a-cost-effective-minimally-invasive-protocol-for-chronic-multi-site

A Cost-Effective and Minimally Invasive Protocol for Chronic Multi-Site Electroencephalography Recording in Freely Moving Mice

Electroencephalography (EEG) serves as a fundamental tool for tracking electrical activity in the brain and has become increasingly important in both ...

Medical College of Georgia at Augusta University

Oxoaldehyde stress has recently emerged as a major source of tissue damage in aging and age-related diseases. The prevailing mechanism involves methylglyoxal production during glycolysis and modification of arginine residues through the formation of methylglyoxal hydroimidazolones (MG-H1). We now tested the hypothesis that oxidation of vitamin C (ascorbic acid or ASA) contributes to this damage when the homeostatic redox balance is disrupted especially in ASA-rich tissues such as the eye lens and brain. MG-H1 measured by liquid chromatography mass spectrometry is several fold increased in the lens and brain from transgenic mice expressing human vitamin C transporter 2 (hSVCT2). Similarly, MG-H1 levels are increased two- to fourfold in hippocampus extracts from individuals with Alzheimer's disease (AD), and significantly higher levels are present in sarkosyl-insoluble tissue fractions from AD brain proteins than in the soluble fractions. Moreover, immunostaining with antibodies against methylglyoxal hydroimidazolones reveals similar increase in substantia nigra neurons from individuals with Parkinson's disease. Results from an in vitro incubation experiment suggest that accumulated catalytic metal ions in the hippocampus during aging could readily accelerate ASA oxidation and such acceleration was significantly enhanced in AD. Modeling studies and intraventricular injection of  C-labeled ASA revealed that ASA backbone carbons 4-6 are incorporated into MG-H1 both in vitro and in vivo, likely via a glyceraldehyde precursor. We propose that drugs that prevent oxoaldehyde stress or excessive ASA oxidation may protect against age-related cataract and neurodegenerative diseases.

Vitamin C is a source of oxoaldehyde and glycative stress in age-related cataract and neurodegenerative diseases.

Age-related cataracts (ARC) are the primary cause of blindness worldwide, and oxidative stress is considered the central pathogenesis of age-related cataractogenesis. Interestingly, ample evidence suggests that there is no remarkable apoptosis present in aged and cataractous human lenses despite the profound disruption of redox homeostasis, raising an essential question regarding the existence of other cell death mechanisms. Here we sought to explore the lens epithelial cell's (LEC) susceptibility to ferroptosis after documentation has concluded that aged and cataractous human lenses manifest with increased reactive oxygen species (ROS) formation, elevated lipid peroxidation, and accumulative intracellular redox-active iron, constituting the three hallmarks of ferroptosis during aging and cataractogenesis. Here we show that very low concentrations of system X inhibitor Erastin (0.5 μM) and glutathione peroxidase 4 (GPX4) inhibitor RSL3 (0.1 μM) can drastically induce human LEC (FHL124) ferroptosis in vitro and mouse lens epithelium ferroptosis ex vivo. Depletion of intracellular glutathione (GSH) in human LECs and mouse lens epithelium significantly sensitizes ferroptosis, particularly under RSL3 challenge. Intriguingly, both human LECs and the mouse lens epithelium demonstrate an age-related sensitization of ferroptosis. Transcriptome analysis indicates that clusters of genes are up-or down-regulated in aged LECs, impacting cellular redox and iron homeostases, such as downregulation of both cystine/glutamate antiporter subunits SLC7A11 and SLC3A2 and iron exporter ferroportin (SLC40A1). Here, for the first time, we are suggesting that LECs are highly susceptible to ferroptosis. Moreover, aged and cataractous human lenses may possess more pro-ferroptotic criteria than any other organ in the human body.

Aging lens epithelium is susceptible to ferroptosis.

The present study aims to understand the mechanism of the lens epithelial cell's strong anti-apoptotic capacity and survival in the mature human lens that, on the one hand, maintains lens transparency over several decades, while on the other hand, increases the risk of posterior capsule opacification (PCO). Here we compared FHL124 cells and HeLa cells, spontaneously immortalized epithelial cell lines derived from the human lens and cervical cancer cells, respectively, of their resistance to TNFα-mediated cell death. TNFα plus cycloheximide (CHX) triggered almost all of HeLa cell death. FHL124 cells, however, were unaffected and able to block caspase-8 activation as well as prevent caspase-3 and PARP-1 cleavage. Interestingly, despite spontaneous NFκB and AP-1 activation and upregulation of multiple cell survival/anti-apoptotic genes in both cell types, only FHL124 cells were able to survive the TNFα challenge. After screening and comparing the cell survival genes, cFLIP was found to be highly expressed in FHL124 cells and substantially upregulated by TNFα stimulation. FHL124 cells with a mild cFLIP knockdown manifested a profound apoptotic response to TNFα stimulus similar to HeLa cells. Most importantly, we confirmed these findings in an ex vivo lens capsular bag culture system. In conclusion, our results show that cFLIP is a critical gene that is regulating lens epithelial cell survival.

