The Glyoxalase System in Age-Related Diseases: Nutritional Intervention As Anti-Ageing Strategy Part 1

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Abstract: The glyoxalase system is critical for the detoxification of advanced glycation end-products (AGEs).AGEs are toxic compounds resulting from the non-enzymatic modification of biomolecules by sugars or their metabolites through a process called glycation. AGEs have adverse effects on many tissues, playing a pathogenic role in the progression of molecular and cellular aging. Due to the age-related decline in different anti-AGE mechanisms, including detoxifying mechanisms and proteolytic capacities, glycated biomolecules are accumulated during normal aging in our body in a tissue-dependent manner. Viewed in this way, anti-AGE detoxifying systems are proposed as therapeutic targets to fight pathological dysfunction associated with AGE accumulation and cytotoxicity. Here we summarize the current state of knowledge related to the protective mechanisms against glycation stress, with a special emphasis on the glyoxalase system as the primary mechanism for detoxifying the reactive intermediates of glycation. This review focuses on glyoxalase 1(GLO1), the first enzyme of the glyoxalase system, and the rate-limiting enzyme of this catalytic process. Although GLO1 is ubiquitously expressed, protein levels and activities are regulated in a tissue-dependent manner. We provide a comparative analysis of GLO1 protein in different tissues. Our findings indicate a role for the glyoxalase system in homeostasis in the eye retina, a highly oxygenated tissue with rapid protein turnover. We also describe modulation of the glyoxalase system as a therapeutic target to delay the development of age-related diseases and summarize the literature that describes the current knowledge about nutritional compounds with properties to modulate the glyoxalase system.

Keywords: glycation stress; glyoxalase system; aging; proteotoxicity

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1. Introduction: Glycative Stress and Unhealthy Aging

A growing body of literature indicates that the accumulation of damaged proteins is a specific hallmark of aging and many age-related diseases, including type 2 diabetes, cancer, neurodegenerative, cardiovascular, and eye-related disorders [1-7]. Aberrant proteins impair cellular homeostasis by forming non-functional and toxic aggregates and this leads to the inactivation of not only the aberrant protein but can also impair the function of other essential proteins due to the stress on—or insufficiency of—the protein quality control machinery in the cell. One prominent mechanism that leads to aberrant molecules is a modification by advanced glycation end-products(AGEs).

Dicarbonyl compounds are generated

from different metabolic pathways (Figure 1)that involve dietary sugar and carbohydrate metabolism to form AGEs. These dicarbonyl compounds interact with biomolecules, such as proteins, lipids, and nucleic acids in a non-enzymatic post-translational modification called glycation. The major glycating dicarbonyl agents are methylglyoxal (MG), glyoxal, or 3-deoxyglucosone [8]. These dicarbonyls are maintained at low levels in homeostatic conditions, but the aging process increases these glycating reagents to pathological levels, enhancing the formation of toxic AGEs and, ultimately, compromising tissue fitness. Given that the formation of AGEs is dependent on glucose concentration, the consumption of high glycemic diets or diabetic conditions leads to a dramatic systemic accumulation of AGEs. This directly correlates with altered metabolism, increased inflammation, and the progression of severe medical conditions. Conversely, the intake of low glycemic diets limits AGE accumulation and is associated with the slower progression of some of these diseases [9-13]. In this context, hyperglycemia imposes additional stress on the age-associated production of glycated proteins and exacerbates the detrimental consequences of AGEs deposits on organ function.

Figure 1. Schematic diagram of α-dicarbonyls formation and detoxification pathways against AGE-derived damage in aging. The formation of highly reactive α-dicarbonyls such as methylglyoxal(MG) is through non-enzymatic degradation of the glycolytic intermediates, including dihydroxyacetone phosphate and glyceraldehyde 3-phosphate and other sources, including amino acid and lipid metabolism. In order to avoid AGE damage, the glyoxalase system is a primary mechanism that limits the synthesis of AGEs, converting high reactive biomolecules, such as MG, into less reactive biomolecules (D-lactate). This process involves the sequential activity of two enzymes GLO1 and GLO2 and the reduced form of glutathione (GSH). Other detoxifying mechanisms imply the activity of DJ-1, aldehyde dehydrogenases(ALDHs), Aldo-keto reductases (AKRs), and acetoacetate degradation enzymes. Once formed, AGEs can be cleared by two proteolytic pathways: the ubiquitin-proteasome (UPS)system and autophagy. These protective mechanisms (highlighted in green) decline under aging and contribute to the onset of age-related diseases such as neurodegeneration, eye-related diseases(AMD, cataract, DR), nephropathies, metabolic syndrome, and cancer. GLO1:glyoxalase 1; GLO2:glyoxalase 2; GSH: glutathione.

