On the Potential Therapeutic Roles of Taurine in Autism Spectrum Disorder

Round 1

Reviewer 1 Report

The authors present their hypothesis about the potential application of taurine as therapeutics for ASD and, to this aim, they describe the state-of-the-art of the mechanisms of action of taurin on macrophage polarization, microglia activation, autophagy, apoptosis and neurogenesis and synaptogenesis, and the implication of these processes in ASD. 

Despite the relevance of the study, the manuscript presents some weaknesses to be addressed.

a) The hypothesis is not fully clear. Indeed, the hypotheses formulated in the abstract (lines 29 - 32), in the main text (lines 209 - 211) and in the conclunsions (lines 301 - 304) are different from each other and target different aspects of taurine function in brain physiology, and thus its relevance in ASD pathophysiology. 

The authors should rephrase their overall hypothesis, based on the collected  experimental evidence, and present it in unambiguous way across the manuscript, moving a clear step forward compared to the current literature.

b) The conclusions part ends with the mechanistic description of mTOR implication in ASD. While the concept is correct, keeping this as the last sentence sounds like the conclusions part is suddenly interrupted. Authors should include a final summarizing message regarding the implications of your hypothesis. 

c) It is not clear why and based on what evidence it is recommended to start the taurin treatment as soon as ASD is diagnosed. This should be better clarified.

d) In the paragraph 2. Microglial activation in ASD, the sentence in lines 70 - 71 has a different syntax compared to the other phrases in the bulletted list and thus it is not clear how microglia affects neuron maturation. Please rephrase this.  

e) The literature is comprehensive of the most relevant former works. However, a couple more would be important to include: 

- Park E, Cohen I, Gonzalez M, Castellano MR, Flory M, Jenkins EC, Brown WT, Schuller-Levis G. Is Taurine a Biomarker in Autistic Spectrum Disorder? Adv Exp Med Biol. 2017;975 Pt 1:3-16. doi: 10.1007/978-94-024-1079-2_1. 

- Kun Liu, Runying Zhu, Hongwei Jiang, Bin Li, Qi Geng, Yanning Li, Jinsheng Qi. Taurine inhibits KDM3a production and microglia activation in lipopolysaccharide-treated mice and BV-2 cells. Molecular and Cellular Neuroscience, Volume 122, 2022, 103759, https://doi.org/10.1016/j.mcn.2022.103759.

Author Response

Response to reviewer 1

We sincerely thank this reviewer for his/her excellent review performed on our work. We provide below point-by-point response to all the observations/suggestions made:

1. a) The hypothesis is not fully clear. Indeed, the hypotheses formulated in the abstract (lines 29 - 32), in the main text (lines 209 - 211) and in the conclusions (lines 301 - 304) are different from each other and target different aspects of taurine function in brain physiology, and thus its relevance in ASD pathophysiology.

Response: We tank reviewer for this important observation. We have modified the abstract, also the text from lines 209-311 and lines 301-304, according with this thoughtful suggestion. In the Conclusion section, we have further explained the microglia function in neuron development… “As mentioned in the introduction, the CNS contains a specialized population of macrophage-like cells known as microglia. Being considered as immune sentinels that are capable of orchestrating a potent inflammatory response, microglia can perform a variety of functions [18]. One of such important microglia functions is related to the phagocytic action during neuronal/synaptic development (most likely reflected in the elimination of the repetitive neurons and synapses) [19]. Microglial activation or malfunction can have a significant impact on the neuron maturation, leading to the neurodevelopmental dysfunction, such as ASD [17].

1. b) The conclusions part ends with the mechanistic description of mTOR implication in ASD. While the concept is correct, keeping this as the last sentence sounds like the conclusions part is suddenly interrupted. Authors should include a final summarizing message regarding the implications of your hypothesis.

Response: This is also a correct observation. We do accept that a final summarizing message was lacking in the original version of our manuscript. We have corrected this issue and added some important obervations and corresponding references.

1. c) It is not clear why and based on what evidence it is recommended to start the taurine treatment as soon as ASD is diagnosed. This should be better clarified.

Response: We suggested that taurine´s treatment should be initiated as soon as the diagnosis would have been made because unfortunately we did not know about the study by Park et al., 2017, where they found that a subgroup of children with ASD had low levels of taurine in their blood. We have removed that part and replaced it with this discussion:

 Experimental research showed that that serum taurine concentrations (<106 μM) from 21 (10 females and 11 males) out of 66 children with ASD were remarkably lower, compared to the average taurine concentrations from their unaffected brothers and sisters where taurine levels were 142.6 ± 10.4 and 150.8 ± 8.4 μM, respectively. These observations allowed the authors to suggest that the taurine levels may be a valid biomarker in a subgroup of ASD patients [111]. Therefore, low serum taurine levels could be used as a diagnostic tool and may further justify the therapeutic strategy we are recommending. Taurine treatment should be initiated in those ASD patients with low serum taurine concentrations. Clinical trials should be performed to test the validity of our hypothesis.

2. d) In the paragraph  Microglial activation in ASD, the sentence in lines 70 - 71 has a different syntax compared to the other phrases in the bulletted list and thus it is not clear how microglia affects neuron maturation. Please rephrase this.

