Introduction
Autism is a neurodevelopmental disorder characterized mainly by impaired social interactions, repetitive behaviors, and limited verbal communications [1]. It is also associated with an increased risk to a host of emotional and psychological conditions, including depression, anxiety, gender dysphoria, schizophrenia, catatonia, and psychosis [2]. While genetics plays a role in autism risk, environmental exposures are likely driving the epidemic. Environmental toxicants induce inflammation, oxidative stress, and mitochondrial dysfunction in the brain, leading to neuropathology [3]. Environmental risk factors include air pollution, nutritional deficiencies, pesticides, toxic metal exposures, infection, and brain trauma [3-8].
Early diagnosis of autism has presented a significant challenge to clinicians for decades [9]. It is generally recognized that autism diagnoses can be divided into two categories: children diagnosed before age two (early-onset autism) and those diagnosed after age two (regressive autism). There is some evidence that children with regressive autism exhibit behaviors prior to regression that are predictive of it [10,11]. That said, another study showed that nearly half of the cases of regressive autism have no such discernible behaviors – the child regresses after a previously normal developmental phase [12].
Through the main body of this paper, we will describe in detail the mechanistic links we believe exist between glyphosate exposure and the myriad pathological changes found in the brain and other systems of children with autism. We will forego a discussion of the early vs regressive autism until near the end, where we will show how our exposure model of autism etiology can shed light on the mystery around autism’s variable childhood age of onset.
The prevalence of autism has increased dramatically over the past two decades in the United States, strongly correlated with the dramatic rise in the use of glyphosate, the active ingredient in the herbicide Roundup, on core crops [13]. While correlation does not prove causation, there is now significant evidence that glyphosate causes neurodevelopmental problems, lending strength to the temporal correlations. In an epidemiological study, children who were born within 2000m of an agricultural source of glyphosate had a significantly increased risk to developing autism [14] While many other factors, both genetic and environmental, contribute to autism, we will show that glyphosate may be the most significant cause of the epidemic [13].
One of the known causes of autism is maternal immune activation (MIA) due to infection during pregnancy. Studies have shown that behavioral abnormalities in the offspring of rodents following MIA are characteristic of schizophrenia and autism [15]. Perturbations of the mother’s gut microbiota during pregnancy can induce autism-like behaviors in mice [16]. Maternal glyphosate exposure at low doses has been shown to cause MIA in pregnant mice, associated with autism-like behaviors in the offspring, through increased expression of soluble epoxide hydrolase (sEH) [17]. Inflammation in the brain upregulates the expression of sEH, an enzyme that oxidizes polyunsaturated fatty acids (PUFAs) [15]. It has been proposed that prophylactic treatment to suppress the expression of sEH could be a beneficial treatment option for autism [17].
The human brain accounts for only 2% of the body mass but consumes 20% of the oxygen. It also has high amounts of PUFAs that are susceptible to oxidative damage through lipid peroxidation, leading to mitochondrial dysfunction [18]. Children naturally have low levels of glutathione, and glutathione deficiency is a common feature associated with autism, along with increased oxidative stress in the brain [19]. Several oxidative stress markers, such as lipid peroxides, malondialdehyde (MDA), protein carbonyls and 3-nitrotyrosine, are elevated in association with autism, and correlated with autism severity [20].
A study comparing children with autism against normal controls found a significant block in cystathionine formation associated with an accumulation of homocysteine and low levels of the essential antioxidant, glutathione. A low level of urinary methionine and S-adenosyl methionine (SAMe) indicates deficiencies in methylation pathways. A significant number of the children who were studied showed deficiencies in three B vitamins: B6 (pyridoxine), B9 (folate), and B12 (cobalamin) [21]. The conversion of homocysteine to cysteine in the transsulfuration pathway depends on pyridoxine as a cofactor [22]. The synthesis of methionine from cysteine depends on both folate and methylcobalamin [23]. Cultured neurons are protected from glutamate-induced neurotoxicity by administration of methylcobalamin, likely through membrane alteration by SAMe [24].
An impaired sulfation pathway was first recognized as a risk factor in autism in a paper published by Waring and Klovrza al. in 2000 [25]. Cystathionine β-synthase converts homocysteine to cystathionine, and this is the rate-limiting step of the transsulfuration pathway, ultimately producing sulfate and phosphoadenosine phosphosulfate (PAPS), the universal sulfate donor. Both ascorbate (vitamin C) and retinoic acid (vitamin A) are cofactors in this pathway, as shown in detail by McCully in 2011 [26]. Both ascorbate and retinoic acid have been shown to have therapeutic value in treating autism. In a study published in 2018, vitamin A deficiency was found in 78% of autistic children, and vitamin A supplementation improved autistic symptoms [27]. In a genetic mouse model of autism, retinoic acid treatment rescued social deficits [28]. In a valproate-induced mouse model of autism, prenatal exposure to ascorbate attenuated the effects of valproate on the autistic behaviors of the pups [29]. A paper published by Alvarez-Moya et al. in 2022 showed conclusively that glyphosate is genotoxic to erythrocytes of salamanders and tilapia, as well as human lymphocytes. They also showed that supplementation with ascorbate and resveratrol (an antioxidant) reduced glyphosate’s genotoxic effects [30].