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Excessive glycation stress promotes protein insolubility, deregulating signaling and protein quality control pathways. AGEs-derived changes in the proteome perturb signaling pathways in tissue physiology (MAP/ERK, JAK-STAT, and PI3K-AKT pathways) that lead to the nuclear translocation of transcription factors involved in multiple cellular functions, including inflammation, apoptosis, ER stress, autophagy, oxidative stress, mitochondrial function, etc. (reviewed in [2,14]). Glycated proteins may also overtax or limit the functionality of proteolytic capacities. These changes ultimately contribute to the onset of multiple age-related disorders.

Multiple studies have demonstrated that the formation of MG and MG-derived AGEs is an important factor in the pathogenesis of diabetes and its complications, such as retinopathy, nephropathy, and neuropathy [15-19]. Dicarbonyl stress is also a contributing mediator of obesity and cardiovascular disease [20,21]. MG may contribute to atherosclerosis through several mechanisms, including the accumulation of MG-derived AGEs in atherosclerotic plaques [22] and MG-induced low-density lipoprotein glycation [23]. The association between MG and hypertension has also been observed in several studies, showing increased MGlevels in the aorta and kidney tissues [24,25]. Several studies have also confirmed that the accumulation of AGEs is correlated with many neurodegenerative disorders, thus affecting brain function, such as Alzheimer's disease, Parkinson's disease, and schizophrenia [26-28]. One of the best examples of the relation between AGE accumulation and aging-related consequences occurs in eye tissues resulting in glycation-induced ocular tissue disorders, such as cataracts, age-related macular degeneration (AMD), and diabetic retinopathy (DR)[29-31]. Regarding cataracts, the leading cause of blindness worldwide, lens crystallins become progressively yellow-brown pigmented with age as a result of the accumulation of AGEs byproducts [31]. As well as the lens, retinal AGEs increase with age and diabetes, especially in the outer retina. AMD is the leading cause of blindness in older individuals in developed nations. Higher AGE levels are found in AMD patients compared to control subjects as well as in AMD mouse models [32-36]. DR is characterized by the accumulation of AGEs in the retina, inducing microvascular damage [37]. micronized purified flavonoid fraction 1000 mg uses These pathological changes result in irreversible damage to the blood-retinal barrier, and macular edema, ultimately resulting in vision loss. In summary, AGEs accumulate throughout the body upon aging, particularly, in diabetic patients. This compromises organismal homeostasis and contributes to the onset and progress of a plethora of age-related diseases.

There are multiple systems to detoxify AGEs. These include the glyoxalase system, the best-characterized mechanism to inhibit the formation of AGEs, and one of the routes able to detoxify intermediates of glycation. However, the anti-AGEs capacities decline with age leading to the accelerated accumulation of AGEs in 'normal older tissues. Although there are different defense mechanisms to limit the accumulation of AGEs in tissues, developing them to prevent the accumulation of AGEs and associated pathologies are still not being exploited [38].In the next section, we summarize the current literature about detoxifying mechanisms focusing on the glyoxalase system to reduce the accumulation of these toxic byproducts in cells and tissues. Finally, we discuss the usefulness of nutritional interventions to boost the glyoxalase system as an anti-aging strategy.

