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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 C into 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 Sc at 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 C β-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 C substrates 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 C were 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 Sc and total PrP Sc levels 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 .

Background The microtubule-associated protein tau forms aggregates in different neurodegenerative diseases called tauopathies. Prior work has shown that a single P301L mutation in tau gene, MAPT, can promote alternative tau folding pathways that correlate with divergent clinical diagnoses. Using progressive chemical denaturation, some tau preparations from the brain featured complex transitions starting at low concentrations of guanidine hydrochloride (GdnHCl) denaturant, indicating an ensemble of differently folded tau species called conformers. On the other hand, brain samples with abundant, tangle-like pathology had simple GdnHCl unfolding profile resembling the profile of fibrillized recombinant tau and suggesting a unitary conformer composition. In studies here we sought to understand tau conformer progression and potential relationships with condensed liquid states, as well as associated perturbations in cell biological processes. Results As starting material, we used brain samples from P301L transgenic mice containing tau conformer ensembles that unfolded at low GdnHCl concentrations and with signatures resembling brain material from P301L subjects presenting with language or memory problems. We seeded reporter cells expressing a soluble form of 4 microtubule-binding repeat tau fused to GFP or YFP reporter moieties, resulting in redistribution of dispersed fluorescence signals into focal assemblies that could fuse together and move within processes between adjacent cells. Nuclear envelope fluorescent tau signals and small fluorescent inclusions behaved as a demixed liquid phase, indicative of liquid-liquid phase separation (LLPS); these droplets exhibited spherical morphology, fusion events and could recover from photobleaching. Moreover, juxtanuclear tau assemblies were associated with disrupted nuclear transport and reduced cell viability in a stable cell line. Staining for thioflavin S (ThS) became more prevalent as tau-derived inclusions attained cross-sectional area greater than 3 μm2, indicating (i) a bipartite composition, (ii) in vivo progression of tau conformers, and (iii) that a mass threshold applying to demixed condensates may drive liquid-solid transitions. Conclusions Tau conformer ensembles characterized by denaturation at low GdnHCl concentration templated the production of condensed droplets in living cells. These species exhibit dynamic changes and develop in vivo, and the larger ThS-positive assemblies may represent a waystation to arrive at intracellular fibrillar tau inclusions seen in end-stage genetic tauopathies.

Tau protein accumulation is a common denominator of major dementias, but this process is inhomogeneous, even when triggered by the same germline mutation. We considered stochastic misfolding of human tau conformers followed by templated conversion of native monomers as an underlying mechanism and derived sensitive conformational assays to test this concept. Assessments of brains from aged TgTauP301L transgenic mice revealed a prodromal state and three distinct signatures for misfolded tau. Frontotemporal lobar degeneration (FTLD)-MAPT-P301L patients with different clinical phenotypes also displayed three signatures, two resembling those found in TgTauP301L mice. As physicochemical and cell bioassays confirmed diverse tau strains in the mouse and human brain series, we conclude that evolution of diverse tau conformers is intrinsic to the pathogenesis of this uni-allelic form of tauopathy. In turn, effective therapeutic interventions in FTLD will need to address evolving repertoires of misfolded tau species rather than singular, static molecular targets. Electronic supplementary material The online version of this article (10.1007/s00401-020-02148-4) contains supplementary material, which is available to authorized users.

