Stable Isotope Labeling Kinetics Research Articles

Choroid plexus protein turnover in human choroid plexus organoids recapitulates turnover in humans measured using stable isotope labeling kinetics (SILK)

AbstractBackgroundThe choroid plexus secretes protein‐rich cerebrospinal fluid (CSF). CSF turnover is a key mechanism for protein clearance. Neurodegenerative diseases such as AD are characterised by accumulation of excess proteins that form pathological inclusions. CSF flow dysregulation could therefore be involved in the pathophysiology of neurodegenerative diseases (ND) such as Alzheimer’s Disease. A method for measuring CSF turnover in vivo may be achieved by quantitating CSF derived protein kinetics, providing a biomarker tool with to interrogate the relationship between CSF flow disequilibrium and neurodegeneration.MethodUsing untargeted proteomics we identify the most abundant proteins in a) stem‐cell derived choroid plexus organoids and b) human CSF. We then develop targeted mass spectrometry methods to quantitate protein specific peptides in both matrices. Using a stable isotope (13C6 leucine) we label a) choroid plexus organoids and b) humans with suspected NPH undergoing a 72 hour lumbar drainage protocol. We collect serial samples of the CSF‐like organoid fluid and CSF. We then measure the ratio of labelled to unlabelled peptides to quantitate the rates of protein labelling (fractional synthesis rate) and label loss (fractional clearance rate), defining the protein turnover rate.ResultHighly‐expressed proteins in human CSF from nine individuals were also present in CSF‐like fluid from CP organoids. Of these common proteins, we were able to quantitate the turnover rates of transthyretin, apolipoprotein E, cystatin C and pigment epithelium‐derived factor, and the synthesis rates of serotransferrin and albumin. We will correlate these findings with clinical measures of CSF production.ConclusionChoroid plexus protein synthesis and clearance can be measured using SILK; choroid plexus organoid fluid protein turnover recapitulates that seen in human CSF.

Do Mouse Data Lie? For Buntanetap, They Totally Predict Human Outcome

AbstractBackgroundOverexpression of neurotoxic proteins drives downstream events that dysregulate axonal transport, lead to inflammation, nerve cell death, and loss of function (Chen et al. 2021). By inhibiting the translation of neurotoxic aggregating proteins ‐ amyloid precursor protein, tau, alpha‐synuclein etc., buntanetap restores axonal transport, lowers inflammation, and protects nerve cells from dying. In three double‐blind, placebo controlled, phase 1/2 clinical studies in both Alzheimer (AD) and Parkinson (PD) patients buntanetap showed statistically significant improvement in movement in PD and cognition in AD (Fang et al. 2023).MethodHere we want to compare our animal data with our human data to evaluate if animal models are predictive of human outcomes. We are comparing data from APP/PS1 AD mice (Teich et al. 2018), Ts65Dn Down Syndrome mice (Chen et al. 2020), and two Phase 1 /2 double‐blind, placebo‐controlled human AD studies: Discover study run by ADCS and AD/PD study run by Annovis.Result1. Buntanetap is a translational inhibitor of APP by pulse‐chase experiment in mice and by stable isotope labeling kinetics (SILK) in humans (Galasko et al. 2022).2. Our drug inhibits APP/Abeta, tau/pTau in a dose‐dependent manner in animals (Lahiri et al. 2007; Teich et al. 2018; Chen et al. 2020) and humans (Maccecchini et al. 2012).3. The biomarker dose‐response is seen at a much higher dose than the efficacy dose‐response.4. We see a statistical improvement in cognition in animals and in humans (Teich et al. 2018; Fang et al. 2023).ConclusionIn all our studies conducted to date in AD mice and humans, we have obtained consistent data on the mechanism of action, consistently lowered levels of neurotoxic aggregating proteins, and consistent improvements in cognition.

