A Proposed Role for Glyphosate in Shared Pathway Disruptions in Autism, Alzheimer’s Disease and Cancer
Perhaps surprisingly, autism and Alzheimer’s disease overlap considerably in symptoms, genetics, and mechanisms, suggesting a similar underlying pathology [105]. PIN1 is normally expressed at very high levels in neurons, but it is inhibited in neurons in Alzheimer’s disease via multiple mechanisms, including downregulation, oxidation, phosphorylation and sequestration [106]. Just as autism rates over time are highly correlated with glyphosate usage on core crops, age-adjusted deaths due to Alzheimer’s disease over time had a correlation coefficient R of 0.917 with glyphosate usage on corn and soy crops in the United States, with a p-value of 2.2E-7 for the probability of a chance occurrence [57].
Also surprisingly, autism and cancer share common features involving mitochondrial dysfunction and dysregulated metabolic pathways, including p53, AKT, mTOR, WNT, NOTCH, and MAPK [107]. There is considerable overlap in the risk genes linked to both conditions [108]. Dysregulated ERK/MAPK signaling can lead to mitochondrial dysfunction upon toxic exposures in both neurons and tumor cells [109]. PIN1 overexpression is a well-known feature of tumor cells, where it facilitates mitosis, proliferation, and metastasis [110].
By contrast, we argue here that PIN1 is suppressed in neurons in association with autism, as is the case for many neurodegenerative diseases, especially Alzheimer’s [111,112]. A neuron is a fully differentiated cell type that is intricately connected to large networks of other neurons. They do not have the option of dividing in the face of stressors. As a consequence, upon toxic exposures, their regulatory pathways have an opposite effect on PIN1 compared to cancer cells, and this drives them toward apoptotic signalling rather than inducing cellular proliferation following severe DNA damage. Oxidative stress can cause severe DNA damage [113]. Much of what is known about PIN1’s function in cells was discovered through studies on cancer cells. In future sections we will reference this literature to help explain the many roles of PIN1 in biology.
Overexpressed mTOR Signaling Pathway: Autism is associated with increased dendritic spine density and reduced developmental spine pruning in neurons. These defects are correlated with overexpressed mTOR signaling and impaired autophagy. This potential for autophagy impairment would result in serious interference in the associated cell death mechanisms that can consequently lead to development of disease [114]. Designer mice have been created with constitutively expressed mTOR activity due to a defect in gene expression of proteins that inhibit mTOR. These mice exhibit autistic behaviors and have the same characteristic spine pruning defects in pyramidal neurons in the temporal lobe, along with impaired autophagy [115]. Microglia play an important role in synaptic pruning, as mice with deficient autophagy in microglia are impaired in synaptic pruning and exhibit behaviors characteristic of autism [116].
Wnt/β-catenin Signaling Pathway: Several of the genes associated with autism converge in the regulation of the Wnt/β-catenin signaling pathway. Both gain and loss of function in this pathway contribute to abnormalities in embryonic brain development associated with autism [117]. Cultivated neurons exposed to glyphosate were impaired in differentiation and growth, eliciting shorter and unbranched axons, and developing less complex dendritic arbors compared to controls [118]. These features are characteristic of impaired neuronal maturation in autism [119]. In the glyphosate study, it was found that both the expression of Wnt5a and the activity of the serine/threonine kinase CaMKII were decreased [118]. Autophosphorylation of CaMKII increases its activity and prolongs the duration of its active state. A missense mutation in CaMKII that results in impaired autophosphorylation and more rapid turnover is associated with autism. When this mutation was introduced in mice, they displayed autistic-like behaviors [120]. Rat pups exposed to glyphosate in utero showed inhibition of the Wnt5a-CaMKII signaling pathway, associated with defects in motor activity and cognitive function [121].
