Influence of Mitochondrial NAD(P) + Transhydrogenase (NNT) on Hypothalamic Inflammation and Metabolic Dysfunction Induced by a High-Fat Diet in Mice.

The mitochondrial protein NAD(P)+ transhydrogenase (NNT) has been implicated in the metabolic derangements observed in obesity. Mice with the C57BL/6J genetic background bear a spontaneous mutation in the Nnt gene and are known to exhibit increased susceptibility to diet-induced metabolic disorders. Most of the studies on NNT in the context of diet-induced obesity have compared C57BL/6J mice with other mouse strains, where differences in genetic background can serve as confounding factors. Moreover, these studies have predominantly employed a high-fat diet (HFD) consisting of approximately 60% of calories from fat, which may not accurately mimic real-world fat-rich diets. In this study, we sought to examine the role of NNT in diet-induced hypothalamic inflammation and metabolic syndrome by using a congenic mice model lacking NNT, along with a HFD providing approximately 45% of calories from fat. Our findings indicate that mice lacking NNT were more protected from HFD-induced weight gain but presented a worse performance on glucose tolerance test, albeit not in insulin tolerance test. Interestingly, the brown adipose tissue of HFD-fed Nnt +/+ mice presented a greater mass and a higher whole-tissue ex-vivo oxygen consumption rate. Also, HFD increased the expression of the inflammatory markers Il1ß, Tlr4 and Iba1 in the hypothalamus of Nnt -/- mice. In conclusion, our study highlights the importance of NNT in the context of diet-induced obesity and metabolic syndrome, indicating its contribution to mitigate hypothalamic inflammation and suggesting its role in the brown adipose tissue increased mass.

Junctional instability in neuroepithelium and network hyperexcitability in a focal cortical dysplasia human model.

Focal cortical dysplasia is a highly epileptogenic cortical malformation with few treatment options. Here, we generated human cortical organoids from patients with focal cortical dysplasia type II. Using this human model, we mimicked some focal cortical dysplasia hallmarks, such as impaired cell proliferation, the presence of dysmorphic neurons and balloon cells, and neuronal network hyperexcitability. Furthermore, we observed alterations in the adherens junctions zonula occludens-1 and partitioning defective 3, reduced polarization of the actin cytoskeleton, and fewer synaptic puncta. Focal cortical dysplasia cortical organoids showed downregulation of the small GTPase RHOA, a finding that was confirmed in brain tissue resected from these patients. Functionally, both spontaneous and optogenetically-evoked electrical activity revealed hyperexcitability and enhanced network connectivity in focal cortical dysplasia organoids. Taken together, our findings suggest a ventricular zone instability in tissue cohesion of neuroepithelial cells, leading to a maturational arrest of progenitors or newborn neurons, which may predispose to cellular and functional immaturity and compromise the formation of neural networks in focal cortical dysplasia.

Interferon-beta induces major histocompatibility complex of class I (MHC-I) expression and a proinflammatory phenotype in cultivated human astrocytes.

Major histocompatibility complex class I (MHC-I) has been implicated in several types of neuroplasticity phenomena. Interferon beta-1b (IFN-ß) increases MHC-I expression by motoneurons after sciatic nerve crush in mice, improving axonal growth and functional recovery. Additionally, IFN-ß induces glial hypertrophy associated with upregulation of glial fibrillary acidic protein (GFAP) and MHC-I in murine astrocytes in vitro. As knowledge about MHC-I and its role in synaptic plasticity in human astrocytes (HAs) is scarce, we investigated these aspects in mature HAs obtained from the neocortex of patients undergoing surgery due to hippocampal sclerosis. Cells were exposed to media in the absence (0 IU/ml) or presence of IFN-ß for 5 days (500 IU/ml). Beta-2 microglobulin (ß2m), a component of the MHC-I, GFAP and vimentin proteins, was quantified by flow cytometry (FC) and increased by 100%, 60% and 46%, respectively, after IFN-ß exposure. We also performed qRT-PCR gene expression analyses for ß2m, GFAP, vimentin, and pro- and anti-inflammatory cytokines. Our data showed that IFN-ß-treated astrocytes displayed ß2m and GFAP gene upregulation. Additionally, they presented a proinflammatory profile with increase in the IL-6 and IL-1ß genes and a tendency to upregulate TNF-α. Moreover, we evaluated the effect of HAs conditioned medium (CM) on the formation/maintenance of neurites/synapses by the PC12 lineage. Synaptophysin protein expression was quantified by FC. The CM of IFN-ß-activated astrocytes was not harmful to PC12 neurites, and there was no change in synaptophysin protein expression. Therefore, IFN-ß activated HAs by increasing GFAP, vimentin and MHC-I protein expression. Like MHC-I modulation and astrocyte activation may be protective after peripheral nerve damage and in some neurodegenerative conditions, this study opens perspectives on the pathophysiological roles of astroglial MHC-I in the human CNS.

