Cheryl Reeves

Evidence for mTOR pathway activation in a spectrum of epilepsy-associated pathologies

IntroductionActivation of the mTOR pathway has been linked to the cytopathology and epileptogenicity of malformations, specifically Focal Cortical Dysplasia (FCD) and Tuberous Sclerosis (TSC). Experimental and clinical trials have shown than mTOR inhibitors have anti-epileptogenic effects in TS. Dysmorphic neurones and balloon cells are hallmarks of FCDIIb and TSC, but similar cells are also occasionally observed in other acquired epileptogenic pathologies, including hippocampal sclerosis (HS) and Rasmussen’s encephalitis (RE). Our aim was to explore mTOR pathway activation in a range of epilepsy-associated pathologies and in lesion-negative cases.Results50 epilepsy surgical pathologies were selected including HS ILAE type 1 with (5) and without dysmorphic neurones (4), FCDIIa (1), FCDIIb (5), FCDIIIa (5), FCDIIIb (3), FCDIIId (3), RE (5) and cortex adjacent to cavernoma (1). We also included pathology-negative epilepsy cases; temporal cortex (7), frontal cortex (2), paired frontal cortical samples with different ictal activity according to intracranial EEG recordings (4), cortex with acute injuries from electrode tracks (5) and additionally non-epilepsy surgical controls (3). Immunohistochemistry for phospho-S6 (pS6) ser240/244 and ser235/236 and double-labelling for Iba1, neurofilament, GFAP, GFAPdelta, doublecortin, and nestin were performed. Predominant neuronal labelling was observed with pS6 ser240/244 and glial labelling with pS6 ser235/236 in all pathology types but with evidence for co-expression in a proportion of cells in all pathologies. Intense labelling of dysmorphic neurones and balloon cells was observed in FCDIIb, but dysmorphic neurones were also labelled in RE and HS. There was no difference in pS6 labelling in paired samples according to ictal activity. Double-labelling immunofluorescent studies further demonstrated the co-localisation of pS6 with nestin, doublecortin, GFAPdelta in populations of small, immature neuroglial cells in a range of epilepsy pathologies.ConclusionsAlthough mTOR activation has been more studied in the FCDIIb and TSC, our observations suggest this pathway is activated in a variety of epilepsy-associated pathologies, and in varied cell types including dysmorphic neurones, microglia and immature cell types. There was no definite evidence from our studies to suggest that pS6 expression is directly related to disease activity.

Hyperphosphorylated tau in patients with refractory epilepsy correlates with cognitive decline: a study of temporal lobe resections

SEE BERNASCONI DOI101093/AWW202 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Temporal lobe epilepsy, the most prevalent form of chronic focal epilepsy, is associated with a high prevalence of cognitive impairment but the responsible underlying pathological mechanisms are unknown. Tau, the microtubule-associated protein, is a hallmark of several neurodegenerative diseases including Alzheimers disease and chronic traumatic encephalopathy. We hypothesized that hyperphosphorylated tau pathology is associated with cognitive decline in temporal lobe epilepsy and explored this through clinico-pathological study. We first performed pathological examination on tissue from 33 patients who had undergone temporal lobe resection between ages 50 and 65 years to treat drug-refractory temporal lobe epilepsy. We identified hyperphosphorylated tau protein using AT8 immunohistochemistry and compared this distribution to Braak patterns of Alzheimers disease and patterns of chronic traumatic encephalopathy. We quantified tau pathology using a modified tau score created specifically for analysis of temporal lobectomy tissue and the Braak staging, which was limited without extra-temporal brain areas available. Next, we correlated tau pathology with pre- and postoperative cognitive test scores and clinical risk factors including age at time of surgery, duration of epilepsy, history of secondary generalized seizures, history of head injury, handedness and side of surgery. Thirty-one of 33 cases (94%) showed hyperphosphorylated tau pathology in the form of neuropil threads and neurofibrillary tangles and pre-tangles. Braak stage analysis showed 12% of our epilepsy cohort had a Braak staging III-IV compared to an age-matched non-epilepsy control group from the literature (8%). We identified a mixture of tau pathology patterns characteristic of Alzheimers disease and chronic traumatic encephalopathy. We also found unusual patterns of subpial tau deposition, sparing of the hippocampus and co-localization with mossy fibre sprouting, a feature of temporal lobe epilepsy. We demonstrated that the more extensive the tau pathology, the greater the decline in verbal learning (Spearman correlation, r = -0.63), recall (r = -0.44) and graded naming test scores (r = -0.50) over 1-year post-temporal lobe resection (P < 0.05). This relationship with tau burden was also present when examining decline in verbal learning from 3 months to 1 year post-resection (r = -0.54). We found an association between modified tau score and history of secondary generalized seizures (likelihood-ratio χ(2), P < 0.05) however there was no clear relationship between tau pathology and other clinical risk factors assessed. Our findings suggest an epilepsy-related tauopathy in temporal lobe epilepsy, which contributes to accelerated cognitive decline and has diagnostic and treatment implications.

