Spatial omics reveals molecular changes in focal cortical dysplasia type II

and indirect functional consequences of these mutations. To comprehensively characterize the impact of mTOR mutations on the brain, we employed here a multimodal approach in a preclinical mouse model of FCD type II (Rheb), focusing on spatial omics techniques to define the proteomic and lipidomic changes. Mass Spectrometry Imaging (MSI) combined with fluorescence imaging and label free proteomics, revealed insight into the brain ’ s lipidome and proteome within the FCD type II affected region in the mouse model. MSI visualized disrupted neuronal migration and differential lipid distribution including a reduction in sulfatides in the FCD type II-affected region, which play a role in brain myelination. MSI-guided laser capture microdissection (LMD) was conducted on FCD type II and control regions, followed by label free proteomics, revealing changes in myelination pathways by oligodendrocytes. Surgical resections of FCD type IIb and postmortem human cortex were analyzed by bulk transcriptomics to unravel the interplay between genetic mutations and molecular changes in FCD type II. Our comparative analysis of protein pathways and enriched Gene Ontology pathways related to myelination in the FCD type II-affected mouse model and human FCD type IIb transcriptomics highlights the animal model ’ s translational value. This dual approach, including mouse model proteomics and human transcriptomics strengthens our understanding of the functional consequences arising from somatic mutations in FCD type II, as well as the identification of pathways that may be used as therapeutic strategies in the future.


Introduction
Focal cortical dysplasia (FCD) refers to a group of various localized abnormalities in the brain's cortex that cause epileptic seizures (Najm et al., 2022).FCD is the most common cause of drug-resistant pediatric epilepsy (60-80% are refractory to current antiepileptic drugs) and accounting for 30% of surgical epilepsy cases in children (Blumcke et al., 2017;Lamberink et al., 2020).For these patients, surgical resection of the epileptic focal region is the only therapeutic option, assuming the epileptic focus is well defined.Nevertheless, even after this invasive surgical treatment, some FCD patients continue having seizures if the epileptic focus is not completely removed.These findings illustrate the need for more effective treatment options (Adler et al., 2017;Guerrini and Barba, 2021;Simona Balestrini et al., 2023;Specchio et al., 2021).
The International League against Epilepsy (ILAE) has divided FCD into three forms (type I, II, and III with sub variants) based on the histopathological characteristics of the lesions (Adler et al., 2017;Muhlebner et al., 2019;Najm et al., 2022;Specchio et al., 2021).FCD type II is one of the most prevalent forms found in patients with drug-resistant epilepsy, with type IIb being the most common (Majolo et al., 2018).
FCD type II shows cortical dyslamination and dysmorphic neurons in isolated lesions.There are two subtypes of FCD type II: FCD IIa, which has dysmorphic neurons but no balloon cells, and FCD IIb, which has both dysmorphic neurons and balloon cells.Dysmorphic neurons are characterized by abnormal morphology and abnormal orientation (Najm et al., 2022).A histological hallmark feature of FCD type IIb is the limited myelination of the lesioned white matter (Gruber et al., 2021).Another important characteristic is the presence of the gray-white matter blurring, which can be seen both in histopathological examinations and on magnetic resonance imaging (MRI) scans (Bernasconi et al., 2019;Muhlebner et al., 2019;Najm et al., 2022;Specchio et al., 2021;Wang et al., 2020).
This study focuses on the investigation of FCD type II.The majority of FCD type II cases are sporadic, suggesting that de novo somatic mutations in genes involved in neuronal cell development and migration represent the underlying disease mechanism (Gerasimenko et al., 2023;Lee et al., 2022;Lim et al., 2015;Muhlebner et al., 2019;Najm et al., 2022).These somatic mutations result in mosaicism in the affected brain, which has led to difficulties in unraveling the functional consequences of the mutations.
Previous literature and research have shown that FCD type II is linked to excessive activation of the mammalian target of rapamycin (mTOR) signaling pathway (Crino, 2016;Lim et al., 2015;Muhlebner et al., 2019).Hyper activation is a connection further confirmed by the discovery of somatic mutations in various genes regulating mTOR signaling (Gerasimenko et al., 2023;Lee et al., 2022).These somatic mutations occur in early neuroprogenitor cells and expand into a mutated clonal neuronal population (Lee et al., 2022).Immunohistochemical studies of FCD type II provide compelling evidence of heightened mTOR signaling.This is corroborated by the robust expression of the phosphorylated form of the mTORC1 downstream target S6 (Curatolo et al., 2018).The serine/threonine protein kinase mechanistic mTOR pathway plays a role in a variety of cellular functions in the brain including cell proliferation, migration, and differentiation throughout brain development as well as metabolism and autophagy (Crino, 2016;Specchio et al., 2021).In further detail, mTORC1 positively modulates lipid metabolism by promoting the expression of many genes involved in fatty acid and cholesterol synthesis (Bockaert and Marin, 2015).These gene expressions are modulated by two transcription factors, sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor-γ (PPARγ) which are activated by mTORC1 (Laplante and Sabatini, 2009).mTORC1 further promotes neuronal cell proliferation and dendritic branching in response to brainderived neurotrophic factor (BDNF).BDNF has been demonstrated to enhance protein synthesis (Takei and Nawa, 2014).Previous studies have also shown the activation of mTORC1 induces protein and lipid synthesis, which aids in the development of the axons and dendrites of a neuron (Lamming and Sabatini, 2013;Takei and Nawa, 2014).mTORC1 is a key signaling pathway for myelination, playing a key role in the differentiation of oligodendrocytes, the cells responsible for producing myelin (Figlia et al., 2018;Gruber et al., 2019).
The activation of the mTOR signaling cascade has been linked to pathological conditions such as cortical malformation that result in neurological disorders (mTORopathies).The term 'mTORopathies' (mTOR pathway-related malformations) is used to describe a range of cortical development abnormalities, including conditions like tuberous sclerosis complex (TSC) and FCD type II.These conditions are characterized by changes in the structure of the cortex, dysmorphic neurons/ glial cells and intractable seizures that result from disruptions in the mTOR signaling pathway (Crino, 2016;Muhlebner et al., 2019).Additionally, experimental preclinical models of epilepsy-associated FCD recapitulate the human pathophysiology of FCD type II (Nguyen et al., 2019;Nguyen, 2024) The abnormalities caused by mTOR activity upregulation (induced by introduction of somatic mutations in this pathway via in utero electroporation) lead to focal cortical disorganization, characterized by disrupted neural migration, cortical dyslamination and dysplastic neurons.Moreover, these preclinical models of FCD Type II represent with spontaneous recurrent seizures (Nguyen et al., 2019).
In this study, we aim to improve the understanding of the etiology of FCD type II and epileptogenesis.The lipidomic research to date has tended to focus on changes in FCD resected tissue from patients and preclinical mouse brain samples by means of bulk liquid chromatography and mass spectrometry (Dentel et al., 2022;Kumar et al., 2018;Kumar et al., 2021).Here, altered lipid classes including triacylglycerols, phosphatidylcholines and phosphatidylethanolamines species were observed in humans.These differential expressions of lipid classes could be linked and indicate the importance of lipids to the pathology of FCD.However, using these techniques the FCD region was not spatially defined.As a result, resected brain tissue or even whole brain homogenates were employed for analysis (Dentel et al., 2022;Kumar et al., 2018;Pires et al., 2021).Mass spectrometry imaging (MSI) can offer great potential to study different molecules such as endogenous metabolites, lipids, proteins or glycans of interest to understand the mechanism of a disease while maintaining their spatial distribution.Moreover, and as previously described by our group, MSI is a valuable tool in drug development as it can be employed in different phases of the drug discovery and development process (Vermeulen et al., 2022).The application of MALDI-MSI is of additional value to improve insight into how these somatic mutations in FCD type II locally modulate the protein/lipid expression in the mouse brain.Therefore, in this study we aim to combine lipid MSI with fluorescence imaging and label free proteomics to study the molecular changes in a preclinical mouse model of FCD Type II.By combining this methodology with human transcriptomics experiments of FCD type IIb resected brain samples, we were able to explore how genetic mutations modulate molecular changes in FCD type II.This spatial omics approach offers insight into the molecular consequences of the local genetic mutation resulting in brain malformations.

