Frontal Cortex In Alzheimer's Disease Research Articles

Discovery of Potential Drug Targeting Key Genes in Alzheimer's Disease: Insights from Transcriptome Analysis and Molecular Docking.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder that presents a significant global health challenge. To explore drugs targeting key genes in AD, R software was used to analyze the data of single nuclei transcriptome from human cerebral frontal cortex in AD, and the differentially expressed genes (DEGs) were screened. Then the gene ontology (GO) analysis, Kyoto gene and genome encyclopedia (KEGG) pathway enrichment and protein-protein interaction (PPI) network were analyzed. The hub genes were calculated by Cytoscape software. Molecular docking and molecular dynamics simulation were used to evaluate and visualize the binding between candidate drugs and key genes. A total of 564 DEGs were screened, and the hub genes were ISG15, STAT1, MX1, IFIT3, IFIT2, RSAD2, IFIT1, IFI44, IFI44L and DDX58. Enrichment terms mainly included response to virus, IFN-γ signaling pathway and virus infection. Diclofenac had good binding effect with IFI44 and IFI44L. Potential drugs may act on key gene targets and then regulate biological pathways such as virus response and IFN-γ-mediated signal pathway, so as to achieve anti-virus, improve immune balance and reduce inflammatory response, and thus play a role in anti-AD.

Effects of Hypertension and Anti-Hypertensive Treatment on Amyloid-β (Aβ) Plaque Load and Aβ-Synthesizing and Aβ-Degrading Enzymes in Frontal Cortex.

Epidemiological data associate hypertension with a predisposition to Alzheimer's disease (AD), and a number of postmortem and in vivo studies also demonstrate that hypertension increases amyloid-β (Aβ) pathology. In contrast, anti-hypertensive medications reportedly improve cognition and decrease the risk of AD, while certain classes of anti-hypertensive drugs are associated with decreased AD-related pathology. We investigated the effects of hypertension and anti-hypertensive treatment on Aβ plaque load in postmortem frontal cortex in AD. Aβ load was significantly increased in hypertensive (n = 20) relative to normotensive cases (n = 62) and was also significantly higher in treated (n = 9) than untreated hypertensives (n = 11). We then looked into mechanisms by which hypertension and treatment might increase Aβ load, focusing on Aβ-synthesizing enzymes, β- and γ-secretase, and Aβ-degrading enzymes, angiotensin-converting enzyme (ACE), insulin-degrading enzyme (IDE) and neprilysin. ACE and IDE protein levels were significantly lower in hypertensive (n = 21) than normotensive cases (n = 64), perhaps translating to decreased Aβ catabolism in hypertensives. ACE level was significantly higher in treated (n = 9) than untreated hypertensives (n = 12), possibly reflecting feedback upregulation of the renin-angiotensin system. Prospective studies in larger cohorts stratified according to anti-hypertensive drug class are needed to confirm these initial findings and to elucidate the interactions between hypertension, anti-hypertensive treatments, and Aβ metabolism.

Altered expression of ZnT10 in Alzheimer's disease brain.

There is an increasing body of evidence suggesting that metal homeostasis is dysregulated in the pathology of Alzheimer's disease (AD). Although expression levels of several transporters belonging the SLC30 family, which comprises predominantly zinc transporters, have been studied in the AD brain, SLC30A10 (ZnT10) has not been studied in this context. To determine if dysregulated expression of ZnT10, which may transport both Zn and Mn, could be a factor that contributes to AD, we investigated if there were differences in ZnT10 mRNA levels in specimens of frontal cortex from AD patients and controls and also if brain tissue from the APP/PS1 transgenic (Tg) mouse model showed abnormal levels of ZnT10 mRNA expression. Our results show that ZnT10 is significantly (P<0.01) decreased in the frontal cortex in AD. Furthermore, we observed a significant decrease in ZnT10 mRNA levels in the APP/PS1-Tg mice compared with wild-type controls (P<0.01). Our results suggest that this dysregulation in ZnT10 could further contribute to disease progression.

