Welcome back! The peer-reviewed literature relevant to Dravet that was published in April focused on investigations at the gene level, with one exception for a zebrafish metabolism study. We are still working on the best format for this blog, so please let us know if you have any feedback!

1. Overview of Models of SCN1A

Schutte, R., et. al. (Jan 2016). Model Systems for Studying Cellular Mechanisms of SCN1A-Related Epilepsy. Journal of Neurophysiology, v. 115. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/26843603

Although published in Jan. 2016, this article was just released in full text last month. It is a comprehensive analysis of the various models researchers use to study SCN1A , including mice, fruit flies, iPSC’s, and a few more. This is a great overview for those not familiar with the methods and models used to study Dravet patients.

Heterologous Systems: These were the first models that showed how SCN1A mutations affected cells. They were made by cloning sodium ion channels expressed in nonexcitable cells (not neurons), and showed that some missense mutations cause loss of function while others show gain of function. They are good for testing whether compounds are sodium channel blockers and showed that the same sodium ion channel can function differently when expressed in different cell types.

Mouse Models: Mice and humans are quite similar genetically and physiologically, making them useful for studying DS. Animals have two copies of each gene, one from each parent. In most cases of DS, the child has one normal copy of SCN1A (often called wild-type, or control), and one mutated copy. It can be difficult to know whether symptoms result from a dysfunctional protein made by a mutated SCN1A, or whether symptoms result simply from having only one healthy copy of the gene. Thus, “knock-out” and “knock-in” mice were created. Knock-in mice have a mutated copy of SCN1A inserted in their DNA. Knock-out mice have one copy SCN1A removed, ideal for studying how a shortage of healthy SCN1A affects the mouse without inserting the variable of a mutated gene.

Mouse models have revealed reduced excitability of GABAergic inhibitory neurons in the hippocampus and cerebellum and have helped scientists understand how genetic background (the genes that are not SCN1A) and development influence DS. For instance, knocking in the same genetic mutation in different strains of mice can result in a mild clinical presentation with no premature death or a severe presentation with premature death, depending on the strain of mouse used and the generation studied. Various theories about why inhibitory neurons are so affected by SCN1A mutations have arisen from mouse models. Some people believe inhibitory neurons express a higher percentage of the mutant sodium channel than excitatory neurons.

Zebrafish: Zebrafish are small vertebrate fish that can be maintained and used in high volume to screen for SCN1A effects more rapidly than mice. Seizures can be induced by treating the water with chemicals such as PTZ. The fish respond with convulsions and electrical discharges that can be recorded. Researchers make sure to validate each model by comparing how the zebrafish react to known anti-seizure medications with how humans and other models react to those medications. High throughput screening on zebrafish identified clemizole as a potential new anti-seizure medication and scientists are currently studying this antihistamine. Other compounds have been screened and further studied as well. Recent advances in CRISPR technology (a method for editing genomes) has enabled more rapid generation of zebrafish models with targeted mutations.

Fruit Fly: The fruit fly has only one gene coding for sodium ion channels (as opposed to humans who have many more), but that one gene can be spliced and edited in different ways to produce a variety of sodium channels. Recently, several knock-in models of human SCN1A mutations have been created and studied, revealing that GEFS+ and DS mutations appear to affect inhibitory neurons differently. 5-HTP (a serotonin precursor) was identified as a seizure suppressor for DS mutant flies, and has brought older studies on serotonin and seizure activity back to the forefront of research.

iPSC-Derived Neurons (induced Pluripotent Stem Cell –Derived Neurons): While animal models allow us to study the effects of mutations in organisms like mice and fish, there are still substantial differences between these animals and humans. One way to get around these differences is to study human cells instead of animal cells. iPSC’s are cells that are taken from the skin or other area of an actual patient, treated to revert back to their undifferentiated form, and then manipulated to develop into neurons. iPSC’s can be created for each of the animal models, as well as for humans.Using iPSC’s allows the researcher to study actual human cells in a culture, without invasive procedures like brain-sectioning or relying on post-mortem donated tissue. The cells appear to behave similarly to neurons in the mouse and fish models with a few exceptions. One drawback is that it is not currently possible to identify neuronal changes specific to human developmental stages.
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Despite these challenges, there is great potential for iPSC’s, both in human cells and in animal cells. For example: Scientists have transplanted GABAergic interneurons into the brains of epileptic mice, where they were able to integrate into the mouse’s existing brain and affect neuronal response. These transplanted cells were even able to suppress seizures. Whether they help us learn about other animals’ neuronal networks or aid us in understanding our own human neuronal networks, iPSC’s are a powerful research tool.

