The Role of Maternal Immune Activation in the Pathogenesis of Autism: A Review of the Evidence, Proposed Mechanisms and Implications for Treatment

Round 1

Reviewer 1 Report

The review manuscript by Zawadzka etal offers a thorough review of maternal immune activation (MIA) and its relationship to autism spectrum disorders (ASD). It is very nicely written and it has a good flow of information from behavior, pathology to cellular and molecular mechanisms underlying MIA and ASD.

I have very minor requests.

On page 2, paragraph reading- “Regardless of many theories on the etiology of ASD, the exact….; line 7 has a few genes mentioned. They are abbreviated and have not been discussed before. Can the authors expand these genes? (TSC etc..). And indicate what these genes are involved in.

Page 3, line 16, please expand VPA. I may have missed it but could not find out what this injection is.

Page 6 and 7, blood-brain barrier is sometimes expanded and sometimes indicated as BBB. The authors may want to stick with one of these.  

Author Response

Response to Reviewer 1

 

Dear Reviewer,

 

Thank you very much for your comprehensive review of our manuscript. We corrected the manuscript according to your suggestions. Please, find below our detail responses to your comments.

 

Best regards,

Magdalena Cieślik and Agata Adamczyk

 

REVIEWER COMMENTS:

Comments and Suggestions for Authors

I have very minor requests.

On page 2, paragraph reading- “Regardless of many theories on the etiology of ASD, the exact….; line 7 has a few genes mentioned. They are abbreviated and have not been discussed before. Can the authors expand these genes? (TSC etc..). And indicate what these genes are involved in.

RESPONSE: Thank you for pointing out our mistakes. According to Reviewer comments the full name of the gene which mutation is closely associated with ASD was added to the manuscript: “Some genes mutations that increase the risk of ASD have been known for a while, such as mutations in TSC1/TSC2 (tuberous sclerosis complex) or FMR1 (fragile X mental re-tardation 1) genes, and new ones are being discovered (e.g. CHD8 - chromodomain helicase DNA-binding protein 8, DYRK1A - dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A, SCN2A - sodium voltage-gated channel alpha subunit 2)“

However, we decided not to include in the manuscript the detailed description of the disorders caused by the described mutations. In our opinion, this could distract the reader. The genetic component in ASD is not the goal of our manuscript. Many studies in the literature thoroughly define this issue. We believe that this corrections increased the value of our article and our explanations you will find adequate.

Our explanation what these genes are involved in:

Tuberous sclerosis complex (TSC) is a genetic disease which is caused by loss-of-function mutations in TSC1 or TSC2 genes. According to the literature data, is estimated 40-50 percent of individuals with TSC have autism spectrum disorder. This making TSC a leading genetic cause of syndromic autism. TSC is an autosomal dominant disorder caused by a mutation in about 70% of individuals with the TSC2 gene, and about 20% in the TSC1 gene. The remainder typically has somatic mosaicism, meaning one copy of the TSC1 or TSC2 gene. The proteins encoded by TSC1 (hamartin) and TSC2 (tuberin) form the TSC protein complex, regulated the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). mTORC1 is involved in major cellular processes including cell growth, proliferation, protein synthesis, and autophagy. When hamartin or tuberin is absent or nonfunctional, the TSC protein complex no longer can inhibit mTORC1 activity. Overactivation of the mTORC1 signaling pathway leads to tumor growth that is characteristic of TSC (Samanta 2020). The core psychiatric and neurological manifestations of TSC are autism, epilepsy, and intellectual disability.

In the case of fragile X syndrome (FXS), data showed that 60% of males meet the criteria for ASD. FXS is the most common inherited single-gene cause of intellectual disability and ASD. It is caused by a mutation on the X chromosome in the fragile X mental retardation 1 (FMR1) gene, typically due to the expansion of the CGG triplet repeat, resulting in disruption to the fragile X mental retardation protein (FMRP) (Marlborough et al. 2021).

