VCP suppresses proteopathic seeding in neurons

Abstract

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.

The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds in a cellular system

“…Although even a small inhibitory effect on disaggregation may contribute to aggregate pathology in neurons over decades, the disease mutations may alternatively affect other steps during aggregate formation, including aggregate seeding and Tau degradation in cooperation with the proteasome. Indeed, an increase in intracellular aggregation is observed when VCP is inhibited in recipient cells at the time of seeding 64 or when VCP D395G is expressed in the recipient cells 21 . Note that we introduced the mutant VCP proteins in cells containing preexistent Tau aggregates to exclude a potential role of VCP in the process of aggregate seeding.…”

“…The mutant proteins, carrying a C-terminal myctag, were transiently overexpressed in Tet-TauRD-Y* cells for 24 h, and then TauRD-Y synthesis was stopped with doxycycline to observe disaggregation. Note that mutant VCP was expressed in cells containing pre-formed aggregates to exclude a potential role of VCP in aggregate seeding 64 . The myc-tagged mutant proteins were present in hexamers that migrated on native PAGE like WT VCP (Supplementary Fig.…”

Network of hotspot interactions cluster tau amyloid folds

“…These concepts can be leveraged using computational protein design to generate protein sequences that can bias these ensembles towards a more defined state compatible with a single fibrillar conformation and not compatible with other conformations. Applications of these designed "restrictive" sequences may allow selective amplification of these structures, using cellular systems of tau and other protein prion-like propagation 71,[78][79][80] offering avenues to diagnose disease based on conformation in premortem samples. These ideas may further inform the capacity of seed to propagate to promote pathology thus relating macroscopic properties such as the shape of protein inclusions in human tissues to molecular properties of the fibrillar structure.…”

Disease-specific selective vulnerability and neuroimmune pathways in dementia revealed by single cell genomics

“…NFE2L1 and MAFG regulons both included genes involved in iron sequestration ( FTH1 and FTL ), and the protective ALS/bvFTD gene SQSTM1 (Figure 7B, C, Table S6). In addition, the MAFG regulon includes VCP , another ALS/bvFTD disease gene that reduces tau proteopathic seeding (Zhu et al, 2022), the PD risk gene SCNA , and genes involved in the regulation of selective autophagy, proteostasis, RNA stabilization, and synaptic vesicle exocytosis (Figure 7D). We confirmed that VCP expression is positively correlated with MAFG expression across EX in the insular cortex (Figure 7E).…”