Anaplastic ganglioglioma-A diagnosis comprising several distinct tumour types.

AIMS: Anaplastic ganglioglioma is a rare tumour, and diagnosis has been based on histological criteria. The 5th edition of the World Health Organization Classification of Tumours of the Central Nervous System (CNS WHO) does not list anaplastic ganglioglioma as a distinct diagnosis due to lack of molecular data in previous publications. We retrospectively compiled a cohort of 54 histologically diagnosed anaplastic gangliogliomas to explore whether the molecular profiles of these tumours represent a separate type or resolve into other entities. METHODS: Samples were subjected to histological review, desoxyribonucleic acid (DNA) methylation profiling and next-generation sequencing. Morphological and molecular data were summarised to an integrated diagnosis. RESULTS: The majority of tumours designated as anaplastic gangliogliomas resolved into other CNS WHO diagnoses, most commonly pleomorphic xanthoastrocytoma (16/54), glioblastoma, isocitrate dehydrogenase protein (IDH) wild type and diffuse paediatric-type high-grade glioma, H3 wild type and IDH wild type (11 and 2/54), followed by low-grade glial or glioneuronal tumours including pilocytic astrocytoma, dysembryoplastic neuroepithelial tumour and diffuse leptomeningeal glioneuronal tumour (5/54), IDH mutant astrocytoma (4/54) and others (6/54). A subset of tumours (10/54) was not assignable to a CNS WHO diagnosis, and common molecular profiles pointing to a separate entity were not evident. CONCLUSIONS: In summary, we show that tumours histologically diagnosed as anaplastic ganglioglioma comprise a wide spectrum of CNS WHO tumour types with different prognostic and therapeutic implications. We therefore suggest assigning this designation with caution and recommend comprehensive molecular workup.

A Novel Multisystem Proteinopathy Caused by a Missense ANXA11 Variant.

OBJECTIVE: Protein misfolding plays a central role not only in amyotrophic lateral sclerosis (ALS), but also in other conditions, such as frontotemporal dementia (FTD), inclusion body myopathy (hIBM) or Paget's disease of bone. The concept of multisystem proteinopathies (MSP) was created to account for those rare families that segregate at least 2 out of these 4 conditions in the same pedigree. The calcium-dependent phospholipid-binding protein annexin A11 was recently associated to ALS in European pedigrees. Herein, we describe in detail 3 Brazilian families presenting hIBM (isolated or in combination with ALS/FTD) caused by the novel p.D40Y change in the gene encoding annexin A11 (ANXA11). METHODS: We collected clinical, genetic, pathological and skeletal muscle imaging from 11 affected subjects. Neuroimaging was also obtained from 8 patients and 8 matched controls. RESULTS: Clinico-radiological phenotype of this novel hIBM reveals a slowly progressive predominant limb-girdle syndrome, but with frequent axial (ptosis/dropped head) and distal (medial gastrocnemius) involvement as well. Muscle pathology identified numerous rimmed vacuoles with positive annexin A11, TDP-43 and p62 inclusions, but no inflammation. Central nervous system was also involved: two patients had FTD, but diffusion tensor imaging uncovered multiple areas of cerebral white matter damage in the whole group (including the corticospinal tracts and frontal subcortical regions). INTERPRETATION: These findings expand the phenotypic spectrum related to ANXA11. This gene should be considered the cause of a novel multisystem proteinopathy (MSP type 6), rather than just ALS. ANN NEUROL 2021;90:239-252.

Magnetic resonance imaging findings and clinical characteristics in mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy in a predominantly adult cohort.

