87

Evaluation of microangiopathy in epilepsy in surgical and post-mortem cohorts

Maria Thom, Tien JIN, Fabio Ribeiro Rodrigues, Hanaa EL HACHAMI, Alicja MRZYGLOD, Joanna Bartkiewicz, Matthias Koepp

UCL, Research Department of Epilepsy

There is growing interest in the role of cerebral small vessel pathology in epilepsy. Although vascular malformations and stroke are well-established causes of epilepsy, accumulating evidence indicates that recurrent, particularly refractory, seizures can also induce microvascular alterations, including blood–brain barrier disruption and acquired vascular remodelling. These vascular changes may impair cerebral perfusion and neuronal function and have been implicated in memory decline in adults. However, the prevalence and distribution of small vessel disease in epilepsy, its distinction from age-related vascular changes, and relationship to glymphatic clearance remain incompletely understood. In a previous study of a small temporal lobe epilepsy (TLE) cohort, we identified age-accelerated hyaline arteriolosclerosis (vascular sclerosis; VS), pericyte alterations, and perivascular space expansion (PVSE) which correlated with measures of cognitive decline on neuropsychological assessment (Liu et al., 2024).

In the present study, we investigated over 165 surgically resected TLE specimens using image analysis methods to quantify PVSE and VS and to assess their relationship with the accumulation of corpora amylacea (CA), an indirect marker of impaired glymphatic clearance, in cortical and white matter regions compared with controls. The relationship to focal tau burden was also explored in a subset. In a post-mortem cohort of over 100 cases representing a range of epilepsy syndromes. VS, PVSE, and perivascular macrophages (PVMs), an indirect marker of glymphatic function, were semi-quantitatively assessed bilaterally in cortical and deep regions. Selected cases were explored with vascular and glymphatic markers, including Claudin-5 (endothelial tight junctions), SLC6A12, PDGFR-beta (pericytes), AQP4 (perivascular astroglia), and LYVE1/PDPN (glymphatic-associated endothelium) to further characterise vascular integrity and structure.

Compared with controls, TLE cases demonstrated greater VS. PVSE was more pronounced in the deep cortical layers and superficial white matter than deep white matter, although its relationship with CA accumulation was less consistent. Similar patterns of preferential subcortical small vessel involvement were identified in the post-mortem cohort including some young adults, together with altered AQP4 distribution, supporting disruption of glymphatic pathways.

These findings provide evidence for degenerative small vessel pathology in a subset of patients with epilepsy, with a regional distribution that differs from normal ageing patterns. As the cerebral vasculature and glymphatic system are dynamic and interdependent, robust and validated neuropathological measures of vascular and glymphatic dysfunction are essential for meaningful correlation with in vivo neuroimaging biomarkers, including MRI-derived perivascular space measures and diffusion tensor imaging analysis along the perivascular space (DTI-ALPS), as well as with neuropsychological measures of cognitive decline.

88

Cellular Senescence in Temporal Lobe Epilepsy: A Neuropathological Study

Joanna Bartkiewicz, Matthias Koepp, Maria Thom

UCL

Purpose: Cellular senescence occurs towards the end of cell cycle. While most damaged cells undergo apoptosis, a programmed cell death, some enter senescence which protects the cell from apoptosis. Although neurons are postmitotic, they have also been shown to undergo senescence1. Senescence is part of normal ageing but has also been implicated in neurodegenerative diseases, as well as epilepsy. In temporal lobe epilepsy (TLE), MRI studies frequently report widespread cortical thinning that appears to progress with age. Previously, on larger cohort, we demonstrated that cortical thinning may reflect altered neuronal composition or disrupted cytoarchitectural organisation, as histological analysis did not reveal corresponding cortical thinning. Here we investigated whether there is a link between senescence and MRI-derived cortical thinning.

Methods: A subgroup of 8 patients with TLE from a cohort of 117 cases, including individuals with hippocampal sclerosis and no additional lesional pathology, were stratified into four groups based on pre-operative MRI measures of cortical thickness: left or right high cortical thinning (LHCT, n = 2; RHCT, n = 2) and left or right low cortical thinning (LLCT, n = 2; RLCT, n = 2). Temporal neocortical sections were immunohistochemically stained for double label: p16/NeuN, p16/Calretinin and p16/Iba1. For p16/Iba1 staining, an additional cohort of 20 patients was analysed (LHCT = 10 and LLCT = 10). These were quantified using cell detection and object classifiers in QuPath. Measures of positive cell counts as well as intensity of the markers were analysed.

