Bioanalytical Measurements for Analysis of Disease Pathways: New Molecules and Methods

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Thursday, April 7
Organizer: Michael Johnson, University of Kansas

Recent improvements in bioanalytical methods have greatly enhanced their sensitivity, selectivity, and applicability to human disease states. This symposium highlights work from investigators who have developed enhanced analytical tools to obtain a greater understanding of problems related to human disease. These topics include the measurement of peptide secretion at sub-second temporal resolution using advanced electrochemical methods to understand Parkinson’s disease and addiction (Sombers), microfluidic analysis of neurotransmitter and hormone secretion to understand neurological and endocrine disorders (Kennedy), application of mass spectrometry to astrocytes and neurons in disease (Sweedler), unraveling the role of serotonin regulation in depression (Hashemi), and nanoparticle- and electrochemical-based approaches for dynamic measurement of metals and neurotransmitters in living brain tissue to understand mechanisms of Alzheimer’s disease (Johnson).

Presentation 1
Real-Time Electrochemical Measurements of Neuropeptide Dynamics: From Single Cells in Culture to the Dorsal Striatum of a Freely-Moving Rat
Leslie Sombers, North Carolina State University
Chronic pain and drug addiction are two of our oldest and biggest public health problems; both exact devastating consequences, a substantial health care burden, and huge costs related to lost work productivity. Rampant opiate abuse, often resulting in tragic loss, has brought the critical need for effective treatment of both disorders into sharp focus. It is well documented that endogenous opioid peptides and dopamine serve as important mediators of reward processing in the basal ganglia, and aberrant activity in both systems is involved in drug addiction and in the pathophysiology of chronic pain. Although dopamine signaling has been intensely studied with respect to both rewarding and aversive stimuli, relatively little is known about opioid peptide signaling in any context. This is largely because few analytical tools are available to directly monitor opioid peptide fluctuations in situ. The Sombers lab has developed a fast-scan voltammetric detection strategy that can reveal the dynamics of endogenous enkephalins in live tissue. This technology has been optimized for sensitivity, selectivity, and stability, and has been used to record real-time neurochemical signals in preparations ranging from single cells in culture to the dorsal striatum of a freely-moving rat engaged in unexpected reward consumption. The experiments reveal the dynamic profile of opioid peptides in the extracellular space, and clarify the physiological conditions required to elicit opioid release. In many cases, the data reveal co-fluctuations of catecholamines and enkephalins with sub-second temporal resolution. These studies are important, because they advance our fundamental understanding of endogenous opioid peptide signaling. A precise determination of how endogenous neurochemical species underpin the hedonic and motivational aspects of reward processing, as well as the motivational and affective aspects of pain and pain relief, is critical to the development of appropriate therapies.

Presentation 2
Monitoring Metabolites and Proteins in the Brain of Living Animals using New Sampling and Analysis Methods
Robert Kennedy, University of Michigan
Measuring chemical dynamics in the brain can provide insight into normal and pathophysiological states. Although sensors provide excellent temporal and spatial resolution, sampling from the brain enables deep chemical analysis of the chemical environment around neurons and glial cells. We have developed improved sampling probes based on microfabrication that greatly improve the spatial resolution over typical microdialysis sampling probes. We have used metabolomics methods to begin identify the chemicals present in the brain extracellular space. So far over 400 compounds have been identified using LC-MS/MS with database matching. We envision that monitoring this large number of compounds will provide new insights into brain function. Finally, we have begun to develop immunoassays suitable for brain proteins. For example, we have developed a homogenous immunoassay for alpha-synuclein, a protein believed to be involved in the pathophysiology of Parkinson’s Disease. We will describe these analytical advances with applications to neuroscience.