OBJECTIVE: We aimed to better characterize the magnetic resonance imaging (MRI) findings of mild malformation of cortical development with oligodendroglial hyperplasia (MOGHE), a rare clinicopathological entity associated with pharmacoresistance recently described in patients with frontal lobe epilepsy. METHODS: We studied 12 patients who underwent epilepsy surgery and whose surgical specimens showed histopathological findings of MOGHE, characterized by preserved cortical lamination, blurred gray-white matter interface due to increased number of oligodendrocytes, and heterotopic neurons in the white matter. The age at MRI evaluation ranged from 11 to 58 years, except for one 4.5-year-old patient. RESULTS: Following a detailed MRI analysis using an in-house protocol, we found abnormalities in all cases. The lesion was circumscribed in the frontal lobe in six (50%) and in the temporal lobe in three (25%) patients. In the remaining three patients (25%), the lesion was multilobar (frontotemporal and temporoparieto-occipital). Cortical thickening was mild in all patients, except in the 4.5-year-old patient, who had pronounced cortical thickening and white matter blurring. We also identified cortical/subcortical hyperintense T2/fluid-attenuated inversion recovery signal associated with gray/white matter blurring in all but one patient. When present, cleft cortical dimple, and deep sulci aided in localizing the lesion. Overall, the MRI findings were like those in focal cortical dysplasia (FCD) Type IIa. Surgical outcome was excellent in five patients (Engel Class I in 25% and II in 17%). The remaining seven patients (58%) had worthwhile seizure reduction (Engle Class III). Incomplete lesion resection was significantly associated with worse outcomes. SIGNIFICANCE: MRI findings associated with MOGHE are similar to those described in FCD Type IIa. Although more frequent in the frontal lobe, MOGHE also occurred in the temporal lobe or involved multiple lobes. Multilobar or extensive MOGHE MRI lesions are associated with less favorable surgical outcomes. Because this is a rare condition, multicenter studies are necessary to characterize MOGHE further.

Transsylvian amygdalohippocampectomy for mesial temporal lobe epilepsy: Comparison of three different approaches.

OBJECTIVE: This study's objective was to compare the transinsular (TI-AH), transuncus (TU-AH), and temporopolar (TP-AH) amygdalohippocampectomy approaches regarding seizure control, temporal stem (TS) damage, and neurocognitive decline. METHODS: We included 114 consecutive patients with unilateral hippocampal sclerosis (HS) who underwent TI-AH, TU-AH, or TP-AH between 2002 and 2017. We evaluated seizure control using Engel classification. We used diffusion tensor imaging and postoperative Humphrey perimetry to assess the damage of the TS. We also performed pre- and postoperative memory performance and intelligence quotient (IQ). RESULTS: There were no significant differences in the proportion of patients free of disabling seizures (Engel IA+IB) among the three surgical approaches in the survival analysis. However, more patients were free of disabling seizures (Engel IA+IB) at 2 years of postsurgical follow-up with TP-AH (69.5%) and TI-AH (76.7%) as compared to the TU-AH (43.5%) approach (p = .03). The number of fibers of the inferior fronto-occipital fasciculus postoperatively was reduced in the TI-AH group compared with the TU-AH and TP-AH groups (p = .001). The rate of visual field defects was significantly higher with TI-AH (14/19, 74%) in comparison to the TU-AH (5/15, 33%) and TP-AH (13/40, 32.5%) approaches (p = .008). Finally, there was a significant postoperative decline in verbal memory in left-sided surgeries (p = .019) and delayed recall for both sides (p < .001) regardless of the surgical approach. However, TP-AH was the only group that showed a significant improvement in visual memory (p < .001) and IQ (p < .001) for both right- and left-sided surgeries. SIGNIFICANCE: The TP-AH group had better short-term seizure control than TU-AH, a lower rate of visual field defects than TI-AH, and improved visual memory and IQ compared to the other groups. Our findings suggest that TP-AH is a better surgical approach for temporal lobe epilepsy with HS than TI-AH and TU-AH.

Toward a better definition of focal cortical dysplasia: An iterative histopathological and genetic agreement trial.