Animal model of FCD type II
All studies were carried out in accordance with internal (UCB Pharma) Ethical Committee and Animal Care Unit guidelines, the European Committee Criteria (Decree 2010/63/CEE) and the Animal Welfare Act (7 USC 2131).All experiments were performed on CD-1 mice of both sexes (Charles River).Timed-pregnant female mice were ordered to the external husbandry unit at gestational day E7-E8.Pregnant females were then housed for a week for adaption to the new environment before any experimentation.In utero electroporation was performed at gestational day E15-16.Briefly, by means of in utero electroporation surgery, pregnant dams were anesthetized with 2.5-3.5% Isoflurane, and a laparotomy was performed to expose the uterine horns and embryos.A DNA plasmid mixture (1-1.5 μL/ 2.5 μg/ μL) with 0.01% Fast Green dye was injected into the ventricle of each embryo using a special needle directly connected to a pneumatic pump.These DNA plasmids were delivered into the ventricle in order to specifically target neural progenitors.Half of the embryos (Control) were injected with an empty plasmid containing a CAG promoter and a red fluorescent reporter (RFP).The other half of the embryos (FCD type II) were injected with a DNA plasmid bearing CAG promoter, Rheb mutant (constitutively active by directed mutagenesis) and green fluorescent reporter (GFP).Following intraventricular injections, tweezer-type electrodes were positioned on the embryo head and a series of six 42 V, 60 msec pulses at 950 msec intervals were applied using a pulse generator (ECM830, BTX) to electroporate the DNA plasmid constructs and transfect neural progenitor cells.The electrodes were oriented to preferentially target medial prefrontal cortex (mPFC).After this procedure, the embryos were placed back into the abdominal cavity to continue with their development until birth.Pregnant dams were provided adequate post-surgical care.Postnatal day (P) 0-2 pups were screened at birth to confirm successful electroporation via expression of GFP reported before recruitment for subsequent studies.Naïve (noninjected), Control and FCD type II mice were grouped housed in ventilated cages on a 12-h light/dark cycle in a temperature-and humiditycontrolled environment, with food and water ad libitum.A total of 12 mice were included in the study.We conducted a comparative analysis involving four mice in each of the three groups: FCD type II mice, naïve mice and control mice.

Brain tissue preparation and IHC staining
Naïve (non-injected), control and FCD type II mice (n = 12, 4 animals per group) were deeply anesthetized and transcardially perfused with ice-cold phosphate buffered saline (PBS, pH 7.4).Whole brains were dissected from the skull and were prepared for cryosectioning.Fresh frozen mouse brain was sectioned at − 21 • C at 12 μm using a Leica cryostat (Leica Microsystems, Wetzlar) on a conductive ITO glass slide (4-8 Ω resistance, Delta Technologies, U.S.A.) and PEN membraneslides.On each ITO slide or PEN membrane slide, one FCD type II, control and naïve brain section were thaw-mounted in randomized order and kept in − 80 • C until further MSI analysis.Each ITO or PEN membrane slide contained 3 brain sections in total (one from each group).ITO slides were used for MSI of lipids.PEN membrane slides were used to perform Laser Capture Microdissection (LMD).
For Immunohistochemistry (IHC), control and FCD type II mice (n = 20, 10 animals per group) were also perfused transcardially with icecold PBS (pH 7.4) followed by ice-cold paraformaldehyde (PFA, 4%).Perfused brains were also postfixed in 4% PFA for an additional 3 h.Fixed brains were then immersed in 30% sucrose/PBS overnight at 4 • C for cryoprotection and sectioned with cryostat into 40 μm thick sections and place onto SuperFrost glass slides.During the staining protocol, sections were blocked for 1 h at room temperature in blocking buffer containing of 2% BSA and 0.1% Triton X-100 in PBS.Sections were then incubated overnight at 4 • C in primary antibody pS6 (S240/244, Cell Signaling, D68F8, 1:1000), diluted in blocking buffer.Following three washes in PBS 1×, sections were then incubated with Alexa Fluor-tagged secondary antibodies at 1:1000 dilution for 2 h at room temperature.For control sections, Alexa Fluor-488 tagged secondary antibody was used and for FCD sections, Alexa Fluor-594 tagged secondary antibody was used to detect the signal of the primary antibody for pS6.

Microscopy and image analysis
Digital acquisitions from IHC were made with using a digital slide scanner (Hamamatsu-Nanozoomer), using a 20× objective.Resulting images and all quantifications were analyzed using a Bayesian algorithm in Visiopharm software (2023.09× 64 version).Representative images were also prepared using Visiopharm software (v 2023.09× 64).The same parameters were used to allow comparison analyses between different images.Quantifications were performed along the rostrocaudal extent of the cingulate cortex in the ipsilateral or contralateral side, based on defined anterior and posterior limits.Two consecutive brain sections were generally analyzed per animal.In each section, a region of interest (ROI) surrounding the electroporated cingulate cortex region in each ipsilateral and contralateral cortex was manually delineated.pS6+ cells total area inside the ROI and pS6+ cells count was determined using a bayesian method.The mean intensity (A.U.) of every single pS6+ cell was also determined using a Bayesian method.pS6+ neurons soma size (μm 2 ) was quantified for each animal by dividing the total area of pS6+ cells in each ROI by pS6+ cells count inside the same ROI.Statistical analyses were performed using GraphPad Prism software (9.2.0 (332) version).Unpaired t-test was performed to determine differences between the two groups with N = 10 for FCD brains and N = for control brains (20 sections in total).The level of significance was defined a P-value <0.0001****.Data were presented as mean ± SEM.

