Toward Omics-Scale Quantitative Mass Spectrometry Imaging of Lipids in Brain Tissue Using a Multiclass Internal Standard Mixture.

Mass spectrometry imaging (MSI) has accelerated our understanding of lipid metabolism and spatial distribution in tissues and cells. However, few MSI studies have approached lipid imaging quantitatively and those that have focused on a single lipid class. We overcome this limitation by using a multiclass internal standard (IS) mixture sprayed homogeneously over the tissue surface with concentrations that reflect those of endogenous lipids. This enabled quantitative MSI (Q-MSI) of 13 lipid classes and subclasses representing almost 200 sum-composition lipid species using both MALDI (negative ion mode) and MALDI-2 (positive ion mode) and pixel-wise normalization of each lipid species in a manner analogous to that widely used in shotgun lipidomics. The Q-MSI approach covered 3 orders of magnitude in dynamic range (lipid concentrations reported in pmol/mm2) and revealed subtle changes in distribution compared to data without normalization. The robustness of the method was evaluated by repeating experiments in two laboratories using both timsTOF and Orbitrap mass spectrometers with an ∼4-fold difference in mass resolution power. There was a strong overall correlation in the Q-MSI results obtained by using the two approaches. Outliers were mostly rationalized by isobaric interferences or the higher sensitivity of one instrument for a particular lipid species. These data provide insight into how the mass resolving power can affect Q-MSI data. This approach opens up the possibility of performing large-scale Q-MSI studies across numerous lipid classes and subclasses and revealing how absolute lipid concentrations vary throughout and between biological tissues.

Glucocerebrosidase activity and lipid levels are related to protein pathologies in Parkinson's disease.

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are progressive neurodegenerative diseases characterized by the accumulation of misfolded α-synuclein in the form of Lewy pathology. While most cases are sporadic, there are rare genetic mutations that cause disease and more common variants that increase incidence of disease. The most prominent genetic mutations for PD and DLB are in the GBA1 and LRRK2 genes. GBA1 mutations are associated with decreased glucocerebrosidase activity and lysosomal accumulation of its lipid substrates, glucosylceramide and glucosylsphingosine. Previous studies have shown a link between this enzyme and lipids even in sporadic PD. However, it is unclear how the protein pathologies of disease are related to enzyme activity and glycosphingolipid levels. To address this gap in knowledge, we examined quantitative protein pathology, glucocerebrosidase activity and lipid substrates in parallel from 4 regions of 91 brains with no neurological disease, idiopathic, GBA1-linked, or LRRK2-linked PD and DLB. We find that several biomarkers are altered with respect to mutation and progression to dementia. We found mild association of glucocerebrosidase activity with disease, but a strong association of glucosylsphingosine with α-synuclein pathology, irrespective of genetic mutation. This association suggests that Lewy pathology precipitates changes in lipid levels related to progression to dementia.

Pyrazole Ureas as Low Dose, CNS Penetrant Glucosylceramide Synthase Inhibitors for the Treatment of Parkinson's Disease.

Parkinson's disease is the second most prevalent progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. Loss-of-function mutations in GBA, the gene that encodes for the lysosomal enzyme glucosylcerebrosidase, are a major genetic risk factor for the development of Parkinson's disease potentially through the accumulation of glucosylceramide and glucosylsphingosine in the CNS. A therapeutic strategy to reduce glycosphingolipid accumulation in the CNS would entail inhibition of the enzyme responsible for their synthesis, glucosylceramide synthase (GCS). Herein, we report the optimization of a bicyclic pyrazole amide GCS inhibitor discovered through HTS to low dose, oral, CNS penetrant, bicyclic pyrazole urea GCSi's with in vivo activity in mouse models and ex vivo activity in iPSC neuronal models of synucleinopathy and lysosomal dysfunction. This was accomplished through the judicious use of parallel medicinal chemistry, direct-to-biology screening, physics-based rationalization of transporter profiles, pharmacophore modeling, and use a novel metric: volume ligand efficiency.

