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Both wild-type and mutant tau proteins can misfold into prions and self-propagate in the central nervous system of animals and people. To extend the work of others, we investigated the molecular basis of tau prion-mediated neurodegeneration in transgenic (Tg) rats expressing mutant human tau (P301S); this line of Tg rats is denoted Tg12099. We used the rat Prnp promoter to drive the overexpression of mutant tau (P301S) in the human 0N4R isoform. In Tg12099(+/+) rats homozygous for the transgene, ubiquitous expression of mutant human tau resulted in the progressive accumulation of phosphorylated tau inclusions, including silver-positive tangles in the frontal cortices and limbic system. Signs of central nervous system dysfunction were found in terminal Tg12099(+/+) rats exhibiting severe neurodegeneration and profound atrophy of the amygdala and piriform cortex. The greatest increases in tau prion activity were found in the corticolimbic structures. In contrast to the homozygous Tg12099(+/+) rats, we found lower levels of mutant tau in the hemizygous rats, resulting in few neuropathologic changes up to 2 years of age. Notably, these hemizygous rats could be infected by intracerebral inoculation with recombinant tau fibrils or precipitated tau prions from the brain homogenates of sick, aged homozygous Tg12099(+/+) rats. Our studies argue that the regional propagation of tau prions and neurodegeneration in the Tg12099 rats resembles that found in human primary tauopathies. These findings seem likely to advance our understanding of human tauopathies and may lead to effective therapeutics for Alzheimer's disease and other tau prion disorders.

Down syndrome (DS) is a common genetic condition caused by trisomy of chromosome 21. Among their complex clinical features, including musculoskeletal, neurological, and cardiovascular disabilities, individuals with DS have an increased risk of developing progressive dementia and early-onset Alzheimer's disease (AD). This dementia is attributed to the increased gene dosage of the amyloid-β (Aβ) precursor protein gene, the formation of self-propagating Aβ and tau prion conformers, and the deposition of neurotoxic Aβ plaques and tau neurofibrillary tangles. Tau amyloid fibrils have previously been established to adopt many distinct conformations across different neurodegenerative conditions. Here, we report the characterization of brain samples from four DS cases spanning 36-63 years of age by spectral confocal imaging with conformation-specific dyes and cryo-electron microscopy (cryo-EM) to determine structures of isolated tau fibrils. High-resolution structures revealed paired helical filament (PHF) and straight filament (SF) conformations of tau that were identical to those determined from AD cases. The PHFs and SFs are made of two C-shaped protofilaments, each containing a cross-β/β-helix motif. Similar to filaments from AD cases, most filaments from the DS cases adopted the PHF form, while a minority (approximately 20%) formed SFs. Samples from the youngest individual with no documented dementia had sparse tau deposits. To isolate tau for cryo-EM from this challenging sample we used a novel affinity-grid method involving a graphene oxide surface derivatized with anti-tau antibodies. This method improved isolation and revealed that primarily tau PHFs and a minor population of chronic traumatic encephalopathy type II-like filaments were present in this youngest case. These findings expand the similarities between AD and DS to the molecular level, providing insight into their related pathologies and the potential for targeting common tau filament folds by small-molecule therapeutics and diagnostics.

The amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC) of Guam is an endemic neurodegenerative disease that features widespread tau tangles, occasional α-synuclein Lewy bodies, and sparse β-amyloid (Aβ) plaques distributed in the central nervous system. Extensive studies of genetic or environmental factors have failed to identify a cause of ALS-PDC. Building on prior work describing the detection of tau and Aβ prions in Alzheimer's disease (AD) and Down syndrome brains, we investigated ALS-PDC brain samples for the presence of prions. We obtained postmortem frozen brain tissue from 26 donors from Guam with ALS-PDC or no neurological impairment and 71 non-Guamanian donors with AD or no neurological impairment. We employed cellular bioassays to detect the prion conformers of tau, α-synuclein, and Aβ proteins in brain extracts. In ALS-PDC brain samples, we detected high titers of tau and Aβ prions, but we did not detect α-synuclein prions in either cohort. The specific activity of tau and Aβ prions was increased in Guam ALS-PDC compared with sporadic AD. Applying partial least squares regression to all biochemical and prion infectivity measurements, we demonstrated that the ALS-PDC cohort has a unique molecular signature distinguishable from AD. Our findings argue that Guam ALS-PDC is a distinct double-prion disorder featuring both tau and Aβ prions.

Accumulation of filamentous aggregates of tau protein in the brain is a pathological hallmark of Alzheimer's disease (AD) and many other neurodegenerative tauopathies. The filaments adopt disease-specific cross-β amyloid conformations that self-propagate and are implicated in neuronal loss. Development of molecular diagnostics and therapeutics is of critical importance. However, mechanisms of small molecule binding to the amyloid core is poorly understood. We used cryo-electron microscopy to determine a 2.7 Å structure of AD patient-derived tau paired-helical filaments bound to the PET ligand GTP-1. The compound is bound stoichiometrically at a single site along an exposed cleft of each protofilament in a stacked arrangement matching the fibril symmetry. Multiscale modeling reveals pi-pi aromatic interactions that pair favorably with the small molecule-protein contacts, supporting high specificity and affinity for the AD tau conformation. This binding mode offers critical insight into designing compounds to target different amyloid folds found across neurodegenerative diseases.

Down syndrome (DS) is caused by the triplication of chromosome 21 and is the most common chromosomal disorder in humans. Those individuals with DS who live beyond age 40 y develop a progressive dementia that is similar to Alzheimer's disease (AD). Both DS and AD brains exhibit numerous extracellular amyloid plaques composed of Aβ and intracellular neurofibrillary tangles composed of tau. Since AD is a double-prion disorder, we asked if both Aβ and tau prions feature in DS. Frozen brains from people with DS, familial AD (fAD), sporadic AD (sAD), and age-matched controls were procured from brain biorepositories. We selectively precipitated Aβ and tau prions from DS brain homogenates and measured the number of prions using cellular bioassays. In brain extracts from 28 deceased donors with DS, ranging in age from 19 to 65 y, we found nearly all DS brains had readily measurable levels of Aβ and tau prions. In a cross-sectional analysis of DS donor age at death, we found that the levels of Aβ and tau prions increased with age. In contrast to DS brains, the levels of Aβ and tau prions in the brains of 37 fAD and sAD donors decreased as a function of age at death. Whether DS is an ideal model for assessing the efficacy of putative AD therapeutics remains to be determined.

The α-synuclein protein can adopt several different conformations that cause neurodegeneration. Different α-synuclein conformers cause at least three distinct α-synucleinopathies: multiple system atrophy (MSA), dementia with Lewy bodies (DLB), and Parkinson's disease (PD). In earlier studies, we transmitted MSA to transgenic (Tg) mice and cultured HEK cells both expressing mutant α-synuclein (A53T) but not to cells expressing α-synuclein (E46K). Now, we report that DLB is caused by a strain of α-synuclein prions that is distinct from MSA. Using cultured HEK cells expressing mutant α-synuclein (E46K), we found that DLB prions could be transmitted to these HEK cells. Our results argue that a third strain of α-synuclein prions likely causes PD, but further studies are needed to identify cells and/or Tg mice that express a mutant α-synuclein protein that is permissive for PD prion replication. Our findings suggest that other α-synuclein mutants should give further insights into α-synuclein prion replication, strain formation, and disease pathogenesis, all of which are likely required to discover effective drugs for the treatment of PD as well as the other α-synucleinopathies.

