Prion Conformation Research Articles

Cryo-EM structures reveal tau filaments from Down syndrome adopt Alzheimer’s disease fold

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.

Guam ALS-PDC is a distinct double-prion disorder featuring both tau and Aβ prions

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.

The human ribosome-associated complex suppresses prion formation in yeast.

Many human diseases are associated with the misfolding of amyloidogenic proteins. Understanding the mechanisms cells employ to ensure the integrity of the proteome is therefore a crucial step in the development of potential therapeutic interventions. Yeast cells possess numerous prion-forming proteins capable of adopting amyloid conformations, possibly as an epigenetic mechanism to cope with changing environmental conditions. The ribosome-associated complex (RAC), which docks near the ribosomal polypeptide exit tunnel and recruits the Hsp70 Ssb to chaperone nascent chains, can moderate the acquisition of these amyloid conformations in yeast. Here we examine the ability of the human RAC chaperone proteins Mpp11 and Hsp70L1 to function in place of their yeast RAC orthologues Zuo1 and Ssz1 in yeast lacking endogenous RAC and investigate the extent to which the human orthologues can perform RAC chaperone activities in yeast. We found that the Mpp11/Hsp70L1 complex can partially correct the growth defect seen in RAC-deficient yeast cells, although yeast/human hetero species complexes were variable in this ability. The proportion of cells in which the Sup35 protein undergoes spontaneous conversion to a [PSI+ ] prion conformation, which is increased in the absence of RAC, was reduced by the presence of the human RAC complex. However, the toxicity in yeast from expression of a pathogenically expanded polyQ protein was unable to be countered by the human RAC chaperones. This yeast system can serve as a facile model for studying the extent to which the human RAC chaperones contribute to combating cotranslational misfolding of other mammalian disease-associated proteins.

The E46K mutation modulates α-synuclein prion replication in transgenic mice.

In multiple system atrophy (MSA), the α-synuclein protein misfolds into a self-templating prion conformation that spreads throughout the brain, leading to progressive neurodegeneration. While the E46K mutation in α-synuclein causes familial Parkinson's disease (PD), we previously discovered that this mutation blocks in vitro propagation of MSA prions. Recent studies by others indicate that α-synuclein adopts a misfolded conformation in MSA in which a Greek key motif is stabilized by an intramolecular salt bridge between residues E46 and K80. Hypothesizing that the E46K mutation impedes salt bridge formation and, therefore, exerts a selective pressure that can modulate α-synuclein strain propagation, we asked whether three distinct α-synuclein prion strains could propagate in TgM47+/- mice, which express human α-synuclein with the E46K mutation. Following intracranial injection of these strains, TgM47+/- mice were resistant to MSA prion transmission, whereas recombinant E46K preformed fibrils (PFFs) transmitted neurological disease to mice and induced the formation of phosphorylated α-synuclein neuropathology. In contrast, heterotypic seeding following wild-type (WT) PFF-inoculation resulted in preclinical α-synuclein prion propagation. Moreover, when we inoculated TgM20+/- mice, which express WT human α-synuclein, with E46K PFFs, we observed delayed transmission kinetics with an incomplete attack rate. These findings suggest that the E46K mutation constrains the number of α-synuclein prion conformations that can propagate in TgM47+/- mice, expanding our understanding of the selective pressures that impact α-synuclein prion replication.

Multiple system atrophy prions transmit neurological disease to mice expressing wild-type human α-synuclein.

