CRISPR screen for protein inclusion formation uncovers a role for SRRD in the regulation of intermediate filament dynamics and aggresome assembly

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.


Figure A. Validation of mClover3-TDP-43
ΔNLS aggregation reporter and screen replicate data.A) Percentage of cells harboring PulSA sensitive TDP-43 foci when C-and N-terminally tagged TDP-43 with WT sequence, NLS mutation, an ALS associated Q331K mutation, or combination of NLS and Q331K mutation are expressed in HEK 293Ts.B) Cell count data of 293Ts 24 hours after transfection.Cells were transfected with N-terminally tagged TDP-43ΔNLS, TDP-43Q331K, TDP-43ΔNLS-Q331K, or mClover3 control, split in duplicate, with one replicate receiving doxycycline to induce transgene expression.C) Mean p-value of replicate screen results plotted against each other indicating reproducible hits that increase (left) or decrease (right) TDP-43 aggregation.D) Entire STRING interaction network of top 100 hits enriched in TDP-43 "more aggregation" dataset after differential expression analysis.Clusters generated with MCL clustering and excludes proteins with no known interactions.

Figure B .
Figure B. Arrayed expression validation of top hits that modify TDP-43 aggregation.A) Histograms of total TDP-43 expression for AAV5 non-targeting control and two sgRNAs targeting UBE3C, BTNL9, and SRRD, respectively.B) Fraction of cells with TDP-43 foci (y-axis) in each TDP-43 expression bin (x-axis) for AAV5 non-targeting control and two sgRNAs targeting UBE3C, BTNL9, and SRRD, respectively.

Figure C .
Figure C. Characterization of SRRD KO lines and SRRD expression pattern.A) Sanger sequencing of SRRD clonal KO A proximal to sgRNA spacer targeting sequence.Two sequenced alleles compared to wild type sequence with corresponding codon sequence beneath.B) Barplot of % of cells harboring TDP-43 aggregates in WT and SRRD KO lines.n= 3 replicates, one way anova with Tukey HSD test.C) Fraction of cells with TDP-43 aggregates (yaxis) in each TDP-43 expression bin (x-axis) for WT and SRRD KO cells.D) Histogram of total TDP-43 expression for WT and SRRD KO lines.E) Barplot of % of cells harboring TDP-43 aggregates in AAV5 targeting controls and SRRD CRISPRi KD lines.n= 3 replicates, one way anova with Tukey HSD test.F) Fraction of cells with TDP-43 aggregates (y-axis) in each TDP-43 expression bin (x-axis) for AAV5 targeting control and two SRRD CRISPRi lines cells.G) Histogram of total TDP-43 expression for AAV5 targeting control and SRRD CRISPRi lines.H) Confocal images of HEK 293Ts stably expressing SRRD-HA, stained for HA and mitochondria (top) and CANX (bottom).

Figure D .
Figure D. Effect of SRRD KO on other aggregation prone proteins.A) Barplot of % of cells harboring mClover3-FUS-P525L aggregates as detected by PulSA in WT and SRRD KO lines.n= 3 replicates, one way anova with Tukey HSD test.And Histogram of total FUS expression for WT and SRRD KO lines.B) Results from an unpublished screen for FUS aggregation showing that SRRD was also a top negative hit in this screen.C) Aggregation of mHTT(Q97) N terminal fragment.Interestingly, SRRD KO does not reduce aggregation of mHTT, the small increase in KO B is explained by increased expression in this line (see right panel).

Figure F .
Figure F. Validation of APEX2 biotinylation activity.A) Western blots probing for streptavidin (biotin) and tagged fusion protein (V5 top blot, FLAG bottom blot) under different combination of APEX2 expression, H2O2 treatment, and Biotin-Phenol treatment.B) Staining for APEX2 fusion protein (V5 top panel, FLAG bottom panel) and streptavidin (probing for biotin) in conditions with or without H2O2 treatment.

Figure G .
Figure G. SRRD interaction network under homeostatic conditions.A) STRING interaction network of top proteins in proximity to SRRD-APEX2 under homeostatic conditions, log fold change cutoff of -1, adjusted p-value cutoff -log10(0.05).Clusters generated with MCL clustering and excludes proteins with no known interactions.

Figure H .
Figure H. VIM organization in SRRD KO cells.A) Representative images of WT, SRRD clonal KO, and SRRD rescue 293Ts stained for VIM.B) Percentage of PulSA detected cells TDP-43 aggregates after 48hr treatment with multiple concentrations of the intermediate filament inhibitor compared to DMSO control.n= 3 replicates, one way anova with Tukey HSD test.

Figure I .
Figure I. SRRD KO depleted protein network.A) STRING interaction network of top depleted proteins, log fold change cutoff of -1, adjusted p-value cutoff -log10(0.05), in SRRD KO quantitative proteomics dataset.Clusters generated with MCL clustering and excludes proteins with no known interactions.

Figure J .
Figure J. NGN2 neuron levels of INA and NEFM.A)Two western blots and B) quantification of NEFM and INA levels in WT, non-targeting (NTC) sgRNA control, and two SRRD CRISPRi NGN2 neuron lines (S2 & S4).n= 2 replicates, one way anova with Tukey HSD test.

Figure
Figure K. SRRD KO reduced VIM cage formation around aggresomes.A) WT, clonal SRRD KO, and SRRD rescue 293Ts treated with 5μM MG132 and stained for VIM and HDAC6.Zoom images of figure 4A.B) WT, clonal SRRD KO, and SRRD rescue 293Ts transfected with mClover3-TDP-43 ΔNLS and stained for VIM.Zoom images of figure 4C.

Figure L .
Figure L. HDAC6 colocalization to TDP-43 aggresomes and altered SQSTM1 in unstressed SRRD KO cells.A) Confocal images of indicated cell lines transfected with mClover3-TDP-43 ΔNLS and stained for HDAC6.B) quantification of TDP-43 ΔNLS and HDCA6 colocalization, with one way anova with Tukey HSD test.C) 60x and zoomed confocal images of indicated cell lines stained for SQSTM1.D) Quantification of the number of large p62 puncta in unstressed WT, SRRD KO, and SRRD rescue cells.

Figure M .
Figure M. SRRD interaction network under proteotoxic stress conditions.A) STRING interaction network of top proteins in proximity to SRRD-APEX2 treated with MG132, log fold change cutoff of -1, adjusted p-value cutoff -log10(0.05).Clusters generated with MCL clustering and excludes proteins with no known interactions.

Figure O .
Figure O. SRRD directly interacts with aggregation prone proteins.A) Yeast growth over time, measured by optical density.Yeast expressed alpha-synuclein alone (blue line), in combination with HSP104-A503S disaggregase (red line), or in combination with SRRD (green line).And area under the curve of each growth curve.Adjusted p-values: Hsp104-A503S rescue of alpha-synuclein toxicity = 0.00217; SRRD rescue of alpha-synuclein toxicity = 0.00725.B) Yeast spotting assay where indicated transgene expression is off under glucose, on under galactose.C) Quantification and representative images of the number of TDP-43-YFP puncta per yeast cell when expressed with empty vector or SRRD.D) SRRD amino acid sequence.E) 293Ts stably expressing -NTD-mRuby3 transfected with mClover3-TDP-43 ΔNLS.