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Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells.

ABSTRACT: Cytoplasmic inclusions containing alpha-synuclein (alpha-Syn) fibrils, referred to as Lewy bodies (LBs), are the signature neuropathological hallmarks of Parkinson's disease (PD). Although alpha-Syn fibrils can be generated from recombinant alpha-Syn protein in vitro, the production of fibrillar alpha-Syn inclusions similar to authentic LBs in cultured cells has not been achieved. We show here that intracellular alpha-Syn aggregation can be triggered by the introduction of exogenously produced recombinant alpha-Syn fibrils into cultured cells engineered to overexpress alpha-Syn. Unlike unassembled alpha-Syn, these alpha-Syn fibrils "seeded" recruitment of endogenous soluble alpha-Syn protein and their conversion into insoluble, hyperphosphorylated, and ubiquitinated pathological species. Thus, this cell model recapitulates key features of LBs in human PD brains. Also, these findings support the concept that intracellular alpha-Syn aggregation is normally limited by the number of active nucleation sites present in the cytoplasm and that small quantities of alpha-Syn fibrils can alter this balance by acting as seeds for aggregation.

SUBMITTER: Luk KC

PROVIDER: S-EPMC2785290 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells.

Luk Kelvin C KC Song Cheng C O'Brien Patrick P Stieber Anna A Branch Jonathan R JR Brunden Kurt R KR Trojanowski John Q JQ Lee Virginia M-Y VM

Proceedings of the National Academy of Sciences of the United States of America 20091105 47

Cytoplasmic inclusions containing alpha-synuclein (alpha-Syn) fibrils, referred to as Lewy bodies (LBs), are the signature neuropathological hallmarks of Parkinson's disease (PD). Although alpha-Syn fibrils can be generated from recombinant alpha-Syn protein in vitro, the production of fibrillar alpha-Syn inclusions similar to authentic LBs in cultured cells has not been achieved. We show here that intracellular alpha-Syn aggregation can be triggered by the introduction of exogenously produced r ...[more]

PMID: 19892735

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Project description:BackgroundConverging evidence suggests a role for microglia-mediated neuroinflammation in Parkinson's disease (PD). Animal models of PD can serve as a platform to investigate the role of neuroinflammation in degeneration in PD. However, due to features of the previously available PD models, interpretations of the role of neuroinflammation as a contributor to or a consequence of neurodegeneration have remained elusive. In the present study, we investigated the temporal relationship of neuroinflammation in a model of synucleinopathy following intrastriatal injection of pre-formed alpha-synuclein fibrils (α-syn PFFS).MethodsMale Fischer 344 rats (N = 114) received unilateral intrastriatal injections of α-syn PFFs, PBS, or rat serum albumin with cohorts euthanized at monthly intervals up to 6 months. Quantification of dopamine neurons, total neurons, phosphorylated α-syn (pS129) aggregates, major histocompatibility complex-II (MHC-II) antigen-presenting microglia, and ionized calcium-binding adaptor molecule-1 (Iba-1) immunoreactive microglial soma size was performed in the substantia nigra. In addition, the cortex and striatum were also examined for the presence of pS129 aggregates and MHC-II antigen-presenting microglia to compare the temporal patterns of pSyn accumulation and reactive microgliosis.ResultsIntrastriatal injection of α-syn PFFs to rats resulted in widespread accumulation of phosphorylated α-syn inclusions in several areas that innervate the striatum followed by significant loss (~ 35%) of substantia nigra pars compacta dopamine neurons within 5-6 months. The peak magnitudes of α-syn inclusion formation, MHC-II expression, and reactive microglial morphology were all observed in the SN 2 months following injection and 3 months prior to nigral dopamine neuron loss. Surprisingly, MHC-II immunoreactivity in α-syn PFF injected rats was relatively limited during the later interval of degeneration. Moreover, we observed a significant correlation between substantia nigra pSyn inclusion load and number of microglia expressing MHC-II. In addition, we observed a similar relationship between α-syn inclusion load and number of microglia expressing MHC-II in cortical regions, but not in the striatum.ConclusionsOur results demonstrate that increases in microglia displaying a reactive morphology and MHC-II expression occur in the substantia nigra in close association with peak numbers of pSyn inclusions, months prior to nigral dopamine neuron degeneration, and suggest that reactive microglia may contribute to vulnerability of SNc neurons to degeneration. The rat α-syn PFF model provides an opportunity to examine the innate immune response to accumulation of pathological α-syn in the context of normal levels of endogenous α-syn and provides insight into the earliest neuroinflammatory events in PD.