In multiple system atrophy (MSA), the protein α-synuclein misfolds into a prion conformation that self-templates and causes progressive neurodegeneration. While many point mutations in the α-synuclein gene, SNCA, have been identified as the cause of heritable Parkinson's disease (PD), none have been identified as causing MSA. To examine whether MSA prions can transmit disease to mice expressing wild-type (WT) human α-synuclein, we inoculated transgenic (Tg) mice denoted TgM20+/- with brain homogenates prepared from six different deceased MSA patients. All six samples transmitted CNS disease to the mice, with an average incubation period of ~280days. Interestingly, TgM20+/- female mice developed disease > 60days earlier than their male counterparts. Brains from terminal mice contained phosphorylated α-synuclein throughout the hindbrain, consistent with the distribution of α-synuclein inclusions in MSA patients. In addition, using our α-syn-YFP cell lines, we detected α-synuclein prions in brain homogenates prepared from terminal mice that retained MSA strain properties. To our knowledge, the studies described here are the first to show that MSA prions transmit neurological disease to mice expressing WT SNCA and that the rate of transmission is sex dependent. By comparison, TgM20+/- mice inoculated with WT preformed fibrils (PFFs) developed severe neurological disease in ~210days and exhibited robust α-synuclein neuropathology in both limbic regions and the hindbrain. Brain homogenates from these animals exhibited biological activities that are distinct from those found in MSA-inoculated mice when tested in the α-syn-YFP cell lines. Differences between brains from MSA-inoculated and WT PFF-inoculated mice potentially argue that α-synuclein prions from MSA patients are distinct from the PFF inocula and that PFFs do not replicate MSA strain biology.

A Novel Connection Between [PSI+] Prion Formation and RNA Splicing in Saccharomyces cerevisiae

Relatively new studies on prions in Saccharomyces cerevisiae show that some protein prion conformations may be beneficial to cell survival in response to environmental stress. Sup35, a translation termination release factor, forms the prion [PSI+]. When [PSI+] forms, translation termination efficiency decreases, which has been shown to result in the alteration of the abundance of cellular proteins, including splicing factor abundance. Therefore, we are testing the hypothesis that [PSI+] prion formation alters splicing efficiency in yeast. First, we employed quantitative RT‐PCR and revealed that splicing efficiency improves upon [PSI+] prion formation. The proposed mechanisms for the effect of [PSI+] on protein abundance include nonsense suppression and non‐stop decay. Non‐stop decay would lead to degradation of the mRNA encoding a splicing protein and therefore would reduce splicing factor protein levels. Non‐sense suppression, a result of translation readthrough, generates a C‐terminally extended protein, which could alter the function of splicing factors, or alter protein levels. We are initiating RT‐PCR and Western Blot analyses to test the contribution of the two mechanisms on splicing factor abundance. To further test the relationship between RNA splicing and [PSI+] prion formation, we used knockout PCR to create splicing factor deletion mutations in a [psi‐] strain, as well as a weak [PSI+]SC37 strain or a strong [PSI+]Str strain. We found that the [PSI+]SC37 conformation can suppress the growth defect in a yeast strain with a deletion of the gene encoding SNU66, a component of the tri‐snRNP. Similarly, the [PSI+]SC37conformation can suppress the growth defect in a yeast strain with a deletion of the MUD2gene, which encodes a gene important for commitment complex formation. In addition, we found that [PSI+]SC37can rescue the splicing defect observed in a mud2Δstrain, consistent with the suppressive growth phenotype. Together, the results show that [PSI+] formation impacts RNA splicing in S. cerevisiae and our future work will focus on determining the mechanisms that underlie this effect.

Chronic Wasting Disease (CWD) in Cervids and the Consequences of a Mutable Protein Conformation.

Chronic wasting disease (CWD) is a prion disease of cervids (deer, elk, moose, etc.). It spreads readily from CWD-contaminated environments and among wild cervids. As of 2022, North American CWD has been found in 29 states, four Canadian provinces and South Korea. The Scandinavian form of CWD originated independently. Prions propagate their pathology by inducing a natively expressed prion protein (PrPC) to adopt the prion conformation (PrPSc). PrPC and PrPSc differ solely in their conformation. Like other prion diseases, transmissible CWD prions can arise spontaneously. The CWD prions can respond to selection pressures resulting in the emergence of new strain phenotypes. Annually, 11.5 million Americans hunt and harvest nearly 6 million deer, indicating that CWD is a potential threat to an important American food source. No tested CWD strain has been shown to be zoonotic. However, this may not be true for emerging strains. Should a zoonotic CWD strain emerge, it could adversely impact the hunting economy and game meat consumers.