2. Detoxifying Mechanisms against Glycative Stress: Major Role of the Glyoxalase System

Multiple detoxifying mechanisms against the accumulation of AGEs have been reported. Figure 1 is a schematic overview of the α-dicarbonyl formation and the different detoxification routes against AGEs-derived damage in aging. The major routes of AGEs synthesis involve the reaction of reactive dicarbonyls derived mainly from the glucose metabolism with primary amines(N-terminal or lysine side chain) or the guanidine group of the arginine side chain [39]. The formation of highly reactive α-dicarbonyls, such as MG, is through the metabolism of the glycolytic intermediates, such as dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, and other sources, including amino acid and lipid metabolism.

AGEs are irreversible and, once formed, can only be eliminated by proteolyticpath-ways [5,9,40,41]. Two major proteolytic capacities are suggested to contribute to the clearance of AGEs: the ubiquitin-proteasome system (UPS) and the autophagic lysosomal proteolytic system(ALPS)[5,9,40,41](Figure 1). The UPS operates mainly on soluble misfolded proteins. In the UPS, substrates are recognized and tagged with ubiquitin and targeted to the proteasome for degradation. The ALPS consists of the targeting of cargo to the lysosomal compartment for degradation. Autophagic cargo can be diverse, including insoluble proteins, proteinaceous aggregates, and even whole organelles. Both proteolytic pathways are functionally cooperative and increasing literature supports crosstalk between the two pathways with reciprocal direct and indirect interactions[42-46]. This crosstalk guarantees a backup mechanism and, in the case of the deficiency of one of the routes, the other proteolytic pathway tends to compensate to maintain a proper and functional proteome [47].

Age-related changes in rates of protein degradation were documented for many tissues more than 3 decades ago, even before the molecular characterization of proteolytic pathways was defined [48]. Nowadays, the molecular and cellular decline of the two major proteolytic routes with age is better understood and there are differences in the degrees of decrease between the UPS and lysosomal systems. Many reports have shown a tissue-dependent decline in UPS, while autophagic decline seems to be universal (reviewed in [49-51). Regarding autophagy, both lysosomal and autophagosomal compartments undergo striking modifications. Changes that contribute to the malfunction of autophagy include a decrease in lysosomal stability, hydrolase activity, accumulation of indigestible material (lipofuscin) in the lysosomal lumen, dysfunctional lysosomal pH, decreased transcriptional level of autophagy-related proteins, decreased stability of the chaperone-mediated autophagy receptor LAMP2A in the lysosomal membrane and decreased association of motor proteins in the autophagic compartments ([49,51,52]). In contrast to autophagy, it is now accepted that changes in proteasome proteolytic abilities with age seem to be more qualitative than quantitative. oteflavonoid Changes in the composition of the proteasomal core catalytic activities and modulatory subunits, decreased proteasome expression, as well as changes in the oxidation state of the proteasome subunits and proteasome substrates, contribute to the age-related inhibition of the UPS capacity (reviewed in [53,54). In some cases, there may just be the insufficient capacity of the proteolytic systems to handle to load. Unfortunately, the efficacy of these two mechanisms declines with age, resulting in the insufficient capacity to recognize and remove damaged proteins and, therefore, the intracellular accumulation of protein aggregates and dysfunctional organelles [55,56]. Net AGEs levels are determined by the balance of the rate of synthesis or formation and rate of removal. The immediate consequence of the decline in proteolytic capacity is the accumulation of long-lived proteins in aged organisms, many of which accumulate glycation-derived damage in their amino acid sequences. The accumulation of AGEs occurs in an age-related and -dependent manner([4,9]) and a recent proteomic analysis in aging research has revealed that AGE biology contains an enriched metabolic pathway associated with age-associated proteomes [57].

Although UPS and ALPS decline with age, there are different protective pathways with the capacity to reduce the synthesis of AGEs. In this review, we focus on these protective mechanisms limiting the biogenesis of AGEs, with a special emphasis on the glyoxalase system, the primary route for detoxifying reactive dicarbonyls [58]. In this section, we will describe the glyoxalase system in detail. We also briefly describe other mechanisms in the detoxification of AGEs: Parkinson-associated protein DJ-1, aldehyde dehydrogenases(ALDHs), Aldo-keto reductases (AKRs), and acetoacetate degradation.

