Soluble receptor for advanced glycation end products as a biomarker of symptomatic vasospasm in subarachnoid hemorrhage.
Impact factor: 3.968
Level of evidence: III
Sample Size/Population: 27 patients after excluding those with significant impairments that could affect the study such as cancer, renal dysfunction, etc. These were then split into non-symptomatic vasospasm and symptomatic vasospasm groups. A rat subarachnoid hemorrhage model was also conducted where the bifurcation of the left anterior and middle cerebral arteries was perforated mimicking a subarachnoid hemorrhage.
Protocol: Blood samples on days 5, 7, 10 and 14 after subarachnoid hemorrhage. Patient assessed on arrival (Hunt and Kosnik system) and discharge (Glasgow Outcome Scale). Blood sRAGE levels were measured using ELISA. In the rat study, neurobehavioral testing was completed and sRAGE levels were tested using ELISA.
Statistical Analysis: Student T-test, chi square test, Mann-Whitney U-test, two-way ANOVA with Tukey-Kramer test with a p-value of .05 being significant.
Study Type: Experimental Results: sRAGE was significantly lower in the SVS group vs non-SVS group, .84 being the cut-off value. In the rat models, there was significantly lower plasma sRAGE in comparison to the sham group.
Limitation: Sample size
Conclusion: sRAGE levels can be used as a potential biomarker for predicting SVS after SAH.
Aida Y, Kamide T, Ishii H, Kitao Y, Uchiyama N, Nakada M, Hori O. Soluble receptor for advanced glycation end products as a biomarker of symptomatic vasospasm in subarachnoid hemorrhage. J Neurosurg. 2019 Nov 1:1-9. doi: 10.3171/2019.8.JNS191269. Epub ahead of print. PMID: 31675694.
Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis
Impact Factor: 3.921
Level of evidence: IV
Study type: Retrospective
Sample size: 11 donors up to 2-5 samples each depending on part of study
Intro: Enhanced oxidative stress and neuroinflammation are key contributors in ALS pathogenesis. RAGE is shown to increase these factors the majority of the time. This study looks at RAGE and its ligands (Advanced glycation end products, HMGB1 and S100/calgranulin family) in a person with ALS vs a normal control. It also discussed sRAGE levels being low in ALS patients due to it being a natural competitor.
Material/Methods: Tissue collection for immunohistochemistry, mRNA analysis and western blot were collected from deceased individuals with ALS and with no neurodegenerative disease diagnosis.
Statistical Analysis: Two-tailed T-test, significant p-value as <.05.
Results: Increased RAGE, increased mRNA expression of AGER (gene encoding RAGE), increased immunostaining of S100B, HMGB1 and CML (AGE prototype) and higher protein levels of RAGE, S100B and HMGB1 in ALS samples in comparison to controls.
Conclusion: There is increased expression of inflammatory RAGE in the human spinal cord affected by ALS. Limitation being this is the end-stage of ALS since these were deceased individuals and since this is the first study of its kind there is no comparison. Overall this shows that RAGE might be a possible mediator in ALS and this is a rational hypothesis.
Juranek, J. K., Daffu, G. K., Wojtkiewicz, J., Lacomis, D., Kofler, J., & Schmidt, A. M. (2015). Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis. Frontiers in cellular neuroscience, 9, 485. https://doi.org/10.3389/fncel.2015.00485
Soluble RAGE Treatment Delays Progression of Amyotrophic Lateral Sclerosis in SOD1 Mice
Impact factor: 3.921
Level of evidence: VII
Study: Experimental
Sample Size: n=3-n=12 depending on experiment
Introduction: The purpose is to see if sRAGE treatment offers protection against premature cell death in motor neurons of SOD1 transgenic mouse (SOD1 being mutated ALS by familial inheritance 15-20% of the time), thereby delaying the onset and progression of ALS and improving overall health status of these mice. sRAGE is a natural competitor of RAGE that sequesters RAGE ligands (such as advanced glycation end products) and blocks their interaction with cell surface RAGE.
Methods: Mice were injected with either sRAGE or murine serum albumin beginning at 8 weeks until termination. Final stage of disease was determined by 20% weight loss or the animal’s inability to right itself within 20 s when placed on its side. Disease onset accompanied by posture and gait impairment, was defined as the time when animals lost ∼10% of their maximal weight, before any noticeable decrease in motor performance tests. Only male mice were used for the sRAGE studies due to the protective effects of estrogen on onset and progression of disease. Immunohistochemistry was performed to see the amount of normal RAGE expression for comparison before injection in the SOD1 mice and were sacrificed at that time. Immunostaining was performed for quantification of lumbar ventral horn motor neurons and glial cells. RNA and RT-PCR was conducted on lumbar spinal samples. Motor function tests were performed using standard well-established procedures and scored by a naive scorer and was handed by a person who was understanding of the treatment codes to differentiate. Muscle strength test was measured by the hanging wire test. Grip strength was measured by manufacturer’s training guidelines.
Statistical analysis: Wilcoxin Signed-Rank test for RNA RT-PCR comparisons. Kaplan-Meier estimate was used to calculate probability of survival over a specific length of time. All values are presented as mean ± standard error (SEM). The statistical significance of differences (p < 0.05) was evaluated by non-parametric ANOVA with Student-Newman–Keuls post-test.
Results: There was increased expression of RAGE in ALS mouse lumbar spinal cord. sRAGE treated mice displayed a higher probability of surviving after disease onset with a p value of 0.007 and in comparison to the MSA-treated group with a p value of 0.01. They had significantly higher body weight at typical disease onset (p=0.01) and days lived (p=0.02) in comparison to MSA mice. sRAGE treated mice also had better grip and muscle strength. sRAGE treated mice had a significantly higher number of neurons at the terminal stage of disease and also a significantly lower amount of astrocytes indicating less progression of disease.
Conclusion: sRAGE treatment could be a potential supplementary candidate for those with ALS. With no significant therapeutic options for this incurable disease, the improvement of lifespan, motor performance and less neuronal death, this could be a potential candidate with further research.
Limitations: Rat model is not equivalent to human model
Juranek, J. K., Daffu, G. K., Geddis, M. S., Li, H., Rosario, R., Kaplan, B. J., Kelly, L., & Schmidt, A. M. (2016). Soluble RAGE Treatment Delays Progression of Amyotrophic Lateral Sclerosis in SOD1 Mice. Frontiers in cellular neuroscience, 10, 117. https://doi.org/10.3389/fncel.2016.00117
The advanced glycation end-product N epsilon-(carboxymethyl)lysine level is elevated in cerebrospinal fluid of patients with amyotrophic lateral sclerosis
Impact factor: 2.274
Level of evidence: IV
Study: Prospective Cohort
Sample Size: 67 patients
Population:
Group 1: patients with sporadic definite and probable ALS according to the revised El Escorial diagnostic criteria (18 male, 7 female; age range 27.5–74.5 years);
Group 2: patients who showed clinical signs only of the lower motoneuron (LMN) representing a possible ALS (7 male, 6 female; age range 27.4–78.2 years); (3) patients with clinically probable and possible AD (3 male, 6 female; age range 52.8–79.6 years); and (4) controls with other neurological diseases not affecting the central nervous system. Routine work up of the control patients revealed normal clinical neurological status and imaging results (8 male, 12 female; age range 33.4–71.2 years.)
Intro: Compare AGE in ALS, other neurological diseases affecting CNS and non affecting CNS. This is different compared to other studies because most are post-mortem, while this study is antemortem.
Methods: Paired lumbar CSF and serum samples were collected from 67 patients. ELISA using a CML-specific monoclonal antibody Statistical analysis: Mann–Whitney U test and Spearman’s rank correlation coefficient (rs). The level of significance was established at p<0.05.
Results: Significantly elevated CML levels in CSF in ALS and LMN disease when compared to control subjects. CSF levels of CML were even higher than in AD patients. Compared to controls, normalized CSF values of CML were increased in ALS, LMN disease and AD by a factor of 1.7, 1.5 and 1.2, respectively. CSF/serum ratio of CML is significantly elevated in both the ALS and LMN disease compared to controls by a factor of 2.3 and 1.9, respectively, whereas for AD an elevation by a factor of 1.4 was seen. For ALS, the CSF/serum ratio was also significantly higher compared to AD (p = 0.029)
Conclusion: significant increases of AGE products in CSF of motor neuron disease patients, especially in ALS, providing further evidence for the involvement of oxidative stress in the pathogenesis of ALS. Results on CML levels in CSF and serum samples suggest that these products are of intrathecal origin, i.e. oxidative stress in ALS patients is most likely confined to the CNS. Furthermore, increased CML CSF levels may provide a novel diagnostic tool as a surrogate marker for glycloxidative mechanisms in ALS.
Limitations: First of its kind, no comparisons.
Kaufmann E, Boehm BO, Süssmuth SD, Kientsch-Engel R, Sperfeld A, Ludolph AC, Tumani H. The advanced glycation end-product N epsilon-(carboxymethyl)lysine level is elevated in cerebrospinal fluid of patients with amyotrophic lateral sclerosis. Neurosci Lett. 2004 Nov 23;371(2-3):226-9. doi: 10.1016/j.neulet.2004.08.071. PMID: 15519762.
Detection of (CML) and non-CML advanced glycation end-products in the anterior horn of amyotrophic lateral sclerosis spinal cord
Impact factor: 3.286
Level of evidence: VI
Study: Observational
Sample Size: 3 ALS + deceased patients and 3 ALS - control deceased patients that were age matched
Intro: The purpose of this paper was to determine if late-stage glycation reaction follows the early glycation reaction these authors had identified previously which leads to the formation of AGEs in the ALS spinal cord.
Methods: The presence of CML and non-CML AGE was examined in ALS spinal cords by using antibodies for CML and non-CML and immunohistochemical staining was performed.
Statistical analysis: Observation only
Results and Conclusion: In the present study, by observation using anti-CML and anti-non-CML AGE antibodies, the presence of AGEs in the anterior horn of the sporadic ALS spinal cord was confirmed. The presence of non-CML AGE in the anterior horn of the ALS spinal cord indicates that the later stage of the glycation reaction is involved in the pathogenesis of ALS. The presence of CML in the anterior horn of the ALS spinal cord was also confirmed. The presence of CML may reflect augmented oxidative stress and not be specific to AGE production only. Therefore, non-CML AGEs were a better indicator of pathogenesis of ALS.
Limitations: One problem is the sensitivity of the antibodies, and the difference in sensitivity may result in different intensity of staining of the target proteins. Some target proteins may be specifically localized in microglia, in which case they would not be able to remain in the early glycation products but would quickly proceed to the formation of non-CML AGE. Alternatively, there may be a special environment in microglial cells that facilitates the rapid progress of the glycation reaction to the later stage. Also it was a relatively small study size.
Kikuchi S, Shinpo K, Ogata A, Tsuji S, Takeuchi M, Makita Z, Tashiro K. Detection of N epsilon-(carboxymethyl)lysine (CML) and non-CML advanced glycation end-products in the anterior horn of amyotrophic lateral sclerosis spinal cord. Amyotroph Lateral Scler Other Motor Neuron Disord. 2002 Jun;3(2):63-8. doi: 10.1080/146608202760196020. PMID: 12215227.
Selective formation of certain advanced glycation end products in spinal cord astrocytes of humans and mice with superoxide dismutase-1 mutation
Impact factor: 14.256
Level of evidence: VI and VII
Study: Experimental + observational
Sample Size: 6 ALS spinal cords at autopsy and 6 SOD1 mice spinal cords + controls Introduction: The aim of the present study was to assess a role for carbonyl stress in motor neuron degeneration associated with superoxide dismutase-1 (SOD1) mutant familial ALS and its transgenic mouse model, using an immunohistochemical investigation of advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs).
Methods: Immunohistochemistry to detect AGEs and ALEs
Statistical analysis: Observation
Results: The most noteworthy finding in this study was the presence of CEL, argpyrimidine, pyrraline and CML in the spinal cord astrocytes of the SOD1 A4V familial ALS patients and the G93A mutant SOD1 transgenic mice. As mentioned above, CEL and argpyrimidine are derived from methylglyoxal, and pyrraline is derived from 3-deoxyglucosone. protein glycation, but not lipid peroxidation, is enhanced in ALS patients with an SOD1 mutation and mutant SOD1 transgenic mice, in which certain AGEs are selectively formed in the spinal cord astrocytes. There was no reactivity in controls.
Shibata N, Hirano A, Hedley-Whyte ET, Dal Canto MC, Nagai R, Uchida K, Horiuchi S, Kawaguchi M, Yamamoto T, Kobayashi M. Selective formation of certain advanced glycation end products in spinal cord astrocytes of humans and mice with superoxide dismutase-1 mutation. Acta Neuropathol. 2002 Aug;104(2):171-8. doi: 10.1007/s00401-002-0537-5. Epub 2002 Apr 18. PMID: 12111360.
Effects of RAGE inhibition on the progression of the disease in hSOD1G93A ALS mice
Impact factor: 2.052
Level of evidence: VII Study: Rat
Introduction: Those with ALS have increased levels of RAGE and many of its ligands, including oxidatively modified nerve growth factor (NGF), high mobility group box 1 protein (HMGB1), S100B and glycated proteins (advanced glycation end products), in the spinal cord of ALS patients and hSOD1G93A mice. HSOD1G93A mice are meant to mimic SOD1 variants in those with familial ALS. The ligands and their binding to RAGE is shown to take part in the neurodegenerative portion of ALS pathogenesis. ALS patients also have less sRAGE in their serum which compete for ligand binding to inhibit the RAGE effects. Previously sRAGE treatment had been used and only extended the life of these mice but did not cross the blood brain barrier which is necessary to prevent neurodegeneration. FPS-ZM1 is a high affinity RAGE-specific inhibitor that does cross the blood brain barrier and they also looked at RAP, a RAGE antagonist peptide that inhibits the interaction of the receptor with multiple ligands.
Methods: PCR and Western Blot
Statistical analysis: Survival, onset, and weight-loss data were analyzed with KaplanMeier curves and log rank test. Groups of four animals were used for biochemical analysis and all data are reported as mean ± SD. Comparisons between two groups were performed with an unpaired t test. Hind-limb grip strength data during the progression of the disease were analyzed by multiple t tests assuming populations with the same standard deviation. Cell culture experiments were repeated in at least three independent primary culture preparations, and values from each independent experiment were combined for data reporting. Multiple group comparisons were performed with one-way ANOVA with Tukey's post-test. Differences were declared statistically significant if P ≤ .05
Results: RAP and FPS-ZM1 both prevented motor neuron death when added to co-cultures and in the ALS spinal cord extracts. In mice, treatment with FPS-ZM1 did not delay onset of the disease but mice showed a significant improvement in hind-limb grip strength. There were significant decreases in mRNA and protein levels of E3 ubiquitin and linked to muscle atrophy. They also had a decrease in astrogliosis and microgliosis as well as an increase in the number of large motor neurons in the ventral horn of the spinal cord.
Conclusion: Inhibition of RAGE signaling has shown to be neuroprotective in models of Alzheimer's, Parkinson's and ALS. Phase 1 and 2 of these trials have been promising but limited to those with impaired glucose intolerance. These mice trials show the benefits in mice and the ability to cross the blood brain barrier. More research is needed but these results show a possible way to have better lifestyles for ALS patients.
Limitations: Rat studies are not equivalent to human studies.
Liu, L., Killoy, K. M., Vargas, M. R., Yamamoto, Y., & Pehar, M. (2020). Effects of RAGE inhibition on the progression of the disease in hSOD1G93A ALS mice. Pharmacology research & perspectives, 8(4), e00636. https://doi.org/10.1002/prp2.636
Advanced glycation end-products produced systemically and by macrophages: A common contributor to inflammation and degenerative diseases
Impact Factor: 10.557
Type of Research: Review Article
Medical: Research on advanced glycation end products (AGEs) and their receptors have led to the indication that AGEs promote host cell death and contribute to organ damage. Elevated levels of AGEs cause formation of reactive oxygen and nitrogen species which leads to further development of AGEs. AGEs have been considered in the pathophysiologies of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and diabetic complications. Activated macrophages often induce inflammatory degeneration of host cells or dysfunction in Alzheimer’s disease, vascular disease, and inflammatory diseases. RAGE ligands, such as AGEs, modulate important biological processes required for macrophage activation. Activated macrophages act as a source of AGE-albumin accumulation in tissue, which may be further used to develop therapeutic agents. There have been developmental efforts of therapeutics against AGEs, including angiotensin II receptor antagonists and angiotensin converting enzyme inhibitors, antioxidants, natural substances, and anti-inflammatory molecules. The interaction of AGEs-RAGE on the plasma membrane triggers the downstream signaling of inflammation, oxidative stress, and apoptosis in many cells, including neurons, endothelial cells, lung cells, and muscle cells. Therapeutic agent development has been focused around suppressing AGEs or RAGE formation and preventing AGEs-RAGE interaction.
Conclusion: More research is required to determine how to prevent, remove, and estimate the AGEs/RAGE interaction in relation to macrophage activation and to understand its pathophysiologic mechanism in many neurodegenerative diseases and diabetic complications.
Byun K, Yoo Y, Son M, Lee J, Jeong GB, Park YM, Salekdeh GH, Lee B. Advanced glycation end-products produced systemically and by macrophages: A common contributor to inflammation and degenerative diseases. Pharmacol Ther. 2017 Sep;177:44-55. doi: 10.1016/j.pharmthera.2017.02.030. Epub 2017 Feb 13. PMID: 28223234.
