EGR3 Deletion Rescues Developmental and Epileptic Encephalopathy in Kcna1 -null Mice

EGR3 Deletion Rescues Developmental and Epileptic Encephalopathy in Kcna1-null Mice | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results EGR3 Deletion Rescues Developmental and Epileptic Encephalopathy in Kcna1 -null Mice View ORCID Profile Arindam Ghosh Mazumder , Saifina Karedia , Nandani Adhyapak , Catharina Schimer , John Samuel Bass , Jessica L Kamen , Miranda J Jankovic , Qinglong Miao , Amelia L Gallitano , Alexander B Saltzman , Antrix Jain , Anna Malovannaya , Edward Glasscock , Isamu Aiba , Jeffrey L Noebels , Vaishnav Krishnan doi: https://doi.org/10.1101/2025.06.07.658422 Arindam Ghosh Mazumder 1 Department of Neurology, Baylor College of Medicine , Houston, TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Arindam Ghosh Mazumder Saifina Karedia 1 Department of Neurology, Baylor College of Medicine , Houston, TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nandani Adhyapak 1 Department of Neurology, Baylor College of Medicine , Houston, TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site Catharina Schimer 1 Department of Neurology, Baylor College of Medicine , Houston, TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site John Samuel Bass 1 Department of Neurology, Baylor College of Medicine , Houston, TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jessica L Kamen 1 Department of Neurology, Baylor College of Medicine , Houston, TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site Miranda J Jankovic 1 Department of Neurology, Baylor College of Medicine , Houston, TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site Qinglong Miao 1 Department of Neurology, Baylor College of Medicine , Houston, TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amelia L Gallitano 3 Department of Basic Medical Sciences, University of Arizona , Phoenix AZ Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alexander B Saltzman 4 Mass Spectrometry Proteomics Core, Advanced Technology Cores, Baylor College of Medicine , Houston TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site Antrix Jain 4 Mass Spectrometry Proteomics Core, Advanced Technology Cores, Baylor College of Medicine , Houston TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anna Malovannaya 4 Mass Spectrometry Proteomics Core, Advanced Technology Cores, Baylor College of Medicine , Houston TX 5 Department of Biochemistry and Molecular Pharmacology, Baylor College of Medicine , Houston TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site Edward Glasscock 2 Department of Biological Sciences, Southern Methodist University , Dallas, TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site Isamu Aiba 1 Department of Neurology, Baylor College of Medicine , Houston, TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jeffrey L Noebels 1 Department of Neurology, Baylor College of Medicine , Houston, TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site Vaishnav Krishnan 1 Department of Neurology, Baylor College of Medicine , Houston, TX Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: vkrish{at}bcm.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract KCNA1 encodes the α-subunit of the voltage-gated potassium channel K V 1.1. Mutations in K V 1.1’s pore domain result in developmental and epileptic encephalopathy (DEE), where early life seizures and a culprit lesion synergistically disrupt neurodevelopmental trajectories, resulting in intellectual disability that often presents with disturbances in sleep, sociability and sensory processing. Abnormalities in the subcellular localization of Kv1.1, via mutations in/autoantibodies against LGI1 and CNTNAP2, also give rise to syndromes of epilepsy and neuropsychiatric impairment. Mice with deletions of Kcna1 (“-/-“) display spontaneous seizures at 2-3 weeks of age and premature mortality. In this study, we applied instrumented home-cage monitoring to examine how aberrations in KCNA1 expression may result in pervasive alterations in spontaneous behavior. Compared to wildtype, Kcna1 -/- mice displayed a robust multifaceted behavioral syndrome featuring marked nocturnal hyperactivity, insomnia, reduced sheltering, fragmented feeding/drinking rhythms, sensory over-responsivity and diminished wheel-running. In identical recordings, Kcna1 +/− mice only displayed increased sheltering, Lgi1 +/− mice displayed mild insomnia and Cntnap2 -/- mice showed home-cage hypoactivity . Kcna1 loss in parvalbumin-positive interneurons (Pv-Cre) resulted in a subtle phenocopy, with mild insomnia accompanied by reduced sheltering behavior, while similar deletions in forebrain pyramidal neurons (Emx1-Cre) or dopaminergic neurons (DAT-Cre) were asymptomatic. Adult-onset conditional deletions of Kcna1 also produced only mild insomnia 6 weeks later. To survey the molecular landscape in Kcna1 -/- mice, we conducted a mass spectrometry proteomic analysis of dissected hippocampal tissue (a predominant seizure onset zone and where astrogliosis is observed). This revealed significant upregulations in BDNF (brain-derived neurotrophic factor) and the immediate early transcription factor EGR3 (early growth response-3), which is necessary for the induction of BDNF following electroconvulsive seizures. Heterozygous or homozygous deletions of Egr3 in Kcna1 -/- mice resulted in significant survival prolongation, a partial neurobehavioral rescue, and a significant improvement in the frequency of spontaneous seizures and spreading depolarization events. These clinical improvements were associated with an amelioration of BDNF induction, hippocampal astrogliosis and proteomic disturbances. Together, these data illustrate how an ion channel that governs excitability at millisecond scales also shapes the spatiotemporal structure of spontaneous behavior at meso- or macroscopic time scales. Our results provide a model and a set of precision endpoints to understand how ictal and interictal features of DEE may be ameliorated by inhibiting the long-term downstream transcriptional alterations imparted by early life seizures. Introduction Developmental and epileptic encephalopathies (DEEs) are characterized by a triad of childhood onset seizures, intellectual disability and neurobehavioral deficits that include disturbances in sleep, communication/social reciprocity, feeding and sensory integration. 1 – 5 Cognitive and psychiatric disturbances in DEE are hypothesized to result from two autonomous but intertwined pathophysiological processes, including (i) an inciting lesion (genetic or structural) capable of itself altering neurodevelopmental trajectories, and (ii) periods of frequent early life seizures (ELS), that further aggravate developmental delays. 3 , 6 – 11 In many genetic DEEs, behavioral deterioration may begin prior to seizure onset and may persist despite complete seizure control or spontaneous remission. 2 – 4 , 10 , 12 The genetic landscape of DEEs 13 includes pathogenic variants in ion channels (e.g., Kcnq2, Scn1a, Gabrb3 ), transcription factors (e.g., Arx ), chromatin remodelers (e.g., Chd2 ), cell adhesion molecules (e.g., Pcdh19 ) and kinases (e.g., Cdkl5 ). Preclinical DEE models play a crucial role in causally linking a genetic diagnosis in an affected patient to a syndrome of seizures and neuropsychiatric disturbances, and ultimately designing a clinically informed precision treatment. Mouse models of DEE offer a short lifespan, the opportunity to dissect circuit-specific mechanisms and a modifiable genome that provides unparalleled construct validity for monogenic DEEs. Achieving adequate face validity has been more challenging. 14 While some models have been shown to faithfully display multiple seizure types 15 , seizures may be difficult to monitor and discern if they arise (and/or result in demise) prior to weaning. 16 , 17 In addition, human syndromes are typically associated with heterozygously expressed variants, while mice may only be seizure-prone in the homozygous state 18 , or remain entirely seizure free during the relatively brief durations of laboratory EEG (electroencephalography) observations. 19 While video-EEG remains the gold-standard technique for clarifying seizure occurrence, attempts to ascertain neurobehavioral phenotypes in these models remain poorly standardized. Separate from tests of cognitive function (e.g., spatial memory, object memory), many laboratories also apply a battery of short behavioral tests to ascertain non-cognitive endpoints (e.g., elevated plus maze [exploratory drive], three-chamber testing [sociability], etc.). The popularity of these testing protocols has recently come under increased scrutiny. 