Alzheimer's Disease Neocortex Research Articles

Isoform-specific upregulation of FynT kinase expression is associated with tauopathy and glial activation in Alzheimer's disease and Lewy body dementias

Cumulative data suggest the involvement of Fyn tyrosine kinase in Alzheimer's disease (AD). Previously, our group has shown increased immunoreactivities of the FynT isoform in AD neocortex (with no change in the alternatively spliced FynB isoform) which associated with neurofibrillary degeneration and reac-tive astrogliosis. Since both the aforementioned neuropathological features are also variably found in Lewy Body dementias (LBD), we investigated potential perturbations of Fyn expression in the post-mortem neocortex of patients with AD, as well as those diagnosed as having one of the two main subgroups of LBD: Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB). We found selective upregulation of FynT expression in AD, PDD, and DLB which also correlated with cognitive impairment. Furthermore, increased FynT expression correlated with hallmark neuropathological lesions, soluble β-amyloid, and phosphorylated tau, as well as markers of microglia and as-trocyte activation. In line with the human post-mortem studies, cortical FynT expression in aged mice transgenic for human P301S tau was upregulated and further correlated with accumulation of aggregated phosphorylated tau as well as with microglial and astrocytic markers. Our findings provide further evi-dence for the involvement of FynT in neurodegenerative dementias, likely via effects on tauopathy and neuroinflammation.

Isoform-specific upregulation of FynT kinase expression is associated with tauopathy and glial activation in Alzheimer's disease and Lewy body dementias.

Cumulative data suggest the involvement of Fyn tyrosine kinase in Alzheimer's disease (AD). Previously, our group has shown increased immunoreactivities of the FynT isoform in AD neocortex (with no change in the alternatively spliced FynB isoform) which associated with neurofibrillary degeneration and reactive astrogliosis. Since both the aforementioned neuropathological features are also variably found in Lewy Body dementias (LBD), we investigated potential perturbations of Fyn expression in the post‐mortem neocortex of patients with AD, as well as those diagnosed as having one of the two main subgroups of LBD: Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB). We found selective upregulation of FynT expression in AD, PDD, and DLB which also correlated with cognitive impairment. Furthermore, increased FynT expression correlated with hallmark neuropathological lesions, soluble β‐amyloid, and phosphorylated tau, as well as markers of microglia and astrocyte activation. In line with the human post‐mortem studies, cortical FynT expression in aged mice transgenic for human P301S tau was upregulated and further correlated with accumulation of aggregated phosphorylated tau as well as with microglial and astrocytic markers. Our findings provide further evidence for the involvement of FynT in neurodegenerative dementias, likely via effects on tauopathy and neuroinflammation.

MicroRNA-34a (miRNA-34a) Mediated Down-Regulation of the Post-synaptic Cytoskeletal Element SHANK3 in Sporadic Alzheimer's Disease (AD).

Integrating a combination of bioinformatics, microRNA microfluidic arrays, ELISA analysis, LED Northern, and transfection-luciferase reporter assay data using human neuronal-glial (HNG) cells in primary culture we have discovered a set of up-regulated microRNAs (miRNAs) linked to a small family of down-regulated messenger RNAs (mRNAs) within the superior temporal lobe neocortex (Brodmann A22) of sporadic Alzheimer's disease (AD) brain. At the level of mRNA abundance, the expression of a significant number of human brain genes found to be down-regulated in sporadic AD neocortex appears to be due to the increased abundance of a several brain-abundant inducible miRNAs. These up-regulated miRNAs—including, prominently, miRNA-34a—have complimentary RNA sequences in the 3′ untranslated-region (3′-UTR) of their target-mRNAs that results in the pathological down-regulation in the expression of important brain genes. An up-regulated microRNA-34a, already implicated in age-related inflammatory-neurodegeneration–appears to down-regulate key mRNA targets involved in synaptogenesis and synaptic-structure, distinguishing neuronal deficits associated with AD neuropathology. One significantly down-regulated post-synaptic element in AD is the proline-rich SH3 and multiple-ankyrin-repeat domain SHANK3 protein. Bioinformatics, microRNA array analysis and SHANK3-mRNA-3′UTR luciferase-reporter assay confirmed the importance of miRNA-34a in the regulation of SHANK3 expression in HNG cells. This paper reports on recent studies of a miRNA-34a-up-regulation coupled to SHANK3 mRNA down-regulation in sporadic AD superior-temporal lobe compared to age-matched controls. These findings further support our hypothesis of an altered miRNA-mRNA coupled signaling network in AD, much of which is supported, and here reviewed, by recently reported experimental-findings in the scientific literature.

