title: Combined enrichment of neuromelanin granules and synaptosomes from human substantia nigra pars compacta tissue for proteomic analysis.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Functional Proteomics, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Plum
authors.affiliation: Department of Functional Proteomics, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Helling
authors.affiliation: Department of Cytology, Institute of Anatomy, Ruhr-Universität Bochum, Bochum, Germany.|Department of Anatomy and Molecular Embryology, Institute of Anatomy, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Theiss
authors.affiliation: Physiopathology in Aging Lab/Brazilian Aging Brain Study Group - LIM22, University of Sao Paulo Medical School, Sao Paulo, Brazil.
authors.forename: R E P
authors.initials: REP
authors.lastname: Leite
authors.affiliation: Department of Medical Proteomics/Bioanalytics, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: C
authors.initials: C
authors.lastname: May
authors.affiliation: Physiopathology in Aging Lab, Geriatrics Discipline, University of Sao Paulo Medical School, Sao Paulo, Brazil.
authors.forename: W
authors.initials: W
authors.lastname: Jacob-Filho
authors.affiliation: Department of Medical Proteomics/Bioanalytics, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Eisenacher
authors.affiliation: Department of Medical Proteomics/Bioanalytics, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Kuhlmann
authors.affiliation: Department of Medical Proteomics/Bioanalytics, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: H E
authors.initials: HE
authors.lastname: Meyer
authors.affiliation: Clinical Neurochemistry, Clinic and Policlinic of Psychiatry, Psychosomatics and Psychotherapy, Bavarian Julius-Maximilians-Universität, Würzburg, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Riederer
authors.affiliation: Physiopathology in Aging Lab/Brazilian Aging Brain Study Group - LIM22, University of Sao Paulo Medical School, Sao Paulo, Brazil.|Department of Neurology, Memory and Aging Center, University of California, San Francisco, USA.
authors.forename: L T
authors.initials: LT
authors.lastname: Grinberg
authors.affiliation: Laboratory of Clinical Neurobiology, Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, Bavarian Julius-Maximilians-Universität, Würzburg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Gerlach
authors.affiliation: Department of Functional Proteomics, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Marcus
mesh_terms: D002499:Centrifugation, Density Gradient / Q000379:methods; D003594:Cytoplasmic Granules / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008543:Melanins; D019636:Neurodegenerative Diseases / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013378:Substantia Nigra / Q000737:chemistry* / Q000378:metabolism; D013574:Synaptosomes / Q000737:chemistry*
chemical_list: D008543:Melanins; C014121:neuromelanin
keywords: Density gradient; NM; Neuromelanin; Parkinson's disease; SN; Substantia nigra pars compacta; Synaptosomes; average g-force in the ultracentrifugation tube; g(av); neuromelanin; substantia nigra pars compacta
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