Browsing by Author "Stankowska, Dorota"
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Item Assessment of WIN 55,212-2 Loaded Reconstituted High-Density Lipoprotein Nanoparticles for Ocular Delivery(2024-03-21) Petty, R. Max; Ranjan, Rajiv; Sabnis, Nirupama; Fudala, Rafal; Lacko, Andras; Gryczynski, Zygmunt; Krishnamoorthy, Raghu; Stankowska, DorotaPurpose: Overcoming challenges in glaucoma therapy, such as biological barriers and retina delivery, led us to develop innovative reconstituted high-density lipoprotein nanoparticles (rHDL NPs) for effective drug delivery. Optic nerve head astrocytes (ONHAs) are vital in maintaining retinal ganglion cell (RGC) axon integrity. This study describes the encapsulation of WIN 55,212-2 (WIN) in rHDL NPs and investigates the delivery mechanism of these nanoparticles in ONHAs. Methods: Using a novel preparation method, a stable rHDL-payload complex was created by combining lipophilic fluorescent dye IR780 or therapeutic agent WIN with phosphatidylcholine and apolipoprotein A-I (Apo A-I). Fluorescent rHDL (rHDL-IR780) was used to assess cellular uptake in human primary ONHAs in vitro. Scavenger receptor class B1 (SR-B1) expression was confirmed in retinal cell lysates by SDS-PAGE followed by western blot analysis. Receptor-mediated payload release through SR-B1 was confirmed by receptor blocking using BLT-1 as a specific SR-B1 receptor-blocking agent. Results: Fluorescent rHDL NPs exhibited an IR780 encapsulation efficiency of 68.7% (103 M), a polydispersity index (PDI) of 0.287 ± 0.013, a size of 14.01 ± 4.36 nm, and a zeta potential of -7.44 ± 0.90 mV. Additionally, drug-loaded rHDL-WIN NPs displayed a WIN encapsulation efficiency of 44.6% (341.4 M), a PDI of 0.280 ± 0.011, a size of 62.04 ± 25.06 nm, and a zeta potential of -20.13 ± 0.86 mV. Western blot analysis on human retinal lysates, ONHA lysates, and RGC lysates indicated the expression of SR-B1 (57/82 kDa (unmodified/glycosylated)). Cellular uptake studies confirmed the ability of rHDL to deliver payloads to ONHAs and RGCs. Receptor blocking with 10 nM BLT-1 highlighted the role of SR-B1 in specific cellular uptake from rHDL to ONHAs (p < 0.01). Conclusions: Our study highlights the role of SR-B1 in facilitating the delivery of rHDL payloads to ONHAs, offering the potential for targeted drug delivery in glaucoma. We anticipate that the cellular uptake by RGCs will follow the same SR-B1-mediated pathway. Successful WIN encapsulation in rHDL NPs suggests a potential avenue for targeting therapies to treat and prevent glaucomatous damage. Further studies are needed to determine the neuroprotective effects of rHDL-WIN and develop the potential of rHDL NPs to be used as an agent to target therapies in glaucoma.Item Bi-functional small molecule with both IOP lowering and neuroprtective activity to treat glaucoma.(2018-03-14) Stankowska, Dorota; Millar, Cameron; Chaphalkar, Renuka; Li, Linya; Ellis, Dorette; Yorio, Thomas; Acharya, SuchismitaPurpose: The multifaceted pathology associated with glaucoma is difficult to treat by monotherapy such as lowering intraocular pressure (IOP). Oxidative stress is involved in IOP elevation and retinal ganglion cell (RGC) death possibly via decreased activity of antioxidant enzymes. Our hypothesis is that, a small hybrid molecule SA-2 and its PLGA encapsulated nanoparticle (NP) drug delivery vehicle (SA-2-NPs) possessing a nitric oxide (NO) donating group and a redox antioxidant moiety will lower IOP as well as possess neuro/cytoprotective potential. Methods: IOP-independent neuroprotection of RGCs: Mice (C57BL6, 12 weeks, n=8) were anesthetized and the optic nerve crush (ONC) was performed on one eye followed by intravitreal injection of 2ml of 2% SA-2 formulated in PBS or vehicle to the crushed eye at day 0 and 3. At day 7, pattern electroretinogram (PERG) was performed, following which retinas were flat mounted. Number of surviving, RBPMS positive RGCs were counted. IOP lowering effect: Nine Brown Norway normotensive rats (n=3 per each group) were used to evaluate the IOP-lowering properties of SA-2-NPs under a masked protocol. Topical eye drops (30 µL/drop) containing: A) Vehicle (PBS), B) Travoprost (0.004%)-positive control or C) SA-2-NPs (100 µg/mL) formulated in PBS were administered in three rats and IOP was measured using the TonoLab rebound tonometer at various time points: 0, 3, 6, 24, 48 and 72 h post-dosing. The entire dosing schedule was repeated 3 times in each rat. Results: Compound SA-2 treatment was significantly (t-test, p 2 vs SA-2 treated group (2100 cells/cm2) and was comparable to the untreated control (2650 cells/cm2). The data are given as the mean ± SEM; n= 3-4. PLGA encapsulated SA-2-NPs (100mg) statistically (t-test, p Conclusion: Taken together, our results are consistent with our hypothesis that this novel class of hybrid compound and its PLGA encapsulated nanoparticles will decrease IOP and protect RGCs from cell death. Further structure optimization of the lead compound, dose optimization, IOP lowering study in ocular hypertensive rodent models are underway.Item Changes in the Expression of SMARCA4 in a Rat Model of Ocular Hypertension(2022) Worley, Josh; Stankowska, Dorota; Kodati, Bindu; Krishnamoorthy, RaghuTitle: Changes in the Expression of SMARCA4 in a Rat Model of Ocular Hypertension Purpose: SMARC4 (BRG1) is an ATP-dependent chromatin remodeling protein belonging to the SWI/SNF family of proteins involved in regulation of gene expression in numerous cell types in the body. The purpose of this study