Image_2_Differential impacts of Cntnap2 heterozygosity and Cntnap2 null homozygosity on axon and myelinated fiber development in mouse.TIF

Over the last decade, a large variety of alterations of the Contactin Associated Protein 2 (CNTNAP2) gene, encoding Caspr2, have been identified in several neuronal disorders, including neurodevelopmental disorders and peripheral neuropathies. Some of these alterations are homozygous but most are heterozygous, and one of the current challenges is to estimate to what extent they could affect the functions of Caspr2 and contribute to the development of these pathologies. Notably, it is not known whether the disruption of a single CNTNAP2 allele could be sufficient to perturb the functions of Caspr2. To get insights into this issue, we questioned whether Cntnap2 heterozygosity and Cntnap2 null homozygosity in mice could both impact, either similarly or differentially, some specific functions of Caspr2 during development and in adulthood. We focused on yet poorly explored functions of Caspr2 in axon development and myelination, and performed a morphological study from embryonic day E17.5 to adulthood of two major brain interhemispheric myelinated tracts, the anterior commissure (AC) and the corpus callosum (CC), comparing wild-type (WT), Cntnap2–/– and Cntnap2+/– mice. We also looked for myelinated fiber abnormalities in the sciatic nerves of mutant mice. Our work revealed that Caspr2 controls the morphology of the CC and AC throughout development, axon diameter at early developmental stages, cortical neuron intrinsic excitability at the onset of myelination, and axon diameter and myelin thickness at later developmental stages. Changes in axon diameter, myelin thickness and node of Ranvier morphology were also detected in the sciatic nerves of the mutant mice. Importantly, most of the parameters analyzed were affected in Cntnap2+/– mice, either specifically, more severely, or oppositely as compared to Cntnap2–/– mice. In addition, Cntnap2+/– mice, but not Cntnap2–/– mice, showed motor/coordination deficits in the grid-walking test. Thus, our observations show that both Cntnap2 heterozygosity and Cntnap2 null homozygosity impact axon and central and peripheral myelinated fiber development, but in a differential manner. This is a first step indicating that CNTNAP2 alterations could lead to a multiplicity of phenotypes in humans, and raising the need to evaluate the impact of Cntnap2 heterozygosity on the other neurodevelopmental functions of Caspr2.</p

Image_1_Differential impacts of Cntnap2 heterozygosity and Cntnap2 null homozygosity on axon and myelinated fiber development in mouse.TIF

Over the last decade, a large variety of alterations of the Contactin Associated Protein 2 (CNTNAP2) gene, encoding Caspr2, have been identified in several neuronal disorders, including neurodevelopmental disorders and peripheral neuropathies. Some of these alterations are homozygous but most are heterozygous, and one of the current challenges is to estimate to what extent they could affect the functions of Caspr2 and contribute to the development of these pathologies. Notably, it is not known whether the disruption of a single CNTNAP2 allele could be sufficient to perturb the functions of Caspr2. To get insights into this issue, we questioned whether Cntnap2 heterozygosity and Cntnap2 null homozygosity in mice could both impact, either similarly or differentially, some specific functions of Caspr2 during development and in adulthood. We focused on yet poorly explored functions of Caspr2 in axon development and myelination, and performed a morphological study from embryonic day E17.5 to adulthood of two major brain interhemispheric myelinated tracts, the anterior commissure (AC) and the corpus callosum (CC), comparing wild-type (WT), Cntnap2–/– and Cntnap2+/– mice. We also looked for myelinated fiber abnormalities in the sciatic nerves of mutant mice. Our work revealed that Caspr2 controls the morphology of the CC and AC throughout development, axon diameter at early developmental stages, cortical neuron intrinsic excitability at the onset of myelination, and axon diameter and myelin thickness at later developmental stages. Changes in axon diameter, myelin thickness and node of Ranvier morphology were also detected in the sciatic nerves of the mutant mice. Importantly, most of the parameters analyzed were affected in Cntnap2+/– mice, either specifically, more severely, or oppositely as compared to Cntnap2–/– mice. In addition, Cntnap2+/– mice, but not Cntnap2–/– mice, showed motor/coordination deficits in the grid-walking test. Thus, our observations show that both Cntnap2 heterozygosity and Cntnap2 null homozygosity impact axon and central and peripheral myelinated fiber development, but in a differential manner. This is a first step indicating that CNTNAP2 alterations could lead to a multiplicity of phenotypes in humans, and raising the need to evaluate the impact of Cntnap2 heterozygosity on the other neurodevelopmental functions of Caspr2.</p

Data_Sheet_1_Differential impacts of Cntnap2 heterozygosity and Cntnap2 null homozygosity on axon and myelinated fiber development in mouse.PDF

