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 ....

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 buffe....

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