CYP46A1 activation by low-dose efavirenz enhances brain cholesterol metabolism in subjects with early Alzheimer’s disease

BackgroundEfavirenz is an anti-HIV drug, and cytochrome P450 46A1 (CYP46A1) is a CNS-specific enzyme that metabolizes cholesterol to 24-hydroxycholesterol (24HC). We have previously shown that allosteric CYP46A1 activation by low-dose efavirenz in a transgenic mouse model of Alzheimer’s disease (AD) enhanced both cholesterol elimination and turnover in the brain and improved animal performance in memory tests. Here, we sought to determine whether CYP46A1 could be similarly activated by a low-dose efavirenz in human subjects. MethodsThis pilot study enrolled 5 subjects with early AD. Participants were randomized to placebo (n = 1) or two daily efavirenz doses (50 mg and 200 mg, n = 2 for each) for 20 weeks and evaluated for safety and CYP46A1 target engagement (plasma 24HC levels). A longitudinal mixed model was used to ascertain the statistical significance of target engagement. We also measured 24HC in CSF and conducted a unique stable isotope labeling kinetics (SILK) study with deuterated water to directly measure CYP46A1 activity changes in the brain.ResultsIn subjects receiving efavirenz, there was a statistically significant within-group increase (P ≤ 0.001) in the levels of plasma 24HC from baseline. The levels of 24HC in the CSF of subjects on the 200-mg dose of efavirenz were also increased. Target engagement was further supported by the labeling kinetics of 24HC by deuterated water in the SILK study. There were no serious adverse effects in any subjects.ConclusionsOur findings suggest efavirenz target engagement in human subjects with early AD. This supports the pursuit of a larger trial for further determination and confirmation of the efavirenz dose that exerts maximal enzyme activation, as well as evaluation of this drug’s effects on AD biomarkers and clinical symptomatology.Trial registrationClinicalTrials.gov, NCT03706885.

Stable Isotope Labeling Kinetics (SILK) tracing of neurofilament light in stem cell‐derived neurons

AbstractBackgroundNeurofilament light polypeptide (NfL) is a basic component of intermediate filaments in neurons that is released from damaged and/or degenerated neuronal axons. As such, NfL levels in CSF and serum are elevated in several neurodegenerative diseases, and have been proposed as a promising biomarker of axonal damage. However, whether NfL’s extracellular release results from passive events or active mechanisms remains largely unknown. Kinetic labeling at the step of protein translation can inform about active translation vs. passive release processes and provide protein metabolism half‐lives and rates of production and clearance.MethodOur aim was to develop a method to study NfL turnover rates using stable isotope labeling kinetics (SILK) in human induced pluripotent stem cell (iPSC)‐derived neurons. We added 13C6‐leucine, the tracer, to the neuron cultures (“pulse”) for four weeks, followed by medium exchange with 12C6‐leucine, the tracee, for an additional four weeks (“chase”). Samples were collected during both the pulse and chase phases of the experiment. 13C6‐leucine incorporation and the levels of labeled NfL in cell lysates and conditioned media from two independent neuronal inductions were analyzed by ultra‐performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) and the tracer‐to‐tracee ratio (TTR) and mole percent excess (MPE) calculated.ResultWe detected ten leucine‐containing peptides unique to NfL in neuronal cell lysates, which are present within the rod‐domain of the protein. These peptides showed different rates of 13C6‐leucine incorporation (reaching up to 69% MPE).ConclusionPreliminary data on the monitoring of these peptides suggest that NfL is turned over at a slow rate in iPSC‐. Tracking of both intracellular and extracellular NfL in neurons derived from healthy control individuals, as well as cells with mutations implicated in neurodegenerative diseases, are expected to shed light into the dynamics of NfL turnover in both physiological and pathological conditions. This will be critical for interpreting NfL in response to disease‐modifying therapies.

The metabolism of human soluble amyloid precursor protein isoforms is quantifiable by a stable isotope labeling-tandem mass spectrometry method

Evidence suggests that β-secretase (BACE1), which cleaves Amyloid Precursor Protein (APP) to form sAPPβ and amyloid-β, is elevated in Alzheimer's disease (AD) brains and biofluids and, thus, BACE1 is a therapeutic target for this devastating disease. The direct product of BACE1 cleavage of APP, sAPPβ, serves as a surrogate marker of BACE1 activity in the central nervous system. This biomarker could be utilized to better understand normal APP processing, aberrant processing in the disease setting, and modulations to processing during therapeutic intervention. In this paper, we present a method for measuring the metabolism of sAPPβ and another APP proteolytic product, sAPPα, in vivo in humans using stable isotope labeling kinetics, paired with immunoprecipitation and liquid chromatography/tandem mass spectrometry. The method presented herein is robust, reproducible, and precise, and allows for the study of these analytes by taking into account their full dynamic potential as opposed to the traditional methods of absolute concentration quantitation that only provide a static view of a dynamic system. A study of in vivo cerebrospinal fluid sAPPβ and sAPPα kinetics using these methods could reveal novel insights into pathophysiological mechanisms of AD, such as increased BACE1 processing of APP.