The cellular prion protein (PrP

Background: Germline mutations in the MAPT gene cause some forms of frontotemporal lobar degeneration (FTLD). Recent studies show that a single mutation in MAPT can promote alternative tau misfolding pathways engendering divergent tau conforms and representing clinical heterogeneity, and that under conditions of cell-free molecular crowding the repertoire of tau forms can include liquid-liquid phase separation (LLPS). Methods: Neuronal nuclear morphologies in FTLD patients and TgTauP301L transgenic mice were analyzed by immunohistochemistry of nuclear lamina. Tau conformers associated with a common behavioral variant of frontotemporal dementia were cloned by endpoint dilution; the cells were assayed for viability and biochemical markers of cell death and were also assessed by video microscopy and photobleaching to determine dynamic aspects of aggregate formation.Results: Analysis of post-mortem tissues from aged neurologically normal controls and other neurodegenerative syndromes indicated that microtubule-associated nuclear clefts were associated with chronological aging and disruptions of the nuclear envelope with FTLD-MAPT. Tau conformers present in FTLD cases and transduced into reporter cells had a high propensity to condense on the nuclear envelope and to disrupt nuclear-cytoplasmic transport. Nuclear envelope fluorescent tau signals and small fluorescent inclusions in a stable clonal line behaved as a demixed liquid state under live cell conditions; indicative of LLPS effects, these droplets exhibited spherical morphology, fusion events and recovery from photobleaching. While pathogenic mutations in some proteins can interfere with physiological functions of membrane-less organelles, a disease-causing MAPT mutation perturbed nuclear-cytoplasmic transport by gain-of-function formation of LLPS on the nuclear envelope, this acting as a molecular cue to trigger regulated cell death. Thioflavin S-positive intracellular aggregates were prevalent in tau-derived inclusions with a size bigger than 3 µm2, inferring that a threshold of critical mass in the liquid state condensation may drive liquid-solid phase transitions. Conclusions: Our findings indicate that within a spectrum of alternative conformers, tau undergoing LLPS is a notably toxic species; demixed droplets on the nuclear envelope hindering nuclear-cytoplasmic transport can serve to trigger cytotoxic pathways and may act as nurseries for the abundant fibrillar structures present at end-stage disease.

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 μm2, 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.

To explore pathogenesis in a young Gerstmann-Sträussler-Scheinker Disease (GSS) patient, the corresponding mutation, an eight-residue duplication in the hydrophobic region (HR), was inserted into the wild type mouse PrP gene. Transgenic (Tg) mouse lines expressing this mutation (Tg.HRdup) developed spontaneous neurologic syndromes and brain extracts hastened disease in low-expressor Tg.HRdup mice, suggesting de novo formation of prions. While Tg.HRdup mice exhibited spongiform change, PrP aggregates and the anticipated GSS hallmark of a proteinase K (PK)-resistant 8 kDa fragment deriving from the center of PrP, the LGGLGGYV insertion also imparted alterations in PrP's unstructured N-terminus, resulting in a 16 kDa species following thermolysin exposure. This species comprises a plausible precursor to the 8 kDa PK-resistant fragment and its detection in adolescent Tg.HRdup mice suggests that an early start to accumulation could account for early disease of the index case. A 16 kDa thermolysin-resistant signature was also found in GSS patients with P102L, A117V, H187R and F198S alleles and has coordinates similar to GSS stop codon mutations. Our data suggest a novel shared pathway of GSS pathogenesis that is fundamentally distinct from that producing structural alterations in the C-terminus of PrP, as observed in other prion diseases such as Creutzfeldt-Jakob Disease and scrapie., Author summary Prion diseases can be sporadic, infectious or genetic. The central event of all prion diseases is the structural conversion of the cellular prion protein (PrPC) to its disease associated conformer, PrPSc. Gerstmann-Sträussler-Scheinker Disease (GSS) is a genetic prion disease presenting as a multi-systemic neurological syndrome. A novel mutation, an eight amino acid insertion, was discovered in a young GSS patient. We created transgenic mice expressing this mutation and found that they recapitulate key features of the disease; namely PrP deposition in the brain and a low molecular weight proteinase K (PK) resistant internal PrP fragment. While structural investigations did not reveal a gross alteration in the conformation of this mutant PrP, the insertion lying at the boundary of the globular domain causes alterations in the unstructured amino terminal portion of the protein such that it becomes resistant to digestion by the enzyme thermolysin. We demonstrate by kinetic analysis and sequential digestion that this novel thermolysin resistant species is a precursor to the pathognomonic PK resistant fragment. Analysis of samples from other GSS patients revealed this same signature, suggesting a common molecular pathway.