was to determine changes in the expression of SMARCA4 in the retina following intraocular pressure (IOP) elevation by the Morrison model in Brown Norway rats. We hypothesize that SMARCA4 expression may modulate the expression of the key genes involved in the neurodegenerative changes seen secondary to IOP elevation. Methods: The Morrison model of ocular hypertension (by injection of hypertonic saline through the episcleral veins) was utilized to unilaterally elevate the IOP in Brown Norway rats. IOP was elevated in the left eye of three retired breeder Brown Norway rats, with the right eye serving as the corresponding contralateral control. Rats were maintained for 2 weeks following IOP elevation and IOP measurements were carried out twice per week. Rats were subsequently euthanized, and retinal sections were obtained from both IOP-elevated and contralateral control eyes. Immunohistochemical analysis of SMARCA4 expression was carried out by immunostaining. Following confocal microscopy imaging, the intensity of immunofluorescence was quantified with the ImageJ software (NIH), and compared between IOP elevated and control eyes. Results: Immunohistochemical analysis revealed an appreciable decrease in the expression of SMARCA4 in retinal sections in two out of three rats, mainly the nerve fiber layer (by 47 to 57%), ganglion cell layer (by 18 to 40%) and inner plexiform layer (by 9 to 19%) in IOP elevated rat eyes compared to control eyes. One out of three tested rats showed a modest increase in immunostaining for SMARCA4 in the nerve fiber layer, ganglion cell layer and inner plexiform layer. Ongoing experiments will replicate these findings in order to generate statistically significant data. Conclusion: Changes in SMARCA4 expression could serve to regulate the expression of gene contributing to neurodegenerative effects due to elevated IOP. Understanding the role of SMARCA4 may allow us to better understand and address the mechanisms involved in glaucomatous neurodegeneration.Item CREB Activation by Mini-Chaperone CPP-P1 Enhances Retinal Ganglion Cell Survival in an Acute Glaucoma Model(2024-03-21) Johnson, Gretchen; Nagaraj, Ram; Stankowska, DorotaPurpose: Alpha-B crystallin is a heat shock protein that has been found to have anti-apoptotic properties and was used to design the novel mini-chaperone called peptain-1 (P1) conjugated with a cell-penetrating peptide (CPP), named CPP-P1. Transcriptomics of primary retinal ganglion cells (RGCs) isolated from adult rats subjected to ocular hypertension and treated with CPP-P1 revealed the activation of CREB signaling as a major pathway activated by the drug. Creb activation by phosphorylation (p-Creb) was previously confirmed in primary RGCs and tested here in a rat model of ocular hypertension. Methods: Adult male Brown Norway rats (N=15, 5 per group) were grouped into naïve, IOP-Vehicle, and IOP-CPP-P1 experimental groups. Silicone oil (20µl) was injected into the anterior chamber, and 2µl of either PBS (vehicle) or CPP-P1 (2µg/µl) was injected intravitreally. On day 7 of elevated IOP, rats were euthanized. Retinal sections obtained were stained with Creb, p-Creb, and DAPI and imaged at 4X for cell counts and at 20X for integrated density measurements. Unpaired t-tests or Mann-Whitney tests in GraphPad Prism were used to calculate statistical significance. Retinal punches from post-mortem human eyes (N=3) were cultured with PBS or CPP-P1 (12.5µg/ml) for 48 hours, and tissue RNA was collected for qPCR. Results: Ganglion cell layer counts obtained from rat retinal sections showed that the silicone oil injury group with no intervention (IOP-Veh) had a 51% decrease in ganglion cells compared to the naïve group (p<0.0001), and the silicone injury with CPP-P1 intervention group (IOP-CPP-P1) showed only a 23% decrease compared to the naïve group (p=0.0016). Integrated density measurements of Creb expression in IOP-Veh was 17% (p=0.615) higher than the naïve group, while in IOP-CPP-P1, it was 136% (p=0.092) higher than the naïve group. Expression of p-Creb showed a decrease in IOP-Veh by 50% (p=0.056) in comparison to the naïve group, while the IOP-CPP-P1 group was significantly higher than the IOP-Veh group (p=0.036). RNA expression of Creb1 in human retinal tissue was increased by 1.7-fold with CPP-P1 treatment. Discussion: IOP-mediated RGC damage was mitigated by CPP-P1 treatment, demonstrating neuroprotective effects when compared to vehicle-treated rats. CREB signaling contributes to CPP-P1-mediated neuroprotection during glaucomatous insults.Item Endothelin B (ETB) receptor selective agonist endothelin 3 (ET-3) andmediated RGC death and implication for glaucoma.(2020) Kodati, Bindu; Krishnamoorthy, Raghu; Stankowska, Dorota; Harris, Payton; Wisniewski, Zoe; Krishnamoorthy, Vignesh; Beall, KallenPurpose: To determine if dietary administration of the dual ETA/ ETB receptor antagonist, macitentan, could protect retinal ganglion cells (RGCs) from cell death following an intravitreal injection of endothelin-1 (ET-1) and endothelin-3 (ET-3) in Brown Norway rats. Methods: Brown Norway rats (n=3 per group) were either untreated or treated with macitentan (5 mg/kg body weight) once a day for 3 days, followed by intravitreal injection of either 4 µl of 500 µM ET-1, ET-3 (2 nmole/eye) or vehicle in one eye. Following the injections, treatments with either macitentan or control gels without macitentan was continued for 7 days. After the treatments, retinal flat mounts were prepared and surviving RGCs were counted in a masked manner. Results: ET-1 produced 46% increase in RGC loss (p< 0.0001), compared to contralateral control eyes in rats 7 days post intravitreal administration. Macitentan treatment modestly protected from RGC loss following intravitreal ET-1 injection (27% RGC loss). ET-3 administration caused a modest increase (23%) in RGC death in this acute model, however, macitentan did not cause any further improvement in RGC survival (25% RGC loss). Conclusion: Both ET-1 and ET-3 mediate RGC loss following intravitreal administration in rats. Endothelin receptor antagonists have the potential to be developed as neuroprotective agents for the treatment of glaucoma.Item Endothelin Mediated Changes in Gene Expression Determined by RNA-sequencing of the Translatome in Primary Retinal Ganglion Cells.