Inflammatory marker levels in children with tobacco smoke exposure.

BACKGROUND: Tobacco smoke exposure (TSE) has inflammatory and immunosuppressive effects which may be associated with altered levels of inflammatory markers and pediatric illnesses. OBJECTIVE: The primary objective was to examine the associations of cotinine-confirmed and parent-reported child TSE patterns and discharge diagnoses with C-reactive protein (CRP), IL-8, and IL-10 in 0-11-year-old pediatric emergency department (PED) patients who lived with ≥ 1 smoker. METHODS: Saliva samples were obtained from 115 children with a mean (SD) age of 3.5 (3.1) years during the PED visit (T0). Saliva was analyzed for cotinine, CRP, IL-8, and IL-10. Parents self-reported their children's TSE patterns; children's medical records were reviewed to identify and categorize discharge diagnoses. Linear regression models were utilized to find T0 associations of cotinine-confirmed and parent-reported child TSE patterns, and PED diagnoses with each inflammatory marker. All models were adjusted for child race/ethnicity, child sex, annual household income, and housing type. The TSE models also adjusted for child discharge diagnosis. RESULTS: At T0, the geometric mean (GeoM) of cotinine was 4.1 ng/ml [95 %CI = 3.2-5.2]; the GeoMs of CRP, IL-8, and IL-10 were 3,326 pg/ml [95 %CI = 2,696-4,105], 474 pg/ml [95 %CI = 386-583], and 1.1 pg/ml [95 %CI = 0.9-1.3], respectively. Parent-reported child TSE patterns were positively associated with ln-transformed CRP levels, while adjusting for the covariates (ß^ = 0.012 [95 %CI:0.004-0.020], p = 0.037). In the parent-reported child TSE pattern model, there were significant positive associations between the covariate of child age with CRP and IL-8 levels (p = 0.028 and p < 0.001, respectively). Children with a bacterial diagnosis had higher IL-8 levels (p = 0.002) compared to the other diagnosis groups. CONCLUSIONS: Results indicate that parent-reported child TSE increases the expression of CRP in ill children and supports prior work demonstrating that IL-8 is higher in children with TSE who have bacterial infections. These findings should be examined in future research with ill children with and without TSE.

A cross-validation based approach for estimating specific gravity in elementary-school aged children using a nonlinear model.

Environmental research often relies on urinary biomarkers which require dilution correction to accurately measure exposures. Specific gravity (SG) and creatinine (UCr) are commonly measured urinary dilution factors. Epidemiologic studies may assess only one of these measures, making it difficult to pool studies that may otherwise be able to be combined. Participants from the National Health and Nutrition Examination Survey 2007-2008 cycle were used to perform k-fold validation of a nonlinear model estimating SG from UCr. The final estimated model was applied to participants from the School Inner-City Asthma Intervention Study, who submitted urinary samples to the Children's Health Exposure Analysis Resource. Model performance was evaluated using calibration metrics to determine how closely the average estimated SG was to the measured SG. Additional models, with interaction terms for age, sex, body mass index, race/ethnicity, relative time of day when sample was collected, log transformed 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and asthma status were estimated and assessed for improvement. The association between monobenzyl phthalate (MBZP) and asthma symptom days, controlling for measured UCr, measured SG, and each estimated SG were compared to assess validity of the estimated SG. The model estimating SG from UCr alone, resulted in a beta estimate of 1.10 (95% CI: 1.01, 1.19), indicating agreement between model-predicted SG and measured SG. Inclusion of age and sex in the model improved estimation (ß = 1.06, 95% CI: 0.98, 1.15). The full model accounting for all interaction terms with UCr resulted in the best agreement (ß = 1.01, 95% CI: 0.93,1.09). Associations between MBZP and asthma symptoms days, controlling for each estimated SG, were within the range of effect estimates when controlling for measured SG and measured UCr (Rate ratios = 1.28-1.34). Our nonlinear modeling provides opportunities to estimate SG in studies that measure UCr or vice versa, enabling data pooling despite differences in urine dilution factors.

Quality assurance and harmonization for targeted biomonitoring measurements of environmental organic chemicals across the Children's Health Exposure Analysis Resource laboratory network.