2.1.Glyoxalase System: The Major Detoxifying Route for Reaction Dicarbonyls

A vast literature supports the glyoxalase system as the major detoxifying route for reactive dicarbonyls in the cytosol of all mammalian cells [58]. The glyoxalase system is the best-characterized pathway for the metabolism of MG. Genes for glyoxalases are evolutionarily conserved and widely distributed in various living systems, such as humans, plants, yeast, bacteria, fungi, and protists. The presence in many diverse taxa points out the high importance of glyoxalase enzymes in the physiological function of biological life. The combined activities of glyoxalases 1 and 2(GLO1, GLO2) catalyze the conversion of reactive, acyclic α-oxoaldehydes into the corresponding α-hydroxy acids [58]. These reactions also require catalytic GSH. In the initial step, GLO1 converts its substrate, hemithioacetal, formed by a spontaneous reaction of the aldehyde of the dicarbonyl MG and GSH, into S-D-lactoylglu throne. Then, GLO2 hydrolyzes S-D-lactoylglu tathione to D-lactate and reforms GSH(Figure 1). puritans vitamin c The activity of GLO1 is directly proportional to GSHconcentration. The GLO1 activity decreases when GSHis removed, such as upon oxidative stress when GSH is converted to GSSG [59].

MG is formed during glycolysis and gluconeogenesis by the degradation of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, as well as by the catabolism of threonine, the oxidation of ketone bodies, and the degradation of glycated proteins. Other substrates, including glyoxal, phenylglyoxal, and hydroxypyru aldehyde, are also metabolized via this pathway [60]. GLO1, the rate-limiting enzyme in the glyoxalase system, catalyzes the primary detoxification step [61], thus the alteration of GLO1 protein is involved in many pathological processes in aging, such as in diabetes, neurodegenerative diseases, cancer, and eye-related diseases [20.

The regulation of GLO1 expression and activity is complex and still not well-understood (Figure 2). The GLO1 promoter sequence contains a metal-response element(MRE), an insulin-response element (IRE), an early gene 2-factor isoform (E2F), and an activating enhancer-binding protein 2α(AP-2α), and an antioxidant-response element (ARE). The function of the IRE and MRE were confirmed in reporter assays where insulin and zinc chloride treatment produced an increased transcriptional response 62]. Similar functional activities were observed for E2F and AP-2α[63,64]. The ARE located in exon 1 of Glo1 serves to join Glo1 to the nuclear factor erythroid 2-related factor 2(NRF2)stress-responsive transcriptional system [65]. Several genes related to MG metabolism and protection against oxidative stress are under the control of the NRF2-ARE pathway [66]. NRF2 is complexed with KEAP1, a substrate adaptor protein for cullin-3-dependent E2 ubiquitin ligase complex, directing NRF2 for degradation by the 26S proteasome upon physiological conditions. Oxidative stress leads to the destabilization of this complex, causing the translocation of NRF2 to the nucleus, and triggering the upregulation of antioxidant genes [67,68]. The binding of NRF2 to the Glo1-ARE increases the basal and inducible expression of GLO1.[65]. NRF2 and antioxidant responses are also upregulated when MG causes the dimerization of KEAP liberating Nrf2 [69].

Several studies show that NRF2 increases GLO1 activity and alleviates intracellular MG stress; thus, the modulation of GLO1 by NRF2 agonists resulted in a decrease in MG and MG-derived protein adducts in both cells and tissues [70-73]. Moreover, hepatic, brain, heart, kidney, and lung Glol mRNA and protein were decreased in NRF2 knockout mice [65]. Altogether, these reports suggest that GLO1 is a downstream target by which the NRF2/KEAP1 pathway performs its protective functions by decreasing MG and dicarbonyl stress. However, the inflammatory activation of the NF-kB (nuclear factor kB)with NRF2 diminishes Glol expression [74]. Glow expression is also negatively regulated by HIFlα(hypoxia-inducible factor lα) under hypoxic conditions, an important physiological driver of dicarbonyl stress [75].

