Beta-endoproteolysis of the cellular prion protein by dipeptidyl peptidase-4 and fibroblast activation protein.

The cellular prion protein (PrPC) converts to alternatively folded pathogenic conformations (PrPSc) in prion infections and binds neurotoxic oligomers formed by amyloid-ß α-synuclein, and tau. ß-Endoproteolysis, which splits PrPC into N- and C-terminal fragments (N2 and C2, respectively), is of interest because a protease-resistant, C2-sized fragment (C2Sc) accumulates in the brain during prion infections, seemingly comprising the majority of PrPSc 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 PrPC ß-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 PrPC's N-terminal domain. For wild-type mouse and human PrPC 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 PrPC 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 C2Sc and total PrPSc 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 PrPC.

Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations.

Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here, we show that macroautophagy requires the Alzheimer's disease (AD)-related protein presenilin-1 (PS1). In PS1 null blastocysts, neurons from mice hypomorphic for PS1 or conditionally depleted of PS1, substrate proteolysis and autophagosome clearance during macroautophagy are prevented as a result of a selective impairment of autolysosome acidification and cathepsin activation. These deficits are caused by failed PS1-dependent targeting of the v-ATPase V0a1 subunit to lysosomes. N-glycosylation of the V0a1 subunit, essential for its efficient ER-to-lysosome delivery, requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the Sec61alpha/oligosaccharyltransferase complex. PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests previously unidentified therapeutic targets.

Prion protein with a mutant N-terminal octarepeat region undergoes cobalamin-dependent assembly into high-molecular weight complexes.

The cellular prion protein (PrPC) 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 (PrPSc). 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 PrPC 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 PrPSc's disordered region, Cbl, and conformational remodeling events may have implications for understanding sporadic prion disease that does not involve exposure to PrPSc.

Prion-like strain effects in tauopathies.

Tau is a microtubule-associated protein that plays crucial roles in physiology and pathophysiology. In the realm of dementia, tau protein misfolding is associated with a wide spectrum of clinicopathologically diverse neurodegenerative diseases, collectively known as tauopathies. As proposed by the tau strain hypothesis, the intrinsic heterogeneity of tauopathies may be explained by the existence of structurally distinct tau conformers, "strains". Tau strains can differ in their associated clinical features, neuropathological profiles, and biochemical signatures. Although prior research into infectious prion proteins offers valuable lessons for studying how a protein-only pathogen can encompass strain diversity, the underlying mechanism by which tau subtypes are generated remains poorly understood. Here we summarize recent advances in understanding different tau conformers through in vivo and in vitro experimental paradigms, and the implications of heterogeneity of pathological tau species for drug development.

Pathologic tau conformer ensembles induce dynamic, liquid-liquid phase separation events at the nuclear envelope.

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.

Application of high-throughput, capillary-based Western analysis to modulated cleavage of the cellular prion protein.

The cellular prion protein (PrPC) is a glycoprotein that is processed through several proteolytic pathways. Modulators of PrPC proteolysis are of interest because full-length PrPC and its cleavage fragments differ in their propensity to misfold, a process that plays a key role in the pathogenesis of prion diseases. PrPC may also act as a receptor for neurotoxic, oligomeric species of other proteins that are linked to neurodegeneration. Importantly, the PrPC C-terminal fragment C1 does not contain the reported binding sites for these oligomers. Western blotting would be a simple end point detection method for cell-based screening of compound libraries for effects on PrPC proteolysis or overall expression level. However, traditional Western blotting methods provide unreliable quantification and have only low throughput. Consequently, we explored capillary-based Western technology as a potential alternative; we believe that this study is the first to report analysis of PrPC using such an approach. We successfully optimized the detection and quantification of the deglycosylated forms of full-length PrPC and its C-terminal cleavage fragments C1 and C2, including simultaneous quantification of ß-tubulin levels to control for loading error. We also developed and tested a method for performing all cell culture, lysis, and deglycosylation steps in 96-well microplates prior to capillary Western analysis. These advances represent steps along the way to the development of an automated, high-throughput screening pipeline to identify modulators of PrPC expression levels or proteolysis.

