Dopamine signaling modulates microglial NLRP3 inflammasome activation: implications for Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is characterized by the loss of nigral dopaminergic neurons leading to impaired striatal dopamine signaling, α-synuclein- (α-syn-) rich inclusions, and neuroinflammation. Degenerating neurons are surrounded by activated microglia with increased secretion of interleukin-1ß (IL-1ß), driven largely by the NLRP3 inflammasome. A critical role for microglial NLRP3 inflammasome activation in the progression of both dopaminergic neurodegeneration and α-syn pathology has been demonstrated in parkinsonism mouse models. Fibrillar α-syn activates this inflammasome in mouse and human macrophages, and we have shown previously that the same holds true for primary human microglia. Dopamine blocks microglial NLRP3 inflammasome activation in the MPTP model, but its effects in this framework, highly relevant to PD, remain unexplored in primary human microglia and in other in vivo parkinsonism models. METHODS: Biochemical techniques including quantification of IL-1ß secretion and confocal microscopy were employed to gain insight into dopamine signaling-mediated inhibition of the NLRP3 inflammasome mechanism in primary human microglia and the SYN120 transgenic mouse model. Dopamine and related metabolites were applied to human microglia together with various inflammasome activating stimuli. The involvement of the receptors through which these catecholamines were predicted to act were assessed with agonists in both species. RESULTS: We show in primary human microglia that dopamine, L-DOPA, and high extracellular K+, but not norepinephrine and epinephrine, block canonical, non-canonical, and α-syn-mediated NLRP3 inflammasome-driven IL-1ß secretion. This suggests that dopamine acts as an inflammasome inhibitor in human microglia. Accordingly, we provide evidence that dopamine exerts its inhibitory effect through dopamine receptor D1 and D2 (DRD1 and DRD2) signaling. We also show that aged mice transgenic for human C-terminally truncated (1-120) α-syn (SYN120 tg mice) display increased NLRP3 inflammasome activation in comparison to WT mice that is diminished upon DRD1 agonism. CONCLUSIONS: Dopamine inhibits canonical, non-canonical, and α-syn-mediated activation of the NLRP3 inflammasome in primary human microglia, as does high extracellular K+. We suggest that dopamine serves as an endogenous repressor of the K+ efflux-dependent microglial NLRP3 inflammasome activation that contributes to dopaminergic neurodegeneration in PD, and that this reciprocation may account for the specific vulnerability of these neurons to disease pathology.

α-Synuclein evokes NLRP3 inflammasome-mediated IL-1ß secretion from primary human microglia.

Synucleinopathies such as Parkinson's disease (PD) are hallmarked by α-synuclein (α-syn) pathology and neuroinflammation. This neuroinflammation involves activated microglia with increased secretion of interleukin-1ß (IL-1ß). The main driver of IL-1ß secretion from microglia is the NLRP3 inflammasome. A critical link between microglial NLRP3 inflammasome activation and the progression of both α-syn pathology and dopaminergic neurodegeneration has been identified in various PD models in vivo. α-Syn is known to activate the microglial NLRP3 inflammasome in murine models, but its relationship to this inflammasome in human microglia has not been established. In this study, IL-1ß secretion from primary mouse microglia induced by α-syn fibrils was dependent on NLRP3 inflammasome assembly and caspase-1 activity, as previously reported. We show that exposure of primary human microglia to α-syn fibrils also resulted in significant IL-1ß secretion that was dependent on inflammasome assembly and involved the recruitment of caspase-1 protein to inflammasome scaffolds as visualized with superresolution microscopy. While canonical IL-1ß secretion was clearly dependent on caspase-1 enzymatic activity, this activity was less clearly involved for α-syn-induced IL-1ß secretion from human microglia. This work presents similarities between primary human and mouse microglia in the mechanisms of activation of the NLRP3 inflammasome by α-syn, but also highlights evidence to suggest that there may be a difference in the requirement for caspase-1 activity in IL-1ß output. The data represent a novel characterization of PD-related NLRP3 inflammasome activation in primary human microglia and further implicate this mechanism in the pathology underlying PD.

