PROJECTS SUPPORTED FOR 2007

INSERM, Institut Pasteur – Lille

Association of Collagen genes with Alzheimer’s disease ?

SUMMARY OF PROPOSED RESEARCH

The genetic of the early-onset forms of Alzheimer’s disease (AD) begins to be well known conversely to that one of the late-onset forms, which appears to be highly more complex. Indeed, the number of genetic factors and the variability of their impact in these late-onset forms clearly complicate their identification. To date, the only recognized risk factor for these forms is the e4 allele of the apolipoprotein E (APOE) gene.

We have brought into the fore that functional polymorphisms within the APOE promoter are associated with an increased risk of developing AD. These data indicate that in addition to the qualitative effect of coding polymorphisms (such as the e4 one) on the AD occurrence, the quantitative variation of expression of a gene (for instance, due to promoter polymorphisms) contribute to the development of the disease. This observation have been extended to other genes implicated in AD such as PS1 and PS2 and for which variations in their expression in the brain of AD cases and controls have been observed. Altogether, these data suggest that the level and the control of expression of involved genes in the AD process may be a possibility of screening to research for new genetic determinants of AD.

From this working hypothesis, we developed a custom microarray in order to study the expression of all the ORFs (open reading frame) located within the chromosomal regions of interest defined by the genome scan studies. A bio-analysis work have been realised to finally keep 2741 ORFs located within 9 loci of interest. We selected for their quality, 12 AD and 12 control samples of total RNA extracted from post-mortem brain tissues and we performed transcriptomic experiments. After computational analysis, we finally identified 106 significantly differentially expressed genes. We already analysed the impact of the ornithine transcarbamylase gene and obtained promising results.

With this background, we propose to evaluate the role of single nucleotide polymorphisms (SNPs) and resulting haplotypes in 4 other highly differentially expressed and positional candidate genes (COL6A1, COL6A2, COL11A1 and COL11A2) with the risk of AD in a collaborative effort.

This project is built to use the state-of-art techniques to identify AD genes : (i) validation of the differential expression in AD brains compared with controls at both mRNA and protein levels; (ii) characterisation of SNPs associated with the risk of developing AD in several independent case-control studies, (ii) evaluation of the functionality of the SNPs yielding significant associations.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

This project is designed for a better understanding of the pathological process of AD by the investigation of its genetic determinants. These genetic factors could be used as new pharmacological targets or be useful for diagnosis.

November 1st, 2007 – October 31th, 2008 (1 year)

39 625€

Institut National Polytechnique de Lorraine, Lipidomix – Nancy

Functional validation of a therapeutic strategy for Alzheimer’s disease

SUMMARY OF PROPOSED RESEARCH

Accumulative evidence emphasizes the key causative role of soluble oligomers of Aβ peptide (sAβ) in neuronal degeneration and early memory loss associated with early AD stages. Therefore, we presume that preventing sAβ toxic effects to neurons might represent a high priority in designing efficient neuroprotective approaches in the treatment of AD. Most of the strategies explored or under evaluation to date propose either to modulate Aβ generation or to increase Aβ clearance from the central nervous system. The project we present here relies on a distinct reasoning, considering that sAβ-induced neurodegeneration and associated synapse loss can be prevented by counteracting sAβ neurotoxic effects through the preservation of active survival (antiapoptotic) signaling pathways in neurons. Accordingly, we have recently observed that humanin (HN), an endogenous 24-aa peptide, protected cerebral cell models from sAβ in vitro, as well as sAβ-induced cognitive impairment in vivo.

Our main goal is to investigate the functional interest of delivering continuously humanin peptides in vivo, in order to prevent sAβ-induced neuronal damages as well as associated neurobehavioral injuries and cognitive impairments in AD mice models. To guaranty long-term administration as well as high local concentrations of HN peptide in particular brain structures including hippocampus, we have chosen to use recombinant cells stably expressing and secreting the therapeutic agent, followed by their encapsulation in alginate beads and selective implantation in mouse brain. In situ secretion of these factors from alginate-embedded cells should provide protection from sAβ-induced toxicity.

Our specific objectives are as follow:

1 – To optimize the implantation of alginate beads, containing recombinant cells designed to stably secrete humanin, in the brain of different mice models of AD (e.g. in AD Tg2576 mice and in stereotaxically sAβ-injected mice) – and to monitor humanin production and diffusion,

2 – To characterize the effects of humanin brain production on both cellular and tissular injuries associated with AD mice models, as well as on the cognitive performances of implanted mice.

