Abstract.

The GAD1 gene produces an enzyme called glutamate decarboxylase (GAD67), which has an important function in the GABAergic systems in the brain (Chatron, et al., 2020)5. GAD67 is vital in maintaining the balance of glutamate (an excitatory) and GABA (an inhibitory) for stimulation processing and GABAergic system function in the brain (Jewett, Sandeep, 2023)15. Recent studies have suggested that individuals diagnosed Autism Spectrum Disorder (ASD), as well as, individuals with schizophrenia and/or epilepsy, have an imbalance in the excitatory and inhibitory ratio in GABAergic systems relating to response to stimulation (Zhao, et al., 2022)43. Further research in 2022, has shown that ASD individuals have abnormal patterns of methylations on the GAD1 gene, when compared to a control group (individuals without ASD) of comparable age and sex (Pearson, et al., 2022)26. It can be theorized that due to the abnormal methylation patterns on this gene, dysfunction or deformation of the GAD67 enzyme could be the cause of the often low levels of GABA and increased glutamate presence in the brains of ASD individuals (Puts, et al., 2017)32, thus, leading to the often overly high sensitivity to stimulation in people with ASD. This though, begs the question, what causes the abnormal methylation patterns? Research from 2012 suggests that exposure in utero to gram negative bacteria, like Escherichia coli (E. coli) and salmonella, containing lipopolysaccharide (LPS) endotoxin causes a drop in glutamate decarboxylase and reelin production (Nouel, et al., 2012)24. Multiple research studies have found that lipopolysaccharide endotoxin exposure alters mitochondrial DNA (mtDNA) methylation patterns and a variety of other methylation patterns on DNA sequences throughout different cell types and species, including human blood cells (Koos, et al., 2020)21, pulmonary artery endothelial cells in humans (Elangovan, et al., 2016)8, as well as, endometrial epithelial cells in bovine species (Jhamat, et al., 2020)16. Given that it is known that LPS endotoxin exposure in utero causes dysfunction in the production of GAD67, we hypothesize that due to exposure of LPS endotoxin, cellular immune responses cause increased production of DNA methyltransferase 1 (DNMT1)(Koos, et al., 2020)21, which directly causes the abnormal methylation patterns found on the GAD1 gene of ASD individuals. This in turn causes a cascade of neuropharmacological events an in E/I imbalance, resulting in either hyper-sensitivity or hypo-sensitivity to stimuli, which is often reported in Autism Spectrum Disorder.

Further research should be conducted on the direct effects of LPS endotoxin exposure in utero on DNA methylation profiles of the GAD1 gene and its promoter regions, as well as, the specific concentration of GABA in multiple regions of the brain in ASD individuals with altered methylation patterns on said gene. It is worth looking into a history of general food poisoning in the mothers during pregnancy with these individuals. It, of course, would not be entirely conclusive that food poisoning would be directly caused by a gram negative bacteria exposure, as food poisoning could come from multiple sources, but it does provide some insight towards possible correlation to LPS endotoxin immune response.

We also suggest that further research be conducted on the efficacy of supplementation of exogenous compounds used in the production of endogenous GABA, such as vitamin B6 (pyridoxine) in conjunction with the amino acid, L-theanine on potential alleviation of ASD symptoms. This is with the hopes of providing more qualitative data to support the hypothesis that ASD symptoms are predominantly caused by or affected by glutamate and GABA imbalance.

Introduction.

The burden of Autism Spectrum Disorder weighs heavy on parents, caretakers, teachers and the diagnosed individuals themselves. In the United States alone, it is estimated that the overall Autism Spectrum Disorder care cost the Country around $268 billion in 2015 and will cost upwards of $461 billion per year by the year, 2025 (Leigh, Du, 2015)22, which is around 1.6% of the total GDP projection for that year. Improvement in the treatment of Autism Spectrum Disorder is imperative. The goal of this research is to get closer to finding the genetic or epigenetic root cause of GABAergic system dysfunction, mainly focusing on the production of endogenous GABA via the conversion of glutamate.