Matrix application
Before matrix application, the samples were dried for 10 min in a desiccator.Tissues were covered with norharmane matrix (7 mg/mL in MeOH/CHCl 3 1:2) using an automated TM-Sprayer (HTX Technologies, LLC, Chapel Hill).15 layers of matrix were sprayed at 30 • C with a flow rate of 0.11 mL/min and a drying time of 30 s between each layer.The velocity was set at 1200 mm/min with a 3 mm tracking space.The gas pressure was set at 10 psi with a flow of 3 L/min.

Lipid data acquisition
Lipid analysis was performed on a timsTOF fleX (Bruker Daltonics Inc.) equipped with the microGRID module.A total of 12 animals were used for MALDI-MSI analysis, with four animals per group.For each animal, two brain sections were imaged, once in positive polarity and once in negative polarity on ITO slides.PEN membrane slides were exclusively measured in negative mode.High spatial resolution imaging on 20 μm and 5 μm raster size were performed using a mass range of m/z 150-1500.Further, data dependent acquisition (DDA) was performed on an Orbitrap Elite hybrid ion trap (Thermo Fisher Scientific) mass spectrometer for lipid identification (25 × 50 μm raster size), in positive and negative mode on an ITO slide.Full-scan Fourier transform mass spectrometry (FTMS) was acquired in parallel with ion trap (IT)-MS/MS.A mass resolution of 240,000 was used to analyze the raw full scan FTMS data, enabling the visualization of all parent ion masses.After, samples were stored at − 80 • C until further analysis for proteomics.

Hematoxylin-Eosin staining
Hematoxylin-Eosin staining (H&E staining) was performed on consecutive sections.Slides were hydrated in distilled tap water for min followed by hematoxylin (0.1% Gill's) staining for 3 min.Next, they were rinsed in running tap water for 3 min followed by a short rinse in distilled water.Slides were then stained with eosin (0.2%) for 30 s followed by a short rinse in 70% ethanol to remove the excess of eosin.Next, the slides were dehydrated in 100% ethanol for 2 min (2×) and equilibrated in xylene for 5 min (2×).The stained sections were mounted with entellan and covered with a glass coverslip, followed by drying at RT.The stained sections were scanned with a digital scanner (Aperio CS2) at 20× magnification.

Data processing
Lipids: The MALDI-MSI data was visualized and processed using Scils lab MVS, version 2021c (Bruker, Bremen, Germany).Mmass (mmass.org) was used for peak picking with a relative intensity threshold of 0.5% and signal to noise ratio of 3 TIC normalization was used for further data analysis with an m/z interval width set to ±0.01 Da.For statistical analysis on the ITO data, unsupervised multivariate probabilistic latent semantic analysis (pLSA) (with random initialization) was performed using 5 components by using the overall peak list on the individual spectra (Verbeeck et al., 2020).These calculated components represent different distributions within the brain tissue.A loading score was calculated for each lipid, indicating its contribution to each component.Further, segmentation by bi-secting k-means with correlation distance (clustering analysis) was performed to obtain data for and define the FCD type II area.This clustering analysis was performed on both positive and negative mode data obtained from ITO slides and negative mode data from PEN membrane slides, revealing two clusters (yellow and purple) (Alexandrov and Kobarg, 2011).Next, receiver operating characteristic (ROC) analysis was performed to evaluate the discriminative m/z values between the two different regions as found by the clustering analysis.Peaks or m/z values that have an AUC ≥ 0.70 or AUC ≤ 0.30 are considered to be specific for the yellow cluster or purple cluster, respectively.The negative mode data from PEN membrane slides was compared with the negative mode ITO slide data, yielding consistent results.Subsequently, the positive and negative mode ROC data obtained from clustering regions from ITO slides were used to compare the regions with the lipids with the highest loading score from the components of the pLSA analysis.The clusters obtained from the negative mode were saved as two new regions in Scils software.These coordinates were then applied to define the FCD type II region for LMD using an in-house build MATLAB script.PEN membrane slides were preferred over ITO slides for LMD, as they allow the use of the same section used for MALDI-MSI for very precise coregistration, resulting in a higher protein yield (Mezger et al., 2021).
High resolution MSI: Clustering analysis was performed on a single FCD type II section.Based on this data, a list of m/z channels was given for the cluster that resembles the migration of individual neurons.From this list, the m/z channel of 848.55 (PC 38:4) was found to contribute the most to this cluster.Using this m/z channel of PC 38:4, the colocalized m/z channels were found.The ROC tool with co-localization is included in the SCiLS program allowing to locate all the m/z channels that are co-localized with a specific channel.This tool employs Pearson's correlation analysis and only includes significant correlations (p = 0.05).This analysis yields a feature list containing m/z channels that are co-localized with the m/z channel 848.55.
Lipid Identification: DDA was utilized to identify structural lipids (Ellis et al., 2018).Parent ion masses with the associated IT-MS/MS spectra were used for lipid identification.Further analysis was done in LipostarMSI after conversion to imzML.Lipids were identified using the database from LipidMaps (July 2020) for [M -H] − and [M + H] + , including Na + and K + adducts with a mass tolerance of 0.00 Da ± 3.00 ppm.MS/MS analyses were conducted on the precursor ions and the results were matched with a m/z tolerance of 0.25 Da ± 0.00 ppm or lower.Further, only lipids with a minimum carbon chain length of 12 were used for identification as these are naturally occurring in animal and/or human physiology.

Fluorescence imaging
Brain sections from the two groups, FCD type II and control (n = 8, 4 animals per group) were imaged using a Leica TCS SP8 STED microscope with a HC PL FLUOTAR 10×/0.3DRY objective in confocal mode.Images of 1024 × 1024 pixels were obtained with an optical zoom of 0.75, which results in a pixel size of 880 × 880 nm.Green fluorescent reporter was excited with a wavelength of 488 nm to define the edges of the FCD type II area and the red fluorescent reporter at 592 nm for the control sections.Emission was detected with a hybrid detector and collected between 721 nm and 773 nm.Images were obtained with a pixel dwell time of 0.3 μs.A pinhole of 1 Airy unit was used.Acquisition and stitching of the images were performed in LAS X Navigator software.Fluorescent images were further evaluated in Qupath 0.2.32.By using the annotation tool "wand tool".Qupath automatically recognized and defined the edges of the fluorescence area.

