Generation and characterization of LANP/pp32 null mice

Abstract

The leucine-rich acidic nuclear protein (LANP) belongs to a family of evolutionarily conserved proteins that are characterized by an amino-terminal domain rich in leucine residues followed by a carboxy-terminal acidic tail. LANP has been implicated in the regulation of a variety of cellular processes including RNA transport, transcription, apoptosis, vesicular trafficking, and intracellular signaling. Abundantly expressed in the developing cerebellum, this protein has also been hypothesized to play a role in cerebellar morphogenesis. LANP has been implicated in disease biology as well, both as a mediator of toxicity in spinocerebellar ataxia type 1 and as a tumor suppressor in cancers of the breast and prostate. To better understand the function of this multifaceted protein, we have generated mice lacking LANP. Surprisingly, these mice are viable and fertile. In addition we could not discern any derangements in any of the major organ systems, including the nervous system, which we have studied in detail. Overall our results point to a functional redundancy of LANP's function, most likely provided by its closely related family members.

FIG. 1.

Generation of LANP null mice. (A) Targeting strategy. The targeting vector was generated by flanking a 2.2-kb region of genomic DNA containing exons 2 and 3 of the LANP gene with loxP sites. The selectable cassette (TK-Neo) containing the genes for neomycin resistance and TK was inserted downstream of the genomic fragment, followed by a third loxP site. Targeted ES clones were subsequently electroporated with Cre recombinase, and the recombination event that deletes exons 2 and 3 and the selection cassette is shown. (B) Southern blotting of DNA isolated from mouse tails using EcoRV-digested DNA and a 5′ probe upstream of the targeted locus. Mice null for LANP display a 4-kb band (arrow) compared to the wild-type band of 6.7 kb. (C) Western blot analysis of cerebellar extracts using an anti-LANP antibody and an antiactin antibody. LANP null mice show no detectable LANP and equal intensity of the actin signal as a loading control.

FIG. 2.

LANP mice display normal growth curves. LANP null mice and wild-type (Wt) and heterozygote littermates show overlapping growth curves. Results are means ± standard errors of the means. Wild type, n = 13; LANP null mice, n = 9; heterozygous mice, n= 14.

FIG. 3.

Morphological analysis of brains of LANP null mice. (A) LANP null mice show depletion of LANP by immunohistochemical staining (right). Note the predominantly nuclear staining (arrow) of LANP in a section from a wild-type littermate. Scale bar = 20 μm. (B) LANP null mice do not show any differences in cerebellar morphology, cell position, or dendritic arborization compared to wild-type littermates based on immunohistochemical analysis using an antibody against the light chain of MAP1B. Scale bar = 50 μm.

FIG. 4.

Behavioral profile of LANP null mice. (A) Open-field test. LANP null mice (n = 17; dark gray bar) are as active as wild-type mice (n = 15; light gray bar) in the open field and display no increase in anxiety. In this test LANP null mice travel distances similar to those traveled by wild-type mice and spend similar times moving; hence, they and wild-type mice exhibit similar speeds during the test. Moreover they and wild-type mice spend approximately similar amounts of time in the center versus the periphery, suggesting no sense of heightened anxiety. Results are means ± standard errors of the means (SEM). (B) Wire test. LANP null mice (n = 20) and wild-type littermates (n = 19) spend similar times suspended on the wire. Results are means ± SEM). (C) Dowel test. LANP null mice (n = 20) and wild-type littermates (n = 23) spend similar times balanced on the dowel. Results are means ± SEM. (D) Rotarod test. LANP null mice (n = 9) perform similarly to wild-type mice (n = 13) on the rotating-rod apparatus. Wild-type and LANP null mice were subjected to Rotarod testing of four trials per day for 4 days at 5 and 11 weeks of age. Results are means ± SEM. (E) Conditioned-fear testing. LANP null mice (n = 8) learn as well as wild-type animals (n = 10) to associate the context or the CS (cue) with a foot shock, as determined by measuring freezing behavior. Mice were tested for both short-term (1 h) and long-term (24 h) fear-conditioned behavior. Results are means ± SEM.

FIG. 4.