(2017-03-14) Krishnamoorthy, Raghu; Stankowska, Dorota; Chaphalkar, Renuka M.Purpose: Endothelin treatment has been shown to produce increased cell death in primary RGCs, however the underlying changes in gene expression are not completely understood. The purpose of the study was to assess endothelin-mediated changes in mRNA expression that occur at the translational level. Methods: Primary RGCs were isolated from post-natal day 5 rat pups by immunopanning with an antibody to Thy1.1. RGCs obtained were allowed to attach and maintained for 7 days for neurite outgrowth to occur. The RGCs were either untreated or treated with endothelin-1 (100 nM) for 24 h in trophic factor-free medium. Following brief incubation with cycloheximide to inhibit protein synthesis, total polysomes were isolated by magnesium precipitation and polysomal RNA was extracted using the Trizol reagent. Libraries for RNA-Seq were prepared with KAPA Stranded RNA-Seq Kit. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina Hiseq3000/4000 for a pair end 150 run. The reads were first mapped to the latest UCSC transcript set using STAR version 2.4.1d and the gene expression level was quantified to annotation model (Partek E/M). Differentially expressed genes were identified using differential gene expression (GSA) algorithm in Partek. Genes showing altered expression with p Results: Analysis of gene ontology of the changes in gene expression revealed a significant increase in expression of several mitochondrial genes including mitochondrial intermediate peptidase (MIPEP) (3.3-fold), cytochrome c oxidase assembly factor (SURF1) (8.7-fold) and Apolipoprotein O Like (APOOL) (7.5-fold). On the other hand, a decrease in expression of mitochondrial genes cytochrome c oxidase subunit 4I2 (8-fold), cytochrome c oxidase 6B2 (8-fold), and DNA polymerase gamma 2 (POLG2) (7-fold) was observed. Conclusions: Analysis of the translatome offers a glimpse into de novo protein synthesis which is an important manifestation of changes in gene expression. Endothelin treatment produced changes in several key regulators of mitochondrial metabolism and bioenergetics which could be indicative of their involvement in neurodegeneration in glaucoma.Item Endothelin-1 Mediated Decrease in Expression of Mitochondrial Proteins ATP5H and COX17 in Retinal Ganglion Cells.(2019-03-05) Stankowska, Dorota; He, Shaoqing; Kodati, Bindu; Krishnamoorthy, Raghu; Chaphalkar, Renuka M.; Chaphalkar, Renuka M.TITLE: Endothelin-1 Mediated Decrease in Expression of Mitochondrial Proteins ATP5H and COX17 in Retinal Ganglion Cells. Purpose: Endothelin-1 (ET-1) treatment has been shown to promote apoptosis of retinal ganglion cells (RGCs), however, the precise mechanisms underlying these effects are still unknown. The purpose of the study was to assess the changes in gene expression at the level of the translatome, occurring during ET-1 mediated neurodegeneration of RGCs. Methods: Primary RGCs isolated from post-natal day 5 rat pups were treated with ET-1 (100 nM) for 24 h in trophic factor-free medium. Polysomal RNA was isolated and libraries for RNA-Seq were prepared. Trimmed mean of M-values (TMM) was used to normalize the gene expression. Genes with expression changes more than 1.5 fold with p Results: STRING network analysis revealed 156 differentially expressed genes, of which 23 genes were identified with known or predicted mitochondrial function. Immunostaining of primary RGCs showed an appreciable decline in expression of COX17, while ATP5H expression was modestly decreased. A decreasing trend (three out of four rats) in immunostaining for ATP5H as well as COX17 was found in retinas of rats intravitreally injected with ET-1 (n=4). Conclusions: ET-1 treatment produced a decrease in expression of key components of mitochondrial electron transport chain. A compromise in bioenergetics could be one mechanism by which ET-1 promotes neurodegeneration of RGCs in glaucoma.Item Enhanced Protection of Retinal Ganglion Cells against Ischemia/Reperfusion Injury and Neurotrophic Factor Deprivation with Compound SA-10(2024-03-21) Pham, Jennifer; Zhang, Wei; Le, Kim-Tuyen; Kodati, Bindu; Amankwa, Charles; Tran, Ashley; Acharya, Suchismita; Stankowska, DorotaPurpose: Glaucoma is one of the leading causes of irreversible blindness worldwide. In glaucoma, the retinal ganglion cells (RGCs), which transmit visual signals to the brain, undergo neurodegeneration, leading to a gradual loss of vision. Oxidative stress is the imbalance between antioxidant activity and free radical production, which has been shown to be associated with glaucomatous RGC degeneration. In this study, we investigated the potential of SA-10, a hybrid nitric oxide donating and sulfone reactive oxygen species (ROS) scavenging