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Two-dimensional difference gel electrophoresis (2D-DE) map showing positions of TAGE-protein spots. Glyceraldehyde (GA) time-dependently induced cell death in SH-SY5Y cells (A). Formation of toxic advanced glycation end products (TAGE) by GA treatment within 12 h. ** p < 0.01 vs. 0 h treatment (n = 6). (B) TAGE were measured by slot blotting analyses with an immunopurified anti-TAGE antibody. Graphical representation of TAGE bands in the slot blot. ** p < 0.01 vs. 0 h treatment (n = 3). Two-dimensional electrophoresis map showing the positions of TAGE proteins from SH-SY5Y cells treated with vehicle control (C) or 1 mM GA for 12 h (D). Differentially expressed proteins marked with black arrows are β-tubulin (MW: 50 kDa, pI: 4.78).
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Nutritional factors can affect the risk of developing neurological disorders and their rate of progression. In particular, abnormalities of carbohydrate metabolism in diabetes mellitus patients lead to an increased risk of neurological disorders such as Alzheimer’s disease (AD). In this study, we investigated the relationship between nervous system...
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... TAGE has been detected in axons and intracellular neuronal cells within the hippocampus and parahippocampal gyrus of patients with AD [12]. Recently, through proteomics analysis, we identified β-tubulin as one of the proteins targeted by TAGE [13]. Microtubules consist of repeating units of heterodimers between α-tubulin and β-tubulin, and their assembly is a crucial event implicated in axon outgrowth in vitro models such as SH-SY5Y, a human neuroblastoma cell line. ...
... Microtubules consist of repeating units of heterodimers between α-tubulin and β-tubulin, and their assembly is a crucial event implicated in axon outgrowth in vitro models such as SH-SY5Y, a human neuroblastoma cell line. As tau phosphorylation diminishes its binding to microtubules, GA-induced tau phosphorylation leads to axonal degeneration via TAGE-mediated abnormal aggregation of β-tubulin in SH-SY5Y cells [13]. However, the impact of TAGE-β-tubulin on axonal elongation in the adult mouse optic nerve, particularly in a model utilizing intraocular injection, remains unexplored. ...
... Our prior work demonstrated that 1 mM GA induced TAGE formation in SH-SY5Y cells [13]. To investigate this further, we examined if intraocular injection of 1 mM GA could trigger TAGE formation. ...
Advanced glycation end-products (AGEs) form through non-enzymatic glycation of various proteins. Optic nerve degeneration is a frequent complication of diabetes, and retinal AGEs accumulation is strongly linked to diabetic retinopathy development. Type 2 diabetes mellitus (T2DM) is a major risk factor for Alzheimer's disease (AD), with patients often exhibiting optic axon degeneration in the nerve fiber layer. Notably, a gap exists in our understanding of how AGEs contribute to neuronal degeneration in the optic nerve within the context of both diabetes and AD. Our previous work demonstrated that glyceraldehyde (GA)-derived toxic advanced glycation end-products (TAGE) disrupt neurite outgrowth through TAGE-β-tubulin aggregation and tau phosphorylation in neural cultures. In this study, we further illustrated GA-induced suppression of optic nerve axonal elongation via abnormal β-tubulin aggregation in mouse retinas. Elucidating this optic nerve degeneration mechanism holds promise for bridging the knowledge gap regarding vision loss associated with DM and AD.
... In our recent studies, we showed that intracellularly accumulated GA-AGEs contributed to the onset/progression of lifestyle-related diseases (LSRDs) by triggering cell In our recent studies, we showed that intracellularly accumulated GA-AGEs contributed to the onset/progression of lifestyle-related diseases (LSRDs) by triggering cell damage [24][25][26][27][28][29][30][31][32][33][34][35][36]. Elevated intracellular levels of GA-AGEs have also been implicated in the pathogenesis of metabolic syndrome (MetS), non-alcoholic steatohepatitis (NASH), cardiovascular diseases (CVD), diabetes mellitus (DM) and its complications, some cancer types, and Alzheimer's disease (AD) [22,23,[37][38][39]. ...
... Previous studies have suggested the involvement of TAGE accumulation in the pathogenesis of NASH, CVD, DM and its complications, some cancer types, and AD [22,23,[37][38][39]55,77]. We recently demonstrated that TAGE were produced by and accumulated in many cell types [24][25][26][27][28][29][30][31][32][33][34][35][36]. Excessive TAGE damage cells, leak into surrounding cells and the circulation, interact with RAGE, and the activation of the TAGE-RAGE axis contributes to the onset/progression of LSRDs [54,55]. ...
