Human Central Nervous System (CNS) ApoE Isoforms Are Increased by Age, Differentially Altered by Amyloidosis, and Relative Amounts Reversed in the CNS Compared with Plasma.

The risk of Alzheimer's disease (AD) is highly dependent on apolipoprotein-E (apoE) genotype. The reasons for apoE isoform-selective risk are uncertain; however, both the amounts and structure of human apoE isoforms have been hypothesized to lead to amyloidosis increasing the risk for AD. To address the hypothesis that amounts of apoE isoforms are different in the human CNS, we developed a novel isoform-specific method to accurately quantify apoE isoforms in clinically relevant samples. The method utilizes an antibody-free enrichment step and isotope-labeled physiologically relevant lipoprotein particle standards produced by immortalized astrocytes. We applied this method to a cohort of well characterized clinical samples and observed the following findings. The apoE isoform amounts are not different in cerebrospinal fluid (CSF) from young normal controls, suggesting that the amount of apoE isoforms is not the reason for risk of amyloidosis prior to the onset of advanced age. We did, however, observe an age-related increase in both apoE isoforms. In contrast to normal aging, the presence of amyloid increased apoE3, whereas apoE4 was unchanged or decreased. Importantly, for heterozygotes, the apoE4/apoE3 isoform ratio was increased in the CNS, although the reverse was true in the periphery. Finally, CSF apoE levels, but not plasma apoE levels, correlated with CSF ß-amyloid levels. Collectively, these findings support the hypothesis that CNS and peripheral apoE are separate pools and differentially regulated. Furthermore, these results suggest that apoE mechanisms for the risk of amyloidosis and AD are related to an interaction between apoE, aging, and the amount of amyloid burden.

Age and amyloid effects on human central nervous system amyloid-beta kinetics.

OBJECTIVE: Age 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 (Aß) kinetics in the central nervous system (CNS) of humans. METHODS: Aß 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 Aß turnover rates (2.5-fold longer half-life over five decades of age). In addition, we found independent effects on Aß42 kinetics specifically in participants with amyloid deposition. Amyloidosis was associated with a higher (>50%) irreversible loss of soluble Aß42 and a 10-fold higher Aß42 reversible exchange rate. INTERPRETATION: These findings reveal a mechanistic link between human aging and the risk of amyloidosis, which may be owing to a dramatic slowing of Aß turnover, increasing the likelihood of protein misfolding that leads to deposition. Alterations in Aß 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.

CNS amyloid-ß, soluble APP-α and -ß kinetics during BACE inhibition.

BACE, a ß-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 ß-secretase pathway and a lowering of CNS amyloid-ß (Aß) levels. The interaction of the ß-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 Aß and soluble APPß (sAPPß), 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, sAPPß, and Aß 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, sAPPß, and Aß 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. sAPPß and sAPPα kinetics were similar, but both significantly slower than Aß. BACE inhibition resulted in decreased labeled sAPPß and Aß in CSF, without observable changes in labeled CSF sAPPα. ELISA concentrations of sAPPß and Aß 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 ß-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.

Amyloid-ß efflux from the central nervous system into the plasma.

OBJECTIVE: The aim of this study was to measure the flux of amyloid-ß (Aß) across the human cerebral capillary bed to determine whether transport into the blood is a significant mechanism of clearance for Aß produced in the central nervous system (CNS). METHODS: Time-matched blood samples were simultaneously collected from a cerebral vein (including the sigmoid sinus, inferior petrosal sinus, and the internal jugular vein), femoral vein, and radial artery of patients undergoing inferior petrosal sinus sampling. For each plasma sample, Aß concentration was assessed by 3 assays, and the venous to arterial Aß concentration ratios were determined. RESULTS: Aß concentration was increased by ∼7.5% in venous blood leaving the CNS capillary bed compared to arterial blood, indicating efflux from the CNS into the peripheral blood (p < 0.0001). There was no difference in peripheral venous Aß concentration compared to arterial blood concentration. INTERPRETATION: Our results are consistent with clearance of CNS-derived Aß into the venous blood supply with no increase from a peripheral capillary bed. Modeling these results suggests that direct transport of Aß across the blood-brain barrier accounts for ∼25% of Aß clearance, and reabsorption of cerebrospinal fluid Aß accounts for ∼25% of the total CNS Aß clearance in humans. Ann Neurol 2014;76:837-844.

Amyloid-beta isoform metabolism quantitation by stable isotope-labeled kinetics.

