The Glyoxalase System in Age-Related Diseases: Nutritional Intervention as Anti-Ageing Strategy

Round 1

Reviewer 1 Report

In this study, Aragones et al provide a comprehensive review of the glyoxalase system as a defense mechanism against accumulation of advanced glycation end-products; and review data on its decline with aging. They also summarize the literature on glyoxalase-1 modulation in diabetes and aging to understand its mechanistic relevance under these stresses. Original data is included on the expression of glyoxalase-1 in various tissues and in various layers of the retina. While overall this review is very informative, the rigor can be further improved and the findings strengthened as follows:

1. Please discuss the current understanding as to why these pathways decline with aging. This is the core focus of the review and there should be a section dedicated to what is known and the key questions that remain to be answered.
2. Figure 1 can be improved by separating the synthesis and degradation pathways for dibarbonyls and AGEs on either side. For example, synthesis pathways can be described on the left and removal pathways on the right. The spelling of 5-D-lactosylglutathione needs to be corrected in the figure.
3. There should be a section on the cellular adverse effects of AGEs. This could be brief, but should delineate the pathways for cellular damage- i.e. do these impair function of specific organelles, energetics, or alter gene expression?
4. If the authors wish to include primary data in Figures 3 and 4, please provide controls for the antibody used or point to prior papers where it has been validated, perhaps against a knockout mouse or another reagent. Also, please show the y-axis on Figure 3A in absolute numbers like in Figure 3F. Also, appropriate controls for immunohistochemistry for GLO1 immunostaining in Figure 4B need to be included.
5. Why does Table 1 indicate that it is a summary of the ‘most relevant’ studies? How did the authors determine as to what is relevant?

Minor:

Page 1,  in the first line of the introduction paragraph, it should read: A growing body of literature…

Author Response

Dear Editor,

We would like to thank the reviewers for the positive comments about our manuscript (“a comprehensive review of the glyoxalase system”, “overall this review is very informative”, “a useful and topical review” and “the scope of paper is well illustrated”) and for giving us the opportunity to submit a revised version of our manuscript. We have modified the manuscript to accommodate the reviewers’ comments. Please find below a detailed response to the reviewer’s comments as well as the changes that we have made in the manuscript.

Reviewer 1. Report Form

In this study, Aragones et al provide a comprehensive review of the glyoxalase system as a defense mechanism against accumulation of advanced glycation end-products; and review data on its decline with aging. They also summarize the literature on glyoxalase-1 modulation in diabetes and aging to understand its mechanistic relevance under these stresses. Original data is included on the expression of glyoxalase-1 in various tissues and in various layers of the retina. While overall this review is very informative, the rigor can be further improved and the findings strengthened as follows:

1. Please discuss the current understanding as to why these pathways decline with aging. This is the core focus of the review and there should be a section dedicated to what is known and the key questions that remain to be answered.

Answer: We have expanded the information about the age-related decline of proteolytic pathways (autophagy and UPS).The new paragraph for proteolytic pathways can be found from line 136 to 155 and from line 161 to 166 (see below). The information regarding glyoxalase system in aging research is summarized in the section 3 and we have included a paragraph about changes of alternative pathways with age (from line 291 to 299).

“Age-related changes in rates of protein degradation were documented for many tissues more than 3 decades ago, even before the molecular characterization of proteolytic pathways was defined (PMID: 6354285). Nowadays the molecular and cellular decline of the 2 major proteolytic routes with age is better understood and there are differences in the degrees of decrease between the UPS and lysosomal system. Many reports have shown a tissue-dependent decline of UPS while autophagic decline seems to be universal (reviewed in PMID: 16125351, PMID: 15325579 and PMID: 24929664). Regarding autophagy, both lysosomal and autophagosomal compartments undergo striking modifications. Changes that  contribute to malfunctioning of autophagy include decrease of lysosomal stability, hydrolase activity, accumulation of indigestible material (lipofusin) in the lysosomal lumen, dysfunctional lysosomal pH, decrease transcriptional level of autophagy-related proteins, decrease stability of the chaperone-mediated autophagy receptor LAMP2A in the lysosomal membrane and decrease association of motor proteins in the autophagic compartments (PMID: 16125351, PMID: 24929664, PMID: 29845728). In contrast to autophagy, it is now accepted that changes in proteasome proteolytic abilities with age seem to be more qualitative than quantitative. Changes in the composition of the proteasomal core catalytic activities and modulatory subunits, decreased proteasome expression, as well as changes in the oxidation state of the proteasome subunits and proteasome substrates contribute to the age-related inhibition of the UPS capacity (reviewed in PMID: 12200039, PMID: 15677694). In some cases, there may just be insufficient capacity of the proteolytic systems to handle to load.”

