immunofluorescence and Confocal imaging

[0127] Purified hRGCs were cultured on MG coated coverslips (thickness 1.5). The cells were seeded at a density of 30,000-40,000 cells per coverslip and allowed to grow for 3 days. Later, the hRGCs w ere treated with the specified molecule (WAY) at specific time points. The culture media was removed after the treatment and the cells were w ashed with IX PBS. The cells were then fixed with 4% paraformaldehyde for 15 minutes at 37 °C. After fixation, the cells were permeabilized using 0.5% Triton-XlOO in PBS for 5 minutes and washed three times for 5 minutes with washingbuffer (1% donkey serum and 0.05% Triton-XlOO in PBS). Afterward, the cells were blocked with a blocking buffer (5% donkey serum and 0.2% Triton-X 100 in PBS) for 1 hour. Antibodies against various proteins, including Tom20 (Santa Cruz), PGCla (Abeam), 5HT1A (Abeam), cAMP (Bio- Techne) were added to the coverslips at a dilution of 1:200 in blocking buffer. The coverslips were then incubated overnight at 4°C and on the next day. washed three times for 5 minutes with the washing buffer and incubated with fluorophore-conjugated secondary antibodies specific to mouse or rabbit (dilution of 1 :500) in the dark at room temperature for 2 hours. Afterward, coverslips were washed three times (5 minutes each) with the washing buffer, the second wash included the addition of 1.43 pM DAPI (a fluorescent nuclear stain). Finally, the coverslips were mounted onto slides using DAKO mounting medium. Confocal immunofluorescence microscopy using Zeiss LSM700 with 63x / 1.4 oil objective visualized the above proteins and nucleus. Analysis was carried out using ImageJ.

[0128] JC1 imaging

[0129] hRGCs were seeded on MG-coated glass bottom (1.5 thickness) MatTek dishes at 40,000 cells per dish and maintained for 3 days. Cells were treated with WAY for the designated time durations. Cells w ere washed with iNS media and then incubated with 250 pl of JC1 media (1 : 100 in iNS) for 30 min in the incubator. The JC1 media was then removed, cells were washed again and 2ml of new iNS was added to the dish. The dish was then transferred to the live cell chamber (5% CO2, 37°C, Tokai Hit) and confocal z-stacks were acquired using Zeiss LSM700 with 63x / 1.4 oil objective. Analysis was carried out using ImageJ with sum projections of red and green channels. The red fluorescence from the tdTomato expressed by the hRGCs was much less intense than the JC1 red staining of mitochondria, thus the red fluorescence measured from the cytoplasm w as considered background and subtracted out of the measurements. For the green fluorescence, the background was measured from outside the cell. Red-to-Green ratios were calculated by dividing the red intensity by the green intensity. These values were then normalized to the control condition, hRGCWTor average DMSO ratio for each cell line.

[0130] Seahorse analysis

[0131] hRGCs were seeded in MG coated 96 well Seahorse plate at 250,000 cells per well and maintained for 2 days. 24h before measurements, media was exchanged with 100 pl iNS with 5 pM WAY or equivalent vehicle control H2O. The day before the experiment the sensor cartridge was hydrated by submerging it in 200 pl of sterile water and kept in a non-CCty 37 °C incubator overnight. The next day, the sterile water was replaced with pre-warmed XF calibrant buffer (Agilent) and the sensors were submerged again and incubated in the same conditions for 45-60minutes. Seahorse media was made by adding stock solutions to XF DMEM to have final concentrations of 21.25mM glucose, 0.36mM sodium pyruvate and 1.25 mM L-glutamine (Agilent), with pH adjusted to 7.38-7.42. Depending on the specific assay being conducted, solutions of 20 pM Oligomycin (Oligo), 20 pM FCCP, 2.5 pg / ml Rotenone plus 5 pM Antimycin A (Rot / AA) and / or 175 mM 2-deoxy -d-glucose (2-DG) were prepared in Seahorse media. In the Seahorse plate with hRGCs, the iNS media was carefully replaced with Seahorse media, by removing 60 pl of iNS from each well and adding 140 pl of Seahorse media. Then, 140 pl of the mixed media was pipetted out and an additional 140 pl of Seahorse media was added to each well to reach a final volume of 180 pl. Any empty wells were filled with 180 pl of Seahorse media. The plate was placed in Incucyte S3 (Sartorius) and one image of each well was taken for cell area normalization using brightfield and red fluorescence (tdTomato) channels. After that, the hRGC Seahorse plate was transferred to a non-CCh, 37°C incubator for at least 45 minutes. The appropriate reagents were added to their respective ports in the cartridge, resulting in final concentrations in the wells as follows: for the ATP rate assay. 2.0 pM Oligo and 0.25 pg / ml Rotenone plus 0.5 pM Antimycin A; for the glycolytic rate assay, 0.25 pg / ml Rotenone plus 0.5 pM Antimycin A and 17.5 mM 2-DG; and for the Mito stress test, 2.0 pM Oligo, 2.0 pM FCCP (optimized concentration determined from FCCP titration) and 0.25 pg / ml Rotenone plus 0.5 pM Antimycin A. Once all the ports were loaded, the cartridge was placed in a non-C02, 37 °C incubator during which the experiment was set up in the WAVE software (Agilent). Next, the cartridge was inserted into the XFe96 analyzer (Agilent) for calibration. After successful calibrations, the hRGC Seahorse plate was placed in the machine and the selected assay (ATP rate assay, glycolytic rate assay, or Mito stress test) was run. Cell area from Incucyte images was measured using Image J and extrapolated for the total cell area in each well for normalization. Finally, the assay results were exported to the appropriate Excel macro using Seahorse Wave Desktop software (Agilent) for analysis.

[0132] Multi-electrode Array (MEA)

[0133] Human stem cell differentiated RGCs (hRGCs) were seeded at 500,000 cells per well of an MEA plate (CytoView MEA 24- White) and cultured for 11 days with new iNS media every other day. On day 11, the media was changed, and the plate was placed into the MEA machine (Maestro edge 384-channel electrode-based assay system from Axion Biosystems) with temperature and CO2 control (37°C, 5% CO2). After 15min of equilibration time a 3 minute recording was taking at every 3 hours for 24 hours. After the 24 hour reading, WAY was carefully added to the wells and mixed gently to give a final concentration of 5 pM. Three-minute recordingswere again taken every 3 hours for 24 hours. Quantification was done by taking the average mean firing rate of each well (from the 16 sensors) for each condition. All timepoints (n = 9) before WAY treatment were averaged to give the control, while all timepoints (n = 9) after the treatment were averaged for WAY.

[0134] Lentiviral (LV) transductions

[0135] Imaging: Approximately 250,000 hRGCs were seeded into a MatTek dish and cultured for 3 days. LV was added at multiplicity of infection (MOI = number of virus particles / number of cells) of 2 for EFla-Cas9 and 8 for EFla-GFP along with polybrene (2 pg / ml). The dish was centrifuged at 800G for 60min and then placed into the incubator (37°C, 5%CO2). Live imaging was done 9 days after transduction.

[0136] Quantitative PCR (qPCR): Approximately 50,000 hRGCs were seeded into 96 well plate and cultured for 3 days. LV was added at the indicated MOIs for Cas9, along with polybrene (2 pg / ml). The dish was centrifuged at 800G for 60min and then placed into the incubator (37°C, 5%CO2). Cell pellets were collected on day 4 after transduction and qPCR was performed for Cas9.

[0137] Western blot: Approximately 50,000 hRGCs were seeded into 96 well plate and cultured for 3 days. LV was added at MOI of 2 for Cas9 and 8 for TBK1, along with polybrene (2 pg / ml). The dish was centrifuged at 800G for 60min and then placed into the incubator (37°C, 5%CO2). Cell lysates were collected on day 8 after transduction and western blot for TBK1 was performed.

[0138] Caspase activity

[0139] hRGCs were plated at a density of 25,000 cells per well in a 96-well clear bottom blackwalled plate and cultured for 3 days. Following this, the cells were treated with WAY at specified time points. The caspase activity was measured using the ApoTox-Glo Triplex assay kit (Promega). 100 pl of Caspase-Gio® 3 / 7 reagent was added to each well and incubated for 30 minutes at room temperature. Luminescence was then measured to quantify caspase activity (referred to as Caspase measurement). The measured values were normalized to the control condition.

[0140] Animals

[0141] All animal studies were approved by the Institutional Animal Care and Use Committee, Indiana University School of Medicine and the "‘Use of Animals in Ophthalmic and Visual Research7’ guidelines of the Association for Research in Vision and Ophthalmology and ARRIVEguidelines were followed. Wild-type C57BL / 6J mice (male, 2-3 months of age) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and housed under standard conditions at the Laboratory7Animal Resource Centre, Indiana University' School of Medicine.

[0142] For ONC, all animals were distributed into 3 groups (n = 3 / set), naive, ONC, ONC+WAY. All mice are acclimatized for 1 week. The ONC+WAY mice group was pretreated with WAY (5mg / kg body weight (bw)) for 5 days daily intraperitoneally. Optic nerve crush (ONC) injury at day 0 was performed on ONC and ONC+WAY mice groups. ONC+WAY mice continued receiving daily WAY treatment post-ONC for varying days according to the experimental plan (5- 25 days post-ONC). After that, all the animals were sacrificed and the eye, optic nerve, kidney and liver were collected. The body weight of all the mice was measured at 5 days before ONC, the day of ONC and the day of sacrifice.

[0143] For microbead (MB) induced high IOP glaucoma, animals were distributed into 3 groups ( n = 3 / set), sham (PBS injection into anterior chamber), MB, MB+WAY, MB+WAY received WAY (5 mg / kg) treatment by IP daily 6 days prior to MB until the day animals are sacrificed for tissue collection (50 - 60 days post MB).

[0144] Optic Nerve CrushThe mice were placed under anesthesia using 2% isoflurane and 0.5% proparacaine hydrochloride ophthalmic drops were administered to the eyes. Ophthalmic ointment was applied to both eyes to prevent dryness of the corneas throughout the procedure. A small incision was made in the upper conjunctiva, allowing for gentle outward retraction of the eyeball using fine forceps. Another pair of forceps w as used to expose the optic nerve by opening the connective tissues covering it. The microscope w as adjusted to focus on the optic nerve and a specific pair of forceps was employed to crush the optic nerve for a duration of 5 seconds, approximately 1 mm behind the eyeball. Following the ONC, the eyeball was carefully placed back into the eye socket and the mouse's head was released. Erythromycin ophthalmic ointment USP, 0.5% (Baush + Lomb, Laval, Canada) was given to prevent infection and lubricate the eye during recovery. After the procedure, the animals were closely monitored until they regained full consciousness.

[0145] Microbead induced high IOP glaucoma modelFor PBS (sham) and polysty rene microbead injections, mice pupils are dilated with a drop of phenylephrine hydrochloride ophthalmic solution (10%) (Akom, Lake Forest, Illinois) followed by local anesthesia with a drop of proparacaine (0.5%) (Bausch and Lomb, Bridgewater, NJ) under whole body anesthesia with isoflurane inhalation (1.5 - 2%) (Akom, Lake Forest, Illinois). Weplace the animal on their right side with their stomach facing out for left eye injection and vice versa. Using two forceps, first we grip the conjunctiva proximal to the globe, then with right hand using thumb, index, and middle finger we grip the 50 pm injection glass needle (World Precision Instruments, Saarasota, FL) to place on the cornea, just anterior to the limbus. Microbead injections are done using glass needle connected by tubing to a Hamilton syringe. We inject of 2 pL of 1 pm, and 2 pL of 6 pm beads (Polyscience, Inc.) followed by 1 pL of the viscoelastic solution Heal on (10 mg / ml sodium hyaluronate, Johnson & Johnson) injection which allows maximum retention of the beads after glass needle is extracted. Injection is done at a rate of 5 pL / min controlled by the experimenter. After bead injection, erythromycin ophthalmic ointment (0.5%) (Bausch and Lomb, Bridgewater, NJ) is applied on eye and the animal is allowed to recover for 24-48 hours prior to resumption of IOP measurements.

