Project Summary


Background


Parkinson Disease (PD) is the second most common progressive neurodegenerative disorder, affecting 1-2% of all individuals above the age of 65. The selective degeneration of subsets of midbrain dopaminergic neurons is believed to be the primary cause for disruption of the ability to control movements. Meaningful hypothesis on the causes of PD and the design of new therapeutics must consider reasons and mechanisms of the selective vulnerability of mesencephalic dopaminergic neurons.


Objective


We propose to apply a highly interdisciplinary approach to construct complex networks consisting of protein coding genes, non-protein-coding genes and cis-regulatory elements within dopaminergic neurons in the brain. This approach will be applied across four chordate organisms (Human, Mouse, Zebrafish and Ciona intestinalis) to identify core network modules that might play key roles in the biology of these neurons as well as differences that might be relevant to the utilization of these organisms as models of PD.


The project will utilize a set of complementary techniques:
  • High throughput expression profiling of genes on single subtypes of dopaminergic neurons via laser microdissection and transgenic lines in Mouse, Ciona and Zebrafish
  • Next generation sequencing of microCAGE assays on dopaminergic neurons, providing quantitative data on transcription start site (TSS) usage as well as a powerful platform for transcript discovery
  • High throughput automated microscopy screening of cis-regulatory elements complemented by co-localization assays of coding and non-coding elements siRNA network perturbation experiments
  • Innovative bioinformatics and systems biology approaches to decipher and define molecular networks (including enhancers, and non-coding RNAs) at play in dopaminergic neurons


The project will be divided in two phases. In the first phase, the focus will be on the generation of expression profiles of dopaminergic neurons based on traditional microarray platforms as well as on innovative microCAGE (CAGE: cap-analysis gene expression) approach. This will be followed by a data integration and data analysis step. The second phase will focus on a subset of coding and non-coding genes chosen in phase 1. These genes will be analyzed by performing siRNA perturbation experiments followed by renewed expression profiling (microarray and CAGE), high throughput microscopy for each siRNA aimed at visualizing the effect of the siRNA, co-localization microscopy assays to determine their relationship with transcription factors (TFs) and other genes identified in the screen and functional screening of cis-regulatory elements predicted in the loci of these transcripts for enhancer function. Finally, all these data will be integrated and models will be derived to decipher the networks at play within dopaminergic neurons. This project relies also on the availability of the data produced until now through the many existing collaborations among consortium partners, such as conserved chordate enhancers and recombinant protein for the DNA binding domain of >200 transcription factors for SELEX-SAGE (TRANSCODE FP6 project); data on the on co-localization of over 120 TFs in zebrafish dopaminergic neurons; ANISEED, a data rich systems biology platform for Ciona embryos; Data of the Fantom3 project and brain transcriptome. Moreover the partners of this consortium have produced a robust set of preliminary data in preparation for this call, including Solexa sequenced microCAGE of laser microdissection neurons and high throughput microscopy cis-regulatory assays.


 
Potential Impact


 
The prevalence of PD in Europe today is roughly 2 million people. Within the next 50 years, the number is expected to rise to 5 Million. In contrast, the population providing care is projected to decrease from a ratio of 60 people of working-age per 1 demented person today to less than 20 by 2050. Thus, the burden placed by Dementia on the working-age population will rise dramatically (Berr, Wancata et al. 2005). This is a great challenge for European society. None of the current available treatments, including LEVODOPA, have been proven to slow the progression of the disease. Deciphering the basic networks of dopaminergic neurons, involved in Parkinson's disease, using an innovative approach that is likely to identify less conventional molecules will generate novel diagnostic and therapeutic candidates of great relevance to this challenge.