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Unfriendly estrogen, GABAₐR disbands
Lake Forest College
Lake Forest, Illinois 60045
Estrogen leads to excitatory neurotransmission through glutamate receptors in epilepsy, but the mechanism behind impaired inhibitory neurotransmission is unknown. GABAA/gephyrin clusters are destabilized within the plasma membrane with E2 treatment.
Is the female sexual developmental hormone, estrogen, implicated in Epilepsy? Estrogen is a hormone essential for animal development and behavior. Its alterations have been associated with Alzheimer’s disease, Schizophrenia, Epilepsy and other psychiatric disorders1,2. Epilepsy is a neurological disorder that affects 65 million people worldwide. The major sign and symptom of epilepsy is seizures which could involve convulsive movement. If the convulsive movement is lacking, it could be an absence seizure3. In Epilepsy, the neurons are hyperexcited due to either mutations or a structural cause (Ex. Injury). Current treatments include anti-epileptic drugs (AEDs), vagal nerve stimulation (VNS), ketogenic diet and surgery4.
Studies have shown that estrogen can increase glutaminergic transmission in hippocampal mice and cause prolonged LTP, long term potentiation5. This would mean that estrogen is likely to increase seizure risks in epileptic patients5,6. Additionally, it has also been shown that AMPA receptor trafficking to the membrane has increased under estrogen treatment (leads to depolarization under glutamate binding)7. It is known that estrogen plays a role in increasing seizure activity by indirectly targeting glutamate G-couple protein receptors, which are receptors that lead to a signaling cascade, eventually depolarizing the neuron8. These results demonstrated mechanisms behind estrogen induced excitatory neurotransmission.
Studies have shown that estrogen acts on the cells via three estrogen receptors (ERα, ERβ, and G protein-coupled ER/GPER)2. Additionally, it is also known that estrogen suppresses inhibitory synaptic transmission and that inhibition is primarily mediated by GABAAR9. GABAA receptors bind to GABA (an inhibitory neurotransmitter) and allow influx of chloride into the postsynaptic membrane which leads to hyperpolarization. However, the mechanism through which estrogen reduces neuronal inhibition through GABAAR’s is not well understood. Mukherjee et al. reported in PNAS that reduced stability of GABAA receptors impairs neuronal inhibition.
Mukherjee et al. studied the effect of E2 or estradiol, analog of estrogen, on GABAA receptors and gephyrin clusters using confocal microscopy in cultured cortical neurons. Gephyrin is a protein that binds the cytoskeleton and intracellular loop domain of GABAA receptors. Its job is to secure GABAA to position. E2 treatment reduced the synaptic puncta (points of clusters) compared to control neurons, which did not receive the E2 treatment. This effect was visible in α2 subunits containing GABAA/gephyrin clusters and in γ2 subunits containing GABAA/gephyrin clusters.
To understand which ER receptor mediates this cluster reduction, the authors exposed the cortical neurons to either an ERα agonist (PPT) or ERβ agonist (070). They found that both agonists decreased the synaptic GABAAR/gephyrin clusters/puncta just like E2 did. This demonstrated that both ER receptors impair the synaptic inhibition and that E2 works through both pathways.
Mukherjee and colleagues next explored whether the cell surface levels or total protein levels of both GABAAR and gephyrin were altered. This is necessary to understand if the number of GABAAR are having an effect on the neuronal inhibition. Using immunoblot, the authors isolated the surface GABAA receptors from the cultured cortical neurons and immunoblotted with antibodies against both types of GABAAR subunits (containing either α2 or γ2). To ensure the surface protein extraction only contained surface GABAAR and not cytosolic GABAAR, GAPDH (a cytosolic enzyme) was used as a control. It was found that there was no difference between control and E2 treatments for total GABAAR and cell surface GABAAR with either subunit (α2 or γ2). A similar result was found with the protein gephyrin. This demonstrated that E2 did not alter the amount of gephyrin or GABAAR expression.
Next, the authors assessed if the binding of GABAAR to gephyrin is modified under E2 treatment. Using immunoprecipitation, antibodies were targeted against α1 subunit of GABAAR (another type of GABAAR) or mouse IgG (control) under control and E2 treatments. Precipitated material was next immunoblotted either against gephyrin antibodies or α1 antibodies. No significant difference was found, indicating that gephyrin’s ability to bind GABAAR was not compromised with E2 treatment.
In a final set of experiments, the researchers assessed the effect of E2 on dynamics of GABAAR. Using single particle tracking method, the authors labelled GABAAR with quantum dots in order to track them. They found that GABAAR had a reduced dwell time in the synapse. That is, the receptors spent less time in the same location. This would explain how E2 could impair neuronal inhibition without reducing the total or surface receptors. Reduced dwell time would make it harder for hyperpolarization to occur since the channel is moving around and would lead to seizures (Figure 1).
