Nienke Timmer

Gray and white matter degeneration revealed by diffusion in an Alzheimer mouse model.

In patients with Alzheimers disease (AD) the severity of white matter degeneration correlates with the clinical symptoms of the disease. In this study, we performed diffusion-tensor magnetic resonance imaging at ultra-high field in a mouse model for AD (APP(swe)/PS1(dE9)) in combination with a voxel-based approach and tractography to detect changes in water diffusivity in white and gray matter, because these reflect structural alterations in neural tissue. We found substantial changes in water diffusion parallel and perpendicular to axonal tracts in several white matter regions like corpus callosum and fimbria of the hippocampus, that match with previous findings of axonal disconnection and myelin degradation in AD patients. Moreover, we found a significant increase in diffusivity in specific hippocampal subregions, which is supported by neuronal loss as visualized with Klüver-Barrera staining. This work demonstrates the potential of ultra-high field diffusion-tensor magnetic resonance imaging as a noninvasive modality to describe white and gray matter structural changes in mouse models for neurodegenerative disorders, and provides valuable knowledge to assess future AD prevention strategies in translational research.

Enoxaparin treatment administered at both early and late stages of amyloid β deposition improves cognition of APPswe/PS1dE9 mice with differential effects on brain Aβ levels.

Enoxaparin (Enox), a low molecular weight heparin, has been shown to lower brain amyloid beta (A beta) load in a mouse model for Alzheimers disease. However, the effect of Enox on cognition was not studied. Therefore, we examined the effect of peripheral Enox treatment on cognition and brain A beta levels in the APPswe/PS1dE9 mouse model by giving injections at an early (starting at 5 months of age) and late (starting at 10 and 12 months of age) stage of A beta accumulation for 3 months. Although Enox had no effect on behaviour in the open field at any age, it improved spatial memory in the Morris water maze in 5-, 10- and 12-month-old mice. Furthermore, Enox treatment seemed to decrease guanidine HCl-extracted brain A beta levels at 5 months of age, but significantly increased guanidine HCl-extracted A beta 42 and A beta 40 levels in both 10- and 12-month-old mice. In vitro, Enox increased aggregation of A beta, even when A beta was pre-aggregated. In conclusion, Enox treatment, either at an early or a late stage of A beta accumulation, could improve cognition in APPswe/PS1dE9 mice. However, since Enox treatment at an early stage of A beta accumulation decreased guanidine HCl-extracted A beta levels and Enox treatment at a late stage enhanced guanidine HCl-extracted A beta levels, it seems that Enox influences A beta deposition differently at different stages of A beta pathology. In any case, our study suggests that enoxaparin treatment has potential as a therapeutic agent for Alzheimers disease.

Aggregation and cytotoxic properties towards cultured cerebrovascular cells of Dutch-mutated Abeta40 (DAbeta(1-40)) are modulated by sulfate moieties of heparin.

Glycosaminoglycans (GAGs), in particular as part of heparan sulfate proteoglycans, are associated with cerebral amyloid angiopathy (CAA). Similarly, GAGs are also associated with the severe CAA found in patients suffering from hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), where the amyloid beta (Abeta) peptide contains the Dutch mutation (DAbeta(1-40)). This suggests a role for GAGs in vascular Abeta aggregation. It was the aim of this study to investigate the effect of different GAGs (heparin, chondroitin sulfate, heparan sulfate), the macromolecule dextran sulfate and, using desulfated heparins, the role of GAG sulfate moieties on the in vitro aggregation of CAA-associated DAbeta(1-40) and on DAbeta(1-40)-induced toxicity of cultured cerebrovascular cells. We also aimed to study the in vivo distribution of various sulfated heparan sulfate GAG epitopes in CAA. Of all GAGs tested, heparin was the strongest inducer of aggregation of DAbeta(1-40) in the different aggregation assays, with both heparin and heparan sulfate reducing Abeta-induced cellular toxicity. Furthermore, (partial) removal of the sulfate moieties of heparin partially abolished the effects of heparin on aggregation and cellular toxicity, suggesting an essential role for the sulfate moieties in heparin. Finally, we demonstrated the in vivo association of sulfated heparan sulfate (HS) GAGs with CAA. We conclude that sulfate moieties within GAGs, like heparin and HS, have an important role in Abeta aggregation in CAA and in Abeta-mediated toxicity of cerebrovascular cells.

Amyloid beta induces cellular relocalization and production of agrin and glypican-1

The major component of senile plaques and vascular amyloid in Alzheimers disease (AD) brains is the amyloid beta protein (Abeta). Besides Abeta, several other proteins have been identified in these lesions, in particular heparan sulfate proteoglycans (HSPG). However, it is still unclear, what causes the excessive accumulation of HSPG in AD brains. Therefore, we investigated if Abeta may influence production and expression of two major Abeta-associated HSPG species, agrin and glypican-1. When human brain pericytes (HBP) were cultured in the presence of Abeta, protein and mRNA expression of both agrin and glypican-1 were increased and more radioactive sulfate was incorporated in the glycosaminoglycan fraction of Abeta-treated HBP. Furthermore, after Abeta treatment, these HSPG were found in association with the amyloid fibrils attached to the cell membrane, in contrast to the intracellular agrin and glypican-1 staining observed in untreated cells. We conclude that Abeta can modulate the cellular expression of agrin and glypican-1, which may contribute to the accumulation of these HSPG in AD lesions.

