PROJECTS SUPPORTED FOR 2008

Centre de recherche J.P. Aubert – Lille

Mechanisms of Tau degradation and neurodegeneration

SUMMARY OF PROPOSED RESEARCH

Accumulation of abnormally phosphorylated Tau proteins is one of the main pathological hallmarks of degenerating neurons in Alzheimer’s disease (AD). Importantly, compelling evidence indicate that the presence of pathological Tau species is closely correlated to the cognitive decline seen in the disease. However, mechanisms underlying their accumulation are still ill-defined. Dysfunctions of the cellular systems regulating protein quality control could be involved since on one hand these latter are defective during ageing and, on the other hand, a number of arguments suggest their involvement in several neurodegenerative diseases. In this context, our working hypothesis is that pathological Tau accumulation results from a deficiency in protein degradation systems.

The present project intends to evaluate this hypothesis by focusing on 2 specific aims:

1) Unraveling mechanisms involved in Tau degradation. We will first investigate how Tau isoform type, phosphorylation and mutation influence its degradation. Studies will be conducted in different cell lines transfected with appropriate expression vectors. Using pharmacological approaches, we will determine the role of both Ubiquitin proteasomal system (UPS) and autophagy/lysosomal pathway (ALP) in this process. Co-immunoprecipitation experiments will attempt to identify protein chaperons involved in Tau degradation.

2) Validating, in cellulo and in vivo the proteins involved in Tau degradation. Relevance of key chaperons regarding Tau accumulation and neuronal death will be tested by using silencing experiments (siRNA and shRNA tools) in established cell models displaying pathological Tau phosphorylation or expressing FTDP-17 Tau mutations. Implication of regulating chaperons will be also validated using a well-established transgenic mouse model reminiscent to AD-Tau pathology that display an age dependent NFD (Thy-Tau22 line) using longitudinal biochemical and immunohistochemical analyses. This latter study will particularly helps to define whether observed dysfunctions precede or occur consequently to neurodegeneration, helping to establish the link to Tau pathology and neuronal loss. Overall, the proposed project will identify key actors involved in Tau abnormal processing and degradation that could be targeted to modulate neurodegeneration linked to Tau pathology.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Determination of mechanisms involved in Tau degradation by an exhaustive study will provide experimental evidences on the role of protein degradation systems in the accumulation of Tau toxic species and likely to identify key actors. As Tau protein is a central player in neurodegeneration linked to AD, the expected data will improve our knowledge of AD etiopathology and open door to new therapeutic strategies based on regulating some players of protein degradation systems.

November 1st, 2008 – October 31th, 2010 (2 years)

80 000€

Institut Pasteur – Paris

Labelling of amyloid plaques by anti-Aβ single chain antibodies

SUMMARY OF PROPOSED RESEARCH

Amyloid aggregates of Aβ peptide are implicated in the pathology of Alzheimer’s disease (AD). Anti-Aβ monoclonal antibodies have been shown to reduce amyloid plaques in vitro and in animal studies. Consequently passive immunization is being considered for treating AD. However these results rely on IgG passing into the CNS and are in contrast to the notion that the brain is an immune-priviledged compartment, since the Blood Brain barrier restricts passage of certain proteins and immunoglobulins.

Besides tetrameric antibodies, camelids produce homodimeric IgGs devoid of light chains. The VHH variable domain of these IgGs is fully capable of antigen binding. We have previously immunized an alpaca against Amyloid β peptide. Anti-Aβ VHHs were able to recognize specifically intraneuronal Aβ oligomers on human brain slices, they prevented Aβ-induced neurotoxicity and inhibited fibril formation. Thanks to their basic pI, these VHH were able to cross in vitro the Blood Brain barrier.

The objective of this study is to obtain homodimeric antibodies specific of Aβ and able to label in vivo amyloid plaques. These antibodies could be good candidates for immunodiagnostic and on long-term for immunotherapy.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Aβ, a 39-42 residue peptide resulting from the proteolytic processing of a membrane bound β -amyloid precursor protein (APP), is one of the major components of the fibrillar deposits observed in Alzheimer patients. Aβ fibril formation is a complex process which involves changes in Aβ conformation and self-association to form oligomers, protofibrils and in fine fibrils. Since the aggregation of soluble Aβ peptide into fibrils is viewed as a critical event in the pathophysiology of AD, preventing, altering or reversing fibril formation may thus be of therapeutic value.