Results: Patients with low cortical thinning in MRI, showed a narrow and localised expression of p16 at the crest of the gyrus, whereas cases with high cortical thinning revealed a ‘pan-cortical’ expression. Moreover, an inverse relationship between p16 and NeuN expression was observed in p16+/NeuN+ cells. 

Conclusion: These preliminary findings indicate that senescence might be a marker of microstructural degeneration that may manifest as cortical thinning in MRI. MRI-derived cortical thinning in TLE does not necessarily correspond to widespread neuronal loss. Instead, cortical thinning may reflect altered neuronal composition in the form of neuropil loss (e.g. from dendritic retraction, spine loss, etc) associated with senescence. Although studies in mouse models of epilepsy have reported increased senescence in microglia2, we observed very few senescent microglia and with the majority of p16+ cells being neurons.

89

Age-dependent coupling of hyperphosphorylated tau and cognitive decline intemporal lobe epilepsy

Isha Puntambekar¹, Alicja MRZYGLOD¹, Joanna Bartkiewicz¹, Gareth Ambler³, Sallie Baxendale^1,2, Maria Thom^1,2, Matthias Koepp^1,2

¹UCL Queen Square Institute Of Neurology, ²National Hospital for Neurology and Neurosurgery, ³UCL Department of Statistical Sciences

Background: Hyperphosphorylated tau (pTau) is increasingly identified in temporal lobe epilepsy (TLE), but its contribution to cognitive function remains uncertain. While pTau accumulation is linked to neurodegeneration and progressive cognitive decline in Alzheimer’s disease, cognitive dysfunction in epilepsy arises from multiple interacting factors.We therefore examined whether pTau contributes to preoperative cognitive function and postoperative cognitive change beyond established clinical covariates.

Methods: Two-hundred and forty-four adults with drug-resistant TLE underwent standardised neuropsychological assessmentbefore and one year after unilateral temporal lobe resection. Resected temporal neocortex was analysed using AT8immunohistochemistry and semi-quantitatively scored for neuronal and dendritic pTau burden (0–5). The contribution of pTau to preoperative cognitive function, and postoperative change in function was modelled using hierarchical linear/logistic regressions adjusting for age, gender, seizure laterality and medication load.

Associations between age, pTau and verbal memory were additionally modelled in separate logistic regressions, either with a linear age term or restricted cubic splines to account for potential non-linearage effects.

Results: pTau burden increased progressively with age at surgery (ρ = 0.50, p < 0.001; range 16-67). An age cut-off of 38.5 years distinguished low (0-2) from high (3-5) pathological burden with 75% sensitivity and 73%specificity. However, pTau did not incrementally contribute to preoperative cognitive performance orearly postoperative cognitive change after accounting for established clinical covariates. Group-level age-trajectory analyses revealed a dissociation between pathology and cognition; the predicted probability ofhigh pTau linearly increased across adulthood. In contrast, the predicted probability of significant postoperative memory decline appeared to follow a non-linear trajectory, mirroring the shape of thepTau trajectory from approximately the fifth decade of life.

Conclusions: Our results suggest that progressive pTau accumulation in TLE may precede age-accelerated cognitivedecline by decades. A ‘tipping point’ for tau-associated cognitive decline may emerge in older ages with failure of compensatory reserve, but could not be identified in the observed age range. These findings position TLE as a human model for studying age-dependent interactions between pathological tauaccumulation, cognitive resilience and neurodegeneration.