... Intracellular GA levels are increased by the frequent and excessive intake of sugarsweetened beverages and/or processed foods, which are prevalent in contemporary daily diets. Reactions between elevated levels of GA and intracellular proteins induce the production of TAGE [79], which exert cytotoxic effects in various cell types, including hepatocytes [24][25][26][27], neuroblastoma cells [28][29][30], cardiomyocytes [31] and cardiac fibroblasts (CFs) [32], pancreatic ductal cells [33], β-cells [34], myoblasts [35], and osteoblasts [36]. Based on these findings, TAGE have been suggested to play a role in apoptosis and/or necrosis, which ultimately lead to cell death and tissue damage. ...
Advanced glycation end-products (AGEs) have recently been implicated in the onset/progression of lifestyle-related diseases (LSRDs); therefore, the suppression of AGE-induced effects may be used in both the prevention and treatment of these diseases. Various AGEs are produced by different biological pathways in the body. Glyceraldehyde (GA) is an intermediate of glucose and fructose metabolism, and GA-derived AGEs (GA-AGEs), cytotoxic compounds that accumulate and induce damage in mammalian cells, contribute to the onset/progression of LSRDs. The following GA-AGE structures have been detected to date: triosidines, GA-derived pyridinium compounds, GA-derived pyrrolopyridinium lysine dimers, methylglyoxal-derived hydroimidazolone 1, and argpyrimidine. GA-AGEs are a key contributor to the formation of toxic AGEs (TAGE) in many cells. The extracellular leakage of TAGE affects the surrounding cells via interactions with the receptor for AGEs. Elevated serum levels of TAGE, which trigger different types of cell damage, may be used as a novel biomarker for the prevention and early diagnosis of LSRDs as well as in evaluations of treatment efficacy. This review provides an overview of the structures of GA-AGEs.
... The chemical structure comprised carbon combined with hydrogen and fluorine atoms ( Figure 2). Nitrocellulose membranes, not PVDF membranes, are generally used to perform slot blot analysis on various proteins [82][83][84][85][86][87][88][89]. Although protein absorption and PVDF membrane durability are superior [33,34], researchers avoid performing slot blot analysis to identify or quantify proteins. ...
... PVDF membranes are commonly used for Western blot analysis [39,40] and can combine with C=O and N-H groups, rendering them superior for protein absorption [33]. However, the appropriate conditions for directly applying Nitrocellulose membranes, not PVDF membranes, are generally used to perform slot blot analysis on various proteins [82][83][84][85][86][87][88][89]. Although protein absorption and PVDF membrane durability are superior [33,34], researchers avoid performing slot blot analysis to identify or quantify proteins. ...
... Previous studies have reported statistical analysis on data obtained from slot blots performed with a nitrocellulose membrane and RIPA buffer (Table 3) [87,88]. However, those with a PVDF membrane and RIPA buffer did not provide data with error bars and the statistically significant difference [102]. ...
Kampo is a Japanese traditional medicine modified from traditional Chinese medicine. Kampo medicines contain various traditional crude drugs with unknown compositions due to the presence of low-molecular-weight compounds and proteins. However, the proteins are generally rare and extracted with high-polarity solvents such as water, making their identification and quantification difficult. To develop methods for identifying and quantifying the proteins in Kampo medicines, in the current study we employ previous technology (e.g., column chromatography, electrophoresis, and membrane chromatography), focusing on membrane chromatography with a polyvinylidene difluoride (PVDF) membrane. Moreover, we consider slot blot analysis based on the principle of membrane chromatography, which is beneficial for analyzing the proteins in Kampo medicines as the volume of the samples is not limited. In this article, we assess a novel slot blot method developed in 2017 and using a PVDF membrane and special lysis buffer to quantify advanced glycation end products-modified proteins against other slot blots. We consider our slot blot analysis superior for identifying and quantifying proteins in Kampo medicines compared with other methods as the data obtained with our novel slot blot can be shown with both error bars and the statistically significant difference, and our operation step is simpler than those of other methods.
... The mechanisms by which intracellularly formed TAGE impair cells have not yet been elucidated in detail. Glycation modifications have been suggested to compromise cellular homeostasis by inducing the loss of function of various biomolecules or by forming toxic aggregates, which inactivate other normal essential proteins [19][20][21][22]. ...
... Another possible cause of cellular damage due to TAGE is proteopathy, a broad term that refers to the crosslinking of glycated proteins between molecules over time, resulting in the formation of larger high-molecular-weight complexes that gradually aggregate and accumulate, disrupting normal protein function [19]. The proper folding of intracellular proteins is crucial for their biological functions, while aberrantly folded proteins may accumulate and aggregate in cells, leading to cellular stress. ...
