Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their toxicity - PubMed (original) (raw)

Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their toxicity

Sarah J Weisberg et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Neurodegenerative diseases constitute a class of illnesses marked by pathological protein aggregation in the brains of affected individuals. Although these disorders are invariably characterized by the degeneration of highly specific subpopulations of neurons, protein aggregation occurs in all cells, which indicates that toxicity arises only in particular cell biological contexts. Aggregation-associated disorders are unified by a common cell biological feature: the deposition of the culprit proteins in inclusion bodies. The precise function of these inclusions remains unclear. The starting point for uncovering the origins of disease pathology must therefore be a thorough understanding of the general cell biological function of inclusions and their potential role in modulating the consequences of aggregation. Here, we show that in human cells certain aggregate inclusions are active compartments. We find that toxic aggregates localize to one of these compartments, the juxtanuclear quality control compartment (JUNQ), and interfere with its quality control function. The accumulation of SOD1G93A aggregates sequesters Hsp70, preventing the delivery of misfolded proteins to the proteasome. Preventing the accumulation of SOD1G93A in the JUNQ by enhancing its sequestration in an insoluble inclusion reduces the harmful effects of aggregation on cell viability.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Aggregates localize to distinct inclusion sites in mammalian cells. (A) VHL and polyQ Htt are models for two types of aggregation-prone proteins. Soluble misfolded proteins accumulate in the JUNQ, whereas amyloidogenic proteins are sequestered in an IPOD-like inclusion. (B) Inclusions were visualized by expression of VHL-GFP and HttQ97-mRFP, respectively; nuclei in all experiments were visualized with NLS-TFP. One-micrometer confocal slices are shown in all panels, and percentage observed phenotype is indicated on the left side of the images. (C) FRAP experiments reveal different mobility properties of VHL in the JUNQ vs. Htt. An area within the inclusion was bleached with full laser power for 2 s. Images represent inclusion fluorescence before, immediately after, and 4 min after bleach (Left to Right). Graphs are average recovery over time from five experiments. Error bars represent standard error.

Fig. 2.

Fig. 2.

Aggregate toxicity accompanies localization of G93A to the JUNQ. (A) Twenty-four hours posttransfection, cells were sorted according to fluorescent protein expression level, allowed to recover, and monitored for viability using an MTS assay. Htt-SOD1G93A is significantly less toxic than G93A. (B) G93A inclusions are JUNQs. G93A colocalizes with VHL but not with Htt. (C) A fusion protein of Htt-SOD1G93A-YFP. (D) Htt-SOD1G93A colocalizes exclusively with Htt (Upper) and is absent from the JUNQ (Lower).

Fig. 3.

Fig. 3.

The JUNQ is a site of cellular quality control. (A) Hsp70-YFP localizes to the JUNQ but not the Htt inclusion. (B) An enrichment of the proteasome is seen in the JUNQ but not in the Htt inclusion. Proteasomes were visualized with LMP2-YFP. (C) p97 colocalizes with VHL in JUNQ but not with Htt. (D) Ubiquitin colocalizes with VHL in the JUNQ. (E) Immunohistochemical analysis of spinal cord sections from an fALS patient harboring a mutation in SOD1(D124V) reveals motor neurons containing inclusions that costain with SOD1 and Hsp70 (Left), and for ubiquitin and Hsp70 (Right). Single staining is shown in F and control (non-ALS) spinal cord sections in G.

Fig. 4.

Fig. 4.

G93A aggregates reduce VHL and Hsp70 mobility in the JUNQ. (A) FRAP (n = 10) reveals that toxic proteins in the JUNQ are less mobile than nontoxic JUNQ substrates. Recovery of G93A in JUNQ was higher than of Htt, but lower than that of VHL. *P ≤ 0.0019; **P ≤ 0.0248; ***P ≤ 0.0044. (B) Toxic proteins in JUNQ decrease the mobility of VHL. JUNQ inclusions containing VHL were analyzed by FRAP in the presence of G93A. Htt is localized to the IPOD and thus has no effect on VHL mobility in the JUNQ. Htt-SOD1G93A does not have any effect on VHL mobility in the JUNQ. *P ≤ 0.001; **P ≤ 0.0067; ***P ≤ 0.0017. (C) G93A aggregates impair the mobility of Hsp70 in the JUNQ. *P ≤ 0.0001; **P ≤ 0.0002; ***P ≤ 0.024. (D) G93A in the JUNQ leads to sequestration of Hsp70. Exchange of Hsp70 between the cytosol and the JUNQ decreased when G93A but not VHL accumulated there.

Fig. 5.

Fig. 5.

G93A inhibits degradation of quality control substrates in the JUNQ. (A) G93A in the JUNQ but not Htt aggregated separately inhibit the degradation of VHL. Cells stably expressing VHL-GFP were transfected with G93A-CFP or Htt-mRFP, and GFP fluorescence was quantified by FACS. Cells were treated with proteasome inhibitor (2 μM MG132) for 6 h. (B) Neither G93A nor Htt directly inhibit the proteasome. Fluorescence levels in cells stably expressing Ub-R-YFP were analyzed as above. (C) Unlike G93A, Htt-SOD1G93A does not inhibit VHL degradation. (D) Model of proteasome function in the JUNQ. When toxic aggregates sequester Hsp70, quality control substrates fail to be delivered to JUNQ proteasomes, which are then stained by MVB151. (E) GFP-expressing cells show diffuse MVB151 staining. (F) VHL JUNQs inclusions do not stain with MVB151. (G) G93A JUNQs are stained with MVB151. (H) MVB151 staining of G93A JUNQs is prevented by washing with MG132. (I) Toxic JUNQ MVB151 staining is also abolished by Hsp70, which allows for substrate delivery to proteasomes.

Fig. 6.

Fig. 6.

Toxic proteins in the JUNQ impair proteostasis. (A) Luciferase accumulation in the JUNQ was observed only if G93A was there as well (Center), indicating that cells with G93A in the JUNQ have a decreased capacity to maintain proteostasis. Fifty cells coexpressing G93A and Luciferase were monitored for IBs. (B) Hsp70 enhances the solubility of VHL in the JUNQ. Hsp70 or empty vector were cotransfected with VHL and G93A. FRAP experiments were conducted as above. *P ≤ 0.0019; **P ≤ 0.024; ***P ≤ 0.0044. (C) Hsp70 reduces the toxic effects of G93A. G93A-CFP was cotransfected with either Hsp70-YFP or SOD1wt-YFP and sorted for YFP fluorescence, and cell viability was assessed as in Fig. 1_D_. *P ≤ 0.0004. (D) Model for the relationship between toxicity and targeting to JUNQ or IPOD.

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