Publications by authors named "Stanley B Prusiner"

171 Publications

How an Infection of Sheep Revealed Prion Mechanisms in Alzheimer's Disease and Other Neurodegenerative Disorders.

Int J Mol Sci 2021 May 4;22(9). Epub 2021 May 4.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California San Francisco, San Francisco, CA 94158, USA.

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.
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http://dx.doi.org/10.3390/ijms22094861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125442PMC
May 2021

Water-Soluble Iridium Photoredox Catalyst for the Trifluoromethylation of Biomolecule Substrates in Phosphate Buffered Saline Solvent.

Org Lett 2021 05 30;23(10):3823-3827. Epub 2021 Apr 30.

Institute for Neurodegenerative Diseases (IND), Weill Institute for Neurosciences, University of California San Francisco, San Francisco, California 94158, United States.

The development of a water-soluble iridium catalyst enables the trifluoromethylation of polar small molecules and peptides in DMSO solution or aqueous media. The reaction was optimized in a microtiter plate format under ambient air, using commercial Langlois reagent as a CF radical source, blue LEDs for excitation, and using DPBS as solvent to provide up to 60% CF- peptide.
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http://dx.doi.org/10.1021/acs.orglett.1c00871DOI Listing
May 2021

Silver Benzoate Facilitates the Copper-Catalyzed C-N Coupling of Iodoazoles with Aromatic Nitrogen Heterocycles.

ACS Omega 2021 Apr 31;6(14):9804-9812. Epub 2021 Mar 31.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California San Francisco, San Francisco, California 94158, United States.

In the literature, C-N coupling methods for the reaction of iodo-oxazole with 2-pyridinone were found to be low yielding. C-N coupling using silver benzoate additives with CuI catalysts and 4,7-dimethoxy-1,10-phenanthroline ligands has been developed to afford synthetically useful yields of the desired heterobicycle product. The reaction conditions are applied to the coupling of a range of iodo-heterocycles with 2-pyridinone. The coupling of a variety of NH-containing heterocycles with 4-iodo-oxazole is also demonstrated. The use of 2-, 4-, or 5-iodo-oxazole allows for the coupling of pyridinone to each oxazole position.
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http://dx.doi.org/10.1021/acsomega.1c00458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8047741PMC
April 2021

Tau aggregates are RNA-protein assemblies that mislocalize multiple nuclear speckle components.

Neuron 2021 05 12;109(10):1675-1691.e9. Epub 2021 Apr 12.

Department of Biochemistry, University of Colorado, Boulder, CO, USA; Howard Hughes Medical Institute, University of Colorado, Boulder, CO, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.neuron.2021.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8141031PMC
May 2021

Prion biology: implications for Alzheimer's disease therapeutics.

Lancet Neurol 2020 10 16;19(10):802-803. Epub 2020 Sep 16.

Institute for Neurodegenerative Diseases, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Neurology, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

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http://dx.doi.org/10.1016/S1474-4422(20)30274-XDOI Listing
October 2020

Expanding spectrum of prion diseases.

Emerg Top Life Sci 2020 09;4(2):155-167

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, U.S.A.

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.
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http://dx.doi.org/10.1042/ETLS20200037DOI Listing
September 2020

Prion protein - mediator of toxicity in multiple proteinopathies.

Nat Rev Neurol 2020 04;16(4):187-188

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA.

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http://dx.doi.org/10.1038/s41582-020-0332-8DOI Listing
April 2020

Discovery of 4-Piperazine Isoquinoline Derivatives as Potent and Brain-Permeable Tau Prion Inhibitors with CDK8 Activity.

ACS Med Chem Lett 2020 Feb 30;11(2):127-132. Epub 2020 Jan 30.

Institute for Neurodegenerative Diseases (IND), UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, California 94518, United States.

Tau prions feature in the brains of patients suffering from Alzheimer's disease and other tauopathies. For the development of therapeutics that target the replication of tau prions, a high-content, fluorescence-based cell assay was developed. Using this high-content phenotypic screen for nascent tau prion formation, a 4-piperazine isoquinoline compound () was identified as a hit with an EC value of 390 nM and 0.04 . Analogs were synthesized using a hypothesis-based approach to improve potency and brain penetration resulting in compound (EC = 15 nM; = 0.63). We investigated the mechanism of action of this series and found that a small set of active compounds were also CDK8 inhibitors.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7025388PMC
February 2020

Kinetics of α-synuclein prions preceding neuropathological inclusions in multiple system atrophy.

PLoS Pathog 2020 02 4;16(2):e1008222. Epub 2020 Feb 4.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, California, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1008222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6999861PMC
February 2020

Replication of multiple system atrophy prions in primary astrocyte cultures from transgenic mice expressing human α-synuclein.