Advanced glycation end products and neurodegenerative diseases: mechanisms and perspective
Impact Factor: 3.115
Type of Study: Review Article Scientist Summary
Purpose: Review the recent advances in studying the link between AGEs and neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s disease
Introduction: Neurodegenerative diseases cause gradual loss of neurons and deposition of misfolded proteins in the brain. Oxidative stress plays a key role in the formation of these misfolded proteins such as tau, α-synuclein, and prions for example. Oxidative stress leads to the formation of AGEs, and AGEs causes further oxidative stress. Hence AGEs contribute to neurodegenerative diseases
Protein Glycation and the formation of AGES: Formation of AGEs is irreversible and causes cross-linking between itself and proteins and peptides. AGEs are formed via reactions involving glucose turning into Schiff base, which turns into Amadori product (fructosamine). Amadori product then turns into AGEs. Alternatively, AGEs can be formed via methylglyoxal (MG) and 3-deoxyglucosone (3-DG).
AGEs mediated signaling pathways: Alzheimer’s causes progressive decline in cognition. AGEs bind to RAGE (receptor for advanced glycation end products). RAGE activation causes activation of NF-kB. Activation of NF-kB can lead to further increased RAGE. Hence ligands for RAGE can increase RAGE such as AGEs. RAGE activates many cellular signaling events, and many are still unknown.
AGEs and Alzheimer’s Disease: AGEs accelerate beta-amyloid plaque formation via protein cross-linking. Another mechanism is that AGEs upregulate amyloid precursor protein (APP) which leads to more beta-amyloid plaques. ApoE4 seems to increase Alzheimer’s risk because AGEs bind to ApoE4 and contributes to plaque formation. AGEs can also induce tau protein hyperphosphorylation which is another marker for Alzheimer’s.
AGEs and Parkinson’s Disease: Parkinson’s causes tremor, shaking, stiff muscles, limited movement, and difficulty in walking and balance. Lewy bodies, which contain α-synuclein, is a marker for Parkinson’s. Aggregation of α-synuclein is induced by AGEs and leads to oxidative stress and cytotoxicity.
Conclusion: AGEs in general lead to accumulation of proteins which causes oxidative stress, inflammatory response, and cells becoming dysfunctional or causing cell death. Possible AGEs could become a marker for neurodegenerative diseases and help in early diagnosis. Possible pharmacological goals can include making AGE inhibitors and RAGE antagonists.
Li J, Liu D, Sun L, Lu Y, Zhang Z. Advanced glycation end products and neurodegenerative diseases: mechanisms and perspective. J Neurol Sci. 2012 Jun 15;317(1-2):1-5. doi: 10.1016/j.jns.2012.02.018. Epub 2012 Mar 11. PMID: 22410257.
High Dietary Advanced Glycation End Products Impair Mitochondrial and Cognitive Function
Impact Factor: 3.909
Level of Evidence: II
Type of Study: RCT on mice
Purpose: Find out the link between dietary AGEs and mitochondrial dysfunction
Materials and Methods: Mice put into AGE+ and AGE- groups. Morris Water Maze to assess learning and memory. Analysis of glycation of BSA. Measured reactive oxygen species. Used immunodetection of AGE-adducts via ELISA. Measured enzymes associated with respiratory chain in cerebral cortex such as complex I and IV. Measured ATP levels via Bioluminescence assay Kit. Nesting behavior was acquired from mice.
Statistics: One-way ANOVA followed with Fischer’s protected least significant difference for post hoc comparisons. A p-value less than 0.05 was considered significant
Results: AGE+ diets increased ROS levels such as superoxide anion, hydrogen peroxide, and hydroxyl radicals compared to AGE- mice. Also had increased Ketoamine Amadori products and glycation intermediates which are associated with AGEs. Indicate AGEs accumulation in brain causing higher ROS levels. AGE+ mice has reduced complex I and IV and decreased ATP levels compared to AGE- mice. Hence diet causes mitochondrial respiratory defects. AGE+ mice had deficits in learning and memory after Morris Water Maze assessment. AGE+ mice scored lower on nest-making task as well.
Discussion: Diet high in AGEs disturbs mitochondrial and cognitive function. This helps in understanding the neuropathology of neurodegenerative diseases. The mechanism for mitochondrial dysfunction seems to be that AGEs cause ROS increase which leads to mitochondrial damage. Mitochondrial damage leads to less ATP production. Hence there is a disturbance in brain metabolism. Looked at hippocampus and hypothesized AGE lead to synaptic plasticity problems linked with the mitochondrial problems. This led to problems in tasks seen in the results section. AGE seems to be a risk factor for deficits in learning, memory, and daily task performance.
Akhter F, Chen D, Akhter A, Sosunov AA, Chen A, McKhann GM, Yan SF, Yan SS. High Dietary Advanced Glycation End Products Impair Mitochondrial and Cognitive Function. J Alzheimers Dis. 2020;76(1):165-178. doi: 10.3233/JAD-191236. PMID: 32444539; PMCID: PMC7581068.
Nutrition and AGE-ing: Focusing on Alzheimer's Disease
Impact Factor: 5.076
Type of Study: Review Article
Introduction: Industrial processing of food causes loss of nutrients and creation of advanced glycation end products (AGEs). Purpose of review is to discuss the link between diet, AGEs, and Alzheimer’s dementia (AD).
Alzheimer’s: Rotterdam study showed in a large prospective study of the lowered risk of AD when taking a statin since statins reduced plasma cholesterol levels. APOE- 𝜀4 is a cholesterol transporter in brain and a risk factor for AD. This may explain the effects of statin
Polyunsaturated Fatty Acids: Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have anti-inflammatory effects in the brain and a diet rich in these polyunsaturated fatty acids suggests an influence in neuroinflammatory pathways. Animal model studies show the decrease in Aβ production when consuming DHA and also with long term omega-3 supplementation
Vitamins: Vitamins are antioxidants. Vitamin E and vitamin C with food have been shown to decrease AD incidence. Giving moderate AD patients Vitamin E was shown to slow disease progression in one study. But no difference was shown in a different study. This difference probably has to do with different vitamin E supplement compositions. Dosage of vitamin E is another issue that needs to be explored. Low vitamin D is risk factor for AD. Polymorphisms in vitamin D receptor have been linked to AD. Older people with hyperhomocysteinemia tend to be have lower vitamin B status and also lower cognitive test scores.
Polyphenols: Polyphenols are found in plants, fruits, vegetables. Epigallocatechin-3-gallate in green tea, 4-O-methyl honokiol in Magnolia officinialis, resveratrol in graphs, and ginkgolid A in ginkgo biloba seem to protect against AD because of antioxidant and anti-inflammatory effects. A interventional study In mice has show that a polyphenol-rich extract from grapes and blueberry showed the mice improved in spatial strategies in adult and middle-aged mice. Curcumin has beta-amyloid reducing effects in animal models with AD.
Dietary-Advanced Glycation End Products (d-AGEs) and Cognitive Decline: In general there is a link between AGEs and AD pathology. Adopting a AGE-restricted diet may help prevent cognitive decline.
Dietary AGEs and Alzheimer’s Disease: Association or Causality: Mediterranean diet and traditional Japanese diet were shown to prevent AD. The traditional Japanese diet is low in meat and dairy products which have high levels of AGEs. The western diet has had an increase in meat eating and total dietary AGEs, and there has been an increase in AD incidence too. Still unsure if other factors are involved such as trace mineral in brain, obesity rates, vitamin D concentrations, physical activity, alcohol consumption rates.
Good Tips for a Healthy Low-AGE Diet: Different studies have shown the average intake of AGEs is 15,000 KU/day in healthy individuals. 7,500 KU/day has been proposed as a realistic daily goal for dietary AGEs. A reduction of this sort has shown reduced circulating AGEs and reduced oxidative stress and inflammatory marks. Broiling, grilling, frying, and roasting have been shown to greatly increase dietary AGEs compared to boiling and stewing. Herbs, condiments, and spices such as curcumin, cinnamon, parsley, thyme, and clove have been shown to reduce cooking-induced AGE production.
Abate G, Marziano M, Rungratanawanich W, Memo M, Uberti D. Nutrition and AGE-ing: Focusing on Alzheimer's Disease. Oxid Med Cell Longev. 2017;2017:7039816. doi: 10.1155/2017/7039816. Epub 2017 Jan 12. PMID: 28168012; PMCID: PMC5266861.
Sulforaphane Inhibits MGO-AGE-Mediated Neuroinflammation by Suppressing NF-κB, MAPK, and AGE-RAGE Signaling Pathways in Microglial Cells
Impact Factor: 5.014
Level of Evidence: VII
Study: Experimental
Intro: Sulforaphane (SFN) is a natural isothiocyanate found in cruciferous vegetables that is known to have antioxidant, anti-inflammatory, anti-apoptotic, cytoprotective, and anti-diabetic effects. This study aims to see the effects of SFN on neuroinflammatory reactions.
Methods: This study looked at Sulforaphane and its effect on AGE inhibition, breakdown, nitrate production, cell viability effects, lactate dehydrogenase production, reactive oxygen species, NF-κB translocation/activation, proinflammatory cytokines, and receptors using western blot, ELISA, and assays.
Statistical analysis: Results are expressed as mean ± standard error of the mean (SEM). One-way analysis of variance followed by Tukey post-hoc test. p < 0.05 was considered statistically significant.
Results/Conclusion: SFN can be used to prevent MGO-AGE formation but cannot be used to alter formation and breakdown of MGO-AGEs. SFN significantly reduced MGO-AGEs nitrate production showing that it could antagonize MGO-AGEs-induced oxidative stress in activated microglia. A dramatically high amount of intracellular AGEs was observed in the MGO-AGEs and SFN treatment significantly lowered the level of AGE inside the microglial cells. Reactive oxygen species produced by MGO-AGEs were significantly reduced in SFN treatment in microglial cells. SFN inhibited NFLP3, decreased activation of GSK3β and p38 phosphorylation and decreased expression of the receptor for AGE all involved in inflammatory processes. SFN also decreased NF-κB activation/translocation which is involved in proinflammatory cytokines. All of these processes were all increased due to AGE-mediated processes. Therefore, SFN may be a strong candidate against neuroinflammation induced by MGO-AGEs or neurodegenerative diseases caused by chronic glycative stress.
Limitations: Preparation of AGEs can vary between batches, unknown concentration of pathological AGEs and need a protocol for AGE development/preparation.
Subedi, L., Lee, J. H., Gaire, B. P., & Kim, S. Y. (2020). Sulforaphane Inhibits MGO-AGE-Mediated Neuroinflammation by Suppressing NF-κB, MAPK, and AGE-RAGE Signaling Pathways in Microglial Cells. Antioxidants (Basel, Switzerland), 9(9), 792. https://doi.org/10.3390/antiox9090792
Sulforaphane inhibits advanced glycation end product-induced pericyte damage by reducing expression of receptor for advanced glycation end products
Impact factor: 2.767
Level of evidence: IV
Sample Size/Population: Bovine Retinal Pericyte Pericyte Cell Culture taken from Bovine Serum Albumin (BSA)
Methods: AGE-Bovine Serum Albumin (BSA) was prepared using D-glyceceraldehyde and 1% protease inhibitor with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and sodium chloride. BSA was incubated for 7 days in sodium phosphate buffer (pH 7.4). Unincorporated sugars were removed by PD-10 column chromatography and dialysis against phosphate-buffered saline. Control nonglycated BSA incubated in same conditions except without reducing sugars. No endotoxin was detected. Bovine retinal pericytes isolated and maintained in Dulbecco Modified Eagle Medium with 20% fetal bovine serum. Pericytes were either treated with either AGE-BSA or Nonglycated BSA, with or without 0.1 or 0.4 µmol/L sulforaphane or 5 µg/mL RAGE-Ab for 2 hrs. ROS was measured after. Pericytes had real-time reverse transcription-PCR done for bovine RAGE, MCP-1, B-actin gene. Western blot was then done to detect the RAGE-Abs. H3 Thymidine incorporation into pericytes was tested with 10% Trichroloacetic acid solution. Apoptotic cell death of pericytes was measured by checking for supernatant.
Statistical Analysis: One-way ANOVA followed by the Tukey. p<.05
Results: 0.4 µM sulforaphane solution completely blocked AGE-induced RAGE protein in pericytes. 0.4 µM sulforaphane and 5µg/mL RAGE-Ab solutions significantly inhibited apoptosis of pericytes due to AGE. Pericyte MCP-1 gene induction by AGE was completely suppressed by 5 µg/mL RAGE-Ab solution.
Conclusion: Sulforphane, a naturally occurring isothiocyanate found in cruciferious vegetables can successfully reduce pericyte damage by AGE through the reduction of RAGE expression, and protect against ROS.
Maeda S, Matsui T, Ojima A, Takeuchi M, Yamagishi S. Sulforaphane inhibits advanced glycation end product-induced pericyte damage by reducing expression of receptor for advanced glycation end products. Nutr Res. 2014 Sep;34(9):807-13. doi: 10.1016/j.nutres.2014.08.010. Epub 2014 Aug 29. PMID: 25241332
Glycation exacerbates the neuronal toxicity of β-amyloid
Impact Factor: 6.304
Level of evidence: VII
Study: Experimental, in vitro
Sample size: Mice, n=3-n=8 depending on portion of study as many studies were conducted in this article
Statistical significance: determined by one-way ANOVA procedure followed by Duncan’s post hoc test with 95% confidence and two-tailed Student’s t-test.
Discussion/conclusion: 𝛃-amyloids (AB) are a major component in neurotoxicity related to Alzheimer's disease. This study aimed to discover the difference in neurotoxic exacerbation between normal 𝛃-amyloids and glycated 𝛃-amyloids (AB-AGE) since it is an ideal substrate for glycation. It was found that AB was glycated to AB-AGE with an age-dependent elevation of AGEs in the brains of mice. It was found that AB-AGE was more toxic by decreasing cell viability, increasing cell apoptosis, inducing tau hyperphosphorylation, and reducing synaptic proteins more than the regular AB. The receptor for advanced glycation end products (RAGE) was increased in hippocampal neurons treated with AB-AGE than AB. When RAGE antibody was used it nearly abolished the AB-AGE effects with the combined above results showing that AB-AGE is likely a more suitable ligand for RAGE causing an increased neurotoxicity over AB. When looking at activity-dependent phosphorylation of GSK-3 there was higher activity with AB-AGE that when inhibited showed higher cell viability, decreased apoptosis and more showing that GSK-3 may mediate the Ab-AGE-induced exacerbation of neural impairments. Aminoguanidine (AG) is a prototype agent that prevents the formation of AGEs. In this study they injected mice for 3 months and measured the levels of AGEs, AB-AGEs and ABs in comparison to controls injected with saline. The results of this showed that this treatment decreased AGE levels, AB, AGE-associated ABs and AB-associated AGEs showing it inhibited the formation of AB-AGE in mice. Mice were then neurologically tested by memory retention and the AG treated groups showed that AG possibly could rescue memory and learning.
Limitation: Rat study
Li XH, Du LL, Cheng XS, Jiang X, Zhang Y, Lv BL, Liu R, Wang JZ, Zhou XW. Glycation exacerbates the neuronal toxicity of β-amyloid. Cell Death Dis. 2013 Jun 13;4(6):e673. doi: 10.1038/cddis.2013.180. PMID: 23764854; PMCID: PMC3698547.
Advanced glycation end products contribute to amyloidosis in Alzheimer disease
Impact factor: 9.412
Level of evidence: IV
Type of study: in vitro
Sample size: 17 brain plaque fractions (10 Alzheimer’s disease brain samples, 7 healthy controls)
Methods: Soluble preparations of synthetic beta-amyloid protein (AP) form fibrillar aggregates that resemble natural amyloid. These are measurable by sedimentation and thioflavin T-based fluorescence. The aggregation of soluble beta AP is enhanced by pre-aggregated beta-amyloid "seed" material which was prepared by a naturally occurring reaction between glucose and protein amino groups resulting in the formation of AGEs.
Statistical analysis: Student’s t-test, significance if P < 0.05
Results: AGE-modified beta AP-nucleation seeds accelerated soluble beta AP aggregation compared to the non-modified "seed" material. Nonenzymatic advanced glycation resulted in accumulation of a set of post-translational covalent adducts on proteins in vivo. In a standardized competitive ELISA, plaque fractions of Alzheimer disease brains had three times more AGE adducts/mg of protein than did samples from healthy, age-matched controls. The addition of preformed aggregates of AGE-modified beta-AP caused a faster aggregation of soluble beta-AP than did the preformed aggregates of unmodified beta-AP during the 2-day incubation.
Conclusion: This suggests that the in vivo half-life of beta-amyloid is prolonged in Alzheimer’s. The prolongation means higher AGE modification accumulation that, in turn, could promote accumulation of additional amyloid. It is possible that the gradual onset of Alzheimer’s symptoms parallels the progressive deposition of beta-amyloid. The acceleration of soluble beta-AP aggregation in AGE-modified beta-AP solutions at physiological pH, concentrations, and plaque fractions likely mimics the process occurring in vivo.
Vitek MP, Bhattacharya K, Glendening JM, Stopa E, Vlassara H, Bucala R, Manogue K, Cerami A. Advanced glycation end products contribute to amyloidosis in Alzheimer disease. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4766-70. doi: 10.1073/pnas.91.11.4766. PMID: 8197133; PMCID: PMC43869.
