The hydrolysis reactions of four 2-deoxy-R-D-glucopyranosyl pyridinium salts exhibit first-order rate constants that are independent of pH in the range of 4.4-10.5 pH units. Derived second-order rate constants for the hydrolysis reactions of 2-deoxy-R-D-glucopyranosyl 4′-bromoisoquinolinium tetrafluoroborate (4d) conducted in the presence of nucleophilic monoanions (µ ) 2.0) including AcO -, Cl -, Br -, and N 3exhibit a Swain-Scott parameter (s) of 0.03 ( 0.10, indicating that these reactions show no sensitivity to the nature of the anion. In the presence of azide ion, a substantial quantity of the 2-deoxy-R-glucopyranosyl 4′-bromoisoquinolinium salt hydrolysis product results from a post rate-limiting reaction of a cationic intermediate with azide. Analysis of the hydrolysis product ratios indicates that the 2-deoxyglucosyl oxocarbenium ion is not solvent-equilibrated in water. Furthermore, the reaction of solvent occurs about 2-fold faster with the cationic intermediate that is formed during solvolysis of the β-anomeric salt than with the corresponding intermediate produced from the reactions of the R-anomer 4d.

SUMMARY The pathogenic mechanism by which dominant mutations in VCP cause multisystem proteinopathy (MSP), a rare neurodegenerative disease that presents as fronto-temporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), remains unclear. To explore this, we inactivate VCP in murine postnatal forebrain neurons (VCP conditional knockout [cKO]). VCP cKO mice have cortical brain atrophy, neuronal loss, autophago-lysosomal dysfunction, and TDP-43 inclusions resembling FTLD-TDP pathology. Conditional expression of a single disease-associated mutation, VCP-R155C, in a VCP null background similarly recapitulates features of VCP inactivation and FTLD-TDP, suggesting that this MSP mutation is hypomorphic. Comparison of transcriptomic and proteomic datasets from genetically defined patients with FTLD-TDP reveal that progranulin deficiency and VCP insufficiency result in similar profiles. These data identify a loss of VCP-dependent functions as a mediator of FTLD-TDP and reveal an unexpected biochemical similarity with progranulin deficiency.

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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 solvolyses of 2-deoxy-alpha- and beta-D-glucopyranosyl 4'-bromoisoquinolinium tetrafluoroborates (1 and 2) were monitored in aqueous methanol, ethanol, trifluoroethanol, and binary mixtures of ethanol and trifluoroethanol. The observed rate constants are consistent with the solvolyses of 1 and 2 proceeding via dissociative (D(N) A(N)) transition states. In comparison to the alpha-anomer, solvolysis of the beta-compound gives a greater transition state charge delocalization onto the ring oxygen atom. Analysis of the solvolysis product ratios indicates that the 2-deoxyglucosyl oxacarbenium ion is not solvent-equilibrated in the solvent mixtures studied. In the solvolysis of compound 1, the solvent trifluoroethanol facilitates diffusional separation of the leaving group and, in so doing, promotes the formation of the retained trifluoroethyl glycoside.