Importance of CSF-based A\u03b2 clearance with age in humans increases with declining efficacy of blood-brain barrier/proteolytic pathways

The kinetics of amyloid beta turnover within human brain is still poorly understood. We previously found a dramatic decline in the turnover of Aβ peptides in normal aging. It was not known if brain interstitial fluid/cerebrospinal fluid (ISF/CSF) fluid exchange, CSF turnover, blood-brain barrier function or proteolysis were affected by aging or the presence of β amyloid plaques. Here, we describe a non-steady state physiological model developed to decouple CSF fluid transport from other processes. Kinetic parameters were estimated using: (1) MRI-derived brain volumes, (2) stable isotope labeling kinetics (SILK) of amyloid-β peptide (Aβ), and (3) lumbar CSF Aβ concentration during SILK. Here we show that changes in blood-brain barrier transport and/or proteolysis were largely responsible for the age-related decline in Aβ turnover rates. CSF-based clearance declined modestly in normal aging but became increasingly important due to the slowing of other processes. The magnitude of CSF-based clearance was also lower than that due to blood-brain barrier function plus proteolysis. These results suggest important roles for blood-brain barrier transport and proteolytic degradation of Aβ in the development Alzheimer’s Disease in humans.

Following spatial Aβ aggregation dynamics in evolving Alzheimer's disease pathology by imaging stable isotope labeling kinetics.

β-Amyloid (Aβ) plaque formation is the major pathological hallmark of Alzheimer's disease (AD) and constitutes a potentially critical, early inducer driving AD pathogenesis as it precedes other pathological events and cognitive symptoms by decades. It is therefore critical to understand how Aβ pathology is initiated and where and when distinct Aβ species aggregate. Here, we used metabolic isotope labeling in APPNL-G-F knock-in mice together with mass spectrometry imaging to monitor the earliest seeds of Aβ deposition through ongoing plaque development. This allowed visualizing Aβ aggregation dynamics within single plaques across different brain regions. We show that formation of structurally distinct plaques is associated with differential Aβ peptide deposition. Specifically, Aβ1-42 is forming an initial core structure followed by radial outgrowth and late secretion and deposition of Aβ1-38. These data describe a detailed picture of the earliest events of precipitating amyloid pathology at scales not previously possible.