OBJECTIVE: Focal cortical dysplasia (FCD) is a major cause of difficult-to-treat epilepsy in children and young adults, and the diagnosis is currently based on microscopic review of surgical brain tissue using the International League Against Epilepsy classification scheme of 2011. We developed an iterative histopathological agreement trial with genetic testing to identify areas of diagnostic challenges in this widely used classification scheme. METHODS: Four web-based digital pathology trials were completed by 20 neuropathologists from 15 countries using a consecutive series of 196 surgical tissue blocks obtained from 22 epilepsy patients at a single center. Five independent genetic laboratories performed screening or validation sequencing of FCD-relevant genes in paired brain and blood samples from the same 22 epilepsy patients. RESULTS: Histopathology agreement based solely on hematoxylin and eosin stainings was low in Round 1, and gradually increased by adding a panel of immunostainings in Round 2 and the Delphi consensus method in Round 3. Interobserver agreement was good in Round 4 (kappa = .65), when the results of genetic tests were disclosed, namely, MTOR, AKT3, and SLC35A2 brain somatic mutations in five cases and germline mutations in DEPDC5 and NPRL3 in two cases. SIGNIFICANCE: The diagnoses of FCD 1 and 3 subtypes remained most challenging and were often difficult to differentiate from a normal homotypic or heterotypic cortical architecture. Immunohistochemistry was helpful, however, to confirm the diagnosis of FCD or no lesion. We observed a genotype-phenotype association for brain somatic mutations in SLC35A2 in two cases with mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy. Our results suggest that the current FCD classification should recognize a panel of immunohistochemical stainings for a better histopathological workup and definition of FCD subtypes. We also propose adding the level of genetic findings to obtain a comprehensive, reliable, and integrative genotype-phenotype diagnosis in the near future.

Interactions between in vivo neuronal-glial markers, side of hippocampal sclerosis, and pharmacoresponse in temporal lobe epilepsy.

OBJECTIVE: To evaluate the interactions of metabolic neuronal-glial changes with the presence and hemispheric-side of hippocampal sclerosis (HS) and its potential role in predicting pharmacoresistance in temporal lobe epilepsy (TLE). METHODS: We included structural magnetic resonance imaging (MRI) and proton magnetic resonance spectroscopy (1 H-MRS) metabolic data for 91 patients with unilateral TLE and 50 healthy controls. We measured the values of total N-acetyl aspartate/total creatine (tNAA/tCr), glutamate/tCr (Glu/tCr), and myo-inositol/tCr (mIns/tCr). To assess the influence of the pharmacoresponse and hemispheric-side of HS on metabolic data, the relationship between clinical and MRI data, and the predictive value of NAA/Cr, we used analysis of variance/covariance and built a logistic regression model. We used bootstrap simulations to evaluate reproducibility. RESULTS: Bilateral tNAA/tCr reduction was associated with pharmacoresistance and with left HS, a decrease of Glu/tCr ipsilateral to the seizure focus was associated with pharmacoresistance, and ipsilateral mIns/tCr increase was related to pharmacoresistance and the presence of left HS. The logistic regression model containing clinical and 1 H-MRS data discriminated pharmacoresistance (area under the curve [AUC] = 0.78). However, the reduction of tNAA/tCr was the main predictor, with the odds 2.48 greater for pharmacoresistance. SIGNIFICANCE: Our study revealed a spectrum of neuronal-glial changes in TLE, which was associated with pharmacoresistance, being more severe in left-sided HS and less severe in MRI-negative TLE. These noninvasive, in vivo biomarkers provide valuable additional information about the interhemispheric differences in metabolic dysfunction, seizure burden, and HS, and may help to predict pharmacoresistance.

Same same but different: A Web-based deep learning application revealed classifying features for the histopathologic distinction of cortical malformations.

OBJECTIVE: The microscopic review of hematoxylin-eosin-stained images of focal cortical dysplasia type IIb and cortical tuber of tuberous sclerosis complex remains challenging. Both entities are distinct subtypes of human malformations of cortical development that share histopathological features consisting of neuronal dyslamination with dysmorphic neurons and balloon cells. We trained a convolutional neural network (CNN) to classify both entities and visualize the results. Additionally, we propose a new Web-based deep learning application as proof of concept of how deep learning could enter the pathologic routine. METHODS: A digital processing pipeline was developed for a series of 56 cases of focal cortical dysplasia type IIb and cortical tuber of tuberous sclerosis complex to obtain 4000 regions of interest and 200 000 subsamples with different zoom and rotation angles to train a neural network. Guided gradient-weighted class activation maps (Guided Grad-CAMs) were generated to visualize morphological features used by the CNN to distinguish both entities. RESULTS: Our best-performing network achieved 91% accuracy and 0.88 area under the receiver operating characteristic curve at the tile level for an unseen test set. Novel histopathologic patterns were found through the visualized Guided Grad-CAMs. These patterns were assembled into a classification score to augment decision-making in routine histopathology workup. This score was successfully validated by 11 expert neuropathologists and 12 nonexperts, boosting nonexperts to expert level performance. SIGNIFICANCE: Our newly developed Web application combines the visualization of whole slide images with the possibility of deep learning-aided classification between focal cortical dysplasia IIb and tuberous sclerosis complex. This approach will help to introduce deep learning applications and visualization for the histopathologic diagnosis of rare and difficult-to-classify brain lesions.

