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, 35 (42), 14286-306

Loss of RAD-23 Protects Against Models of Motor Neuron Disease by Enhancing Mutant Protein Clearance

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Loss of RAD-23 Protects Against Models of Motor Neuron Disease by Enhancing Mutant Protein Clearance

Angela M Jablonski et al. J Neurosci.

Abstract

Misfolded proteins accumulate and aggregate in neurodegenerative disease. The existence of these deposits reflects a derangement in the protein homeostasis machinery. Using a candidate gene screen, we report that loss of RAD-23 protects against the toxicity of proteins known to aggregate in amyotrophic lateral sclerosis. Loss of RAD-23 suppresses the locomotor deficit of Caenorhabditis elegans engineered to express mutTDP-43 or mutSOD1 and also protects against aging and proteotoxic insults. Knockdown of RAD-23 is further neuroprotective against the toxicity of SOD1 and TDP-43 expression in mammalian neurons. Biochemical investigation indicates that RAD-23 modifies mutTDP-43 and mutSOD1 abundance, solubility, and turnover in association with altering the ubiquitination status of these substrates. In human amyotrophic lateral sclerosis spinal cord, we find that RAD-23 abundance is increased and RAD-23 is mislocalized within motor neurons. We propose a novel pathophysiological function for RAD-23 in the stabilization of mutated proteins that cause neurodegeneration.

Significance statement: In this work, we identify RAD-23, a component of the protein homeostasis network and nucleotide excision repair pathway, as a modifier of the toxicity of two disease-causing, misfolding-prone proteins, SOD1 and TDP-43. Reducing the abundance of RAD-23 accelerates the degradation of mutant SOD1 and TDP-43 and reduces the cellular content of the toxic species. The existence of endogenous proteins that act as "anti-chaperones" uncovers new and general targets for therapeutic intervention.

Keywords: ALS; RAD-23; aging; motor neuron disease; neurodegeneration; proteotoxicity.

