Temporal Cortex Tissue Research Articles

Global analysis of synapse‐enriched miRNA in Alzheimer’s disease temporal cortex post‐mortem

AbstractBackgroundSynapse loss is the major neuropathologic correlate of cognitive impairment in Alzheimer’s disease (AD). MicroRNA (miRNA) regulate synaptic plasticity and play a role in AD pathogenesis and therefore represent potential therapeutic targets. The aim of this study is to determine the role of miRNA‐mediated dysfunction at AD synapses.MethodWe prepared homogenates (H) and synaptosomes (SYN) from frozen post‐mortem temporal cortex tissue from 10 AD (mean age = 77.4±4.2) and 10 non‐pathological controls (CN; mean age = 72.3±5.9). We extracted (mirVana) and quantified miRNA using the TaqManR Advanced miRNA Assay, QuantstudioTM and OpenarrayTM system. Samples were normalized (median Ct). DeltaCt values calculated relative to control hsa‐miR‐16‐5p. We performed linear regression to identify differentially expressed (DE) miRNA across groups, controlling the false discovery rate (FDR) using the Benjamini‐Hochberg method. We considered DE miRNA with fold changes <0.8 or >1.2 (adj.p<0.05) in AD vs CN. We searched two predictive databases (TargetScan and miRDB) for gene targets of the DE miRNA and identified overrepresented target genes using the Panther database (PantherDB.org) after controlling the FDR.ResultOf the 751 miRNA included on the Openarray, we detected 411 in H and 432 in SYN, with 89% overlap between AD and CN in SYN. To enrich our analyses for miRNA that are predominantly present at the synapse, we took forward only the 47 miRNA with >1.2‐foldchange (adj.p<0.2) in SYN vs H in either AD or CN. miR‐132‐3p and miR‐132‐5p were under‐expressed (0.17‐fold, p = 0.001 and 0.35‐fold, p = 0.006, respectively) and miR‐181a‐3p was over‐expressed (2.13‐fold, p = 0.04) in AD compared to CN SYN. The 594 gene targets of upregulated synaptic miRNA (miR‐181a‐3p) were overrepresented in pathways regulating synaptic function and mitochondrial bioenergetics and integrity. The 474 and 1230 targets of downregulated synaptic miRNA (miR‐132‐3p and miR‐132‐5p, respectively) were overrepresented in the same pathways as well as pathways related to interleukin production, tau pathology, Abeta production, apoptotic signalling and oxidative stress.ConclusionThree synapse‐specific miRNA are differentially expressed at AD synapses and target genes involved in core AD‐related processes, thus making them promising therapeutic targets. Future studies will seek to validate these findings and determine the specificity for AD over other neurodegenerative diseases.

Gliovascular molecular alterations in Alzheimer’s disease: a cross‐tissue, cross‐species study

AbstractBackgroundInter‐cellular communication within the gliovascular unit (GVU) is critical for cerebral blood flow regulation, and maintenance of the blood‐brain‐barrier (BBB) properties. The breakdown of BBB in Alzheimer’s disease (AD) is well‐established, but precise underlying molecular changes remain unclear. Additionally, whether GVU molecular alterations observed in AD brains are also detected in blood from living patients is unknown. Further, these GVU molecular perturbations require further investigation in different model systems to identify both human brain‐specific and cross‐species conserved alterations. In this study, we investigated prioritized GVU molecules altered in AD brains for their conservation in blood and cross‐species model systems.MethodsWe performed single nucleus RNA sequencing (snRNAseq) of temporal cortex tissue in AD and control brains. We analyzed this data to detect cell‐specific GVU molecular perturbations and their interactions. We investigated molecular interactions between vascular and astrocyte clusters, the major cell types of the GVU of the BBB. To determine whether GVU transcriptional alterations detected in the brain are preserved in the blood, existing blood expression, genetic, and neuroimaging data from two longitudinal antemortem cohorts were analyzed. Using model systems, including mouse, drosophila, and zebrafish, we evaluated the cross‐species conservation of the top GVU alterations detected in AD brains.ResultsBrain snRNAseq revealed transcriptional profiles of 6,541 astrocytes and 2,210 vascular cells. The latter formed three distinct vascular clusters characterized as pericytes, endothelia and perivascular fibroblasts. We identified differentially expressed genes and their enriched pathways within these clusters and observed the highest levels of transcriptional changes within pericytes. Vascular targets that interact with astrocytic ligands have biological functions in cell signaling, angiogenesis, amyloid ß metabolism, and cytoskeletal architecture. We discovered that genetic variants influencing blood expression levels of some of the prioritized GVU genes were associated with neuroimaging burden of cerebrovascular disease in living human cohorts. Our ongoing studies in model systems revealed conservation of some of the top prioritized molecular perturbations across species.ConclusionOur findings prioritized by multiscale, cross‐tissue human data revealed GVU perturbations within interacting pericyte and astrocyte molecules, which are conserved across multiple cross‐species models. These results nominate new molecular targets and mechanistic insights for BBB disruptions in AD.

