Ca2+ ion channel blockers efficiency in suppressing pentylenetetrazol-induced epileptiform seizures in kcnj10a morphant zebrafish

The following essay is part of my masters in neuroscience. It suggests a unique explanation for the quick and multi-systemic spread of epileptiform seizures through astrocytic Ca2+ signaling rather than the commonly researched neuronal origin. The theory here pose a potential for alternative and cheap pharmacological intervention to contain or even prevent epileptic seizures.

Introduction

Epilepsy

Epilepsy is a common neurodevelopmental disorder that manifest itself by recurring epileptic seizures.

The are many causes for epilepsy, yet it is believed, today, that most epilepsy cases have one (monogenic) or more (heterogenic) genetic basis (Thomas & Berkovic, 2014). Following the advances in genome sequencing technology, we are now aware of more than 900 genes associated with epilepsy and 84 are considered to be causal. A majority of these epilepsy causing genes are encoding ion channels (28) and enzyme modulators (25) (Wang, et al., 2016).

Epilepsy manifest itself in a variety of seizure phenotypes and the classification is based on the cortical parts involved and the type of somatic or cognitive impairments associated with the seizure (Wood, 2012):

• Partial seizure – Involves one hemisphere of the brain
• Generalized seizure – Involves both hemispheres of the brain and can be divided into two types
• Absences seizure – Involves some loss of consciousness and interruption of ongoing activities.
• Tonic-clonic – Involves loss of consciousness muscolar contraction and clonic jerking

There are currently twenty three government approved (FDA) drugs for epilepsy (Asadi-Pooya & Sperling, 2015) most of them used to treat one or two specific types of epilepsy by modulating neuronal transmission by altering the action of Glutamate or GABA signalling, ion channels (e.g K+, Ca2+ or Na+) and synaptic protein vesicles. The vast majority of the drugs for epilepsy target the neurons, while a new body of evidence suggest that targeting glia cells might contribute to new pharmacological treatment discoveries. (Devinsky, Vezzani, Najjar, De Lanerolle, & Rogawski, 2013).

Potassium channels dysfunction in epilepsy

Potassium (K+) channels are essential part of the central nervous system and facilitate the resting potential of neurons and enhance depolarization followed by excitatory neurotransmission (Cooper, 2010). There are 3 main types of potassium channels in the CNS: 

• Inward rectifier potassium channels - Kir 
• Two-pore domain leak channels – K2P
• Voltage gated channels - KV 

Today, more than 30 genes encoding for all three types of K+ channels have been associated with epilepsy (Kóhling & Wolfart, 2016).

Excitation of a neuron relies on inward stream of sodium (Na+) cations and outwards release of K+ to the extracellular space, therefore hyperexcitability can occur when one of the K+ channels are dysfunctional ( Sibille, Dao Duc, Holcman, & Rouach, 2015).

Mutations in the KCNQ2 (encoding Kv7.2) and KCNQ3 (encoding Kv7.3) have been associated with benign familial neonatal convulsions (BFNC), Rolandic epilepsy and drug-resistant epilepsy (Brenner & Wilcox, 2012). In mouse models, knocking out KCNQ2 and KCNQ3 resulted in spontaneous seizures along with a reduced threshold to electrically induced seizure while expressing normal hippocampal morphology, suggesting that KCNQ2 and KCNQ3 mutations might be causing seizures due to dysregulation of neurons depolarization (Singh, et al., 2008).

While this is only one example, it is clear that K+ channels have distinct role in regulating neuronal excitability and therefore genes that encode for these channels are in the centre of epilepsy research. (Brenner & Wilcox, 2012).

Astrocytic potassium buffering in the CNS

The majority of research around epilepsy is still neruocentric, yet lately more evidence that glia cells have a role in neurodevelopmental disorders in general and specifically in epilepsy have emerged (Patel, Tweari, Chaunsali, & Sontheimer, 2019).

Astrocytes account for ~20% of the brain and play a crucial role in maintaining the tripartite synaptic homeostasis and information control (Perea, Navarrete, & Araque, 2009), part of this is done by passive, pH, voltage-gated and Ca2+ activated K+ buffering through various K+ channels such as:

• Kir4.1 - inward rectifying channel unique to glia cells 
• K2P – two pores pH modulated K+ channel
• Ca2+ activated channel – activate by intracellular levels of Ca2+
• Kv – Voltage activated K+ have been mainly documented in perinatal or early development stages

Kir4.1, though, has been shown to be the major channel responsible for K+ conductance in astrocytes due to its low input resistance (Seifert, Henneberger, & Steinh?user, 2018).

Kir4.1 dysfunction and mutations in its encoding gene KCNJ10 have been widely associated with epilepsy and EAST syndrome (Nagao, et al., 2013; Mukai, et al., 2018; Dai, et al., 2014) due to the astrocytic channels nature in buffering high synaptic levels of K+ that have signifcant effect in hyperexcitability in epilepsy.

Adding to that, pilocarpine-induced temporal-lobe epilepsy model in rats have shown to increase the expression of Kir4.1 channels in astrocytes suggesting that it plays a significant role in the disease (Nagao, et al., 2013).

The KCNJ10 gene and Kir4.1 channel, therefore, pose a potential research focus to finding new glialcentric pharmacological targets to treat epilepsy and will be the center of this research.

