Single Neuron Ubiquitin-Proteasome Dynamics Accompanying Inclusion Body Formation in Huntington Disease

Andrey S. Tsvetkov,Siddhartha Mitra,Steven Finkbeiner

doi:10.1074/jbc.m806269200

Abstract

The accumulation of mutant protein in intracellular aggregates is a common feature of neurodegenerative disease. In Huntington disease, mutant huntingtin leads to inclusion body (IB) formation and neuronal toxicity. Impairment of the ubiquitin-proteasome system (UPS) has been implicated in IB formation and Huntington disease pathogenesis. However, IBs form asynchronously in only a subset of cells with mutant huntingtin, and the relationship between IB formation and UPS function has been difficult to elucidate. Here, we applied single-cell longitudinal acquisition and analysis to monitor mutant huntingtin IB formation, UPS function, and neuronal toxicity. We found that proteasome inhibition is toxic to striatal neurons in a dose-dependent fashion. Before IB formation, the UPS is more impaired in neurons that go on to form IBs than in those that do not. After forming IBs, impairment is lower in neurons with IBs than in those without. These findings suggest IBs are a protective cellular response to mutant protein mediated in part by improving intracellular protein degradation.

Highlights

Huntington disease (HD)4 is a progressive incurable neurodegenerative disorder caused by the expansion of a polyglutamine stretch in the N-terminal end of the huntingtin protein above a threshold length of ϳ36 [1]
To determine if autophagic activity could be confounding fluorescent reporter measurements of ubiquitin-proteasome system (UPS) function, we examined the activity of the autophagic pathway after proteasome inhibition
UPS Reporter Fluorescence Demonstrates a Graded Response to Proteasome Inhibition—Having validated the use of destabilized fluorescent proteins as reporters of UPS function in primary neurons, we examined the nature of their response to varying levels of proteasome impairment

Summary

EXPERIMENTAL PROCEDURES

Plasmids—mRFP [27], pCS2-Venus [28], and pEGFPCL1(5), pGW1-GFP, pGW1-httQ72-eGFP, pGW1-mRFP [2] have been described. pGW1-httQ72-CFP was generated from pGW1-httQ72-eGFP. pGW1-mRFPu (mRFPu) was generated by subcloning mRFP1 from pcDNA3.1(ϩ) into pEGFP-CL; mRFP1-CL1 was subcloned into pGW1. pGW1-GFPu was constructed by excising EGFP-CL1 from pEGFP-CL1 and inserting it into pGW1. pGW1-Venus-CL1 (Venusu) was generated by subcloning Venus from pCS2-Venus into pcDNA3.1(ϩ). Transfection, Pharmacology, and Colocalization—Primary cultures were transfected 5–7 days in vitro with combinations of pGW1-GFPu and pGW1-mRFP, pGW1-mRFPu, and pGW1GFP, pGW1-Venusu, or pCS2-UbG76V-Venus, and pGW1-CFP, and pGW1-httQ72-eGFP, pGW1-mRFPu, and pGW1-BFP, or pGW1-httQ72-CFP and pYFP-LMP2 in a 1:1 or 1:1:1 molar ratio with 2– 4 ␮g of total plasmid DNA per well. The fraction of positive pixels for CFP-htt IBs that overlapped positive pixels of ubiquitin staining or LMP-YFP fluorescence was calculated with Metamorph for (n ϭ 20 neurons). CFP, Venus, and RFP images were captured using a 86006 beamsplitter and 420/35ϫ; 470/30m, 500/20ϫ; 535/30m, and 580/20ϫ; 630/60m fluorescence filters (Chroma Corp, Rockingham, VT). Algorithms for plate registration, stage movements, filter movements, focusing, and acquisition were generated with Metamorph imaging software (Molecular Devices, Sunnyvale, CA). Survival analysis was performed with the Statview software package (SAS Institute, Cary, NC); t tests for comparisons of means and two-sample Kolmogorov-Smirnov tests for comparisons of distributions were performed with Prism (Graphpad Software, San Diego, CA)

RESULTS

UPS Impairment Decreases after

Though the IBs in our primary