[0146] Retinal flat mounting and RGC count

[0147] Retinal tissues are collected and immersed in ice-cold PBS before being fixed with a 4% paraformaldehyde solution. Then tissues were incubated overnight in guinea pig RBMS antibody at a dilution of 1:200. To detect the primary antibody, an Alexa Fluor 647-labeled goat anti-guinea pig secondary antibody was utilized. Nuclei were stained with DAPI during the final wash step. Eye cups were cut in a flow er shape and flat mounted over the slide using 0.45 pm black MCE membrane. Coverslips were then placed over the slides, securing them with Aqua- Poly / Mount mounting medium. The visualization of the samples was performed using confocal immunofluorescence microscopy, using a Zeiss LSM700 microscope with a 40X / 1.3NA oil objective. A total of eight 40X z-stacked images were captured for each retinal flat mount, with images taken from four quadrants located in both the central and peripheral regions of the retina. Manual quantification of RBPMS -positive retinal ganglion cells (RGCs) was carried out by analyzing the maximum projection of each image using imageJ software.

[0148] Tissue fixation and block-making for cryosection

[0149] The eye cup and optic nerve were fixed in 4% paraformaldehyde in 0.1M phosphate buffer (PB. NaHPCL and Na2HPO4 dissolved in MilliQ H2O. pH7.4) for 6h at RT. After fixation, the tissues were transferred to a 5% sucrose solution in phosphate buffer for 30 minutes. Subsequently, they underwent a series of sucrose w ashes, with increasing concentrations of sucrose (10%, 15% and 20%) in 0.1M PBS. To prepare the tissues for cryosection, they were incubated overnight at 4°C in a solution consisting of 20% sucrose and optimum cutting temperature compound (OCT compound) in a 2: 1 ratio. The following day, tissues were aligned in Tissue-Tekcryo molds and frozen in isopentane cooled by liquid N2. Frozen tissue sections were then obtained using a Leica CM1850 cryostat with a thickness of 12pm for the retina and 14pm for the optic nerve. The sections were collected on microscopy slides. After drying, the slides were stored at - 80°C until further use. When needed, the slides were taken out from the -80°C storage and warmed using a 37°C incubator. The sections were encircled using an ImmEdge pen (Vector Laboratories).

[0150] Eye cup cry osection preparation and staining

[0151] Sections were rehydrated with PBS (in mM: 137 NaCl, 2.7 KC1, 8 Na2HPO4, 2 KH2PO4) containing 0.1% Triton X-100 (PBST) and blocked in a 5% normal goat serum diluted in PBST solution for 30min at RT. After the blocking, the sections were washed three times with PBST for 5 minutes each and incubated with the primary antibody (diluted 1:200) at 4°C for 14- 16 hours. Subsequently, the sections were washed again with PBST (3x5 minutes). Antibodies against 5HT1A was used. Following the washes, the sections were incubated with fluorophore- conjugated anti-mouse or anti-rabbit secondary antibodies (1:500 dilution) and DAPI dye (1: 1000 dilution) in PBST for 1 hour at RT. After this incubation, the sections underwent an additional three washes with PBST for 5 minutes each. To mount the sections, a drop of mounting medium or DAKO was added to a coverslip and the coverslip was carefully placed over the sections. To secure the coverslip, nail polish was used to seal the edges. The sections were allowed to dry at RT for 30 minutes or overnight before they were ready for imaging.

[0152] Optic nerve cryosection preparation and staining

[0153] Sections were first washed with Tris-buffered saline (TBS. pH 7.4) for 5 minutes. Then, they were pre-incubated in 100% methanol for 10 minutes. Subsequently, the sections were blocked using a solution of 10% donkey serum in TBS. The primary antibody against growth - associated protein 43 (GAP43) was applied to the sections (1: 1,000; from sheep). See Benowitz et al., ‘‘Anatomical distribution of the growth-associated protein GAP-43 / B-50 in the adult rat brain,” (1988) J Neurosci 8(1): pp. 339-352; Xie et al.. “Monocyte-derived SDF1 supports optic nerve regeneration and alters retinal ganglion cells' response to Pten deletion,” (2022) Proc Nat Acad Sci USA 119(15): e2113751119; Peterson et al., “Retinal Ganglion Cell Axon Regeneration Requires Complement and Myeloid Cell Activity within the Optic Nerve,” (2021) J Neurosci 41(41): pp. 8508-8531. The antibody was dissolved in solution A [containing TBS2T (300mM NaCl and 0.1% Tween20, pH7.4) with 2% BSA and 10% donkey serum] and incubated overnight at 4°C. On the next day, the sections were rinsed first in TBS with Tween 20 (TBS2T, containing 300 mM NaCl and 0.1% Tween 20, pH 7.4) for 1 hour at 4°C, followed by a rinse in solution A for 1 hour at room temperature. Lastly, the sections were washed again in TBS2T for 1 hour atroom temperature. After washing, sections were incubated with an AlexaFluor-488-conjugated donkey secondary antibody to sheep IgG (1 :500) for 2hr at room temperature, washed in TBS2T (2X5 min, room temperature) and TBS (5min, room temperature). To mount the sections, one drop of DAKO was added to each section and a cover slip was placed over the section. The coverslip was sealed with nail polish and the sections were allowed to dry’ overnight at room temperature before imaging. For quantification, the number of GAP43-positive axons crossing at 0.5 mm from the crush site is counted following published methods by Larry’ Benowitz lab22, 23. In brief, the width of each section w as measured and four sections per nerve were analyzed. The cross-sectional width of the optic nerve was measured at the point at which the counts were taken and was used to calculate the number of axons per millimeter of nerve width. The number of axons per millimeter can then be averaged over the four 14pm sections. Then, the total number of axons present at a distance ‘ ’ (Laa) having a radius ‘r’ can be measured by summing over all sections having thickness t (14|im) (Zaa = 7rr x [ - — - \ / t). See Leon et al.. ' Lens injury stimulates axon regeneration in the mature rat optic nerve,” (2000) J Neurosci 20(12): pp. 4615-4626.

[0154] Visual cliff test

[0155] Mice were pretreated with WAY (5mg / kg) for 5 days daily. After that, unilateral ONC injury' was performed, they were treated with WAY daily for the next 13 days post-ONC and visual cliff tests were done on Day 13. Mice were tested in a visual cliff apparatus (square arena approximately 20cm tall with 62cm sides and a transparent bottom / floor). Half of this arena was placed on a table approximately’ 2 feet tall and the other half was suspended in air to simulate a cliff. A vinyl sheet with a black and w hite checkerboard pattern w as also placed under the apparatus and allowed to drape over the table to enhance the illusion of a steep drop-off. Testing took place under low light conditions (approximately 20 lux), with a white noise machine active to reduce sound disturbances from adjacent rooms. A circular metal platform approximately 2cm tall and 10cm w’ide w’as placed in the center of the apparatus so that half of it was on the ‘cliff side and the other half on the ‘safe’ side. Animals w ere placed on this platform and allowed 120 seconds to step down into the arena. Each animal was tested 5 times with 5 minutes of rest in between trials. Latency to step down and whether the animal made the correct choice (i.e., stepped down into the ‘safe’ zone) were analyzed. If an animal did not step dow n within 120 seconds, it was recorded as “did not choose” and the trial was terminated.

[0156] Optokinetic response test

[0157] After ONC injury (unilateral left crush) on 5-day WAY (5mg / kg) pretreated mice, they were treated with WAY daily for additional 21 days. Optokinetic response (OKR) measurements were done on Day 6 and 21 post ONC. In this test, the mouse is surrounded by 4 computer screens with moving (12deg / sec clockwise or counterclockwise) sinusoidal gratings of either increasing frequency or decreasing contrast separately. A mouse with healthy vision is expected to respond (move head) towards the moving stimulus with higher spatial frequency (high visual acuity (VA)) or decreasing contrast (high contrast sensitivity (CS)) respectively. VA was measured by scoring head movements at increasing spatial frequency but at fixed speed and contrast. CS was measured by scoring head movements at decreasing contrast but at a fixed speed and spatial frequency. CS is presented here as Michelson contrast (max-min / max+min); max, min represents the maximum or minimum contrast that the mouse responded. See Pruskey et al. 2004; Prusky et al., “Enhancement of vision by monocular deprivation in adult mice,” (2006) J Neurosci 26(45): pp. 11554-11561. Quantification of VA and CS in response to both clockwise and counterclockwise along with the combined rotation of the sinusoidal gratings was recorded.

[0158] Histology

[0159] The kidney and liver tissues from the naive, ONC, ONC+WAY mice were collected and fixed in 10% formaldehyde and processed for paraffin sectioning. Paraffin-embedded sections were stained with hematoxylin and eosin to study the histological changes under a light microscope.

[0160] Mass spectroscopic analysis

[0161] To test if WAY crosses the blood-retinal barrier, the inventors performed intraperitoneal (IP) injection at 30mg / kg bw dose (100 pl PBS) to C57BL / 6J mice. Mice were sacrificed and blood and eye tissue were collected at 4hr and 24hr after injection. 75uL of blood was collected into heparinized microcapillary tubes and spun at 5000rpm for 5 minutes. After that, plasma was transferred to a tube and frozen at -80°C. The retina was collected from dissected eye tissue, quick frozen in liquid nitrogen and stored at -80°C. Mass spectrometry was performed from collected serum and retinal tissue.

[0162] Statistics

[0163] For differentiated hRGCs, each treatment and corresponding time points are performed on cells grown on individual wells, hence each group is independent from others. Seven to ten independent biological repeats on hRGCs can be performed for western blots, mitochondrial mass measurements by flow cytometry, cellular apoptosis measurements, qPCR, and seahorse analysis.Thirty to forty hRGC neurons for each condition can be analyzed for high-resolution confocal images and transmission electron microscopy (TEM) acquired over 4-6 biological repeats for each condition. Statistical tests can be done by student’s t-test (for two samples) or ANOVA (three or more conditions) with Tukey's post-hoc tests to adjust for multiple testing among all groups or Dunnett’s tests for comparisons of multiple groups against a control. Nonparametric tests can be used if the data cannot be transformed to meet the t-test / ANOVA assumptions.

[0164] Power calculation for in vivo experiments: The effect size for RGC survival post ONC (FIG 4) or MB injection (FIG. 8) has been determined based on the data presented herein. Using these effect sizes with an a priori power analysis with Bonferroni correction assuming a rejection criterion of 0,05%, 80% power, and uniform variability, yields 8-10 mice per cohort. Both male and female animals can be used. Statistical comparisons can be made across and within sex cohorts. If no significant differences are found between sex cohorts, data from male and female mice can be pooled together. Animals can be randomized to treatments. Investigators can be masked to treatments in all experiments.

[0165] Further reference is made to the following experimental examples.

[0166] EXAMPLES

[0167] The following examples are provided for the purpose of illustrating various embodiments of the invention and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are provided only as examples, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims can occur to those skilled in the art.

[0168] EXAMPLE 1

[0169] High content screening identifies a neuroprotective compound.

