DNAJC7 interacts with RNA binding proteins and regulators of stress response pathways in human MNs.

(A) Schematic of co immunoprecipitation of DNAJC7 from WT MNs.

(B) WB of DNAJC7 from co-IP. Input = 4% of total protein used in co immunoprecipitation.

(C) Venn diagram: all identified proteins from each DNAJC7 IP (n=3), 88 of which persistently identified proteins from each biological replicate. Scatter plot: Gray dots represent proteins not significantly enriched in DNAJC7 IP, red dots represent proteins significantly enriched in DNAJC7 IP fraction (p<0.05) or found exclusive in DNAJC7 IP (61 proteins).

(D and E) Gene ontology of pathways overrepresented within DNAJC7 interactome, filtered by fold enrichment and ranked by number of proteins. FDR = q value.

(F and G) Plot of spectral counts of proteins found in DNAJC7 IP vs IgG IP. Values represent the mean ± standard error of the mean (SEM). Each dot represents a biological replicate (N = 3).

(H) WB of proteins co immunoprecipitated with DNAJC7. Input = 4% of total protein used in co immunoprecipitation. DNAJC7 WB: top band = DNAJC7 protein, lower band = IgG heavy chain visualized at low and high exposure. Note that IgG heavy chain does not appear in panel B because the DNAJC7 antibody was covalently linked to immunoprecipitation beads.

DNAJC7 haploinsufficiency disrupts HNRNPU solubility and target mRNA expression.

(A) Schematic of known ALS causative mutations on DNAJC7 protein marked with *

(B) Schematic of CRISPR-Cas9 knock-in of R156X mutation into healthy control iPSC line.

(C) Left: WB images of DNAJC7 proteins levels from MN lysate derived from isogenic pairs (+/+ = isogenic control, +/- = R156x mutant). Bottom: Values represent the mean ± standard error of the mean (SEM). Four experiments are represented by distinct shaped symbols. N = 4, Unpaired t test (two-tailed): **** P<0.0001.

(D) Schematic of biochemical fractionation of soluble/insoluble material from MN lysates followed by WB analysis. Red labels represent proteins whose solubility change in R156X mutant MNs.

(E) Top: WB images of soluble and insoluble HNRNPU proteins levels from MN lysate derived from isogenic pairs. Bottom: Values represent the mean ± min/max. Experiments are represented by distinct shaped symbols. N = 6, Unpaired t test (two-tailed): **** P<0.0001.

(F) Volcano plot of RNA Sequencing data from R156X vs control. Red or blue colored dots represent transcript significantly upregulated or downregulated, respectively (n = 3, FDR < 0.05). Yellow dots represent transcriptional targets of HNRNPU.

(G) Gene set enrichment of HNRNPU eCLIP targets. Kolmogorov–Smirnov test: N = 3, NES = -1.56, FDR = 0.008.

DNAJC7 haploinsufficiency sensitizes MNs to proteotoxic stress.

(A) Schematic of stress pathways DNAJC7 may influence.

(B) Schematic of live imaging experiments following proteotoxic stress induction.

(C) Representative images of a dying MN co-labeled with SYN1-GFP (green) and propidium iodide (red) and merged. Scale bar, 20 μm.

(D) Kaplan-Meier survival curve of MNs survival following MG132 or DMSO control. 150 cells tracked per condition, Mantel-Cox log-rank test: p<0.0001.

(E) Kaplan-Meier survival curve of MNs survival following Brefeldin or DMSO control. 150 cells tracked per condition, Mantel-Cox log-rank test: p=0.8085.

(F) Kaplan-Meier survival curve of MNs survival following Ganetespib or DMSO control. 129 cells tracked per condition, Mantel-Cox log-rank test: p=0.0001.

(G and H) Schematic of MG132 treatment in MNs followed by tandem mass tag quantitative proteomics.

(I) Violin plot of the average TMT intensities of all proteins for each condition. Two-way ANOVA: no significance

(J) Shrinkage plots of protein level changes in MNs following MG132 treatment for R156X-MNs (right) and isogenic controls (left). Red and blue dots represent significantly increased or decreased proteins, respectively. N = 4 independent differentiations.

(K) Left: bar graph displaying number of proteins changed in response to MG132 for each genotype. Right: Venn diagram showing lack of overlap of altered proteins.

(L) GO enrichment of top reactome pathways upregulated in isogenic MNs following MG132 ranked based on # of genes in pathway. FDR = q value indicated in each bar.

DNAJC7 regulates stress-induced activation of HSF1 and activation of HSF1 rescues the sensitivity of mutant DNAJC7-MN to stress.

(A) Shrinkage plot of basal relative protein abundance between DNAJC7-MNs and isogenic controls. Purple dots represent HSF1 transcriptional targets.

(B) Scatter plot of gene set enrichment analysis of “stress” terms. Colored or gray dots represent significantly (FDR < 0.05) or not significantly (FDR > 0.05) enriched terms, respectively.

(C) GSEA plot of HSF1 targets within the basal proteomic dataset. Kolmogorov–Smirnov test: NES = -1.88, FDR < 1E-7.

(D) Top: WB image of CRYZ and HSPB1 proteins levels from MN lysate derived from isogenic pairs at baseline. Bottom: Values represent the mean ± standard error of the mean (SEM). Experiments are represented by distinct shaped symbols. N = 3, Unpaired t test (two-tailed): CRYZ ***P=0.0002, HSPB1 ****P<0.0001.

(E) Schematic of HSF1 activity assessment in MNs following MG132.

(F) Schematic of HSF1 activation pathway.

(G) Left: WB image of phospho-HSF1 (Ser326) and total HSF1 protein levels in MNs with and without MG132. Right: Values represent ratio of pHSF1/total HSF1 ± standard error of the mean (SEM). Experiments are represented by distinct shaped symbols. N = 3, Two-way ANOVA: *p<0.05, **p<0.01, ***p<0.001.

