α-Synuclein oligomers form by secondary nucleation.

Oligomeric species arising during the aggregation of α-synuclein are implicated as a major source of toxicity in Parkinson's disease, and thus a major potential drug target. However, both their mechanism of formation and role in aggregation are largely unresolved. Here we show that, at physiological pH and in the absence of lipid membranes, α-synuclein aggregates form by secondary nucleation, rather than simple primary nucleation, and that this process is enhanced by agitation. Moreover, using a combination of single molecule and bulk level techniques, we identify secondary nucleation on the surfaces of existing fibrils, rather than formation directly from monomers, as the dominant source of oligomers. Our results highlight secondary nucleation as not only the key source of oligomers, but also the main mechanism of aggregate formation, and show that these processes take place under conditions which recapitulate the neutral pH and ionic strength of the cytosol.

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.

The pathogenesis of Parkinson's disease.

Parkinson's disease is a progressive neurodegenerative condition associated with the deposition of aggregated α-synuclein. Insights into the pathogenesis of Parkinson's disease have been derived from genetics and molecular pathology. Biochemical studies, investigation of transplanted neurons in patients with Parkinson's disease, and cell and animal model studies suggest that abnormal aggregation of α-synuclein and spreading of pathology between the gut, brainstem, and higher brain regions probably underlie the development and progression of Parkinson's disease. At a cellular level, abnormal mitochondrial, lysosomal, and endosomal function can be identified in both monogenic and sporadic Parkinson's disease, suggesting multiple potential treatment approaches. Recent work has also highlighted maladaptive immune and inflammatory responses, possibly triggered in the gut, that accelerate the pathogenesis of Parkinson's disease. Although there are currently no disease-modifying treatments for Parkinson's disease, we now have a solid basis for the development of rational neuroprotective therapies that we hope will halt the progression of this disabling neurological condition.

Meeting Summary of The NYO3 5th NO-Age/AD Meeting and the 1st Norway-UK Joint Meeting on Aging and Dementia: Recent Progress on the Mechanisms and Interventional Strategies.

Unhealthy aging poses a global challenge with profound healthcare and socioeconomic implications. Slowing down the aging process offers a promising approach to reduce the burden of a number of age-related diseases, such as dementia, and promoting healthy longevity in the old population. In response to the challenge of the aging population and with a view to the future, Norway and the United Kingdom are fostering collaborations, supported by a "Money Follows Cooperation agreement" between the 2 nations. The inaugural Norway-UK joint meeting on aging and dementia gathered leading experts on aging and dementia from the 2 nations to share their latest discoveries in related fields. Since aging is an international challenge, and to foster collaborations, we also invited leading scholars from 11 additional countries to join this event. This report provides a summary of the conference, highlighting recent progress on molecular aging mechanisms, genetic risk factors, DNA damage and repair, mitophagy, autophagy, as well as progress on a series of clinical trials (eg, using NAD+ precursors). The meeting facilitated dialogue among policymakers, administrative leaders, researchers, and clinical experts, aiming to promote international research collaborations and to translate findings into clinical applications and interventions to advance healthy aging.

Tau and neurodegeneration.

First identified in 1975, tau was implicated in Alzheimer's disease 10 years later. Filamentous tangle inclusions were known to be made of hyperphosphorylated tau by 1991, with similar inclusions gaining recognition for being associated with other neurodegenerative diseases. In 1998, mutations in MAPT, the gene that encodes tau, were identified as the cause of a dominantly inherited form of frontotemporal dementia with abundant filamentous tau inclusions. While this result indicated that assembly of tau into aberrant filaments is sufficient to drive neurodegeneration and dementia, most cases of tauopathy are sporadic. More recent work in experimental systems showed that filamentous assemblies of tau may first form in one brain area, and then spread to others in a prion-like fashion. Beginning in 2017, work on human brains using high-resolution techniques has led to a structure-based classification of tauopathies, which has opened the door to a better understanding of the significance of tau filament formation.

New SNCA mutation and structures of α-synuclein filaments from juvenile-onset synucleinopathy.

A 21-nucleotide duplication in one allele of SNCA was identified in a previously described disease with abundant α-synuclein inclusions that we now call juvenile-onset synucleinopathy (JOS). This mutation translates into the insertion of MAAAEKT after residue 22 of α-synuclein, resulting in a protein of 147 amino acids. Both wild-type and mutant proteins were present in sarkosyl-insoluble material that was extracted from frontal cortex of the individual with JOS and examined by electron cryo-microscopy. The structures of JOS filaments, comprising either a single protofilament, or a pair of protofilaments, revealed a new α-synuclein fold that differs from the folds of Lewy body diseases and multiple system atrophy (MSA). The JOS fold consists of a compact core, the sequence of which (residues 36-100 of wild-type α-synuclein) is unaffected by the mutation, and two disconnected density islands (A and B) of mixed sequences. There is a non-proteinaceous cofactor bound between the core and island A. The JOS fold resembles the common substructure of MSA Type I and Type II dimeric filaments, with its core segment approximating the C-terminal body of MSA protofilaments B and its islands mimicking the N-terminal arm of MSA protofilaments A. The partial similarity of JOS and MSA folds extends to the locations of their cofactor-binding sites. In vitro assembly of recombinant wild-type α-synuclein, its insertion mutant and their mixture yielded structures that were distinct from those of JOS filaments. Our findings provide insight into a possible mechanism of JOS fibrillation in which mutant α-synuclein of 147 amino acids forms a nucleus with the JOS fold, around which wild-type and mutant proteins assemble during elongation.