In the case of Autism Spectrum Disorder, established research shows that an imbalance of glutamate and GABA in the GABAergic systems of the brain causes hypersensitivity and hyper-reactivity to stimuli (Zhao, et al., 2022)43. This leads to the question, what causes this imbalance?

Epigenetics is the study of changes in gene expression or control, without alteration to the DNA sequence itself. In many cases, the epigenetic alterations to gene expression are caused by methylations or 'marks' (of methyl groups) on the DNA, which changes how DNA polymerase reads the DNA during transcription (Phillips, 2008)28. Based on the methylations patterns, a protein may be expressed entirely, or may be deformed, if a methylation is the wrong spot, causing the protein to consist of the incorrect amino acids. The prefix - 'epi' in Greek, means: above or over. Epigenetics generally supersedes what we understood as traditional genetics.

Disclaimer: The next 2 paragraphs are personal anecdotes.

"From 2021 to present (2023), I've worked at an out of district, severe special needs school as a paraprofessional and a crisis prevention/intervention team member, as well as, worked as a behavioral therapist directly with the parents of ASD individuals. It is very apparent the burden that this disorder places on the caretakers, their families and their peers. It is not to say that ASD individuals are bad people, but rather that they need extensive care, and conditioning via social interactions to function successfully in a neurotypical world due to their disorder. This burden weighs heavy on my heart, even though I do not have a connection by blood to any of these children or adults. This research is extremely important to me, as I have spent extensive amounts of time around ASD individuals during my childhood as well. My neighbor whose front door was 5 steps from my childhood apartment door, suffered from Asperger's Syndrome and severe ADHD. The child which I spent 90% of my time working with at the school suffered from Asperger's syndrome and ADHD. My cousin through marriage also suffers from Asperger's Syndrome and severe ADHD. It's safe to say I understand the detriment that this disorder places on their lives. I have personal investment in genuine treatment of this disorder.

The first time I was exposed to the benefits of exogenous supplementation to potentially increase endogenous production of GABA was via my cousin. My cousin was given 200mg of the amino acid, L-theanine in capsule form, from age 13 to age 16. His behavior and excitability improved dramatically. He went from struggling to communicate and struggling to function in a public school setting, to becoming almost undetectable as an ASD diagnosed individual. He runs track and does well in school. I cannot pin all of the success on L-theanine supplementation, because he also received behavioral and social therapy throughout the years, along with a stringent diet, designed by nutritionists to improve gut microbiome health. It is important to consider all factors when treating any disorder, not just one. I do believe it is important that we continue to look into other causes of ASD, but this experience is what led me down the long path of researching GABAergic system dysfunction and its role in ASD symptoms."

- Christian Teplitzky-Purvis

GABA and it's role in stimulation.

The GABAergic systems of the brain function primarily to regulate overall brain activity related to stimulation by controlling the firing of neurons through an inhibitory neurotransmitter called GABA. The presynaptic neuron in this system releases GABA which binds to specific receptors in the postsynaptic neuron (Jewett, Sandeep, 2023)15. These receptors have different functions. GABAa receptors control the entry of negatively charged chloride ions into the neuron, reducing its action potential or the possibility of producing a nervous effect in response to stimuli. GABAb receptors control the release of potassium ions from the neuron, again reducing its action potential or the possibility of producing a nervous effect in response to stimuli. When a neuron is positively charged, it is more likely that it will produce a nervous effect to respond to stimuli and when it is negatively charged, it is more likely that it will not produce a nervous effect in response to stimuli (Jewett, Sandeep, 2023.; Allen, Sabir, Sharma, 2023.)15, 1. In contrast, glutamate produces the inverse effect, increasing the likelihood of action potential, causing increase response to stimuli (Allen, Sabir, Sharma, 2023)1.