Tau aggregates contribute to neurodegenerative diseases, including frontotemporal dementia and Alzheimer's disease (AD). Although RNA promotes tau aggregation in vitro, whether tau aggregates in cells contain RNA is unknown. We demonstrate, in cell culture and mouse brains, that cytosolic and nuclear tau aggregates contain RNA with enrichment for small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). Nuclear tau aggregates colocalize with and alter the composition, dynamics, and organization of nuclear speckles, membraneless organelles involved in pre-mRNA splicing. Moreover, several nuclear speckle components, including SRRM2, mislocalize to cytosolic tau aggregates in cells, mouse brains, and brains of individuals with AD, frontotemporal dementia (FTD), and corticobasal degeneration (CBD). Consistent with these alterations, we observe that the presence of tau aggregates is sufficient to alter pre-mRNA splicing. This work identifies tau alteration of nuclear speckles as a feature of tau aggregation that may contribute to the pathology of tau aggregates.

Although it is not yet universally accepted that all neurodegenerative diseases (NDs) are prion disorders, there is little disagreement that Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia (FTD), and other NDs are a consequence of protein misfolding, aggregation, and spread. This widely accepted perspective arose from the prion hypothesis, which resulted from investigations on scrapie, a common transmissible disease of sheep and goats. The prion hypothesis argued that the causative infectious agent of scrapie was a novel proteinaceous pathogen devoid of functional nucleic acids and distinct from viruses, viroids, and bacteria. At the time, it seemed impossible that an infectious agent like the one causing scrapie could replicate and exist as diverse microbiological strains without nucleic acids. However, aggregates of a misfolded host-encoded protein, designated the prion protein (PrP), were shown to be the cause of scrapie as well as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker syndrome (GSS), which are similar NDs in humans. This review discusses historical research on diseases caused by PrP misfolding, emphasizing principles of pathogenesis that were later found to be core features of other NDs. For example, the discovery that familial prion diseases can be caused by mutations in PrP was important for understanding prion replication and disease susceptibility not only for rare PrP diseases but also for far more common NDs involving other proteins. We compare diseases caused by misfolding and aggregation of APP-derived Aβ peptides, tau, and α-synuclein with PrP prion disorders and argue for the classification of NDs caused by misfolding of these proteins as prion diseases. Deciphering the molecular pathogenesis of NDs as prion-mediated has provided new approaches for finding therapies for these intractable, invariably fatal disorders and has revolutionized the field.

Prions were initially discovered in studies of scrapie, a transmissible neurodegenerative disease (ND) of sheep and goats thought to be caused by slow viruses. Once scrapie was transmitted to rodents, it was discovered that the scrapie pathogen resisted inactivation by procedures that modify nucleic acids. Eventually, this novel pathogen proved to be a protein of 209 amino acids, which is encoded by a chromosomal gene. After the absence of a nucleic acid within the scrapie agent was established, the mechanism of infectivity posed a conundrum and eliminated a hypothetical virus. Subsequently, the infectious scrapie prion protein (PrPSc) enriched for β-sheet was found to be generated from the cellular prion protein (PrPC) that is predominantly α-helical. The post-translational process that features in nascent prion formation involves a templated conformational change in PrPC that results in an infectious copy of PrPSc. Thus, prions are proteins that adopt alternative conformations, which are self-propagating and found in organisms ranging from yeast to humans. Prions have been found in both Alzheimer's (AD) and Parkinson's (PD) diseases. Mutations in APP and α-synuclein genes have been shown to cause familial AD and PD. Recently, AD was found to be a double prion disorder: both Aβ and tau prions feature in this ND. Increasing evidence argues for α-synuclein prions as the cause of PD, multiple system atrophy, and Lewy body dementia.

Multiple system atrophy (MSA), a progressive neurodegenerative disease characterized by autonomic dysfunction and motor impairment, is caused by the self-templated misfolding of the protein α-synuclein. With no treatment currently available, we sought to characterize the spread of α-synuclein in a transgenic mouse model of MSA prion propagation to support drug discovery programs for synucleinopathies. Brain homogenates from MSA patient samples or mouse-passaged MSA were inoculated either by standard freehand injection or stereotactically into TgM83+/- mice, which express human α-synuclein with the A53T mutation. Following disease onset, brains from the mice were tested for biologically active α-synuclein prions using a cell-based assay and examined for α-synuclein neuropathology. Inoculation studies using homogenates prepared from brain regions lacking detectable α-synuclein neuropathology transmitted neurological disease to mice. Terminal animals contained similar concentrations of α-synuclein prions; however, a time-course study where mice were terminated every five days through disease progression revealed that the kinetics of α-synuclein prion replication in the mice were variable. Stereotactic inoculation into the thalamus reduced variability in disease onset in the mice, although incubation times were consistent with standard inoculations. Using human samples with and without neuropathological lesions, we observed that α-synuclein prion formation precedes neuropathology in the brain, suggesting that disease in patients is not limited to brain regions containing neuropathological lesions.

Glial cytoplasmic inclusions (GCIs) containing aggregated and hyperphosphorylated α-synuclein are the signature neuropathological hallmark of multiple system atrophy (MSA). Native α-synuclein can adopt a prion conformation that self-propagates and spreads throughout the brain ultimately resulting in neurodegeneration. A growing body of evidence argues that, in addition to oligodendrocytes, astrocytes contain α-synuclein inclusions in MSA and other α-synucleinopathies at advanced stages of disease. To study the role of astrocytes in MSA, we added MSA brain homogenate to primary cultures of astrocytes from transgenic (Tg) mouse lines expressing human α-synuclein. Astrocytes from four Tg lines, expressing either wild-type or mutant (A53T or A30P) human α-synuclein, propagated and accumulated α-synuclein prions. Furthermore, we found that MSA-infected astrocytes formed two morphologically distinct α-synuclein inclusions: filamentous and granular. Both types of cytoplasmic inclusions shared several features characteristic of α-synuclein inclusions in synucleinopathies: hyperphosphorylation preceded by aggregation, ubiquitination, thioflavin S-positivity, and co-localization with p62. Our findings demonstrate that human α-synuclein forms distinct inclusion morphologies and propagates within cultured Tg astrocytes exposed to MSA prions, indicating that α-synuclein expression determines the tropism of inclusion formation in certain cells. Thus, our work may prove useful in elucidating the role of astrocytes in the pathogenic mechanisms that feature in neurodegeneration caused by MSA prions.

