Regular articleMice lacking tropomodulin-2 show enhanced long-term potentiation, hyperactivity, and deficits in learning and memory
Introduction
Actin, perhaps the most abundant protein in eukaryotic cells, is involved in most of the dynamic functions at a neuron’s periphery: the motility of growth cones during development, altering the pre- and postsynaptic morphology of the neuron, and generating microdomains on the cell surface. Actin filaments rapidly undergo successive cycles of polymerization and depolymerization, and are thought to play an important role in synaptic plasticity because of their prominent activities in dendritic spines. For example, actin filaments maintain the clustering of both NMDA and AMPA receptors in dendritic spines (Allison et al., 1998). Time-lapse microscopy of hippocampal neurons transfected with GFP-actin shows that spines can undergo submicronmeter changes in shape on the order of seconds (Fischer et al., 1998), and electron microscopy of CA1 pyramidal cells demonstrates changes in spine morphology following induction of long-term potentiation Buchs and Muller 1996, Muller et al 2000, Weeks et al 1999.
The state of actin in the cell (as monomers or polymeric strands) and the length of the filaments are controlled by numerous actin-regulatory proteins. Among these are tropomyosin, which binds along the length of actin filaments and prevents filament depolymerization, and tropomodulin, which binds to the filament’s pointed end, preventing both elongation and depolymerization. In fact, the pointed-end-capping activity of tropomodulin is greatly enhanced by association with tropomyosin, which suggests that the two proteins function as a complex to stabilize the filament and regulate its length. Four vertebrate tropomodulin family members have been cloned in human and mouse: TMOD1 (Tmod1), TMOD2 (Tmod2), TMOD3 (Tmod3), and TMOD4 (Tmod4) Almenar-Queralt et al 1999, Cox and Zoghbi 2000, Ito et al 1995, Kalil et al 2000, Sung et al 1992; Sussman et al., 1994; Watakabe et al., 1996). TMOD1 is expressed in the heart, brain, lens, vasculature, and erythrocytes; TMOD2 in neuronal structures; TMOD3 is ubiquitous; and TMOD4 is highly enriched in skeletal muscle. Studies of vertebrate heart cells and Drosophila indirect flight muscles indicate that tropomodulin regulates actin dynamics at the pointed end and determines filament length Gregorio et al 1995, Littlefield et al 2001, Mardahl-Dumesnil and Fowler 2001, Sussman et al 1998a, Sussman et al 1998b, but tropomodulin’s activities in neurons are not well understood.
Given actin’s crucial role in vertebrate nervous system development and function, we sought to determine the influence of tropomodulins in neuronal development, learning, memory, and behavior. We focused on Tmod2 because of its neuronal-specific expression. Replacing Tmod2 exon 1 with a lacZ reporter gene using homologous recombination in embryonic stem cells, we generated Tmod2lacZ−/− mice that are viable and fertile and exhibit no gross morphological or anatomical abnormalities. We found, however, through behavioral analysis on two different genetic backgrounds, that Tmod2lacZ−/− mice can be hyperactive and suffer deficits in fear conditioning and learning. Our data suggest that Tmod2 plays a role in learning, memory, and synaptic plasticity and that Tmod1 can compensate for the absence of Tmod2 to some extent.
Section snippets
Generation of Tmod2lacZ−/− mice
In order to study the in vivo function of Tmod2, we isolated and characterized the Tmod2 genomic locus and prepared a knock-in deletion construct, Tmod2lacZ. We chose to delete coding exon 1 by replacing it with a β-galactosidase reporter gene (lacZ) in frame to the endogenous ATG (Fig. 1A). Chimeras were obtained from two independent ES cell lines (B3 and A12). These animals were bred to C57BL/6J females to produce F1 mice. In the A12 line, germline transmission was obtained in only one F1
Discussion
We generated two lines of Tmod2 knock-out mice by replacing the exon containing the translational methionine with a lacZ reporter gene that expresses under the endogenous promoter. Examination of total brain protein extracts by Western blot analysis revealed no Tmod2 protein in null animals of either the A12 or the B3 lines. Behavioral analysis of Tmod2lacZ mice demonstrated several abnormalities that were genetic background-dependent. A12 mutant mice were hyperactive; B3 mutant animals showed
Generation of Tmod2 knock-in mice
We used a Tmod2 cDNA (Cox and Zoghbi, 2000) to screen a 129/SvEv genomic DNA library and identified 10 hybridizing clones. Restriction mapping and sequencing revealed that clone 3 contained coding exons 1–4. We used clone 3 to produce a Tmod2 targeting vector (Tmod2lacZ) containing a β-gal/PGK-neo (lacZ-neo) cassette (Friedrich and Soriano, 1991) in frame with the endogenous Tmod2 translational methionine while subsequently deleting the remainder of exon 1. The 5′ and 3′ arms were produced from
Acknowledgements
We thank Edwin Weeber for assistance and advice with hippocampal electrophysiology; Dr. Dawna Armstrong for her neuropathology expertise; Kimberly Fritz-Six in the Fowler lab for assistance with the Western blots with Tropomodulin-3 (TMOD3) antibody; Dr. Mark Sussman for the Tmod1 antibody; Lisa Yuva-Paylor for assistance with behavioral analyses; and Vicky Brandt for insightful editing of the manuscript. This work was supported by the Howard Hughes Medical Institute (H.Y.Z.), the Medical
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