Combined Ex Vivo 9.4T MRI and Quantitative Histopathological Study in Normal and Pathological Neocortical Resections in Focal Epilepsy

High‐resolution magnetic resonance imaging (MRI) may improve the preoperative diagnosis of focal cortical dysplasia (FCD) in epilepsy. Quantitative 9.4T MRI was carried out (T1, T2, T2* and magnetization transfer ratio) on 13 cortical resections, representing pathologically confirmed FCD (five cases) and normal cortex. Quantitative immunohistochemistry for myelination (myelin basic protein/SMI94), neuronal populations [microtubule‐associated protein 2 (MAP2), neurofilament (SMI31, SMI32), synaptophysin, NeuN, calbindin], reactive glia (GFAP), microglia (CD68) and blood–brain barrier permeability (albumin) was carried out in 43 regions of interest (ROI) from normal and abnormal white matter and cortex. MRI was spatially aligned and quantitative analysis carried out on corresponding ROI. Line profile analysis (LPA) of intensity gradients through the cortex was carried out on MRI and immunostained sections. An inverse correlation was noted between myelin/SMI94 and T1, T2 (P < 0.005) and T2* (P < 0.05; Spearmans correlation) and a positive correlation between neuronal MAP2 and T1 (P < 0.005) and T2* (P < 0.05) over all ROI. Similar pathology–MRI correlations were observed for histologically unremarkable white matter ROI only. LPA showed altered gradient contours in regions of FCD, reflecting abnormal cortical lamination and myelo‐architecture, including a preoperatively undetected FCD case. This study demonstrates the ability of quantitative 9.4T MRI to detect subtle differences in neuronal numbers and myelination in histologically normal appearing white matter and LPA in the evaluation of cortical dyslamination. These methods may be translatable to the in vivo detection of mild cortical malformations.

Interictal psychosis following temporal lobe surgery: dentate gyrus pathology.

BACKGROUND De novo interictal psychosis, albeit uncommon, can develop in patients following temporal lobe surgery for epilepsy. Pathological alterations of the dentate gyrus, including cytoarchitectural changes, immaturity and axonal reorganization that occur in epilepsy, may also underpin co-morbid psychiatric disorders. Our aim was to study candidate pathways that may be associated with the development of interictal psychosis post-operatively in patients with hippocampal sclerosis (HS). METHOD A total of 11 patients with HS who developed interictal psychosis (HS-P) post-operatively were compared with a matched surgical HS group without psychosis (HS-NP). Resected tissues were investigated for the extent of granule cell dispersion, mossy fibre sprouting and calbindin expression in the granule cells. We quantified doublecortin, mini-chromosome maintenance protein 2 (MCM2) and reelin-expressing neuronal populations in the dentate gyrus as well as the distribution of cannabinoid type 1 receptor (CBR1). RESULTS The patterns of neuronal loss and gliosis were similar in both groups. HS-P patients demonstrated less mossy fibre sprouting and granule cell dispersion (p < 0.01) and more frequent reduction in calbindin expression in granule cells. There were no group differences in the densities of immature MCM2, doublecortin and reelin-positive cells. CBR1 labelling was significantly lower in Cornu ammonis area CA4 relative to other subfields (p < 0.01); although reduced staining in all hippocampal regions was noted in HS-P compared with HS-NP patients, the differences were not statistically significant. CONCLUSIONS The alterations in dentate gyrus pathology found in HS-P patients could indicate underlying differences in the cellular response to seizures. These mechanisms may predispose to the development of psychosis in epilepsy and warrant further investigation.

Nestin-expressing cell types in the temporal lobe and hippocampus: Morphology, differentiation, and proliferative capacity

Nestin is expressed in immature neuroepithelial and progenitor cell types and transiently upregulated in proliferative neuroglial cells responding to acute brain injury, including following seizures. In 36 temporal lobe (TLobe) specimens from patients with TLobe epilepsy (age range 8–60 years) we studied the number, distribution and morphology of nestin‐expressing cells (NEC) in the pes, hippocampus body, parahippocampal gyrus, amygdala, temporal cortex and pole compared with post mortem control tissues from 26 cases (age range 12 gestational weeks to 76 years). The proliferative fraction of NEC was evaluated in selected regions, including recognized niches, using MCM2. Their differentiation was explored with neuronal (DCX, mushashi, βIII tubulin, NeuN) and glial (GFAP, GFAPdelta, glutamine synthetase, aquaporin4, EAAT1) markers, both in sections or following culture. Findings were correlated with clinical parameters. A stereotypical pattern in the distribution and morphologies of NEC was observed, reminiscent of patterns in the developing brain, with increased densities in epilepsy than adult controls (p < .001). Findings included MCM2‐positive radial glial‐like cells in the periventricular white matter and rows of NEC in the hippocampal fimbria and sulcus. Nestin cells represented 29% of the hippocampal proliferative fraction in epilepsy cases; 20% co‐expressed βIII tubulin in culture compared with 28% with GFAP. Significant correlations were noted between age at surgery, memory deficits and nestin populations. TLobe NEC with ongoing proliferative capacity likely represent vestiges of developmental migratory streams and resident reactive cell populations of potential relevance to hippocampal epileptogenesis, TLobe pathology, and co‐morbidities, including memory decline.