Over the last decade, a large variety of alterations of the Contactin Associated Protein 2 (CNTNAP2) gene, encoding Caspr2, have been identified in several neuronal disorders, including neurodevelopmental disorders and peripheral neuropathies. Some of these alterations are homozygous but most are heterozygous, and one of the current challenges is to estimate to what extent they could affect the functions of Caspr2 and contribute to the development of these pathologies. Notably, it is not known whether the disruption of a single CNTNAP2 allele could be sufficient to perturb the functions of Caspr2. To get insights into this issue, we questioned whether Cntnap2 heterozygosity and Cntnap2 null homozygosity in mice could both impact, either similarly or differentially, some specific functions of Caspr2 during development and in adulthood. We focused on yet poorly explored functions of Caspr2 in axon development and myelination, and performed a morphological study from embryonic day E17.5 to adulthood of two major brain interhemispheric myelinated tracts, the anterior commissure (AC) and the corpus callosum (CC), comparing wild-type (WT), Cntnap2–/– and Cntnap2+/– mice. We also looked for myelinated fiber abnormalities in the sciatic nerves of mutant mice. Our work revealed that Caspr2 controls the morphology of the CC and AC throughout development, axon diameter at early developmental stages, cortical neuron intrinsic excitability at the onset of myelination, and axon diameter and myelin thickness at later developmental stages. Changes in axon diameter, myelin thickness and node of Ranvier morphology were also detected in the sciatic nerves of the mutant mice. Importantly, most of the parameters analyzed were affected in Cntnap2+/– mice, either specifically, more severely, or oppositely as compared to Cntnap2–/– mice. In addition, Cntnap2+/– mice, but not Cntnap2–/– mice, showed motor/coordination deficits in the grid-walking test. Thus, our observations show that both Cntnap2 heterozygosity and Cntnap2 null homozygosity impact axon and central and peripheral myelinated fiber development, but in a differential manner. This is a first step indicating that CNTNAP2 alterations could lead to a multiplicity of phenotypes in humans, and raising the need to evaluate the impact of Cntnap2 heterozygosity on the other neurodevelopmental functions of Caspr2.</p

Organelle and Cellular Abnormalities Associated with Hippocampal Heterotopia in Neonatal <i>Doublecortin</i> Knockout Mice

<div><p>Heterotopic or aberrantly positioned cortical neurons are associated with epilepsy and intellectual disability. Various mouse models exist with forms of heterotopia, but the composition and state of cells developing in heterotopic bands has been little studied. <i>Dcx</i> knockout (KO) mice show hippocampal CA3 pyramidal cell lamination abnormalities, appearing from the age of E17.5, and mice suffer from spontaneous epilepsy. The <i>Dcx</i> KO CA3 region is organized in two distinct pyramidal cell layers, resembling a heterotopic situation, and exhibits hyperexcitability. Here, we characterized the abnormally organized cells in postnatal mouse brains. Electron microscopy confirmed that the <i>Dcx</i> KO CA3 layers at postnatal day (P) 0 are distinct and separated by an intermediate layer devoid of neuronal somata. We found that organization and cytoplasm content of pyramidal neurons in each layer were altered compared to wild type (WT) cells. Less regular nuclei and differences in mitochondria and Golgi apparatuses were identified. Each <i>Dcx</i> KO CA3 layer at P0 contained pyramidal neurons but also other closely apposed cells, displaying different morphologies. Quantitative PCR and immunodetections revealed increased numbers of oligodendrocyte precursor cells (OPCs) and interneurons in close proximity to <i>Dcx</i> KO pyramidal cells. Immunohistochemistry experiments also showed that caspase-3 dependent cell death was increased in the CA1 and CA3 regions of <i>Dcx</i> KO hippocampi at P2. Thus, unsuspected ultrastructural abnormalities and cellular heterogeneity may lead to abnormal neuronal function and survival in this model, which together may contribute to the development of hyperexcitability.</p></div

Morphological features of the two <i>Dcx</i> KO CA3 neuronal layers at P0.

<p>(A–D). Coronal semi-thin sections of the CA3 hippocampal region in wild-type (A, C) and KO (B, D) mice. (A, B) Low magnifications showing the CA3 hippocampal region. The area encompassed by the two vertical white lines indicates the region visualized in C and D. (C, D) Higher magnifications showing nuclei in the CA3 pyramidal layers (delimited by black dotted lines). In WT (C) the pyramidal neurons contain nuclei with a similar aspect. In KO (D), two pyramidal cell layers are visualized, an external layer (SPE) closest to the neuroepithelium bounding the <i>stratum oriens</i> (SO), and an internal layer (SPI) in the <i>stratum pyramidale</i> region but positioned closer to the <i>stratum radiatum</i>. They are separated by an intermediary layer (IN) with a neuropil-like aspect. Each KO pyramidal cell layer contains nuclei with different shapes and sizes. Scale bars A, B: 150 µm; C, D: 15 µm. (E) Measurements of nuclear diameter. Nuclear diameters from the SPI and SPE layers in KO mice are significantly different from each other (Mann-Whitney test, <i>p</i><0.0001). SPE nuclei are significantly smaller than WT (Mann-Whitney test, <i>p</i><0.0001) and SPI nuclei not significantly different. Nuclear diameters from WT and KO (not distinguishing between SPE and SPI), also differ significantly (Mann-Whitney test, <i>p</i><0.0001).</p

<i>Dcx</i> KO CA3 layers show heterogeneous and disorganized cells compared to WT.