A consortium of laboratories established under the Children's Health Exposure Analysis Resource (CHEAR) used a multifaceted quality assurance program to promote measurement harmonization for trace organics analyses of human biospecimens that included: (1) participation in external quality assurance (EQA)/proficiency testing (PT) programs; (2) analyses of a urine-based CHEAR common quality control (QC) pool with each analytical batch across all participating laboratories; (3) method validation against NIST Standard Reference Materials® (SRMs); and (4) analyses of blinded duplicates and other project-specific QC samples. The capability of five CHEAR laboratories in organic chemical analysis increased across the 4-year period, and performance in the external PT program improved over time - recent challenges reporting >90% analytes with satisfactory performance. The CHEAR QC pools were analyzed for several classes of organic chemicals including phthalate metabolites and environmental phenols by the participating laboratories with every batch of project samples, which provided a rich source of measurement data for the assessment of intra- and inter-laboratory variance. Within-laboratory and overall variabilities in measurements across laboratories were calculated for target chemicals in urine QC pools; the coefficient of variation (CV) was generally below 25% across batches, studies and laboratories and indicated acceptable analytical imprecision. The suite of organic chemicals analyzed in the CHEAR QC pool was broader than those reported for commercially available reference materials. The accuracy of each of the laboratories' methods was verified through the analysis of several NIST SRMs and was, for example, 97 ± 5.2% for environmental phenols and 95 ± 11% for phthalates. Analysis of blinded duplicate samples showed excellent agreement and reliability of measurements. The intra-class correlation coefficients (ICC) for phthalate metabolites analyzed in various batches across three CHEAR laboratories showed excellent reliability (typically >0.90). Overall, the multifaceted quality assurance protocols followed among the CHEAR laboratories ensured reliable and reproducible data quality for several classes of organic chemicals. Increased participation in external PT programs through inclusion of additional target analytes will further enhance the confidence in data quality.

Evaluating inter-study variability in phthalate and trace element analyses within the Children's Health Exposure Analysis Resource (CHEAR) using multivariate control charts.

BACKGROUND: The Children's Health Exposure Analysis Resource (CHEAR) program allows researchers to expand their research goals by offering the assessment of environmental exposures in their previously collected biospecimens. Samples are analyzed in one of CHEAR's network of six laboratory hubs with the ability to assess a wide array of environmental chemicals. The ability to assess inter-study variability is important for researchers who want to combine datasets across studies and laboratories. OBJECTIVE: Herein we establish a process of evaluating inter-study variability for a given analytic method. METHODS: Common quality control (QC) pools at two concentration levels (A and B) in urine were created within CHEAR for insertion into each batch of samples tested at a rate of three samples of each pool per 100 study samples. We assessed these QC pool results for seven phthalates analyzed for five CHEAR studies by three different lab hubs utilizing multivariate control charts to identify out-of-control runs or sets of samples associated with a given QC sample. We then tested the conditions that would lead to an out-of-control run by simulating outliers in an otherwise "in-control" set of 12 trace elements in blood QC samples (NIST SRM 955c). RESULTS: When phthalates were assessed within study, we identified a single out-of-control run for two of the five studies. Combining QC results across lab hubs, all of the runs from these two studies were now in-control, while multiple runs from two other studies were pushed out-of-control. In our simulation study we found that 3-6 analytes with outlier values (5xSD) within a run would push that run out of control in 65-83% of simulations, respectively. SIGNIFICANCE: We show how acceptable bounds of variability can be established for a given analytic method by evaluating QC materials across studies using multivariate control charts.

Comparison of Liquid Chromatography Mass Spectrometry and Enzyme-Linked Immunosorbent Assay Methods to Measure Salivary Cotinine Levels in Ill Children.