Functional Interaction Between α-Synuclein and Nurr1 in Dopaminergic Neurons.

Increased expression of alpha-synuclein (ASYN) and decreased expression of Nurr1 are associated with Parkinson's disease (PD) pathogenesis. These two proteins interact functionally and ASYN overexpression suppresses Nurr1 levels. ASYN pan-neuronal overexpression coupled with Nurr1 hemizygosity followed by Nurr1 repression in aging mice results in the manifestation of a typical PD-related phenotype and pathology. Here we investigated in mice the effects of C-terminally truncated ASYN(120) overexpression in dopaminergic (DA-ergic) neurons compounded with Nurr1 hemizygosity ('2-hit-DA'). We report that '2-hit-DA' animals did not manifest a characteristic PD-related phenotype, despite further substantia nigra ASYN-overexpression-dependent and age dependent Nurr1 protein downregulation. However, they displayed increased energy expenditure, reduced striatal dopamine (DA) and prolonged hyperactivity to a novel environment indicating impaired habituation. This DA-ergic dysfunction was observed in young adult '2-hit-DA' mice, persisted throughout life and it was associated with ASYN and Nurr1 synergistic alterations of DAT levels and function. Our experiments indicate that the expression levels of ASYN and Nurr1 are critical in the dysregulation of the nigrostriatal DA system and may be involved in neuropsychiatric aspects of PD.

The Role of Rab Proteins in Parkinson's Disease Synaptopathy.

In patients affected by Parkinson's disease (PD), the most common neurodegenerative movement disorder, the brain is characterized by the loss of dopaminergic neurons in the nigrostriatal system, leading to dyshomeostasis of the basal ganglia network activity that is linked to motility dysfunction. PD mostly arises as an age-associated sporadic disease, but several genetic forms also exist. Compelling evidence supports that synaptic damage and dysfunction characterize the very early phases of either sporadic or genetic forms of PD and that this early PD synaptopathy drives retrograde terminal-to-cell body degeneration, culminating in neuronal loss. The Ras-associated binding protein (Rab) family of small GTPases, which is involved in the maintenance of neuronal vesicular trafficking, synaptic architecture and function in the central nervous system, has recently emerged among the major players in PD synaptopathy. In this manuscript, we provide an overview of the main findings supporting the involvement of Rabs in either sporadic or genetic PD pathophysiology, and we highlight how Rab alterations participate in the onset of early synaptic damage and dysfunction.

Safety, tolerability and pharmacokinetics of the oligomer modulator anle138b with exposure levels sufficient for therapeutic efficacy in a murine Parkinson model: A randomised, double-blind, placebo-controlled phase 1a trial.

BACKGROUND: Synucleinopathies such as Parkinson ́s disease (PD), Dementia with Lewy bodies (DLB) and Multiple System Atrophy (MSA) are characterized by deposition of misfolded and aggregated α-synuclein. Small aggregates (oligomers) of α-synuclein have been shown to be the most relevant neurotoxic species and are targeted by anle138b, an orally bioavailable small molecule compound which shows strong disease-modifying effects in animal models of synucleinopathies. METHODS: Anle138b was studied in a single-centre, double-blind, randomised, placebo-controlled single ascending dose (SAD) and multiple ascending dose (MAD) study in healthy subjects. Eligible participants were randomly assigned (1:1 for sentinel subjects and 1:5 for main group) to placebo or anle138b (dose range 50 mg to 300 mg per day), respectively. In addition, the effect of food on the pharmakokinetics of anle138b in healthy subjects was examined in doses of 150 mg per day. Participants were randomized to treatment sequence (fed→fasted) or (fasted→fed). Treatment was administered orally in hard gelatine capsules containing either 10 mg or 30 mg of anle138b or excipient only. The primary endpoints were safety and tolerability, the secondary endpoint was pharmakokinetics. Data from all randomized individuals were evaluated. CLINICALTRIALS: gov-identifier: NCT04208152. EudraCT-number: 2019-004218-33. FINDINGS: Between December 17th, 2019 and June 27th, 2020 196 healthy volunteers were screened and 68 participants were enrolled. Of these, all completed the study per protocol. There were no major protocol deviations. Adverse events in this healthy volunteer trial were mostly mild and all fully recovered or resolved prior to discharge. From baseline to completion of the trial no medically significant individual changes were observed in any system organ class. Already at multiple doses of 200 mg, exposure levels above the fully effective exposure in the MI2 mouse Parkinson model were observed. INTERPRETATION: The favourable safety and PK profile of anle138b in doses resulting in exposures above the fully effective plasma level in a mouse Parkinson model warrant further clinical trials in patients with synucleinopathies. FUNDING: This study was funded by MODAG GmbH and by the Michael J. Fox foundation for Parkinson's Research.