Behavioral profile of LANP null mice. (A) Open-field test. LANP null mice (n = 17; dark gray bar) are as active as wild-type mice (n = 15; light gray bar) in the open field and display no increase in anxiety. In this test LANP null mice travel distances similar to those traveled by wild-type mice and spend similar times moving; hence, they and wild-type mice exhibit similar speeds during the test. Moreover they and wild-type mice spend approximately similar amounts of time in the center versus the periphery, suggesting no sense of heightened anxiety. Results are means ± standard errors of the means (SEM). (B) Wire test. LANP null mice (n = 20) and wild-type littermates (n = 19) spend similar times suspended on the wire. Results are means ± SEM). (C) Dowel test. LANP null mice (n = 20) and wild-type littermates (n = 23) spend similar times balanced on the dowel. Results are means ± SEM. (D) Rotarod test. LANP null mice (n = 9) perform similarly to wild-type mice (n = 13) on the rotating-rod apparatus. Wild-type and LANP null mice were subjected to Rotarod testing of four trials per day for 4 days at 5 and 11 weeks of age. Results are means ± SEM. (E) Conditioned-fear testing. LANP null mice (n = 8) learn as well as wild-type animals (n = 10) to associate the context or the CS (cue) with a foot shock, as determined by measuring freezing behavior. Mice were tested for both short-term (1 h) and long-term (24 h) fear-conditioned behavior. Results are means ± SEM.

FIG. 4.

Behavioral profile of LANP null mice. (A) Open-field test. LANP null mice (n = 17; dark gray bar) are as active as wild-type mice (n = 15; light gray bar) in the open field and display no increase in anxiety. In this test LANP null mice travel distances similar to those traveled by wild-type mice and spend similar times moving; hence, they and wild-type mice exhibit similar speeds during the test. Moreover they and wild-type mice spend approximately similar amounts of time in the center versus the periphery, suggesting no sense of heightened anxiety. Results are means ± standard errors of the means (SEM). (B) Wire test. LANP null mice (n = 20) and wild-type littermates (n = 19) spend similar times suspended on the wire. Results are means ± SEM). (C) Dowel test. LANP null mice (n = 20) and wild-type littermates (n = 23) spend similar times balanced on the dowel. Results are means ± SEM. (D) Rotarod test. LANP null mice (n = 9) perform similarly to wild-type mice (n = 13) on the rotating-rod apparatus. Wild-type and LANP null mice were subjected to Rotarod testing of four trials per day for 4 days at 5 and 11 weeks of age. Results are means ± SEM. (E) Conditioned-fear testing. LANP null mice (n = 8) learn as well as wild-type animals (n = 10) to associate the context or the CS (cue) with a foot shock, as determined by measuring freezing behavior. Mice were tested for both short-term (1 h) and long-term (24 h) fear-conditioned behavior. Results are means ± SEM.

FIG. 5.

Electrophysiologic assessment of LANP null mutants compared to wild-type mice. (Left) Immunohistochemical analysis using a LANP-specific antibody demonstrates that LANP is abundantly expressed in hippocampal cells of wild-type (WT) mice (top); LANP null mice show no staining and serve as a staining control (bottom). In each image the upper left inset, magnified twofold, is displayed in the lower left corner. Scale bar = 50 μm. (A) The PPF paradigm was used to assess short-term plasticity in LANP null and WT mice. PPF in mutant mice was similar to that in WT mice (LANP null: 134% ± 4.6%, n = 19; WT: 126% ± 4.9%, n = 17; P = 0.1217). (B) Synaptic transmission in the stratum radiatum in area CA1 of the hippocampus was unaltered in LANP null mutants compared to WT mice. No significant differences in the slopes of the lines were seen when a linear regression was calculated with data from all stimulus intensities (LANP null mice: n = 19; slope, 2.68 ± 0.27; r² = 0.8791; WT, n = 16; slope, 2.78 ± 0.24; r² = 0.9107). (C) LTP was induced in LANP null and wild-type mice with two trains, each of 1 s, of 100-Hz stimulation separated by 20 s (arrow). No change in the amount of posttetanic potentiation was seen immediately following stimulation (LANP null: 240% ± 22.8%; n = 14; WT: 263% ± 20.5%, n = 12; P = 0.2350) or 60 min after stimulation (LANP null, 195% ± 19.7%; WT, 182 ± 13.9%; P = 0.3120). (Inset) Representative traces (means of six successive EPSPs) are shown for baseline (0) and 60 min after tetanic potentiation (60) for LANP null mice (gray) and wild types (black). Results (A to C) are means ± standard errors of the means.

FIG. 6.

Normal expression of APRIL/PAL31, an LANP family member, in LANP null mice. Western blotting of mouse cerebellar extracts from three pairs of wild-type and LANP null littermates reveals the absence of LANP staining in LANP null mice (A). Persistence of APRIL/PAL31 is observed with two different antibodies generated against rat (APRIL r) and human (APRIL h) versions of this protein (B and C, respectively). Note that APRIL h recognizes mouse APRIL and LANP, causing a doublet pattern in wild-type lanes (C). (D) A Western blot against GAPDH serves as a loading control. (E) Immunohistochemical localization of APRIL with antibody APRIL r from a LANP null cerebellum shows significant staining in the nuclei of Purkinje cells.

FIG. 7.

Phylogenetic tree of LANP and family members. LANP and representative family members from a variety of species were aligned in clustalV, and a phylogenetic tree was drawn to show their relationships.