Detecting Differences in Prion Protein Conformation by Quantifying Methionine Oxidation.

A prion’s pathogenic character is enciphered in its conformation, which also defines the chemical environments of its amino acids. Differences in chemical environments influence the reactivity of amino acid side chains, in a conformation-dependent manner. Chemical oxidation of susceptible methionines would identify those methionines on the surface of a prion, which would reveal conformation-dependent information. We identified a set of methionine-containing peptides derived from the tryptic, chymotryptic, or tryptic/chymotryptic digestion of recombinant prion protein and the Sc237 strain of hamster-adapted scrapie. We developed a multiple reaction monitoring-based method of quantifying the extent of the methionine oxidation in those peptides. This approach can be used to define a prion’s conformation and to distinguish among prion strains, which is an important component of food safety.

The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer

Conversion of normal prion protein (PrPC) to the pathogenic PrPSc conformer is central to prion diseases such as Creutzfeldt–Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrPC-to-PrPSc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrPSc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrPSc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrPSc mutants were significantly different from each other and from that of WT recPrPSc. Together, our results support that the NPR greatly influences PrPC-to-PrPSc conversion, but it is not essential for the generation of PrPSc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.

Collusion of α-Synuclein and Aβ aggravating co-morbidities in a novel prion-type mouse model

BackgroundThe misfolding of host-encoded proteins into pathological prion conformations is a defining characteristic of many neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and Lewy body dementia. A current area of intense study is the way in which the pathological deposition of these proteins might influence each other, as various combinations of co-pathology between prion-capable proteins are associated with exacerbation of disease. A spectrum of pathological, genetic and biochemical evidence provides credence to the notion that amyloid β (Aβ) accumulation can induce and promote α-synuclein pathology, driving neurodegeneration.MethodsTo assess the interplay between α-synuclein and Aβ on protein aggregation kinetics, we crossed mice expressing human α-synuclein (M20) with APPswe/PS1dE9 transgenic mice (L85) to generate M20/L85 mice. We then injected α-synuclein preformed fibrils (PFFs) unilaterally into the hippocampus of 6-month-old mice, harvesting 2 or 4 months later.ResultsImmunohistochemical analysis of M20/L85 mice revealed that pre-existing Aβ plaques exacerbate the spread and deposition of induced α-synuclein pathology. This process was associated with increased neuroinflammation. Unexpectedly, the injection of α-synuclein PFFs in L85 mice enhanced the deposition of Aβ; whereas the level of Aβ deposition in M20/L85 bigenic mice, injected with α-synuclein PFFs, did not differ from that of mice injected with PBS.ConclusionsThese studies reveal novel and unexpected interplays between α-synuclein pathology, Aβ and neuroinflammation in mice that recapitulate the pathology of Alzheimer’s disease and Lewy body dementia.

COVID-19 RNA Based Vaccines and the Risk of Prion Disease

Development of new vaccine technology has been plagued with problems in the past. The current RNA based SARSCoV-2 vaccines were approved in the US using an emergency order without extensive long term safety testing. In this paper the Pfizer COVID-19 vaccine was evaluated for the potential to induce prion-based disease in vaccine recipients. The RNA sequence of the vaccine as well as the spike protein target interaction were analyzed for the potential to convert intracellular RNA binding proteins TAR DNA binding protein (TDP-43) and Fused in Sarcoma (FUS) into their pathologic prion conformations. The results indicate that the vaccine RNA has specific sequences that may induce TDP-43 and FUS to fold into their pathologic prion confirmations. In the current analysis a total of sixteen UG tandem repeats (ΨGΨG) were identified and additional UG (ΨG) rich sequences were identified. Two GGΨA sequences were found. Potential G Quadruplex sequences are possibly present but a more sophisticated computer program is needed to verify these. Furthermore, the spike protein, created by the translation of the vaccine RNA, binds angiotensin converting enzyme 2 (ACE2), a zinc containing enzyme. This interaction has the potential to increase intracellular zinc. Zinc ions have been shown to cause the transformation of TDP-43 to its pathologic prion configuration. The folding of TDP-43 and FUS into their pathologic prion confirmations is known to cause ALS, front temporal lobar degeneration, Alzheimer’s disease and other neurological degenerative diseases. The enclosed finding as well as additional potential risks leads the author to believe that regulatory approval of the RNA based vaccines for SARS-CoV-2 was premature and that the vaccine may cause much more harm than benefit.