molecule, to promote the survival of RGCs against glaucomatous damage. We investigated its neuroprotective effects following retinal ischemia/reperfusion (I/R) injury in mice and ex vivo following neurotrophic factor (NF) deprivation in human retinal explants (HREs). Methods: Acute I/R injury was induced in C57BL/6J mice (n=2-3 mice/group/sex) through intracameral pressure elevation to 120 mmHg for 1 hour. The mice were pre-treated topically with a PLGA nanosuspension of SA-10 (1% SA-10-NPs) and treated for 14 days (7 doses) after I/R injury. The obtained retinal sections were stained with anti-heme oxygenase-1 antibody (Hmox1, a marker of protective response to oxidative stress) to quantify its expression levels. H&E sections were used to measure retinal thickness. In another set of experiments, biopsy punches from HREs (n=3 donors) were isolated and treated with either SA-10 [10 µM] or vehicle and maintained without NF for 7 days ex vivo (DEV). Four control punches were collected on day 0 (0 DEV). After 7 days, HREs were immunostained with RBPMS (RGC-specific marker), and cell survival was analyzed. Analysis of Variance (ANOVA) was performed for all experiments. Results: In the nerve fiber and ganglion cell layers (ganglion cell complex, GCC), I/R injury caused a significant reduction in Hmox1 expression in female (79.8%, p<0.05) and male (54.5%, p<0.05) mice, compared to the sham control. Additionally, I/R injury led to a decline in GCC thickness by 27.5% (p=0.23) in females and 32.7% (n=2) in males. However, treatment with SA-10-NPs increased Hmox1 expression by 4.2-fold (p<0.05) in females and by 0.5-fold (p=0.43) in males. SA-10-NPs also preserved GCC thickness by 17.6% (p=0.68) in females and 27.1% in males. In human retinal explants, the 7 DEV vehicle-treated group had a significant loss in RGCs by 45.2% (p<0.01) compared to 0 DEV. In contrast, SA-10 treatment enhanced RGC survival at 7 days with an 83.1% (p<0.01) higher RGC counts than the vehicle group. Conclusions: SA-10 and its nanosuspension exhibited significant neuroprotective effects by enhancing Hmox1 expression, preserving retinal thickness, and promoting RGC survival, highlighting its potential as a therapeutic candidate for glaucoma and ischemic stroke.Item Evaluation of Neuroprotective Effect of hybrid compound SA-2 Nanosuspension in Optic Nerve Crush Mouse Model.(2021) Amankwa, Charles E.; Gondi, Sudershan; Funk, Arlene; Debnath, Biddut; Zhang, Wei; Li, Linya; Ferguson, Jonathan; Johnson, Gretchen; Ellis, Dorette; Stankowska, Dorota; Acharya, SuchismitaThis study examines the cytoprotective effects of hybrid antioxidant-nitric oxide (NO) donating compound SA-2 nanosuspension in normal trabecular meshwork-5 cells (NTM-5) and optic nerve crush (ONC) induced-mouse retinal ganglion cell (RGC) death model. SA-2 was encapsulated in poly-lactic-co-glycolic acid (PLGA) nanoparticles and reported earlier. Total nitrite concentrations of SA-2-NPs was determined using Griess assay in both buffer and NTM-5 cell supernatants. NTM-5 cells were treated with tert-butyl hydrogen peroxide (TBHP, EC50 = 5.5mM) at varying concentrations of SA-2 NPs (1, 0.5, 0.25, 0.125 % w/v) and cell viability was measured. 12-week-old C57BL/6J mice were subjected to ONC injury in the left eye and were administered with either vehicle or 1% SA-2-NPs via intravitreal injections (2uL) or eye drops (5uL). Pattern ERG (PERG) was used to assess retinal function and RGC survival was determined using RBPMS-positive RGCs. Data were presented as mean ± SEM, n=3-8. 1% SA-2-NPs dose dependently increased NTM-5 cell viability and total nitrite concentrations (~50%) lasting over 4 days. There was significant improvement in PERG amplitudes (1.7-1.9 times) following SA-2-NP administration as well as increased RGC numbers (by ~20%) relative to ONC-ed eyes. SA-2 nanosuspension shows strong positive trend in protecting RGC's from oxidative stress-induced apoptosis in ONC rodent model. Further investigation is being done for improved dosing and signaling changes in response to SA-2 NPs therapy.Item Hybrid molecule SA-2 improves both mitochondrial respiration and glycolysis in primary human trabecular meshwork cells(2022) Amankwa, Charles E.; Gondi, Sudershan; Stankowska, Dorota; Acharya, SuchismitaPurpose: Oxidative stress (OS) caused by hypoxia/hyperoxia environment results in progressive loss of trabecular meshwork (TM) cells in primary open angle glaucoma (POAG). Our previous report demonstrated; a hybrid nitric oxide (NO) donor-antioxidant molecule SA-2 protect primary human (h) TM cells against t-butyl hydrogen peroxide (TBHP) -induced cell death and increased superoxide dismutase enzyme level. Here we investigated the effect of SA-2 on mitochondrial energy metabolism by measuring the respiration status, glycolysis rate and energy production. Methods: Primary hTM cells obtained from human donor eyes were seeded in 24-well culture plates (Seahorse XFe 24 Cell Mito Stress test kit, Agilent), and starved for 24h before treatment with SA-2 (1 µM,10µM,100µM, and 1mM). In a separate experiment, the cells were pretreated with TBHP (150µM) for 30 minutes, followed by the addition of SA-2 (10µM,100µM). After 24h, the mitochondrial complex inhibitors and uncoupling reagents (oligomycin, FCCP, rotenone/antimycin A) were added. The plate was analyzed for changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XFe24 analyzer following the manufacturer's instructions. Results: The mean OCR was significantly decreased (>70%) followed by increase in the mean