The other mutations described in the manuscript are related to the proteins responsible for:

- chromatin remodeling and regulator of transcription - the chromodomain helicase DNA-binding protein 8 (CHD8) was identified as a leading ASD candidate gene (Wilkinson et al. 2015)

- - various aspects of postnatal neural development by their function as both serine/threonine and tyrosine kinase activities. The dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1 A (DYRK1A) modulates alternative splicing by phosphorylating the splice factor SRSF6 (Dang et al. 2018) as well as plays a role in control the mitochondrial import machinery and biogenesis (Walter et al., 2021).

 - proper neural development and function. The loss-of-function mutations in the Sodium Voltage-Gated Channel Alpha Subunit 2 - SCN2A gene, which encodes the voltage-gated sodium channel NaV1.2, are associated with autism rates up to 50% (Kruth et al. 2020).

 

REVIEWER COMMENTS:

Page 3, line 16, please expand VPA. I may have missed it but could not find out what this injection is.

RESPONSE: Thank you for pointing out this mistake. According to Reviewer comments the abbreviation VPA (valproic acid) was explained on page 2 when it was mentioned for the first time.

REVIEWER COMMENTS:

Page 6 and 7, blood-brain barrier is sometimes expanded and sometimes indicated as BBB. The authors may want to stick with one of these.

RESPONSE: Yes, the manuscript lacked consistency in the use of the abbreviation BBB for the blood-brain barrier. According to Reviewer's suggestion, this error has been corrected. On page 5, the abbreviation is explained and then it is used consistently.

We believe that all corrections increased the value of our article and our explanations you will find adequate. Moreover, bearing in mind the valuable comments of the Editor, we have rewritten the abstract and conclusions to emphasize our hypothesis that MIA induces microglial activation, oxidative stress and mitochondrial dysfunction – a deleterious trio in the brain of offspring leading to neuroinflammation and neurodevelopmental pathologies.

 

REFERENCES:

 

Dang, T., Duan, W.Y., Yu, B., Tong, D.L., Cheng, C., Zhang, Y.F., Wu, W., Ye, K., Zhang, W.X., Wu, M., Wu, B.B., An, Y., Qiu, Z.L. and Wu, B.L. 2018. Autism-associated Dyrk1a truncation mutants impair neuronal dendritic and spine growth and interfere with postnatal cortical development. Molecular psychiatry 23, 747-758.

 

Kruth, K.A., Grisolano, T.M., Ahern, C.A. and Williams, A.J. 2020. SCN2A channelopathies in the autism spectrum of neuropsychiatric disorders: a role for pluripotent stem cells? Mol Autism 11, 23.

 

Marlborough, M., Welham, A., Jones, C., Reckless, S. and Moss, J. 2021. Autism spectrum disorder in females with fragile X syndrome: a systematic review and meta-analysis of prevalence. Journal of neurodevelopmental disorders 13, 28.

 

Samanta, D. 2020. An Updated Review of Tuberous Sclerosis Complex-Associated Autism Spectrum Disorder. Pediatric neurology 109, 4-11.

 

Walter C, Marada A, Suhm T, Ernsberger R, Muders V, Kücükköse C, Sánchez-Martín P, Hu Z, Aich A, Loroch S, Solari FA, Poveda-Huertes D, Schwierzok A, Pommerening H, Matic S, Brix J, Sickmann A, Kraft C, Dengjel J, Dennerlein S, Brummer T, Vögtle FN, Meisinger C. Global kinome profiling reveals DYRK1A as critical activator of the human mitochondrial import machinery. Nat Commun. 2021 Jul 13;12(1):4284. doi: 10.1038/s41467-021-24426-9.

 

Wilkinson, B., Grepo, N., Thompson, B.L., Kim, J., Wang, K., Evgrafov, O.V., Lu, W., Knowles, J.A. and Campbell, D.B. 2015. The autism-associated gene chromodomain helicase DNA-binding protein 8 (CHD8) regulates noncoding RNAs and autism-related genes. Translational psychiatry 5, e568-e568.

