![Modified from Shen [12]: Illustration of MRI pattern from child (left) not destined to develop autism compared to same age child (right) destined to develop autism. Note increased brain size and CSF accumulation on the right side image.](https://www.researchgate.net/profile/Stephanie-Seneff/publication/291391622/figure/fig1/AS:346143788683264@1459538518158/Modified-from-Shen-12-Illustration-of-MRI-pattern-from-child-left-not-destined-to_Q320.jpg)
![Modified from Suzuki [13]: Illustration of MRI image pattern of control brain (left), and autism brain (right); both have same pattern of activated microglia, but degree varies, increased in autism brain.](https://www.researchgate.net/profile/Stephanie-Seneff/publication/291391622/figure/fig2/AS:346143788683265@1459538518179/Modified-from-Suzuki-13-Illustration-of-MRI-image-pattern-of-control-brain-left-and_Q320.jpg)
![Modified from Tang [18]: Normal neuronal axon illustrated on left – autism neuronal axon on right with extra spines, not well 'pruned' .](https://www.researchgate.net/profile/Stephanie-Seneff/publication/291391622/figure/fig3/AS:346143788683266@1459538518201/Modified-from-Tang-18-Normal-neuronal-axon-illustrated-on-left-autism-neuronal-axon_Q320.jpg)
![From Swanson et al. [32], used by permission: Near exact match of tons of glyphosate applied to corn/soy versus number of children with autism as served under IDEA (US Department of Education, Individuals with Disabilities Education Act).](https://www.researchgate.net/profile/Stephanie-Seneff/publication/291391622/figure/fig5/AS:346143788683268@1459538518251/From-Swanson-et-al-32-used-by-permission-Near-exact-match-of-tons-of-glyphosate_Q320.jpg)
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The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic -
A Review of Evidence from the Literature with Analysis
Beecham JE and Stephanie Seneff *
Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge MA 02139, USA
*Corresponding author: Stephanie Seneff, Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge MA 02139, USA,
E-mail: sene@csail.mit.edu
Received date: August 26, 2015, Accepted date: October 06, 2015, Published date: October 13, 2015
Copyright: © 2015 Beecham JE, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
The causes of autism spectrum disorder (ASD) are not well understood. Only a minority of cases are explainable
by specific abnormalities in DNA sequence, whereas the majority are widely assumed to be linked to epigenetic
effects, and/or likely impacted by environmental factors. Here, we postulate autism causation via environmental
and/or dietary sourced toxin acting intermittently in utero on human fetuses to disrupt neurodevelopment in a non-
dose dependent manner. Our theory is informed by a mini-review and correlation of selected studies from the
research literature related to autism, including radiologic, anatomic, metabolic, neurodevelopmental, pharmacologic
and MRI studies. In reviewing and analyzing evidence, we focus on data supporting interaction of the theorized
harmful glycine mimetic at one or more of the following calcium inflow regulatory factors for neurons: the N-methyl
D-aspartate (NMDA) receptor, the glycine receptor (GlyR) and/or the glycine transporter protein 1 (GlyT1).
We postulate this harmful glycine mimetic to act by exerting a direct molecular disruption to calcium regulatory
factors for neurons. This disruption appears to occur in a time sensitive, rather than a strictly dose-dependent
manner, leading to haphazard disorganizations of the normally carefully choreographed steps of early neuronal
migration. Within this analysis, we find support for the contention that a strong candidate for the putative harmful
glycine mimetic is glyphosate, the active ingredient in the pervasive herbicide Roundup®. In addition to glyphosate’s
molecular similarity to glycine, glyphosate is known to have a propensity to avidly bind minerals such as manganese
and magnesium, which minerals are implicated in the normal functioning of several neuronal calcium inflow
regulatory factors. Our theory highlights areas deserving of further study.
Introduction
Since 1980, the number of children known to have autism spectrum
disorder (ASD) has increased dramatically. However this increase is
thought to be at least partly due to changes in diagnostic practices [1].
e risk of autism is known to be associated with advanced paternal
age and with diabetes in the mother during pregnancy [2]. ere is a
well-known male preponderance in ASD cases [1]. Although autism is
believed, by many experts, to be caused by inherited factors, no single
specic DNA sequence alteration has been found to explain more than
a minority of the cases.
A recent report by Yuen et al. [3] has raised questions about the
subject of heritable gene defects in regard to autism causation. Because
autism oen runs in families, experts had assumed that siblings with
the disorder were inheriting the same autism-predisposing genes from
their parents. It now appears this may not be so. ese researchers
sequenced, from 85 families each having two children aected by
autism, 340 whole genomes, including from 170 individuals with ASD.
ey found that the majority of autism sibling pairs (69 percent) had
little to no overlap in abnormal genes. Sibling pairs shared the same
autism-associated gene changes less than one third of the time (31
percent). e majority of autism-aected sibling pairs (69 percent) had
little to no overlap in the gene variations thought to contribute to
autism.
is raises the question whether children are developing autism
from an environmental or dietary toxin for which the DNA alterations
signal presence of the toxin, but wherein the genetic changes are not
conferring all the autism-causing action. e possibility exists that the
putative toxin is also, or perhaps principally, causing damage by
interacting directly within the neurodevelopmental molecular
structures and pathways of the developing brain. A candidate
molecule, as will be discussed in more detail later, is glyphosate. It is
noteworthy that, in a study by Koller et al. [4], human buccal epithelial
cells from cell line TR146 were exposed to glyphosate alone in one test,
and separately in another test using only the glyphosate-containing
formulation known as Roundup®. Each test was at concentrations
representing a 450-fold dilution of concentrations used in spraying
crops in agriculture. Separately, in each of the two test exposures, DNA
damage was found to occur in the exposed epithelial cells.
Experts, when ascribing causation in autism, oen invoke
epigenetic changes, citing unspecied reactions, or perhaps
environmental inuences [5]. Such epigenetic changes, while not
modifying the DNA sequence code, are nevertheless thought to be
heritable and causative, aecting early/fetal neurodevelopment [6].
Environmental causes thus have not been ruled out as causative or
contributory in autism [7], and exposure to air pollution, especially
particulates and heavy metals, are acknowledged to perhaps increase
the risk of autism [8]. Furthermore, some cases of autism have been
Molecular and Genetic Medicine Beecham and Seneff, J Mol Genet Med 2015, 9:4
http://dx.doi.org/10.4172/1747-0862.1000187
Review Article Open Access
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
strongly associated with agents that cause birth defects [9], and these
teratogens are widely believed to have their greatest eect within the
rst 8 weeks from conception.
Rzhetsky et al. [10] reported in 2014 on the results of their analysis
of geographic spatial incidence patterns of ASD and Intellectual
Disability (ID) as reected in insurance claims datasets representing
nearly 1/3 of the entire US population. ey used “the rate of
congenital malformations of the reproductive system as a surrogate for
environmental exposure of parents to unmeasured developmental risk
factors, including toxins” because “70 to 80% of male congenital
malformations of the reproductive system have no clear genetic causes.
Instead, they appear to be driven by specic environmental insults…”
ey found that, “Adjusted for gender, ethnic, socioeconomic, and
geopolitical factors, the ASD incidence rates were strongly linked to
population-normalized rates of congenital malformations of the
reproductive system in males (an increase in ASD incidence by 283%
for every percent increase in incidence of malformations, 95% CI:
[91%, 576%], p<6×10-5)”. eir conclusions state that, in attempting to
ascribe causation in cases of autism, in addition to evaluating possible
chromosomal DNA sequence alterations, “detailed documentation of
environmental factors should be recorded and used… and failure to do
so risks omitting important information.”
To clarify theories of causation in autism, a review of selected
studies was conducted and correlated, as described below.
Selected Magnetic Resonance Imaging (MRI) Studies
Related to Autism
Haari et al. [11] reviewed structural MRI scans from 539 high
functioning adults with autism and compared the images to structural
MRI scans from 573 controls. ese researchers concluded there was
no evidence within their MRI images for signicant between-group
dierences in any measures of gross anatomy or in specic brain
regions, including several which had previously been implicated in
anatomic studies of autism spectrum disorder (ASD), including the
amygdala, hippocampus, most segments of the corpus callosum and
the cerebellum.
Figure 1: Modied from Shen [12]: Illustration of MRI pattern from
child (le) not destined to develop autism compared to same age
child (right) destined to develop autism. Note increased brain size
and CSF accumulation on the right side image.
