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.

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.

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.

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.

The Liver and Kidneys mediate clearance of cardiac troponin in the rat.

Cardiac-specific troponins (cTn), troponin T (cTnT) and troponin I (cTnI) are diagnostic biomarkers when myocardial infarction is suspected. Despite its clinical importance it is still not known how cTn is cleared once it is released from damaged cardiac cells. The aim of this study was to examine the clearance of cTn in the rat. A cTn preparation from pig heart was labeled with fluorescent dye or fluorine 18 (18 F). The accumulation of the fluorescence signal using organ extracts, or the 18 F signal using positron emission tomography (PET) was examined after a tail vein injection. The endocytosis of fluorescently labeled cTn was studied using a mouse hepatoma cell line. Close to 99% of the cTnT and cTnI measured with clinical immunoassays were cleared from the circulation two hours after a tail vein injection. The fluorescence signal from the fluorescently labeled cTn preparation and the radioactivity from the 18F-labeled cTn preparation mainly accumulated in the liver and kidneys. The fluorescently labeled cTn preparation was efficiently endocytosed by mouse hepatoma cells. In conclusion, we find that the liver and the kidneys are responsible for the clearance of cTn from plasma in the rat.

Correlation between Cellular Uptake and Cytotoxicity of Fragmented α-Synuclein Amyloid Fibrils Suggests Intracellular Basis for Toxicity.

Aggregation and intracellular deposition of the protein α-synuclein is an underlying characteristic of Parkinson's disease. α-Synuclein assemblies also undergo cell-cell spreading, facilitating propagation of their cellular pathology. Understanding how cellular interactions and uptake of extracellular α-synuclein assemblies depend on their physical attributes is therefore important. We prepared fragmented fluorescently labeled α-synuclein amyloid fibrils of different average lengths (∼80 nm to >1 µm) and compared their interactions with SH-SY5Y cells. We report that fibrils of all lengths, but not monomers, bind avidly to the cell surface. Their uptake is inversely dependent on their average size, occurs via a heparan sulfate dependent endocytic route, and appears to have a size cutoff of ∼400 nm. The uptake of α-synuclein fibrils, but not monomers, correlates with their cytotoxicity as measured by reduction in metabolic activity, strongly suggesting an intracellular basis for α-synuclein fibril toxicity, likely involving endolysosomes.

Cell surface proteoglycan-mediated uptake and accumulation of the Alzheimer's disease peptide Aß(1-42).

Proteoglycans (PGs) have been found in Alzheimer's disease amyloid-ß (Aß) plaques and their glycosaminoglycan chains reportedly influence Aß aggregation, neurotoxicity and intracellular accumulation in cell and animal models, but their exact pathophysiological role(s) remain unclear. We have studied the cellular uptake of fluorescently labelled Aß(1-42) and Aß(1-40) peptides in normal CHO cells (K1) and the mutant cell line (pgsA-745) which lacks all protein-attached heparan and chondroitin sulfate chains. After 24 h of incubation, CHO-K1 accumulates more Aß(1-42) and Aß(1-40) compared with CHO-pgsA-745, consistent with the suggested role of PGs in Aß uptake. However, after short incubation times (≤3 h) there was no difference; moreover, the time evolution of Aß(1-42) accumulation in CHO-K1 followed an unusual sigmoidal-like trend, indicating a possible involvement of PG-mediated peptide aggregation in Aß endocytosis. Neither Aß(1-42) nor Aß(1-40) could stimulate uptake of a 10 kDa dextran (a general endocytosis marker) suggesting that Aß-induced upregulation of endocytosis does not occur. CHO-K1 cells contained a higher number of Aß(1-42)-positive vesicles, but the intensity difference per vesicle was only marginal suggesting that the superior accumulation of Aß(1-42) stems from a higher number of endocytic events. FRET imaging support that intracellular Aß(1-42) is aggregated in both cell types. We also report that CHO-pgsA-745 cells perform less endocytosis than CHO-K1 and, albeit this does not explain their difference in Aß internalisation, we discuss a general method for data compensation. Altogether, this study contributes new insights into the mechanisms of PG-mediated Aß uptake that may be relevant for our understanding of their role in AD pathology.