Overlay of fluorescence and MSI data
The annotated regions of Qupath were imported into Scils lab.Since the fluorescent images were taken on consecutive sections, manual overlay of the fluorescence images and the MSI clustering data was performed using common morphological features in both tissue sections.This manual overlay had an overall error of <15 μm.
2.5.Fluorescence and MSI-guided LMD followed by label free proteomics

MSI-guided laser microdissection
Laser Capture Microdissection (LMD) was performed using a Leica LMD 7000 Instrument (Leica Microsystems).Overlay of the fluorescence and segmentation data was performed in Scils to select the regions of interest to cut out.Coordinates were transferred using an in-house MATLAB Script to co-register the region of interest with a consecutive section for the LMD.The laser power was set at 35 with an aperture of 18, speed 30, specimen balance 0, line spacing for draw + scan 5, head current 60%, pulse frequency 120 and an offset of 65.Areas of 0.5 mm 2 were ablated from a PEN membrane slide.The ablated regions were captured in 20 μL of ammonium bicarbonate buffer 50 mM (ABC) and stored at − 80 • C until further processing for LC-MS/MS.

Sample preparation for proteomics
Samples were further processed using a protocol, described by Mezger et al. (Mezger et al., 2021) In general, Rapigest (final concentration 0.1%) was added and incubated in a Thermoshaker (Eppendorf, Hamburg, Germany) at 800 rpm at room temperature (RT = 21 • C).After, DTT (final concentration 10 mM) was added to reduce the samples and incubated at 56 • C for 40 min at 800 rpm.The samples were then alkylated by adding IAM (final concentration 20 mM) and incubated at RT for 30 min at 800 rpm.To quench the excess IAM, DTT was added (final concentration 10 mM) and incubated at RT for 10 min at 800 rpm.The first digestion was performed by adding trypsin/Lys C dissolved in a resuspension buffer (50 mM acetic acid (pH < 3))(final concentration 15 μg/mL) and incubated overnight at 37 • C at 800 rpm.The second digestion step was performed by adding 0.3 μL trypsin (final concentration 5 μg/mL) in 80% ACN and incubated at 37 • C for 3 h at 800 rpm.
To terminate the digestion, TFA (final concentration 0.5%) was added and incubated at 37 • C for 45 min at 800 rpm.After centrifugation (15.000 g, 10 min, 4 • C), the supernatant was collected in a tube and concentrated using a Speedvac (Hetovac VR-1, Heto Lab Equipment, Denmark) to obtain a final volume of 30-40 μL.The samples were stored at − 80 • C until LC-MS/MS analysis.

LC-MS/MS analysis
Separation of peptides was performed on a Thermo Fisher Scientific Dionex Ultimate 3000 Rapid Separation ultrahigh-performance liquidchromatography (HPLC) system (Thermo Scientific, Waltham, MA, USA) equipped with an Acclaim PepMap C18 analytical column (2 μm, 75 μm × 500 mm, 100 Å).Aliquots of 5 μL were first desalted using an online installed C18 trapping column.Peptides were then separated with a 240 min linear gradient from 5% to 45% acetonitrile/0.1% formic acid and a flow rate set at 300 nL/min.The HPLC system was coupled online to a high-mass resolution Orbitrap MS Q-Exactive instrument (Thermo Scientific) with a nano electrospray Flex ion source (Proxeon, Thermo Scientific).The mass spectrometer was programmed to run in DDA mode in positive ion polarity with the following parameters: full MS scan of mass range m/z 250-1250 at a resolution of 70,000 at m/z 200 with a maximum injection time of 120 ms, followed by tandem mass spectrometry (MS/MS) scans for the fragmentation of the 10 most intense ions at a resolution of 17,500.Precursors were isolated with a 1.8 m/z window and a maximum injection time of 200 ms.Ions that were already selected for fragmentation were dynamically excluded for 30 s.The instrument was externally calibrated using a standard calibration solution for positive ion mode (Pierce LTQ Velos ESI positive ion calibration solution, Thermo Scientific).
Protein identification was performed in Proteome Discoverer software version 2.5 (Thermo Scientific) in which the raw files were processed by using the search engine Sequest version 2.2 with the Swiss-Prot human database Mus musculus (TaxID 10,090).To perform the database search, the following parameters were used: Carbamidomethylation of cysteine for fixed modifications, methionine oxidation and acetylation of protein N-terminus for variable modifications.Trypsin was used as enzyme with a maximum of two missed cleavages.The precursor mass tolerance was set at 10 ppm and the fragment tolerance at 0.02 Da.The minimum and maximum length of the peptide were set at 6 and 144 amino acids, respectively.The total peptide content was used to normalize the data.False discovery rate (FDR) was used to assess the certainty of the identification and fixed at 1% maximum.For the decoy database search, percolator was used.

Bioinformatic analysis of proteomics data
Proteome Discoverer program was used to identify the proteins with a significant differential expression across the different groups.A oneway background analysis of variance (ANOVA) test was performed, comparing protein abundance in the FCD type II area against the control area or the opposite hemisphere of the FCD type II brain.This statistical analysis computes adjusted p-values, applying the Benjamin Benjamini-Hochberg correction to check whether a proteins' expression differs significantly when compared against background levels (calculated by proteins with similar abundance and abundance ratios across all samples) (Navarro et al., 2014).Proteome Discoverer program was also used to determine the abundance ratios between groups.Protein abundance was calculated as the median of all possible pairwise peptide ratios, derived from peptide intensities.Protein abundance ratios with a foldchange threshold of ±1.5 coupled with an adjusted p-value of ≤0.05, were considered significantly different between the two groups.These differentially expressed proteins were then selected for further analysis by functional enrichment analysis in Metascape with default settings (Zhou et al., 2019).

Subjects
Brain tissues from 33 patients undergoing surgery for intractable epilepsy diagnosed with FCD type IIb and 14 age-and tissue-matched postmortem controls were obtained from the archives of the Departments of Neuropathology of the Amsterdam UMC (Amsterdam, The Netherlands) and the UMC Utrecht (Utrecht, The Netherlands.All cases were reviewed independently by two neuropathologists.The diagnosis of FCD type IIb was confirmed according to the international consensus classification system proposed for grading FCD.(Consortium, T. G. O, 2023) All FCD type IIb samples underwent deep sequencing using DNA extracted from snap-frozen surgical brain tissue targeting 13 genes (FCD panel SoVarGen, Korea).Postmortem control had no history of seizures or other neurological disease and all autopsies were performed within 24 h after death.All procedures received prior approval by the local ethics committee of the contributing medical centers and were in accordance with the guidelines for good laboratory practice of the European Commission.

RNAseq library preparation and sequencing
All library preparation and sequencing were performed at GenomeScan (Leiden, The Netherlands).The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) was used for sample processing.Sample preparation was performed according to the protocol "NEBNext Ultra II Directional RNA Library prep Kit for Illumina" (NEB #E7760S/L).Briefly, mRNA was isolated from total RNA using oligo-dT magnetic beads.After fragmentation of mRNA, cDNA synthesis was performed.Next, sequencing adapters were ligated to the cDNA fragments followed by PCR amplification.Clustering and DNA sequencing was performed using the NovaSeq6000 (Illumina, Foster City, CA, USA) in accordance with manufacturers' guidelines.

