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. 2019 Jul 30:13:75.
doi: 10.3389/fnana.2019.00075. eCollection 2019.

Deprivation of Muscleblind-Like Proteins Causes Deficits in Cortical Neuron Distribution and Morphological Changes in Dendritic Spines and Postsynaptic Densities

Kuang-Yung Lee  1   2 , Ho-Ching Chang  3 , Carol Seah  1 , Li-Jen Lee  3   4   5
Affiliations

Deprivation of Muscleblind-Like Proteins Causes Deficits in Cortical Neuron Distribution and Morphological Changes in Dendritic Spines and Postsynaptic Densities

Kuang-Yung Lee et al. Front Neuroanat. .

Abstract

Myotonic dystrophy (Dystrophia Myotonica; DM) is the most common adult-onset muscular dystrophy and its brain symptoms seriously affect patients' quality of life. It is caused by extended (CTG)n expansions at 3'-UTR of DMPK gene (DM type 1, DM1) or (CCTG)n repeats in the intron 1 of CNBP gene (DM type 2, DM2) and the sequestration of Muscleblind-like (MBNL) family proteins by transcribed (CUG)n RNA hairpin is the main pathogenic mechanism for DM. The MBNL proteins are splicing factors regulating posttranscriptional RNA during development. Previously, Mbnl knockout (KO) mouse lines showed molecular and phenotypic evidence that recapitulate DM brains, however, detailed morphological study has not yet been accomplished. In our studies, control (Mbnl1 +/+; Mbnl2 cond/cond; Nestin-Cre -/-), Mbnl2 conditional KO (2KO, Mbnl1 +/+; Mbnl2 cond/cond; Nestin-Cre +/-) and Mbnl1/2 double KO (DKO, Mbnl1 ΔE3/ΔE3; Mbnl2 cond/cond; Nestin-Cre +/-) mice were generated by crossing three individual lines. Immunohistochemistry for evaluating density and distribution of cortical neurons; Golgi staining for depicting the dendrites/dendritic spines; and electron microscopy for analyzing postsynaptic ultrastructure were performed. We found distributional defects in cortical neurons, reduction in dendritic complexity, immature dendritic spines and alterations of postsynaptic densities (PSDs) in the mutants. In conclusion, loss of function of Mbnl1/2 caused fundamental defects affecting neuronal distribution, dendritic morphology and postsynaptic architectures that are reminiscent of predominantly immature and fetal phenotypes in DM patients.

Keywords: cortical neurons; dendrites; interneurons; muscleblind-like knockouts; myotonic dystrophy; postsynaptic densities.