Abundant evidence suggests a central role for the amyloid-beta (Aß) peptide in Alzheimer's disease (AD) pathogenesis. Production and clearance of different Aß isoforms have been established as targets of proposed disease-modifying therapeutic treatments of AD. However, previous studies used multiple sequential purification steps to isolate the isoforms individually and quantitate them based on a common mid-domain peptide. We created a method to simultaneously purify Aß isoforms and quantitate them by the specific C-terminal peptides in order to investigate Aß isoform physiology in the central nervous system. By using standards generated from in vitro metabolic labeling, the relative quantitation of four peptides representing total amount of Aß (Aß-Total), Aß38, Aß40, and Aß42 were achieved both in cell culture and in human cerebrospinal fluid (CSF). Standard curves for each isoform demonstrated good sensitivity with very low limits of detection and high accuracy. Because the assay does not require antibody development for each Aß isoform peptide, significant improvements in the throughput and accuracy of isoform quantitation were achieved.

Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1.

SphK (sphingosine kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S1P (sphingosine 1-phosphate). S1P promotes cell growth, survival and migration, and is a key regulator of lymphocyte trafficking. Inhibition of S1P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer. In the present paper we describe the discovery and characterization of PF-543, a novel cell-permeant inhibitor of SphK1. PF-543 inhibits SphK1 with a K(i) of 3.6 nM, is sphingosine-competitive and is more than 100-fold selective for SphK1 over the SphK2 isoform. In 1483 head and neck carcinoma cells, which are characterized by high levels of SphK1 expression and an unusually high rate of S1P production, PF-543 decreased the level of endogenous S1P 10-fold with a proportional increase in the level of sphingosine. In contrast with past reports that show that the growth of many cancer cell lines is SphK1-dependent, specific inhibition of SphK1 had no effect on the proliferation and survival of 1483 cells, despite a dramatic change in the cellular S1P/sphingosine ratio. PF-543 was effective as a potent inhibitor of S1P formation in whole blood, indicating that the SphK1 isoform of sphingosine kinase is the major source of S1P in human blood. PF-543 is the most potent inhibitor of SphK1 described to date and it will be useful for dissecting specific roles of SphK1-driven S1P signalling.

Tau Kinetics in Neurons and the Human Central Nervous System.

We developed stable isotope labeling and mass spectrometry approaches to measure the kinetics of multiple isoforms and fragments of tau in the human central nervous system (CNS) and in human induced pluripotent stem cell (iPSC)-derived neurons. Newly synthesized tau is truncated and released from human neurons in 3 days. Although most tau proteins have similar turnover, 4R tau isoforms and phosphorylated forms of tau exhibit faster turnover rates, suggesting unique processing of these forms that may have independent biological activities. The half-life of tau in control human iPSC-derived neurons is 6.74 ± 0.45 days and in human CNS is 23 ± 6.4 days. In cognitively normal and Alzheimer's disease participants, the production rate of tau positively correlates with the amount of amyloid plaques, indicating a biological link between amyloid plaques and tau physiology.

Intra- and Inter-individual Variability of microRNA Levels in Human Cerebrospinal Fluid: Critical Implications for Biomarker Discovery.

MicroRNAs are emerging as promising biomarkers for diagnosis of various diseases. Notably, cerebrospinal fluid (CSF) contains microRNAs that may serve as biomarkers for neurological diseases. However, there has been a lack of consistent findings among CSF microRNAs studies. Although such inconsistent results have been attributed to various technical issues, inherent biological variability has not been adequately considered as a confounding factor. To address this critical gap in our understanding of microRNA variability, we evaluated intra-individual variability of microRNAs by measuring their levels in the CSF from healthy individuals at two time points, 0 and 48 hours. Surprisingly, the levels of most microRNAs were stable between the two time points. This suggests that microRNAs in CSF may be a good resource for the identification of biomarkers. However, the levels of 12 microRNAs (miR-19a-3p, miR-19b-3p, miR-23a-3p, miR-25a-3p, miR-99a-5p, miR-101-3p, miR-125b-5p, miR-130a-3p, miR-194-5p, miR-195-5p, miR-223-3p, and miR-451a) were significantly altered during the 48 hours interval. Importantly, miRNAs with variable expression have been identified as biomarkers in previous studies. Our data strongly suggest that these microRNAs may not be reliable biomarkers given their intrinsic variability even within the same individual. Taken together, our results provide a critical baseline resource for future microRNA biomarker studies.

Associations Between ß-Amyloid Kinetics and the ß-Amyloid Diurnal Pattern in the Central Nervous System.