While emphasizing the interest of in situ production of recombinant proteins in brain from cells encapsulated in transplanted alginate beads, this project will allow establishing the proof of concept that in situ-delivered humanin peptide exhibits neuroprotective capacities able to prevent sAβ-mediated synaptic loss and subsequent cognitive deficit. It is expected to lead to the development of a new therapeutic approach to prevent or to delay the burst of neuronal degeneration responsible for AD. By assessing the usefulness of these treatments in AD mice models exposed to sAβ, this project is expected to confirm the pertinence of the relevant pathways as pharma­cological targets in preventive therapies raised against AD. It should also functionally assess the interest of such neurosurgical approach for in situ delivery of other AD therapeutic proteins.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

Accumulative evidence highlights the critical role played by soluble oligomers of the Aβ peptide in precocious stages of AD development. It is thus crucial to characterize, both in vitro and in vivo, the molecular mechanisms associated with soluble Aβ oligomer-induced neurodegenerative processes in order to identify cellular targets for soluble Aβ as well as factors able to prevent soluble Aβ-induced brain damage and subsequent cognitive impairment. Indeed, the development of therapeutic strategy to treat and/or prevent AD should take soluble Aβ oligomers into account.

In this context and based on our preliminary data, we proposed to make the proof of concept of a potential new therapeutic approach for AD based on continuous production of a neuroprotective factor in the brain of different mice models of AD. This will also allow us to determine whether such approach can be applied for in situ delivery of other therapeutic proteins already identified in our group.

November 1st, 2007 – October 31th, 2008 (1 year)

40 000€

Hôpital de La Salpêtrière – Laboratoire de Neuropathologie Escourolle – Paris

Lipids associated with Aβ peptide

SUMMARY OF PROPOSED RESEARCH

Alzheimer disease (AD) is characterized pathologically by the extracellular accumulation of Aβ peptide and the intracellular accumulation of tau protein. The proteolytic cleavages of APP by β- and g- secretase activities have been thoroughly investigated. We want to focus our research on the mechanisms that allow Aβ to reach the extracellular space once it has been cleaved from APP. The Aβ peptide is, indeed, for one third of its sequence, hydrophobic – and therefore interacts with lipids. Lipids need, on the other hand, transporters, such as apolipoproteins, to be solubilized. We will study lipids and apolipoproteins on tissue sections from AD patients and APPxPS1 transgenic mice. The structure of the senile plaque will be studied with the classical staining methods such as bromine Sudan black B, oil red O (esterified cholesterol), filipin (free cholesterol), Nile red and DiI. We will also use theta-toxin (kindly provided by Sanofi-Aventis), a probe that recognizes free cholesterol in membranes or, after fixation, in the cytoplasm. The fluorescent B subunit of the cholera toxin will be used to label GM1. These classical and morphological methods will be completed by more analytical techniques: we have started to micro-dissect senile plaques on post-mortem frozen samples and on transgenic mice tissue sections. The extracted lipids will be analysed by electrospray mass spectrometry (in collaboration with the Proteomics Platform of the Ecole Supérieure de Physique Chimie Industrielle de la ville de Paris).

The work on the tissue sections will be completed by a study of the secretion of Aβ in an APP transfected SY-5Y cell line (kind gift of Luc Buée, Lille). Our preliminary results have shown that Aβ is recovered, after ultracentrifugation, in a light fraction – indicating that it is associated with lipids and, possibly, apolipoproteins in a complex of high buoyancy. We want to determine the density at which this Aβ complex (which according to our preliminary results may correspond to particles or vesicles) may be isolated. We will study this Aβ complex with electron microscopy (EM) after having isolated it on copper grids covered by Formvar. Immuno EM will enable us to visualize Aβ, apolipoproteins and markers (flotillin, membrane receptors). We will also use standard proteomics methods to study the content in proteins of the isolated fraction and use the Mass Spectrometry to analyze their lipid content. We believe that the study of the mechanisms implicated in the last step of Aβ secretion, by which already cleaved Aβ peptide reaches the extracellular milieu, could open new therapeutic perspectives.

RELEVANCE OF PROPOSED RESEARCH TO ALZHEIMER’S DISEASE

The increase in Aβ secretion and the change in the Aβ42/Aβ40 ratio have been well documented in the rare familial cases of AD. No change in APP metabolism has been evidenced in the common sporadic cases. We formulate the hypothesis that, in these cases, it is the last step of Aβ secretion, which extracts the cleaved Aβ from the cell membrane into the extracellular space, that is altered. We postulate that this step involves both lipids and lipid transporter and believe that a better understanding of the mechanisms will open new way to interact with Aβ secretion leading to the formation of senile plaques.

November 1st, 2007 – October 31th, 2009 (2 years)

80 000€