 

Author Response File: Author Response.docx

Reviewer 2 Report

In their manuscript, Zawadzka, Cieślik and Adamczyk reviewed the known reports on the ways and mechanisms, through which inflammation in pregnant women, termed maternal immune activation (MIA), might lead to the autism spectrum disorders (ASD) in the offspring.

 

The manuscript is prepared in a logically consistent orders and contains the large enough amount of recent data on the subject. However, the text is, unfortunately, abundant in stylistic, typing and grammar errors and needs total proofreading. This is the main concern.

Below I tried to cite some errors in the orders they stay in the text, but the list is not limited to the items stated below – the whole text requires careful rewriting by a professional translator and proof-reader.

1. Line 69: “Many preformed twin studies” – many performed
2. Line 74: “are keep being discovered” - are being discovered, or keep being discovered
3. Line 80: “a child’s neurodevelopment” – the child’s neurodevelopment [or use zero article]
4. Line 86: “with healthy mothers with no diabetes” – with healthy mothers [healthy is a priori with no diabetes]
5. Line 122: “2. . Proinflammatory cytokines” – 2. Proinflammatory cytokines [delete the second dot]
6. 6. Lines 149, 428: “as it was mentioned before” - as mentioned before
7. Lines 163, 180-181, 332 – starting a phrase with “it is/was suggested that” is not a error, but it makes the phrase heavy and entangled. Please consider rephrasing to simplify the style.
8. Line 201: “except for” – besides, or in addition to
9. 9. Lines 193, 243, 273, 312, 332, 414, 578: “what is more” is not a error, but rarely used in scientific texts. Please consider replacing with “moreover”, or “in addition” [at least, in some places].
10. Line 262: “it seems that NF-κB pathways might be” – either “it seems that NF-κB pathways are”, or “NF-κB pathways might be” [“might be” means the same as “it seems that”]
11. Line 393: “is often accompanied by the presence of oxidative stress” - is often accompanied by oxidative stress
12. Line 405: “The grooving body of evidence” – the growing body of evidence [although it’s really grooving for somebody!]
13. Line 518: “infecting the mother or simply activating her immune system” - infecting the maternal animal or simply activating its immune system [“her” is relevant to humans only]

 

The other, minor concern is the review design and the set of facts cited.

1. a) the review is focused at inflammation and MIA. So, are clauses 4 (The role of oxidative stress in autism spectrum disorders), and 5 (Mitochondria dysfunction in MIA-evoked autism spectrum disorders) really pertinent? Of course, oxidative stress and mitochondria dysfunction are associated with the immune response, but there are many other associated areas, such as infections, vaccines and so on. Please, consider exclusion (at your option). In my opinion, they seem excessive.
2. b) Lines 450-451: really 5%? The reference 133 reports ~4% in the abstract (I read the abstract only). To my knowledge, the fraction is 0.5% - 1%, but I I concede that my knowledge could be outdated. Anyway, the statement of 30-80% ASD patients showing different signs of mitochondrial dysfunction looks really too loud. I’d like to recommend rephrasing in a way that 30-80% show different signs that could (or could not) be associated with mitochondrial dysfunction.
3. c) Line 610: “on 50 children for 26-week. A total of 40 children completed the protocol” – how many children did participate, 50 or 40? The abstract reports 40.
4. d) Line 536, Table 2: the flavonoids are more antioxidants than anti-inflammatory agents, and N-acetylcysteine is purely an antioxidant, suppressing the inflammation through its antioxidantive action. Please consider the relevance.

 

My summarising opinion is that the review can be accepted only after full correcting the above-stated and other similar defects.

Author Response

Response to Reviewer 2

 

Dear Reviewer,

 

Thank you very much for your comprehensive review of our manuscript. We corrected the manuscript according to your suggestions. Please, find below our detail responses to your comments.

