Sequestration of TDP-43^216-414 Aggregates by Cytoplasmic Expression of the proSAAS Chaperone

Abstract

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.

Aptamer Reduces Aggregation of Mutant Huntingtin and Rescues Proteostasis Network in Non-Neuronal and Neuronal Cells

“…The effect of this aptamer on aggregation of mutant huntingtin in HEK293 cells was investigated. First, HEK293 cells were co-transfected with pEGFP- Q23 (containing EGFP fused to codons 8–57 inclusive of exon 1 of huntingtin, numbered according to 23Q, Figure S1A) − and pSUPER- pH1-aptamer . The shorter length of huntingtin stretch in pEGFP- Q23 has been shown to be more suitable for transient transfection studies than longer truncations .…”

Tau liquid–liquid phase separation is modulated by the Ca2+‐switched chaperone activity of the S100B protein

“…In the context of neurodegenerative diseases, the importance of biological regulation of LLPS has gained increasing interest given that the formation of liquid droplets of proteins such as tau contributes to increase local protein concentration favoring the formation of rogue oligomers and amyloid fibrils (Boyko et al, 2019; Rai et al, 2021; Wegmann et al, 2018). In this respect, several chaperones have been implicated as important players in the regulation of LLPS by disease‐related proteins, such as TDP‐43, FUS and tau, among others (Darling et al, 2021; Liu et al, 2020; Peinado et al, 2022; Tittelmeier et al, 2020).…”

Structural Variations of Prions and Prion-Like Proteins Associated with Neurodegeneration

“…The tau protein aggregated is seen as neurofibrillary tangles (NFTs), groupings of insoluble ptau, that eventually spread throughout the brain, in a manner similar to PrP Sc [5]. The spread of infectious proteins via aggregation may also be influenced by the presence of chaperones, as inhibition of chaperone proteins may enhance aggregation, while enhancement may be a potential way to prevent the spread of infectious protein aggregates in a homeostasis-maintaining manner [46,47]. Such as the chaperone proSAAS that is neuronally expressed and its function in blocking Aβ undergoing fibrillation [47] or the overexpression of Rnq1 being toxic to [RNQ + ] prions toxicity, thus preventing the conversion of other proteins into amyloid species [42].…”