Xavier Fernàndez-Busquets

Fine structure study of Aβ1-42 fibrillogenesis with atomic force microscopy

One of the hallmarks of Alzheimers disease is the self‐aggregation of the amyloid β peptide (Aβ) in extracellular amyloid fibrils. Among the different forms of Aβ, the 42‐residue fragment (Aβ1–42) readily self‐associates and forms nucleation centers from where fibrils can quickly grow. The strong tendency of Aβ1–42 to aggregate is one of the reasons for the scarcity of data on its fibril formation process. We have used atomic force microscopy (AFM) to visualize in liquid environment the fibrillogenesis of synthetic Aβ1–42 on hydrophilic and hydrophobic surfaces. The results presented provide nanometric resolution of the main structures characteristic of the several steps from monomeric Aβ1–42 to mature fibrils in vitro. Oligomeric globular aggregates of Aβ1–42 precede the appearance of protofibrils, the first fibrillar species, although we have not obtained direct evidence of oligomer‐protofibril interconversion. The protofibril dimensions deduced from our AFM images are consistent with a model that postulates the stacking of the peptide in a hairpin conformation perpendicular to the long axis of the protofibril, forming single β‐sheets ribbon‐shaped. The most abundant form of Aβ1–42 fibril exhibits a nodular structure with a ∼100‐nm periodicity. This length is very similar 1) to the length of protofibril bundles that are the dominant feature at earlier stages in the aggregation process, 2) to the period of helical structures that have been observed in the core of fibrils, and 3) to the distance between regularly spaced, structurally weak fibril points. Taken together, these data are consistent with the existence of a ∼100‐nm long basic protofibril unit that is a key fibril building block.

Inclusion bodies : Specificity in their aggregation process and amyloid-like structure

The accumulation of aggregated protein in the cell is associated with the pathology of many diseases and constitutes a major concern in protein production. Intracellular aggregates have been traditionally regarded as nonspecific associations of misfolded polypeptides. This view is challenged by studies demonstrating that, in vitro, aggregation often involves specific interactions. However, little is known about the specificity of in vivo protein deposition. Here, we investigate the degree of in vivo co-aggregation between two self-aggregating proteins, Abeta42 amyloid peptide and foot-and-mouth disease virus VP1 capsid protein, in prokaryotic cells. In addition, the ultrastructure of intracellular aggregates is explored to decipher whether amyloid fibrils and intracellular protein inclusions share structural properties. The data indicate that in vivo protein aggregation exhibits a remarkable specificity that depends on the establishment of selective interactions and results in the formation of oligomeric and fibrillar structures displaying amyloid-like properties. These features allow prokaryotic Abeta42 intracellular aggregates to act as effective seeds in the formation of Abeta42 amyloid fibrils. Overall, our results suggest that conserved mechanisms underlie protein aggregation in different organisms. They also have important implications for biotechnological and biomedical applications of recombinant polypeptides.

Immunohistochemical analysis of human brain suggests pathological synergism of Alzheimer's disease and diabetes mellitus.

It has been extensively reported that diabetes mellitus (DM) patients have a higher risk of developing Alzheimers disease (AD), but a mechanistic connection between both pathologies has not been provided so far. Carbohydrate-derived advanced glycation endproducts (AGEs) have been implicated in the chronic complications of DM and have been reported to play an important role in the pathogenesis of AD. The earliest histopathological manifestation of AD is the apparition of extracellular aggregates of the amyloid beta peptide (Abeta). To investigate possible correlations between AGEs and Abeta aggregates with both pathologies, we have performed an immuhistochemical study in human post-mortem samples of AD, AD with diabetes (ADD), diabetic and nondemented controls. ADD brains showed increased number of Abeta dense plaques and receptor for AGEs (RAGE)-positive and Tau-positive cells, higher AGEs levels and major microglial activation, compared to AD brain. Our results indicate that ADD patients present a significant increase of cell damage through a RAGE-dependent mechanism, suggesting that AGEs may promote the generation of an oxidative stress vicious cycle, which can explain the severe progression of patients with both pathologies.

Subcellular Localization of Arabidopsis 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase

Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-μm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR-ER. Nevertheless, they represent a previously undescribed subcellular compartment likely capable of synthesizing mevalonate, which provides new evidence for multiorganelle compartmentalization of the isoprenoid biosynthetic pathways in plants.