MULTINODULAR AND VACUOLATING NEURONAL TUMOURS IN EPILEPSY: DYSPLASIA OR NEOPLASIA?

Multinodular and vacuolating neuronal tumor (MVNT) is a new pattern of neuronal tumour included in the recently revised WHO 2016 classification of tumors of the CNS. There are 15 reports in the literature to date. They are typically associated with late onset epilepsy and a neoplastic vs. malformative biology has been questioned. We present a series of ten cases and compare their pathological and genetic features to better characterized epilepsy‐associated malformations including focal cortical dysplasia type II (FCDII) and low‐grade epilepsy‐associated tumors (LEAT). Clinical and neuroradiology data were reviewed and a broad immunohistochemistry panel was applied to explore neuronal and glial differentiation, interneuronal populations, mTOR pathway activation and neurodegenerative changes. Next generation sequencing was performed for targeted multi‐gene analysis to identify mutations common to epilepsy lesions including FCDII and LEAT. All of the surgical cases in this series presented with seizures, and were located in the temporal lobe. There was a lack of any progressive changes on serial pre‐operative MRI and a mean age at surgery of 45 years. The vacuolated cells of the lesion expressed mature neuronal markers (neurofilament/SMI32, MAP2, synaptophysin). Prominent labelling of the lesional cells for developmentally regulated proteins (OTX1, TBR1, SOX2, MAP1b, CD34, GFAPδ) and oligodendroglial lineage markers (OLIG2, SMI94) was observed. No mutations were detected in the mTOR pathway genes, BRAF, FGFR1 or MYB. Clinical, pathological and genetic data could indicate that MVNT aligns more with a malformative lesion than a true neoplasm with origin from a progenitor neuro‐glial cell type showing aberrant maturation.

Early lipofuscin accumulation in Frontal Lobe Epilepsy

This study reports on a novel brain pathology in young patients with frontal lobe epilepsy (FLE) that is distinct from focal cortical dysplasia (FCD).

Characterising subtypes of hippocampal sclerosis and reorganization: correlation with pre and postoperative memory deficit

Neuropathological subtypes of hippocampal sclerosis (HS) in temporal lobe epilepsy (The 2013 International League Against Epilepsy classification) are based on the qualitative assessment of patterns of neuronal loss with NeuN. In practice, some cases appear indeterminate between type 1 (CA1 and CA4 loss) and type 2 HS (CA1 loss) and we predicted that MAP2 would enable a more stringent classification. HS subtypes, as well as the accompanying alteration of axonal networks, regenerative capacity and neurodegeneration have been previously correlated with outcome and memory deficits and may provide prognostic clinical information. We selected 92 cases: 52 type 1 HS, 15 type 2 HS, 18 indeterminate‐HS and 7 no‐HS. Quantitative analysis was carried out on NeuN and MAP2 stained sections and a labeling index (LI) calculated for six hippocampal subfields. We also evaluated hippocampal regenerative activity (MCM2, nestin, olig2, calbindin), degeneration (AT8/phosphorylated tau) and mossy‐fiber pathway re‐organization (ZnT3). Pathology measures were correlated with clinical epilepsy history, memory and naming test scores and postoperative outcomes, at 1 year following surgery. MAP2 LI in indeterminate‐HS was statistically similar to type 2 HS but this clustering was not shown with NeuN. Moderate verbal and visual memory deficits were noted in all HS types, including 54% and 69% of type 2 HS. Memory deficits correlated with several pathology factors including lower NeuN or MAP2 LI in CA4, CA1, dentate gyrus (DG) and subiculum and poor preservation of the mossy fiber pathway. Decline in memory at 1 year associated with AT8 labeling in the subiculum and DG but not HS type. We conclude that MAP2 is a helpful addition in the classification of HS in some cases. Classification of HS subtype, however, did not significantly correlate with outcome or pre‐ or postoperative memory dysfunction, which was associated with multiple pathology factors including hippocampal axonal pathways, regenerative capacity and degenerative changes.