[0170] Many past investigations to identify RGC protective molecules began with a known target pathway. Based on inventors’ findings that promoting mitochondrial biogenesis could be RGC protective the inventors took an unbiased high content screen (HCS) approach using hRGCs differentiated from the H7-ESC (embryonic stem cell, WiCell) reporter line to identify compound(s) that would activate mito-biogenesis. See Surma et al. 2023. Human stem cell reporter lines used here contain a BRN3B-P2A-tdTomato-P2A-Thyl.2 construct inserted into the endogenous RGC specific BRN3B locus by CRISPR / Cas9 gene-editing. Hence, successful RGC differentiation is judged by tdTomato expression and RGC purification is done by CD90 (Thy 1.2)conjugated microbeads and magnetic activated cell sorting (MACS) with 95-99% purity. RGCs differentiated from the method used here show remarkable similarity to human RGCs for geneexpression profiles, electrophysiology, cell morphology' and the ability' to integrate into the mice retina. See Sluch et al., “Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells,” (2017) Stem Cells Transl Med 6(11): pp. 1972-1986; Sluch et al., “Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line,” (2015) Sci Rep 5: P. 16595; Zhang et al., “Role of the Internal Limiting Membrane in Structural Engraftment and Topographic Spacing of Transplanted Human Stem Cell- Derived Retinal Ganglion Cells,” (2021) Stem Cell Reports 16(1): pp. 149-167.

[0171] EXAMPLE 2

[0172] Identification of hRGC neuroprotective compounds which promote mitochondrial biogenesis

[0173] To identify hRGC neuroprotective compounds through promoting mitochondrial biogenesis, the inventors have performed a small molecule screening using purified human stem cell differentiated RGCs (hRGCs) and LOP AC library (Sigma) which contains mostly FDA approved drugs. MitoTracker Deep Red (MTDR) live cell dye binds to the mitochondrial matrix protein through covalent bonding and hence once labelled it reflects the mitochondrial amount. However, MTDR entry into mitochondria depends on the mitochondrial membrane potential which may get affected by the drug treatments leading to erroneous mitochondrial mass measurements. To avoid this complexity, the inventors labeled mitochondria in hRGCs with the MTDR dye first and then treated cells with the drug library' in presence of the dye followed by flow cytometrybased measurements of the mitochondrial mass. Inhibition of mitophagy could also lead to increased mitochondrial mass, however mitophagy inhibition is toxic to hRGCs and causes mitochondrial membrane potential damage which can inhibit MTDR dye binding to mitochondria. See Das et al., “Programmed switch in the mitochondrial degradation pathways during human retinal ganglion cell differentiation from stem cells is critical for RGC survival,” (2020) Redox Biol 34: 101465; Georgakopoulos et al.. “The pharmacological regulation of cellular mitophagy.” (2017) Nat Chem Biol 13: pp. 136-146. Thus, compounds that can promote more MTDR binding to mitochondria are due to the biogenesis of healthy' mitochondria or due to improving membrane potential (health) of existing mitochondrial population.

[0174] Even though several compounds were found that increased mitochondrial mass in hRGCs (FIG. 2A). a secondary screen was performed on those compounds to validate mitochondrial biogenesis induction and discover if they induce pro-survival pathways in hRGCs.

[0175] FIG. 2A, B shows that the small molecule screen identifies compound for increasing mitochondrial biogenesis. (FIG. 2A) Drugs showing >40% change (outside of blue box). (FIG. 2B) Manual validation of WAY- 100635 maleate (WAY) treatment increases mitochondrial mass in H7-hRGCWT. Error bars are SEM. Unpaired student’s t-test, ****, p < 0.0001, n=3.

[0176] Surprisingly, the inventors found WAY- 100635 maleate, an antagonist of serotonin receptor 5-HT1A expressed by HTR1A gene robustly induced mitochondrial biogenesis. (FIG. 2A). This result was reproduced when tested manually (FIG. 2B).

[0177] WAY is a well-characterized antagonist of the 5-HT1 A receptor that is coupled to the inhi bi toiy Gi / o proteins (GPCR)25'33. RGCs are known to secrete serotonin, a 5-HT1A agonist34, which potentially binds to its own 5-HT1 A receptor and activates the coupled Gi / o proteins leading to the suppression of its target adenylyl cyclase (AC) and maintenance a low cAMP levels35, 36. On the contrary, 8-OH-DPAT (DP AT), an agonist of 5-HT1A, is expected to overcome the effects of WAY if a sufficient concentration of this compound is administered (FIG. 3A). In agreement, we see a transient increase in cytosolic cAMP level, particularly in the perinuclear region by WAY (FIG. 3B, C). Perinuclear enrichment of cAMP has been shown to be associated with neuroprotection37'39. This transient activation is the hallmark for GPCR signaling (inhibition arrow from cAMP to AC activation, FIG. 3A) which promotes cellular homeostasis without constitutive activations40'42. WAY mediated cAMP response has been suppressed by DP AT (FIG. 3D, E), confirming that WAY’S effect is mediated by 5-HT1A.

[0178] To test if the transient activation of cAMP leads to RGC protection, the inventors measured hRGC apoptosis by measuring Caspase 3 / 7 activity7using ApoTox-Glo Triplex assay (Promega). The inventors discovered that WAY reduced cellular apoptosis for both the Wt and OPTNE50KhRGCs (FIG. 3F, G). Of note, under the culture conditions, even Wt hRGCs accumulate mitochondrial damage over time with increased apoptosis43 44Thus, reduced apoptosis for both OPTNE50Kand Wt hRGCs by WAY at the basal level suggests its neuroprotective effect in the presence or absence of any genetic disorder. On the contrary, DP AT has no effect on Wt but increased apoptosis for the OPTNE50KhRGCs (FIG. 3H, I).

[0179] The competitive reaction to a GPCR target is a standard method for showing target specificity which we have shown here for WAY and DP AT in hRGCs and was reported for other systems as well45. In addition, WAY binding to 5-HT1 A receptor is well characterized in-vivo32as well as in-vitro46. There are also few reports showing WAY could be an agonist to the dopamine receptor D4 (DRD4)47, 48and DP AT as agonist to the 5-HT749, 50. Based on cAMP modulation by WAY and DP AT, this is unlikely that the above targets are engaged in hRGCs. Since WAY agonist to Gi / o coupled D435would lower cAMP level and DP AT agonist to Gs coupled 5-HT749-50wouldincrease, opposite to what we have observed (FIG. 3B-E). Therefore, D4 and 5-HT7 receptors either have minimal impact or are not engaged in hRGCs. These results suggest that WAY is likely to provide direct RGC protection by transient activation of cAMP.

[0180] FIG. 3A - I depict that WAY transiently activates cyclic AMP (cAMP) level and promotes hRGC survival. (FIG. 3A) Illustration for mechanism of action by antagonist WAY or agonist DPAT through 5-HT1A GPCR. (FIG. 3B) Representative max-projections and central z- plane of confocal IF images of H7-hRGCWTagainst cAMP (anti-cAMP antibody, R&D systems) and DAPI after indicated timepoints of 5 pM WAY treatments. Insets show perinuclear cAMP that increases with WAY treatment. (FIG. 3C) Presents quantification of cAMP intensity per cell area from sum projections, normalized to control, n = 15-18 cells per condition. (FIG. 3D) Representative max projections of H7-hRGCWIagainst cAMP and DAPI after Ih pretreatment with WAY and then 6h WAY with indicated 8-OH-DPAT concentrations, n = 5 images containing an average of 80-150 cells per image. (FIG. 3E) Quantification of cAMP intensity per area from sum projections, normalized to control shows WAY pretreatment increases cAMP level but addition of DPAT suppresses it. DMSO treatment used for control as DPAT solubilizes in DMSO. (FIG. 3F-I) Caspase-3 / 7 activity7for apoptosis was measured using ApoToxGlo-Triplex assay kit in hRGCs normalized to the DMSO control, n=3. Error bars are SEM. One-way ANOVA with Dunnett’s, * p < 0.05, ** p < 0.01. *** p < 0.001, **** p < 0.0001.

[0181] EXAMPLE 3

[0182] WAY as therapeutic drug for optic neuropathy

[0183] Optic nen e crush (PNC) in mouse as a robust optic nen e degeneration model

[0184] The data on stem cell differentiated hRGCs show that WAY works as a RGC protective compound potentially through restoring mitochondrial homeostasis by inhibiting 5-HT1A GPCR. Hence, having discovered here a novel RGC protective mechanism based on cutting-edge cell biology7and screening approaches, the inventors sought to determine if WAY is effective in vivo.

[0185] To achieve these goals, needed was an optic nerve degeneration model that gives robust RGC degeneration over a relatively short duration and associates with disrupted mitochondrial metabolism, so that inventors can measure neuroprotection effects with statistical robustness and mechanistic details. ONC injury provides 50-60% RGC loss within a week which was also observed here (see FIG. 4C, D). See Sanchez-Migallon et al., “Apoptotic Retinal Ganglion Cell Death After Optic Nerve Transection or Crush in Mice: Delayed RGC Loss With BDNF or a Caspase 3 Inhibitor / ’ (2016) Investigative Ophthalmology & Visual Science 57(1): pp. 81-93; Cammalleri, '‘Optic nerve crush as a model of retinal ganglion cell degeneration,” (2022) Annals of Eye Science 7: p. 17; MacNair et al., “Tumor necrosis factor alpha has an early protective effecton retinal ganglion cells after optic nerve crush7(2014) J Neuro inflammation 11(1): p. 194. Since reduced mitochondrial function in retina has been strongly associated with ONC injury in mice, the inventors reasoned that restoring mitochondrial homoeostasis by WAY treatment should provide RGC protection against ONC injury. See Kim et al.. “Drug-induced nephrotoxicity and its biomarkers,” (2012) Biomol Ther (Seoul) 20(3): pp. 268-272. Nigam et al., “Handling of Drugs, Metabolites, and Uremic Toxins by Kidney Proximal Tubule Drug Transporters,’7(2015) Clin J Am Soc Nephrol. 10(1 1): pp. 2039-2049. The inventors have used ONC not as a glaucoma model per se but a robust RGC degeneration model that can be used to test the above neuroprotection strategy. However, in glaucoma axonal degeneration precedes cell body loss, hence the discovery of WAY mediated RGC protection in ONC. which is an acute axonal injury model, is likely to be efficacious in glaucoma as well as for other mitochondrial optic neuropathies such as LHON and DOA. See Howell et al., “Intrinsic axonal degeneration pathways are critical for glaucomatous damage,” (2013) Exp Neurol. 246: pp. 54-61.

[0186] WAY is neuroprotective in vivo

[0187] The inventors tested WAY for neuroprotective activity in vivo using optic nerve crush RGC injury model (FIG. 4 A).

[0188] FIG. 4B - D shows that WAY promotes neuroprotection in mice against ONC injury. (FIG. 4A) Schematic illustration of the daily WAY (5mg / kg) IP injection in C57BL / 6J mouse, unilateral ONC done on DO. (FIG. 4B) Representative tiled confocal image (10X / 0.3NA) of mouse retinal flat mount with immunohistochemistry (IHC) against RGC specific RBPMS protein. Dotted circle and white boxes on the circles represent central and peripheral retinal regions for image acquisitions and quantifications. (FIG. 4C) Representative high-resolution confocal images (40X / 1.3NA) of RGCs from indicated retinas from peripheral and central white boxes (FIG. 4B). (FIG. 4D) Quantification of RGC numbers / mm2of retina under indicated conditions. Error bars are SEM. Two-way ANOVA with Tukey’s, ****, p < 0.0001, not significant (ns), n=16-24 images from 3 mice for each group.

[0189] To test neuroprotective effects, mice were treated with WAY at a low dose of 5mg / kg body weight 5 days pre and post ONC by IP injection and retinas were collected on day 6 post ONC for IHC. RGC numbers were counted both at the peripheral and central retina as marked by white boxes on retinal flat mount IHC image (FIG. 4B). The inventors noted drastic RGC loss in the ONC retina. Remarkably, the inventors discovered that WAY provides robust RGC protection against ONC injury at early (6 dpc) and later time points (14 dpc) establishing its therapeutic potential (FIG. 4C, D). As in glaucoma, symptoms often onset in one eye but eventually startaffecting the other eye, these findings that WAY provides neuroprotection to both the ONC, and contralateral eyes is of high clinical relevance.