Antisense oligonucleotide-based targeting of Tau-tubulin kinase 1 prevents hippocampal accumulation of phosphorylated tau in PS19 tauopathy mice

Tau tubulin kinase-1 (TTBK1), a neuron-specific tau kinase, is highly expressed in the entorhinal cortex and hippocampal regions, where early tau pathology evolves in Alzheimer’s disease (AD). The protein expression level of TTBK1 is elevated in the cortex brain tissues with AD patients compared to the control subjects. We therefore hypothesized that antisense oligonucleotide (ASO) based targeting Ttbk1 could prevent the accumulation of phosphorylated tau, thereby delaying the development of tau pathology in AD. Here we show that in vivo administration of ASO targeting mouse Ttbk1 (ASO-Ttbk1) specifically suppressed the expression of Ttbk1 without affecting Ttbk2 expression in the temporal cortex of PS19 tau transgenic mice. Central administration of ASO-Ttbk1 in PS19 mice significantly reduced the expression level of representative phosphor-tau epitopes relevant to AD at 8 weeks post-dose, including pT231, pT181, and pS396 in the sarkosyl soluble and insoluble fractions isolated from hippocampal tissues as determined by ELISA and pS422 in soluble fractions as determined by western blotting. Immunofluorescence demonstrated that ASO-Ttbk1 significantly reduced pS422 phosphorylated tau intensity in mossy fibers region of the dentate gyrus in PS19 mice. RNA-sequence analysis of the temporal cortex tissue revealed significant enrichment of interferon-gamma and complement pathways and increased expression of antigen presenting molecules (Cd86, Cd74, and H2-Aa) in PS19 mice treated with ASO-Ttbk1, suggesting its potential effect on microglial phenotype although neurotoxic effect was absent. These data suggest that TTBK1 is an attractive therapeutic target to suppress TTBK1 without compromising TTBK2 expression and pathological tau phosphorylation in the early stages of AD.

MicroRNAs as potential biomarkers in temporal lobe epilepsy and mesial temporal lobe epilepsy.

Temporal lobe epilepsy is the most common form of focal epilepsy in adults, accounting for one third of all diagnosed epileptic patients, with seizures originating from or involving mesial temporal structures such as the hippocampus, and many of these patients being refractory to treatment with anti-epileptic drugs. Temporal lobe epilepsy is the most common childhood neurological disorder and, compared with adults, the symptoms are greatly affected by age and brain development. Diagnosis of temporal lobe epilepsy relies on clinical examination, patient history, electroencephalographic recordings, and brain imaging. Misdiagnosis or delay in diagnosis is common. A molecular biomarker that could distinguish epilepsy from healthy subjects and other neurological conditions would allow for an earlier and more accurate diagnosis and appropriate treatment to be initiated. Among possible biomarkers of pathological changes as well as potential therapeutic targets in the epileptic brain are microRNAs. Most of the recent studies had performed microRNA profiling in body fluids such as blood plasma and blood serum and brain tissues such as temporal cortex tissue and hippocampal tissue. A large number of microRNAs were dysregulated when compared to healthy controls and with some overlap between individual studies that could serve as potential biomarkers. For example, in adults with temporal lobe epilepsy, possible biomarkers are miR-199a-3p in blood plasma and miR-142-5p in blood plasma and blood serum. In adults with mesial temporal lobe epilepsy, possible biomarkers are miR-153 in blood plasma and miR-145-3p in blood serum. However, in many of the studies involving patients who receive one or several anti-epileptic drugs, the influence of these on microRNA expression in body fluids and brain tissues is largely unknown. Further studies are warranted with children with temporal lobe epilepsy and consideration should be given to utilizing mouse or rat and non-human primate models of temporal lobe epilepsy. The animal models could be used to confirm microRNA findings in human patients and to test the effects of targeting specific microRNAs on disease progression and behavior.

Single Cell Approaches Reveal Perturbed Brain Vascular Molecules in Alzheimer’s Disease

