Gemma Manich

Early Amyloid Accumulation in the Hippocampus of SAMP8 Mice

Late-onset Alzheimers disease (AD) is the most common form of AD appearing after 65 years of age. To date, however, there are no non-genetically manipulated rodent models that develop a similar sporadic onset of AD with age-related amyloid-beta (Abeta) deposition. Although the senescence accelerated mouse prone 8 (SAMP8) mice have been proposed as a model of AD, the presence of Abeta deposits remains controversial. In this study, we describe the time course of Abeta deposition in SAMP8 mice as well as in control SAMR1 and ICR-CD1 strains of mice. From as early as 6 months onward, SAMP8 mice show Abeta deposition in the hippocampus that increase in number and extent with age. These deposits are comprised of by clustered granules that contain Abeta{42}, Abeta{40}, and other Abeta protein precursor fragments. By marked contrast, control mice show only low numbers of Abeta clusters that do not develop until 15 months of age. The demonstration that SAMP8 mice present with amyloid deposits in their hippocampus makes this animal model a useful tool to understand the mechanisms involved in Abeta deposition in AD.

Cerebral Amyloid Angiopathy, Blood-Brain Barrier Disruption and Amyloid Accumulation in SAMP8 Mice

Cerebrovascular dysfunction and β-amyloid peptide deposition on the walls of cerebral blood vessels might be an early event in the development of Alzheimer’s disease. Here we studied the time course of amyloid deposition in blood vessels and blood-brain barrier (BBB) disruption in the CA1 subzone of the hippocampus of SAMP8 mice and the association between these two variables. We also studied the association between the amyloid deposition in blood vessels and the recently described amyloid clusters in the parenchyma, as well as the association of these clusters with vessels in which the BBB is disrupted. SAMP8 mice showed greater amyloid deposition in blood vessels than age-matched ICR-CD1 control mice. Moreover, at 12 months of age the number of vessels with a disrupted BBB had increased in both strains, especially SAMP8 animals. At this age, all the vessels with amyloid deposition showed BBB disruption, but several capillaries with an altered BBB showed no amyloid on their walls. Moreover, amyloid clusters showed no spatial association with vessels with amyloid deposition, nor with vessels in which the BBB had been disrupted. Finally, we can conclude that vascular amyloid deposition seems to induce BBB alterations, but BBB disruption may also be due to other factors.

Characterization of Amyloid-β Granules in the Hippocampus of SAMP8 Mice

The senescence accelerated mouse-prone 8 (SAMP8) strain of mice is an experimental model of accelerated senescence that has also been proposed as a model of Alzheimers disease as it shares several features with this dementia. We have recently reported amyloid-β (Aβ) granules in the hippocampus of SAMP8 mice, which contain Aβ42 and Aβ40 peptides and other amyloid-β protein precursor fragments. These granules appear clustered mainly in the stratum radiatum of the CA1 region and increase in number and size with age. Here we performed several studies to examine whether the Aβ granules in the hippocampus of SAMP8 mice contain other proteins characteristic of neuropathological aggregates, such as tau, MAP2, and α-synuclein. Moreover, we examined whether the Aβ granules in the hippocampus correspond to heparan sulphate proteoglycan (HSPG) positive granules previously described in this animal model. The results showed that Aβ granules correspond to the HSPG granular structures, being syndecan-2, a protein involved in the remodeling of dendritic spines, the type of HSPG found. Tau and MAP2, but not α-synuclein depositions, were also found in Aβ aggregates. Granules do not appear to have an astrocytic origin, since although some Aβ clusters are associated with astrocyte processes, most clusters are not. On the other hand, the presence of tau, MAP2, and NeuN in Aβ granules suggests a neuronal origin. As the components identified in Aβ granules are characteristic of the aggregates present in some neurodegenerative diseases, the SAMP8 model seems to be appropriate for the study of the processes involved in these pathologies.