Astrocytic calcium signalling in epilepsy

Research from recent years has shown that astrocytes may be involved in the spread of seizures across neural network through a mechanism used to synchronize firing in remote neural networks by Ca2+ waves propagated through astrocyte-to-astrocyte gap junction connection (Tang, Zhang, Ma, Zhang, & Yang, 2017; Parys, C?té, Gallo, DeKoninck, & Sík, 2010) and Ca2+ depndent gliotransmission (Carmignoto & Haydon, 2012).

These mechanisms can facilitate two phenotypes of epilepsy:

• Hyperexcitability of a neuron through gliotransmission (Glutamate)
• Activation of large neural networks through astrocyte to astrocyte Ca2+ signaling

Supporting that, calcium channel blockers like verapamil and amlodipine have shown to suppress epileptiform field potentials and protection against electrical induced convulsions  (Sharma, Gehlot, & Parashar, 2014; K?hling, Str?ub, & Speckmann, 1994; Han, Guan, Wang, Hatzoglou, & Mu, 2015).

There is a growing potential for calcium channel blockers to be used as anticonvulsant medication in epilepsy, yet a lot is unkown regarding the efficacy in different epileptic syndromes.

Research question

Following the previously discussed findings my main question and gene of interest is:

Can Kir4.1 potassium channel dysfunction enhance epileptiform seizures through increased astrocytic Ca2+ signalling?

Previous research suggested that the gene KCNJ10, encoding for the inward rectifying K+ channel, Kir4.1, is involved in epilepsy (Nagao, et al., 2013; Mukai, et al., 2018; Dai, et al., 2014) and that astrocytes Ca2+ waves are involved in inducing epileptoform seizure  (Tang, Zhang, Ma, Zhang, & Yang, 2017).

In this work I would like to explore a possible connection between both.

Hypothesis

The dysfunction of K+ channels in the astrocyte due to KCNJ10 mutations and the increased extracellular positive charge  (Raimondo, Burman, Katz, & Akerman, 2015) can trigger a Ca2+ inward flux through Ca2+ channels in the astrocyte, through T-Type Ca2+ channels, initiating local and remote Ca2+ waves and Ca2+ activated signalling, causing hyper excitability through gliotransmission.

This, presumabley, can be modulated by administration of Ca2+ channel blockers to attenuate the glial signalling.

The hypothesis would then be:

Ca2+ ion channel blockers have higher efficiency supressing pentylenetetrazol-induced epileptiform seizure in kcnj10a morphant zebrafish

Methodological approach

In order to test the suggested hypothesis I have chosen the zebrafish model as it is been used both to model epilepsy in human due to its orthologue nature (Norton, 2013; Sicca, et al., 2016; Zdebik, et al., 2013).

The test will be done by PTZ induced epileptiform activity  (Barabn, Taylor, Castro, & Baier, 2005) in a group of wild type and kcjn10a knock-down zebrafish, both expressing GCaMP as a reporter of synaptic activity  (Burrows, et al., 2020). The seizures will be measured by long-term EEG recording as initialy presented by Zdebik, et al. (2013) and  by calcium imaging as reviewed thoroughly in Burrows, et al. (2020) work.

Both group of WT and kcnj10a-KD will be then given Ca2+ ion channel blocker ethosuximide (ETZ) and remeasured for both spatial (calcium imaging) and magnitude (long-term EEG) of seizures.

Zebrafish:

As previously described, KCNJ10 encodes the Kir4.1 K+ channel, expressed only in glial cells and not in neuron. It will therefore make an easy and proven target to knockdown in the zebrafish and has been established as a model for EAST syndrome in zebrafish by Mahmood, et al. (2013).

For measurement purposes, both WT and KD fish will transgenically express GCaMP to optically measure synaptic activity by calcium imaging.

PTZ Induced seizures

Based on Barabn et al. (2005) model, all fish will be bathed in a medium with 15nM of PTZ that has shown to induce stable pattern of reoccuring seizures.

Ca2+ ion channel blcoker

While many Ca2+ channel blockers have shown to cause heart failures and early death in larval zebrafish, ethosuximide (ETX) have been shown to be effective in ZF model of epilepsy by Gawel, et al.(2020) and will be presented in the embeding medium 45 minutes after PTZ.

Measurement

Two methodologies will be used to measure the effect on both groups:

• Spatial distribution of the ictal discharge by calcium imaging (Burrows, et al., 2020)
• Magnitude of seizures by Long-term EEG magnitude (voltage) of the epileptic activity (Zdebik, et al., 2013).

Predictable outcome

Based on the hypothesis, the expected outcome will be higher magnitude and wider distribution of ictal activity in kcnj10a KD ZF compared to WT but higher efficiency of attenuation and containment of ictal activity  (delta) following ETX in kcnj10a KD ZF compared to WT.

Limitations

kcnj10a ZF model of human KCNJ10

As previously discussed, many of the epilepsies are heterogenic and KCNJ10 mutations in humans can be found in other neurodevelopmental disorders like ASD (Sicca, et al., 2011).

In addition, zebrafish models present ‘duplicated genes and cellular regeneration’ (Phillips & Westerfield, 2014) that do not exist in mammals and can limit the translational value of the findings.

Ca2+ blocker ethosuximide peripheral effect 

ETX has been shown to be effective in low concentrations in ZF, but as it blocks T-type Ca2+ channels throughout the CNS and can have an effect on electrical gradient in synapses and around the CNS, widely affecting locomotive behaviour in ZF (Ji, et al., 2017).

GCaMP

While using GCaMP will pose a powerful measurement tool for the spatial distribution of a seizure in the ZF brain, it may have an effect on Ca2+ gap junction signalling and other Ca2+ based processes and there is little evidence of its effect on glial cells.

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