ObjectiveAge is the single greatest risk factor for Alzheimer's disease (AD), with the incidence doubling every 5 years after age 65. However, our understanding of the mechanistic relationship between increasing age and the risk for AD is currently limited. We therefore sought to determine the relationship between age, amyloidosis, and amyloid‐beta (Aβ) kinetics in the central nervous system (CNS) of humans.MethodsAβ kinetics were analyzed in 112 participants and compared to the ages of participants and the amount of amyloid deposition.ResultsWe found a highly significant correlation between increasing age and slowed Aβ turnover rates (2.5‐fold longer half‐life over five decades of age). In addition, we found independent effects on Aβ42 kinetics specifically in participants with amyloid deposition. Amyloidosis was associated with a higher (>50%) irreversible loss of soluble Aβ42 and a 10‐fold higher Aβ42 reversible exchange rate.InterpretationThese findings reveal a mechanistic link between human aging and the risk of amyloidosis, which may be owing to a dramatic slowing of Aβ turnover, increasing the likelihood of protein misfolding that leads to deposition. Alterations in Aβ kinetics associated with aging and amyloidosis suggest opportunities for diagnostic and therapeutic strategies. More generally, this study provides an example of how changes in protein turnover kinetics can be used to detect physiological and pathophysiological changes and may be applicable to other proteinopathies. Ann Neurol 2015;78:439–453

We developed a stable isotope labeled amino acid administration paradigm, Stable Isotope Labeling Kinetics (SILK). We recently developed and used the tau SILK method to quantify tau protein kinetics in the human central nervous system (CNS). Normal participants (n=6) ingested 13C6-Leucine (10g, 10 days) and its in vivo incorporation kinetics into cerebrospinal fluid (CSF) tau protein were quantified after immunoprecipitation, trypsin digestion, and mass spectrometry analysis of specific Leu-containing tau peptides. In the normal human CNS, tau had a half-life of approximately 20 days. This half-life and turnover rate is much slower than other previously measured CNS proteins, such as amyloid beta (t1/2 ∼8 hours), and Apolipoprotein E (t1/2 ∼12 hours). We have developed a method to measure tau kinetics in human CNS that will be useful to test the hypothesis if tau kinetics are altered in Alzheimer's disease (AD) and other tauopathies. The tau SILK method will enable the field to evaluate the efficacy of tau-targeted therapies, which aim to decrease tau production or enhance tau clearance.

Therapeutic strategies that target disease-associated transcripts are being developed for a variety of neurodegenerative syndromes. Protein levels change as a function of their half-life, a property that critically influences the timing and application of therapeutics. In addition, both protein kinetics and concentration may play important roles in neurodegeneration; therefore, it is essential to understand in vivo protein kinetics, including half-life. Here, we applied a stable isotope-labeling technique in combination with mass spectrometric detection and determined the in vivo kinetics of superoxide dismutase 1 (SOD1), mutation of which causes amyotrophic lateral sclerosis. Application of this method to human SOD1-expressing rats demonstrated that SOD1 is a long-lived protein, with a similar half-life in both the cerebral spinal fluid (CSF) and the CNS. Additionally, in these animals, the half-life of SOD1 was longest in the CNS when compared with other tissues. Evaluation of this method in human subjects demonstrated successful incorporation of the isotope label in the CSF and confirmed that SOD1 is a long-lived protein in the CSF of healthy individuals. Together, the results of this study provide important insight into SOD1 kinetics and support application of this technique to the design and implementation of clinical trials that target long-lived CNS proteins.

BACE, a β-secretase, is an attractive potential disease-modifying therapeutic strategy for Alzheimer's disease (AD) as it results directly in the decrease of amyloid precursor protein (APP) processing through the β-secretase pathway and a lowering of CNS amyloid-β (Aβ) levels. The interaction of the β-secretase and α-secretase pathway-mediated processing of APP in the rhesus monkey (nonhuman primate; NHP) CNS is not understood. We hypothesized that CNS inhibition of BACE would result in decreased newly generated Aβ and soluble APPβ (sAPPβ), with increased newly generated sAPPα. A stable isotope labeling kinetics experiment in NHPs was performed with a (13)C6-leucine infusion protocol to evaluate effects of BACE inhibition on CNS APP processing by measuring the kinetics of sAPPα, sAPPβ, and Aβ in CSF. Each NHP received a low, medium, or high dose of MBI-5 (BACE inhibitor) or vehicle in a four-way crossover design. CSF sAPPα, sAPPβ, and Aβ were measured by ELISA and newly incorporated label following immunoprecipitation and liquid chromatography-mass spectrometry. Concentrations, kinetics, and amount of newly generated APP fragments were calculated. sAPPβ and sAPPα kinetics were similar, but both significantly slower than Aβ. BACE inhibition resulted in decreased labeled sAPPβ and Aβ in CSF, without observable changes in labeled CSF sAPPα. ELISA concentrations of sAPPβ and Aβ both decreased and sAPPα increased. sAPPα increased by ELISA, with no difference by labeled sAPPα kinetics indicating increases in product may be due to APP shunting from the β-secretase to the α-secretase pathway. These results provide a quantitative understanding of pharmacodynamic effects of BACE inhibition on NHP CNS, which can inform about target development.