Impaired Innate Immunity Mechanisms in the Brain of Alzheimer's Disease.

Among environmental factors likely associated with Alzheimer's disease (AD), persistent virus infections, and age-related progressive decline of immune competence might play a pivotal role. However, AD antimicrobial brain immune responses are poorly investigated. The present study focused on genes involved in antimicrobial defenses, especially against virus infections, in the AD brain. In particular, mRNA levels of IRF7, MED23, IL28B, and IFN-α genes were analyzed in hippocampus and temporal cortex brain samples from AD and non-demented controls. All subjects were also genotyped for APOE ε, IRF7, MED23, and IL28B gene polymorphisms. Most AD patients showed decreased mRNA levels of all investigated genes in the hippocampus and temporal cortex. However, a small group of AD patients showed increased hippocampal mRNA expression of MED23, IL28B, and IFN-α. mRNA levels of MED23, IL28B, IFN-α from the hippocampus and those of MED23 from the temporal cortex were further decreased in APOE ε4 allele AD carriers. Moreover, rs6598008 polymorphism of IRF7 was significantly associated with decreased hippocampal expression of IRF7, MED23, IL28B, and IFN-α. These findings suggest that AD brains show impaired innate antimicrobial gene expression profiles, and individual genetic makeup, such as positivity for the APOE ε4 and IRF7 A alleles, might affect brain immune efficiency.

Aß-oligomer uptake and the resulting inflammatory response in adult human astrocytes are precluded by an anti-Aß single chain variable fragment in combination with an apoE mimetic peptide.

An imbalance between production and clearance of soluble amyloid-ß (Aß) initiates the pathological process in sporadic Alzheimer's disease (AD). Aß-specific antibodies seemed promising as therapeutic option in AD mouse models. In patients, however, vascular side-effects and Aß-antibody complex-induced microglial and/or perivascular macrophage inflammatory responses were encountered. To prevent inflammatory reactions, we designed a single chain variable fragment (scFv-h3D6), based on monoclonal antibody bapineuzumab (mAb-h3D6), but lacking the Fc region. ScFv-h3D6 reduced Aß-oligomer burden and prevented AD-associated behavioral and cellular changes in 3xTg-AD mice. As scFv-h3D6 lacks the Fc-tail, it cannot enhance Fc-receptor mediated Aß clearance by microglia and probably exerts its beneficial effects in 3xTg-AD mice through other mechanisms. ScFv-h3D6 restored the increased apoE and apoJ levels in 3xTg-AD brains back to normal. ApoE and apoJ influence cholesterol transport, Aß aggregation and clearance, and their genetic variants are risk factors for sporadic AD. Astrocytes are constitutive scavengers of soluble Aß from the CNS. We previously found apoE and apoJ to inhibit Aß uptake by adult human astrocytes, in vitro, and thus to potentially protect astrocytes from Aß cytotoxicity. In the present study, scFv-h3D6 and mAb-h3D6 inhibited Aß-oligomer uptake by adult human astrocytes. ApoE- and apoJ- mimetic peptides (MP) affected Aß uptake as well as Aß-induced cytokine release similar to intact apoE and apoJ, without interfering with the strong inhibitory effects of scFv-h3D6 on Aß-oligomer uptake. These results suggest that combining Aß-specific scFv and apoE-MP, that inhibits Aß oligomer-induced cytokine release by astrocytes, could offer advantages over currently used therapeutics.

Complement activation by ceramide transporter proteins.

C1q is the initiator of the classical complement pathway and, as such, is essential for efficient opsonization and clearance of pathogens, altered self-structures, and apoptotic cells. The ceramide transporter protein (CERT) and its longer splicing isoform CERTL are known to interact with extracellular matrix components, such as type IV collagen, and with the innate immune protein serum amyloid P. In this article, we report a novel function of CERT in the innate immune response. Both CERT isoforms, when immobilized, were found to bind the globular head region of C1q and to initiate the classical complement pathway, leading to activation of C4 and C3, as well as generation of the membrane attack complex C5b-9. In addition, C1q was shown to bind to endogenous CERTL on the surface of apoptotic cells. These results demonstrate the role of CERTs in innate immunity, especially in the clearance of apoptotic cells.

