Shortened introduction after meeting

manuscript.tex

@@ -196,26 +196,48 @@ Although the genetic nature of ion channel mutations as well as their effects on
 
 \par\null
 
-\section*{Introduction (750 Words Maximum  - Currently 882 ** with additions in \textcolor{red}{red} and deletions in \textit{italics}: 807)} 
+\section*{Introduction (750 Words Maximum  - Currently 702)} 
 %\textit{The Introduction should briefly indicate the objectives of the study and provide enough background information to clarify why the study was undertaken and what hypotheses were tested.}
 
-The properties and combinations of voltage-gated ion channels are vital in determining \textit{action potential generation, neuronal firing properties and excitability} \textcolor{red}{neuronal excitability.} \citep{bernard_channelopathies_2008, carbone_ion_2020, rutecki_neuronal_1992, pospischil_minimal_2008}. \textit{However, ion channel function can be disturbed, for instance through genetic alterations, resulting in altered neuronal firing behavior \citep{carbone_ion_2020}}. In recent years, next generation sequencing has led to an increasing number of clinically relevant \textcolor{red}{ ion channels mutations} \textit{genetic mutations} and has provided the basis for pathophysiological studies of genetic epilepsies, pain disorders, dyskinesias, intellectual disabilities, myotonias, and periodic paralyses \citep{bernard_channelopathies_2008, carbone_ion_2020}. Ongoing efforts of many research groups have contributed to the current understanding of underlying disease mechanism in channelopathies, however a complex pathophysiological landscape has emerged for many channelopathies and is likely a reason for limited therapeutic success with standard care.              
+The properties and combinations of voltage-gated ion channels are vital in determining neuronal excitability \citep{bernard_channelopathies_2008, carbone_ion_2020, rutecki_neuronal_1992, pospischil_minimal_2008}. However, ion channel function can be disturbed, for instance through genetic alterations, resulting in altered neuronal firing behavior \citep{carbone_ion_2020}. In recent years, next generation sequencing has led to an increasing number of clinically relevant ion channel mutations and has provided the basis for pathophysiological studies of genetic epilepsies, pain disorders, dyskinesias, intellectual disabilities, myotonias, and periodic paralyses \citep{bernard_channelopathies_2008, carbone_ion_2020}. Ongoing efforts of many research groups have contributed to the current understanding of underlying disease mechanism in channelopathies, however a complex pathophysiological landscape has emerged for many channelopathies and is likely a reason for limited therapeutic success with standard care.              
+
+Ion channel variants are frequently classified in heterologous expression systems as either a loss of function (LOF) or a gain of function (GOF)  in the respective ionic current \citep{Musto2020, Kullmann2002, Waxman2011, Kim2021}. This LOF/GOF classification is often directly used to predict the effects on neuronal firing \citep{Niday2018, Wei2017, Wolff2017,Masnada2017}, which in turn is important for understanding the pathophysiology of these disorders and for identification of potential therapeutic targets \citep{Orsini2018, Yang2018, Colasante2020, Yu2006}. Experimentally, the effects of channelopathies on neuronal firing are assessed using primary neuronal cultures  \citep{Scalmani2006, Smith2018, Liu2019}  or \textit{in vitro} recordings from slices of transgenic mouse lines \citep{Mantegazza2019, Xie2010,Lory2020, Habib2015, Hedrich14874} but are restricted to limited neuronal types. Different neuron types differ in their composition of ionic currents \citep{yao2021taxonomy, Cadwell2016, BICCN2021, Scala2021} and therefore likely respond differently to changes in the properties of a single ionic current. Expression level of an affected gene \citep{Layer2021} and relative amplitudes of ionic currents \citep{rutecki_neuronal_1992, pospischil_minimal_2008,Kispersky2012, golowasch_failure_2002, barreiro_-current_2012} indeed dramatically influence the firing behavior and dynamics of neurons. Mutations in different sodium channel genes have been experimentally shown to affect firing in a cell-type specific manner based on differences in expression levels of the affected gene \citep{Layer2021}, but also on other cell-type specific mechanisms \citep{Hedrich14874, makinson_scn1a_2016}.
+
+ Cell-type specificity is likely vital for successful precision medicine treatment approaches. For example, Dravet syndrome was identified as the consquence of LOF mutations in \textit{SCN1A} \citep{Claes2001,Fujiwara2003,Ohmori2002}, however limited success in treatment of Dravet syndrome persisted \citep{Claes2001,Oguni2001} until it became evident that inhibitory interneurons and not pyramidal neurons had altered excitability as a result of LOF \textit{SCN1A} mutations  \citep{Yu2006, Colasante2020}. 
+
+Taken together, these examples demonstrate the need to study the effects of ion channel mutations in many different cell types --- a daunting if not impossible experimental challenge. In the context of this diversity, simulations of conductance-based neuronal models are a powerful tool bridging the gap between altered ionic currents and firing in a systematic and efficient way. Furthermore, simlutions potentially allow to predict the effects of drugs needed to alleviate the pathophysiology of the respective mutation \citep{johannesen_genotype-phenotype_2021, lauxmann_therapeutic_2021, Bayraktar}.
+
+In this study, we therefore investigated how the outcome of ionic current kinetic changes on firing depend on neuronal cell type by (1) characterizing firing responses with 2 measures, (2) simulating the response of a repertoire of different neuronal models to changes in single current parameters as well as (3) to more complex changes as they were observed for specific \textit{KCNA1} mutations that are associated with episodic ataxia type~1 \citep{Browne1994, Browne1995, lauxmann_therapeutic_2021}.
+
+%
+%The effects of mutations in ion channel genes on ionic current kinetics are frequently assessed using heterologous expression systems without endogenous currents \citep{Balestrini1044, Noebels2017, Dunlop2008}. 
+
+
+%
+%\textcolor{red}{However, the understanding of these complex genotype-phenotype relationships is still evolving \citep{Wolff2017, johannesen_genotype-phenotype_2021}.} 
+
+%Cell-type specific effects on firing have been experimentally observed \citep{Hedrich14874, makinson_scn1a_2016, Layer2021}. For example, the effects of the R1648H \textit{SCN1A} and R1627H \textit{SCN8A} mutations on firing differ in interneurons and pyramidal neurons  \citep{Hedrich14874, makinson_scn1a_2016}.
+
+% However, the effect of a given channelopathy on the firing behavior of different neuronal types across the brain is often unclear and not feasible to obtain experimentally.
 