The immediate consequence of the decline of proteolytic capacity is the accumulation of long-lived proteins in aged organisms, many of which accumulate glycation-derived damage in their aminoacid sequences. Accumulation of AGEs occurs in an age-related dependent manner (PMID: 21967227; PMID: 20478906) and a recent proteomic analysis in aging research has revealed that AGEs biology is a highly enriched metabolic pathway associated to age-associated proteome (PMID: 33730416)”.

“Although there is no systematic aging analysis of proteins involve in GLO1-independent alternative pathways, age-related changes of those molecular players have been reported. For example, there is a correlation between DJ-1 levels of expression and oxidative stress and different reports showed an increase of DJ-1 with age. DJ-1 mRNA and protein levels increased from 8 to 20 weeks of age in mice (PMID: 22611253) and DJ-1 levels significantly increased as a function of age in human cerebrospinal fluid (PMID: 20157014). In ocular tissues, it has been shown that the DJ-1 is expressed in retinal pigment epithelium and photoreceptors and the expression increased in old eyes (PMID: 22701690). It might reflect a compensatory mechanism due to the decline of glyoxalase system activity”

2. Figure 1 can be improved by separating the synthesis and degradation pathways for dibarbonyls and AGEs on either side. For example, synthesis pathways can be described on the left and removal pathways on the right. The spelling of 5-D-lactosylglutathione needs to be corrected in the figure.

Answer: We have added the changes suggested by the reviewer in New Figure 1.

3. There should be a section on the cellular adverse effects of AGEs. This could be brief, but should delineate the pathways for cellular damage- i.e. do these impair function of specific organelles, energetics, or alter gene expression?

Answer: We have added a brief paragraph about the cellular adverse effects of AGEs, as requested (from line 64 to 71).

“Excessive glycative stress promotes protein insolubility, deregulating signaling and protein quality control pathways. AGEs-derived changes in the proteome perturb signaling pathways in tissue physiology (MAP/ERK, JAK‐STAT and PI3K‐AKT pathways) that lead to nuclear translocation of transcription factors involved in multiple cellular functions including inflammation, apoptosis, ER stress, autophagy, oxidative stress, mitochondrial function, etc (reviewed in PMID: 33506967 and PMID: 30279139). Glycated proteins may also overtax or limit function of the proteolytic capacities. These changes contribute ultimately to the onset of multiple age-related disorders.”

4. If the authors wish to include primary data in Figures 3 and 4, please provide controls for the antibody used or point to prior papers where it has been validated, perhaps against a knockout mouse or another reagent. Also, please show the y-axis on Figure 3A in absolute numbers like in Figure 3F. Also, appropriate controls for immunohistochemistry for GLO1 immunostaining in Figure 4B need to be included.

Answer: We have added changes in Figure 3 and 4, as requested. In addition, we have included a sentence to point to the prior papers where the non-commercial antibody was validated (from line 315 to 317).

“The antibody against GLO1 was previously validated in previous reports and used for the analysis of GLO1 in retinal samples (PMID: 24162587, PMID: 32068111 and PMID: 21056979)”

5. Why does Table 1 indicate that it is a summary of the ‘most relevant’ studies? How did the authors determine as to what is relevant?

Answer: We apologize for the inappropriate term. We have removed the word “most” in the sentence. The goal of Table 1 is to summarize the studies focused on the association between Glo1 genetically modified cells and animal models.

Minor:

Page 1, in the first line of the introduction paragraph, it should read: A growing body of literature…

Answer: We have changed this sentence in the introduction (line 40), as requested.