Lysosomal cathepsin D is upregulated in Alzheimer's disease neocortex and may be a marker for neurofibrillary degeneration.

Alzheimer's disease (AD) is characterized by accumulation of β-amyloid plaques (AP) and neurofibrillary tangles (NFT) in the cortex, together with synaptic loss and amyloid angiopathy. Perturbations in the brain lysosomal system, including the cathepsin family of proteases, have been implicated in AD where they may be involved in proteolytic clearance of misfolded and abnormally aggregated peptides. However, the status of cathepsin D (catD) is unclear in Lewy body dementia, the second most common form of neurodegenerative dementia after AD, and characterized by Lewy bodies (LB) containing aggregated α-synuclein. Furthermore, earlier reports of catD changes in AD have not been entirely consistent. We measured CatD immunoreactivities in the temporal (Brodmann area BA21) and parietal (BA40) cortices of well characterized AD brains as well as two clinical subtypes of Lewy body dementia, namely Parkinson disease dementia (PDD) and dementia with Lewy bodies (DLB), known to show varying degrees of concomitant AD pathology. Increased catD immunoreactivities in AD were found for both neocortical regions measured, where they also correlated with neuropathological NFT scores and phosphorylated pSer396 tau burden, and appeared to co-localize at least partly to NFT-containing neurons. In contrast, catD was increased only in BA40 in DLB and not at all in PDD, did not correlate with LB scores, and did not appreciably co-localize with α-synuclein inclusions. Our study suggests that catD upregulation may be an adaptive response to AD-related processes leading to neurofibrillary degeneration, but may not be directly associated with formation of α-synuclein inclusions in Lewy body dementia.

Cortical amyloid burden and age moderate hippocampal activity in cognitively-normal adults.

Neurodegeneration in the medial temporal lobe, particularly in the hippocampus, is viewed as the primary source of AD-related memory deficits. Yet, in the earliest preclinical phase of Alzheimer's disease (AD), amyloid-beta (Aβ) plaques deposit primarily in the neocortex, not in the medial temporal lobe. Tau tangles, however, do often aggregate in the medial temporal lobe in parallel with amyloid deposition in the neocortex in AD. In the present study, we focused on the relationship between cortical amyloid deposition and hippocampal activity during a memory-encoding task in a sample of cognitively-normal elderly aged 60–89. We hypothesized that age would moderate the Aβ effect on hippocampal activity, and could explain some of the mixed findings in the literature. We report that high cortical Aβ load was associated with lower task-related hippocampal activity during memory encoding. Importantly, this relationship was found more evident in the younger elderly, even after controlling for subsequent recognition memory of the in-scanner task and a general episodic memory construct score. Furthermore, regional cerebrovascular reactivity measured in a subset of participants showed little role in modifying the age-dependent Aβ effect on hippocampal activity. Our findings support the idea that age is an important variable in understanding hippocampal function in preclinical AD.