20 , 21 First, since they are relatively brief (∼5-60 minutes), these tests provide daytime snapshots of emotionality in an exquisitely nocturnal species. Second, test results are often extrapolated into human-like symptoms (e.g., “anxiety-like”, “schizophrenia-like” ), a practice that stems from their originally intended purpose of achieving rapid drug screening. Third, virtually all these assays require a human experimenter, either to manually score event occurrences (e.g., sniffing, grooming) and/or to manually transfer rodent subjects between their home cages and testing arenas. Effective blinding may be impossible when mutants display obvious phenotypic traits (e.g., microcephaly), and human pheromonal cues can markedly impact behavioral results. 22 , 23 Recent advances in videotracking and home-cage instrumentation now permit the collection of more prolonged recordings where multimodal behavioral data is automatically acquired under experimenter-free conditions. 20 , 24 , 25 Using one such commercially available platform, we have previously demonstrated how home-cage monitoring can identify murine-specific features of pervasive neurodevelopment in monogenic epilepsy models 26 , 27 , following prenatal anticonvulsant exposure 28 , and behavioral trajectories associated with aging. 29 Home-cage methods allow for repeated longitudinal testing 29 , 30 and provide datasets that can be analyzed across multiple time scales. 20 In this study, we report a syndrome of home-cage behavioral abnormalities in mice with constitutive deletions of Kcna1 (Kcna1 -/- ). KCNA1 encodes K V 1.1, a voltage-gated potassium channel alpha subunit that assembles as pore-forming homo- or heterotetramers, together with auxiliary beta subunits that modulate channel gating, assembly and trafficking. 31 Pathogenic KCNA1 variants cause disorders of paroxysmal hyperexcitability, both in central (episodic ataxia, epilepsy, paroxysmal kinesogenic dyskinesia) and peripheral nervous systems (myokymia). 31 DEE or DEE-like syndromes have been reported in patients with homozygous or heterozygous loss of function variants in the pore domain. 32 – 38 Kcna1 -/- mice were first described in 1998 39 and have since been employed by numerous laboratories as a model of severe epilepsy and premature mortality. Kcna1 -/- mice are nonataxic and reliably display spontaneous seizures beginning at 2-3 weeks of age 39 with features that suggest a focal onset, beginning with a behavioral pause and head nodding, and progressing to forelimb clonus and tonic-clonic activity 40 . These seizures induce Fos activation within amygdalo-hippocampal circuits 41 , and a proportion of these seizures display a hippocampal onset. 40 Unmedicated Kcna1 -/- mice experience high rates of seizure-related sudden death, with few surviving past 8-10 weeks of age. 42 , 43 Treatment with a ketogenic diet is only able to delay mortality by a few weeks 44 – 46 and many antiseizure medications achieve incomplete seizure control. 47 In contrast, Kcna1 +/− mice are seizure-free, breed successfully and have a normal lifespan. 39 Assessments of behavioral comorbidity in Kcna1 -/- mice have mainly focused on sleep phenotypes: using EEG-EMG recordings, Kcna1 -/- mice exhibit significant deficits in both NREM and REM sleep. 48 , 49 Here, we reveal that Kcna1 -/- mice display a set of robust abnormalities in home-cage behavior that co-occur with spontaneous seizures. In contrast, we identify relatively subtle changes in Kcna1 +/− mice, or in mice with deletions of Lgi1 or Cntnap2 (which encode proteins that are necessary for the accurate subcellular localization of K V 1.1). Using floxed Kcna1 mice 43 , we achieve only a partial phenocopy of Kcna1 -/- behaviors following Kcna1 in parvalbumin-positive interneurons. Through a mass spectrometry proteomic survey of Kcna1 -/- hippocampi, we identify a significantly altered molecular landscape which features upregulations in a range of neuropeptides and the immediate early transcription factor EGR3 (early growth response 3) an immediate early transcription factor known to be induced by electroconvulsive seizures. 50 We show that deletions of Egr3 in Kcna1 -/- mice achieve ameliorations in neurobehavioral abnormalities, proteomic changes, as well as seizure frequency and premature mortality. Methods Animals and Treatments All animal protocols were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee. All data elements as per ARRIVE guidelines are provided. Mice were bred and weaned under standard vivarium conditions with a 12:12h light cycle (1700 lights OFF). Genotyping was conducted at postnatal day 21 using tail DNA samples. Our study includes mice of the following lineages: Kcna1 +/− (Tac:N:NIHS-BC) 39 , 45 , 46 , 51 , floxed Kcna1 ( Kcna1 fl /fl , C57BL/6) 43 , 52 – 54 , Egr3 +/− (C57BL/6) 55 – 57 , Lgi1 +/− (C57BL/6, JAX: 030640) 58 and Cntnap2 +/− (C57BL/6, JAX: 017482) 59 . We utilized the following Cre-expressing mice: CAGGCreER TM (JAX: 004682) 60 , DAT-Cre (dopamine transporter, JAX: 006660) 61 , PV-Cre (parvalbumin, JAX: 017320) 62 and EMX1-Cre (JAX: 005628) 63 . Primer sequences for genotyping are provided in Supplementary Material. Lgi1 +/− mice and Cntnap2 +/− mice were bred to achieve Lgi1 +/− Cntnap2 +/− mice. Double-heterozygous Egr3 +/− Kcna1 +/− mice were bred to compare behavioral profiles for Egr3 +/+ Kcna1 +/+ , Egr3 +/+ Kcna1 -/- , Egr3 +/− Kcna1 -/- and Egr3 -/- Kcna1 -/- mice. Egr3 -/- Kcna1 +/+ were also included in a proteomic survey, To examine the consequence of adult-onset Kcna1 deletion, ∼7-week-old CAGGCre.ER TM +. Kcna1 fl /fl and CAGGCreER- Kcna1 fl /fl littermates received five daily intraperitoneal injections of tamoxifen dissolved in corn oil (0.5ml/kg). 64 Home-cage monitoring Recordings of home-cage behavior were conducted as previously described 26 , 28 , 29 , 65 in 6-8 week old-mice unless otherwise specified. At the start of monitoring, mice were singly housed in Noldus Phenotyper® home-cages (30x30x47cm), fitted with a top unit containing an infrared (IR) camera and IR bulbs), an infrared-lucent shelter, two lickometer-connected water-spouts (providing fresh drinking water with 0% Vs 0.8% sucrose), and a beam-break metered food hopper ( Fig. 1A ). Home-cages were housed within a temperature- and humidity-controlled satellite facility fitted to a 12:12 light cycle (OFF at 1700). Video-recording and tracking was conducted via Ethovision XT 14. We employed a modular design 26 , 28 , 29 , 65 , abbreviated to at least capture (i) at least two 23h-long “baseline recordings” (from 1400 → 1300), (ii) responses to the “light spot test” (1900-2000) 66 and (iii) measurements of voluntary wheel-running on the final day (from 1400 → 1300). CAGGCre.ER TM - Kcna1 fl/fl and CAGGCre.ER TM +. Kcna1 fl/fl did not undergo light spot or wheel running evaluations. Mouse centerpoint X-Y coordinates sampled at 15Hz were automatically tallied to measure horizontal displacement (e.g., cm/min, cm/hour), heatmaps of position probability, shelter engagement time and were also applied to estimate the timing and duration of “sleep” bouts during baseline recordings using established behavioral definitions (≥40s of contiguous immobility 67 ). From lickometers and feeding meters, we extracted both the duration and occurrences of licking and feeding bouts. Wheel rotations/min were tallied using a magnetic wheel sensor (Noldus). Download figure Open in new tab Figure 1. Kcna1 -/- mice display multiple aberrations in home-cage behavior in concert with spontaneous seizures. (A) Mice are studied within square (30x30cm) home-cages featuring an infrared-(IR)lucent shelter, two lickometered water sources, a food hopper, an aerial IR camera and IR bulbs. (B) Raster plot, with every row depicting a single mouse and where every cell depicts the horizontal distances accumulated within a minute of the day. Purple arrow: a generally synchronized rest period is observed in WT mice (n = 19, 10 female) during the mid-active phase, and which is absent in Kcna1 -/- mice (n = 13, 7 female). (C) Kcna1 -/- mice traverse greater distances over baseline recordings (hour x genotype, F21, 798 = 10.89, p<0.0001). (D) This effect is associated with a reduction in behaviorally defined “sleep” (see Methods), fewer “sleep” bouts that were also shorter in duration (KS p<0.0001). (E) Time budgets for sheltering, drinking, feeding and other behaviors. (F) In addition to lower sheltering rates, Kcna1 -/- mice spend less time licking, accumulate fewer sucrose but not water licks and achieve greater feeding