Acta Neuropathol Commun 2019 05 20;7(1):81. Epub 2019 May 20.

Institute for Neurodegenerative Diseases, UCSF Weill Institute for Neurosciences, University of California, San Francisco, Sandler Neurosciences Center, 675 Nelson Rising Lane, San Francisco, CA, 94158, USA.

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.
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http://dx.doi.org/10.1186/s40478-019-0703-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6526619PMC
May 2019

Aβ and tau prion-like activities decline with longevity in the Alzheimer's disease human brain.

Sci Transl Med 2019 05;11(490)

Institute for Neurodegenerative Diseases, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94158, USA.

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.
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http://dx.doi.org/10.1126/scitranslmed.aat8462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640844PMC
May 2019

Multiple system atrophy prions retain strain specificity after serial propagation in two different Tg(SNCA*A53T) mouse lines.

Acta Neuropathol 2019 03 28;137(3):437-454. Epub 2019 Jan 28.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA, USA.

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.
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http://dx.doi.org/10.1007/s00401-019-01959-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6454887PMC
March 2019

A long-lived Aβ oligomer resistant to fibrillization.

Biopolymers 2018 Aug 10;109(8):e23096. Epub 2018 Jan 10.

Department of Pharmaceutical Chemistry, University of California, San Francisco, California.

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.
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http://dx.doi.org/10.1002/bip.23096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039285PMC
August 2018

Structural heterogeneity and intersubject variability of Aβ in familial and sporadic Alzheimer's disease.

Proc Natl Acad Sci U S A 2018 01 8;115(4):E782-E791. Epub 2018 Jan 8.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA 94158;

Point mutations in the amyloid-β (Aβ) coding region produce a combination of mutant and WT Aβ isoforms that yield unique clinicopathologies in familial Alzheimer's disease (fAD) and cerebral amyloid angiopathy (fCAA) patients. Here, we report a method to investigate the structural variability of amyloid deposits found in fAD, fCAA, and sporadic AD (sAD). Using this approach, we demonstrate that mutant Aβ determines WT Aβ conformation through prion template-directed misfolding. Using principal component analysis of multiple structure-sensitive fluorescent amyloid-binding dyes, we assessed the conformational variability of Aβ deposits in fAD, fCAA, and sAD patients. Comparing many deposits from a given patient with the overall population, we found that intrapatient variability is much lower than interpatient variability for both disease types. In a given brain, we observed one or two structurally distinct forms. When two forms coexist, they segregate between the parenchyma and cerebrovasculature, particularly in fAD patients. Compared with sAD samples, deposits from fAD patients show less intersubject variability, and little overlap exists between fAD and sAD deposits. Finally, we examined whether E22G (Arctic) or E22Q (Dutch) mutants direct the misfolding of WT Aβ, leading to fAD-like plaques in vivo. Intracerebrally injecting mutant Aβ40 fibrils into transgenic mice expressing only WT Aβ induced the deposition of plaques with many biochemical hallmarks of fAD. Thus, mutant Aβ40 prions induce a conformation of WT Aβ similar to that found in fAD deposits. These findings indicate that diverse AD phenotypes likely arise from one or more initial Aβ prion conformations, which kinetically dominate the spread of prions in the brain.
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http://dx.doi.org/10.1073/pnas.1714966115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789926PMC
January 2018

Familial Parkinson's point mutation abolishes multiple system atrophy prion replication.

Proc Natl Acad Sci U S A 2018 01 26;115(2):409-414. Epub 2017 Dec 26.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA 94158;

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.
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http://dx.doi.org/10.1073/pnas.1719369115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777081PMC
January 2018

Evidence for sortilin modulating regional accumulation of human tau prions in transgenic mice.

Proc Natl Acad Sci U S A 2017 12 4;114(51):E11029-E11036. Epub 2017 Dec 4.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA 94158;

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.
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http://dx.doi.org/10.1073/pnas.1717193114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754811PMC
December 2017

A novel vector for transgenesis in the rat CNS.

Acta Neuropathol Commun 2017 Nov 21;5(1):84. Epub 2017 Nov 21.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, Sandler Neurosciences Center, 675 Nelson Rising Lane, San Francisco, CA, 94158, USA.