 

Best regards,

Magdalena Cieślik and Agata Adamczyk

 

REVIEWER COMMENTS:

The manuscript is prepared in a logically consistent orders and contains the large enough amount of recent data on the subject. However, the text is, unfortunately, abundant in stylistic, typing and grammar errors and needs total proofreading. This is the main concern.

Below I tried to cite some errors in the orders they stay in the text, but the list is not limited to the items stated below – the whole text requires careful rewriting by a professional translator and proof-reader.

RESPONSE: Thank you for pointing out our mistakes. We carefully corrected whole manuscript in line with Reviewers' comments, performed additional extensive English editing used a professional English MDPI editing service.

REVIEWER COMMENTS:

The other, minor concern is the review design and the set of facts cited.

• a) the review is focused at inflammation and MIA. So, are clauses 4 (The role of oxidative stress in autism spectrum disorders), and 5 (Mitochondria dysfunction in MIA-evoked autism spectrum disorders) really pertinent? Of course, oxidative stress and mitochondria dysfunction are associated with the immune response, but there are many other associated areas, such as infections, vaccines and so on. Please, consider exclusion (at your option). In my opinion, they seem excessive.

RESPONSE: Thank you for this suggestion. While an association between inflammation and ASD has been well-established, the underlying mechanisms by which inflammatory processes may eventually give rise to autistic symptoms are not fully understood.

In this manuscript we propose that MIA-induced immune dysregulation/inflammation, oxidative stress and mitochondrial dysfunction in the brain of offspring as well as linkage between these abnormalities could be the primary molecular pathways downstream of maternal infection. We hypothesized that prenatal immune challenges and pro-inflammatory cytokines crossing the fetus BBB promote microglial cells to pro-inflammatory phenotype. Inflammatory process in the brain induces oxidative stress and mitochondrial dysfunction which, in turn, may exacerbate oxidative stress in a self-perpetuating vicious cycle leading to downstream abnormalities in brain development. Thus, we decided to not remove the chapter concerning the role of oxidative stress and mitochondria dysfunction in MIA-evoked autism. Of course, it is possible that other mechanisms may be involved in MIA-induced abnormalities in brain development. One possibility is that pro-inflammatory cytokines leads to long-lasting changes in the expression of other classes of immune molecules (including major histocompatibility complex I (MHCI) molecules) known to regulate synapse formation, synaptic plasticity and synaptic pruning as well as neural connectivity and function in the brains of offspring. It is also possible that immune signaling may converge upon mammalian target of rapamycin (mTOR) signaling which is altered in the brains of MIA offspring as well as individuals with ASD. Summarizing, while the molecular mechanisms of MIA as a ASD primer requires further research, there is a growing evidence that microglial activation, oxidative stress and mitochondrial dysfunction in the brain of offspring could mediate the neuropathology and abnormal autistic-like behaviors.

In view of your valuable comments, we have rewritten the abstract and conclusions to emphasize our hypothesis that MIA induces microglial activation, oxidative stress and mitochondrial dysfunction – a deleterious trio in the brain of offspring leading to neuroinflammation and neurodevelopmental pathologies.

2. b) Lines 450-451: really 5%? The reference 133 reports ~4% in the abstract (I read the abstract only). To my knowledge, the fraction is 0.5% - 1%, but I I concede that my knowledge could be outdated. Anyway, the statement of 30-80% ASD patients showing different signs of mitochondrial dysfunction looks really too loud. I’d like to recommend rephrasing in a way that 30-80% show different signs that could (or could not) be associated with mitochondrial dysfunction.