90

Neurodegeneration Biomarkers Relate to Hippocampal Tau Signals and Sleep Spindle Activity in Focal Epilepsy

Thaera Arafat^1,2, Tevy Chan^3,4, Jack Lam¹, Katharina Schiller⁵, Nicolas Von Ellenrieder¹, Andrea Bernasconi¹, Neda Bernasconi¹, Jean Gotman¹, Maria Thom², Matthias Koepp², Eliane Kobayashi⁶, Pedro Rosa-Neto^4,6, Boris Bernhardt¹

¹Montreal Neurological Institute-Hospital, McGill University, ²Research Department of Epilepsy, UCL Queen Square Institute of Neurology, University College London, ³Douglas Mental Health University Institute, McGill University, ⁴Research Institute of the McGill University Health Centre, ⁵Hospital Kaufbeuren, Department of Pediatric Neurology, 6Peter O’Donnell Jr. Brain Institute (OBI), University of Texas Southwestern Medical Centre (UTSW)

Rationale: Emerging evidence suggests shared pathophysiological mechanisms between epilepsy and neurodegeneration, with advances in neurodegeneration biomarkers, particularly phosphorylated tau-217 (pTau217), accelerating epilepsy research (Mo et al., 2019; Zawar, 2023). However, associations with in-vivo tau load and sleep spindle activity remain underexplored.

Methods: Seventeen patients with focal epilepsy from a multicentre cohort (mean age 35.9 ± 16.4 years; 9 females; 11 temporal lobe epilepsy) underwent (18F)MK-6240 positron emission tomography (PET) and 3T MRI. Imaging acquisition and analysis were harmonised across Montreal Neurological Institute-Hospital, Canada, and Chalfont Centre for Epilepsy, United Kingdom. Hippocampal tau uptake was quantified as mean bilateral standardized uptake value ratios (SUVRs) using the pons as the reference region. Plasma pTau217, glial fibrillary acidic protein (GFAP), and neurofilament light chain (NfL) were measured using validated immunoassays. Overnight EEG was available in eight patients (47%), with N2 spindle rates quantified using automated detection algorithms.

Results: Mean hippocampal tau uptake showed a strong positive association with plasma GFAP (r=0.71, p=0.001), a moderate positive, non-significant association with pTau217 (r=0.44, p=0.08), and no association with NfL (r=−0.15, p=0.56). In the EEG subgroup, N2 spindle rate showed a strong negative correlation with pTau217 that did not reach statistical significance (r=−0.69, p=0.06), with no associations observed for GFAP (r=0.22, p=0.59) or NfL (r=−0.20, p=0.63).

Conclusion: These findings suggest an association between hippocampal tau uptake and astroglial activation and provide preliminary evidence that reduced sleep spindle activity may reflect early tau-related neurodegenerative processes in focal epilepsy.

91

Loss of excitability homeostasis drives reversible tau accumulation during human brain ageing

Thaera Arafat¹, Alicja MRZYGLOD¹, Luisa Delazer¹, Kai Michael Schubert^1,3, Adolfo Mazzeo², Nicholas Fearns¹, Marian Galovic³, Maria Thom¹, Matthias Koepp¹

¹UCL, Queen Square Institute of Neurology, London, UK, ²Department of Human Neurosciences, Sapienza University of Rome, Rome, Italy, ³Department of Neurology, Clinical Neuroscience Center, University Hospital and University of Zurich, Zurich, Switzerland

Healthy cognitive ageing depends on the ability of neural circuits to maintain homeostatic regulation despite lifelong physiological stress. Progressive failure of this homeostatic reserve is thought to underlie neurodegeneration, yet the biological sequence linking early network dysfunction to later cognitive decline in humans remains poorly understood.

Here, we identify a multiscale pathway connecting impaired brain homeostasis to activity-dependent tau accumulation and cognitive ageing. By integrating magnetic resonance imaging of perivascular spaces, in vivo N-methyl-D-aspartate receptor positron emission tomography, intracranial microdialysis, human brain transcriptomics, histology, quantitative electroencephalography, longitudinal tau positron emission tomography and long-term cognitive follow-up.

We demonstrate convergent evidence that ageing is characterised by progressive disruption of excitatory homeostasis preceding structural degeneration. Reduced astrocytic glutamate transport is associated with elevated extracellular glutamate, persistent receptor overactivation and chronic network hyperexcitability within selectively vulnerable cognitive networks. Preoperative quantitative electroencephalographic markers identify physiological network states associated with subsequent distributed tau accumulation and long-term cognitive decline, whereas asymmetric enlargement of perivascular spaces consistent with altered glymphatic homeostasis is associated with memory trajectories independently of tau pathology, suggesting that impaired interstitial clearance represents an additional early contributor to network vulnerability. These processes localise to regions susceptible to tau accumulation and are markedly amplified in epilepsy, a translational model of accelerated brain ageing. Longitudinal analyses further demonstrate that successful surgical suppression of chronic network hyperexcitability is associated with lower subsequent whole-brain tau burden, whereas persistent seizures are associated with continued tau accumulation, indicating that activity-dependent tau propagation remains biologically modifiable.

