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. (Blood. 2012;120(7):1422-1431) IntroductionDendritic cells (DCs) express TLRs that allow them to detect and respond to pathogen-derived molecules. 1,2 In response to TLR agonists, DCs transition from a resting state to an activated state through a process that that involves the induction of expression of genes encoding a broad array of proteins such as cytokines, chemokines, and costimulatory molecules. 3 Activated DCs play a central role in orchestrating the development of immune responses.Recently, we showed that after exposure to TLR agonists, DCs undergo a striking metabolic transition evident as a pronounced increase in the glycolytic rate. 4 This is highly reminiscent of Warburg metabolism, 5 in which tumor cells preferentially use glycolysis rather than catabolic mitochondrial pathways to conserve and generate metabolic resources to meet the demands of cellular proliferation while still producing sufficient ATP to permit these processes to occur. 6,7 Moreover, the increase in glycolytic rate in DCs was found to be dependent on the PI3K/Akt pathway, 4 which is one of the most commonly mutated signaling pathways in tumors. 8 We reasoned that glycolysis could serve essentially the same purpose in active DCs as it is thought to do in tumors. 4 This view was supported by the fact that glucose restriction inhibits severely the activation and life span of DCs exposed to TLR agonists. 4 However, unlike in most cancers, which continue to consume oxygen at rates comparable to normal tissues despite increased glycolytic rates, 9 activated DCs use significantly less oxygen than do resting DCs. 4 Thus far, the molecular mechanisms underlying mitochondrial impairment in activated DCs, and the metabolic consequences of the loss of mitochondrial function, remain unclear.To address these issues, we have in the present study, undertaken a detailed analysis of mitochondrial function in DCs after TLR stimulation. Using extracellular flux analysis to measure changes in oxygen consumption in real time, we found that 6 hours after stimulation, mitochondrial oxygen consumption was progressively lost due to the ...

Alzheimer’s disease is hypothesized to be caused by an over-production or reduced clearance of amyloid-beta (Aβ) peptide. Autosomal Dominant Alzheimer’s Disease (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 non-carriers 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 non-carriers 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 pre-existing unlabeled peptide was observed in the presence of plaques. These findings support the hypothesis that Aβ42 is overproduced in the CNS of humans with presenilin 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.

Objective Age is the single greatest risk factor for Alzheimer’s disease with the incidence doubling every 5 years after age 65. However, our understanding of the mechanistic relationship between increasing age and the risk for Alzheimer’s disease is currently limited. We therefore sought to determine the relationship between age, amyloidosis, and amyloid-beta kinetics in the central nervous system (CNS) of humans Methods Amyloid-beta kinetics were analyzed in 112 participants and compared to the ages of participants and the amount of amyloid deposition. Results We found a highly significant correlation between increasing age and slowed amyloid-beta turnover rates (2.5-fold longer half-life over five decades of age). In addition, we found independent effects on amyloid-beta42 kinetics specifically in participants with amyloid deposition. Amyloidosis was associated with a higher (>50%) irreversible loss of soluble amyloid-beta42 and a 10-fold higher amyloid-beta42 reversible exchange rate. Interpretation These findings reveal a mechanistic link between human aging and the risk of amyloidosis which may be due to a dramatic slowing of amyloid-beta turnover, increasing the likelihood of protein misfolding that leads to deposition. Alterations in amyloid-beta 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 physiologic and pathophysiologic changes and may be applicable to other proteinopathies.

Serotonin signaling suppresses generation of amyloid-β (Aβ) in vitro and in animal models of Alzheimer’s disease (AD). We show that in an aged transgenic AD mouse model (APP/PS1 plaque-bearing mice), the antidepressant citalopram, a selective serotonin reuptake inhibitor (SSRI), decreased Aβ in brain interstitial fluid (ISF) in a dose-dependent manner. Growth of individual amyloid plaques was assessed in plaque-bearing mice that were chronically administered citalopram. Citalopram arrested the growth of pre-existing plaques and reduced the appearance of new plaques by 78%. In healthy human volunteers, citalopram’s effects on Aβ production and Aβ concentrations in cerebrospinal fluid (CSF) were measured prospectively using stable-isotope labeling kinetics (SILK), with CSF sampling during acute dosing of citalopram. Aβ production in CSF was slowed by 37% in the citalopram group compared to placebo. This change was associated with a 38% decrease in total CSF Aβ concentrations in the drug-treated group. The ability to safely decrease Aβ concentrations is potentially important as a preventive strategy for AD. This study demonstrates key target engagement for future AD prevention trials.

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