entries. Bout durations for sucrose and water licking, as well as feeding are significantly shorter (left shifted, KS p<0.0001 for all). Representative heat maps of position probability are shown. (G) Responses to an hour-long light spot test applied at 1900. (H) Kcna1 -/- mice accumulate fewer wheel rotations (F22, 731 = 2.64, p<0.0001). (I) Wireless EEG achieved within the home-cage by combining two baseplate receivers. During seizure prone days (SP), Kcna1 -/- mice display more pronounced reductions in sheltering compared with seizure-free days. (J) Frequency spectrogram, raw EEG and high-resolution actogram during a representative spontaneous electrographic seizure. On seizure-prone days, Kcna1 -/- mice accumulated significantly greater horizontal distances, lower sheltering rates and lick durations. Mean + s.e.m shown for all. *, **, ***, **** depict p<0.05, <0.01, <0.001 and <0.0001 respectively. KS (Kolmogorov-Smirnov test). Data Visualization and Statistical Analysis We employed sample sizes comparable to previously published results using our home-cage system. Comparisons were always made against littermate controls. When presenting data from “baseline recordings”, the second baseline day is shown. All graphs and statistical comparisons were generated on Prism GraphPad 10, while heatmaps were generated via Matlab. Contrasts between two or among three or more groups were analyzed by unpaired student’s T tests and one-way ANOVA tests, respectively. Post-hoc comparisons for one- way ANOVA were conducted via Tukey’s multiple comparisons test. Significant genotype x time interactions with repeated measures (e.g., Fig. 1C ) were assessed using a mixed model approximating a 2-way ANOVA. 26 – 28 The Kolmogorov-Smirnov (KS) test was employed to compare cumulative frequency distributions of bout lengths (e.g., Fig. 1D ). The log-rank (Mantel-Cox) test was employed to compare survival curves. All graphs depict mean ± SEM. Video-EEG, mass spectrometry proteomics, immunohistochemistry, ELISA and qRT-PCR methods are provided in supplemental material. Results Kcna1 -/- Mice Display Widespread Aberrations in Home-Cage Behavior When compared against wildtype (WT) littermates within our instrumented home-cages ( Fig. 1A ) during baseline recordings (see Methods), 5-week-old Kcna1 -/- mice displayed profound nocturnal hyperactivity ( Fig. 1B, C ), achieving an approximate doubling in daily horizontal distance (∼1000m vs ∼480m/d) within a 30x30cm square home-cage. WT mice displayed a characteristic 24 and relatively synchronized dip in activity during the mid-active phase, which was absent in Kcna1 -/- mice. In conjunction with hyperactivity, Kcna1 -/- mice displayed significant reductions in “sleep” (enquoted, since sleep here is determined behaviorally), and is consistent with previous reports that have employed beam-break actigraphy assessments 44 and traditional EEG/EMG. 48 , 49 “Sleep” bouts were fewer in number and significantly shorter in duration ( Fig. 1D ). Time budgets 68 calculated over the same baseline recordings revealed significant reductions in sheltering and licking durations that could not be explained by increased feeder engagement ( Fig. 1E, F ). Total sucrose licks (but not water licks) were significantly fewer in Kcna1 -/- mice and licking bouts across both liquid sources were significantly shorter in duration ( Fig. 1F ). While Kcna1 -/- mice displayed a greater number of feeding bouts, bout durations of feeding were also significantly shorter ( Fig. 1F ). Next, to examine how Kcna1 -/- mice respond to an aversive stimulus applied within the same home-cage, we presented an hour-long light spot 28 , 66 during the early nocturnal phase. As shown in Fig. 1G , WT mice displayed an initial burst in activity that gradually recedes as shelter engagement becomes more sustained. In contrast, Kcna1 -/- mice responded with brisk hypoactivity and shelter engagement in response to the stimulus, with an equally brisk resumption of hyperactivity following stimulus cessation. The following day, we deployed a running wheel within this home-cage. Kcna1 -/- mice accumulated significantly fewer wheel rotations over this trial ( Fig. 1H ), potentially signaling a hedonic deficit (akin to reduced sucrose drinking). Finally, to broadly examine how inter-daily variations in seizure frequency may be linked to fluctuations in the same home-cage metrics, we deployed wireless EEG implants in a separate cohort of Kcna1 -/- mice and conducted simultaneous home-cage recordings. On average, seizure-prone days (containing at least one spontaneous seizure) featured significantly greater total daily distances and reduced sheltering ( Fig. 1I, J ). EEG implantation itself imposed a marked reduction in home-cage activity levels (compare with Fig. 1C ). Nevertheless, we identified a weak but significant positive correlation between seizure frequency (range 0 −22/d) and total daily distances (r = +0.37, p<0.001). Together, these findings reveal a syndrome of behavioral disturbances that is phenotypically compatible with an encephalopathy, featuring core deficits in arousal and sleep, as well as more diffuse derangements in sensory responsivity, amotivation and fragmented feeding/drinking rhythms. Further, our EEG results suggest that the severity of encephalopathy in Kcna1 -/- mice maybe dynamically modulated by (or modulates) day to day variations in seizure risk. On the Impact of Gene Dosage and Alterations in Subcellular Distribution Heterozygous loss of function variants in KCNA1 can result in either episodic ataxia, myokymia and/or epilepsy in humans. KCNA1 variants within the pore domain are most tightly linked to epilepsy and DEE. 31 . Kcna1 +/− mice are seizure free 39 , but exhibit a lowered seizure threshold when exposed to fluorothyl 39 , and are also more prone than WT mice to develop electrographic seizures following perinatal hypoxia. 69 To examine how reductions in Kcna1 expression may shape home-cage behavior in the absence of spontaneous seizures, we compared a separate cohort of Kcna1 +/− mice and WT littermates ( Fig. 2A , Fig. S1A ). Kcna1 +/− mice exhibited greater sheltering times without an increase in total daily “sleep” or overall activity levels. Light spot testing revealed a more sustained sheltering response in Kcna1 +/− mice, extending for several minutes beyond stimulus cessation ( Fig. 2A ). These results suggest that any potential linear relationships between Kcna1 gene dosage and home-cage behavior abnormalities are either absent or distorted by spontaneous seizures in Kcna1 -/- mice. Download figure Open in new tab Figure 2. Home-cage behavior in Kcna1 +/−, Lgi1 +/−, Cntnap2 +/− and Cntnap2 -/- mice. Each panel depicts raster plots of locomotor activity (top left), hourly distances accumulated (bottom left), total metrics for the second baseline recording (center), and averaged light spot and wheel running responses (right). (A) Compared with WT littermates (n=16, 8 female), Kcna1 +/− mice (n=16, 8 female) display a mild increase in overall sheltering time during baseline recordings. (B) Compared with respective WT littermates (n=16, 8 female), Lgi1 +/− mice (n=16, 8 female) display a mild but significant reduction in total daily “sleep”. (C) Cntnap2 -/- mice (n=19, 6 female) feature significant reductions in total daily distances compared with Cntnap2 +/− (n = 17, 10 female) or littermate WT mice (n=16, 7 female). Mean + s.e.m shown for all. *, *** depict p<0.05 and <0.001 <0.0001 respectively. KS (Kolmogorov-Smirnov). The accurate subcellular localization of KCNA1 is governed in part by two other proteins that are encoded by epilepsy genes: LGI1 and CASPR2. LGI1 is a secreted protein that modulates neuronal excitability by binding its cognate receptors, ADAM22/ADAM23. 70 Heterozygous variants in LGI1 that impair protein secretion or receptor binding give rise to autosomal dominant lateral temporal lobe epilepsy with a penetrance of ∼67%. 71 Auto-antibodies against LGI1 are among the most frequently identified cause of autoimmune encephalitis 72 , where progressive neurocognitive deterioration is accompanied by dystonic seizures. Lgi1 +/− mice have a normal lifespan and fertility, while Lgi1 -/- mice develop spontaneous seizures in the second postnatal week and die by post-natal day 20. 58 , 73 Lgi1 loss is thought to contribute to seizures by regulating excitatory synaptic transmission 74 , 75 and its role in maintaining the expression of KCNA1 at axon terminals 76 and the axon initial segment. 77 Membrane fractions from Lgi1 +/− mice display ∼50% reduction in KCNA1 expression, and patient-derived anti-LGI1 antibodies effectively lower total and synaptic levels of KCNA1 expression. 