Biology subjects

Kashmer, O. M.

Publications and source records attributed to Kashmer, O. M..

4 recordsLinked to original sources

bioRxiv2023

VCP increases or decreases tau seeding using specific cofactors

BackgroundNeurodegenerative tauopathies may progress based on seeding by pathological tau assemblies, whereby an aggregate is released from one cell, gains entry to an adjacent or connected cell, and serves as a specific template for its own replication in the cytoplasm. In vitro seeding reactions typically take days, yet seeding into the complex cytoplasmic milieu can happen within hours. A cellular machinery might regulate this process, but potential players are unknown. MethodsWe used proximity labeling to identify factors that control seed amplification. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity upon seeded intracellular tau aggregation. We identified valosin containing protein (VCP/p97) 5h after seeding. Mutations in VCP underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We utilized tau biosensors, a cellular model for tau aggregation, to study the effects of VCP on tau seeding. ResultsVCP knockdown reduced tau seeding. However, distinct chemical inhibitors of VCP and the proteasome had opposing effects on aggregation, but only when given <8h of seed exposure. ML-240 increased seeding efficiency [~]40x, whereas NMS-873 decreased seeding efficiency by 50%, and MG132 increased seeding [~]10x. We screened VCP co-factors in HEK293 biosensor cells by genetic knockout or knockdown. Reduction of ATXN3, NSFL1C, UBE4B, NGLY1, and OTUB1 decreased tau seeding, as did NPLOC4, which also uniquely increased soluble tau levels. Reduction of FAF2 and UBXN6 increased tau seeding. ConclusionsVCP uses distinct cofactors to determine seed replication efficiency, consistent with a dedicated cytoplasmic processing complex that directs seeds towards dissolution vs. amplification.

cell biology↗

bioRxiv2022

RNA induces unique tau strains and stabilizes Alzheimer disease seeds

Tau aggregation causes neurodegenerative tauopathies, and trans-cellular propagation of tau assemblies of unique structure, i.e. strains, may underlie the diversity of these disorders. Polyanions have been reported to induce tau aggregation in vitro, but the precise trigger to convert tau from an inert to a seed-competent form in disease states is unknown. RNA triggers tau fibril formation in vitro and has been observed in association with neurofibrillary tangles in human brain. We tested whether RNA exerts sequence-specific effects on tau assembly and strain formation. Three RNA homopolymers, polyA, polyU, and polyC all bound tau, but only polyA triggered seed and fibril formation. PolyA:tau seeds and fibrils were sensitive to RNase. The origin of RNA influenced the ability of tau to adopt a structure that would form stable strains. Human RNA potently induced tau seed formation and created tau conformations that preferentially formed stable strains in a HEK293T cell model, whereas other inducers produced strains that sectored. Finally, we found that soluble, but not insoluble, seeds from Alzheimers disease (AD) brain were sensitive to RNase. Thus, RNA specifically induces stable tau strains, and may trigger the formation of dominant pathological assemblies that propagate in AD, and possibly other tauopathies.

biochemistry↗

bioRxiv2022

Dual fates of exogenous tau seeds: lysosomal clearance vs. cytoplasmic amplification

Tau assembly propagation from the extracellular to intracellular space of a cell may underlie neurodegenerative tauopathies. The first step involves tau binding to heparan sulfate proteoglycans on the cell surface, followed by macropinocytosis. Pathological tau assemblies are thought to exit the vesicular compartment as "seeds" for replication in the cytoplasm. Tau uptake is highly efficient, but only [~]1-10% of cells that take up aggregates exhibit seeding. To investigate the basis for this observation, we used fluorescently tagged full-length (FL) tau fibrils added to native U2OS cells, and "biosensor" cells expressing FL tau or repeat domain fused to mClover (Clo). FL tau-Clo bound tubulin, but seeds triggered its aggregation in multiple locations simultaneously in the cytoplasm, generally independent of visible exogenous aggregates. Most exogenous tau trafficked to the lysosome, but imaging revealed a small percentage that slowly and steadily accumulated in the cytosol. Intracellular expression of Gal3-mRuby, which binds intravesicular galactosides and forms puncta upon vesicle rupture, revealed no evidence of vesicle damage following tau exposure. In fact, most seeded cells had no evidence of lysosome rupture. However, live cell imaging indicated that cells with pre-existing Gal3-positive puncta exhibited seeding at a slightly higher rate than the general population, indicating a potential role for vesicle instability as a predisposing factor. Clearance of tau seeds occurred rapidly in both vesicular and cytosolic fractions. Bafilomycin inhibited vesicular clearance, whereas MG132 inhibited cytosolic clearance. Tau seeds that enter the cell thus have at least two fates: lysosomal clearance that degrades most tau, and entry into the cytosol, where seeds replicate, and are cleared by the proteasome.

cell biology↗

bioRxiv2018

Tau monomer encodes strains

Tauopathies have diverse presentation, progression, and neuropathology. They are linked to tau prion strains, self-replicating assemblies of unique quaternary conformation. Strains can be propagated indefinitely in cultured cells, and induce unique patterns of transmissible neuropathology upon inoculation into mice. Aggregates from a single strain reproduce only that strain upon re-inoculation into cells or mice. DS9 and DS10 cell lines propagate distinct synthetic strains. Surprisingly, DS9 monomer inoculated into naive cells encoded an identical \"sub-strain,\" whereas DS10 monomer encoded multiple sub-strains. Sub-strains produced distinct pathology upon inoculation into a tauopathy mouse model (PS19). Brain-derived tau monomer from an Alzheimers brain encoded a single strain. Monomer from a corticobasal degeneration brain encoded three sub-strains in which monomer from each encoded all three upon re-inoculation into cells. Tau monomer thus adopts multiple, stable seed-competent conformations, each of which encodes a limited number of strains. This provides insights into the origins of distinct tauopathies.

biochemistry↗