Analysis of serum of patients with Alzheimer's disease for the level of advanced glycation end products
Impact factor: 1.744
Level of evidence: IV
Study: Retrospective
Sample Size: 30 aged 68-70 years (Had AD or vascular dementia) and 94 controls without signs of cognitive impairment split into 2 groups.
Intro: The purpose is to look for a biochemical marker for AD since AD is based usually on neuro tests. AGEs are involved in beta-amyloids and degeneration of the CNS. Methods: IgG and IgM anti-AGE antibodies in human serum were measured in the ELISA test using the model AGE-protein.
Statistical analysis: Parametric Student t test and 2-way ANOVA
Results: The level of circulating AGE was significantly lower in AD patients than in the 2 groups of controls. The difference between the groups with AD and VaD was not statistically significant, while differences between the group with VaD and control group 1 as well as control group 2 were statistically significant. The difference in mean AGE value between control groups 1 and 2 was also statistically significant. The AD group did not differ in immune complex values from control group 1 or from control group 2 but differed significantly from the VaD group. The VaD group differed from control group 1 but not from control group 2 . The difference in mean IC value between control groups 1 and 2 was not statistically significant.
Conclusion: These initial results of the study suggest a local accumulation of AGEs rather than their presence in the form of soluble circulating AGEs. There were lower levels of circulating serum AGEs in patients with AD in relation to healthy controls.
Limitations: Difficult to compare control groups
Leszek J, Malyszczak K, Bartys A, Staniszewska M, Gamian A. Analysis of serum of patients with Alzheimer's disease for the level of advanced glycation end products. Am J Alzheimers Dis Other Demen. 2006 Oct-Nov;21(5):360-5. doi: 10.1177/1533317506291075. PMID: 17062556.
Advanced glycation endproduct precursor alters intracellular amyloid-beta/A beta PP carboxy-terminal fragment aggregation and cytotoxicity
Impact factor: 3.909
Level of evidence: III
Type of study and any information related to it: in vitro
Sample size: N/A due to in vitro study, all experiments were repeated at least 4 times
Intro: The purpose of this study was to examine the role of carbonyl stress on accumulation of intracellular amyloid-β(Aβ)-mediated cytotoxicity in Alzheimer’s patients. The researchers examined methlygloxyl (MG) and Gloxyl (G) as potential carbonyl stress sources.
Important methods: A human neuroblastoma cell line (MC65) was used as a model for carbonylation due to conditional carbonyl expression. Cells were trypsinized, washed with PBS, and supplemented with or without 1 ug/ml of tetracycline. Cells in these conditions expressed Aβ/CTFs within 3-4 hours of withdrawal from tetracycline. 24-36 hours after tetracycline withdrawal the cells were treated with MG and G for 4 hours with varying dosages. A different set of cells were then treated with α-tocopherol to determine whether lipid peroxidation would affect MG or G treatment. The viability of the cell was determined by a LIVE/DEAD assay as well as using MTT reduction followed by confirmation via spectrometry of solubilized formazan that was produced. Cells were solubilized via sonication which was followed by a BCA microassay to determine protein concentration. The proteins were then electrophoresed in gels containing bicine and 8 M urea which allowed the resolution of Aβ and other low molecular weight species. This was followed by a Western Blot which was probed using anti-mouse secondary antibody and then visualized via chemiluminescence.
Statistical tests ran: Stats were accomplished using Graph Pad Prism software (no other details), p < 0.05
Results: High doses of MG (> 200 uM) was significantly toxic to cells with or without tetracycline treatment. However, once cells were removed from tetracycline treatment there was a significant increase in cell susceptibility to MG toxicity. The EC50 of cells with tetracycline present was 480±3μM while the EC50 once tetracycline was removed was 329±16μM. G was only mildly toxic to cells even at high doses (> 3 mM). While still significant, the highest concentration of G only reduced cell viability by 15% relative to the control. Tetracycline was not shown to significantly modulate these results. Upon removal of tetracycline, Western Blot showed a significant increase in higher molecular weight species with MG treated cells consistent with Aβ/CTF aggregates. MG cells treated with α-tocopherol demonstrated a significant decrease of cytotoxicity in the presence of tetracycline. α-tocopherol also demonstrated ablation of Aβ/CTF aggregate toxicity due to the decrease in toxicity in tetracycline removed cells to the point of toxicity similar to those of tetracycline treated cells. However, α-tocopherol was not able to 100% restore cell viability of MG treated cells. α-tocopherol had no significant effect on G treated cells. α-tocopherol also showed a significant decrease in Aβ/CTF aggregation in MG treated cells to the point of complete loss of high molecular weight bands on the Western blot and a significant reduction of lower molecular weight bands which corresponded to Aβ/CTF-specific toxicity.
Discussion/Conclusion: MG was shown to have shown significant cytotoxicity that was amplified once the tetracycline treatment was stopped. This implicates that MG plays a role in carbonylation that decreases cell viability. G was shown to be mildly cytotoxic but not associated with carbonylation. MG likely contributes to the increase in aggregation of Aβ/CTF which plays a role in cytotoxicity. Decreased MG cytotoxicity due to α-tocopherol in tetracycline treated cells indicates that oxidative stress and lipid peroxidation plays a role separate from Aβ/CTF. Since α-tocopherol was not able to 100% restore cell viability, then this implicates that there are factors besides oxidative stress, lipid peroxidation, or Aβ/CTF aggregate toxicity that is taking part in toxicity. Overall, these findings show that Aβ/CTF aggregation and cytotoxicity was associated with MG exposure and was significantly reduced with treatment of α-tocopherol. Curiously, the AGE precursor G did significantly increase Aβ/CTF aggregation, albeit at a much lower amount than MG, but showed no modulation from treatment of α-tocopherol. This suggests that certain aggregates do not have cytotoxic behaviors but rather may be markers of underlying processes.
Woltjer RL, Maezawa I, Ou JJ, Montine KS, Montine TJ. Advanced glycation end product precursor alters intracellular amyloid-beta/A beta PP carboxy-terminal fragment aggregation and cytotoxicity. J Alzheimers Dis. 2003 Dec;5(6):467-76. doi: 10.3233/jad-2003-5607. PMID: 14757937.
Advanced glycation end products and oxidative stress in Alzheimer's disease
Impact factor: 3.694
Level of evidence: V
Study: Review
Summary: AGEs are present in amyloid plaques in AD, and its extracellular accumulation may be caused by an accelerated oxidation of glycated proteins. AGEs and oxidative stress promote the transformation of soluble proteins into insoluble proteins deposits. Measurement of protein carbonylation is thought to be a good estimation for the extent of oxidative damage of proteins associated with various conditions of oxidative stress, aging, physiological disorders and AD. Aβ induces lipoperoxidation of membranes and lipid peroxidation products. HNE is cytotoxic to neurons and that it impairs the function of membrane proteins including the neuronal glucose transporter GLUT 3, indicating that HNE is a characteristic marker and a toxin leading to neurodegeneration in AD. Increased levels of DNA strand breaks have been found in AD. They were first considered to be part of apoptosis, but it is now widely accepted that oxidative damage is responsible for DNA strand breaks and this is consistent with the increased free carbonyls in the nuclei of neurons and glia in AD. AGEs are formed from a wide variety of reactions afterwards. AGEs are composed of irreversibly cross-linked heterogeneous protein aggregates. There is increasing evidence that the insolubility of Aβ plaques is caused by extensive covalent protein cross-linking. Intracellular proteins deposits including NFTs, Lewy bodies of patients with Parkinson’s disease and Hirano bodies are also crosslinked by AGEs, which may explain their insolubility in detergents and resistance to proteases. Some studies have shown the presence of AGEs in association with two major proteins of AD, Aβ and MAP-tau. This supports that AGEs are involved in the pathogenesis of AD. Aβ and AGE both bind to the receptor for advanced glycation end-products which link both of them to the oxidative process that occurs next in AD. There are new pharmacological approaches that break the cycle of oxidative stress and neurodegeneration used for AD pts. These approaches include AGE-inhibitors, antioxidants, and nonsteroidal antiinflammatory substances. AGE inhibitors might be able to stop formation of AGE-modified Aβ deposits or modify their structure with subsequent loss of AGEs binding to RAGE.
Gella A, Durany N. Oxidative stress in Alzheimer disease. Cell Adh Migr. 2009 Jan-Mar;3(1):88-93. doi: 10.4161/cam.3.1.7402. Epub 2009 Jan 13. PMID: 19372765; PMCID: PMC2675154.
P301 L, an FTDP-17 Mutant, Exhibits Enhanced Glycation in vitro
Impact factor: 3.909
Level of evidence: VI
Type of study and any information related to it: in vitro
Intro: The purpose of this study was to determine the role of glycation in aggregation in a predisposed mutant tau (P301 L, G272 V, and R406 W tau). These mutations are linked to FTDP-17 which is a mutation on chromosome 17 in the MAPT gene linked with dementia and parkinsonism. The subsequent increase in AGEs were also measured.
Important methods: Full length tau and the mutants were expressed in E. Coli BL21 strain. Full length and mutant tau was glycated with 2.5 mM methyl glyoxal (MG) and extrinsic fluorophores (ThS, ThT, and ANS) were attached. A similar mixture was created again except treated with AGEs specific fluorophores. The glycated tau and mutants underwent SDS-PAGE and visualized with transmission electron microscopy. Glycation induced conformational changes were mapped with CD spectroscopy.
Statistical tests ran: unpaired t test, p < 0.05
Results: ThS showed an increased aggregation in G272 V and P301 L mutants. ThT followed a similar trend with an increase in aggregation among P301 L. R406 W showed the least aggregation in both. ANS was used to show hydrophobicity in aggregated proteins. However, there was no significant difference in ANS between the mutants and wild type. P301 L was linked to increased AGEs formation compared to control.
Discussion/Conclusion: This provides evidence that the P301 L mutation, linked to the development of Alzheimer’s, induces aggregation in Tau bodies as well as furthering the formation. This has even more downstream impacts which lead to Alzheimer’s.
Sonawane SK, Chinnathambi S. P301 L, an FTDP-17 Mutant, Exhibits Enhanced Glycation in vitro. J Alzheimers Dis. 2020;75(1):61-71. doi: 10.3233/JAD-191348. PMID: 32250308.
Advanced glycation end products and protein carbonyl levels in plasma reveal sex-specific differences in Parkinson's and Alzheimer's disease
Impact Factor: 9.986
Level of Evidence: III
Type of study and any information related to it: Retrospective
Sample size: PD (n=70), AD (n=40), control (n=30)
Intro: The purpose of this study was to determine if Carboxymethllysine (CML) and Carboxyethyllisine (CEL) levels, two different types of AGEs, varied among male vs female patients with neurodegenerative diseases such as Parkinson’s (PD) and Alzheimer’s (AD)
Important methods: Hoehn and Hoehn and Yahr (HY) and the mini mental state examination (MMSE) were given to participants to assess the disease severity and their mental capabilities. Total serum protein concentration was first analyzed using the Bradford method to give a relative value. Then protein bound CML and CEL was analyzed via centrifugation and Ultra-Performance Liquid Chromatography. Plasma protein carbonyls (PC), which are considered AGE precursors, were analyzed via ELISA.
Statistical tests ran: Significant P < 0.05, nonparametric Mann-Whitney test
Results: CML levels in the plasma were significantly higher in both PD (p < 0.0001) and AD (p < 0.0001) patients compared to the healthy control in both males and females. AD females had significantly lower CEL levels in comparison to female control, PD females, and AD males (Cont vs AD: p < 0.0001, PD vs AD: p < 0.0001, Cont vs PD: p < 0.0113). CEL levels in both PD and AD males were not significantly different compared to the control (p < 0.0001). PC levels were found to be higher in AD males compared to PD males and the control group (Cont vs AD: p < 0.0001; PD vsAD p < 0.0001). AD females were reported to have significantly lower levels of PC than PD females (p = 0.0088).
Discussion/Conclusion: These results suggest there are differences in AGE biomarkers between sexes in neurodegenerative diseases. This should possibly be considered when utilizing AGEs as biomarkers for both PD and AD.
Sharma A, Weber D, Raupbach J, et al. Advanced glycation end products and protein carbonyl levels in plasma reveal sex-specific differences in Parkinson's and Alzheimer's disease. Redox Biol. 2020;34:101546. doi:10.1016/j.redox.2020.101546
Effect of Advanced Glycation End Products on the Progression of Alzheimer's Disease
Impact Factor: 3.909
Level of Evidence: IV
Type of Study: Longitudinal study
Purpose: Examine how concentration of AGEs impacts cognitive performance of patients with Alzheimer’s and Type 2 Diabetes.
Methods: Patients enrolled were interviewed, had cerebral CT scan, and had physical, neurological, and neuropsychiatric exams performed. Excluded patients with cerebrovascular diseases, intracranial space-occupying lesions, mixed-type dementia, and other confounders. Mini-Mental Status Examination (MMSE) and Cognitive Assessment Screening Instrument (CASI), and CDR scale were performed on Alzheimer’s patients to assess cognition. Each patient had blood drawn. AGEs measured using enzyme-linked immunosorbent assay.
Statistics: All statistical tests were two-tailed and p value of less than or equal to 0.05 was considered significant. Normality of data assessed via Shapiro-Wilk test for small samples sizes. Pearson Correlation used between baseline AGEs and changes in MMSE and CASI scores from the beginning to the end of the study. Fischer’s exact test and Student’s t tests used to assess demographic differences and baseline AGEs concentrations between Alzheimer’s patients with and without deterioration. Clinical deterioration based on how much MMSE and CASI scores decreased or by CDR score increases. Regression analysis performed with Alzheimer’s patients with clinical deterioration were the dependent variable.
Results: Total of 25 patients all of Han ethnicity. 5 patients had APOE4 allele. No significant correlation found using Pearson’s correlation between baseline AGEs and change in MMSE. No significant difference in age, sex, APOE4 prevalence, education level, and AGEs concentration between those who deteriorated and those who did not for both CASI and MMSE scores. Pearson correlation shows no statistically significant difference in baseline AGEs and change in CASI scores. Patients without deterioration in CDR scores showed no significant difference with age, sex, education level, and APOE4 prevalence, but patients with deterioration in CDR did have significantly higher AGEs concentration than those without deterioration. This difference remained even after adjusting for age, sex, educational level, and APOE4 status. HbA1c levels changes were assessed from baseline from follow-up to account for diabetes effect on cognition. No significant difference was found in the patient's CDR with and without deterioration when compared. 5 patients HbA1c levels increased but no significant difference between the patients who had no increase in HbA1c and ones who did have an increase.
Conclusion: Showed association between AGEs and decline in cognition in patients with both Alzheimer’s and Type 2 Diabetes after 4 years. Only CDR scores decline significantly in association with AGEs. CDR measures cognitive loss in 6 domains: memory, orientation, judgement, problem solving, community affairs, home, and hobbies. High concentration in AGEs for patients with both Alzheimer’s and Type 2 Diabetes could be a contributing factor for decline in cognition.
Limitations: Small sample size. Detector limitations in distinguishing different AGE types. Longitudinal changes in AGEs were not measured, only HbA1c was. Could not assess for medications and treatments after follow-ups. Physical decline was not measured, only cognitive decline.
Chou PS, Wu MN, Yang CC, Shen CT, Yang YH. Effect of Advanced Glycation End Products on the Progression of Alzheimer's Disease. J Alzheimers Dis. 2019;72(1):191-197. doi: 10.3233/JAD-190639. PMID: 31561370.
Common neurodegenerative pathways in obesity, diabetes, and Alzheimer's disease
Impact Factor: 4.352
Type of Study: Review Scientist Summary
Purpose: Review the mechanisms of obesity and type 2 diabetes and Alzheimer’s.
Obesity and dementia: Obesity increases the risk of dementia. The low grade inflammation associated with obesity can help explain the increase in risk for dementia.
High-fat diet and the brain: High fat diet leads to free-circulating fat which is uptaken by the brain. High fat diet promotes pathogenic events related to Alzheimer’s. Mechanism involves activation of TLR4, which leads to inflammatory responses that lead to Alzheimer’s.
Cognitive decline in diabetes: There is evidence diabetes can lead to cognitive decline. The risk of dementia has been correlated with glycosylated hemoglobin levels. Insulin resistance seems to decrease activation of Akt. GSK3β consequently is more active and phosphorylates tau, and hence leads to neurofibrillary tangles seen in Alzheimer’s. Hyperinsulinemia causes insulin degrading enzyme (IDE) to be sequestered which leads to Aβ peptide accumulation. Get protein misfolding, oxidative stress, and inflamamtin
Neurogenesis in the diabetic brain: Age-dependent cortical and hippocampal atrophy seen in diabetic mice. Decreased neuroprogenitor cells have been seen in type 2 diabetic mice. High-fat diet mice also have impaired neuroprogenitor cells. Increased cytokines in diabetes could interfere with neurogenesis in brain.
Advanced glycation end products (AGEs) and RAGE signaling: AGEs induce RAGE expression. RAGE is increased in Alzheimer’s brains. Increased consumption of high-fat and dry-heat processed foods can increase circulation of AGEs and increase AGEs in brains of obese and diabetic individuals. AGEs is a common mechanism that links diabetes with Alzheimer’s. Feeding a mice rich in methyl glyoxal (AGE) causes metabolic syndrome and cognitive decline.