CSF Biomarkers in Down Syndrome and Autosomal Dominant Alzheimer Disease

Background: Due to trisomy of chromosome 21 and the resultant extra copy of the amyloid precursor protein gene, virtually all adults with Down syndrome (DS) develop Alzheimer disease (AD) pathology by age 40 and are at high risk for dementia given their increased life expectancy. Comparison of cerebrospinal fluid (CSF) biomarker patterns with those of autosomal dominant AD (ADAD) mutation carriers (MC) will enhance our understanding of disease mechanisms in these two genetically defined groups. Methods: CSF samples obtained from adults with DS (n=41) and similarly-aged ADAD MC (n=192) and non-carrier (NC, n=108) siblings (aged 30-61 years) were analyzed for markers of amyloid-β (Aβ40, Aβ42), phosphorylated tau-related processes (pTau181), neuronal/axonal/synaptic injury (total tau, visinin-like protein 1 [VILIP-1], neurofilament light chain [NfL], synaptosomal-associated protein 25 [SNAP-25]), and astrogliosis/neuroinflammation (chitinase-3-like protein 1 [YKL-40]) via immunoassay. Biomarker concentrations were compared as a function of dementia status (asymptomatic/symptomatic), and linear regression was used to evaluate and compare the relationship between biomarker levels and age among groups. Findings: Adults with DS exhibited patterns of AD-related CSF biomarkers remarkably similar to ADAD MC, including reductions in the Aβ42/Aβ40 ratio and increases in markers of phosphorylated tau-related processes, neuronal/axonal/synaptic injury, and astrogliosis/neuroinflammation, with greater degrees of abnormality in those with dementia. Differences included overall higher levels of Aβ and YKL-40 in DS and potential elevations in CSF tau and NfL in the asymptomatic (non-demented) stage. Interpretation: CSF biomarker profiles are useful for identifying and tracking AD-related processes in DS and will likely have utility for clinical care and clinical trial design in this understudied at-risk population. Funding: National Institute on Aging (U01 AG051406, U01 AG051412, UF1AG032438), Eunice Kennedy Shriver National Institute of Child Health and Human Development, German Center for Neurodegenerative Diseases, and Research and Development Grants for Dementia from Japan Agency for Medical Research and Development. Conflict of Interest: AMF has received research funding from the National Institutes of Health /National Institute on Aging, Biogen, Centene, Fujirebio, and Roche Diagnostics. She is a member of the scientific advisory boards for Roche Diagnostics, Genentech, and AbbVie and also consults for Araclon/Grifols, DiademRes, DiamiR, and Otsuka Pharmaceuticals. There are no conflicts. RJB has equity ownership interest in C2N Diagnostics and receives royalty income based on technology (stable isotope labeling kinetics and blood plasma assay) licensed by Washington University to C2N Diagnostics. He receives income from C2N Diagnostics for serving on the scientific advisory board. Washington University, with RJB as co-inventor, has submitted the US nonprovisional patent application “Cerebrospinal fluid (CSF) tau rate of phosphorylation measurement to define stages of Alzheimer’s disease and monitor brain kinases/phosphatases activity.” He has received honoraria from Janssen and Pfizer as a speaker, and from Merck and Pfizer as an advisory board member. He has been an invited speaker, advisory board member, and consultant for F. Hoffman La Roche, Ltd., an invited speaker and consultant for AC Immune and Janssen, and a consultant for Amgen and Eisai. There are no conflicts. AMG has consulted for Eisai, Biogen, Pfizer, AbbVie, Cognition Therapeutics, and GSK. She also served on the Scientific Advisory Board of Denali Therapeutics (2015–2018). There are no conflicts. Ethical Approval: Informed consent was obtained directly from all participants whenever possible; otherwise, assent was obtained, and informed consent obtained from the participant’s legally authorized representative. Institutional Review Board approval was obtained at all sites.

Tau kinetics in Alzheimer disease and primary tauopathies

AbstractBackgroundUnderstanding tau kinetics in the human central nervous system is critical for evaluating the effects of tau‐targeted therapies and designing clinical trials against tau in Alzheimer disease (AD) and other primary tauopathies. We hypothesized that tau production or clearance is altered in tauopathies and measured tau kinetics in AD and in a small set of participants with Progressive Supranuclear Palsy (PSP), Corticobasal Syndrome (CBS), or from Frontotemporal Dementia (FTD)‐MAPT families.Methods In vivo human tau Stable Isotope Labeling Kinetics (SILK) were performed in 65 participants including 10 young normal controls, 23 age‐matched controls, 16 AD and 16 primary tauopathies (2 PSP, 4 CBS, 10 MAPT mutation family). Briefly, participants received 16hr intravenous infusion of 13C6‐leucine and cerebrospinal fluid (CSF) was collected from five lumbar punctures over 120 days. Labeled tau was measured with mass spectrometry to obtain kinetic curves of tau. Production and clearance rates were calculated and compared across different tauopathies. CSF amyloid beta (Aβ), amyloid and tau Positron Emission Tomography (PET) imaging were measured in a subset of participants, and analyzed for correlation with SILK measures.ResultsFirst, we confirmed the reproducibility of previous results (Sato et al., 2018) and demonstrated that tau production rates increased with amyloid measured by PET or CSF Aβ. Second, CSF tau concentration correlated with CSF Aβ40 and Aβ42 in primary tauopathies but only with Aβ40 in those with abnormal Aβ levels. Third, 2 PSP patients had slower clearance of tau with tau half‐lives approximately 1.5‐2.5 times longer than other cohorts. Tau Fractional Synthesis Rate also negatively correlated with tau concentration in primary tauopathies, and CBS and FTD‐MAPT had different slopes, suggesting that they may have different kinetics.ConclusionsThe results from this study provide mechanistic insights into differences between AD and other primary tauopathies, which may be targeted differently when developing therapies against tau metabolism. In vivo tau kinetics can help evaluate the efficacy of tau‐targeted therapies and provide information for designing clinical trials targeting tau.

