Anti-prion drugs do not improve survival in novel knock-in models of inherited prion disease.

Prion diseases uniquely manifest in three distinct forms: inherited, sporadic, and infectious. Wild-type prions are responsible for the sporadic and infectious versions, while mutant prions cause inherited variants like fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). Although some drugs can prolong prion incubation times up to four-fold in rodent models of infectious prion diseases, no effective treatments for FFI and fCJD have been found. In this study, we evaluated the efficacy of various anti-prion drugs on newly-developed knock-in mouse models for FFI and fCJD. These models express bank vole prion protein (PrP) with the pathogenic D178N and E200K mutations. We applied various drug regimens known to be highly effective against wild-type prions in vivo as well as a brain-penetrant compound that inhibits mutant PrPSc propagation in vitro. None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrPSc accumulation in either knock-in mouse model, despite their ability to induce strain adaptation of mutant prions. Our results show that anti-prion drugs originally developed to treat infectious prion diseases do not necessarily work for inherited prion diseases, and that the recombinant sPMCA is not a reliable platform for identifying compounds that target mutant prions. This work underscores the need to develop therapies and validate screening assays specifically for mutant prions, as well as anti-prion strategies that are not strain-dependent.

Upregulated pexophagy limits the capacity of selective autophagy.

Selective autophagy is an essential process to maintain cellular homeostasis through the constant recycling of damaged or superfluous components. Over a dozen selective autophagy pathways mediate the degradation of diverse cellular substrates, but whether these pathways can influence one another remains unknown. We address this question using pexophagy, the autophagic degradation of peroxisomes, as a model. We show in cells that upregulated pexophagy impairs the selective autophagy of both mitochondria and protein aggregates by exhausting the autophagy initiation factor, ULK1. We confirm this finding in cell models of the pexophagy-mediated form of Zellweger Spectrum Disorder, a disease characterized by peroxisome dysfunction. Further, we extend the generalizability of limited selective autophagy by determining that increased protein aggregate degradation reciprocally reduces pexophagy using cell models of Parkinson's Disease and Huntington's Disease. Our findings suggest that the degradative capacity of selective autophagy can become limited by an increase in one substrate.

Anti-prion drugs do not improve survival in knock-in models of inherited prion disease.

Prion diseases uniquely manifest in three distinct forms: inherited, sporadic, and infectious. Wild-type prions are responsible for the sporadic and infectious versions, while mutant prions cause inherited variants like fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). Although some drugs can prolong prion incubation times up to four-fold in rodent models of infectious prion diseases, no effective treatments for FFI and fCJD have been found. In this study, we evaluated the efficacy of various anti-prion drugs on newly-developed knock-in mouse models for FFI and fCJD. These models express bank vole prion protein (PrP) with the pathogenic D178N and E200K mutations. We applied various drug regimens known to be highly effective against wild-type prions in vivo as well as a brain-penetrant compound that inhibits mutant PrP Sc propagation in vitro . None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrP Sc accumulation in either knock-in mouse model, despite their ability to induce strain adaptation of mutant prions. Paradoxically, the combination of Anle138b and IND24 appeared to accelerate disease by 16% and 26% in kiBVI E200K and kiBVI D178N mice, respectively, and accelerated the aggregation of mutant PrP molecules in vitro . Our results show that anti-prion drugs originally developed to treat infectious prion diseases do not necessarily work for inherited prion diseases, and that the recombinant sPMCA is not a reliable platform for identifying compounds that target mutant prions. This work underscores the need to develop therapies and validate screening assays specifically for mutant prions.

The G51D SNCA mutation generates a slowly progressive α-synuclein strain in early-onset Parkinson's disease.

