The inhibition of fibril formation of lysozyme by sucrose and trehalose.

The two disaccharides, trehalose and sucrose, have been compared in many studies due to their structural similarity. Both possess the ability to stabilise and reduce aggregation of proteins. Trehalose has also been shown to inhibit the formation of highly structured protein aggregates called amyloid fibrils. This study aims to compare how the thermal stability of the protein lysozyme at low pH (2.0 and 3.5) is affected by the presence of the two disaccharides. We also address the anti-aggregating properties of the disaccharides and their inhibitory effects on fibril formation. Differential scanning calorimetry confirms that the thermal stability of lysozyme is increased by the presence of trehalose or sucrose. The effect is slightly larger for sucrose. The inhibiting effects on protein aggregation are investigated using small-angle X-ray scattering which shows that the two-component system consisting of lysozyme and water (Lys/H2O) at pH 2.0 contains larger aggregates than the corresponding system at pH 3.5 as well as the sugar containing systems. In addition, the results show that the particle-to-particle distance in the sugar containing systems (Lys/Tre/H2O and Lys/Suc/H2O) at pH 2.0 is longer than at pH 3.5, suggesting larger protein aggregates in the former. Finally, the characteristic distance separating ß-strands in amyloid fibrils is observed for the Lys/H2O system at pH 2.0, using wide-angle X-ray scattering, while it is not clearly observed for the sugar containing systems. This study further shows that the two disaccharides stabilise the native fold of lysozyme by increasing the denaturation temperature. However, other factors, such as a weakening of hydrophobic interactions and hydrogen bonding between proteins, might also play a role in their inhibitory effect on amyloid fibril formation.

Extracellular Vesicles Slow Down Aß(1-42) Aggregation by Interfering with the Amyloid Fibril Elongation Step.

Formation of amyloid-ß (Aß) fibrils is a central pathogenic feature of Alzheimer's disease. Cell-secreted extracellular vesicles (EVs) have been suggested as disease modulators, although their exact roles and relations to Aß pathology remain unclear. We combined kinetics assays and biophysical analyses to explore how small (<220 nm) EVs from neuronal and non-neuronal human cell lines affected the aggregation of the disease-associated Aß variant Aß(1-42) into amyloid fibrils. Using thioflavin-T monitored kinetics and seeding assays, we found that EVs reduced Aß(1-42) aggregation by inhibiting fibril elongation. Morphological analyses revealed this to result in the formation of short fibril fragments with increased thicknesses and less apparent twists. We suggest that EVs may have protective roles by reducing Aß(1-42) amyloid loads, but also note that the formation of small amyloid fragments could be problematic from a neurotoxicity perspective. EVs may therefore have double-edged roles in the regulation of Aß pathology in Alzheimer's disease.

Probing physical properties of single amyloid fibrils using nanofluidic channels.

Amyloid fibril formation is central to the pathology of many diseases, including neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Amyloid fibrils can also have functional and scaffolding roles, for example in bacterial biofilms, and have also been exploited as useful biomaterials. Despite being linear protein homopolymers, amyloid fibrils can exhibit significant structural and morphological polymorphism, making it relevant to study them on the single fibril level. We here introduce the concept of nanofluidic channel analysis to the study of single, fluorescently-labeled amyloid fibrils in solution, monitoring the extension and emission intensity of individual fibrils confined in nanochannels with a depth of 300 nm and a width that gradually increases from 300 to 3000 nm. The change in fibril extension with channel width permitted accurate determination of the persistence length of individual fibrils using Odijk's theory for strongly confined polymers. The technique was applied to amyloid fibrils prepared from the Alzheimer's related peptide amyloid-ß(1-42) and the Parkinson's related protein α-synuclein, obtaining mean persistence lengths of 5.9 ± 4.5 µm and 3.0 ± 1.6 µm, respectively. The broad distributions of fibril persistence lengths indicate that amyloid fibril polymorphism can manifest in their physical properties. Interestingly, the α-synuclein fibrils had lower persistence lengths than the amyloid-ß(1-42) fibrils, despite being thicker. Furthermore, there was no obvious within-sample correlation between the fluorescence emission intensity per unit length of the labelled fibrils and their persistence lengths, suggesting that stiffness may not be proportional to thickness. We foresee that the nanofluidics methodology established here will be a useful tool to study amyloid fibrils on the single fibril level to gain information on heterogeneity in their physical properties and interactions.