Together, these findings support a unified model in which impaired glymphatic and astrocytic homeostasis progressively destabilise excitatory regulation, creating network states that facilitate tau propagation before age-dependent cognitive decompensation becomes clinically manifest. This framework identifies homeostatic failure as a fundamental mechanism of human brain ageing and highlights restoration of glutamate regulation and network activity as potential disease-modifying therapeutic strategies.

92

Neuropathological correlates of clinical progression in childhood Rasmussen encephalitis(RE): a 30-year single-centre cohort study

Eva Ioannidou^1,2, Marios Kaliakatsos², Suresh Pujar², Torsten Baldeweg¹, Sophia Varadkar⁶, Christin Eltze², Robert Robinson², Martin Tisdall^1,7, Zubair Tahir⁷, Ashirwad Merve⁵, Tom Jacques^4,5, Helen Cross³

¹Developmental Neurosciences Department, ²Department of Paediatric Neurology, ³Director’s Office , ⁴Developmental Biology and Cancer Department, ⁵Neuropathology Department, ⁶Director’s Office , ⁷Paediatric Neurosurgery Department

Background: Rasmussen encephalitis (RE) is a rare immune-mediated encephalitis characterised by progressive unilateral hemispheric inflammation, neurological decline and drug-resistant seizures¹. While its pathological hallmarks are well recognised², their relationship with clinical phenotype and disease progression remains poorly understood.

Methods: We performed a retrospective clinico-pathological study of children with RE undergoing epilepsy surgery at Great Ormond Street Hospital over a 30-year period. Original neuropathology reports were systematically reviewed for inflammatory response, microglial nodules, perivascular lymphocytes, neuronophagia, neurodegeneration, hippocampal sclerosis (HS) and the presence of a comment of ‘dual pathology’³ within the sample. These features were correlated with demographic and clinical variables, including age at onset, epilepsia partialis continua (EPC), hemiparesis, cognitive decline and time to surgery.

Results: Fifty-seven children underwent surgery, of whom 52 (91%) had available neuropathology reports. Mean age at seizure onset was 6.3 years and median time to surgery was 3.7 years. Neurodegeneration was the commonest pathological finding (79%), followed by microglial nodules (67%) and neuronophagia (38%). A co-existing ‘dual pathology’ was reported in 29% of cases, while HS was present in 11/31 (35%) cases with available hippocampal tissue. HS was strongly associated with dual pathology (73% vs 15%; p=0.001). Neuronophagia was significantly associated with neurodegeneration (89% vs 66%; p=0.004), supporting active immune-mediated neuronal injury. Although no pathological feature predicted the presence of EPC, hemiparesis or cognitive decline, patients with reported neuronophagia and/or neurodegeneration developed EPC significantly earlier than those without these features (median 0.7 vs 1.6 years, p=0.024; and 1.0 vs 2.3 years, p=0.026, respectively). Greater inflammatory activity also showed a trend towards shorter time to surgery.

Conclusions: This represents the largest clinico-pathological analysis of paediatric RE to date, in the UK. Active neuronal injury was associated with earlier disease progression, while the strong association between HS and dual pathology suggests that consequent structural abnormalities may contribute to the pathological spectrum of RE. These findings provide new insights into the relationship between neuropathology and clinical evolution and generate hypotheses for future mechanistic studies.

References:

1. Varadkar, S. et al. Rasmussen’s encephalitis: clinical features, pathobiology, and treatment advances. Lancet Neurol. 13, 195–205 (2014).

2. Pardo, C. A. et al. The pathology of Rasmussen syndrome: stages of cortical involvement and neuropathological studies in 45 hemispherectomies. Epilepsia 45, 516–526 (2004).

3. Gilani, A. & Kleinschmidt-DeMasters, B. K. How frequent is double pathology in Rasmussen encephalitis? Clin. Neuropathol. 39, 55–63 (2020).