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Figures

Figure 1
Figure 1
Thicknesses of gray matter and white matter in Mbnl mutant mice. The gray matter in the sensorimotor cortex (Cx) and the white matter underneath, the external capsule (EC), were measured in sections collected from adult male control (Con, n = 7), Mbnl2 conditional knockout (CKO; 2KO, n = 7) and Mbnl1/2 double KO (DKO, n = 6) mice (A). Significant differences were not reached in the thicknesses of both Cx (B) and EC (C) among mice of three different genotypes. Bar = 1 mm. Results are mean ± SEM.
Figure 2
Figure 2
Density and distribution of cortical neurons in Mbnl mutant mice. Coronal sections containing the sensorimotor cortex of control (Con, n = 4), Mbnl2 CKO (2KO, n = 4) and Mbnl1/2 DKO mice (DKO, n = 3) mice were immunostained with NeuN (A), Foxp1 (D) and Cux1 (G). To determine the neuronal densities, counting squares of 100 μm × 100 μm were assigned to the upper, middle and lower cortical regions, and the numbers of NeuN-, Foxp1- and Cux1-positive neurons within each counting square were quantified (B,E,H). Next, to measure the distribution of immunolabeled cortical neurons, the thickness of the cortex was equally divided into 10 counting bins with the width of 50 μm, and the percentages of immunolabeled neurons were quantified (C,F,I). Bar = 100 μm. Results are mean ± SEM. Asterisk (*), hash (#) or and (&) indicate significant differences between Con and 2KO mice, Con and DKO, 2KO and DKO, respectively (*,#,&p < 0.05; **,##,&&p < 0.01; ***,###,&&&p < 0.001; two-tailed t-test).
Figure 3
Figure 3
Densities and distribution of GABAergic interneurons in Mbnl mutant mice. Coronal sections containing the sensorimotor cortex of male adult control (Con, n = 4), Mbnl2 CKO (2KO, n = 4) and Mbnl1/2 DKO mice (DKO, n = 3) mice were immunostained with GAD65/67 (A), Parvalbumin, PV (D) and Calretinin, CR (G). To determine the neuronal densities, counting squares of 100 μm × 100 μm were assigned to the upper, middle and lower cortical regions, and the numbers of GAD65/67-, PV- and CR-positive neurons within each counting square were quantified (B,E,H). Next, to measure the distribution of immunolabeled cortical neurons, the thickness of the cortex was equally divided into 10 counting bins with the width of 100 μm and the percentages of immunolabeled neurons were quantified (C,F,I). Bar = 100 μm. Results are mean ± SEM. Asterisk (*), hash (#) or and (&) indicate significant differences between Con and 2KO mice, Con and DKO, 2KO and DKO, respectively (*,#,&, p < 0.05; **,##p < 0.01).
Figure 4
Figure 4
Dendritic architectures of layer II/III pyramidal neurons in Mbnl mutant mice. Golgi-impregnated layers II/III pyramidal neurons were collected from male adult control (Con), Mbnl2 CKO (2KO) and Mbnl1/2 DKO (DKO) mice and reconstructed (A). The dendritic length (B), number of bifurcation nodes (C), number of terminal ends (D) and number of dendritic segments (E) was measured in both apical and basal dendrites. The complexity of the branching pattern in apical and basal dendrites was examined by counting the number of segments in different orders (F), and using Sholl’s concentric ring method (G). Bar = 20 μm. Data is mean ± SEM. For apical dendrites, n = 21 neurons from four Con mice; 9 neurons from three 2KO mice; 13 neurons from three DKO mice. For basal dendrites, n = 11 neurons from three Cons, 12 neurons from three 2KOs; 15 neurons from three DKOs. Asterisk (*) or hash (#) indicate significant differences between Con and 2KO or Con and DKO, respectively (*,#p < 0.05; **,##p < 0.01; ***,###p < 0.001).
Figure 5
Figure 5
Structural analysis of dendritic spines. Golgi-impregnated dendritic spines in the layers II/III pyramidal neurons in the sensorimotor cortex were imaged and measured. Dendritic segments were obtained from male adult control (Con) and Mbnl mutant mice (2KO and DKO). Dendritic spines were classified into stubby, mushroom, thin and filopodia as illustrated with definitions of head/neck and length/width in upper panel. Representative dendrites with different types of dendritic spines (indicated by arrows) are shown in the lower panel (A). By counting the spine numbers, the density of dendritic spines decreased significantly in the DKO group (B). The frequencies of spines with different configurations were calculated. (C) While the lengths of dendritic spines were similar among three groups (D), the widths of dendritic spines were significantly reduced in 2KO and DKO groups (E). Bar = 5 μm. Data is mean ± SEM. In Con group, 1,145 spines were collected from 25 neurons in three mice; in the 2KO group, 947 spines were obtained from 22 neurons in three mice; in the DKO group, 1,652 spines were collected from 27 neurons in three mice. Asterisk (*), hash (#) or and (&) indicate significant differences between Con and 2KO mice, Con and DKO, 2KO and DKO, respectively (&p < 0.01; ***,###p < 0.001).
Figure 6
Figure 6
Electron microscopic analysis of synaptic ultrastructures in Mbnl KO mouse models. Electron micrographs of the postsynaptic densities (PSDs) in the sensorimotor cortex were collected from male adult control (Con, n = 4), Mbnl2 CKO (2KO, n = 4) and Mbnl1/2 DKO (DKO, n = 3) mice (A). By our definition, the PSD thickness is the distance between local maximum and minimum of grayscale values across a synapse; and the PSD width is the electron dense band along the postsynaptic membrane (B). The PSD thickness depicted in cumulative frequency revealed reduced thickness in the DKO mice (C). The PSD width depicted in cumulative frequency showed significant reduction with the curve shifted to the left in the DKO group (D). Bar = 100 nm. Data are mean ± SEM. Hash (#) or and (&) indicate significant differences between Con and DKO, 2KO and DKO, respectively (#p < 0.05).
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