The cellular prion protein (PrP(C)) comprises a natively unstructured N-terminal domain, including a metal-binding octarepeat region (OR) and a linker, followed by a C-terminal domain that misfolds to form PrP(S) (c) in Creutzfeldt-Jakob disease. PrP(C) β-endoproteolysis to the C2 fragment allows PrP(S) (c) formation, while α-endoproteolysis blocks production. To examine the OR, we used structure-directed design to make novel alleles, 'S1' and 'S3', locking this region in extended or compact conformations, respectively. S1 and S3 PrP resembled WT PrP in supporting peripheral nerve myelination. Prion-infected S1 and S3 transgenic mice both accumulated similar low levels of PrP(S) (c) and infectious prion particles, but differed in their clinical presentation. Unexpectedly, S3 PrP overproduced C2 fragment in the brain by a mechanism distinct from metal-catalysed hydrolysis reported previously. OR flexibility is concluded to impact diverse biological endpoints; it is a salient variable in infectious disease paradigms and modulates how the levels of PrP(S) (c) and infectivity can either uncouple or engage to drive the onset of clinical disease.

Widely expressed in the adult central nervous system, the cellular prion protein (PrP(C)) is implicated in a variety of processes, including neuronal excitability. Dipeptidyl aminopeptidase-like protein 6 (DPP6) was first identified as a PrP(C) interactor using in vivo formaldehyde cross-linking of wild type (WT) mouse brain. This finding was confirmed in three cell lines and, because DPP6 directs the functional assembly of K(+) channels, we assessed the impact of WT and mutant PrP(C) upon Kv4.2-based cell surface macromolecular complexes. Whereas a Gerstmann-Sträussler-Scheinker disease version of PrP with eight extra octarepeats was a loss of function both for complex formation and for modulation of Kv4.2 channels, WT PrP(C), in a DPP6-dependent manner, modulated Kv4.2 channel properties, causing an increase in peak amplitude, a rightward shift of the voltage-dependent steady-state inactivation curve, a slower inactivation, and a faster recovery from steady-state inactivation. Thus, the net impact of wt PrP(C) was one of enhancement, which plays a critical role in the down-regulation of neuronal membrane excitability and is associated with a decreased susceptibility to seizures. Insofar as previous work has established a requirement for WT PrP(C) in the Aβ-dependent modulation of excitability in cholinergic basal forebrain neurons, our findings implicate PrP(C) regulation of Kv4.2 channels as a mechanism contributing to the effects of oligomeric Aβ upon neuronal excitability and viability.

The assembly of myosin into higher order structures is dependent upon accessory factors that are often tissue-specific. UNC-45 acts as such a molecular chaperone for myosin in the nematode Caenorhabditis elegans, in both muscle and non-muscle contexts. Although vertebrates contain homologues of UNC-45, their requirement for muscle function has not been assayed. We identified a zebrafish gene, unc45b, similar to a mammalian unc-45 homologue, expressed exclusively in striated muscle tissue, including the somites, heart and craniofacial muscle. Morpholino-oligonucleotide-mediated knockdown of unc45b results in paralysis and cardiac dysfunction. This paralysis is correlated with a loss of myosin filaments in the sarcomeres of the trunk muscle. Morphants lack circulation, heart looping and display severe cardiac and yolk-sac edema and also demonstrate ventral displacement of several jaw cartilages. Overall, this confirms a role for unc45b in zebrafish motility consistent with a function in myosin thick filament assembly and stability and uncovers novel roles for this gene in the function and morphogenesis of the developing heart and jaw. These results suggest that Unc45b acts as a chaperone that aids in the folding of myosin isoforms required for skeletal, cranial and cardiac muscle contraction.

