Overexpression of a conserved HSP40 chaperone reduces toxicity of several neurodegenerative disease proteins

Figures & data

Figure 1. Overexpression of Sis1 mitigates the effects of TDP-43 in yeast and primary rodent cortical neurons. (Adapted from [31) a. Overexpression of TDP-43 is more toxic in the presence of the [PIN⁺] prion and is less toxic in the presence of overexpressed Sis1. The growth of normalized suspensions of [PIN⁺] and [pin⁻] versions of the same yeast strain transformed with pGAL1-TDP-43-YFP (↑TDP-43) and pGAL1-SIS1 (↑SIS1), or empty control plasmids (No ↑), on plasmid selective galactose medium is shown. b. Expression of DNAJB1 reduces toxicity of TDP-43 overexpression in rodent primary cortical neurons. A cumulative risk of death plot is shown for neurons transfected with vectors shown. While DNAJB1 displays some toxicity in control neurons when overexpressed, it also reduces the risk of death in neurons expressing TDP-43. *p < 0.005; **p < 2 × 10⁻¹⁶, by Cox proportional hazards analysis. N = 725-979 neurons per condition, assembled from three separate experiments. c. TDP-43 inhibits proteolysis of cytosolic misfolded proteins and this is reversed by overexpression of Sis1. We used degradation of CG*-GFP, the mutant version of the secretory protein carboxypeptidase Y lacking its signal sequence (ΔssCPY*) tagged with EGFP as a reporter to test for ubiquitin proteasome system (UPS) activity. New protein synthesis was inhibited with cycloheximide, after which levels of CG*-GFP were determined in cell lysates harvested at the times indicated. Data for a [PIN⁺] yeast strain is shown. All cells expressed CG*-EGFP from a GAL1 plasmid and expressed TDP-43 and Sis1 as indicated. Normalized cell lysates run on SDS-PAGE were immunoblotted with anti-GFP for CG*-EGFP level and anti-Pgk1 as an internal loading control. Quantification of three blots showing normalized ratio of CG*-EGFP and Pgk1 is shown with standard error.

Figure 2. [PIN⁺] exacerbates FUS toxicity. a. [PIN⁺] exacerbates FUS toxicity. High [PIN⁺] (L1749) and isogenic [pin⁻] (L2910) cells harboring GAL1-controlled URA3 FUS-YFP (p2043), or YFP (p1752) plasmids were grown in liquid plasmid selective raffinose (2%) medium overnight. Cells were then transferred to plasmid selective Dextrose (2%) and Galactose (2%) media by serial dilution and photographed after 4–5 days. b. The presence of [PIN⁺] does not significantly enhance expression levels of FUS-YFP. Lysates of [pin⁻] or [PIN⁺] cells overexpressing FUS-YFP in 2% Galactose for 1 hour, or 24 hours were analyzed by western blot. Expression levels of FUS-YFP were detected by α-GFP (Roche), while Pgk1 was used as an internal loading control. c. Curing of [PIN⁺] which converts [PIN⁺] cells into [pin⁻] cells, relieves FUS toxicity. [PIN⁺] cells harboring FUS-YFP were grown on YPD+5 mM guanidine hydrochloride (GuHCl) plates for three passages, which causes loss of prions [36]. Two subclones of GuHCl-treated cells (middle two rows), untreated [PIN⁺] (top row) and [pin⁻] cells with FUS-YFP were transferred to plasmid selective galactose plates by serial dilution where they were allowed to grow. d. Cytoduction of [PIN⁺] into [pin⁻] cells exacerbates FUS toxicity. [PIN⁺] or [pin⁻] donor cells (respectively, L1749 and L2910) were mated on YPD with recipient [pin⁻] cells (L2599) harboring FUS-YFP. Representative cytoductants (recipients containing some cytoplasm from the donor) that became [PIN⁺] due to the [PIN⁺] donor (row 1) or remained [pin⁻] due to the [pin⁻] donor (row 2) as well as uncytoduced recipients with FUS-YFP (row 3) or YFP (row 4) are shown for comparison as a serial dilution on plasmid selective galactose plates. e. FUS-YFP forms similar aggregates in [PIN⁺] and [pin⁻] cells. Isogenic [PIN⁺] and [pin⁻] cells transformed with GAL-FUS-YFP plasmid were grown in 2% galactose medium before being imaged.

Figure 3. Excess Sis1 or DNAJB1 respectively relieve FUS toxicity in yeast and HEK cells. a. Overexpressed Sis1 rescues FUS associated toxicity. Isogenic [PIN⁺] and [pin⁻] versions of W303 yeast cells containing GAL1-controlled FUS-YFP (p2043) in addition to GAL1-controlled Sis1 (p1767) or vector (p1768), were grown overnight in plasmid selective raffinose (2%) media. Serial dilutions of cells were spotted and grown on plasmid selective non-inducing raffinose (2%) or inducing galactose (2%) plates. In this experiment [PIN⁺] and [pin⁻] cells were spotted on different plates and are not directly comparable. b. Sis1 overexpression does not enhance FUS protein levels. W303 [pin⁻] cells transformed with different plasmids were grown in plasmid selective galactose for 48 hours. Cells were then lysed and equal amounts of proteins were loaded on SDS-PAGE. Control vectors in lane 1 did not express either FUS or Sis1; FUS-YFP was overexpressed in lane 2; and FUS-YFP and Sis1 were co-overexpressed in lane 3. The blot was probed with anti-GFP (1:10,000, Roche) to detect FUS-YFP, anti-Sis1 (1:1000, Gift from E. Craig) and anti-Pgk1 (1:10,000) as an internal loading control. c. Mammalian DNAJB1 relieves FUS toxicity in human embryonic kidney (HEK) cells. HEK293T cells were transfected with plasmid(s) shown in the horizontal axis of the graph according to the manufacturer's instructions (Life technologies). Cell viability was measured with the MTT assay at 48 h post-transfection. Values represent means ± S.D. (n = 3). Empty vector (pcDNA) was used as a negative control, and eIF4A was used as the positive control since previous studies [28] demonstrated its ability to rescue FUS associated toxicity in HEK293T cells (*p < 0.05 using one way ANOVA with Dunnett's).

Figure 4. Degradation of UPS reporter protein CG*-GFP, is inhibited in yeast in the presence of FUS and overexpression of Sis1 mitigates this effect. Cells were grown in liquid plasmid selective 2% galactose medium for 48 hours to overexpress control (p2039) or FUS (p2049). In b and c GAL1-controlled Sis1 (p1767) or vector (p1768) were also expressed. Cycloheximide (0.5 or 0.7 mg/mL) was added, and cells were collected at the minutes (Min) indicated. Protein was extracted, and run on SDS-PAGE. The blots were probed with anti-GFP (1:10,000, Roche) to detect CG*-GFP levels (left) and anti-Pgk1 (1:10,000) as an internal loading control (right). Strains used were L1749 [PIN⁺] (a and b) and L2910 [pin^-] (c).

Park SK, Hong JY, Arslan F, et al. Overexpression of the essential Sis1 chaperone reduces TDP-43 effects on toxicity and proteolysis. PLoS Genet. 2017;13:e1006805. doi:10.1371/journal.pgen.1006805

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