α-Synuclein fibril-specific nanobody reduces prion-like α-synuclein spreading in mice.
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ABSTRACT:
SUBMITTER: Butler YR
PROVIDER: S-EPMC9296447 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The protein alpha-synuclein (?S) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson's disease. However, the quantitative characterization of the aggregation and spreading of ?S remains challenging to achieve. Previously, we identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-molecule fluorescence measurements and kinetic analysis, we find that the reaction in solution involves two unimolecular structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addition. We have obtained individual rate constants for these key microscopic steps by applying a global kinetic analysis to both the decrease in the concentration of monomeric protein molecules and the increase in oligomer concentrations over a 0.5-140-µM range of ?S. The resulting explicit kinetic model of ?S aggregation has been used to quantitatively explore seeding the reaction by either the compact oligomers or fibrils. Our predictions reveal that, although fibrils are more effective at seeding than oligomers, very high numbers of seeds of either type, of the order of 10(4), are required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular experiments demonstrated that two orders of magnitude lower numbers of oligomers were sufficient to generate high levels of reactive oxygen species, suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.
| S-EPMC4780632 | biostudies-literature
Project description:?-Synuclein is the major component of filamentous inclusions that constitute the defining characteristic of neurodegenerative ?-synucleinopathies. However, the molecular mechanisms underlying ?-synuclein accumulation and spread are unclear. Here we show that intracerebral injections of sarkosyl-insoluble ?-synuclein from brains of patients with dementia with Lewy bodies induced hyperphosphorylated ?-synuclein pathology in wild-type mice. Furthermore, injection of fibrils of recombinant human and mouse ?-synuclein efficiently induced similar ?-synuclein pathologies in wild-type mice. C57BL/6J mice injected with ?-synuclein fibrils developed abundant Lewy body/Lewy neurite-like pathology, whereas mice injected with soluble ?-synuclein did not. Immunoblot analysis demonstrated that endogenous mouse ?-synuclein started to accumulate 3 months after inoculation, while injected human ?-synuclein fibrils disappeared in about a week. These results indicate that ?-synuclein fibrils have prion-like properties and inoculation into wild-type brain induces ?-synuclein pathology in vivo. This is a new mouse model of sporadic ?-synucleinopathy and should be useful for elucidating progression mechanisms and evaluating disease-modifying therapy.
| S-EPMC3613715 | biostudies-literature
Project description:Aggregation of the protein α-Synuclein (αSyn) is of great interest due to its involvement in the pathology of Parkinson's disease. However, under in vitro conditions αSyn is very soluble and kinetically stable for extended time periods. As a result, most αSyn aggregation assays rely on conditions that artificially induce or enhance aggregation, often by introducing rather non-native conditions. It has been shown that αSyn interacts with membranes and conditions have been identified in which membranes can promote as well as inhibit αSyn aggregation. It has also been shown that αSyn has the intrinsic capability to assemble lipid-protein-particles, in a similar way as apolipoproteins can form lipid-bilayer nanodiscs. Here we show that these αSyn-lipid particles (αSyn-LiPs) can also effectively induce, accelerate or inhibit αSyn aggregation, depending on the applied conditions. αSyn-LiPs therefore provide a general platform and additional tool, complementary to other setups, to study various aspects of αSyn amyloid fibril formation.
| S-EPMC6451039 | biostudies-literature
Project description:BackgroundThe accumulation of misfolded proteins appears as a fundamental pathogenic process in human neurodegenerative diseases. In the case of synucleinopathies such as Parkinson's disease (PD) or dementia with Lewy bodies (DLB), the intraneuronal deposition of aggregated alpha-synuclein (?S) is a major characteristic of the disease, but the molecular basis distinguishing the disease-associated protein (?SD) from its normal counterpart remains poorly understood. However, recent research suggests that a prion-like mechanism could be involved in the inter-cellular and inter-molecular propagation of aggregation of the protein within the nervous system.ResultsOur data confirm our previous observations of disease acceleration in a transgenic mouse line (M83) overexpressing a mutated (A53T) form of human ?S, following inoculation of either brain extracts from sick M83 mice or fibrillar recombinant ?S. A similar phenomenon is observed following a "second passage" in the M83 mouse model, including after stereotactic inoculations into the hippocampus or cerebellum. For further molecular analyses of ?SD, we designed an ELISA test that identifies ?SD specifically in sick mice and in the brain regions targeted by the pathological process in this mouse model. ?SD distribution, mainly in the caudal brain regions and spinal cord, overall appears remarkably uniform, whatever the conditions of experimental challenge. In addition to specific detection of ?SD immunoreactivity using an antibody against Ser129 phosphorylated ?S, similar results were observed in ELISA with several other antibodies against the C-terminal part of ?S, including an antibody against non phosphorylated ?S. This also indicated consistent immunoreactivity of the murine ?S protein specifically in the affected brain regions of sick mice.ConclusionsPrion-like behaviour in propagation of the disease-associated ?S was confirmed with the M83 transgenic mouse model, that could be followed by an ELISA test. The ELISA data question their possible relationship with the conformational differences between the disease-associated ?S and its normal counterpart.
| S-EPMC4007641 | biostudies-literature
Project description:Parkinson's disease (PD) is one of many neurodegenerative diseases termed synucleinopathies, neuropathologically defined by inclusions containing aggregated α-synuclein (αS). αS gene (SNCA) mutations can directly cause autosomal dominant PD. In vitro studies demonstrated that SNCA missense mutations may either enhance or diminish αS aggregation but cross-seeding of mutant and wild-type αS proteins appear to reduce aggregation efficiency. Here, we extended these studies by assessing the effects of seeded αS aggregation in αS transgenic mice through intracerebral or peripheral injection of various mutant αS fibrils. We observed modestly decreased time to paralysis in mice transgenic for human A53T αS (line M83) intramuscularly injected with H50Q, G51D or A53E αS fibrils relative to wild-type αS fibrils. Conversely, E46K αS fibril seeding was significantly delayed and less efficient in the same experimental paradigm. However, the amount and distribution of αS inclusions in the central nervous system were similar for all αS fibril muscle injected mice that developed paralysis. Mice transgenic for human αS (line M20) injected in the hippocampus with wild-type, H50Q, G51D or A53E αS fibrils displayed induction of αS inclusion pathology that increased and spread over time. By comparison, induction of αS aggregation following the intrahippocampal injection of E46K αS fibrils in M20 mice was much less efficient. These findings show that H50Q, G51D or A53E can efficiently cross-seed and induce αS pathology in vivo. In contrast, E46K αS fibrils are intrinsically inefficient at seeding αS inclusion pathology. Consistent with previous in vitro studies, E46K αS polymers are likely distinct aggregated conformers that may represent a unique prion-like strain of αS.
| S-EPMC5886210 | biostudies-literature
Project description: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.
| S-EPMC8576194 | biostudies-literature
Project description:Proteinaceous inclusions in neurons, composed primarily of α-synuclein, define the pathology in several neurodegenerative disorders. Neurons can internalize α-synuclein fibrils that can seed new inclusions from endogenously expressed α-synuclein. The factors contributing to the spread of pathology and subsequent neurodegeneration are not fully understood, and different compositions and concentrations of fibrils have been used in different hosts. Here, we systematically vary the concentration and length of well-characterized α-synuclein fibrils and determine their relative ability to induce inclusions and neurodegeneration in different hosts (primary neurons, C57BL/6J and C3H/HeJ mice, and Sprague Dawley rats). Using dynamic-light scattering profiles and other measurements to determine fibril length and concentration, we find that femptomolar concentrations of fibrils are sufficient to induce robust inclusions in primary neurons. However, a narrow and non-linear dynamic range characterizes fibril-mediated inclusion induction in axons and the soma. In mice, the C3H/HeJ strain is more sensitive to fibril exposures than C57BL/6J counterparts, with more inclusions and dopaminergic neurodegeneration. In rats, injection of fibrils into the substantia nigra pars compacta (SNpc) results in similar inclusion spread and dopaminergic neurodegeneration as injection of the fibrils into the dorsal striatum, with prominent inclusion spread to the amygdala and several other brain areas. Inclusion spread, particularly from the SNpc to the striatum, positively correlates with dopaminergic neurodegeneration. These results define biophysical characteristics of α-synuclein fibrils that induce inclusions and neurodegeneration both in vitro and in vivo, and suggest that inclusion spread in the brain may be promoted by a loss of neurons.