An isoform-specific role of FynT tyrosine kinase in Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of dementia in old age and is characterized by the accumulation of β-amyloid plaques and neurofibrillary tangles (NFT). Recent studies suggest that Fyn tyrosine kinase forms part of a toxic triad with β-amyloid and tau in the disease process. However, it is not known whether Fyn is associated with the pathological features of AD in an isoform-specific manner. In this study, we identified selective up-regulation of the alternative-spliced FynT isoform with no change in FynB in the AD neocortex. Furthermore, gene ontology term enrichment analyses and cell type-specific localization of FynT immunoreactivity suggest that FynT up-regulation was associated with neurofibrillary degeneration and reactive astrogliosis. Interestingly, significantly increased FynT in NFT-bearing neurons was concomitant to decreased FynB immunoreactivity, suggesting an involvement of alternative splicing in NFT formation. Furthermore, cultured cells of astrocytic origin have higher FynT to FynB ratio compared to those of neuronal origin. Lastly, primary rat mixed neuron-astrocyte cultures treated with Aβ25-35 showed selective up-regulation of FynT expression in activated astrocytes. Our findings point to an isoform-specific role of FynT in modulating neurofibrillary degeneration and reactive astrogliosis in AD. Fyn kinase is known to interact with β-amyloid and tau, and contributes to Alzheimer's disease pathogenesis. In this study, it is shown that the alternatively spliced FynT isoform is specifically up-regulated in the AD neocortex, with no change in FynB isoform. The increased FynT correlated with markers of neurofibrillary degeneration and reactive astrogliosis. In primary mixed cultures, treatment with amyloid peptides specifically up-regulated FynT in activated astrocytes. This study points to altered alternative splicing as a potential pathogenic mechanism in AD.

Tissue transglutaminase is not a biochemical marker for Alzheimer's disease

Typical hallmarks of Alzheimer's disease (AD) are pathologic deposits in cortical and subcortical regions consisting of self-aggregated proteins such as amyloid-beta (Aβ) or tau. Tissue transglutaminase (tTG) catalyses calcium-dependent cross-linking between proteins (transamidation) resulting in protease-resistant isopeptide bonds. Because of this ability, tTG was suspected to participate in AD pathogenesis. Aβ and tau can be cross-linked by tTG in vitro. In AD neocortex, messenger RNA expression of tTG is increased. However, data on transamidation in AD specimens—activity of not only tTG but also other transglutaminases—are contradictory. The aim of our study was to investigate if tTG is involved in AD development and may be useful as biomarker for AD. We studied human brain samples for tTG concentration, tTG localization, and transamidation activity and cerebrospinal fluid (CSF) for tTG content by novel sensitive and highly specific methods. Neither tTG concentration nor transamidation was increased in AD brain homogenates. Immunohistologically, we found no colocalization of tTG in neocortex sections with tau or Aβ deposits but with blood vessels. Only in rare cases, tTG was detectable in CSF samples. This could be attributed to liberation from erythrocytes. Our data contradict the view that tTG is a potential biochemical marker for AD.

Disruption of neocortical histone H3 homeostasis by soluble Aβ: implications for Alzheimer's disease

Amyloid-β peptide (Aβ) fragment misfolding may play a crucial role in the progression of Alzheimer's disease (AD) pathophysiology as well as epigenetic mechanisms at the DNA and histone level. We hypothesized that histone H3 homeostasis is disrupted in association with the appearance of soluble Aβ at an early stage in AD progression. We identified, localized, and compared histone H3 modifications in multiple model systems (neural-like SH-SY5Y, primary neurons, Tg2576 mice, and AD neocortex), and narrowed our focus to investigate 3 key motifs associated with regulating transcriptional activation and inhibition: acetylated lysine 14, phosphorylated serine 10 and dimethylated lysine 9. Our results in vitro and in vivo indicate that multimeric soluble Aβ may be a potent signaling molecule indirectly modulating the transcriptional activity of DNA by modulating histone H3 homeostasis. These findings reveal potential loci of transcriptional disruption relevant to AD. Identifying genes that undergo significant epigenetic alterations in response to Aβ could aid in the understanding of the pathogenesis of AD, as well as suggesting possible new treatment strategies.

