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Assessment of neuronal ion channels and their alterations in specific mutations of Landau-Kleffner Syndrome

Marisol Gelacio, Sabrina Najibi, Melissa Roshass, Parth Tank
Department of Neuroscience and Biology
Lake Forest College

Epilepsy is characterized by hyperexcitable and hypersynchronous neuronal firing. Genetic causes of epilepsy vary among cases, and due to complex mechanisms involved in epilepsy, no cure exists for this neurological disorder. One type of epilepsy, Landau-Kleffner syndrome (LKS) has recently been associated with two NMDA (glutamate receptor) mutations (D933N and C231Y). These mutations reduce NMDA receptor trafficking and lead to epileptic neurons. The mechanism for this is not well understood. We propose that the excitation in the neurons may be due to compensatory mechanisms through other epilepsy-linked ion channels. We devised five experiments for each of the ion channels. These experiments will assess the expression of these channels, their functional activity, and expression of the interacting proteins in LKS mutations. We expect upregulation of sodium/AMPA channels or a downregulation of potassium/chloride channels. NMDA subunit RNA knockdown will show similar results to LKS NMDA mutations. Finally, we will determine the effect of down regulating sodium/AMPA channels and upregulating potassium/chloride channels in induced LKS glutaminergic neurons. In depth understanding of the mechanism behind this will allow us to have better therapeutic targets.

 

Background

Epilepsy is a brain disorder characterized by two hallmarks: hyperexcitability and hypersynchronous firing in neurons. This type of firing is presented in patients with or without convulsions (1). The first recorded cases of epilepsy can be traced back to almost 2000 BCE (2). Approximately 2.5 million people in the United States, and 65 million worldwide have been diagnosed with epilepsy. Current studies have shown that epilepsy affects mostly younger children and older adults and 1 out of every 26 individuals (3). The spectrum of etiological factors that lead to epileptic brain dysfunction can range from genetic inheritance to symptomatic changes as a result of brain damage. The genetic components of epilepsy are highly variable as causes of seizures and their intensity vary depending on the type of epilepsy (4). While epilepsy is a diverse disease type and currently lacks a cure, treatments do exist to alleviate epileptic symptoms. These include antiepileptic medications (AEDs), surgery, vagus nerve stimulation and ketogenic diet (5,6,7,8).

In this proposal, we will be studying ion channels (Sodium, AMPA, Potassium, and Chloride channels). Ion channels are crucial to look at because they regulate the information being transmitted from one neuron to another. These channels cause the cell to receive information to generate an action potential or not. Mutations on sodium channel (VGSC) subunits, specifically Nav 1.2 and Nav 1.6, give rise to hyperexcitability in epileptic neurons. AMPA receptors are ligand-gated ion channels for glutamate, which binds and allows for an influx of sodium ions. Mutations on the subunits of these receptors lead to hyperexcitability, causing seizures (14,15). The Kv1 ion channel (when activated by internal Na+) allows intracellular K+ to flow out of the cell, and its mutation can reduce efflux and cause hyperexcitability (16). There are many types of chloride channels or co-transporters that are implicated in epilepsy such as CLC and GABAA, however, the hyperpolarization of cells in epilepsy is lacking due to mutations in the channel (17).

 Recent research advancements have led to a greater understanding of the underlying mechanisms of epilepsy. EAS (Epilepsy Aphasia Syndrome), is a form of epilepsy which results in impairment to writing, speech, language comprehension and reading. Although there are many different forms of EAS, the focus of this proposal will be in Landau Kleffner Syndrome (LKS). LKS is a rare neurological disorder that affects young children. Patients with LKS often exhibit seizures and impairment in reading and language comprehension. In LKS, the function of neurons in the Broca’s and Wernicke’s areas of the brain are impaired as the Broca’s area corresponds to express speech and Wernicke’s area corresponds to speech and language comprehension (13). Previous research has suggested that mutations on the GRIN2A gene may give rise to alternative consequences of hyperexcitability in neurons due to decreased GluN2A which is a subunit of the NMDA receptor (18). Addis et al. presented data showing decreased surface receptor expression for NMDA, which resulted in HEK cells becoming hyperexcitable. This is conflicting because one would expect to see a decrease in hyperexcitability following a decrease in NMDA expression, as NMDA provides an influx of sodium and calcium ions to depolarize the postsynaptic cell. However, it is not known how the lack of NMDA receptors induces this epilepsy. Ten mutations of GRIN2A were previously identified in children with EAS, and two of these mutations were associated with LKS: D933N and C231Y on the NMDA protein (9,10,11,12).