The hallmarks of Alzheimer's disease (AD) are the accumulation of Aβ plaques and neurofibrillary tangles composed of hyperphosphorylated tau. We developed sensitive cellular assays using human embryonic kidney-293T cells to quantify intracellular self-propagating conformers of Aβ in brain samples from patients with AD or other neurodegenerative diseases. Postmortem brain tissue from patients with AD had measurable amounts of pathological Aβ conformers. Individuals over 80 years of age had the lowest amounts of prion-like Aβ and phosphorylated tau. Unexpectedly, the longevity-dependent decrease in self-propagating tau conformers occurred in spite of increasing amounts of total insoluble tau. When corrected for the abundance of insoluble tau, the ability of postmortem AD brain homogenates to induce misfolded tau in the cellular assays showed an exponential decrease with longevity, with a half-life of about one decade over the age range of 37 to 99 years. Thus, our findings demonstrate an inverse correlation between longevity in patients with AD and the abundance of pathological tau conformers. Our cellular assays can be applied to patient selection for clinical studies and the development of new drugs and diagnostics for AD.

Previously, we reported that intracranial inoculation of brain homogenate from multiple system atrophy (MSA) patient samples produces neurological disease in the transgenic (Tg) mouse model TgM83+/-, which uses the prion protein promoter to express human α-synuclein harboring the A53T mutation found in familial Parkinson's disease (PD). In our studies, we inoculated MSA and control patient samples into Tg mice constructed using a P1 artificial chromosome to express wild-type (WT), A30P, and A53T human α-synuclein on a mouse α-synuclein knockout background [Tg(SNCA+/+)Nbm, Tg(SNCA*A30P+/+)Nbm, and Tg(SNCA*A53T+/+)Nbm]. In contrast to studies using TgM83+/- mice, motor deficits were not observed by 330-400 days in any of the Tg(SNCA)Nbm mice after inoculation with MSA brain homogenates. However, using a cell-based bioassay to measure α-synuclein prions, we found brain homogenates from Tg(SNCA*A53T+/+)Nbm mice inoculated with MSA patient samples contained α-synuclein prions, whereas control mice did not. Moreover, these α-synuclein aggregates retained the biological and biochemical characteristics of the α-synuclein prions in MSA patient samples. Intriguingly, Tg(SNCA*A53T+/+)Nbm mice developed α-synuclein pathology in neurons and astrocytes throughout the limbic system. This finding is in contrast to MSA-inoculated TgM83+/- mice, which develop exclusively neuronal α-synuclein aggregates in the hindbrain that cause motor deficits with advanced disease. In a crossover experiment, we inoculated TgM83+/- mice with brain homogenate from two MSA patient samples or one control sample first inoculated, or passaged, in Tg(SNCA*A53T+/+)Nbm animals. Additionally, we performed the reverse experiment by inoculating Tg(SNCA*A53T+/+)Nbm mice with brain homogenate from the same two MSA samples and one control sample first passaged in TgM83+/- animals. The TgM83+/- mice inoculated with mouse-passaged MSA developed motor dysfunction and α-synuclein prions, whereas the mouse-passaged control sample had no effect. Similarly, the mouse-passaged MSA samples induced α-synuclein prion formation in Tg(SNCA*A53T+/+)Nbm mice, but the mouse-passaged control sample did not. The confirmed transmission of α-synuclein prions to a second synucleinopathy model and the ability to propagate prions between two distinct mouse lines while retaining strain-specific properties provides compelling evidence that MSA is a prion disease.

The hydrophobic Aβ peptide is highly aggregation prone; it first forms soluble oligomers, which then convert into the amyloid fibrils found in the cerebral plaques of Alzheimer's disease. It is generally understood that as the peptide concentration of Aβ increases, the fibrillization process is accelerated, but we examine the limits on this phenomenon. We found that once a threshold concentration of Aβ is exceeded, a stable oligomer is formed at the expense of fibril formation. The suppression of fibril formation was observed by amyloid-binding dye Thioflavin T and solution nuclear magnetic resonance (NMR). Small-angle X-ray scattering, size exclusion chromatography, and analytical ultracentrifugation demonstrated that Aβ peptides form a range of compact species, with a dimer being an early highly populated oligomer. Solution NMR allowed us to define the secondary structure of this Aβ dimer, which shows interlocking contacts between C-terminal peptide strands. Thus, we present a novel Aβ oligomer that resists conversion to fibrils and remains stable for more than one year.

Multiple system atrophy (MSA) is a rapidly progressive neurodegenerative disease arising from the misfolding and accumulation of the protein α-synuclein in oligodendrocytes, where it forms glial cytoplasmic inclusions (GCIs). Several years of studying synthetic α-synuclein fibrils has provided critical insight into the ability of α-synuclein to template endogenous protein misfolding, giving rise to fibrillar structures capable of propagating from cell to cell. However, more recent studies with MSA-derived α-synuclein aggregates have shown that they have a similar ability to undergo template-directed propagation, like PrP prions. Almost 20 years after α-synuclein was discovered as the primary component of GCIs, α-synuclein aggregates isolated from MSA patient samples were shown to infect cultured mammalian cells and also to transmit neurological disease to transgenic mice. These findings argue that α-synuclein becomes a prion in MSA patients. In this review, we discuss the in vitro and in vivo data supporting the recent classification of MSA as a prion disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease in humans and will pose a considerable challenge to healthcare systems in the coming years. Aggregation of the β-amyloid (Aβ) peptide within the brain is thought to be an initiating event in AD pathogenesis. Many recent studies in transgenic mice have provided evidence that Aβ aggregates become self-propagating during disease, leading to a cascade of protein aggregation in the brain, which may underlie the progressive nature of AD. The ability to self-propagate and the existence of distinct "strains" reveals that Aβ aggregates exhibit many properties indistinguishable from those of prions composed of PrPSc proteins. Here, we review the evidence that Aβ can become a prion during disease and discuss how Aβ prions may be important for understanding the pathobiology of AD.

In the neurodegenerative disease multiple system atrophy (MSA), α-synuclein misfolds into a self-templating conformation to become a prion. To compare the biological activity of α-synuclein prions in MSA and Parkinson's disease (PD), we developed nine α-synuclein-YFP cell lines expressing point mutations responsible for inherited PD. MSA prions robustly infected wild-type, A30P, and A53T α-synuclein-YFP cells, but they were unable to replicate in cells expressing the E46K mutation. Coexpression of the A53T and E46K mutations was unable to rescue MSA prion infection in vitro, establishing that MSA α-synuclein prions are conformationally distinct from the misfolded α-synuclein in PD patients. This observation may have profound implications for developing treatments for neurodegenerative diseases.

In multiple system atrophy (MSA), progressive neurodegeneration results from the protein α-synuclein misfolding into a self-templating prion conformation that spreads throughout the brain. MSA prions are transmissible to transgenic (Tg) mice expressing mutated human α-synuclein (TgM83+/-), inducing neurological disease following intracranial inoculation with brain homogenate from deceased patient samples. Noting the similarities between α-synuclein prions and PrP scrapie (PrPSc) prions responsible for Creutzfeldt-Jakob disease (CJD), we investigated MSA transmission under conditions known to result in PrPSc transmission. When peripherally exposed to MSA via the peritoneal cavity, hind leg muscle, and tongue, TgM83+/- mice developed neurological signs accompanied by α-synuclein prions in the brain. Iatrogenic CJD, resulting from PrPSc prion adherence to surgical steel instruments, has been investigated by incubating steel sutures in contaminated brain homogenate before implantation into mouse brain. Mice studied using this model for MSA developed disease, whereas wire incubated in control homogenate had no effect on the animals. Notably, formalin fixation did not inactivate α-synuclein prions. Formalin-fixed MSA patient samples also transmitted disease to TgM83+/- mice, even after incubating in fixative for 244 months. Finally, at least 10% sarkosyl was found to be the concentration necessary to partially inactivate MSA prions. These results demonstrate the robustness of α-synuclein prions to denaturation. Moreover, they establish the parallel characteristics between PrPSc and α-synuclein prions, arguing that clinicians should exercise caution when working with materials that might contain α-synuclein prions to prevent disease.