Normalization of gene expression using SYBR green qPCR: A case for paraoxonase 1 and 2 in Alzheimer's disease brains

Validating the expression stability of reference genes is crucial for reliable normalization of real-time quantitative PCR (qPCR) data, but relatively few studies have investigated this issue in brain human tissues. The present study thus aimed at identifying in human post-mortem brain tissues a set of suitable endogenous reference genes (ERG) for the expression analysis of potential candidate genes associated with Alzheimer's disease (AD). The mRNA levels of ten common ERGs (ACTB, GAPDH, GPS1, GUSB, M-RIP, PGK1, POL2RF, PPIA, UBE2D2, and YES1) were determined in the frontal cortex of autopsy-confirmed AD and non-demented control cases ( n = 20) using SYBR Green technology. Then, these levels were ranked according to their expression stability using three software applications: geNorm, NormFinder and BestKeeper. Whereas PPIA and UBE2D2 were among the ERGs with the most reliable expression, ACTB was the worst. Subsequently, using PPIA and UBE2D2 as ERGs for normalization, the mRNA levels of paraoxonase 1 (PON1) and paraoxonase 2 (PON2) were quantified in the frontal cortex of AD and control cases ( n = 80) and analyzed using the REST 2009 program. Our results indicate that both paraoxonases are expressed in the human frontal cortex and that PON2 but not PON1 mRNA levels are up-regulated in AD relative to non-demented controls. However, re-analysis of the results by ANCOVA indicated that the significance of the difference between AD and control groups depended upon the ERG used for normalization. The use of a computational method allowing the inclusion of possible confounding factors is thus recommended for the analysis of data.

Neuronal Hemoglobin is Reduced in Alzheimer's Disease, Argyrophilic Grain Disease, Parkinson's Disease, and Dementia with Lewy Bodies

Previous studies have demonstrated the presence of hemoglobin α-chain and β-chain in neurons of the rodent and human brain thus indicating that hemoglobin is a normal component of nerve cells and that hemoglobin may play a role in intraneuronal oxygen homeostasis. Progressing with these studies, hemoglobin expression has been examined in selected cell population in the brains of Alzheimer's disease (AD), argyrophilic grain disease (AGD), Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Double labeling immunofluorescence and confocal microscopy revealed reduced hemoglobin α-chain and β-chain in practically all neurons with small amounts of granular or punctuate hyperphosphorylated tau deposits and in neurons with tangles in the hippocampus and frontal cortex in AD and in the hippocampus in AGD; in ballooned neurons containing αB-crystallin in the amygdala in AD and AGD; and in about 80% of neurons with punctuate α-synuclein deposits and in neurons with Lewy bodies in the substantia nigra pars compacta and in vulnerable neurons of the medulla oblongata in PD and DLB; and in neurons with Lewy bodies in the frontal cortex in DLB. Hemoglobin immunoreactivity was also observed in the core of neuritic plaques and in diffuse plaques, but not in dystrophic neurites. Loss of hemoglobin was specific as neuroglobin was present equally in neurons with and without abnormal protein inclusions, and erythropoietin receptor was expressed equally in neurons without and in neurons with abnormal protein aggregates in AD, AGD, PD, and DLB.

HPA Axis Dysregulation Associated to Apolipoprotein E4 Genotype in Alzheimer's Disease

The present work investigated the involvement of cortisol and its receptors, glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), in Alzheimer's disease (AD). Cortisol was measured in cerebrospinal fluid (CSF) samples from controls, mild cognitive impairment (MCI), progressive MCI evolving to AD, and AD. CSF cortisol levels do not seem to have a prognostic value, as increases in cortisol levels were found only in AD patients. GR expression was decreased while MR expression was increased in the frontal cortex of AD. When considering degeneration (ratio to synaptophysin and the post-synaptic marker PSD95), GR expression was similar between controls and AD, suggesting that GR loss was due to synaptic degeneration in AD. Increases in cortisol levels and MR expression were associated to an apolipoprotein E4 genotype. Cognitive status was negatively associated to CSF cortisol. In apolipoprotein E4 carriers, MR but not GR expression, negatively correlated to Mini-Mental Status Examination score and positively correlated to frontal cortex amyloid-β levels. It is concluded that there is a dysregulation of the hypothalamus-pituitary-adrenal axis in AD that seems to be consequence rather than cause of AD.

