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.

Tuning Hsp104 specificity to selectively detoxify α-synuclein.

Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity and can have unfavorable off-target effects. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress α-synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, α-synuclein-specific Hsp104 variants emerge that mitigate α-synuclein toxicity via distinct ATPase-dependent mechanisms involving α-synuclein disaggregation or detoxification of soluble α-synuclein conformers. Importantly, both types of α-synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinson's disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.

Sequestration of TDP-43216-414 Aggregates by Cytoplasmic Expression of the proSAAS Chaperone.

As neurons age, protein homeostasis becomes less efficient, resulting in misfolding and aggregation. Chaperone proteins perform vital functions in the maintenance of cellular proteostasis, and chaperone-based therapies that promote sequestration of toxic aggregates may prove useful in blocking the development of neurodegenerative disease. We previously demonstrated that proSAAS, a small secreted neuronal protein, exhibits potent chaperone activity against protein aggregation in vitro and blocks the cytotoxic effects of amyloid and synuclein oligomers in cell culture systems. We now examine whether cytoplasmic expression of proSAAS results in interactions with protein aggregates in this cellular compartment. We report that expression of proSAAS within the cytoplasm generates dense, membraneless 2 µm proSAAS spheres which progressively fuse to form larger spheres, suggesting liquid droplet-like properties. ProSAAS spheres selectively accumulate a C-terminally truncated fluorescently tagged form of TDP-43, initiating its cellular redistribution; these TDP-43-containing spheres also exhibit dynamic fusion. Efficient encapsulation of TDP-43 into proSAAS spheres is driven by its C-terminal prion-like domain; spheres must be formed for sequestration to occur. Three proSAAS sequences, a predicted coiled-coil, a conserved region (residues 158-169), and the positively charged sequence 181-185, are all required for proSAAS to form spheres able to encapsulate TDP-43 aggregates. Substitution of lysines for arginines in the 181-185 sequence results in nuclear translocation of proSAAS and encapsulation of nuclear-localized TDP-43216-414. As a functional output, we demonstrate that proSAAS expression results in cytoprotection against full-length TDP-43 toxicity in yeast. We conclude that proSAAS can act as a functional holdase for TDP-43 via this phase-separation property, representing a cytoprotectant whose unusual biochemical properties can potentially be exploited in the design of therapeutic molecules.

Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes.

We describe a multiplex genome engineering technology in Saccharomyces cerevisiae based on annealing synthetic oligonucleotides at the lagging strand of DNA replication. The mechanism is independent of Rad51-directed homologous recombination and avoids the creation of double-strand DNA breaks, enabling precise chromosome modifications at single base-pair resolution with an efficiency of >40%, without unintended mutagenic changes at the targeted genetic loci. We observed the simultaneous incorporation of up to 12 oligonucleotides with as many as 60 targeted mutations in one transformation. Iterative transformations of a complex pool of oligonucleotides rapidly produced large combinatorial genomic diversity >105. This method was used to diversify a heterologous ß-carotene biosynthetic pathway that produced genetic variants with precise mutations in promoters, genes, and terminators, leading to altered carotenoid levels. Our approach of engineering the conserved processes of DNA replication, repair, and recombination could be automated and establishes a general strategy for multiplex combinatorial genome engineering in eukaryotes.