Neuronopathic GBA1L444P Mutation Accelerates Glucosylsphingosine Levels and Formation of Hippocampal Alpha-Synuclein Inclusions.

The most common genetic risk factor for Parkinson's disease (PD) is heterozygous mutations GBA1, which encodes for the lysosomal enzyme, glucocerebrosidase. Reduced glucocerebrosidase activity associates with an accumulation of abnormal α-synuclein (α-syn) called Lewy pathology, which characterizes PD. PD patients heterozygous for the neuronotypic GBA1L444P mutation (GBA1+/L444P) have a 5.6-fold increased risk of cognitive impairments. In this study, we used GBA1+/L444P mice of either sex to determine its effects on lipid metabolism, expression of synaptic proteins, behavior, and α-syn inclusion formation. At 3 months of age, GBA1+/L444P mice demonstrated impaired contextual fear conditioning, and increased motor activity. Hippocampal levels of vGLUT1 were selectively reduced in GBA1+/L444P mice. We show, using mass spectrometry, that GBA1L444P expression increased levels of glucosylsphingosine, but not glucosylceramide, in the brains and serum of GBA1+/L444P mice. Templated induction of α-syn pathology in mice showed an increase in α-syn inclusion formation in the hippocampus of GBA1+/L444P mice compared with GBA1+/+ mice, but not in the cortex, or substantia nigra pars compacta. Pathologic α-syn reduced SNc dopamine neurons by 50% in both GBA1+/+ and GBA1+/L444P mice. Treatment with a GlcCer synthase inhibitor did not affect abundance of α-syn inclusions in the hippocampus or rescue dopamine neuron loss. Overall, these data suggest the importance of evaluating the contribution of elevated glucosylsphingosine to PD phenotypes. Further, our data suggest that expression of neuronotypic GBA1L444P may cause defects in the hippocampus, which may be a mechanism by which cognitive decline is more prevalent in individuals with GBA1-PD.SIGNIFICANCE STATEMENT Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are both pathologically characterized by abnormal α-synuclein (α-syn). Mutant GBA1 is a risk factor for both PD and DLB. Our data show the expression of neuronotypic GBA1L444P impairs behaviors related to hippocampal function, reduces expression of a hippocampal excitatory synaptic protein, and that the hippocampus is more susceptible to α-syn inclusion formation. Further, our data strengthen support for the importance of evaluating the contribution of glucosylsphingosine to PD phenotypes. These outcomes suggest potential mechanisms by which GBA1L444P contributes to the cognitive symptoms clinically observed in PD and DLB. Our findings also highlight the importance of glucosylsphingosine as a relevant biomarker for future therapeutics.

Proteomic Discovery and Validation of Novel Fluid Biomarkers for Improved Patient Selection and Prediction of Clinical Outcomes in Alzheimer's Disease Patient Cohorts.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease characterized by progressive cognitive decline. The two cardinal neuropathological hallmarks of AD include the buildup of cerebral ß amyloid (Aß) plaques and neurofibrillary tangles of hyperphosphorylated tau. The current disease-modifying treatments are still not effective enough to lower the rate of cognitive decline. There is an urgent need to identify early detection and disease progression biomarkers that can facilitate AD drug development. The current established readouts based on the expression levels of amyloid beta, tau, and phospho-tau have shown many discrepancies in patient samples when linked to disease progression. There is an urgent need to identify diagnostic and disease progression biomarkers from blood, cerebrospinal fluid (CSF), or other biofluids that can facilitate the early detection of the disease and provide pharmacodynamic readouts for new drugs being tested in clinical trials. Advances in proteomic approaches using state-of-the-art mass spectrometry are now being increasingly applied to study AD disease mechanisms and identify drug targets and novel disease biomarkers. In this report, we describe the application of quantitative proteomic approaches for understanding AD pathophysiology, summarize the current knowledge gained from proteomic investigations of AD, and discuss the development and validation of new predictive and diagnostic disease biomarkers.

Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes.

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.

Using measures of metabolic flux to align screening and clinical development: Avoiding pitfalls to enable translational studies.