Assessment of activation of the plasma kallikrein-kinin system in frontal and temporal cortex in Alzheimer's disease and vascular dementia

Decreased cerebral blood flow and blood-brain barrier disruption are features of Alzheimer's disease (AD). The plasma kallikrein-kinin system modulates cerebrovascular tone through release of vasoactive bradykinin (BK). Cerebroventricular infusion of Aβ1–40 enhances BK release, suggesting that the activity of this system may be elevated in AD. We investigated the profile of the activating protease of this system, plasma kallikrein (PK), in frontal and temporal brain tissue from postmortem confirmed cases of AD, vascular dementia (VaD), and controls. Measurements of neuron specific enolase messenger ribonucleic acid (mRNA) and protein were used to adjust for neuronal loss. Adjusted PK mRNA was significantly increased in the frontal cortex in AD, and the frontal and temporal cortex in VaD. Similar trends were seen for PK protein level in AD and VaD. PK activity was significantly increased in the frontal and temporal cortex in AD. Increased PK activity in AD is likely to contribute to increased BK release and may thereby influence cerebral blood flow and vascular permeability.

Distribution and expression of picalm in Alzheimer disease.

PICALM, the gene encoding phosphatidylinositol-binding clathrin assembly (picalm) protein, was recently shown to be associated with risk of Alzheimer disease (AD). Picalm is a key component of clathrin-mediated endocytosis. It recruits clathrin and adaptor protein 2 (AP-2) to the plasma membrane and, along with, AP-2 recognizes target proteins. The attached clathrin triskelions cause membrane deformation around the target proteins enclosing them within clathrin-coated vesicles to be processed in lysosomes or endosomes. We examined the distribution of picalm in control and AD brain tissue and measured levels of picalm messenger RNA (mRNA) by real-time polymerase chain reaction. Immunolabeling of brain tissue showed that picalm is predominately present in endothelial cells. This was further supported by the demonstration of picalm in human cerebral microvascular cells grown in culture. Picalm mRNA was elevated in relation to glyceraldehyde-3-phosphate dehydrogenase but not factor VIII-related antigen or CD31 mRNA in the frontal cortex in AD. No change was seen in the temporal cortex or thalamus. The transport of Aβ across vessel walls and into the bloodstream is a major pathway of Aβ removal from the brain and picalm is ideally situated within endothelial cells to participate in this process. Further research is needed to determine whether PICALM expression is influenced by Aβ levels and whether it affects Aβ uptake and transport by endothelial cells.

Regional distribution of TDP‐43 inclusions in Alzheimer disease (AD) brains: Their relation to AD common pathology

Initially, trans activation responsive region (TAR)-DNA-binding protein 43 (TDP-43) was considered to be a disease-specific component of ubiquitin-positive and tau-negative inclusions in the brains of patients with frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS); however, it is now widely known that this protein also abnormally accumulates in neurons in other neurodegenerative diseases. On the basis of observation mainly in the medial temporal lobe, TDP-43-immunoreactive neuronal inclusions have been detected in 20-30% of Alzheimer disease (AD) brains. However, it is controversial whether these cases represent a combined disease, that is, mixed AD/FTLD-U. To address this issue, it is necessary to obtain more knowledge on the region-specific distribution of TDP-43 immunoreactivity and also about its relationship to AD common pathology. Here, we describe abnormal TDP-43 immunoreactivity in the medial temporal lobe in 5/16 AD patients (31%). Most of the depositions were cytoplasmic inclusions, mainly located in the subiculum and parahippocampal gyrus and rarely in dentate granular cells of the hippocampus. TDP-43-positive inclusions and senile plaque/neurofibrillary tangle distribution were not always identical, and intracellular colocalizations of TDP-43 and phospho-tau were also infrequent. The cases showing TDP-43-positive inclusions in the medial temporal lobe also showed abnormally highly dense TDP-43 immunoreactivity in the frontal, but not in the parietal and occipital cortices. Intracellularly, TDP-43-positive inclusions were highly ubiquitinated and colocalized with p62 immunoreactivity as well. Our findings suggest that abnormal TDP-43 deposition and AD pathology (formation of senile plaques and neurofibrillary tangles) might occur independently. However, taken together with the results of previous reports, the distribution of TDP-43 immunoreactivity in the hippocampus and frontal cortex in AD appear to be varying. We consider that it is still too early to determine that the TDP-43 accumulation is a part of AD pathology or result from a completely independent pathology.