AbstractBackgroundInter‐cellular communication within the gliovascular unit (GVU) is critical for cerebral blood flow regulation, maintenance of the blood‐brain‐barrier (BBB) properties, and brain energy metabolism. Although numerous cell biology studies demonstrate the role of the GVU for BBB breakdown in Alzheimer’s disease (AD), the precise molecular changes contributing to its pathophysiology are unclear.MethodWe performed single nucleus RNA sequencing (snRNAseq) of temporal cortex tissue in 24 AD and control brains to understand the transcriptional changes in the cells of GVU. We acquired the snRNAseq profile of 79,751 total brain cells that comprise 6,541 astrocytes and 2,210 vascular cells. The latter formed three distinct vascular clusters characterized as activated pericytes, endothelia and resting pericytes. We identified differentially expressed genes (DEGs) and their enriched pathways in these clusters and detected the most transcriptional changes within activated pericytes. Using our data and a knowledge‐based predictive algorithm, we discovered and prioritized molecular interactions between vascular and astrocyte clusters, the main cell types of the GVU of the BBB.ResultsVascular targets predicted to interact with astrocytic ligands have biological functions in signalling, angiogenesis, amyloid ß metabolism and cytoskeletal structure. Top astrocytic and vascular interacting molecules include both novel and known AD risk genes such as APOE, APP and ECE1. We validated gene expression changes using qPCR. We are in the process of evaluating these gene expression changes in antemortem blood from AD and control participants.ConclusionOur findings provide valuable information into the transcriptional changes in predicted vascular‐astrocytic partners at the GVU, highlighting insights into the molecular mechanisms of BBB breakdown in AD.

PET tracer SMBT‐1 discriminates between BU99008 and Deprenyl binding sites on reactive astrocytes in Alzheimer's disease brains

AbstractBackgroundAstrocytes are the homeostatic cells of the mammalian brain. We recently proposed a “two‐wave” hypothetical model in which reactive astrocytes (RA) are early‐responders to Alzheimer’s disease (AD) pathology and may support a perpetual brain inflammatory state1. Advances in positron emission tomography (PET) imaging biomarkers allow for detecting RA in the early stages of AD (e.g., [11C]‐L‐Deprenyl and [11C]BU99008, which target MAO‐B and the imidazole binding sites 2 – I2Bs, respectively). However, RA assume multiple states, thus, a very heterogenous population of RA exist. Based on a recent consensus, the development of novel PET‐tracers targeting different RA populations is a priority in the field. [18F]‐SMBT‐1, a selective MAO‐B PET‐tracer, was recently designed as an alternative to detect RA in the human brain. However, further studies using [18F]‐SMBT‐1 are needed to evaluate its sensitivity to image RA in AD. We aimed at evaluating the binding properties of SMBT‐1 versus two PET‐tracers targeting RA ‐ [3H]‐L‐Deprenyl and [3H]‐BU99008 ‐ using in vitro human brain techniques.MethodCompetition binding assays for [3H]‐L‐Deprenyl and [3H]‐BU99008 versus SMBT‐1 were performed in postmortem temporal cortex brain homogenate. The number of binding sites and their affinities (IC50 ; half‐maximal inhibitory concentration) as well as percentage were calculated in AD and control brainsResultOur competition binding assays in temporal cortex tissue revealed that SMBT‐1 competes for a single binding site versus [3H]‐L‐Deprenyl in AD (IC50 = 1.141 x 10‐8) and control (IC50 = 1.93 x 10‐8) human brains. Remarkably, SMBT‐1 also competes for [3H]‐BU99008 binding sites in both AD (IC50 = 1.09 x 10‐13 and IC50 = 3.36 x 10‐8, 31% in the high fraction) and control (IC50 = 3.51 x 10‐13 and IC50 = 1.06 x 10‐6, 30% in the high fraction) cases.ConclusionSMBT‐1 competes with [3H]‐L‐Deprenyl for the high affinity MAO‐B binding site and displaces [3H]‐BU99008 binding to the I2Bs. The presence of I2Bs in the entrance channel of MAO‐B may explain this behavior, as SMBT‐1 potentially causes steric hinderance, affecting the availability of this binding site.

Discriminative binding of tau PET tracers PI2620, MK6240 and RO948 in Alzheimer’s disease, corticobasal degeneration and progressive supranuclear palsy brains

Recent mechanistic and structural studies have challenged the classical tauopathy classification approach and revealed the complexity and heterogeneity of tau pathology in Alzheimer’s disease (AD) and primary tauopathies such as corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), progressing beyond distinct tau isoforms. In this multi-tau tracer study, we focused on the new second-generation tau PET tracers PI2620, MK6240 and RO948 to investigate this tau complexity in AD, CBD, and PSP brains using post-mortem radioligand binding studies and autoradiography of large and small frozen brain sections. Saturation binding studies indicated multiple binding sites for 3H-PI2620 in AD, CBD and PSP brains with different binding affinities (Kd ranging from 0.2 to 0.7 nM) and binding site densities (following the order: BmaxAD > BmaxCBD > BmaxPSP). Competitive binding studies complemented these findings, demonstrating the presence of two binding sites [super-high affinity (SHA): IC50(1) = 8.1 pM; and high affinity (HA): IC50(2) = 4.9 nM] in AD brains. Regional binding distribution studies showed that 3H-PI2620 could discriminate between AD (n = 6) and control cases (n = 9), especially in frontal cortex and temporal cortex tissue (p < 0.001) as well as in the hippocampal region (p = 0.02). 3H-PI2620, 3H-MK6240 and 3H-RO948 displayed similar binding behaviour in AD brains (in both homogenate competitive studies and one large frozen hemispherical brain section autoradiography studies) in terms of binding affinities, number of sites and regional patterns. Our small section autoradiography studies in the frontal cortex of CBD (n = 3) and PSP brains (n = 2) showed high specificity for 3H-PI2620 but not for 3H-MK6240 or 3H-RO948. Our findings clearly demonstrate different binding properties among the second-generation tau PET tracers, which may assist in further understanding of tau heterogeneity in AD versus non-AD tauopathies and suggests potential for development of pure selective 4R tau PET tracers.