Clusterin mRNA and Protein in Alzheimer's Disease

Clusterin, a multifunctional lipoprotein is expressed in a number of tissues but expression is particularly high in the brain, where it binds to amyloid-β (Aβ), possibly facilitating Aβ transport into the bloodstream. Its concentration in peripheral blood was identified as a potential biomarker for Alzheimer's disease (AD) and predicted retention of (11)C-Pittsburgh Compound B in the temporal lobe. Single-nucleotide polymorphisms in the clusterin gene, CLU, are associated with the risk of developing AD. We measured clusterin mRNA levels in control and AD brains and investigated the relationship of the clusterin protein to soluble, insoluble, and plaque-associated Aβ. Clusterin mRNA levels were unchanged when normalized to GAPDH but modestly increased in the frontal and temporal cortex in AD in relation to NSE and MAP-2. Levels of NSE and MAP-2 mRNA were reduced in the AD frontal cortex. Clusterin protein concentration was unchanged and did not correlate with the amount of Aβ present. In the frontal cortex, clusterin concentration was higher in APOE ε4-negative brains but no effect of APOE was detected in the temporal cortex or thalamus. Overall clusterin mRNA and protein levels are unaltered in the neocortex in AD and clusterin concentration does not reflect Aβ content. The increase in clusterin noted in peripheral blood in AD may reflect increased passage of this chaperone protein across the blood-brain barrier but further work is needed to determine how CLU variants influence the development of AD.

Decreased rabphilin 3A immunoreactivity in Alzheimer's disease neocortex

Decreased rabphilin 3A immunoreactivity in Alzheimer's disease neocortex

Selective loss of P2Y2 nucleotide receptor immunoreactivity is associated with Alzheimer’s disease neuropathology

The uridine nucleotide-activated P2Y2, P2Y4 and P2Y6 receptors are widely expressed in the brain and are involved in many CNS processes, including those which malfunction in Alzheimer's disease (AD). However, the status of these receptors in the AD neocortex, as well as their putative roles in the pathogenesis of neuritic plaques and neurofibrillary tangles, remain unclear. In this study, we used immunoblotting to measure P2Y2, P2Y4 and P2Y6 receptors in two regions of the postmortem neocortex of neuropathologically assessed AD patients and aged controls. P2Y2 immunoreactivity was found to be selectively reduced in the AD parietal cortex, while P2Y4 and P2Y6 levels were unchanged. In contrast, all three receptors were preserved in the occipital cortex, which is known to be minimally affected by AD neuropathology. Furthermore, reductions in parietal P2Y2 immunoreactivity correlated both with neuropathologic scores and markers of synapse loss. These results provide a basis for considering P2Y2 receptor changes as a neurochemical substrate of AD, and point towards uridine nucleotide-activated P2Y receptors as novel targets for disease-modifying AD pharmacotherapeutic strategies.

Non-tau based neuronal degeneration in Alzheimer's disease — an immunocytochemical and quantitative study in the supragranular layers of the middle temporal neocortex

In Alzheimer's disease (AD), cortical neurons develop neurofibrillary tangles (NFTs) consisting of hyperphosphorylated tau. The neurons eventually die. There are some hints that cortical neurons may also degenerate without the development of cytoskeletal changes. We investigated this possibility by comparing changes in APP staining and neuronal size with respect to the presence or absence of hyperphosphorylated tau. Adjacent sections of the medial temporal neocortex (Brodmann's area 22) of 5 male AD patients aged 60–88 years (Braak V–VI) and 5 age-matched male non-demented control subjects were i) stained with a modified Bielschowsky silver method in order to reveal NFTs and ‘ghost’ tangles, ii) single-stained with anti-APP, and iii) double-labeled with anti-APP and AT8. Anti-APP is directed against the β-amyloid precursor protein and stains virtually all perikarya and proximal neurites of the cortical neurons. AT8 stains pre-tangles, NFTs and extracellular ‘ghost’ tangles due to the recognition of hyperphosphorylated tau. The study was focused on the supragranular cortical layers II–III, since these layers can be clearly delineated from the adjacent molecular and granular cell layers. The results showed that i) APP staining intensity in neurons was variable in the AD cortex, being clearly different from the invariably intense neuronal staining in all controls. Reduced cytoplasmic APP staining was observed, particular in small neurons, while lack of anti-APP staining in proximal neurites, too, was associated with AD. In addition, ii) cross-sectional area measurement on anti-APP-stained neurons revealed that in AD, as compared to controls, a clear decrease in the number of mainly large-sized neurons (> 150 µm 2) was accompanied by a significant increase in the percentage of neurons in the smaller size classes, indicating that many large-sized neurons became smaller in AD. iii) Reduced APP staining and decreased neuronal size were not necessarily associated with the presence or absence of hyperphosphorylated tau in these cells. iv) Twenty-six percent of the neurons contained hyperphosphorylated tau, while the level of NFT-related neuronal loss was low in AD. The present study suggests that non-tau based neuronal degeneration is a major phenomenon in the AD neocortex.