ECAR (~3-fold) after treatment with TBHP compared to oligo/FCCP/rot treated cells, hereafter called as negative control. Treatment with SA-2 at 1 µM,10µM,100µM and 1mM concentrations increased both oligomycin/FCCP induced decrease in ATP production and maximal mitochondrial respiration followed by an increase in the mean ECAR compared to negative control. The mean OCR was higher in SA-2 (100µM) +TBHP treated cells followed by an increase in ECAR in SA-2 (10µM or 100µM) +TBHP treated cells than TBHP and negative control treated cells. N =2-3. Conclusion: Mitochondrial respiration was impaired after TBHP treatment to hTM cells following cell death. While most of the mitochondrial targeting anti-oxidant compounds increase OCR but not ECAR, we found the hybrid NO donor-anti-oxidant compound SA-2 increases ATP production, maximal mitochondrial respiration and increases glycolytic energy production in hTM cells. This finding provides a novel direction for further investigation into the effect of SA-2 and mitochondrial bioenergetics during OS-induced cell death.Item Intravitreal Endothelin-1 (ET-1) Injection Reduces Mitophagy in Retinal Ganglion Cells in MitoQC Mice(2023) Brooks, Calvin D.; Kodati, Bindu; Inman, Denise; Stankowska, Dorota; Krishnamoorthy, RaghuPurpose: The peptide endothelin-1 (ET-1), and its receptors are upregulated in the aqueous humor and retina in animal models of experimentally induced ocular hypertension, and have been shown to have a causative role in retinal ganglion cell (RGC) neurodegeneration. The purpose of this experiment was to assess the role of mitophagy in RGC neurodegeneration following intravitreal ET-1 administration in MitoQC mice. Methods: MitoQC mice (Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl on a C57BL/6 background) at the age of 3 months were used for the study. The mitochondria in these mice display both red and green fluorescence due to expression of a mCherry-GFP tag fused to the mitochondrial targeting sequence of an outer mitochondrial membrane protein, FIS1. When these mitochondria are trafficked to the lysosome for degradation, the green fluorescence is quenched, leaving only the red fluorescence. The MitoQC mice were intravitreally injected in both eyes with either ET-1 (1 nmole) or vehicle (water), and 72 hours following the injections the mice eyes were enucleated and retinal flat mounts were live-imaged using a Zeiss LSM 880 super resolution confocal microscope. Z-stack imaging was used to image the ganglion cell layer. For each Z layer, a threshold algorithm was used to define a region of interest (ROI) that included only areas with red fluorescence, after which red and green fluorescence were quantified for that ROI. Red/green fluorescence intensity was calculated and averaged per image. A red/green ratio larger than 1 is indicative of active mitophagy. This ratio was compared between ET-1 and vehicle-injected mice using a Mann-Whitney test (n=4 eyes per group). Results: At 72 hours after injection with ET-1, the average red/green fluorescence ratio in the RGCs was 0.86, while the vehicle-injected mice had an average red/green ratio of 1.29. These ratios were significantly different (P=0.0003), and the smaller red/green ratio in the ET-1 group indicates lesser mitophagy than the vehicle group. Conclusion: Mitophagy is known to be an important quality control mechanism for neuronal cell survival, and this study provides evidence that mitophagy is impaired by ET-1. The finding indicates that a decline in mitophagy may be associated with endothelin-mediated neurodegeneration in RGCs.Item Involvement of the c-Jun N-terminus kinase (JNK) pathway in Endothelin (ET-1) mediated neurodegeneration of retinal ganglion cells(2021) Kodati, Bindu; Stankowska, Dorota; Krishnamoorthy, Vignesh; Krishnamoorthy, RaghuPURPOSE: Endothelins contribute to neurodegeneration in glaucoma, however, the mechanisms are not completely understood. The goal of this study was to determine if JNK2 plays a causative role in endothelin-1 (ET-1)-mediated loss of RGCs in mice. METHODS: JNK2-/- and wild type (C57BL6) mice (n=4) were intravitreally injected in one eye with 2 nmoles of ET-1, while the contralateral eye was injected with 2 µl of vehicle. The mice were euthanized at 2 h and 24 h post-injection. Retinal sections from the JNK2-/- and wild type (C57BL6) mice were used for immunohistochemical analysis of the phosphorylation of JNK substrate, c-Jun. In a separate set of experiments, JNK2 -/- and wild type mice (n=6) were intravitreally injected with either 2 nmoles of ET-1 or vehicle, and euthanized 7 days post-injection. RGC counts and axonal degeneration were assessed. RESULTS: Intravitreal ET-1 administration produced a significant increase in immunostaining for phospho c-Jun in wild type mice which was appreciably lower in the JNK2 -/- mice. Following ET-1 administration, a significant (p< 0.05) 26% loss of RGCs was found in wild type mice, while JNK2-/- mice showed no significant (p=0.36) loss of RGCs. A significant decrease in the axonal counts and an increase in the collapsed axons was found in ET-1 injected eyes in wild type mice. CONCLUSION: JNK2 appears to play a major role in ET-1 mediated loss of RGCs in mice.Item Mechanisms of peptain-mediated neuroprotection in retinal ganglion cells(2022) Johnson, Gretchen A.; Pham, Jennifer; Kodati, Bindu; Krishnamoorthy, Raghu; Nagaraj, Ram; Stankowska, DorotaPURPOSE: To determine mechanisms underlying neuroprotective effects of the core peptide of alpha-B crystallin, peptain-1 (P1) conjugated