By contrast, an MRI study by Shen et al. [12] reported it was
possible to fairly reliably separate the MRI brain images of children
who will go on to develop autism from controls (Figure 1). Fiy-ve
infants (33 'high-risk' infants having an older sibling with autism
spectrum disorder and 22 'low-risk' infants having no relatives with
autism spectrum disorder) were imaged at 6-9 months; 43 of these (27
high-risk and 16 low-risk) were imaged at 12-15 months; and 42 (26
high-risk and 16 low-risk) were imaged again at 18-24 months. e
children were followed to determine which developed autism. e
presence in the scans of increased brain volume and increased
cerebrospinal uid (CSF) accumulation was reported as predictive of
subsequent development of autism, according to the researchers.
Glial Cells in Autism
Background: e central nervous system is comprised of neurons
and three basic types of glial cells: microglia, astrocytes, and
oligodendrocytes. One primary function of glial cells is to support,
protect, and nurture the neurons, a process that is essential for neural
plasticity and stability. Microglia migrate to the central nervous system
(CNS) during prenatal development and are the resident immune cells
in both the brain and spinal cord. Microglia maintain vigilance for any
type of toxic challenge to the CNS, including injury, infection, and
ischemia. ey respond with activation, into a pro-inammatory state
for example, in response to neuronal cell injury or death.
MRI study of pattern of glial cell activation in autism
Suzuki et al. [13] studied microglial activation using MRI images in
a case-control study design with positron emission tomography and a
radiotracer for microglia; [11C](R)-(1-[2-chrorophynyl]-N-methyl-N-
[1-methylpropyl]-3 isoquinoline carboxamide) ([11C](R)-PK11195)
(Figure 2). ey recruited study participants from the area of
Hamamatsu, Japan. eir study population consisted of Japanese
citizens, twenty men with ASD (age range, 18-31 years; mean [SD] IQ,
95.9 [16.7]) and 20 age- and IQ-matched healthy men as controls.
Figure 2: Modied from Suzuki [13]: Illustration of MRI image
pattern of control brain (le), and autism brain (right); both have
same pattern of activated microglia, but degree varies, increased in
autism brain.
Diagnosis of autism spectrum disorder (ASD) was made in
accordance with the Autism Diagnostic Observation Schedule and the
Autism Diagnostic Interview-Revised. e results focused on the
amount of radiotracer activity discovered in various regions of the test
subject brains as a representative measure of microglial activation in
Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence
from the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187
Page 2 of 16
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
that subject. To their surprise, the pattern of brain region binding
potential was essentially the same in the brains of control subjects as in
the brains of autistic subjects; only the degree varied. In other words,
in the autistic subjects the binding potential was higher, while
maintaining the same general pattern of glial activation within brain
regions as the age and IQ-matched healthy control subjects from the
same area in Japan.
In Japan, glyphosate herbicides are widely used, including for weed
control [14]. In addition, the country of Japan imports wheat from
countries where glyphosate is sprayed onto wheat as a desiccant. Japan
also imports rapeseed, for use as animal feed, from countries spraying
glyphosate onto rapeseed as a desiccant. In the context of the theory
herein advanced, these ndings could t the model of an
environmental/dietary toxin.
Selected Microscopic Studies in Autistic Human Brains
Stoner et al. [15] examined post-mortem brain tissue from 22
children who died between the ages of 2 and 15. Half of the children
studied had been diagnosed with autism, while the other half had not.
e symptoms of those with autism varied from mild to severe. In 10
of the 11 autistic brains, they found patches of cortex in which the
normal pattern of gene expression and cell organization was disrupted.
ese areas were only a few millimeters across (roughly a quarter to a
half inch in size) across the wide expanse of otherwise histologically
normal-appearing cortex.
In some of the abnormal patches, a specic layer was missing. In
other patches, certain expected cells weren’t present. Similar changes
were found in 1 of the 11 children without an autism diagnosis, a child
who had suered from seizures. All areas of cortex sampled from
autistics demonstrated such patches, however the researchers indicated
that the most aected areas of the brain were the prefrontal cortex and
the temporal lobe cortex, while areas such as the optical cortex were
relatively spared. e researchers speculated that the defects could
have resulted from the brain cells in autistics undergoing some sort of
disorganization event or events during the latter part of the rst
trimester or the early part of the second trimester of fetal development.
is study by Stoner et al. le the researchers speculating about
causation of the observed disordered patches. ey mentioned that
their earlier studies had demonstrated that, between the ages of 2 and
16 years, brains from autistic children are heavier than non-autistic
children brains and have a relative increase in prefrontal cortex neuron
numbers of up to a startling 67%. Stoner et al. also discovered that a
decit of markers of excitatory neurons was the most robust indicator
of such a patch of disorganization. Furthermore, they did not believe
this was a result of downregulation of genes. Rather, they speculated
that the patchy areas of disorganization somehow resulted from
neurons ‘failing to reach their intended destination’ or from de novo
changes in early neurodevelopment.
Lopez-Hurtado et al. [16] conducted a microscopic study of post-
mortem brains from 15 age-matched autistic and control subjects
focusing on brain regions associated with the production and
processing of speech. Of the brains studied, 8 were from autism
patients and 7 were controls. ey studied Wernicke’s area (Brodmann
22, speech recognition), Broca’s area (Brodmann 44, speech
production) and the gyrus angularis (Brodmann 39, reading) from
autistic subjects (7-44 years of age) and control subjects (8-56 years of
age). ese researchers found: “Striking dierences in the density of
glial cells, the density of neurons and the number of lipofuscin-
containing neurons in the autistic group compared with the control
group. e mean density of glial cells was greater in the autistic cohort
than controls in area 22 (p<0.001), area 39 (p<0.01) and area 44
(p<0.05). e density of neurons was lesser in autism in area 22
(p<0.01) and area 39 (p<0.01). e autistic group exhibited
signicantly greater numbers of lipofuscin-containing cells in area 22
(p<0.001) and area 39 (p<0.01)”. ey concluded that “the results are
consistent with accelerated neuronal death in association with gliosis
and lipofuscin accumulation in autism aer age seven [17].”
Selected Studies of Neuron Structure in Autism, Link to
Calcium-induced Reduction in Autophagy via mTOR
Hyperactivation
Tang et al. [18] were able to discern dierences in post-mortem
brain samples from children with autism who had died from other
causes (Figure 3). irteen brains came from children ages 2 to 9, and
thirteen brains came from children ages 13 to 20. e controls were
post-mortem brains from children without autism. Tang et al.
measured synapse density in a small section of tissue in each brain,
counting the number of tiny spines that branch from the cortical
neurons. In the control brains, spine density had dropped by about half
during childhood, but by comparison in the autistic brains the density
had dropped by only 16 percent during childhood.
Figure 3: Modied from Tang [18]: Normal neuronal axon
illustrated on le – autism neuronal axon on right with extra spines,
not well ‘pruned’.
Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence
from the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187
Page 3 of 16
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
e researchers then examined brains from mice, comparing
autism-model mouse neurons versus non-autism normal mouse
neurons. ey studied Tsc2+/- ASD mice brains. ese mice have
alteration in the mammalian target of rapamycin (mTOR) such that
the mTOR is constitutively overactive. mTOR is a serine/threonine
kinase, which belongs to the phosphatidylinositol-3 kinase (PI3K)
related kinase (PIKKs) family. It regulates cellular metabolism, growth,
and proliferation. Tang et al.’s study of such autism-model mice with
constitutively hyperactivated mTOR found signs of reduced autophagy.
Autophagy is the process in neurons that, among other things, prunes
neurons of excess spikes. Tang et al. [18] found that the autism-model
mice, similar to the autistic children, had too numerous spikes on
neurons. ey deduced that this excess presence of spikes was due to a
lack of adequate pruning, and postulated this was from a reduction or
blockade of normal autophagy. ese Tsc2+/- mice demonstrated
ASD-like social behaviors.
Aberrant mTOR activation resulting in reduced autophagy is known
to occur in human diseases on a genetic basis. For example, mutations
in either of 2 genes (TSC1 or TSC2) have been determined to cause
tuberous sclerosis complex. Among tuberous sclerosis patients, 20% to
60% are also diagnosed with autism [19,20]. It has been reported that,
of all autism cases, approximately 1% to 4% are tuberous sclerosis
patients [21]. Treatment of tuberous sclerosis patients with rapamycin
can apparently partially correct their inherited defect of reduced
autophagy.
e benecial eect of rapamycin is thought to occur via
rapamycin’s action to inhibit hyperactivated mTOR and thus induce an
increase in autophagy [22]. Rapamycin has been used successfully in
tuberous sclerosis patients to shrink angiomyolipomas [23] and
astrocytomas [24]. Clinical trials are underway to assess the feasibility
and safety of administering rapalogues sirolimus or everolimus in
participants with Tuberous Sclerosis Complex (TSC). e assessment
(Clinical Trial NCT01929642 USA) includes measuring any reduction
in autistic symptoms, such as aggressive behaviors and/or any
improvements in cognitive function.