Misfolding of tau proteins into prions and their propagation along neural circuits are thought to result in neurodegeneration causing Alzheimer's disease, progressive supranuclear palsy, chronic traumatic encephalopathy, and other tauopathies. Little is known about the molecular processes mediating tau prion replication and spreading in different brain regions. Using transgenic (Tg) mice with a neuronal promoter driving expression of human mutant (P301S) tau, we found that tau prion formation and histopathologic deposition is largely restricted to the hindbrain. Unexpectedly, tau mRNA and protein levels did not differ between the forebrain and hindbrain, suggesting that other factors modulating the conversion of tau into a prion exist and are region specific. Using a cell-based prion propagation assay, we discovered that tau prion replication is suppressed by forebrain-derived inhibitors, one of which is sortilin, a lysosomal sorting receptor. We also show that sortilin expression is higher in the forebrain than the hindbrain across the life span of the Tg mice, suggesting that sortilin, at least in part, inhibits forebrain tau prion replication in vivo. Our findings provide evidence for selective vulnerability in mice resulting in highly regulated levels of tau prion propagation, thus affording a model for identification of additional molecules that could mitigate the levels of tau prions in human tauopathies.

Importance:Accumulation of the protein tau is a defining characteristic of several neurodegenerative diseases. Thorough assessment of transgenic (Tg) mouse lines that replicate this process is critical for establishing the models used for testing anti-tau therapeutics in vivo. Objective:To define a consistent mouse model of disease for use in future compound efficacy studies. Design, Setting, and Participants:In this time course study, cohorts of Tg and control mice were euthanized at defined intervals. Collected brains were bisected down the midline. One half was frozen and used to measure the tau prion content, while the other half was fixed for immunostaining with anti-tau antibodies. All mice were maintained at the Hunters Point Animal Facility at the University of California, San Francisco, and all experiments were performed at the Mission Bay Campus of the University of California, San Francisco. Study animals were PS19, homozygous and hemizygous Tg(MAPT*P301S), and B6/J mice. The study dates were August 9, 2010, to October 3, 2016. Main Outcomes and Measures:Tau prions were measured using a cell-based assay. Neuropathology was measured by determining the percentage area positive for immunostaining in defined brain regions. A separate cohort of mice was aged until each mouse developed neurological signs as determined by trained animal technicians to assess mortality. Results:A total of 1035 mice were used in this time course study. These included PS19 mice (51.2% [126 of 246] male and 48.8% [120 of 246] female), Tg(MAPT*P301S+/+) mice (52.3% [216 of 413] male, 43.8% [181 of 413] female, and 3.9% [16 of 413] undetermined), Tg(MAPT*P301S+/-) mice (51.8% [101 of 195] male and 48.2% [94 of 195] female), and B6/J mice (49.7% [90 of 181] male and 50.3% [91 of 181] female). While considerable interanimal variability in neuropathology, disease onset, and tau prion formation in the PS19 mice was observed, all 3 measures of disease were more uniform in the Tg(MAPT*P301S+/+) mice. Comparing tau prion formation in Tg(MAPT*P301S+/+) mice with B6/J controls, the 95% CIs for the 2 mouse lines diverged before age 5 weeks, and significant (P

The inherited prion protein (PrP) prion disorders, which include familial Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease, and fatal familial insomnia, constitute ∼10%-15% of all PrP prion disease cases in humans. Attempts to generate animal models of these disorders using transgenic mice expressing mutant PrP have produced variable results. Although many lines of mice develop spontaneous signs of neurological illness with accompanying prion disease-specific neuropathological changes, others do not. Furthermore, demonstrating the presence of protease-resistant PrP species and prion infectivity-two of the hallmarks of the PrP prion disorders-in the brains of spontaneously sick mice has proven particularly challenging. Here, we review the progress that has been made toward developing accurate mouse models of the inherited PrP prion disorders.

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

The experimental study of prions requires a model for their propagation. However, because prions lack nucleic acids, the simple techniques used to replicate bacteria and viruses are not applicable. For much of the history of prion research, time-consuming bioassays in animals were the only option for measuring infectivity. Although cell models and other in vitro tools for the propagation of prions have been developed, they all suffer limitations, and animal bioassays remain the gold standard for measuring infectivity. A wealth of recent data argues that both β-amyloid (Aβ) and tau proteins form prions that cause Alzheimer's disease, and α-synuclein forms prions that cause multiple system atrophy and Parkinson's disease. Cell and animal models that recapitulate some of the key features of cell-to-cell spreading and distinct strains of prions can now be measured.

The prototypical PrP prion diseases are invariably fatal, and the search for agents to treat them spans more than 30 years, with limited success. However, in the last few years, the application of high-throughput screening, medicinal chemistry, and pharmacokinetic optimization has led to important advances. The PrP prion inoculation paradigm provides a robust assay for testing therapeutic efficacy, and a dozen compounds have been reported that lead to meaningful extension in survival of prion-infected mice. Here, we review the history and recent progress in the field, focusing on studies validated in animal models. Based on screens in cells infected with mouse-passaged PrP prions, orally available compounds were generated that double or even triple the survival of mice infected with the same prion strain. Unfortunately, no compounds have yet shown efficacy against human prions. Nevertheless, the speed of the recent advances brings hope that an effective therapeutic can be developed. A successful treatment for any neurodegenerative disease would be a major achievement, and the growing understanding that the more common neurodegenerative diseases, including Alzheimer's and Parkinson's, progress by an analogous prion mechanism serves to highlight the importance of antiprion therapeutics.

Tau prions are thought to aggregate in the central nervous system, resulting in neurodegeneration. Among the tauopathies, Alzheimer's disease (AD) is the most common, whereas argyrophilic grain disease (AGD), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), Pick's disease (PiD), and progressive supranuclear palsy (PSP) are less prevalent. Brain extracts from deceased individuals with PiD, a neurodegenerative disorder characterized by three-repeat (3R) tau prions, were used to infect HEK293T cells expressing 3R tau fused to yellow fluorescent protein (YFP). Extracts from AGD, CBD, and PSP patient samples, which contain four-repeat (4R) tau prions, were transmitted to HEK293 cells expressing 4R tau fused to YFP. These studies demonstrated that prion propagation in HEK cells requires isoform pairing between the infecting prion and the recipient substrate. Interestingly, tau aggregates in AD and CTE, containing both 3R and 4R isoforms, were unable to robustly infect either 3R- or 4R-expressing cells. However, AD and CTE prions were able to replicate in HEK293T cells expressing both 3R and 4R tau. Unexpectedly, increasing the level of 4R isoform expression alone supported the propagation of both AD and CTE prions. These results allowed us to determine the levels of tau prions in AD and CTE brain extracts.