Mitochondrial dysfunction and inflammasome formation: Glucotoxicity in type 2 diabetes can activate cause mitochondrial injury and lead to inflammasome activation. Inflammasome activation is known to cause neuronal injury. Sirtuins seem a promising therapeutic target for stopping inflammasome formation.
Peripheral and central inflammation connection: Systemic inflammation is involved in insulin resistance caused by type 2 diabetes. Alzheimer patients have higher inflammatory markers peripherally. There seems to be cross-talk between central inflammation and peripheral inflammation. Central inflammation seems to damage the blood brain barrier, which causes immune cells to go into the CNS. Not clear with peripheral inflammation causes central inflammation or vice versa.
Damage to blood brain barrier: Diet induced obesity can cause blood brain barrier damage. When the BBB is damaged, there is chronic low grad inflammation and oxidative stress; when this is seen in obese and diabetic mice, it can lead to central inflammation.
Obesity and Diabetes as risk factors for Alzheimer’s disease: Diabetes decreases the threshold for Aβ needed to cause Alzheimer’s. Higher BMI was associated with greater Alzheimer’s risk. Diet-induced insulin resistance causes higher Aβ production in Tg2576 mice. Layperson Summary
Pugazhenthi S, Qin L, Reddy PH. Common neurodegenerative pathways in obesity, diabetes, and Alzheimer's disease. Biochim Biophys Acta Mol Basis Dis. 2017 May;1863(5):1037-1045. doi: 10.1016/j.bbadis.2016.04.017. Epub 2016 May 6. PMID: 27156888; PMCID: PMC5344771.
Incretin hormones, insulin, glucagon and advanced glycation end products in relation to cognitive function in older people with and without diabetes, a population-based study
Impact Factor: 3.083
Level of Evidence: IV
Type of Study: Cohort Study
Purpose: Study the relationship between biomarkers of glucose metabolism and cognitive function in a cohort study
Methods: Total participants is 3001 people (mean age 72 years). Oral glucose tolerance test, blood pressure, and waist circumference were measured. Type of diabetes not specified. Asked about smoking, exercise level, education level. Plasma glucose, serum insulin, plasma glucagon, serum GIP, and plasma GLP-1 were measured. Skin autofluorescence used to measure AGE levels. Mini-Mental State Examination (MMSE) and A Quick Test of Cognitive Speed (AQT) were used to measure cognitive function. HOMA-IR and ISO used to measure insulin sensitivity/resistance.
Statistics: P-value less than 0.05 is significant. Independent sample t-tests for continuous variables and Chi-squared tests done for categorical variables to compare groups with or without diabetes. Linear associations tested with the biomarkers via multiple regression and were stratified according to diabetes status and glucometabolic status. Two models of adjustments were used: one (Model 1) for age, sex, education, smoking, alcohol, and physical activity, and another (Model 2) for systolic BP, waist circumference, total cholesterol, and drug treatment.
Results: Skin autofluorescence is associated with worse MMSE with Model 1 adjustment and also in people without diabetes. Positive association between 2-hour glucagon and MMSE and negative association between AGEs and MMSE within prediabetics. Post-load incretin levels had positive correlation with MMSE in subgroups with normal glucose tolerance but not within prediabetics. HOMA-IR as a mediator of diabetes and MMSE was significant.
Discussion: Insulin sensitivity and post-load GIP and GLP-1 associated with better cognitive outcomes but had worse cognitive outcomes with AGEs and insulin resistance. Higher glucagon levels are associated with better cognitive outcomes (novel outcome of this study). Most associations are still significant after adjusting for cardiovascular factors. Possible mechanism for glucagon association could be that glucagon crosses the blood-brain barrier and enhances synaptic transmission via cAMP/PKA mechanism. Negative association with fasting levels of GLP-1 and cognitive function. Need interventional study to figure out the mechanism for these results.
Dybjer E, Engström G, Helmer C, Nägga K, Rorsman P, Nilsson PM. Incretin hormones, insulin, glucagon and advanced glycation end products in relation to cognitive function in older people with and without diabetes, a population-based study. Diabet Med. 2020 Jul;37(7):1157-1166. doi: 10.1111/dme.14267. Epub 2020 Feb 26. PMID: 32020688.
The role of inflammation and advanced glycation end products on paratonia in patients with dementia
Impact Factor: 3.376
Level of Evidence: V
Type of Study: Review article
Introduction: Impaired motor function is common with aging. Decreased skeletal muscle function induced by AGEs happens via collagen cross-linking. Get extracellular matrix stiffening and could be involved in paratonia, which is inability to relax muscles. Paratonia is common with dementia patients
Paratonia: Characterized by active, unintentional resistance of muscle against passive movement. Can lead to functional immobility, severe contractures, and pain. Daily living activities are impaired. Often motor function decline precedes cognitive decline in dementia patients.
Role of inflammation in paratonia: Glycation leads to AGE tissue accumulation which damages tissue structures and activates RAGE which leads to inflammation and oxidative stress and hence fibrosis and stiffening. AGEs are removed via enzymes and renal excretion, but these protective functions decline with aging. AGEs also form naturally with aging. AGEs activate production of IL-6, TNF-alpha. Peripheral biochemical changes from AGEs may cause paratonia’s effects. Diabetes mellitus and Alzheimer’s is associated with increased AGEs and patients with diabetes are known to have an increase of paratonia risk.
Therapeutic strategies: Should investigate if reducing AGE levels postpones or improves paratonia. AGE prevention can be done via lifestyle changes such as diet and exercise or pharmacological strategies such as using AGE formation inhibitors, AGE cross-link breakers, RAGE antagonists, AGE absorption inhibitors, and AGE binders. These pharmacological approaches are being studied but so far the results are conflicting. Harmful effects of long term dietary exposure to AGEs is inconclusive. AGE accumulation maybe reduced via glycemic control since uncontrolled glycemic control accelerates AGE formation.
Drenth H, Zuidema S, Bautmans I, Hobbelen H. The role of inflammaging and advanced glycation end products on paratonia in patients with dementia. Exp Gerontol. 2020 Dec;142:111125. doi: 10.1016/j.exger.2020.111125. Epub 2020 Oct 22. PMID: 33132147.
Ribosylation Rapidly Induces α-Synuclein to Form Highly Cytotoxic Molten Globules of Advanced Glycation End Products
Impact factor: 2.74
Level of evidence: III
Type of study and any information related to it: in vitro
Intro: The purpose of this study was to determine if ribosylation via D-ribose of alpha-synuclein contributes to cytotoxic accumulations of AGEs. Alpha-synuclein is the main component of Lewy bodies associated with Parkinson’s. Evidence has shown that glucuronidation contributes to this. D-ribose is present in the CSF and should be examined as well.
Important methods: Human α-synuclein was expressed in E. coli cells and isolated via SDS-PAGE. α-synuclein was incubated with D-ribose for 7 days to induce ribosylation. This was compared with D-glucose. The ribosylated derivative was confirmed using fluorescence at 410 nm. Subsequent AGE formation was confirmed using monoclonal antibody assay with Western Blot. Glycation levels were measured via Fructosamine byproducts which were assayed using nitroblue tetrazolium (NBT). Trypsin residues were tracked to measure lysinyl residue ribosylation in the C-terminal region. Levels of all the measures described were tracked from day 1 to day 7. Atomic force microscopy and thioflavin T (ThT) fluorescence was used to track the aggregation of ribosylated α-Syn. Cytotoxicity of ribosylated on SH-SY5Y cells (in vitro neuronal cells) was determined using MTT assays compared with native α-Syn and D-ribose. Annexin V/PI assays were used to determine if apoptosis or necrosis was induced from incubated ribosylated α-Syn. Lactic acid dehydrogenase (LDH) and Reactive Oxidation Species (ROS) was measured after to confirm if cells are damaged in the presence of ribosylated α-Syn. LDH activity was assayed using a cytotoxicity detection kit and ROS was measured via fluorescence.
Statistical tests ran: two-tailed test, p < 0.05
Results: Glycation end products significantly increased over the 7 days with ribosylated α-synuclein increasing at a first order rate constant of 5.79×10−6⋅s−1, while the first order rate constant was 1.33×10−6⋅s-1 for glucosylated α-Syn. This suggests that α-synuclein in the presence of D-ribose increased at a much faster rate compared to α-Syn in the presence of D-glucose. Glycation levels of ribosylated α-Syn measured using Fructosamine byproducts were also found to be significantly higher than glucosylated α-Syn. Ribosylated α-synuclein showed to be resistant to trypsin digestion (mass ratio of ribosylated α-Syn to protease: 20∶1) which indicated the C-terminal ribosylated faster compared to the N-terminal. Atomic force microscopy and thioflavin T (ThT) fluorescence demonstrated ribosylated aggregation similar to native α-Syn. Cell viability of SH-SY5Y cells were shown to significantly reduce in a concentration dependent pattern with incubation of ribosylated α-Syn. Native α-Syn and ribose incubation did not significantly lower cell viability. Ribosylated α-Syn was shown to significantly activate both cell apoptosis and necrosis. Highly concentrated ribosylated α-Syn demonstrated an increase in LDH activity and ROS compared to native α-Syn and ribose.
Discussion/Conclusion: This study indicates that D-ribose significantly contributes to the glycation of α-synuclein at an even faster rate and higher amount than D-glucose. Ribosylated α-Syn was found to cytotoxic to in vitro neuronal cells. This was likely accomplished by inducing apoptosis and necrosis pathways as well as increasing the amount of LDH activity and ROS levels. The authors also state in their discussion that the ribosylated α-Syn showed a higher cytotoxicity level via LDH and MTT assay compared to glucosylated α-Syn. However, this specific data was not shown. Structural examination and assays showed that ribosylated α-Syn contributed to aggregation, which is characteristic of AGEs.
Chen L, Wei Y, Wang X, He R. Ribosylation rapidly induces alpha-synuclein to form highly cytotoxic molten globules of advanced glycation end products. PLoS One. 2010;5(2):e9052. Published 2010 Feb 4. doi:10.1371/journal.pone.0009052
Oxidative Stress in Parkinson's Disease: Potential Benefits of Antioxidant Supplementation
Impact factor: 5.06
Level of evidence: I
Type of study and any information related to it: Review
Intro: This review covers the oxidative process linked to Parkinson’s and the role antioxidants play in prevention.
Important methods: A plethora of endogenous molecules, vitamins, phenols and polyphenols, terpenes, plant extracts, plant derived molecules, drugs, and other synthetic molecules were examined in relation to their potential antioxidant properties with Parkinson’s.
Results: Results are difficult to summarize as the article is almost more like a list of several dozen molecules. Most of those discussed contributed to relief among certain mechanisms of Parkinson’s.
The following are potentially noteworthy mechanisms in the context of AGEs:
- Iron can contribute to Lewy body formation through accumulation of α-synuclein
- Mitochondrial dysfunction due to heat-shock proteins, such as mortalin, mtHsp70, and GRP75
- NF-kB inflammatory pathway, a molecule that is induced by RAGE
Below are just a few notable molecules that could potentially influence the AGE pathway:
- GSH precursor N-acetyl-L-cysteine (NAC) was linked in rats with a decrease in α-synuclein and had characteristics associated with reduced NF-ĸB
- Ginseng treatment in rats showed suppression of β-amyloid deposition, and inhibition of the NF-ĸB inflammatory pathway
- Hesperidan was linked with reduction in iron levels leading to reduced oxidative stress and mitochondrial dysfunction
Discussion/Conclusion: There is a broad array of potential benefits for developing Parkinson’s related to antioxidation. The authors remarked that there was little evidence of benefit in actual patients, but rather that they were likely neuroprotective in nature. It is still worth to explore in further research for any possible treatment modalities.
Percário S, da Silva Barbosa A, Varela ELP, Gomes ARQ, Ferreira MES, de Nazaré Araújo Moreira T, Dolabela MF. Oxidative Stress in Parkinson's Disease: Potential Benefits of Antioxidant Supplementation. Oxid Med Cell Longev. 2020 Oct 12;2020:2360872. doi: 10.1155/2020/2360872. PMID: 33101584; PMCID: PMC7576349.
S100B is increased in Parkinson’s disease and ablation protects against MPTP-induced toxicity through the RAGE and TNF-α pathway
Impact factor: 11.337
Level of evidence: III
Type of study and any information related to it: in vitro
Sample size: 6 postmortem human midbrain sliced samples with Parkinson’s and 5 control samples were collected. Human serum, CSF and DNA samples of 82 patients with Parkinson’s disease and 64 controls were collected as well. These were compared with MPTP toxin administered rats of S100B ablated mice and wild type.
Intro: S100B is a neurotoxin that can contribute to neurodegenerative processes linked with Parkinson’s. One of the mechanisms is through the RAGE receptor. S100B has also been shown to upregulate the RAGE receptor. This paper analyzed this mechanism in the Substantia Nigra and the result of ablation of S100B.
Important methods: S100B protein, GFAP, and RAGE levels were analyzed using an automated immunoassay and immunostaining. The tyrosine hydroxylase- and Nissl-positive substantia nigra pars compacta neurons were counted using the optical fractionators method with the examiner being blinded to genotype and treatment.
Statistical tests ran: Values stated as means ± SEM, differences between means were analyzed using students t-tests and ANOVA with Newman–Keuls post hoc test. Pearson correlation coefficient was used for correlation analyses, and the Pearson’s chi-square test for comparison of categorical data. p < 0.05
Results: Immunostaining for S100B and RAGE protein in post-mortem midbrain slices of Parkinson’s disease and control subjects revealed significantly higher S100B protein levels in Parkinson’s disease. S100B was found to mostly localized in astrocytes labeled as GFAP positive. There were significantly elevated levels of S100B in the CSF of Parkinson’s patients as well. S100B and RAGE RNA and protein levels were found to be increased in the ventral midbrain region. Ablation of SP100B also showed significantly lower RAGE protein levels in the midbrain of the S100B knockout mice compared to wild type.
Discussion/Conclusion: This study helped confirm that RAGE and S100B expression was localized in the Substantia Nigra, the main brain region linked with Parkinson’s. Ablation of SP100B in mice was shown to lower RAGE levels. This could be an important target of treatment when looking to reduce the impact of AGEs on their receptor in Parkinson’s.
Sathe K, Maetzler W, Lang JD, et al. S100B is increased in Parkinson's disease and ablation protects against MPTP-induced toxicity through the RAGE and TNF-α pathway. Brain. 2012;135(Pt 11):3336-3347. doi:10.1093/brain/aws250
Regulation of striatal astrocytic receptor for advanced glycation end-products variants in an early stage of experimental Parkinson's disease
Impact factor: 4.066
Level of evidence: III
Type of study and any information related to it: Mice Study
Sample size: MPTP mice n = 14, Control mice n = 14
Intro: C-truncated RAGE (sRAGE) is a RAGE variant that is soluble in the extracellular compartment. It can interact with ligands as a decoy for full length RAGE (flRAGE). Increasing the ratio of sRAGE to flRAGE has been observed to decrease the detrimental effects of flRAGE overexpression. The purpose of this study was to examine the differences of these isotypes in the striatum of a 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model. This mouse model is meant to induce a state similar to Parkinson’s.
Important methods: Male adult mice were treated with 4 doses of 20 mg/kg of MPTP hydrochloride in saline solution or with just saline solution via I.P. injections. Each dose was administered every 2 hours. The first set of mice in each MPTP and control (n=10) were decapitated, and the brains dissected and frozen in liquid nitrogen. Five mice within this set had left hemisphere striata stored in RNA later solution RNA analysis via RT-qPCR in order to analyze S100B and RAGE levels. The right hemisphere striata of this group underwent an ELISA analysis and immunoassayed for flRAGE and sRAGE. The other five mice in the first set had their left hemisphere processed with electrical detection for High performance liquid chromatography (HPLC-ED) to analyze levels of dopamine and subsequent metabolites. The right hemisphere underwent a Western Blot analysis. The second set of rats in both MPTP and control (n=4) were anesthetized using pentobarbitol and administered 0.1 M phosphate buffer saline pH 7.4 (PBS) transcardially. This was followed by 4% paraformaldehyde in 0.1 M PBS. The brains were extracted and fixed in 4% paraformaldehyde for 24 hours and then dehydrated using 30% sucrose in 0.1 M PBS for 24 hours in order to undergo immunohistological and fluorescent analysis for activated NF-KB localization.
Statistical tests ran: Student’s t-test test, p < 0.05
Results: MPTP treatment showed significantly increased RAGE mRNA and total protein content (p<0.01). Antibody analysis of Western Blot data did not show increased amount of flRAGE (p > 0.05) but did increase the amount of sRAGE (p < 0.05) in MPTP treated mice. Similar results were demonstrated on immunostaining. Astrocytes were analyzed via double immunofluorescence and showed to have an increase in S100B and inhibitory RAGE variants without any evidence of hypertrophy. NF-KB was shown to be primarily localized in the cytoplasm which indicated a lack of activation in astrocytes.
Discussion/Conclusion: An overall increase RAGE and S100B was observed as expected based on previous literature. However, when comparing sRAGE to flRAGE, only an increase in sRAGE was demonstrated while flRAGE remained at basal levels in MPTP treated mice. The pattern of S100B and inhibitory RAGE localization upon immunofluorescence showed great overlap. This indicates increased astrocytic S100B will induce inhibitory RAGE. The authors believe that this is possibly used to compensate S100B autocrine or paracrine functions. The fact that NF-KB was not active indicates other possible transducing mechanisms at play. Overall this indicates that S100B upregulation in astrocytes induces sRAGE which plays a neuroprotective role in oxidative damage.