Apolipoproteins E and J interfere with amyloid-beta uptake by primary human astrocytes and microglia in vitro.

Defective clearance of the amyloid-ß peptide (Aß) from the brain is considered a strong promoter in Alzheimer's disease (AD) pathogenesis. Astrocytes and microglia are important mediators of Aß clearance and Aß aggregation state and the presence of amyloid associated proteins (AAPs), such as Apolipoproteins E and J (ApoE and ApoJ), may influence Aß clearance by these cells. Here we set out to investigate whether astrocytes and microglia differ in uptake efficiency of Aß oligomers (Aßoligo ) and Aß fibrils (Aßfib ), and whether the Aß aggregation state and/or presence of AAPs affect Aß uptake in these cells in vitro. Adult human primary microglia and astrocytes, isolated from short delay post-mortem brain tissue, were exposed to either Aßoligo or Aßfib alone or combined with a panel of certain AAPs whereafter Aß-positive cells were quantified using flow cytometry. Upon exposure to Aß combined with ApoE, ApoJ, α1-antichymotrypsin (ACT) and a combination of serum amyloid P and complement C1q (SAP-C1q), a clear reduction in astrocytic but not microglial Aßoligo uptake, was observed. In contrast, Aßfib uptake was strongly reduced in the presence of AAPs in microglia, but not in astrocytes. These data provide the first evidence of distinct roles of microglia and astrocytes in Aß clearance. More importantly we show that Aß clearance by glial cells is negatively affected by AAPs like ApoE and ApoJ. Thus, targeting the association of Aß with AAPs, such as ApoE and ApoJ, could serve as a therapeutic strategy to increase Aß clearance by glial cells.

Goodpasture antigen-binding protein/ceramide transporter binds to human serum amyloid P-component and is present in brain amyloid plaques.

Serum amyloid P component (SAP) is a non-fibrillar glycoprotein belonging to the pentraxin family of the innate immune system. SAP is present in plasma, basement membranes, and amyloid deposits. This study demonstrates, for the first time, that the Goodpasture antigen-binding protein (GPBP) binds to human SAP. GPBP is a nonconventional Ser/Thr kinase for basement membrane type IV collagen. Also GPBP is found in plasma and in the extracellular matrix. In the present study, we demonstrate that GPBP specifically binds SAP in its physiological conformations, pentamers and decamers. The START domain in GPBP is important for this interaction. SAP and GPBP form complexes in blood and partly colocalize in amyloid plaques from Alzheimer disease patients. These data suggest the existence of complexes of SAP and GPBP under physiological and pathological conditions. These complexes are important for understanding basement membrane, blood physiology, and plaque formation in Alzheimer disease.

Discriminatory and predictive capabilities of enzyme-linked immunosorbent assay and multiplex platforms in a longitudinal Alzheimer's disease study.

BACKGROUND: Multiplex assays such as xMAP have been proposed for the assessment of Alzheimer's disease (AD) biomarkers amyloid ß 42 (Aß42), tau (Tau), and phosphorylated tau (pTau) in cerebrospinal fluid (CSF). Here, we compared the traditional enzyme-linked immunosorbent assay (ELISA) and xMAP with respect to their: (1) absolute biomarker concentration, (2) ability to distinguish AD from nondemented subjects, (3) ability to monitor AD longitudinally, and (4) ability to predict progression from mild cognitive impairment (MCI) to AD. METHODS: We selected 68 AD, 62 MCI, and 24 nondemented subjects, performed clinical examinations, and obtained CSF at baseline and 2 years later. Aß42, Tau, and pTau were measured with both ELISA and xMAP. RESULTS: Biomarker levels differed considerably between the two assays, and the differences were concentration dependent. No differences were observed in ability to distinguish nondemented subjects from AD patients between ELISA (area under curve of 0.84 for Aß42, 0.79 for Tau, and 0.75 for pTau) and xMAP (area under curve of 0.82 for Aß42, 0.75 for Tau, and 0.73 for pTau), all P < .05. Increased Aß42 levels of AD patients at follow-up compared with baseline were detected with ELISA, whereas increased Tau levels for nondemented subjects and MCI patients were only detected with xMAP. The hazard ratios for progression from MCI to AD did not differ between the assays. CONCLUSION: Both ELISA and multiplex assays can be used to measure AD biomarker levels in CSF to support clinical diagnosis and predict progression from MCI to AD with similar accuracy. Importantly, the assays' output in absolute biomarker concentrations is remarkably different, and this discrepancy cannot be reconciled with simple correction factors.