93

Somatic mosaicism in resected brain tissue: diagnostic rate and implications for patient care

Flavia Santo^1,2, Amy McTague^1,3, Natalie Chandler^2,7, Lara Menzies⁴, Thomas Cullup², Thomas S. Jacques^5,6, Martin M. Tisdall^1,8

¹Developmental Neurosciences, UCL Great Ormond Street Institute of Child Health, ²North Thames Genomic Laboratory Hub, Great Ormond Street NHS Foundation Trust , ³Department of Neurology, Great Ormond Street Hospital, London, UK; Developmental Neurosciences, Zayed Centre for Research into Rare Disease in Children., ⁴Department of Clinical Genetics, Great Ormond Street Hospital for Children, ⁵Department of Histopathology, Great Ormond Street NHS Foundation Trust, London, UK., ⁶Developmental Biology and Cancer Programme, UCL Institute of Child Health, ⁷Genetic and Genomic Medicine, UCL Great Ormond Street Institute of Child Health, ⁸Department of Neurosurgery, Great Ormond Street Hospital for Children

Focal epilepsies may be associated with abnormal structural lesions (brain malformations) visible on MRI that are amenable to surgical resection. Somatic mosaicism, defined as post-zygotic genetic variability affecting only a subgroup of cells or tissues, may be responsible for 30%-40% of epilepsy cases with structural malformations. However, diagnosis remains challenging in routine practice, as mosaicism often occurs at low cellular fractions and may be absent from peripheral blood, limiting detection using standard NHS testing.

We performed deep next generation sequencing (NGS) on resected brain tissue from 50 paediatric patients with focal lesions, including cortical dysplasia (n=32), non-specific epilepsy-related changes (n=7), mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy (MOGHE; n=4), other malformations of cortical development and tuberous sclerosis complex. A targeted panel of 22 genes associated with focal epilepsy and mosaicism was used, achieving a median read depth of 1531× and detection sensitivity of 1% variant allele fraction. All pathogenic variants detected were validated using an orthogonal method and matched blood samples were tested, where available. Complex cases were reviewed in multidisciplinary team meetings consisting of neurology, imaging, histopathology and genomics professionals.

A genetic diagnosis was established in 25/50 patients (50%). Mosaic variants were identified in 14 patients (28%), and germline variants were detected in 10 (20%), including five copy number variants. One further patient harboured both a heterozygous germline and a mosaic variant in DEPDC5, supporting a second-hit mechanism.

Among the patients where follow-up information was available, 21/36 (58%) were seizure-free at 12 months post-surgery. Genetic findings informed various aspects of clinical management and counselling for patients and their families. For example, two patients with MOGHE and SLC35A2 variants are due to now receive D-galactose therapy. Informed reproductive counselling aided pregnancy-related counselling in a further family. In addition, further follow-up and screening was recommended for eight patients with mosaic variants in genes known to cause multi-system disorders e.g. Tuberous Sclerosis Complex, due to the risk of systemic manifestations.

These findings demonstrate that deep sequencing of brain tissue substantially improves diagnostic yield in focal epilepsy and provides clinically actionable information. Broader implementation of this approach within the NHS could reduce the proportion of undiagnosed patients and support precision medicine strategies in epilepsy care.

94

Focal astrocyte Kir4.1 loss drives seizures, spreading depolarisations and postictal impairments

Neela Codadu¹, Yunan Gao^1,2, Olga Tyurikova¹, Zixi Dai², Xueting Ban², Yuyan Weng², Eduard Masvidal-Codina³, Jose Garrido^3,4, Anton Guimera-Brunet^5,6, Dmitri Rusakov¹, Kevan Hashemi⁷, Nicholas Mazarakis², Rob Wykes^1,8

¹Research Department of Epilepsy, Queen Square Institute of Neurology, University College London, ² Gene Therapy, Division of Neuroscience, Department of Brain Sciences, Faculty of Medicine, Imperial College London, ³Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Campus UAB, ⁴Institució Catalana de Recerca i Estudis Avançats (ICREA), ⁵Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), ⁶Institut de Microelectrònica de Barcelona, IMB-CNM (CSIC), ⁷Open Source Instruments Inc, ⁸Division of Neuroscience & Centre for Nanotechnology applied to Medicine, University of Manchester

