New perspectives on corpora amylacea in the human brain

Corpora amylacea are structures of unknown origin and function that appear with age in human brains and are profuse in selected brain areas in several neurodegenerative conditions. They are constituted of glucose polymers and may contain waste elements derived from different cell types. As we previously found on particular polyglucosan bodies in mouse brain, we report here that corpora amylacea present some neo-epitopes that can be recognized by natural antibodies, a certain kind of antibodies that are involved in tissue homeostasis. We hypothesize that corpora amylacea, and probably some other polyglucosan bodies, are waste containers in which deleterious or residual products are isolated to be later eliminated through the action of the innate immune system. In any case, the presence of neo-epitopes on these structures and the existence of natural antibodies directed against them could become a new focal point for the study of both age-related and degenerative brain processes

Study of the transport of substances across the blood-brain barrier with the 8D3 anti-transferrin receptor antibody

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/128014Numerous strategies have been proposed to overcome the blood-brain barrier (BBB) and efficiently deliver therapeutic agents to the brain. One of these strategies consists of linking the pharmacologically active substance to a molecular vector that acts as a molecular Trojan Horse and is capable of crossing the BBB using a receptor-mediated transcellular transport system of the brain capillary endothelial cells (BCECs). The transferrin receptor (TfR) is related to a transcytosis process in these cells, and the 8D3 monoclonal antibody (mAb), directed against the mouse TfR, is able to induce a receptor response. Thus, the 8D3 antibody could be a potential molecular Trojan Horse to transport pharmacologically active substances across the BBB. On these bases, a series of experiments were performed where the 8D3 antibody was conjugated to different cargoes, the resulting constructs were administered in vivo to mice, and the distribution and intracellular mechanisms that these constructs undergo at the BBB were studied. Our results indicated a TfR-mediated and clathrin-dependent internalization process by which the 8D3-cargo constructs enters the BCEC. The resulting endocytic vesicles follow at least two different routes. On one hand, most vesicles enter intracellular processes of vesicular fusion and rearrangement in which the cargo is guided to late endosomes, multivesicular bodies or lysosomes. On the other hand, a small but not negligible percentage of the vesicles follow a different route in which they fuse with the abluminal membrane and open towards the basal lamina, indicating a potential route for the delivery of therapeutic substances. In this route, however, the 8D3−cargo remain fixed to the abluminal membrane, indicating that the 8D3 is maintained linked to the TfR, and the cargo does not go beyond the basal membrane. Altogether, different optimization approaches need to be developed for efficient drug delivery, but receptor-mediated transport (RMT) continues to be one of the most promising strategies to overcome the BBB

Trafficking of Gold Nanoparticles Coated with the 8D3 Anti-Transferrin Receptor Antibody at the Mouse Blood-Brain Barrier

Receptor-mediated transcytosis has been widely studied as a possible strategy to transport neurotherapeutics across the blood-brain barrier (BBB). Monoclonal antibodies directed against the transferrin receptor (TfR) have been proposed as potential carrier candidates. A better understanding of the mechanisms involved in their cellular uptake and intracellular trafficking is required and could critically contribute to the improvement of delivery methods. Accordingly, we studied here the trafficking of gold nanoparticles (AuNPs) coated with the 8D3 anti-transferrin receptor antibody at the mouse BBB. 8D3-AuNPs were intravenously administered to mice and allowed to recirculate for a range of times, from 10 min to 24 h, before brain extraction and analysis by transmission electron microscope techniques. Our results indicated a TfR-mediated and clathrin-dependent internalization process by which 8D3-AuNPs internalize individually in vesicles. These vesicles then follow at least two different routes. On one hand, most vesicles enter intracellular processes of vesicular fusion and rearrangement in which the AuNPs end up accumulating in late endosomes, multivesicular bodies or lysosomes, which present a high AuNP content. On the other hand, a small percentage of the vesicles follow a different route in which they fuse with the abluminal membrane and open to the basal membrane. In these cases, the 8D3-AuNPs remain attached to the abluminal membrane, which suggests an endosomal escape, but not dissociation from TfR. Altogether, although receptor-mediated transport continues to be one of the most promising strategies to overcome the BBB, different optimization approaches need to be developed for efficient drug delivery. Keywords: blood−brain barrier; drug delivery; electron microscopy; monoclonal antibodies; receptor-mediated transport; transferrin receptor

Neo-epitopes emerging in the degenerative hippocampal granules of aged mice can be recognized by natural IgM autoantibodies

Background: Degenerative granular structures appear progressively with age in the hippocampus of most mouse strains. We recently reported that these granules contain a neo-epitope that is recognised by IgM antibodies present as contaminants in many commercial antibodies obtained from mouse ascites and mouse or rabbit serum. We hypothesise that these anti-neo-epitope IgMs are in fact natural auto-antibodies that are generated spontaneously during the foetal stage without previous contact with external antigens and whose repertoire and reactivity pattern have been determined through evolution, being remarkably stable within species and even between species. Findings: In the present work we found that mice from the ICR-CD1, BALB/C and SAMP8 strains have anti-neoepitope IgM antibodies in their plasma at all ages tested and even when maintained under specific opportunistic pathogen-free conditions. Moreover, we determined that these anti-neo-epitope IgMs are also present in rabbit, goat and rat serum. We also found that, in each mouse that presented hippocampal granules, the anti-neo-epitope IgMs contained in its plasma recognised the neo-epitopes in its own granules. Conclusions: This study led to the conclusion that anti-neo-epitope IgMs are widespread natural auto-antibodies contained in the plasma of mice and other species. The presence of these natural auto-antibodies not only explains why they are frequently found as contaminants in commercial antibodies, but also paves the way for a new approach to a treatment and diagnosis of pathological brain processes based on natural IgMs and neo-epitopes