Importance: Recent studies found that the concentration of amyloid-ß (Aß) fluctuates with the sleep-wake cycle. Although the amplitude of this day/night pattern attenuates with age and amyloid deposition, to our knowledge, the association of Aß kinetics (ie, production, turnover, and clearance) with this oscillation has not been studied. Objective: To determine the association between Aß kinetics, age, amyloid levels, and the Aß day/night pattern in humans. Design, Setting, and Participants: We measured Aß concentrations and kinetics in 77 adults aged 60 to 87 years with and without amyloid deposition by a novel precise mass spectrometry method at the Washington University School of Medicine in St Louis, Missouri. We compared findings of 2 orthogonal methods, enzyme-linked immunosorbent assay and mass spectrometry, to validate the day/night patterns and determine more precise estimates of the cosinor parameters. In vivo labeling of central nervous system proteins with stable isotopically labeled leucine was performed, and kinetics of Aß40 and Aß42 were measured. Interventions: Serial cerebrospinal fluid collection via indwelling lumbar catheter over 36 to 48 hours before, during, and after in vivo labeling, with a 9-hour primed constant infusion of 13C6-leucine. Main Outcomes and Measures: The amplitude, linear increase, and other cosinor measures of each participant's serial cerebrospinal fluid Aß concentrations and Aß turnover rates. Results: Of the 77 participants studied, 46 (59.7%) were men, and the mean (range) age was 72.6 (60.4-87.7) years. Day/night patterns in Aß concentrations were more sharply defined by the precise mass spectrometry method than by enzyme-linked immunosorbent assay (mean difference of SD of residuals: Aß40, -7.42 pM; P < .001; Aß42, -3.72 pM; P < .001). Amyloid deposition diminished day/night amplitude and linear increase of Aß42 but not of Aß40. Increased age diminished day/night amplitude of both Aß40 and Aß42. After controlling for amyloid deposition, amplitude of Aß40 was positively associated with production rates (r = 0.42; P < .001), while the linear rise was associated with turnover rates (r = 0.28; P < .05). The amplitude and linear rise of Aß42 were both associated with turnover (r = -0.38; P < .001) and production (r = 0.238; P < .05) rates. Conclusions and Relevance: Amyloid deposition is associated with premature loss of normal Aß42 day/night patterns in older adults, suggesting the previously reported effects of age and amyloid on Aß42 amplitude at least partially affect each other. Production and turnover rates suggest that day/night Aß patterns are modulated by both production and clearance mechanisms active in sleep-wake cycles and that amyloid deposition may impair normal circadian patterns. These findings may be important for the designs of future secondary prevention trials for Alzheimer disease.

Diurnal patterns of soluble amyloid precursor protein metabolites in the human central nervous system.

The amyloid-ß (Aß) protein is diurnally regulated in both the cerebrospinal fluid and blood in healthy adults; circadian amplitudes decrease with aging and the presence of cerebral Aß deposits. The cause of the Aß diurnal pattern is poorly understood. One hypothesis is that the Amyloid Precursor Protein (APP) is diurnally regulated, leading to APP product diurnal patterns. APP in the central nervous system is processed either via the ß-pathway (amyloidogenic), generating soluble APP-ß (sAPPß) and Aß, or the α-pathway (non-amyloidogenic), releasing soluble APP-α (sAPPα). To elucidate the potential contributions of APP to the Aß diurnal pattern and the balance of the α- and ß- pathways in APP processing, we measured APP proteolytic products over 36 hours in human cerebrospinal fluid from cognitively normal and Alzheimer's disease participants. We found diurnal patterns in sAPPα, sAPPß, Aß40, and Aß42, which diminish with increased age, that support the hypothesis that APP is diurnally regulated in the human central nervous system and thus results in Aß diurnal patterns. We also found that the four APP metabolites were positively correlated in all participants without cerebral Aß deposits. This positive correlation suggests that the α- and ß- APP pathways are non-competitive under normal physiologic conditions where APP availability may be the limiting factor that determines sAPPα and sAPPß production. However, in participants with cerebral Aß deposits, there was no correlation of Aß to sAPP metabolites, suggesting that normal physiologic regulation of cerebrospinal fluid Aß is impaired in the presence of amyloidosis. Lastly, we found that the ratio of sAPPß to sAPPα was significantly higher in participants with cerebral Aß deposits versus those without deposits. Therefore, the sAPPß to sAPPα ratio may be a useful biomarker for cerebral amyloidosis.

