Antioxidant System Disturbances, Bioenergetic Disruption, and Glial Reactivity Induced by Methylmalonic Acid in the Developing Rat Brain

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Dalpizolo et al. entitled “Oxidative stress, bioenergetic disruption, inflammation, and glial reactivity induced by methylmalonic acid in the developing rat brain” provides novel insights into the pathogenesis of methylmalonic aciduria focusing on elucidating detrimental effects of MMA, a metabolite accumulated in this disease, on the nervous system using a rat-based in vivo model. The study provides vast experimental evidence on the effects of MMA. However, a more accurate data interpretation would improve the quality of the manuscript.  

 

Title:

• Suggested to rephrase, e.g., Methylmalonic acid induces oxidative stress, bioenergetic disruption, inflammation, and glial reactivity in the developing rat brain. In addition, the term “oxidative stress” implies an imbalance between pro-oxidants and antioxidants. However, ROS levels or markers of lipid peroxidation were not assessed. Thus, it is suggested to consider replacing “oxidative stress” with “the altered antioxidant status” or likewise. Furthermore, the authors did not use the markers of inflammation to mention that inflammation is affected. Inflammation should be confirmed at least by routine staining.

 

Abstract:

• Line 32. Specify which amino acids were statistically significantly affected.
• Lines 38-39. Provide full names for GFAP, GLUT1, and IBA1

Introduction:

• Please provide an in-depth explanation for the selection of markers used in this study.
• Line 85. Add a dot “after administration of MMA in the rat brain”.
• In the last paragraph, specify the amino acids measured and where they were detected
• Line 90. Add a dot

Materials and Methods:

• Indicate the number of animals and provide a more detailed characterizations (weight, sex, etc.).
• Line 97 Ad libitum should be italicized
• Provide a supplier of MMA and specify its purity
• If kits for detection of the antioxidant parameters were commercially available, provide their full names and suppliers.

Author Response

REPLY TO REVIEWER 1

 

We thank the comments and the suggestions that substantially improved the manuscript.

 

Reviewer 1

Comments and Suggestions for Authors

The manuscript by Dalpizolo et al. entitled “Oxidative stress, bioenergetic disruption, inflammation, and glial reactivity induced by methylmalonic acid in the developing rat brain” provides novel insights into the pathogenesis of methylmalonic aciduria focusing on elucidating detrimental effects of MMA, a metabolite accumulated in this disease, on the nervous system using a rat-based in vivo model. The study provides vast experimental evidence on the effects of MMA. However, a more accurate data interpretation would improve the quality of the manuscript.

Title:

Suggested to rephrase, e.g., Methylmalonic acid induces oxidative stress, bioenergetic disruption, inflammation, and glial reactivity in the developing rat brain. In addition, the term “oxidative stress” implies an imbalance between pro-oxidants and antioxidants. However, ROS levels or markers of lipid peroxidation were not assessed. Thus, it is suggested to consider replacing “oxidative stress” with “the altered antioxidant status” or likewise. Furthermore, the authors did not use the markers of inflammation to mention that inflammation is affected. Inflammation should be confirmed at least by routine staining.

Response: We altered the title of the manuscript, as suggested. As for the inflammation markers, we are not able to measure other markers because there are no more samples available. We would have to submit an amendment for our project in order to request more animals for a new treatment, and it would take months to be approved. However, we agree that the measurement of IBA1 is not sufficient to consider an inflammatory response. Therefore, we replaced the term inflammation with glial reactivity throughout the text.

 

Abstract:

Line 32. Specify which amino acids were statistically significantly affected.

Response: The amino acids that were significantly affected are already described in Line 43.

 

Lines 38-39. Provide full names for GFAP, GLUT1, and IBA1

Response: The full names of these proteins have been provided.

 

Introduction:

Please provide an in-depth explanation for the selection of markers used in this study.

Response: We added sentences in the Introduction section that indicate the reasons why we chose the markers evaluated in our investigation.

“MMA was also shown to reduce brain CO₂ production from glucose and acetate, increase lactate production as well as glucose uptake in rat brain, indicating that aerobic glucose oxidation is affected [9].”

