RNA-binding deficient TDP-43 drives cognitive decline in a mouse model of TDP-43 proteinopathy.

TDP-43 proteinopathies including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative disorders characterized by aggregation and mislocalization of the nucleic acid-binding protein TDP-43 and subsequent neuronal dysfunction. Here, we developed endogenous models of sporadic TDP-43 proteinopathy based on the principle that disease-associated TDP-43 acetylation at lysine 145 (K145) alters TDP-43 conformation, impairs RNA-binding capacity, and induces downstream mis-regulation of target genes. Expression of acetylation-mimic TDP-43K145Q resulted in stress-induced nuclear TDP-43 foci and loss of TDP-43 function in primary mouse and human-induced pluripotent stem cell (hiPSC)-derived cortical neurons. Mice harboring the TDP-43K145Q mutation recapitulated key hallmarks of FTLD, including progressive TDP-43 phosphorylation and insolubility, TDP-43 mis-localization, transcriptomic and splicing alterations, and cognitive dysfunction. Our study supports a model in which TDP-43 acetylation drives neuronal dysfunction and cognitive decline through aberrant splicing and transcription of critical genes that regulate synaptic plasticity and stress response signaling. The neurodegenerative cascade initiated by TDP-43 acetylation recapitulates many aspects of human FTLD and provides a new paradigm to further interrogate TDP-43 proteinopathies.

Mediator kinase inhibition suppresses hyperactive interferon signaling in Down syndrome.

Hyperactive interferon (IFN) signaling is a hallmark of Down syndrome (DS), a condition caused by trisomy 21 (T21); strategies that normalize IFN signaling could benefit this population. Mediator-associated kinases CDK8 and CDK19 drive inflammatory responses through incompletely understood mechanisms. Using sibling-matched cell lines with/without T21, we investigated Mediator kinase function in the context of hyperactive IFN in DS. Activation of IFN-response genes was suppressed in cells treated with the CDK8/CDK19 inhibitor cortistatin A, and this occurred through suppression of IFN-responsive transcription factor activity. Moreover, we discovered that CDK8/CDK19 affect splicing, a novel means by which Mediator kinases control gene expression. Kinase inhibition altered splicing in pathway-specific ways and selectively affected IFN-responsive gene splicing in T21 cells. To further probe Mediator kinase function, we completed cytokine screens and untargeted metabolomics experiments. Cytokines are master regulators of inflammatory responses; by screening 105 different cytokine proteins, we show that Mediator kinases help drive IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Metabolomics revealed that Mediator kinase inhibition altered core metabolic pathways, including broad up-regulation of anti-inflammatory lipid mediators. Elevated levels of lipid mediators persisted at least 24hr after Mediator kinase inhibition, and many identified lipids serve as ligands for nuclear receptors (e.g. PPAR, LXR) or G-protein coupled receptors (GPCRs; e.g. FFAR4). Notably, ligand-dependent activation of these GPCRs or nuclear receptors will propagate anti-inflammatory signaling pathways and gene expression programs, and this mechanistic link suggests that metabolic changes caused by CDK8/CDK19 inhibition can durably and independently suppress pro-inflammatory IFN responses. Collectively, our results establish that Mediator kinase inhibition antagonizes IFN signaling through transcriptional, metabolic, and cytokine responses, with implications for DS and other chronic inflammatory conditions.

Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A.

Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.

Pathogenic SPTBN1 variants cause an autosomal dominant neurodevelopmental syndrome.

