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1.
Figure 6

Figure 6. PLP-LMNB1Tg mice exhibit demyelination, aberrant myelin formation, and axonal degeneration. . From: Lamin B1 mediates cell-autonomous neuropathology in a leukodystrophy mouse model.

(A and B) PLP-LMNB1Tg animals exhibited demyelination compared with WT controls, quantified by g ratio of 150–200 axons, P < 0.05. (C and D) PLP-LMNB1Tg animals showed aberrant myelin formation and axonal degeneration. (E and F) Demyelinating axons (e.g., arrow) and axons with normal myelin thickness (e.g., arrowhead) were frequently observed in PLP-LMNB1Tg mice (E) but not in WT controls (F). Myelin-axon structure also appeared irregular and nonuniform in PLP-LMNB1Tg mice compared with WT controls. n = 4 animals per genotype. Scale bars: 2 μm.

Mary Y. Heng, et al. J Clin Invest. 2013 June 3;123(6):2719-2729.
2.
Figure 1

Figure 1. Generation of Lmnb1BAC mice. . From: Lamin B1 mediates cell-autonomous neuropathology in a leukodystrophy mouse model.

(A) Map of the genomic insert in the BAC RP23-460J18 clone (RPCI library, C57BL/6J) used to generate mice with increased dosage of lamin B1 (Lmnb1BAC mice). The genomic insert is 177.20-kb long and contains the entire Lmnb1 locus. Black boxes represent lamin B1–coding sequences, and light gray lines indicate the BAC vector (pBACe3.6) sequence. Primers used for detection of the transgene are shown (T7 side: F1R1; SP6 side: F2, R2). (B) Representative Western blot of lamin B1 and α-tubulin in hemibrain lysates from Lmnb1BAC lines and WT mice at 12 months of age. (C) Quantification of Western blots normalized to α-tubulin. (D) qRT-PCR of lamin B1 transcript levels normalized to GAPDH from corresponding Lmnb1BAC mice hemibrains. Error bars represent mean ± SEM from 4 independent experiments.

Mary Y. Heng, et al. J Clin Invest. 2013 June 3;123(6):2719-2729.
3.
Figure 8

Figure 8. Lamin B1 overexpression results in reduced binding occupancy of YY1 to Plp1 promoter. . From: Lamin B1 mediates cell-autonomous neuropathology in a leukodystrophy mouse model.

(A) Quantitative ChIP coupled to PCR revealed a reduction in YY1 binding to the Plp1 promoter in hindbrains from 12-month-old Lmnb1BAC mice compared with WT controls normalized to 2% input. Primers were designed to amplify 150–200 bp containing either a consensus YY1-binding site or 3′ UTR. 3′ UTR and IgG were used as controls. (B) Representative blot of YY1 total protein levels normalized to α-tubulin in hindbrains of 12-month-old Lmnb1BAC and WT mice. (C) Quantification of YY1 protein level relative to α-tubulin was found not to be markedly different between Lmnb1BAC and WT mice. n = 4 per group of at least 2 independent experiments. P > 0.05.

Mary Y. Heng, et al. J Clin Invest. 2013 June 3;123(6):2719-2729.
4.
Figure 3

Figure 3. Lamin B1 overexpression results in seizures.. From: Lamin B1 mediates cell-autonomous neuropathology in a leukodystrophy mouse model.

Spontaneous EEG activity over the parietal cortex of Lmnb1BAC and WT mice was recorded for 24 hours. (A) EEG recordings from the left and right hemisphere, respectively, are shown for a representative transgenic mouse overexpressing lamin B1 (top traces) or a WT control (bottom traces). Arrowheads mark interictal spikes. (B) Quantification of interictal spikes over 24-hour recordings indicates the occurrence of spontaneous epileptiform activity in Lmnb1BAC mice specifically. n = 10 animals per genotypic group. (C) Histogram quantifying percentage of animals seizing after the administration of 30 mg/kg of PTZ. n = 9 animals per genotypic group. Values are expressed as means ± SEM. *P < 0.05.

Mary Y. Heng, et al. J Clin Invest. 2013 June 3;123(6):2719-2729.
5.
Figure 7

Figure 7. Lamin B1 overexpression results in the decrease of the highly abundant myelin protein PLP.. From: Lamin B1 mediates cell-autonomous neuropathology in a leukodystrophy mouse model.

(A) Quantitative mass spectrometry iTRAQ labeling of hindbrains from 12-month-old Lmnb1BAC mice and WT mice identified PLP at 95% confidence interval of high-quality scoring peptide candidates to be 30% downregulated in Lmnb1BAC animals compared with WT controls. (B) This finding was validated by qRT-PCR. (C) Mice overexpressing lamin B1 specifically in oligodendrocytes (PLP-LMNB1Tg) exhibited a further reduction in Plp1 transcript levels. Values are expressed as means ± SEM. n = 3 per group. **P < 0.01; ***P < 0.001. (D) Transcriptome analysis performed on purified oligodendrocytes validated proteomic changes including decreased Pol II binding to the Plp1 promoter in Lmnb1BAC mice compared with WT controls. Lamin B1 also regulates additional genes. x axis defined as M: log differential-expression ratio (log fold change); y axis defined as A: average log intensity (reads per kb of gene length) from oligodendrocytes between Lmnb1BAC mice and WT controls.

