Name: fractionation for resolution of soluble and insoluble huntingtin species
Uploaded: 2018-02-27T14:30:00
Description: Watch this Scientific Journal Video about Fractionation for Resolution of Soluble and Insoluble Huntingtin Species at JoVE.com

This work was supported by the NIH (RO1-NS090390). We would also like to thank Dr. Joan Steffanfor technical assistance and discussion during the development of this assay.

Animal Ethics Statement - Experiments were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and an approved animal research protocol by the Institutional Animal Care and Use Committee (IACUC) at the University of California, Irvine, an AAALAC accredited institution. All efforts were made to minimize animal suffering.
1. Preparation of Lysis Buffers
<ol>
	<li>Prepare &#34;Soluble&#34; lysis buffer (10 mM Tris pH 7....

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeat+expansion+disease:+progress+and+puzzles+in+disease+pathogenesis.">Repeat expansion disease: progress and puzzles in disease pathogenesis.</a> Nat Rev Genet. 11 (4), 247-258 (2010).</li><li>Reiner, A., Dragatsis, I., Dietrich, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+neuropathology+of+Huntington's+disease.">Genetics and neuropathology of Huntington's disease.</a> Int Rev Neurobiol. 98, 325-372 (2011).</li><li>The Huntington's Disease Collaborative Research Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+gene+containing+a+trinucleotide+repeat+that+is+expanded+and+unstable+on+Huntington's+disease+chromosomes.">A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes.</a> Cell. 72 (6), 971-983 (1993).</li><li>Sassone, J., Colciago, C., Cislaghi, G., Silani, V., Ciammola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington's+disease:+the+current+state+of+research+with+peripheral+tissues.">Huntington's disease: the current state of research with peripheral tissues.</a> Exp Neurol. 219 (2), 385-397 (2009).</li><li>Weydt, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoregulatory+and+metabolic+defects+in+Huntington's+disease+transgenic+mice+implicate+PGC-1alpha+in+Huntington's+disease+neurodegeneration.">Thermoregulatory and metabolic defects in Huntington's disease transgenic mice implicate PGC-1alpha in Huntington's disease neurodegeneration.</a> Cell Metab. 4 (5), 349-362 (2006).</li><li>Schulte, J., Littleton, J. 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P., Housman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrantly+spliced+HTT,+a+new+player+in+Huntington's+disease+pathogenesis.">Aberrantly spliced HTT, a new player in Huntington's disease pathogenesis.</a> RNA Biol. 10 (11), 1647-1652 (2013).</li><li>Neueder, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenic+exon+1+HTT+protein+is+produced+by+incomplete+splicing+in+Huntington's+disease+patients.">The pathogenic exon 1 HTT protein is produced by incomplete splicing in Huntington's disease patients.</a> Sci Rep. 7 (1), 1307 (2017).</li><li>Arndt, J. R., Chaibva, M., Legleiter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+role+of+the+first+17+amino+acids+of+huntingtin+in+Huntington's+disease.">The emerging role of the first 17 amino acids of huntingtin in Huntington's disease.</a> Biomol Concepts. 6 (1), 33-46 (2015).</li><li>Saudou, F., Finkbeiner, S., Devys, D., Greenberg, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntingtin+acts+in+the+nucleus+to+induce+apoptosis+but+death+does+not+correlate+with+the+formation+of+intranuclear+inclusions.">Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions.</a> Cell. 95 (1), 55-66 (1998).</li><li>Kim, S., Kim, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+Approaches+for+Inhibition+of+Protein+Aggregation+in+Huntington's+Disease.">Therapeutic Approaches for Inhibition of Protein Aggregation in Huntington's Disease.</a> Exp Neurobiol. 23 (1), 36-44 (2014).</li><li>O'Rourke, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-2+and+PIAS1+modulate+insoluble+mutant+huntingtin+protein+accumulation.">SUMO-2 and PIAS1 modulate insoluble mutant huntingtin protein accumulation.</a> Cell Rep. 4 (2), 362-375 (2013).</li><li>Shibata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+intracellular+accumulation+of+mutant+Huntingtin+by+Beclin+1.">Regulation of intracellular accumulation of mutant Huntingtin by Beclin 1.</a> J Biol Chem. 281 (20), 14474-14485 (2006).</li><li>Apostol, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-based+assay+for+aggregation+inhibitors+as+therapeutics+of+polyglutamine-repeat+disease+and+validation+in+Drosophila.">A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila.</a> Proc Natl Acad Sci U S A. 100 (10), 5950-5955 (2003).</li><li>Wanker, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+filter+assay+for+detection+of+amyloid-like+polyglutamine-containing+protein+aggregates.">Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates.</a> Methods Enzymol. 309, 375-386 (1999).</li><li>Sontag, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Mutant+Huntingtin+Aggregation+Conformers+and+Modulation+of+SDS-Soluble+Fibrillar+Oligomers+by+Small+Molecules.">Detection of Mutant Huntingtin Aggregation Conformers and Modulation of SDS-Soluble Fibrillar Oligomers by Small Molecules.</a> J Huntingtons Dis. 1 (1), 119-132 (2012).</li><li>Grima, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+Huntingtin+Disrupts+the+Nuclear+Pore+Complex.">Mutant Huntingtin Disrupts the Nuclear Pore Complex.</a> Neuron. 94 (1), 93-107 (2017).</li><li>Sontag, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylene+blue+modulates+huntingtin+aggregation+intermediates+and+is+protective+in+Huntington's+disease+models.">Methylene blue modulates huntingtin aggregation intermediates and is protective in Huntington's disease models.</a> J Neurosci. 32 (32), 11109-11119 (2012).</li><li>Trushina, E., Rana, S., McMurray, C. T., Hua, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricyclic+pyrone+compounds+prevent+aggregation+and+reverse+cellular+phenotypes+caused+by+expression+of+mutant+huntingtin+protein+in+striatal+neurons.">Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons.</a> BMC Neurosci. 10, 73 (2009).</li><li>Shahmoradian, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRiC's+tricks+inhibit+huntingtin+aggregation.">TRiC's tricks inhibit huntingtin aggregation.</a> Elife. 2, e00710 (2013).</li><li>Kim, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteasome+inhibition+induces+alpha-synuclein+SUMOylation+and+aggregate+formation.">Proteasome inhibition induces alpha-synuclein SUMOylation and aggregate formation.</a> J Neurol Sci. 307 (1-2), 157-161 (2011).</li><li>Goldberg, N. R. 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Some precautions are needed for the above protocols to ensure consistent and quantitative results. First, mHTT in both fractions will spontaneously form aggregates over time upon multiple freeze thaw cycles, particularly when in a high concentration. It is thus critical to freeze aliquots of the protein preps, and thaw only the needed volume prior to running the assay as described in the protocol above. Further, if insoluble fraction yields white precipitant upon thawing, reconstitution may be necessary by an additional ...

