Abstract
N-terminal mutant huntingtin (mHTT) fragments with pathogenic polyglutamine (polyQ) tracts spontaneously form stable, amyloidogenic protein aggregates with a fibrillar morphology. Such structures are detectable in brains of Huntington’s disease (HD) patients and various model organisms, suggesting that they play a critical role in pathogenesis. Heat-stable, fibrillar mHTT aggregates can be detected and quantified in cells and tissues using a denaturing filter retardation assay (FRA). Here, we describe step-by-step protocols and experimental procedures for the investigation of mHTT aggregates in complex biosamples using FRAs. The methods are illustrated with examples from studies in cellular, transgenic fly, and mouse models of HD, but can be adapted for any disease-relevant protein with amyloidogenic polyQ tracts.
Anne Ast and Franziska Schindler are co-first authors.
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References
Becher MW, Kotzuk JA, Sharp AH et al (1998) Intranuclear neuronal inclusions in Huntington’s disease and dentatorubral and pallidoluysian atrophy: correlation between the density of inclusions and IT15 CAG triplet repeat length. Neurobiol Dis 397:387–397
Huntington T, Macdonald ME, Ambrose CM et al (1993) A novel gene containing a trinucleotide that is expanded and unstable on huntington’s disease chromosomes. Cell 72:971–983
Davies SW, Turmaine M, Cozens BA et al (1997) Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 90:537–548
Scherzinger E, Lurz R, Turmaine M et al (1997) Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. Cell 90:549–558
Harjes P, Wanker EE (2003) The hunt for huntingtin function: interaction partners tell many different stories. Trend Biochem Sci 28:425–433
Chen S, Ferrone FA, Wetzel R (2002) Huntington’s disease age-of-onset linked to polyglutamine aggregation nucleation. Proc Natl Acad Sci U S A 99:11884–11889
Difiglia M, Sapp E, Chase KO et al (1997) Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277:1990–1993
Scherzinger E, Sittler A, Schweiger K et al (1999) Self-assembly of polyglutamine-containing huntingtin fragments into amyloid-like fibrils: implications for Huntington's disease pathology. Proc Natl Acad Sci U S A 96:4604–4609
Waelter S, Boeddrich A, Lurz R et al (2001) Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation. Mol Biol Cell 12:1393–1407
Ross CA, Poirier MA (2004) Protein aggregation and neurodegenerative disease. Nat Med 10:S10–S17
Jucker M, Walker LC (2013) Self-propagation of pathogenic protein aggregates in neurodegenerative diseases. Nature 501:45–51
Falk HR, Comenzo LR, Skinner M (1997) The systemic amyloidosis. N Engl J Med 337:898–909
Howie AJ, Brewer DB, Howell D et al (2008) Physical basis of colors seen in Congo red-stained amyloid in polarized light. Lab Investig 88:232–242
Pecho-vrieseling E, Rieker C, Fuchs S et al (2014) Transneuronal propagation of mutant huntingtin contributes to non-cell autonomous pathology in neurons. Nat Neurosci 17:1064–1072
Sieradzan KA, Mechan AO, Jones L et al (1999) Huntington’s disease intranuclear inclusions contain truncated, ubiquitinated huntingtin protein. Exp Neurol 99:92–99
Steffan JS, Kazantsev A, Spasic-boskovic O et al (2000) The Huntington’s disease protein interacts with p53 and CREB-binding protein and represses transcription. Proc Natl Acad Sci U S A 97:6763–6768
WMC v R-m, Reid SJ, Jones AL et al (2002) Insoluble TATA-binding protein accumulation in Huntington’s disease cortex. Brain Res Mol Brain Res 109:1–10
Legleiter J, Mitchell E, Lotz GP et al (2010) Mutant Huntingtin fragments form oligomers in a polyglutamine length-dependent manner in vitro and in vivo. J Biol Chem 285:14777–14790