A novel Gerstmann-Sträussler-Scheinker disease mutation defines a precursor for amyloidogenic 8 kDa PrP fragments and reveals N-terminal structural changes shared by other GSS alleles.

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.

Diverse, evolving conformer populations drive distinct phenotypes in frontotemporal lobar degeneration caused by the same MAPT-P301L mutation.

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.

Metabolomic study of disease progression in scrapie prion infected mice; validation of a novel method for brain metabolite extraction.

INTRODUCTION: Prion disease is a form of neurodegenerative disease caused by the misfolding and aggregation of cellular prion protein (PrPC). The neurotoxicity of the misfolded form of prion protein, PrPSc still remains understudied. Here we try to investigate this issue using a metabolomics approach. OBJECTIVES: The intention was to identify and quantify the small-in-size and water-soluble metabolites extracted from mice brains infected with the Rocky Mountain Laboratory isolate of mouse-adapted scrapie prions (RML) and track changes in these metabolites during disease evolution. METHODS: A total of 73 mice were inoculated with RML prions or normal brain homogenate control; brains were harvested at 30, 60, 90, 120 and 150 days post-inoculation (dpi). We devised a high-efficiency metabolite extraction method and used nuclear magnetic resonance spectroscopy to identify and quantify 50 metabolites in the brain extracts. Data were analyzed using multivariate approach. RESULTS: Brain metabolome profiles of RML infected animals displayed continuous changes throughout the course of disease. Among the analyzed metabolites, the most noteworthy changes included increases in myo-inositol and glutamine as well as decreases in 4-aminobutyrate, acetate, aspartate and taurine. CONCLUSION: We report a novel metabolite extraction method for lipid-rich tissue. As all the major metabolites are identifiable and quantifiable by magnetic resonance spectroscopy, this study suggests that tracking of neurochemical profiles could be effective in monitoring the progression of neurodegenerative diseases and useful for assessing the efficacy of candidate therapeutics.

Regulation of Amyloid ß Oligomer Binding to Neurons and Neurotoxicity by the Prion Protein-mGluR5 Complex.

The prion protein (PrPC) has been suggested to operate as a scaffold/receptor protein in neurons, participating in both physiological and pathological associated events. PrPC, laminin, and metabotropic glutamate receptor 5 (mGluR5) form a protein complex on the plasma membrane that can trigger signaling pathways involved in neuronal differentiation. PrPC and mGluR5 are co-receptors also for ß-amyloid oligomers (AßOs) and have been shown to modulate toxicity and neuronal death in Alzheimer's disease. In the present work, we addressed the potential crosstalk between these two signaling pathways, laminin-PrPC-mGluR5 or AßO-PrPC-mGluR5, as well as their interplay. Herein, we demonstrated that an existing complex containing PrPC-mGluR5 has an important role in AßO binding and activity in neurons. A peptide mimicking the binding site of laminin onto PrPC (Ln-γ1) binds to PrPC and induces intracellular Ca2+ increase in neurons via the complex PrPC-mGluR5. Ln-γ1 promotes internalization of PrPC and mGluR5 and transiently decreases AßO biding to neurons; however, the peptide does not impact AßO toxicity. Given that mGluR5 is critical for toxic signaling by AßOs and in prion diseases, we tested whether mGlur5 knock-out mice would be susceptible to prion infection. Our results show mild, but significant, effects on disease progression, without affecting survival of mice after infection. These results suggest that PrPC-mGluR5 form a functional response unit by which multiple ligands can trigger signaling. We propose that trafficking of PrPC-mGluR5 may modulate signaling intensity by different PrPC ligands.

Role of microglial amylin receptors in mediating beta amyloid (Aß)-induced inflammation.