SPTBN1 encodes ßII-spectrin, the ubiquitously expressed ß-spectrin that forms micrometer-scale networks associated with plasma membranes. Mice deficient in neuronal ßII-spectrin have defects in cortical organization, developmental delay and behavioral deficiencies. These phenotypes, while less severe, are observed in haploinsufficient animals, suggesting that individuals carrying heterozygous SPTBN1 variants may also show measurable compromise of neural development and function. Here we identify heterozygous SPTBN1 variants in 29 individuals with developmental, language and motor delays; mild to severe intellectual disability; autistic features; seizures; behavioral and movement abnormalities; hypotonia; and variable dysmorphic facial features. We show that these SPTBN1 variants lead to effects that affect ßII-spectrin stability, disrupt binding to key molecular partners, and disturb cytoskeleton organization and dynamics. Our studies define SPTBN1 variants as the genetic basis of a neurodevelopmental syndrome, expand the set of spectrinopathies affecting the brain and underscore the critical role of ßII-spectrin in the central nervous system.

An HDAC6-dependent surveillance mechanism suppresses tau-mediated neurodegeneration and cognitive decline.

Tauopathies including Alzheimer's disease (AD) are marked by the accumulation of aberrantly modified tau proteins. Acetylated tau, in particular, has recently been implicated in neurodegeneration and cognitive decline. HDAC6 reversibly regulates tau acetylation, but its role in tauopathy progression remains unclear. Here, we identified an HDAC6-chaperone complex that targets aberrantly modified tau. HDAC6 not only deacetylates tau but also suppresses tau hyperphosphorylation within the microtubule-binding region. In neurons and human AD brain, HDAC6 becomes co-aggregated within focal tau swellings and human AD neuritic plaques. Using mass spectrometry, we identify a novel HDAC6-regulated tau acetylation site as a disease specific marker for 3R/4R and 3R tauopathies, supporting uniquely modified tau species in different neurodegenerative disorders. Tau transgenic mice lacking HDAC6 show reduced survival characterized by accelerated tau pathology and cognitive decline. We propose that a HDAC6-dependent surveillance mechanism suppresses toxic tau accumulation, which may protect against the progression of AD and related tauopathies.

A unique tau conformation generated by an acetylation-mimic substitution modulates P301S-dependent tau pathology and hyperphosphorylation.

Abnormal intracellular accumulation of aggregated tau is a hallmark feature of Alzheimer's disease and other tauopathies. Pathological tau can undergo a range of post-translational modifications (PTMs) that are implicated as triggers of disease pathology. Recent studies now indicate that tau acetylation, in particular, controls both microtubule binding and tau aggregation, thereby acting as a central regulator of tau's biochemical properties and providing avenues to exploit for potential therapies. Here, using cell-based assays and tau transgenic mice harboring an acetylation-mimic mutation at residue Lys-280 (K280Q), we evaluated whether this substitution modifies the neurodegenerative disease pathology associated with the aggregate-prone tau P301S variant. Strikingly, the addition of a K280Q-substituted variant altered P301S-mediated tau conformation and reduced tau hyperphosphorylation. We further evaluated neurodegeneration markers in K280Q acetylation-mimic mice and observed reduced neuroinflammation as well as restored levels of N-methyl-d-aspartate receptors and post-synaptic markers compared with the parental mice. Thus, substituting a single lysine residue in the context of a P301S disease-linked mutation produces a unique tau species that abrogates some of the cardinal features of tauopathy. The findings of our study indicate that a complex tau PTM code likely regulates tau pathogenesis, highlighting the potential utility of manipulating and detoxifying tau strains through site-specific tau-targeting approaches.

The Deacetylase HDAC6 Mediates Endogenous Neuritic Tau Pathology.

The initiating events that promote tau mislocalization and pathology in Alzheimer's disease (AD) are not well defined, partly because of the lack of endogenous models that recapitulate tau dysfunction. We exposed wild-type neurons to a neuroinflammatory trigger and examined the effect on endogenous tau. We found that tau re-localized and accumulated within pathological neuritic foci, or beads, comprised of mostly hypo-phosphorylated, acetylated, and oligomeric tau. These structures were detected in aged wild-type mice and were enhanced in response to neuroinflammation in vivo, highlighting a previously undescribed endogenous age-related tau pathology. Strikingly, deletion or inhibition of the cytoplasmic shuttling factor HDAC6 suppressed neuritic tau bead formation in neurons and mice. Using mass spectrometry-based profiling, we identified a single neuroinflammatory factor, the metalloproteinase MMP-9, as a mediator of neuritic tau beading. Thus, our study uncovers a link between neuroinflammation and neuritic tau beading as a potential early-stage pathogenic mechanism in AD.

