Anle138b interaction in α-synuclein aggregates by dynamic nuclear polarization NMR.

Small molecules that bind to oligomeric protein species such as membrane proteins and fibrils are of clinical interest for development of therapeutics and diagnostics. Definition of the binding site at atomic resolution via NMR is often challenging due to low binding stoichiometry of the small molecule. For fibrils and aggregation intermediates grown in the presence of lipids, we report atomic-resolution contacts to the small molecule at sub nm distance via solid-state NMR using dynamic nuclear polarization (DNP) and orthogonally labelled samples of the protein and the small molecule. We apply this approach to α-synuclein (αS) aggregates in complex with the small molecule anle138b, which is a clinical drug candidate for disease modifying therapy. The small central pyrazole moiety of anle138b is detected in close proximity to the protein backbone and differences in the contacts between fibrils and early intermediates are observed. For intermediate species, the 100 K condition for DNP helps to preserve the aggregation state, while for both fibrils and oligomers, the DNP enhancement is essential to obtain sufficient sensitivity.

[11C]MODAG-001-towards a PET tracer targeting α-synuclein aggregates.

PURPOSE: Deposition of misfolded alpha-synuclein (αSYN) aggregates in the human brain is one of the major hallmarks of synucleinopathies. However, a target-specific tracer to detect pathological aggregates of αSYN remains lacking. Here, we report the development of a positron emission tomography (PET) tracer based on anle138b, a compound shown to have therapeutic activity in animal models of neurodegenerative diseases. METHODS: Specificity and selectivity of [3H]MODAG-001 were tested in in vitro binding assays using recombinant fibrils. After carbon-11 radiolabeling, the pharmacokinetic and metabolic profile was determined in mice. Specific binding was quantified in rats, inoculated with αSYN fibrils and using in vitro autoradiography in human brain sections of Lewy body dementia (LBD) cases provided by the Neurobiobank Munich (NBM). RESULTS: [3H]MODAG-001 revealed a very high affinity towards pure αSYN fibrils (Kd = 0.6 ± 0.1 nM) and only a moderate affinity to hTau46 fibrils (Kd = 19 ± 6.4 nM) as well as amyloid-ß1-42 fibrils (Kd = 20 ± 10 nM). [11C]MODAG-001 showed an excellent ability to penetrate the mouse brain. Metabolic degradation was present, but the stability of the parent compound improved after selective deuteration of the precursor. (d3)-[11C]MODAG-001 binding was confirmed in fibril-inoculated rat striata using in vivo PET imaging. In vitro autoradiography showed no detectable binding to aggregated αSYN in human brain sections of LBD cases, most likely, because of the low abundance of aggregated αSYN against background protein. CONCLUSION: MODAG-001 provides a promising lead structure for future compound development as it combines a high affinity and good selectivity in fibril-binding assays with suitable pharmacokinetics and biodistribution properties.

Steller Sea Lion (Eumetopias jubatus) at Tuleny Island, Russia in 2018: Abundance, Composition, and Entanglement.

The Steller sea lion (SSL, Eumetopias jubatus) inhabits the North Pacific Ocean off both the North American and Asian coasts. The abundance of the species in Asia declined by more than half in the second part of the 20th century. Decline recurred in the second decade of the 21th century after a short period of restoration. In contrast with the total dynamics of SSL in Asia, the reproductive aggregation on Tuleny I. (Sea of Okhotsk) has been growing almost continuously since the beginning of its formation in the late 1980s. Long-term monitoring of SSL at Tuleny I. always covered only summer reproductive seasons. We surveyed Tuleny I. in October 2018, and counted 1058 non-pup sea lions and 396 pups. The majority of tagged animals encountered at the rookery were of local origin. About one-third of the summer non-pup sea lions' number could remain at the rookery until the middle of October, which coincides with the seasonal appearance of sea lions off the coast of Japan. The sex-age structure was characterized by total absence of adult males and reduced proportion of subadult males and juveniles. It contrasts with autumn behavior of SSLs in the rookery of the northern Sea of Okhotsk. We observed that 0.7% of the inspected sea lions were entangled in marine debris. The proportion of entangled animals is lower in comparison with that of the whole population of Tuleny I. due to reduction in the ratios of males and juveniles, which entangle in foreign objects more often than adult females.