[0190] EXAMPLE 4

[0191] WAY promotes ganglion cell axon regeneration in vivo.

[0192] The inventors next tested if WAY also promotes axon regeneration after ONC. RGC axon regeneration was measured in the optic nerve by IHC against GAP43 protein on the transverse cryosections followed by confocal imaging as developed by others. See Cheng et al., “Transcription factor network analysis identifies REST / NRSF as an intrinsic regulator of CNS regeneration in mice." (2022) Nature Communications 13(1): p. 4418. GAP43 is a critical player for axonal growth and plasticity discovered by Dr. Benowitz and his colleagues who also developed a robust antibody against GAP43 which has been widely used and shared with the inventors. See Id.; Benowitz et al., “Specific changes in rapidly transported proteins during regeneration of the goldfish optic nerve,’' (1981) J Neuroscience 1(3): pp. 300-307; Benowitz et al., “Transported Proteins in the Regenerating Optic Nerve: Regulation by Interactions with the Optic Tectum,” (1983) Science 222(4620): pp. 185-188; Benowitz et al., “Increased transport of 44,000- to 49,000-dalton acidic proteins during regeneration of the goldfish optic nerve: a two- dimensional gel analysis,” (1983) J Neuroscience 3(11): pp. 2153-2163; Xie et al., “Monocyte- derived SDF1 supports optic nerve regeneration and alters retinal ganglion cells’ response to Pten deletion,” (2022) PN AS 119(15): e2113751119.

[0193] The inventors remarkably found axonal degeneration after the crush site and robust axon regeneration in the treated mouse 25 days after ONC (FIG. 4E, F).

[0194] FIG. 4E, F show that WAY promotes RGC axon regeneration. C57BL / 6J mice were treated with 5mg / kg WAY daily 5 days prior to ONC until 25 days post crush. Confocal IHC image (40x / 1.3NA, tiled) against GAP43 on transverse cr osection of optic nerve.

[0195] Thus, WAY not only protects RGC cell body in the retina but also promotes axon regeneration and maintenance long after acute optic nerve damage.

[0196] EXAMPLE 5

[0197] WAY protects vision in mice

[0198] The inventors next tested if WAY mediated RGC protection and axon regeneration leads to vision protection following ONC injury’ (unilateral left crush) using optokinetic response (OKR) measurements. See Prusky et al., “Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system,” (2004) Invest Ophthalmol Vis Sci. 45(12): pp. 4611-4616; Prusky et al., “Enhancement of Vision by Monocular Deprivation in Adult Mice,” (2006) J Neuroscience 26(45): pp. 11554-11561. Mice were treated with WAY (5mg / kg) daily for27 days, 5 days before ONC, the day of ONC until 21 days post ONC and OKR measurements done on Day 6 and 21 (FIG. 6A).

[0199] FIG. 5A-F shows that WAY maintains visual acuity (VA) in mice after ONC injury. (FIG. 5A) Schematic illustration of the WAY treatment, ONC and subsequent VA measurements. (FIG. 5B) VA measured by scoring head movements at increasing spatial frequency but at fixed speed and contrast. (FIG. 5C, E) Quantification of VA in response to both clockwise and counterclockwise rotation of the sinusoidal gratings. One-way ANOVA with Tukey’s correction. (FIG. 5D, F) VA in response to the individual clockwise or counterclockwise movement of the gratings. Error bars are SEM. Two-way ANOVA with Tukey’s correction, **p < 0.01, ***p < 0.001, ****p < 0.0001. n=4 mice / group

[0200] FIG. 6A-E shows that WAY maintains contrast sensitivity (CS) in mice after ONC injury. CS measurements done on the same mice by OKR as illustrated in FIG. 5A. (FIG. 6A) CS measured by scoring head movements at decreasing contrast but at fixed speed and spatial frequency. (FIG. 6B. D) CS presented here as Michelson contrast (max-min / max+min); max, min represent maximum or minimum contrast that the mouse responded to (Prusky et al. 2006). Data shown combined response to both clockwise and counterclockwise rotations. One-way ANOVA with Tukey’s correction. (FIG. 6C, E) CS data in response to the individual clockwise or counterclockwise movement of the gratings. Error bars are SEM. Two-way ANOVA with Tukey's correction. **p < 0.01. ***p < 0.001, ****p < 0.0001. n=4 mice / group.

[0201] In both VA and CS tests, the mouse is surrounded by 4 computer screens with moving (12deg / sec clockwise or counterclockwise) sinusoidal gratings of either increasing frequency or decreasing contrast separately. A mouse with healthy vision is expected to respond (move head) towards the moving stimulus with higher spatial frequency (high visual acuity (VA)) or decreasing contrast (high contrast sensitivity (CS)) as shown in FIG. 5B and FIG. 6 A respectively. The experiments were done in a masked manner. Remarkably, it was found that WAY treatment protected VA (FIG. 5C, E) and CS (FIG. 6B, D) during early (day 6) and late stage (21 days) of acute ONC injury when compared with the untreated ONC mice. Additionally, more loss of VA (FIG. 5D, F) and CS were observed (FIG.6C, E) in ONC mice when stimulus rotates clockwise, that is from left-to-right. This is likely due to the binocular vision of mice and the left eye being crushed i.e. they have limited capacity7to respond towards the moving gratings at higher frequency from left side. In agreement to the combined response, it was found that WAY treatment maintained both VA (FIG. 5D. F) and CS (FIG. 6C, E) to the level of naive mice for both clockwise and counterclockwise moving stimulus during the period of Day 6-21. Thus, findings of visionmaintenance at 21 days post ONC signifies the robustness and translational potential of WAY mediated neuroprotection.

[0202] In addition to OKR, inventors sought additional evidence that WAY protects vision after acute ONC injury and performed a visual cliff test 13 days post ONC while mice were under daily WAY treatment (5mg / kg) from day -5 to +13 where day 0 is the day of ONC. This experiment was done in a masked manner by the IUSM Behavioral Phenotyping Core as mice were provided with masked identity. For the cliff test, a circular metal platform approximately 2 cm tall and 10 cm wide was placed in the center of the apparatus so that half of it was on the ‘cliff side and the other half on the ‘safe' side as shown in FIG. 7 A.

[0203] FIG. 7A, B shows that WAY maintains visual depth perception after ONC. (FIG. 7A) Animals were tested in a visual cliff apparatus with approximately 2 feet tall cliff (deep) side with similar surface pattern under the glass for optical illusion. (FIG. 7B) Latency time to step down from the platform. Each data point is average from 5 measurements. One-way ANOVA with Dunnetf s, **, p < 0.005. n=3 mice / group.

[0204] Animals were placed on this platform and allowed 120 seconds to step down into the arena. Latency to step down and whether the animal made the correct choice (i.e., stepped down into the ‘safe’ zone) were analyzed. If an animal did not step down within 120 seconds, it was recorded as “did not choose” and the trial was terminated. While most of the animals made the right choice of descending into the safe zone, untreated ONC mice took much longer than the naive mice, but WAY treated ONC mice made the right decision as fast as the naive mice (FIG. 7B). Each animal was tested 5 times. This data makes sense as none of these animals is completely blind, so they are likely to make the right decision. But depth perception depends on the intact binocular vision, which is compromised with the left eye being crushed, leading to a delay in that decision making. See lehisa et al., “Factors affecting depth perception and comparison of depth perception measured by the three-rods test in monocular and binocular vision,” (2020) Heliyon 6(9): e04904. The normal speed of making the safe decision for WAY treated ONC mice suggests maintenance of binocular vision. Hence. WAY showed robust RGC protection, axon regeneration and vision maintenance long after acute ONC injury establishing its strong therapeutic potential.

[0205] EXAMPLE 6

[0206] WAY provides glaucoma neuroprotection

[0207] The inventors next tested if the WAY treatment provides protection to RGCs and vision in mouse under high IOP which is the primary risk factor for glaucoma. Inventors observed that daily WAY treatment protects RGC body loss 57 days after elevating IOP by MB injection (FIG. 8A-C). To test if WAY prevents vision loss, inventors measured visual acuity. They found that thevisual acuity of mice declined within the first week of unilateral MB injection, but it remained stable until 4 weeks. The visual acuity loss was significantly mitigated by WAY treatment during this time course (FIG. 8D). Of note, these data agrees with the published report that showed visual acuity declines in mice post unilateral IOP elevation within first 10 days and then stabilizes until 4-week due to an adaptive metabolic remodeling from fellow eye10.

[0208] FIG. 8 A-C shows WAY prevents RGC and visual acuity loss in glaucoma mouse. (FIG. 8A) Intraocular pressure (IOP) is induced by unilateral (left eye, inset) injection of 2 pL of 1 pm, and 2 pL of 6 pm beads (Polyscience, Inc.) (MB) or PBS (sham) into the anterior chamber of C57 mouse eye. IOP measured by rebound tonometer shows elevation and then reduction. The amplitude and time course of IOP changes with elevated IOP for about a month and then decline to the basal level agrees with published unilateral single bead injection data in C57 mouse24. WAY (5 mg / kg) was treated by IP daily 6 days prior to MB until the day post injection (dpi) of measurements, shows transient faster elevation and reduction of IOP. (FIG. 8B) Confocal IHC image of RBPMS positive RGCs, (FIG. 8C) RGC number counted 57 days after MB injection as in FIG. 4D. (FIG. 8D) Visual acuity measured by OptoMotry (Cerebral Mechanics) where mouse exposed to 4 adjoining LCD monitors with moving sinusoidal gratings (12deg / sec clockwise or counterclockwise) with randomly varying frequency but 100% contrast. Spatial frequency in cycles per degree (c / deg) measured for different condition with mouse identity masked. (FIG. 8C) n = 24 images from 3 mice / group. (FIG. 8D) 3 mice / group. Two-way ANOVA with Tukey’s, ****, p < 0.0001, ***, p < 0.001.

[0209] 5-HT1 A expresses in mouse and human RGCs

[0210] For WAY to directly act upon RGCs via 5-HT1A. this is important to find 5-HT1A expression in human and mouse RGCs. The inventors observed strong expression of 5HT1 A in the plasma membrane of hRGCs (FIG. 9A, B) and in the ganglion cell layer (GCL) of mouse retina (FIG. 9C). The protein concentration in mouse RGCs does not decline with axonal injury as detected by western blot on purified RGCs from mouse retina post ONC injury (FIG. 9D).

[0211] FIG. 9A-D shows robust 5-HT1 A expression in hRGCs and in mouse RGCs. (FIG. 9A) Confocal images show 5-HT1A membrane localization in H7-hRGCs and (FIG. 9C) shows on ganglion cell layer of mouse retinal cryosection. (FIG. 9B) Western blots detect 5-HT1 A protein expression in hRGCs, and (FIG. 9D) detects 5-HT1A expression in RGCs isolated from mouse retina.

[0212] The inventors next undertook a series of studies to demonstrate the mechanisms of 5- HT1 A inhibition mediated mitochondrial biogenesis activation in hRGCs.

[0213] EXAMPLE 7

[0214] WAY restores mitochondrial health and provides protection to glaucoma causing OPTNE50KhRGCs.

[0215] In the examples discussed above, the inventors disclose their HCS screening data showing that 5-HT1 A GPCR antagonist WAY promotes mitochondrial biogenesis and suppresses apoptosis in hRGCs. Of additional relevance, using confocal immunofluorescence (IF) against mitochondria specific Tom20 protein, further observed was a time dependent increase at first, and then a decrease in mitochondrial mass in hRGCs upon WAY treatment (FIG. 10A, B), indicating a transient biogenesis activation like cAMP activation observed under WAY treatment (FIG. 3B, C). This transient activation is often a result of negative-feedback regulation which is the hallmark of GPCR signaling that avoids constitutive activations. See Ruach et al.. “A negative feedback loop in the GPCR pathway underlies efficient coding of external stimuli,” (2022) Mol Syst Biol 18(9): e!0514; Pierce et al., “Seven-transmembrane receptors,” (2002) Nature Reviews Molecular Cell Biology' 3(9): pp. 639-650; Altieri et al., “Rethinking 5-HT1A receptors: emerging modes of inhibitory’ feedback of relevance to emotion-related behavior.” (2013) ACS Chem Neurosci 4(1): pp. 72-83.