Brain ethanolamine phospholipids, neuropathology and cognition: A comparative post-mortem analysis of structurally specific plasmalogen and phosphatidyl species.

Reduced cognition in the elderly is associated with low levels of plasmalogens and high levels of lipid rafts, amyloid plaques, and neurofibrillary tangles in the temporal cortex. A systematic integrative analysis of key indices of these pathologies to determine their collective and independent contributions to cognition was performed. Levels of four phosphatidylethanolamines (PE) and four ethanolamine plasmalogens (PL) of identical sn-1 carbon length and desaturation (stearic, 18:0) and identical sn-2 fatty acid compositions of varying side chain lengths and degrees of unsaturation (oleic, 18:1; linoleic, 18:2; arachidonic, 20:4; docosahexaenoic, 22:6), flotillin-1 expression and amyloid plaque and neurofibrillary tangle densities were measured in inferior temporal cortex tissue from 100 elderly subjects (Rush University Memory and Aging Project, 88.5 ± 5.8 years old). Subjects were evenly distributed with respect to gender (52/48, F/M) and cognitive status (38/24/38, no cognitive impairment/mild cognitive impairment/Alzheimer’s dementia) proximate to death. Multivariate logistic regression analyses were used to determine the relative and collective associations of the neuropathological indices with cognition. Higher levels of tangles, amyloid, or flotillin and lower levels of PL 18:0/22:6 were significantly associated with lower cognition in the base model (adjusted for age, sex, education). Multivariate analysis revealed that only PL 18:0/22:6 (β = 0.506; p < 0.00001), tangles (−0.307; p < 0.01), and flotillin (−0.2027; p < 0.05) were independently associated with reduced cognition. PL 18:0/22:6 and PE 18:0/22:6 levels were independently associated with cognition in the presence of tangles, amyloid, and flotillin, but only PL 18:0/22:6 retained its association with cognition when both PL and PE 18:0/22:6 were included in the model indicating that PE 18:0/22:6 levels were associated with PL 18:0/22:6, not cognition. Only high brain levels of PL 18:0/22:6 (>mean+1SD) was predictive of normal cognition (coef = 1.67, p < 0.05) and non-demented state (coef = −2.73, p < 0.001), whereas low levels of PL 18:0/22:6 and high levels of tangles or flotillin were predictive of dementia. The association of high brain polyunsaturated (PUFA)-PL levels with better cognition was independent of amyloid plaque, neurofibrillary tangle, PE, and flotillin-1 expression. Maintenance or augmentation of brain docosahexaenoic (DHA)-PL levels warrants further investigation as a target for preventing cognitive decline or improving cognition in the elderly, respectively.

Identification and Analysis of BCAS4/hsa-miR-185-5p/SHISA7 Competing Endogenous RNA Axis in Late-Onset Alzheimer's Disease Using Bioinformatic and Experimental Approaches.

Alzheimer’s disease (AD) is a heterogeneous degenerative brain disorder with a rising prevalence worldwide. SHISA7 (CKAMP59) has emerged as one of the most intriguing new members of the SHISA family, in that, unlike other CKAMP counterparts, it exhibits a direct function in inhibitory synaptic GABAAR regulation. We used bioinformatics and experimental methods in this research to explore competing endogenous RNA (ceRNA) regulation of BCAS4 and SHISA7 in tau pathogenesis and their capacity as peripheral biomarkers linked to an abnormal inflammatory response in AD. The Gene Expression Omnibus database included two microarray datasets, including information on mRNAs (GSE106241) and miRNAs (GSE157239) from individuals with AD with different degrees of AD-associated neurofibrillary pathology in the temporal cortex (TC) tissue specimens and corresponding controls were downloaded from the Gene Expression Omnibus database. The limma package in the R software was used to identify differently expressed mRNAs (DEmRNAs) and miRNAs (DEmiRNAs) associated with AD-related neurofibrillary pathology. Additionally, we used the quantitative polymerase chain reaction technique to examine the expression of the BCAS4/hsa-miR-185-5p/SHISA7 ceRNA axis in the peripheral blood (PB) of fifty AD patients and fifty control subjects. BCAS4 was shown to act as a ceRNA to control the SHISA7 expression throughout AD-associated neurofibrillary pathology in TC tissue specimens by sponging hsa-miR-185-5p, based on our bioinformatics study. Furthermore, in PB specimens from individuals suffering from AD and normal controls, we found no substantial differences in BCAS4 expression patterns. SHISA7 expression in AD patients’ PB was found to be reduced, as was the case in the TC. On the other hand, we discovered reduced amounts of hsa-miR-185-5p in AD patients’ PB samples compared to control subjects, unlike in TC tissue, where it had been demonstrated to be overexpressed. BCAS4 and SHISA7 expression levels showed a strong positive correlation, suggesting the presence of an interconnected network, most likely as a result of ceRNA regulation among PB specimens. The present study is the first evidence to highlight the expression of the BCAS4/miR-185-5p/SHISA7 ceRNA axis in the brain and PB of AD patients, and offers a new viewpoint on molecular processes underlying AD pathogenic mechanisms.