A study attempts to replicate the finding that the serotonin reuptake inhibitor citalopram decreases amyloid β concentrations in the cerebrospinal fluid of healthy volunteers.

Alzheimer's disease (AD) is hypothesized to be caused by an overproduction or reduced clearance of amyloid-β (Aβ) peptide. Autosomal dominant AD (ADAD) caused by mutations in the presenilin (PSEN) gene have been postulated to result from increased production of Aβ42 compared to Aβ40 in the central nervous system (CNS). This has been demonstrated in rodent models of ADAD but not in human mutation carriers. We used compartmental modeling of stable isotope labeling kinetic (SILK) studies in human carriers of PSEN mutations and related noncarriers to evaluate the pathophysiological effects of PSEN1 and PSEN2 mutations on the production and turnover of Aβ isoforms. We compared these findings by mutation status and amount of fibrillar amyloid deposition as measured by positron emission tomography (PET) using the amyloid tracer Pittsburgh compound B (PIB). CNS Aβ42 to Aβ40 production rates were 24% higher in mutation carriers compared to noncarriers, and this was independent of fibrillar amyloid deposits quantified by PET PIB imaging. The fractional turnover rate of soluble Aβ42 relative to Aβ40 was 65% faster in mutation carriers and correlated with amyloid deposition, consistent with increased deposition of Aβ42 into plaques, leading to reduced recovery of Aβ42 in cerebrospinal fluid (CSF). Reversible exchange of Aβ42 peptides with preexisting unlabeled peptide was observed in the presence of plaques. These findings support the hypothesis that Aβ42 is overproduced in the CNS of humans with PSEN mutations that cause AD, and demonstrate that soluble Aβ42 turnover and exchange processes are altered in the presence of amyloid plaques, causing a reduction in Aβ42 concentrations in the CSF.

Inhibition of Beta-site APP-cleaving enzyme 1 (BACE1), the β-secretase in the central nervous system (CNS), is an attractive potential disease modifying therapeutic strategy for Alzheimer's disease (AD) as it decreases amyloid precursor protein (APP) processing through the amyloidogenic pathway and CNS β-amyloid peptide levels. The interaction of the β-secretase (amyloidogenic) and α-secretase (non-amyloidogenic) pathway-mediated processing of APP in the rhesus monkey, non-human primate (NHP) CNS is not understood. We hypothesized that CNS inhibition of BACE1 would result in decreased newly-generated amyloid β peptide (Aβ) and sAPPβ, with increased sAPPα. A Stable Isotope Labeling Kinetic (SILK) experiment in NHPs was performed with a 13 C 6 -Leucine infusion protocol to evaluate the effects of a BACE1 inhibitor on CNS processing of APP by measuring the production rates and clearance rates of sAPPα, sAPPβ, and Aβ in cerebrospinal fluid (CSF). Each NHP received vehicle, low dose (10 mg/kg), medium dose (30 mg/kg), and a high dose (125 mg/kg) of the BACE1 inhibitor MBI-5 in a 4-way crossover design. CSF sAPPα, sAPPβ, and Aβ were measured by both ELISA and newly-incorporated label following immunopurification and liquid-chromatography-mass spectrometry. Concentrations, production rates, clearance rates, and total amount of newly-generated APP fragments were calculated. Models were developed to further explore the data and results. Vehicle-treated NHP Aβ production and clearance rates were similar to human measures. sAPPβ and sAPPα production and clearance rates were similar to each other, but much slower than Aβ. The newly-synthesized sAPPα to sAPPβ ratio in NHP CSF is approximately 3:1. Inhibition of BACE1 by MBI-5 resulted in a dose-dependent decrease in newly-labeled CSF sAPPβ and Aβ, without an observable change in newly-labeled CSF sAPPα, while the concentrations of sAPPβ and Aβ both decreased and sAPPα increased as measured by ELISA. While effects on the estimated, newly-synthesized Aβ and sAPPβ levels were directionally consistent to those measured by ELISA, the magnitude of AUC reductions in the newly-synthesized APP fragments, captured during steady state plasma 13 C leucine and peak MBI-5 levels, was greater. These results of BACE1 inhibition in the primate CNS suggest a common pool of APP for both the α-secretase and β-secretase pathways.