Aberrant mTOR hyperactivation is thought to occur in a wide range
of ASD patients with known genetic defects [25,26], such as those
autistics found to have large head size (macrocephaly) early in life [27].
is nding of large head size is reminiscent of the increased brain
volume and increased CSF accumulation found in the children
destined to develop autism as studied by Shen et al. [12], as discussed
above. Additionally, cases of genetic disease patients linked to mTOR
hyperactivation who also relatively oen have a diagnosis of autism
include neurobromatosis type I, Lhermitte-Duclos syndrome, and
Fragile X syndrome [21,26].
It is conceivable however, that mTOR hyperactivation can occur in
autistics for which a gene sequence alteration in DNA is not present.
is type of mTOR hyperactivation is theorized to occur within
neurons exposed intermittently to a toxin from diet and/or
environment. Such a toxin is theorized herein to alter the inux of
calcium into immature neurons in a haphazard and intermittent
manner resulting in reduction of autophagy in such neurons so
exposed, with eects dependent on their stage of development.
Such a haphazard hyperactivation of mTOR has been reproduced
experimentally and linked to the presence of amino acids. Gulati et al.
[28] studied HELA cells in culture and found that mTOR activation
was in relation to the level of amino acids present in the cell culture.
ey found that increased amino acids in the cell culture medium
resulted in an increase of HELA cell intracellular calcium, resulting in
increased calcium binding with calmodulin within the cell, and
subsequent increased activation through the mTOR pathway.
Specically, they state in their report “We demonstrate that the rise in
[Ca(2+)] (i) increases the direct binding of Ca(2+)/calmodulin (CaM)
to an evolutionarily conserved motif in hVps34 that is required for
lipid kinase activity and increased mTOR Complex 1 signaling”.
Glycine is an amino acid that is known to be present in and interact
with human brain cells, in particular during neurodevelopment. A
harmful glycine mimetic is herein theorized to be causative in at least
some cases of autism, and to have an eect in human neurons similar
to that eect of amino acids demonstrated in the cell culture studied by
Guloti [28], i.e. an increased mTOR activation, thus reducing
autophagy and leaving neuron spikes unpruned.
Human Post-mortem Selected Study of Autistic Brains
Finds Markers of Increased Calcium in Autistic Brains
versus Controls
Research on intracellular calcium in autism neurons oers a link to
increased mTOR activation and reduced autophagy. Palmieri et al. [29]
studied temporocortical gray matter from six matched patient-control
pairs of normal and autistic brains to perform post-mortem
biochemical and genetic studies of the mitochondrial aspartate/
glutamate carrier (AGC). e AGC participates in the aspartate/malate
reduced nicotinamide adenine dinucleotide (NAD) shuttle and is
physiologically activated by calcium (Ca(2+)). AGC transport rates
were signicantly higher in tissue homogenates from all six autism
patients, including those with no history of seizures and with normal
electroencephalograms prior to death. is increase was consistently
blunted by the Ca(2+) chelator ethylene glycol tetraacetic acid.
Neocortical Ca(2+) levels were signicantly higher in all six autism
patients compared to controls, according to these researchers.
Furthermore, following removal of the Ca(2+)-containing
postmitochondrial supernatant, these researchers reported they then
observed no subsequent dierence in AGC transport rates in isolated
mitochondria from patients versus controls. is result clearly
demonstrates that Ca(2+) entry provoked mTOR activation, leading to
reduced autophagy in association with autism.
Calcium inux relates to action at neuron membranes
of glycine-related factors
An article by Avila [30] reveals the link between calcium inux into
neurons and the action of ligands at membrane-embedded protein
structures of neurons. For example, Avila describes the glycine
receptor’s (GlyR) action as follows: “GlyR activation during embryonic
and early postnatal development most likely induces a depolarization
of the cell membrane…which in turn may activate calcium inux”.
us, glycine binding at the GlyR appears capable of inuencing the
rate of calcium inux into neurons during early neurodevelopment. As
will be further explored below, we theorize action of a harmful glycine
mimetic in autism causation via its inuence in causing harmful and
haphazard inux of calcium into immature neurons during
neurodevelopment. To further clarify this theory, we identied and
reviewed known facts and selected published literature regarding a
candidate molecule widely dispersed in the environment and present
in certain foods, namely glyphosate.
Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence
from the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187
Page 4 of 16
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
Glyphosate, candidate glycine-mimetic molecule for
comparison to requirements of theory
Glyphosate, a common herbicide, is known to be produced from the
amino acid glycine via addition of a phosphonomethyl group (Figure
4). In 2007, according to the US EPA, glyphosate was the most used
herbicide in the United States agricultural sector, with 180 to 185
million pounds (82,000 to 84,000 tons) applied, and the second-most
used in the home and garden market, where users applied 5 to 8
million pounds (2,300 to 3,600 tons); in addition, industry, commerce,
and government applied 13 to 15 million pounds (5,900 to 6,800 tons).
Figure 4: To glycine, chemist adds a phosphonomethyl group to
make glyphosate.
Glyphosate study, glyphosate from maternal diet found
concentrated in piglet fetal brain
In a study by Kruger et al. [31] of 38 malformed piglets born to sows
fed glyphosate-containing feed, brain tissue samples were obtained
from euthanized 1 day old piglets’ brains. Following appropriate
processing, these samples were tested for glyphosate using ELISA kits
(Abraxis, USA). Glyphosate was found to be present in all piglet brain
samples, at an average of 3.1 micrograms glyphosate per ml of sample.
e authors commented that glyphosate was able to reach the fetal
piglets from maternal feed, i.e. glyphosate from the feed eaten by the
sow was able to pass the placental barrier. e feed, which contained
0.87 to 1.13 parts per million glyphosate, when consumed in the rst
40 days of pregnancy, was associated with a rate of visible gross
malformation in the piglets of 1 per 240 piglets. No microscopic
evaluation of piglet brain tissue for autistic type changes was
conducted.
Glyphosate usage on corn/soy crops versus rate of USA
school children autism
Correlation is not proof of causation, but Swanson et al. [32] point
out the near exact match in correlation between the rise of glyphosate
usage on corn and soy crops in the USA, over the years 1992 to 2010,
and the increase in autism rates over the same period as reported in
the USA public school system (Figure 5). Among the most widely
applied formulations of glyphosate is the pesticide known as
Roundup®. We reviewed studies of glyphosate and Roundup® in
relation to published research regarding brain tissue, including in
relation to calcium inux into neurons.
Figure 5: From Swanson et al. [32], used by permission: Near exact
match of tons of glyphosate applied to corn/soy versus number of
children with autism as served under IDEA (US Department of
Education, Individuals with Disabilities Education Act).
Glycine chelates, Glyphosate, manganese and the
glutamate-glutamine cycle
Glycine, the simplest amino acid, has a molecular weight of 75, and
naturally forms chelates with cations. e strength of glycine chelates
is ideal for biologic processes, for example aiding mineral absorption
from the intestine, while not overly avidly binding minerals which are
needed for use in the body, such as for enzymatic reactions. When
discussing a putative harmful glycine mimetic in the brain, it is useful
to consider chelation in regard to the Glutamate-Glutamine cycle, and
its apparent dysfunction in autism.
We begin with the synapse, the space between two neurons where
chemical signals pass. Receiving the signal are receptors at the post-
synaptic neuron membrane. Among these receptors are the NMDA
receptors, which are neural membrane-embedded protein structures.
When activated, the NMDA receptor opens to allow calcium to enter
the neuron. e activation process, as will be discussed in detail below,
includes the simultaneous binding to the NMDA receptor of both
glutamate and glycine or a glycine mimetic [33].
Studies of autism and glutamate have documented that, in autism
patients, glutamate activity is impaired [34], both in regard to actions
at the synapse [35] where glutamate is released, and in the resultant
calcium signaling, as it relates, for example, to dendritic growth [36]. A
study by Page et al. [37] demonstrated that the ASD patients studied
had a signicantly higher concentration of glutamate in the amygdala-
hippocampal region of the brain than did normal controls. ese
ndings suggest the Glutamate-Glutamine cycle is disrupted in autism.
e cycle can be viewed as starting with the step where glutamate is
released into the synapse by the pre-synaptic neuron. e released
glutamate can participate in binding the NMDA receptors of the post-
synaptic neuron, thus transferring a signal. Such NMDA receptor
activation is subject to other factors, as will be discussed below, but if
Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence
from the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187
Page 5 of 16
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
the activation leads to excess calcium inow into neurons, this can be
problematic. e Glutamate-Glutamine cycle can be viewed as one of
the natural controls to prevent such excess calcium inow into
neurons.