[0216] PGCla is considered to be the master regulator of mitochondrial biogenesis, which, upon translocation to the nucleus, drives the expression of nuclear-encoded mitochondrial electron transport chain (ETC) proteins and mitochondrial transcription factor Tfam51. Previous reports suggest that cAMP can activate PGC la52, 53. Hence WAY mediated transient cAMP activation could lead to transient PGCla activation. To test this, inventors monitored the nuclear to cytoplasmic distribution of PGCla under WAY treatment by confocal IF and observed transient activation (FIG. 10C. D). Of note, both PGCla activation and mitochondrial mass peaked simultaneously at 6h and then declined indicating a strong correlation (FIG. 10B, D). We checked if this is due to WAY degradation in the media by maintaining drug concentration through repeated addition and saw a similar trend (data not shown). This is unlikely that increased mitochondrial mass under WAY treatment is due to mitophagy inhibition, since that would lead to the buildup of damaged mitochondria with reduced membrane potential hence reduced MTDR binding opposite to what we observed in FIG. 2A, B. These data suggest WAY transiently activates mitochondrial biogenesis to promote organelle health.

[0217] FIG. 10A - D depicts that WAY transiently activates mitochondrial biogenesis in hRGCs. (FIG. 10A) Representative confocal images (63X / 1.4NA) of H7-hRGCvlwith Tom20, TdTomato, and DAPI after 5 pM WAY treatment at indicated timepoints. (FIG. 10B) Quantification of Tom20 intensity7per cell area from sum projections of confocal z-stacks, normalized to control, n = 19-30 cells per condition. (FIG. 10C) Representative max projectionsof confocal IF images of H7-hRGCWTagainst PGCla after indicated timepoints of 5 pM WAY treatments. (FIG. 10D) Quantification of PGCla nuclear to cytoplasm intensity ratio, from sum projections, normalized to control, n = 22-33 cells per condition. Error bars are SEM, One-way ANOVA with Dunnett’s, * p < 0.05. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0218] The inventors have recently shown that activation of mitochondrial biogenesis by targeting TBK1 restores mitochondrial homeostasis leading to neuroprotection of hRGCs containing glaucoma causing OPTNE50Kmutation. Since inventors observed that WAY transiently activates mito-biogenesis in the Wt hRGCs, we tested if this also occurs for OPTNE50KhRGCs and found indeed, WAY transiently increases mitochondrial mass as detected by confocal IF against Tom20 protein (FIG. 11 A, B). Interestingly, timepoint (6h) for the peak mitochondrial level also agrees with the Wt hRGCs (FIG. 10A, B) indicating a conserved mechanism of action. Inventors had shown activating mitochondrial biogenesis maintains OXPHOS activity but lowers the ATP production load on mitochondria leading to improved mitochondrial health in the OPTNE50KhRGCs54. They asked if WAY mediated transient biogenesis activation also improves mitochondrial health in the OPTNE50KhRGCs and measured by mitochondrial membrane potential sensitive live JC1 dye. JC1 dye fluoresces green (monomers) when bound to damaged (depolarized) and red (J aggregates) when bound to healthy (polarized) mitochondria55. Indeed, WAY treatment significantly increased JC1 labelled red-to-green mitochondria ratio (FIG. 11C, D) as an indication for restoration of mitochondrial health in the glaucomatous OPTNE50KhRGCs.

[0219] Depolarized mitochondria could lead to excitotoxicity. See Schinder et al., “Mitochondrial dysfunction is a primary event in glutamate neurotoxicity ,” (1996) J Neurosci 16(19): pp. 6125-6133. In agreement, the inventors observed OPTNE50KhRGCs show higher spontaneous firing frequency rate (FIG. HE, F) when measured by multi-electrode array (MEA), supporting prior patch-clamp analysis of OPTNE50KiPSC-hRGCs. See VanderWall et al., “Retinal Ganglion Cells With a Glaucoma OPTN(E50K) Mutation Exhibit Neurodegenerative Phenotypes when Derived from Three-Dimensional Retinal Organoids,” (2020) Stem Cell Reports 15(1): pp. 52-66. However, this excitotoxicity in OPTNE50KhRGCs was mitigated by WAY as an indication for neuroprotection (FIG. 1 IE, F).

[0220] FIG. 11A - F shows that WAY transiently activates mitochondrial biogenesis with neuroprotection effect to OPTNE50K. H7-hRGCs with OPTNE50Kmutation are treated with 5 pM WAY for indicated timepoints. (FIG. 11 A) Images show Mitochondria by confocal IF against Tom20. (FIG. 1 IB) Quantification of Tom20 intensity per cell area from sum projections of confocal z-stacks, normalized to control, n = 23-29 cells per condition. (FIG. 11C) Representative live cell confocal images of JC1 labelled mitochondria in H7-hRGCE50K. (FIG. HD) Quantificationof the red-to-green fluorescence intensity ratios from the sum projections of confocal z-stacks, normalized to the corresponding control values, n = 17-24 cells. (FIG. H E) Mean firing rate averaged from 16 sensors / well over multiple wells, n= 4-5. (FIG. 1 IF) Shown are action potential firing spikes from a single MEA sensor monitored over 3 minutes for respective conditions. (FIG. 1 IB) One-way ANOVA with Dunnett’s. (FIG. 1 ID) Unpaired student’s t-test. (FIG. HE) Two- way ANOVA with Tukey’s. * p < 0.05, ** p < 0.01, **** p < 0.0001.

[0221] This has been reported that transgenic OPTNE50Kmouse retina suffer from excess ATP production load with elevated OXPFIOS and oxidative stress56. To have direct evidence if hRGCs with OPTNE50Kmutation also produce more ATP through OXPHOS, we performed Mito Stress Test by Seahorse analyzer on these cells. Indeed, we observed high mitochondrial APT production hence, high OXPHOS in the OPTNE’0KhRGCs (FIG. 12A). Interestingly, WAY treatment maintained high mitoATP production in the OPTNE50KhRGCs (FIG. 12A) at the same time improved mitochondrial health (FIG. 11C. D). This suggests more healthy mitochondria available to meet the ATP demand lowering OXPHOS burden on individual mitochondrion. Even though WAY maintained high mitoATP production in OPTNE50KhRGCs which is measured from mitochondrial oxygen consumption rate (OCR), we observed WAY restored mitoOCR / glycoPER ratio close to the Wt hRGCs as detected by Seahorse glycolytic rate assay (FIG. 12B). This indicates a metabolic reprograming by WAY that regains energetic and biosynthetic balance between OXPHOS and glycolysis to combat glaucomatous OPTNE50Kstress.

[0222] FIG. 12A, B shows WAY restores OXPHOS and glycolysis balance in OPTNE50KhRGCs. hRGCs are treated with 5 pM WAY for 24 hr followed by seahorse analysis. (FIG. 12A) Seahorse Mito Stress test measures mitochondrial ATP production from change in oxygen consumption rate (OCR) under sequential inhibition of ETC proteins. (FIG. 12B) Glycolytic rate assay measures OCR, and extra cellular acidification rate (ECAR) under sequential inhibition of mitochondrial ETC proteins and glycolysis to calculate mitoOCR and glycolytic proton efflux rate (gly coPER) and their ratio. Measurements are normalized to cell area, n = 4 - 6. Two-w ay ANOVA with Tukey’s correction. * p < 0.05, ** p < 0.01. All error bars are SEM.The inventors have herein showed that WAY treatment transiently activates mitochondrial biogenesis with neuroprotection effects in the OPTNE50KhRGCs. They have also shown WAY treatment to hRGCs transiently activates cAMP which can activate mitobiogenesis master regulator PGCla as well as provide neuroprotection through mitochondria independent mechanisms. If WAY mediated RGC protection is through PGCla then suppressing PGCl activity would suppress the mitochondrial biogenesis and neuroprotection. Based on this rationale, they can first knock out (KO) PGCla in the OPTNE50KhRGCs (as done in FIG. 13A-C but forTBK1) and then measure mitochondrial mass, mitochondrial membrane potential, cellular apoptosis, excitotoxicity at different WAY (5 pM) exposure time points (th to 24h).

[0223] FIG. 13A-C depicts CRISPR mediated gene KO in hRGCs using lentivirus (LV). (FIG. 13A) Confocal image (63x / 1.4NA) of WT H7-hRGCs transduced with the LV containing viral vector with EFla-GFP cassette or no GFP vector (Control LV). (FIG. 13B) Cas9 mRNA level in H7-hRGCs 4 days after LV-EFla-Cas9 transduction after digestion of DNA. Error bars are SEM. One-way ANOVA with Dunnett’s, *, p < 0.05, ****, p < 0.0001, n=3. (FIG. 13C) TBK1 KO in iPSC-hRGCs with OPTNE50Kmutation by LV-U6-gRNA (TBK1) and the LV-EFla-Cas9. 8 days post transduction.

[0224] If the inventors see WAY can promote RGC protective mechanisms in the absence of PGC1 a, they can study if this is through transiently activating cAMP-responsive element binding protein (CREB) transcription factor57. cAMP can directly activate CREB whose transient activation is known to be neuroprotective57.

[0225] WAY is a robust antagonist of serotonin receptor 5-HT1 A and their binding efficiency has been established both biochemically and in vivo. See for example. Fletcher et al., “Electrophysiological, biochemical, neurohormonal and behavioural studies with WAY-100635, a potent, selective and silent 5-HT1A receptor antagonist / ’ (1996) Behav Brain Res 73(1-2): pp. 337-353; Kolan et al., “Growth-inhibition of cell lines derived from B cell lymphomas through antagonism of serotonin receptor signaling,” (2019) Sci Rep 9(1): p. 4276; Hunt et al., “MDMA- induced c-Fos expression in oxytocin-containing neurons is blocked by pretreatment with the 5- HT-1A receptor antagonist WAY 100635.” (2011) Brain Res Bull 86(1-2): pp. 65-73: Hume et al., “Evaluation of [O-methyl-3H]WAY-l 00635 as an in vivo radioligand for 5-HT1A receptors in rat brain,” (1994) Eur J Pharmacol. 271 (2-3): pp. 515-523. As such, it has been show n in rat that WAY binds to 5-HT1 A receptor in brain with >100-fold specificity than other receptors with very strong affinity (IC50 1.35 nM). See Fletcher et al. 1996. WAY is well tolerated in humans, a radiolabeled form of WAY has been used in multiple clinical trials to map the serotonergic system by positron emission tomography (PET) for patients undergoing serotonin receptor targeted treatments (NCT00603018, NCT02810717).

[0226] All of these studies strongly support WAY mediated 5-HT1 A inhibition as an attractive target to pursue for the development of a clinically viable and safe strategy for neuroprotection under inherited genetic disorder or optic nerve injury conditions.

[0227] Further experiments are contemplated as outlined below to resolve the mechanism of action of WAY in human stem cell derived RGCs.

[0228] EXAMPLE 8

[0229] WAY crosses blood-retinal barrier

[0230] Since the inventors observed robust RGC protection against ONC injury by WAY via intraperitoneal injection, they then sought to determine if the effect is direct or indirect. To have a direct effect, WAY needs to cross the blood-retinal barrier and present in the retina. WAY can cross the blood-brain barrier following IP injection in mice. See Markowitz et al., “Drug-induced glomerular disease: direct cellular injury,” (2015) Clin J Am Soc Nephrol 10(7): pp. 1291-1299. To test if WAY crosses the blood-retinal barrier the inventors tested IP injection at 30mg / kg body weight dose (100 pl PBS) to C57BL / 6J mice followed by plasma and retina collection at 4hr and 24hr for detection by mass spectrometry. A significant amount of WAY was measured in the retina and plasma within 4h after injection (FIG. 14).

