AAIC 2017

Joerg Neddens*, Kerstin Beutl*, Stefanie Flunkert, Birgit Hutter-Paier

QPS Austria GmbH, Parkring 12, 8074 Grambach, Austria

*contributed equally

Background:
Alzheimer’s disease (AD) is a neurodegenerative disease characterized by extracellular amyloid plaques and intracellular neurofibrillary tangles. Neuritic plaques often appear in N-terminally truncated forms with a cyclic glutamate residue. pE3 Aβ exhibits cellular toxicity, presents a high self-aggregation predisposition and promotes co-aggregation of non-modified Aβ. The current study is designed to analyze features of AD-related pathological changes in human brain that are spatially associated with pE-Aβ immunoreactive structures, and to verify whether similar changes are present in different animal models of AD.

Methods:
Human paraffin slides of AD patients as well as healthy controls and, cryosections of transgenic mouse models (5xFAD, APPSL) and non-transgenic control mice were labelled by multichannel immunofluorescence to analyze the spatial relations of pE3 Aβ and tau. Therefore, a rabbit polyclonal antibody against pE3 Aβ and a mouse monoclonal antibody (clone AT8) against pSer202/Thr205 tau were used. All samples were digitised and the assessment of immunofluorescent labelling was performed quantitatively by image analysis.

Results:
Depending on genotype and age, mouse models manifest different expressions of pE3 Aβ and tau. 5xFAD mice show a significantly increased pE3 Aβ and tau expression over age, while APPSL mice only show a significant increase of pE3 Aβ at the age of 12 months. Furthermore, expression of pE3 Aβ and tau significantly correlated in both mouse models. Human AD cases showed an increasing pE3 Aβ expression with progressing Braak stages. Expression of tau is only increased in patients of Braak stage V/VI. Additionally, a significant correlation between pE3 Aβ and tau expression in human sections could be observed.

Conclusions:
Correlation between pE3 Aβ and tau expression in human samples as well as in mouse model samples confirms an interdependent expression of pE3 Aβ and tau. Further studies will be required to investigate how these proteins affect each other.

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Joerg Neddens*, Ainara Lopez*, Vera Niederkofler, Stefanie Flunkert, Birgit Hutter-Paier

QPS Austria GmbH, Parkring 12, 8074 Grambach, Austria

*contributed equally

Background:
Alzheimer´s disease (AD) is the most common form of neurodegenerative dementia. Major hallmarks of the disease are extracellular plaque deposits of the β-amyloid peptide (Aβ) and formation of intracellular neurofibrillary tangles, composed of tau protein in the brain tissue. Previous studies have reported the implication of functional impairments of the sensory systems in AD. In fact, retinal structural deficits and visual dysfunctions are often experienced by AD patients providing an opportunity to track this disease in the retina. This study aims to analyze the neuropathological changes occurring both in brain and retina of different AD animal models and address suitable biomarkers for early screening tests for AD.

Methods:
Eyes and brains from various AD animal models (APP rat, TAU rat, TMHT mouse) aged 6 and 12 months are collected. Right eyes and hemibrains are cryosectioned for immunohistochemical labelling, while left eyes and hemibrains are frozen for further biochemical and molecular characterization.

For immunohistochemical analysis, a range of antibodies have been employed to detect and quantitatively analyze different neuropathological markers present in retina, primary and secondary visual cortex (visual system), as well as in the olfactory bulb and piriform cortex (olfactory system).

Results:
AD rodent models show a significant age-dependent shift in neuropathology-related biomarkers when compared to controls, although the results might vary due to the existing biological differences between mice and rats, and retina and brain tissue. In all sections, an overall increase in Aβ and tau protein is expected (Amyloid, Tau). Additional analyses will focus on different neurotransmitters (chAT, GAD67, TH), neuronal cells, glial cells and synapses (NeuN, GFAP, F4/80, PSD95, Piccolo).

Conclusions:
Our results will strengthen the hypothesis that AD manifests in eyes, particularly the retina, and provide evidence for the possibility to track the disease utilizing the appropriate AD biomarkers.

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Barbara Hinteregger^1,2, Tobias Madl², Joerg Neddens¹, Robert Wronski¹, Birgit Hutter-Paier¹

¹QPS Austria GmbH, Parkring 12, 8074 Grambach, Austria
²Medical University Graz, Insitute for Molecular Biology & Biochemstriy, 8010 Graz, Austria

Objectives:
Alzheimer’s Disease (AD) is a severe neurodegenerative disorder and currently affects more than 27 million people worldwide with numbers expected to grow dramatically as the population ages. To investigate Alzheimer-related pathophysiology, several transgenic mouse and rat lines have been established in the recent years. Despite their general applicability in basic and applied research, quantitative tools to monitor pathophysiology as well as associated rewiring of metabolic pathways on a systemic level are lacking. Here we propose to use Nuclear Magnetic Resonance (NMR) spectroscopy-based metabolic phenotyping to provide a quantitative basis for investigation of Alzheimer-related pathophysiology.