Phytochemicals and botanical extracts regulate NF-κB and Nrf2/ARE reporter activities in DI TNC1 astrocytes.

The increase in oxidative stress and inflammatory responses associated with neurodegenerative diseases has drawn considerable attention towards understanding the transcriptional signaling pathways involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Nrf2 (Nuclear Factor Erythroid 2-like 2). Our recent studies with immortalized murine microglial cells (BV-2) demonstrated effects of botanical polyphenols to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and enhance Nrf2-mediated antioxidant responses (Sun et al., 2015). In this study, an immortalized rat astrocyte (DI TNC1) cell line expressing a luciferase reporter driven by the NF-κB or the Nrf2/Antioxidant Response Element (ARE) promoter was used to assess regulation of these two pathways by phytochemicals such as quercetin, rutin, cyanidin, cyanidin-3-O-glucoside, as well as botanical extracts from Withania somnifera (Ashwagandha), Sutherlandia frutescens (Sutherlandia) and Euterpe oleracea (Açaí). Quercetin effectively inhibited LPS-induced NF-κB reporter activity and stimulated Nrf2/ARE reporter activity in DI TNC1 astrocytes. Cyanidin and the glycosides showed similar effects but only at much higher concentrations. All three botanical extracts effectively inhibited LPS-induced NF-κB reporter activity. These extracts were capable of enhancing ARE activity by themselves and further enhanced ARE activity in the presence of LPS. Quercetin and botanical extracts induced Nrf2 and HO-1 protein expression. Interestingly, Ashwagandha extract was more active in inducing Nrf2 and HO-1 expression in DI TNC1 astrocytes as compared to Sutherlandia and Açaí extracts. In summary, this study demonstrated NF-kB and Nrf2/ARE promoter activities in DI TNC1 astrocytes, and further showed differences in ability for specific botanical polyphenols and extracts to down-regulate LPS-induced NF-kB and up-regulate the NRF2/ARE activities in these cells.

Purinergic receptors as potential therapeutic targets in Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of memory and cognitive ability and is a serious cause of mortality. Many of the pathological characteristics associated with AD are revealed post-mortem, including amyloid-ß plaque deposition, neurofibrillary tangles containing hyperphosphorylated tau proteins and neuronal loss in the hippocampus and cortex. Although several genetic mutations and risk factors have been associated with the disease, the causes remain poorly understood. Study of disease-initiating mechanisms and AD progression in humans is inherently difficult as most available tissue specimens are from late-stages of disease. Therefore, AD researchers rely on in vitro studies and the use of AD animal models where neuroinflammation has been shown to be a major characteristic of AD. Purinergic receptors are a diverse family of proteins consisting of P1 adenosine receptors and P2 nucleotide receptors for ATP, UTP and their metabolites. This family of receptors has been shown to regulate a wide range of physiological and pathophysiological processes, including neuroinflammation, and may contribute to the pathogenesis of neurodegenerative diseases like Parkinson's disease, multiple sclerosis and AD. Experimental evidence from human AD tissue has suggested that purinergic receptors may play a role in AD progression and studies using selective purinergic receptor agonists and antagonists in vitro and in AD animal models have demonstrated that purinergic receptors represent novel therapeutic targets for the treatment of AD. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.