MECHANISTIC MATHEMATICAL MODEL OF THE IMPACT OF ANTI-AMYLOID-BETA DRUGS AND BACE INHIBITORS IN ALZHEIMER’S DISEASE

Clinical trials for Alzheimer's Disease (AD) have had a high failure rate (99.6%). The reasons may include insufficient disease understanding, lack of targeting to the right stage of disease or patient population, and insufficient drug exposure in the brain. To maximize the learning from past clinical experience, we established a single mechanistic mathematical model of mild/moderate AD describing molecular mechanisms leading to plaque formation with the help of clinical biomarkers and capture the effects of multiple anti-amyloid beta (Ab) monoclonal antibodies and amyloid targeting β-secretase (BACE) inhibitors. The AD model includes three different pools of Ab species: monomer, soluble oligomer, and plaque and describes Ab production, aggregation, the intercompartment transport as well as antibody-dependent cellular phagocytosis of plaque. The model was informed by data from stable isotope labeling kinetics experiments, molecular binding and clinical biomarker data for four anti-Ab monoclonal antibodies (Aducanumab, Crenezumab, Solanezumab, Bapineuzumab) and three small molecule BACE inhibitors (Elenbecestat, Verubecestat, Atabecestat). The model was calibrated to capture plasma and CSF drug PK, Ab levels and plaque burden from published clinical studies. Because of the complexity of Ab species, binding affinities of antibodies to these species can be difficult to measure, and often a wide range of values is reported for the same antibody. By fitting the model to all available Ab data in different compartments, we were able to estimate apparent in vivo affinities to different Ab species for the four anti-Ab antibodies. Our analyses indicate that for drugs that showed no or minimal plaque reduction in clinical studies, doses that are higher than those tested could lead to plaque reduction. Drugs that induce ADCP can lead to significant plaque reduction at lower doses and with faster kinetics than drugs that do not include this mechanism. We have developed a mechanistic mathematical model to analyze treatment effects of anti-Ab antibodies and BACE inhibitors on soluble Ab species and plaque. The results allow us to compare the effects of various drugs, mechanisms and combinations on plaque burden. In the future, the model will be expanded to capture drug effects at different stages of disease.

Amyloid-β Plaques in Clinical Alzheimer's Disease Brain Incorporate Stable Isotope Tracer In Vivo and Exhibit Nanoscale Heterogeneity.

Alzheimer’s disease (AD) is a neurodegenerative disorder with clinical manifestations of progressive memory decline and loss of executive function and language. AD affects an estimated 5.3 million Americans alone and is the most common form of age-related dementia with a rapidly growing prevalence among the aging population—those 65 years of age or older. AD is characterized by accumulation of aggregated amyloid-beta (Aβ) in the brain, which leads to one of the pathological hallmarks of AD—Aβ plaques. As a result, Aβ plaques have been extensively studied after being first described over a century ago. Advances in brain imaging and quantitative measures of Aβ in biological fluids have yielded insight into the time course of plaque development decades before and after AD symptom onset. However, despite the fundamental role of Aβ plaques in AD, in vivo measures of individual plaque growth, growth distribution, and dynamics are still lacking. To address this question, we combined stable isotope labeling kinetics (SILK) and nanoscale secondary ion mass spectrometry (NanoSIMS) imaging in an approach termed SILK–SIMS to resolve plaque dynamics in three human AD brains. In human AD brain, plaques exhibit incorporation of a stable isotope tracer. Tracer enrichment was highly variable between plaques and the spatial distribution asymmetric with both quiescent and active nanometer sub-regions of tracer incorporation. These data reveal that Aβ plaques are dynamic structures with deposition rates over days indicating a highly active process. Here, we report the first, direct quantitative measures of in vivo deposition into plaques in human AD brain. Our SILK–SIMS studies will provide invaluable information on plaque dynamics in the normal and diseased brain and offer many new avenues for investigation into pathological mechanisms of the disease, with implications for therapeutic development.