As the next step of the cycle, the free glutamate within the synapse
space is normally quickly taken up for recycling by astrocytes. is
removal of glutamate from the synapse helps prevent over-activation of
the NMDA receptors. Within the astrocytes, the enzyme glutamine
synthetase (GS) normally converts glutamate to glutamine. Glutamine
is then returned from the astrocytes to the neuron. Within the pre-
synaptic neuron, glutamine is converted back to glutamate for storage
in internal vesicles. ese vesicles will later move to the pre-synaptic
neuron membrane and subsequently this glutamate will be released
back into the synapse, where the cycle repeats [38].
In essence, GS can be viewed as helping protect neurons from excess
glutamate activity/toxicity. However, GS activity depends on
manganese as a cofactor, and this is where a putative harmful glycine
mimetic could interfere. Glyphosate, as the candidate glycine mimetic,
has a well-known action as an exceptionally avid chelator of cations,
including manganese. us, glyphosate is viewed by some experts as
having the potential to interfere with the free supply of manganese
within astrocytes for use by GS within the Glutamate-Glutamine cycle
[39]. is is theorized as one mechanism by which a putative harmful
glycine mimetic, operating locally in patchy areas of the brain, might
cause damage to neurons by increasing glutamate to toxic levels within
the synapse, thus spiking calcium inow haphazardly into neurons.
is theory appears to correlate to the ndings of Page et al. [37]
regarding excess glutamate in autistic brains in the amygdala-
hippocampal area and the ndings of Stoner et al. [15] regarding
patchy areas of cortical disorganization found in brains of autistic
patients.
As an example of its avidity in binding manganese and magnesium,
glyphosate has been shown to deplete manganese and magnesium
levels in young leaves of non-transgenic soybean plants exposed to
glyphosate [40]. A recent study on dairy cows fed GMO Roundup-
Ready feed showed dramatically reduced levels of serum manganese in
association with glyphosate residues in the urine [41]. Autism has been
linked to reduced manganese levels in the baby teeth of autistics [42].
Seizures, which have been associated with low serum manganese
[43,44], are more prevalent among children with autism [45]. We will
further explore these matters and related factors below.
Round-up’s eects on animal brain slices - calcium
inux, apparently via NMDA receptor activation
Excess calcium entry into neurons is a well-known pathway to
neuronal damage and/or disruption of neurodevelopment. In the study
conducted by Hyrc et al. [46], increasing concentrations of calcium
were applied to in vitro cultures of disassociated embryonic neurons
from 15 to 18 day old mice embryos. Such concentrations of calcium
were found to be predictive of neuronal death during NMDA
stimulation. Cattani et al. [47] studied the eect of glyphosate on
immature neurons of the hippocampus in rats. Maternal rat exposure
to the pesticide involved treating dams orally with 1% Roundup®
(0.38% glyphosate) during pregnancy and lactation (until 15 days old).
Hippocampal slices from 15-day-old rats were acutely exposed to
Roundup(® (0.00005-0.1%) during 30 min, and experiments were
carried out to determine whether glyphosate aects (45)Ca(2+) inux
and cell viability. In this preparation, these researchers investigated the
pesticide’s eects on oxidative stress parameters, (14)C-α-methyl-
amino-isobutyric acid ((14)C-MeAIB) accumulation, as well as
glutamate uptake, release and metabolism. ey found that acute
exposure to Roundup(® (30 min) increases (45)Ca(2+) inux
(apparently by activating NMDA receptors and voltage-dependent
Ca(2+) channels), leading to oxidative stress and neuronal cell death.
Signal transduction pathways involved in the Roundup®-induced
45Ca2+ uptake showed that either AP5 (a NMDA receptor antagonist)
or KN-93 (a Ca2+/calmodulin-dependent protein kinase II selective
inhibitor) prevented Roundup®-induced 45Ca2+ inux.
Cattani et al. [47] also found that such acute exposure of animal
brain slices to glyphosate increased (3)H-glutamate release into the
synaptic cle, decreased glutathione (GSH) content and increased the
lipoperoxidation, characterizing excitotoxicity and oxidative damage.
ey also observed that both acute and chronic exposure to Roundup(®
decreased (3)H-glutamate uptake and metabolism, while inducing
(45)Ca(2+) uptake and (14)C-MeAIB accumulation in immature rat
hippocampus.
In view of the above suggestion that glyphosate has interaction at
neural membrane-embedded receptors, a review was carried out
regarding structure and function of neural membrane-embedded
proteins, particularly as they relate to glycine, calcium inux and
potential sites for interaction of glyphosate and/or glycine mimetics.
Neurotransmitters and membrane-embedded protein
structures in neurons
Glycine is widely recognized as a neurotransmitter, i.e. glycine can,
under specic circumstances, have an eect on the ow of ions across a
neuron’s membrane. e neuron membrane is a lipid bilayer with
embedded proteins. Some of these embedded proteins are pumps, such
as the sodium-potassium ATPase pump, while many embedded
proteins are ion channels. ese ion channels are transmembrane
proteins with a complex conguration that includes a pore through
which ions can pass. Some channels permit passage of ions via opening
of the pore as a result of membrane depolarization, and/or by the
binding to the membrane-embedded protein of certain chemical
ligands. Most channels have selectivity; i.e. when open they allow only
certain ions to pass.
Neural membrane-embedded proteins/receptors: Sites
for putative harmful glycine-mimetic action
Glycine receptor (GlyR)
GlyRs are formed by the assembly of ve subunits which arrange
symmetrically around a central pore. e beta subunit is the only one
which interacts with the anchoring protein gephyrin, making it key to
anchoring GlyRs at the area of the synapse. Of the alpha units, four
types are known. Action of the GlyR, such as in terms of speed of
opening kinetics, depends on which alpha units are present within a
particular GlyR. While the alpha 1 beta combination displays the
fastest kinetics, alpha 2 containing receptors are most abundant during
neurodevelopment.
GlyR’s can be activated by glycine or by taurine or alanine. When
open, chloride can traverse the pore, and ow is according to chloride
gradient. In regard to autism, mutations in genes encoding the alpha 2
subunit have been found in patients who carry a diagnosis of autism
[48].
Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence
from the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187
Page 6 of 16
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
e excellent article by Avila [30] reviews known facts about glycine
receptor activity during neurodevelopment. e brain’s early cortical
development includes formation of mini-columns via migrating
neurons [49]. Interneurons make up only about 15% of neurons, but
are important to establishment of these rst brain circuits [50,51].
GlyRs have been demonstrated to have a role in controlling cortical
interneuron development [30] and migration.
A disruption of migration of neurons can thus be theorized to be
induced when GlyR activation is disrupted. In fact, abnormal halting of
neuron migration can be demonstrated by unopposed action in
application of glycine to neurons in vitro [30]. e ndings of Stoner et
al. of patches of disordered clusters of cells in brains of autistics
suggests migration of neurons is abnormal in autistics. GlyR
malfunction as a result of action of a harmful glycine mimetic is herein
theorized as causative of autism in at least some cases.
Figure 6 illustrates the GlyR ligand binding site. As mentioned,
research has demonstrated that besides glycine, several other
molecules can bind to the GlyR and alter the activation of GlyR. For
example, strychnine is known to be an antagonist of the GlyR, as is
caeine. GlyR activation has an eect on clustering of receptors at
post-synaptic sites [17].
A disruption of GlyR activation theoretically induces alterations in
chloride outow for immature neurons, and thus empowers haphazard
disruptions for calcium inow into immature neurons via the NMDA
receptor channel (subject to occurrence of other coincident actions at
the NMDA receptor as will be discussed below). is putative harmful
glycine mimetic’s action at the GlyR, is envisioned to be via an
exposure(s) from diet and/or environment, dosing the fetus or child in
an intermittent variable-dose manner. e resulting haphazard calcium
inuxes theoretically enable a spectrum of severity in disrupted neuron
migration, reective of the spectrum of symptom severity in ASD cases
clinically.
Figure 6: Glycine receptor (GlyR) viewed with 5th subunit (an
alpha) removed.
e mechanism by which outow of chloride from an immature
neuron aects calcium inow into that immature neuron, has to do
with the eect of chloride outow on the electrical charge at the
neuron’s membrane. A neural membrane usually retains a negative
charge within the cell, and a positive charge on the exterior. However,
at times, such as when chloride outow through the GlyR is high, the
charge of the membrane can change, leading to what is known as
membrane depolarization. Membrane depolarization can then alter
activity at other membrane-embedded protein structures, such as at
the NMDA receptor. Because like charges repel each other, a
depolarization of the membrane, which produces a temporary positive
charge nearer the NMDA receptor, can dislodge the positively charged
magnesium ion from its blocking position within the NMDA receptor,
as illustrated in Figure 7.