Unique strains of α-synuclein aggregates have been postulated to underlie the spectrum of clinical and pathological presentations seen across the synucleinopathies. Whereas multiple system atrophy (MSA) is associated with a predominance of oligodendroglial α-synuclein inclusions, α-synuclein aggregates in Parkinson's disease (PD) preferentially accumulate in neurons. The G51D mutation in the SNCA gene encoding α-synuclein causes an aggressive, early-onset form of PD that exhibits clinical and neuropathological traits reminiscent of both PD and MSA. To assess the strain characteristics of G51D PD α-synuclein aggregates, we performed propagation studies in M83 transgenic mice by intracerebrally inoculating patient brain extracts. The properties of the induced α-synuclein aggregates in the brains of injected mice were examined using immunohistochemistry, a conformational stability assay, and by performing α-synuclein seed amplification assays. Unlike MSA-injected mice, which developed a progressive motor phenotype, G51D PD-inoculated animals remained free of overt neurological illness for up to 18 months post-inoculation. However, a subclinical synucleinopathy was present in G51D PD-inoculated mice, characterized by the accumulation of α-synuclein aggregates in restricted regions of the brain. The induced α-synuclein aggregates in G51D PD-injected mice exhibited distinct properties in a seed amplification assay and were much more stable than those present in mice injected with MSA extract, which mirrored the differences observed between human MSA and G51D PD brain samples. These results suggest that the G51D SNCA mutation specifies the formation of a slowly propagating α-synuclein strain that more closely resembles α-synuclein aggregates associated with PD than MSA.

α-Synuclein Conformational Strains as Drivers of Phenotypic Heterogeneity in Neurodegenerative Diseases.

The synucleinopathies, which include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are a class of human neurodegenerative disorders unified by the presence of α-synuclein aggregates in the brain. Considerable clinical and pathological heterogeneity exists within and among the individual synucleinopathies. A potential explanation for this variability is the existence of distinct conformational strains of α-synuclein aggregates that cause different disease manifestations. Like prion strains, α-synuclein strains can be delineated based on their structural architecture, with structural differences among α-synuclein aggregates leading to unique biochemical attributes and neuropathological properties in humans and animal models. Bolstered by recent high-resolution structural data from patient brain-derived material, it has now been firmly established that there are conformational differences among α-synuclein aggregates from different human synucleinopathies. Moreover, recombinant α-synuclein can be polymerized into several structurally distinct aggregates that exhibit unique pathological properties. In this review, we outline the evidence supporting the existence of α-synuclein strains and highlight how they can act as drivers of phenotypic heterogeneity in the human synucleinopathies.

Somatostatin slows Aß plaque deposition in aged APPNL-F/NL-F mice by blocking Aß aggregation.

The neuroendocrine peptide somatostatin (SST) has long been thought of as influencing the deposition of the amyloid ß peptide (Aß) in Alzheimer's disease (AD). Missing have been in vivo data in a relevant Aß amyloidosis model. Here we crossed AppNL-F/NL-F mice with Sst-deficient mice to assess if and how the presence of Sst influences pathological hallmarks of Aß amyloidosis. We found that Sst had no influence on whole brain neprilysin transcript, protein or activity levels, an observation that cannot be accounted for by a compensatory upregulation of the Sst paralog, cortistatin (Cort), that we observed in 15-month-old Sst-deficient mice. Sst-deficiency led to a subtle but significant increase in the density of cortical Aß amyloid plaques. Follow-on western blot analyses of whole brain extracts indicated that Sst interferes with early steps of Aß assembly that manifest in the appearance of SDS-stable smears of 55-150 kDa in Sst null brain samples. As expected, no effect of Sst on tau steady-state levels or its phosphorylation were observed. Results from this study are easier reconciled with an emerging body of data that point toward Sst affecting Aß amyloid plaque formation through direct interference with Aß aggregation rather than through its effects on neprilysin expression.

Genetically engineered cellular models of prion propagation.