Monovalent cations have different effects on the assembly kinetics and morphology of α-synuclein amyloid fibrils.

Formation of α-synuclein amyloid fibrils is a pathological hallmark of Parkinson's disease and a phenomenon that is strongly modulated by environmental factors. Here, we compared effects of different monovalent cations (Li+, Na+, K+) on the formation and properties of α-synuclein amyloid fibrils. Na+ > Li+ were found to have concentration-dependent catalytic effects on primary nucleation whereas K+ ions acted inhibitory. We discuss this discrepancy in terms of a superior affinity of Na+ and Li+ to carboxylic protein groups, resulting in reduced Columbic repulsion and by considering K+ as an ion with poor protein binding and slight chaotropic character, which could promote random coil protein structure. K+ ions, furthermore, appeared to lower the ß-sheet content of the fibrils and increase their persistence lengths, the latter we interpret as a consequence of lesser ion binding and hence higher line charge of the fibrils. The finding that Na+ and K+ have opposite effects on α-synuclein aggregation is intriguing in relation to the significant transient gradients of these ions across axonal membranes, but also important for the design and interpretation of biophysical assays where buffers containing these monovalent cations have been intermixedly used.

Ginsenoside Rg3 Reduces the Toxicity of Graphene Oxide Used for pH-Responsive Delivery of Doxorubicin to Liver and Breast Cancer Cells.

Doxorubicin (DOX) is extensively used in chemotherapy, but it has serious side effects and is inefficient against some cancers, e.g., hepatocarcinoma. To ameliorate the delivery of DOX and reduce its side effects, we designed a pH-responsive delivery system based on graphene oxide (GO) that is capable of a targeted drug release in the acidic tumor microenvironment. GO itself disrupted glutathione biosynthesis and induced reactive oxygen species (ROS) accumulation in human cells. It induced IL17-directed JAK-STAT signaling and VEGF gene expression, leading to increased cell proliferation as an unwanted effect. To counter this, GO was conjugated with the antioxidant, ginsenoside Rg3, prior to loading with DOX. The conjugation of Rg3 to GO significantly reduced the toxicity of the GO carrier by abolishing ROS production. Furthermore, treatment of cells with GO-Rg3 did not induce IL17-directed JAK-STAT signaling and VEGF gene expression-nor cell proliferation-suggesting GO-Rg3 as a promising drug carrier. The anticancer activity of GO-Rg3-DOX conjugates was investigated against Huh7 hepatocarcinoma and MDA-MB-231 breast cancer cells. GO-Rg3-DOX conjugates significantly reduced cancer cell viability, primarily via downregulation of transcription regulatory genes and upregulation of apoptosis genes. GO-Rg3 is an effective, biocompatible, and pH responsive DOX carrier with potential to improve chemotherapy-at least against liver and breast cancers.

Interaction Kinetics of Individual mRNA-Containing Lipid Nanoparticles with an Endosomal Membrane Mimic: Dependence on pH, Protein Corona Formation, and Lipoprotein Depletion.

Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes.

Single-shot self-supervised object detection in microscopy.

Object detection is a fundamental task in digital microscopy, where machine learning has made great strides in overcoming the limitations of classical approaches. The training of state-of-the-art machine-learning methods almost universally relies on vast amounts of labeled experimental data or the ability to numerically simulate realistic datasets. However, experimental data are often challenging to label and cannot be easily reproduced numerically. Here, we propose a deep-learning method, named LodeSTAR (Localization and detection from Symmetries, Translations And Rotations), that learns to detect microscopic objects with sub-pixel accuracy from a single unlabeled experimental image by exploiting the inherent roto-translational symmetries of this task. We demonstrate that LodeSTAR outperforms traditional methods in terms of accuracy, also when analyzing challenging experimental data containing densely packed cells or noisy backgrounds. Furthermore, by exploiting additional symmetries we show that LodeSTAR can measure other properties, e.g., vertical position and polarizability in holographic microscopy.

Label-free nanofluidic scattering microscopy of size and mass of single diffusing molecules and nanoparticles.