Presentation 3
Towards Ex Vivo Voltammetry Models as Brain Mimics
Parastoo Hashemi, Imperial College London
In vivo voltammetry is used to provide invaluable information about the dynamics of neurotransmitters in real time. A carbon fiber microelectrode is directly implanted into brain tissue and analyte specific waveforms identify and quantify analytes of interest. In our group we are interested in defining the roles that serotonin plays in the brain and have, in recent times, identified in vivo biomarkers of interest to depression pathology in mice. Specifically, we measured changes in serotonin and histamine dynamics that may underlie important specific behavioral phenotypes of depression in mice. Translating this work to humans for diagnostic and therapeutic purposes is challenging because the human brain is not accessible for diagnostic and investigative purposes. In this work we explore new ways to mimic in vivo brain chemistry ex vivo. First, we explore new cellular cultures including human derived stem cells models and skin and hair culture models including fibroblasts and bulge cells. We introduce the rationale behind the new models and the design of new voltametric waveforms, electrodes and microfluidics for the most efficient signal capture. Second, we present novel in vivo measurements from probes designed to measure neurotransmitters in human skin. Via this work we hope to create ex vivo systems that mimic human brain chemistry for diagnostic and therapeutic purposes.

Presentation 4
Chemical Heterogeneity Among Individual Human Pancreatic Islets
Jonathan Sweedler, University of Illinois at Urbana-Champaign
During the transition from health to diabetes, the hormone content of islets is expected to vary. Here we assay transplant quality islets for their cell-cell signaling molecules, including d-amino acids, classical transmitters and peptide hormones. We combine several mass spectrometry platforms for accurate targeting of individual islets from individual donor islet pools to provide a detailed assessment of their chemistry. First, MALDI-TOF MS analyzed hundreds of individual IEQs from different donors. Obtain results on pancreatic islet pools correlated (R2=0.9) with information on sample purity provided by the Islet Core for different batches. MALDI MS sample screening also allowed selection of representative individual islets for liquid chromatography-MS (LC-MS) with trapped ion mobility mass spectrometry (TIMS) for follow-up in-depth chemical characterization. Peptide prohormones known to be expressed by different pancreatic islet cell types (insulin, glucagon, pancreatic prohormone, secretogranins and chromogranins, islet amyloid polypeptide, somatostatin, proSAAS, appetite stimulating hormone) were detected with multiple supporting peptides. The GLP1 processing products gastrointestinal hormones GLP1 and oxyntomodulin unambiguously confirmed non-cannonical islet proglucagon processing products. An alternatively-spliced short form of the insulin precursor was detected as were several hybrid insulin peptide (HIP) conjugates. Our findings support variable cell type composition among individual human islets. We are now comparing the detected cell-cell signaling molecules found in islets from healthy donors to those impacts with type 2 diabetes.

Presentation 5
Tools for Dissecting Neuronal Pathway Function and Their Application to Disease
Michael Johnson, University of Kansas
We will present methods that we have developed to measure free transition metal ions and peptides in living brain tissue on fast timescales. Zinc (Zn2+) dysregulation is implicated in multiple neurodegenerative disorders, including Parkinson’s disease and Alzheimer’s disease. We have developed a method in which we image functionalized gold nanoparticles with two-photon microscopy to measure changes in extracellular Zn2+ levels in whole, living brains. We have employed zebrafish as a model because their whole brains survive in a perfusion chamber outside of the host, enabling access to virtually any pathway. We have measured extracellular Zn2+ levels, altered by release from neuronal terminals evoked by electrical stimulation. Another project has focused on the electrochemical measurement of oxytocin. Oxytocin is a peptide hormone involved in numerous signaling pathways in the brain and peripheral tissues. Biological actions of oxytocin include orgasm, social recognition, and pair bonding, among others. Structurally, oxytocin consists of nine amino acids, including tyrosine and a disulfide bridge. Given previous studies that employed fast-scan cyclic voltammetry to measure met-enkephalin, which also possesses a tyrosine group, we pursued the measurement of oxytocin using a similar approach. Using our optimized voltammetry waveform in which we adjusted the scan rates and peak potentials, we were able to quantify oxytocin in the low µM range. Furthermore, the electrochemical response to oxytocin was linear up to at least 40 µM.

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