Detergent‐Insoluble EAAC1/EAAT3 Aberrantly Accumulates in Hippocampal Neurons of Alzheimer's Disease Patients

Disturbed glutamate homeostasis may contribute to the pathological processes involved in Alzheimer's disease (AD). Once glutamate is released from synapses or from other intracellular sources, it is rapidly cleared by glutamate transporters. EAAC1 (also called EAAT3 or SLC1A1) is the primary glutamate transporter in forebrain neurons. In addition to transporting glutamate, EAAC1 plays other roles in regulating GABA synthesis, reducing oxidative stress in neurons, and is important in supporting neuron viability. Currently, little is known about EAAC1 in AD. To address whether EAAC1 is disturbed in AD, immunohistochemistry was performed on tissue from hippocampus and frontal cortex of AD and normal control subjects matched for age and gender. While EAAC1 immunostaining in cortex appeared comparable to controls, in the hippocampus, EAAC1 aberrantly accumulated in the cell bodies and proximal neuritic processes of CA2-CA3 pyramidal neurons in AD patients. Biochemical analyses showed that Triton X-100-insoluble EAAC1 was significantly increased in the hippocampus of AD patients compared to both controls and Parkinson's disease patients. These findings suggest that aberrant glutamate transporter expression is associated with AD-related neuropathology and that intracellular accumulation of detergent-insoluble EAAC1 is a feature of the complex biochemical lesions in AD that include altered protein solubility.

Association of alleles carried at TNFA -850 and BAT1-22 with Alzheimer's disease

BackgroundInflammatory changes are a prominent feature of brains affected by Alzheimer's disease (AD). Activated glial cells release inflammatory cytokines which modulate the neurodegenerative process. These cytokines are encoded by genes representing several interleukins and TNFA, which are associated with AD. The gene coding for HLA-B associated transcript 1 (BAT1) lies adjacent to TNFA in the central major histocompatibility complex (MHC). BAT1, a member of the DEAD-box family of RNA helicases, appears to regulate the production of inflammatory cytokines associated with AD pathology. In the current study TNFA and BAT1 promoter polymorphisms were analysed in AD and control cases and BAT1 mRNA levels were investigated in brain tissue from AD and control cases.MethodsGenotyping was performed for polymorphisms at positions -850 and -308 in the proximal promoter of TNFA and position -22 in the promoter of BAT1. These were investigated singly or in haplotypic association in a cohort of Australian AD patients with AD stratified on the basis of their APOE ε4 genotype. Semi-quantitative RT-PCR was also performed for BAT1 from RNA isolated from brain tissue from AD and control cases.ResultsAPOE ε4 was associated with an independent increase in risk for AD in individuals with TNFA -850*2, while carriage of BAT1 -22*2 reduced the risk for AD, independent of APOE ε4 genotype. Semi-quantitative mRNA analysis in human brain tissue showed elevated levels of BAT1 mRNA in frontal cortex of AD cases.ConclusionThese findings lend support to the application of TNFA and BAT1 polymorphisms in early diagnosis or risk assessment strategies for AD and suggest a potential role for BAT1 in the regulation of inflammatory reactions in AD pathology.

RESEARCH ARTICLE: Up‐regulation of Adenosine Receptors in the Frontal Cortex in Alzheimer's Disease

AbstractAdenosine receptors are G‐protein coupled receptors which modulate neurotransmitter release, mainly glutamate. Adenosine A1 and A2A receptors were studied in post‐mortem human cortex in Alzheimer's disease (AD) and age‐matched controls. Total adenosine A1 receptor number, determined by radioligand binding assay, using [3H]DPCPX, was significantly increased in AD cases in early and advanced stages without differences with the progression of the disease. A significant increase of A1R (37 kDa) levels was also observed by Western blot in early and advanced stages of AD. In addition, increased numbers of adenosine A2A receptors were observed in AD samples as determined by a binding assay using [3H]ZM 241385 as a radioligand and by Western blot. Increased binding and protein expression levels of adenosine receptors were not associated with increased mRNA levels coding A1 and A2A receptors. Finally, increased A1 and A2A receptor‐mediated response was observed. These results show up‐regulation of adenosine A1 and A2A receptors in frontal cortex in AD, associated with sensitization of the corresponding transduction pathways.