The vertebrate sarcomere is a complex and highly organized contractile structure whose assembly and function requires the coordination of hundreds of proteins. Proteins require proper folding and incorporation into the sarcomere by assembly factors, and they must also be maintained and replaced due to the constant physical stress of muscle contraction. Zebrafish mutants affecting muscle assembly and maintenance have proven to be an ideal tool for identification and analysis of factors necessary for these processes. The still heart mutant was identified due to motility defects and a nonfunctional heart. The cognate gene for the mutant was shown to be smyd1b and the still heart mutation results in an early nonsense codon. SMYD1 mutants show a lack of heart looping and chamber definition due to a lack of expression of heart morphogenesis factors gata4, gata5 and hand2. On a cellular level, fast muscle fibers in homozygous mutants do not form mature sarcomeres due to the lack of fast muscle myosin incorporation by SMYD1b when sarcomeres are first being assembled (19hpf), supporting SMYD1b as an assembly protein during sarcomere formation.

Cellular prion protein (PrP(C)) misfolds to form infectivity-associated scrapie prion protein and generates C-terminal fragments C1 and C2 in healthy and prion-infected animals. C1 cleavage occurs N-terminally of PrP(C)'s hydrophobic domain, whereas the larger C2 fragment is generated by cleavage at the end of the octarepeat region. As the PrP-like proteins Doppel and Shadoo (Sho) have been reported to inhabit similar membrane environments as PrP(C), we investigated endoproteolysis by using a panel of mutant alleles. Doppel undergoes efficient in vivo cleavage at a C1 site mapped to the start of the globular domain, which is a structurally similar cleavage site to that in PrP(C). Sho is processed to C1 and C2 fragments, and proved refractory to mutagenesis to inactivate C1 cleavage. As a reciprocal product of C1 cleavage, Sho also engenders a metabolically stable N1 fragment with a C-terminus after its hydrophobic domain, an observation that may account for N1's association with membrane and/or cellular fractions in vitro and in vivo. Our data indicate that glycosylation status and yet to be identified proteases modulate internal C1 and C2 proteolysis events within the mammalian prion protein family.

The sarcomeres of skeletal and cardiac muscle are highly structured protein arrays, consisting of thick and thin filaments aligned precisely to one another and to their surrounding matrix. The contractile mechanisms of sarcomeres are generally well understood, but how the patterning of sarcomeres is initiated during early skeletal muscle and cardiac development remains uncertain. Two of the most widely accepted hypotheses for this process include the “molecular ruler” model, in which the massive protein titin defines the length of the sarcomere and provides a scaffold along which the myosin thick filament is assembled, and the “premyofibril” model, which proposes that thick filament formation does not require titin, but that a “premyofibril” consisting of non-muscle myosin, α-actinin and cytoskeletal actin is used as a template. Each model posits a different order of necessity of the various components, but these have been difficult to test in vivo. Zebrafish motility mutants with developmental defects in sarcomere patterning are useful for the elucidation of such mechanisms, and here we report the analysis of the herzschlag mutant, which shows deficits in both cardiac and skeletal muscle. The herzschlag mutant produces a truncated titin protein, lacking the C-terminal rod domain that is proposed to act as a thick filament scaffold, yet muscle patterning is still initiated, with grossly normal thick and thin filament assembly. Only after embryonic muscle contraction begins is breakdown of sarcomeric myosin patterning observed, consistent with the previously noted role of titin in maintaining the contractile integrity of mature sarcomeres. This conflicts with the “molecular ruler” model of early sarcomere patterning and supports a titin-independent model of thick filament organization during sarcomerogenesis. These findings are also consistent with the symptoms of human titin myopathies that exhibit a late onset, such as tibial muscular dystrophy.

