Dataset Information

Proteomic characterization of neuromelanin granules isolated from human substantia nigra by laser-microdissection.

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??m 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.

SUBMITTER: Plum S

PROVIDER: S-EPMC5107900 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Proteomic characterization of neuromelanin granules isolated from human substantia nigra by laser-microdissection.

Plum Sarah S Steinbach Simone S Attems Johannes J Keers Sharon S Riederer Peter P Gerlach Manfred M May Caroline C Marcus Katrin K

Scientific reports 20161114

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 studie ...[more]

PMID: 27841354

Similar Datasets

Project description:This article gives a detailed description of a protocol using density gradient centrifugation for the enrichment of neuromelanin granules and synaptosomes from low amounts (?0.15g) of human substantia nigra pars compacta tissue. This has a great advantage compared to already existing methods as it allows for the first time (i) a combined enrichment of neuromelanin granules and synaptosomes and (ii) just minimal amounts of tissue necessary to enable donor specific analysis. Individual specimens were classified as control or diseased according to clinical evaluation and neuropathological examination. For the enrichment of synaptosomes and neuromelanin granules from the same tissue sample density gradient centrifugations using Percoll® and Iodixanol were performed. The purity of resulting fractions was checked by transmission electron microscopy. We were able to establish a reproducible and easy to handle protocol combining two different density gradient centrifugations: using an Iodixanol gradient neuromelanin granules were enriched and in parallel, from the same sample, a fraction of synaptosomes with high purity using a Percoll® gradient was obtained. Our subfractionation strategy will enable a subsequent in depth proteomic characterization of neurodegenerative processes in the substantia nigra pars compacta in patients with Parkinson's disease and dementia with Lewy bodies compared to appropriate controls.Key features of Parkinson's disease are the degeneration of dopaminergic neurons in the substantia nigra pars compacta, an associated loss of the brain pigment neuromelanin and a resulting impairment of the neuronal network. The accumulation of iron binding neuromelanin granules is age- and disease-dependent and disease specific alterations could affect the neuronal iron homeostasis leading to oxidative stress induced cell death. The focus of the described method is the analysis of neuromelanin granules as well as axonal cell-endings of nerve cells (synaptosomes) of individual donors (control and diseased). It is the basis for the identification of disease-relevant changes in the iron homeostasis and the generation of new insight into altered protein compositions or regulations which might lead to disturbed communications between nerve cells resulting in pathogenic processes.

Dataset Information

Proteomic characterization of neuromelanin granules isolated from human substantia nigra by laser-microdissection.

Publications

Proteomic characterization of neuromelanin granules isolated from human substantia nigra by laser-microdissection.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets