Cortical Tissue Samples Research Articles

The cell-type underpinnings of the human functional cortical connectome.

The functional properties of the human brain arise, in part, from the vast assortment of cell types that pattern the cerebral cortex. The cortical sheet can be broadly divided into distinct networks, which are embedded into processing streams, or gradients, that extend from unimodal systems through higher-order association territories. Here using microarray data from the Allen Human Brain Atlas and single-nucleus RNA-sequencing data from multiple cortical territories, we demonstrate that cell-type distributions are spatially coupled to the functional organization of cortex, as estimated through functional magnetic resonance imaging. Differentially enriched cells follow the spatial topography of both functional gradients and associated large-scale networks. Distinct cellular fingerprints were evident across networks, and a classifier trained on postmortem cell-type distributions was able to predict the functional network allegiance of cortical tissue samples. These data indicate that the in vivo organization of the cortical sheet is reflected in the spatial variability of its cellular composition.

The neocortical infrastructure for language involves region-specific patterns of laminar gene expression

The language network of the human brain has core components in the inferior frontal cortex and superior/middle temporal cortex, with left-hemisphere dominance in most people. Functional specialization and interconnectivity of these neocortical regions is likely to be reflected in their molecular and cellular profiles. Excitatory connections between cortical regions arise and innervate according to layer-specific patterns. Here, we generated a gene expression dataset from human postmortem cortical tissue samples from core language network regions, using spatial transcriptomics to discriminate gene expression across cortical layers. Integration of these data with existing single-cell expression data identified 56 genes that showed differences in laminar expression profiles between the frontal and temporal language cortex together with upregulation in layer II/III and/or layer V/VI excitatory neurons. Based on data from large-scale genome-wide screening in the population, DNA variants within these 56 genes showed set-level associations with interindividual variation in structural connectivity between the left-hemisphere frontal and temporal language cortex, and with the brain-related disorders dyslexia and schizophrenia which often involve affected language. These findings identify region-specific patterns of laminar gene expression as a feature of the brain's language network.

Low-level brain somatic mutations in exonic regions are collectively implicated in autism with germline mutations in autism risk genes

Low-level somatic mutations in the human brain are implicated in various neurological disorders. The contribution of low-level brain somatic mutations to autism spectrum disorder (ASD), however, remains poorly understood. Here, we performed high-depth exome sequencing with an average read depth of 559.3x in 181 cortical, cerebellar, and peripheral tissue samples to identify brain somatic single nucleotide variants (SNVs) in 24 ASD subjects and 31 controls. We detected ~2.4 brain somatic SNVs per exome per single brain region, with a variant allele frequency (VAF) as low as 0.3%. The mutational profiles, including the number, signature, and type, were not significantly different between the ASD patients and controls. Intriguingly, when considering genes with low-level brain somatic SNVs and ASD risk genes with damaging germline SNVs together, the merged set of genes carrying either somatic or germline SNVs in ASD patients was significantly involved in ASD-associated pathophysiology, including dendrite spine morphogenesis (p = 0.025), mental retardation (p = 0.012), and intrauterine growth retardation (p = 0.012). Additionally, the merged gene set showed ASD-associated spatiotemporal expression in the early and mid-fetal cortex, striatum, and thalamus (all p < 0.05). Patients with damaging mutations in the merged gene set had a greater ASD risk than did controls (odds ratio = 3.92, p = 0.025, 95% confidence interval = 1.12–14.79). The findings of this study suggest that brain somatic SNVs and germline SNVs may collectively contribute to ASD-associated pathophysiology.

Ferritin is closely associated with microglia in amyotrophic lateral sclerosis.

