Cellular Uptake of Amyloid Forming Proteins Related to Neurodegenerative Disease

Aggregation and deposition of disease-associated protein is a pathological hallmark of several human disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). These diseases are characterized by the formation of amyloid-β (Aβ) and α-synuclein (α-syn) amyloid fibrils, in extracellular and intracellular locations, respectively. Prior to extracellular deposition of Aβ into plaques, Aβ also accumulates within neurons, but the molecular and cellular mechanisms contributing to uptake are not fully understood. Moreover, exact links between disease onset and progression are missing, hindering the development of new disease-modifying therapies.This Thesis describes my research to elucidate how chemical and physical characteristics of Aβ and α-syn, and their ensuing aggregates, influence their cellular uptake. This is important as the endolysosomal system has been implicated as a potential site for onset and progression of disease pathology. Focusing on Aβ uptake I demonstrate that the most aggregation-prone and neurotoxic variant Aβ(1-42) is endocytosed twice as efficiently as Aβ(1-40). I show that the uptake of both variants occurs via clathrin- and dynamin-independent endocytosis, but my work also points to a mechanistic difference; Aβ(1-42) is for example more sensitive to inhibitors of action polymerisation. Further, in studies of Aβ(1-42), I demonstrate that uptake is regulated by small Rho GTPases and highly sensitive to changes in membrane tension, but apparently not via GRAF1-regulated CLIC/GEECs, suggesting the involvement of yet unidentified molecular players. I also show how uptake of pre-formed α-syn fibrils is inversely related to fibril length, and correlates to reductions in metabolic activity, pointing to an important role of cellular uptake and endolysosomal accumulation in toxicity. Lastly, I demonstrate that both monomeric Aβ and fibrillar α-syn are dependent on cell surface proteoglycans for uptake. Importantly, I show that for Aβ this dependency builds up over time, suggesting that local peptide aggregation at the cell surface could precede uptake.Altogether, this Thesis contribute new molecular and mechanistic insights into how cellular uptake contributes to intraneuronal accumulation of amyloidogenic proteins relevant in neurodegenerative disease

Role of Membrane Tension Sensitive Endocytosis and Rho GTPases in the Uptake of the Alzheimer\u27s Disease Peptide Aβ(1-42)

Intraneuronal accumulation of amyloid-β (Aβ) is an early pathological signum of Alzheimer\u27s disease, and compartments of the endolysosomal system have been implicated in both seeding and cell-cell propagation of Aβ aggregation. We have studied how clathrin-independent mechanisms contribute to Aβ 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 Aβ(1-42) into endocytic vesicles and vacuole-like dilations, following relaxation of osmotic pressure-induced cell membrane tension. This indicates Aβ(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 Aβ(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 Aβ(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

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

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

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

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

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

Intraneuronal accumulation of amyloid-? (A?) peptides represent an early pathological feature in Alzheimer\u27s disease. We have therefore utilized flow cytometry and confocal microscopy in combination with endocytosis inhibition to explore the internalisation efficiency and uptake mechanisms of A?(1-40) and A?(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 A?(1-42) compared to A?(1-40); an imaging-based quantification showed that cells treated with 1 ?M peptide for 8 h contained 800,000 peptides of A?(1-42) and 400,000 of A?(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 A?(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 A? variants. By contrast, perturbation of actin polymerisation and inhibition of macropinocytosis reduced A?(1-40) and A?(1-42) uptake considerably. This study clarifies mechanisms of A?(1-40) and A?(1-42) uptake, pinpoints differences between the two variants and highlights a common and putative role of macropinocytosis in the early accumulation of intraneuronal A? in AD. \ua9 2017 The Author(s)

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

Alzheimer\u27s disease is associated with the aggregation of amyloid-? (A?) peptides into oligomers and fibrils. We have explored how model lipid membranes modulate the rate and mechanisms of A?(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 A?(1–42) fibril growth rate by interacting specifically with the growing fibrils. We probe this interaction with help of a purpose-developed F\uf6rster 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 A?(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 A?(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 A?(1–42) into fibrils. This new insight suggests that strategies to reduce fibril-lipid interactions in the brain may have therapeutic value

Cell surface proteoglycan-mediated uptake and accumulation of the Alzheimer\u27s disease peptide Aβ(1–42)

Proteoglycans\ua0(PGs) have been found in Alzheimer\u27s disease\ua0amyloid-β(Aβ) plaques and their\ua0glycosaminoglycan\ua0chains reportedly influence Aβ 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 Aβ(1–42) and Aβ(1–40)\ua0peptides\ua0in normal CHO cells (K1) and the mutant cell line (pgsA-745) which lacks all protein-attached\ua0heparan and chondroitin sulfate\ua0chains. After 24 h of incubation, CHO-K1 accumulates more Aβ(1–42) and Aβ(1–40) compared with CHO-pgsA-745, consistent with the suggested role of PGs in Aβ uptake. However, after short incubation times (≤3 h) there was no difference; moreover, the time evolution of Aβ(1–42) accumulation in CHO-K1 followed an unusual sigmoidal-like trend, indicating a possible involvement of PG-mediated peptide aggregation in Aβ endocytosis. Neither Aβ(1–42) nor Aβ(1–40) could stimulate uptake of a 10 kDa\ua0dextran\ua0(a general endocytosis marker) suggesting that Aβ-induced upregulation of endocytosis does not occur. CHO-K1 cells contained a higher number of Aβ(1–42)-positive\ua0vesicles, but the intensity difference per vesicle was only marginal suggesting that the superior accumulation of Aβ(1–42) stems from a higher number of endocytic events. FRET imaging support that intracellular Aβ(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 Aβ internalisation, we discuss a general method for data compensation. Altogether, this study contributes new insights into the mechanisms of PG-mediated Aβ uptake that may be relevant for our understanding of their role in AD pathology

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\u27s 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