Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

Summary

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

Abstract

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.

Introduction

The disruption of protein quality control networks that ensure proper folding and degradation of cellular proteins is likely central to pathology in Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and other "protein misfolding" disorders^2,3. 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 function^5,6. 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 splicing^8,9 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 β-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 progression^1,13,14. 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.

Protocol

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

1. Prepare "Soluble" lysis buffer (10 mM Tris pH 7.4, 1% Triton-X 100, 150 mM NaCl, 10% glycerol) and sterile filter through 0.22 µm filter.
   1. For a working "Soluble" lysis buffer, measure N-Ethylmaleimide (NEM) and thoroughly dissolve in 100 mM phenylmethysulfonyl flouride (PMSF) dissolved in 100% EtOH.
      NOTE: Lysis Buffer Inhibitors: (i) 20 mM N-ethylmaleimide (NEM); (ii) 0.2 mM PMSF; (iii) 1 mM Sodium orthovanadate (Na₃VO₄); (iv) 1 µg/mL leupeptin; (v) 1 µg/mL aprotinin; (vi) 20 mM Sodium fluoride (NaF).
      CAUTION: Wear a face mask when working with NEM throughout the protocol. Working lysis buffer must be made fresh to ensure active inhibitors.
   2. For soluble lysis buffer with protease inhibitors, add inhibitors in the following order: Sodium Fluoride (NaF), up to volume with cold lysis buffer, Sodium orthovanadate (Na₃VO₄), Aprotinin, and Leupeptin.
2. For "Insoluble" lysis buffer, supplement to 4% SDS by adding 500 µL of 20% SDS to 2 mL of "Soluble" lysis buffer. Leave at room temperature.
   NOTE: SDS will precipitate out when kept at 4 °C ; keep this at room temp. Final Lysis Buffer composition: 10 mM Tris (pH 7.4); 1% Triton X-100; 150 mM NaCl; 10% glycerol; 4% SDS (Insoluble Only).

2. Soluble/Insoluble Fractionation Protocol

NOTE: See Figure 1 for a schematic.

1. Label two sets of tubes for each sample: "Soluble" and "Insoluble."
2. Add X-µL of ice-cold working soluble lysis buffer supplemented with protease inhibitors (see 1.1.1) to tissue.
   NOTE: X = Volume varies depending on tissue amount; See Table 1.
3. For mouse brain tissue (i.e., striatum), dounce slowly 30 times in glass 1 mL douncer and pipet into "Insoluble" labeled tube (keep on ice).
   NOTE: Be careful not to break surface of liquid after starting douncing, as bubbles will form and may yield incomplete homogenization.
4. For cells (i.e., HeLa, HEK293T), rinse cells once with cold, 1x PBS. Remove PBS and apply minimal volume of lysis buffer to cells and lift with cell scraper.
   NOTE: For above cell lines, cells were plated at 5 x 10⁵ cells per 6 well plate and grown to near confluency. One well is comparable to volumes used for mouse striatum.
5. Transfer homogenate or cell lysate into labeled, "Insoluble" tubes and lyse on ice 1 h (Start clock after final tube is processed).
   NOTE: For cell lysates, triturate several times to break up cell clumps prior to starting incubation. Avoid formation of bubbles. Briefly vortex pulse each sample for 1 s halfway through lysing.
6. Centrifuge samples at 4 °C at 15,000 x g for 20 min.
7. Recover supernatant as the "Soluble" fraction.
   1. Remove with pipet, be careful not to disturb pellet/cloudy layer as this will contaminate the fraction. Pipet soluble fraction into "Soluble" labeled tube. Keep on ice.
8. Wash pellet with 500 µL of lysis buffer (x2) and centrifuge at 4 °C at 15,000 x g for 5 min after each wash; it is okay if some of the cloudy layer is pipetted off.
9. After final wash, remove ALL remaining wash buffer leaving only the tissue pellet.
   NOTE: From this point, insoluble samples should now be kept at room temperature.
10. Resuspend pellet with X-µL lysis buffer supplemented with 4% SDS (Volume can be adjusted depending on pellet size; see Table 1).
11. Sonicate each sample for 30 s at room temperature with a probe sonicator (125 Watts, 20 kHz, Pulse, Amplitude 40%, see Table of Materials).
12. Boil samples (rolling boil) for 30 min; Centrifuge briefly (15 s) at 6,000 x g as to not lose any sample.
13. Perform DC (detergent compatible) protein assay (see Table of Materials) or Lowry protein assay to measure protein concentration (recommended started dilutions of 1/5 of sample in respective lysis buffer for cell culture and 1/10 for tissue lysates).
    NOTE: Protein concentrations will have to be read using a DC or Lowry assay due to high detergent concentration.
14. Aliquot samples into 50 µL batches to avoid freeze-thaw issues.
    NOTE: Protocol can be paused here prior to further biochemical analysis by storing both fractions at -20 °C.

3. Western Blot and Quantification

1. Perform western blots as reported elsewhere^1,13 by diluting 30 µg of sample 1:1 in loading buffer (1.6x loading dye, 1x reducing).
   NOTE: 30 µg of protein per fraction is sufficient, but can be adjusted for optimal resolution. 3 - 8% Tris-Acetate poly-acrylamide gels should be used for the insoluble fraction. 4 - 12% Bis-Tris gels are recommended for the soluble fraction.
2. Boil samples (rolling boil) for 5 min. Centrifuge briefly (15 s) at 6,000 x g to collect sample.
3. Analyze fractions by SDS-PAGE and western blot, according to manufacturer's protocol for reduced samples.
   NOTE: Insoluble fraction resolves best when transferred onto 0.45 µM nitrocellulose membrane. 0.45 or 0.2 µM nitrocellulose can be used for the soluble fraction, depending on the size of the protein of interest. Some optimization may be required for best transfer of HMW materials.
4. Stain membrane with whole protein stain (e.g., MEMcode, see the Table of Materials) to visualize protein loading efficiency.
   NOTE: Western blots can be analyzed by mean pixel intensity. Soluble fractions can be normalized to a house-keeping protein best suited for the experimental system. Insoluble fraction as relative protein abundance was verified by reversible protein staining or Histone 3 normalization on separate blot.

Results

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 ...

Discussion

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 ...

Materials

List of materials used in this article

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