Mary Y. Heng, et al. J Clin Invest. 2013 June 3;123(6):2719-2729.
6.
Figure 5

Figure 5. Lamin B1 overexpression in oligodendrocytes results in progressive motor impairment and myelin deficits.. From: Lamin B1 mediates cell-autonomous neuropathology in a leukodystrophy mouse model.

(A) Representative Western blot for lamin B1, FLAG-tagged lamin B1, and α-tubulin in hemibrain lysates from transgenic mouse lines overexpressing lamin B1 selectively in oligodendrocytes, neurons, and astrocytes driven by cell-type–specific promoters (Plp1, Camk2a, and GFAP, respectively) vs. control mice at 12 months of age (lanes are discontinuous). Dual color detection with IR fluorescence for antibody against lamin B1 (green) and antibody against FLAG-tagged lamin B1 (red; top panel). Individual detection against FLAG (second panel), lamin B1 (third panel), and α-tubulin (bottom panel). (B) Quantification of Western blots of total lamin B1 normalized to α-tubulin. (C) qRT-PCR of lamin B1 transcript levels normalized to GAPDH from corresponding cell-specific hemibrains at 12 months of age. Error bars represent mean ± SEM from 3–4 independent experiments. (DF) Animals overexpressing lamin B1 selectively in oligodendrocytes (PLP-LMNB1Tg) exhibited progressive motor deficits on the accelerated rotarod and balance beam. (D) PLP-LMNB1Tg mice had shortened latency to fall on the accelerated rotarod and (E) exhibited increased latency to traverse an 11-mm–wide balance beam with (F) hind limb slips. Values are expressed as means ± SEM, n = 10–12 per group. ***P < 0.001.

Mary Y. Heng, et al. J Clin Invest. 2013 June 3;123(6):2719-2729.
7.
Figure 2

Figure 2. A mouse model of ADLD exhibited cognitive and motor deficits.. From: Lamin B1 mediates cell-autonomous neuropathology in a leukodystrophy mouse model.

Lmnb1BAC and WT mice were behaviorally tested to assess spatial and fear memory in the MWM and PA tests and motor functions in the rotarod and balance beam tests. (A and B) Lmnb1BAC mice exhibited spatial memory deficits in the MWM at 12 month of age. (A) Lmnb1BAC mice spent substantially less time in the target quadrant and (B) made less platform area crossings compared with WT during the probe trial. (C) Lmnb1BAC mice failed to recall an aversive experience made on the previous day on the PA test and exhibited decreased step-through latencies compared with WT mice at 12 months of age. The bars indicate the mean latencies to enter the dark compartment on the training day (white) and 24 hours later on the retention day (black). (DF) Progressive deterioration of motor functions in Lmnb1BAC mice compared with WT mice, (D) Lmnb1BAC mice spent less time on the accelerated rotarod across all 8 trials at 24 but not at 12 months of age compared with WT. (E) Lmnb1BAC mice showed increased latency to traverse a 5-mm–wide balance beam at 12 months and progressively worsened at 24 month. (F) Lmnb1BAC mice also exhibited increased hind limb slips compared with WT controls. Values are expressed as mean ± SEM. n = 10–14 per genotypic group. *P < 0.05; **P < 0.01; ***P < 0.001.

Mary Y. Heng, et al. J Clin Invest. 2013 June 3;123(6):2719-2729.
8.
Figure 4

Figure 4. Lamin B1 overexpression results in aberrant myelin formation, axonal degeneration, and demyelination.. From: Lamin B1 mediates cell-autonomous neuropathology in a leukodystrophy mouse model.

Ultrastructural analysis was performed on pyramidal tracts of 12- and 24-month-old Lmnb1BAC and corresponding WT mice. (A and B) g ratio analyses at 12 and (C and D) 24 months, at which time demyelination is prominent in Lmnb1BAC mice. P < 0.05. (E) WT controls at 12 months. (F and G) Ultrastructural analyses revealed numerous aberrant structures, which included outfoldings, extensions, and invaginations originating from the myelin sheath at 12 months and (H) at 24 months. (I and J) Axonal disintegration and degradation of the entire axon-myelin unit was observed at a significantly higher level at 12 months and (M and N) at 24 months compared with WT controls. (K and L) Hypermyelinated fibers and dying axons were also observed in Lmnb1BAC mice at 12 months of age. (O) Normal oligodendrocyte and (Q) normal compact myelin at 24 months of age in control animals. (P) Representative of frequent dying and degenerating oligodendrocytes in 24-month-old Lmnb1BAC mice. Dying oligodendrocytes correlate with increased (R) irregular and demyelinated fibers in 24-month-old Lmnb1BAC animals. (S and T) Quantification of approximately 150 to 200 axons for 12-month-old Lmnb1BAC mice exhibited significant axonal degeneration and aberrant myelin formations compared with WT controls. n = 3 animals per genotype. Values are expressed as means ± SEM. **P < 0.01, ***P < 0.001. Scale bars: 2 μm.

Mary Y. Heng, et al. J Clin Invest. 2013 June 3;123(6):2719-2729.

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