Extracellular matrix components modulate different stages in β2-microglobulin amyloid formation.

Amyloid deposition of wild-type human β2-microglobulin (WT-hβ2m) in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis (DRA). In vitro, WT-hβ2m does not form amyloid fibrils at physiological pH and temperature unless co-solvents or other reagents are added. Therefore, understanding how fibril formation is initiated and maintained in the joint space is important for elucidating WT-hβ2m aggregation and DRA onset. Here, we investigated the roles of collagen I and the commonly administered anticoagulant, low-molecular-weight (LMW) heparin, in the initiation and subsequent aggregation phases of WT-hβ2m in physiologically relevant conditions. Using thioflavin T (ThT) fluorescence to study the kinetics of amyloid formation, we analyzed how these two agents affect specific stages of WT-hβ2m assembly. Our results revealed that LMW-heparin strongly promotes WT-hβ2m fibrillogenesis during all stages of aggregation. However, collagen I affected WT-hβ2m amyloid formation in contrasting ways: decreasing the lag time of fibril formation in the presence of LMW-heparin and slowing the rate at higher concentrations. We found that in self-seeded reactions, interaction of collagen I with WT-hβ2m amyloid fibrils attenuates surface-mediated growth of WT-hβ2m fibrils, demonstrating a key role of secondary nucleation in WT-hβ2m amyloid formation. Interestingly, collagen I fibrils did not suppress surface-mediated assembly of WT-hβ2m monomers when cross-seeded with fibrils formed from the N-terminally truncated variant ΔN6-hβ2m. Together, these results provide detailed insights into how collagen I and LMW-heparin impact different stages in the aggregation of WT-hβ2m into amyloid which lead to dramatic effects on the time course of assembly

Effect of Poly(propylene imine) Glycodendrimers on beta-Amyloid Aggregation in Vitro and in APP/PS1 Transgenic Mice, as a Model of Brain Amyloid Deposition and Alzheimer's Disease

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Metallosomes for biomedical applications by mixing molybdenum carbonyl metallosurfactants and phospholipids

New supramolecular systems have been prepared by mixing molybdenum organometallic metallosurfactants M(CO)5L and M(CO)4L2 {L = Ph2P(CH2)6SO3Na} with the phospholipid phosphatidylcholine. The analysis of the resulting supramolecular structures using dynamic light scattering and cryo-transmission electron microscopy has shown the formation of different aggregates depending on the metallosurfactant/phospholipid ratio, as well as a significantly different behaviour between the two studied metallosurfactants. Mixed vesicles, with properties very similar to liposomes, can be obtained with both compounds, and are called metallosomes. The formation of the mixed aggregates has also been studied by microfluidics using MeOH and EtOH as organic solvents, and it has been observed that the size of the aggregates is strongly dependent on the organic solvent used. In order to analyse the viability of these mixed systems as CO Releasing Molecules (CORMs) for biomedical applications, the CO release was studied by FT-IR spectroscopy, showing that they behave as photo-CORMs with visible and ultraviolet light. Toxicity studies of the different mixed aggregate systems have shown that metallosomes exhibit a very low toxicity, similar to liposomes that do not contain metallosurfactants, which is well below the results observed for pure metallosurfactants. Micro-FTIR microscopy using synchrotron radiation has shown the presence of metallosurfactants in cells. The results of this study show the influence of the length of the hydrocarbon chain on the properties of these mixed systems, compared with previously reported data

Transcriptomic changes linked to age-dependent neuromelanin accumulation in a new Parkinson's disease mouse model

