Glyphosate Causes Irreversible Abnormal Growth & Delayed Development of Embryonic Rat Neuronal Cells
Primary rat neuronal cultures exposed to glyphosate showed abnormal
morphology, delayed differentiation and decreased activity of a Wnt signalling
pathway involved in embryonic development Dr Eva Sirinathsinghji
new study finds that cultured embryonic rat neurones exposed to sub-lethal
doses of glyphosate show a delay in development characterised by reduced
neuronal polarisation, complexity and morphology. Underlying the abnormalities
was reduced activity of a Wnt protein Wnt5a, a
signalling transduction molecule, as well as its downstream signalling effector
protein kinase II). Effects of glyphosate were rescued through the restoration
of normal Wnt signalling activity, validating its
involvement in glyphosate’s neurotoxic effects.
toxicity of glyphosate herbicides and their formulation products are widely
recognised in the scientific literature, with evidence accumulating on its
neurotoxic effects (see  Banishing Glyphosate, Special
ISIS report). Studies have linked glyphosate to Parkinson’s
disease following acute exposure, increased seizures in epileptic patients, and
deficits in learning and memory; and there is epidemiological evidence of
increased behavioural problems in children such as ADHD (see ). Further, a
recent investigation into neurotoxic mechanisms identified increased oxidative
stress and cell death in the hippocampus of exposed rats linked to increased
levels of extracellular glutamate neurotransmitter . Nevertheless, cellular
mechanisms underlying nervous system damage remained unknown.
The formation of neural circuits during development
depends on many factors including the polarity of neuronal cells. Polarisation
is crucial for the development and correct functioning of various cell types.
Neurones are some of the most highly polarised cells, with functionally and
structurally distinct axons and dendrites extending from the cell body, which
are essential for information processing and the unidirectional signal flow
that characterises neuronal cells. Neural circuit formation therefore depends
on the morphological complexity and differentiation of neurones and the
establishment of proper neural connectivity. Extrinsic factors also modulate
the development of neural circuitry, such as the extracellular Wnt protein
pathways, which among other things, are involved in the development of neuronal
polarity, axon outgrowth and navigation of neuronal outgrowths to form synaptic
connections with other cells. The abnormalities in polarity and neuronal
differentiation observed in this latest study therefore have implications for
the mechanisms behind glyphosate’s teratogenic and neurotoxic properties.
Indeed, Wnt proteins are much involved in embryonic development , including
neural tube, with defects in neural tubes frequently reported in high
glyphosate-use regions such as Argentina (see ).
levels of glyphosate induced abnormal neuronal morphology
study, led by Dr Rosso at the University of Rosario
in Argentina was performed to further understand how sub-lethal doses of
glyphosate (N-phosphonomethyl glycine) may induce neurotoxicity .
the researchers performed MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide]viability assays on cultured primary hippocampal cells
taken from 18 day old
embryos. It assesses the metabolic activity of cellular enzymes, which reflects
the viability of the cell and is a classic in vitro viability test. This was done to determine the range of sub-lethal doses
that could be used in the rest of the research. Adding 2, 4 and 6 mg/ml of
glyphosate to cells, they found no significant difference in viability below 6
mg/ml. At 6 mg/ml, around 25 % of cells died after 4 days exposure.
With sub-lethal doses estimated at 4 mg/ml,
the researchers went on to see whether these low levels were able to
induced neurotoxicity by assessing the extent of polarisation
during the differentiation process of the neural cells. They did this by
measuring the length and morphology of axonal and dendritic branches. Following
1 day of exposure to glyphosate, 38 % of the cells had axons compared
with 70 % of control cells (see Figure 1a). After 48 hour exposure, 70 % of cells
had axons compared to 80 % of control cells, suggesting a developmental delay
in axon development following glyphosate treatment. Control axons were also significantly
longer, with more branching (see Figure 1b). The length and complexity of
dendrites were also significantly lower than control neurones. The effects were
irreversible, as neurones exposed to glyphosate for a
day and then cultured in fresh medium without glyphosate for another day
still showed a significant reduction in axonal length and branching.