Parallel single-nucleus chromatin accessibility and transcriptomic profiling of human late-onset Alzheimer's disease brains.

In the post-GWAS era for late-onset Alzheimer's disease (LOAD), the precise disease-causing genes, the specific causal variants, and molecular mechanisms mediating their pathogenic effects remain unknown. Recent studies using single-nucleus (sn)RNA-seq on human LOAD tissue have achieved unprecedented resolution in identifying cell type-specific gene dysregulation, however the regulatory mechanisms and genetic variability underlying these LOAD-specific transcriptomic signatures remain to be identified. Nuclei were isolated from frozen post-mortem human temporal cortex tissue from LOAD patients and cognitively healthy controls (n = 24, all Caucasian and APOE 3/3). 10X Genomics technology was used to generate single-nucleus (sn)RNA-seq and snATAC-seq libraries in parallel from the same pool of nuclei isolated from each brain sample. Uniform manifold approximation and projection (UMAP) analysis showed a total of 29 clusters assigned to 8 cell types including astrocytes, excitatory neurons, inhibitory neurons, microglia, oligodendrocytes, and oligodendrocyte precursor cells (OPCs) in both snRNA-seq and snATAC-seq datasets. We identified differentially expressed genes (DEGs) and differentially accessible peaks (DAPs) for each cluster and found overlaps with known LOAD GWAS regions (SNP+/- 1Mb) as well as new LOAD loci that were not discovered previously. We next performed co-accessibility analysis and identified co-accessible peaks that were more open in LOAD and corresponded to dysregulation of nearby target genes. This analysis also produced networks of co-accessible LOAD peaks that were enriched in transcription factor (TF) motifs of TFs that are specifically overexpressed in LOAD samples. Integrative multi-omics is a powerful strategy to identify regulatory elements underlying gene dysregulation in LOAD. By integrating single-nucleus transcriptomic and open chromatin profiles from the same pool of nuclei, we are able to identify cell subtype-specific molecular mechanisms contributing to LOAD pathogenesis. This new genetic knowledge will progress the identification of new therapeutic targets.

Cell type-specific gene expression changes in Alzheimer's patients with varying levels of cerebral amyloid angiopathy.

Tau neurofibrillary tangles and senile plaques comprised of insoluble amyloid beta are the major histopathological hallmarks of Alzheimer's disease (AD). More than 85% of autopsy-confirmed AD cases also exhibit some degree of cerebral amyloid angiopathy (CAA), which is characterized by amyloid beta peptide deposits predominantly in blood vessels in the meningeal and intracerebral blood vessels. Consequently, CAA predisposes individuals with AD to cerebral infarction and hemorrhages, accounting for ∼20% of cases in the elderly, which could lead to faster cognitive decline through neurovascular pathway dysfunction. We sought to identify cellular composition changes and characterize cell type-specific expression associated with CAA pathology in AD patients. Using 10x Genomics single nuclei RNA sequencing, we profiled nuclei from frozen post-mortem temporal cortex tissue of 80 individuals with neuropathologic diagnosis of AD and varying levels of CAA, ranging from zero to severe CAA pathology. Braak and Thal measures were also obtained. Downstream analyses were performed in R using Seurat v3.2.3. Quality control filtering based on percentage of genes mapped to the mitochondrial genome and number of genes and UMIs detected resulted in 135,092 nuclei for analysis. Differential gene expression analyses were performed using R package MAST. We identified 30 clusters which were annotated using established cell type marker genes. There were 3 oligodendrocyte, 14 neuronal (2 excitatory and 3 inhibitory), 5 microglial, 3 endothelial, 4 astrocyte and 1 oligodendrocyte precursor cell cluster. We observed a significant increase in microglial cell proportions with increasing CAA severity, for 3 of the microglial clusters. Conversely, the excitatory neuronal proportion were decreased in individuals with more severe CAA. Further investigation is ongoing to identify the genes underlying the dysregulation of these cell types in CAA. We observed cellular proportion changes for multiple cell types with increasing CAA severity. Additional analyses to determine the effects of CAA independent of AD severity will be performed by adjusting for Braak and Thal measures. Deeper characterization of cell type-specific expression profiles is expected to provide novel insights into cell-type specific transcriptome changes associated with vascular risk factors in AD.