Screening campaigns, especially those aimed at modulating enzyme activity, often rely on measuring substrate→product conversions. Unfortunately, the presence of endogenous substrates and/or products can limit one's ability to measure conversions. As well, coupled detection systems, often used to facilitate optical readouts, are subject to interference. Stable isotope labeled substrates can overcome background contamination and yield a direct readout of enzyme activity. Not only can isotope kinetic assays enable early screening, but they can also be used to follow hit progression in translational (pre)clinical studies. Herein, we consider a case study surrounding lipid biology to exemplify how metabolic flux analyses can connect stages of drug development, caveats are highlighted to ensure reliable data interpretations. For example, when measuring enzyme activity in early biochemical screening it may be enough to quantify the formation of a labeled product. In contrast, cell-based and in vivo studies must account for variable exposure to a labeled substrate (or precursor) which occurs via tracer dilution and/or isotopic exchange. Strategies are discussed to correct for these complications. We believe that measures of metabolic flux can help connect structure-activity relationships with pharmacodynamic mechanisms of action and determine whether mechanistically differentiated biophysical interactions lead to physiologically relevant outcomes. Adoption of this logic may allow research programs to (i) build a critical bridge between primary screening and (pre)clinical development, (ii) elucidate biology in parallel with screening and (iii) suggest a strategy aimed at in vivo biomarker development.

A novel glucosylceramide synthase inhibitor attenuates alpha synuclein pathology and lysosomal dysfunction in preclinical models of synucleinopathy.

Mutations in the lysosomal enzyme glucocerebrosidase (GCase, GBA1 gene) are the most common genetic risk factor for developing Parkinson's disease (PD). GCase metabolizes the glycosphingolipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Mutations in GBA1 reduce enzyme activity and the resulting accumulation of glycosphingolipids may contribute to the underlying pathology of PD, possibly via altering lysosomal function. While reduction of GCase activity exacerbates α-synuclein (α-syn) aggregation, it has not been determined that this effect is the result of altered glycosphingolipid levels and lysosome function or some other effect of altering GCase. The glycosphingolipid GlcCer is synthesized by a single enzyme, glucosylceramide synthase (GCS), and small molecule inhibitors (GCSi) reduce cellular glycosphingolipid levels. In the present studies, we utilize a preformed fibril (PFF) rodent primary neuron in vitro model of α-syn pathology to investigate the relationship between glycosphingolipid levels, α-syn pathology, and lysosomal function. In primary cultures, pharmacological inhibition of GCase and D409V GBA1 mutation enhanced accumulation of glycosphingolipids and insoluble phosphorylated α-syn. Administration of a novel small molecule GCSi, benzoxazole 1 (BZ1), significantly decreased glycosphingolipid concentrations in rodent primary neurons and reduced α-syn pathology. BZ1 rescued lysosomal deficits associated with the D409V GBA1 mutation and α-syn PFF administration, and attenuated α-syn induced neurodegeneration of dopamine neurons. In vivo studies revealed BZ1 had pharmacological activity and reduced glycosphingolipids in the mouse brain to a similar extent observed in neuronal cultures. These data support the hypothesis that reduction of glycosphingolipids through GCS inhibition may impact progression of synucleinopathy and BZ1 is useful tool to further examine this important biology.

High-defined quantitative snapshots of the ganglioside lipidome using high resolution ion mobility SLIM assisted shotgun lipidomics.