Cardis et al. has discussed that a lack of GluN2A-containing NMDA receptors can lead to oxidative stress, which leads to epilepsy (42). Considering  the significance of ion channels in regulating voltage inside the postsynaptic neuron, it is also possible that ion channels are involved. Therefore, we hypothesize that individuals with mutations of GRIN2A (LKS) produce a compensatory mechanism of hyperexcitability utilizing alternative ion channels (sodium, AMPA, potassium and chloride). Sodium and AMPA channels will be expected to be upregulated, while potassium and chloride channels will be downregulated. We expect only one channel to be dysfunctional as it would be rare for multiple channels to be dysfunctional in the same cells.

 

Significance

Broader relevance: Approximately 2.5 million people in the United States and 65 million worldwide have been diagnosed with epilepsy. Current studies have shown that epilepsy affects mostly younger children and older adults, with 1 out of every 26 individuals being affected (3). Although multiple drugs and treatments have been developed to address different types of epilepsy, there still remains a lack of understanding relating to the genetics and complex interactions of this disease. Research advancements and assessment of potential therapeutic approaches could increase our understanding of LKS.

Intellectual relevance: Landau-Kleffner syndrome (LKS) is a type of genetic epilepsy that is associated with GRIN2A mutations (19).  Previous research shows that GLUN2A mutations, which lead to a lack of NMDA receptors, can cause epilepsy; however, the mechanism behind this is unknown (20). The compensatory mechanisms behind the excitation of these epileptic neurons may involve four ion channels in maintaining the excitatory input into the cell (21). If AMPA or sodium ion channels are observed to be overactive or if chloride or potassium ion channels are observed to be underactive, it can be concluded that this provides the cause for an activation in these neurons. Additionally, it can be observed that one or multiple channel(s) can be involved in maintaining excitatory input. By identifying the channels that are overactive or underactive, AEDs could be utilized as a translational therapeutic approach to restore their function. Interestingly, a majority of LKS patients do not respond to AEDs; therefore, it is possible for multiple channels (at least two) to be dysfunctional in different groups of cells. Designing drugs that can target multiple specific mechanisms that are dysfunctional can be useful to alleviating aversive symptoms that LKS patients suffer from.

 

Specific aims

The purpose of the specific aims stems from the Addis et. al. study by elucidating the mechanisms behind the hyperexcitability of mutated glutaminergic neurons (22). We will use transgenic mice and induced pluripotent stem cells (iPSCs) to assess the ion channels and their interacting proteins. Additionally, we will knock out the NMDA channel and observe changes in the functional activity of the ion channels. Finally, we will assess a possible therapeutic effect of deleting ion channels involved in compensatory mechanisms.

  1. Study the changes in sodium ion channels in mouse epileptic neurons and iPSCs: To determine the cellular expression and functional activity of the sodium ion channels (VGSCs and ASICs). Additionally, the proteins that interact (CAMKII and PICK1) with the sodium ion channel will be determined and the effect of sodium channel deletion will be assessed.
  2. Study the changes in AMPA receptors in mouse epileptic neurons and iPSCs: To determine the cellular expression and functional activity of the AMPA receptor with its corresponding subunits (GluA1-4). Additionally, the proteins that interact (Map1b and GRIP) with the AMPA receptor will be determined and the effect of AMPA receptor deletion will be assessed.
  3. Study the changes in potassium ion channels in mouse epileptic neurons and iPSCs: To determine the cellular expression and functional activity of the potassium ion channel (Kv1 and Kv7). Additionally, the proteins that interact (ADAM22 and PIP2) with the potassium ion channels will be determined and the effect of potassium channel deletion will be assessed.
  4. Study the changes in chloride channels in mouse epileptic neurons and iPSCs: To determine the cellular expression and functional activity of the chloride ion channel (GABAA and CLC2). Additionally, the proteins that interact (PP1 and gephyrin) with the chloride ion channel will be determined and the effect of chloride channel deletion will be assessed.

 

Research Methods and Design

 

  1. Study the changes in sodium ion channels in mouse epileptic neurons and IPSCs:

Rationale: Voltage gated sodium channels (VGSCs) and acid sensing ion channels (ASICs) contain multiple subunits from which Nav 1.6 and ASIC1 which will be studied, respectively (23,24). Each of these channels with their respective subunits contributes to hyperexcitability and hypersynchrony in epilepsy (24). These associated subunits interacts with CaMKIIβ and PICK1 proteins, which modulate the activity of the sodium channel (25,26). VGSCs and ASICs are known to be overactivated in epilepsy and contribute to seizures. Studying these sodium channels as a compensatory mechanism is necessary to determine if this will cause the neuron to receive hyper-excitatory input.