Brundin P, Melki R, Kopito R (2010) Prion-like transmission of protein aggregates in neurodegenerative diseases. Nat Rev Mol Cell Biol 11:301–307
Pearce MMP, Spartz EJ, Hong W et al (2015) Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain. Nat Commun 6:1–11
Babcock DT, Ganetzky B (2015) Transcellular spreading of huntingtin aggregates in the Drosophila brain. Proc Natl Acad Sci U S A 112(39):E5427–E5433
Wanker EE, Scherzinger E, Heiser V et al (1999) Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates. Methods Enzymol 309:375–386
Kakkar V, Ma C, De Mattos EP et al (2016) The S/T-rich motif in the DNAJB6 chaperone delays polyglutamine aggregation and the onset of disease in a mouse model article the S/T-rich motif in the DNAJB6 chaperone delays polyglutamine aggregation and the onset of disease in a mouse model. Mol Cell 62:272–283
Ehrnhoefer DE, Duennwald M, Markovic P et al (2006) Green tea (−)-epigallocatechin-gallate modulates early events in huntingtin misfolding and reduces toxicity in Huntington’s disease models. Hum Mol Genet 15:2743–2751
Zurawel AA, Kabeche R, Digregorio SE et al (2016) CAG expansions are genetically stable and form nontoxic aggregates in cells lacking endogenous polyglutamine proteins. MBio 7:1–11
Muchowski PJ, Schaffar G, Sittler A et al (2000) Hsp70 and Hsp40 chaperones can inhibit self-assembly of polyglutamine proteins into amyloid-like fibrils. Proc Natl Acad Sci U S A 97:7841–7846
Cohen A, Ross L, Nachman I et al (2012) Aggregation of polyQ proteins is increased upon yeast aging and affected by Sir2 and Hsf1: novel quantitative biochemical and microscopic assays. PLoS One 7:1–10
Melkani GC, Trujillo AS, Ramos R et al (2013) Huntington’s disease induced cardiac amyloidosis is reversed by modulating protein folding and oxidative stress pathways in the Drosophila heart. PLoS Genet 9:e1004024
Kim YE, Hosp F, Frottin F et al (2016) Soluble oligomers of polyq-expanded huntingtin target a multiplicity of key cellular factors. Mol Cell 63:951–964
Heiser V, Scherzinger E, Boeddrich A et al (2000) Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: implications for Huntington’s disease therapy. Proc Natl Acad Sci U S A 97:6739–6744
Heiser V, Engemann S, Bröcker W et al (2002) Identification of benzothiazoles as potential polyglutamine aggregation inhibitors of Huntington’s disease by using an automated filter retardation assay. Proc Natl Acad Sci U S A 99:16400–16406
Acknowledgment
This study received funding from the EC funding initiative ERA-NET NEURON, consortium “ABETA ID” funded by the German Federal Ministry for Education and Research (BMBF), grant no. 01W1301, the Berlin Institute of Health Collaborative Research Grant no. 1.1.2.a.3 “Elucidating the proteostasis network to control Alzheimer’s disease” funded by the German Federal Ministry for Education and Research (BMBF), the Helmholtz Validation Fund grant no. HVF-0013 “Enabling Technologies for Drug Discovery against Protein Misfolding Diseases” funded by the Helmholtz Association, Germany, (to E.E.W.), and the Max Delbrück Center for Molecular Medicine in the Helmholtz Association for application-oriented research (to E.E.W.). Anne Ast and Franziska Schindler contributed equally to this work.
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Ast, A., Schindler, F., Buntru, A., Schnoegl, S., Wanker, E.E. (2018). A Filter Retardation Assay Facilitates the Detection and Quantification of Heat-Stable, Amyloidogenic Mutant Huntingtin Aggregates in Complex Biosamples. In: Precious, S., Rosser, A., Dunnett, S. (eds) Huntington’s Disease. Methods in Molecular Biology, vol 1780. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7825-0_3
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DOI: https://doi.org/10.1007/978-1-4939-7825-0_3
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