Joaquim Duran-Vilaregut

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.

Cell cycle activation in striatal neurons from Huntington's disease patients and rats treated with 3-nitropropionic acid.

This study was undertaken to investigate the potential role of cell cycle re‐entry in an experimental model of Huntingtons disease and in human brain samples. We found that after treatment of rats with the mitochondrial neurotoxin 3‐nitropropionic acid, the expression of cell cycle markers of G1 phase measured by immunohistochemistry was induced in the striatal brain region. Furthermore, we detected an increase in the nuclear and also cytoplasmatic E2F‐1 expression, suggesting that this protein could activate the apoptotic cascade in rat brain. Western blot analysis of post‐mortem brain samples from patients also showed an increase in the expression of E2F‐1 and cyclin D1 in comparison with control samples. These results indicate that cell cycle re‐entry is activated in Huntingtons disease and may contribute to the neurodegenerative process.

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.

Blood-brain barrier disruption in the striatum of rats treated with 3-nitropropionic acid.

3-nitropropionic acid (3-NPA) is a natural toxin that is used to induce models of Huntingtons disease (HD) in experimental animals. Here we injected 3-NPA into Sprague-Dawley rats in order to evaluate its effects on the blood-brain barrier (BBB). Evans blue (EB) extravasation was used to identify injured areas in the brains of the treated animals and immunostainings of endothelial brain barrier antigen (EBA), zona occludens-1 (ZO-1) and laminin were used as markers to characterize the effects of the neurotoxin on the BBB. Treated rats had a significant loss of body weight compared to controls, and a correlation between motor affectation and body weight loss was observed in the former. The lateral part of the striatum was specifically injured in treated animals and the BBB almost disappeared in the core of the injured areas, as evidenced by a high EB extravasation and severe alterations of the immunostainings of the three BBB integrity markers compared to those of control animals. We conclude that the BBB is severely affected in the 3-NPA rat model of HD and that disruption of this barrier is a crucial event during the development of this disease.

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.

Expression pattern of ataxia telangiectasia mutated (ATM), p53, Akt, and glycogen synthase kinase-3β in the striatum of rats treated with 3-nitropropionic acid

3‐Nitropropionic acid (3‐NPA) is a mitochondrial toxin used in the laboratory to replicate neurodegenerative conditions that are accompanied by degeneration of the caudate‐putamen. 3‐NPA induces depletion in ATP production, reactive oxygen species production, and secondary excitotoxicity mediated by activation of N‐methyl‐D‐aspartate receptors that culminates in the triggering of cell death mechanisms, including apoptosis. We here examined by immunohistochemical methods whether cellular expression of phosphoSer1981‐ataxia telangiectasia mutated (ATM), phosphoSer15‐p53, phosphoSer473‐Akt, and phosphoSer9‐glycogen synthase kinase‐3β (GSK3β), which are key signal molecules that play a critical role in regulating cellular processes related to cell survival and demise, were involved in the striatal neurodegeneration in the brains of rats treated with 3‐NPA. Our results indicate that the toxin induced the activation of ATM and p53 only in astrocytes, and a role for these proteins in neuronal degeneration was ruled out. On the other hand, striatal neurons lost the active form of Akt as soon as they began to appear pyknotic, indicating impairment of the PI3K/Akt/GSK3 pathway in their degenerative process. The inactive form of GSK3β was detected extensively, mainly in the rim of the striatal lesions around degenerating neurons, which could be attributed to a cell death or cell survival response.

Neuronal apoptosis in the striatum of rats treated with 3-nitropropionic acid is not triggered by cell-cycle re-entry.

Although terminally differentiated neurons lack the capacity to undergo cell division, they retain the capacity to reactivate the cell cycle. This reactivation, however, has been linked to the degeneration of neurons in many experimental models of neurodegenerative disease and in post-mortem brains of affected patients. Expression of markers of the G1 phase and apoptotic neurons has been detected in the striatal lesion of rats treated with 3-nitropropionic acid (3-NPA). Here we examined whether neuronal apoptosis induced by 3-NPA was mediated by the reactivation of the cell cycle. To this end, we studied whether TUNEL-positive neurons expressed the G1-phase markers cyclin-dependent kinase 4 (CDK4) and cyclin D (CyD). In addition, we also evaluated the neuronal expression of pRb and Ki67 antigens, both of which are involved in the regulation of cell-cycle progression. In 3-NPA-treated rats, CDK4 and CyD were not detected in TUNEL-positive neurons, but they were expressed in neurons in the core of the lesion, which were assumed to be in a more advanced stage of degeneration, since they had weaker NeuN staining and lacked Hoechst staining. In addition, injured neurons in the striatal lesion of 3-NPA-treated rats had lost the constitutive expression of pRb and Ki67 that we had detected in control animals. Taken together, these results indicate that neuronal apoptosis in the striatal lesion of 3-NPA-treated rats was not triggered by cell-cycle re-entry, and we conclude that expression of G1 markers may be considered an aberrant survival response, with no relation to the mechanisms of apoptosis.