Cellular FLICE-like inhibitory protein (cFLIP) critically maintains apoptotic resistance in human lens epithelial cells.

Crystallins, the most prevalent lens proteins, have no turnover throughout the entire human lifespan. These long-lived proteins are susceptible to post-synthetic modifications, including oxidation and glycation, which are believed to be some of the primary mechanisms for age-related cataractogenesis. Thanks to high glutathione (GSH) and ascorbic acid (ASA) levels as well as low oxygen content, the human lens is able to maintain its transparency for several decades. Aging accumulates substantial changes in the human lens, including a decreased glutathione concentration, increased reactive oxygen species (ROS) formation, impaired antioxidative defense capacity, and increased redox-active metal ions, which induce glucose and ascorbic acid degradation and protein glycation. The glycated lens crystallins are either prone to UVA mediated free radical production or they attract metal ion binding, which can trigger additional protein oxidation and modification. This vicious cycle is expected to be exacerbated with older age or diabetic conditions. ASA serves as an antioxidant in the human lens under reducing conditions to protect the human lens from damage, but ASA converts to the pro-oxidative role and causes lens protein damage by ascorbylation in high oxidation or enriched redox-active metal ion conditions. This review is dedicated in honor of Dr. Frank Giblin, a great friend and superb scientist, whose pioneering and relentless work over the past 45 years has provided critical insight into lens redox regulation and glutathione homeostasis during aging and cataractogenesis.

Protein posttranslational modification (PTM) by glycation: Role in lens aging and age-related cataractogenesis.

Ultraviolet B (UVB) has been well documented to induce capsular cataracts; however, the mechanism of the lens epithelial cell-mediated repair process after UVB irradiation is not fully understood. The purpose of this study was to better understand lens epithelial cell repair after UVB-induced epithelium damage.

Mitotic Activation Around Wound Edges and Epithelialization Repair in UVB-Induced Capsular Cataracts.

Posterior capsule opacification (PCO) is a frequent complication after cataract surgery, and advanced PCO requires YAG laser (Nd: YAG) capsulotomy, which often gives rise to more complications. Lens epithelial cell (LEC) proliferation and transformation (i.e., epithelial-mesenchymal transition (EMT)) are two critical elements in PCO initiation and progression pathogenesis. While PCO marginally impacts aged cataract surgery patients, PCO incidences are exceptionally high in infants and children undergoing cataract surgery. The gene expression of lens epithelial cell aging and its role in the discrepancy of PCO prevalence between young and older people have not been fully studied. Here, we conducted a comprehensive differentially expressed gene (DEG) analysis of a cell aging model by comparing the early and late passage FHL124 lens epithelial cells (LECs). , TGFβ2, cell treatment, and  mouse cataract surgical models were used to validate our findings. We found that aged LECs decelerated rates of cell proliferation accompanied by dysregulation of cellular immune response and cell stress response. Surprisingly, we found that LECs systematically downregulated epithelial-mesenchymal transition (EMT)-promoting genes. The protein expression of several EMT hallmark genes, e.g., fibronectin, αSMA, and cadherin 11, were gradually decreased during LECs aging. We then confirmed these findings  and found that aged LECs markedly alleviated TGFβ2-mediated EMT. Importantly, we explicitly confirmed the  findings from the  mouse cataract surgery studies. We propose that both the high proliferation rate and EMT-enriched young LECs phenotypic characteristics contribute to unusually high PCO incidence in infants and children.

Aged Lens Epithelial Cells Suppress Proliferation and Epithelial-Mesenchymal Transition-Relevance for Posterior Capsule Opacification.