Quantifying the Role of Lysine in Prion Replication by Nano-LC Mass Spectrometry and Bioassay.

Prions propagate by a template driven process, inducing the normal cellular isoform (PrPC) to adopt the prion (PrPSc) conformation. In PrPC, the positions of lysines are highly conserved and strongly influence prion propagation. In this study, covalent modification was used to quantitate the role of lysines in the PrPSc template that drives prion replication. The ε-amino group of lysines in the PrPSc (hamster-adapted scrapie Sc237) template was acetylated by either acetic anhydride (Ac2O) or the N-hydroxysuccinimide ester of acetic acid (Ac-NHS). The extent of lysine acetylation in PrPSc was quantitated by mass spectrometry or Western blot-based analysis. Identical samples were bioassayed to quantitate the loss of infectivity associated with lysine acetylation. The reduction of infectivity at the highest reagent concentration was approximately 90% (∼10-fold). Ten of the eleven prion lysines were acetylated to a greater extent (25−400-fold) than the observed loss of infectivity. Only one lysine, at position 220 (K220), had a reactivity that is consistent with the loss of infectivity. Although lysines are highly conserved and play a crucial role in converting PrPC into the PrPSc conformation, once that conformation is adopted, the lysines present in the PrPSc template play only a limited role in prion replication. In principle, this approach could be used to clarify the role of other amino acids in the replication of prions and other prion-like protein misfolding diseases.

Prion biology: implications for Alzheimer's disease therapeutics

The deposition of extracellular amyloid β is one of the hallmarks of Alzheimer's disease and cerebral amyloid angiopathy. The formation of amyloid fibrils is one manifestation of the misfolding and aggregation of amyloid β into a prion conformation, which then induces the misfolding of additional copies of amyloid β in a self-propagating process. Prions were first discovered for the PrP protein,1,2 and are the cause of Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. In 1994, soon after the discovery of PrP-prions, self-propagating forms of unrelated proteins in yeast and other fungi were found to have beneficial, rather than pathological, roles in these organisms.

Prions: Roles in Development and Adaptive Evolution.

Prions are often considered as anomalous proteins associated primarily with disease rather than as a fundamental source of diversity within biological proteomes. Whereas this longstanding viewpoint has its genesis in the discovery of the original namesake prions as causative agents of several complex diseases, the underlying assumption of a strict disease basis for prions could not be further from the truth. Prions and the spectrum of functions they comprise, likely represent one of the largest paradigm shifts concerning molecular-encoded phenotypic diversity since identification of DNA as the principle molecule of heredity. The ability of prions to recruit similar proteins to alternate conformations may engender a reservoir of diversity supplementing the genetic diversity resulting from stochastic mutations of DNA and subsequent natural selection. Here we present several currently known prions and how many of their functions as well as modes of transmission are intricately linked to adaptation from an evolutionary perspective. Further, the stability of some prion conformations across generations indicates that heritable prion-based adaptation is a reality.

A General Mass Spectrometry-Based Method of Quantitating Prion Polymorphisms from Heterozygous Chronic Wasting Disease-Infected Cervids.