| S-EPMC5701756 | biostudies-literature
Project description:Reducing α-synuclein pathology constitutes a plausible strategy against Parkinson's disease. As we recently demonstrated, the β-wrapin protein AS69 binds an N-terminal region in monomeric α-synuclein, interferes with fibril nucleation, and reduces α-synuclein aggregation in vitro and in a fruit fly model of α-synuclein toxicity. The aim of this study was to investigate whether AS69 also reduces α-synuclein pathology in mammalian neurons. To induce α-synuclein pathology, primary mouse neurons were exposed to pre-formed fibrils (PFF) of human α-synuclein. PFF were also injected into the striatum of A30P-α-synuclein transgenic mice. The extent of α-synuclein pathology was determined by phospho-α-synuclein staining and by Triton X-100 solubility. The degeneration of neuronal somata, dendrites, and axon terminals was determined by immunohistochemistry. AS69 and PFF were taken up by primary neurons. AS69 did not alter PFF uptake, but AS69 did reduce PFF-induced α-synuclein pathology. PFF injection into mouse striatum led to α-synuclein pathology and dystrophic neurites. Co-injection of AS69 abrogated PFF-induced pathology. AS69 also reduced the PFF-induced degeneration of dopaminergic axon terminals in the striatum and the degeneration of dopaminergic dendrites in the substantia nigra pars reticulata. AS69 reduced the activation of astroglia but not microglia in response to PFF injection. Collectively, AS69 reduced PFF-induced α-synuclein pathology and the associated neurodegeneration in primary neurons and in mouse brain. Our data therefore suggest that small proteins binding the N-terminus of α-synuclein monomers are promising strategies to modify disease progression in Parkinson's disease.
| S-EPMC8313869 | biostudies-literature
Project description:BACKGROUND: ?-Synuclein (?-syn) plays a central role in the pathogenesis of synucleinopathies, a group of neurodegenerative disorders that includes Parkinson disease, dementia with Lewy bodies and multiple system atrophy. Several findings from cell culture and mouse experiments suggest intercellular ?-syn transfer. RESULTS: Through a methodology used to obtain synthetic mammalian prions, we tested whether recombinant human ?-syn amyloids can promote prion-like accumulation in neuronal cell lines in vitro. A single exposure to amyloid fibrils of human ?-syn was sufficient to induce aggregation of endogenous ?-syn in human neuroblastoma SH-SY5Y cells. Remarkably, endogenous wild-type ?-syn was sufficient for the formation of these aggregates, and overexpression of the protein was not required. CONCLUSIONS: Our results provide compelling evidence that endogenous ?-syn can accumulate in cell culture after a single exposure to exogenous ?-syn short amyloid fibrils. Importantly, using ?-syn short amyloid fibrils as seed, endogenous ?-syn aggregates and accumulates over several passages in cell culture, providing an excellent tool for potential therapeutic screening of pathogenic ?-syn aggregates.
| S-EPMC4064824 | biostudies-other
Project description:α-Synuclein (α-syn) and tau aggregates are the neuropathological hallmarks of Parkinson's disease (PD) and Alzheimer's disease (AD), respectively, although both pathologies co-occur in patients with these diseases, suggesting possible crosstalk between them. To elucidate the interactions of pathological α-syn and tau, we sought to model these interactions. We show that increased accumulation of tau aggregates occur following simultaneous introduction of α-syn mousepreformed fibrils (mpffs) and AD lysate-derived tau seeds (AD-tau) both in vitro and in vivo. Interestingly, the absence of endogenous mouse α-syn in mice reduces the accumulation and spreading of tau, while the absence of tau did not affect the seeding or spreading capacity of α-syn. These in vivo results are consistent with our in vitro data wherein the presence of tau has no synergistic effects on α-syn. Our results point to the important role of α-syn as a modulator of tau pathology burden and spreading in the brains of AD, PDD, and DLB patients.
| S-EPMC7588140 | biostudies-literature