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.