Abstract
Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are characterized by intracellular inclusions composed mostly of α-synuclein (Baba et al., Am J Pathol 152:879–884, 1998). How inclusion formation impacts neuronal function prior to death is key to understanding disease progression and identifying therapeutic windows. In the α-synuclein fibril model, exposure of primary neurons to α-synuclein fibrils induces endogenously expressed α-synuclein to form inclusions which closely resembles pathologic mechanisms in humans with PD and DLB (Volpicelli-Daley et al., Neuron 72, 57–71, 2011). In this model, at 7 days after exposure of neurons to fibrils, when there is no neuron death, inclusions in the axon selectively impair axonal transport of endosomes carrying the TrkB receptor and LC3-positive autophagosomes (Volpicelli-Daley et al., Mol Biol Cell 25:4010–4023, 2014). In addition, the frequency and amplitude of spontaneous Ca2+ transients are reduced in neurons 7 days after fibril exposure. Here we discuss protocols for plating primary hippocampal neurons, generating fibrils and measuring axonal transport and Ca2+ transients. These assays provide additional assays of neurotoxicity allowing researchers to determine if a therapeutic intervention can prevent neuronal defects before intractable neurodegeneration.
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References
Baba M, Nakajo S, Tu PH, Tomita T, Nakaya K, Lee VM, Trojanowski JQ, Iwatsubo T (1998) Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy bodies. Am J Pathol 152:879–884
Volpicelli-Daley LA, Luk KC, Patel TP, Tanik SA, Riddle DM, Stieber A, Meaney DF, Trojanowski JQ, Lee VM (2011) Exogenous alpha-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron 72:57–71
Volpicelli-Daley LA, Gamble KL, Schultheiss CE, Riddle DM, West AB, Lee VM (2014) Formation of alpha-synuclein Lewy neurite-like aggregates in axons impedes the transport of distinct endosomes. Mol Biol Cell 25:4010–4023
Chen TW, Wardill TJ, Sun Y, Pulver SR, Renninger SL, Baohan A, Schreiter ER, Kerr RA, Orger MB, Jayaraman V, Looger LL, Svoboda K, Kim DS (2013) Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499:295–300
Volpicelli-Daley LA, Luk KC, Lee VM (2014) Addition of exogenous alpha-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous alpha-synuclein to Lewy body and Lewy neurite-like aggregates. Nat Protoc 9:2135–2146
Polinski NK, Volpicelli-Daley LA, Sortwell CE, Luk KC, Cremades N, Gottler LM, Froula J, Duffy MF, Lee VM, Martinez TN, Dave KD (2018) Best practices for generating and using alpha-synuclein pre-formed fibrils to model Parkinson’s disease in rodents. J Parkinsons Dis 8(2):303–322
Seibenhener ML, Wooten MW (2012) Isolation and culture of hippocampal neurons from prenatal mice. J Vis Exp. https://doi.org/10.3791/3634
Bousset L, Brundin P, Bockmann A, Meier B, Melki R (2016) An efficient procedure for removal and inactivation of alpha-synuclein assemblies from laboratory materials. J Parkinsons Dis 6:143–151
Roy S, Winton MJ, Black MM, Trojanowski JQ, Lee VM (2008) Cytoskeletal requirements in axonal transport of slow component-b. J Neurosci 28:5248–5256
Dotti CG, Sullivan CA, Banker GA (1988) The establishment of polarity by hippocampal neurons in culture. J Neurosci 8:1454–1468
Kalmar B, Innes A, Wanisch K, Kolaszynska AK, Pandraud A, Kelly G, Abramov AY, Reilly MM, Schiavo G, Greensmith L (2017) Mitochondrial deficits and abnormal mitochondrial retrograde axonal transport play a role in the pathogenesis of mutant Hsp27-induced Charcot Marie Tooth Disease. Hum Mol Genet 26:3313–3326
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Volpicelli-Daley, L.A. (2019). Assays for Neuronal Defects Caused by Early Formation of α-Synuclein Inclusions in Primary Cultured Neurons. In: Bartels, T. (eds) Alpha-Synuclein. Methods in Molecular Biology, vol 1948. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9124-2_1
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DOI: https://doi.org/10.1007/978-1-4939-9124-2_1
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