to a cell-permeable peptide CPP (P1-CPP) in retinal ganglion cells (RGCs) in a rodent model of glaucoma. METHODS: Intraocular pressure (IOP) was elevated in Brown Norway (BN) rats and intravitreally injected with 2 µl of either P1-CPP or vehicle, once a week for a period of 2 weeks. Rats were euthanized, primary adult RGCs were isolated by the immunopanning method. Total RNA was isolated using the Trizol/column method. RNA-sequencing was performed using an Illumina platform. The resulting FASTQ files were uploaded into Galaxy for analysis with FASTQC, RNASTAR, feature counts, and finally DESeq2. The results from DESeq2 were then assessed with Qiagen's Ingenuity Pathway Analysis (IPA) to identify significantly upregulated pathways. Relative Creb-1 expression normalized to reference gene GAPDH was determined in IOP-P1-CPP and IOP-vehicle treated rat RGCs. Briefly, quantitative Polymerase Chain Reaction (qPCR) was performed using BioRad's PrimePCR Assay and SsoAdvanced Universal SYBR Green Supermix on the BioRad's CFX96 Real-Time System C1000 Touch Thermal Cycler. RESULTS: RNA-seq analysis from rat RGCs isolated following 2 weeks of IOP-elevation revealed that P1-CPP treated groups had several differentially expressed (DEGs), compared to vehicle-treated groups, including 6343 significantly upregulated and 5960 significantly downregulated. Some significantly upregulated pathways following P1-CPP treatment include phagosome formation, synaptic long-term depression, and CREB signaling in neurons. The IOP and vehicle-treated groups, when compared to the naïve group, demonstrated a decreased expression of members of the CREB signaling pathway (Creb-1, c-RAF, MEK1/2, ERK1/2, and p90RSK). This decline was prevented by P1-CPP treatment. Quantitative PCR further confirmed the RNA-seq findings of the increased expression of Creb-1 in P1-CPP treated rats compared to that of vehicle-treated group. CONCLUSIONS: Mechanism of action of P1-CPP in a rodent model of glaucoma includes the activation of the pro-survival CREB signaling pathway, phagosome formation, and long-term synaptic depression to prevent cell death and vision loss.Item Multifunctional small molecule TLR4 antagonist for treating ocular neovascularization(2017-03-14) Panda, Santosh; Cai, Jiyang; Stankowska, Dorota; Ufret-Vincenty, Rafael; Acharya, SuchismitaPurpose: The multifactorial pathological challenge of ocular neovascularization is difficult to address so far only by anti-VEGF therapy. We have tested our hypothesis that, a novel class of natural product derived compound with toll like receptor 4 (TLR4) antagonist activity can ameliorate the hyper-inflammation produced by macrophage/macroglia over activation as well as decrease choroidal neovascularization (CNV) size in mice. Methods: Inhibition of cytokines: Mouse bone marrow derived macrophages were treated with high mobility group box1 (HMGB1, 100 ng/mL), an endogenous TLR4 ligand for 8 hours, with or without 100 mg/mL of test compounds. The mRNA levels of TNF-a, iNOS were measured by real-time RT-PCR, and normalized to the control cells. Inhibition of VEGF production: ARPE-19 cells were treated either with 100ng/mL or without HMGB1 along with the compounds (50μg/mL) for 24 h. Supernatants were collected and assayed using human VEGF ELISA kit according to manufacturer’s instructions. Experiments were repeated three times and one way ANOVA was used for statistical analysis. Inhibition of CNV: Laser CNV was induced in C57BL/6 mice (male, 10-12 weeks, n = 5). Each eye received 4 laser burns. The compounds (200 μg/mL) or BSS (vehicle) were administered by IP injection once before and once daily up to 10 days following laser injury. Fundus fluorescein angiography and optical coherence tomography was used to visualize the CNV lesions. RPE/choroid/sclera flat mounts were prepared and stained with both FITC conjugated isolectin B4 and anti-ICAM-2 antibody to quantitatively measure the size of CNV lesion. Results: Compound treatment significantly (p Conclusions: Our results are consistent with our hypothesis that this novel class of compounds will decrease ocular inflammation and neovascularization. Further structure optimization of the lead compound and TLR4 dependent and independent mechanistic investigation are underway.Item Neuroprotection of human and rodent retinal ganglion cells by a hybrid antioxidant-nitric oxide donor small molecule, SA-2(2022) Pham, Jennifer; Johnson, Gretchen A.; Acharya, Suchismita; Stankowska, DorotaPURPOSE: Current treatments of glaucoma are aimed at lowering intraocular pressure (IOP), which is a key driver of retinal ganglion cell (RGC) death. Another contributing factor to RGC death is exposure to reactive oxygen species (ROS). At present, there is no FDA-approved neuroprotective treatment to prevent glaucomatous optic neuropathy and loss of RGCs. Our novel hybrid molecule, SA-2, contains both a nitric oxide (NO) donating group to lower IOP and a ROS scavenging group to protect RGCs. We hypothesize that SA-2 will inhibit the death of RGCs in an in vitro and an ex vivo neurotrophic factor deprivation model. METHODS: Retinal punches from human explants (n=4 donors/experiments) were isolated and treated with either SA-2 [1 mM] or vehicle and maintained without neurotrophic factors for 7 days ex vivo. In each experiment, 4 baseline retinal explants were collected on day 0. At the end of the experiment, explants were immunostained with RBPMS and Brn-3a (RGC-specific markers) and cell survival was analyzed. In three biological replicates, primary