78 When compared against their respective WT littermates within home-cages, Lgi1 +/− mice displayed mild insomnia without hyperactivity ( Fig. 2B , Fig.S1B), but were otherwise indistinguishable during baseline trials, light spot and wheel running trials. CASPR2, encoded by CNTNAP2 , is a trans-synaptic membrane protein that is necessary for the clustering of KCNA1 subunits to the juxtaparanodal region of myelinated axons 79 , 80 and for normal patterns of neuronal migration. 59 , 81 , 82 In humans, biallelic loss of function variants of CNTNAP2 give rise to DEE, featuring autism and focal cortical dysplasia, while the pathogenicity of Cntnap2 haploinsufficiency remains controversial. 83 , 84 Given strong peripheral and central nervous system expression, auto-antibodies against Caspr2 result in autoimmune encephalitis that is frequently accompanied by neuromyotonia and dysautonomia. 72 , 85 While Cntnap2 -/- mice exhibit normal nerve conduction velocities, they display reductions in social interactions/vocalizations and develop spontaneous seizures beginning at ∼6 months of age. 59 , 79 , 81 Mice with deletions of Cntnap2 and/or infusions of patient-derived anti-Caspr2 display hypersensitivity to noxious stimuli through the downregulation of K V 1.1 in membranes of dorsal root ganglion neurons 86 . Compared to WT mice within home-cages, Cntnap2 -/- mice accumulated significantly lower total horizontal displacement during baseline recordings, replicating prior results 87 ( Fig. 2C , Fig. S1C ). This effect came with more prolonged drinking and feeding durations ( Fig. S1C ), but without changes in “sleep” amount and timing. WT, Cntnap2 +/− and Cntnap2 -/- mice displayed similar initial responses to light spot testing, while Cntnap2 -/- mice featured early shelter exit, suggesting a state of lowered risk aversion. WT and double-heterozygous Cntnap2 +/− Lgi +/− mice were also indistinguishable across our home-cage metrics ( Fig. S1D ). Together, these results demonstrate that Kcna1 haploinsufficiency or genetic lesions that disturb the subcellular localization of KCNA1 can also perturb metrics of home-cage behavior in directions that are quite distinct from Kcna1 -/- mice. Effects of Temporal and Spatially Precise Deletions of Kcna1 Next, we explored whether widespread deletions of Kcna1 achieved beyond early developmental timepoints are sufficient to recapitulate the behavioral abnormalities in Kcna1 -/- mice. We adopted an established strategy that utilizes the expression of tamoxifen-inducible Cre- recombinase under the control of a ubiquitous promoter 64 crossed with floxed Kcna1 mice ( Kcna1 fl /fl ) 43 , 88 . As shown in Fig. 3A , CAGGCre.ER TM positive and negative Kcna1 fl /fl mice displayed similar home-cage metrics prior to tamoxifen exposure. Six weeks later, tamoxifen exposure produced a significant, > 90% reduction in Kcna1 mRNA levels ( Fig. S2A ). At this time point, CAGGCre.ER TM + Kcna1 fl /fl mice displayed a mild but significant reduction in total daily “sleep” with subtle reductions in sleep bout durations, but were otherwise indistinguishable from CAGGCre.ER TM -negative littermates ( Fig. 3A , Fig. S2A ). Thus, a deletion of Kcna1 delayed until adulthood is likely to elicit compensatory mechanisms that maintain behavioral homeostasis. Download figure Open in new tab Figure 3. Effects of delayed or cell-type specific genetic deletions of Kcna1 . (A) Prior to tamoxifen induction (“Pre-TAM”), CAGGCre.ERTM+ Kcna1fl /fl (n=19, 9 female) and CAGGCre.ERTM- Kcna1fl /fl mice (n=21, 11 female) were indistinguishable on baseline recordings. 6 weeks following tamoxifen exposure, compared with CAGGCre.ERTM- Kcna1fl /fl mice (n=23, 10 female), CAGGCre.ERTM+. Kcna1fl /fl mice (n=24, 12 female) displayed a mild but significant reduction in total daily “sleep”, as well as a subtle reduction in “sleep” bout durations (KS p<0.05). (B) Selective deletions of Kcna1 from PV+ interneurons result in Kcna1 -/- like reductions in sheltering and “sleep”. All remaining home-cage metrics were unchanged across groups. Cre-negative mice (grey) were always compared against respective Cre-positive littermates. Sample sizes: Emx1Cre- (n=22, 6 female), Emx1Cre+ (n=20, 13 female), PVCre- (n=13, 10 female), PVCre+ (n=25, 13 female), DATCre- (n=18, 10 female) and DATCre+ (n=21, 8 female). Mean + s.e.m shown for all. *depicts p<0.05. KS (Kolmogorov Smirnov test) Conditional deletions of Kcna1 in excitatory forebrain neurons of the neocortex, hippocampus, amygdala and certain vagal circuits 89 , achieved via the Emx1-Cre driver are sufficient to give rise to spontaneous seizures and premature mortality, albeit with a milder phenotype compared with Kcna1 -/- mice. 54 To examine how Kcna1 loss in specific neuronal populations may phenocopy aspects of the Kcna1 -/- neurobehavioral syndrome, we recorded the same home-cage metrics in Kcna1 fl /fl mice with or without the concurrent expression of Cre-recombinase in Emx1-expressing, parvalbumin (PV)-expressing or dopamine-transporter (DAT)-expressing cells. KCNA1 plays a key role in controlling the excitability of fast-spiking interneurons. 90 – 92 Similarly, Kv1 channels also function to restrain dopamine release from mesencephalic dopaminergic neurons 93 , 94 where their expression is necessary for the central stimulatory effects of amphetamine. 95 , 96 Emx1- and PV-Cre positive mice displayed a marked reduction in hippocampal Kcna1 mRNA levels ( Fig. S2B, C ). Mice with Kcna1 deletions in Emx1+ or DAT+ neurons, were indistinguishable from their respective Cre-negative littermates. In contrast, mice with PV-specific deletions of Kcna1 displayed reductions in total daily “sleep” and sheltering rates, together with a significant increase in sucrose drinking ( Fig.3B , Fig. S2B-D ). Responses to light spot stimulation or wheel-running were similar in all three conditions ( Fig. S2B-D ). We confirmed a significant downregulation of hippocampal Kcna1 mRNA in CAGGCre.ER TM, Emx1Cre and PVCre expressing mice ( Fig. S2A-C ). Together, these findings suggest that loss of Kcna1 in excitatory cortical and limbic circuits may serve to selectively enhance seizure risk, while similar deletions of Kcna1 in PV interneurons may drive alterations in sleep and sheltering behavior in Kcna1 -/- mice. Kcna1 deletions in either cell type alone were insufficient to recapitulate the robust hyperactivity and bout fragmentation observed with constitutive deletions of Kcna1 . Changes to the Hippocampal Proteome in Kcna1 -/- Mice Next, to reveal insights into the molecular mechanisms that may directly mediate all or parts of the Kcna1 -/- neurobehavioral syndrome, we conducted microscale label-free proteome profiling of snap-frozen dissected whole hippocampal tissue. After correcting for multiple comparisons, we identified 150 proteins that were significantly up-(60) or down-regulated (90) in expression ( Fig. 4A , Supplemental table 1)). Downregulated proteins included KCNA1, as well as several proteins where reduced expression or loss of function has also been linked to DEE, including WDR45 97 , GRIN2A 98 , CASPR2 84 , SYNGAP1 99 , KCND2 100 and GRIA2. 101 Among upregulated proteins, we identified a series of neuropeptides that have been linked to temporal lobe epilepsy, including several members of the granin family (SCG2, SCG3, CHGB) 102 – 104 , preproenkephalin (PENK) 105 , NPY 106 , CCK 107 and BDNF. 108 Increases in the hippocampal expression of enkephalin and NPY have been previously reported in the megencephaly ( mceph) mouse, which has a frameshift deletion that truncates KCNA1. 109 While each of these targets are independently worthy of validation and follow up, we focused on an upregulated transcription factor, EGR3, as a potential direct link between spontaneous seizures and long-term maladaptive transcriptional changes. Download figure Open in new tab Figure 4. Hippocampal tissues in Kcna1 -/- mice display an induction of EGR3 and BDNF. (A) Z-normalized intensity-based absolute quantification (iBAQ) scores for proteins that are significantly downregulated (top) and upregulated (bottom) after correcting for false discoveries. N=3 biological replicates are shown for each group. See Supplemental Table 1 for full protein lists. (B) Confirmation by immune- histochemistry of EGR3 and GFAP induction in both ventral and dorsal hippocampal fields (n=5/genotype, scale bar = 50um). (C) Quantification of B. (D) Confirmation of BDNF induction via ELISA (n=5/group). (E) Compared with WT mice (n=25, 13 female) and Egr3 +/− mice (n=30, 14 female), Egr3 -/- mice (n=18, 8 female) displayed significantly lower sheltering rate and increased sucrose licks during baseline recordings, without significant increases in total daily “sleep”. Light-spot and wheel- running responses were similar. Mean + s.e.m shown for all. **, ***, **** depict p<0.01, <0.001 and <0.0001 respectively. EGR3 belongs to a family of immediate early genes (EGR1-4) with three zinc-finger motifs that bind to a canonical EGR DNA response element. 