PROJECTS SUPPORTED FOR 2010

Université Bordeaux II – Bordeaux

Nutrition and neuroimaging markers of Alzheimer’s Disease

SUMMARY OF PROPOSED RESEARCH

Late-onset Alzheimer’s Disease (AD) is a multi-factorial disease resulting from the effect of aging and a complex interaction of both genetic and environmental risk factors. In the absence of etiologic treatment for AD, the identification of modifiable environmental factors that could slow down cognitive decline preceding dementia or AD, such as nutritional factors, has aroused increasing interest. While fundamental research has suggested a protective role of several nutrients, such as polyunsaturated fatty acids, anti-oxidant micro-nutrients and folates against AD, randomized clinical trials using nutritional supplements have been disappointing to date. Several reasons may be proposed. First, the protective nutrient(s) may not have been accurately targeted. Dietary patterns, which represent a combination of foods, may be better associated to disease risk than single foods or nutrients. Second, the protective mechanism of selected nutrients, such as long-chain omega-3 fatty acids (n-3 PUFA), may have not been well identified. Advances in neuroimaging now allow preclinical detection of atrophy and alterations of microstructure integrity in the brain. The identification of Magnetic Resonance Imaging (MRI) markers associated with nutritional factors would enable to evidence morphological mechanisms supporting their role in AD. The main objective is therefore to study the association between nutritional factors and MRI morphological markers of AD. Nutritional factors will be studied at two levels: (a) at the global diet level, by identifying dietary patterns; (b) at the biomarker nutrient level, with a particular emphasis on long-chain n-3 PUFA measured in plasma. MRI markers will be studied at two levels: (a) at the macroscopic morphological level, by studying MRI markers of brain atrophy and small-vessel disease; (b) at the microscopic morphological level, by studying brain grey matter and white matter microstructure integrity. The subjects are from the Bordeaux sample (n=2104) of the Three-City (3C) study, an epidemiological population-based cohort which started in 1999-2000 to identify vascular risk factors of dementia. MRI were performed at baseline and at 4-yr follow-up. The 10-yr MRI acquisition is currently starting. In this project, the principal investigator will be in charge of the study of the relationships between baseline plasma long-chain n-3 PUFA or patterns of nutritional data and MRI markers of AD assessed at baseline, 4y or 10y (grey matter, hippocampal and white matter lesions volumes, total fractional anisotropy and apparent diffusion coefficient maps in white or grey matter).

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Overall, this project would enable to explore morphological mechanisms of a potential preventive role of nutritional factors in AD, using new neuroimaging techniques which enable to detect brain alterations in the preclinical phase of AD. To our knowledge, the relationship between long-chain n-3 PUFA and MRI markers of AD has never been explored, which may place this project at a high international level of innovation.

November 1st, 2010 – October 31th, 2012 (2 years)