Continuous 24-hour leptin, proopiomelanocortin, and amino acid measurements in human cerebrospinal fluid: correlations with plasma leptin, soluble leptin receptor, and amino acid levels.

CONTEXT: In order to characterize diurnal changes in central leptin and its target neuropeptide, proopiomelanocortin (POMC), we measured leptin and POMC in cerebrospinal fluid (CSF) as related to changes in plasma leptin and soluble leptin receptor (sOB-R) levels. CSF and plasma levels of 20 amino acids (AA) were also measured because AA can affect brain POMC. DESIGN AND PARTICIPANTS: Stored CSF and plasma samples obtained from eight healthy subjects who served as controls for a previous study were evaluated. CSF was collected hourly over 33 h via indwelling subarachnoid catheter. Leptin, sOB-R, and POMC were measured by sensitive ELISA and AA by gas chromatography-mass spectrometry. RESULTS: There was a diurnal rhythm for plasma leptin with a peak at 2200 h (144% of baseline) and there was a similar diurnal rhythm for CSF leptin with a peak (117%) 3-5 h after the plasma peak. Plasma sOB-R was lowest at 0300 h and correlated negatively with plasma and CSF leptin. A diurnal rhythm for POMC in CSF was also detected with a peak (125%) at 0100 h. A positive correlation existed between CSF POMC and leptin in individual subjects over time. CSF levels of many AA increased at night. There was a significant correlation between CSF POMC and 10 AA, including leucine, isoleucine, tryptophan, and tyrosine. CONCLUSIONS: Diurnal changes occur in leptin and POMC in human CSF that likely reflect changes in central leptin and melanocortin activity. Our results suggest that nocturnal elevations in leptin, AA, and POMC may help to suppress appetite and feeding at night.

Increased in vivo amyloid-ß42 production, exchange, and loss in presenilin mutation carriers.

Alzheimer's disease (AD) is hypothesized to be caused by an overproduction or reduced clearance of amyloid-ß (Aß) peptide. Autosomal dominant AD (ADAD) caused by mutations in the presenilin (PSEN) gene have been postulated to result from increased production of Aß42 compared to Aß40 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 Aß 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 Aß42 to Aß40 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 Aß42 relative to Aß40 was 65% faster in mutation carriers and correlated with amyloid deposition, consistent with increased deposition of Aß42 into plaques, leading to reduced recovery of Aß42 in cerebrospinal fluid (CSF). Reversible exchange of Aß42 peptides with preexisting unlabeled peptide was observed in the presence of plaques. These findings support the hypothesis that Aß42 is overproduced in the CNS of humans with PSEN mutations that cause AD, and demonstrate that soluble Aß42 turnover and exchange processes are altered in the presence of amyloid plaques, causing a reduction in Aß42 concentrations in the CSF.

Disruption of the sleep-wake cycle and diurnal fluctuation of ß-amyloid in mice with Alzheimer's disease pathology.

Aggregation of ß-amyloid (Aß) in the brain begins to occur years before the clinical onset of Alzheimer's disease (AD). Before Aß aggregation, concentrations of extracellular soluble Aß in the interstitial fluid (ISF) space of the brain, which are regulated by neuronal activity and the sleep-wake cycle, correlate with the amount of Aß deposition in the brain seen later. The amount and quality of sleep decline with normal aging and to a greater extent in AD patients. How sleep quality as well as the diurnal fluctuation in Aß change with age and Aß aggregation is not well understood. We report a normal sleep-wake cycle and diurnal fluctuation in ISF Aß in the brain of the APPswe/PS1δE9 mouse model of AD before Aß plaque formation. After plaque formation, the sleep-wake cycle markedly deteriorated and diurnal fluctuation of ISF Aß dissipated. As in mice, diurnal fluctuation of cerebrospinal fluid Aß in young adult humans with presenilin mutations was also markedly attenuated after Aß plaque formation. Virtual elimination of Aß deposits in the mouse brain by active immunization with Aß(42) normalized the sleep-wake cycle and the diurnal fluctuation of ISF Aß. These data suggest that Aß aggregation disrupts the sleep-wake cycle and diurnal fluctuation of Aß. Sleep-wake behavior and diurnal fluctuation of Aß in the central nervous system may be functional and biochemical indicators, respectively, of Aß-associated pathology.

Effects of age and amyloid deposition on Aß dynamics in the human central nervous system.

