Huntingtin as a regulator of APP/Abeta trafficking in Alzheimer's disease – HuntAbeta

1) We will study the contribution of transport regulator in axonal trafficking of APP. We will use state of the art imaging techniques and microfluidic devices that discriminate axons, dendrites and cell bodies of neurons. Neurons from mice which are mutants for transport regulators will be seeded into this microfluidic devices.APP axonal transport will be assessed by confocal videomicroscopy.

2) We will analyze the contribution of transport regulators on synapse formation/maintenance and Abeta production using co-cultures in microfluidic chambers. Abeta production will be quantified by ultrasensitive ELISA dosage on supernatants of neurons.

3) We will investigate the consequences of transport regulation on Abeta production in vivo by crossing AD mice (TgAPPswe,PS1L166P) with mice inducibly deleted for transport regulator. We will then investigate these mice at the neuropathological and behavioral levels using cognitive tests. Abeta production, amyloid formation and synaptic markers will be assessed.

Microfluidic devices have been developed to visualize axons and synapses. These microchambers were used to investigate the APP trafficking and the dosage of Abeta in mutant axons and at the synapses of these neurons.
Experiments using cortical neurons transduced with fluorescent APP lentivirus and seeded into microfluidic devices permit to track APP transport in the distal part of the axons, from 5 days in vitro to 21 days later. APP-containing vesicles present a fast axonal transport as previously observed with neurons transfected with APP. After 11 days in vitro, APP vesicle velocity is modified into axons of neurons that are mutant for transport regulator. Moreover the production of Abeta is also modified. These results suggest that regulation APP axonal trafficking could modulate Abeta production.

The main outstanding feature of this project is to identify new therapeutic targets for Alzheimer's disease.

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Alzheimer’s disease (AD) is characterized by the abnormal trafficking and processing of the Amyloid Precursor Protein, APP whose function is still poorly understood. As APP is the precursor of Aß whose levels are crucial for AD, APP processing has been extensively studied. However, little is known on how APP is trafficked from the cell bodies to the synapses. In particular, what are the mechanisms that control APP transport in axons and dendrites? What are their impacts on Aß production and synapse maintenance? Can we reduce Aß production in AD models by modulating APP transport?

Our laboratory as well as other groups have identified huntingtin (htt) -the protein that when mutated causes Huntington’s disease (HD)- as a key protein that regulates intracellular transport (Gauthier et al. Cell 2004). We found that htt regulates the transport of APP and that htt phosphorylation at Serine 421 dictates the directionality of its transport within axons. (Colin et al. EMBO J 2008). Our expertise in htt function in axonal transport, our know-how in the study of molecular mechanisms of transport within neurons and their alterations in disease, as well as the analysis of the consequences on neuronal function and survival, put us in a privileged position to study the influence of htt on APP trafficking in health and AD.

1) We will study the contribution of htt in axonal transport of APP. We will use state of the art imaging techniques and microfluidic devices to investigate the role of htt and its phosphorylation on the axonal and dendritic transport of APP in hippocampal neurons. We will analyze the contribution of htt and its phosphorylation on synapse formation/maintenance and Abeta production using co-cultures in microfluidic chambers.

2) Huntingtin is also known to regulate endocytosis. We will investigate whether htt and its phosphorylation could impact on APP endocytosis and processing. We will study the role of htt on APP endocytosis and its association with secretases.

3) We will investigate the consequences of htt expression and phosphorylation on Abeta production in vivo by crossing AD mice (TgAPPswe,PSEN1DE9) with mice inducibly deleted for htt in the hippocampus (showing reduced transport) as well as with htt mice HdhS421A and HdhS421D mice. We will then investigate these mice at the neuropathological and behavioral levels using cognitive tests. Abeta production, amyloid formation and synaptic markers will be assessed.

We believe this proposal will lead to a better knowledge of the cellular and molecular mechanisms that control APP trafficking from intracellular compartments to the plasma membranes at synapses and, from the membrane to endocytic compartments. These approaches could therefore reveal new mechanisms for the regulation of Aß production and identify potential therapeutic strategies. Finally, our work may also reveal an unexpected link between AD and HD and open new research avenues on neurodegenerative processes.