Florian Tribl

“Subcellular Proteomics” of Neuromelanin Granules Isolated from the Human Brain

“Subcellular proteomics” is currently the most effective approach to characterize subcellular compartments. Based on the powerful combination of subcellular fractionation and protein identification by LC-MS/MS we were able for the first time to 1) isolate intact neuromelanin granules from the human brain and 2) establish the first protein profile of these granules. This compartment containing neuromelanin (NM) is primarily located in the primate’s substantia nigra, one of the main brain regions that severely degenerates in Parkinson disease. We used mechanic tissue disaggregation, discontinuous sucrose gradient centrifugation, cell disruption, and organelle separation to isolate NM granules from human substantia nigra. Using transmission electron microscopy we demonstrated that the morphological characteristics of the isolated NM granules are similar to those described in human brain tissue. Fundamentally we found numerous proteins definitely demonstrating a close relationship of NM-containing granules with lysosomes or lysosome-related organelles originating from the endosome-lysosome lineage. Intriguingly we further revealed the presence of endoplasmic reticulum-derived chaperones, especially the transmembrane protein calnexin, which recently has been located in lysosome-related melanosomes and has been suggested to be a melanogenic chaperone.

Identification of L-ferritin in Neuromelanin Granules of the Human Substantia Nigra A TARGETED PROTEOMICS APPROACH

In the pigmented dopaminergic neurons of the human substantia nigra pars compacta the system relevant in iron storage is the polymer neuromelanin (NM). Although in most cells this function is mainly accomplished by ferritin, this protein complex appears not to be expressed in NM-containing neurons. Nevertheless the conceivable presence of iron-storing proteins as part of the NM granules has recently been discussed on the basis of Mössbauer spectroscopy and synchrotron x-ray microspectroscopy. Intriguingly by combining subcellular fractionation of NM granules, peptide sequencing via tandem mass spectrometry, and the additional confirmation by multiple reaction monitoring and immunogold labeling for electron microscopy, L-ferritin could now be unambiguously identified and localized in NM granules for the first time. This finding not only supports direct evidence for a regulatory role of L-ferritin in neuroectodermal cell pigmentation but also integrates a new player within a complicated network governing iron homeostasis in the dopamine neurons of the human substantia nigra. Thus our finding entails far reaching implications especially when considering etiopathogenetic aspects of Parkinson disease.

Tyrosinase is not detected in human catecholaminergic neurons by immunohistochemistry and Western blot analysis

Catecholaminergic neurons of the primate substantia nigra (SN) pars compacta (SNc) and the locus coeruleus contain neuromelanin (NM) granules as characteristic structures underlying the pigmentation of these brain areas. Due to a phylogenetic appearance NM granules are absent in the rodent brain, but gradually become present in primates until they reach a maximal expression in humans. Although a possible mechanism of pigment formation may be autoxidation of the NM precursors dopamine or noradrenalin, several groups have suggested an enzymatic formation of NM mediated by tyrosinase or a related enzyme. Since tyrosinase mRNA is suggested to be expressed in the SN of mice and humans, we reinvestigated the expression of tyrosinase in the human SNc and the locus coeruleus at the protein level by immunohistochemistry and Western blot analysis, but could not detect tyrosinase in these brain regions.

Towards multidimensional liquid chromatography separation of proteins using fluorescence and isotope‐coded protein labelling for quantitative proteomics

HPLC has emerged as a valuable tool for separating proteins. To address the analysis of complex proteomes and quantitative changes of proteins therein, we developed a multidimensional LC (MDLC)‐based approach followed by large gel 1‐D SDS‐PAGE. Here we present a novel strategy that allows for simultaneously identifying and quantifying differentially regulated proteins following three separation and fractionation steps. This MDLC platform integrates advantages of dual protein labelling using both fluorescence and isotope‐coded tags for subsequent detection and quantification of abundance ratios of proteins by MS.

Analysis of organelles within the nervous system: impact on brain and organelle functions

Currently, neuroproteomic approaches aimed at the profiling of total brain areas generally mirror the expression of the most abundant proteins, but fail to uncover less abundant proteins. By contrast, the focus on typical brain subproteomes, (e.g., synaptic vesicles, synaptic terminal membranes or the postsynaptic density), may give a more specific insight into brain function. Subproteomes are accessible via several strategies, including subcellular fractionation or affinity-based pull-down approaches. Combined with mass spectrometric quantification approaches, subcellular proteomics is expected to reveal differences in the protein constitution of related cellular organelles. Focusing on novel functions and mechanistic models, we review recent data on the analysis of brain-derived organelles and subproteomes, including presynaptic termini, synaptic vesicles, neuronal plasma membranes, postsynaptic density and neuromelanin granules, which were identified as novel lysosome-related organelles within the human brain.