Objective: Cotinine is the preferred biomarker to validate levels of tobacco smoke exposure (TSE) in children. Compared to enzyme-linked immunosorbent assay methods (ELISA) for quantifying cotinine in saliva, the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) has higher sensitivity and specificity to measure very low levels of TSE. We sought to compare LC-MS/MS and ELISA measures of cotinine in saliva samples from children overall and the associations of these measures with demographics and TSE patterns. Method: Participants were nonsmoking children (N = 218; age mean (SD) = 6.1 (5.1) years) presenting to a pediatric emergency department. Saliva samples were analyzed for cotinine using both LC-MS/MS and ELISA. Limit of quantitation (LOQ) for LC-MS/MS and ELISA was 0.1 ng/ml and 0.15 ng/ml, respectively. Results: Intraclass correlations (ICC) across methods = 0.884 and was consistent in sex and age subgroups. The geometric mean (GeoM) of LC-MS/MS = 4.1 (range: < LOQ - 382 ng/mL; 3% < LOQ) which was lower (p < 0.0001) than the ELISA GeoM = 5.7 (range: < LOQ - 364 ng/mL; 5% < LOQ). Similar associations of cotinine concentrations with age ( < -0.10, p < 0.0001), demographic characteristics (e.g., income), and number of cigarettes smoked by caregiver ( > 0.07, p < 0.0001) were found regardless of cotinine detection method; however, cotinine associations with sex and race/ethnicity were only found to be significant in models using LC-MS/MS-derived cotinine. Conclusions: Utilizing LC-MS/MS-based cotinine, associations of cotinine with sex and race/ethnicity of child were revealed that were not detectable using ELISA-based cotinine, demonstrating the benefits of utilizing the more sensitive LC-MS/MS assay for cotinine measurement when detecting low levels of TSE in children.

Combined exposure to lead, cadmium, mercury, and arsenic and kidney health in adolescents age 12-19 in NHANES 2009-2014.

BACKGROUND: Occupational and environmental exposures to toxic metals are established risk factors for the development of hypertension and kidney disease in adults. There is some evidence of developmental metal nephrotoxicity in children and from animal studies; however, to our knowledge no previous studies have examined associations between co-exposure to nephrotoxic environmental metals and children's kidney health. OBJECTIVE: The objective of this study was to assess the association between co-exposure to lead (Pb), cadmium (Cd), mercury (Hg), and arsenic (As), measured in urine and blood, and kidney parameters in US adolescents. METHODS: We performed a cross-sectional analysis of a subsample of 2709 children aged 12-19 participating in the National Health and Nutrition Examination Survey (NHANES) between 2009 and 2014. We analyzed urine levels of 4 nephrotoxic metals selected a priori (As, Cd, Pb and Hg), Umix, and 3 nephrotoxic metals in blood (Cd, Pb, and Hg), Bmix, using a weighted quantile sum (WQS) approach. We applied WQS regression to analyze the association of Bmix and Umix with estimated glomerular filtration rate (eGFR), serum uric acid (SUA), urine albumin, blood urea nitrogen (BUN), and systolic blood pressure (SBP), adjusting for sex, race/ethnicity, age, head of household's education level, height, BMI, serum cotinine, and NHANES cohort year. Umix and urine albumin models were also adjusted for urine creatinine, and Bmix models were also adjusted for fish consumption. Subanalyses included stratification by sex and an arsenic-only model including six speciated forms of As measured in urine. RESULTS: In WQS regression models, each decile increase of Umix was associated with 1.6% (95% CI: 0.5, 2.8) higher BUN, 1.4% (95% CI: 0.7, 2.0) higher eGFR, and 7.6% (95% CI: 2.4, 13.1) higher urine albumin. The association between Umix and BUN was primarily driven by As (72%), while the association with eGFR was driven by Hg (61%), and Cd (17%), and the association with urine albumin was driven by Cd (37%), Hg (33%), and Pb (25%). There was no significant relationship between Umix and SUA or SBP. In WQS models using the combined blood metals, Bmix, each decile increase of Bmix was associated with 0.6% (95% CI: 0.0, 1.3) higher SUA; this association was driven by Pb (43%), Hg (33%), and Cd (24%) and was marginally significant (p = 0.05). No associations were observed between Bmix and urine albumin, eGFR, BUN, or SBP. CONCLUSIONS: The findings suggest metals including As, Pb, Hg, Cd and their combinations may affect renal parameters, although potential reverse causation cannot be ruled out due to the cross-sectional study design. Implications of early life low-level exposure to multiple metals on kidney function may have far-reaching consequences later in life in the development of hypertension, kidney disease, and renal dysfunction. Longitudinal studies should further evaluate these relationships.

