Biology subjects

Chrenek, M. A.

Publications and source records attributed to Chrenek, M. A..

7 recordsLinked to original sources

bioRxiv2026

Transcriptomic Profiling of High vs. Low Flow Regions of Mouse and Human Trabecular Meshwork

PurposeAqueous humor outflow through the trabecular meshwork (TM) is segmental, demonstrating high flow (HF) and low flow (LF) regions. Here, we investigate transcriptomic differences between flow regions in naive mouse and non-glaucomatous human TM tissue to better understand intraocular pressure (IOP) regulation. MethodsHuman eyes (<6 hours postmortem) from two donors were perfused with a fluorescent tracer to identify HF and LF regions and fixed. In parallel, one pair of 8-month-old C57Bl/6J mouse eyes were similarly processed. Sagittal sections from HF and LF regions underwent whole-transcriptome spatial profiling. Differential expression and gene set variation analysis were conducted to identify transcript and pathway-level differences between HF vs. LF TM. Selected targets were validated by immunolabeling. ResultsGenes with relevance to TM outflow were identified as significantly differentially expressed in the human dataset, including ADAM metallopeptidase domain 15 (ADAM15), vimentin (VIM), chitinase 3-like 1 (CHI3L1), and several transcription factors (e.g., FOS, JUNB, ESR1). Pathways related to epigenetic modifications were also differentially enriched in human eyes. In both human and mouse eyes, myocilin was significantly upregulated in HF regions, despite greater protein labeling in LF regions of human eyes. In both species, rho-kinase signaling pathways showed increased enrichment in LF regions, while cell stress pathways, and TNF- signaling were increased in HF TM. ConclusionsHF regions maintain a more active stress response that facilitates greater outflow, whereas LF regions exhibit more matrix accumulation and contractility. This characterization of segmental flow regions can inform future studies to target trabecular outflow and lower IOP.

bioengineering↗

bioRxiv2025

Long-Term Functional Rescue of Trauma-Induced Vision Loss by a Novel, Small Molecule TrkB Activator

Brain-derived neurotrophic factor (BDNF) signaling through the tropomyosin-related kinase B (TrkB) receptor promotes neuronal growth and survival following an injury. However, its short half-life and pleiotropic effects limit the clinical use of BDNF as a therapy in neurodegenerative disorders. Identification of novel and selective TrkB activators may ameliorate the damage caused to retinal neurons during eye-related injuries, and may reduce adverse visual outcomes associated with visual trauma. We previously described a selective TrkB agonist, N-[2-(5-hydroxy-1H-indol-3-yl) ethyl]-2-oxopiperidine-3-carboxamide (HIOC), that reduces the decline in visual function in a mouse model of ocular trauma (1). Using the lead optimization approach, we subsequently synthesized a fluoropyridine analog of HIOC, 2-fluoro-N-(2-(5-hydroxy-1H-indol-3-yl) ethyl) nicotinamide (HIFN), which also successfully activates TrkB. HIFN is a more potent TrkB activator than the parent compound, HIOC. Further, treatment with HIFN demonstrated neuroprotection in an animal model of overpressure ocular blast injury, ameliorating blast-related visual functional decline. Mice treated with HIFN had better visual acuity, contrast sensitivity, and retinal function supported by enhanced survival of retinal ganglion cells compared to vehicle-treated animals. Moreover, HIFN exhibited better protective effects than HIOC. The therapeutic effects of HIFN were attributed to TrkB activation, as blocking the receptor with a selective receptor antagonist (ANA-12) abrogated the neuroprotection. Together, our results identify HIFN, a novel TrkB receptor activator, as a strategy for decreasing retinal degeneration and progressive vision loss associated with traumatic ocular injury. In addition, this compound may have broader applications treating other diseases with altered TrkB activity.

neuroscience↗

bioRxiv2024

Oral supplementation with Nicotinamide Riboside treatment protects RGCs in DBA/2J mouse model