“Additionally, MMA has been shown to induce oxidative stress via increased production of reactive oxidative species (ROS) and disruption of antioxidant defenses in rat brain [3,7,10].”

 

We also stress that a main focus of our study was to compare the vulnerability of the cerebral cortex and striatum to the effects caused by MMA. Therefore, we altered the last paragraph of the Introduction as follows.

“We aimed to compare the vulnerability of the cerebral cortex and striatum, two regions particularly affected in methylmalonic aciduria, to the toxic effects of MMA using different animal models. Since mounting evidence shows that MMA induces oxidative stress and bioenergetic impairment [3,6-10], we investigated here the effects of MMA on antioxidant defenses, citric acid cycle (CAC) activity, as well as neural damage. The levels of the amino acids (threonine, tryptophan, valine, tyrosine, serine, proline, methionine, lysine, hydroxyproline, histidine, leucine, isoleucine, glutamine, alanine, arginine, asparagine, phenylalanine, glutamate and aspartate) were also measured in the cerebral cortex and striatum by LC-MS/MS.”

 

Line 85. Add a dot “after administration of MMA in the rat brain”.

Response: The correction was performed.

 

In the last paragraph, specify the amino acids measured and where they were detected.

Response: The amino acids have been specified.

 

Line 90. Add a dot

Response: The correction was performed.

 

Materials and Methods:

Indicate the number of animals and provide a more detailed characterizations (weight, sex, etc.).

Response: The correction was performed.

 

Line 97 Ad libitum should be italicized:

Response: The correction was performed.

 

Provide a supplier of MMA and specify its purity.

Response: The correction was performed.

 

If kits for detection of the antioxidant parameters were commercially available, provide their full names and suppliers.

Response: We have not used kits.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript investigates important neuropathological mechanisms underlying methylmalonic aciduria (MMA), employing both intraperitoneal and intracerebroventricular administration methods in Wistar rats. While the study significantly contributes to understanding the pathological impact of MMA on neural tissues, numerous opportunities exist to enhance the manuscript’s scientific rigor, methodological transparency, and clinical relevance.

Improvement suggestions:

1. The introduction provides a succinct description of methylmalonic aciduria but lacks broader context. Describe specific enzymes inhibited by MMA and their functional significance within metabolic pathways. Incorporating current hypotheses regarding cellular damage, neuronal vulnerability, and how these biochemical disruptions translate into clinical manifestations would better frame the study’s significance.
2. The manuscript provides brief information about the use of Wistar rats but lacks an in-depth justification for choosing this specific model. Clarifying the appropriateness of the developmental stage selected (30 days old) concerning the human developmental period impacted by MMA would strengthen methodological rigor.
3. The methodological section briefly describes intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of MMA. It is necessary to elaborate on the rationale for choosing these doses and the timing intervals between administrations. Discussing the pharmacokinetics and pharmacodynamics of MMA administration would significantly enhance methodological transparency. Outlining the criteria for dosage selection, including prior dose-response studies or literature-based justifications, would reinforce the validity.
4. The methods section briefly outlines the assays used to measure antioxidant defenses. Provide comprehensive descriptions of the underlying principles, sensitivities, and potential limitations of these assays. Clarifying potential sources of variability or interference in enzyme activity assays and strategies employed to minimize these would further strengthen the scientific quality.
5. The manuscript currently lists antibodies and detection methods but lacks detail about procedural consistency, normalization strategies, and validation of antibody specificity. Providing information on blot quantification techniques, normalization methods (e.g., total protein normalization versus normalization using housekeeping proteins), and justifying the choice of normalization markers, such as β-actin, is essential.
6. The manuscript shows region-specific differences in antioxidant and bioenergetic responses. A more profound discussion of the biological significance of these regional differences, possibly related to metabolic rate, neuronal density, receptor distribution, or susceptibility to oxidative stress, is recommended.
7. The manuscript briefly touches on impaired citric acid cycle (CAC) activities. To enhance the scientific depth, expanding the discussion to describe precisely how reductions in specific enzymes, such as SDH and MDH, affect cellular metabolism, energy production, and redox balance would be valuable. Connecting these biochemical alterations explicitly to neuronal health, synaptic function, or neurotransmitter synthesis would elevate the clinical relevance of the bioenergetic disruptions described.
8. The discussion highlights increased glutamate and co-agonists, such as glycine and serine. Clarify the mechanisms by which MMA exposure modulates amino acid metabolism and NMDA receptor activation.
9. The findings indicate altered levels of amino acids. It would be beneficial to delve deeper into how these changes potentially relate to MMA-induced neuronal damage, neurotransmitter imbalance, or excitotoxicity.
10. GFAP and Iba1 elevations suggest glial activation. A nuanced discussion distinguishing between the potentially protective and harmful roles of activated glia in the MMA context would improve interpretative clarity.
11. GLUT-1 expression increases without detailed exploration. Expanding on potential compensatory metabolic adaptations due to impaired bioenergetics or increased energy demands would significantly clarify physiological implications. Discussing how these changes might reflect blood-brain barrier dynamics or astrocytic metabolic shifts would further contextualize the data.
12. Though limitations are briefly acknowledged, a detailed exploration of how experimental design choices affect translatability to human disease would enhance critical appraisal. Suggesting specific follow-up studies, such as genetic animal models, chronic MMA exposure paradigms, or translational clinical studies, would guide subsequent research efforts, demonstrating clear scientific foresight.
13. Incorporating recent and relevant literature to support novel insights or to highlight contradictory findings would substantially strengthen the manuscript’s contribution.
14. The conclusion could explicitly relate findings to potential clinical management or prognostic strategies for MMA, emphasizing the relevance and practical implications of the research outcomes to clinical neuroscience and metabolic disorder management.