TNFR-associated factor-2 (TRAF-2) in Alzheimer's disease

Levels of tumor necrosis factor-α (TNF-α) are increased in the brain in Alzheimer's disease (AD). The TNF-α/TNF-R signaling pathways involve complex interactions between several proteins, including TNF-receptor-associated factor-2 (TRAF-2). We have examined the distribution and levels of TRAF-2 in AD and control brains and also whether single nucleotide polymorphisms (SNPs) in the TRAF-2 gene are associated with AD and influence TRAF-2 expression. Immunohistochemistry demonstrated TRAF-2 in AD and control cortex in neurons, within plaque-associated neurites and some neurofibrillary tangles. Western blots revealed a band of the expected apparent molecular mass (∼50 kDa) for TRAF-2, in homogenates of AD and control cortex. RT-PCR showed the levels of TRAF-2 mRNA to be significantly higher in the frontal cortex of AD than control brains ( p = 0.015). TRAF-2 mRNA expression was not linked to any SNPs. The 3′ UTR SNP (rs7852970) GG allele was significantly protective against AD ( p = 0.030). Our findings suggest that the TRAF-2 pathway is involved AD. The mechanisms are currently unclear and need further examination.

MMP‐2, ‐3 and ‐9 levels and activity are not related to Aβ load in the frontal cortex in Alzheimer's disease

Matrix metalloproteinases (MMPs) -2, -3 and -9 are up-regulated in several cell types on exposure to amyloid beta peptide (Abeta) and have Abeta-degrading activity in vitro. The aims of this study were to determine (i) the distribution of MMP-2, -3 and -9 in the cerebral cortex in Alzheimer's disease (AD) and control brains; (ii) whether the levels and activity of these proteases are increased in AD; and (iii) whether their activity is related to Abeta load. In addition, we examined whether promoter polymorphisms in the MMP-3 and -9 genes are associated with AD in the study cohort. Paraffin sections of frontal lobe from AD and control cases were immunostained for MMP-2, -3 and -9 and tissue homogenates used for MMP activity assays. DNA from these cases was genotyped for the MMP-3 5A/6A (-1171) and MMP-9 C-1562T promoter polymorphisms. Immunohistochemistry revealed MMP-3 in plaques and both MMP-3 and -9 around scattered neurones. The levels and activity of all three MMPs were similar in AD and control brains and bore no relationship to Abeta load. Analysis of MMP-3 -1171 5A/6A allele frequencies showed that the 6A allele (with reduced promoter activity) was associated with AD; the MMP-9 C-1562T polymorphism was not. The levels and activities of MMP-2, -3 and -9 are not increased in the frontal cortex in AD and are not related to Abeta load. Our findings suggest that altered expression of these proteases does not make a significant contribution to the accumulation of Abeta in AD.

Occludin is overexpressed in Alzheimer's disease and vascular dementia

The tight junctions (TJs) are key players in the control of blood-brain barrier (BBB) properties, the most complex TJs in the vascular system being found in the endothelial cells of brain capillaries. One of the main TJs proteins is occludin, which anchors plasma membranes of neighbour cells and is present in large amounts in the brain endothelia. Previous studies demonstrated that disruption of BBB in various pathological situations associates with changes in occludin expression, and this change could be responsible for malfunction of BBB. Therefore in this study, applying an immunohistochemical approach, we decided to explore the occludin expression in frontal cortex (FC) and basal ganglia in ageing control, Alzheimer's disease (AD), and vascular dementia (VD) brains, as far as all these pathologies associate microangiopathy and disruption of BBB. Strikingly, we found selected neurons, astrocytes and oligodendrocytes expressing occludin, in all cases studied. To estimate the number of occludin-expressing neurons, we applied a stereological approach with random systematic sampling and the unbiased optical fractionator method. We report here a significant increase in ratio of occludin-expressing neurons in FC and basal ganglia regions in both AD and VD as compared to ageing controls. Within the cerebral cortex, occludin was selectively expressed by pyramidal neurons, which are the ones responsible for cognitive processes and affected by AD pathology. Our findings could be important in unravelling new pathogenic pathways in dementia disorders and new functions of occludin and TJs.