Brain-Wide Insulin Resistance, Tau Phosphorylation Changes, and Hippocampal Neprilysin and Amyloid-ß Alterations in a Monkey Model of Type 1 Diabetes.

Epidemiological findings suggest that diabetic individuals are at a greater risk for developing Alzheimer's disease (AD). To examine the mechanisms by which diabetes mellitus (DM) may contribute to AD pathology in humans, we examined brain tissue from streptozotocin-treated type 1 diabetic adult male vervet monkeys receiving twice-daily exogenous insulin injections for 8-20 weeks. We found greater inhibitory phosphorylation of insulin receptor substrate 1 in each brain region examined of the diabetic monkeys when compared with controls, consistent with a pattern of brain insulin resistance that is similar to that reported in the human AD brain. Additionally, a widespread increase in phosphorylated tau was seen, including brain areas vulnerable in AD, as well as relatively spared structures, such as the cerebellum. An increase in active ERK1/2 was also detected, consistent with DM leading to changes in tau-kinase activity broadly within the brain. In contrast to these widespread changes, we found an increase in soluble amyloid-ß (Aß) levels that was restricted to the temporal lobe, with the greatest increase seen in the hippocampus. Consistent with this localized Aß increase, a hippocampus-restricted decrease in the protein and mRNA for the Aß-degrading enzyme neprilysin (NEP) was found, whereas various Aß-clearing and -degrading proteins were unchanged. Thus, we document multiple biochemical changes in the insulin-controlled DM monkey brain that can link DM with the risk of developing AD, including dysregulation of the insulin-signaling pathway, changes in tau phosphorylation, and a decrease in NEP expression in the hippocampus that is coupled with a localized increase in Aß. SIGNIFICANCE STATEMENT: Given that diabetes mellitus (DM) appears to increase the risk of developing Alzheimer's disease (AD), understanding the mechanisms by which DM promotes AD is important. We report that DM in a nonhuman primate brain leads to changes in the levels or posttranslational processing of proteins central to AD pathobiology, including tau, amyloid-ß (Aß), and the Aß-degrading protease neprilysin. Additional evidence from this model suggests that alterations in brain insulin signaling occurred that are reminiscent of insulin signaling pathway changes seen in human AD. Thus, in an in vivo model highly relevant to humans, we show multiple alterations in the brain resulting from DM that are mechanistically linked to AD risk.

Endogenous murine Aß increases amyloid deposition in APP23 but not in APPPS1 transgenic mice.

Endogenous murine amyloid-ß peptide (Aß) is expressed in most Aß precursor protein (APP) transgenic mouse models of Alzheimer's disease but its contribution to ß-amyloidosis remains unclear. We demonstrate ∼ 35% increased cerebral Aß load in APP23 transgenic mice compared with age-matched APP23 mice on an App-null background. No such difference was found for the much faster Aß-depositing APPPS1 transgenic mouse model between animals with or without the murine App gene. Nevertheless, both APP23 and APPPS1 mice codeposited murine Aß, and immunoelectron microscopy revealed a tight association of murine Aß with human Aß fibrils. Deposition of murine Aß was considerably less efficient compared with the deposition of human Aß indicating a lower amyloidogenic potential of murine Aß in vivo. The amyloid dyes Pittsburgh Compound-B and pentamer formyl thiophene acetic acid did not differentiate between amyloid deposits consisting of human Aß and deposits of mixed human-murine Aß. Our data demonstrate a differential effect of murine Aß on human Aß deposition in different APP transgenic mice. The mechanistically complex interaction of human and mouse Aß may affect pathogenesis of the models and should be considered when models are used for translational preclinical studies.

Chronic anti-murine Aß immunization preserves odor guided behaviors in an Alzheimer's ß-amyloidosis model.