Digital analysis of hormonal immunostaining in pituitary adenomas classified according to WHO 2017 criteria and correlation with preoperative laboratory findings.

OBJECTIVE: The authors sought to evaluate clinical and laboratory data from pituitary adenoma (PA) patients with functioning PA (associated with acromegaly [n = 10] or Cushing disease [n = 10]) or nonfunctioning PA (NFPA; n = 10) that were classified according to 2017 WHO criteria (based on the expression of the transcription factors pituitary-specific positive transcription factor 1 [Pit-1], a transcription factor member of the T-box family [Tpit], and steroidogenic factor 1 [SF-1]) and to assess the immunostaining results for growth hormone (GH) and adrenocorticotropic hormone (ACTH) in the corresponding tumors. METHODS: Clinical and laboratory data were collected retrospectively. The percentage of tumoral cells positive for Pit-1, Tpit, or SF-1 was assessed and ImageJ software was used to evaluate immunopositivity in PAs with 2 different antibodies against GH (primary antibody 1 [AbGH-1] and primary antibody 2 [AbGH-2]) and 2 different antibodies against ACTH (primary antibody 1 [AbACTH-1] and primary antibody 2 [AbACTH-2]). RESULTS: Cells with positive Pit-1 staining were more frequently observed in lesions from patients with acromegaly (acromegaly group) than in lesions from patients with Cushing disease (Cushing group; p < 0.001) and those from patients with NFPA (NFPA group; p < 0.001). The percentage of Tpit-positive cells was higher in the Cushing group than in the acromegaly (p < 0.001) and NFPA (p < 0.001) groups. No difference was detected regarding SF-1 frequency among all groups (p = 0.855). In acromegalic individuals, GH immunostaining levels varied depending on the antibody employed, and only one of the antibodies (AbGH-2) yielded higher values in comparison with the values for NFPA patients (p < 0.001). For all of the antibodies employed, no significant correlations were detected between GH tissue expression and the laboratory data (serum GH vs AbGH-1, p = 0.933; serum GH vs AbGH-2, p = 0.853; serum insulin-like growth factor-1 [IGF-1] vs AbGH-1, p = 0.407; serum IGF-1 vs AbGH-2, p = 0.881). In the Cushing group data, both antibodies showed similar ACTH tissue expression, which was higher than that obtained in the NFPA group (p < 0.001). There were no significant associations between ACTH immunohistochemical findings and ACTH serum levels (serum ACTH vs AbACTH-1, p = 0.651; serum ACTH vs AbACTH-2, p = 0.987). However, ACTH immunostaining evaluated with AbACTH-1 showed a significant correlation with 24-hour urinary cortisol (24-hour cortisol vs AbACTH-1, p = 0.047; 24-hour cortisol vs AbACTH-2, p = 0.071). CONCLUSIONS: Immunostaining for Pit-1 and Tpit accurately identified lesions associated with acromegaly and Cushing disease, respectively. Conversely, SF-1 did not differentiate NFPA from lesions of the other two groups. Regarding hormonal tissue detection, results of the current investigation indicate that different antibodies may lead not only to divergent immunohistochemical results but also to lack of correlation with laboratory findings. Finally, PA classification based on transcription factor expression (Pit-1, Tpit, and SF-1), as proposed by the 2017 WHO classification of pituitary tumors, may avoid the limitations of PA classification based solely on digital immunohistochemical detection of hormones.

Dysregulation of NEUROG2 plays a key role in focal cortical dysplasia.

