James G. Bollinger

Washington University in St. Louis | WUSTL , Wash U · Department of Neurology

Objective: In Alzheimer's disease, hyperphosphorylated tau is associated with formation of insoluble paired helical filaments that aggregate as neurofibrillary tau tangles and are associated with neuronal loss and cognitive symptoms. Dual orexin receptor antagonists decrease soluble amyloid-β levels and amyloid plaques in mouse models over-express...

Introduction: Continuous measures of amyloid burden as measured by positron emission tomography (PET) are being used increasingly to stage Alzheimer's disease (AD). This study examined whether cerebrospinal fluid (CSF) and plasma amyloid beta (Aβ)42/Aβ40 could predict continuous values for amyloid PET. Methods: CSF Aβ42 and Aβ40 were measured wi...

Introduction: Sleep deprivation increases cerebrospinal fluid (CSF) amyloid beta (Aβ) and tau levels; however, sleep's effect on Aβ and tau in plasma is unknown. Methods: In a cross-over design, CSF Aβ and tau concentrations were measured in five cognitively normal individuals who had blood and CSF collected every 2 hours for 36 hours during sle...

Although the APOE ε4 allele is the strongest genetic risk factor for sporadic Alzheimer’s disease (AD), the relationship between apolipoprotein (apoE) and AD pathophysiology is not yet fully understood. Relatively little is known about the apoE protein species, including post-translational modifications, that exist in the human periphery and CNS. T...

The extracellular buildup of amyloid beta (Aβ) plaques in the brain is a hallmark of Alzheimer’s disease (AD). Detection of Aβ pathology is essential for AD diagnosis and for identifying and recruiting research participants for clinical trials evaluating disease-modifying therapies. Currently, AD diagnoses are usually made by clinical assessments,...

Introduction: This report details the approach taken to providing a dataset allowing for analyses on the performance of recently developed assays of amyloid beta (Aβ) peptides in plasma and the extent to which they improve the prediction of amyloid positivity. Methods: Alzheimer's Disease Neuroimaging Initiative plasma samples with corresponding...

Objective To determine the diagnostic accuracy of a plasma Aβ42/Aβ40 assay in classifying amyloid PET status across global research studies using samples collected by multiple centers that utilize different blood collection and processing protocols. Methods Plasma samples (n=465) were obtained from three large Alzheimer’s Disease (AD) research coh...

The National Institute on Aging and the Alzheimer’s Association framework for classifying Alzheimer’s disease (AD) utilizes measures of pathology for amyloid, tau, and neurodegeneration (ATN), that can identify participants for clinical trials. Currently, amyloid pathology is determined by costly PET or invasive CSF measurements. When applied to pa...

Recent advances in the development of novel Alzheimer’s disease (AD) measures of amyloid, tau, and neurodegeneration in cerebrospinal fluid (CSF) and blood have enabled a better understanding of the links between amyloid‐beta (Aβ), tau species and neurodegeneration in the brain, CSF, and blood. The discoveries of novel tau species in CSF and blood,...

Introduction: Blood-based assays to measure brain amyloid beta (Aβ) deposition are an attractive alternative to the cerebrospinal fluid (CSF)-based assays currently used in clinical settings. In this study, we examined different blood-based assays to measure Aβ and how they compare among centers and assays. Methods: Aliquots from 81 plasma sampl...

In vivo gold standard for the ante-mortem assessment of brain β-amyloid pathology is currently β-amyloid PET or cerebrospinal fluid measures of β-amyloid42 or the β-amyloid42/β-amyloid40 ratio. The widespread acceptance of a biomarker classification scheme for the Alzheimer’s disease continuum has ignited interest in more affordable and accessible...

Recent advances in understanding the links between amyloid‐beta (Aβ) and specific tau isoforms in brain, cerebrospinal fluid (CSF), and blood indicate that a cascade of events of Alzheimer’s disease (AD) pathophysiology begin with detection of altered CSF and blood Aβ 42/40 ratio, followed by increases in amyloid plaques by Positron Emission Tomogr...

Skyline is a freely available, open-source Windows client application for accelerating targeted proteomics experimentation, with an emphasis on the proteomics and mass spectrometry community as users and as contributors. This review covers the informatics encompassed by the Skyline ecosystem, from computationally assisted targeted mass spectrometry...

Objective: We examined whether plasma β-amyloid (Aβ)42/Aβ40, as measured by a high-precision assay, accurately diagnosed brain amyloidosis using amyloid PET or CSF p-tau181/Aβ42 as reference standards. Methods: Using an immunoprecipitation and liquid chromatography-mass spectrometry assay, we measured Aβ42/Aβ40 in plasma and CSF samples from 158...

