Soluble α-synuclein-antibody complexes activate the NLRP3 inflammasome in hiPSC-derived microglia.

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-1ß (IL-1ß) secretion, indicating these complexes are neuroinflammatory in a human context. A further increase in inflammation was observed with addition of oligomerized amyloid-ß peptide (Aß) 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.

α-Synuclein Oligomers Induce Glutamate Release from Astrocytes and Excessive Extrasynaptic NMDAR Activity in Neurons, Thus Contributing to Synapse Loss.

Synaptic and neuronal loss are major neuropathological characteristics of Parkinson's disease. Misfolded protein aggregates in the form of Lewy bodies, comprised mainly of α-synuclein (αSyn), are associated with disease progression, and have also been linked to other neurodegenerative diseases, including Lewy body dementia, Alzheimer's disease, and frontotemporal dementia. However, the effects of αSyn and its mechanism of synaptic damage remain incompletely understood. Here, we show that αSyn oligomers induce Ca2+-dependent release of glutamate from astrocytes obtained from male and female mice, and that mice overexpressing αSyn manifest increased tonic release of glutamate in vivo In turn, this extracellular glutamate activates glutamate receptors, including extrasynaptic NMDARs (eNMDARs), on neurons both in culture and in hippocampal slices of αSyn-overexpressing mice. Additionally, in patch-clamp recording from outside-out patches, we found that oligomerized αSyn can directly activate eNMDARs. In organotypic slices, oligomeric αSyn induces eNMDAR-mediated synaptic loss, which can be reversed by the drug NitroSynapsin. When we expose human induced pluripotent stem cell-derived cerebrocortical neurons to αSyn, we find similar effects. Importantly, the improved NMDAR antagonist NitroSynapsin, which selectively inhibits extrasynaptic over physiological synaptic NMDAR activity, protects synapses from oligomeric αSyn-induced damage in our model systems, thus meriting further study for its therapeutic potential.SIGNIFICANCE STATEMENT Loss of synaptic function and ensuing neuronal loss are associated with disease progression in Parkinson's disease (PD), Lewy body dementia (LBD), and other neurodegenerative diseases. However, the mechanism of synaptic damage remains incompletely understood. α-Synuclein (αSyn) misfolds in PD/LBD, forming Lewy bodies and contributing to disease pathogenesis. Here, we found that misfolded/oligomeric αSyn releases excessive astrocytic glutamate, in turn activating neuronal extrasynaptic NMDA receptors (eNMDARs), thereby contributing to synaptic damage. Additionally, αSyn oligomers directly activate eNMDARs, further contributing to damage. While the FDA-approved drug memantine has been reported to offer some benefit in PD/LBD (Hershey and Coleman-Jackson, 2019), we find that the improved eNMDAR antagonist NitroSynapsin ameliorates αSyn-induced synaptic spine loss, providing potential disease-modifying intervention in PD/LBD.

Conversion of Synthetic Aß to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.

UNLABELLED: The aggregation of amyloid-ß peptide (Aß) in brain is an early event and hallmark of Alzheimer's disease (AD). We combined the advantages of in vitro and in vivo approaches to study cerebral ß-amyloidosis by establishing a long-term hippocampal slice culture (HSC) model. While no Aß deposition was noted in untreated HSCs of postnatal Aß precursor protein transgenic (APP tg) mice, Aß deposition emerged in HSCs when cultures were treated once with brain extract from aged APP tg mice and the culture medium was continuously supplemented with synthetic Aß. Seeded Aß deposition was also observed under the same conditions in HSCs derived from wild-type or App-null mice but in no comparable way when HSCs were fixed before cultivation. Both the nature of the brain extract and the synthetic Aß species determined the conformational characteristics of HSC Aß deposition. HSC Aß deposits induced a microglia response, spine loss, and neuritic dystrophy but no obvious neuron loss. Remarkably, in contrast to in vitro aggregated synthetic Aß, homogenates of Aß deposits containing HSCs induced cerebral ß-amyloidosis upon intracerebral inoculation into young APP tg mice. Our results demonstrate that a living cellular environment promotes the seeded conversion of synthetic Aß into a potent in vivo seeding-active form. SIGNIFICANCE STATEMENT: In this study, we report the seeded induction of Aß aggregation and deposition in long-term hippocampal slice cultures. Remarkably, we find that the biological activities of the largely synthetic Aß aggregates in the culture are very similar to those observed in vivo This observation is the first to show that potent in vivo seeding-active Aß aggregates can be obtained by seeded conversion of synthetic Aß in a living (wild-type) cellular environment.