Author Response

REPLY TO REVIEWER 2

Comments and Suggestions for Authors

The manuscript investigates important neuropathological mechanisms underlying methylmalonic aciduria (MMA), employing both intraperitoneal and intracerebroventricular administration methods in Wistar rats. While the study significantly contributes to understanding the pathological impact of MMA on neural tissues, numerous opportunities exist to enhance the manuscript’s scientific rigor, methodological transparency, and clinical relevance.

Improvement suggestions:

1. The introduction provides a succinct description of methylmalonic aciduria but lacks broader context. Describe specific enzymes inhibited by MMA and their functional significance within metabolic pathways. Incorporating current hypotheses regarding cellular damage, neuronal vulnerability, and how these biochemical disruptions translate into clinical manifestations would better frame the study’s significance.

Response: As suggested by the Reviewer, we inserted sentences that describe specific enzymes and processes inhibited by MMA and their functional significance within metabolic pathways. We also inserted sentences to better clarify that we aimed to study the vulnerability of the cerebral cortex and striatum to MMA effects.

“It is important to note that MMA is an inhibitor of succinate dehydrogenase (SDH) activity and of the mitochondrial dicarboxylate that transports succinate [3,6-9]. MMA was also shown to inhibit the activities of complexes I, I+III, and II+III, CO2 production from glucose and acetate, increase lactate production as well as glucose uptake in rat brain, indicating that aerobic glucose oxidation is affected [3,9]. These findings suggest the presence of bioenergetic dysfunction in the brain and other tissues and may explain the increased lactate levels observed in patients [2,3].”

 

“We aimed to compare the vulnerability of the cerebral cortex and striatum, two regions particularly affected in methylmalonic aciduria, to the toxic effects of MMA using different animal models. Since mounting evidence shows that MMA induces oxidative stress and bioenergetic impairment [3,6-10], we investigated here the effects of MMA on antioxidant defenses, citric acid cycle (CAC) activity, and neural damage. The levels of the amino acids (threonine, tryptophan, valine, tyrosine, serine, proline, methionine, lysine, hy-droxyproline, histidine, leucine, isoleucine, glutamine, alanine, arginine, asparagine, phenylalanine, glutamate and aspartate) were also measured in the cerebral cortex and striatum by LC-MS/MS.”

 

2. The manuscript provides brief information about the use of Wistar rats but lacks an in-depth justification for choosing this specific model. Clarifying the appropriateness of the developmental stage selected (30 days old) concerning the human developmental period impacted by MMA would strengthen methodological rigor.