Olfaction is often impaired in Alzheimer's disease (AD) and is also dysfunctional in mouse models of the disease. We recently demonstrated that short-term passive anti-murine-Aß immunization can rescue olfactory behavior in the Tg2576 mouse model overexpressing a human mutation of the amyloid precursor protein (APP) after ß-amyloid deposition. Here we tested the ability to preserve normal olfactory behaviors by means of long-term passive anti-murine-Aß immunization. Seven-month-old Tg2576 and non-transgenic littermate (NTg) mice were IP-injected biweekly with the m3.2 murine-Aß-specific antibody until 16 mo of age when mice were tested in the odor habituation test. While Tg2576 mice treated with a control antibody showed elevations in odor investigation times and impaired odor habituation compared to NTg, olfactory behavior was preserved to NTg levels in m3.2-immunized Tg2576 mice. Immunized Tg2576 mice had significantly less ß-amyloid immunolabeling in the olfactory bulb and entorhinal cortex, yet showed elevations in Thioflavin-S labeled plaques in the piriform cortex. No detectable changes in APP metabolite levels other than Aß were found following m3.2 immunization. These results demonstrate efficacy of chronic, long-term anti-murine-Aß m3.2 immunization in preserving normal odor-guided behaviors in a human APP Tg model. Further, these results provide mechanistic insights into olfactory dysfunction as a biomarker for AD by yielding evidence that focal reductions of Aß may be sufficient to preserve olfaction.

Immunization targeting a minor plaque constituent clears ß-amyloid and rescues behavioral deficits in an Alzheimer's disease mouse model.

Although anti-human ß-amyloid (Aß) immunotherapy clears brain ß-amyloid plaques in Alzheimer's disease (AD), targeting additional brain plaque constituents to promote clearance has not been attempted. Endogenous murine Aß is a minor Aß plaque component in amyloid precursor protein (APP) transgenic AD models, which we show is ∼3%-8% of the total accumulated Aß in various human APP transgenic mice. Murine Aß codeposits and colocalizes with human Aß in amyloid plaques, and the two Aß species coimmunoprecipitate together from brain extracts. In the human APP transgenic mouse model Tg2576, passive immunization for 8 weeks with a murine-Aß-specific antibody reduced ß-amyloid plaque pathology, robustly decreasing both murine and human Aß levels. The immunized mice additionally showed improvements in two behavioral assays, odor habituation and nesting behavior. We conclude that passive anti-murine Aß immunization clears Aß plaque pathology--including the major human Aß component--and decreases behavioral deficits, arguing that targeting minor endogenous brain plaque constituents can be beneficial, broadening the range of plaque-associated targets for AD therapeutics.

Early endosomal abnormalities and cholinergic neuron degeneration in amyloid-ß protein precursor transgenic mice.

Early endosomal changes, a prominent pathology in neurons early in Alzheimer's disease, also occur in neurons and peripheral tissues in Down syndrome. While in Down syndrome models increased amyloid-ß protein precursor (AßPP) expression is known to be a necessary contributor on the trisomic background to this early endosomal pathology, increased AßPP alone has yet to be shown to be sufficient to drive early endosomal alterations in neurons. Comparing two AßPP transgenic mouse models, one that contains the AßPP Swedish K670N/M671L double mutation at the ß-cleavage site (APP23) and one that has the AßPP London V717I mutation near the γ-cleavage site (APPLd2), we show significantly altered early endosome morphology in fronto-parietal neurons as well as enlargement of early endosomes in basal forebrain cholinergic neurons of the medial septal nucleus in the APP23 model, which has the higher levels of AßPP ß-C-terminal fragment (ßCTF) accumulation. Early endosomal changes correlate with a marked loss of the cholinergic population, which is consistent with the known dependence of the large projection cholinergic cells on endosome-mediated retrograde neurotrophic transport. Our findings support the idea that increased expression of AßPP and AßPP metabolites in neurons is sufficient to drive early endosomal abnormalities in vivo, and that disruption of the endocytic system is likely to contribute to basal forebrain cholinergic vulnerability.

Aß measurement by enzyme-linked immunosorbent assay.