Response: This is again an excellent observation. We have removed unnecessary text and now we suggest the following:

The central nervous system (CNS) contains a specialized population of macrophage-like cells known as microglia. Being considered as immune sentinels that are capable of orchestrating a potent inflammatory response, microglia can perform a variety of activities at various periods of life, both in normal and pathological conditions [18]. Microglia have a multitude of roles [19] throughout CNS maturation, such as

phagocytic action during neuronal/synaptic development (most likely represented by the elimination of repetitive neurons and synapses); this process is also known as “ synaptic pruning” [17].

Appealing scientific proof implies that microglial activation or malfunction can have a significant impact on neuron maturation by reducing synaptic pruning, leading to the neurodevelopmental dysfunction, such as ASD[17].

1. e) The literature is comprehensive of the most relevant former works. However, a couple more would be important to include:

Response: We have added several important references

Page 8: To investigate the probable function of taurine in ASD, serum was taken from 66 children with ASD (males: 45; females: 21, ages 1.5 to 11.5 years, average age, 5.2 +/- 1.6). Children with ASD did not significantly differ in taurine concentration from their siblings who were not impacted by the disease. However, 21 out of 66 ASD patients exhibited low taurine levels (<106 M). According to this research, taurine may serve as a reliable biomarker in a subpopulation of ASD children [111].  Park et al., 2017

Page 9: Finally, in a recent work, taurine was administered to lipopolysaccharide (LPS)-treated mice and microglial (BV-2) cells. Taurine inhibited the LPS-induced increase in lysine demethylase 3a (KDM3a), a promoter of inflammation and microglia activation, and improved the sociability of LPS-treated mice, inhibited microglia activation in the hippocampus, and reduced generation of brain inflammatory factors, such as interleukin-6, tumor necrosis factor-α, inducible nitric oxide synthase, and cyclooxygenase-2 [117]. Liu et al., 2022

Reviewer 2 Report

In the present hypothesis, Alberto et al. have very nicely summarized the potential of a sulfur containing amino acid 'Taurine' as a potential therapeutic candidate for the autism spectrum disorder. I really support the idea and eco their rationale that there is a need to look for the treatments for the kids/adults affected by autism. I have minor suggestions:

1. It will be nice to include a schematic of how taurine can regulate the populations of microglia and autophagy pathway. 

2. Make a flowchart of the hypothesis covering each sections.

3. Authors could also discuss the role of astrocytes in this context since they also are know to be a key player along with microglia in autism.

Author Response

Response to the reviewer 2

We sincerely thank reviewer 2 for his/her insightful suggestions:

In the present hypothesis, Alberto et al. have very nicely summarized the potential of a sulfur containing amino acid 'Taurine' as a potential therapeutic candidate for the autism spectrum disorder. I really support the idea and eco their rationale that there is a need to look for the treatments for the kids/adults affected by autism. I have minor suggestions:

1. It will be nice to include a schematic of how taurine can regulate the populations of microglia and autophagy pathway.

Response: We have included a new figure depicting the putative role of taurine in the inhibition of the excessive microglia activation process that characterizes ASD.

Figure 3. Autism spectrum disorder (ASD) results from the exposure to environmental factors + genetic susceptibility. After that exposure, microglia cells in the brain become activated and induce a neuro-inflammatory state characterized by autophagy impairment, which consists in a defective synaptic pruning (the extra synapses are not removed by microglia cells, so the neurons from ASD brains have an excessive amount of dendritic spines. The "intense world syndrome," which describes the autistic brain as hyper-reactive with a hyper-connectivity of local neural circuits, is similar to this phenotype. Due to a greater number of synaptic connections and increased spine density, such complex connections are characterized by heightened neuronal information processing and storage inside the brain microcircuits (for review see [68]. Modified from [68]. Created with https://biorender.com/. The Creative Commons CC-BY license governs the usage of this image, which allows for free use, sharing, and copying in every format only when the original project is correctly acknowledged. Reproduced with permission from: Angrand, L.; Masson, J.-D.; Rubio-Casillas, A.; Nosten-Bertrand, M.; Crépeaux, G. Inflammation and Autophagy: A Convergent Point between Autism Spectrum Disorder (ASD)-Related Genetic and Environmental Factors: Focus on Aluminum Adjuvants. Toxics 2022, 10, 518. https://doi.org/10.3390/toxics10090518.

We welcome the reviewer to analyze the figure and the corresponding text. If further changes are needed, please let us know.

2. Make a flowchart of the hypothesis covering each sections.

Response: We believe that the new figure shown above contains all the taurine´s protective effects we have mentioned in our hypothesis.

3. Authors could also discuss the role of astrocytes in this context since they also are know to be a key player along with microglia in autism.

Response: We thank the reviewer 2 for this important suggestion.  We have added a short explanatory text in page 9.

Excessive glutamate release through the mTOR pathway can negatively impact the autophagy process [114]. In this regard it is important to mention that astrocytes are responsible for the clearance and transport of glutamate, which is possible due to the presence of glutamate transport proteins (GLAST) and glutamate transporter 1 (GLT-1) on the astrocytic membranes. Abnormalities of glial cells regarding glutamate metabolism may lead to the behavioral impairments in animals (for review see [117]).

117. Gzielo, K.; Nikiforuk, A. Astroglia in Autism Spectrum Disorder. Int J Mol Sci. 2021, 21, 11544. doi: 10.3390/ijms222111544.

Author Response File: Author Response.docx