Figures

Figure 1.
Figure 1.
Locomotor defects of C. elegans models of ALS and their modification by the loss of ERAD and UPS genes. A, Pronounced defects in locomotion were seen in the animals overexpressing mutSOD1, wtTDP-43, and mutTDP-43 in the nervous system in comparison with N2 (WT) (F(4,20) = 36.75, p < 0.0001). *p < 0.05 (Dunnett's multiple-comparison test). ****p < 0.001 (Dunnett's multiple-comparison test). B, Locomotion of mutTDP-43 animals in the background of various mutant ERAD and UPS alleles. Average speed of animals of indicated genotypes in a 30 s forced swimming assay in M9. Both suppressors and enhancers were found. No change, There was no significant change in the locomotor deficit of that background (p > 0.05, Student's t test). Enhancer, There was a significant decrease in the average speed of the animals (p < 0.05, Student's t test). Suppressor, There was a significant increase in the average locomotor speed of those animals (p < 0.05, Student's t test). cdc-48.1 (tm544); cdc-48.2 (tm659) mutants are synthetic lethal; therefore, the effect of both mutants together cannot be tested in this study. C, Loss of rad-23 and ufd-2 suppresses mutTDP-43 toxicity likely via independent pathways. Average speed of animals of indicated genotypes in a 30 s forced swimming assay in M9. A one-way ANOVA reveals group differences in the average speed of mutTDP-43, mutTDP-43; rad-23 (tm3690), mutTDP-43; ufd-2 (tm1380), and mutTDP-43; rad-23 (tm3690); ufd-2 (tm1380) (F(3,16) = 13.41; p = 0.0001). *p < 0.05. ****p < 0.001. D, Loss of rad-23 and ufd-3 suppresses mutTDP-43 toxicity via likely independent pathways. Average speed of animals of indicated genotypes in a 30 s forced swimming assay in M9. A one-way ANOVA reveals group differences in the average speed of mutTDP-43, mutTDP-43; rad-23 (tm3690), mutTDP-43; ufd-3 (tm2915), and mutTDP-43; rad-23 (tm3690); ufd-3 (tm2915) (F(3,16) = 60.55; p < 0.0001). *p < 0.05. **p < 0.01. ****p < 0.001.
Figure 2.
Figure 2.
Loss of rad-23 in C. elegans protects against models of ALS via an effect in the nervous system. A, Schematic of C. elegans RAD-23 gene with predicted protein shown. Lines indicate areas with insertions and/or deletions in the used mutants. B, C, Loss of rad-23 (tm2595) (B) or (tm3690) (C) causes a twofold suppression in the locomotor deficit of mutTDP-43 but has no effect on wtTDP-43 (p = 0.108 for tm2595 and p = 0.473 for tm3690). D, E, Loss of rad-23 (tm2595) (D) or (tm3690) (E) leads to a twofold suppression in the locomotor deficit of mutSOD1 animals but has no effect on the wtSOD1 animals (p = 0.615 for tm2595 and p = 0.274 for tm3690). F, Neither rad-23 allele is different from WT speed in the swimming assay. F(2,12) = 0.006579; p = 0.9934. G, mutSOD1 fed rad-23 RNAi for two generations shows no change (p = 0.7748), but mutSOD1; sid-1; Punc-119::SID-1 animals show a significant improvement in the swimming assay. H, Outcrossing the rad-23 (tm3690) allele from mutTDP-43; tm3690 leads to a rescue of the mutTDP-43 locomotor deficit. I, Overexpression of RAD-23 in the nervous system of mutTDP-43; rad-23 (tm3690) is sufficient to rescue the mutTDP-43 locomotor deficit. J, There is no change in the average locomotor speed of the mutTDP43 animals when RAD-23 is overexpressed in the nervous system using the Punc-119 promoter (F(5,24) = 1.112; p = 0.3801). *p < 0.05 (Student's t test). **p < 0.01 (Student's t test). ***p < 0.005 (Student's t test). ns, Not significant.
Figure 3.
Figure 3.
Loss of rad-23 in C. elegans protects against neurodegeneration following expression of mutTDP-43 in vivo. We generated animals with GABAergic motor neurons labeled with GFP using Punc-25::GFP in WT (N2; “control”), mutTDP-43, and mutTDP-43; rad-23 (tm3690) backgrounds. A, Loss of rad-23 reduces the percentage of C. elegans with mutTDP-43 that display broadening of axons. B, mutTDP-43 causes an increase in the number of gaps within the ventral nerve cord of animals compared with control. ***p < 0.005 (Dunnett's multiple-comparison test following one-way ANOVA). ns, Not significant. Loss of rad-23 reduces the number of ventral nerve cord gaps in the mutTDP-43 animals to WT. There is no significant difference in the number of gaps in mutTDP-43; rad-23 animals compared with control (WT). C, Representative images of WT, mutTDP-43, and mutTDP-43; rad-23 (tm3690) animals labeled with Punc-25::GFP.