 

  1. Evaluate levels of sodium channels (Nav 1.6 and ASIC1) using northern blot, western blot, and IFA:

Human fibroblast cells will be obtained from patients with LKS GRIN2A (NMDA subunit) mutations: C231Y and D933N (12). The cells will be converted to induced pluripotent stem cells (iPSCs) by using four known chemical inducers: Oct-3/4, Sox2, c-Myc, and KLF4 (27). iPSCs will be transformed to glutaminergic neurons by inhibiting the SHH-mediated pathway and applying FGF-1 (28). Ten transgenic mice of each condition (wild-type, C231Y and D933N mutants) will be donated by the Addis et al. lab (12). This will allow us to make wild-type mice without the sodium ion channel. Northern blot will be used to measure and detect mRNA levels of the sodium channel subunit, Nav 1.6. Each model will be examined independently. Western blot will be used to scan for the presence of Nav 1.6 protein levels in transgenic mice and induced glutaminergic cells expressing the GRIN2A mutations(12). Actin will act as the loading control. The mRNA and protein levels will be examined for the wild-type (positive control), C231Y mutant, D933N mutant, and a WT mouse with the knockdown of the sodium ion channel (negative control), which will be done by using CRISPR. CRISPR-Cas9 will remove the gene that encodes the ion channel, where Cas9 will attach to the gRNA, align with the target DNA sequence. Cas9 will then cut both strands of the DNA double helix, which can then ligate with another DNA insertion or deletion (41). To visualize the data, IFA will be used, DAPI will stain the nucleus of the cells and Alexa Fluor 594 will stain the Nav 1.6/ASIC1 subunits. Factorial ANOVA will be used to confirm the statistical difference between the models (iPSC/mouse) and three groups (WT, C231Y, D933N).

Prediction: There will be an overexpression of mRNA levels and proteins of the sodium ion channel subunits, Nav 1.6/ASIC1, in northern, western blot, and IFA compared to the WT. Alternatively, there will be a downregulation and underexpression of mRNA and protein levels in the sodium channel subunits.

 

  1. Assess functional activity of the sodium channels using cellular electrophysiology:

Sodium channel activity will be assessed using current clamp and voltage cell attached patch clamp. Both experiments will be done in the WT, C231Y and D933N in both models The current clamp will be used to measure the changes in voltage recorded intracellularly from the cells. The change in voltage will be translated as a generation of an EPSP and action potential frequency. Cell attached patch clamp will be used to extracellularly assess the changes of a single ion channel, and will measure the change in current, while holding the voltage constant. The average opening time, average conductance, and opening frequency of VGSCs and ASICs will be tested. Both electrophysiology tests will be repeated 10 times. Between subjects t-test and ANOVA will be used to analyze the difference between the WT, D933N, C231Y, where p<0.05 will be considered statistically significant. Factorial ANOVA will be used to confirm the statistical difference between the models (iPSC/mouse) and the three groups: WT, C231Y and D933N.

Prediction: There will be an increase in EPSP amplitude and frequency in both the mutants (C231Y and D933N) in both models compared to WT, since sodium channels, especially VGSCs render the cell to become more depolarized. Both sodium channels will exhibit high average opening time and higher average conductance. These channels will be open for a longer period of time causing high influx of sodium ions into the membrane, which increases the possibility of an action potential. Alternatively, there could be less opening time and less conductance, which would conclude that the sodium channel is not being used as a protective mechanism.

 

  1. Assess the interaction between sodium channels and related proteins using laser-capture microdissection (LCM)/mass spectrometry (MS) and coimmunoprecipitation (CoIP):

LCM is a method used to dissect and obtain a specific section of the tissue. Further on, this tissue is analyzed for protein levels using MS. We expect similar results in each brain region, each mutation and both models. This will help identify protein interaction of the sodium channel with interacting proteins, to see if interacting protein levels are upregulated or downregulated in the area of interest mentioned above (29). LCM will be used only on mice, as the IPSC-derived glutaminergic cells can be directly forwarded to MS. Multiple slices from each mice brain will be used to increase statistical significance. The masses obtained from MS will be compared to WT masses. GAPDH will be used as a positive control. To specify a direct interaction, CoIP will be used. CoIP isolates the ion channel using primary antibody and the interacting protein is extracted along with the ion channel. The protein lysate is placed through western blot to detect the interacting protein using secondary antibody. The interaction is being detected between the VGSC/ASIC and CAMKII/PICK1, respectively. Factorial ANOVA will be used to observe a statistical significance between WT, C231Y and D933N for each ion channel.