The neuritic plaque in the brain of Alzheimer's disease patients consists of an amyloid composed primarily of Aß, an approximately 4-kDa peptide derived from the amyloid precursor protein. Multiple lines of evidence suggest that Aß plays a key role in the pathogenesis of the disease, and potential treatments that target Aß production and/or Aß accumulation in the brain as ß-amyloid are being aggressively pursued. Methods to quantitate the Aß peptide are, therefore, invaluable to most studies aimed at a better understanding of the molecular etiology of the disease and in assessing potential therapeutics. Although other techniques have been used to measure Aß in the brains of AD patients and ß-amyloid-depositing transgenic mice, the enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used, reliable, and sensitive methods for quantitating the Aß peptide. Here we describe methods for the recovery of both soluble and deposited Aß from brain tissue and the subsequent quantitation of the peptide by sandwich ELISA.

Calpastatin modulates APP processing in the brains of ß-amyloid depositing but not wild-type mice.

We report that neuronal overexpression of the endogenous inhibitor of calpains, calpastatin (CAST), in a mouse model of human Alzheimer's disease (AD) ß-amyloidosis, the APP23 mouse, reduces ß-amyloid (Aß) pathology and Aß levels when comparing aged, double transgenic (tg) APP23/CAST with APP23 mice. Concurrent with Aß plaque deposition, aged APP23/CAST mice show a decrease in the steady-state brain levels of the amyloid precursor protein (APP) and APP C-terminal fragments (CTFs) when compared with APP23 mice. This CAST-dependent decrease in APP metabolite levels was not observed in single tg CAST mice expressing endogenous APP or in younger, Aß plaque predepositing APP23/CAST mice. We also determined that the CAST-mediated inhibition of calpain activity in the brain is greater in the CAST mice with Aß pathology than in non-APP tg mice, as demonstrated by a decrease in calpain-mediated cytoskeleton protein cleavage. Moreover, aged APP23/CAST mice have reduced extracellular signal-regulated kinase 1/2 (ERK1/2) activity and tau phosphorylation when compared with APP23 mice. In summary, in vivo calpain inhibition mediated by CAST transgene expression reduces Aß pathology in APP23 mice, with our findings further suggesting that APP metabolism is modified by CAST overexpression as the mice develop Aß pathology. Our results indicate that the calpain system in neurons is more responsive to CAST inhibition under conditions of Aß pathology, suggesting that in the disease state neurons may be more sensitive to the therapeutic use of calpain inhibitors.

In vivo turnover of tau and APP metabolites in the brains of wild-type and Tg2576 mice: greater stability of sAPP in the beta-amyloid depositing mice.

The metabolism of the amyloid precursor protein (APP) and tau are central to the pathobiology of Alzheimer's disease (AD). We have examined the in vivo turnover of APP, secreted APP (sAPP), Abeta and tau in the wild-type and Tg2576 mouse brain using cycloheximide to block protein synthesis. In spite of overexpression of APP in the Tg2576 mouse, APP is rapidly degraded, similar to the rapid turnover of the endogenous protein in the wild-type mouse. sAPP is cleared from the brain more slowly, particularly in the Tg2576 model where the half-life of both the endogenous murine and transgene-derived human sAPP is nearly doubled compared to wild-type mice. The important Abeta degrading enzymes neprilysin and IDE were found to be highly stable in the brain, and soluble Abeta40 and Abeta42 levels in both wild-type and Tg2576 mice rapidly declined following the depletion of APP. The cytoskeletal-associated protein tau was found to be highly stable in both wild-type and Tg2576 mice. Our findings unexpectedly show that of these various AD-relevant protein metabolites, sAPP turnover in the brain is the most different when comparing a wild-type mouse and a beta-amyloid depositing, APP overexpressing transgenic model. Given the neurotrophic roles attributed to sAPP, the enhanced stability of sAPP in the beta-amyloid depositing Tg2576 mice may represent a neuroprotective response.

Age-dependent dysregulation of brain amyloid precursor protein in the Ts65Dn Down syndrome mouse model.