Figure S3. Correlation between all hSOD1 peptides measured. Linear regression analysis comparing peptides shows strong correlations exist between all peptide measurements. All P values < 0.0001 for each analysis.

Figure S5. No changes in peripheral hSOD1 after 1000 ug hSOD1 ASO intracerebroventricular injection. A. hSOD1 protein levels relative to N15‐labeled recombinant standard by LC‐MS in plasma. No changes in plasma hSOD1 protein levels were observed (Student's T‐test). B. 13C6‐Leu labeled hSOD1 in plasma by LC‐MS. No changes in plasma 13C6‐Leu labeled...

Figure S2. Correlation between all hTau peptides measured. Linear regression analysis comparing peptides shows strong correlations exist between all three peptide measurements. All p values < 0.0001 for each analysis.

Figure S4. Characterizing 13C6‐Leucine incorporation into hTau protein in hTauWT transgenic mice. hTau mice were acclimated to leucine‐free chow for 7 days, and labeled with 5 mg/ml 13C6‐leucine for 7 days. Mice were euthanized at 6 (n = 2), 10 (n = 3), 14 (n = 3), and 17 (n = 2) days post‐end of label. Because of the high label incorporation (near...

Table S2. Transition ions used for hSOD1 tandem LC‐MS/MS. GLHGFHVHE peptide measurements were used as representative data in figures.

Figure S1. No differences in 13C6‐Leucine oral labeling between treatment groups in all experiments performed. Plasma 13C6‐Leucine values were analyzed in all samples analyzed and compared between groups. A. hTau ICV ASO plasma 13C6‐Leucine at Day 23 post surgery, as seen in Figure 2. No differences were observed (Student's T‐test, P = 0.4496). B....

Table S1. Primers and probes used for genotyping and qPCR analysis of hTau (MAPT), hSOD1 (SOD1) and mouse GAPDH (GAPDH) DNA.

Figure S6. No protein concentration pharmacodynamics observed in CSF after 1000 ug hSOD1 ASO intracerebroventricular injection at 50 days post treatment. A. hSOD1 mRNA levels in the CNS by qPCR. hSOD1 mRNA levels are significantly lowered in spinal cord and frontal cortex by ~70% and 50%, respectively, after ASO treatment relative to inactive ASO c...

Table S3. Transition ions used for hTau tandem LC‐MS/MS. TPSLPTPPTR peptide measurements were used as representative data in figures.

Objectives Clinical trials for progressive neurodegenerative disorders such as Alzheimer's Disease and Amyotrophic Lateral Sclerosis have been hindered due to the absence of effective pharmacodynamics markers to assay target engagement. We tested whether measurements of new protein production would be a viable pharmacodynamics tool for RNA‐targeted...

OBJECTIVES/SPECIFIC AIMS: We aimed to develop an assay to measure new protein synthesis after Antisense Oligonucleotide treatment, which we hypothesized to be the earliest biochemical identification of RNA-targeting therapy efficacy. METHODS/STUDY POPULATION: We treated 2 transgenic animal models expressing proteins implicated in neurodegenerative...

Data-independent acquisition (DIA) is an emerging mass spectrometry (MS)-based technique for unbiased and reproducible measurement of protein mixtures. DIA tandem mass spectrometry spectra are often highly multiplexed, containing product ions from multiple cofragmenting precursors. Detecting peptides directly from DIA data is therefore challenging;...

Protein phosphorylation, one of the most common types of post-translational modifications, is the central regulatory mechanism of cellular signaling networks. In human cells, thousands of proteins are continuously and dynamically phosphorylated and dephosphorylated at specific sites and times in response to external and internal stimuli. Reversible...

Purpose: Proteomic analysis of blood proteins in DBS is gaining attention as a possible replacement for measurements in plasma/serum collected by venipuncture. We aimed to develop and provisionally validate a nanoflow LC-PRM-MS method for clinical use. Experimental design: We used Skyline to develop a nanoflow LC-PRM-MS method to quantify glycat...

As compared to conventional high performance liquid chromatography (HPLC) techniques, nanoflow HPLC exhibits increased sensitivity and limits of detection. However, nanoflow HPLC suffers from low throughput due to instrument failure (e.g. fitting fatigue and trapping column failure), limiting the utility of the technique for clinical and industrial...

The risk of Alzheimers 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 risk for AD. In order to address the hypothesis that amounts of ApoE isoforms...

