Oligomeric amyloid structures are crucial therapeutic targets in Alzheimer's and other amyloid diseases. However, these oligomers are too small to be resolved by standard light microscopy. We have developed a simple and versatile tool to image amyloid structures by using thioflavin T without the need for covalent labeling or immunostaining. The dynamic binding of single dye molecules generates photon bursts that are used for fluorophore localization on a nanometer scale. Thus, photobleaching cannot degrade image quality, allowing for extended observation times. Super-resolution transient amyloid binding microscopy promises to directly image native amyloid by using standard probes and record amyloid dynamics over minutes to days. We imaged amyloid fibrils from multiple polypeptides, oligomeric, and fibrillar structures formed during different stages of amyloid-β aggregation, as well as the structural remodeling of amyloid-β fibrils by the compound epi-gallocatechin gallate.

Background Neuronal uptake and subsequent spread of proteopathic seeds, such as αS (alpha-synuclein), Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery participating in this process is poorly understood. One proteinopathy called multisystem proteinopathy (MSP) is associated with dominant mutations in Valosin Containing Protein (VCP). MSP patients have muscle and neuronal degeneration characterized by aggregate pathology that can include αS, Tau and TDP-43. Methods We performed a fluorescent cell sorting based genome-wide CRISPR-Cas9 screen in αS biosensors. αS and TDP-43 seeding activity under varied conditions was assessed using FRET/Flow biosensor cells or immunofluorescence for phosphorylated αS or TDP-43 in primary cultured neurons. We analyzed in vivo seeding activity by immunostaining for phosphorylated αS following intrastriatal injection of αS seeds in control or VCP disease mutation carrying mice. Results One hundred fifty-four genes were identified as suppressors of αS seeding. One suppressor, VCP when chemically or genetically inhibited increased αS seeding in cells and neurons. This was not due to an increase in αS uptake or αS protein levels. MSP-VCP mutation expression increased αS seeding in cells and neurons. Intrastriatal injection of αS preformed fibrils (PFF) into VCP-MSP mutation carrying mice increased phospho αS expression as compared to control mice. Cells stably expressing fluorescently tagged TDP-43 C-terminal fragment FRET pairs (TDP-43 biosensors) generate FRET when seeded with TDP-43 PFF but not monomeric TDP-43. VCP inhibition or MSP-VCP mutant expression increases TDP-43 seeding in TDP-43 biosensors. Similarly, treatment of neurons with TDP-43 PFFs generates high molecular weight insoluble phosphorylated TDP-43 after 5 days. This TDP-43 seed dependent increase in phosphorlyated TDP-43 is further augmented in MSP-VCP mutant expressing neurons. Conclusion Using an unbiased screen, we identified the multifunctional AAA ATPase VCP as a suppressor of αS and TDP-43 aggregate seeding in cells and neurons. VCP facilitates the clearance of damaged lysosomes via lysophagy. We propose that VCP’s surveillance of permeabilized endosomes may protect against the proteopathic spread of pathogenic protein aggregates. The spread of distinct aggregate species may dictate the pleiotropic phenotypes and pathologies in VCP associated MSP.

Desmin-associated myofibrillar myopathy (MFM) has pathologic similarities to neurodegeneration-associated protein aggregate diseases. Desmin is an abundant muscle-specific intermediate filament, and disease mutations lead to its aggregation in cells, animals, and patients. We reasoned that similar to neurodegeneration-associated proteins, desmin itself may form amyloid. Desmin peptides corresponding to putative amyloidogenic regions formed seeding-competent amyloid fibrils. Amyloid formation was increased when disease-associated mutations were made within the peptide, and this conversion was inhibited by the anti-amyloid compound epigallocatechin-gallate. Moreover, a purified desmin fragment (aa 117 to 348) containing both amyloidogenic regions formed amyloid fibrils under physiologic conditions. Desmin fragment-derived amyloid coaggregated with full-length desmin and was able to template its conversion into fibrils in vitro. Desmin amyloids were cytotoxic to myotubes and disrupted their myofibril organization compared with desmin monomer or other nondesmin amyloids. Finally, desmin fragment amyloid persisted when introduced into mouse skeletal muscle. These data suggest that desmin forms seeding-competent amyloid that is toxic to myofibers. Moreover, small molecules known to interfere with amyloid formation and propagation may have therapeutic potential in MFM.