Defects in sphingolipid metabolism have emerged as a common link across neurodegenerative disorders, and a deeper understanding of the lipid content in preclinical models and patient specimens offers opportunities for development of new therapeutic targets and biomarkers. Sphingolipid metabolic pathways include the formation of glycosphingolipid species that branch into staggeringly complex structural heterogeneity within the globoside and ganglioside sub-lipidomes. Characterization of these sub-lipidomes has typically relied on liquid chromatography-mass spectrometry-based (LC-MS) approaches, but such assays are challenging and resource intensive due to the close structural heterogeneity, the presence of isobaric and isomeric species, and broad dynamic range of endogenous glycosphingolipids. Here, we apply Structures for Lossless Ion Manipulations (SLIM)-based High Resolution Ion Mobility (HRIM)-MS to enable rapid, repeatable, quantitative assays with deep structural information sufficient to resolve endogenous brain gangliosides at the level of individual molecular species. Analyses were performed using a prototype SLIM-MS instrument equipped with a 13-m serpentine path which enabled resolution of closely related isomeric analytes such as GD1a d36:1 and GD1b d36:1 based on recorded mass-to-charge (m/z) and arrival times. To demonstrate the power of our methodology, brain extracts derived from wild-type mice hemi-brains were analyzed by HRIM-MS using flow injection analyses (FIA) without the need for additional separation by liquid chromatography. Endogenous ganglioside species were readily resolved, identified, and quantified by FIA-SLIM-MS analyses within 2 min per sample. Thus, the FIA-SLIM-MS platform enables robust quantification across a broad range of lipid species in biological specimens in a standardized assay format that is readily scalable to support studies with large sample numbers.

LRRK2 inhibition prevents endolysosomal deficits seen in human Parkinson's disease.

LRRK2 has been implicated in endolysosomal function and likely plays a central role in idiopathic Parkinson's disease (iPD). In iPD, dopaminergic neurons within the substantia nigra are characterized by increased LRRK2 kinase activity, endolysosomal deficits, and accumulation of autophagic vesicles with incompletely degraded substrates, including α-synuclein. Although LRRK2 has been implicated in endolysosomal and autophagic function, it remains unclear whether inhibition of LRRK2 kinase activity can prevent endolysosomal deficits or reduce dopaminergic neurodegeneration. In this study, we characterized the endolysosomal and autophagic defects in surviving dopaminergic neurons of iPD patient brain tissue. We next showed that these defects could be reproduced reliably in vivo using the rotenone model of iPD. Results suggested that there was impaired endosomal maturation, resulting in lysosomal dysfunction and deficits in protein degradation. A highly selective, brain-penetrant LRRK2 kinase inhibitor not only improved apparent endosomal maturation and lysosomal function, but also prevented rotenone-induced neurodegeneration in vivo. The fact that a LRRK2 kinase inhibitor was capable of preventing the neuropathological and endolysosomal abnormalities observed in human iPD suggests that LRRK2 inhibitors may have broad therapeutic utility in iPD, not only in those who carry a LRRK2 mutation.

cAMP, Ca2+, pHi, and NO Regulate H-like Cation Channels That Underlie Feeding and Locomotion in the Predatory Sea Slug Pleurobranchaea californica.

A systems approach to regulation of neuronal excitation in the mollusc Pleurobranchaea has described novel interactions of cyclic AMP-gated cation current (INa,cAMP), Ca2+, pHi, and NO. INa,cAMP appears in many neurons of feeding and locomotor neuronal networks. It is likely one of the family of hyperpolarization-activated, cyclic-nucleotide-gated currents (h-current) of vertebrate and invertebrate pacemaker networks. There are two isoforms. Ca2+ regulates both voltage dependence and depolarization-sensitive inactivation in both isoforms. The Type 1 INa,cAMP of the feeding network is enhanced by intracellular acidification. A direct dependence of INa,cAMP on cAMP allows the current to be used as a reporter on cAMP concentrations in the cell, and from there to the intrinsic activities of the synthetic adenyl cyclase and the degradative phosphodiesterase. Type 2 INa,cAMP of the locomotor system is activated by serotonergic inputs, while Type 1 of the feeding network is thought to be regulated peptidergically. NO synthase activity is high in the CNS, where it differs from standard neuronal NO synthase in not being Ca2+ sensitive. NO acidifies pHi, potentiating Type 1, and may act to open proton channels. A cGMP pathway does not mediate NO effects as in other systems. Rather, nitrosylation likely mediates its actions. An integrated model of the action of cAMP, Ca2+, pHi, and NO in the feeding network postulates that NO regulates proton conductance to cause neuronal excitation in the cell body on the one hand, and relief of activity-induced hyperacidification in fine dendritic processes on the other.