The Sprn gene encodes Shadoo (Sho), a glycoprotein with biochemical properties similar to the unstructured region of cellular prion protein (PrP C ). Sho has been considered a candidate for the hypothetical π protein that supplies a PrP C -like function to maintain the viability of Prnp 0/0 mice lacking the PrP C protein. To understand these relationships more clearly we probed the cell biology of Sho and created knockout mice. Besides full-length and a “C1” C-terminal fragment, we describe a 6-kDa N-terminal Sho neuropeptide, “N1,” which is present in membrane-enriched subcellular fractions of wild-type mice. Sprn null alleles were produced that delete all protein coding sequences yet spare the Mtg 1 gene transcription unit that overlaps the Sprn 3′ UTR; the resulting mice bred to homozygosity were viable and fertile, although Sprn 0/0 mice maintained in two genetic backgrounds weighed less than wild-type mice. Lack of Sho protein did not affect prion incubation time. Contrasting with lethality reported for knockdown of expression in Prnp 0/0 embryos using lentiviruses targeted against the Sprn 3′ UTR, we established that double-knockout mice deficient in both Sho and PrP C are fertile and viable up to 690 d of age. Our data reduce the impetus for equating Sho with the notional π protein and are not readily reconciled with hypotheses wherein expression of PrP C and Sho are both required for completion of embryogenesis. Alternatively, and in accord with some reports for PrP C , we infer that Sho’s activity will prove germane to the maintenance of neuronal viability in postnatal life.

An almost unique place within protein databases, twenty-five years of study has underscored the enigmatically subtle role of PrP(C) in normal cell biology. It seems that PrP has evolved (and survived) to perform a function that does not have a precedent amongst transmembrane cell-surface proteins, perhaps representing a new type of plasma membrane ecosystem. In a context where we await a clarifying insight to unify a panoply of PrP(C) data into logical molecular framework, the GPI-anchored N-glycosylated Doppel and Sho proteins are tantalizing in that they correspond roughly to the front and back halves of PrP(C) itself. These molecules may be simpler - and more "understandable" - entities that can be pursued in parallel to PrP(C), and could open up the biology of mammalian prion proteins from fresh directions. Dpl has a profound role in successful gametogenesis that warrants close scrutiny and a case for deeper study can be made for Sho, a recently discovered CNS-expressed protein with many parallels to established facets of PrP biochemistry. In an aerial view of biomedical research, Sho and Dpl can be considered as adjacent islands in a prion protein archipelago. As such, the coming years of molecular exploration should be extremely interesting.

An almost unique place within protein databases, twenty-five years of study has underscored the enigmatically subtle role of PrP(C) in normal cell biology. It seems that PrP has evolved (and survived) to perform a function that does not have a precedent amongst transmembrane cell-surface proteins, perhaps representing a new type of plasma membrane ecosystem. In a context where we await a clarifying insight to unify a panoply of PrP(C) data into logical molecular framework, the GPI-anchored N-glycosylated Doppel and Sho proteins are tantalizing in that they correspond roughly to the front and back halves of PrP(C) itself. These molecules may be simpler - and more "understandable" - entities that can be pursued in parallel to PrP(C), and could open up the biology of mammalian prion proteins from fresh directions. Dpl has a profound role in successful gametogenesis that warrants close scrutiny and a case for deeper study can be made for Sho, a recently discovered CNS-expressed protein with many parallels to established facets of PrP biochemistry. In an aerial view of biomedical research, Sho and Dpl can be considered as adjacent islands in a prion protein archipelago. As such, the coming years of molecular exploration should be extremely interesting.