The HUPO Brain Proteome Project Wish List – Summary of the 9th HUPO BPP Workshop 9–10 January 2008, Barbados

The Human Brain Proteome Project (HUPO BPP) aims at advancing knowledge and the understanding of neurodiseases and aging with the purpose of identifying prognostic and diagnostic biomarkers, as well as to push new diagnostic approaches and medications. The participating groups meet in semi‐annual workshops to discuss the progress, as well as the needs, within the field of proteomics. The 9th HUPO BPP workshop took place in Barbados from 9–10 January, 2008. Discussing the future HUPO BPP Roadmap, the attendees drafted the so called HUPO BPP wish list containing timelines, suggestions and missions. This wish list will be updated regularly and will serve as a guideline for the next phase.

Sub-Proteome Processing: Isolation of Neuromelanin Granules from the Human Brain

The sub-proteome analysis of organelles is a field of high relevance for molecular biology, because it provides detailed insights into the protein composition of cellular compartments. This approach not only results in a catalogue of organellar proteins, but in fact holds the potential to uncover the enzymatic armament engaged in biochemical reactions and to identify novel mechanisms of organelle biogenic pathways. Knowledge about protein localization may be a first step towards extensive functional analyses of specific target proteins engaged in development, aging, or disease. Moreover, several disorders of the human brain include aberrant protein function in specific compartments. Thus, a closer look at cellular organelles will allow for advancing our current perceptions of pathogenic processes. This chapter aims to provide a methodological workflow given by the isolation of neuromelanin granules from the human midbrain. This approach encompasses several modular steps that can easily be adjusted to any other organelle of interest and follows the sequence of (1) organelle isolation, (2) isolation quality controls by transmission electron microscopy and Western immuno blotting, and (3) gel-based protein separation towards protein identification by mass spectrometry.

Isolation of Neuromelanin Granules

Neuromelanin granules are pigmented organelles in the human midbrain that give name to a brain area, substantia nigra pars compacta, which macroscopically appears as a dark brown region in the midbrain due to the insoluble pigment neuromelanin. The substantia nigra pars compacta massively degenerates in Parkinsons disease and gives rise to severely disabling movement symptoms. It has been suggested that neuromelanin granules play an important role in the neurodegenerative events in Parkinsons disease: redox‐active iron is bound to neuromelanin and thereby retained within this compartment, but in Parkinsons disease it is thought to be increasingly released into the cytosol, promoting oxidative stress. This unit includes a methodological workflow for the isolation of neuromelanin granules from the human midbrain. This top‐down approach (describes an approach that reduces the complexity of the sample stepwise from the level of tissue to cell, and from cell to organelle) encompasses the organelle isolation by sequential density gradient centrifugation and the assessment of the isolation efficacy by western blotting. Curr. Protoc. Cell Biol. 41:3.31.1‐3.31.15.

Neuromelanin, ein Pigment mit unbekannter Funktion

Zusammenfassung Neuromelanin ist ein polymeres, nahezu unlösliches Pigment, das nur in bestimmten Regionen des menschlichen Gehirns und des einiger Säugetiere (Primaten, Kühe, Pferde, einige Schafrassen) vorkommt. Es wird hauptsächlich in der Substantia nigra (SN) und im Locus coeruleus (LC) gefunden, in denen die katecholaminergen Neurotransmitter Dopamin (SN) und Noradrenalin (LC) synthetisiert werden. Die verschiedenen Farben der Neuromelanine (SN: braun bis schwarz; LC: bläulich) weisen auf Unterschiede in der Biosynthese und in der chemischen Zusammensetzung hin. Bisher sind keine Enzyme bekannt, die an der Biosynthese der Neuromelanine beteiligt sind. Tyrosinase oxidiert L-Tyrosin zu L-DOPA (L-3,4-Dihydroxyphenylalanin, Vorstufe von Dopamin) und Folgeprodukten und ist das wesentliche an der Biosynthese von Melanin beteiligte Enyzm; im menschlichen Gehirn konnte es jedoch nicht zweifelsfrei nachgewiesen werden. Deshalb gehen viele Wissenschaftler davon aus, dass Neuromelanine unspezifisch durch Autoxidation von Dopamin und Noradrenalin gebildet werden und nur funktionslose Abfallprodukte katecholaminerger Neurone sind. Es gibt jedoch verschiedene Hinweise dafür, dass die Synthese der Neuromelanine durch Enzyme kontrolliert wird und die Neuromelanine eine physiologische Funktion als Eisenspeicher haben.