Chronic wasting disease (CWD) is the only prion disease naturally transmitted among farmed and free-ranging cervids (deer, elk, moose, etc.). These diseases are always fatal and have long asymptomatic incubation periods. By 2019, CWD-infected cervids had been detected in 26 states, three Canadian provinces, South Korea, Norway, Finland, and Sweden. Prions (PrPSc) replicate by inducing a normal cellular prion protein (PrPC) to adopt the prion conformation. This prion templated conformational conversion is influenced by PrPC polymorphisms. Cervid PrPC contains at least 20 different polymorphic sites. By using chymotrypsin, trypsin, or trypsin followed by chymotrypsin to digest denatured cervid PrP, 19 peptides suitable for multiple reaction monitoring (MRM)-based analysis and spanning positions 30-51, 61-112, and 114-231 of cervid PrP were identified. Ten of these peptides span polymorphism-containing regions of cervid PrP. The other nine contain no polymorphisms, so they can be used as internal standards. Calibration curves relating the area ratios of MRM signals from polymorphism-containing peptides to appropriate internal standard peptides were linear and had excellent correlation coefficients. Samples from heterozygous (G96/S96) white-tailed deer orally dosed with CWD from homozygous (G96/G96) deer were analyzed. The G96 polymorphism comprised 75 ± 5% of the total PrP from the G96/S96 heterozygotes. Heterozygous animals facilitate conversion of different PrPC polymorphisms into PrPSc. This approach can be used to quantitate the relative amounts of the polymorphisms present in other animal species and even humans.

Transient multimers modulate conformer abundances of prion protein monomer through conformational selection

Prions are known to be involved in neurodegenerative pathologies such as Creutzfeld-Jakob disease. Current models point to a molecular event which rely on a transmissible structural change that leads to the production of β-sheet-rich prion conformer (PrPSc). PrPSc itself has the capability to trigger the structural rearrangement of the ubiquitously present prion (PrPc) substrate in a self-perpetuating cascade. In this article, we demonstrate that recombinant PrPc exists in a conformational equilibrium. The conformers’ abundances were shown to be dependent on PrPc concentration through the formation of transient multimers leading to conformational selection. The study of PrPc mutants that follow dedicated oligomerization pathways demonstrated that the conformers’ relative abundances are modified, thus reinforcing the assertion that the nature of conformers’ interactions orient the oligomerization pathways. Further this result can be viewed as the “signature” of an aborted oligomerization process. This discovery sheds a new light on the possible origin of prion protein diseases, namely that a change in prion protein structure could be transmitted through the formation of transient multimers having different conformer compositions. This could explain the selection of a transient multimeric type that could be viewed as the precursor of PrPSc responsible for structural information transmission, and strain apparition.

Fibril-induced glutamine-/asparagine-rich prions recruit stress granule proteins in mammalian cells.

Prions of lower eukaryotes are self-templating protein aggregates that replicate by converting homotypic proteins into stable, tightly packed beta-sheet-rich protein assemblies. Propagation is mediated by prion domains, low-complexity regions enriched in polar and devoid of charged amino acid residues. In mammals, compositionally similar domains modulate the assembly of dynamic stress granules (SGs) that associate via multivalent weak interactions. Dysregulation of SGs composed of proteins with prion-like domains has been proposed to underlie the formation of pathological inclusions in several neurodegenerative diseases. The events that drive prion-like domains into transient or solid assemblies are not well understood. We studied the interactors of the prototype prion domain NM of Saccharomyces cerevisiae Sup35 in its soluble or fibril-induced prion conformation in the mammalian cytosol. We show that the interactomes of soluble and prionized NM overlap with that of SGs. Prion induction by exogenous seeds does not cause SG assembly, demonstrating that colocalization of aberrant protein inclusions with SG components does not necessarily reveal SGs as initial sites of protein misfolding.