RGCs were isolated from rat pups and treated with either SA-2 (1 mM, 100 µM) or vehicle with or without neurotrophic factors for 48 h. Active caspase 3 and 7 assay was performed and apoptotic cell counts were analyzed. In another set of experiments, rat retinal explants were isolated and incubated with tert-Butyl hydroperoxide (TBHP) along with either SA-2 [1 mM] or vehicle for 2 h (n=2-4 explants/group). Production of superoxide by mitochondria was assessed using MitoSOX reagent according to manufacturer instructions. All cell counts were performed in a masked manner using ImageJ Software. One-way ANOVA or nonparametric Kruskal-Wallis was used for statistical analysis by GraphPad Prism 9 Software. RESULTS: In ex vivo human retinal explants, there was a significant increase in RGC survival by 39% in the SA-2 treated group compared to the vehicle group at day 7 (p< 0.0001). In rodent primary RGCs, SA-2 mediated a significant decrease in apoptotic cells by 30% (p< 0.01) and a 67% (p< 0.05) decrease in dead cell count. In rodent retinal explants, there was a significant decrease (by 59%, p< 0.0001) in the production of superoxide by mitochondria in the TBHP and SA-2 treated group, compared to the TBHP vehicle group. CONCLUSION: SA-2 was shown to be effective at preserving retinal ganglion cell survival in human retinal explants, rat retinal explants and primary rat RGCs by preventing apoptosis and protecting the cells from oxidative stress.Item Novel Compound SA-21 with Antioxidant Capability - The Prospect for Neuroprotection in Glaucoma.(2021) Ferguson, Jonathan; Johnson, Gretchen; Amankwa, Charles E.; Zhang, Wei; Li, Linya; Gondi, Sudershan; Funk, Arlene; Ellis, Dorette; Acharya, Suchismita; Stankowska, DorotaPurpose: Current treatments for glaucoma do not fully address neurodegeneration of retinal ganglion cells (RGCs). Our objective was to determine if compound SA-21, a hybrid superoxide dismutase and glutathione mimetic, could inhibit death of trabecular meshwork cells (NTM5), RGCs and rescue the functional decline of RGCs in an optic nerve crush (ONC) model. Methods: The structure of SA-21 was confirmed by magnetic resonance (NMR) spectroscopy and mass spectrometry. Reactive oxygen species (ROS) release was performed using pyrogallol assay. NTM5 cells were oxidatively stressed with TBHP (5.5mM) or vehicle in the presence of SA-21 (1mM, 100µM, 10µM, 1µM) for 24h. Cell survival was assessed by MTT assay. C57BL6 male mice (12-weeks old, n=4-5) were anesthetized, underwent ONC surgery, and at day 0 and 3 were intravitreally injected with 1% SA-21 (2µl) or vehicle. On day 7, pattern electroretinogram (PERG) was performed, animals were euthanized, and the number of surviving RBPMS-positive RGCs were counted. Results: SA-21 (1mM) treatment significantly decreased ROS production (~50%) measured by pyrogallol assay and increased NTM5 cell viability (~20%, p< 0.0094) following TBHP treatment compared to cells treated with the vehicle. ONC produced a 48% loss of RGCs, which was decreased in SA-21 treated mice (by ~10%) and demonstrated a trend in increase in PERG amplitude. Conclusions: SA-21 compound has antioxidant capability and protects NTM5 cells from oxidative stress. Intravitreally injected SA-21 at the selected dose in mice demonstrated trend in neuroprotection but further investigation is required.Item Novel Peptain for Neuroprotection in Glaucoma(2020) Stankowska, Dorota; Krishnamoorthy, Vignesh; Krishnamoorthy, Raghu; Chaphalkar, Renuka; Nagaraj, Ram; Nahomi, Rooban; Nam, Mi-hyun; Beall, Kallen; Brodrick, Ashley; Kodati, BinduPurpose: To determine if intravitreal administration of the core peptide of [alpha]-B crystallin, peptain-1 (P1) conjugated to a cell permeable peptide (CPP) (named P1-CPP) could inhibit retinal ganglion cell (RGC) death and functional decline in a rodent model of glaucoma. Methods: Primary RGCs were treated with endothelin-3 (ET-3) in the presence of either P1-CPP (12.5 µg/ml) or vehicle, following which RGC survival was assessed. In a different set of experiments, intraocular pressure (IOP) was elevated in one eye of Brown Norway (BN) rats and intravitreally injected with 2 µl of either P1-CPP or vehicle, once in a week for 6 weeks. RGC function was assessed by the pattern electroretinogram (PERG) amplitude. Retinal flat mounts were imaged and surviving RGCs were counted. Results: ET-3 treatment lead to 24% of RGCs loss compared to vehicle treated cells (p< 0.0001). P1-CPP treatment significantly lowered the ET-3-mediated cell loss (7% cell death, p< 0.001). IOP elevation in vehicle injected animals produced 11% and 27% loss of RGCs, in central and peripheral retina respectively, which was significantly lower in P1-CPP treated rats (7% loss in both eccentricities, **p< 0.01). P1-CPP treatment also promoted axonal protection during IOP elevation. IOP elevation caused 63% decline in the PERG amplitude (*p< 0.03) in comparison with naïve rats, which was sustained by P1-CPP treatment. Conclusion:The intravitreally injected P1-CPP provides cellular as well as functional protection of RGCs, which could facilitate neuroprotection against glaucomatous insults.Item P1-CPP promotes Foxo1 and Creb signaling and reduces apoptosis in Neurotrophic Factor-Deprived Primary Retinal Ganglion Cells(2023) Johnson, Gretchen; Pham, Jennifer; Krishnamoorthy, Raghu; Nagaraj, Ram; Stankowska, DorotaPurpose: To elucidate the intracellular mechanisms underlying neuroprotective effects of the core peptide of a-B crystallin, peptain-1 (P1) conjugated to a