Sulfated Polysaccharides Promote the Assembly of Amyloid β1–42 Peptide into Stable Fibrils of Reduced Cytotoxicity

The histopathological hallmarks of Alzheimer disease are the self-aggregation of the amyloid β peptide (Aβ) in extracellular amyloid fibrils and the formation of intraneuronal Tau filaments, but a convincing mechanism connecting both processes has yet to be provided. Here we show that the endogenous polysaccharide chondroitin sulfate B (CSB) promotes the formation of fibrillar structures of the 42-residue fragment, Aβ1–42. Atomic force microscopy visualization, thioflavin T fluorescence, CD measurements, and cell viability assays indicate that CSB-induced fibrils are highly stable entities with abundant β-sheet structure that have little toxicity for neuroblastoma cells. We propose a wedged cylinder model for Aβ1–42 fibrils that is consistent with the majority of available data, it is an energetically favorable assembly that minimizes the exposure of hydrophobic areas, and it explains why fibrils do not grow in thickness. Fluorescence measurements of the effect of different Aβ1–42 species on Ca2+ homeostasis show that weakly structured nodular fibrils, but not CSB-induced smooth fibrils, trigger a rise in cytosolic Ca2+ that depends on the presence of both extracellular and intracellular stocks. In vitro assays indicate that such transient, local Ca2+ increases can have a direct effect in promoting the formation of Tau filaments similar to those isolated from Alzheimer disease brains.

Amyloid-dependent triosephosphate isomerase nitrotyrosination induces glycation and tau fibrillation

Alzheimers disease neuropathology is characterized by neuronal death, amyloid beta-peptide deposits and neurofibrillary tangles composed of paired helical filaments of tau protein. Although crucial for our understanding of the pathogenesis of Alzheimers disease, the molecular mechanisms linking amyloid beta-peptide and paired helical filaments remain unknown. Here, we show that amyloid beta-peptide-induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neuroblastoma cells. Consequently, nitro-triosephosphate isomerase was found to be present in brain slides from double transgenic mice overexpressing human amyloid precursor protein and presenilin 1, and in Alzheimers disease patients. Higher levels of nitro-triosephosphate isomerase (P < 0.05) were detected, by Western blot, in immunoprecipitates from hippocampus (9 individuals) and frontal cortex (13 individuals) of Alzheimers disease patients, compared with healthy subjects (4 and 9 individuals, respectively). Triosephosphate isomerase nitrotyrosination decreases the glycolytic flow. Moreover, during its isomerase activity, it triggers the production of the highly neurotoxic methylglyoxal (n = 4; P < 0.05). The bioinformatics simulation of the nitration of tyrosines 164 and 208, close to the catalytic centre, fits with a reduced isomerase activity. Human embryonic kidney (HEK) cells overexpressing double mutant triosephosphate isomerase (Tyr164 and 208 by Phe164 and 208) showed high methylglyoxal production. This finding correlates with the widespread glycation immunostaining in Alzheimers disease cortex and hippocampus from double transgenic mice overexpressing amyloid precursor protein and presenilin 1. Furthermore, nitro-triosephosphate isomerase formed large beta-sheet aggregates in vitro and in vivo, as demonstrated by turbidometric analysis and electron microscopy. Transmission electron microscopy (TEM) and atomic force microscopy studies have demonstrated that nitro-triosephosphate isomerase binds tau monomers and induces tau aggregation to form paired helical filaments, the characteristic intracellular hallmark of Alzheimers disease brains. Our results link oxidative stress, the main etiopathogenic mechanism in sporadic Alzheimers disease, via the production of peroxynitrite and nitrotyrosination of triosephosphate isomerase, to amyloid beta-peptide-induced toxicity and tau pathology.

The blood-brain barrier: Structure, function and therapeutic approaches to cross it

Abstract The blood-brain barrier (BBB) is constituted by a specialized vascular endothelium that interacts directly with astrocytes, neurons and pericytes. It protects the brain from the molecules of the systemic circulation but it has to be overcome for the proper treatment of brain cancer, psychiatric disorders or neurodegenerative diseases, which are dramatically increasing as the population ages. In the present work we have revised the current knowledge on the cellular structure of the BBB and the different procedures utilized currently and those proposed to cross it. Chemical modifications of the drugs, such as increasing their lipophilicity, turn them more prone to be internalized in the brain. Other mechanisms are the use of molecular tools to bind the drugs such as small immunoglobulins, liposomes or nanoparticles that will act as Trojan Horses favoring the drug delivery in brain. This fusion of the classical pharmacology with nanotechnology has opened a wide field to many different approaches with promising results to hypothesize that BBB will not be a major problem for the new generation of neuroactive drugs. The present review provides an overview of all state-of-the-art of the BBB structure and function, as well as of the classic strategies and these appeared in recent years to deliver drugs into the brain for the treatment of Central Nervous System (CNS) diseases.