Bioinformatic analysis of transcriptomics
The Bestus Bioinformaticus Decontamination Using Kmers (BBDuk) tool from the BBTools suite was used for adapter removal, quality trimming and removal of contaminant sequences (ribosomal/bacterial) (Bushnell et al., 2017).A phred33 score of 20 was used to assess the quality of the read; any read shorter than 31 nucleotides in length was excluded from the downstream analysis.Reads were aligned directly to the human GRCh38 reference transcriptome (Gencode version 33) using Salmon v0.11.3 (Harrow et al., 2012;Patro et al., 2017).All analyses were performed using R statistical software (Team, R. C, 2023).Transcript counts were summarized to the gene level and scaled used library size and average transcript length using the R package tximport (Soneson et al., 2015).Genes detected in <20% of the samples in any diagnosis and with counts less than six across all samples were filtered out.The gene counts were than normalized using the weighted trimmed mean of M values (TMM) method using the R package edgeR (Robinson et al., 2010).The normalized counts were than log2 transformed using the voom function from the R package limma (Ritchie et al., 2015).The subsequent differential expression was carried out using the R package limma using a Benjamini-Hochberg adjusted p value to account for multiple testing.Gene-set enrichment using GO ontology was performed using fsgea package (Ashburner et al., 2000;Consortium, T. G. O, 2023;Korotkevich, 2021).

Development of a preclinical Focal Cortical Dysplasia type II mouse model
As described in the methods, FCD type II mice were generated using in utero electroporation technique to mimic brain mosaic mutations.The mutation is induced in the constitutive activator of mTOR protein, resulting in enhanced mTOR pathway activity via phosphorylation.The model recapitulates the human pathophysiology of FCD type II.The abnormalities caused by mTOR hyperactivity led to focal cortical disorganization, characterized by disrupted neural migration, cortical dyslamination and dysplastic neurons (Fig. 1A and B).A quantitative assessment of cell soma size in FCD brains was performed and compared to control brains to validate our observations.Our analysis revealed a significant increase in soma size in cells in the FCD brain compared to those in control brains (p-value <0.0001) (Fig. 1E).The expression of phosphorS6 (pS6) was quantified through immunofluorescence staining to verify mTOR pathway hyperactivation in FCD brains compared to control brains (Fig. 1C and D).Our analysis showed a significant increase in pS6 expressions levels in the FCD brains compared to control brains (Fig. 1F).These data confirm the mTOR pathway hyperactivation in FCD type II phenotype, as evidenced by the increase in pS6 expression levels (Patil et al., 2016) Additionally, the model consistently shows expression of in vivo epileptogenesis with manifestation of spontaneous recurrent seizures starting at postnatal day 20 (Data not shown).

Fluorescence imaging validates the localization of the Focal Cortical Dysplasia affected area
Fluorescence imaging was used to verify the precise localization of the affected neurons.Injection of the plasmid tagged with a fluorescent reporter, GFP for FCD type II, was able to visualize the FCD type II region (Supplementary Fig. S1).In the fluorescent images, GFP labeled cortical neurons were visible scattered throughout all cortical layers of the brain.Comparing these images to the control plasmid, where cortical neurons were labeled with RFP, showed a clear difference -RFP-labeled neurons were more concentrated in the superficial layers (Fig. 1D).These fluorescent images also show the heterogeneity between each animal (Supplementary Fig. S1).To precisely outline the edges of each FCD type II region we used fluorescence positivity.caused by the mTOR mutation.We were able to detect differential lipid distribution of phosphatidylinositols (PI), phosphatidylethanolamines (PE), sterols (ST), sulfatides, phosphatidylserines (PS), phosphatidylcholines (PC), sphingomyelins (SM) and phosphatic acid (PA) (Supplementary Table S1 and S2).

MALDI-MSI was used to investigate the local changes in the lipids
Two statistical approaches were used to categorize brain sections based on their lipid composition, revealing distinct anatomical regions.Overlay and verification with H&E staining confirmed that the yellow cluster corresponds to the corpus callosum, and the purple cluster to the cortex.Further, the yellow cluster aligned with the boundaries of the FCD type II lesion (indicating downregulated lipids) and the center of the FCD type II lesion matched the purple colour (indicating upregulated lipids) (Fig. 2A).This specific location was unique to the FCD type II lesion and not observed in control and naïve brains.
Further, unsupervised pLSA analysis also identified distinct anatomical regions within the brain in negative mode.Component 3 described the corpus callosum, while component 4 described the cortex, with component 4 matching the center of the FCD type II lesion (Fig. 2B) a distinction absent in control and naïve brain sections.For negative mode, we focused on pLSA components 3 and 4, representing downregulated and upregulated lipids in the FCD type II lesion, respectively.This dual statistical approach allowed us to compile and validate a list of discriminative lipids for the FCD type II lesion (Fig. 2C).
The same dual statistical approach was used for the positive mode, employing clustering analysis and unsupervised pLSA analysis to categorize two distinct anatomical regions.Clustering analysis identified these regions with the yellow cluster corresponding to the corpus callosum, and the purple cluster to the cortex.Unsupervised pLSA analysis allowed us to select components 1 and 2 (Supplementary Fig. S3) where component 1 described the corpus callosum, while component 2 described the cortex.In detail, component 1 represented the downregulated lipids in the FCD type II lesion, while component 2 represented the upregulated lipids in the FCD type II lesion.
The FCD type II lesion was described by component 2. Various PCs exhibited differential abundance in the FCD type II lesion.For instance, PC 38:1 was specific to the FCD type II area, concentrated at the core of the localized FCD type II lesion whereas PC 32:0 is less abundant in this inner area.SM were also detected, such as SM 42:2 in the inner region the FCD type II area (Supplementary Table S2).
In our initial analysis, we explored FCD type II lesions without any prior knowledge of their specific location.Subsequently, we employed fluorescence microscopy for a more precise co-registration.This allowed us to accurately delineate the FCD type II lesion.This refined approach facilitated a detailed statistical comparison with corresponding regions in other groups, including the opposite hemisphere of the FCD type II brain, as well as in control and naïve brains.We identified several lipids that were either upregulated or downregulated, with the majority overlapping with the lipids identified in our initial unsupervised analysis.Similar lipids such as ShexCer and PE were found.Notably, two lipids  were exclusively found to be downregulated in the FCD type II lesion compared to other regions.

