MRX is a disorder characterized by cognitive impairment without any other distinctive clinical features. A major challenge has been to uncover the molecular causes of MRX and the underlying cellular mechanisms responsible for reduced cognitive function. Eleven genes involved in MRX have been identified to date and notably, three of these encode regulators or effectors of the Rho subfamily of small GTP-binding proteins 1–3 . Members of this family, including RhoA, Rac and Cdc42, are key regulators of the actin cytoskeleton and affect many aspects of neuronal development and morphogenesis 3–7 . The three Rho-linked MRX genes encode (i) oligophrenin-1, a Rho- GTPase activating protein (Rho-GAP) 8 , (ii) PAK3 (p21-activated kinase-3), a serine/threonine kinase downstream of Rac and Cdc42 (ref. 9) and (iii) ARHGEF6, a Rac GTPase exchange factor also known as αPIX or Cool-2 (ref. 10). The association between mutations in Rho-linked genes and MRX highlights the importance of Rho pro- teins in neuronal function and has led to the hypothesis that abnor- malities in Rho signaling may be a cause of MRX 3 . Studies examining the effects of these mutations on neuronal signaling and development are therefore critical for the elucidation of cellular mechanisms underlying normal cognitive function and disease. Here we focus on the functional characterization of oligophrenin-1 (encoded by the OPHN1 gene in humans; Ophn-1 in mice), a protein with a Rho-GAP domain shown to negatively regulate RhoA, Rac and Cdc42 in vitro and in non-neuronal cells 8,11 . OPHN1 was identified by the analysis of a balanced translocation t(X;12) observed in a female patient with mild mental retardation. Its involvement in MRX was established by the identification of a mutation within the OPHN1 coding sequence in a family with MRX (MRX 60). In these two cases, the OPHN1 mutation is associated with a loss of, or dramatic reduc- tion in, mRNA product 8 . Recent studies show that oligophrenin-1 is present in neuronal and astroglial cells and that it colocalizes with actin at the tip of growing neurites 11 . However, the function of oligophrenin-1 in the brain is unknown and it remains to be seen how mutations in OPHN1 affect neuronal development and function, and contribute to MRX. To begin to understand how oligophrenin-1 deficiency affects neu- ronal function, we examined the effects of reducing oligophrenin-1 levels on the morphology of developing hippocampal (CA1) neurons in organotypic slices. We focused on dendritic spines, the main sites of excitatory synapses in the brain 12 , because changes in spine dimen- sions and density have been associated with synaptic plasticity 13–16 and learning 17 , as well as with neurological disorders including men- tal retardation 18–20 . Using RNA interference (RNAi) and antisense RNA approaches, we found that downregulation of oligophrenin-1 in CA1 neurons resulted in a significant shortening of dendritic spines. We showed that this spine length phenotype was mediated by the RhoA/Rho-kinase signaling pathway, acting downstream of oligophrenin-1. Furthermore, we identified an interaction between oligophrenin-1 and Homer, placing oligophrenin-1 within a post- synaptic complex that potentially links oligophrenin-1 to glutamate receptor signaling. Our findings suggest how OPHN1 mutations may compromise cognitive function. RESULTS Oligophrenin-1 in the brain We began the characterization of oligophrenin-1 by determining its presence, distribution and subcellular localization in the brain. Immunoblot experiments of rat tissues showed that oligophrenin-1 protein levels were highest in the brain, but were also detectable to variable extents in other tissues (Fig. 1a). Oligophrenin-1 levels were 1 Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA. 2 Molecular and Cellular Biology Program, State University of New York at Stony Brook, Stony Brook, New York 11794, USA. 3 These authors contributed equally to this work. Correspondence should be addressed to L.V.A. (vanaelst@cshl.org). Published online 14 March 2004; doi:10.1038/nn1210 The X-linked mental retardation protein oligophrenin-1 is required for dendritic spine morphogenesis Eve-Ellen Govek 1–3 , Sarah E Newey 1,3 , Colin J Akerman 1 , Justin R Cross 1 , Lieven Van der Veken 1 & Linda Van Aelst 1,2 Of 11 genes involved in nonspecific X-linked mental retardation (MRX), three encode regulators or effectors of the Rho GTPases, suggesting an important role for Rho signaling in cognitive function. It remains unknown, however, how mutations in Rho-linked genes lead to MRX. Here we report that oligophrenin-1, a Rho-GTPase activating protein that is absent in a family affected with MRX, is required for dendritic spine morphogenesis. Using RNA interference and antisense RNA approaches, we show that knock-down of oligophrenin-1 levels in CA1 neurons in rat hippocampal slices significantly decreases spine length. This phenotype can be recapitulated using an activated form of RhoA and rescued by inhibiting Rho-kinase, indicating that reduced oligophrenin-1 levels affect spine length by increasing RhoA and Rho-kinase activities. We further demonstrate an interaction between oligophrenin-1 and the postsynaptic adaptor protein Homer. Our findings provide the first insight into how mutations in a Rho-linked MRX gene may compromise neuronal function. ARTICLES 364 VOLUME 7 | NUMBER 4 | APRIL 2004 NATURE NEUROSCIENCE © 2004 Nature Publishing Group http://www.nature.com/natureneuroscience