Age-dependent formation of TMEM106B amyloid filaments in human brains.

Many age-dependent neurodegenerative diseases, such as Alzheimer's and Parkinson's, are characterized by abundant inclusions of amyloid filaments. Filamentous inclusions of the proteins tau, amyloid-ß, α-synuclein and transactive response DNA-binding protein (TARDBP; also known as TDP-43) are the most common1,2. Here we used structure determination by cryogenic electron microscopy to show that residues 120-254 of the lysosomal type II transmembrane protein 106B (TMEM106B) also form amyloid filaments in human brains. We determined the structures of TMEM106B filaments from a number of brain regions of 22 individuals with abundant amyloid deposits, including those resulting from sporadic and inherited tauopathies, amyloid-ß amyloidoses, synucleinopathies and TDP-43 proteinopathies, as well as from the frontal cortex of 3 individuals with normal neurology and no or only a few amyloid deposits. We observed three TMEM106B folds, with no clear relationships between folds and diseases. TMEM106B filaments correlated with the presence of a 29-kDa sarkosyl-insoluble fragment and globular cytoplasmic inclusions, as detected by an antibody specific to the carboxy-terminal region of TMEM106B. The identification of TMEM106B filaments in the brains of older, but not younger, individuals with normal neurology indicates that they form in an age-dependent manner.

Synapsin III gene silencing redeems alpha-synuclein transgenic mice from Parkinson's disease-like phenotype.

Fibrillary aggregated α-synuclein (α-syn) deposition in Lewy bodies (LB) characterizes Parkinson's disease (PD) and is believed to trigger dopaminergic synaptic failure and a retrograde terminal-to-cell body neuronal degeneration. We described that the neuronal phosphoprotein synapsin III (Syn III) cooperates with α-syn to regulate dopamine (DA) release and can be found in the insoluble α-syn fibrils composing LB. Moreover, we showed that α-syn aggregates deposition, and the associated onset of synaptic deficits and neuronal degeneration occurring following adeno-associated viral vectors-mediated overexpression of human α-syn in the nigrostriatal system are hindered in Syn III knock out mice. This supports that Syn III facilitates α-syn aggregation. Here, in an interventional experimental design, we found that by inducing the gene silencing of Syn III in human α-syn transgenic mice at PD-like stage with advanced α-syn aggregation and overt striatal synaptic failure, we could lower α-syn aggregates and striatal fibers loss. In parallel, we observed recovery from synaptic vesicles clumping, DA release failure, and motor functions impairment. This supports that Syn III consolidates α-syn aggregates, while its downregulation enables their reduction and redeems the PD-like phenotype. Strategies targeting Syn III could thus constitute a therapeutic option for PD.

In Vivo 18F-Flortaucipir PET Does Not Accurately Support the Staging of Progressive Supranuclear Palsy.

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder characterized by neuroglial tau pathology. A new staging system for PSP pathology postmortem has been described and validated. We used a data-driven approach to test whether postmortem pathologic staging in PSP can be reproduced in vivo with 18F-flortaucipir PET. Methods: Forty-two patients with probable PSP and 39 controls underwent 18F-flortaucipir PET. Conditional inference tree analyses on regional binding potential values identified absent/present pathology thresholds to define in vivo staging. Following the postmortem staging approach for PSP pathology, we evaluated the combinations of absent/present pathology (or abnormal/normal PET signal) across all regions to assign each participant to in vivo stages. ANOVA was applied to analyze differences among means of disease severity between stages. In vivo staging was compared with postmortem staging in 9 patients who also had postmortem confirmation of the diagnosis and stage. Results: Stage assignment was estimable in 41 patients: 10, 26, and 5 patients were classified in stage I/II, stage III/IV, and stage V/VI, respectively, whereas 1 patient was not classifiable. Explorative substaging identified 2 patients in stage I, 8 in stage II, 9 in stage III, 17 in stage IV, and 5 in stage V. However, the nominal 18F-flortaucipir--derived stage was not associated with clinical severity and was not indicative of pathology staging postmortem. Conclusion:18F-flortaucipir PET in vivo does not correspond to neuropathologic staging in PSP. This analytic approach, seeking to mirror in vivo neuropathology staging with PET-to-autopsy correlational analyses, might enable in vivo staging with next-generation tau PET tracers; however, further evidence and comparisons with postmortem data are needed.