The disruption of protein quality control networks that ensure proper folding and degradation of cellular proteins is likely central to pathology in Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and other &#34;protein misfolding&#34; disorders2,3. A detailed understanding of the proteostasis network components and their contributions to pathology are therefore crucial to developing improved therapeutic interventions. HD is caused by the abnormal expansion of a CAG repeat within the HD gene resulting in an expanded stretch of polyglutamines (polyQ) in the huntingtin (HTT) protein4. A consistent pathological feature of HD pathogenesis is the resulting misfolding, accumulation, and aggregation of HTT in regions of the brain and in peripheral tissues, which has been shown to interfere in several ways with normal cell homeostasis and function5,6. While HTT is ubiquitously expressed, medium spiny neurons in the striatum are selectively vulnerable and most overtly affected with notable cortical atrophy also associated with HD pathogenesis.
The formation of aggregates of HTT in the brain of HD patients and animal models has typically served as a proxy marker for disease progression and dominant-negative gain of function for mHTT7. Although the precise mechanisms by which protein misfolding and aggregation may contribute to mHTT-induced toxicity remain unclear, the formation of these inclusions in the brain of HD patients and various animal models is an invariant and inevitable feature. Inclusions appear to be comprised largely of accumulated HTT fragments containing the N-terminal expanded polyQ, indicating that proteolysis and processing of full-length HTT as well as alternative splicing8,9 may play an important role in the pathogenesis of HD. N-terminal HTT fragments may constitute a pathologic form of HTT that can aggregate rapidly, nucleate, and accelerate or propagate the aggregation process10.
However, the presence of these inclusions does not necessarily correlate with HTT-induced toxicity or cell death11. HTT has been proposed to undergo an aggregation process from a soluble monomer through soluble oligomeric species and &#946;-sheet fibrils to insoluble aggregates and inclusions12. Resolving these various protein species via standard biochemical assays has been a challenge in the field due to their stability in low-detergent lysis buffer and difficulty in visualizing using standard biochemical assays. Thus, methodological considerations are critical in detecting the degree and pattern of accumulated and aggregated mHTT.
The protocol presented here provides a method to visualize several intermediates of HTT, specifically the formation of an insoluble HMW HTT species that appears to closely track with HD pathogenesis and disease progression1,13,14. Being able to resolve and track multiple species of mHTT provides researchers a biochemical tool to study disease pathogenesis and evaluate potential therapeutic interventions through their modulation and impact on disease pathogenesis.

