CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

Identification of molecular mechanisms of X-Linked Mental Retardation associated to Oligophrenin-1 and TM4SF2

Introduction

Non Syndromic X-Linked Mental Retardation (NS-XLMR) is a disorder characterized by significant limitations both in intellectual functioning and in adaptive behavior without any other distinctive clinical feature. A major challenge is to uncover the molecular causes of NS-XLMR and the underlying cellular mechanisms responsible for reduced cognitive function.

Recently mutations in a series of genes have been associated to NS-XLMR. We concentrated on two of these: TM4SF2 (Tetraspanin-7) (Fig. 1) and oligophrenin-1.

 

Oligophrenin-1 (OPHN1) encodes a synaptic RhoGTPase-activating protein (Oligophrenin-1) that regulates dendritic spine shape and outgrowth of axons in brain (by its interaction with F-actin and Homer) (Khelfaoui et al 2009). Oligophrenin-1 is composed of two different domains, namely an amino-terminal BAR domain,a Pleckstrin homology domain and a central RhoGAP domain followed by three carboxyl-terminal proline-rich sequences, which are putative SH3-binding sites. In mature neurons, the protein is detected on both sides of the synapse suggesting that it may somehow participate to synaptic formation and/or function. However the role of Oligophrenin-1 in the brain is unknown and it remains to be seen how mutations in OPHN1 affect neuronal development and function and contribuite to MRX. By using the C-terminal fragment of Oligophrenin 1 as bait in a two-hybrid screening of a human fetal brain cDNA library we identified as an interactor Rev-erbAalpha, an orphan nuclear receptor that constitutively suppresses gene transcription and regulates the circadian clock in the CNS . We confirmed the interaction in vitro and in vivo by co-immunoprecipitation and GST-pull down. In COS-7 cells we observed that over-expressed Oligophrenin-1 is able to recruit Rev-erbAalpha (normally localized in the nucleus) to the cytoplasm. Furthermore, over-expression of Oligophrenin-1 induces an accumulation of endogenous Rev-erbAalpha in dendritic spines in hippocampal neurons. The Oligophrenin-1ΔC (deleted of C-terminus), mutant that mimics the mutation present in the XLMR patients, wasn't able to induce this effect in COS7 and in hippocampal neurons. More remarkably, synaptic activity mediated by AMPA receptors induces translocation of Rev-erbAalpha to the dendritic spines. RNAi experiments showed that Oligophrenin-1 is required for the translocation of Rev-erbAalpha in spines induced by synaptic activity.

Moreover we observed an alteration of Rev-erbAalpha expression in Oligophrenin-1 KO mouse: there is a reduction of Rev-erbAalpha in hippocampus. Our results demonstrate, for the first time, the interaction between an orphan receptor (Rev-erbAalpha) and a synaptic protein (Oligophrenin-1). This interaction regulates the localization of Rev-erbAalpha between the synapse and the nucleus induced by synaptic activation. We can hypothesize that the absence of Oligophrenin-1 (in patients affected by MR), followed by the loss of its interaction with Rev-erbAalpha, may be associated to a possible dysfunction of Rev-erbAalpha.

IL1RAPL1 (3) is a protein that belongs to a novel class of the IL-1/Toll receptor family characterized by the presence of the 150 amino acid carboxy terminus that has no significant homology with any protein of known function. TM4SF2 is a member of the Tetraspanins, evolutionary conserved membrane proteins that tend to cluster dynamically with numerous partner proteins in membrane microdomains and are involved various intracellular and intercellular processes. Consistent with a role in cognitive function, there is ubiquitously high expression of TM4SF2 in brain neuron. It is yet not known whether TM4SF2 is involved in neurite outgrowth and synaptic plasticity as seen for other tetraspanins. By a two-hybrid screening of a human fetal brain cDNA library performed with the C-terminal intracellular domain of TM4SF2 as bait, we identified PICK1 and hnRNP K as possible interactors of TM4SF2. PICK1 is a scaffold protein present at excitatory synapses while hnRNP K is a component of the heterogeneous nuclear ribonucleoprotein complex recently found to be involved in the regulation of actin dynamics. Now, we are going to evaluate the role of TM4SF2 and of its interactors in the regulation of signaling pathways that seem to be crucial for cognitive functions according to clinical data. These projects are addressing fundamental problems in neuroscience.

The goal of these projects is to provide new important information on the molecular mechanisms that are involved in brain functions and they also shed light on pathogenesis of spines and synaptic related diseases associated to major cognitive disorders.

Publications

  • Khelfaoui M, Pavlowsky A, Powell AD, Valnegri P, Cheong KW, Blandin Y, Passafaro M, Jefferys JG, Chelly J, Billuart P (2009) Inhibition of RhoA pathway rescues the endocytosis defects in Oligophrenin1 mouse model of mental retardation. Hum. Mol. Genet. 18:2575-83.

Grants

Italian Ministry of University and Scientific Research PRIN, 2007-2009

Compagnia San Paolo, 2007-2009

Fondazione Cariplo, 2009-2011

Fondazione Mariani, 2009-2011

Collaborations

  • P. Billuart, Institut Cochin, Faculté de Medicine, Paris, France.
  • C. Sala, CNR Institute of Neuroscience, Milano.

 

PI photo

Maria Passafaro

Contact information

email  E-mail

email  +39 02 5031 7102

Participating staff
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