Anaplastic Lymphoma Kinase and Leukocyte Tyrosine Kinase: Functions and genetic interactions in learning, memory and adult neurogenesis
Highlights
► Alk and Ltk are expressed throughout the mouse hippocampus. ► Mice lacking Alk have enhanced spatial memory. ► The enhanced spatial memory of Alk mutant mice is Ltk dependent. ► Alk and Ltk have partially redundant functions in adult neurogenesis.
Introduction
The two closely related receptor tyrosine kinases, Anaplastic Lymphoma Kinase (Alk) and Lymphocyte Tyrosine Kinase (Ltk), are expressed in a variety of neural tissues during mammalian development and in adults (Bernards and de la Monte, 1990, Haase et al., 1991, Iwahara et al., 1997, Morris et al., 1997, Hurley et al., 2006, Degoutin et al., 2009). There are only a few functional studies of both Alk and Ltk in mammals, with just one published study addressing functions of Alk in mammalian behavior and adult neurogenesis and no publications addressing the functions of Ltk (Bilsland et al., 2008). In Drosophila, Alk and its ligand, Jelly belly (Jeb), play roles in axon targeting of photoreceptors, neuromuscular junction growth and function, as well as early mesoderm development (Rohrbough and Broadie, Weiss et al., 2001, Englund et al., 2003, Lee et al., 2003, Bazigou et al., 2007). Similar neural functions are documented for Hen1, a C. elegans Jeb homolog, and Scd2, a Caenorhabditis elegans Alk homolog (Ishihara et al., 2002, Liao et al., 2004, Reiner et al., 2008). Though neither Hen1 nor Scd2 are required for C. elegans development, the secreted signaling molecule Hen1 is required for integration of conflicting sensory inputs (Ishihara et al., 2002). Scd2 functions in C. elegans neuromuscular junction formation and in the dauer response to environmental stress (Liao et al., 2004, Reiner et al., 2008).
In three recent publications Drosophila Alk regulates 1) larval neurogenesis, 2) adult responses to ethanol and 3) body size and adult learning (Cheng et al., 2011, Gouzi et al., 2011, Lasek and Lim, 2011). Drosophila Alk regulates the tumor suppressor Neurofibromin 1 (Nf-1) in control of body size and adult learning. In this study, we assessed whether mammalian Alk has similar functions with respect to learning and neurogenesis.
In Drosophila and C. elegans there is a single homologous receptor that most closely resembles Alk. In vertebrates, Alk and Ltk are found, presumably as a consequence of duplication and divergence of a common ancestral receptor. Ltk is structurally very similar to Alk and differs from it in its extracellular domain where Ltk lacks a region homologous to the amino-terminal portion of Alk (Fig. 2). In zebrafish, the development of one class of pigment cells not found in mammals, iridophores, is dependent on Ltk function (Lopes et al., 2008). Both Alk and Ltk belong to the insulin receptor superfamily of receptor tyrosine kinases, though both have diverged from other insulin receptor family members. Alk and Ltk structurally constitute a small family of receptor tyrosine kinases distinct from the insulin and insulin-related receptors (Robinson et al., 2000). The structural similarity between Alk and Ltk, along with their inferred common evolutionary origin, suggests that in mammals they may have related or even interlocking functions.
We explored the functional interaction between Alk and Ltk by analyzing behavioral and neurogenic phenotypes in mice harboring targeted single mutations in Alk and Ltk as well as double-mutant mice lacking both Alk and Ltk. Our objective was to determine if there is a genetic interaction between the two receptors that would support any of three possible functional relationships: 1) independent, 2) redundant or 3) antagonistic functional interactions. Our analysis focused on hippocampal neurogenesis and hippocampus-dependent behaviors because both receptors are expressed throughout the adult hippocampus.
Section snippets
Alk and Ltk expression in the hippocampus
We analyzed first the expression of Alk and Ltk mRNAs in the brains of adult mice using in situ hybridization. Alk mRNA is expressed throughout the hippocampus, including the dentate gyrus (Fig. 1A–C). We also employed anti-Alk antisera and anti-doublecortin, a specific marker of committed neural progenitors, to determine whether adult born neural progenitors express Alk. Alk protein is clearly found in doublecortin-positive newborn neurons (Fig. 1D–F). Alk mRNA and protein expression is
Discussion
In this study, we analyzed hippocampal mRNA expression, behavioral phenotypes, a marker of adult neurogenesis and mRNA levels in mice with targeted mutations in the two highly related receptor tyrosine kinases, Alk and Ltk. Our results are strikingly similar to recently published papers concerning the functions of Alk in Drosophila. Specifically we found that loss of Alk function in mammals enhances retention of spatial memory, just as inhibition of Alk function in adult Drosophila enhances
Knockout mice
Targeted mutations in Alk and Ltk were induced by homologous recombination at each locus in mouse ES cells. The targeting vectors encode neomycin resistance and their integration results in deletion of essential coding sequences. In the Alk locus exons 20 and 21 are deleted, a total of 93 amino acids. The deleted exons encode the entire intracellular juxtamembrane domain and the initial portion of the tyrosine kinase catalytic domain. In the Ltk locus exons 10 and 11 are deleted, a total of 66
Behavioral tests
Mice were first tested for exploratory behavior and measures of anxiety in the open field and the elevated zero maze. Subsequently, mice were tested for sensorimotor function using the rotorod. Finally, spatial learning and memory was assessed using in the water maze.
Acknowledgments
We thank Jessica Siegel, Reid Olsen, and Jenna Rosenberg for their assistance with the behavioral testing and data analyses.
Supported in part by NCI R01 CA69129 (S.W.M) and by the American Lebanese Syrian Associated Charities (ALSAC), and St. Jude Children's Research Hospital (L.X., S.W.M.).
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