RESPONSE: Extensive research has been done regarding the ASD-mitochondria connections. New investigations are in progress and novel findings are being discovered. Indeed, the publication from last year we cited indicates that a subset of children with ASD (c.a. 4%) could be diagnosed with a definite mitochondrial disease. Also, previous publications give the same values (c.a. 4%), so we have revised this information in the manuscript as suggested by the Reviewer. However, previous research shows that up to 5% of children with autism have classical mitochondrial disease while 10–20% of patients with classic mitochondrial disease demonstrate ASD features. The coexistence of ASD with mitochondrial disease is higher than the prevalence of either ASD or mitochondrial disease in the general population (Griffiths and Levy, 2017). Consequently, we have mitigated the information regarding the occurrence of mitochondrial dysfunction in autism as Reviewer suggested, and in accordance with the latest literature we indicated that: “However, the latest research examining biomarkers of mitochondrial dysfunction suggest that abnormalities of mitochondrial function could affect a much higher number of children with ASD, perhaps up to 80%.”

3. c) Line 610: “on 50 children for 26-week. A total of 40 children completed the protocol” – how many children did participate, 50 or 40? The abstract reports 40.

RESPONSE: We have corrected this in the Table 2, 40 children participated in these investigations

• d) Line 536, Table 2: the flavonoids are more antioxidants than anti-inflammatory agents, and N-acetylcysteine is purely an antioxidant, suppressing the inflammation through its antioxidantive action. Please consider the relevance.

RESPONSE: Luteolin and quercetin are plant-derived flavonoids, that show a broad range of effects, including antioxidant, anti-inflammatory, anticancer, and neuroprotective properties (Tsilioni et al., 2015). Luteolin is capable of inhibiting pro-inflammatory cytokines expression, NF‐kB signaling, and TLR4 signaling, as well as weaken microglial activation (Nabavi et al., 2015). In series of trials, the flavonoid properties were analyzed in the context of ASD treatment. Therefore, we decided to add these compounds as a potential immunomodulatory therapy. It remains of course unclear whether any improvement associated with their use is driven by their proposed anti-inflammatory actions, either alone or in combination with other effects.

According to N-acetyl cysteine (NAC) – as you suggested we have decided to remove this compound from the Table 2. Though this precursor of glutathione has antioxidant as well as anti-inflammatory actions arising from inhibition of TNF-α, IL-1β and IL-6 (Palacio et al., 2011, Pinar et al., 2016).

 

We believe that all corrections increased the value of our article and our explanations you will find adequate.

 

REFERENCES:

 

Griffiths KK, Levy RJ. Evidence of Mitochondrial Dysfunction in Autism: Biochemical Links, Genetic-Based Associations, and Non-Energy-Related Mechanisms. Oxid Med Cell Longev. 2017;2017:4314025. doi: 10.1155/2017/4314025.

 

Nabavi SF, Braidy N, Gortzi O, Sobarzo-Sanchez E, Daglia M, Skalicka-Woźniak K, Nabavi SM. Luteolin as an anti-inflammatory and neuroprotective agent: A brief review. Brain Res Bull. 2015 Oct;119(Pt A):1-11. doi: 10.1016/j.brainresbull.2015.09.002.

 

Palacio JR, Markert UR, Martínez P. Anti-inflammatory properties of N-acetylcysteine on lipopolysaccharide-activated macrophages. Inflamm Res. 2011 Jul;60(7):695-704. doi: 10.1007/s00011-011-0323-8.

 

Pinar Karapinar S, Ulum YZ, Ozcelik B, Dogan Buzoglu H, Ceyhan D, Balci Peynircioglu B, Aksoy Y. The effect of N-acetylcysteine and calcium hydroxide on TNF-α and TGF-β1 in lipopolysaccharide-activated macrophages. Arch Oral Biol. 2016 Aug;68:48-54. doi: 10.1016/j.archoralbio.2016.03.017.

 

Tsilioni I, Taliou A, Francis K, Theoharides TC. Children with autism spectrum disorders, who improved with a luteolin-containing dietary formulation, show reduced serum levels of TNF and IL-6. Transl Psychiatry. 2015 Sep 29;5(9):e647. doi: 10.1038/tp.2015.142.

Author Response File: Author Response.docx