P2Y receptors in Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia, affecting more than 10% of people over the age of 65. Age is the greatest risk factor for AD, although a combination of genetic, lifestyle and environmental factors also contribute to disease development. Common features of AD are the formation of plaques composed of beta-amyloid peptides (Aß) and neuronal death in brain regions involved in learning and memory. Although Aß is neurotoxic, the primary mechanisms by which Aß affects AD development remain uncertain and controversial. Mouse models overexpressing amyloid precursor protein and Aß have revealed that Aß has potent effects on neuroinflammation and cerebral blood flow that contribute to AD progression. Therefore, it is important to consider how endogenous signalling in the brain responds to Aß and contributes to AD pathology. In recent years, Aß has been shown to affect ATP release from brain and blood cells and alter the expression of G protein-coupled P2Y receptors that respond to ATP and other nucleotides. Accumulating evidence reveals a prominent role for P2Y receptors in AD pathology, including Aß production and elimination, neuroinflammation, neuronal function and cerebral blood flow.

Beneficial effects of dietary EGCG and voluntary exercise on behavior in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is a progressive, age-dependent neurodegenerative disorder affecting specific brain regions that control memory and cognitive functions. Epidemiological studies suggest that exercise and dietary antioxidants are beneficial in reducing AD risk. To date, botanical flavonoids are consistently associated with the prevention of age-related diseases. The present study investigated the effects of 4 months of wheel-running exercise, initiated at 2-months of age, in conjunction with the effects of the green tea catechin (-)-epigallocatechin-3-gallate (EGCG) administered orally in the drinking water (50 mg/kg daily) on: (1) behavioral measures: learning and memory performance in the Barnes maze, nest building, open-field, anxiety in the light-dark box; and (2) soluble amyloid-ß (Aß) levels in the cortex and hippocampus in TgCRND8 (Tg) mice. Untreated Tg mice showed hyperactivity, relatively poor nest building behaviors, and deficits in spatial learning in the Barnes maze. Both EGCG and voluntary exercise, separately and in combination, were able to attenuate nest building and Barnes maze performance deficits. Additionally, these interventions lowered soluble Aß1-42 levels in the cortex and hippocampus. These results, together with epidemiological and clinical studies in humans, suggest that dietary polyphenols and exercise may have beneficial effects on brain health and slow the progression of AD.

Loss of P2Y2 nucleotide receptors enhances early pathology in the TgCRND8 mouse model of Alzheimer's disease.

Neuroinflammation is a prominent feature in Alzheimer's disease (AD) and activation of the brain's innate immune system, particularly microglia, has been postulated to both retard and accelerate AD progression. Recent studies indicate that the G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is an important regulator of innate immunity by assisting in the recruitment of monocytes to injured tissue, neutrophils to bacterial infections and eosinophils to allergen-infected lungs. In this study, we investigated the role of the P2Y2R in progression of an AD-like phenotype in the TgCRND8 mouse model that expresses Swedish and Indiana mutations in amyloid precursor protein (APP). Our results indicate that P2Y 2 R expression is upregulated in TgCRND8 mouse brain within 10 weeks of age and then decreases after 25 weeks of age, as compared to littermate controls expressing low levels of the P2Y 2 R. TgCRND8 mice with homozygous P2Y 2 R deletion survive less than 5 weeks, whereas mice with heterozygous P2Y 2 R deletion survive for 12 weeks, a time point when TgCRND8 mice are fully viable. Heterozygous P2Y 2 R deletion in TgCRND8 mice increased ß-amyloid (Aß) plaque load and soluble Aß1-42 levels in the cerebral cortex and hippocampus, decreased the expression of the microglial marker CD11b in these brain regions and caused neurological deficits within 10 weeks of age, as compared to age-matched TgCRND8 mice. These findings suggest that the P2Y2R is important for the recruitment and activation of microglial cells in the TgCRND8 mouse brain and that the P2Y2R may regulate neuroprotective mechanisms through microglia-mediated clearance of Aß that when lost can accelerate the onset of an AD-like phenotype in the TgCRND8 mouse.

Up-regulation and activation of the P2Y(2) nucleotide receptor mediate neurite extension in IL-1ß-treated mouse primary cortical neurons.