Prediction: MS will show higher levels of CamKIIβ or PICK1 in one or both mutants compared to control.  In addition, CoIP will confirm MS and show presence of CAMKIIβ or PICK1. Alternatively, it is possible both proteins will be found to be interacting. If no bands are present, this will indicate that there is no interaction between these proteins and the subunits of the channels.

 

  1. Assess the effect of NMDA gene knock out on WT induced glutamatergic cells

WT iPSC cells will be induced into glutaminergic neurons, causing these cells to be epileptic and similar to the C231Y/D933N mutations (28). RNAi will be used to inhibit the NMDA subunit genes reducing NMDA protein expression compared to the WT. Factorial ANOVA will be used to quantify a statistical significance between the WT and NMDA-Rnai VGSC and ASICs. The changes in the ion channel expression, ion channel functionality, and their interacting proteins will be assessed using the same methods mentioned in 1A,1B, and 1C.

Prediction: Northern and western blot, and IFA will show the same results as 1A. Voltage cell attached patch clamp will show the same results as 1B. CoIP analysis will show the same results in 1C.

 

  1. Examine the effect of sodium channel gene knockdown the epileptic neurons using patch clamp.

The VGSC genes along with the ASIC genes will be knockdown in both models to decrease sodium channels using CRISPR (41). This will be done only if subaims 1A-D indicate that sodium channels are overactivated. Within both models, WT, C231Y and D933N and treatment condition, which consists of the knockdown of VGSC and ASIC, will be tested. The functional activity will be assessed using voltage cell-attached patch clamp recordings. Factorial ANOVA will be used to confirm the statistical difference between the model and the four groups: WT, C231Y, D933N, and treatment condition.

Prediction: A knockdown of the sodium channels will cause less conductance, less opening time and less opening frequency due to lack of sodium ions coming into the cell, causing an absence of hyperexcitation. Alternatively, other ion channels, such as an efflux of potassium and/or an influx of chloride channel could be further inactivated as a result of another compensatory mechanism.

 

  1. Study the changes in AMPA receptor in mice epileptic neurons and IPSCs:

Rationale: AMPA receptors are highly concentrated in excitatory synapses that are linked to the postsynaptic density (32). The contribution of AMPA receptors and its subunits: GluA1-4, indicate a transient role on the acquisition of the ‘kindling’ phenomenon as well as suggesting an involvement of AMPA receptors in the maintenance of a kindled state (33). Thus, we expect to observe an overactivation of these subunits contributing to hyperexcitability.

 

  1. Evaluate levels of AMPA receptor using northern blot, western blot and IFA.

The mRNA levels of the AMPA receptor will be measured and detected by northern blot, as well as the presence of GluA1 specific protein levels by using Western Blot, as described in section 1A. Finally, to visualize the data, IFA will be used to stain the AMPA ion channel as described in section 1A. Factorial ANOVA will be used to confirm the statistical difference between the model (iPSC/mouse) and the three groups: WT, C231Y and D933N.

Prediction: There will be an underexpression of the mRNA levels and specific proteins of the AMPAr subunit, GluA, in northern, western blot, and IFA compared to WT. Alternatively, there will be an overexpression of mRNA and protein levels in the AMPA receptor subunit.

 

  1. Assess functional activity of the AMPA receptor using cellular electrophysiology

The same electrophysiology records will be done as described in section 1B. AMPA receptors will be studied to record average opening time and conductance. Factorial ANOVA will be used to confirm the statistical difference between the model  and the three groups.

Prediction: An increase in EPSPs and action potentials will be generated in C231Y/D933N compared to WT. Increase in opening time and conductance of the AMPA receptor in the mutants will be expected as well. Alternatively, there would be no increase in opening time or conductance, causing AMPA to be a protective mechanism.

 

C.Assess the interaction between AMPA receptors and related proteins using LCM/MS and CoIP

Interaction between ion channel and proteins can be measured only if abnormal AMPA expression or functionality is observed in sections 2A and 2B. This applies to other ion channels as well. Levels of interacting proteins of AMPA channel, CaMKIIβ and GRIP, will be measured using LCM and MS described in section 1C (29). If the interacting proteins directly bind with the AMPA channel, there will be a band visible on the blot (30).