Selective effects of the APOE ε4 allele on presynaptic cholinergic markers in the neocortex of Alzheimer's disease

The effects of the APOE epsilon4 allele on a range of pre- and postsynaptic cholinergic markers were studied in a cohort of community-based Alzheimer's disease (AD) patients. Compared with age-matched controls, the postmortem AD neocortex showed decreased choline acetyltransferase (ChAT) and acetyl cholinesterase activities, lower muscarinic M2, and nicotinic alpha4beta2 receptor densities, as well as reduced M1 receptor coupling to G-proteins. However, the epsilon4 allele was dose-dependently correlated only with higher losses of ChAT activities. AD patients with two epsilon4 alleles also had more beta-amyloid containing senile plaques in the temporal cortex compared to patients with 0/1 epsilon4. This study suggests that APOE epsilon4 selectively affects presynaptic cholinergic function which may contribute to the clinical and neuropathological features of AD.

Nuclear Pore Complex Proteins in Alzheimer Disease

Ultrastructural studies of neurofibrillary tangles in Alzheimer disease (AD) have demonstrated a close relationship between nuclear pores and the cytoplasmic paired helical filaments comprising the tangles, as well as nuclear irregularity in many tangle-bearing neurons; nuclear pore aggregation has been observed in nearby neurons. These observations prompted examination of the nuclear pore complex (NPC) and proteins critical to nucleocytoplasmic transport in neurons with and without tangles in AD and control cases. Light microscopic study of hippocampus and neocortex in AD and controls revealed that all nuclei were labeled by antibodies to NPC proteins, including the central transporter nucleoporin Nup62. Nucleoporin and tau label revealed significantly more nuclear irregularity in AD, often associated with neurofibrillary tangles. Double label of Nup62 with apoptotic markers (TUNEL and active caspase-3) and a cell-cycle protein (cyclin B1) revealed no clear relationship of nuclear irregularity to apoptosis or cell-cycle protein expression. However, cytoplasmic accumulation of nuclear transport factor 2 (NTF2), a protein that transports cargo from the cytoplasm into the nucleus, was observed in a subset of hippocampal neurons with and without tangles in AD but not control cases. Further investigation of the NPC and nucleocytoplasmic transport in AD is warranted.

Impaired coupling of muscarinic M 1 receptors to G-proteins in the neocortex is associated with severity of dementia in Alzheimer's disease

Impaired transmission of acetylcholine-mediated signaling by postsynaptic muscarinic M 1 receptors has been postulated to underlie the limited efficacy of cholinergic replacement therapies in Alzheimer's disease (AD). However, a clear relationship between the functionality of M 1 receptors and dementia severity has not been demonstrated. The present study aims to measure M 1 coupling to its nucleotide binding (G-) protein in the AD neocortex, and to correlate neurochemical findings with clinical features. A cohort of dementia patients was longitudinally assessed for cognitive decline, with postmortem neuropathological confirmation of AD diagnosis. Measures of M 1 receptor density, M 1/G-protein coupling and choline acetyltransferase (ChAT) activities were performed in the frontal and temporal cortex of 24 AD patients as well as in 12 age-matched controls. We found that M 1 receptor densities were unchanged in AD, which contrasted with significantly reduced M 1 coupling to G-proteins in severely demented AD patients. Loss of M 1/G-protein coupling in the frontal cortex, but not the temporal cortex, also correlated with the rate of cognitive decline. Additionally, correlations between M 1/G-protein coupling and ChAT activities were demonstrated in both regions. These results suggest that defective coupling of neocortical M 1 receptors to G-proteins is a neurochemical substrate of cognitive decline in AD. Based on its associations with ChAT deficits and dementia severity, we propose that M 1/G-protein uncoupling may have a significant role in the disease mechanism of AD and thus may be considered to be a potential therapeutic target.

