Single neuron ubiquitin-proteasome dynamics accompanying inclusion body formation in huntington disease

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.

Figures

FIGURE 1.

Levels of proteasome reporters increase after inhibition of proteasome. A, after transfection with GFPu and mRFP, striatal neurons were treated with 50 μm MG132 for 12 h. GFP fluorescence (A) and the ratio of GFPu/mRFP fluorescence (B) both increase after treatment relative to control. C, after transfection with mRFPu and GFP, striatal neurons were treated with 50 μm MG132 for 12 h. The mRFPu/GFP ratio is significantly greater than the control (p < 0.02). D, after transfection with Venusu and CFP, striatal neurons were treated with 2 μm epoxomycin (solid lines) or vehicle (broken lines) for 10 h. Both mean change in Venusu/CFP fluorescence (D) and single-cell distributions of Venusu/CFP fluorescence (E) are increased relative to control (p < 0.05, p < 0.05). F, after transfection with UbG76V-Venus and CFP, striatal neurons were treated with 2 μm epoxomycin for 10 h. Both mean change in UbG76V-Venus/CFP fluorescence (F) and single-cell distributions of UbG76V-Venus/CFP fluorescence (G) are increased (p < 0.05, p < 0.01). Experiments were repeated twice with over 50 cells analyzed in each condition.

FIGURE 2.

Limited interaction between the UPS and autophagic pathways in neurons. A, 24 h after cotransfection with UbG76V-Venus and CFP, striatal neurons were treated with vehicle or 50 nm BafA1. BafA1 treatment caused a significant amount of toxicity above control (p < 0.03, top line). Mean UbG76V-Venus/CFP ratio (B) and the distribution of the single-cell changes in UbG76V-Venus/CFP (C) in these cells did not increase above control in 20 h after BafA1 addition. D, neurons or HEK293 cells (E) were treated with BafA1 or epoxomycin, followed by Western blotting with an LC3 antibody. While BafA1 caused accumulation of LC3-II in both neurons and HEK293 cells, epoxomycin increased LC3-II levels only in HEK293 cells. Unlabeled lanes in E are lysates from cells transfected with LC3.

FIGURE 3.

Inhibition of proteasome activity is toxic in a dose-dependent fashion. A, UPS reporter fluorescence shows a dose-dependent response to MG132 treatment. MG132 at the indicated doses was added to striatal neurons 24 h after transfection with mRFPu and GFP. The change in mRFPu/GFP ratio over the first 2.5 h after MG132 administration is shown. B, UPS reporter fluorescence continues to increase up to 12 h after the addition of MG132. Note the difference in scale with A. Measurements from 80 μm were excluded due to noticeable toxicity. C, MG132 is toxic to neurons in a dose-dependent fashion. The same neurons shown in A and B were observed with the risk of death as shown. Longitudinal analysis was repeated twice on different transfections, with n > 50 for each treatment in each experiment.

FIGURE 4.

IB formation and UPS function in primary neurons. A, GFP-htt, BFP, and mRFPu were imaged over the course of days to follow htt IB formation, UPS impairment, and neuronal survival. Single-cell distributions (B) or population means (C) of the change in mRFPu/GFP fluorescence in the interval preceding IB formation at 54 h. The increase in mRFPu/GFP ratio was higher in neurons that went on to form IBs (p < 0.05, p < 0.05). D, in a parallel experiment, single-cell distributions of the change in mRFPu/GFP fluorescence in the interval preceding IB formation at 76 h also show higher UPS impairment in those neurons that will go on to form IBs (p < 0.05). After 54 h, single-cell distributions (E) or population means (F) show a greater increase in mRFPu/GFP fluorescence in those cells that did not form IBs (p < 0.05, p < 0.05). The survival of those neurons that formed htt IBs at 18 h (G) or 27 h (H) was better than the survival of neurons that survived at least that long but never formed IBs (p < 0.01, p < 0.03). Longitudinal analysis was repeated twice in different experiments with over 300 cells analyzed in each experiment, with n > 30 for each cohort.