BACKGROUND: Neuroinflammation in the brain consequent to activation of microglia is viewed as an important component of Alzheimer's disease (AD) pathology. Amyloid beta (Aß) protein is known to activate microglia and unleash an inflammatory cascade that eventually results in neuronal dysfunction and death. In this study, we sought to identify the presence of amylin receptors on human fetal and murine microglia and determine whether Aß activation of the inflammasome complex and subsequent release of cytokines is mediated through these receptors. METHODS: The presence of dimeric components of the amylin receptor (calcitonin receptor and receptor activity modifying protein 3) were first immunohistochemically identified on microglia. Purified human fetal microglial (HFM) cultures were incubated with an in vivo microglial marker, DyLight 594-conjugated tomato lectin, and loaded with the membrane-permeant green fluorescent dye, Fluo-8L-AM for measurements of intracellular calcium [Ca2+]i. HFM and BV-2 cells were primed with lipopolysaccharide and then exposed to either human amylin or soluble oligomeric Aß1-42 prior to treatment with and without the amylin receptor antagonist, AC253. Changes in the inflammasome complex, NLRP3 and caspase-1, were examined in treated cell cultures with Western blot and fluorometric assays. RT-PCR measurements were performed to assess cytokine release. Finally, in vivo studies were performed in transgenic mouse model of AD (5xFAD) to examine the effects of systemic administration of AC253 on markers of neuroinflammation in the brain. RESULTS: Acute applications of human amylin or Aß1-42 resulted in an increase in [Ca2+]i that could be blocked by the amylin receptor antagonist, AC253. Activation of the NLRP3 and caspase-1 and subsequent release of cytokines, TNFα and IL-1ß, was diminished by AC253 pretreatment of HFMs and BV2 cells. In vivo, intraperitoneal administration of AC253 resulted in a reduction in microglial markers (Iba-1 and CD68), caspase-1, TNFα, and IL-1ß. These reductions in inflammatory markers were accompanied by reduction in amyloid plaque and size in the brains of 5xFAD mice compared to controls. CONCLUSION: Microglial amylin receptors mediate Aß-evoked inflammation, and amylin receptor antagonists therefore offer an attractive therapeutic target for intervention in AD.

Bioenergetic mechanisms in astrocytes may contribute to amyloid plaque deposition and toxicity.

Alzheimer disease (AD) is characterized neuropathologically by synaptic disruption, neuronal loss, and deposition of amyloid ß (Aß) protein in brain structures that are critical for memory and cognition. There is increasing appreciation, however, that astrocytes, which are the major non-neuronal glial cells, may play an important role in AD pathogenesis. Unlike neurons, astrocytes are resistant to Aß cytotoxicity, which may, in part, be related to their greater reliance on glycolytic metabolism. Here we show that, in cultures of human fetal astrocytes, pharmacological inhibition or molecular down-regulation of a main enzymatic regulator of glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB3), results in increased accumulation of Aß within and around astrocytes and greater vulnerability of these cells to Aß toxicity. We further investigated age-dependent changes in PFKFB3 and astrocytes in AD transgenic mice (TgCRND8) that overexpress human Aß. Using a combination of Western blotting and immunohistochemistry, we identified an increase in glial fibrillary acidic protein expression in astrocytes that paralleled the escalation of the Aß plaque burden in TgCRND8 mice in an age-dependent manner. Furthermore, PFKFB3 expression also demonstrated an increase in these mice, although at a later age (9 months) than GFAP and Aß. Immunohistochemical staining showed significant reactive astrogliosis surrounding Aß plaques with increased PFKFB3 activity in 12-month-old TgCRND8 mice, an age when AD pathology and behavioral deficits are fully manifested. These studies shed light on the unique bioenergetic mechanisms within astrocytes that may contribute to the development of AD pathology.

Toll-like receptor-mediated immune response inhibits prion propagation.