Constitutive Dyrk1A is abnormally expressed in Alzheimer disease, Down syndrome, Pick disease, and related transgenic models

DYRK1A, dual-specificity tyrosine-regulated kinase 1A, maps to human chromosome 21 within the Down syndrome (DS) critical region. Dyrk1 phosphorylates the human microtubule-associated protein tau at Thr212 in vitro, a residue that is phosphorylated in fetal tau and hyper-phosphorylated in Alzheimer disease (AD) and tauopathies, including Pick disease (PiD). Furthermore, phosphorylation of Thr212 primes tau for phosphorylation by glycogen synthase kinase 3 (GSK-3). The present study examines Dyrk1A in the cerebral cortex of sporadic AD, adult DS with associated AD, and PiD. Increased Dyrk1A immunoreactivity has been found in the cytoplasm and nuclei of scattered neurons of the neocortex, entorhinal cortex, and hippocampus in AD, DS, and PiD. Dyrk1A is found in sarkosyl-insoluble fractions which are enriched in phosphorylated tau in AD brains, thus suggesting a possible association of Dyrk1A with neurofibrillary tangle pathology. Yet, no clear relationship has been observed between tau phosphorylation at Thr212, and GSK-3 and Dyrk1A expression in diseased brains. Transgenic mice bearing a triple tau mutation (G272V, P301L, and R406W) and expressing hyper-phosphoyrylated tau in neurons of the entorhinal cortex, hippocampus, and cerebral neocortex show increased expression of Dyrk1A in individual neurons in the same regions. However, transgenic mice over-expressing Dyrk1A do not show increased phosphorylation of tau at Thr212, thus suggesting that Dyrk1A over-expression does not trigger per se hyper-phosphorylation of tau at Thr212 in vivo. The present observations indicate modifications in the expression of constitutive Dyrk1A in the cytoplasm and nuclei of neurons in various neurodegenerative diseases associated with tau phosphorylation.

Dephosphorylation of Tau by Protein Phosphatase 5: IMPAIRMENT IN ALZHEIMER'S DISEASE

Protein phosphatase (PP) 5 is highly expressed in the mammalian brain, but few physiological substrates have yet been identified. Here, we investigated the kinetics of dephosphoryation of phospho-tau by PP5 and found that PP5 had a K(m) of 8-13 microm toward tau, which is similar to that of PP2A, the major known tau phosphatase. This K(m) value is within the range of intraneuronal tau concentration in human brain, suggesting that tau could be a physiological substrate of both PP5 and PP2A. PP5 dephosphorylated tau at all 12 Alzheimer's disease (AD)-associated abnormal phosphorylation sites studied, with different efficiency toward each site. Thr(205), Thr(212), and Ser(409) of tau were the most favorable sites; Ser(199), Ser(202), Ser(214), Ser(396), and Ser(404) were less favorable sites; and Ser(262) was the poorest site for PP5. Overexpression of PP5 in PC12 cells resulted in dephosphorylation of tau at multiple phosphorylation sites. The activity but not the protein level of PP5 was found to be decreased by approximately 20% in AD neocortex. These results suggest that tau is probably a physiological substrate of PP5 and that the abnormal hyperphosphorylation of tau in AD might result in part from the decreased PP5 activity in the diseased brains.

Beta-secretase protein and activity are increased in the neocortex in Alzheimer disease.