Secreted phospholipase A2s (sPLA2s) regulate eicosanoid formation and have been implicated in asthma. Although sPLA2s function as enzymes, some of the sPLA2s bind with high affinity to a C-type lectin receptor, called PLA2R1, which has functions in both cellular signaling and clearance of sPLA2s. We sought to examine the expression of PLA2R1 in the...

In targeted proteomics, the development of robust methodologies is dependent upon the selection of a set of optimal peptides for each protein-of-interest. Unfortunately, predicting which peptides and respective product ion transitions provide the greatest signal-to-noise ratio in a particular assay matrix is complicated. Using in vitro synthesized...

Head and neck cancers, including oral squamous cell carcinoma (OSCC), are the sixth most common malignancy in the world and are characterized by poor prognosis and a low survival rate. Saliva is oral fluid with intimate contact with OSCC. Besides non-invasive, simple, and rapid to collect, saliva is a potential source of biomarkers. In this study,...

Significance On activation, blood platelets package components from their cytoplasm into microparticles (MPs), tiny vesicles released by cytoplasmic membrane budding and shedding. Given that MPs can impact other cellular lineages on internalization, we aimed to decipher the mechanisms promoting MP internalization by cellular recipients. We modeled...

Targeted mass spectrometry is an essential tool for detecting quantitative changes in low abundant proteins throughout the proteome. Although Selected Reaction Monitoring (SRM) is the preferred method for quantifying peptides in complex samples, the process of designing SRM assays is laborious. Peptides have widely varying signal responses dictated...

We have previously shown that secreted phospholipases A2 (sPLA2s) from animal venoms inhibit the in vitro development of P. falciparum, the agent of malaria. In addition, the inflammatory-type human group IIA (hGIIA) sPLA2 circulates at high levels in the serum of malaria patients. However, the role of the different human sPLA2s in host defense aga...

Key Points When activated and in platelet storage bags, platelets release respiratory-competent mitochondria, a recognized damage-associated molecular pattern. Mitochondria, descendant of Rickettsia prowazekii, serve as substrate for bactericidal sPLA2-IIA to promote inflammation.

Statistical process control (SPC) is a robust set of tools that aids in the visualization, detection, and identification of assignable causes of variation in any process that creates products, services, or information. A tool has been developed termed Statistical Process Control in Proteomics (SProCoP) which implements aspects of SPC (e.g., control...

The mammalian epidermis provides both an interface and a protective barrier between the organism and its environment. Lipid, processed into water-impermeable bilayers between the outermost layers of the epidermal cells, forms the major barrier that prevents water from exiting the organism, and also prevents toxins and infectious agents from enterin...

Quantitative analysis of fatty acids (FAs) is an important area of analytical biochemistry. Ultra high sensitivity FA analysis usually is done with gas chromatography of pentafluorobenzyl esters coupled to an electron capture detector. With the popularity of electrospray ionization mass spectrometers coupled to liquid chromagraphy, it would be conv...

Resolution of inflammation is an active process that is mediated in part by antiinflammatory lipid mediators. Although phospholipase A2 (PLA2) enzymes have been implicated in the promotion of inflammation through mobilizing lipid mediators, the molecular entity of PLA2 subtypes acting upstream of antiinflammatory lipid mediators remains unknown. He...

Rationale: Indirect airway hyperresponsiveness (AHR) is a fundamental feature of asthma that is manifest as exercise-induced bronchoconstriction (EIB). Secreted phospholipase A2 group X (sPLA2-X) plays a key role in regulating eicosanoid formation and the development of inflammation and AHR in murine models. Objectives: We sought to examine sPLA...

Previous work has shown that disruption of the gene for group X secreted phospholipase A (sPLA-X) markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLAs in the mammalian genome, the involvement of other sPLAs in the asthma model is possible - in particular, the group V sPLA (...

The mammalian epidermis provides both an interface and a protective barrier between the organism and its environment. Lipid, processed into water-impermeable bilayers between the outermost layers of the epidermal cells, forms the major barrier that prevents water from exiting the organism, and also prevents toxins and infectious agents from enterin...

Attempts to characterize, quantify, and/or modulate the activity of the secreted phospholipase A(2) family of enzymes result from the diversity of physiological roles for which these enzymes have been implicated. The 1-palmitoyl-2-(10-pyrenedecanoyl)-phosphatidylglycerol (pyrenePG)-based fluorometric assay is a sensitive and readily adaptable metho...

Group V-secreted phospholipase A2 (GV sPLA2) hydrolyzes bacterial phospholipids and initiates eicosanoid biosynthesis. Here, we elucidate the role of GV sPLA2 in the pathophysiology of Escherichia coli pneumonia. Inflammatory cells and bronchial epithelial cells both express GV sPLA2 after pulmonary E. coli infection. GV−/− mice accumulate fewer po...