Visualization of individual neurons with MSI
High spatial resolution MALDI-MSI experiments were performed to examine individual cells within the FCD type II area.By employing high spatial resolution (5 μm raster size), we could not only visualize the translocation of single cells within this area but also map out the distribution of specific lipids, such as PC 38:4, a phosphatidylcholine (Fig. 3A).The choice of PC 38:4 was deliberate because its spatial distribution mirrors the migration pattern of cells, more specifically individual neurons, within the FCD type II area, as confirmed by clustering analysis (Supplementary Fig. S4).
Selecting PC 38:4 allowed us to further investigate which other lipids shared a similar distribution pattern.This allowed us to identify the lipids associated with the individual neurons, aiding in the identification of a unique lipid pattern present in these migrating neurons.Our MALDI-MSI experiments revealed several different PC species present in these cells (Supplementary Table S3).

Overlay of the fluorescence images with MSI data
The fluorescence data together with the regions defined by MSI clustering analysis were combined and aligned using common anatomical features in the sections (Fig. 3B, C and D.).This multimodal visualization was used to identify and delineate the FCD type II lesion with high precision for further LMD analysis (Supplementary Fig. S5 and S6).

Proteomic profile of the FCD type II area
To enhance our understanding of the molecular changes induced by the mTOR mutation, we employed spatially resolved label-free proteomics.This approach served as a complementary analysis to the lipidome findings, providing additional insight into the relative abundance of proteins within the FCD type II lesion.To determine which proteins were up and down regulated in the FCD type II area, a comparison was made with selected corresponding areas in both control brains as well as the opposite hemisphere of the FCD type II brain (Supplementary Fig. S5e).A total of 874 unique proteins were identified in both control and FCD type II samples together (Fig. 4A).61 of the 874 unique proteins were found to be differently expressed in FCD type II brain areas compared to control brain regions (adjusted p-value 0.05; fold change (FC) threshold set at 1.5-fold) (Supplementary Fig. S7).Clustering analysis, as depicted in the heatmap, successfully differentiated FCD type II samples from control samples (Fig. 4B).The same analysis was conducted to compare the FCD type II area to the same region in the contralateral hemisphere of the FCD type II brain (referred to as non-FCD in the paper).61 proteins were identified to be significantly different from the 849 uniquely found proteins.(adjusted p-value 0.05; FC threshold set at 1.5-fold).
Functional enrichment analysis for the combined up-and down regulated proteins was performed in Metascape to analyze the biological processes that are changed in FCD type II, compared to the control and non-FCD samples.Enriched processes when comparing the FCD type II with control included ensheathment of neurons, regulation of leukocyte migration, synaptic vesicle cycle and negative regulation of cellular component organization/neuron death and adaptive immune system (Fig. 4 C-E).Detailed annotations and p-values can be found in the supplementary material (Supplementary Table S4-S7).Four compact myelin related proteins showed a downregulation in FCD type II (e.g., Mbp, Cldn11, Tspan2 and PLP1).Downregulation of proteins involved in non-compact myelin was observed in FCD type II and included JAM3, CD9.Mag and Opalin found in the membrane of the myelin whereas CNP, Sirt2, Rhog are found in the cytoskeletal and vesicular components of tubulin (Jahn et al., 2009).Several of these genes (including Cnp, Mag, Plp1, and Tspan2) are involved in oligodendrocyte differentiation.Glial cell development (Mag, Plp1, Sirt2, and Tspan2) and differentiation (Cnp, Mag, Plp1, Sirt2, and Tspan2) are also involved in this process of ensheathment and downregulated in FCD type II.In the cellular component organization pathway, two GTPase activators for example were discovered to be increased in FCD type II (Arhgap1, Rap1gap).The pathway of neuron ensheathment was also enriched when comparing FCD type II vs non-FCD revealing several myelin related proteins such as Mpp, Cldn11 and Plp1, which were also found in the comparison with the control samples.(Supplementary Fig. S8 and Tables S8-S11) Certain synaptic vesicle cycle processes, such as neutral amino acid transport, amino acid, carboxylic acid, and organic acid transmembrane transport, and vesicle mediated transport in synapse, are altered in both comparisons, according to enrichment analysis.The FCD type II area was shown to have lower levels of three vesicular inhibitory amino acid transporters: Slc32a1, Slc1a4, and Slc6a5.Only one unique route was identified in the FCD type II vs non-FCD comparison, fatty acid betaoxidation, which included overexpression of mitochondrial isovaleryl-CoA dehydrogenase (Ivd) and trifunctional enzyme subunit beta (Hadhb).

Commonly up and down regulated proteins
To evaluate commonly modulated proteins between the naïve/control groups and FCD type II group, we focused on the proteins that showed consistent changes across all conditions.Specifically, we concentrated on proteins that were significantly different in the FCD type II group when compared with the other conditions.Out of all the previous mentioned comparisons, 13 proteins were identified that were significantly different in each comparison involving FCD type II (adjusted p-value 0.05; FC threshold set at 1.5-fold).Seven of these proteins were found to be more abundant in the FCD type II area, whereas six were found to be less abundant in the FCD type II area (Table 1).

Transcriptome of tissue from FCD type IIb patients confirms impact on myelination
As first level validation of the findings from the FCD type IIb lesion, transcriptomic data from tissue of FCD type IIb patients compared to autopsy control tissue was used.In total, 1975 upregulated and 1090 downregulated genes were identified.Performing gene-set enrichment using GO ontology identified 116 pathways enriched in the significantly up-and down-regulated genes (Supplementary Table S12).Among these genes related to myelination were found to be significantly enriched in the upregulated genes (GO terms: myelination, adjusted p-value <0.001 and central nervous system myelination, adjusted p-value <0.05) (Fig. 5A).In addition, correlation analyses showed that for both pathways there is a substructure in the gene coexpression pattern, which can support pathways activity in tissue.Sub clusters show anti-correlation to each other but strong positive correlation in gene expression within the sub cluster (Fig. 5B and C).