Functional Peptidomics: Stimulus- and Time-of-Day-Specific Peptide Release in the Mammalian Circadian Clock.

Daily oscillations of brain and body states are under complex temporal modulation by environmental light and the hypothalamic suprachiasmatic nucleus (SCN), the master circadian clock. To better understand mediators of differential temporal modulation, we characterize neuropeptide releasate profiles by nonselective capture of secreted neuropeptides in an optic nerve horizontal SCN brain slice model. Releasates are collected following electrophysiological stimulation of the optic nerve/retinohypothalamic tract under conditions that alter the phase of the SCN activity state. Secreted neuropeptides are identified by intact mass via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We found time-of-day-specific suites of peptides released downstream of optic nerve stimulation. Peptide release was modified differentially with respect to time-of-day by stimulus parameters and by inhibitors of glutamatergic or PACAPergic neurotransmission. The results suggest that SCN physiology is modulated by differential peptide release of both known and unexpected peptides that communicate time-of-day-specific photic signals via previously unreported neuropeptide signatures.

Internalized Tau Oligomers Cause Neurodegeneration by Inducing Accumulation of Pathogenic Tau in Human Neurons Derived from Induced Pluripotent Stem Cells.

Neuronal inclusions of hyperphosphorylated and aggregated tau protein are a pathological hallmark of several neurodegenerative tauopathies, including Alzheimer's disease (AD). The hypothesis of tau transmission in AD has emerged from histopathological studies of the spatial and temporal progression of tau pathology in postmortem patient brains. Increasing evidence in cellular and animal models supports the phenomenon of intercellular spreading of tau. However, the molecular and cellular mechanisms of pathogenic tau transmission remain unknown. The studies described herein investigate tau pathology propagation using human neurons derived from induced pluripotent stem cells. Neurons were seeded with full-length human tau monomers and oligomers and chronic effects on neuronal viability and function were examined over time. Tau oligomer-treated neurons exhibited an increase in aggregated and phosphorylated pathological tau. These effects were associated with neurite retraction, loss of synapses, aberrant calcium homeostasis, and imbalanced neurotransmitter release. In contrast, tau monomer treatment did not produce any measureable changes. This work supports the hypothesis that tau oligomers are toxic species that can drive the spread of tau pathology and neurodegeneration. SIGNIFICANCE STATEMENT: Several independent studies have implicated tau protein as central to Alzheimer's disease progression and cell-to-cell pathology propagation. In this study, we investigated the ability of different tau species to propagate pathology in human neurons derived from induced pluripotent stem cells, which to date has not been shown. We demonstrated that tau oligomers, but not monomers, induce accumulation of pathological, hyperphosphorylated tau. This effect was accompanied with neurite degeneration, loss of synapses, aberrant calcium homeostasis, imbalanced neurotransmitter release, and ultimately with neuronal death. This study bridges various tau pathological phenotypes into a single and relevant induced pluripotent stem cell neuronal model of human disease that can be applied to the discovery of the mechanisms of tau-induced neurodegeneration.

In vivo isotopically labeled atherosclerotic aorta plaques in ApoE KO mice and molecular profiling by matrix-assisted laser desorption/ionization mass spectrometric imaging.