[0231] FIG. 14 shows that LC-MS chromatograms detect WAY in mice retina. Plasma and retina isolated from mice 4h after IP injection. The blue filled peak is the analyte of interest. WAY retention time is 5.5 minutes. Representative chromatogram for treated retinal tissue is from 4 independent experiments.

[0232] WAY concentration in retina was maximum after 4h (2006.41+ / - 263.75 ng / gm) and reduced (17.05+ / -3.03 ng / gm) 24h after the I.P injection at 30mg / kg dose. Thus, to maintain a steady retinal concentration, the inventors used once a daily IP injection, however, since they observed robust RGC protection against ONC at a much lower dose, 5mg / kg body weight was chosen. This suggests WAY could be efficacious even at a much lower dose and / or capable of maintaining neuroprotective effect with intermittent treatments which can be further tested.

[0233] EXAMPLE 9

[0234] WAY causes no toxicity in mice

[0235] The detection of any systemic toxicity of a drug candidate is always a clinical concern. To evaluate systemic toxicity, the inventors treated mice daily with WAY (5mg / kg) for 30 days and performed hematoxylin and eosin (H&E) staining on kidney and liver sections. Drug induced kidney toxicity often leads to different glomerular lesions58. However, the inventors did not see any structural damage or lesions in the glomerulus in WAY treated mice compared to the control (FIG. 22A).

[0236] FIG. 15 A, B shows that WAY does not show systemic toxicity in mice. H&E-stained histological images (40x / 0.6NA) of (FIG. 15A) kidney tissue and (FIG. 15B) liver tissue sections, from naive or WAY treated (5 mg / kg body weight) C57BL / 6J mice by IP injection once daily for 30 days.

[0237] For drug induced liver toxicity, necroinflammatory patterns are found around the central hepatic vein. See Kleiner, Recent Advances in the Histopathology of Drug-Induced LiverInjury,’7(2018) Surg Pathol Clin 11(2): pp. 297-311; Kleiner et al., “Drug-induced Liver Injurs- : The Hepatic Pathologist's Approach,” (2017) Gastroenterol Clin North Am 46(2): pp. 273-296. However, no noticeable necroinflammatory patterns were observed in WAY treated mice (FIG. 22B). In addition, no sign of stress, behavioral change, or body weight loss were observed. On the contrary, the inventors observed significant vision maintenance 21 days after ONC injurs’ (see above) which suggests high safety and efficacy of WAY. Furthermore, radio isotope labeled WAY is already in use for multiple serotonin receptor targeted clinical trials (NCT00603018, NCT02810717) for mapping the interaction of drug of interest with serotonin receptors in the brain. Data presented here on the differentiated hRGCs and on animal model provides a unique situation where a mechanistic understanding of restoring mitochondrial homeostasis successfully translated to vision protection. By performing above experiments inventors can resolve if 1) WAY mediated RGC protection in mice occurs through restoring mitochondrial homeostasis; and 2) optimum dose and frequency required for prolonged neuroprotection as is desired for any small molecule therapy. Further experiments to resolve mechanism of action of WAY in vivo are described below.

[0238] EXAMPLE 10

[0239] Resolve molecular mechanisms for WAY-mediated neuroprotection in human stem cell derived RGCs

[0240] Identify the specific Gi / o subty pes and adenylyl cyclase (s) (ACs) involved in WAY- mediated RGC protection

[0241] The inventors intend to identify 5-HT1 A s cognate G protein and its target AC involved in WAY response as to resolve their direct effect on cAMP activation and RGC protection, and if in the absence of corresponding G protein and AC, WAY loses its beneficial effects.

[0242] The Gi / o family’ includes Gai 1. Gai2. Gai3, and Gao proteins which have been reported to couple with 5-HT1A receptor36. The activation of any of these G proteins could be suppressed by the antagonist (WAY) binding to 5-HT1 A GPCR. Loss of specific inhibitory G protein involved in WAY signaling can disrupt the transient cAMP activation. Inventors can test this prediction by knocking out (KO) individual Gail, Gai2, Gai3, and GaO protein in hRGCs then measure cAMP level to compare between WT and KO in the absence and presence of WAY. Inventors can perform CRISPR / Cas9 based knockout in hRGCs by LV-gRNA as shown for TBK1 knockout for feasibility’ (FIG. 13).

[0243] Upon detection of involved G protein, they can measure if the loss of this G protein abolishes WAY’S neuroprotective responses in hRGCs such as transient activation of mitochondrial biogenesis and suppression of cellular apoptosis.

[0244] Identify ACs involved

[0245] There are nine transmembrane ACs (tmACs) and one soluble AC (sAC) in mammals59, that may be involved in WAY -mediated cAMP modulation. This is important to identify the specific AC involved as it has been reported that sAC activation can promote RGC survival and axon growth39. Active sAC can also directly translocate to mitochondria and maintain organelle health independent of cytosolic mechanisms60’61. To identify which type is involved, we can treat hRGCs with pan-tmAC inhibitor dDADO62and two sAC specific inhibitor KH763and 2HE63in 1 - 10 mM range and measure if this blocks WAY mediated transient activation of cAMP level as measured in FIG. 3B-E. These AC inhibitors are commercially available from Sigma-Aldrich and are well characterized for their inhibition effect in cultured cells with above concentration range64. Once we identify the involved class of ACs, we can next inhibit its activity and test if that suppresses WAY effect on cellular apoptosis and mito-biogenesis. We can recapitulate the pharmacological inhibition effect by CRISPR / Cas9 based gene knockout of corresponding ACs in hRGCs. Since, there is only one sAC, the knockout approach for this is straightforward. However, in case of tmACs are involved, we can knockout individually or in combination if more than one tmAC(s) partially blocks WAY mediated transient cAMP activation in hRGCs.

[0246] High quality pre-packaged LV-gRNA for human ACs and Gi / 0 family genes are available from ThermoFisher and can be used here. These proteins are widely studied with literature information on commercially available robust antibodies that we can use for above.

[0247] Mitochondria dependent and -independent mechanisms for WAY mediated RGC protection.

[0248] Inventors’ data shows WAY transiently activates mito-biogenesis and cAMP level with improved mitochondrial health and reduced hRGC apoptosis. They intend to resolve three aspects of WAY-mediated effects in hRGCs: (a) mechanisms for WAY-mediated transient mito- biogenesis activation, (b) They can test whether WAY maintains mitochondrial health independent of its effect on mito-biogenesis. (c) They can test if WAY exerts its neuroprotective effects in the absence of mitochondrial

[0249] Mechanisms underlying WAY-mediated activation of mitochondrial biogenesis in hRGCs:

[0250] Biogenesis master regulator PGClcc activation by cAMP could occur via few mechanisms. cAMP can bind and activate CaMKII (pThr286)65, 66through autophosphorylation. Active CaMKII either can phosphorylate and activate P38 MAPK (pThr!80)52’67, 68or Sirtl (pSer47)69. Next, active P38 MAPK can phosphorylate PGC1 a for activation, or active SIRT1 can deacetylate PGCla to block proteasomal degradation leading to activation70'72. Inventors can measure the activation of above proteins in hRGCs treated with WAY over a time course (Ih, 6h,18h, 24h) by western bloting against the native and phosphorylated (active) forms. Based on activation of above molecular players, they can suppress the involved protein such as by CRISPR knockout as in FIG. 13 and test if that blocks WAY effect on biogenesis. We can measure mitochondrial mass by confocal IF as in FIG. 10A, B and by mtDNA copy number relative to nuclear DNA by qPCR as we routinely measure54, 73. For Sirtl involvement, we can measure the acetylation status of PGC1 a following published method and antibody against acetylated-Lys (Cell Signaling Tech)70. Robust antibodies against the above proteins to detect total and phosphorylated forms are commercially available with strong literature evidence

[0251] PGCla independent contribution of WAY mediated maintenance of mitochondrial health in hRGCs:

[0252] This is possible that WAY activates sAC for translocating it into the mitochondria for a local cAMP dependent mitochondrial membrane potential (health) maintenance60, 61independent of PGCla dependent mechanisms. To test this, inventors can knockout PGCla in hRGCs by transducing LV-U6-gRNA (PGCla) (ThermoFisher) and LV-Cas9. Then can measure mitochondrial membrane potential in hRGCs under WAY treatment by live cell mitochondrial membrane potential dye JC1 (demonstrated in FIG. 11C, D and the reference54from our lab). Inventors can validate the findings by another live cell mito-membrane potential dye TMRM (ThermoFisher) which enters mitochondria if membrane potential is high or healthy. They can analyze mitochondrial ultrastructure in PGCla knockout hRGCs to study if WAY rescues cristae area, morphology’ and mitochondrial shape (elongation, sphericity) as done by them previously54which directly reflects organelle health and OXPHOS competency74. The inventors have extensive experience in analyzing mitochondrial ultrastructure54. Restoration of mitochondrial health by WAY independent of PGCla would indicate a biogenesis independent mechanism such as byreduced oxidative stress or by direct effect of sAC60, 61. In that case, inventors can test if sAC translocates to mitochondria in hRGCs by confocal IF (anti-sAC antibody. Millipore Sigma) under WAY treatment and whether the protective effect is abolished by' sAC inhibitor KH763

[0253] RGC protection by WAY in the absence of mitochondrial function:

[0254] Inhibition of mitochondrial OXPHOS by electron transport chain blockers, rotenone, oligomycin-antimycin (OA) or by proton uncoupler CCCP have been shown to cause RGC death by inventors and others18, 73. Inventors can inhibit mitochondrial function in hRGCs by above mitochondrial stressors individually and in presence of WAY. Under continuous treatment by these molecules, they can measure cellular apoptosis at 24h, 48h and 72h and cAMP level at shorter exposures such as 30min, 6h and 24h as done in FIG. 3B, C. These experiments can reveal if WAYrescues cellular apoptosis and maintains transient cAMP activation in the absence of mitochondrial function potentially through activating mitochondria independent neuroprotective pathways.

[0255] Expected results and alternatives:

[0256] The inventors expect to identify the specific AC and Gi / o involved in WAY mediated RGC protection. Based on neuroprotection effect of sAC39, they expect that WAY can activate sAC and suppression of sAC activity can suppress WAY effect on cAMP activation, mitobiogenesis and cellular apoptosis.

[0257] They expect that loss of involved Gi / o protein can constitutively activate AC hence, cAMP level in hRGCs disrupting the transient effect of WAY. Constitutive rise of cAMP may cause hRGC death as has been shown to be neurotoxic75. This is possible that we can identify Gai 1 as the involved G protein, which has been shown to be activated upon 5-HT1A agonist treatment in HEK293 cells76.

[0258] For transient biogenesis activation, inventors expect WAY to activate both CaMKII and Sirtl and knockout of these genes to suppress PGCla mediated biogenesis. Since CaMKII and Sirtl are bona fide PGCl a activators and have been shown to be RGC protective66, 71, 77.

[0259] Given PGCla is critical for mitochondrial health and neuroprotection78, 79, and WAY- cAMP response can activate multiple neuroprotective pathways beyond PGC1 a, inventors expect PGCla loss can disrupt mitochondrial health (membrane potential) and ultrastructure which can be partially rescued by WAY. Based on this rationale we also expect partial rescue of hRGC survival by WAY under mitochondrial function loss.