Response: We utilized 30-day-old rats because this period represents a critical period of neurodevelopment, corresponding to early adolescence in humans. It is characterized by fundamental processes such as intense synaptogenesis, neural circuit remodeling, mitochondrial maturation, and high cerebral energy demand, all of which are highly susceptible to metabolic and oxidative insults (Semple et al., Progress in Neurobiology, 106-107, 1-16, 2013). Therefore, at this stage of development, the enzymatic system associated with energy production is well-organized and structured, allowing for accurate measurement of bioenergetics homeostasis. Furthermore, previous studies investigating metabolic disorders associated with mitochondrial dysfunction and oxidative stress in models of organic acidemias have shown that using juvenile animals reveals pronounced alterations in mitochondrial function and oxidative biomarkers (Viegas et al., Free Radical Research, 48(6), 659–669, 2014; da Rosa-Junior et al., Neurotox Res. 35(4):809-822, 2019; Brondani et al., Biomed Pharmacother. 187:118123, 2025). This reinforces the relevance of studying such effects in this age range. In addition, 30-day-old rats are considered young animals, and methylmalonic aciduria presentation occurs in young children.

The following sentences were added to the manuscript:

“We utilized 30-day-old rats for both models described in the following sections because this period represents a critical period of neurodevelopment, corresponding to early adolescence in humans. It is characterized by fundamental processes such as intense synaptogenesis, neural circuit remodeling, mitochondrial maturation, and high cerebral energy demand, all of which are highly susceptible to metabolic and oxidative insults [11].  In addition, 30-day-old rats are considered young animals, and methylmalonic aciduria presentation occurs in young children [2,4].”

 

3. The methodological section briefly describes intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of MMA. It is necessary to elaborate on the rationale for choosing these doses and the timing intervals between administrations. Discussing the pharmacokinetics and pharmacodynamics of MMA administration would significantly enhance methodological transparency. Outlining the criteria for dosage selection, including prior dose-response studies or literature-based justifications, would reinforce the validity.

Response: We used similar concentrations of MMA as those employed in previous studies from our research group when investigating the effects of MMA or organic acids accumulated in other inherited metabolic disorders on various biochemical parameters. Therefore, we added sentences in the manuscript to clarify this point. However, we are not able to discuss the pharmacokinetics and pharmacodynamics of MMA because such parameters were not measured.

“This experimental protocol, including the doses and the time intervals between administrations, was adapted from a previous study that investigated the effects of organic acids structurally related to MMA [12]. No animal suffering (respiratory distress and convulsions) or mortality was observed.”

 

“We used this dose of MMA because it was shown to cause no animal suffering (respiratory distress and convulsions) or mortality and was adequate to disturb critical CNS systems in a previous study [6].”

 

4. The methods section briefly outlines the assays used to measure antioxidant defenses. Provide comprehensive descriptions of the underlying principles, sensitivities, and potential limitations of these assays. Clarifying potential sources of variability or interference in enzyme activity assays and strategies employed to minimize these would further strengthen the scientific quality.

Response: We have provided more details about the techniques used to measure the antioxidant defenses.

 

5. The manuscript currently lists antibodies and detection methods but lacks detail about procedural consistency, normalization strategies, and validation of antibody specificity. Providing information on blot quantification techniques, normalization methods (e.g., total protein normalization versus normalization using housekeeping proteins), and justifying the choice of normalization markers, such as β-actin, is essential.

Response: We have updated Topic 2.6 (Western blotting) to meet the request.

 

6. The manuscript shows region-specific differences in antioxidant and bioenergetic responses. A more profound discussion of the biological significance of these regional differences, possibly related to metabolic rate, neuronal density, receptor distribution, or susceptibility to oxidative stress, is recommended.

Response: We thank the Reviewer for the suggestion. We have not investigated the exact mechanisms involved in the differential vulnerability of striatum and cerebral cortex to MMA effects. However, the higher striatum susceptibility may be related to several aspects described in previous studies, including large amounts of cyclophilin-D that favor the induction of mitochondrial permeability transition, increased calcium sensitivity and massive glutamatergic inputs received from the cortex.

Therefore, the following paragraph was added to the manuscript.