Limited expression of heparan sulphate proteoglycans associated with Aβ deposits in the APPswe/PS1dE9 mouse model for Alzheimer's disease

N. M. Timmer, M. K. Herbert, J. W. Kleinovink, A. J. Kiliaan, R. M. W. de Waal and M. M. Verbeek (2010) Neuropathology and Applied Neurobiology36, 478–486
Limited expression of heparan sulphate proteoglycans associated with Aβ deposits in the APPswe/PS1dE9 mouse model for Alzheimers disease

Do Amyloid β-associated Factors Co-deposit with Aβ in Mouse Models for Alzheimer's Disease?

Senile plaques and cerebral amyloid angiopathy in Alzheimers disease (AD) patients not only consist of the amyloid-β protein (Aβ), but also contain many different Aβ-associated factors, such as heparan sulfate proteoglycans, apolipoproteins, and complement factors. These factors may all influence Aβ deposition, aggregation, and clearance and therefore seem important in the development of human Aβ deposits. To study AD pathology and test new therapeutic agents, many different mouse models have been created. By transgenic expression of the amyloid-β protein precursor, frequently in combination with other transgenes, these animals develop Aβ deposits that morphologically resemble their human counterparts. Whether this resemblance also applies to the presence of Aβ-associated factors is largely unclear. In this review, the co-deposition of factors known to associate with human Aβ deposits is summarized for several different AD mouse models.

Low glycine levels in brain homogenates of TgSwDI mice compared to wild-type mice

Background: In Alzheimer’s disease (AD), accumulation of amyloid b (Ab) protein can disturb normal glutamatergic neurotransmission. Glutamate is an important neurotransmitter in learning and memory but, when present in high amounts, can also be excitotoxic, resulting in neuronal loss. Indeed, the loss of glutamatergic neurons can be linked to the cognitive impairments seen in AD patients. As disturbances in the glutamatergic system are closely linked to AD, it is our aim to search for potential biomarkers of the glutamatergic system. To this end, we characterized levels of glutamate-related metabolites in the TgSwDI mouse model for AD. Methods: Male and female TgSwDI mice with mild (3 months old) and severe (9 months old) Ab pathology and non-transgenic (C57Bl/6) age-matched controls, were sacrificed by cervical dislocation. Afterwards, their brains were dissected into different brain regions (i.e. hippocampus, cortex) and snapfrozen until analysis. Distilled water was used to dissolve metabolites from tissue. Using an amino acid analyzer, concentrations of various glutamate-related amino acids (i.e. glutamate, glutamine, GABA, glycine, aspartate, asparagine, alanine) were determined. Results: Glutamate levels were hardly changed in TgSwDI mice. However, a consistent significant decrease in glycine concentration was observed in TgSwDI mice compared to agematched non-transgenic mice, independent of age, sex or the brain area tested. Furthermore, in most of the brain samples, aspartate levels were also (significantly) decreased. Levels of other amino acids were unaltered or inconsistently changed in the various brain areas. Conclusions: In TgSwDI mice, glutamate levels remain unchanged, but brain glycine levels, and to a lesser extent aspartate levels, are decreased. This result suggests that these metabolites may be candidate biomarkers reflecting disturbances in glutamatergic neurotransmission.

Enoxaparin improves cognition of APPswe/PS1dE9 and TgSwDI mice with minor effect on amyloid β levels

bapineuzumab reached maximum concentrations within one hour after each infusion in most subjects. At the four doses assessed, the average volume of distribution at steady state, total body clearance, and apparent terminal half-life of bapineuzumab ranged from 49-80 mL/kg, 0.07-0.09 mL/hr/kg, and 20-33 days, respectively. Exposure to serum bapineuzumab in terms of maximum concentration and area under the concentration-time curve (AUC) increased dose proportionally with the average AUC ratios between the third and first infusions ranging from 1.0-1.6. Plasma levels of Ab40 gradually reached maximum after approximately one day, and the exposure to plasma Ab40 increased less than dose proportionally. CSF bapineuzumab concentrations increased with dose (4.5 to 39.3 ng/mL), while the average ratios of CSF to serum bapineuzumab concentrations were consistent at 0.0020.003. No anti-bapineuzumab antibodies were detected in any subject. Conclusions: The pharmacokinetics of bapineuzumab can be characterized by small volume of distribution, slow clearance, and long terminal half-life. Exposure to bapineuzumab was dose proportional at the range observed with minimal-to-modest accumulation per current dosing interval. Plasma levels of Ab40 increased in response to bapineuzumab, reaching maximum approximately one day after each infusion. Consistent ratios of CSF to serum bapineuzumab concentrations indicated dose-independent flow from serum to CSF. The absence of anti-bapineuzumab antibodies indicated a low likelihood of immunogenicity. The observed pharmacokinetic profile of bapineuzumab supports the dose regimen currently tested in phase 3 clinical trials.