The pro-inflammatory cytokine interleukin-1ß (IL-1ß), whose levels are elevated in the brain in Alzheimer's and other neurodegenerative diseases, has been shown to have both detrimental and beneficial effects on disease progression. In this article, we demonstrate that incubation of mouse primary cortical neurons (mPCNs) with IL-1ß increases the expression of the P2Y2 nucleotide receptor (P2Y2R) and that activation of the up-regulated receptor with UTP, a relatively selective agonist of the P2Y2R, increases neurite outgrowth. Consistent with the accepted role of cofilin in the regulation of neurite extension, results indicate that incubation of IL-1ß-treated mPCNs with UTP increases the phosphorylation of cofilin, a response absent in PCNs isolated from P2Y2R(-/-) mice. Other findings indicate that function-blocking anti-αv ß3/5 integrin antibodies prevent UTP-induced cofilin activation in IL-1ß-treated mPCNs, suggesting that established P2Y2R/αv ß3/5 interactions that promote G12 -dependent Rho activation lead to cofilin phosphorylation involved in neurite extension. Cofilin phosphorylation induced by UTP in IL-1ß-treated mPCNs is also decreased by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), suggesting a role for P2Y2R-mediated and Gq-dependent calcium mobilization in neurite outgrowth. Taken together, these studies indicate that up-regulation of P2Y2Rs in mPCNs under pro-inflammatory conditions can promote cofilin-dependent neurite outgrowth, a neuroprotective response that may be a novel pharmacological target in the treatment of neurodegenerative diseases.

P2 receptors for extracellular nucleotides in the central nervous system: role of P2X7 and P2Y2 receptor interactions in neuroinflammation.

Extracellular nucleotides induce cellular responses in the central nervous system (CNS) through the activation of ionotropic P2X and metabotropic P2Y nucleotide receptors. Activation of these receptors regulates a wide range of physiological and pathological processes. In this review, we present an overview of the current literature regarding P2X and P2Y receptors in the CNS with a focus on the contribution of P2X7 and P2Y(2) receptor-mediated responses to neuroinflammatory and neuroprotective mechanisms.

Neuroprotective roles of the P2Y(2) receptor.

Purinergic signaling plays a unique role in the brain by integrating neuronal and glial cellular circuits. The metabotropic P1 adenosine receptors and P2Y nucleotide receptors and ionotropic P2X receptors control numerous physiological functions of neuronal and glial cells and have been implicated in a wide variety of neuropathologies. Emerging research suggests that purinergic receptor interactions between cells of the central nervous system (CNS) have relevance in the prevention and attenuation of neurodegenerative diseases resulting from chronic inflammation. CNS responses to chronic inflammation are largely dependent on interactions between different cell types (i.e., neurons and glia) and activation of signaling molecules including P2X and P2Y receptors. Whereas numerous P2 receptors contribute to functions of the CNS, the P2Y(2) receptor is believed to play an important role in neuroprotection under inflammatory conditions. While acute inflammation is necessary for tissue repair due to injury, chronic inflammation contributes to neurodegeneration in Alzheimer's disease and occurs when glial cells undergo prolonged activation resulting in extended release of proinflammatory cytokines and nucleotides. This review describes cell-specific and tissue-integrated functions of P2 receptors in the CNS with an emphasis on P2Y(2) receptor signaling pathways in neurons, glia, and endothelium and their role in neuroprotection.

Nucleotides released from Aß1₋42 -treated microglial cells increase cell migration and Aß1₋42 uptake through P2Y2 receptor activation.

