Parkinson’s disease is characterized by accumulation of α-synuclein (αSyn). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal death in part via microglial activation. Heretofore, it remained unknown if oligomeric/fibrillar αSyn could activate the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome in human microglia and whether anti-αSyn antibodies could prevent this effect. Here, we show that αSyn activates the NLRP3 inflammasome in human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) via dual stimulation involving Toll-like receptor 2 (TLR2) engagement and mitochondrial damage. In vitro, hiMG can be activated by mutant (A53T) αSyn secreted from hiPSC-derived A9-dopaminergic neurons. Surprisingly, αSyn–antibody complexes enhanced rather than suppressed inflammasome-mediated interleukin-1β (IL-1β) secretion, indicating these complexes are neuroinflammatory in a human context. A further increase in inflammation was observed with addition of oligomerized amyloid-β peptide (Aβ) and its cognate antibody. In vivo, engraftment of hiMG with αSyn in humanized mouse brain resulted in caspase-1 activation and neurotoxicity, which was exacerbated by αSyn antibody. These findings may have important implications for antibody therapies aimed at depleting misfolded/aggregated proteins from the human brain, as they may paradoxically trigger inflammation in human microglia.
The protein α-synuclein (αsyn) forms pathologic aggregates in a number of neurodegenerative diseases including Lewy body dementia (LBD) and Parkinson’s disease (PD). It is unclear why diseases such as LBD may develop widespread αsyn pathology, while in Alzheimer’s disease with amygdala restricted Lewy bodies (AD/ALB) the αsyn aggregates remain localized. The amygdala contains αsyn aggregates in both LBD and in AD/ALB; to understand why αsyn pathology continues to progress in LBD but not in AD/ALB, tissue from the amygdala and other regions were obtained from 14 cases of LBD, 9 cases of AD/ALB, and 4 controls for immunohistochemical and biochemical characterization. Utilizing a panel of previously characterized αsyn antibodies, numerous unique pathologies differentiating LBD and AD/ALB were revealed; particularly the presence of dense neuropil αsyn aggregates, astrocytic αsyn, and αsyn-containing dystrophic neurites within senile plaques. Within LBD, these unique pathologies were predominantly present within the amygdala. Biochemically, the amygdala in LBD prominently contained specific carboxy-truncated forms of αsyn which are highly prone to aggregate, suggesting that the amygdala may be prone to initiate development of αsyn pathology. Similar to carboxy-truncated αsyn, it was demonstrated herein that the presence of aggregation prone A53T αsyn is sufficient to drive misfolding of wild-type αsyn in human disease. Overall, this study identifies within the amygdala in LBD the presence of unique strain-like variation in αsyn pathology that may be a determinant of disease progression. Electronic supplementary material The online version of this article (10.1186/s40478-019-0787-2) contains supplementary material, which is available to authorized users.
Tauopathies are a group of neurodegenerative disorders, including Alzheimer’s disease, defined by the presence of brain pathological inclusions comprised of abnormally aggregated and highly phosphorylated tau protein. The abundance of brain tau aggregates correlates with disease severity and select phospho-tau epitopes increase at early stages of disease. We generated and characterized a series of novel monoclonal antibodies directed to tau phosphorylated at several of these phospho-epitopes, including Ser396/Ser404, Ser404 and Thr205. We also generated phosphorylation independent antibodies against amino acid residues 193–211. We show that most of these antibodies are highly specific for tau and strongly recognize pathological inclusions in human brains and in a transgenic mouse model of tauopathy. They also reveal epitope-specific differences in the biochemical properties of Alzheimer’s disease sarkosyl-insoluble tau. These new reagents will be useful for investigating the progression of tau pathology and further as tools to target the cellular transmission of tau pathology.
Immunotherapies designed to treat neurodegenerative tauopathies that primarily engage extracellular tau may have limited efficacy as tau is primarily intracellular. We generated tau-targeting single-chain variable fragments (scFvs) and intrabodies (iBs) from the phosphorylated tau-specific antibodies CP13 and PHF1 and the pan-tau antibody Tau5. Recombinant adeno-associated virus (rAAV) was utilized to express these antibody fragments in homozygous JNPL3 P301L tau mice. Two iBs (CP13i, PHF1i) and one scFv (PHF1s) abrogated tau pathology and delayed time to severe hindlimb paralysis. In a second tauopathy model (rTg4510), CP13i and PHF1i reduced tau pathology, but cognate scFvs did not. These data demonstrate that (1) disease-modifying efficacy does not require antibody effector functions, (2) the intracellular targeting of tau with phosphorylated tau-specific iBs is more effective than extracellular targeting with the scFvs, and (3) robust effects on tau pathology before neurodegeneration only resulted in modest disease modification as assessed by delay of severe motor phenotype.
Accumulation of the tau protein in fibrillar intracellular aggregates is a defining feature of multiple neurodegenerative diseases collectively referred to as tauopathies. Despite intensive study of tau, there is limited information on the formation and clearance dynamics of tau inclusions. Using rAAV vectors to mediate expression of Dendra2-tagged human wild-type, P301L and pro-aggregant P301L/S320F tau proteins, with and without the addition of exogenous tau fibrillar seeds, we evaluated tau inclusion dynamics in organotypic brain slice culture (BSC) models using long-term optical pulse labeling methodology. Our studies reveal that tau inclusions typically form in 12–96 h in tauopathy BSC models. Unexpectedly, we demonstrate appreciable turnover of tau within inclusions with an average half-life of ~ 1 week when inclusions are newly formed. When BSCs with inclusions are aged in culture for extended periods, tau inclusions continue to turnover, but their half-lives increase to ~ 2 weeks and ~ 3 weeks after 1 and 2 months in culture, respectively. Individual tau inclusions can be long-lived structures that can persist for months in these BSC models and for even longer in the human brain. However, our data indicate that tau inclusions, are not ‘tombstones’, but dynamic structures with appreciable turnover. Understanding the cellular processes mediating this inclusion turnover may lead to new therapeutic strategies that could reverse pathological tau inclusion formation.
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