Resumen del trabajo presentado en el 50th Annual Meeting Society for Neuroscience, celebrado de forma virtual del 8 al 11 de noviembre de 2021In Parkinson's disease (PD) there is a preferential degeneration of neuromelanin (NM)-containing neurons, especially neurons from the Substantia Nigra (SN) but also from the Ventral Tegmental Area (VTA) and Locus Coeruleus (LC). We generated a new NM-producing mouse model, based on the tissue-specific constitutive expression of human tyrosinase (hTyr) under the tyrosine hydroxylase (TH) promoter (tgNM), that mimics the distribution and age-dependent accumulation of NM in the human brain (i.e. catecholaminergic groups A1-A14). TgNM mice exhibited major PD features, including both motor and non-motor behavioral alterations, inclusion body formation, neuronal degeneration in lower brainstem areas (LC) together with neuronal dysfunction in higher brainstem areas (SN and VTA). In order to understand the mechanisms by which NM accumulation in specific brain areas ultimately interferes with the normal functioning of cells, we characterized genome-wide transcriptomic changes linked to the intracellular presence and progressive accumulation of NM in two NM-accumulating neuronal subpopulations (SN and VTA) that are known to be differentially susceptible to PD pathology. We selectively isolated single dopaminergic NM-containing neurons by laser capture microdissection from male and female wild-type and tgNM animals at 3 months, 12 months and 20 months of age (n=4-6 mice per group). We performed differential expression analysis, resulting in statistically significant differentially expressed genes at all ages (p-value<0.5). Gene-set enrichment analysis (GSEA) with Reactome Pathway Database led to the identification of altered biological pathways in tgNM related to neuroinflammation, vesicle-mediated transport and lipid metabolism, transcription and translation, mitochondrial function and cell cycle (senescence) (False Discovery Rate<0.05). Targeted-based validation of candidate RNA species was performed in microdissected samples by quantitative real-time PCR and candidate biological pathways were validated at the protein level by western blot in dissected ventral midbrain tissues from biological replicates. The transcriptomic profiles identified in this project contribute to our understanding of selective vulnerability in PD and brain aging, and points to key biological pathways and molecular targets in prodromal and early PD

Calcium Ions Promote Formation of Amyloid β-Peptide (1–40) Oligomers Causally Implicated in Neuronal Toxicity of Alzheimer's Disease

Amyloid β-peptide (Aβ) is directly linked to Alzheimer's disease (AD). In its monomeric form, Aβ aggregates to produce fibrils and a range of oligomers, the latter being the most neurotoxic. Dysregulation of Ca2+ homeostasis in aging brains and in neurodegenerative disorders plays a crucial role in numerous processes and contributes to cell dysfunction and death. Here we postulated that calcium may enable or accelerate the aggregation of Aβ. We compared the aggregation pattern of Aβ(1–40) and that of Aβ(1–40)E22G, an amyloid peptide carrying the Arctic mutation that causes early onset of the disease. We found that in the presence of Ca2+, Aβ(1–40) preferentially formed oligomers similar to those formed by Aβ(1–40)E22G with or without added Ca2+, whereas in the absence of added Ca2+ the Aβ(1–40) aggregated to form fibrils. Morphological similarities of the oligomers were confirmed by contact mode atomic force microscopy imaging. The distribution of oligomeric and fibrillar species in different samples was detected by gel electrophoresis and Western blot analysis, the results of which were further supported by thioflavin T fluorescence experiments. In the samples without Ca2+, Fourier transform infrared spectroscopy revealed conversion of oligomers from an anti-parallel β-sheet to the parallel β-sheet conformation characteristic of fibrils. Overall, these results led us to conclude that calcium ions stimulate the formation of oligomers of Aβ(1–40), that have been implicated in the pathogenesis of AD

Detection of PLGA-based nanoparticles at a single-cell level by synchrotron radiation FTIR spectromicroscopy and correlation with X-ray fluorescence microscopy.

Poly-lactide-co-glycolide (PLGA) is one of the few polymers approved by the US Food and Drug Administration as a carrier for drug administration in humans; therefore, it is one of the most used materials in the formulation of polymeric nanoparticles (NPs) for therapeutic purposes. Because the cellular uptake of polymeric NPs is a hot topic in the nanomedicine field, the development of techniques able to ensure incontrovertible evidence of the presence of NPs in the cells plays a key role in gaining understanding of their therapeutic potential. On the strength of this premise, this article aims to evaluate the application of synchrotron radiation-based Fourier transform infrared spectroscopy (SR-FTIR) spectromicroscopy and SR X-ray fluorescence (SR-XRF) microscopy in the study of the in vitro interaction of PLGA NPs with cells. To reach this goal, we used PLGA NPs, sized around 200 nm and loaded with superparamagnetic iron oxide NPs (PLGA-IO-NPs; Fe\u2083O\u2084; size, 10-15 nm). After exposing human mesothelial (MeT5A) cells to PLGA-IO-NPs (0.1 mg/mL), the cells were analyzed after fixation both by SR-FTIR spectromicroscopy and SR-XRF microscopy setups. SR-FTIR-SM enabled the detection of PLGA NPs at single-cell level, allowing polymer detection inside the biological matrix by the characteristic band in the 1,700-2,000 cm(-1) region. The precise PLGA IR-signature (1,750 cm(-1) centered pick) also was clearly evident within an area of high amide density. SR-XRF microscopy performed on the same cells investigated under SR-FTIR microscopy allowed us to put in evidence the Fe presence in the cells and to emphasize the intracellular localization of the PLGA-IO-NPs. These findings suggest that SR-FTIR and SR-XRF techniques could be two valuable tools to follow the PLGA NPs' fate in in vitro studies on cell cultures