For over three decades, cultured cells have been a useful tool for dissecting the molecular details of prion replication and the identification of candidate therapeutics for prion disease. A major issue limiting the translatability of these studies has been the inability to reliably propagate disease-relevant, non-mouse strains of prions in cells relevant to prion pathogenesis. In recent years, fueled by advances in gene editing technology, it has become possible to propagate prions from hamsters, cervids, and sheep in immortalized cell lines originating from the central nervous system. In particular, the use of CRISPR-Cas9-mediated gene editing to generate versions of prion-permissive cell lines that lack endogenous PrP expression has provided a blank canvas upon which re-expression of PrP leads to species-matched susceptibility to prion infection. When coupled with the ability to propagate prions in cells or organoids derived from stem cells, these next-generation cellular models should provide an ideal paradigm for identifying small molecules and other biological therapeutics capable of interfering with prion replication in animal and human prion disorders. In this review, we summarize recent advances that have widened the spectrum of prion strains that can be propagated in cultured cells and cutting-edge tissue-based models.

A single protective polymorphism in the prion protein blocks cross-species prion replication in cultured cells.

The bank vole (BV) prion protein (PrP) can function as a universal acceptor of prions. However, the molecular details of BVPrP's promiscuity for replicating a diverse range of prion strains remain obscure. To develop a cultured cell paradigm capable of interrogating the unique properties of BVPrP, we generated monoclonal lines of CAD5 cells lacking endogenous PrP but stably expressing either hamster (Ha), mouse (Mo), or BVPrP (M109 or I109 polymorphic variants) and then challenged them with various strains of mouse or hamster prions. Cells expressing BVPrP were susceptible to both mouse and hamster prions, whereas cells expressing MoPrP or HaPrP could only be infected with species-matched prions. Propagation of mouse and hamster prions in cells expressing BVPrP resulted in strain adaptation in several instances, as evidenced by alterations in conformational stability, glycosylation, susceptibility to anti-prion small molecules, and the inability of BVPrP-adapted mouse prion strains to infect cells expressing MoPrP. Interestingly, cells expressing BVPrP containing the G127V prion gene variant, identified in individuals resistant to kuru, were unable to become infected with prions. Moreover, the G127V polymorphic variant impeded the spontaneous aggregation of recombinant BVPrP. These results demonstrate that BVPrP can facilitate cross-species prion replication in cultured cells and that a single amino acid change can override the prion-permissive nature of BVPrP. This cellular paradigm will be useful for dissecting the molecular features of BVPrP that allow it to function as a universal prion acceptor.

Alpha-synuclein seeding shows a wide heterogeneity in multiple system atrophy.

BACKGROUND: Multiple system atrophy (MSA) is a neurodegenerative condition characterized by variable combinations of parkinsonism, autonomic failure, cerebellar ataxia and pyramidal features. Although the distribution of synucleinopathy correlates with the predominant clinical features, the burden of pathology does not fully explain observed differences in clinical presentation and rate of disease progression. We hypothesized that the clinical heterogeneity in MSA is a consequence of variability in the seeding activity of α-synuclein both between different patients and between different brain regions. METHODS: The reliable detection of α-synuclein seeding activity derived from MSA using cell-free amplification assays remains challenging. Therefore, we conducted a systematic evaluation of 168 different reaction buffers, using an array of pH and salts, seeded with fully characterized brain homogenates from one MSA and one PD patient. We then validated the two conditions that conferred the optimal ability to discriminate between PD- and MSA-derived samples in a larger cohort of 40 neuropathologically confirmed cases, including 15 MSA. Finally, in a subset of brains, we conducted the first multi-region analysis of seeding behaviour in MSA. RESULTS: Using our novel buffer conditions, we show that the physicochemical factors that govern the in vitro amplification of α-synuclein can be tailored to generate strain-specific reaction buffers that can be used to reliably study the seeding capacity from MSA-derived α-synuclein. Using this novel approach, we were able to sub-categorize the 15 MSA brains into 3 groups: high, intermediate and low seeders. To further demonstrate heterogeneity in α-synuclein seeding in MSA, we conducted a comprehensive multi-regional evaluation of α-synuclein seeding in 13 different regions from 2 high seeders, 2 intermediate seeders and 2 low seeders. CONCLUSIONS: We have identified unexpected differences in seed-competent α-synuclein across a cohort of neuropathologically comparable MSA brains. Furthermore, our work has revealed a substantial heterogeneity in seeding activity, driven by the PBS-soluble α-synuclein, between different brain regions of a given individual that goes beyond immunohistochemical observations. Our observations pave the way for future subclassification of MSA, which exceeds conventional clinical and neuropathological phenotyping and considers the structural and biochemical heterogeneity of α-synuclein present. Finally, our methods provide an experimental framework for the development of vitally needed, rapid and sensitive diagnostic assays for MSA.