<p>Electron micrographs of the hippocampus from WT (A–B) and KO mice (C–E). (A) In WT, the broad layer of pyramidal neurons (delimited by dashed black lines) shows a more regular arrangement of adjacent columns of neurons (marked by circles). A layer bordering the <i>stratum oriens</i> (SO) displays neurons with occasional oval elongated shaped nuclei. All nuclei are surrounded by a light cytoplasm. (B) High magnification of pyramidal neurons displaying rounded or oval nuclei, with a homogeneous aspect of the nuclear membrane. (C) In the KO, the SPE bounds the SO (lower dashed black line) and the SPI is limited by the <i>stratum radiatum</i> (SR) (upper dashed black line). In both the SPE and SPI layers two types of cells are shown. One exhibits a clear cytoplasm and large nuclei (black asterisks) with round, elongated or lobulated shapes, enclosing light chromatin with peripherally located nucleoli, similar in aspect to the neuronal chromatin observed in WT. The second type of cell (white asterisks) has a darker cytoplasm, occasional tapered processes and encloses nuclei with dense chromatin. They are intermingled and often closely juxtaposed with neuronal-like cells. (D) In the SPI, two types of cells are shown, pyramidal neuronal-like cells with a clear cytoplasm and peripheral nucleoli (black asterisk) and a second cell type with a darker cytoplasm whose nucleoli are rarely peripheral (white asterisk). (E) In the SPE similar cell types with light (black asterisk) and dark cytoplasm (white asterisks) are also identified. Arrows in D and E indicate the irregular shape of the nuclear membrane. Scale bars: A, C: 5 µm; B, D, E: 2.5 µm.</p

qPCR results (mean expression values, expressed in relative units) for <i>Olig</i>1 and <i>Npy</i>.

<p>qPCR results (mean expression values, expressed in relative units) for <i>Olig</i>1 and <i>Npy</i>.</p

Ultrastructural organization of the intermediary layer (IN).

<p>(A) Low magnification showing the IN delimited by black dashed lines. Cells from the SPI (black symbols #) and SPE (black asterisks) flank numerous neuritic profiles enclosed in the IN layer. The arrows, arrowhead and ‘1’ correspond to zones enlarged in C–E respectively. (B) Higher magnification of the IN. Numerous dense cellular profiles are visualized, corresponding to neuronal processes (likely to be immature dendrites and axons) and synaptic contacts (see ‘E’). A neurite from a cell located in the SPE (black asterisk) extending to and juxtaposing (arrows) the soma from a cell in the SPI (black symbol #). (C) High magnification of the neuropil-like region indicated by two arrows in A and B. Note the close apposition between a probable dendritic profile from an SPE cell and an SPI soma (black symbol #). (D) Higher magnification of arrowhead in A. The cellular profiles of the IN display heterogeneous aspects: some of them with a light cytoplasm correspond to transversal sections of neurites, and contain tubulo-vesicular structures and regions without cytoskeletal elements (arrowhead). (E) is a detail of an axo-somatic synaptic contact identified in A and B as ‘1’. Some synaptic vesicles are observed (arrow). (F, G) Neurites of hippocampal pyramidal neurons. (F) A neurite from a WT pyramidal cell, longitudinally sectioned, contains numerous adjacent microtubules closely arranged in parallel fascicles (arrowhead). (G) In KO, a neurite from an SPI pyramidal neuron shows sparser, more widely-spaced microtubules (arrowhead). Scale bars: A, 2 µm; B, C, D: 1 µm; E–G: 0.5 µm.</p

<i>Sst</i> interneurons in the region of the <i>stratum pyramidale</i> of WT and <i>Dcx</i> KO hippocampi.

<p>(A–H) Immunoreactivity to <i>Sst</i> transcripts in WT (A, C, E, G) and KO (B, D, F, H) mouse brain coronal sections. Representative images showing <i>in situ</i> hybridization results at two levels of the dorsal hippocampus (A–D; E–H). C, D and G, H are higher magnifications of A, B and E, F respectively. In WT, <i>Sst</i>-positive interneurons are mainly found outside the <i>stratum pyramidale</i> in the <i>stratum oriens</i> and <i>radiatum</i> at this age. In the <i>Dcx</i> KO, some <i>Sst</i>-positive cells are observed associated with the SPI, IN and occasionally the SPE. Scale bars, A (for A, B, E, F) 100 µm; C (for C, D, G, H) 50 µm.</p