Phenotype of mice following oligodendrocyte-specific deletion of <i>Npc1</i>.

<p>(A, B) Weight curves for male (A) and female (B) mice. Data are mean +/− SD. (C) Age-dependent performance on balance beam. Data are mean +/− SD. * <i>p</i><0.05, *** <i>p</i><0.001.</p

Loss of myelin proteins and Purkinje cell degeneration in aged oligodendrocyte-specific null mutants.

<p>(A) Western blots of myelin-specific proteins from brainstem, cerebral cortex and cerebellar homogenates of 23-week-old <i>Npc1^flox/−, CNP^Cre/+</i> mice and controls. GAPDH controls for loading. (B) MBP immunofluorescence in cerebellar lobules III–VI of <i>Npc1^flox/−, CNP^Cre/+</i> and control mice at 7 & 23 weeks. Bar, 500 µm. (C, D) (C) Olig2 immunofluorescence (to mark oligodendrocyte lineage cells) in the cerebellar white matter of <i>Npc1^flox/−, CNP^Cre/+</i> mice and controls at 23 weeks. Schematic is shown on the left. Quantification of Olig2⁺ cell number is shown in (D). Data are mean +/− SD. n.s., not significant. Bar, 200 µm. (E) Calbindin immunofluorescence in the cerebellum of <i>Npc1^flox/−, CNP^Cre/+</i> mice and controls at 7 & 23 weeks. Roman numerals indicate cerebellar lobules. Bar, 500 µm. (F) Western blots of calbindin from cerebellar homogenates of <i>Npc1^flox/−, CNP^Cre/+</i> mice and controls at 7 & 23 weeks. GAPDH controls for loading. (G) Quantification of Purkinje cell density in midline cerebellar lobules at 23 weeks. Data are mean +/− SD. * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001, n.s., not significant.</p

Forebrain dysmyelination in mice following neuron-specific deletion of <i>Npc1</i>.

<p>(A) Schematic of midline sagittal section of the mouse brain, with the area shown in panel B highlighted by the black rectangle. Illustration is from <a href="http://www.gensat.org" target="_blank">www.gensat.org</a>. (B) (Top three panels) MBP immunofluorescence in forebrain sagittal sections of <i>Npc1^flox/−, Syn1-Cre⁺</i> and control mice at P16, and at 7 & 16 weeks. Bar, 500 µm. Area highlighted by white rectangle is enlarged in the inset in the upper right of each panel. (Bottom panel) FluoroMyelin staining of the corpus callosm of <i>Npc1^flox/−, Syn1-Cre⁺</i> and control mice at 16 weeks. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (C) Western blots of myelin-specific proteins and neurofilament protein (NF-200) from brainstem and cerebral cortex homogenates of P16 <i>Npc1^flox/−, Syn1-Cre⁺</i> mice and controls. GAPDH controls for loading. (D) MBP and neurofilament protein (NF) co-staining of P16 <i>Npc1^flox/−, Syn1-Cre⁺</i> and littermate control mice. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (E) Electron microscopy of the corpus callosum of P16 <i>Npc1^flox/−, Syn1-Cre</i> and control mice. Bar, 500 nm.</p

Npc1 Acting in Neurons and Glia Is Essential for the Formation and Maintenance of CNS Myelin

<div><p>Cholesterol availability is rate-limiting for myelination, and prior studies have established the importance of cholesterol synthesis by oligodendrocytes for normal CNS myelination. However, the contribution of cholesterol uptake through the endocytic pathway has not been fully explored. To address this question, we used mice with a conditional null allele of the <i>Npc1</i> gene, which encodes a transmembrane protein critical for mobilizing cholesterol from the endolysosomal system. Loss of function mutations in the human <i>NPC1</i> gene cause Niemann-Pick type C disease, a childhood-onset neurodegenerative disorder in which intracellular lipid accumulation, abnormally swollen axons, and neuron loss underlie the occurrence of early death. Both NPC patients and <i>Npc1</i> null mice exhibit myelin defects indicative of dysmyelination, although the mechanisms underlying this defect are incompletely understood. Here we use temporal and cell-type-specific gene deletion in order to define effects on CNS myelination. Our results unexpectedly show that deletion of <i>Npc1</i> in neurons alone leads to an arrest of oligodendrocyte maturation and to subsequent failure of myelin formation. This defect is associated with decreased activation of Fyn kinase, an integrator of axon-glial signals that normally promotes myelination. Furthermore, we show that deletion of <i>Npc1</i> in oligodendrocytes results in delayed myelination at early postnatal days. Aged, oligodendocyte-specific null mutants also exhibit late stage loss of myelin proteins, followed by secondary Purkinje neuron degeneration. These data demonstrate that lipid uptake and intracellular transport by neurons and oligodendrocytes through an Npc1-dependent pathway is required for both the formation and maintenance of CNS myelin.</p></div

Oligodendrocyte-specific deletion of <i>Npc1</i> leads to blockade of oligodendrocyte maturation.

<p>(A) NG2 (to mark OPCs) and CC1 (to mark mature oligodendrocytes) staining in the cortex and corpus callosum of P16 <i>Npc1^flox/−, CNP^Cre/+</i> and control mice. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (B) Western blot of NG2 expression levels from cerebral cortex homogenates of P16 <i>Npc1^flox/−, CNP^Cre/+</i> mice and controls. GAPDH controls for loading. (C) Quantification of CC1⁺ cell number in the corpus callosum of P16 <i>Npc1^flox/−, CNP^Cre/+</i> mice and controls. Data are mean +/− SD. * <i>P</i><0.05.</p

Forebrain dysmyelination in mice with oligodendrocyte-specific deletion of <i>Npc1</i>.