Propagation of Aß pathology: hypotheses, discoveries, and yet unresolved questions from experimental and human brain studies.

In brains of patients with Alzheimer's disease (AD), Aß peptides accumulate in parenchyma and, almost invariably, also in the vascular walls. Although Aß aggregation is, by definition, present in AD, its impact is only incompletely understood. It occurs in a stereotypical spatiotemporal distribution within neuronal networks in the course of the disease. This suggests a role for synaptic connections in propagating Aß pathology, and possibly of axonal transport in an antero- or retrograde way-although, there is also evidence for passive, extracellular diffusion. Striking, in AD, is the conjunction of tau and Aß pathology. Tau pathology in the cell body of neurons precedes Aß deposition in their synaptic endings in several circuits such as the entorhino-dentate, cortico-striatal or subiculo-mammillary connections. However, genetic evidence suggests that Aß accumulation is the first step in AD pathogenesis. To model the complexity and consequences of Aß aggregation in vivo, various transgenic (tg) rodents have been generated. In rodents tg for the human Aß precursor protein, focal injections of preformed Aß aggregates can induce Aß deposits in the vicinity of the injection site, and over time in more distant regions of the brain. This suggests that Aß shares with α-synuclein, tau and other proteins the property to misfold and aggregate homotypic molecules. We propose to group those proteins under the term "propagons". Propagons may lack the infectivity of prions. We review findings from neuropathological examinations of human brains in different stages of AD and from studies in rodent models of Aß aggregation and discuss putative mechanisms underlying the initiation and spread of Aß pathology.

Multiple factors contribute to the peripheral induction of cerebral ß-amyloidosis.

Deposition of aggregated amyloid-ß (Aß) peptide in brain is an early event and hallmark pathology of Alzheimer's disease and cerebral Aß angiopathy. Experimental evidence supports the concept that Aß multimers can act as seeds and structurally corrupt other Aß peptides by a self-propagating mechanism. Here we compare the induction of cerebral ß-amyloidosis by intraperitoneal applications of Aß-containing brain extracts in three Aß-precursor protein (APP) transgenic mouse lines that differ in levels of transgene expression in brain and periphery (APP23 mice, APP23 mice lacking murine APP, and R1.40 mice). Results revealed that beta-amyloidosis induction, which could be blocked with an anti-Aß antibody, was dependent on the amount of inoculated brain extract and on the level of APP/Aß expression in the brain but not in the periphery. The induced Aß deposits in brain occurred in a characteristic pattern consistent with the entry of Aß seeds at multiple brain locations. Intraperitoneally injected Aß could be detected in blood monocytes and some peripheral tissues (liver, spleen) up to 30 d after the injection but escaped histological and biochemical detection thereafter. These results suggest that intraperitoneally inoculated Aß seeds are transported from the periphery to the brain in which corruptive templating of host Aß occurs at multiple sites, most efficiently in regions with high availability of soluble Aß.

A fluorogenic aryl fluorosulfate for intraorganellar transthyretin imaging in living cells and in Caenorhabditis elegans.

Fluorogenic probes, due to their often greater spatial and temporal sensitivity in comparison to permanently fluorescent small molecules, represent powerful tools to study protein localization and function in the context of living systems. Herein, we report fluorogenic probe 4, a 1,3,4-oxadiazole designed to bind selectively to transthyretin (TTR). Probe 4 comprises a fluorosulfate group not previously used in an environment-sensitive fluorophore. The fluorosulfate functional group does not react covalently with TTR on the time scale required for cellular imaging, but does red shift the emission maximum of probe 4 in comparison to its nonfluorosulfated analogue. We demonstrate that probe 4 is dark in aqueous buffers, whereas the TTR·4 complex exhibits a fluorescence emission maximum at 481 nm. The addition of probe 4 to living HEK293T cells allows efficient binding to and imaging of exogenous TTR within intracellular organelles, including the mitochondria and the endoplasmic reticulum. Furthermore, live Caenorhabditis elegans expressing human TTR transgenically and treated with probe 4 display TTR·4 fluorescence in macrophage-like coelomocytes. An analogue of fluorosulfate probe 4 does react selectively with TTR without labeling the remainder of the cellular proteome. Studies on this analogue suggest that certain aryl fluorosulfates, due to their cell and organelle permeability and activatable reactivity, could be considered for the development of protein-selective covalent probes.