Amyloid β concentrations and stable isotope labeling kinetics of human plasma specific to central nervous system amyloidosis

IntroductionCerebrospinal fluid analysis and other measurements of amyloidosis, such as amyloid-binding positron emission tomography studies, are limited by cost and availability. There is a need for a more practical amyloid β (Aβ) biomarker for central nervous system amyloid deposition. MethodsWe adapted our previously reported stable isotope labeling kinetics protocol to analyze the turnover kinetics and concentrations of Aβ38, Aβ40, and Aβ42 in human plasma. ResultsAβ isoforms have a half-life of approximately 3 hours in plasma. Aβ38 demonstrated faster turnover kinetics compared with Aβ40 and Aβ42. Faster fractional turnover of Aβ42 relative to Aβ40 and lower Aβ42 and Aβ42/Aβ40 concentrations in amyloid-positive participants were observed. DiscussionBlood plasma Aβ42 shows similar amyloid-associated alterations as we have previously reported in cerebrospinal fluid, suggesting a blood-brain transportation mechanism of Aβ. The stability and sensitivity of plasma Aβ measurements suggest this may be a useful screening test for central nervous system amyloidosis.

The effect of angiotensin receptor neprilysin inhibitor, sacubitril/valsartan, on central nervous system amyloid-β concentrations and clearance in the cynomolgus monkey

Sacubitril/valsartan (LCZ696) is the first angiotensin receptor neprilysin inhibitor approved to reduce cardiovascular mortality and hospitalization in patients with heart failure with reduced ejection fraction. As neprilysin (NEP) is one of several enzymes known to degrade amyloid-β (Aβ), there is a theoretical risk of Aβ accumulation following long-term NEP inhibition. The primary objective of this study was to evaluate the potential effects of sacubitril/valsartan on central nervous system clearance of Aβ isoforms in cynomolgus monkeys using the sensitive Stable Isotope Labeling Kinetics (SILK™)-Aβ methodology.The in vitro selectivity of valsartan, sacubitril, and its active metabolite sacubitrilat was established; sacubitrilat did not inhibit other human Aβ-degrading metalloproteases. In a 2-week study, sacubitril/valsartan (50mg/kg/day) or vehicle was orally administered to female cynomolgus monkeys in conjunction with SILK™-Aβ. Despite low cerebrospinal fluid (CSF) and brain penetration, CSF exposure to sacubitril was sufficient to inhibit NEP and resulted in an increase in the elimination half-life of Aβ1-42 (65.3%; p=0.026), Aβ1-40 (35.2%; p=0.04) and Aβtotal (29.8%; p=0.04) acutely; this returned to normal as expected with repeated dosing for 15days. CSF concentrations of newly generated Aβ (AUC(0–24h)) indicated elevations in the more aggregable form Aβ1-42 on day 1 (20.4%; p=0.039) and day 15 (34.7%; p=0.0003) and in shorter forms Aβ1-40 (23.4%; p=0.009), Aβ1-38 (64.1%; p=0.0001) and Aβtotal (50.45%; p=0.00002) on day 15. However, there were no elevations in any Aβ isoforms in the brains of these monkeys on day 16. In a second study cynomolgus monkeys were administered sacubitril/valsartan (300mg/kg) or vehicle control for 39weeks; no microscopic brain changes or Aβ deposition, as assessed by immunohistochemical staining, were present. Further clinical studies are planned to address the relevance of these findings.

A randomized controlled study to evaluate the effect of bexarotene on amyloid-β and apolipoprotein E metabolism in healthy subjects

IntroductionWe conducted a phase Ib proof of mechanism trial to determine whether bexarotene (Targretin) increases central nervous system (CNS) apolipoprotein E (apoE) levels and alters Aβ metabolism in normal healthy individuals with the APOE ε3/ε3 genotype. MethodsWe used stable isotope labeling kinetics (SILK-ApoE and SILK-Aβ) to measure the effect of bexarotene on the turnover rate of apoE and Aβ peptides and stable isotope spike absolute quantitation (SISAQ) to quantitate their concentrations in the cerebrospinal fluid (CSF). Normal subjects were treated for 3 days with bexarotene (n = 3 women, 3 men, average 32 years old) or placebo (n = 6 women, average 30.2 years old) before administration of C13-leucine and collection of plasma and CSF over the next 48 hours. Bexarotene concentrations in plasma and CSF were also measured. ResultsOral administration of bexarotene resulted in plasma levels of 1 to 2 μM; however, only low nM levels were found in CSF. Bexarotene increased CSF apoE by 25% but had no effect on metabolism of Aβ peptides. DiscussionBexarotene has poor CNS penetration in normal human subjects. Drug treatment resulted in a modest increase in CSF apoE levels. The absence of an effect on Aβ metabolism is likely reflective of the low CNS levels of bexarotene achieved. This study documents the utility of SILK-ApoE technology in measuring apoE kinetics in humans. Trial RegistrationThis trial is registered at clinicaltrials.gov (NCT02061878).