LRRK2 Kinase Inhibition Attenuates Astrocytic Activation in Response to Amyloid ß1-42 Fibrils.

Intracerebral accumulation of amyloid-ß in the extracellular plaques of Alzheimer's disease (AD) brains represents the main cause of reactive astrogliosis and neuroinflammatory response. Of relevance, leucine-rich repeat kinase 2 (LRRK2), a kinase linked to genetic and sporadic Parkinson's disease (PD), has been identified as a positive mediator of neuroinflammation upon different inflammatory stimuli, however its pathogenicity in AD remains mainly unexplored. In this study, by using pharmacological inhibition of LRRK2 and murine primary astrocytes, we explored whether LRRK2 regulates astrocytic activation in response to amyloid-ß1-42 (Aß1-42). Our results showed that murine primary astrocytes become reactive and recruit serine 935 phosphorylated LRRK2 upon Aß1-42 fibril exposure. Moreover, we found that pharmacological inhibition of LRRK2, with two different kinase inhibitors, can attenuate Aß1-42-mediated inflammation and favor the clearance of Aß1-42 fibrils in astrocytes. Overall, our findings report that LRRK2 kinase activity modulates astrocytic reactivity and functions in the presence of Aß1-42 deposits and indicate that PD-linked LRRK2 might contribute to AD-related neuroinflammation and pathogenesis.

Small heat shock proteins induce a cerebral inflammatory reaction.

More than 80% of Alzheimer's disease (AD) patients have some degree of cerebral amyloid angiopathy (CAA). In addition to arteries and veins, capillaries can also be affected. Capillary CAA (capCAA), rather than CAA in larger vessels, is associated with flame-like amyloid-beta (Aß) deposits that may extend beyond the vessel wall and radiate into the neuropil, a phenomenon also known as "dyshoric angiopathy." Aß deposits in AD, parenchymal as well as (cap)CAA and dyshoric angiopathy, are associated with a local inflammatory reaction, including activation of microglial cells and astrocytes that, among others, produce cytokines and reactive oxygen species. This neuroinflammatory reaction may account for at least part of the cognitive decline. In previous studies we observed that small heat shock proteins (sHsps) are associated with Aß deposits in AD. In this study the molecular chaperones Hsp20, HspB8 and HspB2B3 were found to colocalize with CAA and capCAA in AD brains. In addition, Hsp20, HspB8 and HspB2B3 colocalized with intercellular adhesion molecule 1 (ICAM-1) in capCAA-associated dyshoric angiopathy. Furthermore, we demonstrated that Hsp20, HspB8 and HspB2B3 induced production of interleukin 8, soluble ICAM-1 and monocyte chemoattractant protein 1 by human leptomeningeal smooth muscle cells and human brain astrocytes in vitro and that Hsp27 inhibited production of transforming growth factor beta 1 and CD40 ligand. Our results suggest a central role for sHsps in the neuroinflammatory reaction in AD and CAA and thus in contributing to cognitive decline.

The potential convergence of NLRP3 inflammasome, potassium, and dopamine mechanisms in Parkinson's disease.