Viana SD, Valero J, Rodrigues-Santos P, Couceiro P, Silva AM, Carvalho F, Ali SF, Fontes-Ribeiro CA, Pereira FC. Regulation of striatal astrocytic receptor for advanced glycation end-products variants in an early stage of experimental Parkinson's disease. J Neurochem. 2016 Aug;138(4):598-609. doi: 10.1111/jnc.13682. Epub 2016 Jun 13. PMID: 27221633.
RAGE Silencing Ameliorates Neuroinflammation by Inhibition of p38-NF-κB Signaling Pathway in Mouse Model of Parkinson's Disease
Impact factor: 5.3
Level of evidence: III
Type of study and any information related to it: Mice Study
Sample size: 78 mice divided into 6 groups: control, MPTP, negative control of lentiviral vectors of RAGE (RAGE-NC), lentivirus small interference RAGE (RAGE-siRNA), MPTP + RAGE-NC, and MPTP + RAGE-siRNA.
Intro: The purpose of this study was to look at the role of RAGE in an MPTP induced Parkinson’s mouse model and the effect of siRNA RAGE treatment
Important methods: MPTP and normal control mice were given two I.P. injections of MPTP (30mg/kg) or saline every 2 days for 5 weeks. RAGE-NC and RAGE-siRNA groups were infected with lentivirus via brain surgery. The MPTP + RAGE-NC group and the MPTP + RAGE-siRNA group were infected with lentivirus via brain surgery and then followed a similar MPTP injection regimen 1 week later. During the seventh week a rotarod test was used before the brains were harvested to measure motor balance and coordination. Dopamine (DA), the 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) metabolites were measured from the striatum using high pressure liquid chromatography with electrochemical detection (HPLC-ECD). Substantia Nigra tissues went through Western Blot, separated with SDS gels, and transferred to polyvinylidene membrane. These proteins were then incubated with primary rabbit antibody to track phospho-p38, p38, phospho-IκB, IκB, RAGE, Tyrosine Hydroxylase (TH), COX-2, and B-actin. SN samples also went through a fluorescence immunohistochemistry targeted toward TH.
Statistical tests ran: ANOVA, Student-Neuman-Keuls, unpaired t-test, p < 0.05
Results: The rotarod test confirmed that the MPTP treated mice reduced motor function. The RAGE-NC and RAGE-siRNA groups were not significantly different compared to control with the motor function. Motor deficits of MPTP + RAGE siRNA significantly improved compared to the MPTP + RAGE-NC group. Western Blot analysis showed a significant increase of RAGE in MPTP mice compared to control. RAGE expression was significantly suppressed in the RAGE siRNA + MPTP treated mice compared to the MPTP + RAGE-NC group. The DA, DOPAC, and HVA analysis showed decreased levels in MPTP treated mice. Administration of RAGE siRNA significantly reversed this depletion to a degree after MPTP treatment in RAGE siRNA + MPTP mice group. TH immunofluorescence indicated that the RAGE suppressed MPTP group protected the dopaminergic neurons and mitigated the neurodegeneration caused by MPTP. Immunoblotting also showed that MPTP treatment significantly reduced TH expression which was antagonized via the RAGE siRNA treatment. RAGE suppression also decreased the expression of COX2, p38 phosphorylation, and NF-kB activation which prevented inflammatory deficits from MPTP treated mice.
Discussion/Conclusion: Having the motor deficits of the MPTP + RAGE siRNA group improve compared to the MPTP + RAGE-NC group suggests that the RAGE receptor plays a role in the pathology of MPTP induced PD motor dysfunction. Western blot analysis suggests that siRNA can inhibit RAGE mRNA transcription and expression. Results show that p38 MAPK activation was responsible for inducing the NF-kB inflammatory pathway leading to degeneration of DA and TH neurons. siRNA of RAGE significantly inhibited downstream inflammatory effects of RAGE.
Wang X, Sun X, Niu M, Zhang X, Wang J, Zhou C, Xie A. RAGE Silencing Ameliorates Neuroinflammation by Inhibition of p38-NF-κB Signaling Pathway in Mouse Model of Parkinson's Disease. Front Neurosci. 2020 Apr 29;14:353. doi: 10.3389/fnins.2020.00353. PMID: 32410941; PMCID: PMC7201072.
Reducing oxidative toxicity of L-dopa in combination with two different antioxidants: an essential oil isolated from Rosa Damascena Mill., and vitamin C
Impact factor: 5.9
Level of evidence: II
Type of study and any information related to it: Mice study
Sample size: 48 male albino mice split into 4 groups
Intro: The purpose of this study was to track oxidative stress related to L-dopa treatment and potential anti-oxidative treatments such as Rose Oil and vitamin C. Oxidation was measured by tracking malondialdehyde (MDA) levels, protein carbonyl content (PCC), and advanced glycation end products (AGEs) in blood plasma. This summary will focus on AGEs.
Important methods: Control mice received two i.p. injections of solvent 45 minutes apart. All other mice groups received two i.p. injections of L-dopa (100 mg/kg) 45 minutes apart followed by benserazide (10 mg/kg). The combination therapy group was pretreated 1 hour prior with an i.p. injection of Ascorbic acid (400 mg/kg) and rose oil (400 mg/kg). Mice were sacrificed via Nembutal anesthesia after 30 minutes followed by plasma collection via centrifuge. AGEs levels were measured via OxiSelect AGE competitive ELISA kit using anti-AGE polyclonal antibody followed by HRP conjugated secondary antibody.
Statistical tests ran: All data is expressed as mean ± SE, One way ANOVA, LSD post hoc test, p < 0.05
Results: In mice treated with L-dopa alone as well as all antioxidant pretreated mice, the AGEs in blood plasma were significantly higher compared to the control. Both the Rose Oil group and the ascorbic acid pretreated group showed a significant decrease in AGE accumulation relative to the group treated with L-dopa alone.
Discussion/Conclusion: Phenols and flavonoids are the key ingredients in rose oil that have been reported to have antioxidant effects. Essential oil also contains natural polyphenols and terpenes which cross the BBB that have exhibited a protective effect in neurodegenerative disease, also likely due to antioxidation. These results suggest that administration of antioxidants prior to exposure of AGEs may have a beneficial effect at reducing neurodegeneration.
Nikolova G, Karamalakova Y, Gadjeva V. Reducing oxidative toxicity of L-dopa in combination with two different antioxidants: an essential oil isolated from Rosa Damascena Mill., and vitamin C. Toxicol Rep. 2019 Mar 22;6:267-271. doi: 10.1016/j.toxrep.2019.03.006. PMID: 30984563; PMCID: PMC6444129.
Replication of enhanced carbonyl stress in a subpopulation of schizophrenia
Impact factor: 3.351
Level of evidence: IV
Study: Retrospective
Sample Size: 156 patients with schizophrenia, 221 age-matched controls excluding DM and CKD
Intro: Pentosidine is a known marker for AGEs. This study looked at the serum level of pentosidine in schizophrenic patients and compared them to age matched controls.
Statistical analysis: T-Test
Results/Conclusion: Mean pentosidine levels were 1.6-fold higher in schizophrenics compared with controls. This showed schizophrenics had a higher rate of AGEs in comparison to controls and therefore higher carbonyl stress.
Limitations: It is possible that antipsychotic medication may affect plasma pentosidine levels. Second, clinical information was unavailable and therefore not considered when evaluating pentosidine levels.
Miyashita M, Arai M, Yuzawa H, Niizato K, Oshima K, Kushima I, Hashimoto R, Fukumoto M, Koike S, Toyota T, Ujike H, Arinami T, Kasai K, Takeda M, Ozaki N, Okazaki Y, Yoshikawa T, Amano N, Miyata T, Itokawa M. Replication of enhanced carbonyl stress in a subpopulation of schizophrenia. Psychiatry Clin Neurosci. 2014 Jan;68(1):83-4. doi: 10.1111/pcn.12081. Epub 2013 Oct 28. PMID: 24393354.
Advanced glycation end products and schizophrenia: A systematic review
Impact Factor: 3.745
Level of Evidence: I
Type of Study: Systematic Review
Introduction: Symptoms of schizophrenia include delusions, hallucinations, social withdrawal, anhedonia, problems with memory and attentions. Much of the pathophysiology of schizophrenia is unknown but studies show oxidative stress mediated by AGEs plays a role.
Method: Literature search up until July 2014. Included English-language peer-reviewed journal articles and had studies where patients had schizophrenia or schizoaffective disorder and the diagnosis of these disorders was based on ICD or DSM criteria. Excluded articles not written in English, review articles, opinions, or hypothesis articles.
Results: 6 articles met the criteria. 2 groups made: AGEs and pathophysiology of schizophrenia, and AGEs and cardiovascular risk in schizophrenia. Possible that creation of free radicals damages the nervous system to give rise to schizophrenia. Another study showed depleted vitamin B6, which schizophrenic patients had, was associated with elevated oxidative stress. Pentosidine levels in a third study 1.6 times higher than healthy control and pyridoxal values were significantly lower in schizophrenic patients. Known that schizophrenic increases cardiovascular risk and also known that AGE accumulation is important for cardiovascular disease pathogenesis. AGEs promote damaging vascular and increases atherosclerotic process.
Discussion: Agents that inhibits AGE formation or trap ROS could benefit schizophrenic patients. Vitamin B6 and vitamin B1 and AGE-inhibitors need to be studied more to see if they are clinically beneficial. AGEs binding to RAGE is a key step in atherosclerosis; interventions exploring the blockage of the binding need to be explored. Diet and smoking are exogenous sources of AGEs. Schizophrenic patients tend to have unhealthy diets and lifestyles. Need to see if diet and smoking intervention can help with schizophrenia. Possible confounders for the association between schizophrenia with oxidative stress and chronic inflammation are diabetes, chronic renal failure, antipsychotic medication, smoking; however, in these 6 studies, these variables were not associated with AGE levels.
Limitations: Small samples sizes. Skin autofluorescence AGE measurements used in these studies is a reliable measure of plasma AGE levels however it is unreliable in dark skin since it affects light absorption.
Kouidrat Y, Amad A, Arai M, Miyashita M, Lalau JD, Loas G, Itokawa M. Advanced glycation end products and schizophrenia: A systematic review. J Psychiatr Res. 2015 Jul-Aug;66-67:112-7. doi: 10.1016/j.jpsychires.2015.04.023. Epub 2015 May 8. PMID: 26001588.
Increased advanced glycation end-products (AGEs) assessed by skin autofluorescence in schizophrenia
Impact factor: 3.745
Level of evidence: IV
Study: Prospective
Sample Size: 55 schizophrenia patients, 55 healthy controls. Study excluded those with alcohol dependence, drug dependence, mental retardation, diabetes, renal dysfunction or hx of head injury.
Methods: Measure AGEs through skin autofluorescence. Hypothesized that schizophrenia pts will have higher AGEs in comparison to controls and that a subgroup of patients with a more severe course of disease will be even higher. This is due to higher oxidative stress in schizophrenia.
Statistical analysis: Schizophrenia patients and controls were compared using Mann Whitney tests for quantitative variables and Fisher’s exact tests for qualitative variables. The relationships between skin AF and clinical variables were investigated in the schizophrenic group using Spearman’s rho correlation coefficients. Contributions of possible confounding variables were examined by multivariate analysis of covariance (MANCOVA) and logistic regression. p<0.05 is considered significant.
Results/Conclusion: AGEs were significantly elevated in schizophrenia patients in comparison to controls even when controlling for glomerular filtration rate, HDL and BMI. Skin AF in Kraepelinian patients (more severe course of disease) was significantly higher than in non-Kraepelinian patients. Kraepelinian patients were older than non-Kraepelinian patients and had a longer mean duration of disease than non-Kraepelinian patients. Therefore, when controlling for these it was no longer significant. Other interesting variables and AGEs were that they were higher with age and smoking.
Limitations: This study only included caucasian participants as dark skin can influence autofluorescence. A variety of skin compounds other than AGEs may also affect skin AF, and some AGEs (e.g., carboxymethyl-lysine) are not fluorescent. However, skin AF is considered to be a validated marker of the AGE pool.
Kouidrat Y, Amad A, Desailloud R, Diouf M, Fertout E, Scoury D, Lalau JD, Loas G. Increased advanced glycation end-products (AGEs) assessed by skin autofluorescence in schizophrenia. J Psychiatr Res. 2013 Aug;47(8):1044-8. doi: 10.1016/j.jpsychires.2013.03.016. Epub 2013 Apr 21. PMID: 23615188.
Association between skin autofluorescence of advanced glycation end products and affective disorders in the lifelines cohort study
Impact factor: 4.839
Level of evidence: IV
Study: Cross sectional case control study of the lifelines cohort study
Sample Size: 81,041 (41.7% male, aged 18–91 years), 6676 (8.2%) were cases with an affective disorder. Participants reporting a diagnosis of bipolar disorder, schizophrenia, dementia or Parkinson's disease were excluded.
Introduction: Looks at affective disorders and their association with oxidative stress. Affective disorder patients typically have lower life spans that have corresponded with higher rates of cardiovascular disease and in turn relationships with higher oxidative stress. Previously this could not be measured but with advanced glycation end products and skin autofluorescence oxidative stress and disorders like depression, their relationship can be quantified because AGEs are considered stable biomarkers for oxidative stress.
Methods: The presence of an affective disorder was assessed using the Dutch translation of the MINI. The following classifications were assessed and used as outcome measures: major depressive episode, dysthymia, generalized anxiety disorder, panic disorder (with or without agoraphobia), or social phobia. Then SAF was measured. Information about somatic morbidities, lifestyle, work and education was obtained by self-administered questionnaires. Routine serum measures (including glucose, HbA1c, lipids, renal function) were made from fasting state blood samples. Anthropometric measurements were taken by a trained research nurse. Blood pressure was measured using an automated Dinamap Monitor.
Statistical analysis: Comparisons between individuals with versus without a current affective disorder regarding sociodemographic and lifestyle factors, cardiometabolic parameters and history of somatic morbidities were conducted using unpaired t-tests, Mann-Whitney U tests and X2 tests for normal distributed, non-normal distributed and categorical data, respectively. Associations between presence of affective disorders and SAF were analyzed through logistic regression analyses.
Results and Conclusion: There is a cross-sectional association between affective disorders and AGE concentration measured by SAF, the strongest being major depressive disorder. It was also stronger for those with more than one affective disorder showing that there is cumulative oxidative stress in affective disorders. There was no association between SAF and dysthymia. For generalized anxiety disorder, panic disorder or social phobia, the associations lost significance when adjusting for cardiometabolic factors and somatic morbidities.
Limitations: Persons of non-Western descent and severely (mentally or physically) ill individuals were underrepresented.
Hagen JM, Sutterland AL, da Fonseca Pereira de Sousa PAL, Schirmbeck F, Cohn DM, Lok A, Tan HL, Zwinderman AH, de Haan L. Association between skin autofluorescence of advanced glycation end products and affective disorders in the lifelines cohort study. J Affect Disord. 2020 Oct 1;275:230-237. doi: 10.1016/j.jad.2020.06.040. Epub 2020 Jul 14. PMID: 32734913.
Skin autofluorescence of advanced glycation end products and mortality in affective disorders in the lifelines cohort study: A mediation analysis
Impact factor: 4.839
Level of evidence: IV
Study: Prospective Cohort
Sample Size: 81,041 participants from the lifelines cohort study. Exclusion criteria were schizophrenia or bipolar disorder or severe physical illness as assessed by the general practitioner, not being able to visit the general practitioner, to fill in the questionnaires
Introduction: Study looked at how psychiatric disorders are associated with premature death and how oxidative stress is linked to this. It stated how a cause of oxidative stress has been shown in skin autofluorescence (SAF) levels of advanced glycation end products and this is how it can be measured. AGEs promote inflammation, amplify reactive oxygen species, etc. and therefore are linked with an increase in morbidity and mortality in the general healthy population as they increase as we age and in severely ill patients. Increases in AGEs has been linked to psychiatric disorders and the purpose of this study is to see if SAF is associated with mortality and morbidity in affective disorders such as depressive and anxiety disorders.
Methods: Measured SAF on the volar forearm and did the MINI which is a short interview done by a research nurse to classify psychiatric disorders using DSM-IV. They were assessed to find major depressive episodes, dysthymia, generalized anxiety disorder, panic disorder and social phobia.
Statistical analysis: AF was assessed as mediator in Cox proportional hazards models for all-cause or natural-cause mortality. Partial mediation of SAF of the association between affective disorders and mortality was shown to be significant but attenuated by cardiometabolic panels but those with major depressive disorder continued to remain significant even after adjustment
Results and Conclusion: Increased mortality of those with major depressive disorder. This was also shown with increased SAF of AGEs in this population even after adjustments for comorbidities. Increased SAF was also shown to have mediated mortality in other affective disorders but was attenuated by adjustments for comorbidities. This still shows that oxidative stress measured by SAF of AGEs can be a predictor of premature death in affective disorders.
Limitations: It is complex with so many comorbidities in populations with affective disorders and is difficult to account for them and how they can have an effect on the study. Short duration of follow up was also a limitation. Those recruited by their general practitioners were asked to only approach relatively healthy patients and this results in bias of the more severely ill individuals.