-The effects of mutations in ion channel genes on ionic current kinetics are frequently assessed using heterologous expression systems \textcolor{red}{not expressing} \textit{which do not express} endogenous currents \citep{Balestrini1044, Noebels2017, Dunlop2008}. Ion channel variants are frequently classified as either a loss of function (LOF) or a gain of function (GOF) with respect to changes in \textcolor{red}{ionic current} \textit{gating of the altered ion channels} \citep{Musto2020, Kullmann2002, Waxman2011, Kim2021} \textit{. This classification of the effects on ionic currents} \textcolor{red}{This LOF/GOF classification} is often directly used to predict the effects on neuronal firing \citep{Niday2018, Wei2017, Wolff2017,Masnada2017}, which in turn is important for understanding the pathophysiology of these disorders and for identification of potential therapeutic targets \citep{Orsini2018, Yang2018, Colasante2020, Yu2006}. \textcolor{red}{However, the understanding of these complex genotype-phenotype relationships is still evolving \citep{Wolff2017, johannesen_genotype-phenotype_2021}.} \textit{Genotype-phenotype relationships are complex and the understanding of the relationships between these is still evolving \citep{Wolff2017, johannesen_genotype-phenotype_2021}.} Experimentally, the effects of channelopathies on neuronal firing can be assessed using primary neuronal cultures  \citep{Scalmani2006, Smith2018, Liu2019}  or \textit{in vitro} recordings from slices of transgenic mouse lines \citep{Mantegazza2019, Xie2010,Lory2020, Habib2015, Hedrich14874}. However, the effect of a given channelopathy on the firing behavior of different neuronal types across the brain is often unclear and not feasible to obtain experimentally. Different neuron types differ in their composition of ionic currents \citep{yao2021taxonomy, Cadwell2016, BICCN2021, Scala2021} and therefore likely respond differently to changes in the properties of a single ionic current. \textit{For example, the expression level of an affected gene can correlate with firing behavior \textit{in the simplest case} \citep{Layer2021} and altering relative amplitudes of ionic currents can dramatically influence the firing behavior and dynamics of neurons \citep{rutecki_neuronal_1992, pospischil_minimal_2008,Kispersky2012, golowasch_failure_2002, barreiro_-current_2012}.}\textcolor{red}{Expression level of an affected gene \citep{Layer2021} and relative amplitudes of ionic currents \citep{rutecki_neuronal_1992, pospischil_minimal_2008,Kispersky2012, golowasch_failure_2002, barreiro_-current_2012} can dramatically influence the firing behavior and dynamics of neurons.} Cell-type specific effects on firing have been experimentally observed. For example, the firing effects of the R1648H \textit{SCN1A} and R1627H \textit{SCN8A} mutations are different in interneurons and pyramidal neurons  \citep{Hedrich14874, makinson_scn1a_2016}.
 %However, if gating kinetics are affected this can have complex consequences on the firing behavior of a specific cell type and the network activity within the brain.
 