BACKGROUNDGastric cancer is increasingly recognized in Zambia. Although nutritional factors contribute to gastric cancer risk, their effect in Zambia is unknown.OBJECTIVEThe objective was to investigate the association between intake of dietary antioxidants, urinary 8-iso prostaglandin F2α (8-iso PGF2α) as a marker of oxidative stress, and gastric cancer.DESIGNThis was a case-control study at the University Teaching Hospital in Zambia. Gastric cancer cases were compared with age- and sex-matched controls. Urine 8-iso PGF2α was measured primarily by ELISA, and by gas chromatography-mass spectrometry in a subset, expressed as a ratio to creatinine. Blood was collected for Helicobacter pylori, HIV serology, gastrin-17, and pepsinogen 1 and 2 concentrations. Clinical and dietary data were collected by using questionnaires. Food items were broadly classified into 7 major categories (fruit, vegetables, fish, meat, insects, cereals, and starches).RESULTSFifty cases with gastric cancer (mean age: 61 y; n = 31 males) and 90 controls (mean age: 54 y; n = 41 males) were enrolled. Median urinary 8-iso PGF2α excretion was higher in cases (0.014; IQR: 0.008-0.021) than in controls (0.011; IQR: 0.006-0.018; P = 0.039). On univariate analysis, habitual fruit intake was lower in cases than in controls during the dry season (P = 0.02). On multivariate analysis, smoking (OR: 7.22; IQR: 1.38-37.9) and gastric atrophy (OR: 2.43; IQR: 1.12-5.13) were independently associated with cancer, and higher fruit intake was protective (OR: 0.44; IQR: 0.20-0.95). Isoprostane excretion was inversely correlated with total fruit intake (ρ = -0.23; n = 140; P = 0.006).CONCLUSIONUrinary 8-iso PGF2α excretion was associated with the risk of gastric cancer, as were smoking and gastric atrophy, but increased fruit intake conferred protection. This trial was registered at www.pactr.org as ISRCTN52971746.

, 189ra77 (2013); 5 Sci Transl Med et al. Rachel Potter Presenilin Mutation Carriers 42 Production, Exchange, and Loss in β Increased in Vivo AmyloidEditor's Summary gauge target engagement for therapeutic trials. kinetics and provide a metric to β quantify the effects of proposed disease-modifying drugs to normalize altered A mutation carriers that indicated both a reversible exchange pool and increased irreversible loss. Future studies could 42 kinetics in β noncarriers. In addition to increased production rates, the authors unexpectedly found altered A 42 production rate in AD mutation carriers that was ~25% higher than that in β authors found an increased A 42 peptides in individuals carrying autosomal dominant AD mutations and related noncarriers. The β 40, and A β 38, A β isoform kinetics to test specific hypotheses regarding the production rates of the A β (SILK) approach to measure A have now used a stable isotope labeling kinetics et al. 42, can recapitulate amyloidosis. Potter β peptides, especially A β model studies based on human autosomal dominant mutations have shown that increasing the production of A 42 peptide causes deposition of amyloid in plaques, nerve destruction, and ultimately AD dementia. Animal β of A The amyloid hypothesis of Alzheimer's disease (AD) proposes that increased production or impaired clearance 42: A Cycle of Gain and Loss β A

Participants in prospective primary prevention trials will be exposed to a compound for multiple years; thus the compound would need to have a proven safety record, such as is true for SSRIs, particularly in a clinically asymptomatic population. In this study we examined the hypothesis that as in transgenic mouse and retrospective human studies (Cirrito et al., 2011) there would be an effect of citalopram on prospectively lowering Amyloid beta levels in humans. A randomized double-blind placebo controlled study was performed to determine the central nervous system effect of 60 mg of the SSRI antidepressant citalopram on Abeta protein production and levels. Healthy 18–50 year old male and female volunteers received either placebo or 60 mg of citalopram prior to the start of the central nervous system stable isotope labeling kinetics study of Abeta, as previously described. A lumbar catheter was placed and hourly sampling of blood and CSF was conducted during and after administration of a stable-isotope labeled amino acid (13C6-leucine). Metabolism of Abeta was measured using stable isotope labeling kinetics (SILK-Aβ®) assay (as in Bateman et al., 2006) with the addition of stable isotope spike absolute quantitation (SISAQ™) to allow for quantitation of Abeta concentrations (C2N Diagnostics). Placebo (n = 9) and citalopram (n = 11) treated subjects did not differ on any demographic variables. Citalopram-treated subjects had significantly lower mean total Abeta concentrations (36.7 +/- 10.5) ng/mL than placebo-treated (54.6 +/- 15.5) ng/mL (p = 0.007) over the hours 5–36. During the same period there was a trend towards lower newly generated Abeta (p = 0.06) in the citalopram group (7.5 +/- 1.3) ng/mL vs placebo (9.3 +/- 1.4) ng/mL however the groups did not differ in their fractional synthesis or clearance rates. We corroborate our prior findings in a prospective human study showing that citalopram lowers Abeta concentrations compared with placebo. We had also expected to see a lower rate of Abeta production and a decrease in newly generated Abeta, thus we are currently replicating and extending our results. The ability to decrease Abeta concentrations is potentially important as a preventive strategy for AD.