Iron deposition is a hallmark of amyotrophic lateral sclerosis (ALS) and has been strongly implicated in its pathogenesis. As a byproduct of cellular oxidative stress, iron dysregulation modifies basal levels of the regulatory iron-binding protein ferritin. Examination of thoracic and lumbar spinal cord tissues found increased ferritin immunostaining in white matter axons that corresponded to areas of increased microgliosis in 8 ALS patients versus 8 normal subjects. Gray matter areas containing the motor neurons also demonstrated increased ferritin and microglia in ALS compared to controls but at lower levels than in the white matter. Motor neurons with or without TDP-43 inclusions did not demonstrate either increased ferritin or associated microglial activation. We also observed an association of ferritin with microglia in cerebral cortical tissue samples of ALS cases and in the spinal cord tissues of transgenic mice expressing the SOD1G93A mutation. Elevated ferritin levels were detected in the insoluble fraction from spinal cord tissues of individuals with ALS. These findings suggest that activated microglia and increased ferritin may play significant roles in ALS progression since they are found closely associated in areas of axonal and cortical degeneration.

Reprogramming of Energy Metabolism in Human PKD1 Polycystic Kidney Disease: A Systems Biology Analysis.

Multiple alterations of cellular metabolism have been documented in experimental studies of autosomal dominant polycystic kidney disease (ADPKD) and are thought to contribute to its pathogenesis. To elucidate the molecular pathways and transcriptional regulators associated with the metabolic changes of renal cysts in ADPKD, we compared global gene expression data from human PKD1 renal cysts, minimally cystic tissues (MCT) from the same patients, and healthy human kidney cortical tissue samples. We found gene expression profiles of PKD1 renal cysts were consistent with the Warburg effect with gene pathway changes favoring increased cellular glucose uptake and lactate production, instead of pyruvate oxidation. Additionally, mitochondrial energy metabolism was globally depressed, associated with downregulation of gene pathways related to fatty acid oxidation (FAO), branched-chain amino acid (BCAA) degradation, the Krebs cycle, and oxidative phosphorylation (OXPHOS) in renal cysts. Activation of mTORC1 and its two target proto-oncogenes, HIF-1α and MYC, was predicted to drive the expression of multiple genes involved in the observed metabolic reprogramming (e.g., GLUT3, HK1/HK2, ALDOA, ENO2, PKM, LDHA/LDHB, MCT4, PDHA1, PDK1/3, MPC1/2, CPT2, BCAT1, NAMPT); indeed, their predicted expression patterns were confirmed by our data. Conversely, we found AMPK inhibition was predicted in renal cysts. AMPK inhibition was associated with decreased expression of PGC-1α, a transcriptional coactivator for transcription factors PPARα, ERRα, and ERRγ, all of which play a critical role in regulating oxidative metabolism and mitochondrial biogenesis. These data provide a comprehensive map of metabolic pathway reprogramming in ADPKD and highlight nodes of regulation that may serve as targets for therapeutic intervention.

Maximum likelihood estimation of renal transporter ontogeny profiles for pediatric PBPK modeling.

Optimal treatment of infants with many renally cleared drugs must account for maturational differences in renal transporter (RT) activity. Pediatric physiologically-based pharmacokinetic (PBPK) models may incorporate RT activity, but this requires ontogeny profiles for RT activity in children, especially neonates, to predict drug disposition. Therefore, RT expression measurements from human kidney postmortem cortical tissue samples were normalized to represent a fraction of mature RT activity. Using these data, maximum likelihood estimated the distributions of RT activity across the pediatric age spectrum, including preterm and term neonates. PBPK models of four RT substrates (acyclovir, ciprofloxacin, furosemide, and meropenem) were evaluated with and without ontogeny profiles using average fold error (AFE), absolute average fold error (AAFE), and proportion of observations within the 5-95% prediction interval. Novel maximum likelihood profiles estimated ontogeny distributions for the following RT: OAT1, OAT3, OCT2, P-gp, URAT1, BCRP, MATE1, MRP2, MRP4, and MATE-2 K. Profiles for OAT3, P-gp, and MATE1 improved infant furosemide and neonate meropenem PBPK model AFE from 0.08 to 0.70 and 0.53 to 1.34 and model AAFE from 12.08 to 1.44 and 2.09 to 1.36, respectively, and improved the percent of data within the 5-95% prediction interval from 48% to 98% for neonatal ciprofloxacin simulations, respectively. Even after accounting for other critical population-specific maturational differences, novel RT ontogeny profiles substantially improved neonatal PBPK model performance, providing validated estimates of maturational differences in RT activity for optimal dosing in children.

Clustering Schizophrenia Genes by Their Temporal Expression Patterns Aids Functional Interpretation.