Project description:Autosomal dominant variants in LRP10 have been identified in patients with Lewy body diseases (LBDs), including Parkinson's disease (PD), Parkinson's disease-dementia (PDD), and dementia with Lewy bodies (DLB). Nevertheless, there is little mechanistic insight into the role of LRP10 in disease pathogenesis. In the brains of control individuals, LRP10 is typically expressed in non-neuronal cells like astrocytes and neurovasculature, but in idiopathic and genetic cases of PD, PDD, and DLB, it is also present in α-synuclein-positive neuronal Lewy bodies. These observations raise the questions of what leads to the accumulation of LRP10 in Lewy bodies and whether a possible interaction between LRP10 and α-synuclein plays a role in disease pathogenesis. Here, we demonstrate that wild-type LRP10 is secreted via extracellular vesicles (EVs) and can be internalised via clathrin-dependent endocytosis. Additionally, we show that LRP10 secretion is highly sensitive to autophagy inhibition, which induces the formation of atypical LRP10 vesicular structures in neurons in human-induced pluripotent stem cells (iPSC)-derived brain organoids. Furthermore, we show that LRP10 overexpression leads to a strong induction of monomeric α-synuclein secretion, together with time-dependent, stress-sensitive changes in intracellular α-synuclein levels. Interestingly, patient-derived astrocytes carrying the c.1424 + 5G > A LRP10 variant secrete aberrant high-molecular-weight species of LRP10 in EV-free media fractions. Finally, we show that this truncated patient-derived LRP10 protein species (LRP10splice) binds to wild-type LRP10, reduces LRP10 wild-type levels, and antagonises the effect of LRP10 on α-synuclein levels and distribution. Together, this work provides initial evidence for a possible functional role of LRP10 in LBDs by modulating intra- and extracellular α-synuclein levels, and pathogenic mechanisms linked to the disease-associated c.1424 + 5G > A LRP10 variant, pointing towards potentially important disease mechanisms in LBDs.

Project description:The distinct neuropathological features of the different α-Synucleinopathies, as well as the diversity of the α-Synuclein (α-Syn) intracellular inclusion bodies observed in post mortem brain sections, are thought to reflect the strain diversity characterizing invasive α-Syn amyloids. However, this "one strain, one disease" view is still hypothetical, and to date, a possible disease-specific contribution of non-amyloid factors has not been ruled out. In Multiple System Atrophy (MSA), the buildup of α-Syn inclusions in oligodendrocytes seems to result from the terminal storage of α-Syn amyloid aggregates first pre-assembled in neurons. This assembly occurs at the level of neuronal cytoplasmic inclusions, and even earlier, within neuronal intranuclear inclusions (NIIs). Intriguingly, α-Syn NIIs are never observed in α-Synucleinopathies other than MSA, suggesting that these inclusions originate (i) from the unique molecular properties of the α-Syn fibril strains encountered in this disease, or alternatively, (ii) from other factors specifically dysregulated in MSA and driving the intranuclear fibrillization of α-Syn. We report the isolation and structural characterization of a synthetic human α-Syn fibril strain uniquely capable of seeding α-Syn fibrillization inside the nuclear compartment. In primary mouse cortical neurons, this strain provokes the buildup of NIIs with a remarkable morphology reminiscent of cat's eye marbles (see video abstract). These α-Syn inclusions form giant patterns made of one, two, or three lentiform beams that span the whole intranuclear volume, pushing apart the chromatin. The input fibrils are no longer detectable inside the NIIs, where they become dominated by the aggregation of endogenous α-Syn. In addition to its phosphorylation at S129, α-Syn forming the NIIs acquires an epitope antibody reactivity profile that indicates its organization into fibrils, and is associated with the classical markers of α-Syn pathology p62 and ubiquitin. NIIs are also observed in vivo after intracerebral injection of the fibril strain in mice. Our data thus show that the ability to seed NIIs is a strain property that is integrally encoded in the fibril supramolecular architecture. Upstream alterations of cellular mechanisms are not required. In contrast to the lentiform TDP-43 NIIs, which are observed in certain frontotemporal dementias and which are conditional upon GRN or VCP mutations, our data support the hypothesis that the presence of α-Syn NIIs in MSA is instead purely amyloid-strain-dependent.

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Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells.

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Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells.

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