The pathology of Parkinson's disease (PD) is characterized by α-synuclein aggregation, microglia-mediated neuroinflammation, and dopaminergic neurodegeneration in the substantia nigra with collateral striatal dopamine signaling deficiency. Microglial NLRP3 inflammasome activation has been linked independently to each of these facets of PD pathology. The voltage-gated potassium channel Kv1.3, upregulated in microglia by α-synuclein and facilitating potassium efflux, has also been identified as a modulator of neuroinflammation and neurodegeneration in models of PD. Evidence increasingly suggests that microglial Kv1.3 is mechanistically coupled with NLRP3 inflammasome activation, which is contingent on potassium efflux. Potassium conductance also influences dopamine release from midbrain dopaminergic neurons. Dopamine, in turn, has been shown to inhibit NLRP3 inflammasome activation in microglia. In this review, we provide a literature framework for a hypothesis in which Kv1.3 activity-induced NLRP3 inflammasome activation, evoked by stimuli such as α-synuclein, could lead to microglia utilizing dopamine from adjacent dopaminergic neurons to counteract this process and fend off an activated state. If this is the case, a sufficient dopamine supply would ensure that microglia remain under control, but as dopamine is gradually siphoned from the neurons by microglial demand, NLRP3 inflammasome activation and Kv1.3 activity would progressively intensify to promote each of the three major facets of PD pathology: α-synuclein aggregation, microglia-mediated neuroinflammation, and dopaminergic neurodegeneration. Risk factors overlapping to varying degrees to render brain regions susceptible to such a mechanism would include a high density of microglia, an initially sufficient supply of dopamine, and poor insulation of the dopaminergic neurons by myelin.

The pattern of inflammatory markers during electroconvulsive therapy in older depressed patients.

OBJECTIVES: An association is found between changes in cytokine levels and antidepressant treatment outcome. Also, a proinflammatory profile is associated with a favourable electroconvulsive therapy (ECT) outcome. This paper investigates the pattern of inflammatory markers during a course of ECT in older depressed patients and whether this pattern is associated with ECT outcome. We hypothesised that ECT has an anti-inflammatory effect. METHODS: The pattern of CRP, IL-6, IL-10, and TNF-α during a course of ECT was examined using longitudinal mixed model analyses. Serum samples were collected in 99 older depressed patients (mean age: 72.8 ± 8.3 years, MADRS score 33.8 ± 9.0). RESULTS: After Bonferroni correction, there were no statistically significant alterations in levels of inflammatory markers during and after ECT. Effect sizes (Cohen's d) were -0.29 for CRP, -0.13 for IL-6, -0.06 for IL-10, and -0.07 for TNF-α. Changes in CRP or cytokine levels did not differ between remitters and non-remitters. Median baseline levels of CRP were significantly higher in remitters. CONCLUSIONS: A small to medium effect size towards decreased CRP and IL-6 levels was observed. An anti-inflammatory effect of ECT could not be confirmed. However, the findings may suggest that patients with an inflammatory profile benefit more from ECT than other patients. Further studies are needed to confirm these findings.

Inflammation and Cognitive Functioning in Depressed Older Adults Treated With Electroconvulsive Therapy: A Prospective Cohort Study.

Objective: Despite the effectiveness of electroconvulsive therapy (ECT), patients and practitioners are often reluctant to start it due to the risk of transient cognitive side effects, particularly in older patients. Inflammatory processes may be associated with the occurrence of these effects. This study assessed whether inflammatory markers prior to ECT are associated with cognitive functioning in depressed patients treated with ECT.Methods: Between 2011 and 2013, 97 older patients (mean [SD] age = 73.1 [8.1] years) with severe unipolar depression (according to DSM-IV) referred for ECT were included. Mini-Mental State Examination (MMSE) scores were used to determine cognitive functioning prior to, weekly during, and in the first week after a course of ECT. Serum levels of C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) were assessed prior to ECT.Results: In fully adjusted models, there was an association between TNF-α and cognitive functioning (ß = -1.05; 95% CI, -2.04 to -0.06; f2 = 0.06). An association was also found between baseline levels of IL-10 and TNF-α and lower MMSE scores during ECT (IL-10: ß = -2.08; 95% CI, -3.22 to -0.95; TNF-α: ß = -0.65; 95% CI, -1.07 to -0.22). In addition, an association was found between baseline CRP and lower MMSE scores directly after a course of ECT (ß = -0.51; 95% CI, -0.93 to -0.09; f2 = 0.10). Associations with IL-6 did not reach significance.Conclusions: This study suggests that inflammatory processes are associated with lower cognitive functioning prior to ECT and predispose for further cognitive dysfunction during and after a course of ECT.Trial registration: ClinicalTrials.gov identifier: NCT02667353.