Quantitative phosphoproteomics uncovers dysregulated kinase networks in Alzheimer's disease.

Alzheimer's disease (AD) is a form of dementia characterized by amyloid-β plaques and tau neurofibrillary tangles that progressively disrupt neural circuits in the brain. The signaling networks underlying AD pathological changes are poorly characterized at the phosphoproteome level. Using mass spectrometry, we analyzed the proteome and tyrosine, serine and threonine phosphoproteomes of temporal cortex tissue from patients with AD and aged-matched controls. We identified cocorrelated peptide clusters that were linked to varying levels of phospho-tau, oligodendrocyte, astrocyte, microglia and neuron pathologies. We found that neuronal synaptic protein abundances were strongly anti-correlated with markers of microglial reactivity. We also observed that phosphorylation sites on kinases targeting tau and other new signaling factors were correlated with these peptide modules. Finally, we used data-driven statistical modeling to identify individual peptides and peptide clusters that were predictive of AD histopathologies. Together, these results build a map of pathology-associated phosphorylation signaling events occurring in AD.

High-Throughput Data of Circular RNA Profiles in Human Temporal Cortex Tissue Reveals Novel Insights into Temporal Lobe Epilepsy

Background/Aims: Circular RNAs (circRNAs) are a class of long noncoding RNAs with a closed loop structure that regulate gene expression as microRNA sponges. CircRNAs are more enriched in brain tissue, but knowledge of the role of circRNAs in temporal lobe epilepsy (TLE) has remained limited. This study is the first to identify the global expression profiles and characteristics of circRNAs in human temporal cortex tissue from TLE patients. Methods: Temporal cortices were collected from 17 TLE patients and 17 non-TLE patients. Total RNA was isolated, and high-throughput sequencing was used to profile the transcriptome of dysregulated circRNAs. Quantitative PCR was performed for the validation of changed circRNAs. Results: In total, 78983 circRNAs, including 15.29% known and 84.71% novel circRNAs, were detected in this study. Intriguingly, 442 circRNAs were differentially expressed between the TLE and non-TLE groups (fold change≥2.0 and FDR≤0.05). Of these circRNAs, 188 were up-regulated, and 254 were down-regulated in the TLE patient group. Eight circRNAs were validated by real-time PCR. Remarkably, circ-EFCAB2 was intensely up-regulated, while circ-DROSHA expression was significantly lower in the TLE group than in the non-TLE group (P<0.05). Bioinformatic analysis revealed that circ-EFCAB2 binds to miR-485-5p to increase the expression level of the ion channel CLCN6, while circ-DROSHA interacts with miR-1252-5p to decrease the expression level of ATP1A2. Conclusions: The dysregulations of circRNAs may reflect the pathogenesis of TLE and circ-EFCAB2 and circ-DROSHA might be potential therapeutic targets and biomarkers in TLE patients.

Loss of constitutive functional γ-aminobutyric acid type A-B receptor crosstalk in layer 5 pyramidal neurons of human epileptic temporal cortex.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in adult central nervous system, and profound alterations of GABA receptor functions are linked to temporal lobe epilepsy (TLE). Here we describe the functional relationships between GABA receptors type B (GABAB R) and type A (GABAA R) in human temporal cortex and how TLE affects this aspect of GABAergic signaling. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded by patch-clamp techniques from human L5 pyramidal neurons in slices from temporal cortex tissue obtained from surgery. We describe a constitutive functional crosstalk between GABAB Rs and GABAA Rs in human temporal layer 5 pyramidal neurons, which is lost in epileptic tissues. The activation of GABAB Rs by baclofen, in addition to the expected reduction of mIPSC frequency, produced, in cortex of nonepileptic patients, the prolongation of mIPSC rise and decay times, thus increasing the inhibitory net charge associated with a single synaptic event. Block of K+ channels did not prevent the increase of decay time and charge. Protein kinase A (PKA) blocker KT5720 and pertussis toxin inhibited the action of baclofen, whereas 8Br-cAMP mimicked the GABAB R action. The same GABAB R-mediated modulation of GABAA Rs was observed in pyramidal neurons of rat temporal cortex, with both PKA and PKC involved in the process. In cortices from TLE patients and epileptic rats, baclofen lost its ability to modulate mIPSCs. Our results highlight the association of TLE with functional changes of GABAergic signaling that may be related to seizure propagation, and suggest that the selective activation of a definite subset of nonpresynaptic GABAB Rs may be therapeutically useful in TLE.