The larger brain of the rat enables a much greater repertoire of complex behaviors than mice, likely making rats preferential for investigating neurodegeneration. Because molecular tools for specific expression of transgenes in the rat brain are sparse, we chose Prnp encoding the prion protein (PrP) to develop a novel vector to drive transgene expression in the rat brain. We compared the rat Prnp sequence with mouse and Syrian hamster Prnp sequences, identifying conserved genetic elements and hypothesizing that these elements would be able to drive neuronal transgene expression. We investigated this by generating a vector termed RaPrnp that encompasses portions of the rat Prnp gene. Importantly, we replaced the rat Prnp open reading frame (ORF) with a cloning site for rapid and seamless In-Fusion cloning. To validate the in vivo neuronal specificity of the RaPrnp vector in rats, we generated stable RaPrnp-LacZ/enhanced green fluorescent protein (EGFP) transgenic (Tg) rat lines, which led to robust LacZ activity and high EGFP fluorescence in the central nervous system of embryos and adult animals. Next, we restored the rat Prnp ORF and generated multiple Tg(RaPrnp-PrP) lines, demonstrating that overexpression of Prnp accelerates the onset of scrapie. While the incubation time in wild-type (WT) rats was 175 ± 3 days post inoculation (dpi), one line, Tg2919, overexpressed RaPrP at 4.4-fold and exhibited a reduced incubation time of 149 ± 2 dpi. The second line, Tg2922, overexpressed RaPrP at 9.7-fold compared with WT animals and had an incubation time of 112 ± 0 dpi. Tg2922 rats inoculated with rat RML showed extensive vacuolation of the brainstem in contrast to WT and Tg2919 animals in which vacuolation was most prominent in the hippocampus and striatum as well as the motor and sensory cortices. It is possible that construction of Tg rats with modified phenotypes will prove more advantageous than mice for neurodegeneration studies.
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http://dx.doi.org/10.1186/s40478-017-0484-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697436PMC
November 2017

Kinetics of Human Mutant Tau Prion Formation in the Brains of 2 Transgenic Mouse Lines.

JAMA Neurol 2017 12;74(12):1464-1472

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco.

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 < .05) neuropathology in the hindbrain of 24-week-old mice was quantifiable.

Conclusions And Relevance: The assessment of disease progression using 3 criteria showed that disease onset in PS19 mice is too variable to obtain reliable measurements for drug discovery research. However, the reproducibility of tau prion formation in young Tg(MAPT*P301S+/+) mice establishes a rapid assay for compound efficacy in vivo.
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http://dx.doi.org/10.1001/jamaneurol.2017.2822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5822201PMC
December 2017

MSA prions exhibit remarkable stability and resistance to inactivation.

Acta Neuropathol 2018 01 28;135(1):49-63. Epub 2017 Aug 28.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, 675 Nelson Rising Lane, San Francisco, CA, 94158, USA.

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 (PrP) prions responsible for Creutzfeldt-Jakob disease (CJD), we investigated MSA transmission under conditions known to result in PrP 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 PrP 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 PrP and α-synuclein prions, arguing that clinicians should exercise caution when working with materials that might contain α-synuclein prions to prevent disease.
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http://dx.doi.org/10.1007/s00401-017-1762-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5756500PMC
January 2018

A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells.

Nat Chem 2017 09 3;9(9):874-881. Epub 2017 Apr 3.

Institute for Neurodegenerative Diseases, University of California, San Francisco, California 94143, USA.

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 tau 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 tau 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 tau in cells is packaged in a remarkably small peptide.
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http://dx.doi.org/10.1038/nchem.2754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5759337PMC
September 2017

Bioassays and Inactivation of Prions.

Cold Spring Harb Perspect Biol 2017 Aug 1;9(8). Epub 2017 Aug 1.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, California 94158.

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.
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http://dx.doi.org/10.1101/cshperspect.a023499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538415PMC
August 2017

α-Synuclein: Multiple System Atrophy Prions.

Cold Spring Harb Perspect Med 2018 07 2;8(7). Epub 2018 Jul 2.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, California 94158.

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.
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http://dx.doi.org/10.1101/cshperspect.a024588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5561534PMC
July 2018

β-Amyloid Prions and the Pathobiology of Alzheimer's Disease.

Cold Spring Harb Perspect Med 2018 05 1;8(5). Epub 2018 May 1.

Institute for Neurodegenerative Diseases, Departments of Neurology and of Biochemistry and Biophysics, Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, California 94143.

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 PrP 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.
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http://dx.doi.org/10.1101/cshperspect.a023507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554751PMC
May 2018

Experimental Models of Inherited PrP Prion Diseases.

Cold Spring Harb Perspect Med 2017 Nov 1;7(11). Epub 2017 Nov 1.

Institute for Neurodegenerative Diseases, Departments of Neurology and Biochemistry and Biophysics, Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, California 94143.

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.
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http://dx.doi.org/10.1101/cshperspect.a027151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513788PMC
November 2017

Developing Therapeutics for PrP Prion Diseases.

Cold Spring Harb Perspect Med 2017 Apr 3;7(4). Epub 2017 Apr 3.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, California 94143.