Treatment with diphenyl-pyrazole compound anle138b/c reveals that α-synuclein protects melanoma cells from autophagic cell death.

Recent epidemiological and clinical studies have reported a significantly increased risk for melanoma in people with Parkinson's disease. Because no evidence could be obtained that genetic factors are the reason for the association between these two diseases, we hypothesized that of the three major Parkinson's disease-related proteins-α-synuclein, LRRK2, and Parkin-α-synuclein might be a major link. Our data, presented here, demonstrate that α-synuclein promotes the survival of primary and metastatic melanoma cells, which is the exact opposite of the effect that α-synuclein has on dopaminergic neurons, where its accumulation causes neuronal dysfunction and death. Because this detrimental effect of α-synuclein on neurons can be rescued by the small molecule anle138b, we explored its effect on melanoma cells. We found that treatment with anle138b leads to massive melanoma cell death due to a major dysregulation of autophagy, suggesting that α-synuclein is highly beneficial to advanced melanoma because it ensures that autophagy is maintained at a homeostatic level that promotes and ensures the cell's survival.

Depopulation of dense α-synuclein aggregates is associated with rescue of dopamine neuron dysfunction and death in a new Parkinson's disease model.

Parkinson's disease (PD) is characterized by the presence of α-synuclein aggregates known as Lewy bodies and Lewy neurites, whose formation is linked to disease development. The causal relation between α-synuclein aggregates and PD is not well understood. We generated a new transgenic mouse line (MI2) expressing human, aggregation-prone truncated 1-120 α-synuclein under the control of the tyrosine hydroxylase promoter. MI2 mice exhibit progressive aggregation of α-synuclein in dopaminergic neurons of the substantia nigra pars compacta and their striatal terminals. This is associated with a progressive reduction of striatal dopamine release, reduced striatal innervation and significant nigral dopaminergic nerve cell death starting from 6 and 12 months of age, respectively. In the MI2 mice, alterations in gait impairment can be detected by the DigiGait test from 9 months of age, while gross motor deficit was detected by rotarod test at 20 months of age when 50% of dopaminergic neurons in the substantia nigra pars compacta are lost. These changes were associated with an increase in the number and density of 20-500 nm α-synuclein species as shown by dSTORM. Treatment with the oligomer modulator anle138b, from 9 to 12 months of age, restored striatal dopamine release, prevented dopaminergic cell death and gait impairment. These effects were associated with a reduction of the inner density of large α-synuclein aggregates and an increase in dispersed small α-synuclein species as revealed by dSTORM. The MI2 mouse model recapitulates the progressive dopaminergic deficit observed in PD, showing that early synaptic dysfunction is associated to fine behavioral motor alterations, precedes dopaminergic axonal loss and neuronal death that become associated with a more consistent motor deficit upon reaching a certain threshold. Our data also provide new mechanistic insight for the effect of anle138b's function in vivo supporting that targeting α-synuclein aggregation is a promising therapeutic approach for PD.

Anle138b modulates α-synuclein oligomerization and prevents motor decline and neurodegeneration in a mouse model of multiple system atrophy.

