Abstract
Astrocytes are heterogeneous cells of the central nervous system whose uptake of neurotransmitters and neuromodulators can influence synaptic signaling. Any malfunction in this process can lead to serious defects in synaptic transmission found in, for example, neurodegenerative diseases like Alzheimer’s or epilepsy.
Here we describe how to visualize the uptake of an extracellularly located protein by in vitro cultured astrocytes on the example of tissue plasminogen activator, a serine protease tightly involved in long-term potentiation and seizure generation.
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References
Verkhratsky A, Zorec R, Parpura V (2017) Stratification of astrocytes in healthy and diseased brain. Brain Pathol 27(5):629–644
Fernández-Monreal M, López-Atalaya JP, Benchenane K, Leveille F, Cacquevel M, Plawinski L, MacKenzie ET, Bu G, Buisson A, Vivien D (2004) Is tissue-type plasminogen activator a neuromodulator? Mol Cell Neurosci 25:594–601
Cassé F, Bardou I, Danglot L, Briens A, Montagne A, Parcq J, Alahari A, Galli T, Vivien D, Docagne F (2012) Glutamate controls tPA recycling by astrocytes, which in turn influences glutamatergic signals. J Neurosci 32(15):5186–5199
Yepes M, Sandkvist M, Moore EG, Bugge TH, Strickland DK, Lawrence DA (2003) Tissue-type plasminogen activator induces opening of the blood-brain barrier via the LDL receptor-related protein. J Clin Invest 112:1533–1540
Nicole O, Docagne F, Ali C, Margaill I, Carmeliet P, MacKenzie ET, Vivien D, Buisson A (2001) The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat Med 7(1):59–64
Tsirka SE, Gualandris A, Amaral DG, Strickland S (1997) Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature 377(6547):340–344
Louessard M, Lacroix A, Martineau M, Mondielli G, Montagne A, Lesept F, Lambolez B, Cauli B, Mothet JP, Vivien D, Maubert E (2016) Tissue plasminogen activator expression is restricted to subsets of excitatory pyramidal glutamatergic neurons. Mol Neurobiol 53(7):5000–5012
Jeanneret V, Wu F, Merino P, Torre E, Diaz A, Cheng L, Yepes M (2016) Tissue-type plasminogen activator (tPA) modulates the postsynaptic response of cerebral cortical neurons to the presynaptic release of glutamate. Front Mol Neurosci 9:121
Wu F, Torre E, Cuellar-Giraldo D, Cheng L, Yi H, Bichler EK, GarcĂa PS, Yepes M (2015) Tissue-type plasminogen activator triggers the synaptic vesicle cycle in cerebral cortical neurons. J Cereb Blood Flow Metab 35(12):1966–1976
Parcq J, Bertrand T, Montagne A, Baron AF, Macrez R, Billard JM, Briens A, Hommet Y, Wu J, Yepes M, Lijnen HR, Dutar P, Anglés-Cano E, Vivien D (2012) Unveiling an exceptional zymogen: the single-chain form of tPA is a selective activator of NMDA receptor-dependent signaling and neurotoxicity. Cell Death Differ 19(12):1983–1991
Bertrand T, Lesept F, Chevilley A, Lenoir S, Aimable M, Briens A, Hommet Y, Bardou I, Parcq J, Vivien D (2015) Conformations of tissue plasminogen activator (tPA) orchestrate neuronal survival by a crosstalk between EGFR and NMDAR. Cell Death Dis 6:e1924
Samson AL, Nevin ST, Croucher D, Niego B, Daniel PB, Weiss TW, Moreno E, Monard D, Lawrence DA, Medcalf R (2008) Tissue-type plasminogen activator requires a co-receptor to enhance NMDA receptor function. J Neurochem 107(4):1091–1101
Samson AL, Medcalf RL (2006) Tissue-type plasminogen activator: a multifaceted modulator of neurotransmission and synaptic plasticity. Neuron 50(5):673–678
Gottschling C, Dzyubenko E, Geissler M, Faissner A (2016) The indirect neuron-astrocyte coculture assay: an in vitro set-up for the detailed investigation of neuron-glia interactions. J Vis Exp (117):e54757. https://doi.org/10.3791/54757
Schildge S, Bohrer C, Beck K, Schachtrup C (2013) Isolation and culture of mouse cortical astrocytes. J Vis Exp (71):50079. https://doi.org/10.3791/50079
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682
Braganza O, Bedner P, Hüttmann K, von Staden E, Friedman A, Seifert G, Steinhäuser C (2012) Albumin is taken up by hippocampal NG2 cells and astrocytes and decreases gap junction coupling. Epilepsia 53(11):1898–1906
Ivens S, Kaufer D, Flores LP, Bechmann I, Zumsteg D, Tomkins O, Seiffert E, Heinemann U, Friedman A (2007) TGF-β receptor-mediated albumin uptake into astrocytes is involved in neocortical epileptogenesis. Brain 130:535–547
Acknowledgments
We thank the IGSN and the DFG, SPP1757 Fa 159/20-1,2 for providing financial support. We also thank Egor Dzyubenko for valuable discussions and help with establishing the astrocytic cultures and Sandra Lata and David Wegrzyn for performing the “test runs” of the protocol and their valuable feedback.
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Bres, E.E., Faissner, A. (2019). Detection of Protein Uptake in In Vitro Cultured Astrocytes Exemplified by the Uptake of the Serine Protease, Tissue Plasminogen Activator. In: Di Benedetto, B. (eds) Astrocytes. Methods in Molecular Biology, vol 1938. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9068-9_14
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DOI: https://doi.org/10.1007/978-1-4939-9068-9_14
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