Reviewer 2 Report

This is a useful and topical review that focuses on nutritional intervention to modulate the glyoxalase system as a therapeutic target to delay the development of age-related diseases. The authors summarize a complex and expansive literature that describes the current knowledge about nutritional compounds with properties to modulate the glyoxalase system. Although the authors focus on the retina, they also address the nervous system and its disorders associated with old age but the references for the latter are rather thin. Additionally, the authors should address up front a comprehensive Nov. 2020 review (He et al. Glyoxalase system: A systematic review of its biological activity, related-diseases, screening methods and small molecule regulators, in Biomed. Pharmacotherap 131 https://www.sciencedirect.com/science/article/pii/S0753332220308568  to make clear the new/novel aspects of the present paper that are not covered by He et al. (2020) or which clarify/disagree/build on the review by He et al. (2020). Finally, it would be helpful if the authors could briefly note the presence or abence of any toxic effects from prolonged human/animal treatment with the various nutritional factors they discuss.

 

The scope of paper is well illustrated by Figure 1 but the Section 2.2. issue discussed below may require the authors to modify their diagram.

 

Section 2.2. In relation to “scavenging by acetoacetate to form 3-hydroxyhexane-2,5-dione”, the authors should take account of a recent paper in Toxics 2021, 9, 98. https://doi.org/10.3390/toxics9050098 that discusses the relationship between gamma-diketones and diabetic neuropathy.  This may help clarify “The physiological relevance of these systems remains unclear and it has been questioned whether these enzymes are crucial for the detoxification of AGEs in tissues due to 230 the high activity of the glyoxalase system.” In regard to the second paragraph, note that external exposure to n-hexane (which is metabolized to 2,5-hexanedione) not only is an established cause of peripheral neuropathy but has also been linked to parkinsonism in isolated cases (https://pubmed.ncbi.nlm.nih.gov/7651443/   https://pubmed.ncbi.nlm.nih.gov/12736734/   https://pubmed.ncbi.nlm.nih.gov/29352205/)

In sum, the alternative “detoxication” pathway can generate a hydroxy gamma-diketone that will react with proteins throughout the body and, based on extensive studies, will (a) promote distal retrograde axonal degeneration in elongate peripheral nerves and spinal pathways https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1637239/   and (b) testicular injury  https://pubmed.ncbi.nlm.nih.gov/12471174/  , tissues that are required to transport proteins over long distances during which they can complex with gamma-diketones to form oxidized pyrroles. Thus, this “detoxication” pathway is actually a potential auto-entoxication pathway. Does this and alternative pathways color the lack of MG increase in tissues of Glo1 KO organisms? (line 515)

 

Section 2.3. “Retinal activity was the highest value while that liver, kidney, brain and heart only represented 46%, 27%, 22% and 11% of detoxifying retinal capacity, respectively.”  Are the data on the differential tissue distribution of pGLO1?

 

Section 2.3. The retinal results are very interesting, given as the authors point out, the tissue is composed of post-mitotic cells. Can this be extended to the central nervous where the vast majority of neurons are post-mitotic?

 

Section 3.1. “In humans, several studies have investigated the impact of aging on GLO1. These studies found a reduction of GLO1 activity in multiple tissues such as arterial tissues, lens, brain and red blood cells with age [98-101].”  These references refer to red blood cells (98) and lens (99,100); which reference refers to brain?  Specify what is known concerning the “reduction of GLO1 activity in multiple tissues such as arterial tissues, lens, brain and red blood cells with age”.  Should a reference such as the following also be cited here? https://pubmed.ncbi.nlm.nih.gov/16427160/

 

Section 4.2. There is also literature on polyphenols and neurodegenerative diseases, for example: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180342/   Given coverage of AD and PD in this article, it would seem relevant briefly to summarize this literature in one or two sentences and references,

 

Cyanidin/genistein.  This section should be expanded somewhat to incorporate evidence from studies of other tissues (notably the nervous system) that support the protective effect and mechanisms of cyanidin.e.g.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680520/  and  10.1016/j.biopha.2020.110663 Similarly, for xanthohumol, e.g. doi: 10.3389/fphar.2018.00199

 

Author Response

Dear Editor,

We would like to thank the reviewers for the positive comments about our manuscript (“a comprehensive review of the glyoxalase system”, “overall this review is very informative”, “a useful and topical review” and “the scope of paper is well illustrated”) and for giving us the opportunity to submit a revised version of our manuscript. We have modified the manuscript to accommodate the reviewers’ comments. Please find below a detailed response to the reviewer’s comments as well as the changes that we have made in the manuscript.