IDENTIFICATION OF AN ITGA7 VARIANT ASSOCIATED WITH ALZHEIMER’S DISEASE AND MULTIPLE OTHER NEURODEGENERATIVE DISEASES

The genetic causes of mixed Alzheimer's Disease (AD) and Dementia with Lewy Bodies (DLB) pathology remain unclear. In the current study we searched for exonic variants contributing to dual pathology in younger patients. We analyzed whole exome sequencing data collected through Stanford University and the Alzheimer's Disease Sequencing Project (ADSP). High impact (missense or nonsense) variants found in autopsy-confirmed mixed AD/DLB cases (age < 75 years, n=143) were screened against older, autopsy-confirmed pure AD controls (age > 85 years, n=274) and clinically-defined healthy controls (age > 80 years, n=4572). Variants seen in at least two dual path cases and none of the older, pure AD controls or healthy controls were examined further. For the remaining variants, we searched other neurodegenerative exome databases to determine their specificity to mixed AD/DLB pathology. For our top variant we performed immunohistochemical staining of autopsy-confirmed AD/DLB mixed pathology brain tissue. Finally, we explored potential functional effects of the variant by examining gene expression in post-mortem temporal cortex RNA-seq data from AD cases (n=82) and controls (n=80). The initial screening process yielded eleven variants present in at least two dual pathology cases and none of the healthy controls or older pure AD cases. Of these, we focused on rs76938320-A (NP_001138468.1:p. Ser700Leu, MAF=0.0002) on exon 18 of ITGA7 because of its predicted probable damaging effect using in silico prediction tools, its presence in three younger AD cases (age < 80 years) in ADSP, its high frequency in the ALS exome database, and the finding of a homozygous carrier with TDP43 and tau pathology. ITGA7 transcript isoforms that contain exon 18 show significantly higher expression in AD temporal cortex tissue when compared to controls (P < 0.001) in the Mayo Clinic Transcriptomics data set. Immunohistochemical staining of AD, AD/DLB, DLB, and vascular dementia brain tissue suggest that AD and AD/DLB tissue express higher levels of ITGA7 than vascular controls and pure DLB. Convergent data from exome sequencing, immunohistochemical stains, and transcriptomic analysis suggest that this ITGA7 variant may predispose not only to mixed AD/DLB pathology but to the development of multiple proteinopathies. Normalized FPKM values of the three ITGA7 transcript isoforms encoding rs76938420 are significantly higher in AD cases (n = 82) than in controls (n = 80).ITGA7 transcript expression values in post-mortem brain tissue were corrected for subject age at death, gender, RIN, source, and cell type markers. *** P < 0.001. ITGA7 neuronal cytoplasmic immunoreactivity in post-mortem brain tissue. Mixed DLB/AD and AD cases demonstrate stronger neuronal cytoplasmic immunoreactivity for ITGA7 when compared to vascular dementia and DLB cases. ITGA7 immunoreactivity is strongest within neurons containing neurofibrillary tangles.

Gene expression analysis reveals the dysregulation of immune and metabolic pathways in Alzheimer's disease.

In recent years, several pathway analyses of genome-wide association studies reported the involvement of metabolic and immune pathways in Alzheimer's disease (AD). Until now, the exact mechanisms of these pathways in AD are still unclear. Here, we conducted a pathway analysis of a whole genome AD case-control expression dataset (n=41, 25 AD cases and 16 controls) from the human temporal cortex tissue. Using the differently expressed AD genes, we identified significant KEGG pathways related to metabolism and immune processes. Using the up- and down- regulated AD gene list, we further found up-regulated AD gene were significantly enriched in immune and metabolic pathways. We further compare the immune and metabolic KEGG pathways from the expression dataset with those from previous GWAS datasets, and found that most of these pathways are shared in both GWAS and expression datasets.

Evaluation of the Nicotinic Acetylcholine Receptor-Associated Proteome at Baseline and Following Nicotine Exposure in Human and Mouse Cortex.

Nicotinic acetylcholine receptors (nAChRs) support the initiation and maintenance of smoking, but the long-term changes occurring in the protein complex as a result of smoking and the nicotine in tobacco are not known. Human studies and animal models have also demonstrated that increasing cholinergic tone increases behaviors related to depression, suggesting that the nAChR-associated proteome could be altered in individuals with mood disorders. We therefore immunopurified nAChRs and associated proteins for quantitative proteomic assessment of changes in protein–protein interactions of high-affinity nAChRs containing the β2 subunit (β2*-nAChRs) from either cortex of mice treated with saline or nicotine, or postmortem human temporal cortex tissue from tobacco-exposed and nonexposed individuals, with a further comparison of diagnosed mood disorder to control subjects. We observed significant effects of nicotine exposure on the β2*-nAChR-associated proteome in human and mouse cortex, particularly in the abundance of the nAChR subunits themselves, as well as putative interacting proteins that make up core components of neuronal excitability (Na/K ATPase subunits), presynaptic neurotransmitter release (syntaxins, SNAP25, synaptotagmin), and a member of a known nAChR protein chaperone family (14-3-3ζ). These findings identify candidate-signaling proteins that could mediate changes in cholinergic signaling via nicotine or tobacco use. Further analysis of identified proteins will determine whether these interactions are essential for primary function of nAChRs at presynaptic terminals. The identification of differences in the nAChR-associated proteome and downstream signaling in subjects with various mood disorders may also identify novel etiological mechanisms and reveal new treatment targets.