Figure 7: NMDA receptor, note the magnesium ion is dislodged
from within the NMDA channel by the depolarization caused by
chloride outow at the GlyR when glycine binds there.
However, the NMDA receptor channel will not open to calcium
inow just because the magnesium ion exits the channel of the NMDA
receptor. e two binding sites of the NMDA receptor must also be
properly lled. A molecule capable of agonist binding at the so-called
glycine binding site of the NMDA receptor must be in place. Also, a
molecule of glutamate must be bound at the glutamate binding site of
the NMDA receptor. Only when these three events occur at the same
time, i.e. the magnesium ion’s exit from the NMDA channel, the
binding of an activating molecule to the glycine site of the NMDA
receptor, and the binding of glutamate to the glutamate site of the
NMDA receptor, only then will the NMDA channel allow calcium to
ow into the neuron.
From this complex set of requirements it is clear how carefully
neurodevelopment guards against haphazard or excess entry of
calcium into immature neurons. Conversely, as theorized herein, when
a harmful glycine mimetic is able to disrupt this carefully
choreographed system, the normal neurodevelopment sequence does
not occur. Recall how calcium excess within a mouse brain model of
autism was associated with reduced autophagy and malformed poorly
‘pruned’ neurons. It seems that proper neurodevelopment is critically
dependent on the correct and benecial control of calcium entry into
immature neurons.
Taurine, an amino acid, is also capable of binding the GlyR. When
taurine binds to the GlyR, such taurine binding, like the binding of
glycine, opens the GlyR ion channel to gradient directed ow of
chloride. However, taurine binding appears to induce only
Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence
from the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187
Page 7 of 16
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
approximately half as much chloride ow as compared to glycine
binding. In eect, taurine is a glycine mimetic which reduces chloride
ow compared to glycine binding of the GlyR. is implies a reduced
opportunity of chloride ow to spur depolarization of the immature
neuron’s membrane. us, taurine is seen as a partial agonist of the
GlyR.
Taurine tends to modulate the action of glycine at the GlyR, i.e.
taurine’s presence can serve as a natural competition with glycine. is
natural competition results in fewer membrane depolarization events.
In eect, taurine’s binding at the GlyR during neurodevelopment is a
partial interference with glycine’s ability to boost calcium inow events
for the neuron. is has profound potential to inuence neuron
migration during neurodevelopment.
Because taurine, as mentioned above, only permits approximately
half the chloride ow at the GlyR when compared to glycine binding
the GlyR, it is thus less likely that a membrane depolarization will
occur with taurine bound to the GlyR (Figure 8). In fact, the amino
acid taurine is reported [52-54] to serve to counteract glutamate-
induced elevations in intracellular calcium ions within neurons, and
thus taurine confers protection against neurodegeneration. e
concentration of taurine compared to the concentration of glycine in
the vicinity of the GlyR results in a choreographed competition.
Taurine, in theory, will likely similarly compete for binding versus
glyphosate, and therefore taurine likely would tend to protect from any
theorized harm from glyphosate binding at the GlyR.
Studies by Leon et al. [54] demonstrated a neuron-protective eect
for taurine thought to operate via modulating the calcium inux to
neurons which otherwise would occur due to glutamate at the NMDA
receptor. Glutamate applied without taurine was able to drive
apoptosis (neuron cell death) via release of cytochrome C. Taurine
application along with glutamate was shown to prevent such apoptosis.
Figure 8: Taurine binding to GlyR reduces chloride ow by
approximately 50% compared to ow from glycine binding of GlyR,
so no depolarization occurs with taurine bound to GlyR, thus
magnesium ion remains within NMDA receptor channel,
continuing to block calcium from entering immature neuron.
e level of taurine present in the brain is known to progressively
increase during embryogenesis. At the same time, the dierent GlyR
assemblies, from incorporation of dierent alpha subunits, means that
another factor is present beyond taurine concentration. Dierent
GlyRs vary in their sensitivity to taurine. Homomeric GlyRa2, for
example, is 10 times less sensitive to taurine [30] compared to the most
sensitive GlyR.
us the choreography of neurodevelopment is tuned to genetic
distribution of dierent membrane-embedded receptors/proteins over
time, and to eects of local concentrations of competing receptor
agonists. In regard to the theory herein advanced, we theorize the
harmful action of a putative glycine mimetic acting at the GlyR to
disrupt such delicate choreography. is damaging eect on
neurodevelopment is notably a local and time-sensitive eect, rather
than dependent solely on overall dose of toxin to the fetus or mother.
Another balanced mechanism during neurodevelopment by which
neurons and axons perform coordinated migration is the frequency of
spontaneous waves within a neuronal circuit, i.e. waves of depolarizing
or ‘bursting’ activity. e eects within the brain of a disruptive
decrease of the local frequency of such spontaneous bursting activity
via toxins that act at the GlyR was demonstrated by Hansen et al.
[55,56].
ey examined white leghorn chick embryos’ spinal neuron
migration in ovo by cutting a square hole through the egg shell and
placing a pipette via which they administered GlyR antagonists,
strychnine or picrotoxin. By thereby decreasing calcium transient
inuxes locally, and thus decreasing the normal spontaneous rhythmic
bursting activity (RBA) of developing neuron circuits, they noted
migration abnormalities. Such GlyR antagonists, when administered at
certain days during gestation, also altered gene expression locally of
genes which normally code for neuron axon guidance/adhesion
proteins.
Glycine as ligand, binding to NMDA receptor
As discussed above, the NMDA receptor has a binding site for both
glutamate and glycine (D-serine also can bind the glycine site of the
NMDA receptor). Only when both of these binding sites of the NMDA
receptor, the glutamate site and the glycine site, are agonistically lled,
and only if the magnesium ion is simultaneously displaced from the
NMDA channel, only then will the NMDA channel allow calcium to
enter the neuron. As mentioned, this tight control of calcium entry has
import for neurodevelopment. e amount of calcium entering via the
neuron’s NMDA receptors is known to be further modulated by pH
and zinc.
If a molecule of the herein theorized putative harmful glycine
mimetic binds more avidly than glycine at the glycine binding site of
the NMDA receptor, then the chances of calcium inow into immature
neurons could theoretically be increased. is is because the theorized
more avid binding of the putative harmful glycine mimetic to the
NMDA receptor’s glycine site, would extend the time for the
simultaneous occurrence of the other two events, i.e. for the
simultaneous exit of the magnesium ion from the NMDA channel and
the simultaneous binding of glutamate to the NMDA receptor’s
glutamate binding site.
Glycine concentration at synapse, glycine transporter protein
T1 (GlyT1)
Another mechanism by which a spectrum of severity of autism is
envisioned to occur, from the action of the putative harmful glycine
mimetic, relates to glycine transport proteins. Neurotransmitter
glycine participates in a recycling mechanism of uptake of glycine from
Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence
from the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187
Page 8 of 16
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
the synapse by cells in the brain. Storage of glycine in the pre-synaptic
neurons occurs for a time, followed by re-release of the stored
neurotransmitter back into the synapse (Figure 9).
Figure 9: Neurotransmitter glycine is taken up from synapse by
GlyT1 transporter protein (not shown), stored, then re-released into
synapse to bind glycine receptor of post-synaptic neuron.
Glycine transporter proteins GlyT1 and GlyT2 are membrane-
embedded proteins specic for glycine handling. e primary role of
GlyT1 is thought to be to maintain glycine concentrations below
saturation level at postsynaptic NMDA receptors, thus modulating
inux of calcium into neurons. GlyT1 is known to be subject to
inhibition by glycine mimetics. For example, sarcosine, one of the
naturally occurring N-methyl analogues of glycine, is a known
inhibitor of GlyT1. Sarcosine is also one of the possible metabolic
breakdown products of glyphosate.
We theorize that glyphosate directly, or sarcosine from glyphosate
breakdown, act via GlyT1 inhibition to locally and intermittently
inhibit the uptake of glycine from the synapse. We theorize that,
during neurodevelopment, such inhibition of GlyT1 permits excess
synaptic glycine to accumulate. us glycine could bind to saturation
the NMDA receptor of an immature neuron, thus increasing calcium
entry via the NMDA receptors into the immature neuron, damaging
neurodevelopment.
In regard to GlyT1 inhibition and neurodevelopment, the work of
Schmitz et al. [57] is of interest. ey performed unilateral intrastriatal
injection in mice of toxin 6-hydroxydopamine and studied the re-
enervation response with and without GlyT1 inhibition. In the studied
mice whose glycine transporter protein1 function was inhibited
pharmacologically for 4 weeks, the axon sprouting of the neurons
responding to the denervation was, at 7 weeks, twice as dense as
controls. is ‘over-sprouting’ occurred via action of the NMDA
receptors, according to Schmitz et al.