PurposeThe aim of this study was to test whether oral administration of nicotinamide riboside (NR), the nicotinamide adenine dinucleotide (NAD+) precursors, protect retina ganglion cells (RGCs) from neurodegeneration in DBA/2J (D2) mice, which is a widely used mouse model of age-related inherited glaucoma. MethodOral NR or NAM administration (NR low dose: 1150mg/kg; NR high dose: 4200mg/kg; NAM low dose group: 500mg/kg; NAM high dose: 2000mg/kg of body weight per day) essentially started when D2 mice were 4 or 9 months old and continued up to 12 months old. Control cohort identically received food/water without NAM or NR. Intraocular pressure (IOP) was measured every month until experiment completion. Pattern electroretinography (PERG) was recorded. Retinas were harvested for whole mount immunofluorescence staining with RGCs marker Brn3a and imaged by fluorescent confocal microscopy. Optic nerves were harvested for axon staining and quantification. Retinal NAD+ levels were enzymatically assayed. ResultsNR oral supplementary treatment started at 4 months old robustly increased retinal NAD+ levels in D2 mice (NRHigh vs. vehicle: 273.7{+/-}23.59% vs. 108.70{+/-}12.10%, p<0.001). In aged vehicle group (12 months old), there was significantly diminution of the P1 and N2 components of PERG response compare with naive group (naive vs. vehicle: P1: 7.82{+/-} 0.70uV vs 1.63{+/-} 0.17uV, p<0.0001; N2: -13.29{+/-} 0.83uV vs. -3.22{+/-} 0.27uV, p<0.0001; Kruskal-Wallis test with Dunn s multiple comparison test). NR treatment preserved aged D2 visual function when mice were 9 and 12 months old. In addition, long-term NR high dose treatment significantly protected against total RGCs loss and optic nerve atrophy (RGC: NRHigh vs. vehicle: 1412{+/-}62.00vs 475.2{+/-}94.68 cells/field, p<0.00001; axon numbers: NRHigh vs. vehicle: 23990{+/-}1159 vs 8573{+/-}1160, n=41-53, p<0.0001). Furthermore, long-term NR supplementation prevent iris depigmentation and delayed IOP elevation. ConclusionNR oral supplementary treatment significantly preserved RGC and axon numbers, potentially preserves retinal function via elevated retinal NAD+ level in aged D2 mice. Interestingly, NR treatment also prevented iris atrophy, delayed IOP elevation associated with this glaucoma model. NR oral supplementation thus treated several aspects of murine pigment dispersion glaucoma. Given parallels between this model and glaucoma in human, out data indicate that NR is worth exploring as a therapeutic candidate in treatment of glaucoma.

neuroscience↗

bioRxiv2024

Loss of Pigment Epithelium Derived Factor Sensitizes C57BL/6J Mice to Light-Induced Retinal Damage

5.1PurposePigment epithelium-derived factor (PEDF) is a neurotrophic glycoprotein secreted by the retinal pigment epithelium (RPE) that supports retinal photoreceptor health. Deficits in PEDF are associated with increased inflammation and retinal degeneration in aging and diabetic retinopathy. We hypothesized that light-induced stress in C57BL/6J mice deficient in PEDF would lead to increased retinal neuronal and RPE defects, impaired expression of neurotrophic factor Insulin-like growth factor 1 (IGF-1), and overactivation of Galectin-3-mediated inflammatory signaling. MethodsC57BL/6J mice expressing the RPE65 M450/M450 allele were crossed to PEDF KO/KO and wildtype (PEDF +/+) littermates. Mice were exposed to 50,000 lux light for 5 hours to initiate acute damage. Changes in visual function outcomes were tracked via electroretinogram (ERG), confocal scanning laser ophthalmoscopy(cSLO), and spectral domain optical coherence tomography (SD-OCT) on days 3, 5, and 7 post-light exposure. Gene and protein expression of Galectin-3 were measured by digital drop PCR (ddPCR) and western blot. To further investigate the role of galectin-3 on visual outcomes and PEDF expression after damage, we also used a small-molecule inhibitor to reduce its activity. ResultsFollowing light damage, PEDF KO/KO mice showed more severe retinal thinning, impaired visual function (reduced a-, b-, and c-wave amplitudes), and increased Galectin-3 expressing immune cell infiltration compared to PEDF +/+. PEDF KO/KO mice had suppressed damage-associated increases in IGF-1 expression. Additionally, baseline Galectin-3 mRNA and protein expression were reduced in PEDF KO/KO mice compared to PEDF +/+. However, after light damage, Galectin-3 expression decreases in PEDF +/+ mice but increases in PEDF KO/KO mice without reaching PEDF +/+ levels. Galectin-3 inhibition worsens retinal degeneration, reduces PEDF expression in PEDF +/+ mice, and mimics the effects seen in PEDF knockouts. ConclusionsLoss of PEDF alone does not elicit functional defects in C57BL/6J mice. However, under light stress, PEDF deficiency significantly increases severe retinal degeneration, visual deficits, Galectin-3 expression, and suppression of IGF-1 than PEDF +/+. PEDF deficiency reduced baseline expression of Galectin-3, and pharmacological inhibition of Galectin-3 worsens outcomes and suppresses PEDF expression in PEDF +/+, suggesting a novel co-regulatory relationship between the two proteins in mitigating light-induced retinal damage.

