Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Summary

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

Introduction

Protein aggregation is a biological process by which misfolded proteins group up and may act as causative agents in neurodegenerative diseases (amyloidosis). Characterizing protein aggregation is essential in understanding the role of aggregates in cellular dysfunction as well as in facilitating the discovery of new factors that influence the onset of the pathology. The visualization of fluorescence-tagged proteins in living cells is a powerful method which may aid in the development of assays applicable to high content screening (HCS)^1,2,3,4

Protocol

1. Lentivirus production

NOTE: The production and manipulation of lentiviral vectors was carried out according to the National Institutes of Health (NIH) guidelines for research involving recombinant DNA. The plasmid encoding the wild-type and A4V mutant SOD1 tagged with enhanced YFP (SOD1WT-YFP and SOD1A4V-YFP) are described in Kim et al.¹¹ Both gene fusion products were amplified using the PCR primer pair 5′-ATCGTCTAGACACCATGGCGACGAAGGTCGTGTGC-3′ and 5′-TAGCGG CCGCTACTTGTACAGCTCGTCCATGCC-3′and inserted into the pTRIP-delta U3 CMV plasmid¹⁹ using XhoI and BsrGI ....

Results

Generating stable cell line using lentivirus: The overall strategy for monitoring SOD1 protein aggregates is illustrated in Figure 1. In a first step, we generated a lentiviral expression vector for SOD1 stable gene delivery into cell lines (Step 1). Two lentiviral vectors encoding YFP-tagged SOD1 WT and SOD1 A4V (SOD1WT-YFP and SOD1A4V-YFP, respectively) with packing and envelope plasmids were prepared (Figure 2A

Discussion

There are two main approaches for generating stable cell lines. The first takes several weeks and requires transient transfection and resistance selection of the genomic integrated plasmid DNA vectors. The second takes a matter of hours through the use of lentivirus, making this protocol amenable to the effective expression of target protein in multiple cell lines with limited effort. The TRIP-CMV vector¹⁹ was a sustained vector to use, with conserved transduction efficiency and stable transgene e.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ALLN (C20H37N3O4)	Millipore	208719
MG132 (C26H41N3O5)	Sigma-Aldrich	C2211
Epoxomicin (C28H50N4O7)	Sigma-Aldrich	E3652
Hoechst 33342	Invitrogen	H-3570
Opera	Perkin Elmer	OP-QEHS-01
Opera EvoShell software	Perkin Elmer	Ver 1.8.1
Operetta	Perkin Elmer	OPRT1288
Harmony Imaging software	Perkin Elmer	Ver 3.0.0
Columbus Image analysis software	Perkin Elmer	Ver 2.3.2
CyBi Hummingwell liquid handling	CyBio AG	OL 3387 3 0110