Mouse models are valuable tools in studying lens biology and biochemistry, and the Cre-loxP system is the most used technology for gene targeting in the lens. However, numerous genes are indispensable in lens development. The conventional knockout method either prevents lens formation or causes simultaneous cataract formation, hindering the studies of their roles in lens structure, growth, metabolism, and cataractogenesis during lens aging. An inducible Cre-loxP mouse line is an excellent way to achieve such a purpose. We established a lens-specific Cre ERT2 knock-in mouse (LCEK), an inducible mouse model for lens-specific gene targeting in a spatiotemporal manner. LCEK mice were created by in-frame infusion of a P2A-CreER at the C-terminus of the last coding exon of the gene alpha A crystallin (Cryaa). LCEK mice express tamoxifen-inducible Cre recombinase uniquely in the lens. Through ROSA and two endogenous genes (Gclc and Rbpj) targeting, we found no Cre recombinase leakage in the lens epithelium, but 50-80% leakage was observed in the lens cortex and nucleus. Administration of tamoxifen almost completely abolished target gene expression in both lens epithelium and cortex but only mildly enhanced gene deletion in the lens nucleus. Notably, no overt leakage of Cre activity was detected in developing LCEK lens when bred with mice carrying loxP floxed genes that are essential for lens development. This newly generated LCEK line will be a powerful tool to target genes in the lens for gene functions study in lens aging, posterior capsule opacification (PCO), and other areas requiring precision gene targeting.

A tamoxifen-inducible Cre knock-in mouse for lens-specific gene manipulation.

A sharp drop in lenticular glutathione (GSH) plays a pivotal role in age-related cataract (ARC) formation. Despite recognizing GSH's importance in lens defense for decades, its decline with age remains puzzling. Our recent study revealed an age-related truncation affecting the essential GSH biosynthesis enzyme, the γ-glutamylcysteine ligase catalytic subunit (GCLC), at aspartate residue 499. Intriguingly, these truncated GCLC fragments compete with full-length GCLC in forming a heterocomplex with the modifier subunit (GCLM) but exhibit markedly reduced enzymatic activity. Crucially, using an aspartate-to-glutamate mutation knock-in (D499E-KI) mouse model that blocks GCLC truncation, we observed a notable delay in ARC formation compared to WT mice: Nearly 50% of D499E-KI mice remained cataract-free versus ~20% of the WT mice at their age of 20 months. Our findings concerning age-related GCLC truncation might be the key to understanding the profound reduction in lens GSH with age. By halting GCLC truncation, we can rejuvenate lens GSH levels and considerably postpone cataract onset.

Prevention of age-related truncation of γ-glutamylcysteine ligase catalytic subunit (GCLC) delays cataract formation.

The class 3 phosphatidylinositol 3-kinase (Pik3c3) plays critical roles in regulating autophagy, endocytosis, and nutrient sensing, but its expression profile in the kidney remains undefined. Recently, we validated a Pik3c3 antibody through immunofluorescence staining of kidney tissues from cell type-specific Pik3c3 knockout mice. Immunohistochemistry unveiled significant disparities in Pik3c3 expression levels across various kidney cell types. Notably, renal interstitial cells exhibit minimal Pik3c3 expression. Further, coimmunofluorescence staining, utilizing nephron segment- or cell type-specific markers, revealed nearly undetectable levels of Pik3c3 expression in glomerular mesangial cells and endothelial cells. Intriguingly, although podocytes exhibit the highest Pik3c3 expression levels among all kidney cell types, the renal proximal tubule cells (RPTCs) express the highest level of Pik3c3 among all renal tubules. RPTCs are known to express the highest level of the epidermal growth factor receptor (EGFR) in adult kidneys; however, the role of Pik3c3 in EGFR signaling within RPTCs remains unexplored. Therefore, we conducted additional cell culture studies. The results demonstrated that Pik3c3 inhibition significantly delayed EGF-stimulated EGFR degradation and the termination of EGFR signaling in RPTCs. Mechanistically, Pik3c3 inhibition surprisingly did not affect the initial endocytosis process but instead impeded the lysosomal degradation of EGFR. In summary, this study defines, for the first time, the expression profile of Pik3c3 in the mouse kidney and also highlights a pivotal role of Pik3c3 in the proximal tubule cells. These findings shed light on the intricate mechanisms underlying Pik3c3-mediated regulation of EGFR signaling, providing valuable insights into the role of Pik3c3 in renal cell physiology.  This is the first report defining the class 3 phosphatidylinositol 3-kinase (Pik3c3) expression profile in the kidney. Pik3c3 is nearly absent in renal interstitial cells, glomerular mesangial cells, and endothelial cells. Remarkably, glomerular podocytes express the highest Pik3c3 level in the kidney. However, the proximal tubule exhibits the highest expression level among all renal tubules. This study also unveils the pivotal role of Pik3c3 in regulating EGFR degradation and signaling termination in RPTCs, furthering our understanding of Pik3c3 in renal cell physiology.