Figure 4.
Figure 4.
RAD-23 protein is expressed throughout the worm, including the worm nervous system. A–C, A RAD-23 protein translational fusion with GFP driven by the endogenous rad-23 promoter (PRAD-23::RAD-23::GFP) is expressed throughout the worm in young adult animals. D, RAD-23 protein is expressed in the cell body and processes of a mechanosensory neuron (arrowhead), (E) vulva of the worm, (F) nerve cord (arrowhead), and (G) the head.
Figure 5.
Figure 5.
Loss of RAD-23 in C. elegans protects against aging decline and proteotoxicity. A–D, Biomechanical profiling of N2 (WT) and rad-23 (tm3690) animals (n ≥ 10/group). There is no difference between WT and rad-23 (tm3690) in speed (A), force (B), and power (C) at L4 through 3 d after L4. At 4 and 5 d after L4, rad-23 animals perform better in speed (A), force (B), and power (C) measurements than WT. D, Rad-23 animals show a more coordinated biomechanical profile at 5 d after L4 than WT. E, The lifespan of rad-23 animals is extended by 22.2% compared with WT on tunicamycin (p < 0.0001), but the lifespans of WT and rad-23 are identical on vehicle (p = 0.646). F, Rad-23 animals show an ∼25% lifespan extension during heat stress compared with WT (p < 0.0001). G, There are no group differences in survival among WT, wtTDP-43, mutTDP-43, wtSOD1, and mutSOD1 with increasing doses of UV irradiation (F(4,15) = 0.1285; p = 0.9697). There are group differences among WT, rad-23 (tm2595 and tm3690), and mutTDP-43 and mutSOD1 in the rad-23 (tm3690) background (F(6,21) = 2.961, p < 0.05). As predicted, loss of rad-23 (tm2595 and tm3690) causes a hypersensitivity to UV stress compared with WT animals (p < 0.001). MutTDP-43 and mutSOD1 in the loss of rad-23 background show a synthetic hypersensitivity to UV stress (p < 0.001). *p < 0.05 (Student's t test). **p < 0.01 (Student's t test). ***p < 0.005 (Student's t test). ****p < 0.001 (Student's t test).
Figure 6.
Figure 6.
Loss of rad-23 does not suppress the C. elegans mutTDP-43 locomotor deficit via known pathways. Loss of (A) png-1 (cy9), or (B) xpa-1, or (C) ercc-1 (C), or (D) csb-1 does not protect against the locomotor deficit caused by mutTDP-43 in the swimming assay (p > 0.05, Student's t test). One representative experiment is shown. Description of alleles: ok698, 913 bp deletion; tm2073, 804 bp deletion; ok2335, 1620 bp deletion; cy9, single nucleotide polymorphism. n.s, Not significant.
Figure 7.
Figure 7.
Knockdown of RAD-23 orthologs in mammalian motor neurons protects against toxicity of SOD1 or TDP-43. A–E, hR23A or hR23B miRNA knocks down endogenous target in HEK293 cells (n = 3/group) but does not knock down the expression of a cotransfected GFP plasmid. *p < 0.05. **p < 0.01. F, HSV infection of miRNA to hR23A or hR23B in mixed spinal cord cultures leads to specific knockdown of target protein compared with control. G, Infection of mixed spinal cord cultures with LacZ or an hR23A, hR23B, or control miRNA has no effect on motor neuron survival (F(3,12) = 0.8132, p = 0.3166). Knockdown of hR23A or hR23B protects against motor neuron death caused by wtSOD1 (F(3,12) = 17.39, p = 0.0001) and mutSOD1 (F(3,12) = 54.64, p < 0.0001). mutSOD1 infection causes a greater degree of motor neuron death than wtSOD1 (p = 0.0078). H, Infection of mixed spinal cord cultures with LacZ or a scrambled, hR23A, or hR23B miRNA has no effect on motor neuron survival (F(3,12) = 0.8875, p = 0.2100). Knockdown of hR23A or hR23B protects against motor neuron death caused by wtTDP-43 (F(3,12) = 16.66, p = 0.0001) and mutTDP-43 (F(3,12) = 48.07; p < 0.0001). **p < 0.01. ***p < 0.005. ****p < 0.001. ns, n.s, Not significant. MutTDP-43 infection causes a greater degree of motor neuron death than wtTDP-43 (p = 0.0255). I, Representative bright-field images of mixed spinal cord cultures infected as indicated stained for SMI-32 motor neuron marker. Coinfection of mutSOD1 cultures with HSV-miRNA to hR23A or hR23B increases motor neuron survival. J, Knockdown of hR23A or hR23B protects against motor neuron death caused by mutSOD1 (F(2,6) = 13.99, p = 0.0055) but has no effect on survival in motor neurons infected with LacZ (F(2,6) = 4.745, p = 0.0581) in cultures treated with vehicle. Knockdown of hR23B protects cultures infected with HSV-LacZ (F(2,6) = 11.82, p = 0.0083) or HSV-mutSOD1 (F(2,6) = 101.9, p < 0.0001) from toxicity of ER stress induced with tunicamycin.