Discussion
The purpose of this study was to gain a better understanding of the underlying molecular changes in Focal Cortical Dysplasia type II by means of a multimodal omics approach in an experimental model.We were able to visualize and characterize local functional changes unique to the FCD type II region in the lipidome and proteome by integrating MALDI-MSI, fluorescence imaging, and MSI-guided LC-MS/MS and compare this with human transcriptomics data.
In this study, we used an animal model employing the insertion of a plasmid with a mutation into the brain of the embryos to generate an FCD type II lesion during development.As a result, each animal has a unique presentation of the affected region.The increase in soma size observed together with the elevated pS6 level in our study further supports the mTOR pathway hyperactivation, corresponding to FCD type II phenotype.Furthermore, the FCD type II-affected area is very heterogeneous.With fluorescence imaging, we were able to recognize not only the FCD type II affected area in each animal, but also the disrupted migration of the cortical neurons.
As previously described in the introduction, mTOR activation has an influence on lipid synthesis, but little is known about the impact on adjacent cells or spatial characteristics (Bockaert and Marin, 2015).Our MALDI-MSI lipidomics revealed that both SHexCer levels and PS levels in the FCD type II brain are locally lower than in control and naïve groups.Sulfatide species (SHexCer) and phosphatidylserines (PS) are known to play a role in the myelination process in the brain.Abnormalities in the process of myelination have been observed in both surgically removed tissue from patients with epilepsy and in epilepsy animal models (Donkels et al., 2020).Numerous studies have investigated the myelin pathology in FCD type IIb and tuberous sclerosis complex (TSC).This myelin pathology is attributed to the activation of the mTOR pathway, which hinders the transformation of oligodendrocyte progenitor cells (OPC) into mature myelin-producing  oligodendrocytes (Gruber et al., 2019;Gruber et al., 2021;Muhlebner et al., 2020;Muhlebner et al., 2016;Scholl et al., 2017).
The observed reduction in SHexCer lipids, which not only aid in preservation of the myelin structure but also plays a role in regulating oligodendrocyte development, supports that there are changes in myelination occur in epileptic tissue and may be linked to impairment of oligodendrocytes (Maganti et al., 2019;Scholl et al., 2017).Furthermore, it has been previously reported that long chain (>40) sulfatides are predominantly found in mature oligodendrocytes, supporting the hypothesis that the reduction of sulfatides, particularly the long chain variants, are relevant (Hirahara et al., 2017).
PS, on the other hand, is necessary for neuron survival, growth, and synapse formation.Translocation of PS from the inner to the outer cytoplasmic leaflet occurs during oxidative stress and apoptosis (Glade and Smith, 2015;Kim and Park, 2020).A recent study has identified PS on abnormal myelin profiles and even shed myelin fragments (Djannatian et al., 2023).Other research demonstrated that low PS levels in the brain have a neurodegenerative impact.It was shown in a pentylenetetrazol seizure-induced rat model that supplements of PS have an antioxidant role in the brain (Liu et al., 2012).
Our study's observation of a decrease in PS and SHexCer aligns with previous research that reported a reduction in oligodendrocyte count through immunohistochemistry staining.This finding suggests potential impairment of oligodendrocytes in FCD type II, as discussed previously (Blumcke et al., 2021;Donkels et al., 2020).One of the studies has also mentioned a time-dependent correlation between myelin pathology and mTOR activation (Scholl et al., 2017).Another hypothesis states that demyelination, along with impaired potassium buffering in unmyelinated axons, contributes to the dysfunctional neural networks sustaining epileptogenesis, providing a possible explanation for the timedependent relationship (de Curtis et al., 2021).Our results do not only strengthen existing findings in the literature but also clarifies on the lipid-related aspects of FCD type II (Donkels et al., 2020).So far, no research has been performed on the effect of PS or SHexCer supplements in FCD type II animal models.Our findings might open up new possibilities for potential therapeutics in the treatment of FCD type II.
Precise co-registration of fluorescence data and MSI data allowed us to have a more in detailed look at the FCD type II lesion.Next to finding similar results as discussed above, a number of PC species that have not been described before were found in the FCD type II area.PCs are known to form the primary component of the cell membrane in neurons, implying that these cells reflect cortical neuron dispersion over all cortical layers.With high spatial resolution, MSI was able to highlight these optical characteristics in the damaged FCD type II area.
The strength of this spatial omics approach allowed us next to only take the affected FCD type II lesion into account for the proteomics analysis and to investigate the affected pathways specifically in the FCD type II area and not in the whole brain.
FCD type II revealed mechanisms associated with a negative regulation of cellular organization impacting processes such as formation, composition or breakdown of cell structures.In FCD type II, two GTPase activators were discovered to be increased.(Arhgap1, Rap1gap) The activation of the PI3K-Akt signaling pathway has been associated to RAP1gap (Pei et al., 2019).Other research suggests that Lrpap1 functions as an anti-apoptotic signal in neurons by activating the Akt pathway.This route is activated by phosphorylation and upregulates mTOR activity, which is in line with the inserted mutation in FCD type II mice (Porta et al., 2014).
Further, proteomics analysis comparing all conditions revealed an enrichment in vesicular inhibitory amino acid transporter (Slc32a1) (VGAT) and neutral amino acid transporter A (Slc1a4).GABA and glycine are transported to the brain via VGAT.Increased GABA transport in the brain has previously been observed resulting in increased synaptic transmission that cause seizures (Banerjee et al., 2020).ASCT1, the neutral amino acid transporter, is encoded by Slc1a4.ASCT1 transports L-serine to the neurons where it functions as a building block as well as a precursor for production of L-cysteine, phosphatidyl-L-serine, sphingolipids, nucleotides and the neuromodulators D-serine and glycine.Several studies in humans with intractable seizures have reported a mutation in Slc1a4 (Damseh et al., 2015;Sedlackova et al., 2021).Furthermore, ASCT1 knock out (ASCT1-KO) mice had elevated glycine expression (Kaplan et al., 2018).Higher glycine levels in ASCT1-KO mice may be due to impaired L-serine transport.In ASCT1-KO astrocytes, a lack of L-serine exchange may enhance L-serine conversion to glycine by the serine hydroxymethyl transferase enzyme.Because this binding site is normally not saturated (Kaplan et al., 2018;Sherwood et al., 2021), extracellular glycine increases can amplify impulsedependent NMDAR activation, resulting in pro-cognitive effects.Glycine transporter 1 (Gly1) regulates synaptic availability of glycine in the hippocampus.Increased GlyT1 expression in the epileptogenic hippocampus leads to decreased extracellular glycine during the interictal phase.In chronic epilepsy, pre-convulsive glycine receptors might contribute to seizure production, and prospective anticonvulsant therapy methods are already reported in the literature (Lai et al., 2022;Shen et al., 2015;Zhao et al., 2016).
Our proteomic findings support our lipidomics data indicating a decrease in demyelination.Several proteins including Myelin associated glycoprotein (MAG), 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP), which is involved in the RNA metabolism in the myelin cells, and myelin basic protein (MBP), have been identified to be downregulated in the FCD type II region when compared to the control group and naïve group in our study (Gravel et al., 2009).Myelin-associated glycoprotein (MAG) typically interacts with gangliosides on axons and this interaction is required for proper axon-myelin cell-cell interaction but it also prevents axon outgrowth after damage (Schnaar and Lopez, 2009).In addition, these findings are supported by available transcriptomic data from FCD type IIb patients highlighting the upregulation of myelinationassociated pathway in FCD type IIb tissue as well as strong gene coexpression patterns, which support the activity of these pathways in FCD type IIb tissue.The observed increased (co)expression of