[0260] Potential Pitfalls and Alternatives:

[0261] This is likely that inventors can identify the specific ACs involved given the robust changes in cAMP level that they observed in hRGCs upon WAY treatment. How ever, if they see no effect of Gi / o knockout on WAY mediated cAMP changes this would indicate a non-specific target to stimulator GPCR. In that case, they can knockout Gas in hRGCs and study if the WAY mediated cAMP activation is blocked.

[0262] If CRISPR KO of a gene in hRGCs causes cell death, inventors can take gene knockdown approach by lentiviral shRNA delivery.

[0263] If they see WAY can protect RGCs in the absence of PGCl a or mitochondrial function, they can study if this is through activating Nrf2 mediated anti-oxidant response pathw ay and / or by activating cAMP-responsive element binding protein (CREB) transcription factor57. cAMP can directly activate both these transcription factors which are known to be neuroprotective80 57

[0264] EXAMPLE 11

[0265] Identification of the optimum WAY dose and frequency for longest neuroprotection

[0266] The inventors have observed both short as well as longer term vision maintenance in post ONC mice with no toxicity for 30 days of WAY treatments (5mg / kg). This suggests it is possible to test even longer treatments with lower doses to see the maximum protection range which is critical for clinical considerations. For this, the inventors can perform daily IP injection of 0.5, 2.5 and 5mg / kg WAY from day-5 to +56 with ONC done on day 0. Then they can perform visual acuity and contrast sensitivity test by OKR and depth perception test by visual cliff test. Following the vision tests, they can collect retina for RGC count and optic nerve for axonal measurements by GAP43 1HC on day +14. +28, +42 and +56. The inventors can identify the lowest dose required for longer vision, RGC and optic nen e protection. Next, they can test how long the drug efficacy remains by only pretreating mice 5 days before ONC with above identified low dose and then performing vision tests, retinal tissue collection for above experiments on day +7 and +14. This can help a determination as how frequently treatment is needed for optic nerve and vision protection.

[0267] Since systemic toxicity' of WAY for 30 days treatment is not observed, the inventors cannot initially perform intravitreal injection (IV) to study the local effect as this is an invasive and stressful procedure for animals. However, this may be contemplated if unexpected toxicity and / or behavioral changes occur for prolonged (>30 days) treatment.

[0268] Thus, based on the data and from the above experiments can resolve the mechanisms of WAY mediated RGC protection. In addition, the studies can identify a safe, optimum WAY dose and treatment conditions for effective therapy. Hence, WAY and or its derivates provide strong candidate(s) for optic neuropathy therapy development.

[0269] The remaining examples can demonstrate other novel RGC protective compounds examined for their utility. In addition to WAY, from the MTDR screening above the inventors have discovered several other compounds such as L-glutamic acid, 4-Imidazoleacetic acid hydrochloride (I4AA) that increased mitochondrial mass as potential activator of biogenesis, and Ouabain that reduced mitochondrial mass as potential activator of mitophagy (FIG. 16).

[0270] FIG. 26 shows that the small molecule screen identifies compound for activator of mitobiogenesis and mitophagy'. Drugs showing >40% change (outside of blue box) in H7- hRGCWT. Error bars are SEM. Red arrows indicate compounds that show reduced apoptosis in H7- hRGCs in the secondary screen.

[0271] Although, the inventors have found several other compounds with more prominent effect in modulating mito mass in hRGCs, above compounds (red arrows in FIG. 26) some haveshown robust reduction of Caspase 3 / 7 activity in both WT and glaucomatous OPTNE^0KhRGCs. Reduction of caspase activity is the bona fide readout for reduced cellular apoptosis, hence a potential neuroprotection effect. The inventors discovered that WAY provides similar robust reduction of caspase activity in human stem cell differentiated hRGCs and successfully showed strong RGC protection, axon regeneration and vision maintenance in mice against ONC injury’. Thus, the above compounds, like WAY, present strong feasibility for RGC protection and for neuroprotection therapies for optic neuropathy’ including glaucoma. The results are described below.

[0272] EXAMPLE 12

[0273] L-Glutamic acid as a therapeutic drug for optic neuropathy

[0274] The inventors have found that an agonist of glutamate neurotransmitter (L-Glutamic acid hydrochloride, “L-Glut”) increased mitochondrial mass in hRGCs from the screening (FIG. 16) and the result was reproduced when tested manually (FIG. 17A).

[0275] FIG. 17 A, B shows that L-Glutamic acid (L-Glut) promotes mitochondrial mass and lowers apoptosis in hRGCs. (FIG. 17A) Manual validation of L-Glut treatment at 5 pM for 24h shows increase in mitochondrial mass compared to the untreated control (CTR) in H7-hRGClthat was identified from high throughput screen in FIG. 16. L-Glut is water soluble hence we used untreated cells as control. Error bars are SEM. Unpaired student's t-test. (FIG. 17B) Apoptosis measurements by Apo-Tox Gio Triplex assay in presence of L-Glut (5 pM) at the indicated time points for hRGCs differentiated from H7 WT, H7-OPTNE50K, patient derived iPSCs with OPTNE50Kmutation and iPSCs with OPTNE50Kmutation corrected cells. Error bars are SEM. One- Way ANOVA with Dunnett's. Error bars are SEM.* p<0.05, ** p<0.01, ***p<0.001, **** p<0.000I, n=3

[0276] This suggests that like WAY, L-Glut also activates mitobiogenesis in hRGCs. As with WAY, the inventors further observed robust reduction of cellular apoptosis in a time dependent manner for the WT and glaucoma causing OPTNE50KhRGCs (FIG. 17B). Of note, glutamate mediated overstimulation of the N-methyl-D-aspartate receptor (NMD AR) induces excitotoxic cell death in stroke, trauma, epilepsy, Huntington’s disease and amyotrophic lateral sclerosis. See Choi et al., “Glutamate neurotoxicity and diseases of the nervous system,” (1988) Neuron 1(8): pp. 623- 634. Excess glutamate was also observed in the vitreous of glaucoma patients and is thought to be the reason for RGC excitotoxic death. See Cheung et al., “Neuroprotection in glaucoma: drugbased approaches,” (2008) Optom Vis Sci 85(6): pp. 406-416. However, this observation was later called into question with conflicting evidence. See Lotery et al., “Glutamate excitotoxicity in glaucoma: truth or fiction?,” (2005) Eye (Lond). 19(4): pp. 369-370; Salt et al., “Glutamateexcitotoxicity in glaucoma: throwing the baby out with the bathwater?,’7(2006) Eye (Lond) 20(6): pp. 730-731 ; Osborne et al., “Glutamate excitotoxicity in glaucoma: truth or fiction? By AJ Lotery — Reply,” (2006) Eye (Lond) 20(12): pp. 1392-1394. The current discovery’ that L-Glutamic acid reduces apoptosis in human RGCs is novel. Thus, based on the inventor’s data a carefully tested low dose of L-Glut could provide strong RGC protection for optic neuropathy’ therapy development.

[0277] EXAMPLE 13

[0278] 4-Imidazoleacetic acid hydrochloride (MAA) as a therapeutic drug for optic neuropathy

[0279] Like WAY and L-Glut. MAA increased mitochondrial mass in hRGCs from the screening (FIG. 16) and the result was reproduced when tested manually (FIG. 18 A).

[0280] FIG. 18 A, B shows that MAA promotes mitochondrial mass and lowers apoptosis in hRGCs. (FIG. 18A) Manual validation of MAA treatment at 5 pM for 24h shows increase in mitochondrial mass compared to the untreated control (CTR) in H7-hRGCWTthat was identified from high throughput screen in FIG. 16. MAA is water soluble hence we used untreated cells as control. (FIG. 18B) Apoptosis measurements by Apo-Tox Gio Triplex assay in presence of MAA (5 pM) after 24h treatments to the indicated WT hRGCs. Error bars are SEM. Unpaired student's t-test. .* p<0.05, ** p<0.0I, ***p<0.001, n=3.

[0281] This suggests that MAA also activates mitobiogenesis in hRGCs with potential neuroprotection effect. Indeed, it was observed that robust reduction of cellular apoptosis occurred for the WT and E5 OK-corrected hRGCs when treated with MAA (FIG. 18B). MAA has been shown to antagonize the GAB Ac receptors in white perch retinal neurons. See Tunni cliff et al., “Pharmacology and function of imidazole 4-acetic acid in brain,” (1998) Gen Pharmacol 31(4): pp. 503-509. To date, there has been no study indicating that MAA could serve as RGC protective compound. Thus, the current discovery7that MAA reduces apoptosis in hRGCs is novel, hence MAA and / or its derivatives that target GAB Ac receptor provide novel therapeutic agent(s) for optic neuropathy therapy development.

[0282] EXAMPLE 14

[0283] Ouabain as a therapeutic drug for optic neuropathy

[0284] From the MTDR screening the inventors identified ouabain among a group of compounds that reduced mitochondrial mass in the hRGCs (FIG. 16) and the result was reproduced when tested manually (FIG. 19A). Reduction of mitochondrial mass could be due to increased mitophagy caused by mitochondrial damage such as for mitochondrial membrane uncoupler CCCP.

[0285] However, the compound could also enhance mitophagy of existing damaged mitochondria without causing any additional damage. This would help RGCs to restore mitochondrial homeostasis with a neuroprotective effect. Indeed, ouabain showed robust reduction of apoptosis for both the WT and glaucoma causing OPTNE50KhRGCs (FIG. 19B).

[0286] FIG. 19A, B shows that ouabain promotes mitochondrial degradation and lowers apoptosis in hRGCs. (FIG. 19A) Manual validation of ouabain treatment at 5 pM for 24h shows reduced mitochondrial mass in H7-hRGCWTcompared to the DMSO vehicle control (CTR) that was identified from the high throughput screen in figure 16. (FIG. 19B) Apoptosis measurements in the WT and in OPTNE50KhRGCs by Apo-Tox Gio Triplex assay in presence of ouabain (5 pM) for 24h. Error bars are SEM. Unpaired Student’s t-test. * p<0.05. **** p<0.0001, n=3.

[0287] Ouabain is a steroid hormone which is a classic inhibitor of Na+ / K+-ATPase membrane channel and upon binding ouabain activates cell growth, proliferation and cell survival pathw ays. See Xie et al., “Na(+) / K(+)-ATPase as a signal transducer.” (2002) Eur J Biochem 269(10): pp. 2434-2439. It has been shown that in primary rat RGC culture ouabain treatment increased cell survival by suppressing inflammatory pathways when isolated from naive as well as from retinas following optic nerve axotomy. See Salles von-Held-Ventura et al., “The trophic effect of ouabain on retinal ganglion cells is mediated by IL-1 (3 and TNF-a,” (2016) Biochemical and Biophysical Research Communications 478(1): pp. 378-384; Mazala-de-Oliveira et al., “Ouabain-Na(+) / K(+)- ATPase Signaling Regulates Retinal Neuroinflammation and ROS Production Preventing Neuronal Death by an Autophagy-Dependent Mechanism Following Optic Nerve Axotomy In Vitro,” (2022) Neurochem Res 47(3): pp. 723-738. However, it is still unknown if ouabain is neuroprotective for human RGCs and if in vivo under optic nerve damage conditions. The inventor’s data showing ouabain reduces apoptosis in human hRGCs is novel. Further studies can include identifying the optimum dose, treatment duration and the mechanism of RGC protection in mice ONC and in high IOP glaucoma conditions. Thus, based on these data a carefully tested dose of ouabain and / or its derivatives could provide strong RGC protection for optic neuropathy therapy development which is an unmet area.