Schizophrenia is a highly heritable brain disorder with a typical symptom onset in early adulthood. The 2-hit hypothesis posits that schizophrenia results from differential early neurodevelopment, predisposing an individual, followed by a disruption of later brain maturational processes that trigger the onset of symptoms. We applied hierarchical clustering to transcription levels of 345 genes previously linked to schizophrenia, derived from cortical tissue samples from 56 donors across the lifespan. We subsequently calculated clustered-specific polygenic risk scores for 743 individuals with schizophrenia and 743 sex- and age-matched healthy controls. Clustering revealed a set of 183 genes that was significantly upregulated prenatally and downregulated postnatally and 162 genes that showed the opposite pattern. The prenatally upregulated set of genes was functionally annotated to fundamental cell cycle processes, while the postnatally upregulated set was associated with the immune system and neuronal communication. We found an interaction between the 2 scores; higher prenatal polygenic risk showed a stronger association with schizophrenia diagnosis at higher levels of postnatal polygenic risk. Importantly, this finding was replicated in an independent clinical cohort of 3233 individuals. We provide genetics-based evidence that schizophrenia is shaped by disruptions of separable biological processes acting at distinct phases of neurodevelopment. The modeling of genetic risk factors that moderate each other's effect, informed by the timing of their expression, will aid in a better understanding of the development of schizophrenia.

Intracellular Iron Accumulation Induces Inflammatory and Oxidative Status of the Host After Japanese Encephalitis Viral Infection.

The factors mitigating the microglia/macrophage activation and inflammatory damage in Japanese encephalitis (JE)virus infected CNS are still being ascertained. We aim to characterize the changes in iron transporter and iron storage proteins along with inflammatory and oxidative stress-mediated signaling during the JE viral infection. Cortical tissue samples from mice with JE viral infection were processed for biochemical, histological, and molecular analysis. Iron storage protein, i.e., ferritin, was found significantly increased post-JE viral infection, and iron accumulation was noted in cortical tissue. Key proinflammatory associated markers, such as TNF-α, IL-6, and its regulator TLR4, were found tobe increased, while SOCS1 (anti-inflammatory regulator) transcription decreased with increased levels of oxidative stress markers NOX2-mediated NF-ΚB/p65 and protein carbonyl. Furthermore, it is noted that hepcidin level increased and ferroportin level decreased, and iron transporter gene expression got imbalanced after JE viral infection. This observation was further confirmed by deferoxamine (DFO) treatment to JE viral infection mice model, where the decline in hepcidin transcription level and iron load in cortical tissue of JEviralinfected animals was noted. However, no change was found in the ferroportin level compared to JEviralinfected animals. Together, these findings suggest that iron overload and hepcidin-ferroportin regulation are involved in JE viral infection disease pathologies and associated with the inflammatory and oxidative status of the host during infection.

Global Energy Metabolism Deficit in Alzheimer Disease Brain

BackgroundThe understanding of Alzheimer’s disease (AD) has been dominated by the amyloid hypothesis. However, therapies targeting beta-amyloid have largely failed, generating interest in other potential pathogenic factors including energy metabolism.ObjectivesTo interrogate canonical energy metabolism pathways from human prefrontal cortical tissue samples obtained from necropsy comparing AD and control.Design, Setting, and ParticipantsPostmortem pre-frontal cortical tissue from 10 subjects histologically diagnosed with AD and 10 control (CTRL) subjects was subjected to untargeted metabolomics to interrogate energy metabolism pathways. The samples were matched by age, sex, and postmortem interval.Metabolite MeasurementsUntargeted metabolomics analyses were via Metabolon®.ResultsGlucose-derived energy metabolites in the glycolytic and pentose phosphate pathway and the ketone body β-hydroxybutyrate were uniformly decreased in AD brain vs. CTRL brain.ConclusionThis pilot study aimed to identify energy metabolism abnormalities using untargeted brain metabolomics in two independent subject cohorts. Our study revealed a pattern of global energy deficit in AD brain, supporting a growing body of evidence of deficient energy metabolism in AD.

The effects of excess salt intake on the kidney metabolism in normal Sprague Dawley rats.