40 000€

INSERM, Faculté de médecine – Strasbourg

Epigenetics in AD-related cognitive impairments

SUMMARY OF PROPOSED RESEARCH

Alzheimer’s Disease (AD) is a neurodegenerative disease which, besides its two histopathological hallmarks (amyloid plaques and tauopathy), is also characterized by neuronal loss and memory impairments. This project is based on the recent evidence, including ours, that epigenetic regulations are dynamically implicated in higher brain functions such as plasticity and memory formation. We will focus on epigenetic regulations implicating histone acetylation-related processes. Histone acetyltransferases (HATs) act on gene expression by changing chromatin structure at specific promoters. It was recently shown that HAT activity is a critical component for hippocampus-dependent long-term memory. A primary Alzheimer’s disease symptom, namely the impairment of declarative memory, can be modeled in rodents by subjecting animals to a hippocampal-dependent task in which the animals have to establish a spatial memory. Our latest experiments in normal young adult rats, point to several original findings showing that, not only the expression of different HATs (CBP, p300 and PCAF) is induced as spatial memory forms and consolidates, but their activation converge to the epigenetic tagging of the genome on specific histones in physiological conditions. In this project, we propose to extent these recent findings to the physiopathology of Alzheimer’s disease by using two mutated mice strains bearing AD-related mutations (APPxPS and Tau). We have preliminary data suggesting that the CBP-dependent pathway, as well as histone acetylation, are altered in an APP- and Tau-dependent pathological context. We will ask the following questions : 1) To which extend these epigenetic modifications are altered during spatial memory formation in Alzheimer’s disease transgenic mouse models? 2) Is it possible to counteract these regulations and enhance memory functions with epigenetic modulators (HDAC inhibitors and HAT activators)? Molecules inducing a histone hyper-acetylated state, like HDAC inhibitors potentiate memory formation in normal and HAT-deficient mice. Recently, the HDAC2 isoform has been linked to memory functions and we can test more specific inhibitors than those previously tested. Moreover, we have an ongoing collaboration with Dr. Kundu (Bangalore, India), who has developped the only known small-molecule activator of HAT. By linking it to carbon nanospheres, it is able to cross the blood-brain barrier and acetylate brain histones after a systemic injection. We will test whether such molecules display promnesic functions in vivo. Experiments require classical biochemical techniques such as western-blot, RT-qPCR, as well as behavioral studies leaded on transgenic mouse models.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

November 1st, 2010 – October 31th, 2012 (2 years)

79 340€

Université de Poitiers, GReVIC – Poitiers

Links between inflammation and autophagy in Alzheimer

SUMMARY OF PROPOSED RESEARCH

Alterations in autophagy and inflammation occur at the early stages of Alzheimer’s disease (AD). Autophagy is a major system for protein and organelle degradation in the lysosomes. Inflammation is an immune process to eliminate any foreign substance from the host. Several data indicate that autophagy is altered in AD, with the accumulation of autophagic vacuoles (AVs). In addition, autophagy has been shown to be connected to inflammation in peripheral tissues. Both autophagy and inflammation are currently recognized to interfere with the amyloidogenic pathway, switching the cleavage of amyloid peptide precursor (APP) to β-amyloid peptide (Aβ) which is the major component of senile plaques in AD. However, the links between autophagy and inflammation in AD remain to be discovered. Therefore, it will be interesting to study these links at the central level to bring a new therapeutic strategy limiting the progression of this dementia. The specific aims of our project are (1) study inflammatory responses in vitro and in vivo models where autophagy is suppressed and (2) analyze autophagic responses in vitro and in vivo models under inflammatory conditions. Co-cultures of neurons/astrocytes/microglia prepared from neural-cell-specific Atg5^-/- mice (Atg5^flox/flox/nestin-Cre) or wild-type mice will be exposed to an inflammatory stress (LPS or Aβ42). Groups of these mice will be also treated or not with LPS in order to examine the inflammatory responses in vivo under cerebral autophagy KO. Inflammatory factors will be downregulated either by siRNA gene silencing or by the treatment with the C16 compound, a specific inhibitor of PKR involved in inflammation. Autophagy monitoring will be investigated in the double APPswePS1dE9 transgenic mouse model of AD treated or not with the C16 compound after 3, 9 and 15 months of treatment. Beclin-1, p62/sequestosome-1 and the conversion of MAP-LC3-I to MAP-LC3-II will be used as markers for autophagy. The number of autophagosomes/cells will be determined by counting LC3-brightly fluorescent dots in GFP-LC3#53 mice and electron microscopy will be investigated for the ultratructure of AVs and lysosomes. Inflammatory factors (interleukins, chemokines, NF-κB) will be measured by Luminex® X-map^® technology and Western blot. The APP metabolism will be investigated by measuring secretase activities and by detection of specific APP fragments by western blot. Apoptosis will be assessed by measuring caspase activities and TUNEL procedure. This work will allow to elucidate the crosstalks between autophagy and inflammation in AD and to elaborate new therapeutic strategies aimed at modulating autophagy and/or inflammation.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Increasing evidences from studies in human and different models indicate that alterations in autophagy and inflammation may contribute to the development of AD-like neuropathology hallmarks, namely tau and amyloid aggregations and neurodegeneration. Clear-cut links between autophagy and inflammation were recently established in peripheral tissues (liver and intestine). Targeting inflammation in AD is a well recognized therapeutic strategy. Since autophagy constitutes a defense mechanism, it was proposed that its induction might represent a therapeutic tool to cope with abnormal protein accumulation and neuronal death. Study of the possible links between autophagy and inflammation in brain, especially in AD-like pathological conditions, will allow the elaboration of novel and original therapeutic strategies based on the inhibition of inflammation and/or the activation of autophagy.