[0288] EXAMPLE 15

[0289] BX795 as a therapeutic drug for optic neuropathy

[0290] As discussed herein, the inventors have discovered that in glaucoma causing OPTNE50KhRGCs mitochondrial metabolism is in fact higher than the wild-type neurons but with less mitochondrial mass to meet the metabolic demand. This causes mitochondrial bum out leading to mitochondrial swelling, damage, and ultimately dismpted mitochondrial homeostasis and neurodegeneration. However, activating mitochondrial biogenesis through targeting TBK1 by itspotent inhibitor BX795 led to activation of mito biogenesis which restored the organelle homeostasis ultimately leading to neuroprotection of E50K hRGCs against a mitochondrial stressor CCCP. See Surma et al. 2023. Since activating mitobiogenesis, like with WAY, showed robust optic nerve and vision maintenance against ONC in mice, BX795 and or its derivatives present potential for robust therapeutic agents for optic neuropathy treatment.

[0291] EXAMPLE 16

[0292] RTA-408 (Omaveloxolone) as a therapeutic drug for optic neuropathy

[0293] Damaged mitochondria are the primary source for cellular ROS (mtROS). See Murphy, “How mitochondria produce reactive oxygen species,” (2009) Biochem J 417(1): pp. 1-13. Increased ROS level have been widely found in the primary open-angle glaucoma (POAG) patients’ retina with high eye pressure. Mitochondrial biogenesis and antioxidant response pathways involve a common player, Nrf2. Mitochondrial biogenesis requires mtDNA transcription and activated PGCla stimulates Nrfl and Nrf2 which drive the expression of mitochondrial transcription factor Tfam. See Piantadosi et al.. “Redox regulation of mitochondrial biogenesis.” (2012) Free Radic Biol Med 53(11): pp. 2043-2053. Nrf2 is also the master regulator for ROS removal pathways. See Holstrom et al. 2016. In basal conditions, Nrf2 is rapidly degraded by the Keapl-Cullin3 (Cul3) / Rbx E3 ubiquitin ligase system and proteasome pathway. Under elevated ROS. cysteine sensors of Keapl react with the ROS electrophiles leading to Nrf2 release and stabilization. See Dinkova-Kostova et al., “Keapl, the cysteine-based mammalian intracellular sensor for electrophiles and oxidants,” (2017) Arch Biochem Biophys 617: pp. 84-93. Several of Nrf2 activators are under FDA clinical trials for mitochondrial dysfunction related neurodegenerative conditions such as Parkinson’s, Alzheimer’s and Huntington’s disease. See Robledinos-Anton et al., “Activators and Inhibitors of NRF2: A Review of Their Potential for Clinical Development,” (2019) Oxid Med Cell Longev 2019: p. 9372182. Over-expression ofNrf2 have been shown to be RGC protective in the mouse optic nerve crush model. See Yang et al., “Antioxidant Treatment Limits Neuroinflammation in Experimental Glaucoma,” (2016) Invest Ophthalmol Vis Sci 57(4): pp. 2344-2354. Pharmacological Nrf2 activators bardoxolone (RTA- 402) and sulforaphane have been shown to be RGC protective against anterior ischemic optic neuropathy while CDDO-Im and dimethyl fumarate (DMF) have been shown to protect RGCs against ONC in mouse. See Chien et al., “The Effects of Two Nrf2 Activators, Bardoxolone Methyl and Omaveloxolone, on Retinal Ganglion Cell Survival during Ischemic Optic Neuropathy,” (2021) Antioxidants (Basel) 10(9): epub 20210915; Pan et al., “Sulforaphane Protects Rodent Retinas against Ischemia-Reperfusion Injury through the Activation of the Nrf2 / HO-l Antioxidant Pathway,” (2014) PLOS ONE 9(12): el 14186.; Himori et al., “Criticalrole of Nrf2 in oxidative stress-induced retinal ganglion cell death / ’ (2013) J Neurochem 127(5): pp. 669-680; Mori et al., ‘'Dimethyl Fumarate Promotes the Survival of Retinal Ganglion Cells after Optic Nerve Injury, Possibly through the Nrf2 / HO-l Pathway,” (2020) Int J Mol Sci 22(1): epub 20201230. While pro-survival effects of Nrf2 activation on mouse RGCs are well studied, the specific activator that is efficacious for human RGCs is yet to be discovered. This is important as ROS electrophile toxicity (such as superoxides, peroxides, glutathione, alkyl hydroperoxides, and peroxinitrite) varies in different cell ty pes under different disease conditions and specific ROS removal requires activation of the corresponding antioxidant protein. See Forman et al., “Targeting oxidative stress in disease: promise and limitations of antioxidant therapy,” (2021) Nat Rev Drug Discov 20(9): pp. 689-709. This is presumably a reason why a majority of clinical trials targeting oxidative stress in general with non-specific compounds fail81.

[0294] Damaged mitochondria are the primary source for cellular reactive oxygen species (ROS) which at excess levels cause cellular apoptosis. The inventors’ findings that glaucomatous OPTNE50KhRGCs possess more damaged mitochondria makes them vulnerable to having elevated ROS. The transcription factor, nuclear factor erythroid-2 related factor 2 (Nrf2) protein is the master regulator of mitochondrial ROS (mtROS) clearance through activation of Antioxidant Response Element (ARE) gene. See Holmstrom et al., “The multifaceted role of Nrf2 in mitochondrial function,” (2016) Curr Opin Toxicol 1: pp. 80-91. There is crosstalk between the mito biogenesis master regulator PGCla and oxidative stress response master regulator Nrf2 signaling pathways. See Gureev et al., “Regulation of Mitochondrial Biogenesis as a Way for Active Longevity: Interaction Between the Nrf2 and PGC-la Signaling Pathways,” (2019) Front Genet. 10: p. 435. As such, PGCla regulates Nrf2 activity and vice-versa.

[0295] The inventors have shown that mitochondrial damage by CCCP activates PGCl mediated mitochondrial biogenesis. See Surma et al. 2023. Thus, they asked if CCCP damage also activates Nrf2 in hRGCs. Phosphorylation at Ser40 is critical for Nrf2 activation. See Huang et al., “Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription.” (2002) J Biol Chem. 277(45): pp. 42769-42774. Indeed, the inventors observed increased p-Nrf2Ser40for the WT hRGCs under CCCP treatment (FIG. 20 A, B) but not for OPTNE50KhRGCs (FIG. 20A, C) suggesting a defect in Nrf2 activation for the glaucomatous hRGCs with potential oxidative stress.

[0296] FIG. 20A-C shows that Nrf2 activation in OPTNE50KhRGCs is defective. (FIG. 20A) Western blot images ofNrf2 and p-Nrf2 in presence of 10 pM CCCP. (FIG. 20B, C) Protein band intensity normalized with-respect-to the corresponding GAPDH and successively with the DMSO vehicle control to identify changes over time. Error bars are SEM. n=3.

[0297] Based on above discoveries inventors asked if at the basal level glaucoma causing OPTNE50KhRGCs suffer more oxidative stress and whether that can be mitigated by activating Nrf2. Indeed, the inventors have shown glaucomatous hRGCs with OPTNE50Kmutation suffer oxidative stress by the live cell ROS sensor DCFDA (FIG. 21 A, B). Thus, they seek to determine if specific Nrf2 activators can reduce oxidative stress and promote pro-survival pathway in the OPTNE50KhRGCs and tested several Nrf2 activators such as sulphorafane, DMF and RTA-408. The inventors have discovered that RTA-408, showed robust reduction of ROS level as revealed by reduced DCFDA green signal in both the Wt and in OPTNE50KhRGCs (FIG. 21C, D).

[0298] FIG. 21A-D shows that the Nrf2 activator RTA-408 reduces ROS level in hRGCs. (FIG. 21A) Max projection of confocal z-stacks, of hRGCs treated with 100 nM RTA-408 or vehicle control DMSO for 24h. ROS level detected in the live cells by DCFDA which fluoresces green upon oxidation by ROS. (FIG. 21B-D) Quantification of ROS intensity from sum projections of confocal z-stacks. normalized to cell area. Error bars are SEM. Student's t-test, *, p-value < 0.09, **, <0.01. n=13-16.

[0299] Furthermore, RTA-408 shows robust reduction of cellular apoptosis in both the WT and glaucomatous OPTNE’0KhRGCs (FIG. 22), establishing its novel therapeutic application for glaucoma and or mitochondrial dysfunction related optic neuropathy. Of note, RTA-408 is an FDA approved drug used for Friedreich ataxia treatment (Omav el oxoIone).

[0300] FIG. 22 shows that Nrf2 activation by RTA-408 reduces apoptosis in hRGCs. Caspase- 3 / 7 activity was measured in hRGCs after 24h DMSO or 100 nM RTA-408 using ApoToxGlo- Triplex assay kit. Error bars are SEM. Student’s t-test, **, p-value < 0.01, n=3.

[0301] While embodiments of the present disclosure have been described herein, it is to be understood by those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions can now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

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Claims

CLAIMS1. A method of treating optic neuropathy in a patient, comprising: administering a therapeutically effective amount of WAY-100635 maleate to the patient, wherein the administration of the WAY-100635 maleate restores mitochondrial homeostasis in retinal ganglion cells.

2. The method of claim 1, wherein the patient has glaucoma.

3. The method of claim 1, wherein the patient has an inherited optic neuropathy.

4. The method of claim 1, further comprising the step of activating mitochondrial biogenesis in the retinal ganglion cells through TBK1 mediated activation of PGC-la / B.

5. The method of claim 1, wherein the administration is performed intraperitoneally.

6. The method of claim 1, wherein the administration further leads to suppression of neuronal apoptosis in the retinal ganglion cells.

7. A pharmaceutical composition for treating optic neuropathy, comprising: a therapeutically effective amount of WAY-100635 maleate, a salt thereof, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7, further comprising: L-Glutamic acid hydrochloride.

9. The pharmaceutical composition of claim 7, further comprising: 4-Imidazoleacetic acid hydrochloride.

10. The pharmaceutical composition of claim 7, further comprising one or more of BX-795 hydrochloride, RTA-408, or Ouabain.

11. A method of promoting retinal ganglion cell axon regeneration in a patient, comprising: administering a therapeutically effective amount of WAY- 100635 maleate to the patient.

12. The method of claim 11, further comprising the step of visual inspection, vision test, or MRI scans of patient’s brain to detect if visual signal is being properly processed in transmitted the brain.

13. A method of reducing oxidative stress in retinal ganglion cells, comprising: administering a therapeutically effective amount of WAY-100635 maleate to the patient.

14. The method of claim 13, wherein the patient has normal tension glaucoma with a prevalence of OPTNE50Kmutation.

15. The method of claim 13, further comprising the step of reducing neuronal apoptosis in the retinal ganglion cells.

16. A method of improving visual acuity- in a patient following optic nerve injury, comprising: administering a therapeutically effective amount of WAY-100635 maleate to the patient.

17. A method for the treatment or prevention of an eye disease or condition in a patient in need thereof, wherein the eye disease or condition is the result of mitochondrial dysfunction comprising the administration of pharmaceutical composition that includes an agent or that restores mitochondrial homeostasis.

18. The method of claim 17, wherein the eye disease or conditions is selected from the group comprising glaucoma and optic neuropathy.

19. The method of claim 18. wherein the optic neuropathies are selected from the group consisting of ischemic optic neuropathy, anterior ischemic optic neuropathy, posterior ischemic optic neuropathy, radiation optic neuropathy, optic neuritis, compressive optic neuropathy, infiltrative optic neuropathy, traumatic optic neuropathy, mitochondrial optic neuropathy, nutritional optic neuropathy, toxic optic neuropathy, and hereditary- or inherited optic neuropathy.

20. The method of claim 17, wherein the agent that restores mitochondrial homeostasis is selected from the group consisting of WAY- 100635 maleate or other pharmaceutically acceptable salt, L-Glutamic acid hydrochloride, Ouabain, 4-Imidazoleacetic acid hydrochloride (MAA). BX-795 hydrochloride and RTA-408 (Omaveloxolone).

21. The method of any of claims 17-20, wherein the agent that restores mitochondrial homeostasis provides neuroprotection, axon regeneration and maintenance of vision in the patient.

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