Group X (GX) phospholipase A2, a member of a large group of secreted phospholipases A2 (sPLA2s), has recently been demonstrated to play an important in vivo role in the release of arachidonic acid and subsequent formation of eicosanoids. In a Th2 cytokine-driven mouse asthma model, deficiency of mouse GX (mGX)-sPLA2 significantly impairs developmen...

The cytosolic (group IV) phospholipase A2 (cPLA2s) family contains six members. We have prepared recombinant proteins for human α, mouse β, human γ, human δ, human ϵ, and mouse ζ cPLA2s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA2β action on phosphatidylcholine vesicles is activated by anionic phosphoinosi...

Secreted phospholipase A(2) group X (sPLA(2)-X) has recently been identified in the airways of patients with asthma and may participate in cysteinyl leukotriene (CysLT; C(4), D(4), and E(4)) synthesis. We examined CysLT synthesis and arachidonic acid (AA) and lysophospholipid release by eosinophils mediated by recombinant human sPLA(2)-X. We found...

Combined liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is a powerful method for the analysis of oxygenated metabolites of polyunsaturated fatty acids including eicosanoids. Here we describe the synthesis of a new derivatization reagent N-(4-aminomethylphenyl)pyridinium (AMPP) that can be coupled to eicosanoid...

LC/ESI-MS/MS has been previously demonstrated to be a powerful method to detect and quantify molecular species of glycerophospholipids including lysophospholipids. In this study, we provide an improved pre-mass spectrometry lipid extraction procedure that avoids the acid-catalyzed decomposition of plasmenyl phospholipids that is problematic with pr...

Among mammalian secreted phospholipases A2 (sPLA(2)s), the group X enzyme has the most potent hydrolyzing capacity toward phosphatidylcholine, the major phospholipid of cell membrane and lipoproteins. This enzyme has recently been implicated in chronic inflammatory diseases such as atherosclerosis and asthma and may also play a role in colon tumori...

Group IVA cytosolic phospholipase A2 (cPLA2α) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA2α (IMLF-/-) were reconstituted with wild type and cPLA2α mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate...

Increased lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA(2) is a causative agent. Here we show that selective inhibition of Lp-PLA(2) with darapladib reduced development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic sw...

With the recent ability to use combined liquid chromatography/electrospray tandem mass spectrometry to analyze for several eicosanoids in biological samples in a single and rapid experiment, heavy isotope-labeled eicosanoids are needed as internal standards in order to quantify eicosanoid analytes. The present study describes a practical preparatio...

The C-terminal "CaaX"-motif-containing proteins usually undergo three sequential post-translational processing steps: (1) attachment of a prenyl group to the cysteine residue; (2) proteolytic removal of the last three amino acids "aaX"; (3) methyl esterification of the exposed alpha-carboxyl group of the prenyl-cysteine residue. The Trypanosoma bru...

Arachidonic acid metabolites, the eicosanoids, are key mediators of allergen-induced airway inflammation and remodeling in asthma. The availability of free arachidonate in cells for subsequent eicosanoid biosynthesis is controlled by phospholipase A(2)s (PLA(2)s), most notably cytosolic PLA(2)-alpha. 10 secreted PLA(2)s (sPLA(2)s) have also been id...

The prevailing hypothesis that a signalling pathway involving cPLA(2)alpha is required to enhance the gating of the voltage-gated proton channel associated with NADPH oxidase was tested in human eosinophils and murine granulocytes. This hypothesis invokes arachidonic acid (AA) liberated by cPLA(2)alpha as a final activator of proton channels. In hu...

To date, 12 secreted phospholipases A2 (sPLA2s) have been identified in the mouse species and divided into three structural collections (I/II/V/X, III, and XII). On the basis of their different molecular properties and tissue distributions, each sPLA2 is likely to exert distinct functions by acting as an enzyme or ligand for specific soluble protei...

Platelet Activating Factor (PAF) is a potent mediator of inflammation whose biological activity depends on the acetyl group esterified at the sn-2 position of the molecule. PAF-acetylhydrolase (PAF-AH), a secreted calcium-independent phospholipase A(2), is known to inactivate PAF by formation of lyso-PAF and acetate. However, PAF-AH deficient patie...

Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacologica...

Secreted phospholipases A(2) (sPLA(2)) are enzymes released in plasma and extracellular fluids during inflammatory diseases. Because human group IB and X sPLA(2)s are expressed in the lung, we examined their effects on primary human lung macrophages (HLM). Both sPLA(2)s induced TNF-alpha and IL-6 release in a concentration-dependent manner by incre...