Seeded strain-like transmission of ß-amyloid morphotypes in APP transgenic mice.

The polymorphic ß-amyloid lesions present in individuals with Alzheimer's disease are collectively known as cerebral ß-amyloidosis. Amyloid precursor protein (APP) transgenic mouse models similarly develop ß-amyloid depositions that differ in morphology, binding of amyloid conformation-sensitive dyes, and Aß40/Aß42 peptide ratio. To determine the nature of such ß-amyloid morphotypes, ß-amyloid-containing brain extracts from either aged APP23 brains or aged APPPS1 brains were intracerebrally injected into the hippocampus of young APP23 or APPPS1 transgenic mice. APPPS1 brain extract injected into young APP23 mice induced ß-amyloid deposition with the morphological, conformational, and Aß40/Aß42 ratio characteristics of ß-amyloid deposits in aged APPPS1 mice, whereas APP23 brain extract injected into young APP23 mice induced ß-amyloid deposits with the characteristics of ß-amyloid deposits in aged APP23 mice. Injecting the two extracts into the APPPS1 host revealed a similar difference between the induced ß-amyloid deposits, although less prominent, and the induced deposits were similar to the ß-amyloid deposits found in aged APPPS1 hosts. These results indicate that the molecular composition and conformation of aggregated Aß in APP transgenic mice can be maintained by seeded conversion.

Soluble Aß seeds are potent inducers of cerebral ß-amyloid deposition.

Cerebral ß-amyloidosis and associated pathologies can be exogenously induced by the intracerebral injection of small amounts of pathogenic Aß-containing brain extract into young ß-amyloid precursor protein (APP) transgenic mice. The probable ß-amyloid-inducing factor in the brain extract has been identified as a species of aggregated Aß that is generated in its most effective conformation or composition in vivo. Here we report that Aß in the brain extract is more proteinase K (PK) resistant than is synthetic fibrillar Aß, and that this PK-resistant fraction of the brain extract retains the capacity to induce ß-amyloid deposition upon intracerebral injection in young, pre-depositing APP23 transgenic mice. After ultracentrifugation of the brain extract, <0.05% of the Aß remained in the supernatant fraction, and these soluble Aß species were largely PK sensitive. However, upon intracerebral injection, this soluble fraction accounted for up to 30% of the ß-amyloid induction observed with the unfractionated extract. Fragmentation of the Aß seeds by extended sonication increased the seeding capacity of the brain extract. In summary, these results suggest that multiple Aß assemblies, with various PK sensitivities, are capable of inducing ß-amyloid aggregation in vivo. The finding that small and soluble Aß seeds are potent inducers of cerebral ß-amyloidosis raises the possibility that such seeds may mediate the spread of ß-amyloidosis in the brain. If they can be identified in vivo, soluble Aß seeds in bodily fluids also could serve as early biomarkers for cerebral ß-amyloidogenesis and eventually Alzheimer's disease.

The presence of Aß seeds, and not age per se, is critical to the initiation of Aß deposition in the brain.

The deposition of the ß-amyloid (Aß) peptide in senile plaques and cerebral Aß-amyloid angiopathy can be seeded in ß-amyloid precursor protein (APP)-transgenic mice by the intracerebral infusion of brain extracts containing aggregated Aß. Previous studies of seeded ß-amyloid induction have used relatively short incubation periods to dissociate seeded ß-amyloid induction from endogenous ß-amyloid deposition of the host, thus precluding the analysis of the impact of age and extended incubation periods on the instigation and spread of Aß lesions in brain. In the present study using R1.40 APP-transgenic mice (which do not develop endogenous Aß deposition up to 15 months of age) we show that: (1) seeding at 9 months of age does not induce more Aß deposition than seeding at 3 months of age, provided that the incubation period (6 months) is the same; and (2) very long-term (12 months) incubation after a focal application of the seed results in the emergence of Aß deposits throughout the forebrain. These findings indicate that the presence of Aß seeds, and not the age of the host per se, is critical to the initiation of Aß aggregation in the brain, and that Aß deposition, actuated in one brain area, eventually spreads throughout the brain.

Induction of cerebral beta-amyloidosis: intracerebral versus systemic Abeta inoculation.