November 1st, 2010 – October 31th, 2012 (2 years)

80 000€

CNRS, Aix Marseille II – Marseille

MMPs and TIMPs in Alzheimer’s disease pathogenesis 

SUMMARY OF PROPOSED RESEARCH

The prevailing view of Alzheimer’s disease (AD) pathogenesis posits that accumulation of neurotoxic beta-amyloid peptide (Aβ), resulting from an imbalance between its generation and degradation, is the central event triggering neurodegeneration. Aβ results from the sequential cleavage of its transmembrane beta amyloid precursor protein (βAPP) by β and g secretases. Alternatively, the βAPP can be cleaved by a-secretases to generate a neuroprotective soluble fragment (sAPPa, precluding the production of Aβ) Recent evidence suggest that adamalysins (A Disintegrin And Metalloprotease, ADAM) are the physiological a-secretases and matrix metalloproteinases (MMPs) degrade the oligomeric and fibrillar forms of Aβ. Both, ADAMs and MMPs are inhibited by the endogenous tissue inhibitors of metalloproteinases (TIMPs), which are upregulated in amyloid plaques and in the cerebrospinal fluid of AD patients. Our working hypothesis is that the accumulation of TIMPs in the AD brain impairs metalloproteinase activity and favours the accumulation of Aβ. Three laboratories have joined forces to test this hypothesis in the context of ongoing collaborations. The objectives are to investigate: 1) How the metalloproteinase/TIMP system affects the sAPPa/Aβ balance and distribution and how in turn Aβ affects the expression/activity of metalloproteinases and TIMPs; 2) How the modulation of metalloproteinases by TIMPs influences synaptotoxicity, inflammation, neurodegeneration and learning behaviour. In order to achieve these goals, the partners in the consortium will share interdisciplinary skills in cell and molecular biology, biochemistry, cell imaging and animal behaviour. We will use cell lines that carry the βAPPsw mutation and organotypic cultures of hippocampal slices from 5XFAD transgenic mice carrying the same mutation and known to accumulate high levels of Aβ and exhibit learning impairment much earlier than any other transgenic mice. In all cases, we will specifically modulate the activity of different metalloproteinases using pharmacological and molecular tools (ie, recombinant WT and mutated inactive TIMPs) to ascertain the importance of the interplay between the metalloproteinase/TIMP system and their putative substrates βAPP/Aβ in the pathogenesis of AD and learning impairment.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

We expect to improve our basic knowledge and provide new insights on the molecular and cellular mechanisms of Aβ accumulation/clearance and the impact on the pathogenesis of AD. By studying the influence of Aβ in the organisation of the cytoskeleton we expect to provide new evidences of neurotoxic events upstream of plaque formation that affect synaptic plasticity, and learning behaviour in the end. The main originality and novelty of our project lies on the study of endogenous inhibitors of metalloproteinases as factors that contribute to AD through the control of Aβ degrading metalloproteinases. We expect to identify and validate new targets (the TIMPs and/or the MMPs) and molecular tools (recombinant fluorescent TIMPs) that pave the way to design both medical imaging probes for the analysis and the detection of metalloproteinase activities in the diseased brain, and novel therapeutic strategies in AD via the modulation of metalloproteinase activity.

November 1st, 2010 – October 31th, 2012 (2 years)

75 000€