Figure 8.
Figure 8.
hR23A expression is increased in the spinal cord of mutSOD1 mice at P90 and P120. A, B, Representative Western blot (A) and quantification (n = 3–5/group) (B) of hR23A expression in the spinal cord and brain of age-matched males. hR23A expression is increased in the spinal cord of mutSOD1 mice compared with WT (C57BL6) at P90 and P120, but not at P60. C, D, Representative Western blot (C) and quantification (n = 3–5/group) (D) of hR23B expression in the spinal cord and brain of age-matched males. There is no change in hR23B expression in the spinal cord or brain at P60, P90, or P120. *p < 0.05 (Student's t test). **p < 0.01 (Student's t test).
Figure 9.
Figure 9.
Manipulations of RAD-23 expression change TDP-43 and SOD1 abundance and solubility. A, Representative Western blots of the total amount of wtTDP-43 and mutTDP-43 following overexpression or knockdown of hR23A or hR23B. B, The quantification of the amount of total wtTDP-43 or mutTDP-43 following overexpression or knockdown of hR23A or hR23B. There is an ∼90% decrease (p < 0.005) in the abundance of mutTDP-43 following knockdown of hR23A or hR23B. C, There is no change in the amount of hTDP-43 mRNA normalized to actin in HEK293 cells following hR23A or hR23B knockdown (n = 3/group). hTDP-43 mRNA from untransfected cells was subtracted from all values. D, Representative Western blots of wtTDP-43 or mutTDP-43 expression for the indicated time points following CHX treatment with either a control or hR23A miRNA. F(2,8) = 0.1380; p = 0.8732. E, Quantification (n = 4 independent experiments) of percentage of starting wtTDP-43 or mutTDP-43 remaining. F, Representative Western blots of wtSOD1 or mutSOD1 expression for the indicated time points following CHX with either a control or hR23A miRNA. G, Quantification (n = 3 independent experiments) of percentage of starting wtSOD1 or mutSOD1 remaining. H, I, Knockdown of hR23A further reduces percentage of starting mutTDP-43 remaining after 270 min of CHX (n = 3 independent experiments). *p < 0.5. H, Representative Western blot and (I) quantification of mutTDP-43 remaining after 270 min of CHX in the presence of the indicated miRNA. J, Western blot image; hR23A miRNA knocks down endogenous hR23A compared with control miRNA but does not knock down RNAi-resistant hR23A (hR23Ares) cDNA. hR23B miRNA knocks down endogenous hR23B compared with control miRNA but does not knock down RNAi-resistant hR23B (hR23Bres) cDNA. K, Cotransfection of an hR23A or hR23B miRNA dramatically decreases total mutTDP-43 protein compared with control. This is blocked by cotransfection with hR23Ares or hR23Bres cDNA. L, Representative Western blot of CPY*-GFP expression for the indicated time points following CHX treatment with indicated miRNA. *p < 0.05 (Student's t test). **p < 0.01 (Student's t test).
Figure 10.
Figure 10.
Reducing R23 abundance accelerates SOD1 and TDP-43 turnover in MEFs and primary spinal cord neurons. A, Amount of mutTDP-43 remaining is reduced after 180 min of CHX in hR23A- and hR23B-null MEFs compared with WT. B, WT or hR23B-null (hR23B−/−) MEFs were cotransfected with mutTDP-43 construct and LacZ or full-length (FL) hR23B construct. Media was changed 24 h later, and cells were replated at equal densities another 24 h later. CHX was then added 24 h after replating for the indicated periods of time. Samples were processed for Western blot and probed with myc and actin antibodies. C, Representative Western blot of wtSOD1 and mutSOD1 abundance in neurons after 0 and 180 min of CHX. Mixed rat spinal cord culture neurons were infected with HSV to express wtSOD1 or mutSOD1 along with HSV expressing the hR23A, hR23B, or control miRNA. D, Representative Western blot of wtTDP-43 and mutTDP-43 abundance in neurons after 0 and 180 min of CHX. Mixed rat spinal cord culture neurons were infected with HSV to express wtTDP-43 or mutTDP-43 along with HSV expressing the hR23A, hR23B, or control miRNA.