Prion diseases are progressive neurodegenerative disorders affecting humans and various mammals. The prominent neuropathological change in prion diseases is neuroinflammation characterized by activation of neuroglia surrounding prion deposition. The cause and effect of this cellular response, however, is unclear. We investigated innate immune defenses against prion infection using primary mixed neuronal and glial cultures. Conditional prion propagation occurred in glial cultures depending on their immune status. Preconditioning of the cells with the toll-like receptor (TLR) ligand, lipopolysaccharide, resulted in a reduction in prion propagation, whereas suppression of the immune responses with the synthetic glucocorticoid, dexamethasone, increased prion propagation. In response to recombinant prion fibrils, glial cells up-regulated TLRs (TLR1 and TLR2) expression and secreted cytokines (tumor necrosis factor-α, interleukin-1ß, interleukin-6, granulocyte-macrophage colony-stimulating factor, and interferon-ß). Preconditioning of neuronal and glial cultures with recombinant prion fibrils inhibited prion replication and altered microglial and astrocytic populations. Our results provide evidence that, in early stages of prion infection, glial cells respond to prion infection through TLR-mediated innate immunity.

Prion Infectivity Plateaus and Conversion to Symptomatic Disease Originate from Falling Precursor Levels and Increased Levels of Oligomeric PrPSc Species.

UNLABELLED: In lethal prion neurodegenerative diseases, misfolded prion proteins (PrP(Sc)) replicate by redirecting the folding of the cellular prion glycoprotein (PrP(C)). Infections of different durations can have a subclinical phase with constant levels of infectious particles, but the mechanisms underlying this plateau and a subsequent exit to overt clinical disease are unknown. Using tandem biophysical techniques, we show that attenuated accumulation of infectious particles in presymptomatic disease is preceded by a progressive fall in PrP(C) level, which constricts replication rate and thereby causes the plateau effect. Furthermore, disease symptoms occurred at the threshold associated with increasing levels of small, relatively less protease-resistant oligomeric prion particles (oPrP(Sc)). Although a hypothetical lethal isoform of PrP cannot be excluded, our data argue that diminishing residual PrP(C) levels and continuously increasing levels of oPrP(Sc) are crucial determinants in the transition from presymptomatic to symptomatic prion disease. IMPORTANCE: Prions are infectious agents that cause lethal brain diseases; they arise from misfolding of a cell surface protein, PrP(C) to a form called PrP(Sc). Prion infections can have long latencies even though there is no protective immune response. Accumulation of infectious prion particles has been suggested to always reach the same plateau in the brain during latent periods, with clinical disease only occurring when hypothetical toxic forms (called PrP(L) or TPrP) begin to accumulate. We show here that infectivity plateaus arise because PrP(C) precursor levels become downregulated and that the duration of latent periods can be accounted for by the level of residual PrP(C), which transduces a toxic effect, along with the amount of oligomeric forms of PrP(Sc).

Interaction between Shadoo and PrP Affects the PrP-Folding Pathway.

UNLABELLED: Prion diseases are characterized by conformational changes of a cellular prion protein (PrP(C)) into a ß-sheet-enriched and aggregated conformer (PrP(Sc)). Shadoo (Sho), a member of the prion protein family, is expressed in the central nervous system (CNS) and is highly conserved among vertebrates. On the basis of histoanatomical colocalization and sequence similarities, it is suspected that Sho and PrP may be functionally related. The downregulation of Sho expression during prion pathology and the direct interaction between Sho and PrP, as revealed by two-hybrid analysis, suggest a relationship between Sho and prion replication. Using biochemical and biophysical approaches, we demonstrate that Sho forms a 1:1 complex with full-length PrP with a dissociation constant in the micromolar range, and this interaction consequently modifies the PrP-folding pathway. Using a truncated PrP that mimics the C-terminal C1 fragment, an allosteric binding behavior with a Hill number of 4 was observed, suggesting that at least a tetramerization state occurs. A cell-based prion titration assay performed with different concentrations of Sho revealed an increase in the PrP(Sc) conversion rate in the presence of Sho. Collectively, our observations suggest that Sho can affect the prion replication process by (i) acting as a holdase and (ii) interfering with the dominant-negative inhibitor effect of the C1 fragment. IMPORTANCE: Since the inception of the prion theory, the search for a cofactor involved in the conversion process has been an active field of research. Although the PrP interactome presents a broad landscape, candidates corresponding to specific criteria for cofactors are currently missing. Here, we describe for the first time that Sho can affect PrP structural dynamics and therefore increase the prion conversion rate. A biochemical characterization of Sho-PrP indicates that Sho acts as an ATP-independent holdase.