Astrocytic A beta 1-42 uptake is determined by A beta-aggregation state and the presence of amyloid-associated proteins.

Intracerebral accumulation of amyloid-beta (A beta) leading to A beta plaque formation, is the main hallmark of Alzheimer's disease and might be caused by defective A beta-clearance. We previously found primary human astrocytes and microglia able to bind and ingest A beta 1-42 in vitro, which appeared to be limited by A beta 1-42 fibril formation. We now confirm that astrocytic A beta-uptake depends on size and/or composition of A beta-aggregates as astrocytes preferably take up oligomeric A beta over fibrillar A beta. Upon exposure to either fluorescence-labelled A beta 1-42 oligomers (A beta(oligo)) or fibrils (A beta(fib)), a larger (3.7 times more) proportion of astrocytes ingested oligomers compared to fibrils, as determined by flow cytometry. A beta-internalization was verified using confocal microscopy and live-cell imaging. Neither uptake of A beta(oligo) nor A beta(fib), triggered proinflammatory activation of the astrocytes, as judged by quantification of interleukin-6 and monocyte-chemoattractant protein-1 release. Amyloid-associated proteins, including alpha1-antichymotrypsin (ACT), serum amyloid P component (SAP), C1q and apolipoproteins E (ApoE) and J (ApoJ) were earlier found to influence A beta-aggregation. Here, astrocytic uptake of A beta(fib) increased when added to the cells in combination with SAP and C1q (SAP/C1q), but was unchanged in the presence of ApoE, ApoJ and ACT. Interestingly, ApoJ and ApoE dramatically reduced the number of A beta(oligo)-positive astrocytes, whereas SAP/C1q slightly reduced A beta(oligo) uptake. Thus, amyloid-associated proteins, especially ApoJ and ApoE, can alter A beta-uptake in vitro and hence may influence A beta clearance and plaque formation in vivo.

APOE ε4 genotype-dependent cerebrospinal fluid proteomic signatures in Alzheimer's disease.

BACKGROUND: Aggregation of amyloid ß into plaques in the brain is one of the earliest pathological events in Alzheimer's disease (AD). The exact pathophysiology leading to dementia is still uncertain, but the apolipoprotein E (APOE) ε4 genotype plays a major role. We aimed to identify the molecular pathways associated with amyloid ß aggregation using cerebrospinal fluid (CSF) proteomics and to study the potential modifying effects of APOE ε4 genotype. METHODS: We tested 243 proteins and protein fragments in CSF comparing 193 subjects with AD across the cognitive spectrum (65% APOE ε4 carriers, average age 75 ± 7 years) against 60 controls with normal CSF amyloid ß, normal cognition, and no APOE ε4 allele (average age 75 ± 6 years). RESULTS: One hundred twenty-nine proteins (53%) were associated with aggregated amyloid ß. APOE ε4 carriers with AD showed altered concentrations of proteins involved in the complement pathway and glycolysis when cognition was normal and lower concentrations of proteins involved in synapse structure and function when cognitive impairment was moderately severe. APOE ε4 non-carriers with AD showed lower expression of proteins involved in synapse structure and function when cognition was normal and lower concentrations of proteins that were associated with complement and other inflammatory processes when cognitive impairment was mild. Repeating analyses for 114 proteins that were available in an independent EMIF-AD MBD dataset (n = 275) showed that 80% of the proteins showed group differences in a similar direction, but overall, 28% effects reached statistical significance (ranging between 6 and 87% depending on the disease stage and genotype), suggesting variable reproducibility. CONCLUSIONS: These results imply that AD pathophysiology depends on APOE genotype and that treatment for AD may need to be tailored according to APOE genotype and severity of the cognitive impairment.