The microglial P2Y6 receptor mediates neuronal loss and memory deficits in neurodegeneration.

Microglia are implicated in neurodegeneration, potentially by phagocytosing neurons, but it is unclear how to block the detrimental effects of microglia while preserving their beneficial roles. The microglial P2Y6 receptor (P2Y6R) - activated by extracellular UDP released by stressed neurons - is required for microglial phagocytosis of neurons. We show here that injection of amyloid beta (Aß) into mouse brain induces microglial phagocytosis of neurons, followed by neuronal and memory loss, and this is all prevented by knockout of P2Y6R. In a chronic tau model of neurodegeneration (P301S TAU mice), P2Y6R knockout prevented TAU-induced neuronal and memory loss. In vitro, P2Y6R knockout blocked microglial phagocytosis of live but not dead targets and reduced tau-, Aß-, and UDP-induced neuronal loss in glial-neuronal cultures. Thus, the P2Y6 receptor appears to mediate Aß- and tau-induced neuronal and memory loss via microglial phagocytosis of neurons, suggesting that blocking this receptor may be beneficial in the treatment of neurodegenerative diseases.

Tau aggregation and its relation to selected forms of neuronal cell death.

How neurons die in neurodegenerative diseases is still unknown. The distinction between apoptosis as a genetically controlled mechanism, and necrosis, which was viewed as an unregulated process, has blurred with the ever-increasing number of necrotic-like death subroutines underpinned by genetically defined pathways. It is therefore pertinent to ask whether any of them apply to neuronal cell death in tauopathies. Although Alzheimer's disease (AD) is the most prevalent tauopathy, tauopathies comprise an array of over 30 diseases in which the cytoplasmic protein tau aggregates in neurons, and also, in some diseases, in glia. Animal models have sought to distil the contribution of tau aggregation to the cell death process but despite intensive research, no one mechanism of cell death has been unequivocally defined. The process of tau aggregation, and the fibrillar structures that form, touch on so many cellular functions that there is unlikely to be a simple linear pathway of death; as one is blocked another is likely to take the lead. It is timely to ask how far we have advanced into defining whether any of the molecular players in the new death subroutines participate in the death process. Here we briefly review the currently known cell death routines and explore what is known about their participation in tau aggregation-related cell death. We highlight the involvement of cell autonomous and the more recent non-cell autonomous pathways that may enhance tau-aggregate toxicity, and discuss recent findings that implicate microglial phagocytosis of live neurons with tau aggregates as a mechanism of death.

Microglia become hypofunctional and release metalloproteases and tau seeds when phagocytosing live neurons with P301S tau aggregates.

The microtubule-associated protein tau aggregates in multiple neurodegenerative diseases, causing inflammation and changing the inflammatory signature of microglia by unknown mechanisms. We have shown that microglia phagocytose live neurons containing tau aggregates cultured from P301S tau mice due to neuronal tau aggregate-induced exposure of the "eat me" signal phosphatidylserine. Here, we show that after phagocytosing tau aggregate-bearing neurons, microglia become hypophagocytic while releasing seed-competent insoluble tau aggregates. These microglia express a senescence-like phenotype, demonstrated by acidic ß-galactosidase activity, secretion of paracrine senescence-associated cytokines, and maturation of matrix remodeling enzymes, results that are corroborated in P301S mouse brains and ex vivo brain slices. In particular, the nuclear factor κB­dependent activation of matrix metalloprotease 3 (MMP3/stromelysin1) was replicated in brains from patients with tauopathy. These data show that microglia that have been activated to ingest live tau aggregates-bearing neurons behave hormetically, becoming hypofunctional while acting as vectors of tau aggregate spreading.

Mapping the binding site topology of amyloid protein aggregates using multivalent ligands.

A key process in the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases is the aggregation of proteins to produce fibrillary aggregates with a cross ß-sheet structure, amyloid. The development of reagents that can bind these aggregates with high affinity and selectivity has potential for early disease diagnosis. By linking two benzothiazole aniline (BTA) head groups with different length polyethylene glycol (PEG) spacers, fluorescent probes that bind amyloid fibrils with low nanomolar affinity have been obtained. Dissociation constants measured for interaction with Aß, α-synuclein and tau fibrils show that the length of the linker determines binding affinity and selectivity. These compounds were successfully used to image α-synuclein aggregates in vitro and in the post-mortem brain tissue of patients with Parkinson's disease. The results demonstrate that multivalent ligands offer a powerful approach to obtain high affinity, selective reagents to bind the fibrillary aggregates that form in neurodegenerative disease.