Infectious prions accumulate to high levels in non proliferative C2C12 myotubes.

Prion diseases are driven by the strain-specific, template-dependent transconformation of the normal cellular prion protein (PrP(C)) into a disease specific isoform PrP(Sc). Cell culture models of prion infection generally use replicating cells resulting in lower levels of prion accumulation compared to animals. Using non-replicating cells allows the accumulation of higher levels of PrP(Sc) and, thus, greater amounts of infectivity. Here, we infect non-proliferating muscle fiber myotube cultures prepared from differentiated myoblasts. We demonstrate that prion-infected myotubes generate substantial amounts of PrP(Sc) and that the level of infectivity produced in these post-mitotic cells, 10(5.5) L.D.50/mg of total protein, approaches that observed in vivo. Exposure of the myotubes to different mouse-adapted agents demonstrates strain-specific replication of infectious agents. Mouse-derived myotubes could not be infected with hamster prions suggesting that the species barrier effect is intact. We suggest that non-proliferating myotubes will be a valuable model system for generating infectious prions and for screening compounds for anti-prion activity.

Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP(C)-deficiency.

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 Mtg1 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.

Development of isotope labeling liquid chromatography mass spectrometry for mouse urine metabolomics: quantitative metabolomic study of transgenic mice related to Alzheimer's disease.

Because of a limited volume of urine that can be collected from a mouse, it is very difficult to apply the common strategy of using multiple analytical techniques to analyze the metabolites to increase the metabolome coverage for mouse urine metabolomics. We report an enabling method based on differential isotope labeling liquid chromatography mass spectrometry (LC-MS) for relative quantification of over 950 putative metabolites using 20 µL of urine as the starting material. The workflow involves aliquoting 10 µL of an individual urine sample for ¹²C-dansylation labeling that target amines and phenols. Another 10 µL of aliquot was taken from each sample to generate a pooled sample that was subjected to ¹³C-dansylation labeling. The ¹²C-labeled individual sample was mixed with an equal volume of the ¹³C-labeled pooled sample. The mixture was then analyzed by LC-MS to generate information on metabolite concentration differences among different individual samples. The interday repeatability for the LC-MS runs was assessed, and the median relative standard deviation over 4 days was 5.0%. This workflow was then applied to a metabolomic biomarker discovery study using urine samples obtained from the TgCRND8 mouse model of early onset familial Alzheimer's disease (FAD) throughout the course of their pathological deposition of beta amyloid (Aß). It was showed that there was a distinct metabolomic separation between the AD prone mice and the wild type (control) group. As early as 15-17 weeks of age (presymptomatic), metabolomic differences were observed between the two groups, and after the age of 25 weeks the metabolomic alterations became more pronounced. The metabolomic changes at different ages corroborated well with the phenotype changes in this transgenic mice model. Several useful candidate biomarkers including methionine, desaminotyrosine, taurine, N1-acetylspermidine, and 5-hydroxyindoleacetic acid were identified. Some of them were found in previous metabolomics studies in human cerebrospinal fluid or blood samples. This work illustrates the utility of this isotope labeling LC-MS method for biomarker discovery using mouse urine metabolomics.