Functional Analysis of the X-linked Mental Retardation Protein, Oligophrenin-1, in the Brain

Linda Van Aelst, Ph.D.

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Grant Program:

David Mahoney Neuroimaging Program

Funded in:

March 2003, for 2 years

Funding Amount:

$100,000

Abstract

Functional Analysis of the X-linked Mental Retardation Protein, Oligophrenin-1, in the Brain

Non-specific X-linked mental retardation (MRX) is characterized by mental impairment without any other distinctive clinical features. More than a dozen loci have been implicated in this condition, and importantly, of the eight genes that have been identified to date, three encode regulators or effectors of members of the Rho family of GTPases. These findings have led to the hypothesis that abnormal Rho GTPase signaling may be a prominent cause of MRX. However, how alterations in Rho signaling result in changes in neuronal connectivity and/or plasticity that give rise to MRX remain unknown. In this proposal, we will focus on the functional characterization of oliophrenin-1, a putative Rho GTPase activating protein (RHO GAP) that is lacking in unrelated MRX families.

Our goal is to elucidate how a lack of oligophrenin-1 results in a mental retardation phenotype and how its interaction(s) with Rho family GTPases (which include RhoA, Rac and Cdc42) contributes to this phenotype. To achieve this goal, we will employ complementary cellular and molecular experimental approaches. First, we will determine what effect(s) absence and ectopic expression of oligophrenin-1 has on the cellular morphology of developing neurons. This will involve performing live cell imaging of biolistically-transfected pyramidal neurons in hippocampal slices using two-photon miscroscopy. Second, we will investigate the signaling pathways in which oligophrenin-1 participates, by determining which target Rho GTPase(s) it acts upon in neuronal cells. To this end, we will perform RhoA, Rac1 and Cdc42 activity assays in vivo and compare the phenotypes of dominant negative and activated mutant forms of the Rho GTPases with those generated by gain-of and loss-of function conditions for oligophrenin-1.

Finally, we will further define the molecular and signaling functions of oligophrenin-1 by identifying new interacting proteins. The effects of selected oligophrenin-1 interacting proteins on dendritic arborization and spine formation will be examined in the context of oligophrenin-1 signaling using the above hippocampal slice system.

We believe that studies on oligophrenin-1 will correlate basic cellular and molecular biology with the disease phenotype of mental retardation. These studies will advance our understanding of neuronal development and determine how the disruption of proteins intimately associated with Rho GTPases contributes to MRX.

Hypothesis

Hypothesis:
We hypothesize that the Rho-linked MRX protein, oligophrenin-1, will act as a negative regulator of the Rho GTPases via its GAP activity, and that increased Rho GTPase activity due to a loss of oligophrenin-1 protein will result in abnormal neuronal morphology by interfering with dendrite and spine formation in hippocampal pyramidal cells. Any change in dendritic length and number or spine formation may ultimately result in aberrant targeting of processes and improper synapse formation, and in this way contribute to mental retardation.

Goals:
The overall goal of this study is to elucidate the molecular and cellular mechanism(s) by which a lack of oligophrenin-1 results in mental retardation. Our specific aims are:
1. To determine what effect absence and ectopic expression of oligophrenin-1 has on dendritic arborization and spine formation of pyramidal cells in hippocampal slices.
2. To investigate the signaling pathways in which oligophrenin-1 participates, by determining which Rho GTPase(s) it acts upon in neuronal cells.
3. To define the molecular and signaling functions of oligophrenin-1 by identifying interacting proteins.

Methods:
We will investigate the cellular function of oligophrenin-1 by manipulating its expression in developing hippocampal neurons. To interfere with expression of the endogenous oligophrenin-1 gene, we will make use of antisense RNA and RNA interference (RNAi) approaches. The full-length sense and antisense oligophrenin-1 constructs as well as the siRNA duplexes and hairpin constructs will be biolistically transfected (along with a GFP-expressing vector, for visualization) into postnatal day 4, 7 and 14 hippocampal slices. The dendritic structure of transfected CA1 hippocampal neurons will then be imaged by two-photon microscopy, and the effects on dendritic arborization and spine formation will be analyzed. To identify which Rho GTPase(s) oligophrenin-1 acts upon in neurons, we will 1) perform RhoA, Rac and Cdc42 activity assays in cultured hippocampal neurons and 2) compare the phenotypes generated by oligophrenin-1 gain- and loss- of function conditions with those triggered upon expression of dominant negative and constitutively active mutants of RhoA, Rac1 and Cdc42. To identify oligophrenin-1 interacting proteins in the developing rat hippocampus, we will use the yeast two-hybrid (YTH) system.

Findings:
Our research has focused on determining the function of oligophrenin-1 (OPHN1), a gene located on chromosome Xq12 that codes for a negative regulator of Rho GTPases. Mutations in OPHN1 (which result in OPHN1 loss of function) have been reported in families with X-linked mental retardation. In order to study the effects of loss of OPHN1 on neurons, we have used a novel technique (RNA interference) to down-regulate, or "knock-down," the levels of this protein in normal nerve cells. This allows us to mimic the disease state and study what happens to neurons lacking this protein during development. Our results have shown that loss of OPHN-1 changes the shape and function of neurons, which in turn affects how these cells communicate with each other. This neuron-to-neuron communication underlies normal cognitive functions and learning. Therefore, these findings give us insights into the cellular changes that may occur in the brains of boys with mental retardation, as well as mechanisms that contribute to learning and memory in normal individuals.

Selected Publications

Janas, J.A., Skowronski, J., Van Aelst, L. Lentiviral Delivery of RNAi in Hippocampal Neurons. Methods Enzymol. 2006;406:593-605.

Newey S.E., Velamoor V., Govek E-E., and Van Aelst L. The Rho GTPases, dendritic structure and mental retardation. J Neurobiol. 2005 Jul;64(1):58-74 .

Govek E-E., Newey S.E., and Van Aelst L. The role of the Rho GTPases in neuronal development. Genes Dev. 2005 Jan 1;19(1):1-49 .

Govek E.-E., Newey S.E., Akerman C.J., Cross J.R., Van der Veken L. and Van Aelst L. The X-linked mental retardation protein oligophrenin-1 is required for dendritic spine morphogenesis. Nat Neurosci. 2004 Apr;7(4):364-72 .

Nadif Kasri, N. and Van Aelst, L. (2007). Spines, Rho GTPases and mental retardation. In the New Encyclopedia of Neuroscience – Synaptic Organization and Structure. (In press).