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.

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.

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.

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

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-neo-epitope 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.

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.

Clustered granules present in the hippocampus of aged mice result from a degenerative process affecting astrocytes and their surrounding neuropil.

Clusters of pathological granular structures appear and progressively increase in number with age in the hippocampus of several mice strains, markedly in the senescence-accelerated mouse prone 8 mice. In the present work, we performed an ultrastructural study of these granules paying special attention to the first stages of their formation, which have not been previously explored. The analysis of the immature granules allowed concluding that granules are not simple accumulations of molecular waste but the result of a degenerative process involving principally astrocytic processes, although nearby neuronal structures can be also affected. The granule generation includes the instability of the plasmatic membranes and the appearance of abnormal membranous structures that form intracellular bubbles or blebs of variable sizes and irregular shapes. These structures and some organelles degenerate producing some membranous fragments, and an assembly process of the resulting fragments generates the dense-core nucleus of the mature granule. Moreover, we found out that the neo-epitope recently described in the mature granules and localised abundantly in the membranous fragments of their dense-core nucleus emerges in the first stages of the granule formation. On the other hand, with this study, we increase the evidences that each cluster of granules is formed by the granules comprised in one astrocyte. A better knowledge of the causes of the granule formation and the function of the neo-epitope will help in both the interpretation of the physiological significance of the granules and their contribution to the degenerating processes in aging brain.

Presence of a neo-epitope and absence of amyloid beta and tau protein in degenerative hippocampal granules of aged mice.

Clustered pathological granules related to a degenerative process appear and increase progressively with age in the hippocampus of numerous mouse strains. We describe herein the presence of a neo-epitope of carbohydrate nature in these granules, which is not present in other brain areas and thus constitutes a new marker of these degenerative structures. We also found that this epitope is recognised by a contaminant IgM present in several antibodies obtained from mouse ascites and from both mouse and rabbit sera. These findings entail the need to revise the high number of components that are thought to be present in the granules, such as the controversial ß-amyloid peptides described in the granules of senescence-accelerated mouse prone-8 (SAMP8) mice. Characterisation of the composition of SAMP8 granules, taking into account the presence of the neo-epitope and the contaminant IgM, showed that granules do not contain either ß-amyloid peptides or tau protein. The presence of the neo-epitope in the granules but not in other brain areas opens up a new direction in the study of the neurodegenerative processes associated with age. The SAMP8 strain, in which the progression of the granules is enhanced, may be a useful model for this purpose.

Study of the transcytosis of an anti-transferrin receptor antibody with a Fab' cargo across the blood-brain barrier in mice.

One strategy used to transport pharmacologically active substances across the blood-brain barrier (BBB) is to link the substance to a molecule capable of crossing the BBB using a receptor-mediated transcellular transport system in brain capillary endothelial cells. The transferrin receptor (TfR) is related to a transcytosis process in these cells, and the 8D3 antibody, directed against mouse TfR, is able to induce a receptor response. In this work, the potential of 8D3 to carry molecules across the BBB was investigated. This antibody (either unlabeled or FITC-labeled) was intravenously administered to ICR-CD1 mice. Immunocomplexes (ICs) consisting of 8D3 antibody (carrier) and Fab' fragments (simulated cargo), in some cases directly fluorolabeled with FITC, were also administered to ICR-CD1 mice. At different time-points of recirculation, the IC components were studied using immunohistochemical procedures and fluorescence resonance energy transfer (FRET). The results suggested that 8D3 alone or in an IC with Fab' fragments entered the endothelial cells. FRET analysis indicated that the colocalization of their fluorescent signals inside the endothelial cells decreased with time, indicating that ICs can be processed and Fab' fragments probably separated from 8D3. However, neither 8D3 alone nor the IC components crossed the BBB.