<table>
	<tbody>
		<tr>
			<td>Sterile Filter</td>
			<td>Millipore</td>
			<td>SCGP505RE</td>
			<td>Screw cap, sterile vaccum filter</td>
		</tr>
		<tr>
			<td>1 mL Tissue Grinder, Dounce</td>
			<td>Wheaton</td>
			<td>357538</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Qsonica</td>
			<td>Model Q125</td>
			<td></td>
		</tr>
		<tr>
			<td>DC Protein Assay</td>
			<td>Biorad</td>
			<td>5000111</td>
			<td>Comparable to Lowry assay</td>
		</tr>
		<tr>
			<td>Tris 1M buffer solution</td>
			<td>Alfa Aesar</td>
			<td>J60636</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 5M solution</td>
			<td>Teknova</td>
			<td>S0251</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>20% SDS solution</td>
			<td>Teknova</td>
			<td>S0295</td>
			<td></td>
		</tr>
		<tr>
			<td>N-ethylmaleimide</td>
			<td>Sigma</td>
			<td>E1271</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethylsulfony flouride</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td>create 100mM stock solution in 100%EtOH, store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Sodium orthovanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
			<td>Create 0.5M stock solution in water</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td>Create 10mg/ml stock solution in water</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
			<td>Create 10mg/ml stock solution in water</td>
		</tr>
		<tr>
			<td>Sodium Fluoride</td>
			<td>Sigma</td>
			<td>S4504</td>
			<td>Create 500mM stock solution in water</td>
		</tr>
		<tr>
			<td>Anti-HTT</td>
			<td>Millipore</td>
			<td>MAB5492</td>
			<td>Use 1:1000 for western blot, 1:500 for filter retardation assay</td>
		</tr>
		<tr>
			<td>Anti-GAPDH</td>
			<td>Novus Biologicals</td>
			<td>NB100-56875</td>
			<td>Use 1:1000 for soluble western blot</td>
		</tr>
	</tbody>
</table>

fractionation for resolution of soluble and insoluble huntingtin species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

Resolution of soluble and insoluble cell lysates following fractionation can be detected using western analysis and filter retardation assays (Figure 2). As an example, HEK293T cells were transfected using transfection reagent (e.g., lipofectamine 2000), with HTT exon 1 encoding cDNA containing 97 glutamine repeats15 followed by the poly proline rich region, and these cells were allowed to express for 44 h. Cells were treated ...

Watch this Scientific Journal Video about Fractionation for Resolution of Soluble and Insoluble Huntingtin Species at JoVE.com