cell-permeable peptide CPP (P1-CPP) in primary retinal ganglion cells (RGCs). Targets of the investigation were limited to Creb1, Bak1/Bad, and Foxo1, based upon RNA sequencing data obtained from RGCs of IOP-elevated rats treated with P1-CPP in comparison with the vehicle. Methods: Primary RGCs isolated from Sprague Dawley rat pups were deprived of neurotrophic factors (NT) namely, BDNF, CNTF, and Forskolin for 48 hours, either in the presence or absence of P1-CPP (4µM). After the treatments, RNA isolation was carried out using Trizol reagent. Subsequently, cDNA synthesis and qPCR analysis of the target genes expression, including Creb1 (n=2), Foxo1 (n=3), and Bak1 (n=3), was performed. Another set of RGCs subjected to the same treatments was fixed with 4% paraformaldehyde for 20 minutes and used for immunocytochemical analyses of p-CREB (n=3), FOXO1 (n=3), and BAD (n=3) protein expression. Immunostaining with an RBPMS antibody was used as an RGC marker. N indicates experimental repeats. Results: Following NT deprivation, there was an increase in mRNA expression of Creb1 (2-fold) in RGCs treated with P1-CPP, compared to the vehicle-treated RGCs. Moreover, the phosphorylated (active) form, p-CREB, was increased (by 102%; p=0.04) in primary RGCs treated with P1-CPP, compared to the vehicle-treated group. Pro-apoptotic Bak1 mRNA expression was not changed in the P1-CPP-treated RGCs compared to the vehicle-treated group. Primary RGCs stained for BAD protein showed a decrease (by 62%; p=0.08) in the P1-CPP treated group compared to the vehicle-treated RGCs. Foxo1 mRNA levels were increased by more than 2-fold in the P1-CPP treated RGCs, compared to the vehicle-treated RGCs. FOXO1 protein was also elevated in primary RGCs treated with P1-CPP compared to the vehicle group (by 59%). Conclusions: P1-CPP is neuroprotective against neurotrophic factor deprivation through multiple mechanisms, including early changes in the expression of mitochondrial homeostasis regulator Foxo1, activation of the pro-survival CREB pathway, and inhibition of pro-apoptotic members of the BCL-2 family of proteins.Item SMALL MOLECULE SA-2 USEFUL FOR TREATING ALZHEIMER'S DISEASE(2020) Weston, Courtney; Mahapatra, Vasu; Stankowska, Dorota; Acharya, Suchismita; Amankwa, Charles E.Purpose: Alzheimer's disease (AD) is a neurodegenerative disorder affecting mainly the aged population. Oxidative stress is known to play important role in the pathogenesis of AD and is increased in the brain with aging. Reactive oxygen species (ROS) are released in excess which leads to neuronal atrophy and long-term tissue damage. Low levels of nitric oxide (NO) is beneficial for neurogenesis and has antioxidant activity. Our goal is to synthesize novel hybrid molecules with dual antioxidant-nitric oxide donating activity against oxidative stress-induced hippocampal neuronal HT-22 cell deaths. Method: The compounds (SA-2, SA-9 and SIN-1) were synthesized and encapsulated into nanoparticles (NP). Structure of the compounds and size of nanoparticles were confirmed using 1HNMR and DLS. Mouse HT-22 cells were seeded in 96 well plates, tert-butyl hydrogen peroxide (TBHP,10µM- 5mM) was added and incubated at 2, 4, 6 and 12 hours. MTT assay (Promega) was performed to determine the effective concentration (EC50) of TBHP. For cell viability study cells were treated with/without TBHP and compounds (SA-2, SA-2 NP, SA-9, SIN-1, 1µM, 10 µM and 100 µM). After 6 hours, MTT assay was performed to assess cell proliferation. Results: We found the EC50 of TBHP is 80µM (6h). Compound SA-2 (100µM) and SA-2 NP (0.1%, equivalent to 25µM of free SA-2) provided significant protection to TBHP induced HT-22 cell death. Conclusion: SA-2 NP is highly effective than free SA-2. Further assessments are ongoing.Item Synthesis and in vitro study of dual acting hybrid compound for treating Glaucoma(2020) Stankowska, Dorota; Acharya, Suchismita; Ellis, Dorette; Weston, Courtney; Li, Linya; Gondi, SudershanPurpose: Glaucoma presents with high intraocular pressure (IOP) due to decreased aqueous humor outflow and impaired trabecular meshwork (TM). Increased IOP affects the optic nerve head leading to degeneration of retinal ganglion cells (RGC's). Currently many therapies aim to reduce IOP, however they do not address the damage done to the RGCs. Our goal is to find a multifunctional molecule that can lower IOP while also being neuroprotective. For this project, we planned to synthesize a series of compounds and evaluate their cellular activity in primary human TM cells. Methods: Compounds SA-2, PLGA encapsulated SA-2 nanoparticles (NP) and SA-24 were synthesized. Proton NMR and dynamic light scattering methods were used to determine structure and particle size. Greiss assay was used to assess total nitrite and reactive oxygen species (ROS) scavenging assay was performed following the manufacturer's protocol. Next, primary hTM-80 cells were seeded in 96 well plates to confluency, and exposed to tert-butyl hydrogen peroxide (TBHP, 300uM) for 15 min. followed by treatment with the compounds. The cell proliferation was measured using MTT assays (Promega). The experiments were done in quadruplicates. Results: SA-2, SA-2NP and SA-24 were equally effective in scavenging ROS at 250 µM and SA-2 provided highest amount of NO. Compound SA-2 (200uM) and SA-2NP (1%) significantly protect TBHP induced decreased cell proliferation. Conclusion: Compound SA-2 has both NO releasing and ROS scavenging activity. As expected, SA-2NP has slow release profile and better proliferative activity.