Amyloid ß-protein (Aß) deposits in brains of Alzheimer's disease patients generate proinflammatory cytokines and chemokines that recruit microglial cells to phagocytose Aß. Nucleotides released from apoptotic cells activate P2Y(2) receptors (P2Y(2) Rs) in macrophages to promote clearance of dead cells. In this study, we investigated the role of P2Y(2) Rs in the phagocytosis and clearance of Aß. Treatment of mouse primary microglial cells with fibrillar (fAß(1-42) ) and oligomeric (oAß(1-42) ) Aß(1-42) aggregation solutions caused a rapid release of ATP (maximum after 10 min). Furthermore, fAß(1-42) and oAß(1-42) treatment for 24 h caused an increase in P2Y(2) R gene expression. Treatment with fAß(1-42) and oAß(1-42) aggregation solutions increased the motility of neighboring microglial cells, a response inhibited by pre-treatment with apyrase, an enzyme that hydrolyzes nucleotides. The P2Y(2) R agonists ATP and UTP caused significant uptake of Aß(1-42) by microglial cells within 30 min, which reached a maximum within 1 h, but did not increase Aß(1-42) uptake by primary microglial cells isolated from P2Y(2) R(-/-) mice. Inhibitors of α(v) integrins, Src and Rac decreased UTP-induced Aß(1-42) uptake, suggesting that these previously identified components of the P2Y(2) R signaling pathway play a role in Aß phagocytosis by microglial cells. Finally, we found that UTP treatment enhances Aß(1-42) degradation by microglial cells, but not in cells isolated from P2Y(2) R(-/-) mice. Taken together, our findings suggest that P2Y(2) Rs can activate microglial cells to enhance Aß clearance and highlight the P2Y(2) R as a therapeutic target in Alzheimer's disease.

Pro-inflammatory cytokines and lipopolysaccharide induce changes in cell morphology, and upregulation of ERK1/2, iNOS and sPLA2-IIA expression in astrocytes and microglia.

BACKGROUND: Activation of glial cells, including astrocytes and microglia, has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including Alzheimer's and Parkinson's diseases. Although cultured astrocytes and microglia are capable of responding to pro-inflammatory cytokines and lipopolysaccharide (LPS) in the induction and release of inflammatory factors, no detailed analysis has been carried out to compare the induction of iNOS and sPLA2-IIA. In this study, we investigated the effects of cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma to induce temporal changes in cell morphology and induction of p-ERK1/2, iNOS and sPLA2-IIA expression in immortalized rat (HAPI) and mouse (BV-2) microglial cells, immortalized rat astrocytes (DITNC), and primary microglia and astrocytes. METHODS/RESULTS: Cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma induced a time-dependent increase in fine processes (filopodia) in microglial cells but not in astrocytes. Filopodia production was attributed to IFN-gamma and was dependent on ERK1/2 activation. Cytokines induced an early (15 min) and a delayed phase (1 ~ 4 h) increase in p-ERK1/2 expression in microglial cells, and the delayed phase increase corresponded to the increase in filopodia production. In general, microglial cells are more active in responding to cytokines and LPS than astrocytes in the induction of NO. Although IFN-gamma and LPS could individually induce NO, additive production was observed when IFN-gamma was added together with LPS. On the other hand, while TNF-alpha, IL-1beta, and LPS could individually induce sPLA2-IIA mRNA and protein expression, this induction process does not require IFN-gamma. Interestingly, neither rat immortalized nor primary microglial cells were capable of responding to cytokines and LPS in the induction of sPLA2-IIA expression. CONCLUSION: These results demonstrated the utility of BV-2 and HAPI cells as models for investigation on cytokine and LPS induction of iNOS, and DITNC astrocytes for induction of sPLA2-IIA. In addition, results further demonstrated that cytokine-induced sPLA2-IIA is attributed mainly to astrocytes and not microglial cells.

Probing the amyloid-beta(1-40) fibril environment with substituted tryptophan residues.

A signature feature of Alzheimer's disease is the accumulation of plaques, composed of fibrillar amyloid-beta protein (Abeta), in the brain parenchyma. Structural models of Abeta fibrils reveal an extensive beta-sheet network with a hydrophobic core extending throughout the fibril axis. In this study, phenylalanines in the Abeta(1-40) sequence were substituted with tryptophan residues at either position 4 (F4W) or 19 (F19W) to probe the fibril environment. The F4W substitution did not alter self-assembly kinetics, while the F19W change slightly lengthened the lag phase without hindering fibril formation. The tryptophan fluorescence of Abeta(1-40) F19W, but not Abeta(1-40) F4W, underwent a marked blue shift during fibril formation and this shift was temporally correlated with thioflavin T binding. Isolated Abeta(1-40) F19W fibrils exhibited the largest fluorescence blue shifts consistent with W19 insertion into the Abeta(1-40) fibril inner core and direct probing of the substantially hydrophobic environment therein.