Hagen JM, Sutterland AL, Liefers T, Schirmbeck F, Cohn DM, Lok A, Tan HL, Zwinderman AH, de Haan L. Skin autofluorescence of advanced glycation end products and mortality in affective disorders in the lifelines cohort study: A mediation analysis. J Affect Disord. 2021 Mar 1;282:1082-1089. doi: 10.1016/j.jad.2020.12.202. Epub 2021 Jan 6. PMID: 33601681.
Right treatment for the right schizophrenic patients based on carbonyl stress pathophysiology
Impact factor: 3.351
Level of evidence: VI
Study: Review
Sample Size: n/a
Introduction: To look at schizophrenia and carbonyl stress with its pathophysiology.
Results/Conclusion: This review concluded that AGEs are high in a lot of chronic diseases including psychiatric disorders like schizophrenia. There is a subgroup of schizophrenia with low activity of glyoxalase I that is used in AGE detoxification of pentoside and low pyridoxamine that inhibits AGE formation. This subgroup had a more severe course of disease with treatment resistant schizophrenia, longer hospitalizations, higher doses of antipsychotic meds and more. It is hypothesized that high dose pyridoxamine should help this subgroup of patients.
Limitations: Limited information, short article
Ozaki N. Right treatment for the right schizophrenic patients based on carbonyl stress pathophysiology. Psychiatry Clin Neurosci. 2018 Jan;72(1):2. doi: 10.1111/pcn.12620. PMID: 29314398.
Design and Feasibility of a Randomized Controlled Pilot Trial to Reduce Exposure and Cognitive Risk Associated With Advanced Glycation End Products in Older Adults With Type 2 Diabetes
Impact factor: 3.365
Level of evidence: IV
Type of study: in vivo, cross sectional
Sample size: 52 subjects with mild cognitive impairment (MCI), 23 cognitively normal patients with subjective memory complaints (SMCs)
Methods: To assess AGE consumption, subjects were given an AGEs eating habits questionnaire (frequency of meat, fish, poultry, cheese, egg yolk, fats, fast foods, convenience breakfast and snack foods consumption in the last 7 days; portion sizes; methods of cooking). In order to convert the questionnaire into quantitative values, each food was assigned a numerical value, multiplied by the number of entries. The sum of entries per day provided an AGEs score. Subjects were randomly assigned to one of two diets and were asked to maintain the instruction for the study for 6 months. At baseline, both groups were guided by a registered dietitian to follow guidelines to maintain good glycemic control. All subjects were monitored weekly by telephone to ascertain and encourage adherence with their diet. The following criteria were measured before and after the 6 month study: blood tests, AGEs levels, nutritional assessment, comprehensive neuropsychological assessment, neuroimaging, and anthropometrics.
Statistical analysis: AGEs and other biomarkers reduction in serum from the beginning to 6 months were analyzed using two-sample t-test or Mann–Whitney. Chi-squared test was used for categorical outcomes. Significance was defined p < 0.05.
Results: There was no difference in age or sex between the recruitment strategies of social media and websites. Cognition status was not associated with adherence to either of the dietary guidelines. All participants demonstrated adherence to the dietary guidance.
Conclusion: This study approached the challenges in recruiting MCI participants from the general population. It proved mass mailing and social media advertising effective in recruiting candidates. Using an informant and an objective global cognition tool (MoCA) instead of relying on subjective complaints alone was important for MCI screening. This study also shows that an intensive follow-up during the intervention encourages consistency in the patient.
Lotan R, Ganmore I, Livny A, Shelly S, Zacharia M, Uribarri J, Beisswenger P, Cai W, Schnaider Beeri M, Troen AM. Design and Feasibility of a Randomized Controlled Pilot Trial to Reduce Exposure and Cognitive Risk Associated With Advanced Glycation End Products in Older Adults With Type 2 Diabetes. Front Nutr. 2021 Feb 15;8:614149. doi: 10.3389/fnut.2021.614149. PMID: 33659267; PMCID: PMC7917071.
Skin autofluorescence is associated with diabetic peripheral neuropathy in Chinese Patients with type 2 diabetes: a cross-sectional study.
Sample Size: 820 Chinese patients with Type II Diabetes.
Protocol: Skin autofluorescence (SAF) and nerve conduction studies (NCS) were performed. Patients were divided into three groups based on the first and third quartiles of their SAF values. They were also divided into two groups based on their NCS: diabetic peripheral neuropathy (DPN) and non- diabetic peripheral neuropathy (non-DPN).
Statistical Analysis: Student’s t-test, Chi-squared test; For all, p < 0.01.
Study Type: Cross-sectional
Results: The DNP group had significantly higher SAF values than the non-DPN group. SAF was also positively correlated with risk of DPN, even after adjusting for confounders. A receiver operating characteristic analysis showed that an SAF value above 2.57 ng/mL corresponded to a threefold increase in risk of DPN (p < 0.01).
Conclusion: SAF is shown to be a risk factor for developing peripheral neuropathy is Chinese patients with Type II Diabetes.
Wan L, Qin G, Yan W, Sun T. Skin Autofluorescence Is Associated with Diabetic Peripheral Neuropathy in Chinese Patients with Type 2 Diabetes: A Cross-Sectional Study. Genet Test Mol Biomarkers. 2019 Jun;23(6):387-392. doi: 10.1089/gtmb.2018.0328. PMID: 31161820; PMCID: PMC6555182.
Protective effects and possible mechanisms of ergothioneine and hispidin against methylglyoxal-induced injuries in rat pheochromocytoma cells.
Impact factor: 5.076
Level of evidence: Level VI
Type of study and any information related to it: This paper investigated if Ergothioneine (EGT) and hispidin (HIP), both antioxidants, can protect against advanced glycation end product (AGEs) neuronal cell damage. Specifically, the researchers were interested if EGT and HIP could help prevent diabetic encephalopathy (DE) in Alzheimer’s patients. Rat pheochromocytoma (PC12) cells were used in this study and Methylglyoxal (MGO) was used as a precursor to AGEs.
Sample size: Cells were seeded at a density of 1*105 cells/well in a 24 well plate.
Important methods: PC12 cells were purchased from the Food Industry Research and Development Institute. Intracellular reactive oxygen levels (ROS) were measured by using a fluorogenic probe to monitor H2O2 levels. Reagents were added at 24, 48, and 72 hours. Protein carbonyl levels were measured as described by Reznick and Packer. AGE and RAGE levels were measured using ELISA. NF-κB levels were measured using Western Blotting.
Statistical tests ran: A one-way ANOVA test followed by Fisher’s protected LSD test was used to compare group means. A p-value of less than 0.05 was considered statistically significant.
Results: This study demonstrated that treating PC12 cells with MGO and glucose together decreased cell viability to 45%. When the cells were treated with MGO and glucose followed by EGT and HIP the cell viability increased to 82-90% after 24, 48 and 72 hours of incubation. MGO and glucose were found to significantly increase protein carbonyl levels in PC12 cells. When the cells were treated with EGT alone protein carbonyl levels were significantly decreased. When the cells were treated with HIP alone protein carbonyl levels were significantly decreased at 48 and 72 hours but not at 24 hours. EGT and HIP did not have a significant synergistic benefit when used as a combined treatment to decrease protein carbonyl levels. MGO and glucose (36% and 43.6%) were shown to have significantly increased ROS at 48 and 72 hours in PC12 cells. Cells treated with EGT (28.1%), HIP (25.2%) and EGT and HIP (23.4%) together had significantly decreased ROS levels. Treating PC12 cells with MGO and glucose significantly increased nuclear NF-κB levels. EGT, HIP and EGT and HIP treatments were found to significantly decrease nuclear NF-κB levels. Treating PC12 cells with MGO and glucose significantly increased AGE and RAGE levels. Cells treated with EGT, HIP and EGT and HIP together had significantly lower AGE and RAGE levels compared to cells treated with MGO and glucose, but significantly higher levels when compared to the control group. Treatment with EGT alone significantly decreased AGE and RAGE levels by 72 hours. Treatment with HIP alone did not significantly decrease AGE and RAGE levels until 96 hours. The combined treatment of EGT and HIP significantly decreased AGE levels by 96 hours.
Discussion/Conclusion: This study suggests that EGT and HIP have neuroprotective effects through the inhibition of the AGE/RAGE/ NF-κB toxic pathway, with EGT showing an ability to limit AGE formation. In vivo studies are needed to establish if EGT and HIP can be used to protect against DE.
Song TY, Yang NC, Chen CL, Thi TLV. Corrigendum to "Protective Effects and Possible Mechanisms of Ergothioneine and Hispidin against Methylglyoxal-Induced Injuries in Rat Pheochromocytoma Cells". Oxid Med Cell Longev. 2019;2019:2029096. Published 2019 Aug 6. doi:10.1155/2019/2029096
Accumulation rate of advanced glycation end products in recent onset psychosis: A longitudinal study
Impact factor: 2.118
Level of Evidence: Level IV
Study type: longitudinal cohort study/ longitudinal observational study
Sample size: 111 patients included at baseline, 78 could be retrieved and invited for a follow-up measurement. 286 healthy controls that had previously participated, 160 were successfully approached for a follow-up measurement.
Intro: Measurement of oxidative stress is challenging. However, a validated approach to measure oxidative stress. Measuring the concentration of advanced glycation end products (AGEs) in the skin. Increased skin AGE-levels compared to healthy controls have been demonstrated at cross-sectional level in chronic schizophrenia and in recent onset psychosis. It has also been speculated that these findings reflect increased cardiovascular disease risk. This study aims to observe the accumulation rate of AGE in patients suffering from recent onset psychosis versus healthy controls corrected for several possible confounders. This study hypothesized that AGE-accumulation is increased in patients compared to controls.
Methods: Advanced Glycation End products (AGE) levels in recent onset psychosis were invited for a follow-up measurement 12–24 months after baseline measurement. All participants were subjected to three consecutive skin autofluorescence measurements (intraclass correlation coefficient for absolute agreement was 0.906) using a calibrated AGE-reader. Cardiovascular (CVD) risk assessment including an interviewer-assisted questionnaire, compiling the evaluation of body mass index (BMI), waist circumference, blood pressure, cumulative tobacco exposure since first assessment, level of physical activity, and familial risk of CVD. Autofluorescence measurements were conducted from the volar side of the dominant forearm.
Statistical Analysis: Within group differences of characteristics in patients and healthy controls were analyzed using a paired t-test. Between group differences used unpaired t-test for continuous data. Pearson's chi-squared or Fisher exact test for dichotomous data. AGE-accumulation rate with general subject characteristics (ethnicity, criteria for metabolic syndrome, familial risk of CVD, exposure to tobacco smoking, cannabis use, weight gain) was investigated in patients and controls through hierarchical multiple regression analyses
Results: The current study showed a significant association of exposure to tobacco smoking and ethnicity with AGE-accumulation rate in healthy controls. An indication of an increased AGE-accumulation rate was shown in patients suffering from recent onset psychosis compared to healthy controls, independent of ethnicity and tobacco smoking but not independent of cannabis use.
Conclusion: The result of the study shows that there is a markedly (not significant) increase of AGE accumulation observed in patients suffering from recent onset of psychosis compared to healthy controls. This is independent of tobacco smoking and ethnicity but not of cannabis use.
Julia M. Hagen, Arjen L. Sutterland, Sarah Edrisy, Hanno L. Tan, Lieuwe de Haan, Accumulation rate of advanced glycation end products in recent onset psychosis: A longitudinal study, Psychiatry Research, Volume 291,2020,113192, ISSN 0165-1781.
Type 2 Diabetes, skin autofluorescence and brain atrophy
Impact Factor: 2.4
Type of Study: Cross Sectional Study
Detailed Summary: This study was conducted at the axis of Type 2 Diabetes Mellitus, Advanced AGE glycation products, and also neurodegeneration. One of the effects seen in people you suffer from Type 2 Diabetes is atrophy of the brain and there is thought to be a link between this brain atrophy and the accumulation of Advanced Age glycation products. The study was done using many different modes of measuring the level of AGE which included brain magnetic resonance imaging, cognitive testing, and also non invasive skin autofluorescence. The study was conducted on people who were 55 years and above and the groups were split by whether they were suffering from Type 2 Diabetes or not (the control). The sample size was about 486 people who participated in the study. The tests that were run were using multivariable linear regression which was used to analyze the relationships between the amount of gray matter, the presence of absence of Type 2 Diabetes, and the amount of SFA which is a marker for the presence of AGE. The results of the study showed that there was a greater amount of SAF in people who were suffering from Type 2 Diabetes. Each of the variables were independently associated with a lower amount of gray matter volume however the study mentions that further analysis is required for the relationship of GMV, SAF, and Type 2 Diabetes.
Moran C, Münch G, Forbes JM, Beare R, Blizzard L, Venn AJ, Phan TG, Chen J, Srikanth V. Type 2 diabetes, skin autofluorescence, and brain atrophy. Diabetes. 2015 Jan;64(1):279-83. doi: 10.2337/db14-0506. Epub 2014 Jul 22. PMID: 25053588.
Methylglyoxal-Derived Advanced Glycation Endproducts Accumulate in Multiple Sclerosis Lesions
Impact factor: 5.085
Level of evidence: IV
Type of study: prospective cohort
Sample size: 18 MS patients (9 relapsing remitting MS, 8 secondary progressive MS, and 1 clinically isolated syndrome patient).
Methods: The treatment group consisted of 6 patients: Tysabri® (n = 1), Gilenya® combined with Methotrexate® (n = 1), Endoxan® (n = 1), Copaxone® (n = 1), and Methotrexate® (n = 2). 12 MS patients were untreated. CSF and paired plasma samples of the MS patients were obtained and analyzed using ultra-performance liquid chromatography tandem mass spectrometry. MS lesions were analyzed with immunohistochemistry. GLO1 activity was measured using spectrophotometry analysis.
Statistical analysis: Baseline characteristics of post-mortem samples from NDCs and MS patients were analyzed using one-way ANOVA with Tukey's multiple comparisons test . α-dicarbonyl and AGE levels of the post-mortem material were analyzed using one-sided unpaired t-test. Partial correlation analysis was used to determine CSF α-dicarbonyls and AGEs and disease parameters expanded disability scale score (EDSS), number of relapses and disease duration. Linear regression analysis was used to determine associations between plasma α-dicarbonyls and AGEs and CSF α-dicarbonyls and AGEs. P ≤ 0.05 was considered statistically significant.
Results: MG-H1 is increased in the lesion area of MS patients and MGO-derived AGEs are present in astrocytes in MS lesions. α-dicarbonyls and AGEs in CSF do not correlate with disease parameters of MS.
Conclusion: MGO-derived MG-H1 is significantly increased in MS lesions and mostly present in astrocytes. AGE levels and α-dicarbonyl in the CSF do not correlate with disease parameters, but do correlate with plasma levels.
Wetzels S, Vanmierlo T, Scheijen JLJM, van Horssen J, Amor S, Somers V, Schalkwijk CG, Hendriks JJA, Wouters K. Methylglyoxal-Derived Advanced Glycation Endproducts Accumulate in Multiple Sclerosis Lesions. Front Immunol. 2019 Apr 24;10:855. doi: 10.3389/fimmu.2019.00855. PMID: 31068938; PMCID: PMC6491451.
Methylglyoxal-Derived Advanced Glycation Endproducts Accumulate in Multiple Sclerosis Lesions
Impact Factor: 6.429
Level of Evidence: IV
Type of Study: Case Control Study
Introduction: Multiple sclerosis (MS) is a central nervous system inflammatory disease. Cause is unknown, but disease involves immune response against myelin sheaths. Methylglyoxal (MGO) and glyoxal (GO) are precursors for advanced glycation end products (AGEs). AGEs lead to oxidative damage.
Purpose: Hypothesized that in MS, AGE levels are increased because of inflammatory activation of macrophages and astrocytes. Asked if α-dicarbonyls levels and AGEs in CSF correlated with plasma levels.
Materials and Methods: 18 MS patients gathered for study for CSF and plasma samples. 12 of them were untreated. 15 post-mortem tissue samples were obtained from MS patients as well for immunohistochemistry analysis. 10 white matter tissue samples were collected from non-demented controls (NDCs) The controls were age and gender matched. α-dicarbonyl and AGEs were measured in plasma, CSF, and post-mortem tissue samples using ultra-performance liquid chromatography tandem mass spectrometry. GLO1 activity was measured using spectrophotometry to determine S-D-Lactoylglutathione from MGO.
Statistical Analysis: One-way ANOVA with Tukey’s multiple comparison’s test for baseline characteristics from postmortem NDCs and MS patients. One-sided unpaired t-test for α-dicarbonyl and AGE levels. Linear regression analysis between plasma α-dicarbonyl and AGEs and CSF α-dicarbonyl and AGEs. P values less than or greater than 0.05 is significant.
Results: MG-H1, a major MGO-derived AGE, was increased in MS lesions compared to controls. The MGO-derived AGEs in general were found to be in the GFAP+ astrocytes. No correlation between CSF AGE levels and the number of relapses in MS, duration of MS, and EDSS (Expanded Disability scale score). Negative correlation between MG-H1 in CSF and disease duration and also between protein bound MG-H1 and EDSS.