The biochemical and neuropathological properties of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) prions are faithfully maintained upon transmission to guinea pigs. However, primary and secondary transmissions of BSE and vCJD in guinea pigs result in long incubation periods of ∼450 and ∼350 days, respectively. To determine if the incubation periods of BSE and vCJD prions could be shortened, we generated transgenic (Tg) mice expressing guinea pig prion protein (GPPrP). Inoculation of Tg(GPPrP) mice with BSE and vCJD prions resulted in mean incubation periods of 210 and 199 days, respectively, which shortened to 137 and 122 days upon serial transmission. In contrast, three different isolates of sporadic CJD prions failed to transmit disease to Tg(GPPrP) mice. Many of the strain-specified biochemical and neuropathological properties of BSE and vCJD prions, including the presence of type 2 protease-resistant PrPSc, were preserved upon propagation in Tg(GPPrP) mice. Structural modeling revealed that two residues near the N-terminal region of α-helix 1 in GPPrP might mediate its susceptibility to BSE and vCJD prions. Our results demonstrate that expression of GPPrP in Tg mice supports the rapid propagation of BSE and vCJD prions and suggest that Tg(GPPrP) mice may serve as a useful paradigm for bioassaying these prion isolates.ImportanceVariant Creutzfeldt-Jakob disease (vCJD) and bovine spongiform encephalopathy (BSE) prions are two of the prion strains most relevant to human health. However, propagating these strains in mice expressing human or bovine prion protein has been difficult because of prolonged incubation periods or inefficient transmission. Here, we show that transgenic mice expressing guinea pig prion protein are fully susceptible to vCJD and BSE prions but not to sporadic CJD prions. Our results suggest that the guinea pig prion protein is a better, more rapid substrate than either bovine or human prion protein for propagating BSE and vCJD prions.

The amyloid-β (Aβ) peptide of Alzheimer's disease (AD) forms polymorphic fibrils on the micrometer and molecular scales. Various fibril growth conditions have been identified to cause polymorphism, but the intrinsic amino acid sequence basis for this polymorphism has been unclear. Several single-site mutations in the center of the Aβ sequence cause different disease phenotypes and fibrillization properties. The E22G (Arctic) mutant is found in familial AD and forms protofibrils more rapidly than wild-type Aβ. Here, we use solid-state NMR spectroscopy to investigate the structure, dynamics, hydration and morphology of Arctic E22G Aβ40 fibrils. (13)C, (15)N-labeled synthetic E22G Aβ40 peptides are studied and compared with wild-type and Osaka E22Δ Aβ40 fibrils. Under the same fibrillization conditions, Arctic Aβ40 exhibits a high degree of polymorphism, showing at least four sets of NMR chemical shifts for various residues, while the Osaka and wild-type Aβ40 fibrils show a single or a predominant set of chemical shifts. Thus, structural polymorphism is intrinsic to the Arctic E22G Aβ40 sequence. Chemical shifts and inter-residue contacts obtained from 2D correlation spectra indicate that one of the major Arctic conformers has surprisingly high structural similarity with wild-type Aβ42. (13)C-(1)H dipolar order parameters, (1)H rotating-frame spin-lattice relaxation times and water-to-protein spin diffusion experiments reveal substantial differences in the dynamics and hydration of Arctic, Osaka and wild-type Aβ40 fibrils. Together, these results strongly suggest that electrostatic interactions in the center of the Aβ peptide sequence play a crucial role in the three-dimensional fold of the fibrils, and by inference, fibril-induced neuronal toxicity and AD pathogenesis.

Because no drug exists that halts or even slows any neurodegenerative disease, developing effective therapeutics for any prion disorder is urgent. We recently reported two compounds (IND24 and IND81) with the 2-aminothiazole (2-AMT) chemical scaffold that almost doubled the incubation times in scrapie prion-infected, wild-type (wt) FVB mice when given in a liquid diet. Remarkably, oral prophylactic treatment with IND24 beginning 14 days prior to intracerebral prion inoculation extended survival from ∼120 days to over 450 days. In addition to IND24, we evaluated the pharmacokinetics and efficacy of five additional 2-AMTs; one was not followed further because its brain penetration was poor. Of the remaining four new 2-AMTs, IND114338 doubled and IND125 tripled the incubation times of RML-inoculated wt and Tg4053 mice overexpressing wt mouse prion protein (PrP), respectively. Neuropathological examination of the brains from untreated controls showed a widespread deposition of self-propagating, β-sheet-rich "scrapie" isoform (PrP(Sc)) prions accompanied by a profound astrocytic gliosis. In contrast, mice treated with 2-AMTs had lower levels of PrP(Sc) and associated astrocytic gliosis, with each compound resulting in a distinct pattern of deposition. Notably, IND125 prevented both PrP(Sc) accumulation and astrocytic gliosis in the cerebrum. Progressive central nervous system dysfunction in the IND125-treated mice was presumably due to the PrP(Sc) that accumulated in their brainstems. Disappointingly, none of the four new 2-AMTs prolonged the lives of mice expressing a chimeric human/mouse PrP transgene inoculated with Creutzfeldt-Jakob disease prions.

ObjectiveMutations in the gene encoding the prion protein (PrP) are responsible for approximately 10 to 15% of cases of prion disease in humans, including Creutzfeldt-Jakob disease (CJD). Here, we report on the discovery of a previously unreported C-terminal PrP mutation (A224V) in a CJD patient exhibiting a disease similar to the rare VV1 subtype of sporadic (s) CJD and investigate the role of this mutation in prion replication and transmission.MethodsWe generated transgenic (Tg) mice expressing human PrP with the V129 polymorphism and A224V mutation, denoted Tg(HuPrP,V129,A224V) mice, and inoculated them with different subtypes of sCJD prions.ResultsTransmission of sCJD VV2 or MV2 prions was accelerated in Tg(HuPrP,V129,A224V) mice, compared to Tg(HuPrP,V129) mice, with incubation periods of ∼110 and ∼210 days, respectively. In contrast, sCJD MM1 prions resulted in longer incubation periods in Tg(HuPrP,V129,A224V) mice, compared to Tg(HuPrP,V129) mice (∼320 vs. ∼210 days). Prion strain fidelity was maintained in Tg(HuPrP,V129,A224V) mice inoculated with sCJD VV2 or MM1 prions, despite the altered replication kinetics.InterpretationOur results suggest that A224V is a risk factor for prion disease and modulates the transmission behavior of CJD prions in a strain-specific manner, arguing that residues near the C-terminus of PrP are important for controlling the kinetics of prion replication.