Figure 11.
Figure 11.
Inhibition of the proteasome and autophagy blocks the enhanced clearance of mutTDP-43 by loss of RAD-23. A, Timeline of CHX experiment performed with inhibitors. HEK293 cells were transfected with mutTDP-43 and hR23A, hR23B, or control miRNA. CHX was added for 4.5 h and indicated drug was added 30 min after CHX. B, Representative Western blots for +hR23B miRNA experiment. Treatment with MG-132, epoxomicin, or 3-MA blocks the clearance of mutTDP-43 seen after 270 min of CHX treatment in the vehicle (DMSO) group. C, Quantification of Western blots. Treatment with MG-132, epoxomicin (“epox”), and 3-MA blocked the turnover of mut-TDP43 by ∼25%. n = 5 independent experiments for +scrambled miRNA; n = 3 independent experiments for +hR23A miRNA; n = 3 independent experiments for +hR23B miRNA. F(8,20) = 3.156; p = 0.0176. *p < 0.05. **p < 0.01. D, Total polyubiquitinated (polyUb) substrate load in indicated MEF lines following −MG-132 (DMSO) or +MG-132 treatment for 12 h. MG-132 induction ratio: +MG-132/−MG-132 (all normalized to actin). E, F, Representative Western blot of polyUb-mutTDP-43 following immunoprecipitation (IP) of myc-wtTDP-43 (E) or myc-mutTDP-43 (F) in HEK293 cells cotransfected with hR23A, hR23B, or control miRNA. Knockdown of hR23A or hR23B inhibits the ubiquitination of mutTDP-43 but has no effect on the ubiquitination status of wtTDP-43. G, Representative Western blot showing total polyUb-load in HEK293 cells transfected with mutTDP-43 and indicated miRNA. Cells were treated with DMSO (−MG-132) or MG-132 (+MG-132) 12 h before cell lysis as indicated. H, Myc-mutTDP-43 was immunoprecipitated and immunoblotted for ubiquitin and myc; representative Western blot is shown. I, Quantification (n = 3 independent experiments) of Western blots shown in H. Data were normalized to average +control miRNA condition. There is an increase in ubiquitinated mutTDP-43 and its flux through the proteasome following RAD-23 knockdown.
Figure 12.
Figure 12.
Loss of rad-23 reduces TDP-43 and SOD1 insolubility. A, Representative Western blot of hTDP-43 protein abundance following a sequential detergent extraction of C. elegans overexpressing mutTDP-43 in WT or rad-23 backgrounds. Loss of rad-23 causes a shift of TDP-43 to a more soluble (TX) fraction over insoluble (SARK, UREA) fractions. B, There is a decrease of phospho-TDP-43 in the loss of rad-23 background in soluble and insoluble fractions in C. elegans. C, Total wtTDP-43 levels are indistinguishable in the loss of rad-23 (tm3690) background compared with the control WT TDP-43 background in C. elegans. D, Representative Western blot showing that there is an increase in the abundance of insoluble mutTDP-43 in HEK293 cells following cotransfection with the hR23A cDNA. E, qPCR (n = 3/group) measuring hTDP-43 mRNA abundance normalized to tba-1 in indicated C. elegans strains. #Undetected. **p < 0.01. ***p < 0.01. F, Rad-23 (tm3690) and ufd-2 (tm1380) cause an additive effect in reducing mutTDP-43 and phosophorylated-TDP43 abundance in C. elegans. G, H, FRAP on mutSOD1-YFP aggregates within the C. elegans nervous system. mutSOD1-YFP has a higher rebound with loss of rad-23 (tm3690). G, Representative images of FRAP assay. H, Quantification of FRAP (n = 14/group). *p < 0.05. Data are normalized to starting fluorescence intensity.
Figure 13.
Figure 13.
hR23A and hR23B are aberrantly expressed in human ALS tissue. A, B, There is mislocalization of hR23A and hR23B in motor neurons of the spinal cord in ALS cases. A, Representative images of motor neurons within the gray matter of spinal cord from control and ALS cases. Insets within ALS Case 2, Granular cytoplasmic and nuclear staining often found in ALS cases. B, Distribution of hR23A and hR23B cytoplasm and nuclear staining found in control (n = 9) and ALS cases (n = 12). C, There is an increase in hR23A and hR23B protein expression in ALS (n = 5) spinal cord sections compared with controls (n = 5). **p < 0.01 (Student's t test). D, Proposed model. Loss of rad-23 destabilizes aggregates, allowing them to become more soluble and more easily degraded.

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