Introduction: This study aimed to determine the effects of a high salt diet upon the metabolism of O 2 and substances in the kidney in normal Sprague Dawley (SD) rats. Methods were developed enabling the intermittent collection of renal arteriovenous (A-Vr) blood and urine samples and to measure renal blood flow (RBF) and arterial pressure (BP) in conscious freely moving rats with GFR determined in a separate group of conscious rats. Other groups were studied to obtain cortical (Cx) and outer medullary (OM) tissue samples for associated transcriptomic and metabolomic profiles. Methods: SD rats were instrumented with a renal arterial ultrasonic flow probe, a femoral arterial and renal venous catheters, and RBF and BP measured continuously (24/7). Arterial (A) and renal venous (Vr) blood and urine were collected repeatedly before and 7, 14, and 21 days after increasing the salt diet from 0.4% (LS) to 4% (HS) NaCl. GFR was measured in age-matched rats and used as control shams for normalization. Blood O 2 content was determined by radiometry. A global metabolomic analysis was performed on plasma and Cx and OM tissue samples at JAX laboratories (Thermo Q-Exactive Orbitrap coupled to Vanquich UPLC system) and together with mRNAseq analysis for Cx and OM by Novogene Inc. Results: BP increased less than 5 mmHg by day 3 of the HS diet while RBF rose nearly 20% (P&lt;0.05) and both were sustained at this level throughout the study. O 2 delivery, O 2 consumption (VO 2 ) and O 2 extraction rose over the 21 days of the HS diet (P&lt;0.05). Total GFR increased from 0.64 to 0.83 mL/min/100 g bw (P&lt;0.05) at HS 7 and remained elevated at HS 21. Whole kidney Na + reabsorption and kidney VO 2 were highly correlated positively (r=0.99). A sparse partial least squares discriminant analysis (sPLS-DA) of metabolomics for each of the 4 modes (C18 +/- and HILIC +/-) revealed clear distinctions between LS, HS14, and HS21 within Cx and OM. The mRNAseq tissue analysis found that the HS diet resulted in reduced expression of genes related to the TCA cycle and metabolism of amino acids and fatty acids. This was consistent with the metabolomic data showing a reduction of tissue citrate and succinate levels. In contrast, glycolysis-related genes including hexokinase, pyruvate kinase and lactate dehydrogenase were upregulated in the Cx tissue by HS. Minimal changes in metabolic pathways were observed in the OM tissue. A-Vr x RBF enabled metabolomic flux determination which showed the release of glucose on LS fed rats which was not evident with the HS diet. Uptake of citrate which was seen on LS was less evident on HS but there was a clear tendency observed for increased release of lactate, pyruvate, and α-ketoglutarate. Conclusions: Normal SD rats fed a HS underwent changes of RBF, O 2 extraction and utilization of metabolic substrates, most notably transitioning ATP production predominantly driven from TCA cycle to non-TCA cycle pathways which we are currently exploring more deeply. HL-116264, HL-151587 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dominance status is associated with a variation in cannabinoid receptor 1 expression and amphetamine reward

The rewarding effects of psychostimulants appear to be distinct between dominant and subordinate individuals. In turn, the endocannabinoid system is an important modulator of drug reward in the nucleus accumbens and medial prefrontal cortex, however the connection with social dominance is yet to be established. Male rats were classified as dominant or subordinate on the basis of their spontaneous agonistic interactions and drug reward was assessed by means of conditioned place preference with amphetamine (AMPH). In addition, the expression of CB1R, CB2R, FAAH1, and DAGLa was quantified from accumbal and cortical tissue samples. Our findings demonstrate that dominant rats required a lesser dose of AMPH to acquire a preference for the drug-associated compartment, thereby suggesting a higher sensitivity to the rewarding effects of AMPH. Furthermore, dominants exhibited a lower expression of CB1R in the medial prefrontal cortex and nucleus accumbens. This study illustrates how CBR1 expression could differentiate the behavioral phenotypes associated to social dominance.

Single-cell dissection of the human brain vasculature.