BACKGROUND: MSA is a fatal neurodegenerative disease characterized by autonomic failure and severe motor impairment. Its main pathological hallmark is the accumulation of α-synuclein in oligodendrocytes, leading to glial and neuronal dysfunction and neurodegeneration. These features are recapitulated in the PLP-hαSyn mouse model expressing human α-synuclein in oligodendrocytes. At present, there is no effective disease-modifying therapy. Previous experiments have shown that the aggregation inhibitor, anle138b, reduces neurodegeneration and behavioral deficits in mouse models of other proteinopathies. OBJECTIVES: To test the therapeutic potential of anle138b in a mouse model of MSA. METHODS: Two-month-old PLP-hαSyn mice were fed over a period of 4 months with pellets containing anle138b at two different doses (0.6 and 2 g/kg) and compared to healthy controls and PLP-hαSyn mice fed with placebo pellets. At the end of the treatment, behavioral and histological analyses were performed. RESULTS: We observed a reversal of motor function to healthy control levels when PLP-hαSyn mice were treated with both doses of anle138b. Histological and molecular analyses showed a significant reduction in α-synuclein oligomers and glial cytoplasmic inclusions in animals fed with anle138b compared to nontreated mice. These animals also present preservation of dopaminergic neurons and reduction in microglial activation in SN correlating with the α-synuclein reduction observed. CONCLUSIONS: Anle138b reduces α-synuclein accumulation in PLP-hαSyn mice, leading to neuroprotection, reduction of microglial activation, and preservation of motor function supporting the use of anle138b in a future clinical trial for MSA. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Photophysics of diphenyl-pyrazole compounds in solutions and α-synuclein aggregates.

BACKGROUND: Recently diphenyl-pyrazole (DPP) compounds and especially anle138b were found to reduce the aggregation of α-synuclein or Tau protein in vitro as well as in a mouse model of neurodegenerative diseases [1,2]. Direct interaction of the DPPs with the fibrillar structure was identified by fluorescence spectroscopy. Thereby a strong dependence of the fluorescence on the surroundings could be identified [3]. METHODS: Stationary and time-resolved emission experiments were performed on DPP compounds substituted by different halogens. RESULTS: The compounds reveal a pronounced dependence of the fluorescence on the surrounding solvent. In non-polar solvents they show strong emission in the blue part of the spectrum while in polar and proton donating solvents, such as water or acetic acid a dual fluorescence can be observed where a red-shifted emission points to a charge transfer in the excited state with large dipole moment. Non-radiative processes including photochemical reactions are observed for DPP substituted with heavy halogens. Upon binding of anle138b and its derivatives to protein fibrils in aqueous buffer, strong enhancement of the fluorescence at short wavelengths is found. CONCLUSION: The investigations of the DPPs in different surroundings lead to a detailed model of the fluorescence characteristics. We propose a model for the binding in fibrils of different proteins, where the DPP is located in a hydrophobic groove independent of the specific sequence of the amino acids. GENERAL SIGNIFICANCE: These investigations characterize the binding site of the DPP anle138b in protein aggregates and contribute to the understanding of the therapeutic mode of action of this compound.

The bacterial SRP receptor, FtsY, is activated on binding to the translocon.

Proteins are inserted into the bacterial plasma membrane cotranslationally after translating ribosomes are targeted to the translocon in the membrane via the signal recognition particle (SRP) pathway. The targeting pathway involves an interaction between SRP and the SRP receptor, FtsY. Here we focus on the role of FtsY and its interaction with the translocon in controlling targeting. We show that in unbound FtsY the NG and A domains interact with one another. The interaction involves the membrane-targeting region at the junction between A and N domain. The closed form of FtsY is impaired in binding to SRP. Upon binding to the phospholipid-embedded translocon the domains of FtsY move apart. This enhances the docking of the FtsY NG domain to the homologous NG domain of the SRP protein Ffh. Thus, FtsY binding to the translocon has a central role in orchestrating the formation of a quaternary transfer complex in which the nascent peptide is transferred to the translocon. We propose that FtsY activation at the translocon ensures that ribosome-SRP complexes are directed to available translocons. This way sequestering SRP in futile complexes with unbound FtsY can be avoided and efficient targeting to the translocon achieved.

Anle138b and related compounds are aggregation specific fluorescence markers and reveal high affinity binding to α-synuclein aggregates.