Methods:
In this study, we carried out untargeted NMR-based metabolic phenotyping of rat (wild-type, APPsi) and mouse (wild-type, Tg4-42) biofluids and tissues to define a metabolite biomarker panel. NMR is excellently suited to monitor the perturbations in a large pool of metabolites in biofluids and tissues and reflects changes downstream of genomic, transcriptomic and proteomic fluctuations. Therefore, metabolic phenotyping represents an accurate biochemical profile of an organism in health and disease aiding to further understanding of alterations in complex biological networks involved in AD. The measurement of a defined biomarker panel for AD in these transgenic animal models allows for a quantitative read-out of AD pathophysiology which can be exploited in a wide range of applications such as drug evaluation and monitoring.

Results:
We will present, for the first time, a NMR-based metabolite biomarker panel of rat (APPsi) and mouse (Tg4-42) biofluids and tissues. This give further help to understand the devastating neurodegenerative disease, related complex biochemical pathways and pathophysiological processes of AD.

Discussions:
Using NMR-based metabolic phenotyping we defined a quantitative readout of transgenic animal models in the form of a biomarker panel. These biomarkers not only contribute to the understanding of this devastating neurodegenerative disease and the related pathophysiological processes on a systemic level, but set the base for a wide range of biomedical applications. Our approach can be easily extended to other tissues, matrices, or disease models and translated across species since metabolic pathways are conserved through evolution, and are essentially similar in rodents and humans.

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Meritxell Aguiló^1,2, Balázs Dobrovich¹, Joerg Neddens¹, Stefanie Flunkert¹, Nicole Taub¹, Robert Zimmermann², Birgit Hutter-Paier¹

¹QPS Austria GmbH, Parkring 12, 8074 Grambach, Austria
²Institute of Molecular Biosciences, University of Graz, Graz, Austria.

Objectives:
Alzheimer’s disease (AD) is an irreversible neurodegenerative process in which memory functions, intellectual abilities and reasoning skills are progressively lost. AD brains are diagnosed under the co-existence of two main pathological hallmarks: (1) Neurofibrillary tangles (NFT) composed of hyperphosphorylated Tau aggregates, and (2) senile plaques comprising of insoluble β-amyloid (Aβ). Mentioned pathological events do not develop in the same brain region; while NFT production starts in the entorhinal and transentorhinal cortex, Aβ plaque loading begins in the neocortex. Moreover, the onset of both pathologies is not triggered at the same time. Therefore, a mouse model reproducing both spatio-temporal conditions is mandatory in order to obtain trustworthy results in AD preclinical trials.

Methods:
To generate such a model, we virally induced the expression of P301L human Tau, which is the most prevailing mutation in various types of 4R tauopathies, in the entorhinal cortex of the APPSL transgenic mouse by stereotactic injection.

Results:
Biochemical and histological analysis revealed stable expression of P301L Tau in entorhinal cortex and connected brain areas for at least 9 months after injection. Furthermore, besides the high density of senile plaques, several epitopes of aberrant and hyperphosphorylated Tau were characterized in AD-related regions including NFT biomarkers. Ongoing experiments aim to investigate the occurrence of synergistic effects between Aβ and Tau in addition to the behavioral characterization of these mice.

Discussions:
To summarize, we show that our inducible mouse model is a promising new tool which mimics spatio-temporal patterns of AD-related pathology. This model might be valuable to reliably test new compounds, and may potentially help decreasing clinical trial failures.

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Alvarez^1,2, I. Alvarez¹, O. Iglesias², M. Aleixandre³, C. Linares⁴, J. Figueroa^1,5

¹Medinova Institute of Neurosciences, Clinica RehaSalud, A Coruña, Spain.
²Clinical Research Department, QPS Holdings, A Coruña, Spain.
³Faculty of Psychology, University of Granada, Granada, Spain.
⁴Faculty of Medicine, University of Malaga, Malaga, Spain.
⁵Santiago de Compostela University Hospital, Santiago de Compostela, Spain.

Objectives:
To assess severity- and APOE4-related variations in serum vascular endothelial growth factor (VEGF) levels and in its cognitive correlates in patients with Alzheimer disease (AD).

Methods:
Serum VEGF levels and cognitive performance were evaluated in AD, amnestic mild cognitive impairment (MCI) and control subjects. VEGF levels were measured in baseline serum samples by using specific ELISA kits for VEGF¹⁶⁵.

Results:
VEGF levels were higher in AD patients than in MCI cases (p<0.05) and controls (p<0.01), and showed a progressive increase with clinical severity in the whole study population (p<0.001) and in the subgroup of ApoE4 carriers (p<0.05). Increased VEGF levels correlated with impairments of memory, language, praxis and executive function in the total AD sample; whereas in the subgroup of ApoE4 cases showed only significant correlations with the decline of praxis and executive function. These associations seem to reflect the progressive increase with AD severity of both VEGF levels and cognitive deterioration. However, higher VEGF values were associated to better memory and language performance in ApoE4 carriers with moderately-severe AD.

Discussions:
VEGF serum levels are elevated and correlate with cognitive impairment in AD patients; show a significant severity-related increase in ApoE4 carriers; and are associated to better memory and language performance in moderately-severe ApoE4 cases. These findings are pointing to VEGF as a relevant molecular target in AD pathology and therapy; and suggest that increases in VEGF levels might represent an endogenous response driven by pathological factors and could entail cognitive benefits, particularly in AD ApoE4 carriers.