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The aggregation of specific proteins within the nervous system is generally assumed to be a crucial element in the pathogenesis of a plethora of age-related neurodegenerative diseases. These diseases are often referred to as proteinopathies, with Alzheimer’s disease (AD) and Parkinson’s disease (PD) being the two most common neurodegenerative diseases of the elderly. In AD, amyloid-β peptides (Aβ) are the main component of insoluble aggregates forming senile plaques; in PD the α-synuclein protein (α-syn) is the main component of Lewy bodies (LBs) and Lewy neurites (LNs). Recent findings have shown that Aβ aggregates as well as α-syn aggregates, can initiate the conversion of the same properly-folded protein into the misfolded, disease-associated aggregated state. This has led to the suggestion of a prion-like mechanism of disease pathogenesis. Moreover, abnormally folded proteins may adopt a range of structurally different morphologies. Such protein conformers (strains) have been described to harbor different biological activities. The objective of this thesis was to elucidate the prion-like aspects of Aβ in mouse models and in organotypic slice cultures.
This thesis is divided into three sections: The first section describes the results of a study aiming to elucidate the strain-like transmission of Aβ morphotypes in mouse models of cerebral β-amyloidosis. In the second section, organotypic slice cultures were developed as a versatile tool to model cerebral β-amyloidosis and gather mechanistic insights into prion-like misfolding of Aβ. The last section is dedicated to our effort to adopt the organotypic slice cultures to model LB and cell-to-cell transmission of misfolded α-syn aggregates.
The first study of this thesis was initiated to further examine the hypothesis that different Aβ morphotypes (Aβ strains) may exist. The strain-like properties of misfolded Aβ aggregates after seeded transmission were investigated in two Aβ-precursor protein (APP) transgenic (tg) mouse models, APP23 and APPPS1. Influenced by differences in the ratios of the two main Aβ isoforms, Aβ40 and Aβ42, the two models develop Aβ plaques of different morphology. Amyloid-laden brain extracts of either an aged APP23 or an aged APPPS1 mouse were intracerebrally injected into young pre-depositing APP23 or APPPS1 recipient mice. Similar to the strain concept described in prion diseases, the intrinsic characteristics of the source material could be stably propagated in the recipient mice, confirming a mechanism of seeded strain-like transmission. This study was performed in collaboration with K.P.R. Nilsson from the University of Linköping in Sweden and was published in EMBO Reports (Heilbronner et al., 2013).
In the second study of this thesis, we established a new hippocampal slice culture (HSC) model of cerebral β-amyloidosis. The Aβ plaque formation in HSCs was induced by a single treatment of Aβ-laden brain extract from aged APP tg mice on top of the culture and the continuous supply of synthetic Aβ into the culture medium. This HSC model reproduces many aspects of cerebral β-amyloidosis such as Aβ-deposit-induced microglia response and neuronal alterations. Notably, the morphological appearance and the conformational structure of the induced Aβ deposits in HSCs depended again on both, the extract and the synthetic Aβ species used, resembling the results found in APP tg mice. The new HSC model of cerebral β-amyloidosis hence demonstrates the seeded conversion of synthetic Aβ in HSCs. To provide evidence for the synthetic nature of induced Aβ aggregates on HSCs, we used in addition HSCs from App-null mice and showed that endogenously expressed Aβ is not necessary for the Aβ plaque formation. However, induced Aβ plaque formation requires a living cellular environment since fixation of the HSC prior to treatment largely prevented amyloid plaque formation. Given that synthetic Aβ aggregates generated in vitro show less seeding activity in vivo and are therefore distinct from biological Aβ, we tested the biological efficiency of HSC-derived synthetic Aβ aggregates in vivo. Remarkably, we were the first to succeed in converting synthetic Aβ into biologically functional seeds by using the HSC model. Our result leads to the inference that the diversity of factors in the HSC influences the intrinsic characteristics of the synthetic peptide, rendering it more biologically-active than in vitro-generated synthetic Aβ aggregates. This study was performed in collaboration with Prof. B. Heimrich from the University of Freiburg in Germany and is in press in The Journal of Neuroscience (Novotny et al., 2016) .
The success of the HSC model to mimic cerebral β-amyloidosis encouraged us to utilize HSCs to investigate other proteinopathies. The third part of this thesis outlines that LB- and LN-like α-syn inclusions can be induced in HSCs. Indeed, we succeeded in inducing α-syn lesions in cultures from wildtype mice and mice overexpressing human mutated α-syn by one-time application of tg brain extract containing α-syn aggregates on top of the HSCs. The same treatment yielded no inclusions on HSCs derived from α-syn-deficient mice, indicating that culture-derived α-syn is necessary for the induction of α-syn inclusions. This study is ongoing and results are presented as unpublished data in the last part of this thesis.
In summary, the results of the first two studies presented here support the concept of prion-like-templated misfolding of Aβ. By using HSCs we succeeded for the first time to convert synthetic Aβ into in vivo seeding-active seeds. Furthermore, we emphasize that the involvement of cellular processes in a living HSC is crucial for the conversion to biologically-active synthetic Aβ aggregates. Consequently this new HSC model for cerebral β-amyloidosis is suitable to explore the contribution of cellular and molecular cofactors to achieve seeding-active Aβ aggregates. Additionally, our last study shows that the HSC model could also be applicable to other proteinopathies. We are the first to show inducible LB- and LN-like α-syn inclusions in slice cultures. This model can be used to investigate the cell-to-cell transmission of α-syn and could shed light on how misfolded α-syn spreads to healthy cells. |
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