Purpose: Gastric cancer is the ninth leading cause of cancer death in Zambia, yet there are no studies evaluating risk factors. Decreased antioxidant intake and increased oxidative stress have been associated with carcinogenesis. Aim: To investigate the association of gastric cancer with dietary antioxidant intake and oxidative stress as measured by urinary 8-iso-prostaglandin F2α (8-iso PGF2α). Methods: Patients with new endoscopic diagnosis of gastric adenocarcinoma (n=50, age 61 ±15.3 yr, 38% F) were compared to controls with normal endoscopy in a 2:1 ratio (n=90, age 54 ± 14.9 yr, 54% F) at the University Teaching Hospital in Lusaka, Zambia in this prospective case control study. A detailed food frequency questionnaire specific to the Zambian diet created a priori for this study was utilized where food items were classified into 7 categories (fruits, vegetables, fish, meat, insects, cereals and starches). Blood was collected for Helicobacter pylori and HIV serology, gastrin 17, pepsinogen 1 and 2 concentrations. Fasting spot urine samples were collected, stored in butylated hydroxytoluene at -80C, and urine 8-iso PGF2α quantified using ELISA and normalized to creatinine; this was validated in a subset (14 cases, 24 controls) using gas chromatography/mass spectrometry (GC/MS). Univariate and multivariate regression analyses were performed to determine the role of dietary antioxidants in gastric cancer. Results: Of the 50 cases of gastric adenocarcinoma, 30 (60%) were located in antrum, 17 (34%) in gastric body and 10 (20%) in cardia, while remainder extended across more than one site. When segregated by the Lauren classification, 39 (78%) were intestinal type adenocarcinoma, 7 (14%) were diffuse and 2 (4%) were mixed; 2 were not classified. 76% of cases and 87% of controls were H. pylori seropositive (p=0.10). Median urinary 8-iso PGF2α/creatinine ratio was higher in patients with gastric cancer (0.014, IQR 0.008-0.021) than in controls (0.011, IQR 0.006-0.018; p=0.039). There was significant correlation between ELISA and GC/MS in measurement of urinary isoprostanes (Spearman's ρ = 0.35; p=0.03). Fruit intake during the dry season was lower in cases than in controls in univariate analysis (p=0.02), and smoking (OR 7.22, CI 1.38-3.96, p=0.019) and gastric atrophy (OR 2.43, CI 1.12-5.13, p=0.024) were independently associated with cancer in multivariate analysis. Isoprostane excretion was inversely correlated with total fruit intake (ρ= -0.23; n=140; p=0.006). Conclusion: Gastric adenocarcinoma is associated with decreased seasonal fruit intake and increased urinary 8-iso PGF2α excretion (a marker of oxidative stress) as well as smoking and gastric atrophy, but not with H. pylori seropositivity.

TLR agonists initiate a rapid activation program in dendritic cells (DCs) that requires support from metabolic and bioenergetic resources. We found previously that TLR signaling promotes aerobic glycolysis and a decline in oxidative phosphorylation (OXHPOS) and that glucose restriction prevents activation and leads to premature cell death. However, it remained unclear why the decrease in OXPHOS occurs under these circumstances. Using real-time metabolic flux analysis, in the present study, we show that mitochondrial activity is lost progressively after activation by TLR agonists in inflammatory blood monocyte-derived DCs that express inducible NO synthase. We found that this is because of inhibition of OXPHOS by NO and that the switch to glycolysis is a survival response that serves to maintain ATP levels when OXPHOS is inhibited. Our data identify NO as a profound metabolic regulator in inflammatory monocyte-derived DCs.