Table 1
Significant proteins in FCD type II: Significantly upregulated (gray) and downregulated (white) proteins with their accession number (UniProtKB) and gene name found in the FCD type II area compared to control, naïve and non-FCD areas.(Protein discoverer, cut-off>1.5-foldfor up and < − 0.5-fold for down regulation).myelination-related genes in human samples, together with a decrease in the abundance of myelin-related proteins in the animal model, may suggest the brain's attempt to compensate for the underlying pathology found in FCD type II.This dynamic interaction may highlight that the elevated genes are part of an adaptive response in which the brain attempts to restore normal brain function in the presence of FCD type II.Further, it shows that the epileptic features develop with the maturing of the brain in this specific FCD type II animal model.These results aligns with other studies reporting a link between the reduction of myelin in and around the cortical lesions of patients with confirmed mTOR hyperactivation and impairment of oligodendroglial turnover, proliferation and maturation due to aberrant mTOR activation in patients and animal models (Bercury et al., 2014;Blumcke et al., 2021;Donkels et al., 2020;Donkels et al., 2017;Elbaz and Popko, 2019;Gruber et al., 2021;Ishii et al., 2019;Lebrun-Julien et al., 2014;Lee et al., 2022;Muhlebner et al., 2012;Muhlebner et al., 2020;Scholl et al., 2017;Shepherd et al., 2013).
The decrease in oligodendroglial renewal can be attributed by the disruption of this nervous system development process, which involves some identified proteins such as tetrasparin2 (tspan2) and Opalin, of which both are associated to oligodendrocyte differentiation.These pathways were also detected in patients with FCD type IIa (Gruber et al., 2021).
It is relevant to highlight that one of the identified enriched pathways is the adaptive immune system.In a study conducted in the context of TSC, a negative correlation was observed between inflammatory markers and abnormal myelin (Muhlebner et al., 2020).Based on our results, future research should not only explore the possibility of investigating oligodendrocyte differentiation but also consider the potential involvement of the adaptive immune system as promising strategies for new therapeutic interventions.
In conclusion, our data expanded our knowledge of FCD type II with respect to the molecular changes in the lipids and proteins by only taking the FCD type II area into account.Our findings reinforce the current knowledge there is about FCD type II and provide new insights into the functional consequences of mTOR somatic mutations.With this multimodal approach, these findings did not only offer spatial insight into FCD type II but also present new potential strategies for therapeutic options through a better understanding of the interplay between molecular and genetic changes underlying the disease mechanism.

Fig. 1 .
Fig. 1.Hematoxylin and eosin staining and immunofluorescence staining of FCD type II mouse brain and control mouse brain.A Hematoxylin and eosin (H&E) stained mice brain slides from FCD type II animals showing the lesion with focal cortical disorganization, characterized by disrupted neural migration, cortical dyslamination and dysplastic enlarged neurons in the lesion and B a control brain slide.C Immunofluorescence staining of phosphoS6 (pS6) from FCD type II affected brains with the Green Fluorescent Reporter (GFP) D and a control brain slide with the Red Fluorescent Reporter (RFP) E Quantification of soma size (total cells area∕total cells number (a.u)) in control brain (pS6 + ∕RFP + FCD-) and FCD brain (pS6 + ∕GFP+ (FCD+)).Unpaired t-test used with control mice sections (N = 10) and FCD mice sections (N = 10).Each datapoint is the average of two consecutive brain sections.Data is shown as mean + ∕-SEM.P-value <0.0001 **** F Quantification of pS6 kinase (red reporter from Alexa Fluor-594 tagged secondary antibody signal)) in FCD lesion (ipsilateral side) versus non-lesional areas (contralateral side) in FCD mice (GFP+(FCD+)) Unpaired t-test used with FCD mice sections (N = 10).Each datapoint is the average of image quantifications of two consecutive brain sections.Data is shown as mean + ∕-SEM.P-value <0.0001 ****.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2 .
Fig. 2. MALDI-MSI Imaging of FCD type II brain revealed changes in lipid composition in negative mode.MALDI experiments were performed in negative mode at 20 μm raster size.A Clustering data of the three groups (FCD type II, Control and Naïve).The corpus callosum is represented by the yellow cluster and the cortex by the purple cluster.The center of the FCD type II lesion matches the purple cluster.B Unsupervised pLSA analysis with random initialization.The corpus callosum is represented by component 3 and the cortex is presented by component 4. The center of the FCD type II lesion matches component 4. C List of distinctive lipids for the yellow cluster and purple cluster for the negative mode data.D MSI-images (20 μm raster size) of selected lipids showing the different distribution in the FCD type II area.MSI-images of SHexCer t36:1, SHexCer t38:1 and SHexCer d42:2 t42:2 reveal their decreased intensity in the FCD type II area while MSI-images of PA 40:6, PE 38:6 and PE 40:6 show their preferred location in the center of the FCD type II area.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3 .
Fig. 3. High spatial resolution MALDI-MSI showed migrating neurons in FCD type II lesion and overlay of MALDI-MSI data with fluorescent data defined edges of FCD type II lesion.A MALDI-MSI image, measured in positive mode at 5 μm raster size shows differences in spatial distribution of a specific lipid in the FCD type II area (left) compared to the other hemisphere (control).PC 38:4 (m/z value of 848.52 ± 0.1 Da) shows the cortical dyslamination caused by FCD type II, B MALDI-MSI image of a selected mass m/z 888.62 ± 0.1 Da (SHexCer d42:2) (20 μm raster size) selected to represent the cluster data.C Fluorescent image of green fluorescent reporter (GFP) fluorescence signal of a consecutive section, D overlay of the MSI image (20 μm) with the fluorescent image showing the localization of the FCD type II area.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4 .
Fig. 4. Pathway analysis of FCD type II brain proteome compared to control brain.A Venn diagram depicting the 874 distinct proteins discovered in FCD type II and Control.B Heatmap of the 61 differentially expressed proteins indicated sample grouping into FCD type II and Control conditions.C p-value-colored heatmap of enriched terms across the input differently expressed gene lists.D Network of enriched terms colored by cluster identity, in which nodes with the same cluster identification are typically adjacent to one another.E Network of enriched terms colored by p-value, with terms containing more genes having a higher p-value.

Fig. 5 .
Fig. 5. Transcriptomic analysis of human FCD type IIb samples.Transcriptome changes in FCD type IIb cortex tissue highlight changes in myelination-related genes.A Volcano plot of FCD type IIb cortex compared to control postmortem tissue highlighting upregulation of GO terms for myelination.B Correlation analysis of genes involved in central nervous system myelination (GO identifier: 0022010) revealed a substructure with strong positive correlations between the genes of this pathway, which can indicate pathway activity in the tissue.C Correlation analysis of genes involved in myelination (GO identifier: 0042552) also highlighted two subgroups with strong positive correlations between the genes support the activity of the pathway in FCD type IIb tissue.