Label-free characterization of single biomolecules aims to complement fluorescence microscopy in situations where labeling compromises data interpretation, is technically challenging or even impossible. However, existing methods require the investigated species to bind to a surface to be visible, thereby leaving a large fraction of analytes undetected. Here, we present nanofluidic scattering microscopy (NSM), which overcomes these limitations by enabling label-free, real-time imaging of single biomolecules diffusing inside a nanofluidic channel. NSM facilitates accurate determination of molecular weight from the measured optical contrast and of the hydrodynamic radius from the measured diffusivity, from which information about the conformational state can be inferred. Furthermore, we demonstrate its applicability to the analysis of a complex biofluid, using conditioned cell culture medium containing extracellular vesicles as an example. We foresee the application of NSM to monitor conformational changes, aggregation and interactions of single biomolecules, and to analyze single-cell secretomes.

Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides.

The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.

Delivery of Oligonucleotide Therapeutics: Chemical Modifications, Lipid Nanoparticles, and Extracellular Vesicles.

Oligonucleotides (ONs) comprise a rapidly growing class of therapeutics. In recent years, the list of FDA-approved ON therapies has rapidly expanded. ONs are small (15-30 bp) nucleotide-based therapeutics which are capable of targeting DNA and RNA as well as other biomolecules. ONs can be subdivided into several classes based on their chemical modifications and on the mechanisms of their target interactions. Historically, the largest hindrance to the widespread usage of ON therapeutics has been their inability to effectively internalize into cells and escape from endosomes to reach their molecular targets in the cytosol or nucleus. While cell uptake has been improved, "endosomal escape" remains a significant problem. There are a range of approaches to overcome this, and in this review, we focus on three: altering the chemical structure of the ONs, formulating synthetic, lipid-based nanoparticles to encapsulate the ONs, or biologically loading the ONs into extracellular vesicles. This review provides a background to the design and mode of action of existing FDA-approved ONs. It presents the most common ON classifications and chemical modifications from a fundamental scientific perspective and provides a roadmap of the cellular uptake pathways by which ONs are trafficked. Finally, this review delves into each of the above-mentioned approaches to ON delivery, highlighting the scientific principles behind each and covering recent advances.

Fluorescent base analogues in gapmers enable stealth labeling of antisense oligonucleotide therapeutics.

To expand the antisense oligonucleotide (ASO) fluorescence labeling toolbox beyond covalent conjugation of external dyes (e.g. ATTO-, Alexa Fluor-, or cyanine dyes), we herein explore fluorescent base analogues (FBAs) as a novel approach to endow fluorescent properties to ASOs. Both cytosine and adenine analogues (tC, tCO, 2CNqA, and pA) were incorporated into a 16mer ASO sequence with a 3-10-3 cEt-DNA-cEt (cEt = constrained ethyl) gapmer design. In addition to a comprehensive photophysical characterization, we assess the label-induced effects on the gapmers' RNA affinities, RNA-hybridized secondary structures, and knockdown efficiencies. Importantly, we find practically no perturbing effects for gapmers with single FBA incorporations in the biologically critical gap region and, except for pA, the FBAs do not affect the knockdown efficiencies. Incorporating two cytosine FBAs in the gap is equally well tolerated, while two adenine analogues give rise to slightly reduced knockdown efficiencies and what could be perturbed secondary structures. We furthermore show that the FBAs can be used to visualize gapmers inside live cells using fluorescence microscopy and flow cytometry, enabling comparative assessment of their uptake. This altogether shows that FBAs are functional ASO probes that provide a minimally perturbing in-sequence labeling option for this highly relevant drug modality.

Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.

A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery.

RNA-based therapies have great potential to treat many undruggable human diseases. However, their efficacy, in particular for mRNA, remains hampered by poor cellular delivery and limited endosomal escape. Development and optimisation of delivery vectors, such as lipid nanoparticles (LNPs), are impeded by limited screening methods to probe the intracellular processing of LNPs in sufficient detail. We have developed a high-throughput imaging-based endosomal escape assay utilising a Galectin-9 reporter and fluorescently labelled mRNA to probe correlations between nanoparticle-mediated uptake, endosomal escape frequency, and mRNA translation. Furthermore, this assay has been integrated within a screening platform for optimisation of lipid nanoparticle formulations. We show that Galectin-9 recruitment is a robust, quantitative reporter of endosomal escape events induced by different mRNA delivery nanoparticles and small molecules. We identify nanoparticles with superior escape properties and demonstrate cell line variances in endosomal escape response, highlighting the need for fine-tuning of delivery formulations for specific applications.