RATIONALE: The ability to quantify rates of formation, regression and/or remodeling of atherosclerotic plaque should facilitate a better understanding of the pathogenesis and management of cardiovascular disease. In the current study, we coupled a stable isotope labeled tracer protocol with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to examine spatial and temporal lipid dynamics in atherosclerotic plaque. METHODS: To promote plaque formation in the aorta region, ApoE KO mice were fed a high cholesterol diet (0.15% cholesterol) and orally dosed with (2,2,3,4,4,6-d(6))-cholesterol over several weeks. Tissue sections of ~10 µm thickness were analyzed by MALDI-MSI using matrix deposition by either chemical sublimation or acoustic droplet ejection. RESULTS: MALDI-MSI yielded distinct spatial distribution information for a variety of lipid classes including specific lysophosphatidylcholines typically associated with atherosclerosis-related tissue damage such as phospholipase 2 (Lp-PLA(2)) that mediate chemotactic responses to inflammation (e.g. LPC 16:0, LPC 18:0 and LPC 18:1) as well as free cholesterol and cholesteryl esters that contribute to atheroma formation. MALDI mass spectra acquired from aorta tissue sections clearly distinguished non-esterified and esterified versions of (2,2,3,4,4,6-d(6))-cholesterol within aortic plaque regions and showed distinct spatial accumulation of the cholesterol tracer. CONCLUSIONS: The ability to couple stable isotope based protocols with MALDI-MSI enables a novel strategy to characterize the effects of therapeutic treatments on atherosclerotic plaque formation, regression and potential remodeling of the complex lipid components with high chemical specificity and spatiotemporal information.

Neuromodulatory control of a goal-directed decision.

Many cost-benefit decisions reduce to simple choices between approach or avoidance (or active disregard) to salient stimuli. Physiologically, critical factors in such decisions are modulators of the homeostatic neural networks that bias decision processes from moment to moment. For the predatory sea-slug Pleurobranchaea, serotonin (5-HT) is an intrinsic modulatory promoter of general arousal and feeding. We correlated 5-HT actions on appetitive state with its effects on the approach-avoidance decision in Pleurobranchaea. 5-HT and its precursor 5-hydroxytryptophan (5-HTP) augmented general arousal state and reduced feeding thresholds in intact animals. Moreover, 5-HT switched the turn response to chemosensory stimulation from avoidance to orienting in many animals. In isolated CNSs, bath application of 5-HT both stimulated activity in the feeding motor network and switched the fictive turn response to unilateral sensory nerve stimulation from avoidance to orienting. Previously, it was shown that increasing excitation state of the feeding network reversibly switched the turn motor network response from avoidance to orienting, and that 5-HT levels vary inversely with nutritional state. A simple model posits a critical role for 5-HT in control of the turn network response by corollary output of the feeding network. In it, 5-HT acts as an intrinsic neuromodulatory factor coupled to nutritional status and regulates approach-avoidance via the excitation state of the feeding network. Thus, the neuromodulator is a key organizing element in behavioral choice of approach or avoidance through its actions in promoting appetitive state, in large part via the homeostatic feeding network.

Generic automated method for liquid chromatography-multiple reaction monitoring mass spectrometry based monoclonal antibody quantitation for preclinical pharmacokinetic studies.

Quantitation of therapeutic monoclonal antibodies (mAb) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for pharmacokinetic (PK) studies is becoming an essential complement to traditional antibody-based ligand binding assays (LBA). Here we show an automated method to perform LC-MS/MS-based quantitation, with IgG1 conserved peptides, a heavy isotope labeled mAb internal standard, and anti-human Fc enrichment. All reagents in the method are commercially available with no requirement to develop novel assay-specific reagents. The method met traditional quantitative LC-MS/MS assay analytical characteristics in terms of precision, accuracy, and specificity. The method was applied to the pharmacokinetic study of a mAb dosed in cynomolgus monkey, and the results were compared with the immunoassay data. This methodology has the potential to benefit and accelerate the early biopharmaceutical development process, particularly by enabling PK analysis across species and candidate molecules with minimal method development.

Fibrinogen α-chain-derived peptide is upregulated in hippocampus of rats exposed to acute morphine injection and spontaneous alternation testing.