Primary structural differences at residue 226 of deer and elk PrP dictate selection of distinct CWD prion strains in gene-targeted mice

Although the unifying hallmark of prion diseases is CNS neurodegeneration caused by conformational corruption of host prion protein (PrP) to its infective counterpart, contagious transmission of chronic wasting disease (CWD) results from shedding of prions produced at high titers in the periphery of diseased cervids. While deer and elk PrP primary structures are equivalent except at residue 226, which is glutamate in elk and glutamine in deer, the effect of this difference on CWD pathogenesis is largely unknown. Using a gene-targeting approach where the mouse PrP coding sequence was replaced with elk or deer PrP, we show that the resulting GtE226 and GtQ226 mice had distinct kinetics of disease onset, prion conformations, and distributions of prions in the brains of diseased mice following intracerebral CWD challenge. These findings indicate that amino acid differences at PrP residue 226 dictate the selection and propagation of divergent strains in deer and elk with CWD. Because prion strain properties largely dictate host-range potential, our findings suggest that prion strains from elk and deer pose distinct risks to sympatric species or humans exposed to CWD. GtE226 and GtQ226 mice were also highly susceptible to CWD prions following intraperitoneal and oral exposures, a characteristic that stood in stark contrast to previously produced transgenic models. Remarkably, disease transmission was effective when infected mice were cohoused with naïve cagemates. Our findings indicate that gene-targeted mice provide unprecedented opportunities to accurately investigate CWD peripheral pathogenesis, CWD strains, and mechanisms of horizontal CWD transmission.

Replication of multiple system atrophy prions in primary astrocyte cultures from transgenic mice expressing human \u03b1-synuclein

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.

Investigating the Roles of the NAC and RAC in Stress Induced Prion Formation in Yeast

Many mammalian neurodegenerative diseases and the prion‐transmitted spongiform encephalopathies are associated with the misfolding of proteins into amyloid conformations. In Saccharomyces cerevisiae, there exist a number of proteins capable of folding into prion forms, yet the acquisition of prion phenotypes can occasionally result in increased fitness, possibly by serving as epigenetic switches that can rapidly confer beneficial heritable phenotypes. Thus, the possible control mechanisms yeast use to modulate the acquisition of prion conformations makes yeast an important model system for understanding amyloid formation. Eukaryotes have two conserved ribosome‐associated chaperone complexes that interact with nascent polypeptide chains to promote correct folding of newly synthesized proteins: the nascent polypeptide‐associated complex (NAC), a heterodimeric complex consisting of an α subunit, paired with one of two possible β subunit paralogs; and the ribosome‐associated complex (RAC), consisting of the Hsp70 chaperone Ssz1 and the Hsp40 chaperone Zuo1, which anchors the complex onto ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. Both of these complexes associate with ribosomes near the polypeptide exit tunnel and both have roles in co‐translational protein homeostasis and ribosome biogenesis. Previous work in our lab has shown that yeast cells lacking Zuo1, and thus lacking RAC chaperone function on ribosomes, exhibit higher frequencies of spontaneous and induced formation of the [PSI+] prion, the self‐propagating amyloid form of Sup35, a translation termination factor. These findings are consistent with a role for the RAC in chaperoning nascent Sup35 to prevent misfolding of its N‐terminal prion domain as it emerges from the ribosome. An alternative role for Zuo1 is as a transcriptional co‐activator for genes in the pleiotropic drug response pathway (PDR), which enhances resistance to various drugs and environmental toxins. Zuo1's role in this pathway requires unfolding of its C‐terminal domain, which prevents Zuo1's association with ribosomes. Since a variety of environmental stressors are known to induce prion formation, interactions that promote RAC's dissociation from ribosomes under those conditions, may result in increased prion formation. We conducted a screen for Zuo1 dependent enhancers of PDR that revealed a possible role for the NAC minor β subunit Btt1. The contributions of ribosome‐associated processes in prion formation is a critical step in understanding the regulation and physiological impacts of amyloid formation. Our current work examines the role of RAC and NAC as stress‐responsive modulators of protein folding and seeks to understand the role of the NAC in facilitating a RAC dependent stress‐response.Support or Funding InformationNational Institutes of Health, NIGMS Award Number R15GM119081 to DMC and by Ursinus CollegeThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.