Amyloid plaques, a major pathological feature of Alzheimer disease (AD), are composed of an internal fragment of amyloid precursor protein (APP): the 4-kd amyloid-beta protein (Abeta). The metabolic processing of APP that results in Abeta formation requires 2 enzymatic cleavage events, a gamma-secretase cleavage dependent on presenilin, and a beta-secretase cleavage by the aspartyl protease beta-site APP-cleaving enzyme (BACE). To test the hypothesis that BACE protein and activity are increased in regions of the brain that develop amyloid plaques in AD. We developed an antibody capture system to measure BACE protein level and BACE-specific beta-secretase activity in frontal, temporal, and cerebellar brain homogenates from 61 brains with AD and 33 control brains. In the brains with AD, BACE activity and protein were significantly increased (P<.001). Enzymatic activity increased by 63% in the temporal neocortex (P =.007) and 13% in the frontal neocortex (P =.003) in brains with AD, but not in the cerebellar cortex. Activity in the temporal neocortex increased with the duration of AD (P =.008) but did not correlate with enzyme-linked immunosorbent assay measures of insoluble Abeta in brains with AD. Protein level was increased by 14% in the frontal cortex of brains with AD (P =.004), with a trend toward a 15% increase in BACE protein in the temporal cortex (P =.07) and no difference in the cerebellar cortex. Immunohistochemical analysis demonstrated that BACE immunoreactivity in the brain was predominantly neuronal and was found in tangle-bearing neurons in AD. The BACE protein and activity levels are increased in brain regions affected by amyloid deposition and remain increased despite significant neuronal and synaptic loss in AD.

Cyclooxygenase-2 and Presenilin-1 Gene Expression Induced by Interleukin-1β and Amyloid β42 Peptide Is Potentiated by Hypoxia in Primary Human Neural Cells

Lipid messengers and amyloid beta (Abeta) peptides generated by cyclooxygenase-2 (COX-2) and presenilin-1 (PS1) mediate pro-inflammatory signaling and neural degeneration in Alzheimer's disease (AD) brain. This study provides data showing that the COX-2 and PS1 genes each transcribe rare, highly labile RNA species that display early response gene behavior in human neural (HN) cells in primary culture, down-regulation during human neural development, and up-regulation in AD neocortex and hippocampal CA1. Together, interleukin-1beta and amyloid beta42 peptide [IL-1beta+Abeta42] synergistically activated COX-2 and PS1 gene expression preceded by increases in AP1-, STAT1alpha-, and in particular NF-kappaBp50/p65- and HIF-1alpha-DNA binding. These events were markedly potentiated by hypoxia and blocked by the antioxidant alpha-phenyl-N-tert-butyl nitrone. Broad transcription profiling further indicated that hypoxia-induced, [IL-1beta+Abeta42]-treated HN cells display robust induction of COX-2 and PS1 as well as a pro-inflammatory gene family that includes NF-kappaBp50/p105, IL-1beta precursor, and cytosolic phospholipase A2 genes. These findings indicate a novel [IL-1beta+Abeta42]-mediated, hypoxia-enhanced, free radical-triggered gene program that drives inflammatory gene signaling and suggest a mechanism by which hypoxia during aging contributes episodically to amyloidogenesis, inflammation, and AD pathophysiology.

Hyperphosphorylation and accumulation of neurofilament proteins in Alzheimer disease brain and in okadaic acid-treated SY5Y cells

We investigated the role of neurofilament (NF) proteins in Alzheimer disease (AD) neurofibrillary degeneration. The levels and degree of phosphorylation of NF proteins in AD neocortex were determined by Western blots developed with a panel of phosphorylation-dependent NF antibodies. Levels of all three NF subunits and the degree of phosphorylation of NF-H and NF-M were significantly increased in AD as compared to Huntington disease brains used as control tissue. The increase in the levels of NF-H and NF-M was 1.7- and 1.5-fold ( P<0.01) as determined by monoclonal antibody SMI33, and was 1.6-fold ( P<0.01) in NF-L using antibody NR4. The phosphorylation of NF-H and NF-M in AD was increased respectively at the SMI31 epitope by 1.6- and 1.9-fold ( P<0.05) and at the SMI33 epitope by 2.7- and 1.3-fold ( P<0.01 and P<0.05). Essentially similar effects were observed in SY5Y human neuroblastoma cells when treated with okadaic acid, an inhibitor of protein phosphatase (PP)-2A and -1. This is the first biochemical evidence which unambiguously demonstrates the hyperphosphorylation and the accumulation of NF subunits in AD brain, and shows that the inhibition of PP-2A/PP-1 activities can lead to the hyperphosphorylation of NF-H and NF-M subunits.