Abstract
Neuromelanin is a complex polymer pigment found primarily in the dopaminergic neurons of human substantia nigra. Neuromelanin pigment is stored in granules including a protein matrix and lipid droplets. Neuromelanin granules are yet only partially characterised regarding their structure and function. To clarify the exact function of neuromelanin granules in humans, their enrichment and in-depth characterization from human substantia nigra is necessary. Previously published global proteome studies of neuromelanin granules in human substantia nigra required high tissue amounts. Due to the limited availability of human brain tissue we established a new method based on laser microdissection combined with mass spectrometry for the isolation and analysis of neuromelanin granules. With this method it is possible for the first time to isolate a sufficient amount of neuromelanin granules for global proteomics analysis from ten 10 μm tissue sections. In total 1,000 proteins were identified associated with neuromelanin granules. More than 68% of those proteins were also identified in previously performed studies. Our results confirm and further extend previously described findings, supporting the connection of neuromelanin granules to iron homeostasis and lysosomes or endosomes. Hence, this method is suitable for the donor specific enrichment and proteomic analysis of neuromelanin granules.
Highlights
The analysis revealed 166 proteins significantly overrepresented in NM granules compared to control
Analysis of different age groups could lead to deeper insights in NM granules genesis and function
Performing differential studies comparing proteins isolated from human subjects with no history of neurological or neurodegenerative diseases and lacking neuropathological abnormalities and subjects with neurodegenerative diseases could lead to a better understanding of the role of NM granules during neurodegeneration
Summary
The use of human post mortem brain tissue was approved by the ethics committees of the Ruhr-University Bochum, Germany (file number 4760-13) and the University of Würzburg, Germany (file number 78/99) accordance with German guidelines and regulations. Tissue from the Newcastle Brain Tissue Resource was obtained in accordance with the approval of the joint Ethics Committee of Newcastle, United Kingdom, and North Tyneside Health Authority, United Kingdom, with written informed consent and following Newcastle Brain Tissue Resource brain banking procedures. Cryopreserved postmortem SN sections from five human subjects with no history of neurological or neurodegenerative diseases and lacking neuropathological abnormalities were provided by the Brain Bank Center Würzburg, the Newcastle Brain Tissue Resource, UK and the Netherlands Brain Bank, Netherlands Institute for Neuroscience, Amsterdam. Neuropathological diagnoses were assigned using accepted international neuropathological criteria including neuritic Braak stages, Thal amyloid phases, CERAD scores, NIA-AA scores and McKeith criteria
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