Recent Structural and Computational Insights into Conformational Diseases

Protein aggregation correlates with the development of several deleterious human disorders such as Alzheimers disease, Parkinsons disease, prion-associated transmissible spongiform encephalopathies and type II diabetes. The polypeptides involved in these disorders may be globular proteins with a defined 3D-structure or natively unfolded proteins in their soluble conformations. In either case, proteins associated with these pathogeneses all aggregate into amyloid fibrils sharing a common structure, in which beta-strands of polypeptide chains are perpendicular to the fibril axis. Because of the prominence of amyloid deposits in many of these diseases, much effort has gone into elucidating the structural basis of protein aggregation. A number of recent experimental and theoretical studies have significantly increased our understanding of the process. On the one hand, solid-state NMR, X-ray crystallography and single molecule methods have provided us with the first high-resolution 3D structures of amyloids, showing that they exhibit conformational plasticity and are able to adopt different stable tertiary folds. On the other hand, several computational approaches have identified regions prone to aggregation in disease-linked polypeptides, predicted the differential aggregation propensities of their genetic variants and simulated the early, crucial steps in protein self-assembly. This review summarizes these findings and their therapeutic relevance, as by uncovering specific structural or sequential targets they may provide us with a means to tackle the debilitating diseases linked to protein aggregation.

Modulation of Aβ42 fibrillogenesis by glycosaminoglycan structure

The role of amyloid β (Aβ) peptide in the onset and progression of Alzheimers disease is linked to the presence of soluble Aβ species. Sulfated glycosaminoglycans (GAGs) promote Aβ fibrillogenesis and reduce the toxicity of the peptide in neuronal cell cultures, but a satisfactory rationale to explain these effects at the molecular level has not been provided yet. We have used circular dichroism, Fourier transform infrared spectroscopy, fluorescence microscopy and spectroscopy, protease digestion, atomic force microscopy (AFM), and molecular dynamics simulations to characterize the association of the 42‐residue fragment Aβ42 with sulfated GAGs, hyaluronan, chitosan, and poly(vinyl sulfate) (PVS). Our results indicate that the formation of stable Aβ42 fibrils is promoted by polymeric GAGs with negative charges placed in‐frame with the 4.8‐Å separating Aβ42 monomers within protofibrillar β‐sheets. Incubation of Aβ42 with excess sulfated GAGs and hyaluronan increased amyloid fibril content and resistance to proteolysis 2‐ to 5‐fold, whereas in the presence of the cationic polysaccharide chitosan, Aβ42 fibrillar species were reduced by 25% and sensitivity to protease degradation increased ∼3‐fold. Fibrils of intermediate stability were obtained in the presence of PVS, an anionic polymer with more tightly packed charges than GAGs. Important structural differences between Aβ42 fibrils induced by PVS and Aβ42 fibrils obtained in the presence of GAGs and hyaluronan were observed by AFM, whereas mainly precursor protofibrillar forms were detected after incubation with chitosan. Computed binding energies per peptide from −11.2 to −13.5 kcal/mol were calculated for GAGs and PVS, whereas a significantly lower value of −7.4 kcal/mol was obtained for chitosan. Taken together, our data suggest a simple and straightforward mechanism to explain the role of GAGs as enhancers of the formation of insoluble Aβ42 fibrils trapping soluble toxic forms.—Valle‐Delgado, J. J., Alfonso‐Prieto, M., de Groot, N. S., Ventura, S., Samitier, J., Rovira, C., Fernàndez‐Busquets, X. Modulation of Aβ42 fibrillogenesis by glycosaminoglycan structure. FASEB J. 24, 4250–4261 (2010). www.fasebj.org

A nanovector with complete discrimination for targeted delivery to Plasmodium falciparum-infected versus non-infected red blood cells in vitro

Current administration methods of antimalarial drugs deliver the free compound in the blood stream, where it can be unspecifically taken up by all cells, and not only by Plasmodium-infected red blood cells (pRBCs). Nanosized carriers have been receiving special attention with the aim of minimizing the side effects of malaria therapy by increasing drug bioavailability and selectivity. Liposome encapsulation has been assayed for the delivery of compounds against murine malaria, but there is a lack of cellular studies on the performance of targeted liposomes in specific cell recognition and on the efficacy of cargo delivery, and very little data on liposome-driven antimalarial drug targeting to human-infecting parasites. We have used fluorescence microscopy to assess in vitro the efficiency of liposomal nanocarriers for the targeted delivery of their contents to pRBCs. 200-nm liposomes loaded with quantum dots were covalently functionalized with oriented, specific half-antibodies against P. falciparum late form-infected pRBCs. In less than 90min, liposomes dock to pRBC plasma membranes and release their cargo to the cell. 100.0% of late form-containing pRBCs and 0.0% of non-infected RBCs in P. falciparum cultures are recognized and permeated by the content of targeted immunoliposomes. Liposomes not functionalized with antibodies are also specifically directed to pRBCs, although with less affinity than immunoliposomes. In preliminary assays, the antimalarial drug chloroquine at a concentration of 2nM, ≥10 times below its IC(50) in solution, cleared 26.7±1.8% of pRBCs when delivered inside targeted immunoliposomes.