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. 2010 Sep 9;5(9):e12649.
doi: 10.1371/journal.pone.0012649.

Cpd-1 null mice display a subtle neurological phenotype

Rupinder K Kular  1 , Rocky G Gogliotti, Puneet Opal
Affiliations

Cpd-1 null mice display a subtle neurological phenotype

Rupinder K Kular et al. PLoS One. .

Abstract

Background: CPD1 (also known as ANP32-E) belongs to a family of evolutionarily conserved acidic proteins with leucine rich repeats implicated in a variety of cellular processes regulating gene expression, vesicular trafficking, intracellular signaling and apoptosis. Because of its spatiotemporal expression pattern, CPD1 has been proposed to play an important role in brain morphogenesis and synaptic development.

Methodology/principal findings: We have generated CPD1 knock-out mice that we have subsequently characterized. These mice are viable and fertile. However, they display a subtle neurological clasping phenotype and mild motor deficits.

Conclusions/significance: CPD1 is not essential for normal development; however, it appears to play a role in the regulation of fine motor functions. The minimal phenotype suggests compensatory biological mechanisms.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of CPD1 null mice.
A. Loss of CPD1 message in CPD1 null mice. RNA extracted from brains of CPD1 +/+, CPD1 +/−, and CPD−/− mice show loss of CPD1 transcripts by RT-PCR analysis. GAPDH is used as an internal control. Reduction in CPD1 transcript level is evident in CPD1 +/− mice. B. Antibody to CPD1 does not cross-react against ANP homologues. FLAG-tagged versions of CPD1, LANP and APRIL were transfected into cells and immunoprecipitaed with anti-FLAG antibody. The antibody to CPD1 specifically recognizes CPD1 and not the other ANP homologues. Western blot with FLAG demonstrates the transfection and pull-down of the relevant proteins. C. CPD1 null mice do not express CPD1 protein. Protein extracted from brains of CPD1 +/+, CPD1 +/−, and CPD−/− mice show levels of CPD1 protein by western blot analysis. Actin is used as loading control. Asterisks denote non-specific bands. D. Expression of endogenous CPD1. Immunostaining of neuro2a cells with α-CPD1 antibody displays localization of CPD1 protein. DAPI staining marks the nucleus. E. Immunostaining on brains dissected from CPD1 +/+ and CPD1 −/− mice shows reduced CPD1 staining in CPD1 null mice.
Figure 2
Figure 2. Analysis of motor deficits in CPD1 null mice.
A. Accelerating rotarod analysis: 4 month old CPD1 +/+ (n = 6) and CPD1 −/− (n = 12) were tested for a fall from a rotating rod. Average of 4 trails on each day is plotted. Error bars = SEM; p = 0.067, suggesting no significant changes. B. Dowel test: 4 month old CPD1 null mice require more time to traverse a balance beam. CPD1 +/+ (n = 6) and CPD1−/− (n = 12) were made to traverse a beam placed on two platforms for 8 trials on 8 days. CPD1−/− mice took longer time to traverse the beam (p<0.005). C. CPD1 null mice display clasping behavior. CPD1 +/+ (n = 3) and CPD1 −/− (n = 16) were held from their tails and tested for clasping of the limbs. CPD1 null mice clasp their limbs more times and for longer duration than age matched control (p<0.005).
Figure 3
Figure 3. CPD1 null mice do not depict any memory related behavioral changes tested by Y maze.
4 month old CPD1 +/+ (n = 3) and CPD1−/− (n = 8) mice were tested on Y maze for spontaneous alterations to enter arms of Y maze. Mice were tested for 5 minutes or 22 entries (whichever came first) in arms of a Y maze. No significant difference in % of alterations were observed (p>0.05).
Figure 4
Figure 4. Expression levels of LANP and APRIL do not alter in CPD1 null mice.
A. Brain lysates from CPD1 +/+, CPD1 +/−, and CPD1 −/− mice were subjected to western blot analysis with an anti-LANP antibody to check for protein levels of LANP. Lysate from LANP null mice served as control with actin as loading control. B. Brain lysates from CPD1 +/+, CPD1 +/−, and CPD1 −/− mice were subjected to western blot analysis to check for protein levels of APRIL with an APRIL antibody. C. mRNA expression analysis of CPD-1, LANP and APRIL was performed using quantitative PCR. GAPDH served as an internal control and the fold difference is relative to expression in wild type mice.
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