Despite the importance of the cerebrovasculature in maintaining normal brain physiology and in understanding neurodegeneration and drug delivery to the central nervous system1, human cerebrovascular cells remain poorly characterized owing to their sparsity and dispersion. Here we perform single-cell characterization of the human cerebrovasculature using both ex vivo fresh tissue experimental enrichment and post mortem in silico sorting of human cortical tissue samples. We capture 16,681 cerebrovascular nuclei across 11 subtypes, including endothelial cells, mural cells and three distinct subtypes of perivascular fibroblast along the vasculature. We uncover human-specific expression patterns along the arteriovenous axis and determine previously uncharacterized cell-type-specific markers. We use these human-specific signatures to study changes in 3,945 cerebrovascular cells from patients with Huntington's disease, which reveal activation of innate immune signalling in vascular and glial cell types and a concomitant reduction in the levels of proteins critical for maintenance of blood-brain barrier integrity. Finally, our study provides a comprehensive molecular atlas of the human cerebrovasculature to guide future biological and therapeutic studies.

Histological and Immunohistochemical Assessment of Ovarian Tissue and Endometrium in Patients with Diminished Ovarian Reserve

<h3>Study Objective</h3> To analyze the histological and immunohistochemical features of ovarian tissue and endometrium in women with diminished ovarian reserve and to investigate the correlation between these parameters and the effectiveness of infertility treatment <h3>Design</h3> Prospective study, Level II. <h3>Setting</h3> National Medical Research Center. <h3>Patients or Participants</h3> 70 patients with diminished ovarian reserve who underwent drug-free in vitro activation using a modified surgical technique. Two bilateral ovarian cortical tissue samples and one endometrial sample were obtained from each patient during the surgery. 40 patients out of 70 underwent ovarian stimulation after at least a one-month postoperative period. <h3>Interventions</h3> Bilateral ovarian cortex biopsy via laparoscopy, ovarian tissue fragmentation in aliquots of 1 mm², and hysteroscopy with endometrial biopsy were performed in all patients, followed by histological and immunohistochemical analysis (expression of proteins of the Hippo signaling pathway (YAP, MST-1, LATS-1) and PTEN (inhibitor of PI3K signaling pathway). The Human Protein Atlas datasets were used as a comparison group. <h3>Measurements and Main Results</h3> We revealed an impaired functioning of intracellular signaling pathways (primarily YAP- and PTEN-dependent) both in ovarian tissue and endometrial samples. Moreover, the expression of the proteins targeted in the study was significantly reduced in patients who responded to stimulation (n=31). Histological analysis of tissue samples of patients with diminished ovarian reserve indicated profound changes in various structures of the ovaries: surface epithelium, vascular component, stroma of ovarian cortex. In 25% of patients, chronic endometritis was also determined. <h3>Conclusion</h3> Specific histological and immunohistochemical features of ovarian tissue and endometrium associated with diminished ovarian reserve and the outcome of ovarian stimulation were shown.

Does duration of HIV infection substitute for age as a risk factor for amyloid deposition?

Increasing access to more tolerable ARV treatments has vastly extended the expected lifespan of People with HIV (PWH), with some models predicting that in developed nations approximately two-thirds of PWH will be older than 50 years by 2030. Longer lifespans increase the risk of age-related neuropathologies, such as the deposition of amyloid beta plaques (Aβ42), regarded as an important prognostic marker of Alzheimer’s Disease (AD). Frequently first detected during mid-life, Aβ42 may predate the manifestation of neurocognitive symptoms associated with AD by 1-2 decades. The etiological significance of Aβ42 deposition in PWH is an active area of investigation. There is little clarity thus far in the clinical and research literature on whether the presence of Aβ42 in PWH leads to elevated risk of developing AD in those with and without HIV Associated Neurocognitive Disorders (HAND). Biological measures of neuropathology, such as CSF serum assays and brain tissue staining, have great potential in determining the extent to which Aβ42 deposition patterns in PWH are HAND-specific, and in linking these deposits to clinical outcomes. In this way, they might provide insight into otherwise seemingly paradoxical findings, such as the observation that neither age nor HIV clinical variables (including ARV status and duration of HIV infection) were able to explain the roughly 5 times greater incidence of mild cognitive impairment (MCI) over a period of 14 months in neurocognitively asymptomatic PWH compared to seronegative controls. In the study by Morgello and colleagues in this issue of AIDS, the authors examined hippocampal and frontal cortical tissue samples from a large cohort of 197 PWH and 63 HIV seronegative controls who donated brain tissue to the Manhattan HIV Brain Bank. Using this approach, they were able to quantify the association of HIV status and the presence of amyloid deposits in brain regions that are typically affected in AD. Despite the study being relatively well-powered, they were unable to identify between- 3 group differences in the occurrence of Aβ42 deposits. Rather, within the PWH cohort, duration of HIV infection, and, somewhat paradoxically, undetectable HIV viral load, emerged as significant predictors of the likelihood of detecting Aβ42.