Despite the importance of the aberrant polymerization of Abeta in the early pathogenic cascade of Alzheimer's disease, little is known about the induction of Abeta aggregation in vivo. Here we show that induction of cerebral beta-amyloidosis can be achieved in many different brain areas of APP23 transgenic mice through the injection of dilute Abeta-containing brain extracts. Once the amyloidogenic process has been exogenously induced, the nature of the induced Abeta-deposition is determined by the brain region of the host. Because these observations are reminiscent of a prion-like mechanism, we then investigated whether cerebral beta-amyloidosis also can be induced by peripheral and systemic inoculations or by the intracerebral implantation of stainless steel wires previously coated with minute amounts of Abeta-containing brain extract. Results reveal that oral, intravenous, intraocular, and intranasal inoculations yielded no detectable induction of cerebral beta-amyloidosis in APP23 transgenic mice. In contrast, transmission of cerebral beta-amyloidosis through the Abeta-contaminated steel wires was demonstrated. Notably, plasma sterilization, but not boiling of the wires before implantation, prevented the induction of beta-amyloidosis. Our results suggest that minute amounts of Abeta-containing brain material in direct contact with the CNS can induce cerebral beta-amyloidosis, but that systemic cellular mechanisms of prion uptake and transport to the CNS may not apply to Abeta.

E22Q-mutant Abeta peptide (AbetaDutch) increases vascular but reduces parenchymal Abeta deposition.

Patients that have hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) generate both wild-type beta-amyloid (Abetawt) and E22Q-mutant beta-amyloid (AbetaDutch). Postmortem analysis of HCHWA-D brains reveals severe cerebral amyloid angiopathy with very little parenchymal amyloid deposition. To investigate amyloidosis in the presence of both Abetawt and AbetaDutch variants, transgenic (tg) APP23 mice were crossed with APPDutch mice. Although single-tg APP23 mice deposited Abetawt with aging, double-tg APP23/APPDutch mice co-deposited AbetaDutch (mainly AbetaDutch1-40) and Abetawt at twofold higher total Abeta levels. Vascular Abeta deposits and hemorrhages were twice as high in APP23/APPDutch mice compared with APP23 mice. Surprisingly, parenchymal Abeta deposition was reduced in the double-tg mice compared with the single-tg APP23 mice. Our findings suggest that AbetaDutch1-40 inhibits parenchymal amyloidosis but exacerbates vascular amyloid, hence explaining the compartment-specific distribution of cerebral amyloid in HCHWA-D patients.

Gleevec increases levels of the amyloid precursor protein intracellular domain and of the amyloid-beta degrading enzyme neprilysin.

Amyloid-beta (Abeta) deposition is a major pathological hallmark of Alzheimer's disease. Gleevec, a known tyrosine kinase inhibitor, has been shown to lower Abeta secretion, and it is considered a potential basis for novel therapies for Alzheimer's disease. Here, we show that Gleevec decreases Abeta levels without the inhibition of Notch cleavage by a mechanism distinct from gamma-secretase inhibition. Gleevec does not influence gamma-secretase activity in vitro; however, treatment of cell lines leads to a dose-dependent increase in the amyloid precursor protein intracellular domain (AICD), whereas secreted Abeta is decreased. This effect is observed even in presence of a potent gamma-secretase inhibitor, suggesting that Gleevec does not activate AICD generation but instead may slow down AICD turnover. Concomitant with the increase in AICD, Gleevec leads to elevated mRNA and protein levels of the Abeta-degrading enzyme neprilysin, a potential target gene of AICD-regulated transcription. Thus, the Gleevec mediated-increase in neprilysin expression may involve enhanced AICD signaling. The finding that Gleevec elevates neprilysin levels suggests that its Abeta-lowering effect may be caused by increased Abeta-degradation.

Molecular Imaging of Cardiac Amyloidosis.

Transthyretin and light-chain amyloidosis are the 2 main causes of cardiac amyloidosis. Recent developments in molecular imaging have transformed our ability to diagnose transthyretin cardiac amyloidosis noninvasively and unmasked a hitherto unrecognized prevalence of the disease. This review summarizes the current and evolving imaging approaches, their molecular structural basis, and the gaps in imaging capabilities that have arisen as a result of parallel developments in pharmacotherapy delivering the first effective treatment options for this condition.