If a re-enervation sequence can be conceptualized as similar to the
original enervation sequences of neurodevelopment, then the study of
Schmitz et al. might provide a relevant theoretical explanation. A spike
of putative harmful glycine-mimetic molecules in a localized area of
cortex in a developing human fetal brain might produce a disruptive
eusion of neuron spikes due to local blockade of GlyT1 by the
harmful glycine mimetic (or its metabolite). Such patches of putative
over-exuberant neuron spikes are reminiscent of the ndings of Tang
et al. [18], and reminiscent of the increased neuron density found in
patches in brains of autistic children by Stoner et al. [15].
In fascinating studies [56,58] by Hansen et al., these researchers
administered the GlyT1 inhibitor sarcosine via in ovo method during
neurodevelopment of white leghorn chick embryos. ey documented
that sarcosine caused an increase in the frequency of calcium
transients, which translated into an increase of the frequency of
spontaneous rhythmic neuronal bursting activity. ey found this
produced abnormalities in neuron/axon migration.
In summary, Hansen et al. conrmed that rhythmic bursting
activity (RBA) of neuron circuits is key for early pathnding decisions
by neurons. Moderate slowing of the frequency of RBA causes
“motoneurons to make dorsoventral (D-V) pathnding errors and to
alter the expression of molecules involved in that decision.” Conversely,
moderate speeding up of RBA in neuron circuits strongly perturbs “the
anteroposterior (A-P) pathnding process by which motoneurons
fasciculate into pool-specic fascicles at the limb base and then
selectively grow to muscle targets.” In their studies, these researchers
found that resumption of normal frequency of RBA sometimes allowed
axons to correct the A-P pathnding errors, perhaps leaving aberrant
nerves.
Aberrant connectivity has, in fact, been documented in the brains of
autistic children [59] using resting state functional magnetic resonance
(fMRI) imaging. Di Martino et al., studying autistic children using
fMRI methods, concluded that their examination of functional
connectivity (FC) of striatal networks in children with ASD revealed
“abnormalities in circuits involving early developing areas, such as the
brainstem and insula, with a pattern of increased FC in ectopic circuits
that likely reects developmental derangement rather than immaturity
of functional circuits.” is pattern also ts our theory of a dietary/
environmental sourced toxin acting locally as a harmful glycine
mimetic, having patchy eects in the CNS beyond the cortex.
Neuron migration has been visualized. Avila et al. [60]
demonstrated in vivo, using cultured brain slices from mice embryos,
that actomyosin contractions alter neuron migration. ese
researchers recorded an example of neuron migration in video format
and have made the recording available for viewing online as movie S2
of their open access report [60].
In summary, this delicately choreographed neurodevelopmental
process in humans, including the precise frequency of waves of
depolarization in synchronized neurons within circuits, this pattern of
neurons which alternately sprout, prune and migrate, this
choreography can be locally disrupted, we theorize, by a relatively
small number of molecules of a harmful glycine mimetic. From the
available research literature, such disruption, for example via the GlyR
and/or the GlyT1 and/or the NMDA receptor, can reasonably be
theorized to produce neuronal/axonal migration defects reminiscent of
those found in studies of brains of autistic patients.
Next, we will address the neuron membrane-embedded protein
structures known as Na-K-Cl cotransporter 1 (NKCC1) and K-Cl
cotransporter 2 (KCC2).
Glycine, NKCC1 versus KCC2 in neurons, brain weight
Within the brain during normal neurodevelopment, the types and
numbers of membrane-embedded proteins will change as neurons
Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence
from the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187
Page 9 of 16
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
mature. As illustrated in Figure 10, in immature neurons the
membrane will typically have more NKCC1 proteins embedded, and
fewer KCC2 proteins. One result is that immature neurons tend to
keep internal chloride concentration higher.
Figure 10: In normal circumstances, the membranes of immature
neurons include numerous NKCC1, fewer KCC2; intracellular
chloride concentration tends to be higher in normal immature
neurons as compared to mature neurons.
As neurons proceed through normal fetal development, they will
begin to manufacture fewer of the NKCC1 proteins and instead
manufacture more of the KCC2 proteins. erefore, mature neurons
will have more KCC2 proteins embedded in their membrane and fewer
NKCC1 proteins embedded, as illustrated in Figure 11. e result, in
normal circumstances, is that mature neurons tend to have a lower
concentration of chloride internally.
Figure 11: In normal circumstances, mature neurons have fewer
NKCC1 and more KCC2; intracellular chloride concentration tends
to be lower in normal mature neurons as compared to immature
neurons.
In both the immature neuron and the mature neuron, the glycine
receptors (GlyR) aid in keeping the internal chloride concentration
within expected bounds (Figure 12). e programmed normal change
of neuron membrane-embedded proteins predominance, from
predominately NKCC1 (immature neurons) to predominately KCC2
(mature neurons), is commonly referred to as the ‘GABA switch.’ is
reects the fact that gamma amino butyric acid (GABA), in normal
circumstances, acts to inhibit hyperactivity in mature neurons, such as
during birth and post-natally [61]. e GABA switch is oen found to
be impaired in autism patients, a nding that we theorize might be
related to action of a harmful glycine mimetic during
neurodevelopment, as further described below.
Figure 12: Under normal circumstances, chloride ows out of an
immature neuron when GlyR is open. Under normal circumstances,
chloride ows into a mature neuron when GlyR is open.
Interestingly, a partial agonist of the GlyR is caeine. It is
noteworthy that autistic children receiving a low dose of daily caeine
over weeks or months have been reported [62] to sometimes
experience improvement of autism symptoms as a result of the
caeine.
As previously mentioned, it is well known that the brains of autistic
children, in their early childhood years, are heavier than controls. We
postulate that this weight increase is caused by increased water content
of the brain, such as in neuropil, glial cells and/or neurons. We believe
such water retention could be a response to excess cell-internal
chloride, caused by impaired chloride exit following glyphosate
binding to glycine receptors. Chloride retention has been implicated in
the brain swelling associated with edema following brain trauma [63].
Furthermore, sarcosine, a breakdown product of glyphosate, has been
shown to activate glycine receptors, but with a reduced eect on the
chloride channel [64]. Hence, chloride can be expected to accumulate
over time within the cell due to reduced eux in response to sarcosine
or glyphosate binding.
Glycine and the choroid plexus
With regard to cerebrospinal uid (CSF), production is typically
balanced to resorption. e average adult produces approximately 500
mL of CSF per day, but because CSF is constantly resorbed, only
100-160 mL is present in adults at any one time under normal
circumstances. It has long been thought that CSF returns to the
vascular system by entering the venous sinuses of the dura via the
arachnoid granulations (also known as villi). However, recent research
[65] has suggested that CSF ow along the cranial nerves and spinal
nerve roots allows at least some CSF to ow into the brain’s lymphatic
Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence
from the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187
Page 10 of 16
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
channels. Such ow may play a substantial role in CSF resorption, in
particular in the neonate or fetus, in which arachnoid granulations are
sparsely distributed.
is apparent interplay between CSF and lymphatic uid opens the
door to new discoveries in brain metabolism and immune activity. For
example, the possibility of harmful/reactive substances entering the
CSF from lymph might be considered. Lymph is the uid that bathes
lymph nodes, interacts with thymus-derived cells [66] and ows
throughout the body using lymph channels. Lymph re-enters the blood
via the lymphatic duct in the chest. ese factors may be explored in
future studies to explain the immune alterations in autism.
With regard to the present theory, the precise details of glycine’s
action with respect to the choroid plexus are, as yet, not well studied or
understood. Nevertheless, the excess CSF accumulation visible on the
MRI images reported by Shen et al. [12] in children destined to
develop autism is perhaps tentatively linkable to glycine, and/or a
theorized harmful glycine mimetic.
For example, presence of a glycine receptor site in sheep choroid
plexus was established by Preston [67]. A co-transporter of GABA and
glycine known as GAT-2 has been reported within human choroid
plexus cells. Studies by Schlessinger et al. [68] on the function of the
GAT-2 transporter have revealed that, in addition to transporting
GABA and glycine, the GAT-2 transporter also has a strong interaction
with a glycine mimetic known as glycylglycine. us, it is theorized
herein that a harmful glycine mimetic could act via glycine sites in the
choroid plexus to increase CSF production. e details of such action
are, as yet, unclear.
Relevant Treatment results in ASD patients
In order to further evaluate the theory herein advanced, selected
pharmacologic treatment successes in autism are considered in light of
neural membrane-embedded proteins and neurotransmitters.