Binding and uptake of A beta1-42 by primary human astrocytes in vitro.

Clearance of the amyloid-beta peptide (A beta) as a remedy for Alzheimer's disease (AD) is a major target in on-going clinical trials. In vitro studies confirmed that A beta is taken up by rodent astrocytes, but knowledge on human astrocyte-mediated A beta clearance is sparse. Therefore, by means of flow cytometry and confocal laser scanning microscopy (CLSM), we evaluated the binding and internalization of A beta1-42 by primary human fetal astrocytes and adult astrocytes, isolated from nondemented subjects (n = 8) and AD subjects (n = 6). Furthermore, we analyzed whether alpha1-antichymotrypsin (ACT), which is found in amyloid plaques and can influence A beta fibrillogenesis, affects the A beta uptake by human astrocytes. Upon over night exposure of astrocytes to FAM-labeled A beta1-42 (10 microM) preparations, (80.7 +/- 17.7)% fetal and (52.9 +/- 20.9)% adult A beta-positive astrocytes (P = 0.018) were observed. No significant difference was found in A beta1-42 uptake between AD and non-AD astrocytes, and no influence of ApoE genotype on A beta1-42 uptake was observed in any group. There was no difference in the percentage of A beta-positive cells upon exposure to A beta1-42 (10 microM) combined with ACT (1,000:1, 100:1, and 10:1 molar ratio), versus A beta1-42 alone. CLSM revealed binding of A beta1-42 to the cellular surfaces and cellular internalization of smaller A beta1-42 fragments. Under these conditions, there was no increase in cellular release of the proinflammatory chemokine monocyte-chemoattractant protein 1, as compared with nontreated control astrocytes. Thus, primary human astrocytes derived from different sources can bind and internalize A beta1-42, and fetal astrocytes were more efficient in A beta1-42 uptake than adult astrocytes.

C4b-binding protein in Alzheimer's disease: binding to Abeta1-42 and to dead cells.

In the Alzheimer's disease (AD) brain, binding of Clq within the Cl complex, the initiating molecule of the classical complement pathway, to apoptotic cells, DNA and amyloid-beta (Abeta), the major constituent of senile plaques, can initiate complement activation. However, the extent of activation is determined by the balance between activation and inhibition. Fluid-phase complement inhibitor C4b-binding protein (C4BP) was immunohistochemically detected in Abeta plaques and on apoptotic cells in AD brain. In vitro, C4BP bound apoptotic and necrotic but not viable brain cells (astrocytes, neurons and oligodendrocytes) and limited complement activation on dead brain cells. C4BP also bound Abeta1-42 peptide directly, via the C4BP alpha-chain, and limited the extent of complement activation by Abeta. C4BP levels in cerebrospinal fluid (CSF) of dementia patients and controls were low compared to levels in plasma and correlated with CSF levels of other inflammation-related factors. In conclusion, C4BP binds to dead brain cells and Abeta peptide in vitro, is present in CSF and possibly protects against excessive complement activation in AD brains.

Decoration of Fibrin with Extracellular Chaperones.