Reviewer 2. Form

This is a useful and topical review that focuses on nutritional intervention to modulate the glyoxalase system as a therapeutic target to delay the development of age-related diseases. The authors summarize a complex and expansive literature that describes the current knowledge about nutritional compounds with properties to modulate the glyoxalase system. Although the authors focus on the retina, they also address the nervous system and its disorders associated with old age but the references for the latter are rather thin. Additionally, the authors should address up front a comprehensive Nov. 2020 review (He et al. Glyoxalase system: A systematic review of its biological activity, related-diseases, screening methods and small molecule regulators, in Biomed. Pharmacotherap 131 https://www.sciencedirect.com/science/article/pii/S0753332220308568  to make clear the new/novel aspects of the present paper that are not covered by He et al. (2020) or which clarify/disagree/build on the review by He et al. (2020). Finally, it would be helpful if the authors could briefly note the presence or absence of any toxic effects from prolonged human/animal treatment with the various nutritional factors they discuss.

The scope of paper is well illustrated by Figure 1 but the Section 2.2. issue discussed below may require the authors to modify their diagram.

Answer: We have included a paragraph about the review by He et al. (2020) in section 4 in order to clarify the different aspects that our review covers (from line 572 to 581). He et al. focused on drug discovery of glyoxalase system modulators from a biochemical perspective, emphasizing the different classes, chemical structure and structure-activity relationship between GLO1 protein and modulators. Our review is a comprehensive review of the glyoxalase system written from a physiological angle, stressing the changes of GLO1 associated to disease and including original data (comparative analysis in tissues with special emphasis in ocular tissues). Also, we summarize how different dietary compounds can be a safe source of enhancers of GLO1 activity. Thus the reviews are complementary.

“There is an increasing interest in the discovery of small molecule regulators with the capacity to modulate glyoxalase system. He et al. recently summarized high-throughput microplate assays to identify novel regulators, chemical structures and structure-activity relationship between GLO1 protein and modulators.  (reviewed in PMID: 32858501). These studies are useful for drug design and the optimization of therapeutic doses for these compounds will require human clinical trials. However, dietary interventions based on supplements or changes in diet may be an alternative source of multiple glyoxalase activity enhancers to prevent or alleviate glycation-derived damage with age. In this section, we summarize the current status of knowledge about the anti-glycative activities of different nutritional compounds.”

Section 2.2. In relation to “scavenging by acetoacetate to form 3-hydroxyhexane-2,5-dione”, the authors should take account of a recent paper in Toxics 2021, 9, 98. https://doi.org/10.3390/toxics9050098 that discusses the relationship between gamma-diketones and diabetic neuropathy.  This may help clarify “The physiological relevance of these systems remains unclear and it has been questioned whether these enzymes are crucial for the detoxification of AGEs in tissues due to 230 the high activity of the glyoxalase system.” In regard to the second paragraph, note that external exposure to n-hexane (which is metabolized to 2,5-hexanedione) not only is an established cause of peripheral neuropathy but has also been linked to parkinsonism in isolated cases (https://pubmed.ncbi.nlm.nih.gov/7651443/   https://pubmed.ncbi.nlm.nih.gov/12736734/   https://pubmed.ncbi.nlm.nih.gov/29352205/)

In sum, the alternative “detoxication” pathway can generate a hydroxy gamma-diketone that will react with proteins throughout the body and, based on extensive studies, will (a) promote distal retrograde axonal degeneration in elongate peripheral nerves and spinal pathways https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1637239/   and (b) testicular injury  https://pubmed.ncbi.nlm.nih.gov/12471174/  , tissues that are required to transport proteins over long distances during which they can complex with gamma-diketones to form oxidized pyrroles. Thus, this “detoxication” pathway is actually a potential auto-entoxication pathway. Does this and alternative pathways color the lack of MG increase in tissues of Glo1 KO organisms? (line 515)

 

Answer: We thank the reviewer for the comments. We have added a new paragraph to indicate that alternative pathways to glyoxalase system could generate toxic molecules (from line 286 to 289).

“Alternative pathways that might compensate the deficiency of glyoxalase system could potentially generate toxic molecules such as γ-diketones, which are associated with peripheral axonal degeneration and testicular injury (PMC8146965; PMID: 12471174)”

Section 2.3. “Retinal activity was the highest value while that liver, kidney, brain and heart only represented 46%, 27%, 22% and 11% of detoxifying retinal capacity, respectively.”  Are the data on the differential tissue distribution of pGLO1?