In layman's terms the more positively charged the neuron, the more likely it is to fire and cause a nervous effect in response to stimuli. The more negatively charged the neuron is, the less likely it is to fire and cause a nervous effect in response to stimuli. GABA acts as an inhibitory and decreases the excitability of neurons, by allowing negatively charged chloride ions to flow into them and positively charged potassium ions to flow out of them. On the other hand, glutamate acts as an excitatory and allows for positively charged ions like sodium and calcium to flow into the neuron.

The balance of excitatory (glutamate) and inhibitory (GABA) neurotransmitters in crucial in maintaining homeostasis in the stimuli response centers of the brain. This could potentially be a cause for the recorded salt cravings in children diagnosed with Autism Spectrum Disorders (Good, 2011)11.

In relation to Autism Spectrum Disorder, it is known via past research that ASD diagnosed individuals tend to possess low levels of GABA and have an increased presence of glutamate in GABAergic systems. Increased cortical excitability has been shown in ASD diagnosed individuals, which have been tested to repetitive behaviors in comparison to a control group of neurotypical individuals (Puts, et al., 2017)32. On the side, a different study noted the opposite observation, that in individuals with reduced glutamate levels in the striatum exhibited deficiencies in social skills generally associated with ASD (Horder, et al., 2018)13. Considering the fact that imbalances in the direction of increased inhibitory presence causes dysfunction in response to stimuli concurrent with ASD symptoms, just like the inverse polarization via imbalance in the direction of increased excitatory presence, the homeostasis and balance of excitatory and inhibitory neurotransmitters in GABAergic systems is crucial to response to stimuli in neurotypical function (Sarawagi, Soni, Patel, 2021)34.

Thus, it is important to question why many individuals diagnosed with Autism have imbalances in GABA and glutamate, with a general trend towards increased glutamate levels and decreased GABA levels.

Endogenous GABA production & GAD67.

GABA, the inhibitory neurotransmitter involved in depolarization of neurons in GABAergic systems is created endogenously from conversion of glutamate (Jewett, Sandeep, 2023.; Allen, Sabir, Sharma, 2023.)15, 1. The process which converts glutamate into GABA involves a reaction catalyzed by the enzyme: Glutamate Decarboxylase (GAD1). GAD1 uses pyridoxine, commonly referred to as vitamin B6 to synthesize GABA from glutamate (Chatron, et al., 2020; Burd, et al., 2000)5, 4.

The key enzyme in this process, glutamate decarboxylase is found on a gene called GAD1, found on chromosome 2 in the human genome. Studies have also shown that GABA is also important outside of simple inhibitory function. GABA has been suggested to help with in utero synaptogenesis and neuronal development (Nouel, et al., 2012)24.

Many anecdotal accounts have shown that probiotic supplementation for ASD individuals ameliorates some of the symptoms. Unsurprisingly, recent research now shows that Bifidobacterium dentium and multiple species of lactic acid bacteria, commonly found in probiotic supplements, either produce GABA and/or stimulate GABA production depending on the species (Pokusaeva, et al., 2017; Yogeswara, Maneerat, Haltrich, 2020)30, 41. In rat models, it has been shown that the administration of these types of Bifidobacterium, alleviates visceral hypersensitivity in the gut, potentially reducing chronic or recurring abdominal pain due to GI disorders like irritable bowel disease (Pokusaeva, et al., 2017)30, which is a common occurrence among individuals with autism. Ironically, these bacteria are all gram positive bacteria  (Mokoena, 2017)23, unlike the gram negative bacteria, which we will elaborate on, that are theorized to cause disruptions in glutamate decarboxylase production.

It is clear that normal production and function of GAD67 is crucial neurotypical brain function and development, hence, why it is a key focus of this research and of many others in the search for the cause of ASD symptoms.

Epigenetics and Methylation.

As stated in the introduction, epigenetics is the study of changes in gene expression or control, without disruption to the physical DNA sequence. This is unlike mutation, which does cause a change in the physical sequence of the DNA, which in many cases can cause the incorrect amino acid to be translated via the mutated mRNA, potentially causing deformation of produced proteins.