“Our results indicate that the striatum is more vulnerable than the cerebral cortex to the deleterious effects provoked by MMA. This differential vulnerability may be explained by a number of factors. Previous studies have shown that the striatum contains high levels of cyclophilin-D, which may promote mitochondrial permeability transition, and that mitochondrial membrane potential in the striatum is more sensitive to calcium [61, 62]. In addition, the presence of massive glutamatergic inputs in the striatum [63] reinforces the hypothesis that MMA-induced toxic effects are mediated by NMDA receptor activation, which leads to increased ROS generation. Consistent with this, high levels of iron are also found in the striatum, which possibly further contributes to ROS formation [64].”

 

7. The manuscript briefly touches on impaired citric acid cycle (CAC) activities. To enhance the scientific depth, expanding the discussion to describe precisely how reductions in specific enzymes, such as SDH and MDH, affect cellular metabolism, energy production, and redox balance would be valuable. Connecting these biochemical alterations explicitly to neuronal health, synaptic function, or neurotransmitter synthesis would elevate the clinical relevance of the bioenergetic disruptions described.

Response: We have improved the discussion to describe how the reduction of the activities of specific enzymes affects cellular metabolism. In this regard, previous studies demonstrated that MMA inhibits the activity of SDH. Although Okun et al. (J. Biol. Chem. 277(17):14674–14680, 2002) did not verify a direct inhibition of complex II activity by MMA, they found a decrease of the activity of this complex elicited by 2-methylcitrate and malonate, which can be generated in neurons by MMA loading.  Therefore, the bioenergetic impairment can be attributed to a synergic inhibition of SDH caused by MMA, malonate and 2-methylcitrate. Furthermore, Okun et al. also suggested that oxalacetate regeneration is impaired because 2-methylcitrate is generated by the reaction between propionyl-CoA and oxalacetate, and MMA inhibits the transmitochondrial malate shuttle and pyruvate carboxylase. Altogether, these mechanisms may cause gluconeogenesis impairment and ketogenesis activation.

The following paragraph was added to the Discussion:

“We cannot rule out that 2-methylcitrate and malonate also mediate complex II activity inhibition, as Okun et al. [35] showed that MMA loading in striatal neurons causes the formation of these acids. Therefore, we speculate that, in our animal models, the CAC impairment may be due to a synergic effect caused by MMA, malonate and 2-methylcitrate. Interestingly, the same study suggested that oxalacetate regeneration is impaired by these acids since 2-methylcitrate is formed from propionyl-CoA and oxalacetate, while MMA inhibits the malate shuttle and pyruvate carboxylase. Thus, it is conceivable that these mechanisms, which may lead to gluconeogenesis impairment and ketogenesis activation, also occur in our models.”

 

8. The discussion highlights increased glutamate and co-agonists, such as glycine and serine. Clarify the mechanisms by which MMA exposure modulates amino acid metabolism and NMDA receptor activation.

Response: We believe that the Discussion about the altered levels of amino acids observed in our study has been substantially improved, as follows:

“In line with this mechanism, we observed increased levels of the NMDA receptor agonist glutamate, as well as of the co-agonists glycine and serine. Glycine levels are elevated possibly by methylmalonylation and consequent inhibition of the glycine cleavage pathway [43], whereas glutamate may be derived from a-ketoglutarate that is accumulated due to CAC impairment. However, further investigations to determine the metabolism of glutamate in the brain are needed since recent works demonstrated no alterations in the pool of this amino acid in MUT HEK293 cells [44] and patient-derived neurons [45]. As for serine, it was shown that the de novo synthesis and transport of this amino acid were increased in MUT HEK293 cells due to enrichment of proteins involved in serine metabolism [44]. Although we did not establish the sources of these amino acids, our findings suggest that the increase in these amino acids induced by MMA may contribute to NMDA receptor overactivation and subsequent ROS generation.”

 

9. The findings indicate altered levels of amino acids. It would be beneficial to delve deeper into how these changes potentially relate to MMA-induced neuronal damage, neurotransmitter imbalance, or excitotoxicity.

Response: Regarding the effects of MMA on glutamate, glycine and serine levels, we believe that an appropriate response was given in question 8 (see above). As for the neurotransmitter imbalance, the following paragraph has been modified.