50 Intronic variants in EGR3 have been linked to schizophrenia, but have not yet met genome-wide significance. 110 – 113 Reductions in EGR3 expression have also been observed in postmortem samples of prefrontal cortex and hippocampus from patients with schizophrenia. 114 Following electroconvulsive seizures (ECS), EGR3 expression is induced in hippocampal dentate granule cells 115 , 116 and is necessary for ECS-induced increases in BDNF expression 56 and dendritic remodeling. 117 Egr3 -/- mice display a mild gait ataxia due to muscle spindle agenesis 57 and other behavioral phenotypes that have been extrapolated as “schizophrenia-like” : heightened aggression 118 , open-field hyperactivity 119 , exaggerated startle responses 119 , mild insomnia 55 and memory impairments that correlate with deficits in both hippocampal long term depression and potentiation. 119 , 120 Based on these observations, we hypothesized early onset seizures in Kcna1 -/- mice result in a vicious cycle of inductions in EGR3 and the pro-epileptic neurotrophin BDNF 108 , 121 , 122 that could sustain seizure risk and EGR3 levels. If EGR3 hypofunction is capable of broadly disrupting neurocognitive function, we reasoned that pathological increases in EGR3 activity may also exert deleterious effects on behavior, as shown in models of Mecp2 123 or Kctd13 124 mutations. We validated the induction of EGR3 on a separate cohort of WT and Kcna1 -/- mice using immunohistochemistry, confirming a ∼3-fold increase in EGR3 positive cells in dentate granule cells in both dorsal (anterior) and ventral (posterior) hippocampal regions ( Fig. 4B, C ). GFAP staining, as a marker of astrogliosis 51 , was also elevated ( Fig. 4B, C ) and was also identified in our unbiased proteomic screen (supplemental table 1). Using ELISA in separate mice, we confirmed that whole hippocampal lysates Kcna1 -/- mice contained significantly increased levels of BDNF ( Fig. 4D ). In recordings of home-cage behavior ( Fig. 4E , Fig. S3A ), Egr3 +/− and Egr3 -/- mice exhibited levels of activity and “sleep” during baseline recordings that were comparable to WT littermates. Egr3 -/- mice spent a significantly lower time in the shelter and a significantly elevated number of sucrose licks. Responses to the light spot test were qualitatively similar across all three genotypes. On average, Egr3 -/- mice accumulated fewer wheel rotations, but this effect was not statistically significant ( Fig. 4E ). Heterozygous or Homozygous Deletions of Egr3 Ameliorate Ictal and Interictal Abnormalities in Kcna1 -/- mice To directly test our hypothesis, we asked whether Egr3 deletions can rescue epileptic and/or encephalopathic features in Kcna1 -/- mice. We bred double heterozygous Egr3 +/− Kcna1 +/− mice and studied the following four (out of 9 possible) genotypes: Egr3 +/+ Kcna1 +/+ (WT), Egr3 +/+ Kcna1 -/- , Egr3 +/− Kcna1 -/- and Egr3 -/- Kcna1 -/- mice. On this modified background, we observed that the loss of one or two copies of Egr3 significantly prolonged survival ( Fig. 5A ). This effect was associated with an amelioration of overall hyperactivity levels ( Fig. 5B, C ). We did not observe reduced sheltering in Egr3 +/+ Kcna1 -/- compared with Egr3 +/+ Kcna1 +/+ mice, and Egr3 -/- Kcna1 -/- displayed the lowest amount of sheltering overall ( Fig. 5B , Fig. S3B ). Egr3 deletions also improved the duration of sucrose and water drinking bouts, without impacting overall lick numbers ( Fig. 5D ). Unlike hyperactivity patterns, behaviorally measured deficits in “sleep” were not restored by Egr3 deletion, although Egr3 -/- Kcna1 -/- mice did display more normalized “sleep” bout distributions ( Fig. 5E ). Egr3 deletion did not alter responses to light spot stimulation ( Fig. 5F ), but did improve overall wheel running ( Fig. 5G ). Homozygous reductions in Egr3 also facilitated significant reductions in overall GFAP immunoreactivity and hippocampal BDNF levels ( Fig. 5H ). To examine whether these neurobehavioral and histopathological changes were associated with improvements in seizure frequency, we conducted tethered EEG recordings that also permit measurements of spreading depolarization events in Kcna1 -/- mice. 125 , 126 Over a 12-day recording period, heterozygous deletions of Egr3 lowered the frequency of spontaneous seizures, and Egr3 -/- Kcna1 -/- mice were seizure free ( Fig. 5I ). Finally, to examine whether improvements in ictal and interictal behavioral features would be associated with a rescue in protein expression patterns, we conducted tandem mass tag proteomics on hippocampal samples from Egr3 +/+ Kcna1 +/+ (WT), Egr3 +/+ Kcna1 -/- , Egr3 +/− Kcna1 -/- and Egr3 -/- Kcna1 -/- mice. In a hierarchical clustering tree revealed from protein expression patterns, samples from Egr3 -/- Kcna1 +/+ and Egr3 -/- Kcna1 -/- mice were most similar, and together, these genotypes most closely resembled expression patterns in WT ( Egr3 +/+ Kcna1 +/+ mice). Download figure Open in new tab Figure 5. Egr3 Deletion Ameliorates the Ictal and Several Interictal Abnormalities in Kcna1 -/- Mice. (A) Compared with Egr3 +/+ Kcna1 -/- mice (n=32), Egr3 +/− Kcna1 -/- mice (n=56) and Egr3 -/- Kcna1 -/- mice (n=24) displayed significantly prolonged survival. (B) As assessed through hourly distances, Egr3 +/+ Kcna1 -/- mice (n=23, 13 female) were significantly more hyperactive compared with WT ( Egr3 +/+ Kcna1 +/+, n=19, 5 female), Egr3 +/− Kcna1 -/- (n=36, 19 female) or Egr3 -/- Kcna1 -/- mice (n=16, 6 female, F66, 1980 = 3.42, p<0.0001). Total distances for B (F3,90 = 4.44, p<0.001) and total sheltering (F3,90 = 8.36, p<0.0001). (C) Locomotor activity raster plot for all mice shown in B. (D) Total number of feeding, sucrose and water licking bouts for all four genotypes, together with cumulative frequency distribution histograms shown. Compared with Egr3 +/+ Kcna1 -/- (teal), Egr3 -/- Kcna1 -/- mice (red) displayed more prolonged bouts across all three categories (KS p<0.0001). (E) Egr3 deletion did not improve reductions in total daily “sleep” in Kcna1 -/- mice, but did partially improve “sleep” bout durations (KS p<0.0001). (F) Responses to light spot testing. (G) Egr3 deletion rescued reductions in wheel-running in Kcna1 -/- mice assessed in hourly bins (F69, 1734 = 1.31, p<0.01) or total wheel rotations (F3,79 = 4.52, p<0.001). (H) Egr3 deletion also improved astrogliosis, as measured by GFAP immunoreactivity and total hippocampal BDNF levels. (I) Using conventional tethered equipped with DC band recording facilities, Egr3 -/- Kcna1 -/- mice and to a lesser extent, Egr3 +/− Kcna1 -/- mice featured a significant improvement in the frequency of spontaneous seizures and spreading depolarization events (n=3-4/group). (J) Hippocampal proteomic profiles from ∼7-week old mice visualized as heatmaps of relative Z-normalized expression (averaged across biological replicates), ranked in order of increasing fold change in Egr3 -/- Kcna1 -/- mice compared with Egr3 +/+ Kcna1 -/- mice. The dendrogram shown was constructed by hierarchical clustering of protein expression profiles using average linkage on correlation-based pairwise distances between biological samples. Mean + s.e.m shown for all. *, **, ***, **** depict p<0.05, <0.01, <0.001 and <0.0001 respectively. KS (Kolmogorov-Smirnov test). Discussion In this study, we leveraged the advantages of home-cage monitoring to elaborately visualize a syndrome of neurobehavioral abnormalities that co-occur with spontaneous seizures in mice lacking Kcna1 /Kv1.1. 