Along with transcriptional regulation, there is also post-translational regulation of GLO1 protein (Figure2). GLO1 is acetylated by cytosolic sirtuin-2[76,77], and its expression may be decreased by activation of RAGE (receptor for advanced glycation end-products); however, these mechanisms are not clearly understood [78]. sistanche A recent study showed that GLO1 protein can be modified by the phosphorylation of threonine 107 (T107) and the nitrosylation of cysteine 139 [79]. In this study, the phosphorylation of T107 by calmodulin-dependent kinase II delta in the GLO1 protein was reported as a precise mechanism regulating the glyoxalase system. Specifically, the phosphorylation of GLO1 at T107 affects the kinetic efficiency of MG detoxification and proteasomal degradation rate. Thus, its altered status is associated with the development of age-related diseases [79].

Figure 2. Mechanisms of glyoxalase 1(GLO1)regulation. GLO1 activity can be regulated through multiple mechanisms, including transcriptional regulation and post-translational modifications. The Glo1 promoter contains various regulatory elements, such as antioxidant response (ARE), metal-response(MRE), and insulin-response (IRE)elements, and binding sites for AP-2α and E2F.In normal conditions, the nuclear factor erythroid 2-related factor 2 (NRF2) is complexed with KEAP1, a substrate adaptor protein for cullin-3-dependent E2 ubiquitin ligase complex, directing NRF2 for degradation by the ubiquitin-proteasome system (UPS). Oxidative stress leads to the destabilization of the complex NRF2-KEAP1, causing the detachment of NRF2 that is translocated to the nucleus which triggers the upregulation of different antioxidant genes. The binding of NRF2 to the Glo1-ARE increases the expression of GLO1. Under hypoxia conditions, Glow expression is inversely regulated by hypoxia-inducible factor 1α(HIFlx). Different post-translational modifications in the cytosol can impact GLO1 stability.

2.2. Alternative Detoxification Mechanisms as Putative Backup Systems to Compensate the Lack of Glyoxalase Activity

While the primary mechanism for detoxifying reactive dicarbonyls in the glyoxalase system, there are alternative routes with the capacity to detoxify dicarbonyls formed during sugar metabolism. These include ALDHs, AKRs, the Parkinson-associated protein DJ-1, and scavenging by acetoacetate to form 3-hydroxy hexane-2,5-dione (3-HHD)[80]. The physiological relevance of these systems remains unclear and it has been questioned whether or not these enzymes are crucial for the detoxification of AGEs in tissues due to the high activity of the glyoxalase system. They seem to be components of backup systems that operate in the absence of glyoxalase activity although a tissue-dependent role of these routes cannot be discounted.

DJ-1, also known as Parkinson's disease protein 7(PARK7), plays an essential role in Parkinson's disease (PD). The lack of functional DJ-1 protein has been shown to cause autosomal recessive PD [81,82]. DJ-1 was reported to have two different activities:(1) glyoxalase activity in vitro, converting MG into lactate and preventing MG-induced tissue damage in Caenorhabditis elegans [83], and (2) deglycase activity in vitro, reducing early-stage MG byproducts [84]. Recently, other studies have also shown that DJ-1 plays a relevant role in DNA deglycase [85-87]. what is cistanche The detoxifying capacity of DJ-1 in the absence of glutathione(GSH) makes this an alternative route to the glyoxalase system, which requires the presence of GSH. However, Pfaff et al.using both DJ-1 knockdowns in Drosophila cells and DJ-1 knockout in the whole organism observed no differences in the accumulation of MG protein adducts [88].

AKRs are a superfamily of proteins able to reduce aldehydes and ketones into primary and secondary alcohols. AKRs metabolize MG to hydroxy acetone or lactaldehyde. Some studies showed that the transgenic expression of both human and mouse Aldo-keto reductases in rodent fibroblast cells protects against MG-induced damage, suggesting that AKRs can participate in MG detoxification and a reduction in AGEs levels [89-91]. High AKR1B3 activity was detected in Glo1 knockout mouse Schwann cells as well as an increased expression during MG exposure, suggesting that it could be a compensatory mechanism induced by lack of the glyoxalase system or excessive glycation stress [92]. Interestingly, the lack of AKR1B3 resulted in higher levels of MG and AGEs in the hearts of diabetic mice [91].

