Rapid replication in vitro implicates distinct populations of misfolded hippocampal 4R TAU protein in accelerated progression of Alzheimer’s disease.

The cellular prion protein (PrP^C) converts to alternatively folded pathogenic conformations (PrP^Sc) in prion infections and binds neurotoxic oligomers formed by amyloid-β α-synuclein, and tau. β-Endoproteolysis, which splits PrP^Cinto N- and C-terminal fragments (N2 and C2, respectively), is of interest because a protease-resistant, C2-sized fragment (C2^Sc) accumulates in the brain during prion infections, seemingly comprising the majority of PrP^Scat disease endpoint in mice. However, candidates for the underlying proteolytic mechanism(s) remain unconfirmed in vivo. Here, a cell-based screen of protease inhibitors unexpectedly linked type II membrane proteins of the S9B serine peptidase subfamily to PrP^Cβ-cleavage. Overexpression experiments in cells and assays with recombinant proteins confirmed that fibroblast activation protein (FAP) and its paralog, dipeptidyl peptidase-4 (DPP4), cleave directly at multiple sites within PrP^C’s N-terminal domain. For wild-type mouse and human PrP^Csubstrates expressed in cells, the rank orders of activity were human FAP ~ mouse FAP > mouse DPP4 > human DPP4 and human FAP > mouse FAP > mouse DPP4 >> human DPP4, respectively. C2 levels relative to total PrP^Cwere reduced in several tissues from FAP-null mice, and, while knockout of DPP4 lacked an analogous effect, the combined DPP4/FAP inhibitor linagliptin, but not the FAP-specific inhibitor SP-13786, reduced C2^Scand total PrP^Sclevels in two murine cell-based models of prion infections. Thus, the net activity of the S9B peptidases FAP and DPP4 and their cognate inhibitors/modulators affect the physiology and pathogenic potential of PrP^C.

The accumulation of tau fibrils is associated with neurodegenerative diseases, which are collectively termed tauopathies. Cryo-EM studies have shown that the packed fibril core of tau adopts distinct structures in different tauopathies, such as Alzheimer's disease, corticobasal degeneration, and progressive supranuclear palsy. A subset of tauopathies are linked to missense mutations in the tau protein, but it is not clear whether these mutations impact the structure of tau fibrils. To answer this question, we developed a high-throughput protein purification platform and purified a panel of 37 tau variants using the full-length 0N4R splice isoform. Each of these variants was used to create fibrils in vitro, and their relative structures were studied using a high-throughput protease sensitivity platform. We find that a subset of the disease-associated mutations form fibrils that resemble wild-type tau, while others are strikingly different. The impact of mutations on tau structure was not clearly associated with either the location of the mutation or the relative kinetics of fibril assembly, suggesting that tau mutations alter the packed core structures through a complex molecular mechanism. Together, these studies show that single-point mutations can impact the assembly of tau into fibrils, providing insight into its association with pathology and disease.

The cellular prion protein (PrP^C) has a C-terminal globular domain and a disordered N-terminal region encompassing five octarepeats (ORs). Encounters between Cu(II) ions and four OR sites produce interchangeable binding geometries; however, the significance of Cu(II) binding to ORs in different combinations is unclear. To understand the impact of specific binding geometries, OR variants were designed that interact with multiple or single Cu(II) ions in specific locked coordinations. Unexpectedly, we found that one mutant produced detergent-insoluble, protease-resistant species in cells in the absence of exposure to the infectious prion protein isoform, scrapie-associated prion protein (PrP^Sc). Formation of these assemblies, visible as puncta, was reversible and dependent upon medium formulation. Cobalamin (Cbl), a dietary cofactor containing a corrin ring that coordinates a Co³⁺ ion, was identified as a key medium component, and its effect was validated by reconstitution experiments. Although we failed to find evidence that Cbl interacts with Cu-binding OR regions, we instead noted interactions of Cbl with the PrP^C C-terminal domain. We found that some interactions occurred at a binding site of planar tetrapyrrole compounds on the isolated globular domain, but others did not, and N-terminal sequences additionally had a marked effect on their presence and position. Our studies define a conditional effect of Cbl wherein a mutant OR region can act in cis to destabilize a globular domain with a wild type sequence. The unexpected intersection between the properties of PrP^Sc's disordered region, Cbl, and conformational remodeling events may have implications for understanding sporadic prion disease that does not involve exposure to PrP^Sc.

Recent studies show that a single MAPT gene mutation can promote alternative tau misfolding pathways engendering divergent forms of frontotemporal dementia and that under conditions of molecular crowding, the repertoire of tau forms can include liquid-liquid phase separation (LLPS). We show here that following pathogenic seeding, tau condenses on the nuclear envelope (NE) and disrupts nuclear-cytoplasmic transport (NCT). Interestingly, NE fluorescent tau signals and small fluorescent inclusions behaved as demixed liquid droplets in living cells. Thioflavin S-positive intracellular aggregates were prevalent in tau-derived inclusions with a size bigger than 3 μm ² , indicating that a threshold of critical mass in the liquid state condensation may drive liquid-solid phase transitions. Our findings indicate that tau undergoing LLPS is more toxic amongst a spectrum of alternative conformers; LLPS droplets on the NE that disrupt NCT serve to trigger cell death and can act as nurseries for fibrillar structures abundantly detected in end-stage disease.

Prion diseases are progressive neurodegenerative disorders affecting humans and various mammals. The prominent neuropathological change in prion-affected brains is neuroinflammation, histopathologically characterized by reactive gliosis surrounding prion deposition. The cause and effect of these cellular responses are still unclear. Here we investigate the impact of innate immune responses on prion replication using in vitro cell culture models. Hamster-adapted transmissible mink encephalopathy prions, hyper (HY) and drowsy (DY) strains, were assayed for accumulation of pathogenic prion protein (PrP^Sc) in primary glial cultures derived from 8-day-old hamster pups. The kinetics of PrP^Sc accumulation largely depended on prion strain and brain regions from where glial cells originated. Glial cells derived from the cerebellum were susceptible to HY, but resistant to DY strain as determined by western blot analysis, immunocytochemistry, and animal bioassay. Glial cells from the cerebral cortex were, however, refractory to both strains. PrP^Sc accumulation was affected by innate immune modulators. Priming glial cells with lipopolysaccharide decreased prion replication, whereas pre-treatment with dexamethasone, inhibiting innate immunity, increased susceptibility to DY infection. Our results suggest that neuroinflammation resulting from prion infection is a response to resolve and/or prevent prion propagation in the brain. It implies a therapeutic potential of innate immune modulation in the early stages of prion disease.