39 Under task-free conditions, Kcna1 -/- mice displayed marked hyperactivity and hyperarousal, together with excessively fragmented bouts of sleep, feeding and drinking. In response to light- spot stimulation within their own home-cages 26 – 29 , Kcna1 -/- mice displayed a relatively abrupt reduction in home-cage exploration, potentially reflecting enhanced risk aversion (paralleling states of heightened anxiety 66 ). Kcna1 -/- mice also displayed fewer sucrose licks and accumulated fewer wheel rotations, pointing to alterations in natural reward sensitivity. Importantly, these quantitative phenotypes were acquired digitally through (i) prolonged recordings, (ii) conducted under experimenter-free conditions and (iii) after the habituation to a novel standardized cage that the mouse adopts as its home-cage. 20 We propose that this constellation of changes in sleep/arousal, sensory responsivity, amotivation and behavioral fragmentation effectively models the encephalopathy of DEE. In search for the upstream determinants of these encephalopathy-like behavioral changes, we sought to separate the neurobehavioral consequences of early widespread Kcna1 loss from those purely downstream of spontaneous seizures (caused by the loss Kv1.1). Using wireless EEG deployed within home-cages, we found that daily measures of home-cage hyperactivity were positively correlated with daily variations in seizure frequency. Rather than infer any true causal relationship (i.e., seizures cause hyperactivity, or vice versa), this suggests that biological mechanisms which dynamically modulate seizure risk in Kcna1 -/- mice may pleiotropically alter peri-ictal home-cage behavior. To further isolate the contributions of seizures, we examined how variations in KCNA1 expression that do not result in spontaneous seizures impact home-cage behavior. Under identical home-cage conditions, mice that were haploinsufficient for Kcna1 and which are seizure free 39 were effectively asymptomatic. Mice with deletions of Lgi1 (which express 50% less KCNA1 at axon initial segments 77 ) only displayed mild insomnia without hyperactivity. In contrast, those with homozygous deletions of Cntnap2 (which lack KCNA1 at juxtaparanodal regions 79 ) featured hypoactivity without hypersomnia, and double heterozygous ( Lgi1 +/− Cntnap2 +/− ) mice were also behaviorally asymptomatic. By utilizing Kcna1 fl /fl mice 43 , we found that tamoxifen-induced 64 adult-onset deletions of Kcna1 also only produced mild insomnia without impacting activity levels and bout structure. We further conducted a limited survey of cell-type specific Kcna1 deletions: only PVCre+ Kcna1 fl /fl mice displayed a mild behavioral phenocopy of Kcna1 -/- mice, featuring significant reductions in sheltering and total daily “sleep”, while deletions of Kcna1 in Emx1-Cre or DAT-Cre expressing neurons did not alter home-cage metrics. Taken together, these results highlight how widespread KCNA1 loss occurring in early neurodevelopmental windows and early onset spontaneous seizures are both necessary to establish aberrations in home-cage behavior in Kcna1 -/- mice. These results provide a preclinical example of how the encephalopathy of this DEE has both lesional developmental and epileptic contributions. As a corollary, these data suggest that gene replacement strategies designed to alleviate DEE-like features in patients with KCNA1 DEE must be administered early in disease course and successfully transduce a wide population of cell types to be efficacious. Next, we pivoted to exploring candidate molecular mechanisms downstream of Kcna1 loss and seizures that might perpetuate a state of pervasive seizure risk and encephalopathy. We chose to conduct a proteomic exploration of the hippocampus, a nidus for seizure generation in Kcna1 -/- mice, and where prominent astrogliosis 51 and mossy fiber sprouting is also evident following status epilepticus. 40 While several significantly regulated proteins were worthy of follow up, our attention was drawn to the induction of EGR3 and BDNF, given the extensive and highly corroborated studies demonstrating upregulations of EGR3 following electroconvulsive seizures. 50 , 56 , 115 , 117 , 127 Increases in Egr3 mRNA levels (also measured cross-sectionally) have been observed following amygdala kindling 128 , and in three distinct rodent models of audiogenic seizures (the tremor mouse 129 , the Wistar Audiogenic Rat and the Genetic Audiogenic Seizure Hamster 130 , 131 ). BDNF may function both upstream and downstream of EGR3: while Egr3 deletions severely attenuate BDNF inductions following electroconvulsive seizures 56 , 117 , BDNF has been shown to induce EGR3 in ex vivo cortical neuron culture models. 132 , 133 Using continuous video-EEG in rats rendered epileptic through pilocarpine injections, one previous study showed that mRNA levels for both BDNF and Egr3 were increased 3h following spontaneous seizures, and which both returned to baseline levels 24h later. 134 To examine whether Egr3 deletion may ameliorate either neurobehavioral or epileptic features in Kcna1 -/- mice, we crossed double- heterozygous Egr3 +/− Kcna1 +/− mice. Compared to WT mice in this modified background, ( Egr3 +/+ ) Kcna1 -/- mice still displayed spontaneous seizures, premature mortality, hyperactivity, insomnia, reduced wheel-running and fragmented sleep and licking bouts ( Fig. 5 ), but did not display reduced sheltering, reduced sucrose-licking or abnormal light-spot responses ( Fig. 1 ). Deleting one or two Egr3 alleles led to improved seizure frequency, prolonged survival, attenuated hyperactivity and restored wheel-running interest in Kcna1 -/- mice but did not improve reductions in total daily “sleep”. Ablating Egr3 also improved markers of astrogliosis and reduced BDNF levels, suggesting that at least in the context of Kcna1 loss, BDNF induction may indeed be downstream of Egr3 induction. Multiple lines of evidence implicate a proconvulsant/pro-epileptogenic role for BDNF. 108 Chemically induced status epilepticus dramatically raises BDNF levels and the activation of BDNF’s receptor, tropomysin-related kinase B (TrkB), whereas inhibiting TrkB post-status epilepticus reduces the frequency of spontaneous seizures and deficits in exploratory behavior. 135 Mice with transgenic overexpressions of BDNF or TrkB display hippocampal CA3 hyperexcitability and experience more clinically severe kainate-induced seizures. 121 , 122 Direct hippocampal infusions of BDNF have also been reported to elicit spontaneous limbic seizures. 136 Finally, Egr3 deletion also improved the frequency of spreading depolarization events which may correlate with improved survival in Egr3 -/- Kcna1 -/- compared with Egr3 +/+ Kcna1 -/- mice. 137 While transcription factors have been traditionally considered to be ‘undruggable’ targets 138 , inhibitors of Egr1 (whose zinc finger domains are 90% homologous to Egr3 139 ) are currently being developed to treat atopic dermatitis, where Egr1 plays a crucial role in modulating local inflammation and cell replication. 140 , 141 Since we observed significant disease modifying effects with deletion of only a single copy of Egr3 , and since Egr3 +/− mice were phenotypically indistinguishable from WT mice under home- cage conditions, we expect that partial pharmacological inhibition of Egr3 that is instituted in early developmental windows could offer disease modifying effects that may adjunct routine antiseizure medications use. We acknowledge four main limitations to this study. First, while our home-cage metrics within each experiment were consistently compared between littermate subjects, we did not back-cross our mice to ensure that all mice were studied on the same background. We transparently report these background effects: for example, Cre-negative Kcna1 fl/fl mice in our PV-Cre experiment were hyperactive compared with Cre-negative Kcna1 fl/fl mice within Emx1 comparisons. Similarly, WT littermates to Lgi1 +/− mice behaved distinctly when compared to WT littermates of Kcna1 -/- mice. This may have introduced potential floor or ceiling effects when examining U-shaped continuous variables such as distances/day, licks/day, etc. Second, we did not systematically conduct wired or tethered EEG recordings for most of our behavioral comparisons. We focused on behavioral metrics as a primary endpoint and therefore cannot rule out whether spontaneous seizures in any single mouse either disrupted or normalized our selected metrics. Third, while our home-cage approach provides a detailed analysis of the spatiotemporal structure of spontaneous behavior over a representative day, they still provide only a snapshot of behavior in singly housed mice. Solutions to achieve individual subject-level kinematic data from group housed mice over multi-day or multi-week recordings remains an active area of technological innovation. Fourth, while our home-cage survey provides quantitative metrics regarding several dimensions of mouse behavior (rest and activity patterns, risk aversion, natural reward, appetitive behaviors), it does not directly or indirectly measure aspects of learning and memory, which is itself a broad multidimensional set of constructs (fear learning, spatial learning, object learning, etc.). Nevertheless, home-cage behavioral measurements provide a valuable backdrop with which to appraise learning and memory deficits, if present. While memory impairments in Kcna1 -/- mice 142 might not be caused by poor sleep or hyperactivity, pharmacological treatments to improve sleep function may improve memory function. In summary, our results validate the use of home-cage monitoring as an ethologically sound approach to measuring neuropsychiatric comorbidity in a monogenic mouse model of DEE. We provide a set of precision endpoints that can be employed to test future therapies aimed at alleviating seizures and encephalopathy-like behavioral changes, and which can be compared across mouse models. 