Persistent organic pollutants and the size of ovarian reserve in reproductive-aged women

Industrial chemicals such as persistent organic pollutants (POPs) have been associated with reduced fertility in women, including longer time-to-pregnancy (TTP), higher odds for infertility, and earlier reproductive senescence. Fertility is highly dependent on the ovarian reserve, which is composed of a prenatally determined stock of non-growing follicles. The quantity and quality of the follicles decline with age, thereby eventually leading to menopause. In the clinical setting, assessing ovarian reserve directly through the histological analysis of follicular density in ovaries is not practical. Therefore, surrogate markers of ovarian reserve, such as serum anti-Müllerian hormone (AMH) are typically used. Here, we studied associations between chemical exposure and ovarian reserve in a cohort of pregnant women undergoing elective caesarean section (n = 145) in Stockholm, Sweden. Full data (histological, clinical, serum) were available for 50 women. We estimated the size of the reserve both directly by determining the density of follicles in ovarian cortical tissue samples, and indirectly by measuring AMH in associated serum samples. Concentrations of 9 organochlorine pesticides (OCPs), 10 polychlorinated biphenyls (PCBs), 3 polybrominated diphenylethers (PBDEs) and 9 perfluoroalkyl substances (PFAS) were determined in serum, and clinical data were retrieved from electronic medical records. Healthy follicle densities (median 0, range 0–193 follicles/mm3) and AMH levels (median 2.33 ng/mL, range 0.1–14.8 ng/mL) varied substantially. AMH correlated with the density of growing follicles. Twenty-three chemicals detected in more than half of the samples were included in the analyses. None of the chemicals, alone or as a mixture, correlated with AMH, growing or atretic follicles. However, HCB, transnonachlor, PCBs 74 and 99 were associated with decreased non-growing follicle densities. HCB and transnonachlor were also negatively associated with healthy follicle density. Further, mixture of lipophilic POPs (PBDE 99, p,p’-DDE, and PCB 187) was associated with lower non-growing follicle densities. In addition, exposure to HCB, p,p’-DDE, and mixture of OCPs were significantly associated with higher odds of infertility. The results suggest that exposure to chemicals may reduce the size of ovarian reserve in humans, and strongly encourage to study mechanisms behind POP-associated infertility in women in more detail.

Paramagnetic Centers of Mineral Component in Annealed Bone Tissue

The mineral components of powdered samples of cortical dense bone tissue of domestic animals (pig and cow) were studied by electron paramagnetic resonance (EPR). Bone tissue was heated in a temperature range 600-1000 ºС in an oven and then held for more than one year at room temperature. This was done to allow short-lived defects to decay and to let the mineral structures stabilize. In these heat-treated samples, within the limits of the sensitivity of the experiments, no EPR signals were detected, but after irradiation with X-rays, multicomponent EPR signals appeared. Spectra vary depending on the heating temperatures. Paramagnetic centers related to РО32–, NO42–, CO2–, CO33– and O– groups are observed. The g-factor of spectroscopic splitting and amplitude of ultrafine interaction of the indicated paramagnetic centers in the spectra were determined. Due to different relaxation behavior of the paramagnetic centers, and hence their saturation effects, the form of the total EPR signals significantly depends on the microwave power level at which the spectra are recorded. Therefore, EPR signals were recorded at high (5 mW) and low (0.13 mW) microwave power levels. The temperature dependency of EPR signal amplitude was determined for some paramagnetic centers in the range of 600-1000 ºС. The EPR signal properties of the heated bone samples and synthetic hydroxylapatites were compared. The spectra indicate that phosphates in bone tissue have a more complex structure than simple synthetic hydroxylapatite. The results of this work can be used to create synthetic analogs of bone tissue, to help in the manufacturing of implants that are used to treat bone tissue, and to study the processes related to the assimilation of mineralogic-based implants by living bone tissue.

