Region-specific bioconversion of dynorphin neuropeptide detected by in situ histochemistry and MALDI imaging mass spectrometry

Abstract

Brain region-specific expression of proteolytic enzymes can control the biological activity of endogenous neuropeptides and has recently been targeted for the development of novel drugs, for neuropathic pain, cancer, and Parkinson's disease. Rapid and sensitive analytical methods to profile modulators of enzymatic activity are important for finding effective inhibitors with high therapeutic value. Combination of in situ enzyme histochemistry with MALDI imaging mass spectrometry allowed developing a highly sensitive method for analysis of brain-area specific neuropeptide conversion of synthetic and endogenous neuropeptides, and for selection of peptidase inhibitors that differentially target conversion enzymes at specific anatomical sites. Conversion and degradation products of Dynorphin B as model neuropeptide and effects of peptidase inhibitors applied to native brain tissue sections were analyzed at different brain locations. Synthetic dynorphin B (2pmol) was found to be converted to the N-terminal fragments on brain sections whereas fewer C-terminal fragments were detected. N-ethylmaleimide (NEM), a non-selective inhibitor of cysteine peptidases, almost completely blocked the conversion of dynorphin B to dynorphin B(1-6; Leu-Enk-Arg), (1-9), (2-13), and (7-13). Proteinase inhibitor cocktail, and also incubation with acetic acid displayed similar results. Bioconversion of synthetic dynorphin B was region-specific producing dynorphin B(1-7) in the cortex and dynorphin B (2-13) in the striatum. Enzyme inhibitors showed region- and enzyme-specific inhibition of dynorphin bioconversion. Both phosphoramidon (inhibitor of the known dynorphin converting enzyme neprilysin) and opiorphin (inhibitor of neprilysin and aminopeptidase N) blocked cortical bioconversion to dynorphin B(1-7), wheras only opiorphin blocked striatal bioconversion to dynorphin B(2-13). This method may impact the development of novel therapies with aim to strengthen the effects of endogenous neuropeptides under pathological conditions such as chronic pain. Combining histochemistry and MALDI imaging MS is a powerful and sensitive tool for the study of inhibition of enzyme activity directly in native tissue sections.

Spatial Distribution of Endogenous Tissue Protease Activity in Gastric Carcinoma Mapped by MALDI Mass Spectrometry Imaging

“…This is the prerequisite to assure no overlaying endogenous substance P signal. Generated peptides have been observed for specific substrate-tissue-combinations like dynorphin B on rat brain (18). The observed time-dependent and transient generation of such products suggest that substance P is not merely sequestered into the tissue, but truly digested.…”

“…MSI is frequently used for analysis of the in situ distribution of proteins (11,12), lipids (13,14), drugs (14), as well as peptides (15), and N-glycans (16,17). A recent study with a neuropeptide, dynorphin, spotted onto rat brain slices suggested that it may be possible to detect the levels of substrate and peptide bioconversion products simultaneously by MSI (18).…”

Methods of Measuring Enzyme Activity Ex Vivo and In Vivo

“…Magnetic resonance spectroscopy (MRS) and imaging (MRI) based on 31 P permits the measurement of creatine kinase activity (2) because the signal from ATP and phosphocreatine occur at different chemical shifts. Techniques that acquire substrates and products from tissue, such as microdialysis (e.g., 11β-hydroxysteroid dehydrogenase type 1 (3–5)), electroosmotic push-pull perfusion (e.g., aminopeptidases (6)), or MALDI-MS (e.g., related to dynorphins (7)) may use or measure natural substrates and products. There are many reasons to use natural substrates.…”

“…In terms of studying enzymatic processes for understanding biochemistry and neurochemistry, perhaps the greatest power of MALDI is high spatial resolution. This is especially important in neurological applications since the function of a particular enzyme is often tied to its location in a specific brain region (7). Furthermore, this can rely on natural substrates and products making it more amenable to tissue-based studies (7).…”

Multiplex enzyme activity imaging by MALDI-IMS of substrate library conversions

“…Previous studies show that MALDI imaging is a valuable tool for investigating spatial enzyme activities. However, these studies were limited to the exploration of single natural peptide substrates and frozen tissues 15 – 17 or phospholipids 18 . Here we explore the potential of MALDI-IMS for mapping multiple enzyme activities in zinc-fixated, paraffin-embedded tissues by spray applying enzyme substrate libraries of naturally derived, but artificial peptides.…”