molecular biology↗

bioRxiv2020

THE CIRCADIAN CLOCK IN THE RETINAL PIGMENT EPITHELIUM CONTROLS THE DIURNAL RHYTHM OF PHAGOCYTIC ACTIVITY

The diurnal peak of phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segments (POS) is under circadian control, and it is believed that this process involves interactions from both the retina and RPE. Previous studies have demonstrated that a functional circadian clock exists within multiple retinal cell types and RPE cells. Thereby, the aim of the current study was to determine whether the circadian clock in the retina and or RPE controls the diurnal phagocytic peak of photoreceptor outer segments and whether selective disruption of the circadian clock in the RPE would affect RPE cells function and the viability during aging. To that aim, we first generated and validated an RPE tissue-specific KO of the essential clock gene, Bmal1, and then we determined the daily rhythm in phagocytic activity by the RPE in mice lacking a functional circadian clock in the retina or RPE. Then using electroretinography, spectral domain-optical coherence tomography, and optomotor response measurements of visual function we determined the effect of Bmal1 removal in young (6-month old) and old (18-month old) mice. RPE morphology and lipofuscin accumulation was also determined in young and old mice. Our data show that the circadian clock in the RPE controls the daily diurnal phagocytic peak of POS. Surprisingly, the lack of a functional RPE circadian clock or the diurnal phagocytic peak does not result in any detectable age-related degenerative phenotype in the retina or RPE. Thus, our results demonstrate that the loss of the circadian clock in the RPE or the lack of the daily peak in phagocytosis of POS does not result in deterioration of photoreceptors or the RPE during aging.

neuroscience↗

bioRxiv2019

Systemic Treatment with Nicotinamide Riboside is Protective in Three Mouse Models of Retinal Degeneration

Purpose: The retina is highly metabolically active, suggesting that metabolic dysfunction could underlie many retinal degenerative diseases. Nicotinamide adenine dinucleotide (NAD+) is a cofactor and a co-substrate in several cellular energetic metabolic pathways. Maintaining NAD+ levels may be therapeutic in retinal disease since retinal NAD+ levels decline with age and during retinal damage or degeneration. The purpose of this study was to investigate whether systemic treatment with nicotinamide riboside (NR), a NAD+ precursor, is protective in disparate models of retinal damage or degeneration. MethodsThree mouse models of retinal degeneration were tested: an albino mouse model of light-induced retinal degeneration (LIRD) and two models of retinitis pigmentosa (RP), including a mouse line deficient in interphotoreceptor binding protein (IRBP) gene expression (IRBP KO), and a naturally-occuring cGMP phosphodiesterase 6b mutant mouse model of RP (the Pde6brd10 mouse). Mice were intraperitoneally (IP) injected with PBS or NR at various times relative to damage or degeneration onset. One to two weeks later, retinal function was assessed by electroretinograms (ERGs) and retinal morphology was assessed by optical coherence tomography (OCT). Afterwards, retina sections were H&E stained for morphological analysis or by terminal deoxynucleiotidyl transferase dUTP nick and labeling (TUNEL). Retinal NAD+/NADH levels were enzymatically assayed. ResultsThe retinal degeneration models exhibited significantly suppressed retinal function, and where examined, severely disrupted photoreceptor cell layer and significantly decreased numbers of nuclei and increased accumulation of DNA breaks as measured by TUNEL-labeled cells in the outer nuclear layer (ONL). These effects were prevented by various NR treatment regimens. IP treatment with NR also resulted in increased levels of NAD+ in retina. ConclusionsThis is the first study to report protective effects of NR treatment in mouse models of retinal degeneration. The positive outcomes in several models, coupled with human tolerance to NR dosing, suggest that maintaining retinal NAD+ via systemic NR treatment should be further explored for clinical relevance.

molecular biology↗

bioRxiv2019

Dopamine 2 receptor signaling controls the daily burst in phagocytic activity in the mouse retinal pigment epithelium

PurposeA burst in phagocytosis of spent photoreceptor outer fragments by retinal pigment epithelium (RPE) is a rhythmic process occurring 1-2 hours after the onset of light. This phenomenon is considered crucial for the health of the photoreceptors and RPE. We have recently reported that dopamine, via dopamine 2 receptor (D2R), shifts the circadian rhythm in the RPE.\n\nMethodsHere, we first investigated the impact of the removal of D2R on the daily peak of phagocytosis by RPE and then we analyzed the function and morphology of retina and RPE in the absence of D2R.\n\nResultsD2R KO mice do not show a daily burst of phagocytic activity after the onset of light. Also, in contrast to control, phosphorylation of FAK did not increase significantly in KO mice at ZT1. RNA sequencing revealed a total of 394 differentially expressed genes (DEGs) between ZT23 and ZT1 in the control mice, whereas in D2R KO mice, we detected 1054 DEGs. Pathway analysis of the gene expression data implicated integrin signaling to be one of the upregulated pathways in control but not in D2R KO mice. No difference in retinal thickness, visual function, or morphology of RPE cells was observed between WT and D2R KO mice at the age of 3 and 12 months.\n\nConclusionsOur data suggest that removal of D2R prevents the burst in phagocytosis and a related increase in the phosphorylation of FAK after light onset. The pathway analysis points towards a putative role of D2R in controlling integrin signaling, which is known to play an important role in the control of the daily burst of phagocytosis by the RPE. Our data also indicate that the absence in the burst of phagocytic activity in the early morning does not produce any apparent deleterious effect on the retina or RPE up to one year of age.

cell biology↗