Co-expression of cancer driver genes: IDH-wildtype glioblastoma-derived tumorspheres

BackgroundDriver genes of GBM may be crucial for the onset of isocitrate dehydrogenase (IDH)-wildtype (WT) glioblastoma (GBM). However, it is still unknown whether the genes are expressed in the identical cluster of cells. Here, we have examined the gene expression patterns of GBM tissues and patient-derived tumorspheres (TSs) and aimed to find a progression-related gene.MethodsWe retrospectively collected primary IDH-WT GBM tissue samples (n = 58) and tumor-free cortical tissue samples (control, n = 20). TSs are isolated from the IDH-WT GBM tissue with B27 neurobasal medium. Associations among the driver genes were explored in the bulk tissue, bulk cell, and a single cell RNAsequencing techniques (scRNAseq) considering the alteration status of TP53, PTEN, EGFR, and TERT promoter as well as MGMT promoter methylation. Transcriptomic perturbation by temozolomide (TMZ) was examined in the two TSs.ResultsWe comprehensively compared the gene expression of the known driver genes as well as MGMT, PTPRZ1, or IDH1. Bulk RNAseq databases of the primary GBM tissue revealed a significant association between TERT and TP53 (p < 0.001, R = 0.28) and its association increased in the recurrent tumor (p < 0.001, R = 0.86). TSs reflected the tissue-level patterns of association between the two genes (p < 0.01, R = 0.59, n = 20). A scRNAseq data of a TS revealed the TERT and TP53 expressing cells are in a same single cell cluster. The driver-enriched cluster dominantly expressed the glioma-associated long noncoding RNAs. Most of the driver-associated genes were downregulated after TMZ except IGFBP5.ConclusionsGBM tissue level expression patterns of EGFR, TERT, PTEN, IDH1, PTPRZ1, and MGMT are observed in the GBM TSs. The driver gene-associated cluster of the GBM single cells were enriched with the glioma-associated long noncoding RNAs.

Deep multilayer brain proteomics identifies molecular networks and Netrin‐1 accumulation in Alzheimer’s disease progression

AbstractBackgroundAlzheimer’s disease (AD) displays a long asymptomatic stage prior to development of dementia. Identification of molecular alterations during AD progression would provide insight into temporal alterations associated with AD.MethodAD stage‐associated molecular networks were characterized by mass spectrometry in 90 frontal cortical tissue samples in five groups, including controls with low/high amyloid plaque and tau tangle pathology (LPC and HPC, respectively), cases with high Aβ pathology and slight mild cognitive impairment (MCI), late‐stage AD with high plaque/tangle pathology, and samples with progressive supranuclear palsy (PSP). We additionally performed whole proteome comparisons between human AD and the 5XFAD and Tau P301S mouse models. Protein targets implicated as altered by AD progression in the proteomic analyses were validated by affinity assays and immunostaining in human tissue and mouse models.ResultsAD stage‐associated molecular networks were characterized by profiling 14,513 proteins and 34,173 phosphosites in human brain with mass spectrometry, highlighting 173 protein alterations in 17 pathways. In total 16,128 proteins in 5xFAD and wild‐type mouse cortex were profiled, and share 97, 89, and 169 protein alterations with HPC, MCI, and AD, respectively, whereas Tau P301S mice share less than 15 protein alterations with each group. Neither mouse model shares more than 15 non‐tau phosphopeptide alterations with each AD sub‐group, although Tau P301S shares up to 78 phospho‐Tau peptides with human AD. During validation of proteins implicated in the proteome characterization, Netrin‐1 levels were found to increase in AD stage progression by immunoblotting but was not augmented in any other neurodegenerative disease cases tested. Additionally, affinity assays demonstrated a direct Aβ‐Netrin‐1 interaction, and immunostaining demonstrated Netrin‐1 and amyloid plaque colocalization in human AD and 5X‐FAD brain tissues, but not in Tau P301S.ConclusionOur proteomic analyses reveal altered proteins in AD and related mouse models. Human‐mouse comparisons suggest 5xFAD mice resemble symptomatic AD and that Tau P301S mice reproduce only AD‐related Tau phosphorylation. Netrin‐1 accumulates in AD, shows direct Aβ binding, and colocalizes with amyloid plaques in human AD and mouse 5XFAD brain tissue, and may be vital to AD pathogenesis. These results provide novel insights into AD‐associated proteins and pathways.