Figure 1 Sub-lethal
doses (4mg/ml) of glyphosate induce abnormal morphology of embryonic rat
neurones and delayed differentiation; (A) Confocal microscope images of immuno-labelled
neurones with the axonal marker Tau1 (green) and β-tubulin (red) following
1, 2 and 4 days in vitro (DIV) culture; (B) Number of polarised cells, total
axon length, and total axonal branch tips in glyphosate treated cells following
2, 4 DIV; (C) Total dendritic length and total dendritic branching tip number
(TDBTN) in glyphosate-treated neurones following 2 and 4 DIV
Analysis of growth cones (situated on the
very tips of developing dendrites and axons that seeks synaptic targets with
other neurones) showed that glyphosate caused a significant reduction in their
size. Staining for markers of growth cones of minor processes as well as
prospective neurones revealed a 70 % and 54 % reduction respectively. Furthermore,
the organization of actin (a structural protein abundant in neurones) around the
growth cones was disrupted. Neuronal polarisation depends on the organisation of the actin cytoskeleton. Application
of a chemical that destabilises actin, cytochalasin D, to neurones was able to
abolish the effects of glyphosate, showing that glyphosate neurotoxicity works
via disrupting actin cytoskeleton organisation and stability.
the role of Wnt in the development of neural circuitry well known, the
researchers went on to see if any Wnt pathways were dysregulated by glyphosate
treatment. First they looked at expression levels of different Wnt proteins.
They found that glyphosate treatment reduced the mRNA expression
(by approximately 25 %) of one Wnt gene, Wnt5a, after
24 hours of 4 mg/ml glyphosate treatment.
After 48 hours, there was also a significant reduction in Wnt5a protein levels.
Wnt5 - previously been shown to be involved in axonal branching and growth,
axon guidance and neurite development, as well as actin organisation - was
disrupted following glyphosate treatment [5-8].
To confirm the involvement of Wnt5a in
glyphosate-induced neurotoxicity, recombinant Wnt5a proteins were added to
neuronal cultures along with glyphosate; this resulted in neurones with similar
morphology to controls (see Figure 2). After 2 days of glyphosate treatment
alongside recombinant Wnt5a expression, neurones showed normal
axon branching and lengths as well as normal dendritic branching complexity.
Figure 2 Glyphosate
neurotoxicity rescued by addition of Wnt5a protein to neuronal cells; (A) Confocal
microscope images of neurones treated with glyphosate and recombinant Wnt5
following 2 days in vitro (DIV) culture; (B) Axonal length and number of axonal
branch tips in cells treated with glyphosate with and without Wnt5a; (C) Dendritic
length and total dendritic branching tip number (TDBTN) in cells treated with
glyphosate with and without Wnt5a
Wnt proteins can signal through different pathways, referred to as the canonical and non-canonical
pathways. The canonical Wnt pathway causes the accumulation of β-catenin
in cells that act as a transcriptional activator. There are two non-canonical
pathways: the non-canonical planar cell polarity
pathway and the non-canonical Wnt/calcium dependent pathway. The latter helps
regulate calcium release from the endoplasmic reticulum in order to control
intracellular calcium levels. Increased calcium levels leads to activation of
signalling molecules such as Ca2+/calmodulin-dependent protein
kinase II (CaMKII), which is involved in many pathways including those
important for learning and memory, development of
neural circuits and synaptic plasticity, calcium homeostasis, and reuptake in
cardiomyocyte heart cells among other things. The researchers found that
following 24 hours of glyphosate exposure, cultured hippocampal neurones showed
reduced levels of phosphorylated CaMKII levels by 25 %. The canonical pathways
were not altered by glyphosate treatment. Knocking down CaMKII expression using
RNA interference techniques abolished glyphosate’s neurotoxic effects on the
neurones, indicating that Wnt5a requires CaMKII to elicit its effects on axon
growth and elongation. Further, knocking down CaMKII in untreated neurones
caused abnormalities similar to that seen under glyphosate treatment,
suggesting that glyphosate’s effects work via a reduction in CaMKII activity.
Though glyphosate has been shown to affect both the health of
people and planet, new research is helping to elucidate the underlying
mechanisms of its toxic effects. This work serves to further confirm the
dangers of widespread glyphosate use.