The cellular prion protein interacts with and promotes the activity of Na,K-ATPases.

The prion protein (PrP) is best known for its ability to cause fatal neurodegenerative diseases in humans and animals. Here, we revisited its molecular environment in the brain using a well-developed affinity-capture mass spectrometry workflow that offers robust relative quantitation. The analysis confirmed many previously reported interactions. It also pointed toward a profound enrichment of Na,K-ATPases (NKAs) in proximity to cellular PrP (PrPC). Follow-on work validated the interaction, demonstrated partial co-localization of the ATP1A1 and PrPC, and revealed that cells exposed to cardiac glycoside (CG) inhibitors of NKAs exhibit correlated changes to the steady-state levels of both proteins. Moreover, the presence of PrPC was observed to promote the ion uptake activity of NKAs in a human co-culture paradigm of differentiated neurons and glia cells, and in mouse neuroblastoma cells. Consistent with this finding, changes in the expression of 5'-nucleotidase that manifest in wild-type cells in response to CG exposure can also be observed in untreated PrPC-deficient cells. Finally, the endoproteolytic cleavage of the glial fibrillary acidic protein, a hallmark of late-stage prion disease, can also be induced by CGs, raising the prospect that a loss of NKA activity may contribute to the pathobiology of prion diseases.

Viral alpha-synuclein knockdown prevents spreading synucleinopathy.

The accumulation of aggregated alpha-synuclein (α-syn) in Parkinson's disease, dementia with Lewy bodies and multiple system atrophy is thought to involve a common prion-like mechanism, whereby misfolded α-syn provides a conformational template for further accumulation of pathological α-syn. We tested whether silencing α-syn gene expression could reduce native non-aggregated α-syn substrate and thereby disrupt the propagation of pathological α-syn initiated by seeding with synucleinopathy-affected mouse brain homogenates. Unilateral intracerebral injections of adeno-associated virus serotype-1 encoding microRNA targeting the α-syn gene reduced the extent and severity of both the α-syn pathology and motor deficits. Importantly, a moderate 50% reduction in α-syn was sufficient to prevent the spread of α-syn pathology to distal brain regions. Our study combines behavioural, immunohistochemical and biochemical data that strongly support α-syn knockdown gene therapy for synucleinopathies.

The aminoglycoside G418 hinders de novo prion infection in cultured cells.

The study of prions and the discovery of candidate therapeutics for prion disease have been facilitated by the ability of prions to replicate in cultured cells. Paradigms in which prion proteins from different species are expressed in cells with low or no expression of endogenous prion protein (PrP) have expanded the range of prion strains that can be propagated. In these systems, cells stably expressing a PrP of interest are typically generated via coexpression of a selectable marker and treatment with an antibiotic. Here, we report the unexpected discovery that the aminoglycoside G418 (Geneticin) interferes with the ability of stably transfected cultured cells to become infected with prions. In G418-resistant lines of N2a or CAD5 cells, the presence of G418 reduced levels of protease-resistant PrP following challenge with the RML or 22L strains of mouse prions. G418 also interfered with the infection of cells expressing hamster PrP with the 263K strain of hamster prions. Interestingly, G418 had minimal to no effect on protease-resistant PrP levels in cells with established prion infection, arguing that G418 selectively interferes with de novo prion infection. As G418 treatment had no discernible effect on cellular PrP levels or its localization, this suggests that G418 may specifically target prion assemblies or processes involved in the earliest stages of prion infection.