November 1st, 2008 – October 31th, 2010 (2 years)

23 000€

Hôpital de La Salpêtrière – Laboratoire de Neuropathologie Escourolle – Paris

Dyslipidemia as a risk factor in Alzheimer’s disease

SUMMARY OF PROPOSED RESEARCH

Dysregulation of lipid homeostasis and modified lipid status are key elements in the etiology of Alzheimer’s disease (AD) that are influenced to a significant manner by the intake of dietary lipids. We have in our laboratory a novel mouse model for dyslipidemia, in which the animals display a reduced expression of the lipolysis stimulated receptor, LSR (LSR^-/+), a hepatic receptor for chylomicrons, the lipoproteins that transport dietary lipids to the different tissues. LSR^-/+ animals display a significantly lower capacity for the processing of dietary lipids. In this dyslipemia model, preliminary studies of different brain structures revealed a lower ratio of the omega 3 polyunsaturated fatty acid (PUFA), docohexaenoic acid (DHA) versus the omega 6 PUFA, arachidonic acid in the hippocampus and cortex of LSR^-/+ animals on an LDL-receptor^-/- background, as compared to LDL-R^-/- controls. Dietary omega 3 polyunsaturated fatty acids are important determinants in minimizing the predisposition towards Alzheimer’s disease. Pilot studies performed in our laboratory suggest that LSR^-/+ exhibit a potential problem in short-term memory. Our current working hypothesis is that disruption of lipid homeostasis can lead to a modification of lipid status with a significant impact on normal neuronal function and predisposition towards neurodegeneration and ultimately Alzheimer’s disease. The objective of this research project is to study the relationship between dyslipidemia and changes in cognitive capacity using the LSR^-/+ animal model. Combined methodologies using whole animal studies of cognitive capacity for short-term and spatial memory as well as in vitro studies using isolated synaptosomes will allow us to relate changes in lipid status in the CNS with behavioral modifications. A nutritional approach using diets supplemented with omega 3 PUFA and DHA in particular, will be used in order to attempt to ‘rescue’ the CNS and re-establish the lipid homeostasis necessary for normal neuronal function. Indeed, preliminary studies indicate that DHA is able to increase LSR activity, suggesting that LSR expression is amenable to regulation by dietary lipid composition providing a new strategy towards preventing AD development.

The specific aims of this project are the following:

1. To determine if dyslipidemic LSR mice present cognitive impairments correlated with an altered lipid status and metabolism.
2. To explore structural and functional synaptic markers and investigate the potential link between these parameters and lipid homeostasis alteration in LSR mice.
3. To validate potential nutritional approaches aimed towards prevention of AD.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

The proposed research focuses on the molecular mechanisms involved in maintaining lipid homeostasis in the central nervous system with the aim in identifying the key regulatory pathways that promote normal neuronal function. A novel mouse model of dyslipidemia that has been recently characterized in our laboratory will be used to study the relationship between disrupted lipid homeostasis, modified lipid status and cognitive capacity. The results obtained will allow us to identify novel nutritionally-based strategies for the maintenance of proper lipid homeostasis, most particularly during the aging process, in order to prevent or slow down the detrimental cellular processes that predispose towards neurodegeneration and Alzheimer’s disease.

November 1st, 2008 – October 31th, 2010 (2 years)

80 000€

CNRS, Ecole Supérieur de Physique et Chimie de Paris – Paris

Aneuploidies in the brain of AD patients (ANEUAD)

SUMMARY OF PROPOSED RESEARCH

Differentiated mature neurons continuously hold their cell cycle in check to avoid re-entry in a mitotic process. However the system which controls the neuronal cell cycle is jeopardized by some neurodegenerative diseases most noticeably by Alzheimer disease (AD). The re-entry of neurons in the cell cycle constitutes the basis of the “mitotic hypothesis” of AD pathogenesis. Re-activation of cell division-like phenomena could explain the co-occurrence of amyloid deposits and of neurofibrillary tangles, thus providing the relationship between tau and amyloid β(Aβ) pathology. On the other hand, studies performed on lymphocytes of AD patients show that they are more often aneuploid.

We want to test the hypothesis that specific aneuploidies that increase the copy number of the APP gene in a significant proportion of neurons are regularly present in AD brains and could explain the over-expression of APP and the increase of Aβ production. In order to test this hypothesis we propose to use Comparative Genomic Hybridization (CGH-array) on genomic DNA extracted from AD brains. We will systematically look for amplification and/or deletion of chromosomal segments, hoping to detect duplication of the APP chromosomal region in a significant number of cases. This pilot study also aims at testing the feasibility of CGH-array in post-mortem brains and the sensitivity of the technique.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

The proposed pilot research is relevant for determining the cause of AD. It will determine weather specific aneuploidies (particularly in the chromosomal segment including the APP gene) are found in a significant proportion of neurons in the brains of AD patients.

November 1st, 2008 – October 31th, 2009 (1 year)

32 500€