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.

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.

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.

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.

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.

Application of CRISPR genetic screens to investigate neurological diseases.

The adoption of CRISPR-Cas9 technology for functional genetic screens has been a transformative advance. Due to its modular nature, this technology can be customized to address a myriad of questions. To date, pooled, genome-scale studies have uncovered genes responsible for survival, proliferation, drug resistance, viral susceptibility, and many other functions. The technology has even been applied to the functional interrogation of the non-coding genome. However, applications of this technology to neurological diseases remain scarce. This shortfall motivated the assembly of a review that will hopefully help researchers moving in this direction find their footing. The emphasis here will be on design considerations and concepts underlying this methodology. We will highlight groundbreaking studies in the CRISPR-Cas9 functional genetics field and discuss strengths and limitations of this technology for neurological disease applications. Finally, we will provide practical guidance on navigating the many choices that need to be made when implementing a CRISPR-Cas9 functional genetic screen for the study of neurological diseases.

Discriminating Strains of Self-Propagating Protein Aggregates Using a Conformational Stability Assay.

Prions and other self-propagating protein aggregates can exist as distinct strains, which are thought to represent different conformations of aggregates. There is growing evidence that protein aggregate strains may be important for understanding the biology of common neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. While methodology for discriminating prion strains is in widespread use, there is a paucity of tools for comparing the conformational properties of aggregates composed of ß-amyloid (Aß) peptide or α-synuclein protein, particularly when present in complex samples such as brain extracts. The conformational stability assay (CSA) is a simple technique that measures the relative resistance of protein aggregates to chemical denaturation. While originally developed to differentiate prion strains, the CSA has since been adapted for use with other protein aggregates. Here, we describe the CSA in detail and outline its utility for distinguishing prion strains as well as unique conformational states of Aß and α-synuclein aggregates.

Prion-like propagation of ß-amyloid aggregates in the absence of APP overexpression.

The amyloid cascade hypothesis posits that the initiating event in Alzheimer's disease (AD) is the aggregation and deposition of the ß-amyloid (Aß) peptide, which is a proteolytic cleavage product of the amyloid precursor protein (APP). Mounting evidence suggests that the formation and spread of prion-like Aß aggregates during AD may contribute to disease progression. Inoculation of transgenic mice that overexpress APP with pre-formed Aß aggregates results in the prion-like induction of cerebral Aß deposition. To determine whether Aß deposition can also be induced when physiological APP levels are present in the brain, we inoculated AppNL-F mice, a knock-in model of AD that avoids potential artifacts associated with APP overexpression, with Aß aggregates derived from the brains of AD patients or transgenic mice. In all cases, induced Aß deposition was apparent in the corpus callosum, olfactory bulb, and meningeal blood vessels of inoculated mice at 130-150 days post-inoculation, whereas uninoculated and buffer-inoculated animals exhibited minimal or no Aß deposits at these ages. Interestingly, despite being predominantly composed of protease-resistant Aß42 aggregates, the induced parenchymal Aß deposits were largely diffuse and were unreactive to an amyloid-binding dye. These results demonstrate that APP overexpression is not a prerequisite for the prion-like induction of cerebral Aß deposition. Accordingly, spreading of Aß deposition may contribute to disease progression in AD patients.