Prions are proteins that adopt alternative conformations that become self-propagating; the PrPSc 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.

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.neurodegeneration | bioluminescence imaging | seeding | proteinopathies

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.

The clinical and pathological differences between synucleinopathies such as Parkinson's disease and multiple system atrophy have been postulated to stem from unique strains of α-synuclein aggregates, akin to what occurs in prion diseases. Here, we demonstrate that inoculation of transgenic mice with different strains of recombinant or brain-derived α-synuclein aggregates produces clinically and pathologically distinct diseases. Strain-specific differences were observed in the signs of neurological illness, time to disease onset, morphology of cerebral α-synuclein deposits, and the conformational properties of the induced aggregates. Moreover, different strains targeted distinct cellular populations and cell types within the brain, recapitulating the selective targeting observed between human synucleinopathies. Strain-specific clinical, pathological, and biochemical differences were faithfully maintained upon serial passaging, implying that αsynuclein propagates via prion-like conformational templating. Thus, pathogenic α-synuclein exhibits key hallmarks of prion strains, providing evidence that disease heterogeneity among the synucleinopathies is caused by distinct α-synuclein strains.Parkinson's disease (PD) and related diseases, including dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), are progressive neurodegenerative disorders. The brains of PD, DLB, and MSA patients contain intracellular inclusions composed of aggregated α-synuclein (α-syn). Thus, these diseases are commonly referred to as αsynucleinopathies, or simply synucleinopathies 1 . α-Syn is a 140-amino acid cytoplasmic protein that is found within presynaptic nerve terminals and is involved in the assembly of SNARE complexes 2 . In disease, α-syn polymerizes into insoluble β-sheet-rich protein aggregates that become phosphorylated at residue Ser129 and deposit within the central nervous system 3,4 . α-Syn is believed to play a central pathogenic role in the synucleinopathies since mutation of the gene encoding α-syn causes early-onset PD 5 .There is mounting evidence that α-syn becomes "prion-like" during disease, leading to a progressive cell-to-cell spreading of protein aggregates within the brain 6 . Prions are selfpropagating protein aggregates that cause neurodegenerative disorders such as Creutzfeldt-Jakob disease in humans and scrapie in sheep. Prion replication and spreading is thought to occur via a template-directed refolding mechanism, in which aggregated prion protein (PrP) catalyzes the conformational conversion of properly-folded PrP into additional copies of the misfolded form 7 . Similar to the experimental transmission of prion disease, injection of mice with pre-formed α-syn aggregates induces the aggregation and deposition of α-syn within the brain and, in some instances, accelerates the onset of neurological illness [8][9][10][11][12][13] . The prionlike behavior of α-syn aggregates provides a potential molecular explanation for the progressive nature of PD and related synucleinopathies.The synucleinopathies ar...

Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing selfpropagation. 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.

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 ADor 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.neurodegeneration | bioluminescence imaging | seeding | proteinopathies

The cellular prion protein, PrPC, is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS‐expressed Doppel or internally deleted PrP (‘ΔPrP’). This paradigm has facilitated mapping of activity determinants in PrPC and implicated a cryptic PrPC‐like protein, ‘π’. Shadoo (Sho) is a hypothetical GPI‐anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N‐terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrPC, but is low in cerebellar granular neurons (CGNs) containing PrPC and high in PrPC‐deficient dendritic processes. In Prnp0/0 CGNs, Sho transgenes were PrPC‐like in their ability to counteract neurotoxic effects of either Doppel or ΔPrP. Additionally, prion‐infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for π, and since it engenders a PrPC‐like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology.

Significance As people live longer, the prevalence and economic impact of neurodegenerative diseases rise. No cures or effective treatments exist for any of these fatal disorders, so identifying potential therapeutics that extend survival in animal models is vital. Many neurodegenerative illnesses have been shown to be caused by the accumulation of self-propagating misfolded proteins—the hallmark of prion diseases. We report the efficacy of 2-aminothiazoles, which were identified in cell-based screens as antiprion compounds, in extending the lives of prion-infected animals. Efficacy was limited by the development of drug-resistant prions, which is likely to have important implications for creating therapeutics in many different neurodegenerative diseases.