 %Altering relative amplitudes of ionic currents can dramatically influence the firing behavior and dynamics of neurons \citep{rutecki_neuronal_1992, pospischil_minimal_2008,Kispersky2012, golowasch_failure_2002, barreiro_-current_2012}, however other properties of ionic currents impact neuronal firing as well. 
 %In addition, cell-type specific effects on firing are possible. For instance, the R1648H mutation in \textit{SCN1A} increases firing in inhibitory interneurons but not pyramidal neurons \citep{Hedrich14874}. In extreme cases, a mutation can have opposite effects on different neuron types. For example, the homologous mutation R1627H in \textit{SCN8A} is associated which increased firing in interneurons, but decreases pyramidal neuron excitability \citep{makinson_scn1a_2016}.
 %For instance, the R1648H mutation in \textit{SCN1A} increases firing in inhibitory interneurons but not pyramidal neurons \citep{Hedrich14874}. In extreme cases, a mutation can have opposite effects on different neuron types. For example, the homologous mutation R1627H in \textit{SCN8A} is associated which increased firing in interneurons, but decreases pyramidal neuron excitability \citep{makinson_scn1a_2016}.
 
-Despite this evidence of cell-type specific effects of ion channel mutations on firing, the dependence of firing outcomes in ion channel mutations on cell-type is generally not known. Cell-type specificity is likely vital for successful precision medicine treatment approaches. For example, Dravet syndrome was identified as the consquence of LOF mutations in \textit{SCN1A} \citep{Claes2001,Fujiwara2003,Ohmori2002}, however limited success in treatment of Dravet syndrome persisted \citep{Claes2001,Oguni2001}. \textcolor{red}{until it became evident that inhibitory interneurons and not pyramidal neurons had altered excitability as a result of LOF \textit{SCN1A} mutations  \citep{Yu2006, Colasante2020}}. \textit{Once it became evident that inhibitory interneurons and not pyramidal neurons had altered excitability as a result of LOF \textit{SCN1A} mutations  \citep{Yu2006}, alternative approaches based on this understanding began to show promise \citep{Colasante2020}.}
+
+%\textit{Once it became evident that inhibitory interneurons and not pyramidal neurons had altered excitability as a result of LOF \textit{SCN1A} mutations  \citep{Yu2006}, alternative approaches based on this understanding began to show promise \citep{Colasante2020}.}
 %Once it became evident that only inhibitory interneurons and not pyramidal neurons had altered excitability as a result of LOF \textit{SCN1A} mutations  \citep{Yu2006} alternative approaches, based on this understanding such as gene therapy, began to show promise \citep{Colasante2020}. 
 
-Due to the high clinical relevance of understanding cell-type dependent effects of channelopathies, computational modelling approaches are used to assess the impacts of altered ionic current properties on firing behavior, bridging the gap between changes in the biophysical properties induced by mutations, firing and clinical symptoms. Conductance-based neuronal models enable insight into the effects of ion channel mutations with specific effects of the resulting ionic current as well as enabling \textit{in silico} assessment of the relative effects of changes in biophysical properties of ionic currents on neuronal firing. Furthermore, modelling approaches enable predictions of the effects of a specific mutation and drug induced biophysical property changes \citep{Layer2021,Liu2019,johannesen_genotype-phenotype_2021, lauxmann_therapeutic_2021}.
+
+%modelling approaches enable predictions of the effects of specific mutations and 
+%induced biophysical property changes \citep{johannesen_genotype-phenotype_2021, lauxmann_therapeutic_2021}.
+
 %Computational modelling approaches can be used to assess the impacts of altered ionic current properties on firing behavior, bridging the gap between changes in the biophysical properties induced by mutations, firing and clinical symptoms. Conductance-based neuronal models enable insight into the effects of ion channel mutations with specific effects of the resulting ionic current as well as enabling \textit{in silico} assessment of the relative effects of changes in biophysical properties of ionic currents on neuronal firing. Furthermore, modelling approaches enable predictions of the effects of specific mutation and drug induced biophysical property changes \citep{Layer2021,Liu2019,johannesen_genotype-phenotype_2021, lauxmann_therapeutic_2021}. 
 