OBJECTIVE: Focal cortical dysplasias (FCDs) are an important cause of drug-resistant epilepsy. In this work, we aimed to investigate whether abnormal gene regulation, mediated by microRNA, could be involved in FCD type II. METHODS: We used total RNA from the brain tissue of 16 patients with FCD type II and 28 controls. MicroRNA expression was initially assessed by microarray. Quantitative polymerase chain reaction, in situ hybridization, luciferase reporter assays, and deep sequencing for genes in the mTOR pathway were performed to validate and further explore our initial study. RESULTS: hsa-let-7f (p = 0.039), hsa-miR-31 (p = 0.0078), and hsa-miR34a (p = 0.021) were downregulated in FCD type II, whereas a transcription factor involved in neuronal and glial fate specification, NEUROG2 (p < 0.05), was upregulated. We also found that the RND2 gene, a NEUROG2-target, is upregulated (p < 0.001). In vitro experiments showed that hsa-miR-34a downregulates NEUROG2 by binding to its 5'-untranslated region. Moreover, we observed strong nuclear expression of NEUROG2 in balloon cells and dysmorphic neurons and found that 28.5% of our patients presented brain somatic mutations in genes of the mTOR pathway. INTERPRETATION: Our findings suggest a new molecular mechanism, in which NEUROG2 has a pivotal and central role in the pathogenesis of FCD type II. In this way, we found that the downregulation of hsa-miR-34a leads to upregulation of NEUROG2, and consequently to overexpression of the RND2 gene. These findings indicate that a faulty coupling in neuronal differentiation and migration mechanisms may explain the presence of aberrant cells and complete dyslamination in FCD type II. Ann Neurol 2018;83:623-635.

ATP Synthase Subunit Beta Immunostaining is Reduced in the Sclerotic Hippocampus of Epilepsy Patients.

Epilepsy is a common disease presenting with recurrent seizures. Hippocampal sclerosis (HS) is the commonest histopathological alteration in patients with temporal lobe epilepsy (TLE) undergoing surgery. HS physiopathogenesis is debatable. We have recently studied, by using mass spectrometry-based proteomics, an experimental model of TLE induced by electrical stimulation. Specifically, protein expressions of both the beta subunit of mitochondrial ATP synthase (ATP5B) and of membrane ATPases were found to be reduced. Here, we investigated tissue distribution of ATP5B and sodium/potassium-transporting ATPase subunit alpha-3 (NKAα3), a protein associated with neuromuscular excitability disorders, in human hippocampi resected "en bloc" for HS treatment (n = 15). We used immunohistochemistry and the stained area was digitally evaluated (increase in binary contrast of microscopic fields) in the hippocampal sectors (CA1-CA4) and dentate gyrus. All HS samples were classified as Type 1, according to the International League Against Epilepsy (ILAE) 2013 Classification (predominant cell loss in CA1 and CA4). ATP5B was significantly decreased in all sectors and dentate gyrus of HS patients compared with individuals submitted to necropsy and without history of neurological alterations (n = 10). NKAα3 expression showed no difference. Moreover, we identified a negative correlation between frequency of pre-operative seizures and number of neurons in CA1. In conclusion, our data showed similarity between changes in protein expression in a model of TLE and individuals with HS. ATP5B reduction would be at least in part due to neuronal loss. Future investigations on ATP5B activity could provide insights into the process of such cell loss.

Toxic effects of phytol and retinol on human glioblastoma cells are associated with modulation of cholesterol and fatty acid biosynthetic pathways.

Glioblastoma (GBM) is the most common primary brain tumor. Genetic mutations may reprogram the metabolism of neoplastic cells. Particularly, alterations in cholesterol and fatty acid biosynthetic pathways may favor biomass synthesis and resistance to therapy. Therefore, compounds that interfere with those pathways, such as phytol (PHY) and retinol (RET), may be appropriate for cytotoxic approaches. We tested the effect of PHY or RET on the viability of human GBM cell lines (U87MG, A172 and T98G). Since the compounds showed a dose-dependent cytotoxic effect, additional analyses were performed with IC50 values. Transcriptome analyses of A172 cells treated with PHY IC50 or RET IC50 revealed down-regulated genes involved in cholesterol and/or fatty acid biosynthetic pathways. Thus, we investigated the expression of proteins required for cholesterol and/or fatty acid synthesis after treating all lineages with PHY IC50 or RET IC50 and comparing them with controls. Sterol regulatory element-binding protein 1 (SREBP-1) expression was reduced by PHY in U87 and T98G cells. However, fatty acid synthase (FAS) protein expression, which is regulated by SREBP-1, was down-regulated in all lineages after both treatments. Moreover, farnesyl-diphosphate farnesyltransferase (FDFT1) levels, a protein associated with cholesterol synthesis, were reduced in all lineages by PHY and in U87MG and A172 cells by RET. Our results suggest that SREBP-1, FAS and FDFT1 are potential target(s) for future in vivo approaches against GBM and support the use of inhibitors of their synthesis, including PHY and RET, for such approaches.

Evaluation of mitochondrial respiratory function in highly glycolytic glioma cells reveals low ADP phosphorylation in relation to oxidative capacity.