Prions are proteins that adopt alternative conformations that become self-propagating; the PrP(Sc) prion causes the rare human disorder Creutzfeldt-Jakob disease (CJD). We report here that multiple system atrophy (MSA) is caused by a different human prion composed of the α-synuclein protein. MSA is a slowly evolving disorder characterized by progressive loss of autonomic nervous system function and often signs of parkinsonism; the neuropathological hallmark of MSA is glial cytoplasmic inclusions consisting of filaments of α-synuclein. To determine whether human α-synuclein forms prions, we examined 14 human brain homogenates for transmission to cultured human embryonic kidney (HEK) cells expressing full-length, mutant human α-synuclein fused to yellow fluorescent protein (α-syn140*A53T-YFP) and TgM83(+/-) mice expressing α-synuclein (A53T). The TgM83(+/-) mice that were hemizygous for the mutant transgene did not develop spontaneous illness; in contrast, the TgM83(+/+) mice that were homozygous developed neurological dysfunction. Brain extracts from 14 MSA cases all transmitted neurodegeneration to TgM83(+/-) mice after incubation periods of ∼120 d, which was accompanied by deposition of α-synuclein within neuronal cell bodies and axons. All of the MSA extracts also induced aggregation of α-syn*A53T-YFP in cultured cells, whereas none of six Parkinson's disease (PD) extracts or a control sample did so. Our findings argue that MSA is caused by a unique strain of α-synuclein prions, which is different from the putative prions causing PD and from those causing spontaneous neurodegeneration in TgM83(+/+) mice. Remarkably, α-synuclein is the first new human prion to be identified, to our knowledge, since the discovery a half century ago that CJD was transmissible.

Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)-YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson's disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.

Prions are proteins that adopt self-propagating aberrant folds. The self-propagating properties of prions are a direct consequence of their distinct structures, making the understanding of these structures and their biophysical interactions fundamental to understanding prions and their related diseases. The insolubility and inherent disorder of prions have made their structures difficult to study, particularly in the case of the infectious form of the mammalian prion protein PrP. Many investigators have therefore preferred to work with peptide fragments of PrP, suggesting that these peptides might serve as structural and functional models for biologically active prions. We have used x-ray fiber diffraction to compare a series of different-sized fragments of PrP, to determine the structural commonalities among the fragments and the biologically active, self-propagating prions. Although all of the peptides studied adopted amyloid conformations, only the larger fragments demonstrated a degree of structural complexity approaching that of PrP. Even these larger fragments did not adopt the prion structure itself with detailed fidelity, and in some cases their structures were radically different from that of pathogenic PrP(Sc).

An increasing number of studies argues that self-propagating protein conformations (i.e., prions) feature in the pathogenesis of several common neurodegenerative diseases. Mounting evidence contends that aggregates of the amyloid-β (Aβ) peptide become self-propagating in Alzheimer's disease (AD) patients. An important characteristic of prions is their ability to replicate distinct strains, the biological information for which is enciphered within different conformations of protein aggregates. To investigate whether distinct strains of Aβ prions can be discerned in AD patients, we performed transmission studies in susceptible transgenic mice using brain homogenates from sporadic or heritable (Arctic and Swedish) AD cases. Mice inoculated with the Arctic AD sample exhibited a pathology that could be distinguished from mice inoculated with the Swedish or sporadic AD samples, which was judged by differential accumulation of Aβ isoforms and the morphology of cerebrovascular Aβ deposition. Unlike Swedish AD- or sporadic AD-inoculated animals, Arctic AD-inoculated mice, like Arctic AD patients, displayed a prominent Aβ38-containing cerebral amyloid angiopathy. The divergent transmission behavior of the Arctic AD sample compared with the Swedish and sporadic AD samples was maintained during second passage in mice, showing that Aβ strains are serially transmissible. We conclude that at least two distinct strains of Aβ prions can be discerned in the brains of AD patients and that strain fidelity was preserved on serial passage in mice. Our results provide a potential explanation for the clinical and pathological heterogeneity observed in AD patients.

An increasing number of studies continue to show that the amyloid β (Aβ) peptide adopts an alternative conformation and acquires transmissibility; hence, it becomes a prion. Here, we report on the attributes of two strains of Aβ prions formed from synthetic Aβ peptides composed of either 40 or 42 residues. Modifying the conditions for Aβ polymerization increased both the protease resistance and prion infectivity compared with an earlier study. Approximately 150 d after intracerebral inoculation, both synthetic Aβ40 and Aβ42 prions produced a sustained rise in the bioluminescence imaging signal in the brains of bigenic Tg(APP23:Gfap-luc) mice, indicative of astrocytic gliosis. Pathological investigations showed that synthetic Aβ40 prions produced amyloid plaques containing both Aβ40 and Aβ42 in the brains of inoculated bigenic mice, whereas synthetic Aβ42 prions stimulated the formation of smaller, more numerous plaques composed predominantly of Aβ42. Synthetic Aβ40 preparations consisted of long straight fibrils; in contrast, the Aβ42 fibrils were much shorter. Addition of 3.47 mM (0.1%) SDS to the polymerization reaction produced Aβ42 fibrils that were indistinguishable from Aβ40 fibrils produced in the absence or presence of SDS. Moreover, the Aβ amyloid plaques in the brains of bigenic mice inoculated with Aβ42 prions prepared in the presence of SDS were similar to those found in mice that received Aβ40 prions. From these results, we conclude that the composition of Aβ plaques depends on the conformation of the inoculated Aβ polymers, and thus, these inocula represent distinct synthetic Aβ prion strains.

Prions are self-propagating protein conformers that cause a variety of neurodegenerative disorders in humans and animals. Mouse models have played key roles in deciphering the biology of prions and in assessing candidate therapeutics. The development of transgenic mice that form prions spontaneously in the brain has advanced our understanding of sporadic and genetic prion diseases. Furthermore, the realization that many proteins can become prions has necessitated the development of mouse models for assessing the potential transmissibility of common neurodegenerative diseases. As the universe of prion diseases continues to expand, mouse models will remain crucial for interrogating these devastating illnesses.

Bank voles are uniquely susceptible to a wide range of prion strains isolated from many different species. To determine if this enhanced susceptibility to interspecies prion transmission is encoded within the sequence of the bank vole prion protein (BVPrP), we inoculated Tg(M109) and Tg(I109) mice, which express BVPrP containing either methionine or isoleucine at polymorphic codon 109, with 16 prion isolates from 8 different species: humans, cattle, elk, sheep, guinea pigs, hamsters, mice, and meadow voles. Efficient disease transmission was observed in both Tg(M109) and Tg(I109) mice. For instance, inoculation of the most common human prion strain, sporadic Creutzfeldt-Jakob disease (sCJD) subtype MM1, into Tg(M109) mice gave incubation periods of ∼200 days that were shortened slightly on second passage. Chronic wasting disease prions exhibited an incubation time of ∼250 days, which shortened to ∼150 days upon second passage in Tg(M109) mice. Unexpectedly, bovine spongiform encephalopathy and variant CJD prions caused rapid neurological dysfunction in Tg(M109) mice upon second passage, with incubation periods of 64 and 40 days, respectively. Despite the rapid incubation periods, other strain-specified properties of many prion isolates--including the size of proteinase K-resistant PrPSc, the pattern of cerebral PrPSc deposition, and the conformational stability--were remarkably conserved upon serial passage in Tg(M109) mice. Our results demonstrate that expression of BVPrP is sufficient to engender enhanced susceptibility to a diverse range of prion isolates, suggesting that BVPrP may be a universal acceptor for prions.