Toxic advanced glycation end-products (TAGE), formed by glyceraldehyde (GA) as an intermediate in the non-enzymatic reaction with intracellular proteins, are highly cytotoxic and have been implicated in the pathogenesis of various diseases. However, the mechanisms underlying the degradation and removal of TAGE remain largely unknown. In the present study, we identified the checkpoint kinase-1 (CHK1) mutant, d270KD, which was rapidly degraded intracellularly by GA, and showed that its degradation was mainly mediated by the ubiquitin-proteasome pathway. The high-molecular-weight complexes formed by the GA stimulation of d270KD were abundant in the RIPA-insoluble fraction, which also contained high levels of TAGE. The knockdown of p62/SQSTM1 reduced the amount of high-molecular-weight complexes in the RIPA-insoluble fraction, indicating its involvement in the formation of TAGE aggregates. The present results suggest that the ubiquitin-proteasome pathway and p62 play a role in the degradation and aggregation of intracellular TAGE formed by GA. This study provides new insights into the mechanisms underlying TAGE metabolism and may lead to the development of novel therapeutic strategies for diseases associated with TAGE accumulation.
... Therefore, researchers cannot compare the AGE content of samples with that calculated using other methods. In addition, nitrocellulose membranes were selected for the other experiments, because PVDF membranes may have been unsuitable for use in slot blotting [14,52,53], although some In this examination, only the component (A Right − Blank Right ) was multiplied with the calibration-corrected luminance value, which was used for the bands of HRP marker solution on both right and left sides of the PVDF membrane. ...
... TAGE and N ε -carboxymethy-lysine (CML) were semi-quantified, and the data were statistically analyzed [14,52,53]. Because the value was calculated as the luminance of AGEs per β-actin, this method can be normalized with a loading control (i.e., β-actin). ...
... Therefore, researchers cannot compare the AGE content of samples with that calculated using other methods. In addition, nitrocellulose membranes were selected for the other experiments, because PVDF membranes may have been unsuitable for use in slot blotting [14,52,53], although some studies still report their use [13,54]. Bronowicka-Szydełko et al. blotted serum samples onto PVDF membranes and used anti-MGO-derived AGEs as primary and secondary antibodies or only secondary antibodies for probing the proteins [13]. ...
Various types of advanced glycation end-products (AGEs) have been identified and studied. I have reported a novel slot blot analysis to quantify two types of AGEs, glyceraldehyde-derived AGEs, also called toxic AGEs (TAGE), and 1,5-anhydro-D-fructose AGEs. The traditional slot blot method has been used for the detection and quantification of RNA, DNA, and proteins since around 1980 and is one of the more commonly used analog technologies to date. However, the novel slot blot analysis has been used to quantify AGEs from 2017 to 2022. Its characteristics include (i) use of a lysis buffer containing tris-(hydroxymethyl)-aminomethane, urea, thiourea, and 3-[3-(cholamidopropyl)-dimetyl-ammonio]-1-propane sulfonate (a lysis buffer with a composition similar to that used in two-dimensional gel electrophoresis-based proteomics analysis); (ii) probing of AGE-modified bovine serum albumin (e.g., standard AGE aliquots); and (iii) use of polyvinylidene difluoride membranes. In this review, the previously used quantification methods of slot blot, western blot, immunostaining, enzyme-linked immunosorbent assay, gas chromatography–mass spectrometry (MS), matrix-associated laser desorption/ionization–MS, and liquid chromatography–electrospray ionization–MS are described. Lastly, the advantages and disadvantages of the novel slot blot compared to the above methods are discussed.
... Importantly, AGEs from glucose did not have this effect. It was proposed that elucidation of the mechanism of GA action in neurodegeneration may contribute to the development of new neuroprotective therapies (Nasu et al. 2020). In another study on the role of tau, the effects of its full length (Tau 441) and a shortened, pathologic form (Tau 421) on cytoskeleton and plasma membrane functions were evaluated. ...
Neurodegenerative diseases represent a large group of disorders characterized by gradual loss of neurons and functions of the central nervous systems. Their course is usually severe, leading to high morbidity and subsequent inability of patients to independent functioning. Vast majority of neurodegenerative diseases is currently untreatable, and only some symptomatic drugs are available which efficacy is usually very limited. To develop novel therapies for this group of diseases, it is crucial to understand their pathogenesis and to recognize factors which can influence the disease course. One of cellular structures which dysfunction appears to be relatively poorly understood in the light of neurodegenerative diseases is tubulin cytoskeleton. On the other hand, its changes, both structural and functional, can considerably influence cell physiology, leading to pathological processes occurring also in neurons. In this review, we summarize and discuss dysfunctions of tubulin cytoskeleton in various neurodegenerative diseases different than primary tubulinopathies (caused by mutations in genes encoding the components of the tubulin cytoskeleton), especially Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, prion diseases, and neuronopathic mucopolysaccharidoses. It is also proposed that correction of these disorders might attenuate the progress of specific diseases, thus, finding newly recognized molecular targets for potential drugs might become possible.