Introduction: Astrocytes regulate neuronal excitability through potassium buffering mediated by Kir4.1 inwardly-rectifying potassium channels. Reduced astrocytic Kir4.1 expression has been reported in human temporal lobe epilepsy, yet its causal role in epileptogenesis remains unclear. In epilepsy, spreading depolarisations (SDs), slowly propagating waves of neuronal and glial depolarisation, are temporally associated with seizures (SzSD) and have been implicated in postictal (period immediately following seizure termination) dysfunction and Sudden Unexpected Death in Epilepsy. However, whether focal astrocytic Kir4.1 loss acquired in adult brain networks is sufficient to drive seizures and SDs, and how SDs influence the postictal state, is unknown.

Methods: We generated focal Kir4.1 knockout mice (Kir4.1-cKO) by deleting the Kcnj10 gene encoding Kir4.1 channels in hippocampal astrocytes of adult Kir4.1-floxed mice using viral Cre recombinase. Ex vivo validation of Kir4.1 loss was performed using western blotting. Extracellular potassium dynamics were examined in hippocampal slices by measuring genetically encoded potassium indicator (GINKO2) fluorescence. To test seizure and SD susceptibility in vivo, optogenetics and DC-coupled graphene micro-transistor arrays were used in awake head-fixed mice. Continuous AC-coupled and video-DC-coupled iEEG telemetry recordings were employed to characterise the emergence of spontaneous seizures and SD.

Results: Kir4.1-cKO showed significantly reduced Kir4.1 protein expression (controls vs cKO: P=0.03). Electrical stimulation of Schaffer collaterals evoked larger GINKO2 fluorescence responses in CA1 stratum radiatum, indicating impaired potassium buffering. Chronic AC-coupled telemetry in Kir4.1-cKO revealed the emergence of spontaneous seizures 7–10 days post-viral injection. In awake head-fixed mice, Kir4.1-cKO hippocampal networks showed increased susceptibility to optogenetic stimulation-induced seizures-alone and SzSD. SzSD profoundly altered the postictal state, resulting in significantly prolonged postictal depression and delayed recovery compared with seizures-alone (P=0.016). Chronic video-DC-coupled telemetry (n = 6 mice, 3 weeks) revealed spontaneous seizures-alone and SzSD events. Compared with seizures-alone, SzSD exhibited higher power (P<0.0001) and greater postictal depression (P<0.0001). Video-DC-coupled iEEG analysis showed that SD-associated seizures exhibited a severe behavioural phenotype (maximum Racine score: 5-6), and postictal behaviour during the120s following seizure termination was characterised by behavioural arrest, loss of posture, and body jerks for ~87% of the postictal period, compared with ~1.7% following seizures-alone (P<0.0001), consistent with delayed functional recovery.

Conclusion: Focal hippocampal astrocytic Kir4.1 loss is sufficient to drive spontaneous seizures accompanied by frequent SDs, which markedly exacerbate postictal dysfunction. These findings provide mechanistic insight into how impaired astrocytic potassium buffering promotes epileptogenesis and seizure-associated pathology.

95

Modelling Depdc5 loss of function in rat primary cortical neurons

Angus Gane^1,2,3,4, Madeleine Marshall1, Marie Pronot^1,3,4, Alfredo Gonzalez-Sulser^1,3,4, Michael Cousin^1,3,4

¹University of Edinburgh, ²NHS Lothian, ³Simons Initiative for the Developing Brain, ⁴Muir Maxwell Epilepsy Centre

Background: Genetic variants in DEPDC5 are the most common cause of genetic focal epilepsy. DEPDC5 is known to form a complex with NPRL2 and NPRL3 termed GATOR1 which inhibits the mTOR signalling pathway. A rat model of Depdc5 loss of function induced by in utero electroporation produces focal seizures making the rat an exciting model to elucidate the mechanisms underlying DEPDC5-related epilepsy. We have developed a model of Depdc5 knockdown in cortical neuronal cultures to investigate the role of Depdc5 in neurons.

