Participants involved: Paris.
Collaborating institutes: Amsterdam (responsible).

Objectives

1. establish the specific roles of the Rho-linked MRX genes oligophrenin1, RhoGEF and PAK3 in neurite outgrowth and branching
2. to establish their role in the formation of synaptic connections, in particular spine synapses
3. to determine their specific role in signal transduction pathways that modulate the actin cytoskeleton through Rho proteins. Together with the studies in transgenic mice, these investigations should establish the molecular and cellular mechanisms by which mutations in Rho-linked MRX genes lead to mental retardation

Methodology and study materials

Primary cultures of rat and mouse neurons will be used to investigate how mutated oligophrenin1, αPIX, and PAK3 alter neuronal development. Comparisons will be made between neurons derived from control and mutant mice (work package 5), and between neurons transfected with either normal or mutant expression constructs (both participants).

The effects on growth cone morphology, neurite outgrowth and branching will be determined by confocal microscopy of fluorescent labelled neurons, followed by quantitative image analysis and video-time-lapse microscopy of the dynamics of actin and Rho-linked MRX proteins (participant Amsterdam).

Investigated will be how mutated oligophrenin1, PAK3 and αPIX alter the formation of synapses and the morhology of spines. Neurons from genetically modified mice will be immunostained for spine and synapse markers and quantitatively compared with control neurons. Together with the method mentioned above, any deviations in normal neuronal morphogenesis or synapse formation due to mutation of the Rho-linked MRX genes should be established (both participants).

Subsequently, we will investigate how wild type oligophrenin1, αPIX, and PAK3 are activated in response to various extracellular signals by studying their phosphorylation status and cellular localisation. Effects of the mutant oligophrenin1, PAK3 and αPIX on the (actin) cytoskeleton will be investigated by determining the co-localisation with cytoskeletal proteins and by analysing the dynamics of the actin cytoskeleton with fluorescent time-lapse microscopy (participant Amsterdam).