A cautionary note in the interpretation of human papillomavirus E6 immunohistochemistry in focal cortical dysplasia

Chen et al reported the remarkable finding of high-risk human papillomavirus 16 (HPV16) in focal cortical dysplasia type IIB (FCDIIB) in epilepsy. In addition to the molecular identification with reverse transcriptase polymerase chain reaction and in situ hybridization (ISH), they demonstrated E6 protein with immunohistochemistry (IHC) in balloon cells that was both highly specific and sensitive for FCDIIB and not observed in the cells within tuberous sclerosis lesions, temporal lobe epilepsy specimens, gangliogliomas, or postmortem normal controls. Their interpretation was that HPV infection was relevant to both the developmental pathogenesis of FCDIIB and functionally linked to mammalian target of rapamycin (mTOR) pathway activation in FCDIIB. Another group subsequently confirmed cytoplasmic HPV16 in dysmorphic neurons and balloon cells in FCDIIB. We have recently shown that mTOR pathway activation, as assessed by IHC, is present in a wide variety of acquired or developmental lesions in focal epilepsy, including hippocampal sclerosis, ganglioglioma, and Rasmussen encephalitis, and is not limited to FCDIIB or the mTORopathies. Following this, we explored whether IHC for the HPV16 E6 antigen might represent a more specific biomarker for FCDIIB, potentially applicable in diagnostic practice to distinguish FCDIIB from other dysplasia types and FCD mimics. We undertook IHC for HPV16/18 E6 antigen (ab30716; Abcam, Cambridge, MA; 1:100) in 21 cases representing a spectrum of pathologies in focal epilepsy and control cases as detailed in our previous study. We also utilized IHC for HPV major capsid protein L1 (M3528; Dako, Carpinteria, CA; 1:50) in selected cases. We confirmed distinct cytoplasmic labeling of balloon cells and some dysmorphic neurons in FCDIIB with E6 and L1 antibodies (Fig ). However, we also observed labeling of neurons in FCDIIIA, some neurons in nondysplastic superficial cortex, Rasmussen encephalitis, dysmorphic neurons in ganglioglioma, and reactive astroglia with E6 antibodies. Furthermore, ISH for HPV in 3 FCDIIB cases (Ventana INFORM HPV Probes; Roche Diagnostics, Mannheim, Germany) was negative. The pathogenic localization and potential role of HPV z(an epidermotropic oncogenic virus) in neuroepithelium and FCDIIB, particularly in regard to its latency, immunogenicity, influence on cell cycle dynamics, and mTOR activation, remain to be fully understood. Meanwhile, we provide a cautionary note that HPV16 demonstration by IHC, although dramatically highlighting balloon cells in FCDIIB, may not be specific for this pathology and may represent a cross-reaction with an as yet unidentified neuroglial protein. Potential Conflicts of Interest Nothing to report.

Doublecortin-expressing cell types in temporal lobe epilepsy

Doublecortin (DCX) is widely regarded as a marker of immature and migrating neurons during development. While DCX expression persists in adults, particularly in the temporal lobe and neurogenic regions, it is unknown how seizures influence its expression. The aim of the present study was to explore the distribution and characteristics of DCX-expressing cells in surgical and postmortem samples from 40 adult and paediatric patients, with epilepsy and with or without hippocampal sclerosis (HS), compared to post mortem controls. The hippocampus (pes and body), parahippocampal gyrus, amygdala, temporal pole and temporal cortex were examined with DCX immunohistochemistry using four commercially-available DCX antibodies, labelled cells were quantified in different regions of interest as well as their co-expression with cell type specific markers (CD68, Iba1, GFAP, GFAP∂, nestin, SOX2, CD34, OLIG2, PDGFRβ, NeuN) and cell cycle marker (MCM2). Histological findings were compared with clinical data, as well as gene expression data obtained from the temporal cortex of 83 temporal lobe epilepsy cases with HS. DCX immunohistochemistry identified immature (Nestin−/NeuN−) neurons in layer II of the temporal neocortex in patients with and without epilepsy. Their number declined significantly with age but was not associated with the presence of hippocampal sclerosis, seizure semiology or memory dysfunction. DCX+ cells were prominent in the paralaminar nuclei and periamygdalar cortex and these declined with age but were not significantly associated with epilepsy history. DCX expressing cells with ramified processes were prominent in all regions, particularly in the hippocampal subgranular zone, where significantly increased numbers were observed in epilepsy samples compared to controls. DCX ramified cells co-expressed Iba1, CD68 and PDGFRβ, and less frequently MCM2, OLIG2 and SOX2, but no co-localization was observed with CD34, nestin or GFAP/GFAP ∂. Gene expression data from neocortical samples in patients with TLE and HS supported ongoing DCX expression in adults. We conclude that DCX identifies a range of morphological cell types in temporal lobe epilepsy, including immature populations, glial and microglial cell types. Their clinical relevance and biological function requires further study but we show some evidence for alteration with age and in epilepsy.