Role of matrix metalloproteinase‐9 (MMP‐9) in striatal blood–brain barrier disruption in a 3‐nitropropionic acid model of Huntington's disease

J. Duran‐Vilaregut, J. del Valle, G. Manich, A. Camins, M. Pallàs, J. Vilaplana and C. Pelegrí (2011) Neuropathology and Applied Neurobiology37, 525–537

Time-course of blood–brain barrier disruption in senescence-accelerated mouse prone 8 (SAMP8) mice

Senescence of the cerebrovascular system and an abnormal function of the blood–brain barrier have been related with Alzheimers disease. We studied here the time‐course of blood–brain barrier disruption in senescence‐accelerated mouse prone 8 (SAMP8) mice, which is a murine model of senescence and is also considered a model of Alzheimers disease. We used a previously described method that allows evaluating blood–brain barrier integrity by observing Evans blue extravasation from brain blood vessels. Three brain regions (cortex, hippocampus and hippocampal fissure) of SAMP8 brains were analyzed at 3, 6, 9, 12 and 15 months of age. Moreover, genetically related senescence‐accelerated mouse resistant 1 (SAMR1) and ICR‐CD1 mice were studied. Results indicate that Evans blue permeability in SAMP8 and SAMR1 increases from 6 to 15 months in the three studied regions. At 15 months of age, SAMP8 and SAMR1 mice showed higher Evans blue extravasation in CA1 and Fissure than ICR‐CD1 mice. Further studies are required to understand the senescence process in SAMR1 mice, as blood–brain barrier alterations in old age have unexpectedly been observed. On the other hand, as blood–brain barrier permeability in SAMP8 mice increases with age, blood–brain barrier alterations may contribute to the cerebral pathology observed in this strain.

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.

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 β-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 β-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.

Aβ increases neural stem cell activity in senescence-accelerated SAMP8 mice.

Neurogenesis persists in the adult brain as a form of plasticity due to the existence of neural stem cells (NSCs). Alterations in neurogenesis have been found in transgenic Alzheimers disease (AD) mouse models, but NSC activity and neurogenesis in sporadic AD models remains to be examined. We herein describe a remarkable increase in NSC proliferation in the forebrain of SAMP8, a non-transgenic mouse strain that recapitulates the transition from healthy aging to AD. The increase in proliferation is transient, precedes AD-like symptoms such as amyloid beta 1-42 [Aβ(1-42)] increase or gliosis, and is followed by a steep decline at later stages. Interestingly, in vitro studies indicate that secreted Aβ(1-42) and PI3K signaling may account for the early boost in NSC proliferation. Our results highlight the role of soluble Aβ(1-42) peptide and PI3K in the autocrine regulation of NSCs, and further suggest that over-proliferation of NSCs before the appearance of AD pathology may underlie neurogenic failure during the age-related progression of the disease. These findings have implications for therapeutic approaches based on neurogenesis in AD.

Systemic administration of 3-nitropropionic acid points out a different role for active caspase-3 in neurons and astrocytes

The intraperitoneal administration of 3-nitropropionic acid, which is commonly used to induce toxicity models of Huntingtons disease in experimental animals, produces severe brain injury in the lateral part of the striatum. We studied the presence of active caspase-3 in neurons and astrocytes from brains of rats treated with 3-nitropropionic acid following a subacute administration protocol. Active caspase-3 was almost absent in the core of the striatal lesion. However, it was expressed, albeit weakly, in the neurons present in the rim of the lesion. In cortex and non-injured striatal areas, and in the cortex and striatum of control animals, active caspase-3 staining was widely distributed and vivid, but localized in the cell bodies of astrocytes rather than in neurons. In treated animals, some of the active caspase-3 positive neurons localized in the rim of the lesion were also positive for TUNEL staining. This indicates the presence of a caspase-mediated apoptotic process. TUNEL was not present in control animals or in the astrocytes of treated animals. Thus, the presence of active caspase-3 in astrocytes may be merely constitutive.

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

BackgroundDegenerative 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.FindingsIn 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.ConclusionsThis 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.