Lipid membranes catalyse the fibril formation of the amyloid-ß (1-42) peptide through lipid-fibril interactions that reinforce secondary pathways.

Alzheimer's disease is associated with the aggregation of amyloid-ß (Aß) peptides into oligomers and fibrils. We have explored how model lipid membranes modulate the rate and mechanisms of Aß(1-42) self-assembly, in order to shed light on how this pathological reaction may occur in the lipid-rich environments that the peptide encounters in the brain. Using a combination of in vitro biophysical experiments and theoretical approaches, we show that zwitterionic DOPC lipid vesicles accelerate the Aß(1-42) fibril growth rate by interacting specifically with the growing fibrils. We probe this interaction with help of a purpose-developed Förster resonance energy transfer assay that monitors the proximity between a fibril-specific dye and fluorescent lipids in the lipid vesicle membrane. To further rationalise these findings we use mathematical models to fit the aggregation kinetics of Aß(1-42) and find that lipid vesicles alter specific mechanistic steps in the aggregation reaction; they augment monomer-dependent secondary nucleation at the surface of existing fibrils and facilitate monomer-independent catalytic processes consistent with fibril fragmentation. We further show that DOPC vesicles have no effect on primary nucleation. This finding is consistent with experiments showing that Aß(1-42) monomers do not directly bind to the lipid bilayer. Taken together, our results show that plain lipid membranes with charge and composition that is representative of outer cell membranes can significantly augment autocatalytic steps in the self-assembly of Aß(1-42) into fibrils. This new insight suggests that strategies to reduce fibril-lipid interactions in the brain may have therapeutic value.

Endocytic uptake of monomeric amyloid-ß peptides is clathrin- and dynamin-independent and results in selective accumulation of Aß(1-42) compared to Aß(1-40).

Intraneuronal accumulation of amyloid-ß (Aß) peptides represent an early pathological feature in Alzheimer's disease. We have therefore utilized flow cytometry and confocal microscopy in combination with endocytosis inhibition to explore the internalisation efficiency and uptake mechanisms of Aß(1-40) and Aß(1-42) monomers in cultured SH-SY5Y cells. We find that both variants are constitutively internalised via endocytosis and that their uptake is proportional to cellular endocytic rate. Moreover, SH-SY5Y cells internalise consistently twice the amount of Aß(1-42) compared to Aß(1-40); an imaging-based quantification showed that cells treated with 1 µM peptide for 8 h contained 800,000 peptides of Aß(1-42) and 400,000 of Aß(1-40). Both variants co-localised to >90% with lysosomes or other acidic compartments. Dynasore and chlorpromazine endocytosis inhibitors were both found to reduce uptake, particularly of Aß(1-42). Overexpression of the C-terminal of the clathrin-binding domain of AP180, dynamin2 K44A, or Arf6 Q67L did however not reduce uptake of the Aß variants. By contrast, perturbation of actin polymerisation and inhibition of macropinocytosis reduced Aß(1-40) and Aß(1-42) uptake considerably. This study clarifies mechanisms of Aß(1-40) and Aß(1-42) uptake, pinpoints differences between the two variants and highlights a common and putative role of macropinocytosis in the early accumulation of intraneuronal Aß in AD.

Binding of Thioflavin-T to Amyloid Fibrils Leads to Fluorescence Self-Quenching and Fibril Compaction.

Thioflavin-T binds to and detects amyloid fibrils via fluorescence enhancement. Using a combination of linear dichroism and fluorescence spectroscopies, we report that the relation between the emission intensity and binding of thioflavin-T to insulin fibrils is nonlinear and discuss this in relation to its use in kinetic assays. We demonstrate, from fluorescence lifetime recordings, that the nonlinearity is due to thioflavin-T being sensitive to self-quenching. In addition, thioflavin-T can induce fibril compaction but not alter fibril structure. Our work underscores the photophysical complexity of thioflavin-T and the necessity of calibrating the linear range of its emission response for quantitative in vitro studies.