Prediction: We expect high levels of CamKIIβ or GRIP, protein in MS suggesting its possible interaction with AMPA channel. CoIP will present a direct evidence of interaction between CamKIIβ or GRIP and the AMPA receptor. The interaction will be visible through a band present for one or both interacting proteins. Alternatively, there will not display any change in LCM/MS for mutants compared to WT as well as no interaction in CoIP.

 

  1. Assess the effect of NMDA gene knock out on WT induced glutamatergic cells

WT iPSC cells will be induced into glutaminergic neurons, causing these cells to be epileptic and similar to the C231Y/D933N mutations as described in 1D (28). Factorial ANOVA will be used to quantify a statistical significance between the WT and NMDA-Rnai AMPA receptors.

Prediction: Northern and western blot, and IFA will show the same results as 2A. Voltage cell attached patch clamp will show the same results as 2B. CoIP analysis will show the same results in 2C.

 

  1. Examine the effect of AMPA receptor gene knockdown on the epileptic neurons using patch clamp.

The AMPA receptor associated genes will be knockdown to decrease AMPA receptor activity using CRISPR as described in section 1E. This will be done only if subaims 2A-D indicate an overactivation of AMPA receptors. Factorial ANOVA will be used to confirm the statistical difference between the model and the three groups.

Prediction: A knockdown of AMPA receptors, will cause a lack sodium ions coming into the cell, causing an absence of hyperexcitation. Alternatively, if there are depolarizations, other ion channels, such as an efflux of potassium and/or an influx of chloride ions could occur.

 

  1. Study the changes in potassium channels in mice epileptic neurons and IPSCs:

Rationale: Studying the function of neuronal potassium channels is necessary to understand the effects of potassium channel inactivation on neuronal function and membrane potential. This has implications for epilepsy, as downregulation in potassium channels can lead to epileptic-like hyperactivity. The main potassium subunits that are associated with epilepsy are Kv1.1 and the Kv7.2 (35). Potassium channel may contribute to neuronal hyperexcitability in LKS.  In addition to that, ADAM22/PIP2 have been shown to interact with Kv1/Kv7 channels, respectively. (43,44).

 

  1. Evaluate levels of potassium channels (Kv1 and Kv7) using northern blot, western blot and IFA

Northern and western blot will be performed in mice models and glutaminergic cells to scan for the quantitative presence of mRNA, specific protein levels in the potassium subunits Kv1.1 and Kv7.2, and staining of the potassium channels will be examined as described in section 1A. Factorial ANOVA will be used to confirm the statistical difference between the model and the three groups (WT, C231Y, D933N).

Prediction: There will be an underexpression of the mRNA levels and specific proteins of the potassium ion channel subunits, Kv1.1 and Kv7.2, in northern, western blot, and IFA compared to WT. Alternatively, there will be an overexpression of mRNA and protein levels in the potassium ion channel subunits.

 

  1. Assess functional activity of the potassium channel using cellular electrophysiology

The same electrophysiology records will be done as described in section 1B. Potassium channels Kv1 and Kv7 will be studied to record average opening time and conductance. Factorial ANOVA will be used to confirm the statistical difference between the model  and the three groups.

Prediction: An increase in EPSPs and action potentials will be generated in C231Y/D933N compared to WT. Decrease in opening time and conductance of these potassium channels in the mutants will be expected. Alternatively, there would be an increase in opening time or conductance in the mutant groups, causing potassium channels to be a protective mechanism.

 

  1. Assess the interaction between potassium channels and related proteins using LCM/MS and CoIP

Using LCM/MS and CoIP, the interaction among neuronal potassium channels and its interacting proteins will be measured as described in 1C. Kv1 and Kv7 channels have a known interaction with ADAM22 and PIP2, respectively (43,44).

Prediction: We expect to observe low levels of ADAM22 and/or PIP2 in MS. Additionally, CoIP results will confirm that there is less binding and explain the compensatory mechanism. Alternatively, we might see interaction, suggesting a different pathway of activation.

 

  1. Assess the effect of NMDA gene knock out on WT induced glutamatergic cells

WT iPSC cells will be induced into glutaminergic neurons, causing these cells to be epileptic and similar to the C231Y/D933N mutations as described in 1D (28). Factorial ANOVA will be used to quantify a statistical significance between the WT and NMDA-Rnai potassium channels.