Aß43 aggregates exhibit enhanced prion-like seeding activity in mice.

When injected into genetically modified mice, aggregates of the amyloid-ß (Aß) peptide from the brains of Alzheimer's disease (AD) patients or transgenic AD mouse models seed cerebral Aß deposition in a prion-like fashion. Within the brain, Aß exists as a pool of distinct C-terminal variants with lengths ranging from 37 to 43 amino acids, yet the relative contribution of individual C-terminal Aß variants to the seeding behavior of Aß aggregates remains unknown. Here, we have investigated the relative seeding activities of Aß aggregates composed exclusively of recombinant Aß38, Aß40, Aß42, or Aß43. Cerebral Aß42 levels were not increased in AppNL-F knock-in mice injected with Aß38 or Aß40 aggregates and were only increased in a subset of mice injected with Aß42 aggregates. In contrast, significant accumulation of Aß42 was observed in the brains of all mice inoculated with Aß43 aggregates, and the extent of Aß42 induction was comparable to that in mice injected with brain-derived Aß seeds. Mice inoculated with Aß43 aggregates exhibited a distinct pattern of cerebral Aß pathology compared to mice injected with brain-derived Aß aggregates, suggesting that recombinant Aß43 may polymerize into a unique strain. Our results indicate that aggregates containing longer Aß C-terminal variants are more potent inducers of cerebral Aß deposition and highlight the potential role of Aß43 seeds as a crucial factor in the initial stages of Aß pathology in AD.

Sensitive protein misfolding cyclic amplification of sporadic Creutzfeldt-Jakob disease prions is strongly seed and substrate dependent.

Unlike variant Creutzfeldt-Jakob disease prions, sporadic Creutzfeldt-Jakob disease prions have been shown to be difficult to amplify in vitro by protein misfolding cyclic amplification (PMCA). We assessed PMCA of pathological prion protein (PrPTSE) from 14 human sCJD brain samples in 3 substrates: 2 from transgenic mice expressing human prion protein (PrP) with either methionine (M) or valine (V) at position 129, and 1 from bank voles. Brain extracts representing the 5 major clinicopathological sCJD subtypes (MM1/MV1, MM2, MV2, VV1, and VV2) all triggered seeded PrPTSE amplification during serial PMCA with strong seed- and substrate-dependence. Remarkably, bank vole PrP substrate allowed the propagation of all sCJD subtypes with preservation of the initial molecular PrPTSE type. In contrast, PMCA in human PrP substrates was accompanied by a PrPTSE molecular shift during heterologous (M/V129) PMCA reactions, with increased permissiveness of V129 PrP substrate to in vitro sCJD prion amplification compared to M129 PrP substrate. Combining PMCA amplification sensitivities with PrPTSE electrophoretic profiles obtained in the different substrates confirmed the classification of 4 distinct major sCJD prion strains (M1, M2, V1, and V2). Finally, the level of sensitivity required to detect VV2 sCJD prions in cerebrospinal fluid was achieved.

The existence of Aß strains and their potential for driving phenotypic heterogeneity in Alzheimer's disease.

Reminiscent of the human prion diseases, there is considerable clinical and pathological variability in Alzheimer's disease, the most common human neurodegenerative condition. As in prion disorders, protein misfolding and aggregation is a hallmark feature of Alzheimer's disease, where the initiating event is thought to be the self-assembly of Aß peptide into aggregates that deposit in the central nervous system. Emerging evidence suggests that Aß, similar to the prion protein, can polymerize into a conformationally diverse spectrum of aggregate strains both in vitro and within the brain. Moreover, certain types of Aß aggregates exhibit key hallmarks of prion strains including divergent biochemical attributes and the ability to induce distinct pathological phenotypes when intracerebrally injected into mouse models. In this review, we discuss the evidence demonstrating that Aß can assemble into distinct strains of aggregates and how such strains may be primary drivers of the phenotypic heterogeneity in Alzheimer's disease.