In epigenetics there are multiple different factors which can contribute to modification of gene translation, including but not limited to: non-coding RNAs, like siRNA, miRNA and piRNA, etc., histone modification and most importantly DNA methylation, all of which are commonly referred to as epigenetic marks. These all modify the expression of proteins without changing the structure of the DNA itself.

The focus on this research is mainly centered around DNA methylations and their modification of the GAD1 gene.

Methylations are added methyl groups on the DNA, usually in the promoter region of gene sequences which code for specific proteins. When DNA is methylated, it generally causes close-by histones to become de-acetylated. When a histone becomes de-acetylated, the chromatin (strands containing DNA and proteins), wraps tighter around the histone protein, preventing RNA polymerase III from transcribing and copying information from further regions of the DNA. In other words, methylations prevent certain gene sequences from being transcribed and translated, without altering the DNA itself (Phillips, 2008)28.

Different gene sequences have different methylation patterns, which allow for only specific sections of the DNA to be transcribed into mRNA for protein synthesis. This is important for ensuring that the correct amino acids are produced to form a functioning protein.

Conclusion

Considering that exposure to the LPS endotoxin in utero causes a decreased concentration of GAD67 in the hippocampus of effected individuals (Nouel, et al., 2012)24, and the tendency of cells to produce increased amounts of DNA methyltransferase in response to exposure to the LPS endotoxin, causing methylation pattern changes in other cell types (Koos, et al., 2020; Elangovan, et al., 2016; Jhamat, et al., 2020)21, 8, 16, we hypothesize that the variance in methylation patterns on the GAD1 gene of individuals diagnosed with Autism Spectrum Disorder is specifically due to prenatal exposure to lipopolysaccharide endotoxin.

Future Research & Recommendations

Further research should be conducted on the direct effects of LPS endotoxin exposure in utero on DNA methylation profiles of the GAD1 gene and its promoter regions, as well as, the specific concentration of GABA in multiple regions of the brain in ASD individuals with altered methylation patterns on said gene. Future studies should collective quantitative data in relation to the E/I imbalance of glutamate/GABA in people

It is worth examining the history of general food poisoning in the mothers during pregnancy with peoples diagnosed with autism, schizophrenia and/or epilepsy. It, of course, would not be entirely conclusive that food poisoning would be directly caused by a gram negative bacteria exposure, as food poisoning could come from multiple sources, but it does provide some insight towards possible correlation to LPS endotoxin immune response.

We also suggest that further research be conducted on the efficacy of supplementation of exogenous compounds used in the production of endogenous GABA, such as vitamin B6 (pyridoxine) in conjunction with the amino acid, L-theanine (which prevents l-glutamic acid from binding to glutamate receptors and increasing the endogenous production of GABA) (Ota, et al., 2015)25 on potential alleviation of ASD symptoms, which has shown promise for some form of amelioration of symptoms (Rizzo, et al., 2022; Kimura, et al., 2007; Ota, et al., 2015; Hannant, et al., 2021)33, 20, 25, 12. This is with the hopes of providing more qualitative data to support the hypothesis that ASD symptoms are predominantly caused by or affected by glutamate and GABA imbalance.

Conflicts of Interest and Funding

There were no financial conflicts of interests involved in this review. This review was entirely internally funded.

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Final Statement

Unironically, spinach is high in vitamin b6, which is required to synthesize GABA, so it can be inferred that eating high amounts of spinach would not cause autism, but rather could possibly increase endogenous GABA production. This website was named 'spinachcausesautism.com' to intentionally attract more attention. One could say it is "clickbait"... and they wouldn't be incorrect. It was designed as "clickbait" so that it could hopefully drive more traffic to the study, and free resources which could potentially provide immense benefit to future researchers, parents, caretakers and individuals themselves who are diagnosed with GABAergic system dysfunction related disorders. This entire project and study is out of good will, to hopefully do a good deed.

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