Prediction: Northern and western blot, and IFA will show the same results as 3A. Voltage cell attached patch clamp will show the same results as 3B. CoIP analysis will show the same results in 3C.

 

  1. Examine the effect of potassium channel gene insertion on the epileptic neurons using patch clamp

The potassium associated genes for Kv1 and Kv7 will be knockin to increase potassium channel activity using CRISPR as described in section 1E (41). This will be done only if subaims 3A-D indicate that potassium channels are underactivated. Factorial ANOVA will be used to confirm the statistical difference between the model and the three groups.

Prediction: A knockin of potassium channels will cause an efflux of potassium ions rendering the cell to be hyperpolarized. Alternatively, if there are depolarizations, other ion channels, such as an influx of sodium and/or an influx of chloride ions could occur.

 

  1. Study the changes in chloride ion channels in mice epileptic neurons and IPSCs:

Rationale: Previous studies have shown that chloride ion channels are involved in mediating hyperpolarization in postsynaptic neurons (37,38). Mutation in GABAA receptor (a chloride channel) subunit can reduce hyperpolarization leading to seizures (38). Therefore, the mutations highlighted by Addis et al, can be explained by chloride channel downregulation. The following chloride channels will be used as they have high implication in epilepsy to understand the compensatory mechanism: CIC2 and GABAA (37,38).

 

  1. Evaluate levels of chloride channels (ClC2 and GABAA) using northern blot, western blot and IFA

The mRNA levels of the chloride channels, ClC2 and GABAA will be measured and detected through Northern Blot, the presence of its specific protein levels through Western Blot, and the visualization of the stained chloride channels, as described in section 1A. Factorial ANOVA will be used to confirm the statistical difference between the model and the three groups: WT, C231Y and D933N.

Prediction: Northern and Western blot analysis will show a decrease in levels of ClC2 and/or GABAA which would explain the depolarization in LKS patients. IFA will show a decreased levels of chloride channels compared to WT. This decrease could be due to either decreased total protein levels or decrease receptor trafficking.

 

  1. Assess functional activity of the chloride channels using cellular electrophysiology

The same electrophysiology records will be done as described in section 1B. Chloride channels ClC2 and GABAA will be studied to record average opening time and conductance. Factorial ANOVA will be used to confirm the statistical difference between the model (iPSC/mouse) and the three groups: WT, C231Y and D933N. Factorial ANOVA will be used to confirm the statistical difference between the model and the three groups.

Prediction: An increase in EPSPs and action potentials will be generated in C231Y/D933N compared to WT from the current clamp recordings. Decrease in opening time and conductance of these chloride channels in the mutants will be expected, due to reduced influx of chloride ions. Alternatively, there would be an increase in opening time or conductance in the mutant groups, causing chloride channels to be a protective mechanism.

 

  1. Assess the interaction between chloride channels and related proteins using LCM/MS and CoIP

GABAA receptor is known to interact with Gephyrin, a receptor stabilizing protein (39). CIC-2 ion channels have shown interaction with phosphatase-1 (40). LCM/MS and CoIP will be used again as described in 1C.
Prediction: We predict that the isolated tissue will contain low levels of interacting proteins mentioned above. This would explain the downregulation of chloride channels as there would be less regulation. Additionally, the CoIP analysis would show low levels of the interacting proteins binding to the chloride channels. Alternatively, there would be interaction observed. As well as, it is possible that none of the ion channels are found to be responsible for LKS.

 

  1. Assess the effect of NMDA gene knock out on WT induced glutamatergic cells

WT iPSC cells will be induced into glutaminergic neurons, causing these cells to be epileptic and similar to the C231Y/D933N mutations as described in 1D (28). Factorial ANOVA will be used to quantify a statistical significance between the WT and NMDA-Rnai chloride channels.

Prediction: Northern and western blot, and IFA will show the same results as 4A. Voltage cell attached patch clamp will show the same results as 4B. CoIP analysis will show the same results in 4C.

 

  1. Examine the effect of chloride channel gene insertion on the epileptic neurons using patch clamp.

Assuming subaims 4A and 4B showed a decreased activity of the chloride channels, knockin of chloride channel associated genes will be done using CRISPR as described in section 1E. Factorial ANOVA will be used to confirm the statistical difference between the model and the three groups

Prediction:  A knockin of chloride channels will cause an influx of chloride ions rendering the cell to be hyperpolarized. Alternatively, if there are depolarizations, other ion channels, such as an influx of sodium and/or an efflux of potassium ions would be involved.

 

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