... Cell clusters contained more than 5 cells were excluded [21]. Otherwise, the neurite length was obtained by the measurement of the longest neurite in each image using the software Motic Images Plus 3.0 [22]. ...
... The activity of ACH on neurite outgrowth formation in high glucose-treated cells was investigated. To create the neurite outgrowth model, SH-SY5Y was cultured in serum starvation, which was low glucose DMEM supplemented with 1% FBS to avoid overgrowth of cells; 10 µM retinoic acid (RA) was added for 24 h [22]. In response to serum starvation and retinoic acid, SH-SY5Y will begin neurite outgrowth formation. ...
Hyperglycemia is one of the important causes of neurodegenerative disorders and aging. Aquilaria crassna Pierre ex Lec (AC) has been widely used to relieve various health ailments. However, the neuroprotective and anti-aging effects against high glucose induction have not been investigated. This study aimed to investigate the effects of hexane extract of AC leaves (ACH) in vitro using human neuroblastoma SH-SY5Y cells and in vivo using nematode Caenorhabditis elegans. SH-SY5Y cells and C. elegans were pre-exposed with high glucose, followed by ACH treatment. To investigate neuroprotective activities, neurite outgrowth and cell cycle progression were determined in SH-SY5Y cells. In addition, C. elegans was used to determine ACH effects on antioxidant activity, longevity, and healthspan. In addition, ACH phytochemicals were analyzed and the possible active compounds were identified using a molecular docking study. ACH exerted neuroprotective effects by inducing neurite outgrowth via upregulating growth-associated protein 43 and teneurin-4 expression and normalizing cell cycle progression through the regulation of cyclin D1 and SIRT1 expression. Furthermore, ACH prolonged lifespan, improved body size, body length, and brood size, and reduced intracellular ROS accumulation in high glucose-induced C. elegans via the activation of gene expression in the DAF-16/FoxO pathway. Finally, phytochemicals of ACH were analyzed and revealed that β-sitosterol and stigmasterol were the possible active constituents in inhibiting insulin-like growth factor 1 receptor (IGFR). The results of this study establish ACH as an alternative medicine to defend against high glucose effects on neurotoxicity and aging.
... In this review, the content that overlaps with the previous paper has been simplified, and novel findings have been added. In our recent study, the intracellular generation/accumulation of TAGE was detected in many cell types [30][31][32][33][34][35][36][37][38][39][40][41][42]. Elevated levels of TAGE damage cells, which results in the leakage of TAGE into the circulation and to the surrounding cells; the damage induced promotes the onset and progression of lifestyle-related diseases (LSRD) [9][10][11]. ...
... We found that TAGE induced similar alterations to those observed in AD. GA induced dysfunctional neurite outgrowth via TAGE-β-tubulin aggregation, which resulted in the TAGE-dependent abnormal aggregation of β-tubulin and tau phosphorylation in SH-SY5Y cells [40]. These findings indicated that the formation of TAGE-β-tubulin contributed to the production of paired helical filaments, one of the components of NFT. ...
The habitual and excessive consumption of sugar (i.e., sucrose and high-fructose corn syrup, HFCS) is associated with the onset and progression of lifestyle-related diseases (LSRD). Advanced glycation end-products (AGEs) have recently been the focus of research on the factors contributing to LSRD. Approaches that inhibit the effects of AGEs may be used to prevent and/or treat LSRD; however, since the structures of AGEs vary depending on the type of reducing sugars or carbonyl compounds to which they respond, difficulties are associated with verifying that AGEs are an etiological factor. Cytotoxic AGEs derived from glyceraldehyde, a triose intermediate in the metabolism of glucose and fructose, have been implicated in LSRD and are called toxic AGEs (TAGE). A dietary imbalance (the habitual and excessive intake of sucrose, HFCS, or dietary AGEs) promotes the generation/accumulation of TAGE in vivo. Elevated circulating levels of TAGE have been detected in non-diabetics and diabetics, indicating a strong relationship between the generation/accumulation of TAGE in vivo and the onset and progression of LSRD. We herein outline current findings on “TAGE as a new target” for human health.