Novel clearance of muscle proteins by muscle cells.

Blood levels of cardiac troponins (cTn) and myoglobin are analysed when myocardial infarction (MI) is suspected. Here we describe a novel clearance mechanism for muscle proteins by muscle cells. The complete plasma clearance profile of cTn and myoglobin was followed in rats after intravenous or intermuscular injections and analysed by PET and fluorescence microscopy of muscle biopsies and muscle cells. Compared with intravenous injections, only 5 % of cTnT, 0.6 % of cTnI and 8 % of myoglobin were recovered in the circulation following intramuscular injection. In contrast, 47 % of the renal filtration marker FITC-sinistrin and 81 % of cTn fragments from MI-patients were recovered after intramuscular injection. In addition, PET and biopsy analysis revealed that cTn was taken up by the quadriceps muscle and both cTn and myoglobin were endocytosed by cultured muscle cells. This local clearance mechanism could possibly be the dominant clearance mechanism for cTn, myoglobin and other muscle damage biomarkers released by muscle cells.

Amyloid formation of fish ß-parvalbumin involves primary nucleation triggered by disulfide-bridged protein dimers.

Amyloid formation involves the conversion of soluble protein species to an aggregated state. Amyloid fibrils of ß-parvalbumin, a protein abundant in fish, act as an allergen but also inhibit the in vitro assembly of the Parkinson protein α-synuclein. However, the intrinsic aggregation mechanism of ß-parvalbumin has not yet been elucidated. We performed biophysical experiments in combination with mathematical modeling of aggregation kinetics and discovered that the aggregation of ß-parvalbumin is initiated by the formation of dimers stabilized by disulfide bonds and then proceeds via primary nucleation and fibril elongation processes. Dimer formation is accelerated by H2O2 and hindered by reducing agents, resulting in faster and slower aggregation rates, respectively. Purified ß-parvalbumin dimers readily assemble into amyloid fibrils with similar morphology as those formed when starting from monomer solutions. Furthermore, addition of preformed dimers accelerates the aggregation reaction of monomers. Aggregation of purified ß-parvalbumin dimers follows the same kinetic mechanism as that of monomers, implying that the rate-limiting primary nucleus is larger than a dimer and/or involves structural conversion. Our findings demonstrate a folded protein system in which spontaneously formed intermolecular disulfide bonds initiate amyloid fibril formation by recruitment of monomers. This dimer-induced aggregation mechanism may be of relevance for human amyloid diseases in which oxidative stress is often an associated hallmark.

Graphene oxide sheets and quantum dots inhibit α-synuclein amyloid formation by different mechanisms.

Aggregation and amyloid formation of the 140-residue presynaptic and intrinsically disordered protein α-synuclein (α-syn) is a pathological hallmark of Parkinson's disease (PD). Understanding how α-syn forms amyloid fibrils, and investigations of agents that can prevent their formation is therefore important. We demonstrate herein that two types of graphene oxide nanoparticles (sheets and quantum dots) inhibit α-syn amyloid formation by different mechanisms mediated via differential interactions with both monomers and fibrils. We have used thioflavin-T fluorescence assays and kinetic analysis, circular dichroism, dynamic light scattering, fluorescence spectroscopy and atomic force microscopy to asses the kinetic nature and efficiency of this inhibitory effect. We show that the two types of graphene oxide nanoparticles alter the morphology of α-syn fibrils, disrupting their interfilament assembly and the resulting aggregates therefore consist of single protofilaments. Our results further show that graphene oxide sheets reduce the aggregation rate of α-syn primarily by sequestering of monomers, thereby preventing primary nucleation and elongation. Graphene quantum dots, on the other hand, interact less avidly with both monomers and fibrils. Their aggregation inhibitory effect is primarily related to adsorption of aggregated species and reduction of secondary processes, and they can thus not fully prevent aggregation. This fine-tuned and differential effect of graphene nanoparticles on amyloid formation shows that rational design of these nanomaterials has great potential in engineering materials that interact with specific molecular events in the amyloid fibril formation process. The findings also provide new insight into the molecular interplay between amyloidogenic proteins and graphene-based nanomaterials in general, and opens up their potential use as agents to manipulate fibril formation.

Independent Size and Fluorescence Emission Determination of Individual Biological Nanoparticles Reveals that Lipophilic Dye Incorporation Does Not Scale with Particle Size.