Calcium channel blockers/modulators, verapamil and
ketamine
In reviewing the literature regarding calcium channel blocker use in
treatment of autism, no randomized clinical trials were found. e
authors of the present article were struck, however, by an online blog
series of reports by the non-scientist father of an autistic son. We
hesitate to mention this anecdote here, but feel that even anecdotal
evidence can sometimes point the way to clues of causation. erefore,
we present the story of an autistic boy, Anthony (not his real name).
e father’s blog post (at Epiphanyasd.blogspot.com) indicates that
during one summer, Anthony was dealing with allergies and was
struggling with ‘aggressive’ symptoms of autism, as per his father’s
reports online. As the blog describes, Anthony was saying ‘Be nice’ and
‘to hit your head’ associated with attempts to self-control his ares of
aggressive autistic symptoms.
e father reports giving Anthony an anti-histamine during that
summer, but found it was only eective for Anthony for a couple of
hours per dose. e father, as he mentioned in his posts, had
researched the work of Italian professor and clinician Antonio Persico
[69] in regards to calcium and the autistic brain. e father reports he
researched the calcium channel blocker verapamil. Satised that a low
dose of verapamil would be safe for his son, and seeing Anthony
continuing to demonstrate aggressive symptoms, the father began
Anthony on a dose of 20 milligrams verapamil. e father recorded
online the circumstances regarding his son as follows: “One aernoon,
I decided to give a very small dose (20 mg) of Verapamil, and before
my eyes, the anger and agitation began to fade and was replaced by
calm. It was the most amazing experiment that I have witnessed and
within 20 minutes there was complete calm”.
While anecdotal reports clearly have obvious limits, they may
sometimes serve as a clue to areas deserving of further study under
application of a validated scientic method. In addition, although the
authors of the present article do not recommend that parents
independently apply non-prescribed medical regimens for their
autistic children, it is heartening to read the follow-up note in the blog
concerning Anthony. As the father described it: “In the following
weeks, I would still hear Anthony say `be nice,’ but this was no longer
followed by any aggressive behavior. e trigger was still there to
energize these channels, but they had been blocked by Verapamil. It
was like ring a gun, but with no ammunition; there was a `click,’ but
no `bang.’ “
Another calcium channel blocker, ketamine, has been reported in
the scientic literature to have value in treating autism. Ketamine is a
known antagonist of the NMDA receptor, capable of reducing the
inux of calcium into neurons. Successful anesthesia of 5 autistic
children has been reported by an anesthesiology group using oral
ketamine preoperatively [70]. e anesthesiology group indicated that
they found the symptoms of autism were less likely to interfere with
the surgery if the patient was given ketamine orally in the preoperative
period. is report of ketamine success in reducing symptoms of
autism appears to link reduction of calcium entry into neurons with
reduction in autism symptoms.
NKCC1 Chloride channel and bumetanide
In the brain, the diuretic bumetanide blocks action of the
membrane embedded protein NKCC1, and thus can alter internal
chloride concentration within neurons. Bumetanide, when given to
pregnant mice with models of autism, has been reported to prevent or
reduce autistic behavior in ospring [61]. In humans, Lemonnier et al.
[71] reported successful use of bumetanide in a 10 month treatment
study in adolescents/young adults with autism. ese researchers
stated that their treatment was based on their understanding that
bumetanide decreases the level of intra-neuronal chloride (Cl-)i and
reinforces the natural GABA inhibition of excitation of neurons in
autistic patients. ey report reduced severity of autism symptoms and
indicate that bumetanide treatment improves emotion recognition and
enhances the activation of brain regions involved in social and
emotional perception during the perception of emotional faces by
bumetanide-treated autistics.
e theory herein advanced proposes the harmful action of a
putative glycine mimetic to over-concentrate chloride within immature
neurons. Bumetanide, by blocking such chloride-concentrating action,
might at least temporarily relieve such excess chloride concentrations
within immature neurons.
Glutamate and the Multi-system nature of autism, food
choices linked to putative harmful glycine-mimetic
Certain chemistry ndings are typical of autistic patients, such as
elevated plasma and brain glutamate [72] and an altered amino acid
prole in the blood [73]. In fact, autism is widely recognized to be a
multi-system disorder [74]. For example, pediatricians have described
Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence
from the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187
Page 11 of 16
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
in autistics a somewhat characteristic pattern of signs and symptoms
including smelly bowel movements, bloated bellies, dry skin and
frequent colds and ear infections. Such multi-system manifestations in
autism will be very briey addressed below to indicate their linkage,
within the theory herein advanced, to a putative causative harmful
glycine mimetic.
Modern processed food is well known to contain excess ammonia
due to the methods of food processing. For example, as far back as
1973, elevated ammonia content of gelatin, cheese, breakfast cereal,
bacon, corn, peas, numerous other vegetables, cheddar cheese,
buttermilk and other foods was reported in the scientic literature
[75]. Injecting ammonia into meat as a means to control E. coli has
been accepted practice for years, though only recently widely
publicized. Part of the reason the public was not generally aware of the
elevated ammonia content of hamburger and what is known as ‘pink
slime’ as a meat component, was that the amount of ammonia used
was not required to be listed as an ingredient on the label due to the
fact that the government viewed the ammonia as a processing
chemical.
Under normal circumstances, excess ammonia would be a burden
for normal human digestion. We theorize this burden is made more
dicult by the presence of a harmful glycine mimetic within such high
ammonia food. We theorize that the combination in food of excess
ammonia and a harmful glycine mimetic having biocide capability
alters the gut microbiome, the gut lining, the blood chemistry, and has
implications for neurodevelopment.
In order to better describe this theory, it is instructive to begin by
reviewing the ndings of a study of gobie sh by Peh et al. [76]. ey
studied ammonia detoxication in the gobie sh euryhaline
Bostrychus sinensis exposed to excess ammonia in a hyperosmotic
environment, whereby drinking was essential for osmoregulation. ey
found alterations in intestine/contents, which alterations they linked to
action of the enzyme system the sh uses to de-toxify ammonia. is
enzyme system is the well-known glutamate dehydrogenase (GDH) -
glutamine synthetase (GS) system. e expected products of the GDH-
GS system in detoxifying ammonia include glutamine and glutamate,
with perhaps un-detoxied ammonia le over if the ammonia
substrate was in excess.
In their study of the gobie sh, Peh et al. did nd in intestine/
contents a signicantly elevated glutamine level aer ammonia
exposure, a sign the GDH-GS system was in operation and detoxifying
ammonia. But they did not nd elevated glutamate in the intestine/
contents. ey reasoned that the glutamate must have been generated
from the GDH-GS system, but had apparently been absorbed into the
blood stream of the sh, likely so that this relatively toxic glutamate
could be either detoxied within other organs, or changed into other
amino acids.
Turning now to studies in autistics, Aldred et al. [73] studied blood
samples from patients with autism or Asperger syndrome and blood
samples from their siblings and parents. ey found that all family
members had the same pattern, with raised glutamate levels in their
plasma, but reduced plasma glutamine. is is reminiscent of the
pattern of glutamine in the intestine and glutamate absorbed into the
circulation from ammonia detoxication as found by Peh et al. in the
gobie sh. is pattern in the autism families studied by Aldred et al.,
we believe, is due to the detoxication of excess dietary ammonia by
their human gut microbiome.
But, autistics it seems, also have other markers suggesting
detoxication of excess food ammonia. In addition to the decreased
plasma glutamine and increased plasma glutamate, Aldred et al. also
measured plasma levels of other amino acids. ey found autistic
patients and their family members had elevated plasma levels of
alanine, phenylalanine, asparagine, tyrosine and lysine when compared
to controls.
ese ndings suggest to us that autistics and their family members
were eating, generally speaking, a very similar diet of high-ammonia
foods as compared to controls, food that also putatively contained a
harmful glycine mimetic. is pattern of plasma amino acid elevations
in autistics and their family members, we believe, ts the pattern of
excess food ammonia with detoxication via GDH-GS. Our view is
that some of the excess glutamate absorbed from the intestine of the
autistics and the intestine of their family members became substrate.
We believe, for example, some glutamate was transaminated by alanine
aminotransferase into lactate-derived pyruvate to form alanine.
Elevated plasma alanine was found by Aldred et al. [73] in autistics and
their family members.
Autism, gut microbiota
Damage to neurodevelopment within the fetus is herein theorized
from maternal exposure to a putative harmful glycine mimetic.
