TY - JOUR
T1 - VCP suppresses proteopathic seeding in neurons
AU - Zhu, Jiang
AU - Pittman, Sara
AU - Dhavale, Dhruva
AU - French, Rachel
AU - Patterson, Jessica N.
AU - Kaleelurrrahuman, Mohamed Salman
AU - Sun, Yuanzi
AU - Vaquer-Alicea, Jaime
AU - Maggiore, Gianna
AU - Clemen, Christoph S.
AU - Buscher, William J.
AU - Bieschke, Jan
AU - Kotzbauer, Paul
AU - Ayala, Yuna
AU - Diamond, Marc I.
AU - Davis, Albert A.
AU - Weihl, Conrad
N1 - Funding Information:
We thank Genome engineering and iPSC center, particularly Dr. Monica Sentmanat, for generated the Cas9 transduced cell line. We thank the Alvin J. Siteman Cancer Center at WUSM in St. Louis, MO, for the use of a flow sorter. We thank Hope center at WUSM in St. Louis, MO, for providing machines, including Nanozoomer and flow cytometer, and for the lentivirus package service performed by Dr. Mingjie Li’s group. Finally, we thank. Dr. Hemmo Meyer from the University of Duisburg-Essen for the VCP mcherry vectors; Dr. Yang Shi for troubleshooting the flow cytometer; the Mouse Genetics Core at WUSM in St. Louis, MO and their staffs for handling and breeding mice.
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - 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.
AB - 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.
KW - Alpha-synuclein
KW - CRISPR screen
KW - Frontotemporal dementia
KW - Seeding
KW - TDP-43
UR - http://www.scopus.com/inward/record.url?scp=85128146878&partnerID=8YFLogxK
U2 - 10.1186/s13024-022-00532-0
DO - 10.1186/s13024-022-00532-0
M3 - Article
C2 - 35414105
AN - SCOPUS:85128146878
SN - 1750-1326
VL - 17
JO - Molecular Neurodegeneration
JF - Molecular Neurodegeneration
IS - 1
M1 - 30
ER -