-In this study, we therefore investigated how the outcome of ionic current kinetic changes on firing depend on neuronal cell type by (1) characterizing firing responses with 2 measures, (2) simulating the response of a repertoire of different neuronal models to changes in single current parameters as well as (3) to more complex changes as they were observed for \textit{specific mutations. For this task we chose mutations in the \textit{KCNA1} gene, encoding  the potassium channel subunit \Kv, that are associated with episodic ataxia type~1 \citep{Browne1994, Browne1995, lauxmann_therapeutic_2021}.} \textcolor{red}{ specific \textit{KCNA1} mutations that are associated with episodic ataxia type~1 \citep{Browne1994, Browne1995, lauxmann_therapeutic_2021}}
+%\textit{specific mutations. For this task we chose mutations in the \textit{KCNA1} gene, encoding  the potassium channel subunit \Kv, that are associated with episodic ataxia type~1 \citep{Browne1994, Browne1995, lauxmann_therapeutic_2021}.} 
-%\par\null
+%%\par\null
 
 \section*{Materials and Methods}
 % \textit{The materials and methods section should be brief but sufficient to allow other investigators to repeat the research (see also Policy Concerning Availability of Materials). Reference should be made to published procedures wherever possible; this applies to the original description and pertinent published modifications. }

ref.bib

@@ -1,4 +1,22 @@
  
+ 
+@Article{Bayraktar,
+  author   = {Bayraktar, Erva and Liu, Yuanyuan and Sonnenberg, Lukas and Hedrich, Ulrike B. S. and Sara, Yildirim and Eltokhi, Ahmed and Lyu, Hang and Lerche, Holger and Wuttke, Thomas V. and Lauxmann, Stephan},
+  journal  = {British Journal of Pharmacology},
+  title    = {In vitro effects of {S}-{Licarbazepine} as a potential precision therapy on {SCN8A} variants causing neuropsychiatric disorders},
+  issn     = {1476-5381},
+  number   = {n/a},
+  volume   = {n/a},
+  abstract = {Background and Purpose Variants in SCN8A, the coding gene for NaV1.6 channels, are characterized by a variety of symptoms including, intractable epileptic seizures, psychomotor delay, progressive cognitive decline, autistic features, ataxia or dystonia. Standard anticonvulsant treatment has only a limited impact on the course of disease. Experimental Approach We investigated the therapeutic potential of S-Licarbazepine, an enhancer of slow inactivation of voltage gated sodium channels, on two variants with biophysical and neuronal gain-of-function (G1475R, M1760I) and one variant with biophysical gain-of-function but neuronal loss-of-function (A1622D) in neuroblastoma cells and in murine primary hippocampal neuron cultures. These three variants cover the broad spectrum of NaV1.6-associated disease and are linked to representative phenotypes of mild to moderate epilepsy (G1475R), developmental and epileptic encephalopathy (M1760I) and intellectual disability without epilepsy (A1622D). Key Results Similar to known effects on NaV1.6 wildtype channels, S-Licarbazepine predominantly enhances slow inactivation on all tested variants, irrespective of their particular biophysical mechanisms. However, beyond that, S-Licarbazepine exhibits variant-specific effects including a partial reversal of pathologically slowed fast inactivation dynamics (A1622D, M1760I) and a trend to reduce enhanced persistent Na+ current by A1622D variant channels. Furthermore, our data in primary transfected neurons reveal that not only variant-associated hyperexcitability (M1760I and G1475R) but also hypoexcitability (A1622D) can be modulated by S-Lic. Conclusion and Implications S-Licarbazepine not only owns substance- but also variant-specific effects. Personalized treatment regimens optimized to achieve such variant-specific pharmacological modulation may help to reduce adverse side effects and improve the overall therapeutic outcome of SCN8A-related disease.},
+  doi      = {10.1111/bph.15981},
+  file     = {Full Text PDF:https\://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/bph.15981:application/pdf},
+  keywords = {epilepsy, developmental and epileptic encephalopathy, sodium channel blocker, S-Licarbazepine, NaV1.6, SCN8A, precision medicine},
+  language = {en},
+}
+
+ 
+ 
+ 
 @Article{Clerx2019,
   author   = {Clerx, Michael and Beattie, Kylie A. and Gavaghan, David J. and Mirams, Gary R.},
   journal  = {Biophysical Journal},