The cellular prion protein PrP(C) refolds into a beta-sheet enriched, infectivity-associated form called PrP(Sc). Shadoo (Sho) is a newly discovered glycoprotein that is also expressed in the adult brain. Wild type (wt) mouse Sho consists of an arginine-rich region, a hydrophobic central domain of five tandem A/LAAG amino acid repeats R1-R5 with similarity to the hydrophobic domain of PrP(C), and a C-terminal domain with one N-linked carbohydrate. As some alanine-rich proteins and PrP with a shortened C-terminal domain form amyloid we investigated conformational properties of wt Sho and polymorphic variants with insertion/deletions centered on R3. Recombinant mouse and sheep Sho converted to an amyloid-like form without recourse to chemical denaturation or acidification. For wt proteins this transition was marked by increased thioflavin T binding, Congo red staining, presence of fibrillar structures by electron microscopy, formation of sodium dodecyl sulfate-resistant complexes and the generation of a C-terminal proteinase K resistant core of 5-8 kDa. Variant Sho proteins differing within the R1-R5 region exhibited most but not all of these properties. Our studies define a proteinase K -resistant signature fragment for the amyloid fold of Sho and raise the question of a physiological role for this form of the wt protein.

BACKGROUND: The cellular prion protein PrP(C) is encoded by the Prnp gene. This protein is expressed in the central nervous system (CNS) and serves as a precursor to the misfolded PrP(Sc) isoform in prion diseases. The prototype prion disease is scrapie in sheep, and whereas Prnp exhibits common missense polymorphisms for V136A, R154H and Q171R in ovine populations, genetic variation in mouse Prnp is limited. Recently the CNS glycoprotein Shadoo (Sho) has been shown to resemble PrP(C) both in a central hydrophobic domain and in activity in a toxicity assay performed in cerebellar neurons. Sho protein levels are reduced in prion infections in rodents. Prompted by these properties of the Sho protein we investigated the extent of natural variation in SPRN. PRINCIPAL FINDINGS: Paralleling the case for ovine versus human and murine PRNP, we failed to detect significant coding polymorphisms that alter the mature Sho protein in a sample of neurologically normal humans, or in diverse strains of mice. However, ovine SPRN exhibited 4 missense mutations and expansion/contraction in a series of 5 tandem Ala/Gly-containing repeats R1-R5 encoding Sho's hydrophobic domain. A Val71Ala polymorphism and polymorphic expansion of wt 67(Ala)(3)Gly70 to 67(Ala)(5)Gly72 reached frequencies of 20%, with other alleles including Delta67-70 and a 67(Ala)(6)Gly73 expansion. Sheep V71, A71, Delta67-70 and 67(Ala)(6)Gly73 SPRN alleles encoded proteins with similar stability and posttranslational processing in transfected neuroblastoma cells. SIGNIFICANCE: Frequent coding polymorphisms are a hallmark of the sheep PRNP gene and our data indicate a similar situation applies to ovine SPRN. Whether a common selection pressure balances diversity at both loci remains to be established.

Genetic and biochemical mechanisms linking onset or progression of Alzheimer Disease and prion diseases have been lacking and/or controversial, and their etiologies are often considered independent. Here we document a novel, conserved and specific genetic interaction between the proteins that underlie these diseases, amyloid-β precursor protein and prion protein, APP and PRP, respectively. Knockdown of APP and/or PRNP homologs in the zebrafish (appa, appb, prp1, and prp2) produces a dose-dependent phenotype characterized by systemic morphological defects, reduced cell adhesion and CNS cell death. This genetic interaction is surprisingly exclusive in that prp1 genetically interacts with zebrafish appa, but not with appb, and the zebrafish paralog prp2 fails to interact with appa. Intriguingly, appa & appb are largely redundant in early zebrafish development yet their abilities to rescue CNS cell death are differentially contingent on prp1 abundance. Delivery of human APP or mouse Prnp mRNAs rescue the phenotypes observed in app-prp-depleted zebrafish, highlighting the conserved nature of this interaction. Immunoprecipitation revealed that human APP and PrP(C) proteins can have a physical interaction. Our study reports a unique in vivo interdependence between APP and PRP loss-of-function, detailing a biochemical interaction that considerably expands the hypothesized roles of PRP in Alzheimer Disease.