Discussion: α-dicarbonyl and AGE levels in CSF do not correlate with disease characteristics but do correlate with plasma levels. Activity of GLO1 (major MGO detoxifying enzyme) activity was same between white matter in MS patients and controls. Means increase in MG-H1 in MS patients due to increased MGO formation and not lowered degradation by GLO1. Since MGO-derived AGEs found in astrocytes, suggest activated astrocytes might exert effect by binding to RAGE on microglia/macrophages in MS patients and play a part in the inflammatory environment of MS lesions. The negative correlations in the results section could be explained by the notion that people with longer disease duration and higher EDSS scores get less relapses and hence lower amount of inflammation.
Limitations: Many of the MS patients were on anti-inflammatory drugs.
Wetzels S, Vanmierlo T, Scheijen JLJM, van Horssen J, Amor S, Somers V, Schalkwijk CG, Hendriks JJA, Wouters K. Methylglyoxal-Derived Advanced Glycation Endproducts Accumulate in Multiple Sclerosis Lesions. Front Immunol. 2019 Apr 24;10:855. doi: 10.3389/fimmu.2019.00855. PMID: 31068938; PMCID: PMC6491451.
Diagnostic potential of plasma carboxymethyllysine and carboxyethyllysine in multiple sclerosis
Impact Factor: 6.304
Level of evidence: V
Type of study: case control study
Sample size: 99 patients with AD (Alzheimer's disease) (n=99) and 43 healthy controls non (n=43)
Summary: The purpose of this study was to compare the levels of advanced glycation end products (AGEs) AND nN-(Carboxymethyl)lysine (CML) and N-(Carboxyethyl)lysine (CEL) in patients with multiple sclerosis with healthy controls and correlating these results with indicators of MS disease severity.
Important methods: The CML and CEL levels were analyzed in 99 patients with MS and 43 healthy controls by tandem mass spectrometry (LC/MS/MS). Patients were stratified based on drug modifying therapies (MDTs) including interferon beta, glatiramer acetate and natalizumab.
Statistical tests ran: Significant P < 0.05, CML and CEL plasma levels were examined using a linear regression model. ANOVA examined the differences in CML and CEL plasma levels among MS patients at different disease stages. A simple T test assessed the differences in CML and CEL plasma levels between clinically stable MS patients and MS patients in clinical relapse.
Results: Significantly higher plasma levels of CEL in MS patients compared to healthy controls. CML plasma levels were not different in MS patients compared to HC but similar to CEL, CML plasma levels correlated with the rate of clinical relapse. Higher levels of AGEs correlate with high inflammatory and predisposes patients to higher rate of yearly clinical relapse.
Conclusion: This study demonstrates CML and CEL have inflammatory effects through the RAGE receptor. The study also concluded that AGEs may provide a simple diagnostic tool in MS. Specifically with CEL levels, 91 percent of patients with MS were identified of having higher than normal levels of AGEs.
Limitations: The sample size is small.
Sternberg, Z., Hennies, C., Sternberg, D., Wang, P., Kinkel, P., Hojnacki, D., Weinstock-Guttmann, B., & Munschauer, F. (2010). Diagnostic potential of plasma carboxymethyllysine and carboxyethyllysine in multiple sclerosis. Journal of neuroinflammation, 7, 72. https://doi.org/10.1186/1742-2094-7-72
Soluble receptor for advanced glycation end products in multiple sclerosis: a potential marker of disease severity
Impact Factor: 5.280
Level of Evidence: IV
Type of Study: Cohort Study Scientist Summary
Introduction: Soluble RAGE (sRAGE) is RAGE that lacks transmembrane domain and is secreted in plasma. sRAGE believed to bind to RAGE ligands and present effects of AGE-RAGE signaling. This is a pilot study assessing whether serum sRAGE levels differ between healthy controls and patients with multiple sclerosis (MS). Severity of MS is assessed on whether it correlates with sRAGE levels using Kurtzke Expanded Disability Status Scale (EDSS).
Methods: 37 clinically stable MS patients (24 females and 13 males) with mean age of 44.3 ± 9.1 years. MS patients recruited from Baird MS center, Department of Neurology, Jacobs Neurological Institute, and from Buffalo, New York in the United States. EDSS ranged from a mean of 2.81 ± 1.99 (range of 0 to 8.5 years) with disease duration ranging from 1 year to a maximum of 30 years. Had 31 relapsing-remitting (RR) cases and 6 secondary progressive (SP) patients. Had 22 healthy controls (12 females and 10 males) with mean age of 40.2 ± 11.2 years. Healthy patients were from the Neurology Department staff. Used ELISA to measure sRAGE levels.
Statistics: Groups differences in ages measured using ANOVA and chi-squared test used to measure gender differences. sRAGE levels measured using 2 by 2 factorial analysis of covariance with 2 between sex and group (MS vs controls) and age, age of disease onset, and progression (EDSS / disease duration) as covariates. Spearman correlation used to relate EDSS, rate of relapse, and sRAGE levels. A p value that was equal to or less than 0.05 was considered significant.
Results: Serum levels of sRAGE in MS patients were significantly lower compared to healthy controls. No significant difference was seen in gender distribution and age distribution in MS and the control group. Female MS patients had lower sRAGE levels compared to male MS patients (p=0.05). No significant difference seen between sRAGE and age or sRAGE and age of disease onset. Inverse relationship seen between sRAGE and EDSS (R2=0.166, p=0.012) in regress analysis. Each EDSS unit was equivalent to 6.3% reduction in sRAGE which accounted for 16.6% of the variance. Inverse relation seen between sRAGE and rate of MS clinical relapse (p=0.0126).
Discussion: Results show relationship between sRAGE and disease severity show potential role of RAGE in MS pathology. Hypothesize MS patients with lower serum sRAGE have more inflammatory cells going to CNS. Gender differences possibly explained by 17beta-estradiol upregulates RAGE receptor and also females had more pro-inflammatory cytokines compared to male MS patients may explain the gender difference too. Increasing sRAGE maybe promising therapeutic target.
Limitation: CSF of MS patients were not measured.
Sternberg Z, Weinstock-Guttman B, Hojnacki D, Zamboni P, Zivadinov R, Chadha K, Lieberman A, Kazim L, Drake A, Rocco P, Grazioli E, Munschauer F. Soluble receptor for advanced glycation end products in multiple sclerosis: a potential marker of disease severity. Mult Scler. 2008 Jul;14(6):759-63. doi: 10.1177/1352458507088105. Epub 2008 May 27. PMID: 18505774.
Soluble Receptor for Advanced Glycation End Products (sRAGE) is Up-Regulated in Multiple Sclerosis Patients Treated with Interferon β-1a
Impact Factor: 1.303
Level of Evidence: IV
Type of Study: Case control study
Purpose: Study the link between RAGE and Multiple Sclerosis (MS)
Methods: 50 patients with sporadically relapsing-remitting MS were recruits and 5- healthy match controls as well. Excluded people with low vitamin D levels and presence of HLA-DRB1*15. Patients given IFNβ-1a treatment intramuscular with a dose of 20 µg, 3 times a week over 2 years of follow up. Real-time PCR was performed to assess RAGE expression in patients and controls. RAGE plasma concentration evaluated using sRAGE (human soluble receptor for AGEs) and ELISA to assess sRAGE via double antibody technology.
Statistics: Kruschke’s Bayesian estimation to fit 2 sample Bayesian t-test. Bayesian independent t-test also used. Spearman correlation coefficient used for demographics and RAGE expression correlation. Less than 0.05 was considered significant.
Results: Real-time PCR showed significant upregulation of RAGE in MS patients. sRAGE was also significantly higher in MS patients. RAGE transcript levels were significantly correlated with age of disease onset and disease duration. RAGE transcript levels were not significant with expanded disability status scale (EDSS) scores. EDSS showed significant inverse correlation with sRAGE plasma concentration. Each unit increase in sRAGE levels showed 0.308 increase in EDSS scores. All MS patients were responsive to IFNβ-1a therapy and had increased sRAGE levels.
Discussion: Propose up-regulation of sRAGE as a possible clinical response to IFNβ-1a therapy for MS patients. Previous studies have shown significant decrease of sRAGE in MS patients and controls. Suggests sRAGE has anti-inflammatory effects.
Limitations: This study did not measure endogenous secretory RAGE (esRAGE). sRAGE analysis on CSF samples were not performed because lacked CFS samples from healthy subjects. Possible other factors increased sRAGE levels.
Rahimi M, Aghabozorg Afjeh SS, Omrani MD, Arsang-Jang S, Ganji M, Noroozi R, Taheri M, Ghafouri-Fard S. Soluble Receptor for Advanced Glycation End Products (sRAGE) is Up-Regulated in Multiple Sclerosis Patients Treated with Interferon β-1a. Cell Physiol Biochem. 2018;46(2):561-567. doi: 10.1159/000488622. Epub 2018 Mar 28. PMID: 29617694.
Disease modifying drugs modulate endogenous secretory receptor for advanced glycation end-products, a new biomarker of clinical relapse in multiple sclerosis
Impact Factor: 3.125
Level of Evidence: IV
Type of Study: Longitudinal Observational Study
Purpose: Study endogenous secretory RAGE (receptor for advanced glycation end products) serum levels in multiple sclerosis (MS) patients compared with healthy controls. Also study esRAGE (endogenous secretory RAGE) serum levels and chronic use of disease modifying therapies (DMDs). Also study if esRAGE correlates with MS severity.
Methods: 98 MS patients recruited (24 males, 70 relapsing remitting (RR), 22 secondary progressive, 6 primary progressive (PP)). 42% of these MS patients were treated with DMD (disease-modifying therapy). Serum esRAGE measured via ELISA.
Statistics: ANOVA used to analyze patients vs healthy controls (HC) and also males vs females, DMD-naïve vs DMD-treated, relapsing remitting vs secondary progressive vs primary progressive, and clinically stable vs relapsing patients. Regression analysis used between esRAGE and MS disease severity indicators such as EDSS, MS severity scale, disease duration, rate of clinical relapse within 2 years from blood draw date.
Results: esRAGE serum higher in MS patients then HCs but not statistically significant. DMD naïve MS patients had higher esRAGE levels than DMD treated patients and HCs. DMD-treated patients with MS has lower esRAGE than HCs. In DMD-naïve group, primary progressive MS (PPMS) patients had more esRAGE than relapsing remitting MS patients (RRMS) and were higher in esRAGE than secondary progressive MS patients (SPMS) patients. In DMD-treated, esRAGE higher in SPMS than RRMS. In DMD-naïve patients, esRAGE inversely correlated with rate of clinical relapse and EDSS in univariate regression but in multivariate regression, the relationship only held with clinical relapse.
Discussion: DMDs may exert beneficial effect via modulating esRAGE since DMD-treated patients had higher esRAGE levels. One mechanisms maybe that esRAGE reduces RAGE ligand production. The MS patients used many drugs such as antihypertensives, hypolipidemic, anxiolytic, antidepressants, antispastics, anticoagulants. These drugs are known to interfere with RAGE axis in some way. Low esRAGE may play a role in MS disease pathology. esRAGE serum shows higher sensitivity and specificity for MS clinical relapse compared to sRAGE, MHGB1, CEL, and pentosidine. But esRAGE serum levels shows low sensitivity/specificity for MS disease progression. Helps show esRAGE can be used as a clinical marker when combined with other markers.
Limitations: Small sample size. Could not difference effects of specific DMDs on esRAGE levels.
Sternberg Z, Sternberg D, Drake A, Chichelli T, Yu J, Hojnacki D. Disease modifying drugs modulate endogenous secretory receptor for advanced glycation end-products, a new biomarker of clinical relapse in multiple sclerosis. J Neuroimmunol. 2014 Sep 15;274(1-2):197-201. doi: 10.1016/j.jneuroim.2014.07.005. Epub 2014 Jul 15. PMID: 25064498.
Advanced glycation endproducts are increased in the animal model of multiple sclerosis but cannot be reduced by pyridoxamine treatment or glyoxalase 1 overexpression
Impact Factor: 4.556
Level of Evidence: VII
Sample Size: 8 case/8 control mice in experiment 1, 10 case/11control mice in experiment 2, 5 case/5 control mice in experiment 3.
Protocol: Experiment 1 tested the effect of experimental autoimmune encephalitis (EAE) on AGE production. 8 mice were subjected to EAE and the level of AGE production in the case mice was compared to 8 control mice in the same setting. Experiment 2 tested the effect of pyroxidine treatment on AGE production. 11 mice were were subjected to EAE and treated with pyroxidine. Levels of AGE were compared in the case mice to 10 control mice subjected to EAE but without pyroxidine treatment. Experiment 3 tested the effect of Glo-1 expression on AGE production. 5 mice overexpressing Glo-1 were subjected to EAE and compared to 5 control mice without Glo-1 that were subjected to EAE. AGEs were measured from the plasma, spinal cord and brain.
Statistical Analysis: Unpaired t-test, two way ANOVA, Sidak’s multiple comparison.
Study Type: Animal study- mouse model.
Results: Methylglyoxal (MGO), glyoxal (GO) and 3-deoxyglucosone (3DG) were all significantly decreased and free MGH-1was significantly increased in the plasma of EAE mice in the plasma of mice subjected to EAE. MGO, free CML, free CEL and free MGH-1 levels were significantly increased and Glo-1 was significantly decreased in the spinal cords of EAE mice. MGO, GO, free CML and Glo-1 were significantly increased in the brains of EAE mice. There was no significant difference in the AGE production between control EAE mice and EAE mice that were treated with pyroxidine or mice with overexpressed Glo-1.
Conclusion: EAE in mice successfully mimics the pathology of multiple sclerosis in humans. Increased AGEs are produced when mice are subjected to EAE, but pyroxidine treatment and Glo-1 expression that results in decreased AGE in humans does not result in decreased AGE and improvement of EAE in mice.
Wetzels S, Wouters K, Miyata T, et. al. Advanced glycation endproducts are increased in the animal model of multiple sclerosis but cannot be reduced by pyridoxamine treatment or glyoxalase 1 overexpression. Int J Mol Sci. 2018;19(5):1311. doi: 10.3390/ijms19051311.
Advanced Glycation Endproducts Are Increased in the Animal Model of Multiple Sclerosis but Cannot Be Reduced by Pyridoxamine Treatment or Glyoxalase 1 Overexpression
Impact Factor: 4.37
Level of evidence: VII
Study: Experimental, in vitro
Sample size: Mice, n=8
Introduction: MS is a demyelinating autoimmune disease. The immune response in MS patients leads to the infiltration of immune cells in the CNS and their subsequent activation. Immune cell activation induces glycolysis, one of the end products of glycolysis is methylglyoxal (MGO.) MGO can rapidly form advanced glycation end products. AGEs continue to induce inflammatory responses. The purpose of this study was to determine whether AGE levels are induced in experimental autoimmune encephalitis in an animal model of MS.
Method: Researchers investigated whether AGE levels are induced in the experimental autoimmune encephalitis (EAE) animal model of MS. Then researchers used pyridoxamine treatment and the Glo-1 overexpression animal model to reduce AGE levels in the plasma and the CNS of the mice subjected EAE
Results: Researchers investigated the AGE plasma levels in mice with induced EAE and compared them to healthy controls. Researchers observed significantly higher levels of AGEs in mice with EAE than controls. GLO1 (the MGO detoxification enzyme) activity was significantly decreased in the spinal cord of mice subjected to EAE compared to their healthy wild type. In the experiment, researchers concluded pyridoxamine intervention did not reduce AGE endproduct levels in the plasma and spinal cord during EAE.
Discussion: Researchers concluded that dicarbonyl and AGE levels are significantly increased in experiment models of MS and mice induced with EAE. This suggests that similar pathways are activated in the progression of MS. However, researchers were unable to reduce AGE levels by pyridoxamine treatment and Glo-1 overexpression in EAE model.
Limitation: Mice study
Statistical significance: data presented as mean +/- standard error of the mean (SEM) . Data is analyzed using unpaid t-test and two way ANOVA with SIdak's multiple comparisons post tes. A p<.05 was considered statistically significant
Wetzels, S., Wouters, K., Miyata, T., Scheijen, J., Hendriks, J., Schalkwijk, C. G., & Vanmierlo, T. (2018). Advanced Glycation Endproducts Are Increased in the Animal Model of Multiple Sclerosis but Cannot Be Reduced by Pyridoxamine Treatment or Glyoxalase 1 Overexpression. International journal of molecular sciences, 19(5), 1311. https://doi.org/10.3390/ijms19051311
Increased tau phosphorylation and receptor for advanced glycation endproducts (RAGE) in the brain of mice infected with Leishmania amazonensis
Impact factor: 6.633
Level of evidence: VII
Study: Experimental/Rat
Introduction: Neurological symptoms have been observed in humans with parasitic infection of leishmania, but the neurodegenerative process has not been studied. This study looked to measure tau phosphorylation, the receptor for advanced glycation end products along with other markers to compare them with controls who had not been infected with leishmaniasis.
Methods: Mice were injected with leishmaniasis. Four months post-infection, mice were euthanized, and brain cortex removed. Another group received antioxidant therapy for 5 days before euthanization. To estimate parasite burden in the lesions, the entire infected footpads were removed, and amastigotes were recovered from the lesions and counted. Inflammatory parameters in liver and serum (TNF-a and IL-1b) were measured to confirm systemic inflammation. Western blot was used to estimate tau phosphorylation. The inflammatory markers were tested for by ELISA. TRAP assay is an antioxidant assay that was used to show antioxidant capacity.