BACKGROUND: The amyloid hypothesis predicts that increased production or decreased clearance of ß-amyloid (Aß) leads to amyloidosis, which ultimately culminates in Alzheimer disease (AD). OBJECTIVE: To investigate whether dynamic changes in Aß levels in the human central nervous system may be altered by aging or by the pathology of AD and thus contribute to the risk of AD. DESIGN: Repeated-measures case-control study. SETTING: Washington University School of Medicine in St Louis, Missouri. PARTICIPANTS: Participants with amyloid deposition, participants without amyloid deposition, and younger normal control participants. MAIN OUTCOME MEASURES: In this study, hourly cerebrospinal fluid (CSF) Aß concentrations were compared with age, status of amyloid deposition, electroencephalography, and video recording data. RESULTS: Linear increases were observed over time in the Aß levels in CSF samples obtained from the younger normal control participants and the older participants without amyloid deposition, but not from the older participants with amyloid deposition. Significant circadian patterns were observed in the Aß levels in CSF samples obtained from the younger control participants; however, circadian amplitudes decreased in both older participants without amyloid deposition and older participants with amyloid deposition. Aß diurnal concentrations were correlated with the amount of sleep but not with the various activities that the participants participated in while awake. CONCLUSIONS: A reduction in the linear increase in the Aß levels in CSF samples that is associated with amyloid deposition and a decreased CSF Aß diurnal pattern associated with increasing age disrupt the normal physiology of Aß dynamics and may contribute to AD.

ß-amyloid dynamics in human plasma.

OBJECTIVES: To investigate dynamic changes in human plasma ß-amyloid (Aß) concentrations, evaluate the effects of aging and amyloidosis on these dynamics, and determine their correlation with cerebrospinal fluid (CSF) Aß concentrations. DESIGN: A repeated plasma and CSF sampling study. SETTING: The Washington University School of Medicine in St Louis, Missouri. PARTICIPANTS: Older adults with amyloid deposition (Amyloid+), age-matched controls without amyloid deposition (Amyloid-), and younger normal controls (YNCs) were enrolled for the study. MAIN OUTCOME MEASURES: Hourly measurements of plasma Aß were compared between groups by age and amyloidosis. Plasma Aß and CSF Aß concentrations were compared for correlation, linear increase, and circadian patterns. RESULTS: Circadian patterns were observed in plasma Aß, with diminished amplitudes with aging. Linear increase of Aß was only observed for CSF Aß in the YNC and Amyloid- groups, but not in the Amyloid+ group. No linear increase was observed for plasma Aß. No significant correlations were found between plasma and CSF Aß concentrations. CONCLUSIONS: Plasma Aß, like CSF, demonstrates a circadian pattern that is reduced in amplitude with increasing age but is unaffected by amyloid deposition. However, we found no evidence that plasma and CSF Aß concentrations were related on an hourly or individual basis.

In vivo human apolipoprotein E isoform fractional turnover rates in the CNS.

Apolipoprotein E (ApoE) is the strongest genetic risk factor for Alzheimer's disease and has been implicated in the risk for other neurological disorders. The three common ApoE isoforms (ApoE2, E3, and E4) each differ by a single amino acid, with ApoE4 increasing and ApoE2 decreasing the risk of Alzheimer's disease (AD). Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aß) peptide deposition. Therefore, quantifying ApoE isoform production and clearance rates may advance our understanding of the role of ApoE in health and disease. To measure the kinetics of ApoE in the central nervous system (CNS), we applied in vivo stable isotope labeling to quantify the fractional turnover rates of ApoE isoforms in 18 cognitively-normal adults and in ApoE3 and ApoE4 targeted-replacement mice. No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain. However, CNS and peripheral ApoE isoform turnover rates differed substantially, which is consistent with previous reports and suggests that the pathways responsible for ApoE metabolism are different in the CNS and the periphery. We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis.

Decreased clearance of CNS beta-amyloid in Alzheimer's disease.

Alzheimer's disease is hypothesized to be caused by an imbalance between ß-amyloid (Aß) production and clearance that leads to Aß accumulation in the central nervous system (CNS). Aß production and clearance are key targets in the development of disease-modifying therapeutic agents for Alzheimer's disease. However, there has not been direct evidence of altered Aß production or clearance in Alzheimer's disease. By using metabolic labeling, we measured Aß42 and Aß40 production and clearance rates in the CNS of participants with Alzheimer's disease and cognitively normal controls. Clearance rates for both Aß42 and Aß40 were impaired in Alzheimer's disease compared with controls. On average, there were no differences in Aß40 or Aß42 production rates. Thus, the common late-onset form of Alzheimer's disease is characterized by an overall impairment in Aß clearance.