Exogenous induction of synucleinopathy in transgenic mice - An experimental study on the prion-like properties of alpha-synuclein

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Aufrufstatistik

URI: http://hdl.handle.net/10900/80642
http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-806420
http://dx.doi.org/10.15496/publikation-22036
Dokumentart: Dissertation
Date: 2020-02-09
Language: English
Faculty: 4 Medizinische Fakultät
Department: Medizin
Advisor: Jucker, Mathias (Prof. Dr.)
Day of Oral Examination: 2017-06-23
DDC Classifikation: 570 - Life sciences; biology
610 - Medicine and health
Keywords: Parkinson-Krankheit
Other Keywords:
alpha-Synuclein
Parkinson's Disease
Transgenic Mice
Seeding
Prion-like
Mutation
Cerebrospinal fluid
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Inhaltszusammenfassung:

Dissertation ist gesperrt bis 09. Februar 2020 !

Abstract:

The accumulation of misfolded proteins into insoluble aggregates is a common feature in a variety of neurodegenerative diseases, and it is thought that the aggregation process plays a central role in the pathogenesis. Parkinson´s disease (PD) is the most common move-ment disorder and characterised by a progressive and selective degeneration of dopamin-ergic neurons of the substantia nigra. Histopathologically, the hallmark feature of PD is the intracellular deposition of aggregated α-synuclein (αS) protein in Lewy bodies. In PD pa-tients, Lewy pathology occurs in a stereotypic pattern, originating in lower brain regions before spreading to higher areas of the brain. Despite a significant amount of research, the mechanism underlying the formation of αS lesions is still poorly understood. However, recent findings highlight that a nucleation-dependent aggregation mechanism, initially de-scribed for prion diseases may also contribute to the propagation of protein aggregation in other neurodegenerative disorders. According to this concept, corrupted protein particles can act as nuclei (or ‘seeds’) of aggregation and further convert endogenous proteins into their pathological isoforms. The main objective of this thesis was to study the ‘prion-like’ properties of αS to gain fur-ther insight into the pathogenesis of PD. In our studies we used different transgenic mouse models of synucleinopathy, which are based on mutations identified in human disease and replicate some aspects of the PD pathology, to probe this theoretical explanation of dis-ease. These mouse lines harbor either the A53T (Tg-9813[A53T]αS and Tg-M83[A53T]αS) or A30P human αS transgene (Tg-[A30P]αS). Tg-9813[A53T]αS mice de-velop a severe and early-onset synucleinopathy. On the other hand, both Tg-M83[A53T]αS and Tg-[A30P]αS mice (both being homozygous for the transgene) have a delayed-onset of motor symptoms, whereas the latter tend to exhibit a milder disease phe-notype. As shown previously by other research groups, synucleinopathy can be induced by exogenous αS seeds in vivo. Accordingly, we performed a series of inoculation experi-ments to investigate the seeding effect of pathological αS. In a first set of experiments, we assessed whether αS seeds were still capable of inducing fatal synucleinopathy when treated with formaldehyde. In a previous study, our lab had shown that Aβ seeds resist the inactivation by formaldehyde fixation similar to prions. Therefore, we intracerebrally inoculated young pre-symptomatic mice with extracts from formaldehyde-fixed and fresh-frozen brainstem tissue. Remarkably, in Tg-9813[A53T]αS mice we found that fixed αS seeds were able to induce synucleinopathy lesions after 30 days of incubation. Stimulated by these results, we repeated the experiment in Tg-[A30P]αS mice and incubated until motor symptoms presented. Intriguingly, we found that the pathogenicity of αS seeds was retained even after formaldehyde fixation prior to the injection. Remarkably, the extract from formaldehyde-fixed brainstem tissue was almost as potent as the fresh-frozen tissue-derived extract with regard to both survival time and pathological αS load. The second part of this thesis was focused on investigating whether CSF from mice with synucleinopathy is seeding active. While our group and others had already shown that extracts from mouse brain tissue containing aggregated αS are potent inducers of synucle-inopathy, it is still unclear if extracellular αS from a bodily fluid is also pathogenic. Our first results revealed an elevated level of αS in the CSF of symptomatic Tg-[A30P]αS mice. Therefore, we intracerebrally inoculated young presymptomatic Tg-[A30P]αS mice with CSF of spontaneously ill donors. To compare the putative seeding potential of CSF-derived αS, we additionally performed intracerebral inoculations of the PBS-soluble and PBS-insoluble fractions of brainstem-derived extracts. Our results showed that CSF was not able to induce a synucleinopathy. Likewise, the PBS-soluble fraction failed to induce αS lesions in Tg-[A30P]αS mice, suggesting a lack of pathogenicity. Conversely, we found a severe induction of synucleinopathy lesions and a significantly reduced life span after inoculation with PBS-insoluble αS seeds in comparison to the non-tg control inoculum. Although less than 4% of the αS remained in this fraction, PBS-insoluble αS seeds were highly seeding-active when compared to the original extract that was diluted to match with the αS level of both the soluble and insoluble fractions. In addition, we found that the seeded induction of αS lesions occurred in a concentration-dependent manner. Finally, in a last set of experiments we have investigated the cross-seeding effect of two mutant αS pathogens, since in vitro studies have indicated that there are indications for structural and functional differences among fibrils of either the mutant A53T or A30P hu-man αS. When injected into the hippocampus of young presymptomatic Tg-M83[A53T]αS and Tg-[A30P]αS mice, we found that both extracts induced an accelerated disease phe-notype with reduced survival times. Moreover, we observed seeded synucleinopathy le-sions in each of the recipient mouse strains. However, we found that both the incubation time and the pathology were not different between the tg extract-injected mice in either of the lines indicative of a strong host effect. Therefore, to determine whether the two αS extracts have a differential cross-seeding capacity, we injected hemizygous Tg-M83[A53T]αS and Tg-[A30P]αS mice that – uninoculated – remain healthy until late adult-hood. Intriguingly, both extracts were capable of inducing a de novo synucleinopathy in both mouse strains. While we did not find a significant difference in the incubation periods between the two αS extracts in the hemizygous Tg-M83[A53T]αS mice, the A30P-derived extract was markedly more potent in reducing the survival time than A53T in hemizygous Tg-[A30P]αS mice. In summary, we were able to study the exogenous seeding mechanism of αS in different mouse models of synucleinopathy. Our data provide new insight into the persistent nature of αS seeds that is reminiscent of prions. Moreover, the results of this thesis indicate that PD-linked αS mutants may dictate functional characteristics in vivo. Thus, our findings support the concept that synucleinopathies share several common features with prion dis-eases. Consequently, Insight into the seeding aspect of the disease could lead to a better understanding of the misfold initiation and spread of the pathogenic protein, ultimately pav-ing the way to therapeutical strategies targeting this particular attribute of PD.

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