Answer: There is no currently commercial antibody available against the phosphorylated form of GLO1. Morgenstern et al. analyzed exclusively cytosolic extract from 6 week and 28 week mouse liver samples using a Phos-Tag-Gel (Zinc) approach (reference 70). Based on the reviewer’s comment we have modified the sentence to include that possibility for future studies (from line 383 to 385). In addition, we included a Supp Fig. 1 that shows that total levels of GLO1 correlate with differential tissue GLO1 activity.

“Interestingly, the observed decline of GLO1 activity in liver was linked to a loss of GLO1 phosphorylation upon aging. GLO1 phosphorylation was not examined in other tissues. It remains to be clarified if tissue-dependent differences in the ratio of this posttranscriptional modification could be behind differences in GLO1 activity”

Section 2.3. The retinal results are very interesting, given as the authors point out, the tissue is composed of post-mitotic cells. Can this be extended to the central nervous where the vast majority of neurons are post-mitotic?

Answer: An ongoing project is currently evaluating that possibility. Based on the reviewer’s comment we have included this sentence to include that possibility for future studies (from line 359 to 362)

“A similar scenario might occur in other tissues composed of cells with low- regeneration capacity such central nervous system where the vast majority of neurons are post-mitotic. Evaluation of GLO1 levels along with cell-specific markers might allow to evaluate the cell-to cell variation within a given tissue.”

Section 3.1. “In humans, several studies have investigated the impact of aging on GLO1. These studies found a reduction of GLO1 activity in multiple tissues such as arterial tissues, lens, brain and red blood cells with age [98-101].”  These references refer to red blood cells (98) and lens (99,100); which reference refers to brain?  Specify what is known concerning the “reduction of GLO1 activity in multiple tissues such as arterial tissues, lens, brain and red blood cells with age”.  Should a reference such as the following also be cited here? https://pubmed.ncbi.nlm.nih.gov/16427160/

Answer: We have included a paragraph to provide more information about GLO1 in brain and have included the reference, as requested (from line 464 to 466).

“GLO1 levels decreased in old aged human cortices compared to young brain. Elevated GLO1 levels were found in Alzheimer’s patients and GLO1 amount also decrease with age. PMID: 16427160”

Section 4.2. There is also literature on polyphenols and neurodegenerative diseases, for example: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180342/   Given coverage of AD and PD in this article, it would seem relevant briefly to summarize this literature in one or two sentences and references.

Answer: We have included a paragraph to provide more information about polyphenols and neurodegenerative diseases and have included the reference, as requested (from line 616 to 619).

“Recent reports have focused on the therapeutic potential of polyphenols in neurodegenerative disorders. Resveratrol, curcumin, capsaicin and epigallocatechin gallate induce neuroprotective effects through NRF2-induction in experimental models of Alzheimer’s and Parkinson’s disease (reviewed in PMCID: PMC6514598, PMCID: PMC7180342”

Cyanidin/genistein.  This section should be expanded somewhat to incorporate evidence from studies of other tissues (notably the nervous system) that support the protective effect and mechanisms of cyanidin.e.g.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680520/  and  10.1016/j.biopha.2020.110663 Similarly, for xanthohumol, e.g. doi: 10.3389/fphar.2018.00199

Answer: We have included several paragraphs to expand the information about cyanidin and xanthohumol with special stress on nervous system, as requested by the reviewer (from line 631 to 634; from line 679 to 683).

“Anthocyanin have been reported to induce therapeutic activities in a wide range of disorders in the nervous systems such as cerebral ischemia, Alzheimer's disease and Parkinson's disease (PMID: 29080525. Anthocyanins prevent neurotoxocity but there is limited information about the impact of anthocyanins on glyoxalase systems.”

“Xanthohumol, a prenylated flavonoid found in hops and beer, attenuated glycative stress in osteoblastic MC3T3-E1 cells by increasing NRF2 and GLO1 activity [182]. Different reports have shown neuroprotective properties of xanthohumol in murine neuroblastoma N2a cells stably expressing human Swedish mutant amyloid precursor protein and in ischemic stroke animal model PMCID: PMC5893754. However, it was not explored if upregulation of GLO1 activity contributes to the neuroprotective effect”

 

Round 2

Reviewer 1 Report

Thank you for addressing my comments