<p>(A) (Top panel) FluoroMyelin staining of the corpus callosum of <i>Npc1^flox/−, CNP^Cre/+</i> and control mice at P16. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (Bottom two panels) MBP immunofluorescence in forebrain sagittal sections of <i>Npc1^flox/−, CNP^Cre/+</i> mice and controls at P16 and 7 weeks. Bar, 500 µm. Area highlighted by white rectangle is enlarged in the inset in the upper right of each panel. (B) Western blots of myelin-specific proteins and neurofilament protein (NF-200) from brainstem and cerebral cortex homogenates of P16 <i>Npc1^flox/−, CNP^Cre/+</i> mice and controls. GAPDH controls for loading. (C) MBP and neurofilament protein (NF) co-staining in the corpus callosum of P16 <i>Npc1^flox/−, CNP^Cre/+</i> and control mice. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 200 µm. (D) Electron microscopy of the corpus callosum of a P16 <i>Npc1^flox/−, CNP^Cre/+</i> and control mice. Bar, 50 nm.</p

Comparison of Strength Reduction Method for Slope Stability Analysis Based on ABAQUS FEM and FLAC(3D) FDM

Strength reduction method is widely used in the slope stability analysis. However, it is short of unified instability evaluation standard at present. And the different numerical calculation methods also influence the safety factor of strength reduction method. Taking a typical slope in this paper, the same model meshes are established in ABAQUS FEM and FLAC3D FDM by using the self-compiled model transformation program ABAQUS-FLAC3D. Then the same elastic-plastic constitutive and yield criterion are both employed in ABAQUS FEM and FLAC3D FDM. The safety factors obtained from the two numerical calculation methods are compared, and the results are also compared with that of Spencer limit equilibrium method. It is observed the safety factors calculated by ABAQUS FEM are slightly higher than that of FLAC3D FDM for the same instability evaluation standards. Moreover, the safety factors obtained from the run-through of plastic zone and the saltation of the displacement at characteristic point are much closer to that of Spencer limit equilibrium method. Hence the combination of the run-through of plastic zone and the saltation of the displacement at characteristic point as the slope instability evaluation standard is suggested in this paper. Meanwhile, the correctness of self-compiled improved strength reduction method is verified by comparing with the result of FLAC3D built-in strength reduction method

Neuron-specific deletion of <i>Npc1</i> leads to blockade of oligodendrocyte maturation.

<p>(A) NG2 (to mark OPCs) and CC1 (to mark mature oligodendrocytes) staining in the corpus callosum of P16 <i>Npc1^flox/−, Syn1-Cre⁺</i> and control mice. Bar, 200 µm. (B) Western blot of NG2 expression levels from cerebral cortex homogenates of P16 <i>Npc1^flox/−, Syn1-Cre⁺</i> mice and controls. GAPDH controls for loading. (C) Quantification of CC1⁺ cell number in the corpus callosum of P16 <i>Npc1^flox/−, Syn1-Cre⁺</i> mice and controls. Data are mean +/− SD. *** <i>P</i><0.001. (D) Sip1 and CC1 co-staining in the corpus callosum of a P16 <i>Npc1^flox/−, Syn1-Cre⁺</i> and control mice. Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 100 µm. (E) Cerebral cortex homogenates of P16 control (lane 1) and <i>Npc1^flox/−, Syn1-Cre⁺</i> mice (lane 2) were subject to immunoprecipitation with an anti-Fyn antibody. The resulting lysates were probed for total Fyn, and for Fyn phosphorylated at tyrosine 420 (active form) or tyrosine 531 (inactive form).</p

The effect of timing of <i>Npc1</i> deletion on CNS myelination.

<p>(A) MBP immunofluorescence in brain midline sagittal sections of 7-week-old WT (top), 7-week-old <i>Npc1^Δ/−</i> (middle), and 22-week-old <i>Npc1^flox/−, Cre-ER^{TM +}</i> mice following tamoxifen injections at 6 weeks (bottom). Bar, 1 mm. (B) FluoroMyelin staining of forebrain regions of 20-week-old <i>Npc1^flox/+, Cre-ER^{TM +}</i> control following tamoxifen injections at 6 weeks (top), 7-week-old <i>Npc1^Δ/−</i> (middle), and 22-week-old <i>Npc1^flox/−, Cre-ER^{TM +}</i> mice following tamoxifen injections at 6 weeks (bottom). Ctx, cortex; CC, corpus callosum; Hp, hippocampus. Bar, 500 µm. (C, D) Western blots of CNP, MBP and neurofilament protein (NF-200) expression levels from cerebral cortex (C) and cerebellar (D) homogenates of 22-week-old <i>Npc1^flox/−, Cre-ER^{TM +}</i> mice and their littermate controls following tamoxifen injections at 6 weeks. GAPDH controls for loading.</p

Raman Study on the G Mode of Graphene for Determination of Edge Orientation

We report a confocal Raman study on edges of single-layer graphene. It is found that edge orientations could be identified by G mode in addition to D mode. We observe that G mode at the edges of single-layer graphene exhibits polar behaviors and different edges such as zigzag- or armchair-dominated responses differently than the polarization of the incident laser. Moreover, G mode shows stiffening at zigzag-dominated edges, while it is softened at armchair-dominated ones. Our observations are in good agreement with recent theory (Sasaki, K. et al. J. Phys. Soc. Jpn. 2010, 79, 044603) and could be well-explained by the unique properties of pseudospin at graphene edges, which lead to asymmetry of Raman active modes and non-adiabatic processes (Kohn Anomaly) at different types of edges. This work could be useful for further study on the properties of the graphene edge and development of graphene-based devices