Isolation of Neuromelanin Granules

Florian Tribl1

1 Medizinisches Proteom‐Center, Ruhr‐Universitaet Bochum, Bochum, Germany
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.31
DOI:  10.1002/0471143030.cb0331s41
Online Posting Date:  December, 2008
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Abstract

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 Parkinson's 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 Parkinson's disease: redox‐active iron is bound to neuromelanin and thereby retained within this compartment, but in Parkinson's 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. © 2008 by John Wiley & Sons, Inc.

Keywords: human brain; neuromelanin; lysosome‐related organelle; organelle isolation; density gradient

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Neuromelanin Granules from Human Substantia Nigra Pars Compacta Tissue
  • Support Protocol 1: Preparation of a Discontinuous Sucrose Gradient
  • Support Protocol 2: Quality Control by Immunoblot Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Neuromelanin Granules from Human Substantia Nigra Pars Compacta Tissue

  Materials
  • Disinfecting agents for dissecting instruments and surfaces (e.g., any liquid formulation used to clean metal surgical instruments is appropriate)
  • Dissected, unfixed human substantia nigra pars compacta, prepared at the Brain Bank (stored at −80°C)
  • Separation buffer (see recipe), cooled to 4°C
  • Ice
  • 20‐ml discontinuous sucrose gradients in 50‐ml conical polyethylene screw‐cap centrifuge tubes (see protocol 2)
  • Isolation buffer (see recipe)
  • 80% (v/v) Percoll solution (see recipe)
  • Wash buffer (see recipe)
  • Liquid nitrogen
  • Cooling plate
  • Plastic sack (e.g., an autoclave bag)
  • 250‐ and 500‐ml beakers
  • Petri dishes, 100‐mm in diameter
  • Scissors
  • 150‐µm nylon mesh
  • Balance accurate to ±0.1 g
  • Dissecting instruments including:
    • Scalpels or single‐edge razor blades
    • Forceps
  • 10‐ml syringes (B. Braun Melsungen AG), single‐use
  • 15‐ and 50‐ml conical polyethylene screw‐cap centrifuge tubes
  • Styropor boxes
  • Refrigerated Rotanta 96 RS centrifuge (Hettich GmbH) with swing‐out rotor inserts for 15‐ml and 50‐ml conical polyethylene screw‐cap centrifuge tubes
  • 1.5‐ml microcentrifuge tubes (Eppendorf)
  • 1‐ml syringe equipped with a 26‐G needle
  • −80°C freezer
  • Additional reagents and equipment for preparing a discontinuous sucrose gradient ( protocol 2)
NOTE: It is highly recommended to use human substantia nigra pars compacta that is prepared by experienced pathologists. Substantia nigra pars compacta should be dissected from transverse brain tissue plates of the brain stem (see Fig. A, B) and should be kept intact. NOTE: For biosafety reasons, during the tissue preparation steps use personal protective equipment including a laboratory coat, a surgical mask, safety glasses, and gloves to avoid dermal and mucous membrane contact with blood and brain tissue. Wear a laboratory coat, safety glasses, and gloves throughout the other procedures. All steps should be performed at 4°C.

Support Protocol 1: Preparation of a Discontinuous Sucrose Gradient

  Materials
  • 1.0 M sucrose gradient solution, ice cold (see recipe)
  • 1.2 M sucrose gradient solution, ice cold (see recipe)
  • 1.4 M sucrose gradient solution, ice cold (see recipe)
  • 1.6 M sucrose gradient solution, ice cold (see recipe)
  • Ice
  • 50‐ml conical polyethylene screw‐cap centrifuge tubes
  • Test tube rack
  • 150‐mm glass Pasteur pipets with a straight tip, constricted, but unplugged
  • Styropor box

Support Protocol 2: Quality Control by Immunoblot Analysis

  Materials
  • 2× tricine/SDS sample buffer (Novex)
  • Polyacrylamide gels, e.g., precast 10% to 20% gradient tricine gels (Novex), or equivalent gels (unit 6.1)
  • 10 × tricine/SDS running buffer (Novex)
  • 10× reducing agent (Novex)
  • Isolated neuromelanin granules sample (see protocol 1)
  • Molecular weight markers
  • Ice
  • Tris‐glycine transfer buffer, 20× (Novex)
  • Ponceau red solution (Pierce)
  • Tris‐buffered saline (TBS; appendix 2A)
  • Blocking solution (see recipe)
  • Primary antibodies
  • Secondary antibodies (HRP‐conjugated)
  • Tris‐buffered saline‐Triton X‐100 (TBS‐T; see recipe)
  • ECL solutions (Pierce)
  • Heater
  • Power supply
  • XCell II Mini Gel Electrophoresis System (Novex)
  • XCell II blot module, blotting cassette, and sponges (Novex)
  • Benchtop centrifuge
  • Gel knife
  • Precut nitrocellulose membranes (Invitrogen GmbH)
  • Sponges
  • Whatman 3MM Chr paper (Schleicher & Schuell)
  • Rocking platform
  • Gel documentation system
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Figures

Videos

Literature Cited

Literature Cited
   Fedorow, H., Tribl, F., Halliday, G., Gerlach, M., Riederer, P., and Double, K.L. 2005. Neuromelanin in human dopamine neurons: Comparison with peripheral melanins and relevance to Parkinson's disease. Prog. Neurobiol. 75:109‐124.
   Gahl, W.A., Potterf, B., Durham‐Pierre, D., Brilliant, M.H., and Hearing, V.J. 1995. Melanosomal tyrosine transport in normal and pink‐eyed dilution murine melanocytes. Pigment Cell Res. 8:229‐233.
   Graham, J.M. and Rickwood, D. 1997. Subcellular Fractionation: A Practical Approach. pp. 339. Oxford University Press, New York, N.Y.
   Halliday, G.M., Fedorow, H., Rickert, C.H., Gerlach, M., Riederer, P., and Double, K.L. 2006. Evidence for specific phases in the development of human neuromelanin. Neurobiol. Aging. 27:506‐512.
   Hynd, M.R., Lewohl, J.M., Scott, H.L., and Dodd, P.R. 2003. Biochemical and molecular studies using human autopsy brain tissue. J. Neurochem. 85:543‐562.
   Marsden, C.D. 1961. Pigmentation in the nucleus substantiae nigrae of mammals. J. Anat. 95:256‐261.
   Rogers, S.L., Tint, I.S., and Gelfand, V.I. 1998. In vitro motility assay for melanophore pigment organelles. Methods Enzymol. 289:361‐372.
   Seiji, M., Shimao, K., Birbeck, M.S.C., and Fitzpatrick, T.B. 1963. Subcellular localization of melanin biosynthesis. Ann. NY Acad. Sci. 100:497‐533.
   Tribl, F., Gerlach, M., Marcus, K., Asan, E., Tatschner, T., Arzberger, T., Meyer, H. E., Bringmann, G., and Riederer, P. 2005. Subcellular proteomics of neuromelanin granules isolated from the human brain. Mol. Cell. Proteomics 4:945‐957.
   Tribl, F., Marcus, K., Meyer, H.E., Bringmann, G., Gerlach, M., and Riederer, P. 2006. Subcellular proteomics reveals neuromelanin granules to be a lysosome‐related organelle. J. Neural Transm. 113:741‐749.
   Whittaker, V.P. 1965. The application of subcellular fractionation techniques to the study of brain function. Prog. Biophys. Mol. Biol. 15:39‐96.
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