Statistical analysis: Student's T Test (two-tailed) was applied for simple comparisons between control and infected animals in each assay. For Comparison of multiple means, ANOVA with Tukey’s post hoc was performed. The results of measurements were expressed as mean ± standard error of the mean (SEM). Significant when p< 0.05. Results and
Conclusion: Tau phosphorylation was increased and not the total number of tau in infected mice. RAGE was significantly increased in infected animals. These two together as tau being able to become a ligand for RAGE is strongly implicated in neuronal death and pro-inflammation. Although an increase in TNF-a levels was detected in other organs, such as liver, no significant changes in the levels of both TNF-a and IL-1b were detected in the brain cortex of infected animals indicating that tau phosphorylation and RAGE upregulation are not associated with TNF-a and IL-1b in leishmaniasis. There was diminished antioxidant capacity in comparison to controls from the TRAP assay. Mice that received antioxidant therapy had less phosphorylated tau. A possible conclusion of this information is that the neurologic symptoms associated with leishmaniasis are associated with disruptions in the homeostasis of CNS proteins, such as tau and RAGE, as a consequence of oxidative stress.
Limitations: Rat studies are not equivalent to human studies.
Gasparotto J, Senger MR, Kunzler A, Degrossoli A, de Simone SG, Bortolin RC, Somensi N, Girardi CS, de Souza Cda S, Calabrese Kda S, Dal-Pizzol F, Moreira JC, Silva FP Jr, Gelain DP. Increased tau phosphorylation and receptor for advanced glycation endproducts (RAGE) in the brain of mice infected with Leishmania amazonensis. Brain Behav Immun. 2015 Jan;43:37-45. doi: 10.1016/j.bbi.2014.06.204. Epub 2014 Jul 8. PMID: 25014011.
Methyl-CpG binding protein 2, receptors of innate immunity and receptor for advanced glycation end-products in human viral meningoencephalitis
Impact Factor: 1.160
Level of Evidence: Level VI
Type of study and any information related to it: retrospective histological examination of human brain samples
Sample size: Viral Encephalitis group (n = 24), control (n = 22)
Intro: The purpose of this study was to inspect the cause of microglial cell nodule formation as well as their potential role in inflammatory modulation.
Important methods: Brain samples were histologically examined using paraffin slides. The brain samples contained regions of cerebellum, brain stem, spinal cord, and cerebral cortex of parietal, frontal and temporal lobes. Cresyl violet dye was used to stain. Samples were incubated overnight in solutions of human primary antibodies targeting both the n-terminus and c-terminus of RAGE. These reactions were visualized using biotinylated secondary antibodies and ABComplex/HRP conjugate. For the negative control samples, an appropriate mouse antibody was used as a primary antibody.
Statistical tests ran: None -- Samples were only examined using microscopy
Results: The negative control samples produced negative results. In the experimental samples, there was a large amount of immunoreactivity to RAGE receptors in brain regions with widespread necrotic tissue, hypertrophied glial cells, and cells confined near blood vessels.
Discussion/Conclusion: This implicates that RAGE plays a role in neuronal cell death and inflammation during viral encephalitis. Since there were no reported statistical tests, it is hard to say how significant of a difference there was to the control samples. However, the images shown in the paper very clearly show the localization and accumulation of RAGE as described in the results.
Maslinska D, Laure-Kamionowska M, Maslinski S. Original articleMethyl-CpG binding protein 2, receptors of innate immunity and receptor for advanced glycation end-products in human viral meningoencephalitis. Folia Neuropathologica. 2014;52(4):428-435. doi:10.5114/fn.2014.47844.
Receptor for Advanced Glycation End Products (RAGE) Mediates Cognitive Impairment Triggered by Pneumococcal Meningitis
Impact factor: 6.035
Level of evidence: II
Type of study and any information related to it: Rat study
Sample size: 24 brain samples of viral meningoencephalitis cadavers, 22 brain samples of control, age matched cadavers
Intro: The purpose of this study was to examine the role of RAGE and blood brain barrier damage in pneumococcal meningitis.
Important methods: Serotype III S. Pneumoniae was injected into 8-week-old male Wistar rats via the cisterna magna. Control rats received an injection of artificial CSF (aCSF) and S. Pneumoniae suspension. Eighteen hours later the meningitis was confirmed from incubating CSF via sheep blood agar. The rats then received 100 mg/kg of Ceftriaxone via i.p. injections for 7 days, twice a day. Buprenorphine was used as an analgesic. From this point there were 4 groups the rats were split into: control/saline, control/FPS-ZM1, meningitis/saline, and meningitis/FPS-ZM1. FPS-ZM1 (RAGE-specific inhibitor) or saline were injected i.p. immediately after meningitis confirmation. Evan’s blue was then injected to determine the BBB integrity one hour before being euthanized. BBB rats were euthanized at 12, 18, and 24h after meningitis confirmation and did not receive any antibiotics. The permeability of the BBB was measured at each of these time frames. Another experiment was run with rats euthanized after 10 days. These rats were administered FPS-ZM1 or saline for 5 days and ceftriaxone for 7 days. These rats were subjected to an Open-field test for memory retention, a novel object recognition task for nonaversive and nonspatial memory, and a Morris Water Maze test for spatial memory. After euthanization, the prefrontal cortex and hippocampus was removed. TNF-alpha, Il-1B, IL-6 and IL-10 levels were analyzed using ELISA assays. Western blot analysis with primary antibodies were used for RAGE, Aβ1–42, and CD68. Immunohistochemistry with primary antibodies targeting RAGE was also performed.
Statistical tests ran: Shapiro-Wilk normality test, paired student’s t-test, ANOVA, Tukey’s Post hoc, Kruskal-wallis-H, Wilcoxin, p < 0.05
Results: RAGE inhibition using FPS-ZM1 was significantly associated with a prevention of BBB breakdown at 12, 18, and 24h in the cerebral cortex and hippocampus. RAGE inhibited rat significantly avoided habituation memory impairment caused by meningitis. This group also demonstrated significant ability of recognition memory where the non-RAGE inhibited group did not. Spatial memory was also significantly improved in the RAGE inhibited group. Rage inhibition significantly decreased IL-1B and TNF-alpha levels in the prefrontal cortex which were increased in the meningitis group compared to control. IL-10 levels in the RAGE inhibited group were still increased compared to control in the hippocampus. RAGE inhibition significantly decreased the amount of RAGE, Aβ1–42, and CD68 which were increased in the non inhibited meningitis group. Microglia indicators were also prevented from increasing in the RAGE inhibited group. RAGE immunostaining showed that inhibition prevented an increase in RAGE and Aβ1–42 staining in both the hippocampus and prefrontal cortex.
Discussion/Conclusion: RAGE inhibition with ZPS-ZM1 showed significant prevention of increases in cytokine activity and microglia activation linked with inflammation in the meningitis model. BBB structural integrity was also preserved with RAGE inhibition throughout the first 24 hours of meningitis. Behavioral models showed maintenance of spatial memory, recognition memory, and habituation memory with RAGE inhibition meningitis survivor rats. These benefits have possible long-term implications.
Giridharan VV, Generoso JS, Collodel A, Dominguini D, Faller CJ, Tardin F, Bhatti GS, Petronilho F, Dal-Pizzol F, Barichello T. Receptor for Advanced Glycation End Products (RAGE) Mediates Cognitive Impairment Triggered by Pneumococcal Meningitis. Neurotherapeutics. 2020 Sep 4. doi: 10.1007/s13311-020-00917-3. Epub ahead of print. PMID: 32886341.
Advanced glycation end products, oxidative stress and metalloproteinases are altered in the cerebral microvasculature during aging
Impact Factor: 1.649
Level of Evidence: VII
Type of study and any information related to it: Retrospective
Sample size: young (3-6 months old) (n=15) and aged (18-24 months) (n=15) male Wistar rats
Intro: The purpose of this study was to examine the role of the microvasculature of the brain in relation to aging processes in regards to protein carbonyls (PC) (AGE precursor) production.
Important methods: Wistar rats were kept in standard housing and food accommodations and anesthetized with intraperitoneal injections of 5% chloral hydrate (1 ml/100g weight body). Once incised, the blood was washed away using phosphate-buffered saline (PBS) containing 3 mmol/L CaCl2. The brain was then removed and frozen using liquid nitrogen. Microvessels were then isolated from the brains. In order to determine PC production, the microvessels reacted with 10 nM DNPH in 2N chloride acid. Proteins were precipitated in 10% trichloroacetic acid and measured using spectrophotometry.
Statistical tests ran: Significant P < 0.05, results presented as mean ± standard error of the mean, Student's t-test was used to compare groups
Results: The PC concentration in the aged rats was 5.7 ± 0.45 nmol/mg which was a 106.7% significant increase (p < 0.05) compared to the young rats (2.7 ± 0.02 nmol/mg protein)
Discussion/Conclusion: This suggests that aged microvasculature may play a role in the accumulation of AGE precursors, which is linked to neuronal cell death and neurodegenerative diseases.
Safciuc F, Constantin A, Manea A, Nicolae M, Popov D, Raicu M, Alexandru D, Constantinescu E. Advanced glycation end products, oxidative stress and metalloproteinases are altered in the cerebral microvasculature during aging. Curr Neurovasc Res. 2007 Nov;4(4):228-34. doi: 10.2174/156720207782446351. PMID: 18045147.
Receptor for Advanced Glycation Endproducts is upregulated in temporal lobe epilepsy and contributes to experimental seizures
Impact Factor: 5.332
Level of Evidence: III
Type of Study: Experimental study on mice
Purpose: Study the effect of how HMGB1’s effects mediated by RAGE and how RAGE contributes to epilepsy in mice
Methods: Had 2 mice models: an acute epilepsy model and a chronic epilepsy model. The acute model used 10 wild type mice, 8 RAGE -/- mice, and 8 TLR4 -/- mice. The chronic model used 6 mice in each the 3 groups. Kianic acid was injected intracranially to induce seizures. EEG was used to measure seizure activity. In the chronic epilepsy model, EEG was used 8 weeks later to establish chronic epilepsy. All mice were male and age matched. HMGB1 was also injected into these 3 groups of mice with there being 10 mice in the wild type, RAGE -/-, and TLR4 -/- mice groups. Immunohistochemistry was used to analyze the chronic epileptic mice and the sham controls for the chronic epileptic mice model. Immunohistochemistry was used to measure RAGE immunoreactivity. Immunohistochemistry used Nissl staining. Western blot was also performed. Real-time quantitative PCR performed to analyze RNA as well. Novel Objection Recognition Test (NORT) used to measure cognitive function of mice.
Results: Both TLR4 -/- and RAGE -/- mice had reduced seizures compared to wild type mice. Both average time spent in seizures and the number of seizures were reduced in both TLR4 -/- and RAGE -/- mice compared to wild type mice. When 5.5 μg HMGB1 was injected into wild type mice, number and durations of seizures increased. Injecting 5.5 μg HMGB1 into RAGE -/- caused similar results, but injecting 5.5 μg into TLR4 -/- caused no change. Number of chronic hippocampal paroxysmal discharges were reduced in the knockout mice compared to wild type. Spontaneous epileptic activity also decreased in knockout mice compared to wild type, but TLR4 -/- showed a greater decrease compared to RAGE -/-. NORT showed epileptic wild type mice showed impaired memory compared to non-epileptic wild type mice. Epileptic knockout mice groups showed similar results to epileptic wild type mice. Granule cell dispersion in epileptic wild type and knockout mice showed increased width of granule cell layer. Had neurodegeneration in CA1 pyramidal cell layer in epileptic wild type mice and epileptic TLR4 -/- mice. CA1 pyramidal cells were more damaged in the epileptic RAGE -/- mice compared to epileptic wild type and epileptic TLR4 -/- mice. Had reduced CA3-CA4 pyramidal cells in the wild type epileptic mice. Doublecortin (DCX) neurons were reduced in epileptic wild type and epileptic RAGE -/- mice, but were present in epileptic TLR4 -/- mice. DCX did not differ in the naïve mice in all 3 groups. In RAGE immunoreactivity, RAGE was expressed in neurons in pyramidal cells, granule cells, and hilar interneurons for the control mice. Mice with acute seizures showed increased RAGE expression in hippocampal astrocytes an vessels.
Statistics: ANOVA followed by Tukey’s test used.
Discussion: RAGE contribution to seizures is less than that of TLR4. Study supports notion that HMGB1/RAGE signaling contributes to seizures.
Iori V, Maroso M, Rizzi M, Iyer AM, Vertemara R, Carli M, Agresti A, Antonelli A, Bianchi ME, Aronica E, Ravizza T, Vezzani A. Receptor for Advanced Glycation Endproducts is upregulated in temporal lobe epilepsy and contributes to experimental seizures. Neurobiol Dis. 2013 Oct;58:102-14. doi: 10.1016/j.nbd.2013.03.006. Epub 2013 Mar 21. PMID: 23523633.
Polymorphisms in the receptor for advanced glycation end products gene are associated with susceptibility to drug-resistant epilepsy
Impact Factor: 2.274
Level of Evidence: IV
Type of Study: Cohort study
Introduction: There is a link between epilepsy and CNS inflammation. RAGE is involved in sustaining inflammatory reactions. This study looked at genetic variations of RAGE in the Chinese population to find polymorphisms that affect epilepsy.
Materials and Methods: 280 epilepsy patients and 274 healthy controls from 2 hospitals in China. Patients were divided into 2 groups: drug-resistant epilepsy (DRE) and drug-responsive epilepsy (no-DRE). DRE means no seizures or 50% reduced seizure frequency after a minimum of a year of antiepileptic drugs. No-DRE defined as no change or less than 50% reduction in seizure frequency after a minimum of one year with 2 or more antiepileptic drugs
Results: Significant difference observed between epilepsy patients and controls in with G82S genetic variant for RAGE (p=0.031). No significant difference between epilepsy patients and controls for -374A/T and -425/G genetic variants
Discussion: G82S polymorphism for RAGE was associated with DRE, and the variant was more common in DRE patients than controls. 82S allele is hence a risk factor for DRE. Hypothesize that 82S genotype has higher ligand-binding affinity in RAGE and so causes increase NF-kB mediated inflammatory mediators which contributes to epilepsy. 4 haplotypes revealed in study: C-A-A more frequent in epileptic patients than controls
Limitations: Limited ethnic groups (China only) and small sample size. Need to test RAGE G82S contributing to DRE in larger and ethnically diverse population
Guo M, Wang J, Qi H, Liu F, Yao L, Zhang S, Li K. Polymorphisms in the receptor for advanced glycation end products gene are associated with susceptibility to drug-resistant epilepsy. Neurosci Lett. 2016 Apr 21;619:137-41. doi: 10.1016/j.neulet.2016.01.043. Epub 2016 Jan 30. PMID: 26828298.
Levels of Soluble Receptor for Advanced Glycation End Products in Acute Ischemic Stroke without a Source of Cardioembolism
Impact Factor: 2.630
Level of Evidence: IV
Type of Study: Cohort Study
Background: sRAGE, which is a shortened form of the extracellular domain of RAGE, it can stope AGE-RAGE mediated vascular damage. sRAGE can bind to RAGE and prevent ligand binding to it. Purpose is to study the link between acute ischemic stroke (AS) and sRAGE
Methods: 259 people with a first case of AS made up the experimental group. Experimental group 394 age matched volunteers were acquired for the control group. Analysis of sRAGE was compared between these two groups. sRAGE levels were measured against 3 risk factors for AS: diabetes mellitus, hypertension, and hypercholesteremia
Statistics: p value less than 0.05 is significant. Student t-test was used to compare controls and AS group in terms of sRAGE. ANOVA and Scheffe post-hoc test was used among 4 groups defined as having the 3 risk factors and having AS. Chi2 test were performed too. Plasma sRAGE levels were turned into log graphs since the data was not normally distributed. Odds ratio was used for AS and sRAGE levels.
Results: sRAGE was higher in people who did not have the 3 risk factors for AS compared to people who did have the 3 risk factors. sRAGE was also significantly higher in AS regardless of if the 3 risk factors were present or not. Odds ratio showed sRAGE can be used as a biomarker for AS risk.
Discussion: Can use sRAGE levels as a risk factor for AS even with patients who do not have other risk factors. sRAGE in this case is a risk factor independent of the conventional vascular risk factors. Correlation between sRAGe and NIHSS scores (measures neurological deficit severity from strokes) was significant in AS patients who did not have the 3 risk factors. Possible sRAGE levels were affected by medications. Possible that vascular risk factors had a bigger impact on sRAGE levels than neurological deficit severity. Also sRAGE can be released from endothelial cells or dying cells. Hence the group hypothesized that in a certain range, decreased sRAGE levels show severity of AS, but above a certain level, the injured cells releasing sRAGE will hide the negative correlation between sRAGE and AS severity.
Limitations: Causation could not be established due to study design. Did not analyze how medications affected sRAGE levels. Could not determine if sRAGE can predict severity of strokes and morbidity in those who had the 3 risk factors but had not had a stroke yet.
Park HY, Yun KH, Park DS. Levels of Soluble Receptor for Advanced Glycation End Products in Acute Ischemic Stroke without a Source of Cardioembolism. J Clin Neurol. 2009 Sep;5(3):126-32. doi: 10.3988/jcn.2009.5.3.126. Epub 2009 Sep 30. PMID: 19826563; PMCID: PMC2760717.
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