BACKGROUND: Special diphenyl-pyrazole compounds and in particular anle138b were found to reduce the progression of prion and Parkinson's disease in animal models. The therapeutic impact of these compounds was attributed to the modulation of α-synuclein and prion-protein aggregation related to these diseases. METHODS: Photophysical and photochemical properties of the diphenyl-pyrazole compounds anle138b, anle186b and sery313b and their interaction with monomeric and aggregated α-synuclein were studied by fluorescence techniques. RESULTS: The fluorescence emission of diphenyl-pyrazole is strongly increased upon incubation with α-synuclein fibrils, while no change in fluorescence emission is found when brought in contact with monomeric α-synuclein. This points to a distinct interaction between diphenyl-pyrazole and the fibrillar structure with a high binding affinity (Kd=190±120nM) for anle138b. Several α-synuclein proteins form a hydrophobic binding pocket for the diphenyl-pyrazole compound. A UV-induced dehalogenation reaction was observed for anle138b which is modulated by the hydrophobic environment of the fibrils. CONCLUSION: Fluorescence of the investigated diphenyl-pyrazole compounds strongly increases upon binding to fibrillar α-synuclein structures. Binding at high affinity occurs to hydrophobic pockets in the fibrils. GENERAL SIGNIFICANCE: The observed particular fluorescence properties of the diphenyl-pyrazole molecules open new possibilities for the investigation of the mode of action of these compounds in neurodegenerative diseases. The high binding affinity to aggregates and the strong increase in fluorescence upon binding make the compounds promising fluorescence markers for the analysis of aggregation-dependent epitopes.

Reducing tau aggregates with anle138b delays disease progression in a mouse model of tauopathies.

Pathological tau aggregation leads to filamentous tau inclusions and characterizes neurodegenerative tauopathies such as Alzheimer's disease and frontotemporal dementia and parkinsonism linked to chromosome 17. Tau aggregation coincides with clinical symptoms and is thought to mediate neurodegeneration. Transgenic mice overexpressing mutant human P301S tau exhibit many neuropathological features of human tauopathies including behavioral deficits and increased mortality. Here, we show that the di-phenyl-pyrazole anle138b binds to aggregated tau and inhibits tau aggregation in vitro and in vivo. Furthermore, anle138b treatment effectively ameliorates disease symptoms, increases survival time and improves cognition of tau transgenic PS19 mice. In addition, we found decreased synapse and neuron loss accompanied by a decreased gliosis in the hippocampus. Our results suggest that reducing tau aggregates with anle138b may represent an effective and promising approach for the treatment of human tauopathies.

Interfering with aggregated α-synuclein in advanced melanoma leads to a major upregulation of MHC class II proteins.

Melanoma is the most serious and deadly form of skin cancer and with progression to advanced melanoma, the intrinsically disordered protein α-synuclein is upregulated to high levels. While toxic to dopaminergic neurons in Parkinson's disease, α-synuclein is highly beneficial for primary and metastatic melanoma cells. To gain detailed insights into this exact opposite role of α-synuclein in advanced melanoma, we performed proteomic studies of high-level α-synuclein-expressing human melanoma cell lines that were treated with the diphenyl-pyrazole small-molecule compound anle138b, which binds to and interferes with the oligomeric structure of α-synuclein. We also performed proteomic and transcriptomic studies of human melanoma xenografts that were treated systemically with the anle138b compound. The results reveal that interfering with oligomerized α-synuclein in the melanoma cells in these tumor xenografts led to a substantial upregulation and expression of major histocompatibility complex proteins, which are pertinent to enhancing anti-melanoma immune responses.

Anle138b: a novel oligomer modulator for disease-modifying therapy of neurodegenerative diseases such as prion and Parkinson's disease.

In neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and prion diseases, deposits of aggregated disease-specific proteins are found. Oligomeric aggregates are presumed to be the key neurotoxic agent. Here we describe the novel oligomer modulator anle138b [3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole], an aggregation inhibitor we developed based on a systematic high-throughput screening campaign combined with medicinal chemistry optimization. In vitro, anle138b blocked the formation of pathological aggregates of prion protein (PrP(Sc)) and of α-synuclein (α-syn), which is deposited in PD and other synucleinopathies such as dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Notably, anle138b strongly inhibited all prion strains tested including BSE-derived and human prions. Anle138b showed structure-dependent binding to pathological aggregates and strongly inhibited formation of pathological oligomers in vitro and in vivo both for prion protein and α-synuclein. Both in mouse models of prion disease and in three different PD mouse models, anle138b strongly inhibited oligomer accumulation, neuronal degeneration, and disease progression in vivo. Anle138b had no detectable toxicity at therapeutic doses and an excellent oral bioavailability and blood-brain-barrier penetration. Our findings indicate that oligomer modulators provide a new approach for disease-modifying therapy in these diseases, for which only symptomatic treatment is available so far. Moreover, our findings suggest that pathological oligomers in neurodegenerative diseases share structural features, although the main protein component is disease-specific, indicating that compounds such as anle138b that modulate oligomer formation by targeting structure-dependent epitopes can have a broad spectrum of activity in the treatment of different protein aggregation diseases.

Mercury content in the fur of sea otters (Enhydra lutris) from the Commander Islands.

The sea otter (Enhydra lutris) is a keystone species in the ecosystem which is currently in depression in Russia. The objectives of this study were to: (1) establish if the sea otters from the Commander Islands have hazardous levels of mercury (Hg) in their fur; (2) assess Hg pollution in sea otters during a period of high abundance and population depression; (3) identify the age and sex differences in sea otters by Hg content. The sea otters were classified from no to low risk for Hg health effects. Differences in Hg content during periods of low and high population size were not statistically significant. Hg concentrations in adult sea otters were significantly higher than in the young, and higher in males than in females. This study presents the first data on Hg content in sea otters' fur and the first estimate of Hg contamination for the Commander Islands population.

First Satellite Tagging of the Northern Fur Seals (Callorhinus ursinus) on the Tyuleniy Island, the Sea of Okhotsk.

In October 2018, three Northern fur seals (two adult females and one juvenile male) were deployed with satellite tags on the Tyuleniy Island in the Sea of Okhotsk. The operational time of the tags ranged from 33 to 203 days. The adult females started their winter migration in the first half of November; the initial stage of their winter migration occurred in the Japan/East Sea, which they entered through the La Perouse Strait. The juvenile male left the rookery in mid-October, crossed the Sea of Okhotsk in a north-western direction and returned to the south. The male had reached the coastal areas of Hokkaido Island, Japan by the end of November. From the Sea of Okhotsk, the male entered the Pacific Ocean through the Yekaterina Strait and subsequently entered the Japan/East Sea via the Tsugaru Strait. The winter foraging of the male occurred within the north-eastern part of the Japan/East Sea just off the Tsugaru Strait. After 3 months, the male returned to the Pacific and remained off the Sanriku Coast (Honshu Island).

The clinical drug candidate anle138b binds in a cavity of lipidic α-synuclein fibrils.

Aggregation of amyloidogenic proteins is a characteristic of multiple neurodegenerative diseases. Atomic resolution of small molecule binding to such pathological protein aggregates is of interest for the development of therapeutics and diagnostics. Here we investigate the interaction between α-synuclein fibrils and anle138b, a clinical drug candidate for disease modifying therapy in neurodegeneration and a promising scaffold for positron emission tomography tracer design. We used nuclear magnetic resonance spectroscopy and the cryogenic electron microscopy structure of α-synuclein fibrils grown in the presence of lipids to locate anle138b within a cavity formed between two ß-strands. We explored and quantified multiple binding modes of the compound in detail using molecular dynamics simulations. Our results reveal stable polar interactions between anle138b and backbone moieties inside the tubular cavity of the fibrils. Such cavities are common in other fibril structures as well.

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.

Correction: Migrations of young spotted seals (Phoca largha) from Peter the Great Bay, Sea of Japan/East Sea, and the pattern of their use of seasonal habitats.

