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Les astrocytes et la maladie de Alzheimer

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Background & general objectives
Understanding the molecular mechanisms underlying the pathological manifestations and progression of Alzheimer’s disease (AD) is crucial for developing effective, targeted therapies for those afflicted with this devastating disease. While much progress has been made, there remain significant questions regarding the generalizability of genetic risk factors and the potential efficacy of current therapeutic targets. There is a great need for the identification of novel therapeutic targets to prevent early disruptions. Deepened understanding of the early effects of Aβ will direct the search for novel genetic and environmental risk factors. Additionally, targeting a mechanism impacted at an early stage of the disease has great potential to reduce the need for long-term therapeutic intervention, thereby increasing quality of life for AD patients and reducing societal burden simultaneously. The goal of our proposal is to better understand one such early disruption : amyloid-beta (Aβ) mediated impairment of postsynaptic dendritic spines and its consequences on subsequent functional alterations of the synapses. Our proposed study is designed to address whether and how Aβ oligomers disrupt the morphology and function of neurons and glia.

Astrocytes implication in the synaptic toxicity observed in the early AD.

Recent studies suggest that astrocytes may play a major role in synaptic dysfunction (Henneberger and Rusakov, 2010). Indeed, astrocytic processes invade the heart of most of synapses and actively contribute to morphological and functional changes in synaptic transmission (Perea et al., 2009). This contribution includes fine regulation of neurotransmitter content in the synapse through mechanisms of uptake together with the release of gliotransmitters that modulate synaptic transmission. Astrocytes, unlike neurons, do not possess the ability to communicate each other via electrical phenomena. They possess their own excitability, based on intracellular calcium movements (Henneberger and Rusakov, 2010). These calcium signals, slow and compartmentalized, superimpose and modulate the fast neuronal transmission.
In this context, we aim at investigate the astrocyte-neuron communication nearby synapse. Development of new highly-resolutive tools will allow us to study the involvement of astrocytes in the phenomenon of synaptotoxicity observed in the early stages of Alzheimer’s disease. These functional adaptations will be correlated to structural plasticity known to affect astrocyte at the level of tripartite synapse (Haber et al., 2006).

By combining structural, electrophysiological (single or dual patch-clamp) and confocal calcium imaging approaches we want to investigate :
- The astrocytes contribution to the functional and structural deleterious effects of Aβ on dendritic spine and the molecular actors involved in this partnership in pathological conditions.

The light shit experiments gave us the opportunity to further investigate the astrocytes morphological changes in a transgenic animal model of AD. For this purpose, we used clarified tissue samples.

Passive Clarity protocol :

1.- Prepare the hydrogel monomer solution

40% Acrylamide 19.8 mL (final concentration : 4% Hydrogel network monomer)
TMED 60 microL
APS 300 microL
32% Paraformaldehyde 18.75 mL (final concentration : 4%)
10X PBS 15 mL (final concentration:1X Salt buffer)
Deionized water 96 mL - Aqueous solvent

2.- 12 month-old mice were perfused transcardially perfusion with 15 ml PBS and 50 ml of the hydrogel solution. Extract the brain from the skull. We stored the sample in 15 mL hydrogel solution at 4°C shaking during 2 days.

3.-Add TMED 40 microL and APS 15 microL. De-gas the sample container adding mineral oil the top of the tube to remove oxygen from inhibiting polymerization. Incubate the sample at 37°C for about 3 hours to polymerize and crosslink the hydrogel matrix.

4.- Extract the brain and remove excess gel from the sample surface and immediatelly cut the brain in 3 mm blocks or using a vibratom 200-400 micrometres slices.

5.- Place sample in 50 mL of clearing solution
Sodium dodecyl sulfate 40 g (final concentration:4%, Lipid clearing surfactant)
Boric acid 12.366 g (final concentration : 200 mM, pH buffer)
Sodium hydroxide To pH 8.5 - pH adjustment ions
Deionized water Fill to 1 L - Aqueous solvent

6.- Incubate sample in clearing solution overnight to wash out excess unreacted monomers from the brain (37°C or room temperature, shaking preferable). Replace 50 mL clearing solution and continue incubation at 37°C or room temperature Note : Washes from the first 2 days contain toxic hydrogel monomers and must be disposed of as waste. Afterwards, clearing solution is safe for sink disposal.

7.- Repeat clearing solution washing everyday during one month until the tissue becomes translucid. The tissue will clear slowly from passive diffusion of SDS micelles. The duration of passive clearing is dependent on factors such as the size/thickness of the tissue sample and the incubation temperature. Samples can be stored indefinitely in the clearing solution.

8.- Immunostaining : Place sample in PBST (0.1% TritonX in 1X PBS) After that, the samples were incubated in a wet chamber in PBST 1% BSA during 24h

9.- Then, they were incubated in a solution PBST 0,03% BSA of primary antibody (29 microg/mL, anti GFAP on Rabbit, DAKO) for 48h at room temperature

10.- Wash sample with buffer 24h

11.-Incubate sample 48h in secondary antibody (15 microg/mL, anti rabbit Cy3, Jackson immunoreserch) solution at room temperature with shaking protected of the light.

12.-Wash sample with buffer 24h

13.-Mount and observe

The images (single slice or stacks) below, at various scale, are encouraging preliminary results, STED-SPIM will be the following step.

Z projection of a 100 µm thick (2mmX2mm) block of mouse brain
Clarified block of mouse brain immuno stained anti-GFAP showing amyloïd plaques (bright zones).
3D from a 100 µm thick Stack of mouse cortex
Astrocytes are antiGFAP / Cy3 stained. Fluorescence is excited by the 532 nm beam alone. The stack dimensions are : 140X100X100 µm3 in XYZ
The sample is cleared and embedded in a pluronic gel.
Astrocytes in a mouse brain block
The 3D animation comes from a stack of 100, 1µm thick optical slices (600µm X 600µm) in a block of a mouse brain embedded in a pluronic gel. The astrocytes are anti-GFAP/CY3 immunostained.