Advancements in nanoparticle characterization techniques are critical for improving the understanding of how biological nanoparticles (BNPs) contribute to different cellular processes, such as cellular communication, viral infection, as well as various drug-delivery applications. Since BNPs are intrinsically heterogeneous, there is a need for characterization methods that are capable of providing information about multiple parameters simultaneously, preferably at the single-nanoparticle level. In this work, fluorescence microscopy was combined with surface-based two-dimensional flow nanometry, allowing for simultaneous and independent determination of size and fluorescence emission of individual BNPs. In this way, the dependence of the fluorescence emission of the commonly used self-inserting lipophilic dye 3,3'-dioctadecyl-5,5'-di(4-sulfophenyl)oxacarbocyanine (SP-DiO) could successfully be correlated with nanoparticle size for different types of BNPs, including synthetic lipid vesicles, lipid vesicles derived from cellular membrane extracts, and extracellular vesicles derived from human SH-SY5Y cell cultures; all vesicles had a radius, r, of ∼50 nm and similar size distributions. The results demonstrate that the dependence of fluorescence emission of SP-DiO on nanoparticle size varies significantly between the different types of BNPs, with the expected dependence on membrane area, r2, being observed for synthetic lipid vesicles, while a significant weaker dependence on size was observed for BNPs with more complex composition. The latter observation is attributed to a size-dependent difference in membrane composition, which may influence either the optical properties of the dye and/or the insertion efficiency, indicating that the fluorescence emission of this type of self-inserting dye may not be reliable for determining size or size distribution of BNPs with complex lipid compositions.

Redox-Dependent Copper Ion Modulation of Amyloid-ß (1-42) Aggregation In Vitro.

Plaque deposits composed of amyloid-ß (Aß) fibrils are pathological hallmarks of Alzheimer's disease (AD). Although copper ion dyshomeostasis is apparent in AD brains and copper ions are found co-deposited with Aß peptides in patients' plaques, the molecular effects of copper ion interactions and redox-state dependence on Aß aggregation remain elusive. By combining biophysical and theoretical approaches, we here show that Cu2+ (oxidized) and Cu+ (reduced) ions have opposite effects on the assembly kinetics of recombinant Aß(1-42) into amyloid fibrils in vitro. Cu2+ inhibits both the unseeded and seeded aggregation of Aß(1-42) at pH 8.0. Using mathematical models to fit the kinetic data, we find that Cu2+ prevents fibril elongation. The Cu2+-mediated inhibition of Aß aggregation shows the largest effect around pH 6.0 but is lost at pH 5.0, which corresponds to the pH in lysosomes. In contrast to Cu2+, Cu+ ion binding mildly catalyzes the Aß(1-42) aggregation via a mechanism that accelerates primary nucleation, possibly via the formation of Cu+-bridged Aß(1-42) dimers. Taken together, our study emphasizes redox-dependent copper ion effects on Aß(1-42) aggregation and thereby provides further knowledge of putative copper-dependent mechanisms resulting in AD.

Role of Membrane Tension Sensitive Endocytosis and Rho GTPases in the Uptake of the Alzheimer's Disease Peptide Aß(1-42).

Intraneuronal accumulation of amyloid-ß (Aß) is an early pathological signum of Alzheimer's disease, and compartments of the endolysosomal system have been implicated in both seeding and cell-cell propagation of Aß aggregation. We have studied how clathrin-independent mechanisms contribute to Aß endocytosis, exploring pathways that are sensitive to changes in membrane tension and the regulation of Rho GTPases. Using live cell confocal microscopy and flow cytometry, we show the uptake of monomeric Aß(1-42) into endocytic vesicles and vacuole-like dilations, following relaxation of osmotic pressure-induced cell membrane tension. This indicates Aß(1-42) uptake via clathrin independent carriers (CLICs), although overexpression of the bar-domain protein GRAF1, a key regulator of CLICs, had no apparent effect. We furthermore report reduced Aß(1-42) uptake following overexpression of constitutively active forms of the Rho GTPases Cdc42 and RhoA, whereas modulation of Rac1, which is linked to macropinosome formation, had no effect. Our results confirm that uptake of Aß(1-42) is clathrin- and dynamin-independent and point to the involvement of a new and distinct clathrin-independent endocytic mechanism which is similar to uptake via CLICs or macropinocytosis but that also appear to involve yet uncharacterized molecular players.