Children with autism commonly have sleep disorders and dysregulated circadian rhythms, suggesting disturbances in melatonin metabolism [31]. Melatonin is enzymatically degraded in the liver to 6-hydroxymelatonin and excreted in the urine as 6-sulfatoxymelatonin [32]. Several studies have shown that nocturnal levels of 6-sulfatoxymelatonin are significantly reduced in association with autism [33,34], likely due to impaired melatonin synthesis in the pineal gland. Melatonin is synthesized in the pineal gland from serotonin, through the addition of both an acetyl group and a methyl group. Serotonin is derived from the amino acid tryptophan, a product of the shikimate pathway in plants and microbes. Glyphosate’s primary mechanism of toxicity in plants is believed to be its suppression of the shikimate pathway [35,36]. Therefore, glyphosate likely interferes with tryptophan synthesis by the gut microbes. Mice with a null mutation in the enzyme tryptophan hydroxylase (and therefore severely deficient in brain serotonin) displayed multiple characteristic features of autism [37]. S-adenosyl methionine is the source of the methyl group, so its deficiency would likely also lead to a deficiency in melatonin [38]. Importantly, glyphosate exposure to rats prenatally and perinatally caused a 43% reduction in serum melatonin levels measured after the rat pups had matured, likely through epigenetic effects [39].
A remarkable postmortem study showed disturbingly low levels of various cobalamin conjugates in autistic brains [40]. The authors measured levels of five different cobalamin species: hydroxycobalamin, methylcobalamin, adenosylcobalamin, cyanocobalamin, and glutathionylcobalamin. All of the conjugated cobalamins were significantly reduced in association with autism, particularly glutathionylcobalamin, which could be due in part to a deficiency in glutathione. Glutathionylcobalamin is an intermediate in the formation of methylcobalamin [41]. Methylcobalamin injections increase production of melatonin by the rat pineal gland, indicating a dependency on methylcobalamin to catalyze melatonin synthesis [42].
Proline is one of the 20 coding amino acids, and it has unique properties that play a powerful role in biology. Proline is the only coding amino acid that exists in two different isomers (cis- and trans-), which contain the exact same molecular formula but with different arrangements in space. Interestingly, proline can spontaneously switch back and forth between cis- and trans- isomers when it is embedded in a peptide sequence, although this happens infrequently. There is a class of enzymes called peptidylprolyl isomerases (PPIases) that catalyze the switch, and, when active, they can increase the rate of flipping from a cis- to a trans- isoform of proline by a factor of 1,000 [43].
PPIase NIMA-Interacting 1 (PIN1) is a member of the class of PPIases, and it has diverse roles in human development and the response to cellular stressors. It acts as a molecular switch by inducing conformational changes in the affected protein by isomerizing prolines [44]. Prolyl isomerization is involved in many cellular processes, including apoptosis, mitosis, cell signaling, ion channel gating, amyloidogenesis and neurodegeneration [45]. PIN1 regulates the function of several powerful signaling proteins, including catalytic activity, phosphorylation status, protein interactions, subcellular location and protein stability [46,47]. It plays a central role in phosphorylation pathways, which regulate many aspects of cellular activities in response to stressors, including the DNA damage response, antioxidant defenses, and programmed death. It is also involved in the balance between excitatory and inhibitory neurotransmitter responses, and in the maturation of neurons during early life [48]. PIN1 is overexpressed in association with many cancers [49]. By contrast, it is under-expressed in association with many neurogenerative diseases [48].
PIN1 has a very specific function, which is to switch a proline residue preceded by serine or threonine from the cis- to the trans- isomer. It is only active when the preceding serine or threonine is phosphorylated. Furthermore, enzymes that dephosphorylate the preceding residue, particularly protein phosphatase 2A (PP2A), are only active when the proline is in the trans- state [50]. Thus, removal of the phosphate anion depends upon PIN1’s activity in maintaining the proline residue in the trans- configuration. This remarkably complex epigenetic effect has powerful influences on both the activity of the altered protein and its localization within the cell (e.g., nucleus or mitochondria). Protein phosphorylation is probably the most common post-translational modification in proteins, and 96% of the phosphorylations are applied to the (Ser/Thr)-Pro motif [51].
In this paper, we will develop the argument that autism can be characterized as a PIN1 deficiency syndrome. We will show that many of the neurodevelopmental defects and morphological features of autism are linked to PIN1 deficiency. We also argue that the disruptions in synaptic signaling linked to autism can be explained by PIN1 deficiency. Many of the genetic links to autism involve proteins that are regulated by PIN1. We provide substantial evidence from the research literature that glyphosate’s mechanisms of toxicity can be expected to suppress PIN1 activity, either directly through cysteine oxidation by reactive oxygen species induced by glyphosate, or via suppression of melatonin synthesis, resulting in DAPK1 overexpression. This effect can explain its link to the autism epidemic. Finally, we hypothesize that autistic children are likely to be highly sensitive to the mRNA vaccines, as many of the toxic effects of the vaccine induce metabolic disruptions that are already ongoing in the autistic brain.