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, 7 (3), e32970

Restricted Morphological and Behavioral Abnormalities Following Ablation of β-Actin in the Brain

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Restricted Morphological and Behavioral Abnormalities Following Ablation of β-Actin in the Brain

Thomas R Cheever et al. PLoS One.

Abstract

The local translation of β-actin is one mechanism proposed to regulate spatially-restricted actin polymerization crucial for nearly all aspects of neuronal development and function. However, the physiological significance of localized β-actin translation in neurons has not yet been demonstrated in vivo. To investigate the role of β-actin in the mammalian central nervous system (CNS), we characterized brain structure and function in a CNS-specific β-actin knock-out mouse (CNS-ActbKO). β-actin was rapidly ablated in the embryonic mouse brain, but total actin levels were maintained through upregulation of other actin isoforms during development. CNS-ActbKO mice exhibited partial perinatal lethality while survivors presented with surprisingly restricted histological abnormalities localized to the hippocampus and cerebellum. These tissue morphology defects correlated with profound hyperactivity as well as cognitive and maternal behavior impairments. Finally, we also identified localized defects in axonal crossing of the corpus callosum in CNS-ActbKO mice. These restricted defects occurred despite the fact that primary neurons lacking β-actin in culture were morphologically normal. Altogether, we identified novel roles for β-actin in promoting complex CNS tissue architecture while also demonstrating that distinct functions for the ubiquitously expressed β-actin are surprisingly restricted in vivo.

Conflict of interest statement

Competing Interests: James Ervasti, the corresponding author, is currently an academic editor. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Conditional ablation of β-actin in the mouse central nervous system.
(A–B) Quantitative Western blot analysis of actin isoform expression in control and CNS-ActbKO brains at embryonic day (E)13.5, 18.5 and in adults (6–9 months). Data plotted as mean ± standard error of the mean with an n≥3 per genotype per age point. (C) Immunofluorescence with actin isoform-specific antibodies on saggital sections of the hippocampus at E18.5. β-actin staining was dramatically reduced in CNS-ActbKO brain sections. γ-actin staining gave a similar localization pattern to β-actin on co-labeled control sections and was still present in CNS-ActbKO embryos. αsm-actin was restricted to blood vessels in control brains (arrows) but was prominent in select cells in CNS-ActbKO brain sections (see inset, inset scale bar 25 µm). Scale bar 100 µm. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.
Figure 2
Figure 2. Gross characterization of CNS-ActbKO mice.
(A) While the expected number of CNS-ActbKO embryos were present at E18.5, nearly 2/3's died between birth and weaning at P21. (B) Surviving CNS-ActbKO mice were significantly smaller than control littermates from weaning at P21 through adulthood, although they followed a similar trend in growth. (C) Despite their reduced mass, CNS-ActbKO mice had a significantly higher caloric intake than control mice when normalized to body weight. (D) Hind-limb clasping/contractures were present in CNS-ActbKO mice indicating neurological dysfunction. (E) Brains from CNS-ActbKO mice appeared grossly normal and were proportionate in size to the smaller body mass of CNS-ActbKO mice. n≥3 mice for each genotype. Data plotted as mean ± standard error of the mean. * indicates p<0.05, **indicates p<0.01.
Figure 3
Figure 3. Histological abnormalities in CNS-ActbKO brains.
(A) Midsaggital sections through the cerebellum revealed variable abnormalities in the foliation pattern of the cerebellum. Aberrantly positioned folia are marked by asterisks. Scale bar 1 mm. (B) Morphological abnormalities in the hippocampus as seen in saggital sections. Arrows indicate the position of the hippocampal fissure normally found between the dentate gyrus and CA1 neuronal layer. In CNS-ActbKO mice, the hippocampal fissure was displaced (arrows) which correlated with an abnormal invagination of the dentate gyrus. The region corresponding to this displacement is indicated by dashed lines which also reveals a dramatic decrease in cell body staining in the CA1 neuronal layer. Scale bar 0.5 mm. (C) The abnormal morphology of the hippocampus in CNS-ActbKO mice is further illustrated in coronal sections where the dentate gyrus displacement (black arrows) and decrease in CA1 region staining (red arrows) can also be seen. Scale bar 0.5 mm. (D) Axons of the corpus callosum crossed the midline normally in rostral sections from CNS-ActbKO brains (arrows) but failed to cross the midline in more caudal sections (arrows in E). Scale bars in D–E, 0.5 mm. (F) The lamination of cortical layers 1–6 was preserved in 6 month old control and CNS-ActbKO mice. Scale bar 0.5 mm.
Figure 4
Figure 4. Maternal behavior deficit in CNS-ActbKO mice.
(A) Pups born to CNS-ActbKO females did not survive longer than one day. (B) CNS-ActbKO mothers exhibited a pup retrieval deficit. While control mothers retrieved pups into a nest, pups born to CNS-ActbKO females were scattered throughout the cage. The pup retrieval deficit was independent of the pups however, as pups born to CNS-ActbKO females were retrieved normally by foster control females.
Figure 5
Figure 5. CNS-ActbKO mice exhibited hyperactivity and decreased performance in the Morris water maze.
(A) CNS-ActbKO mice performed comparably to controls in a four day Rotarod assay experiment. Control and CNS-ActbKO animals were age matched and between 4–6 months old. Data plotted as mean ± standard error of the mean. (B) Five minute traces from control and CNS-ActbKO mice in an Open Field Activity Assay. (C–D) CNS-ActbKO mice traveled significantly greater distances and at significantly greater velocities than control mice. (E) Representative 30 second traces from control and CNS-ActbKO mice during the probe trial of a Morris water maze test. (F) CNS-ActbKO mice performed similarly to control mice with a cued platform and followed a similar learning curve until the final day of the hidden platform learning phase, at which time controls found the platform significantly faster. (G–H) In the probe trial, CNS-ActbKO mice spent a significantly smaller percentage of time in the platform quadrant and traveled significantly less distance in that quadrant. (I) CNS-ActbKO mice also crossed an area 50% larger than that of the actual platform significantly less times than control mice. n = 4 male mice for each genotype. * indicates p<0.05, ** p<0.01, *** p<0.001.
Figure 6
Figure 6. CNS-ActbKO primary neurons exhibit normal morphology.
(A) Hippocampal neurons derived from E15.5 control and CNS-ActbKO embryos and cultured for 3 days in vitro. Neurons were stained with antibodies specific for β- and γ-actin. β-actin was efficiently ablated in hippocampal neuronal cell bodies and growth cones (B) from CNS-ActbKO embryos. Arrows in the bottom panels in (A) indicates a non-neuronal cell expressing β-actin as a positive control. (C) Representative images of control and CNS-ActbKO neurons stained with a βIII-tubulin antibody for morphological characterization (D). Scale bars 20 µm in (B), 50 µm in all other panels.

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