BACKGROUND: Many proteins bind to fibrin during clot formation in plasma. We previously identified by mass spectrometry the most abundant proteins that noncovalently bind to fibrin clots. Several of these proteins (e.g., apolipoprotein J/clusterin, haptoglobin, α2-macroglobulin, α1-antitrypsin) can act as extracellular chaperones. OBJECTIVE: We hypothesize that clot-binding proteins may interact with fibrin as chaperones. The goal of this study is to test this hypothesis and to investigate the origin of the cross-ß or amyloid structures in fibrin clots, which are associated with protein unfolding. METHODS AND RESULTS: A thioflavin T assay was used to detect cross-ß structures. A steadily increasing amount was measured in the fibrinogen fraction of plasma during heat stress, a standard treatment to induce unfolding of proteins. Heat-stressed plasma was clotted and clot-bound proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results showed that the amounts of the clot-bound proteins were related to the duration of the heat stress. This indicates that cross-ß structures in unfolded fibrin(ogen) are involved in clot binding of the proteins, which supports our chaperone hypothesis. A contributing role of fibrin formation itself was studied by clotting purified fibrinogen with thrombin in the presence of thioflavin T. The fluorescence intensity increased in time in the presence of thrombin, but did not increase in its absence. This provides evidence for the generation of cross-ß structures during fibrin formation. CONCLUSION: Fibrin clots generated in plasma are decorated with extracellular chaperones. The binding of these chaperones involves cross-ß structures originating both from unfolded fibrinogen and from fibrin formation.

Inflammation and remission in older patients with depression treated with electroconvulsive therapy; findings from the MODECT study✰.

BACKGROUND: Compelling evidence links elevated levels of C-reactive protein (CRP) and other inflammatory markers to poor treatment outcome of antidepressant medication. Little is known about the contribution of low-grade inflammation to treatment response to electroconvulsive therapy (ECT) in severely depressed patients. METHOD: Associations between serum levels of CRP, interleukin-6, interleukin-10, and tumour necrosis factor-α as well as remission of depression, time to remission, and speed of decline of depressive symptoms were examined in 95 older (mean age: 73.1 years) depressed patients treated with ECT. RESULTS: Moderately elevated levels of CRP at baseline (3 to 10 mg/L), but no other inflammatory markers, were associated with higher remission rates. In patients with moderately elevated CRP levels, the odds ratio for remission was 3.62 (95% confidence interval [CI], 1.09-11.97; p = 0.04). Time to remission was shorter in those with moderately elevated CRP levels (p = 0.05). Speed of decline was higher in patients with moderately elevated CRP levels as compared with those with low CRP levels (decline of 3.2 Montgomery Åsberg Depression Rating Scale points per administration vs. 2.3 points per administration, p = 0.03). LIMITATIONS: Because of the observational design, residual confounding through other lifestyle or demographic factors cannot be ruled out. CONCLUSIONS: Although earlier studies showed that low-grade inflammation contributes to poor treatment response in those treated with antidepressants, our study provides clues that low-grade inflammation does not have such a detrimental effect on the treatment response to ECT. This is underscored by our finding that moderately elevated CRP levels were associated with increased remission rates in depressed patients treated with ECT. Replication studies are warranted.

ApoE and clusterin CSF levels influence associations between APOE genotype and changes in CSF tau, but not CSF Aß42, levels in non-demented elderly.

Apolipoprotein E (APOE) ε4 genotype is associated with increased cerebral amyloid beta (Aß) deposition in nondemented elderly and suggested to influence ApoE as well as ApoJ (clusterin [Clu]) and ApoA1 expression. We aimed to assess whether APOE affects early Alzheimer's disease pathophysiology via these apolipoproteins. Cerebrospinal fluid (CSF) ApoE, Clu, ApoA1, and CSF amyloid beta1-42 (Aß42) and tau levels were assessed in 403 individuals with subjective cognitive decline and mild cognitive impairment using enzyme-linked immunosorbent assay. Whether CSF apolipoprotein levels mediated APOEε4 allele frequency effects on CSF Aß42 and tau in nondemented elderly was investigated using mediation analysis, with age- and gender-adjusted linear regression analyses. CSF ApoE mediated 48% of the association between APOEε4 and CSF tau, whereas Clu and ApoA1 did not. In addition, CSF Clu partially mediated the relation between CSF ApoE and tau (12%). CSF apolipoproteins did not mediate the inverse relation between APOEε4 and CSF Aß42, despite a strong association between the latter 2 biomarkers. In summary, our findings suggest that ApoE and Clu are involved in Aß-independent pathways as part of the cascade leading to Alzheimer pathology.