07 Dec Neuroactive Steroids Evoke Inhibition Through Protein Kinase C Mechanism

MedicalResearch.com Interview with:

Dr. Paul Davies

Dr. Paul Davies PhD
Tufts University School of Medicine
Department of Neuroscience
Boston, MA 02111

MedicalResearch.com: What is the background for this study? What are the main findings?

Response: Inhibition in the brain regulates neuronal action potential generation, too little inhibition can directly cause conditions such as epileptic seizures, neurodevelopment disorders, and neurodegenerative disorders. The main type of inhibition in the mammalian brain occurs when the neurotransmitter γ-aminobutyric acid (GABA) binds to the GABA type A receptors (GABAARs), a ligand-gated ion channel. Once bound with GABA, the receptor changes shape to open the ion channel allowing negative charged chloride ions to flow through into the cell and inhibiting excitation steaming from positive charged ions flowing through opposing excitatory ion channels. GABAARs mediate both synaptic (phasic) and extrasynaptic (tonic) inhibitory neurotransmission in the CNS and are the sites of action of benzodiazepines, barbiturates, general anesthetics and neuro-active steroids. We have been focused on the extrasynaptic GABAARs that mediate tonic inhibition. In the dentate gyrus of the hippocampus, neocortex, striatum and the thalamus tonic inhibition is largely dependent on GABAARs composed of α4, β2/3, and δ subunits. Neuro-active steroids play a central role in regulating behavior via their ability to allosterically enhance GABAARs, particularly extrasynaptic α4-containing GABAARs. Allosteric enhancement means that neuro-active steroids bind to GABAARs and cause a further change in the structure of the ion channel allowing it to remain open for longer.

For the last few decades, allosteric enhancement of GABAARs by neuro-active steroids has been the prevailing explanation for how the steroids increase inhibition in the brain. However, recently we described a new mechanism where the neuro-active steroid, THDOC, increased the association of protein kinase C (PKC) with extrasynaptic α4β3 subunit-containing GABAARs. The increase in PKC-mediated phosphorylation of α4 and β3 subunits leads to an increase in membrane insertion from intracellular stores, an increase in GABAAR stability in the membrane, and a prolonged increase of tonic inhibition, even after when the neuro-active steroids have been removed.
For this present study we asked whether other neuro-active steroids demonstrated the same metabotropic activity. We tested another endogenous neuro-active steroid, allopregnanolone (ALLO), and the synthetic neuro-active steroid, ganaxolone. In collaboration with SAGE Therapeutics, we also tested another synthetic neuro-active steroid, SGE-516.

We found that all the neuro-active steroids tested were able to allosterically potentiate both synaptic and, to a lesser degree, extrasynaptic GABAARs. Short 15-minute exposures to neuro-active steroids resulted in significantly increase in phosphorylation of β3 subunits, and long lasting enhancement of tonic current. These increases were metabotropic in nature, being dependent upon PKC mediated phosphorylation. Following this short 15-minute exposure we saw a change in synaptic currents only with SGE-516 suggesting a selectivity of this metabotropic pathway to extrasynaptic GABAARs. Although ganaxolone was an effective allosteric modulator, it did not produce a metabotropic enhancement of tonic current suggesting that not all neuroactive steroids work through this pathway.

MedicalResearch.com: What should readers take away from your report?

Response: The main point is that the membrane expression of extrasynaptic GABAARs can be increased following exposure to neuro-active steroids. This increase in GABAAR expression is through a metabotropic, PKC-dependent, mechanism which is independent of the well characterized properties of neuro-active steroids to allosterically modulate the ion channel. Extrasynaptic GABAARs are responsible for mediating the tonic inhibition that regulates the excitability of neurons and the activity of neuronal circuits. Reduction in tonic inhibition is implicated in numerous neurological, psychiatric, and neurodevelopmental disorders that include anxiety, epilepsy, depression, premenstrual syndrome, autism spectrum disorders, and schizophrenia. Therefore, boosting the level of extrasynaptic GABAARs with neuro-active steroids would boost tonic inhibition. This metabotropic mechanism could be a therapeutic target for many conditions.

Additionally, we show that both endogenous and synthetic neuro-active steroids have this metabotropic, PKC-dependent, mechanism for boosting tonic current. However, the synthetic neuro-active steroid ganaxolone did not possess this metabotropic mechanism on tonic current demonstrating that not all neuro-active steroids work through this pathway or have the same efficacies, pointing to potential structure-function dependent pathways.

MedicalResearch.com: What recommendations do you have for future research as a result of this study?

Response: The most obvious aim for future research is determining the exact mechanism behind the increase in PKC mediated phosphorylation. We have previously shown that the increase in phosphorylation of α4 and β3 subunits is not due to a direct activation of PKC by neuro-active steroids. Therefore, an intermediary protein may be involved that promotes the phosphorylation of the GABAAR subunits.

Furthermore, the phosphorylation is apparently specific to certain GABAAR subunits, we detected no changes in phosphorylation or trafficking of GABAAR α1 or α5 subunits. Our future research will be directed towards answering the question of why specific subunits are targeted by this metabotropic mechanism.

MedicalResearch.com: Is there anything else you would like to add?

Response: Utilizing the information from our research to form a basis for therapy is very important. A reduction in tonic inhibition has been noted in several pathological conditions. Such conditions include epilepsy, and Autism Spectrum Disorders. We have begun examining if the anti-epileptic properties of some neuro-active steroids could be mediated by a metabotropic pathway. Additionally, we have recently observed a deficit of tonic inhibition in a mouse model for Fragile X syndrome, the most common form of inherited intellectual disability and a major cause of a diagnosis for autism spectrum disorders. We have observed a significant decrease in tonic inhibition in dentate gyrus granule cells in fragile X mice compared to wild-type mice. A 10 min exposure to a neuro-active steroid induced a >3 fold increase in tonic current in the fragile X mice which was prevented with PKC inhibition. We believe that some detrimental behavioral phenotypes in fragile X syndrome and other neurodevelopmental disorders may be treated by neuro-active steroids working to increase the trafficking of extrasynaptic GABAARs.

MedicalResearch.com: Thank you for your contribution to the MedicalResearch.com community.

Citation:

Neuropharmacology. 2016 Oct 12;113(Pt A):314-322. doi: 10.1016/j.neuropharm.2016.10.010. [Epub ahead of print]
Endogenous and synthetic neuroactive steroids evoke sustained increases in the efficacy of GABAergic inhibition via a protein kinase C-dependent mechanism.
Modgil A1, Parakala ML1, Ackley MA2, Doherty JJ2, Moss SJ3, Davies PA4.

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Last Updated on December 8, 2016 by Marie Benz MD FAAD