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

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.

Pre-symptomatic functional brain changes in PS1 E280A mutation carriers compared to other biomarkers: Pilot data from the Alzheimer's Prevention Initiative Biomarker project

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-Ab ) 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). Results: Placebo (n1⁄4 9) and citalopram (n1⁄4 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 1⁄4 0.007) over the hours 5-36. During the same period there was a trend towards lower newly generated Abeta (p 1⁄4 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.Conclusions: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.

A Semi‐Physiological Model of Amyloid‐β Biosynthesis and Clearance in Human Cerebrospinal Fluid: A Tool for Alzheimer's Disease Research and Drug Development

Stable isotope labeling kinetics (SILK) was successfully applied to quantify endogenous amyloid-β (Aβ) metabolism in human cerebrospinal fluid (CSF). A semi-physiological model describing Aβ biosynthesis and degradation in human CSF and the impact of the γ-secretase inhibitor semagacestat should be developed and validated based on digitized data from three published SILK studies. Aβ biosynthesis was adequately characterized by six transit compartments. At each transition step, a first-order degradation process was implemented. A two-compartment model best described semagacestat CSF concentration-time profiles. Semagacestat concentrations were linked to the Aβ production by an inhibitory Emax model. For model validation, three individual Aβ profiles from literature were successfully predicted. Model application demonstrated a 35% decreased Aβ elimination rate constant in Alzheimer's disease (AD) patients. Study design optimization revealed that SILK studies could be conducted with significant less sampling points compared to the standard protocol without losing information about the Aβ metabolism, if analyzed by the presented model. In conclusion, the analysis outlined the advantages and opportunities of integrating all available data and knowledge into a semi-physiological model. The model can serve as valuable tool for researchers and clinicians interested in the pathology of AD as well as in the development of new therapeutics for AD.

Measurement of apolipoprotein E and amyloid β clearance rates in the mouse brain using bolus stable isotope labeling

BackgroundAbnormal proteostasis due to alterations in protein turnover has been postulated to play a central role in several neurodegenerative diseases. Therefore, the development of techniques to quantify protein turnover in the brain is critical for understanding the pathogenic mechanisms of these diseases. We have developed a bolus stable isotope-labeling kinetics (SILK) technique coupled with multiple reaction monitoring mass spectrometry to measure the clearance of proteins in the mouse brain.ResultsCohorts of mice were pulse labeled with 13 C6-leucine and the brains were isolated after pre-determined time points. The extent of label incorporation was measured over time using mass spectrometry to measure the ratio of labeled to unlabeled apolipoprotein E (apoE) and amyloid β (Aβ). The fractional clearance rate (FCR) was then calculated by analyzing the time course of disappearance for the labeled protein species. To validate the technique, apoE clearance was measured in mice that overexpress the low-density lipoprotein receptor (LDLR). The FCR in these mice was 2.7-fold faster than wild-type mice. To demonstrate the potential of this technique for understanding the pathogenesis of neurodegenerative disease, we applied our SILK technique to determine the effect of ATP binding cassette A1 (ABCA1) on both apoE and Aβ clearance. ABCA1 had previously been shown to regulate both the amount of apoE in the brain, along with the extent of Aβ deposition, and represents a potential molecular target for lowering brain amyloid levels in Alzheimer's disease patients. The FCR of apoE was increased by 1.9- and 1.5-fold in mice that either lacked or overexpressed ABCA1, respectively. However, ABCA1 had no effect on the FCR of Aβ, suggesting that ABCA1 does not regulate Aβ metabolism in the brain.ConclusionsOur SILK strategy represents a straightforward, cost-effective, and efficient method to measure the clearance of proteins in the mouse brain. We expect that this technique will be applicable to the study of protein dynamics in the pathogenesis of several neurodegenerative diseases, and could aid in the evaluation of novel therapeutic agents.

Effect of gamma-secretase modulating drugs on Abeta isoform production rates from cultured cells using stable isotope labeling kinetics and mass spectrometry

Effect of gamma-secretase modulating drugs on Abeta isoform production rates from cultured cells using stable isotope labeling kinetics and mass spectrometry