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

The overall goal of this procedure is to describe a useful tool for visualization, characterization, and quantification of huntingtin protein flux for the analysis of protein homeostasis in Huntington's disease model systems. This method can help us understand questions in protein homeostasis and neurodegenerative disease fields such as understanding the impact of certain therapeutic interventions on disease pathogenesis. The main advantage of this technique is that it allows investigators to isolate and resolve insoluble protein species for quantitative analysis.Though this method can provide insight into biochemical changes during Huntington's disease pathogenesis, it can also be applied to other protein misfolding diseases such as Parkinson's and Alzheimer's disease. To begin this procedure, label two sets of tubes for each sample as soluble and insoluble. Prepare a lysis buffer immediately prior to sample lysis in the soluble tube by adding the appropriate amount of inhibitors.Next, add ice cold working soluble lysis buffer supplemented with protease inhibitors to the tissue. For mouse brain tissue, slowly dounce 30 times in a one milliliter glass douncer. Be careful not to break the surface of liquid as bubbles will form and may yield incomplete homogenization.Then pipette it into the insoluble-labeled tube on ice. For cell lines, cells were plated at five times 10 to the fifth cells in each six-well plate and grown to near confluency. Afterward, rinse the cells once with cold 1X PBS and remove it.Then apply minimal volume of lysis buffer to the cells and lift them with a cell scraper. Next, transfer homogenate or cell lysate into the labeled insoluble tube and lyse on ice for one hour. For cell lysates, triturate several times to break up cell clumps prior to starting incubation and avoid forming bubbles.Briefly pulse vortex each sample for one second halfway through lysing. After that, centrifuge the samples at four degrees Celsius at 15, 000 x g for 20 minutes. Then remove the supernatant.Be careful not to disturb the pellet or cloudy layer as this will contaminate the fraction. Pipette the supernatant as soluble fraction into the soluble-labeled tube and keep it on ice. Keeping track of the soluble and insoluble lysis buffers is key.Switching the buffers or placing the insoluble buffer or samples on ice will be detrimental to the outcome of the protocol. Wash the pellet with 500 microliters of lysis buffer and centrifuge at four degrees Celsius at 15, 000 x g for five minutes. Repeat the wash one more time.Subsequently, remove all the remaining wash buffer leaving only the tissue pellet. Next, resuspend the pellet with lysis buffer supplemented with 4%SDS. Sonicate each sample for 30 seconds at room temperature with a probe sonicator.Sonicating small volumes for a long period of time is technically challenging. Be careful not to lose any sample by emulsifying or spraying while completing this step. Afterward, boil the samples for 30 minutes.Then centrifuge them briefly at 6, 000 x g. Subsequently, perform detergent compatible protein assay or Lowry protein assay to measure protein concentration. Following that, aliquot the samples into 50 microliter batches to avoid freeze/thaw issues.In this step, perform western blots by diluting 30 micrograms of sample at a ratio of one to one in loading buffer. Boil the samples for five minutes and centrifuge them briefly at 6, 000 x g. Next, analyze the fractions by SDS page and western blot.Stain the membrane with whole protein stain to visualize protein loading efficiency. Here are the soluble and insoluble fractions from HEK 293T cell lysates resolved on a 4-12%Bis-Tris and 3-8%Tris-Acetate page gels respectively. Soluble fraction normalized to GAPDH loading control shows fluctuation of mutant huntingtin exon 1 soluble monomer based on exposure to protease inhibitor MG132.Insoluble fraction shows that high molecular weight accumulated species of mutant huntingtin is also modulated by MG132 treatment. And here are the samples of soluble and insoluble fractions from HeLA cell lysates using western blot and filter retardation analysis. Data shows that the presence of an over expressed SUMO isoform increases both high molecular weight mutant and fibrillary insoluble mutant huntingtin in the insoluble fraction.This figure shows the regulation of high molecular weight mutant huntingtin accumulation after modulating a potential therapeutic in the insoluble fractionation from HeLa cells over expressing 97Q huntingtin exon 1. Therapeutic target PIAS1 was either silenced with an siRNA towards PIAS1 or over expressed. Insoluble fractions from mouse striata treated with microRNA against PIAS1 and resolved on 3-8%Tris-Acetate page gels show modulation of high molecular weight insoluble huntingtin.Don't forget that working with NEM can be extremely hazardous and taking precautions such as wearing a face mask should always be taken while performing this protocol. Once mastered, this technique from lysate to end of fractionation can be done in about three to four hours depending on the number of samples. An additional hour should be allowed for protein quantification and aliquoting.Following this procedure, other methods such as agarose gel electrophoresis or filter retardation can be performed in order to detect soluble oligomers or insoluble fibrils respectively. After watching this video, you should have a good understanding of how to isolate and resolve insoluble misfolded proteins for quantitative analysis.

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