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

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

Tau filaments from multiple cases of sporadic and inherited Alzheimer's disease adopt a common fold.

The ordered assembly of tau protein into abnormal filaments is a defining characteristic of Alzheimer's disease (AD) and other neurodegenerative disorders. It is not known if the structures of tau filaments vary within, or between, the brains of individuals with AD. We used a combination of electron cryo-microscopy (cryo-EM) and immuno-gold negative-stain electron microscopy (immuno-EM) to determine the structures of paired helical filaments (PHFs) and straight filaments (SFs) from the frontal cortex of 17 cases of AD (15 sporadic and 2 inherited) and 2 cases of atypical AD (posterior cortical atrophy). The high-resolution structures of PHFs and SFs from the frontal cortex of 3 cases of AD, 2 sporadic and 1 inherited, were determined by cryo-EM. We also used immuno-EM to study the PHFs and SFs from a number of cortical and subcortical brain regions. PHFs outnumbered SFs in all AD cases. By cryo-EM, PHFs and SFs were made of two C-shaped protofilaments with a combined cross-β/β-helix structure, as described previously for one case of AD. The higher resolution structures obtained here showed two additional amino acids at each end of the protofilament. The immuno-EM findings, which indicated the presence of repeats 3 and 4, but not of the N-terminal regions of repeats 1 and 2, of tau in the filament cores of all AD cases, were consistent with the cryo-EM results. These findings show that there is no significant variation in tau filament structures between individuals with AD. This knowledge will be crucial for understanding the mechanisms that underlie tau filament formation and for developing novel diagnostics and therapies

Cleaved TMEM106B forms amyloid aggregates in central and peripheral nervous systems

Filaments made of residues 120-254 of transmembrane protein 106B (TMEM106B) form in an age-dependent manner and can be extracted from the brains of neurologically normal individuals and those of subjects with a variety of neurodegenerative diseases. TMEM106B filament formation requires cleavage at residue 120 of the 274 amino acid protein; at present, it is not known if residues 255-274 form the fuzzy coat of TMEM106B filaments. Here we show that a second cleavage appears likely, based on staining with an antibody raised against residues 263-274 of TMEM106B. We also show that besides the brain TMEM106B inclusions form in dorsal root ganglia and spinal cord, where they were mostly found in non-neuronal cells. We confirm that in the brain, inclusions were most abundant in astrocytes. No inclusions were detected in heart, liver, spleen or hilar lymph nodes. Based on their staining with luminescent conjugated oligothiophenes, we confirm that TMEM106B inclusions are amyloids. By in situ immunoelectron microscopy, TMEM106B assemblies were often found in structures resembling endosomes and lysosomes.</p

Prominent microglial inclusions in transgenic mouse models of α-synucleinopathy that are distinct from neuronal lesions.

Alpha-synucleinopathies are a group of progressive neurodegenerative disorders, characterized by intracellular deposits of aggregated α-synuclein (αS). The clinical heterogeneity of these diseases is thought to be attributed to conformers (or strains) of αS but the contribution of inclusions in various cell types is unclear. The aim of the present work was to study αS conformers among different transgenic (TG) mouse models of α-synucleinopathies. To this end, four different TG mouse models were studied (Prnp-h[A53T]αS; Thy1-h[A53T]αS; Thy1-h[A30P]αS; Thy1-mαS) that overexpress human or murine αS and differed in their age-of-symptom onset and subsequent disease progression. Postmortem analysis of end-stage brains revealed robust neuronal αS pathology as evidenced by accumulation of αS serine 129 (p-αS) phosphorylation in the brainstem of all four TG mouse lines. Overall appearance of the pathology was similar and only modest differences were observed among additionally affected brain regions. To study αS conformers in these mice, we used pentameric formyl thiophene acetic acid (pFTAA), a fluorescent dye with amyloid conformation-dependent spectral properties. Unexpectedly, besides the neuronal αS pathology, we also found abundant pFTAA-positive inclusions in microglia of all four TG mouse lines. These microglial inclusions were also positive for Thioflavin S and showed immunoreactivity with antibodies recognizing the N-terminus of αS, but were largely p-αS-negative. In all four lines, spectral pFTAA analysis revealed conformational differences between microglia and neuronal inclusions but not among the different mouse models. Concomitant with neuronal lesions, microglial inclusions were already present at presymptomatic stages and could also be induced by seeded αS aggregation. Although nature and significance of microglial inclusions for human α-synucleinopathies remain to be clarified, the previously overlooked abundance of microglial inclusions in TG mouse models of α-synucleinopathy bears importance for mechanistic and preclinical-translational studies

Cryo-EM structures of amyloid-beta filaments with the Arctic mutation (E22G) from human and mouse brains