Individuals with Down syndrome develop beta-amyloid deposition characteristic of early-onset Alzheimer's disease (AD) in mid-life, presumably because of an extra copy of the chromosome 21-located amyloid precursor protein (App) gene. App mRNA and APP metabolite levels were assessed in the brains of Ts65Dn mice, a mouse model of Down syndrome, using quantitative PCR, western blot analysis, immunoprecipitation, and ELISAs. In spite of the additional App gene copy, App mRNA, APP holoprotein, and all APP metabolite levels in the brains of 4-month-old trisomic mice were not increased compared with the levels seen in diploid littermate controls. However starting at 10 months of age, brain APP levels were increased proportional to the App gene dosage imbalance reflecting increased App message levels in Ts65Dn mice. Similar to APP levels, soluble amino-terminal fragments of APP (sAPPalpha and sAPPbeta) were increased in Ts65Dn mice compared with diploid mice at 12 months but not at 4 months of age. Brain levels of both Abeta40 and Abeta42 were not increased in Ts65Dn mice compared with diploid mice at all ages examined. Therefore, multiple mechanisms contribute to the regulation towards diploid levels of APP metabolites in the Ts65Dn mouse brain.

Modulation of Abeta generation by small ubiquitin-like modifiers does not require conjugation to target proteins.

The sequential processing of the APP (amyloid precursor protein) by the beta- and gamma-secretase and generation of the Abeta (amyloid-beta) peptide is a primary pathological factor in AD (Alzheimer's disease). Regulation of the processing or turnover of these proteins represents potential targets for the development of AD therapies. Sumoylation is a process by which SUMOs (small ubiquitin-like modifiers) are covalently conjugated to target proteins, resulting in a number of functional consequences. These include regulation of protein-protein interactions, intracellular trafficking and protein stability, which all have the potential to impact on several aspects of the amyloidogenic pathway. The present study examines the effects of overexpression and knockdown of the major SUMO isoforms (SUMO1, 2 and 3) on APP processing and the production of Abeta peptides. SUMO3 overexpression significantly increased Abeta40 and Abeta42 secretion, which was accompanied by an increase in full-length APP and its C-terminal fragments. These effects of SUMO3 were independent of its covalent attachment or chain formation, as mutants lacking the motifs responsible for SUMO chain formation or SUMO conjugation led to similar changes in Abeta. SUMO3 overexpression also up-regulated the expression of the transmembrane protease BACE (beta-amyloid-cleaving enzyme), but failed to affect levels of several other unrelated proteins. Suppression of SUMO1 or combined SUMO2+3 by RNA interference did not affect APP levels or Abeta production. These findings confirm a specific effect of SUMO3 overexpression on APP processing and the production of Abeta peptides but also suggest that endogenous sumoylation is not essential and likely plays an indirect role in modulating the amyloid processing pathway.

Dissociated phenotypes in presenilin transgenic mice define functionally distinct gamma-secretases.

Gamma-secretase depends on presence of presenilins (PS), Nct, Aph-1, and PEN-2 within a core complex. This endoproteolytic activity cleaves within transmembrane domains of amyloid-beta precursor protein (APP) and Notch, and familial Alzheimer's disease (FAD) mutations in PS1 or PS2 genes shift APP cleavage from production of amyloid-beta (Abeta) 40 peptide to greater production of Abeta42. Although studies in PS1/PS2-deficient embryonic cells define overlapping activities for these proteins, in vivo complementation of PS1-deficient animals described here reveals an unexpected spectrum of activities dictated by PS1 and PS2 alleles. Unlike PS1 transgenes, wild-type PS2 transgenes expressed in the mouse CNS support little Abeta40 or Abeta42 production, and FAD PS2 alleles support robust production of only Abeta42. Although wild-type PS2 transgenes failed to rescue Notch-associated skeletal defects in PS1 hypomorphs, a "gained" competence in this regard was apparent for FAD alleles of PS2. The range of discrete and divergent processing activities in mice reconstituted with different PS genes and alleles argues against gamma-secretase being a single enzyme with intrinsically relaxed substrate and cleavage site specificities. Instead, our studies define functionally distinct gamma-secretase variants. We speculate that extrinsic components, in combination with core complexes, may tailor functional variants of this enzyme to their preferred substrates.