Fourier transform infrared spectroscopy (FTIR) characterization of the interaction of anti-cancer photosensitizers with dendrimers

International audienceThe systemic or local administration of a photosensitizer for photodynamic therapy is highly limited by poor selectivity, rapid deactivation and long-lasting skin toxicity due to unfavorable biodistribution. Drug delivery systems based on nanocarriers may help specific and effective delivery of photosensitizers. In the present paper, the interaction of two photosensitizers, methylene blue and rose bengal, with phosphorous cationic and anionic dendrimers as potential nanocarriers, has been characterized. A novel method is presented based on the analysis of the infrared spectra of mixtures of photosensitizer and dendrimer. The capacity of dendrimers to bind the photosensitizers has been evaluated by obtaining the corresponding binding curves. It is shown that methylene blue interacts with both cationic and anionic dendrimers, whereas rose bengal only binds to the cationic ones. Dendrimers are shown to be potential nanocarriers for a specific delivery of both photosensitizers

Effect of Poly(propylene imine) Glycodendrimers on β-Amyloid Aggregation in Vitro and in APP/PS1 Transgenic Mice, as a Model of Brain Amyloid Deposition and Alzheimer’s Disease

Poly(propylene imine) (PPI) glycodendrimers are promising candidates as drug carriers and antiamyloidogenic and antiprionic agents. In this study the anti-β-amyloid capacity of PPI glycodendrimers of the fourth and fifth generations was investigated in vitro and in vivo. We assessed distinct PPI glycodendrimers including G4mDS and G5mDS, with electroneutral maltose shell, and G4mOS and G4m-IIIOS, with cationic maltose or maltotriose shell. Our results show that in vitro PPI maltose dendrimers reduce the toxicity of Aβ(1-42). However, only the electroneutral maltose dendrimers G4mDS and G5mDS reduce the toxicity of Alzheimer's disease brain extracts in SH-SY5Y neuroblastoma cells. PPI maltose dendrimers with electroneutral or cationic surface penetrate the cytoplasm of cultured cells, and they reach the brain when administered intranasally. Both cationic G4mOS and electroneutral G4mDS are able to modify the total burden of β-amyloid in APP/PS1 mice. The studied dendrimers did not reverse memory impairment in APP/PS1 mice following chronic administration; moreover, cationic G4mOS caused cognitive decline in nontransgenic mice. In spite of the capacity of G4mDS and G4mOS to cross the blood-brain barrier and modulate Aβ aggregation in APP/PS1 mice, further studies are needed to learn how to reduce the harmful effects of maltose dendrimers in vivo

Age-dependent neuromelanin accumulation in a novel humanized transgenic mouse model for Parkinson's disease and brain aging

Trabajo presentado en Neuroscience, celebrado en Chicago (Estados Unidos), del 19 al 23 de octubre de 2019Parkinson's disease (PD) is characterized by a selective and progressive loss of neurons that contain the dark brown pigment neuromelanin (NM), especially neurons from the substantia nigra (SN) and the locus coeruleus (LC), as well as, to a lesser extent, neurons from the ventral tegmental area (VTA) and the dorsal motor nucleus of the vagus nerve (DMNV). In humans, NM accumulates with age, the latter being the main risk factor for PD. The contribution of NM to PD pathogenesis remained unknown because, unlike humans, common laboratory animals lack NM. To overcome this major limitation, we have recently generated a new humanized rodent model based on the overexpression of human tyrosinase in the SN (AAV-hTyr). These animals show an age-dependent production of human-like NM, up to levels reached in elderly humans, and an age-dependent parkinsonian phenotype. We have now generated a humanized transgenic mouse model (Tg-TH-hTyr) that represents the first model that recapitulates the age-dependent accumulation and distribution of NM in all catecholaminergic neuronal cell groups in the brain, including SN, VTA, LC, and DNV. Using this unique animal model, we have assessed the functional, morphological and molecular implications of progressive NM accumulation in cellular functions. Our results show that NM accumulation affects neuronal function and viability in different catecholaminergic neuronal groups, which is relevant to PD pathogenesis and to the manifestation of both motor and non-motor symptoms of the disease