Serial block-face scanning electron microscopy applied to study the trafficking of 8D3-coated gold nanoparticles at the blood-brain barrier

Due to the physical and physiological properties of the blood-brain barrier (BBB), the transport of neurotherapeutics from blood to brain is still a pharmaceutical challenge. We previously conducted a series of experiments to explore the potential of the anti-transferrin receptor 8D3 monoclonal antibody (mAb) to transport neurotherapeutics across the BBB. In that study, gold nanoparticles (AuNPs) were coated with the 8D3 antibody and administered intravenously to mice. Transmission electron microscopy was used and a two-dimensional (2D) image analysis was performed to detect the AuNPs in the brain capillary endothelial cells (BCECs) and brain parenchyma. In the present work, we determined that serial block-face scanning electron microscopy (SBF-SEM) is a useful tool to study the transcytosis of these AuNPs across the BBB in three dimensions and we, therefore, applied it to gain more knowledge of their transcellular trafficking. The resulting 3D reconstructions provided additional information on the endocytic vesicles containing AuNPs and the endosomal processing that occurs inside BCECs. The passage from 2D to 3D analysis reinforced the trafficking model proposed in the 2D study, and revealed that the vesicles containing AuNPs are significantly larger and more complex than described in our 2D study. We also discuss tradeoffs of using this technique for our application, and conclude that together with other volume electron microscopy imaging techniques, SBF-SEM is a powerful approach that is worth of considering for studies of drug transport across the BBB

Astrocytes and neurons produce distinct types of polyglucosan bodies in Lafora disease

Lafora disease (LD), the most devastating adolescence‐onset epilepsy, is caused by mutations in the EPM2A or EPM2B genes, which encode the proteins laforin and malin, respectively. Loss of function of one of these proteins, which are involved in the regulation of glycogen synthesis, induces the accumulation of polyglucosan bodies (PGBs) known as Lafora bodies (LBs) and associated with neurons in the brain. Ageing and some neurodegenerative conditions lead to the appearance of another type of PGB called corpora amylacea , which are associated with astrocytes and contain neo‐epitopes that can be recognized by natural antibodies. Here we studied the PGBs in the cerebral cortex and hippocampus of malin knockout mice, a mouse model of LD. These animals presented not only LBs associated with neurons but also a significant number of PGBs associated with astrocytes. These astrocytic PGBs were also increased in mice from senescence‐accelerated mouse‐prone 8 (SAMP8) strain and mice with overexpression of Protein Targeting to Glycogen (PTGOE), indicating that they are not exclusive of LD. The astrocytic PGBs, but not neuronal LBs, contained neo‐epitopes that are recognized by natural antibodies. The astrocytic PGBs appeared predominantly in the hippocampus but were also present in some cortical brain regions, while neuronal LBs were found mainly in the brain cortex and the pyramidal layer of hippocampal regions CA2 and CA3. Our results indicate that astrocytes, contrary to current belief, are involved in the etiopathogenesis of LD

Estudio del transporte de sustancias a través de la barrera hematoencefálica mediante el anticuerpo 8D3 dirigido contra el receptor de transferrina

[eng

New perspectives on corpora amylacea in the human brain

Corpora amylacea are structures of unknown origin and function that appear with age in human brains and are profuse in selected brain areas in several neurodegenerative conditions. They are constituted of glucose polymers and may contain waste elements derived from different cell types. As we previously found on particular polyglucosan bodies in mouse brain, we report here that corpora amylacea present some neo-epitopes that can be recognized by natural antibodies, a certain kind of antibodies that are involved in tissue homeostasis. We hypothesize that corpora amylacea, and probably some other polyglucosan bodies, are waste containers in which deleterious or residual products are isolated to be later eliminated through the action of the innate immune system. In any case, the presence of neo-epitopes on these structures and the existence of natural antibodies directed against them could become a new focal point for the study of both age-related and degenerative brain processes

Periodic acid-Schiff granules in the brain of aged mice: from amyloid aggregates to degenerative structures containing neo-epitopes

Brain ageing in mice leads to the progressive appearance and expansion of degenerative granular structures frequently referred as "PAS granules" because of their positive staining with periodic acid-Schiff (PAS). PAS granules are present mainly in the hippocampus, although they have also been described in other brain areas such as piriform and entorhinal cortices, and have been observed in other mammals than mice, like rats and monkeys. PAS granules have been identified as a wide range of brain deposits related to numerous neurodegenerative diseases, such as amyloid deposits, neurofibrillary tangles, Lafora bodies, corpora amylacea and polyglucosan bodies, and these identifications have generated controversy and particular theories about them. We have recently reported the presence of a neo-epitope in mice hippocampal PAS granules and the existence of natural IgM auto-antibodies directed against the neo-epitope in the plasma of the animals. The significance of the neo-epitope and the autoantibodies is discussed in this review. Moreover, we observed that the IgM anti-neo-epitope is frequently present as a contaminant in numerous commercial antibodies and is responsible of a considerable amount of false positive immunostainings, which may produce misinterpretations in the identification of the granules. Now that this point has been clarified, this article reviews and reconsiders the nature and physiopathological significance of these degenerative granules. Moreover, we suggest that neo-epitopes may turn into a useful brain-ageing biomarker and that autoimmunity could become a new focus in the study of age-related degenerative processes