LEDs are another group of α-dicarbonyl metabolizing enzymes that oxidize MG to pyruvate. ALDH expression was increased in mouse Schwann wild-type cells upon MG treatment [92]. In a zebrafish model,glo1 knockout fish showed that induced ALDH activity compensates for the lack of GLO1 [93]. However, at least in mice, the compensatory mechanisms are tissue-dependent, as the increased expression of AKRs and ALDHs were observed in liver tissue but only AKRs were reported in kidneys in Glo1 knockout mice [94]. In human studies, the 3-DG metabolite produced by aldehyde dehydrogenase 1A1(ALDH1A1) activity was increased in plasma and erythrocytes of diabetic patients [92]. Recently, it was also shown that ketone body acetoacetate reduced MG concentration by a non-enzymatic reaction during diabetic and dietary ketosis [95,96]. They found that this metabolic route involves a non-enzymatic aldol-reaction between MG and the ketone body acetoacetate, leading to 3-hydroxy hexane-2,5-dione, which is present in the blood of insulin-starved patients. Alternative pathways that might compensate for the deficiency of the glyoxalase system could potentially generate toxic molecules such as y-diketones, which are associated with peripheral axonal degeneration and testicular injury [97,98].

Although there is no systematic aging analysis of proteins involved in GLO1-independent alternative pathways, age-related changes in those molecular players have been reported. For example, there is a correlation between D]-1 levels of expression and oxidative stress, and different reports showed an increase in DJ-1 with age. DJ-1 mRNA and protein levels increased from 8 to 20 weeks of age in mice [99] and DJ-1 levels significantly increased as a function of age in human cerebrospinal fluid [100]. In ocular tissues, it has been shown that the DJ-1 is expressed in retinal pigment epithelium and photoreceptors and the expression increased in old eyes [101]. It might reflect a compensatory mechanism due to the decline in glyoxalase system activity.

2.3. Tissue-Dependent Activity of Glyoxalase System

Although GLO1 is a ubiquitous protein, the levels of this enzyme are regulated in a tissue-dependent manner. In order to evaluate the role of the glyoxalase system in different tissues, we examined the expression and activity of GLO1 in non-ocular(liver, brain, heart, and kidney)and ocular tissues (retina, RPE/choroid, and lens)from wild type C57BL/6] mice. Using antibodies that specifically recognize GLO1, Western blotting and immunohistochemistry were performed to quantify protein levels. GLO1 activity in cytosolic extracts was determined spectrophotometrically as the initial rate of formation of S-D-lactoylglutathione, as previously reported [30,102]. These results are summarized in Figure 3.

Figure 3. A comparative analysis of GLO1 protein and activity in ocular and non-ocular tissues. (A) GLO1 activity was assayed in non-ocular tissues and retinal tissues from WT mice as previously described [29] and activity was expressed as a percentage(%) compared to liver. (B)Liver and(C) retina representative Western blot analysis of WT and Glow overexpression transgenic mice(Glo1 Tg+/+)using a monoclonal antibody (non-commercial) and polyclonal antibody for Glol (commercial, GeneTex)[36,103,104]. (D)RepresentativeWestern blot analysis of non-ocular tissue extracts (50ug) of WT mice using a monoclonal antibody for Glo1 (non-commercial) and (E) protein quantification of GLO1 normalized to control loading (Ponceau staining). (F) GLO1 activity was performed in ocular tissues (Retina, RPE/Choroid, and Lens)from WT mice as previously described [29] and activity was expressed as milliunits per milligram of protein. Values are mean ± SEM. The sample size is n=4from the GLO1 protein and activity assays.