Figure 1: ERα and ERβ mediated reduced dwell time of GABAA receptors. Estradiol treatment is known to suppress synaptic inhibition leading to seizures. Mukherjee et al.2 report that E2 activates both of its receptors (ERα and ERβ) leading to destabilization of GABAAR/gephyrin complex and reducing dwell time of the complex. The total number of GABAAR or gephyrin on cell surface is similar to control. However, the destabilization of the complex leads to reduced neuronal inhibition which can contribute to seizures.
Mukherjee and colleagues’ study is important because it provides a deeper understanding of how estrogen affects GABAAR, eventually leading to suppression of neuronal inhibition. Another reason is that this study helps highlight possible therapeutic targets. If we are aware that estradiol reduces neuronal inhibition through a post synaptic membrane where it targets the estrogen receptors and reduces the stability of gephyrin scaffold, then drug targeting to increase stability could be researched in the future. If the gephyrin scaffold is stabilized on the membrane, the GABAAR will be stabilized, allowing for chloride influx and normal neuronal inhibition. Although this study shows great results, it lacks a few experimental parts which could help in understanding the results. The sexual specific effect of estradiol on neuronal inhibition has been noted, however, the authors have not included any such results with this study. Another issue of this study is that there are inconsistencies with the subunits measured (such as α1 or α2) and estrogen receptors targeted. If all subunits are analyzed for each part, a better understanding could be obtained.
Past research has focused on understanding the estrogen mediated excitatory neurotransmission5,7,8. There has been growing research in understanding the impaired synaptic inhibition due to E2 treatment. The current study provides a deeper understanding of the mechanism behind the epileptic pathology of reduced inhibition. However, further research is required to elucidate the precise mechanism by which E2 affects the stability of the proteins. Estrogen has been known to modulate MAPK signaling and GSK3B protein. Additionally, these proteins are known to phosphorylate gephyrin2. A further understanding of how phosphorylation is mediated could help uncover treatment for epilepsy.
- Hughes, Z. A., Liu, F., Marquis, K., Muniz, L., Pangalos, M. N., Ring, R. H., … & Brandon, N. J. (2009). Estrogen receptor neurobiology and its potential for translation into broad spectrum therapeutics for CNS disorders. Current molecular pharmacology, 2(3), 215-236.
- Mukherjee, J., Cardarelli, R. A., Cantaut-Belarif, Y., Deeb, T. Z., Srivastava, D. P., Tyagarajan, S. K., … & Moss, S. J. (2017). Estradiol modulates the efficacy of synaptic inhibition by decreasing the dwell time of GABAA receptors at inhibitory synapses. Proceedings of the National Academy of Sciences, 114(44), 11763-11768.
- Moshé, S. L., Perucca, E., Ryvlin, P., & Tomson, T. (2015). Epilepsy: new advances. The Lancet, 385(9971), 884-898.
- Elliott, R. E., Morsi, A., Kalhorn, S. P., Marcus, J., Sellin, J., Kang, M., … & Devinsky, O. (2011). Vagus nerve stimulation in 436 consecutive patients with treatment-resistant epilepsy: long-term outcomes and predictors of response. Epilepsy & Behavior, 20(1), 57-63.
- Kramár, E. A., Chen, L. Y., Brandon, N. J., Rex, C. S., Liu, F., Gall, C. M., & Lynch, G. (2009). Cytoskeletal changes underlie estrogen’s acute effects on synaptic transmission and plasticity. Journal of Neuroscience, 29(41), 12982-12993.
- Velíšková, J., De Jesus, G., Kaur, R., & Velíšek, L. (2010). Females, their estrogens, and seizures. Epilepsia, 51(s3), 141-144.
- Zadran, S., Qin, Q., Bi, X., Zadran, H., Kim, Y., Foy, M. R., … & Baudry, M. (2009). 17-β-estradiol increases neuronal excitability through MAP kinase-induced calpain activation. Proceedings of the National Academy of Sciences, 106(51), 21936-21941.
- Boulware, M. I., Weick, J. P., Becklund, B. R., Kuo, S. P., Groth, R. D., & Mermelstein, P. G. (2005). Estradiol activates group I and II metabotropic glutamate receptor signaling, leading to opposing influences on cAMP response element-binding protein. Journal of Neuroscience, 25(20), 5066-5078.
- Huang, G. Z., & Woolley, C. S. (2012). Estradiol acutely suppresses inhibition in the hippocampus through a sex-specific endocannabinoid and mGluR-dependent mechanism. Neuron, 74(5), 801-808.
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