The Arctic mutation, encoding E693G in the amyloid precursor protein (APP) gene [E22G in amyloid-β (Aβ)], causes dominantly inherited Alzheimer’s disease. Here, we report the high-resolution cryo-EM structures of Aβ filaments from the frontal cortex of a previously described case (AβPParc1) with the Arctic mutation. Most filaments consist of two pairs of non-identical protofilaments that comprise residues V12–V40 (human Arctic fold A) and E11–G37 (human Arctic fold B). They have a substructure (residues F20–G37) in common with the folds of type I and type II Aβ42. When compared to the structures of wild-type Aβ42 filaments, there are subtle conformational changes in the human Arctic folds, because of the lack of a side chain at G22, which may strengthen hydrogen bonding between mutant Aβ molecules and promote filament formation. A minority of Aβ42 filaments of type II was also present, as were tau paired helical filaments. In addition, we report the cryo-EM structures of Aβ filaments with the Arctic mutation from mouse knock-in line AppNL−G−F. Most filaments are made of two identical mutant protofilaments that extend from D1 to G37 (AppNL−G−F murine Arctic fold). In a minority of filaments, two dimeric folds pack against each other in an anti-parallel fashion. The AppNL−G−F murine Arctic fold differs from the human Arctic folds, but shares some substructure

Microglial inclusions and neurofilament light chain release follow neuronal α-synuclein lesions in long-term brain slice cultures.

BACKGROUND: Proteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms. Thus, understanding of the initiation and propagation of such lesions is key for developing therapeutics to delay or halt disease progression. METHODS: Alpha-synuclein (αS) inclusions were induced in long-term murine and human slice cultures by seeded aggregation. An αS seed-recognizing human antibody was tested for blocking seeding and/or spreading of the αS lesions. Release of neurofilament light chain (NfL) into the culture medium was assessed. RESULTS: To study initial stages of α-synucleinopathies, we induced αS inclusions in murine hippocampal slice cultures by seeded aggregation. Induction of αS inclusions in neurons was apparent as early as 1week post-seeding, followed by the occurrence of microglial inclusions in vicinity of the neuronal lesions at 2-3 weeks. The amount of αS inclusions was dependent on the type of αS seed and on the culture's genetic background (wildtype vs A53T-αS genotype). Formation of αS inclusions could be monitored by neurofilament light chain protein release into the culture medium, a fluid biomarker of neurodegeneration commonly used in clinical settings. Local microinjection of αS seeds resulted in spreading of αS inclusions to neuronally connected hippocampal subregions, and seeding and spreading could be inhibited by an αS seed-recognizing human antibody. We then applied parameters of the murine cultures to surgical resection-derived adult human long-term neocortical slice cultures from 22 to 61-year-old donors. Similarly, in these human slice cultures, proof-of-principle induction of αS lesions was achieved at 1week post-seeding in combination with viral A53T-αS expressions. CONCLUSION: The successful translation of these brain cultures from mouse to human with the first reported induction of human αS lesions in a true adult human brain environment underlines the potential of this model to study proteopathic lesions in intact mouse and now even aged human brain environments

Assembly of α-synuclein and neurodegeneration in the central nervous system of heterozygous M83 mice following the peripheral administration of α-synuclein seeds.

Peripheral administration (oral, intranasal, intraperitoneal, intravenous) of assembled A53T α-synuclein induced synucleinopathy in heterozygous mice transgenic for human mutant A53T α-synuclein (line M83). The same was the case when cerebellar extracts from a case of multiple system atrophy with type II α-synuclein filaments were administered intraperitoneally, intravenously or intramuscularly. We observed abundant immunoreactivity for pS129 α-synuclein in nerve cells and severe motor impairment, resulting in hindlimb paralysis and shortened lifespan. Filaments immunoreactive for pS129 α-synuclein were in evidence. A 70% loss of motor neurons was present five months after an intraperitoneal injection of assembled A53T α-synuclein or cerebellar extract with type II α-synuclein filaments from an individual with a neuropathologically confirmed diagnosis of multiple system atrophy. Microglial cells changed from a predominantly ramified to a dystrophic appearance. Taken together, these findings establish a close relationship between the formation of α-synuclein inclusions in nerve cells and neurodegeneration, accompanied by a shift in microglial cell morphology. Propagation of α-synuclein inclusions depended on the characteristics of both seeds and transgenically expressed protein

Cryo-EM structures of amyloid-β 42 filaments from human brains

Alzheimer’s disease is characterized by a loss of memory and other cognitive functions and the filamentous assembly of Aβ and tau in the brain. The assembly of Aβ peptides into filaments that end at residue 42 is a central event. Yang et al. used electron cryo–electron microscopy to determine the structures of Aβ42 filaments from human brain (see the Perspective by Willem and Fändrich). They identified two types of related S-shaped filaments, each consisting of two identical protofilaments. These structures will inform the development of better in vitro and animal models, inhibitors of Aβ42 assembly, and imaging agents with increased specificity and sensitivity. —SM

Formaldehyde-fixed brain tissue from spontaneously ill α-synuclein transgenic mice induces fatal α-synucleinopathy in transgenic hosts

In summary, the present results indicate that a-synuclein seeds in brain tissue resist formaldehyde fixation as previously reported for Aβ. It is likely, albeit to be proven, that this is also true for aggregated tau other self-propagating pathogenic protein aggregates. These findings can now be exploited to further establish the relationship between the molecular architecture of a-synuclein lesions and individual pathogenesis and thereby exploit archived formalin-fixed brain material