Efficient 1-Hour Technetium-99 m Pyrophosphate Imaging Protocol for the Diagnosis of Transthyretin Cardiac Amyloidosis.

BACKGROUND: Technetium-99 m pyrophosphate protocols for transthyretin cardiac amyloidosis diagnosis have variably used 1- and 3-hour imaging time points. We investigated whether imaging at 1 hour with superior efficiency had comparable diagnostic accuracy as 3-hour imaging. METHODS: This is a registry analysis of patients with suspected transthyretin cardiac amyloidosis referred for technetium-99 m pyrophosphate at a single tertiary center from June 2015 through January 2019. Patients underwent planar and single-photon emission computed tomography (SPECT) imaging at 1 and 3 hours. A positive Tc-99m pyrophosphate study was defined by the presence of diffuse myocardial tracer uptake on SPECT. For planar imaging, visual semiquantitative (grades 0-3, ≥2 considered positive) and quantitative heart to contralateral ratios (≥1.5 considered positive) were used. RESULTS: Two hundred thirty-three patients (69% men; median age, 77 [69-83] years) underwent the study protocol. There were 60 (25.8%) patients with diffuse myocardial uptake, 1 (0.4%) with regional uptake, and 172 (73.8%) with no myocardial uptake. Results of SPECT were identical at 1 and 3 hours. Planar imaging at 1 hour had 98% sensitivity and 96% specificity. Planar grade 0 uptake or heart to contralateral ratio ≤1.2 and planar grade 3 uptake or heart to contralateral ratio ≥2.0 were always associated with negative and positive SPECT, respectively. For planar grades 1 and 2 uptake and heart to contralateral ratio 1.3 to 1.9, SPECT was needed to make a diagnosis. No patient with light-chain cardiac amyloidosis had positive SPECT. CONCLUSIONS: An efficient 1-hour technetium-99 m pyrophosphate protocol had comparable diagnostic performance to a 3-hour protocol.

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.

Peptide probes detect misfolded transthyretin oligomers in plasma of hereditary amyloidosis patients.

Increasing evidence supports the hypothesis that soluble misfolded protein assemblies contribute to the degeneration of postmitotic tissue in amyloid diseases. However, there is a dearth of reliable nonantibody-based probes for selectively detecting oligomeric aggregate structures circulating in plasma or deposited in tissues, making it difficult to scrutinize this hypothesis in patients. Hence, understanding the structure-proteotoxicity relationships driving amyloid diseases remains challenging, hampering the development of early diagnostic and novel treatment strategies. We report peptide-based probes that selectively label misfolded transthyretin (TTR) oligomers circulating in the plasma of TTR hereditary amyloidosis patients exhibiting a predominant neuropathic phenotype. These probes revealed that there are much fewer misfolded TTR oligomers in healthy controls, in asymptomatic carriers of mutations linked to amyloid polyneuropathy, and in patients with TTR-associated cardiomyopathies. The absence of misfolded TTR oligomers in the plasma of cardiomyopathy patients suggests that the tissue tropism observed in the TTR amyloidoses is structure-based. Misfolded oligomers decrease in TTR amyloid polyneuropathy patients treated with disease-modifying therapies (tafamidis or liver transplant-mediated gene therapy). In a subset of TTR amyloid polyneuropathy patients, the probes also detected a circulating TTR fragment that disappeared after tafamidis treatment. Proteomic analysis of the isolated TTR oligomers revealed a specific patient-associated signature composed of proteins that likely associate with the circulating TTR oligomers. Quantification of plasma oligomer concentrations using peptide probes could become an early diagnostic strategy, a response-to-therapy biomarker, and a useful tool for understanding structure-proteotoxicity relationships in the TTR amyloidoses.

Targeting protein aggregation for the treatment of degenerative diseases.

The aggregation of specific proteins is hypothesized to underlie several degenerative diseases, which are collectively known as amyloid disorders. However, the mechanistic connection between the process of protein aggregation and tissue degeneration is not yet fully understood. Here, we review current and emerging strategies to ameliorate aggregation-associated degenerative disorders, with a focus on disease-modifying strategies that prevent the formation of and/or eliminate protein aggregates. Persuasive pharmacological and genetic evidence now supports protein aggregation as the cause of postmitotic tissue dysfunction or loss. However, a more detailed understanding of the factors that trigger and sustain aggregate formation and of the structure-activity relationships underlying proteotoxicity is needed to develop future disease-modifying therapies.