High-grade gliomas are aggressive and intensely glycolytic tumors. In the present study, we evaluated the mitochondrial respiratory function of glioma cells (T98G and U-87MG) and fresh human glioblastoma (GBM) tissue. To this end, measurements of oxygen consumption rate (OCR) were performed under various experimental conditions. The OCR of T98G and U-87MG cells was well coupled to ADP phosphorylation based on the ratio of ATP produced per oxygen consumed of ~2.5. In agreement, the basal OCR of GBM tissue was also partially associated with ADP phosphorylation. The basal respiration of intact T98G and U-87MG cells was not limited by the supply of endogenous substrates, as indicated by the increased OCR in response to a protonophore. These cells also displayed a high affinity for oxygen, as evidenced by the values of the partial pressure of oxygen when respiration is half maximal (p 50). In permeabilized glioma cells, ADP-stimulated OCR was only approximately 50% of that obtained in the presence of protonophore, revealing a significant limitation in oxidative phosphorylation (OXPHOS) relative to the activity of the electron transport system (ETS). This characteristic was maintained when the cells were grown under low glucose conditions. Flux control coefficient analyses demonstrated that the impaired OXPHOS was associated with the function of both mitochondrial ATP synthase and the adenine nucleotide translocator, but not the phosphate carrier. Altogether, these data indicate that the availability and metabolism of respiratory substrates and mitochondrial ETS are preserved in T98G and U-87MG glioma cells even though these cells possess a relatively restrained OXPHOS capability.

Sulfasalazine intensifies temozolomide cytotoxicity in human glioblastoma cells.

Temozolomide (TMZ) is an alkylating agent used to treat glioblastoma. This tumor type synthesizes the antioxidant glutathione through system X c (-) , which is inhibited by sulfasalazine (SAS). We exposed A172 and T98G human glioblastoma cells to a presumably clinically relevant concentration of TMZ (25 µM) and/or 0.5 mM SAS for 1, 3, or 5 days and assessed cell viability. For both cell lines, TMZ alone did not alter viability at any time point, while the coadministration of TMZ and SAS significantly reduced cell viability after 5 days. The drug combination exerted a synergistic effect on A172 cells after 3 and 5 days. Therefore, this particular lineage was subjected to complementary analyses on the genetic (transcriptome) and functional (glutathione and proliferating cell nuclear antigen (PCNA) protein) levels. Cellular pathways containing differentially expressed genes related to the cell cycle were modified by TMZ alone. On the other hand, SAS regulated pathways associated with glutathione metabolism and synthesis, irrespective of TMZ. Moreover, SAS, but not TMZ, depleted the total glutathione level. Compared with the vehicle-treated cells, the level of PCNA protein was lower in cells treated with TMZ alone or in combination with SAS. In conclusion, our data showed that the association of TMZ and SAS is cytotoxic to T98G and A172 cells, thus providing useful insights for improving TMZ clinical efficacy through testing this novel drug combination. Moreover, the present study not only reports original information on differential gene expression in glioblastoma cells exposed to TMZ and/or SAS but also describes an antiproliferative effect of TMZ, which has not yet been observed in A172 cells.

Intrasellar xanthogranuloma mimicking macroadenoma.

Intracranial xanthogranulomas (XGs) have been found at various sites, but xanthogranuloma of the sellar region is extremely rare. We report about a case of sellar XG in a 34-year-old female. Magnetic resonance imaging showed a solid-cystic mass located at the sella turcica. The cystic component was hyperintense on the T1-weighted image (WI) and T2WI. The solid component was hyperintense on T1WI and hypointense on T2WI. There was peripheral enhancement after gadolinium administration. The diagnosis of cystic macroadenoma was considered before surgery. Final diagnosis of XG was confirmed by histopathological examination after surgical resection. Gross total resection of the lesion was achieved using the microscope through endoscopic endonasal transsphenoidal approach. The patient had a good outcome and no symptom of diabetes insipidus, hormonal evaluation did not show any alterations compatible with hypopituitarism and prolactin levels were normal XG should receive diagnostic consideration for the sellar mass lesions with cystic components hyperintense on T1WI and T2WI, solid components hyperintense on T1WI and hypointense on T2WI, and CT without evidence of calcifications. It is important to consider the possibility of XG when pertinent, as it facilitates a proper surgical approach strategy.

A rare report of a metastatic lung large-cell neuroendocrine carcinoma in palatine tonsil.