Methods: Lentiviruses expressing three short hairpin RNA (shRNA) systems and a scrambled control were synthesised. Rat primary cortical neuronal cultures were prepared from E16-18 embryos transduced after one day in vitro (DIV1). RNA was extracted for cDNA synthesis and quantitative PCR at 72 hours post transduction to determine the extent of knockdown. Two pairs of primers targeting the C and N termini of Depdc5 were normalised to the reference gene Tbp. Protein extraction and Western blot analysis was performed at DIV10 and normalised to total protein and βIII-Tubulin. Cells were fixed at DIV5 for immunocytochemical analysis of soma size, pS6/S6 intensity and neurite complexity using antibodies against MAP2 to determine if previously reported phenotypes are detectable in culture.

Results: Lentiviruses expressing shRNA2 and shRNA3 reduced both Depdc5 transcript expression and protein expression. No change was observed in soma size, pS6/S6 or dendritic complexity at DIV5, despite a high transduction efficiency. Excitatory and inhibitory synapse quantification, calcium imaging and multielectrode array recordings to determine culture excitability are ongoing.

Conclusion and future directions: We have established a rat primary cortical neuronal culture model of Depdc5 loss of function. At the timepoints analysed, we found no evidence of previously reported phenotypes associated with Depdc5 loss of function and mTORC1 activation. Future work will assess these phenotypes at later time points and examine whether a network level hyperexcitability phenotype using calcium imaging and multielectrode array recordings can be identified. Establishment of this model will reveal insights into the function of Depdc5 in neurons and potentially provide a platform for screening possible therapeutic targets.

96

A Gut Feeling About Epilepsy: A Systematic Review and Meta-Analysis of Gut Microbiota Alterations in People with Epilepsy

Pooja Goswami¹, Prem Jareonsettasin^2,3, Josemir W. Sander^2,3,4

¹Faculty of Health, Medicine and Life Sciences, Maastricht University, ²UCL Queen Square Institute of Neurology, ³Social Determinants of Health in Epileptic Disorders (SdHIELD) Research Group, ⁴Department of Neurology, West China Hospital, Sichuan University

Background: Epilepsy is a common neurological disorder associated with significant clinical and social burden. It is a symptom complex with multiple possible underlying mechanisms. Emerging work suggests that the gut microbiota may play a role in epileptogenesis through the microbiota-gut-brain axis. However, findings on gut microbiota alterations in epilepsy compared to controls remain inconsistent.

Objective: To explore gut microbiota diversity and composition between individuals with epilepsy and healthy controls, focusing on Shannon α-diversity, β-diversity, and exploratory phylum-level relative abundance. Secondary analyses explored whether gut microbiota patterns differed across subgroups, including drug-resistant epilepsy, age group, and geographical location.

Methods: A systematic search was conducted to identify studies investigating gut microbiota composition in people with epilepsy. Eligible studies comparing people with epilepsy to controls were included in meta-analysis when sufficient α-diversity data were available. Standardised mean differences (SMD) were estimated for Shannon α-diversity using a random-effects model. Reported beta diversity findings were summarised descriptively, while the relative abundance of taxa was explored using comparative statistical analyses. Subgroup and sensitivity analyses explored potential sources of heterogeneity, including age group and influential studies.

Results: Included studies (n= 31) varied substantially in population characteristics, epilepsy type, and control-group selection. Qualitative synthesis of taxonomic findings suggested alterations in major bacterial phyla, particularly Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria, although findings were inconsistent across studies. β-diversity more consistently suggested distinct clustering between epilepsy and control samples. Meta-analysis of Shannon α-diversity showed no significant overall difference between epilepsy and control groups (SMD = −0.02, 95% CI −0.92 to 0.88, p = 0.96), with substantial heterogeneity (I² = 92%). Exploratory analysis of phylum-level relative abundance showed no significant differences between epilepsy and control groups after adjustment for multiple testing. However, Proteobacteria was numerically higher in people with epilepsy. Subgroup analyses by age group, drug sensitivity and geographical location showed no significant difference.

Conclusion: No significant differences in Shannon w-diversity or phylum-level relative abundance were identified between epilepsy and controls. β-diversity findings suggested differences in overall microbial community composition, indicating that epilepsy may be associated with compositional shifts. Subgroup analyses reported no statistical differences. However, reverse causality cannot be ruled out, as seizure burden, long-term ASM use, and disease- or lifestyle-related factors may also contribute to alterations in microbiota. Current evidence remains heterogeneous, and larger standardised studies with detailed participant data are needed to identify epilepsy-associated microbiome signals.