[This corrects the article DOI: 10.1371/journal.pone.0244232.].

Migrations of young spotted seals (Phoca largha) from Peter the Great Bay, Sea of Japan/East Sea, and the pattern of their use of seasonal habitats.

We studied the migrations of young spotted seals during their annual cycle. In May 2017, we attached satellite tags (SPOT-293A) to three individuals (two underyearlings and one yearling) captured at their breeding ground in Peter the Great Bay, western Sea of Japan/East Sea. The operational time of the installed tags ranged from 207 to 333 days; a total of 27195 locations were uploaded. All three seals migrated east and further north along the coast of the mainland. The average daily migration speed of the seals ranged between 70 and 135 km/day. The yearling moved faster than the underyearlings. During early August, they arrived at their summer habitats, which were located in the northern part of the Tatar Strait (Sea of Japan/East Sea) for the underyearling seals and in Aniva Bay (Sea of Okhotsk) for the yearling seal. While moving from the place of tagging to the summer feeding grounds, the seals covered a distance of 2300 to 3100 km. From August to October, each seal permanently stayed within the same isolated area. The reverse migration of all three seals began in November. When the seals traveled south, they used the same routes by which they had moved north in the spring, but they moved at a faster speed. By December, two seals returned to their natal islands, where both stayed until their transmitters stopped sending signals (in March 2018).

11 C Radiolabeling of anle253b: a Putative PET Tracer for Parkinson's Disease That Binds to α-Synuclein Fibrils in†vitro and Crosses the Blood-Brain Barrier.

There is an urgent clinical need for imaging of α-synuclein (αSyn) fibrils, the hallmark biomarker for Parkinson's disease, in neurodegenerative disorders. Despite immense efforts, promising tracer candidates for nuclear imaging of αSyn are rare. Diphenyl pyrazoles are known modulators of αSyn aggregation and thus bear potential for non-invasive detection of this biomarker in vivo. Here we demonstrate high-affinity binding of the family member anle253b to fibrillar αSyn and present a high-yielding site-selective radiosynthesis route for 11 C radiolabeling using in-situ generated [11 C]formaldehyde and reductive methylation. Radio-HPLC of the tracer after incubation with rat serum in vitro shows excellent stability of the molecule. Positron emission tomography in healthy animals is used to assess the pharmacokinetics and biodistribution of the tracer, showing good penetration of the blood-brain barrier and low background binding to the non-pathological brain.

Tau-induced mitochondrial membrane perturbation is dependent upon cardiolipin.

Misfolding and aggregate formation by the tau protein has been closely related with neurotoxicity in a large group of human neurodegenerative disorders, which includes Alzheimer's disease. Here, we investigate the membrane-active properties of tau oligomers on mitochondrial membranes, using minimalist in vitro model systems. Thus, exposure of isolated mitochondria to oligomeric tau evoked a disruption of mitochondrial membrane integrity, as evidenced by a combination of organelle swelling, efflux of cytochrome c and loss of the mitochondrial membrane potential. Tau-induced mitochondrial dysfunction occurred independently of the mitochondrial permeability transition (mPT) pore complex. Notably, mitochondria were rescued by pre-incubation with 10-N-nonyl acridine orange (NAO), a molecule that specifically binds cardiolipin (CL), the signature phospholipid of mitochondrial membranes. Additionally, NAO prevented direct binding of tau oligomers to isolated mitochondria. At the same time, tau proteins exhibited high affinity to CL-enriched membranes, whilst permeabilisation of lipid vesicles also strongly correlated with CL content. Intriguingly, using single-channel electrophysiology, we could demonstrate the formation of non-selective ion-conducting tau nanopores exhibiting multilevel conductances in mito-mimetic bilayers. Taken together, the data presented here advances a scenario in which toxic cytosolic entities of tau protein would target mitochondrial organelles by associating with their CL-rich membrane domains, leading to membrane poration and compromised mitochondrial structural integrity.