Glycyrrhizin Blocks the Detrimental Effects of HMGB1 on Cortical Neurogenesis After Traumatic Neuronal Injury.

Despite medical advances, neurological recovery after severe traumatic brain injury (TBI) remains poor. Elevated levels of high mobility group box protein-1 (HMGB1) are associated with poor outcomes; likely via interaction with receptors for advanced-glycation-end-products (RAGE). We examined the hypothesis that HMGB1 post-TBI is anti-neurogenic and whether this is pharmacologically reversible. Post-natal rat cortical mixed neuro-glial cell cultures were subjected to needle-scratch injury and examined for HMGB1-activation/neuroinflammation. HMGB1-related genes/networks were examined using genome-wide RNA-seq studies in cortical perilesional tissue samples from adult mice. Post-natal rat cortical neural stem/progenitor cell cultures were generated to quantify effects of injury-condition medium (ICM) on neurogenesis with/without RAGE antagonist glycyrrhizin. Needle-injury upregulated TNF-α/NOS-2 mRNA-expressions at 6 h, increased proportions of activated microglia, and caused neuronal loss at 24 h. Transcriptome analysis revealed activation of HMGB1 pathway genes/canonical pathways in vivo at 24 h. A 50% increase in HMGB1 protein expression, and nuclear-to-cytoplasmic translocation of HMGB1 in neurons and microglia at 24 h post-injury was demonstrated in vitro. ICM reduced total numbers/proportions of neuronal cells, but reversed by 0.5 μM glycyrrhizin. HMGB1 is activated following in vivo post mechanical injury, and glycyrrhizin alleviates detrimental effects of ICM on cortical neurogenesis. Our findings highlight glycyrrhizin as a potential therapeutic agent post-TBI.

Comparative Enhancement of Motor Function and BDNF Expression Following Different Brain Stimulation Approaches in an Animal Model of Ischemic Stroke

Background Combinatory intervention such as high-frequency (50-100 Hz) excitatory cortical stimulation (ECS) given concurrently with motor rehabilitative training (RT) improves forelimb function, except in severely impaired animals after stroke. Clinical studies suggest that low-frequency (≤1 Hz) inhibitory cortical stimulation (ICS) may provide an alternative approach to enhance recovery. Currently, the molecular mediators of CS-induced behavioral effects are unknown. Brain-derived neurotrophic factor (BDNF) has been associated with improved recovery and neural remodeling after stroke and thus may be involved in CS-induced behavioral recovery. Objective To investigate whether inhibitory stimulation during RT improves functional recovery of severely impaired rats, following focal cortical ischemia and if this recovery alters BDNF expression (study 1) and depends on BDNF binding to TrkB receptors (study 2). Methods Rats underwent ECS + RT, ICS + RT, or noCS + RT treatment daily for 3 weeks following a unilateral ischemic lesion to the motor cortex. Electrode placement for stimulation was either placed ipsilateral (ECS) or contralateral (ICS) to the lesion. After treatment, BDNF expression was measured in cortical tissue samples (study 1). In study 2, the TrkB inhibitor, ANA-12, was injected prior to treatment daily for 21 days. Results ICS + RT treatment significantly improved impaired forelimb recovery compared with ECS + RT and noCS + RT treatment. Conclusion ICS given concurrently with rehabilitation improves motor recovery in severely impaired animals, and alters cortical BDNF expression; nevertheless, ICS-mediated improvements are not dependent on BDNF binding to TrkB. Conversely, inhibition of TrkB receptors does disrupt motor recovery in ECS + RT treated animals.