26 , 27 , 29 By combining these endpoints with conventional EEG recordings, we show that targeted deletions of a seizure-induced transcription factor (EGR3) can achieve therapeutic improvements in both ictal and interictal features of disability. Competing Interests VK is a paid consultant for the Digital In Vivo Alliance (Jackson Labs) and serves on the Scientific Advisory Board for Enliten AI. JLN serves on the Scientific Advisory Board for Rapport Therapeutics and Ovid Therapeutics. Data Availability All raw data from this study are available from the corresponding author upon reasonable request. Mass spectrometry proteomics data has been deposited publicly (see supplemental methods). Funding This work was supported by grants from the NIH to VK (K08NS110924, R01NS131399), EG (R01NS099188, R01NS129643) and JLN (R01NS029709). The BCM Mass Spectrometry Proteomics Core is supported by CPRIT Core Facility Award (RP210227), P30 Cancer Center Support Grant (NCI CA125123), Intellectual Developmental Disabilities Research Center Award (P50 HD103555), Gulf Coast Center for Precision Environmental Health (P30 ES030285) and the S10 High End Instrument Award (OD026804). Supplemental Material Supplemental Methods Mass Spectrometry Proteomics Bilateral whole hippocampi were rapidly dissected from mouse brains between 1000-1400h, frozen in dry ice, stored at −80C and provided to the BCM Mass Spectrometry Proteomics Core Facility. We applied microscaled label-free proteome profiling to first assess proteomic disturbances between WT and Kcna1 -/- mice (n = 3/group, Fig. 4A /Supplemental Table 1), 143 . Frozen tissues were pulverized on a cold metal block with mechanical action. Tissue powder was dissolved in 50mM ammonium bicarbonate and digested using LysC enzyme for 2 hours at room temperature followed by trypsin at 37 0 C overnight. Enzyme reaction was neutralized using 10% formic acid and peptides were measured using the Pierce™ Quantitative Colorimetric Peptide Assay (Thermo Scientific 23275). Offline high pH STAGE peptide fractionation (15 fractions combined to 5 peptide pools) for 50µg peptide was carried out as described previously 143 . LC-MS/MS analysis was carried out using a nano-LC 1000 system (Thermo Fisher Scientific, San Jose, CA) coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Peptides were eluted using a 110min gradient of 2-30% acetonitrile/0.1% formic acid at a flow rate of 200nl/min onto the mass spectrometer operating in data-dependent acquisition mode with 3 second cycle time and a 5 second dynamic exclusion. MS1 was acquired in Orbitrap (120,000 resolution, 350-1400m/z) followed by MS2 in IonTrap (HCD 30%, AGC 5E4, 30ms ion injection). Raw data were processed with Proteome Discoverer 2.1 software (Thermo Scientific) using Mascot 2.4 (Matrix Science 144 ) against the mouse NCBI RefSeq database updated on 03/24/2020. Search settings included Trypsin/P enzymatic digestion to form peptides from 7-50 amino acids in length, a parent ion charge between 2-6, a maximum of 2 missed cleavages, and cleave protein n-term methionine enabled. Precursor and product ion tolerances were set to 20ppm and 0.5Da respectively. Dynamic modifications included oxidation on methionine, carbamidomethyl on cysteine, and protein N- terminal acetylation. Identified peptides were validated at 5% false discover rate (FDR) with Percolator 145 . The gene product inference and iBAQ-based quantitation was carried out using the gpGrouper algorithm 143 . Expression data was median-normalized before downstream data analyses. Differentially expressed proteins were calculated using a moderated t-test to calculate p-values and log 2 fold change in the R package limma with multiple hypotheses correction by Benjamini-Hochberg method 146 . To examine how proteomic changes may be restored by Egr3 deletion ( Fig. 5J /Supplemental Table 2), we simultaneously profiled identically collected hippocampal samples from Egr3 +/+ Kcna1 +/+ (n=5), Egr3 +/+ Kcna1 -/- (n=4), Egr3 +/− Kcna1 -/- (n=3), Egr3 -/- Kcna1 -/- (n=4) and Egr3 -/- Kcna1 +/+ mice (n=4). Given the more complex group design, tandem mass tag (TMT) labeling was chosen to reduce technical variability, as multiplexing allows for samples to be analyzed simultaneously, controlling for batch effects. Protein extraction, digestion and peptide fractionation was carried out as previously described 147 . Briefly, pulverized tissue was lysed in 8M urea and digested using LysC and Trypsin proteases as before, and 25µg peptide per sample were labeled with TMT10 plex isobaric label reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. High-pH offline fractionation was performed, combined into 24 peptide pools, and acidified to a final concentration of 0.1% formic acid. Deep-fractionated peptide samples were separated on a nano-LC 1200 system coupled to the Orbitrap Exploris480 mass spectrometer (Thermo Fisher Scientific). Peptides were eluted using a 110min gradient of 2-30% acetonitrile/0.1% formic acid at a flow rate of 200nl/min onto the mass spectrometer operating in data-dependent acquisition mode, with a 2 second cycle time and a dynamic exclusion of 20 seconds. MS1 was measured in the Orbitrap at 120000 resolution, scan range 375-1500m/z and 50ms Injection time, followed by MS2 in the Orbitrap at 30000 resolution (HCD 38%) with TurboTMT algorithm. Dynamic exclusion was set to 20 seconds and the isolation width was set to 0.7m/z. MS raw data processing and differential analysis was carried out as described previously 148 , with precursor AUCs and reporter ion intensities extracted with Masic 149 ]. Reverse decoys and common contaminants were added to the GENCODE mouse protein database using Philosopher 150 . Mass spectra were searched using MSFragger (v3.5) with mass calibration 151 . Fixed modifications included carbamidomethylation of cystine (+57.021465), and TMT reagent on lysine (+229.1629). Variable modifications include oxidation of methionine (+15.9949), peptide N-term pyroGlu formation from Q (−17.02560) or E (−18.01060), peptide N-term 2-oxo-propanoic acid formation from C (−17.00329, approximated as – 17.02560), protein N-term acetylation (+42.01567), and peptide N-term TMT (+229.1629). Database search was performed with strict trypsin rule (cleave after K/R) for peptides between 7 and 50 amino acids, charge 2-6, between 350-10,000 Da, up to 2 missed cleaves, and cleave protein n-term methionine enabled. Precursor mass tolerance was set to 20 ppm, a fragment mass tolerance of 0.02 Da. The top 300 peaks were used, with a minimum of 15. Peptide validation was performed with Percolator and filtered to 1% PSM FDR 145 . Gene product inference and iBAQ-based quantification was carried out using the gpGrouper algorithm 143 . Expression data was median- normalized and batch corrected for TMT multiplex with ComBat 152 before downstream data analyses. Differentially expressed proteins were calculated using the moderated t-test to calculate p-values and log fold changes as implemented in limma 146 . Electroencephalography Our study employed both wireless and wired/tethered EEG. To enable simultaneous home-cage monitoring and EEG recording and permit unhindered access to shelters ( Fig. 1I, J ), we implanted ∼5.5-week-old Kcna1 -/- mice with EMKA easyTEL S-ETA devices ( wireless ; under sterile precautions and isoflurane anesthesia), that included (i) two biopotential leads that will be affixed subdurally in right frontal and left posterior parietal regions using dental cement, tunneled to a (ii) a wireless transponder positioned in the left flank. Following a ∼96h recovery period (featuring meloxicam analgesia and saline boluses as needed), we transferred mice into home-cages with two wireless receivers placed underneath. At least 8 complete days of wireless EEG was acquired at 1000Hz through IOX2 (EMKA’s bio- potential signal acquisition interface) and reviewed using LabChart reader using a bandpass filter (1-30Hz). We tallied electrographic seizures, which were easily recognizable epochs of rhythmic EEG activity with evolutions in frequency and morphology. 