Previously published data indicated that the retina and liver display the highest activity of GLO1([30]; Figure 3A). Note that retinal activity was the highest value while the liver, kidney, brain, and heart only represented 46%, 27%, 22%, and 11% of detoxifying retinal capacity, respectively. We evaluated if the activity of GLO1 correlated with the level of the enzyme by assessment of GLO1 protein levels by Western blotting. The antibody against GLO1 was previously validated in previous reports and used for the analysis of GLO1 in retinal samples [36,103,104]. As a positive control, a comparative analysis was also performed in retina and liver tissues from transgenic mice overexpressing GLO1 on C57BL/6J (B6) background [105]. To examine the levels of GLO1, we used two different antibodies: a polyclonal rabbit antibody (commercial antibody from GeneTex)and a monoclonal mouse antibody (non-commercial antibody) reported in different animal models for the study of GLO1 biology [103,106]. We were able to detect GLO1 protein in liver and retina wild-type tissues by Western blotting, and we found the highest expression in transgenic mice in both tissues (Figure 3B, C, and Supplementary Figure S1). Two bands were recognized for both antibodies. The differential electrophoretic profiles of these GLO1-positives suggest that posttranscriptional changes could be vital in the role of the protein. Accordingly, a recent study indicated that phosphorylated GLO1 is more efficient and more stable, supporting these post-transcriptional alterations as a precise mechanism regulating GLO1 activity [79]. However, there is scant information about how post-transcriptional modifications modulate glyoxalase 1 activity.

As expected, we found GLO1 protein in all non-ocular tissues analyzed, with the liver showing the highest expression. The relative order of GLO1 expression was liver>kidney>brain>heart(Figure 3D, E). This corroborates the results of a previous study[30]. There is limited information about the role of GLO1 in ocular tissues. As we previously reported, the enzymatic assay revealed that GLO1 activity is~10 fold higher in the retina compared to the lens or RPE/choroid(Figure 3F,[30]). The overexpression of glyoxalase I improve human retinal pericyte survival under hyperglycemic conditions [107] and an angiotensin receptor blocker that restores GLO1 in diabetic rats was shown to reduce retinal acellular capillaries [18]. Additionally, the lack of GLO1 in Zebrafish impacts the adult retina vessel architecture, although increased angiogenic sprout formation is only observed in glo1-/-overfed zebrafish but not in normal feeding [93].

The retina is a highly complex, very dynamic tissue with diverse cell types (Figure 4A). Blood flow, and consequent exposure to xenobiotics and other stressors, are among the highest in the body. Each morning, 10 percent of the outer tips of retina photoreceptors are shed and must be removed by adjacent retinal pigmented epithelial cells. We performed immunohistochemical analysis to characterize for the first time the spatial differences of GLO1 in the retina. GLO1 protein was present in all cell types within the retina, with high levels within cell bodies of the inner nuclear layer and ganglion cell laver. Photoreceptor cell bodies in the outer nuclear layer had lower levels. In photoreceptors, most GLO1 proteins was found within the inner and outer segments. The RPE also had high levels of GLO1 protein, whereas the choroid and sclera had a lower amount of GLO1 protein (Figure 4B, C).

Figure 4. Immunohistochemistry of GLO1 in mouse retinal tissues. (A) Cross-sectional, cellular schematic of the retina illustrating its three primary layers comprised of the ganglion cell layer(GCL), containing retinal ganglion cells (RGC), inner nuclear layer(INL), hosting interneurons of amacrine, bipolar and horizontal cells as well as Müller glial cells, and outer nuclear layer(ONL), housing rod and cone photoreceptors. The sensory tissue, or neuroretina, is connected to the retinal pigmented epithelium(RPE). Red arrows indicated the RPE layer. (B) Representative picture of GLO1 immunostaining in retinal samples from WT mice. (C) Mean intensity fluorescence of GLO1 normalized to the value in the RPE. Data shown are mean ± standard errors of the means (SEM). Our results in the retina are relevant because the retina is a highly differentiated post-mitotic tissue, where glycation-derived damage cannot be reduced by cellular division [5,9]. Furthermore, changes in GLO1 have been associated with retinal damage [108]. A similar scenario might occur in other tissues composed of cells with low regeneration capacity, such as the central nervous system, where the vast majority of neurons are post-mitotic. The evaluation of GLO1 levels along with cell-specific markers might allow us to evaluate the cell-to-cell variation within a given tissue. Our results suggest that the high level of retinal GLO1 protein and activity might play an important protective role against AGE-derived damage with age.

This article is extracted from Cells 2021, 10, 1852. https://doi.org/10.3390/cells10081852 https://www.mdpi.com/journal/cells