Age-dependent decline of neprilysin in Alzheimer's disease and normal brain: Inverse correlation with Aβ levels

Brain deposition of amyloid-β (Aβ) is a pathological hallmark of Alzheimer disease (AD) but Aβ is also detected in non-demented elderly individuals. Neprilysin has been shown to be an important enzyme to degrade Aβ in brain. We investigated whether decreased neprilysin levels contributes to the accumulation of Aβ in AD and in normal aging. No difference in neprilysin protein and mRNA levels were found between AD subjects and age-matched controls. Protein levels of neprilysin were reduced with age in the temporal and frontal cortex of AD and normal brain. A significant positive correlation between insoluble Aβ 40 and Aβ 42 with age was found in cortex of normal brain whereas in AD brain the correlation between age and Aβ was weaker. Our findings of an inverse correlation between neprilysin and insoluble Aβ levels in both groups suggest that neprilysin is involved in the clearance of Aβ. The observed age-dependent decline in neprilysin may be related to the increased Aβ levels during normal aging. The similar rate of decline in neprilysin with age may not be the major cause of the high levels of Aβ associated with AD but is likely to be a trigger of AD pathology.

Transglutaminase bonds in neurofibrillary tangles and paired helical filament tau early in Alzheimer's disease

Transglutaminase-catalyzed ε(γ-glutamyl)lysine cross-links exist in Alzheimer's disease (AD) paired helical filament (PHF) tau protein but not normal soluble tau. To test the hypothesis that these cross-links could play a role in the formation of neurofibrillary tangles (NFT), we used single- and double-label immunofluorescence confocal microscopy and immunoaffinity purification and immunoblotting to examine ε(γ-glutamyl)lysine cross-links in AD and control brains. The number of neurons that are immunoreactive with an antibody directed at the ε-(γ-glutamyl)lysine bond was significantly higher in AD cortex compared with age-matched controls and schizophrenics. PHF tau-directed antibodies AT8, MC-1 and PHF-1 co-localized with ε(γ-glutamyl)lysine immunolabeling in AD NFT. Immunoaffinity purification and immunoblotting experiments demonstrated that PHF tau contains ε(γ-glutamyl)lysine bonds in parietal and frontal cortex in AD. In control cases with NFT present in the entorhinal cortex and hippocampus, indicative of Braak and Braak stage II, ε(γ-glutamyl)lysine bonds were present in PHF tau in parietal and frontal cortex, despite the lack of microscopically detectable NFT or senile plaques in these cortical regions. The presence of PHF tau with ε(γ-glutamyl)lysine bonds in brain regions devoid of NFT in stage II (but regions, which would be expected to contain NFT in stage III) suggests that these bonds occur early in the formation of NFT.

Spatial distribution of diffuse, primitive, and classic amyloid-beta deposits and blood vessels in the upper laminae of the frontal cortex in Alzheimer disease.

The spatial distribution of the diffuse, primitive, and classic amyloid-beta deposits was studied in the upper laminae of the superior frontal gyrus in cases of sporadic Alzheimer disease (AD). Amyloid-beta-stained tissue was counterstained with collagen IV to determine whether the spatial distribution of the amyloid-beta deposits along the cortex was related to blood vessels. In all patients, amyloid-beta deposits and blood vessels were aggregated into distinct clusters and in many patients, the clusters were distributed with a regular periodicity along the cortex. The clusters of diffuse and primitive deposits did not coincide with the clusters of blood vessels in most patients. However, the clusters of classic amyloid-beta deposits coincided with those of the large diameter (>10 microm) blood vessels in all patients and with clusters of small-diameter (< 10 microm) blood vessels in four patients. The data suggest that, of the amyloid-beta subtypes, the clusters of classic amyloid-beta deposits appear to be the most closely related to blood vessels and especially to the larger-diameter, vertically penetrating arterioles in the upper cortical laminae.