96 days of combined home-cage wireless EEG recordings from Kcna1 -/- mice (n=15) were divided into seizure free or seizure prone days (containing at least one spontaneous seizure). To measure seizure occurrence in Kcna1 -/- mice with or without Egr3 deletions, we employed a tethered approach positioning 36 gauge Teflon coated silver wires subdurally over mouse cortex and neck muscles, followed by a cement cap 125 , 126 . Following a five-day recovery period, EEG recordings were collected within observation cages furnished with bedding, ad libitum chow/water. EEG was recorded and analyzed using Bioamp DC amplifiers and LabChart. An aerial infrared camera permitted continuous video recording. Immunohistochemistry (IHC) and Enzyme linked Immunosorbent Assay (ELISA) IHC against EGR3 and GFAP was conducted using published protocols 153 with slight modifications. Mice were deeply anaesthetized with an intraperitoneal injection of Beuthanasia-D [0.1ml of pentobarbital (390mg/L) and phenytoin (50mg/L)] and then transcardially perfused with cold phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA)/ PBS. Brains were extracted and post-fixed in 4% PFA/PBS for 24h, and then dehydrated in 30% sucrose/PBS for 24h. After cryo-protection, brains were cautiously dipped in cryo-molds containing Tissue Plus optimal cutting temperature embedding medium (Fisher Healthcare) and frozen at −80°C. 20µm free- floating coronal cryosections were cut using a Leica CM1950 cryostat (Leica Biosystems) at −20°C. Prior to staining, sections mounted on Superfrost slides were allowed to warm to room temperature and washed with PBS [10 min], dipped in Liberate Antibody Binding solution (L.A.B. Polysciences Inc.) for antigen retrieval [5 min], followed by 3 x 5 min PBS washes, permeabilized with 0.5% TritonX- 100/PBS [30 min/37°C/water bath] and underwent an additional three 10 minute PBS washes. After drawing a PAP pen hydrophobic barrier, sections were blocked for an hour in 5% donkey serum/PBS and incubated in primary antibodies for 1 hour (37°C), followed by overnight room temperature incubations. Primary antibodies used were rabbit-anti-NeuN [Millipore #ABN78; 1:250; overnight/4°C], mouse-anti-GFAP (Clone N206A/8) [Neuromab #75-240; 1:500; overnight/4°C] and rabbit-anti-Egr3 115 ; 1:1000; 48h/4°C]. Following this, sections were washed with PBS [10 min], incubated in Alexa Fluor 488 donkey anti-rabbit, Alexa Fluor 555 donkey anti-mouse and Alexa Fluor 568 donkey anti-mouse [Invitrogen; 1h (except for anti-Egr3 which was 24h/4°C) /RT/dark; 1:400]. Next, sections were thoroughly washed, incubated in DAPI [5 minutes, dark] and mounted with Prolong Diamond Antifade mountant [Invitrogen]. Coverslipped sections were imaged on a Keyence BZ-X800 fluorescent microscope. All images were captured at 20X and/or 4X magnification, and analyzed using FIJI software (ImageJ, NIH). For cell counting, images at 20X magnification were taken and cells were counted from 4-6 sections/mouse from a set of 4-5 mice/group. For ELISA measurements of BDNF protein levels, we employed an established commercially available Quantikine ELISA kit (R&D Systems) and followed the protocol as per manufacturer’s instructions 154 , 155 . Bilateral whole hippocampal samples were snap frozen and stored in −80°C. For preparing tissue lysates, hippocampi were thawed on ice, rinsed with 1X PBS, homogenized with equal volume of Lysis Buffer 17 (with Protease inhibitor cocktail [1:10]) and centrifuged at 13000g for 15 min at 4°C. Sample absorbance at 595nm wavelength was measured using microplate reader. Quantitative Reverse Transcriptase Polymerase Chain Reaction qRT-PCR measurements of Kcna1 mRNA levels was performed as described previously 156 with minor modifications. Dissected bilateral hippocampal specimens were snap frozen in dry ice and stored at −80°C. Tissues were thawed and homogenized in Trizol reagent (Sigma Aldrich, USA). Chloroform was added and centrifuged at 12,000 g/15min/4°C. Next, the aqueous layer was exposed to 100% 2- propanol and centrifuged at 12,000 g/10min/4°C. The pellet formed was washed in 75% ethanol twice, centrifuged 7500g/5min/4°C, dissolved in RNAse-free water and quantified through NanoDrop One (Thermo Scientific). Reverse transcription was conducted using a high-capacity cDNA-RT kit (Applied Biosystems), as per manufacturer protocols. Subsequently, quantitative real-time PCR (qRT- PCR) analysis was accomplished using Power SYBR Green PCR Master Mix (Applied Biosystems) on a Quant Studio 3 real-time PCR system (Applied Biosystems). We employed the ΔΔCT method, utilizing Rplp0 as the reference transcript 157 . Download figure Open in new tab Supplemental Figure 1. (A) Time budgets (of sheltering, feeding, drinking and “other” behaviors) and cumulative frequency distribution histograms of bout duration for WT and Kcna1 +/− mice in Fig. 2A . (B) Time budgets and cumulative frequency distribution histograms of bout duration for WT and Lgi1 +/− mice in Fig. 2B . (C) Time budgets and cumulative frequency distribution histograms of bout duration for WT, Cntnap2 +/− and Cntnap2 -/- mice in Fig. 2C . (D) Cntnap2 +/− Lgi +/− mice double heterozygous mice behaved similarly to littermate WT mice under baseline conditions, light spot and wheel-running trials. Mean + s.e.m shown for all. Download figure Open in new tab Supplemental Figure 2: (A) Time budgets (of sheltering, drinking, feeding and “other” behaviors for CAGGCre.ERTM-positive and CAGGCre.ERTM-negative Kcna1fl /fl mice ( Fig. 3A ) before and 6 weeks after tamoxifen injections. Cumulative frequency distribution histograms of bout duration (center), and qRT-PCR validation of Kcna1 mRNA knockdown (right). (B) Time budgets, cumulative frequency distribution histograms of bout duration, light spot, wheel-running trial and qRT-PCR validation of Kcna1 mRNA knockdown in Emx1Cre-positive and Emx1Cre-negative Kcna1fl /fl mice ( Fig. 3B ). (C) Time budgets, cumulative frequency distribution histograms of bout duration, light spot, wheel-running trial and qRT-PCR validation of Kcna1 mRNA knockdown in PVCre-positive and PVCre-negative Kcna1fl /fl mice ( Fig. 3B ). (D) Time budgets, cumulative frequency distribution histograms of bout duration, light spot and wheel-running trial in PVCre-positive and PVCre-negative Kcna1fl /fl mice ( Fig. 3B ). Mean + s.e.m shown for all. *** depicts p<0.001 Download figure Open in new tab Supplemental Figure 3: (A) Time budgets (of sheltering, drinking, feeding and “other” behaviors) and cumulative frequency distribution histograms of bout duration in Egr3 +/+, Egr3 +/− and Egr3 -/- mice ( Fig. 4E ). Egr3 -/- mice displayed more prolonged feeding bout durations (KS p<0.0001). (B) Time budgets for Egr3 +/+ Kcna1 -/- mice, Egr3 +/+ Kcna1 +/+, Egr3 +/− Kcna1 -/- and Egr3 -/- Kcna1 -/- mice. 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Share EGR3 Deletion Rescues Developmental and Epileptic Encephalopathy in Kcna1 -null Mice Arindam Ghosh Mazumder , Saifina Karedia , Nandani Adhyapak , Catharina Schimer , John Samuel Bass , Jessica L Kamen , Miranda J Jankovic , Qinglong Miao , Amelia L Gallitano , Alexander B Saltzman , Antrix Jain , Anna Malovannaya , Edward Glasscock , Isamu Aiba , Jeffrey L Noebels , Vaishnav Krishnan bioRxiv 2025.06.07.658422; doi: https://doi.org/10.1101/2025.06.07.658422 Share This Article: Copy Citation Tools EGR3 Deletion Rescues Developmental and Epileptic Encephalopathy in Kcna1 -null Mice Arindam Ghosh Mazumder , Saifina Karedia , Nandani Adhyapak , Catharina Schimer , John Samuel Bass , Jessica L Kamen , Miranda J Jankovic , Qinglong Miao , Amelia L Gallitano , Alexander B Saltzman , Antrix Jain , Anna Malovannaya , Edward Glasscock , Isamu Aiba , Jeffrey L Noebels , Vaishnav Krishnan bioRxiv 2025.06.07.658422; doi: https://doi.org/10.1101/2025.06.07.658422 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7633) Biochemistry (17680) Bioengineering (13889) Bioinformatics (41928) Biophysics (21445) Cancer Biology (18585) Cell Biology (25491) Clinical Trials (138) Developmental Biology (13374) Ecology (19897) Epidemiology (2067) Evolutionary Biology (24308) Genetics (15606) Genomics (22496) Immunology (17736) Microbiology (40385) Molecular Biology (17175) Neuroscience (88583) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4822) Physiology (7641) Plant Biology (15149) Scientific Communication and Education (2045) Synthetic Biology (4293) Systems Biology (9822) Zoology (2271)