The corpus callosum is the largest white matter tract in the brain, and it connects the two cerebral hemispheres together. Wnt5a-evoked CaMKII signaling instructs specific growth and guidance behaviors in the corpus callosum, controlling its development [122]. Intriguingly, the corpus callosum is thin and underdeveloped in PIN1 knockout mice [123]. Many neurodevelopmental disorders have been linked to malformation of the corpus callosum, including autism, ADHD, and schizophrenia [124]. BTBR mice, a well-established model for mouse autism, exhibit complete agenesis of the corpus callosum [125]. A large percentage of humans suffering from agenesis of the corpus callosum display many traits associated with autism, including deficits in communication and social skills and repetitive behaviors [126].
The DAPK1 regulatory function, which inhibits PIN1 activity, is central to CaMKII autophosphorylation of threonine at position 286 (Thr286), which is required for synaptic long-term potentiation (LTP) and depression (LTD) [127,128]. Both LTP and LTD, which oppose each other, are involved in synaptic plasticity. The autophosphorylation of CaMKII results in the binding of the molecule to the N-methyl-D-aspartate (NMDA) receptor (NMDAR) subunit GluN2B and accumulation of CaMKII during LTP. DAPK1, through activating calcineurin (CaN), blocks CaMKII from binding to GluN2B by competitive inhibition. This creates a fine balance, regulated by Ca2+/CaM influx, which determines whether CaMKII will become attached to GluN2B (establishing LTP), or not (establishing LTD), a balance that is deregulated in autism. Specifically, DAPK1 overexpression, which we have argued can happen due to glyphosate’s inhibition of melatonin synthesis, inhibits the accumulation of CaMKII in the synapse following LTP stimuli [128].
Wnt/β-catenin signaling increases both mRNA expression and protein synthesis of the cell adhesion molecule neuroligin-3, which in turn is essential for the maturation of synapses. Genetic mutations in neuroligin-3 are associated with autism [129]. β-catenin is a major substrate for PIN1 in neural progenitor cells. The developing brain of PIN1 knockout mice shows reduced expression of β-catenin during differentiation, leading to significantly fewer upper layer neurons in the motor cortex [130].
The Integrin β1/FAK/SRC Signaling Pathway: Tetraspanin 7 (TSPAN7) is a master regulator of morphological changes that take place during cell differentiation through cytoskeleton remodeling [131]. Focal adhesion kinase (FAK) is a tyrosine kinase that regulates cellular adhesion, motility, proliferation, and survival, and it interacts with TSPAN7 through the Integrin β1/FAK/SRC signaling pathway. FAK plays an important role in neural migration, dendritic morphology, axonal branching, and synapse formation, all of which are dysregulated in autism [132]. Activation and autophosphorylation of FAK promote neurite formation in neurons [133]. TSPAN7 knockout rats exhibit autism-like behaviors, and this has been linked to impairments in this pathway, which is critical for neurite outgrowth [134].
One of the major pathogenic features of autism is reduced cell migration. Lymphoblasts share features with neurons, and it has been discovered that growing them in culture can serve a useful role to identify systemic pathologies in pathways linked to specific diseases. The protein expression level of FAK is significantly decreased in autistic lymphoblasts, and this was associated with increased adhesion properties and decreased migration, attributed to impaired FAK function in the lymphoblasts [132]. PIN1 isomerizes two prolines in FAK adjacent to Ser-910 and Ser-571, and this leads to dephosphorylation of FAK Tyr-397 [110]. PIN1 isomerization is required to recruit the phosphatase to FAK. Dephosphorylation of FAK at Tyr397 inhibits FAK kinase activity, promoting the disassembly of focal adhesion and enhancing tumor cell metastasis [135]. Experimentally, FAK Tyr-397 dephosphorylation causes cells to round up and lose their attachment to focal adhesions, promoting cellular migration [136]. Thus, low expression of PIN1 would explain the pathology observed in autism, because it leads to suppression of FAK Tyr-397 dephosphorylation. Autistic neurons have reduced migratory capabilities due to increased adhesion complexes that lead to migratory defects, a process in which PIN1 inactivation and DAPK1 over-activation are major participators. This defect interferes with the maturation process of neurons in the brain during neurodevelopment.