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.
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http://dx.doi.org/10.1101/cshperspect.a023747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378016PMC
April 2017

Tau prions from Alzheimer's disease and chronic traumatic encephalopathy patients propagate in cultured cells.

Proc Natl Acad Sci U S A 2016 12 28;113(50):E8187-E8196. Epub 2016 Nov 28.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, CA 94143;

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.
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http://dx.doi.org/10.1073/pnas.1616344113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167200PMC
December 2016

Guinea Pig Prion Protein Supports Rapid Propagation of Bovine Spongiform Encephalopathy and Variant Creutzfeldt-Jakob Disease Prions.

J Virol 2016 11 14;90(21):9558-9569. Epub 2016 Oct 14.

Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, California, USA Department of Neurology, University of California, San Francisco, San Francisco, California, USA Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, USA

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 PrP, 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.

Importance: Variant 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.
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http://dx.doi.org/10.1128/JVI.01106-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068510PMC
November 2016

Structural Polymorphism of Alzheimer's β-Amyloid Fibrils as Controlled by an E22 Switch: A Solid-State NMR Study.

J Am Chem Soc 2016 08 28;138(31):9840-52. Epub 2016 Jul 28.

Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.

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.
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http://dx.doi.org/10.1021/jacs.6b03715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5149419PMC
August 2016

FoxO3 regulates neuronal reprogramming of cells from postnatal and aging mice.

Proc Natl Acad Sci U S A 2016 07 11;113(30):8514-9. Epub 2016 Jul 11.

Institute for Stem Cell Biology and Regenerative Medicine and Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305;

We and others have shown that embryonic and neonatal fibroblasts can be directly converted into induced neuronal (iN) cells with mature functional properties. Reprogramming of fibroblasts from adult and aged mice, however, has not yet been explored in detail. The ability to generate fully functional iN cells from aged organisms will be particularly important for in vitro modeling of diseases of old age. Here, we demonstrate production of functional iN cells from fibroblasts that were derived from mice close to the end of their lifespan. iN cells from aged mice had apparently normal active and passive neuronal membrane properties and formed abundant synaptic connections. The reprogramming efficiency gradually decreased with fibroblasts derived from embryonic and neonatal mice, but remained similar for fibroblasts from postnatal mice of all ages. Strikingly, overexpression of a transcription factor, forkhead box O3 (FoxO3), which is implicated in aging, blocked iN cell conversion of embryonic fibroblasts, whereas knockout or knockdown of FoxO3 increased the reprogramming efficiency of adult-derived but not of embryonic fibroblasts and also enhanced functional maturation of resulting iN cells. Hence, FoxO3 has a central role in the neuronal reprogramming susceptibility of cells, and the importance of FoxO3 appears to change during development.
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http://dx.doi.org/10.1073/pnas.1607079113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968717PMC
July 2016

Towards authentic transgenic mouse models of heritable PrP prion diseases.

Acta Neuropathol 2016 10 28;132(4):593-610. Epub 2016 Jun 28.

Institute for Neurodegenerative Diseases, University of California, San Francisco, 675 Nelson Rising Lane, San Francisco, CA, 94143-0518, USA.

Attempts to model inherited human prion disorders such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS) disease, and fatal familial insomnia (FFI) using genetically modified mice have produced disappointing results. We recently demonstrated that transgenic (Tg) mice expressing wild-type bank vole prion protein (BVPrP) containing isoleucine at polymorphic codon 109 develop a spontaneous neurodegenerative disorder that exhibits many of the hallmarks of prion disease. To determine if mutations causing inherited human prion disease alter this phenotype, we generated Tg mice expressing BVPrP containing the D178N mutation, which causes FFI; the E200K mutation, which causes familial CJD; or an anchorless PrP mutation similar to mutations that cause GSS. Modest expression levels of mutant BVPrP resulted in highly penetrant spontaneous disease in Tg mice, with mean ages of disease onset ranging from ~120 to ~560 days. The brains of spontaneously ill mice exhibited prominent features of prion disease-specific neuropathology that were unique to each mutation and distinct from Tg mice expressing wild-type BVPrP. An ~8-kDa proteinase K-resistant PrP fragment was found in the brains of spontaneously ill Tg mice expressing either wild-type or mutant BVPrP. The spontaneously formed mutant BVPrP prions were transmissible to Tg mice expressing wild-type or mutant BVPrP as well as to Tg mice expressing mouse PrP. Thus, Tg mice expressing mutant BVPrP exhibit many of the hallmarks of heritable prion disorders in humans including spontaneous disease, protease-resistant PrP, and prion infectivity.
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http://dx.doi.org/10.1007/s00401-016-1585-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5152593PMC
October 2016
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