(H) RT-qPCR quantification of HSP70 transcript levels, fold change relative to WT + DMSO control. Experiments are represented by distinct shaped symbols. N = 2-3, Two-way ANOVA: *p<0.05.

(I) Kaplan-Meier survival curve of MNs survival following 10 μM or 20 μM DTHIB. 144 cells tracked per condition, Mantel-Cox log-rank test: WT vs R156X (10 μM) p<0.0001, WT vs R156X (20 μM) p<0.0001.

(J) Kaplan-Meier survival curve of MNs survival following MG132 with ORF-LV or HSF1-LV. 150 cells tracked per condition, Mantel-Cox log-rank test: WT+ORF vs R156X+ORF p<0.0001, R156X+HSF1 vs R156X+ORF p<0.0001, R156X+HSF1 vs WT+HSF1 p=0.177.

(K) Schematic of DNAJC7-MN increased stress resistance following HSF1 activation and selective degeneration after HSF1 inhibition.

(L) Schematic of single cell RNA-Seq dataset from Pineda et al.,46 used for analysis.

(M) Left: scatter plot of gene set enrichment analysis of HSF1 Targets (MSigDB#: M19734) by cell type. Each dot represents a different cell type. Colored or gray dots represent significant (FDR < 0.05) or not significant (FDR > 0.05) in ALS vs Control samples. Inhib = Inhibitory, Ex = Excitatory, Oligo = Oligodendrocyte. Right: GSEA plots of HSF1 Targets (MSigDB#: M19734) in ALS vs Control in excitatory neurons or inhibitory. Labeled ticks indicate specific HSF1 targets also significantly reduced in DNAJC7-MNs. Kolmogorov–Smirnov test: Ex. (NES = -1.28, FDR = 0.031), Inhib. (NES = -1.41, FDR = 0.006).

(A) Schematic of 14-day small-molecular iPSC differentiation to lower motor neurons.

(B) Venn diagram of 88 proteins redundantly identified across n = 3 experiments. Of those 88, 61 proteins were significantly enriched in DNAJC7 IP or exclusively identified within the DNAJC7 IP.

(C) STRING analysis of DNAJC7 interactome. Colors correspond to relevant identified GO enrichment pathways.

DNAJC7 haploinsufficiency disrupts HNRNPU solubility and target mRNA expression.

(A) Schematic of iPSC differentiation into lower motor neurons followed by immunofluorescence (IF), WB or RNA Sequencing following maturation of 50 days in culture.

(B) Confocal images of MNs derived from DNAJC7 isogenic pair immunolabeled with anti ISL1/2, anti MAP2, and Hoechst 33342. Scale bar, 25 μm.

(C) Quantification of B, values represent the mean ± standard error of the mean (SEM). Experiments are represented by distinct shaped symbols. N = 4.

(D) Heat map of differentially expressed genes (FDR < 0.05) in RNA Seq of R156X vs isogenic control, N = 3.

(E) WB images of soluble and insoluble MATR3 and HNRNPL proteins levels from MN lysate derived from isogenic pairs.

(F and G) Non-significant gene set enrichment of MATR3 and HNRNPL eCLIP targets (Kolmogorov–Smirnov test).

DNAJC7 haploinsufficiency sensitizes MNs to proteotoxic stress.

(A) Kaplan-Meier survival curve of MNs survival following MG132 or DMSO control. Three independent differentiations separated. 50 cells per condition per experiment, Mantel-cox log-rank test: p=0.0003, p<0.0001, p=0.0002.

(B) Kaplan-Meier survival curve of MNs survival following Brefeldin or DMSO control. Three independent differentiations separated. 43 cells per condition per experiment, Mantel-cox log-rank test: p=0.9105, p=0.4659, p=0.9033.

(C) Kaplan-Meier survival curve of MNs survival following Ganetespib or DMSO control. Three independent differentiations separated. 50 cells per condition per experiment, Mantel-cox log-rank test: p=0.0137, p=0.0372, p=0.0403.

(D) Heat map of hierarchically clustered (k-means) of average relative protein abundance for each group from TMT-MS. N = 4 independent differentiations per condition.

DNAJC7 regulates stress-induced activation of HSF1 and activation of HSF1 rescues the sensitivity of mutant DNAJC7-MN to stress.

(A) GSEA of non-enriched “stress” related pathways in baseline proteomic dataset. DNA damage response (MSigDB#: M13636), unfolded protein response (MSigDB#: M5922), cellular response to oxidative stress (MSigDB#: M45123), and cellular response to chemical stress (MSigDB#: M29264).

(B) GSEA of HSF1 transcriptional targets (MSigDB#: M19734) de-enrichment in RNA transcriptional profile of DNAJC7-MNs. Kolmogorov–Smirnov test: NES = -1.36, FDR = 0.02.

(C and D) Violin plots of relative expression of HSF1 transcriptional targets (MSigDB#: M19734) as a group in human cortical (left) or spinal (right) tissue. Subtypes are categorized into Neuron, Glia, Vascular (Vasc.), or Immune cellular subgroups.

(E) Kaplan-Meier survival curve of MNs survival following MG132 with ORF-LV escalating doses of ORF-LV. 36- 50 cells tracked per condition.

(F) Superplots of relative expression of HSF1 following HSF1-LV or ORF-LV transduction. Experiments are represented by distinct shaped symbols, individual cell values plotted in background. N = 2, Sidak’s multiple comparisons test: WT vs R156X (ORF) p=0.9898, WT vs R156X (HSF1) p=0.9718.

Results of LC-MS/MS DNAJC7 IP vs IgG IP.