INTRODUCTION: Neuroendocrine carcinoma is a rare form of cancer originating from neuroendocrine cells, with the lungs being the most common site of occurrence. These tumors have the potential to metastasize to the head and neck region. CASE REPORT: A 57-year-old man, with a smoking history of 74 pack-years, presented with complaints of hoarseness, dry cough, dysphagia, and significant weight loss over a two-month period. During oral examination, a submucosal nodule in the left palatine tonsil was discovered. Histological analysis confirmed a poorly differentiated tumor consisting of large cells with nuclear pleomorphism and abundant cytoplasm. The tumor tested positive for CD56, chromogranin, synaptophysin, and EMA. Further imaging revealed a substantial endobronchial lesion in the upper segment of the left lower lobe. Biopsy results from this lesion were morphologically and immunohistochemically consistent with those from the oral lesion. A diagnosis of metastatic large-cell neuroendocrine carcinoma originating from the lung and involving the oral mucosa was established. CONCLUSION: This case highlights the metastatic potential of pulmonary neuroendocrine carcinoma and its occurrence in atypical dissemination sites. Additionally, our findings underscore the importance of early detection of oral metastases to ensure accurate diagnosis and expedite appropriate treatment.

Identifying cellular markers of focal cortical dysplasia type II with cell-type deconvolution and single-cell signatures.

Focal cortical dysplasia (FCD) is a brain malformation that causes medically refractory epilepsy. FCD is classified into three categories based on structural and cellular abnormalities, with FCD type II being the most common and characterized by disrupted organization of the cortex and abnormal neuronal development. In this study, we employed cell-type deconvolution and single-cell signatures to analyze bulk RNA-seq from multiple transcriptomic studies, aiming to characterize the cellular composition of brain lesions in patients with FCD IIa and IIb subtypes. Our deconvolution analyses revealed specific cellular changes in FCD IIb, including neuronal loss and an increase in reactive astrocytes (astrogliosis) when compared to FCD IIa. Astrogliosis in FCD IIb was further supported by a gene signature analysis and histologically confirmed by glial fibrillary acidic protein (GFAP) immunostaining. Overall, our findings demonstrate that FCD II subtypes exhibit differential neuronal and glial compositions, with astrogliosis emerging as a hallmark of FCD IIb. These observations, validated in independent patient cohorts and confirmed using immunohistochemistry, offer novel insights into the involvement of glial cells in FCD type II pathophysiology and may contribute to the development of targeted therapies for this condition.

Transcriptome analyses of the cortex and white matter of focal cortical dysplasia type II: Insights into pathophysiology and tissue characterization.

Introduction: Focal cortical dysplasia (FCD) is a common cause of pharmacoresistant epilepsy. According to the 2022 International League Against Epilepsy classification, FCD type II is characterized by dysmorphic neurons (IIa and IIb) and may be associated with balloon cells (IIb). We present a multicentric study to evaluate the transcriptomes of the gray and white matters of surgical FCD type II specimens. We aimed to contribute to pathophysiology and tissue characterization. Methods: We investigated FCD II (a and b) and control samples by performing RNA-sequencing followed by immunohistochemical validation employing digital analyses. Results: We found 342 and 399 transcripts differentially expressed in the gray matter of IIa and IIb lesions compared to controls, respectively. Cholesterol biosynthesis was among the main enriched cellular pathways in both IIa and IIb gray matter. Particularly, the genes HMGCS1, HMGCR, and SQLE were upregulated in both type II groups. We also found 12 differentially expressed genes when comparing transcriptomes of IIa and IIb lesions. Only 1 transcript (MTRNR2L12) was significantly upregulated in FCD IIa. The white matter in IIa and IIb lesions showed 2 and 24 transcripts differentially expressed, respectively, compared to controls. No enriched cellular pathways were detected. GPNMB, not previously described in FCD samples, was upregulated in IIb compared to IIa and control groups. Upregulations of cholesterol biosynthesis enzymes and GPNMB genes in FCD groups were immunohistochemically validated. Such enzymes were mainly detected in both dysmorphic and normal neurons, whereas GPNMB was observed only in balloon cells. Discussion: Overall, our study contributed to identifying cortical enrichment of cholesterol biosynthesis in FCD type II, which may correspond to a neuroprotective response to seizures. Moreover, specific analyses in either the gray or the white matter revealed upregulations of MTRNR2L12 and GPNMB, which might be potential neuropathological biomarkers of a cortex chronically exposed to seizures and of balloon cells, respectively.