PurposePrevious studies showed that lowering PrP(C) concomitantly reduced PrP(Sc) in the brains of mice inoculated with prions. We aimed to develop assays that measure PrP(C) on the surface of human T98G glioblastoma and IMR32 neuroblastoma cells. Using these assays, we sought to identify chemical hits, confirmed hits, and scaffolds that potently lowered PrP(C) levels in human brains cells, without lethality, and that could achieve drug concentrations in the brain after oral or intraperitoneal dosing in mice.MethodsWe utilized HTS ELISA assays to identify small molecules that lower PrP(C) levels by ≥30% on the cell surface of human glioblastoma (T98G) and neuroblastoma (IMR32) cells.ResultsFrom 44,578 diverse chemical compounds tested, 138 hits were identified by single point confirmation (SPC) representing 7 chemical scaffolds in T98G cells, and 114 SPC hits representing 6 scaffolds found in IMR32 cells. When the confirmed SPC hits were combined with structurally related analogs, >300 compounds (representing 6 distinct chemical scaffolds) were tested for dose-response (EC₅₀) in both cell lines, only studies in T98G cells identified compounds that reduced PrP(C) without killing the cells. EC₅₀ values from 32 hits ranged from 65 nM to 4.1 μM. Twenty-eight were evaluated in vivo in pharmacokinetic studies after a single 10 mg/kg oral or intraperitoneal dose in mice. Our results showed brain concentrations as high as 16.2 μM, but only after intraperitoneal dosing.ConclusionsOur studies identified leads for future studies to determine which compounds might lower PrP(C) levels in rodent brain, and provide the basis of a therapeutic for fatal disorders caused by PrP prions.

ObjectiveTo determine whether oral quinacrine increases survival in sporadic Creutzfeldt-Jakob disease (sCJD).MethodsThis NIH/National Institute on Aging-funded, double-blinded, placebo-controlled, stratified randomization treatment trial was conducted at the University of California, San Francisco from February 2005 through May 2009 (ClinicalTrials.gov, NCT00183092). Subjects were randomized (50:50) to quinacrine (300 mg daily) or placebo with inpatient evaluations at baseline, and planned for months 2, 6, and 12. Subjects returning for their month-2 visit were offered open-label quinacrine. The primary outcome was survival from randomization to month 2.ResultsOf 425 patients referred, 69 subjects enrolled, 54 subjects were randomized to active drug or placebo, and 51 subjects with sCJD were included in survival analyses. Survival for the randomized portion of the trial (first 2 months) showed no significant difference between the 2 groups (log-rank statistic, p = 0.43; Cox proportional relative hazard = 1.43, quinacrine compared with placebo, 95% confidence interval = 0.58, 3.53). The quinacrine-treated group, however, declined less on 2 of 3 functional scales, the modified Rankin and Clinical Dementia Rating, than the placebo group during the first 2 months.ConclusionThis interventional study provides Class I evidence that oral quinacrine at 300 mg per day does not improve 2-month survival of patients with sCJD, compared with placebo. Importantly, this study shows that double-blinded, placebo-controlled, randomized treatment trials are possible in prion disease. Furthermore, the quantitative data collected on the course of sCJD will be useful for future trials.Classification of evidenceThis study provides Class I evidence that quinacrine does not improve survival for people with sCJD when given orally at a dose of 300 mg per day for 2 months.

Prions are proteins that adopt alternative conformations, which become self-propagating. Increasing evidence argues that prions feature in the synucleinopathies that include Parkinson's disease, Lewy body dementia, and multiple system atrophy (MSA). Although TgM83(+/+) mice homozygous for a mutant A53T α-synuclein transgene begin developing CNS dysfunction spontaneously at ∼10 mo of age, uninoculated TgM83(+/-) mice (hemizygous for the transgene) remain healthy. To determine whether MSA brains contain α-synuclein prions, we inoculated the TgM83(+/-) mice with brain homogenates from two pathologically confirmed MSA cases. Inoculated TgM83(+/-) mice developed progressive signs of neurologic disease with an incubation period of ∼100 d, whereas the same mice inoculated with brain homogenates from spontaneously ill TgM83(+/+) mice developed neurologic dysfunction in ∼210 d. Brains of MSA-inoculated mice exhibited prominent astrocytic gliosis and microglial activation as well as widespread deposits of phosphorylated α-synuclein that were proteinase K sensitive, detergent insoluble, and formic acid extractable. Our results provide compelling evidence that α-synuclein aggregates formed in the brains of MSA patients are transmissible and, as such, are prions. The MSA prion represents a unique human pathogen that is lethal upon transmission to Tg mice and as such, is reminiscent of the prion causing kuru, which was transmitted to chimpanzees nearly 5 decades ago.

Prions are proteins that acquire alternative conformations that become self-propagating. Transformation of proteins into prions is generally accompanied by an increase in β-sheet structure and a propensity to aggregate into oligomers. Some prions are beneficial and perform cellular functions, whereas others cause neurodegeneration. In mammals, more than a dozen proteins that become prions have been identified, and a similar number has been found in fungi. In both mammals and fungi, variations in the prion conformation encipher the biological properties of distinct prion strains. Increasing evidence argues that prions cause many neurodegenerative diseases (NDs), including Alzheimer's, Parkinson's, Creutzfeldt-Jakob, and Lou Gehrig's diseases, as well as the tauopathies. The majority of NDs are sporadic, and 10% to 20% are inherited. The late onset of heritable NDs, like their sporadic counterparts, may reflect the stochastic nature of prion formation; the pathogenesis of such illnesses seems to require prion accumulation to exceed some critical threshold before neurological dysfunction manifests.

There is not a single pharmaceutical that halts or even slows any neurodegenerative disease. Mounting evidence shows that prions cause many neurodegenerative diseases, and arguably, scrapie and Creutzfeldt-Jakob disease prions represent the best therapeutic targets. We report here that the previously identified 2-aminothiazoles IND24 and IND81 doubled the survival times of scrapie-infected, wild-type mice. However, mice infected with Rocky Mountain Laboratory (RML) prions, a scrapie-derived strain, and treated with IND24 eventually exhibited neurological dysfunction and died. We serially passaged their brain homogenates in mice and cultured cells. We found that the prion strain isolated from IND24-treated mice, designated RML[IND24], emerged during a single passage in treated mice. Although RML prions infect both the N2a and CAD5 cell lines, RML[IND24] prions could only infect CAD5 cells. When passaged in CAD5 cells, the prions remained resistant to high concentrations of IND24. However, one passage of RML[IND24] prions in untreated mice restored susceptibility to IND24 in CAD5 cells. Although IND24 treatment extended the lives of mice propagating different prion strains, including RML, another scrapie-derived prion strain ME7, and chronic wasting disease, it was ineffective in slowing propagation of Creutzfeldt-Jakob disease prions in transgenic mice. Our studies demonstrate that prion strains can acquire resistance upon exposure to IND24 that is lost upon passage in mice in the absence of IND24. These data suggest that monotherapy can select for resistance, thus intermittent therapy with mixtures of antiprion compounds may be required to slow or stop neurodegeneration.