... We previously reported that intracellular TAGE were generated by hepatocytes [16,17], cardiomyocytes [8], skeletal muscle myoblasts [13], pancreatic islet β-cells [12], pancreatic ductal cells [18], and neuroblastoma cells [19], and are responsible for inducing cell death. Therefore, intracellular TAGE is speculated to promote LSRD via cell death and organ dysfunction [2,20]. ...
Cardiovascular disease (CVD) is a lifestyle-related disease (LSRD) induced by the dysfunction and cell death of cardiomyocytes. Cardiac fibroblasts are activated and differentiate in response to specific signals, such as transforming growth factor-β released from injured cardiomyocytes, and are crucial for the protection of cardiomyocytes, cardiac tissue repair, and remodeling. In contrast, cardiac fibroblasts have been shown to induce injury or death of cardiomyocytes and are implicated in the pathogenesis of diseases such as cardiac hypertrophy. We designated glyceraldehyde-derived advanced glycation end-products (AGEs) as toxic AGEs (TAGE) due to their cytotoxicity and association with LSRD. Intracellular TAGE in cardiomyocytes decreased their beating rate and induced cell death in the absence of myocardial ischemia. The TAGE levels in blood were elevated in patients with CVD and were associated with myocardial ischemia along with increased risk of atherosclerosis in vascular endothelial cells in vitro. The relationships between the dysfunction or cell death of cardiac fibroblasts and intracellular and extracellular TAGE, which are secreted from certain organs, remain unclear. We examined the cytotoxicity of intracellular TAGE by a slot blot analysis, and TAGE-modified bovine serum albumin (TAGE-BSA), a model of extracellular TAGE, in normal human cardiac fibroblasts (HCF). Intracellular TAGE induced cell death in normal HCF, whereas TAGE-BSA did not, even at aberrantly high non-physiological levels. Therefore, only intracellular TAGE induced cell death in HCF under physiological conditions, possibly inhibiting the role of HCF.
... We also showed that the neurotoxicity of TAGE was stronger than that of well-known AGEs, including Glu-AGEs, in neurons (Takeuchi et al., 2000a. We recently performed a two-dimensional immunoblot analysis, which revealed that βtubulin was a target of TAGE (Nasu et al., 2020). Microtubules comprise repeating units of α/β-tubulin heterodimers, and their assembly is an important event that has been implicated in neurite outgrowth. ...
... Microtubules comprise repeating units of α/β-tubulin heterodimers, and their assembly is an important event that has been implicated in neurite outgrowth. In SH-SY5Y cells, GA was found to induce TAGE-β-tubulin formation and the abnormal aggregation of βtubulin and suppress neurite outgrowth (Nasu et al., 2020). However, the effects of AGE inhibitors on the TAGEassociated inhibition of neurite outgrowth through the abnormal aggregation of β-tubulin in neurons remain unknown. ...
... Our previous immunohistochemical findings revealed staining for β-tubulin in growing neurites and cell bodies, with strong staining being detected in axons and growth cones. However, βtubulin staining was also observed in the cytosol and axon hillock area of GA-treated neurons, which appeared to be the sites at which neurite outgrowth was initiated (Nasu et al., 2020). To examine the effects of AG, PM, and PA on the inhibition of neurite outgrowth by GA, we performed immunocytochemistry for α-internexin, an axon marker protein, in order to measure neurite lengths (Yuan et al., 2006). ...
Diabetes mellitus (DM) has been identified as a risk factor for the onset and progression of Alzheimer’s disease (AD). In our previous study, we demonstrated that glyceraldehyde (GA)-derived toxic advanced glycation end-products (toxic AGEs, TAGE) induced similar alterations to those observed in AD. GA induced dysfunctional neurite outgrowth via TAGE-β-tubulin aggregation, which resulted in the TAGE-dependent abnormal aggregation of β-tubulin and tau phosphorylation in human neuroblastoma SH-SY5Y cells. However, the effects of inhibitors of AGE formation on dysfunctional neurite outgrowth caused by GA-induced abnormalities in the aggregation of β-tubulin and tau phosphorylation remain unknown. Aminoguanidine (AG), an AGE inhibitor, and pyridoxamine (PM), a natural form of vitamin B6 (VB6), are effective AGE inhibitors. Therefore, the present study investigated whether AG or PM ameliorate TAGE-β-tubulin aggregation and the suppression of neurite outgrowth by GA. The results obtained showed that AG and PM inhibited the formation of TAGE-β-tubulin, mitigated the GA-induced suppression of neurite outgrowth, and reduced GA-mediated increases in tau phosphorylation levels. Collectively, these results suggest the potential of AG and PM to prevent the DM-associated onset and progression of AD.