“MMA also reduced tyrosine levels in the striatum of rats. A decrease in this amino acid in the brain has been associated with reduced catecholamine synthesis, which may help explain the neurological dysfunction observed in methylmalonic aciduria [46]. Impairment of dopaminergic neurotransmission, particularly in the striatum, potentially contributes to motor dysfunction. Supporting this hypothesis, previous studies have shown that tyrosine and phenylalanine depletion impairs memory and cognitive performance [47].

10. GFAP and Iba1 elevations suggest glial activation. A nuanced discussion distinguishing between the potentially protective and harmful roles of activated glia in the MMA context would improve interpretative clarity.

Response: As requested, the following sentences have been added to the Discussion.

“Activation of astrocytes and microglia can function either as proinflammatory, causing the release of molecules that trigger neuroinflammatory response, or as anti-inflammatory [48]. Although we did not determine which glial phenotype is involved in the MMA effects shown here, previous studies have demonstrated that MMA increases pro-inflammatory cytokine levels and causes oxidative stress in vitro in C6 astroglial cells and in vivo in rat brain [49, 50]. Furthermore, earlier data suggest that accumulation of toxic metabolites in glutaric aciduria type I, a metabolic disorder related to methylmalonic aciduria, can alter astrocyte phenotype and initiate neuronal degeneration [51, 52]. Similar findings indicative of inflammation were also observed in the plasma of patients [53] as well as in models of other inherited metabolic disorders characterized by oxidative stress and bioenergetic dysfunction [54-56]. Therefore, we speculate that the activation of glia mediated by MMA observed in our present study elicits a pro-inflammatory response.”

 

11. GLUT-1 expression increases without detailed exploration. Expanding on potential compensatory metabolic adaptations due to impaired bioenergetics or increased energy demands would significantly clarify physiological implications. Discussing how these changes might reflect blood-brain barrier dynamics or astrocytic metabolic shifts would further contextualize the data.

Response: As suggested, we added the following sentences to describe the potential adaptations resulting from MMA-induced GLUT1 increase.

“In this regard, GLUT-1 expression is frequently upregulated under metabolic stress conditions such as hypoxia or mitochondrial dysfunction, aiming to enhance glucose uptake [58]. Additionally, glucose entry may sustain the pentose phosphate pathway, generating NADPH essential for redox support of neurons [59]. Increasing GLUT-1 may help restore the bioenergetic status impaired by MMA and boost NADPH, thus enhancing GSH recycling reduced by MMA.”

 

12. Though limitations are briefly acknowledged, a detailed exploration of how experimental design choices affect translatability to human disease would enhance critical appraisal. Suggesting specific follow-up studies, such as genetic animal models, chronic MMA exposure paradigms, or translational clinical studies, would guide subsequent research efforts, demonstrating clear scientific foresight.

Response: We thank the Reviewer for the suggestion. An additional limitation of our study is the inability to evaluate the consequences of chronic MMA accumulation. Therefore, the following sentences were inserted.

“On the other hand, our models fail to evaluate the chronic effects caused by sustained high levels of MMA observed in patients. Thus, further studies should be conducted in genetic and chronic MMA exposure-based animal models. Such models are also important to elucidate novel mechanisms that seem to be independent of MMA accumulation [60].”

 

13. Incorporating recent and relevant literature to support novel insights or to highlight contradictory findings would substantially strengthen the manuscript’s contribution.

Response: We believe that incorporating sentences and paragraphs to answer the other questions has substantially strengthened the manuscript.

 

14. The conclusion could explicitly relate findings to potential clinical management or prognostic strategies for MMA, emphasizing the relevance and practical implications of the research outcomes to clinical neuroscience and metabolic disorder management.

Response: We believe that our findings must be taken with caution, as the animal models have limitations. Thus, the following sentence was inserted in the manuscript.

“Additional studies are essential to confirm whether glial reactivity and disturbances in amino acid metabolism may be considered novel targets for therapeutic investigation.”

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed the comments

Reviewer 2 Report

Comments and Suggestions for Authors

In this second review, having seen the manuscript improvements and the authors' detailed responses to previous comments, I recommend acceptance for publication.