The utility of bank voles for studying prion disease.

The transmission of prions between species is typically an inefficient process due to the species barrier, which represents incompatibility between prion seed and substrate molecules. Bank voles (Myodes glareolus) are an exception to this rule, as they are susceptible to a diverse range of prion strains from many different animal species. In particular, bank voles can be efficiently infected with most types of human prions and have played a critical role in validating variably protease-sensitive prionopathy (VPSPr) and certain forms of Gerstmann-Sträussler-Scheinker (GSS) disease as bona fide prion disorders rather than non-transmissible proteinopathies. The bank vole prion protein (BVPrP) confers a "universal prion acceptor" phenotype when expressed in mice and when used as a substrate for in vitro prion amplification assays, indicating that the unique prion transmission properties of bank voles are mediated by BVPrP. Over-expression of BVPrP in mice can also promote the spontaneous development of prion disease, indicating that BVPrP is intrinsically prone to both spontaneous and template-directed misfolding. Here, we discuss the utility of bank voles and BVPrP for prion research and how they have provided new tools for establishing rapid animal bioassays, modeling spontaneous prion disease, standardizing prion diagnostics, and understanding the molecular basis of the species barrier.

Identification of a homology-independent linchpin domain controlling mouse and bank vole prion protein conversion.

Prions are unorthodox pathogens that cause fatal neurodegenerative diseases in humans and other mammals. Prion propagation occurs through the self-templating of the pathogenic conformer PrPSc, onto the cell-expressed conformer, PrPC. Here we study the conversion of PrPC to PrPSc using a recombinant mouse PrPSc conformer (mouse protein-only recPrPSc) as a unique tool that can convert bank vole but not mouse PrPC substrates in vitro. Thus, its templating ability is not dependent on sequence homology with the substrate. In the present study, we used chimeric bank vole/mouse PrPC substrates to systematically determine the domain that allows for conversion by Mo protein-only recPrPSc. Our results show that that either the presence of the bank vole amino acid residues E227 and S230 or the absence of the second N-linked glycan are sufficient to allow PrPC substrates to be converted by Mo protein-only recPrPSc and several native infectious prion strains. We propose that residues 227 and 230 and the second glycan are part of a C-terminal domain that acts as a linchpin for bank vole and mouse prion conversion.

Cellular models for discovering prion disease therapeutics: Progress and challenges.

Prions, which cause fatal neurodegenerative disorders such as Creutzfeldt-Jakob disease, are misfolded and infectious protein aggregates. Currently, there are no treatments available to halt or even delay the progression of prion disease in the brain. The infectious nature of prions has resulted in animal paradigms that accurately recapitulate all aspects of prion disease, and these have proven to be instrumental for testing the efficacy of candidate therapeutics. Nonetheless, infection of cultured cells with prions provides a much more powerful system for identifying molecules capable of interfering with prion propagation. Certain lines of cultured cells can be chronically infected with various types of mouse prions, and these models have been used to unearth candidate anti-prion drugs that are at least partially efficacious when administered to prion-infected rodents. However, these studies have also revealed that not all types of prions are equal, and that drugs active against mouse prions are not necessarily effective against prions from other species. Despite some recent progress, the number of cellular models available for studying non-mouse prions remains limited. In particular, human prions have proven to be particularly challenging to propagate in cultured cells, which has severely hindered the discovery of drugs for Creutzfeldt-Jakob disease. In this review, we summarize the cellular models that are presently available for discovering and testing drugs capable of blocking the propagation of prions and highlight challenges that remain on the path towards developing therapies for prion disease.

α-Synuclein strains target distinct brain regions and cell types.

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 among human synucleinopathies. Strain-specific clinical, pathological and biochemical differences were faithfully maintained after serial passaging, which implies that α-synuclein propagates via prion-like conformational templating. Thus, pathogenic α-synuclein exhibits key hallmarks of prion strains, which provides evidence that disease heterogeneity among the synucleinopathies is caused by distinct α-synuclein strains.