2. GABA Expression and SUDEP in Mice

Xia, G. (Apr 2016). Altered GABAA receptor expression in brainstem nuclei and SUDEP in Gabrg2+/Q390X mice associated with epileptic encephalopathy. Epilepsy Research, volume 123. http://www.epires-journal.com/article/S0920-1211(16)30049-3/fulltext

GABRG2 mutations are associated with GEFS+ (Generalized Epilepsy with Febrile Seizures and other seizures) and sometimes with Dravet syndrome. Mouse models with these mutations (“knock in”) display SUDEP (Sudden Unexplained Death in Epilepsy Patients) throughout the lifespan of the mouse. Interestingly, though, “knock out” mouse models, which only contain one copy of the functional gene (and an absence of the other copy, rather than a mutation in the second copy), display absence seizures and generally mild cases of epilepsy without increased risk of SUDEP. This study examined the differences between how the neurons functioned in each of these models to try to determine how the first model contributes to SUDEP. They found that GABAA receptors (inhibitory receptors) were decreased and mutated receptors were increased in the knock in (mutated) model, but not in the knock out (one copy missing) model. Because GABA receptors play such an important role in cardiorespiratory (heart rate and breathing) function, and changes in these receptors were observed in the high SUDEP knock in model, they are possibly involved in the heart rate and breathing problems associated with SUDEP.

3. Zebrafish Metabolism

Kumar, M., et. al. (Apr 2016) Altered Glycolysis and Mitochondrial Respiration in a Zebrafish Model of Dravet Syndrome. eNeuro, volume 3(2). http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820792/

Zebrafish with SCN1A mutations exhibit most of the characteristics of Dravet syndrome we see in humans. Scientists use the zebrafish to study seizures, of course, but they also use them to study various bodily processes that may be altered in Dravet syndrome, such as metabolism (the chemical processes inside the body that maintain the chemical environment). One component of metabolism is the breakdown of food molecules, converting energy from food to a usable energy source for the body. Much of this breakdown is done in the mitochondria of cells, small structures often termed the “powerhouses” of the cell. Glycolysis and mitochondrial respiration are two chemical processes in the mitochondria that help convert sugar and fat to chemical energy, which the cell can use for other necessary functions.

In this study, SCN1A-mutated zebrafish showed decreased glycolytic rates and oxygen consumption rates compared to the controls. Treating the mutated fish with a ketogenic (high fat, low carbohydrate) diet returned the rates to control-level. Chemically exciting the neurons in the controls made the glycolytic rate rise quickly and slightly increased the oxygen consumption rate, but both quickly returned to baseline. The mutated zebrafish, however, experienced a slower and exaggerated increase in both rates.

Examining the expression of various glycolytic genes showed 5 genes that were downregulated in the mutated zebrafish, suggesting that lowered metabolism may play a role in Dravet syndrome.

4. Endocannabinoid Receptors

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4804326/

This article was summarized in the February review, and the full text just came out last month.

5. PCDH19 Epilepsy

Liu, AJ, et. al. (May 2016). Genotype and phenotype of female Dravet syndrome with PCDH19 mutations. Chinese Journal of Pediatrics, volume 54(5). Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/27143072

The authors studied 75 female patients with Dravet syndrome in China who did not have an SCN1A mutation. They found PCDH19 mutations in 6 of the 75 SCN1A-negative Dravet syndrome patients (8%), and evaluated the clinical features of these PCDH19-positive patients. This is important to the Dravet community because it highlights the need for routine PCDH19 testing in females with Dravet syndrome who do not have an SCN1A mutations. PCDH19 epilepsy and Dravet syndrome have many features in common, but PCDH19 epilepsy includes generalized tonic clonic seizures and focal seizures, seizures occur in clusters and are fever-sensitive, short seizure duration, rare status epilepticus, varying development delay, and autism spectrum characteristics. For more information on PCDH19 epilepsy, click here: http://www.pcdh19info.org/