Fibrinogen is a secreted glycoprotein that is synthesized in the liver, although recent in situ hybridization data support its expression in the brain. It is involved in blood clotting and is released in the brain upon injury. Here, we report changes in the extracellular levels of fibrinogen α-chain-derived peptides in the brain after injections of saline and morphine. More specifically, in order to assess hippocampus-related working memory, an approach pairing in vivo microdialysis with mass spectrometry was used to characterize extracellular peptide release from the hippocampus of rats in response to saline or morphine injection coupled with a spontaneous alternation task. Two fibrinopeptide A-related peptides derived from the fibrinogen α-chain-fibrinopeptide A (ADTGTTSEFIEAGGDIR) and a fibrinopeptide A-derived peptide (DTGTTSEFIEAGGDIR)-were shown to be consistently elevated in the hippocampal microdialysate. Fibrinopeptide A was significantly upregulated in rats exposed to morphine and spontaneous alternation testing compared with rats exposed to saline and spontaneous alternation testing (P < 0.001), morphine alone (P < 0.01), or saline alone (P < 0.01), respectively. The increase in fibrinopeptide A in rats subjected to morphine and a memory task suggests that a complex interaction between fibrinogen and morphine takes place in the hippocampus.

A sensitive aß oligomer assay discriminates Alzheimer's and aged control cerebrospinal fluid.

A hallmark of Alzheimer's disease (AD) brain is the amyloid ß (Aß) plaque, which is comprised of Aß peptides. Multiple lines of evidence suggest that Aß oligomers are more toxic than other peptide forms. We sought to develop a robust assay to quantify oligomers from CSF. Antibody 19.3 was compared in one-site and competitive ELISAs for oligomer binding specificity. A two-site ELISA for oligomers was developed using 19.3 coupled to a sensitive, bead-based fluorescent platform able to detect single photons of emitted light. The two-site ELISA was >2500× selective for Aß oligomers over Aß monomers with a limit of detection ∼ 0.09 pg/ml in human CSF. The lower limit of reliable quantification of the assay was 0.18 pg/ml and the antibody pairs recognized Aß multimers comprised of either synthetic standards, or endogenous oligomers isolated from confirmed human AD and healthy control brain. Using the assay, a significant 3- to 5-fold increase in Aß oligomers in human AD CSF compared with comparably aged controls was demonstrated. The increase was seen in three separate human cohorts, totaling 63 AD and 54 controls. CSF oligomers ranged between 0.1 and 10 pg/ml. Aß oligomer levels did not strongly associate with age or gender, but had an inverse correlation with MMSE score. The C statistic for the Aß oligomer ROC curve was 0.86, with 80% sensitivity and 88% specificity to detect AD, suggesting reasonable discriminatory power for the AD state and the potential for utility as a diagnostic marker.

Synthesis, accumulation, and release of d-aspartate in the Aplysia californica CNS.

d-Aspartate (d-Asp) is an endogenous molecule that is often detected in CNS and endocrine tissues. Using capillary electrophoresis and a variety of radionuclide detection techniques, we examine the synthesis, release, and uptake/accumulation of d-Asp in the CNS of the marine mollusk Aplysia californica. We observe the preferential synthesis and accumulation of d-Asp over l-aspartate (l-Asp) in neuron-containing ganglia compared to surrounding sheath tissues. Little conversion of d-Asp to l-Asp is detected. The Ca(2+) ionophore ionomycin and elevated extracellular potassium stimulates release of d-Asp from the cerebral ganglia. Lastly, radioactive d-Asp in the extracellular media is efficiently taken up and accumulated by individual F-cluster neurons. These observations point to a role for d-Asp in cell-to-cell signaling with many characteristics similar to classical transmitters.

Nitric oxide potentiates cAMP-gated cation current by intracellular acidification in feeding neurons of pleurobranchaea.

A pH-sensitive cAMP-gated cation current (I(Na,cAMP)) is widely distributed in neurons of the feeding motor networks of gastropods. In the sea slug Pleurobranchaea this current is potentiated by nitric oxide (NO), which itself is produced by many feeding neurons. The action of NO is not dependent on either cGMP or cAMP signaling pathways. However, we found that NO potentiation of I(Na,cAMP) in the serotonergic metacerebral cells could be blocked by intracellular injection of MOPS buffer (pH 7.2). In neurons injected with the pH indicator BCECF, NO induced rapid intracellular acidification to several tenths of a pH unit. Intracellular pH has not previously been identified as a specific target of NO, but in this system NO modulation of I(Na,cAMP) via pH(i) may be an important regulator of the excitability of the feeding motor network.