Pik3c3 expression profiling in the mouse kidney and its role in proximal tubule cell physiology.

The human lens is composed of a monolayer of lens epithelial cells (LECs) and elongated fibers that align tightly but are separated by the plasma membrane. The integrity of the lens plasma membrane is crucial for maintaining lens cellular structure, homeostasis, and transparency. Glutathione peroxidase 4 (GPX4), a selenoenzyme, plays a critical role in protecting against lipid peroxidation. This study aims to elucidate the role of GPX4 in lens plasma membrane stability during lens development using in vitro, ex vivo, and in vivo systems. Our findings reveal that GPX4 deficiency triggers lens epithelial apoptosis-independent but ferroptosis-mediated cell death. Blocking lens GPX4 activity during ex vivo culture induces lens opacification, LEC death, and disruption of lens fiber cell arrangement. Deletion of lens-specific Gpx4 results in significant unsaturated phospholipid loss and an increase in oxidized phospholipids. Consequently, lenses with Gpx4 deficiency exhibit massive disruption of lens fiber cell structure, significant loss of LECs via ferroptosis, and formation of newborn cataracts. Remarkably, administering the lipid peroxidation inhibitor, liproxstatin-1, to pregnant mothers at embryonic days 9.5 significantly prevents lipid peroxidation, LEC death, and lens developmental defects. Our study unveils the crucial role of GPX4 in lens development and transparency, and also provides a successful intervention approach to prevent lens developmental defects through lipid peroxidation inhibition.

Deficiency in glutathione peroxidase 4 (GPX4) results in abnormal lens development and newborn cataract.

There is currently limited information regarding inflammation and cytokine levels in the aqueous humor (AH) of adult patients with cataract who have undergone phacoemulsification cataract extraction without other ocular comorbidities.

Elevated Inflammatory Cytokines Persist in the Aqueous Humor Years After Cataract Surgery.

Cataract surgery is the most commonly performed ophthalmic procedure worldwide, yet long-term molecular adaptations of lens epithelial cells (LECs) following surgery remain poorly understood.

Transcriptomic Analysis of Human Lens Epithelium Tissue With and Without Cataract Surgery: Uncovering Novel Pathways of Post-Surgical Lens Epithelium Remodeling.

Article	Year
Vitamin C is a source of oxoaldehyde and glycative stress in age-related cataract and neurodegenerative diseases. Aging cell\| PubMed ID: 32567221	2020
Aging lens epithelium is susceptible to ferroptosis. Free radical biology & medicine\| PubMed ID: 33722625	2021
Cellular FLICE-like inhibitory protein (cFLIP) critically maintains apoptotic resistance in human lens epithelial cells. Cell death & disease\| PubMed ID: 33837174	2021
Protein posttranslational modification (PTM) by glycation: Role in lens aging and age-related cataractogenesis. Experimental eye research\| PubMed ID: 34297945	2021
Mitotic Activation Around Wound Edges and Epithelialization Repair in UVB-Induced Capsular Cataracts. Investigative ophthalmology & visual science\| PubMed ID: 34967856	2021
Aged Lens Epithelial Cells Suppress Proliferation and Epithelial-Mesenchymal Transition-Relevance for Posterior Capsule Opacification. Cells\| PubMed ID: 35805085	2022
A tamoxifen-inducible Cre knock-in mouse for lens-specific gene manipulation. Experimental eye research\| PubMed ID: 36372215	2023
Prevention of age-related truncation of γ-glutamylcysteine ligase catalytic subunit (GCLC) delays cataract formation. Science advances\| PubMed ID: 38669339	2024
Pik3c3 expression profiling in the mouse kidney and its role in proximal tubule cell physiology. American journal of physiology. Cell physiology\| PubMed ID: 39250817	2024
Deficiency in glutathione peroxidase 4 (GPX4) results in abnormal lens development and newborn cataract. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 39560644	2024
Elevated Inflammatory Cytokines Persist in the Aqueous Humor Years After Cataract Surgery. Investigative ophthalmology & visual science\| PubMed ID: 40183733	2025
Transcriptomic Analysis of Human Lens Epithelium Tissue With and Without Cataract Surgery: Uncovering Novel Pathways of Post-Surgical Lens Epithelium Remodeling. Investigative ophthalmology & visual science\| PubMed ID: 40643341	2025

Xingjun Fan

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