The microtubule-associated protein (MAP) tau has been implicated in the pathology of numerous neurodegenerative diseases. In the past decade, the hyperphosphorylated and aggregated states of tau protein have been important targets in the drug discovery field for the potential treatment of Alzheimer's disease. Although several compounds have been reported to reduce the hyperphosphorylated state of tau or impact the stabilization of tau, their therapeutic activities are remain to be validated. Recently, reduction of total cellular tau protein has emerged as an alternate intervention point for drug development and a potential treatment of tauopathies. We have developed and optimized homogenous assays, using the AlphaLISA and HTRF assay technologies, for the quantification of total cellular tau protein levels in the SH-SY5Y neuroblastoma cell line. The signal-to-basal ratios were 375 and 5.3, and the Z' factors were 0.67 and 0.60 for the AlphaLISA and HTRF tau assays, respectively. The clear advantages of these homogeneous tau assays over conventional total tau assays, such as ELISA and Western blot, are the elimination of plate wash steps and miniaturization of the assay into 1536-well plate format for the ultra-high-throughput screening of large compound libraries.

Recently, we described the aminothiazole lead (4-biphenyl-4-ylthiazol-2-yl)-(6-methylpyridin-2-yl)-amine (1), which exhibits many desirable properties, including excellent stability in liver microsomes, oral bioavailability of ∼40 %, and high exposure in the brains of mice. Despite its good pharmacokinetic properties, compound 1 exhibited only modest potency in mouse neuroblastoma cells overexpressing the disease-causing prion protein PrP(Sc) . Accordingly, we sought to identify analogues of 1 with improved antiprion potency in ScN2a-cl3 cells while retaining similar or superior properties. Herein we report the discovery of improved lead compounds such as (6-methylpyridin-2-yl)-[4-(4-pyridin-3-yl-phenyl)thiazol-2-yl]amine and cyclopropanecarboxylic acid (4-biphenylthiazol-2-yl)amide, which exhibit brain exposure/EC50 ratios at least tenfold greater than that of compound 1.

PurposeTo discover drugs lowering PrP(Sc) in prion-infected cultured neuronal cells that achieve high concentrations in brain to test in mouse models of prion disease and then treat people with these fatal diseases.MethodsWe tested 2-AMT analogs for EC50 and PK after a 40 mg/kg single dose and 40-210 mg/kg/day doses for 3 days. We calculated plasma and brain AUC, ratio of AUC/EC50 after dosing. We reasoned that compounds with high AUC/EC50 ratios should be good candidates going forward.ResultsWe evaluated 27 2-AMTs in single-dose and 10 in 3-day PK studies, of which IND24 and IND81 were selected for testing in mouse models of prion disease. They had high concentrations in brain after oral dosing. Absolute bioavailability ranged from 27-40%. AUC/EC50 ratios after 3 days were >100 (total) and 48-113 (unbound). Stability in liver microsomes ranged from 30->60 min. Ring hydroxylated metabolites were observed in microsomes. Neither was a substrate for the MDR1 transporter.ConclusionsIND24 and IND81 are active in vitro and show high AUC/EC50 ratios (total and unbound) in plasma and brain. These will be evaluated in mouse models of prion disease.

Prions, or infectious proteins, cause a class of uniformly fatal neurodegenerative diseases. Prions are composed solely of an aberrantly folded isoform (PrP(Sc)) of a normal cellular protein (PrP(C)). Shared sequence identity of PrP(Sc) with PrP(C) has limited the detection sensitivity of immunochemical assays, as antibodies specific for the disease-causing PrP(Sc) isoform have not been developed. Here we report the generation of three new monoclonal antibodies (MAbs) to PrP, which were isolated following immunization of Prnp(0/0) Balb/cJ mice with highly purified PrP(Sc) isolated from brain lipid rafts. Epitope mapping using synthetic PrP peptides revealed that the three MAbs bind different epitopes of PrP. The DRM1-31 MAb has a conformational epitope at the proposed binding site for the putative prion conversion co-factor "protein X." The DRM1-60 MAb binds a single linear epitope localized to the β2-α2 loop region of PrP, whereas DRM2-118 binds an epitope that includes sequences within the octarepeat region and near the site of N-terminal truncation of PrP(Sc) by proteinase K. Our novel anti-PrP MAbs with defined PrP epitopes may be useful in deciphering the conformational conversion of PrP(C) into PrP(Sc).

The aggregation and deposition of amyloid-β (Aβ) peptides are believed to be central events in the pathogenesis of Alzheimer's disease (AD). Inoculation of brain homogenates containing Aβ aggregates into susceptible transgenic mice accelerated Aβ deposition, suggesting that Aβ aggregates are capable of self-propagation and hence might be prions. Recently, we demonstrated that Aβ deposition can be monitored in live mice using bioluminescence imaging (BLI). Here, we use BLI to probe the ability of Aβ aggregates to self-propagate following inoculation into bigenic mice. We report compelling evidence that Aβ aggregates are prions by demonstrating widespread cerebral β-amyloidosis induced by inoculation of either purified Aβ aggregates derived from brain or aggregates composed of synthetic Aβ. Although synthetic Aβ aggregates were sufficient to induce Aβ deposition in vivo, they exhibited lower specific biological activity compared with brain-derived Aβ aggregates. Our results create an experimental paradigm that should lead to identification of self-propagating Aβ conformations, which could represent novel targets for interrupting the spread of Aβ deposition in AD patients.

A profound change in thinking about the etiologies of many neurodegenerative diseases has far-reaching implications for developing therapeutics.

A profound change in thinking about the etiologies of many neurodegenerative diseases has far-reaching implications for developing therapeutics.

We report here the transmission of human prions to 18 new transgenic (Tg) mouse lines expressing 8 unique chimeric human/mouse prion proteins (PrP). Extracts from brains of two patients, who died of sporadic Creutzfeldt-Jakob disease (sCJD), contained either sCJD(MM1) or sCJD(VV2) prion strains and were used for inocula. Mice expressing chimeric PrP showed a direct correlation between expression level and incubation period for sCJD(MM1) prions irrespective of whether the transgene encoded methionine (M) or valine (V) at polymorphic residue 129. Tg mice expressing chimeric transgenes encoding V129 were unexpectedly resistant to infection with sCJD(VV2) prions, and when transmission did occur, it was accompanied by a change in strain type. The transmission of sCJD(MM1) prions was modulated by single amino acid reversions of each human PrP residue in the chimeric sequence. Reverting human residue 137 in the chimeric transgene from I to M prolonged the incubation time for sCJD(MM1) prions by more than 100 days; structural analyses suggest a profound change in the orientation of amino acid side chains with the I→M mutation. These findings argue that changing the surface charge in this region of PrP greatly altered the interaction between PrP isoforms during prion replication. Our studies contend that strain-specified replication of prions is modulated by PrP sequence-specific interactions between the prion precursor PrP(C) and the infectious product PrP(Sc).