Serum MicroRNA-4521 is a Potential Biomarker for Focal Cortical Dysplasia with Refractory Epilepsy.

Early biomarker-based diagnosis of focal cortical dysplasia (FCD) represents a major clinical challenge. The aim of this study was to identify novel brain microRNAs (miRNAs) in patients with refractory epilepsy and FCD as potential biomarkers. We evaluated serum hsa-miR-4521 as a promising novel biomarker in patients with FCD. Tissue for microarray was obtained from nine patients with temporal lobe refractory epilepsy who underwent surgery to remove epileptic foci identified by cortical video electroencephalogram monitoring. Control tissue was collected from eight patients with hypertension who required emergency surgery to remove an intracranial hematoma. The Affymetrix® GeneChip® Command Console® Software (Affymetrix miRNA 4.0) was used to compare miRNA expression in the cerebral cortex of experimental and control patients. Temporal cortex tissue and serum samples were taken from the same patients for verification of hsa-miR-4521 expression by real-time quantitative polymerase chain reaction (RT-qPCR). The experimental and control patients did not differ significantly in terms of age and gender. 19.4 % (148/764) of the total miRNAs were differentially expressed in experimental and control tissue, which is in agreement with the existing literature. We selected miRNA-4521 for further analysis; the fold-change in expression was 14.4707 and the q value was almost 0, which confirmed up-regulation. Significant up-regulation of hsa-miR-4521 was further validated by RT-qPCR. miRNA microarrays can efficiently and conveniently identify differentially expressed miRNAs in epilepsy brain tissue. This is the first study to identify differential expression of hsa-miR-4521 in brain tissue and serum of refractory epilepsy patients and suggests that serum hsa-miR-4521 may represent a potential diagnostic biomarker for FCD with refractory epilepsy.

Brain Amyloid-Beta Fragment Signatures in Pathological Ageing and Alzheimer's Disease by Hybrid Immunoprecipitation Mass Spectrometry

Background: Senile plaques in Alzheimer's disease (AD) are composed of amyloid-β (Aβ), especially N-truncated forms including Aβ_4-42. These are thought to be neurotoxic. However, individuals may live for decades with biomarker evidence of cerebral β-amyloidosis (positive amyloid PET imaging and/or low cerebrospinal fluid levels of the 42 amino acid form of Aβ) without cognitive impairment. This condition may be termed pathological ageing (PA). Objective: To investigate whether there is a difference in the cerebral Aβ fragment pattern in brain specimens from non-demented (PA) and demented (AD) individuals expressing the full neuropathological triad of AD (senile plaques, neurofibrillary tangles and neurodegeneration). Methods: We extracted Aβ using formic acid and hybrid (6E10 and 4G8) immunoprecipitation from fresh-frozen temporal cortex tissue of 6 elderly individuals (mean age ± SD: 89 ± 3.5 years) with PA and 10 patients with AD (mean age ± SD: 72 ± 8.5 years). The full spectrum of Aβ peptides was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Results: AD patients had generally more N-terminally truncated and pyroglutamate-modified Aβ than PA patients, whereas PA patients had on average more Aβ_1-40 than AD patients. Conclusion: Senile plaques in AD may have an Aβ fragment composition distinct from PA with more N-terminally and pyroglutamate-modified Aβ peptides that may be linked to neurotoxicity.

Monoubiquitination Promotes Calpain Cleavage of the Protein Phosphatase 2A (PP2A) Regulatory Subunit α4, Altering PP2A Stability and Microtubule-associated Protein Phosphorylation

Multiple neurodegenerative disorders are linked to aberrant phosphorylation of microtubule-associated proteins (MAPs). Protein phosphatase 2A (PP2A) is the major MAP phosphatase; however, little is known about its regulation at microtubules. α4 binds the PP2A catalytic subunit (PP2Ac) and the microtubule-associated E3 ubiquitin ligase MID1, and through unknown mechanisms can both reduce and enhance PP2Ac stability. We show MID1-dependent monoubiquitination of α4 triggers calpain-mediated cleavage and switches α4's activity from protective to destructive, resulting in increased Tau phosphorylation. This regulatory mechanism appears important in MAP-dependent pathologies as levels of cleaved α4 are decreased in Opitz syndrome and increased in Alzheimer disease, disorders characterized by MAP hypophosphorylation and hyperphosphorylation, respectively. These findings indicate that regulated inter-domain cleavage controls the dual functions of α4, and dysregulation of α4 cleavage may contribute to Opitz syndrome and Alzheimer disease.