Amyloid-beta(1-42) fibrillar precursors are optimal for inducing tumor necrosis factor-alpha production in the THP-1 human monocytic cell line.

Pathological studies have determined that fibrillar forms of amyloid-beta protein (Abeta) comprise the characteristic neuritic plaques in Alzheimer's disease (AD). These studies have also revealed significant inflammatory markers such as activated microglia and cytokines surrounding the plaques. Although the plaques are a hallmark of AD, they are only part of an array of Abeta aggregate morphologies observed in vivo. Interestingly, not all of these Abeta deposits provoke an inflammatory response. Since structural polymorphism is a prominent feature of Abeta aggregation both in vitro and in vivo, we sought to clarify which Abeta morphology or aggregation species induces the strongest proinflammatory response using human THP-1 monocytes as a model system. An aliquot of freshly reconstituted Abeta(1-42) in sterile water (100 microM, pH 3.6) did not effectively stimulate the cells at a final Abeta concentration of 15 microM. However, quiescent incubation of the peptide at 4 degrees C for 48-96 h greatly enhanced its ability to induce tumor necrosis factor-alpha (TNFalpha) production, the level of which surprisingly declined upon further aggregation. Imaging of the Abeta(1-42) aggregation solutions with atomic force microscopy indicated that the best cellular response coincided with the appearance of fibrillar structures, yet conditions that accelerated or increased the level of Abeta(1-42) fibril formation such as peptide concentration, temperature, or reconstitution in NaOH/PBS at pH 7.4 diminished its ability to stimulate the cells. Finally, depletion of the Abeta(1-42) solution with an antibody that recognizes fibrillar oligomers dramatically weakened the ability to induce TNFalpha production, and size-exclusion separation of the Abeta(1-42) solution provided further characterization of an aggregated species with proinflammatory activity. The findings suggested that an intermediate stage Abeta(1-42) fibrillar precursor is optimal for inducing a proinflammatory response in THP-1 monocytes.

Oligomeric amyloid-beta(1-42) induces THP-1 human monocyte adhesion and maturation.

Amyloid-beta (Abeta) is a naturally occurring 40- or 42-residue peptide fragment with a primary role in Alzheimer's disease (AD). Aggregated Abeta accumulates as both dense core plaques and diffuse deposits in the brains of AD patients. Abeta plaques are surrounded by activated microglia, some of which are believed to be derived from peripheral blood monocytes that have infiltrated the central nervous system and differentiated into phagocytes in response to Abeta. We have modeled this process using THP-1 human monocytes and found Abeta(1-42) to be as effective as phorbol myristate acetate at differentiating THP-1 monocytes based on cell adhesion, fibronectin binding, CD11b cell-surface expression, and morphological changes. Cell adhesion studies and atomic force microscopy imaging revealed an inverse correlation between Abeta(1-42)-induced monocyte maturation and aggregation progression. Freshly reconstituted Abeta(1-42) solutions were the most effective, yet continued aggregation reduced, and eventually abolished, the ability to induce monocyte adhesion. Abeta(1-40), lower aggregation concentrations of Abeta(1-42), and an aggregation-restricted Abeta(1-42) L34P mutant had little effect on monocyte adhesion under the same conditions as Abeta(1-42). These findings implicated an oligomeric, but not monomeric or fibrillar, Abeta(1-42) aggregation species in the monocyte maturation process. The rapidly-formed Abeta(1-42) oligomers were distinct from Abeta-derived diffusible ligands which did not elicit significant THP-1 monocyte adhesion. These data demonstrate that a specific oligomeric Abeta(1-42) aggregation species can potently initiate the THP-1 monocyte maturation process.