Abstract
Humans age and the ageing process affects cells in all areas of the human body, including nerve cells within the brain. With advancing age there is also a rise in the probability of developing a neurodegenerative disorder such as, e.g., amyotrophic lateral sclerosis, Huntington’s disease, Parkinson’s disease, or Alzheimer’s disease. In all these age-related neurodegenerative disorders, distinct neuron populations within specific brain regions are primarily affected. For example, Parkinson’s disease is characterized by a slowly progressive degeneration of dopaminergic neurons in the substantia nigra whereas the entorhinal cortex is first affected in Alzheimer’s disease. In patients suffering from Huntington’s disease, neurons in both striatum and cortex undergo substantial cell loss and in amyotrophic lateral sclerosis the neurodegeneration arises from the spinal cord and the motor cortex. For the investigation of the differences in neuronal vulnerability, it is important to examine the protein expression pattern in these specific neural populations. By this, conclusions about the origination process of these diseases can be achieved. In order to obtain this objective, specific isolation of distinct neurons from the surrounding brain tissue is indispensable. However, discrimination as well as isolation of distinct types of neurons can be challenging, due to the brain tissue’s complexity. With traditional methods such as the homogenization of tissue samples, a specific isolation of single neuron populations is not feasible because homogenization results into a mixture containing all cell types. Laser microdissection can overcome this technical limitation. First, this method enables visualization of tissues via a microscopic unit and therefore an enhanced discrimination of different brain cells. Second, a laser device guarantees a contact-free and consequently a contamination-free separation of distinct neurons from the surrounding brain tissue. In the following, we present a detailed protocol that includes a workflow for the isolation and analysis of neurons from freshly frozen post mortem human brain tissue samples. During this procedure, the brain tissue is sectioned, stained, laser microdissected, and ultimately analyzed by high-performance liquid chromatography—mass spectrometry.
This is a preview of subscription content, log in via an institution.
References
Mattson MP, Magnus T (2006) Ageing and neuronal vulnerability. Nat Rev Neurosci 7:278–294
Bartels AL, Leenders KL (2009) Parkinson's disease: the syndrome, the pathogenesis and pathophysiology. Cortex 45:915–921
Rangel-Barajas C, Rebec GV (2016) Dysregulation of corticostriatal connectivity in huntington's disease: a role for dopamine modulation. J Huntingtons Dis 5:303–331
Molina M, Steinbach S, Park YM, Yun SY, Di Lorenzo Alho AT, Heinsen H, Grinberg LT, Marcus K, Leite RE, May C (2015) Enrichment of single neurons and defined brain regions from human brain tissue samples for subsequent proteome analysis. J Neural Transm (Vienna) 122:993–1005
Bowen RL, Atwood CS (2004) Living and dying for sex. A theory of aging based on the modulation of cell cycle signaling by reproductive hormones. Gerontology 50:265–290
Dillin A, Gottschling DE, Nyström T (2014) The good and the bad of being connected: the integrons of aging. Curr Opin Cell Biol 26:107–112
Kalia LV, Lang AE (2015) Parkinson's disease. Lancet 386:896–912
Plum S, Steinbach S, Abel L, Marcus K, Helling S, May C (2015) Proteomics in neurodegenerative diseases: Methods for obtaining a closer look at the neuronal proteome. Proteomics Clin Appl 9:848–871
Datta S, Malhotra L, Dickerson R, Chaffee S, Sen CK, Roy S (2015) Laser capture microdissection: Big data from small samples. Histol Histopathol 30:1255–1269
Mukherjee S, Rodriguez-Canales J, Hanson J, Emmert-Buck MR, Tangrea MA, Prieto DA, Blonder J, Johann DJ (2013) Proteomic analysis of frozen tissue samples using laser capture microdissection. Methods Mol Biol 1002:71–83
Murray GI (2007) An overview of laser microdissection technologies. Acta Histochem 109:171–176
Acknowledgment
This work was financially supported by the Bundesministerium für Bildung und Forschung (WTZ FKZ 01DN14023), Germany, by the HUPO Brain Proteome Project (HBPP), PURE, a project of Nordrhein-Westfalen, a federal German state, Germany, and by the Deutsche Parkinson Gesellschaft.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Aring, L., Steinbach, S., Marcus, K., May, C. (2018). Isolation of Distinct Types of Neurons from Fresh Brain Tissue Using Laser Microdissection in Combination with High-Performance Liquid Chromatography—Mass Spectrometry. In: Murray, G. (eds) Laser Capture Microdissection. Methods in Molecular Biology, vol 1723. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7558-7_14
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7558-7_14
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7557-0
Online ISBN: 978-1-4939-7558-7
eBook Packages: Springer Protocols