However, neurodevelopment continues aer birth. is raises the
possibility of damage to developing neurons occurring from a harmful
glycine mimetic in the child aer birth. For example, once the
newborn begins developing his or her own gut microora, exposure to
a harmful glycine mimetic with biocide capabilities could alter gut
microora, contributing to excess glutamate in blood and in the brains
of autistics if excess ammonia is present. Gut ora alterations have
been well documented in autism [77-79], as well as leaky gut syndrome
[80,81], excess fecal content of short chain fatty acids and elevated
ammonia [82]. In vivo studies on rats have shown that ammonia
administered via injection of ammonium acetate activates NMDA
receptors in the brain, leading to increased intracellular calcium,
calcium uptake into mitochondria, and initiation of neuronal death
[83].
Regarding gut microbiota, the excellent review and analysis by
Krajmalnik-Brown et al. (65) found a ‘hyper-Westernization’ of the gut
microbiota of children with ASD. ey speculated this alteration
“could indicate that gut microbiota dierences that are driven by
unique aspects of the Western lifestyle compared to the developing
world lead to the association of unique gut microbiota composition
with ASD.” If indeed it is present, the nature of that unique microbiota
composition in ASD still eludes science. As these researchers
concluded, “e complexity of the symptoms and the etiology of ASD
coupled with the complexity of the microbiota and its functions has
presented challenges in establishing the nature of an association
between gut microbiota and ASD, pinning down whether a link even
exists and for which individuals with ASD, and in producing a
mechanistic understanding of the nature of this association.”
Some of the reported changes in gut ora in autistics are thought to
be related to frequent use in autistics of antibiotics, such as in
treatment of otitis media. However, studies [84,85] have so far found
no signicant dierence in gut microbiome between autistics and their
neurotypical non-autistic siblings. As one study concluded, “Results
did not indicate clinically meaningful dierences between groups.”
Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence
from the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187
Page 12 of 16
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
Nevertheless, glyphosate does appear to have the capability to alter
the gut microbiome, Shehata et al. [79] tested bacteria in culture
medium against various strengths of herbicide-formulated glyphosate.
ey found that most of the pathogenic bacteria they tested were
resistant to glyphosate, while most of the benecial bacteria were
found to be moderately to highly susceptible. us, glyphosate appears
to have a tendency to reduce benecial species, perhaps contributing to
the hyper-Westernized gut microora described by researchers
studying the subject of gut microora in autism.
ere are studies which hint at dierences in gut microbiota
between autistics and non-autistic non-siblings. Finegold et al. [78] in
2002 reported on a study of gastric, small bowel and stool samples
from late onset autism patients compared to non-sibling controls.
Special care was taken to capture and culture anaerobes. Many of these
autism patients were on a gluten-free, casein-free diet. All had
gastrointestinal symptoms, primarily diarrhea and/or constipation. All
patients had received no antibacterial agents for at least 1 month prior
to the study. Results indicated a denite dierence between groups, i.e.
“Children with autism had 9 species of Clostridium not found in
controls, whereas controls yielded only 3 species not found in children
with autism. In all, there were 25 dierent clostridial species found. In
gastric and duodenal specimens; the most striking nding was total
absence of non-spore-forming anaerobes and microaerophilic bacteria
from control children and signicant numbers of such bacteria from
children with autism.”
Limitations of study design in earlier autism research
Autism appears to be associated with relatively minor and dicult
to visualize histologic manifestations, such as patchy disruptions of
mini-column organization in brain development [86,87], as discussed
above. Studies focused on larger scale defects in neurodevelopment
such as malformations may miss such key localized ndings. A study
of sub-lethal dosing of rats with glyphosate [88] did document
vacuolar changes in brain tissue but claimed to nd no other changes,
without referencing neuronal clustering or orderliness of mini-column
arrangement of neurons or the like. In order to answer the question of
whether a putative glycine-mimetic candidate molecule, such as
glyphosate, might play a role in autism causation, it will be useful to
design studies which have the potential to shed light on the relevant
issues.
Risperidone, NMDA receptors, AMPA receptors
e rst drug approved by the FDA for treatment of autism,
risperidone, is the most widely used. Risperidone can have severe side
eects, but can also prove eective in reducing tantrums, aggression
and self-injury. e improvement is generally seen in approximately
half the patients and can be dramatic, taking eect in a matter of
weeks. Symptoms oen return, however, when the drug is
discontinued. Side eects of weight gain, sleepiness and high prolactin
eects limit the use of risperidone.
For the purpose of the theory herein advanced, the mechanism of
action of risperidone is relevant. Current theories of action tend to
focus on risperidone and blockage of D2 and 5-HT2A receptors;
however, the study by Choi et al. [89] suggests an action of risperidone
in regard to other neuron receptors. ese researchers compared 3
weeks of dosing at 3 dierent levels of risperidone in juvenile rats as
compared to the same dosing in adults rats. ey found “Risperidone
(at 1.0 and 3.0 mg/kg/day) signicantly decreased NMDA binding in
caudate-putamen of juvenile and adult animals.” In contrast, the same
two doses of risperidone “decreased NMDA receptors in nucleus
accumbens of juveniles and not adults.”
ey also found that “risperidone (at 1.0 and 3.0 mg/kg/day)
increased AMPA receptors in medial prefrontal cortex and caudate-
putamen of juvenile animals, whereas risperidone (at 3.0 mg/kg)
increased AMPA receptors in caudate-putamen and hippocampus of
adults.” ese ndings t the theory herein advanced that haphazard
calcium inow into neurons is key to autism. For example, such
haphazard calcium inow should theoretically be reduced when
NMDA receptor activity is decreased. Risperidone success, in the
study, was associated with a reduction of NMDA receptors. Also, the
increase of AMPA receptors documented in the Choi et al. study of
risperidone mechanism of action ts the theory herein advanced,
because AMPA receptors within the brain are generally of the
GluR2(R) type. e GluR2(R) receptor is known to admit sodium and
potassium, while not permitting calcium to inow.
Discussion
e available evidence appears to link research ndings in autism to
defects in early (including in utero) neurodevelopment [15]. Scientic
studies are still ongoing to determine which governing factors for
human neurodevelopment act at which steps to inuence neuron
migration and/or gene expression. However, in view of the rising
incidence of autism diagnosis, we hold that it is time to consider the
theory herein advanced that human fetal neurodevelopment might be
altered by exposure to intermittent doses of a relatively small number
of molecules of a putative harmful glycine-mimetic.
e theory that such a toxin is originating from environmental
and/or dietary sources is supported by the nding that the brains of at
least some of the control subjects in the studies by Suzuki et al. [13]
and Stoner et al. [15] had ndings similar to the ndings in the brains
of autistics. Similarly, the plasma amino acid changes documented by
Aldred et al. [73] in autistics were present also in their family
members. Similarly, the fact that studies of gut microbiota of autistics
compared to gut microbiota of their non-autistic siblings [84] have not
shown signicant dierences could be explained by the presence in
food for both groups of a harmful glycine mimetic acting as a biocide.
Importantly, the theory herein advanced holds that an
environmental or dietary sourced exposure to a human fetus of such a
putative harmful glycine mimetic would likely not follow a standard
dose response curve. We believe evidence [30] supports the view that
the eects on neurodevelopment in the fetus of such theorized harmful
glycine mimetic exposure are dependent on factors such as timing of
exposure, and on location of molecular interactions. is theory, if
proven, might have far reaching implications, such as precluding, at
least for pregnant women, the supposed utility of safe allowable limits
in food or on crops of the putative harmful glycine mimetic.
In essence, neurodevelopment is unlike other natural processes, in
that a small number of molecules disrupting neurodevelopment at just
the wrong time could have devastating autism-causing and long lasting
outsized eects [9]. is viewpoint is valid, we believe, because fetal
neurodevelopment is not a steady state process [9]. Rather, fetal
neurodevelopment, especially in its early stages, has delicate and
naturally choreographed steps [9,30] which appear inordinately
sensitive to small disruptive insults. Such small disruptive intermittent
events coming from a harmful glycine mimetic during
Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence
from the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187
Page 13 of 16
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
neurodevelopment could explain previously puzzling autism features
such as the patchy cortical foci of disorganization [15] or the autistic
brain’s functional connectivity alterations, or the failure of proper
development of the GABA switch [61], or the wide spectrum of
symptoms in ASD.
We recognize that this paper is speculative, but we hope it will
inspire others to conduct research to test the validity of our proposed
hypothesis. If our ideas are validated, it is imperative for governments
to take regulatory action against the practice of widespread glyphosate
usage on food crops.
Acknowledgement
is research is supported in part by Quanta Computers, Taiwan,
under the auspices of the Qmulus program.
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Citation: Beecham JE, Seneff S (2015) The Possible Link between Autism and Glyphosate Acting as Glycine Mimetic - A Review of Evidence
from the Literature with Analysis. J Mol Genet Med 9: 187. doi:10.4172/1747-0862.1000187
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ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 4 • 1000197
