Journal: The Journal of Neuroscience

Article Title: Defects of Lipid Synthesis Are Linked to the Age-Dependent Demyelination Caused by Lamin B1 Overexpression

doi: 10.1523/JNEUROSCI.1668-15.2015

Figure Lengend Snippet: Plp-FLAG-LMNB1 TG mice show accurately targeted exogenous lamin B1 expression and age-dependent motor dysfunction. A, Representative immunoblot of TG and WT protein lysates from different CNS regions showed the presence of the FLAG tagged LMNB1 band (arrow) that migrated slightly higher than the endogenous LMNB1 protein. This higher band and the band in the FLAG immunoblot were present only in the TG samples. FB, Forebrain; BS, brainstem; CB, cerebellum; SC, spinal cord. B, Quantitation of immunoblot data revealed overexpression of LMNB1 was highest in the spinal cord. The ratio of the top lamin B1 band intensity to the total lamin B1 (both top and bottom) band intensities were plotted for the TG samples. Data plotted were mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; n = 3 TG animals per CNS region. C, IHC from brain sections of TG mice showed the colocalization of FLAG (red) and LMNB1 (green) staining. Colocalization was also observed with FLAG (red) and the oligodendrocyte maker, CC1 (green). However, no colocalization is observed between FLAG (red) with the neuronal marker, NeuN (green), and astrocyte marker GFAP (green). Scale bar, 50 μm. D, WT 13-month-old mice showed no obvious phenotype, whereas TG littermate showed kyphosis (arrow) and forelimb atrophy (arrowhead). E, Open-field activity analysis shows a significant reduction in average velocity in the 13-month-old TG mice. Data plotted are mean ± SEM; ***p < 0.001; n = 3–4 animal per genotype per time point.

Article Snippet: The following antibodies were used in this study: FLAG (F7425, F4042 both from Sigma-Aldrich), Lamin B1 (ab16048, Abcam; 33-2000, Invitrogen; sc-6217, Santa Cruz Biotechnology), APC/CC-1 (OP 80, Millipore), GFAP (MAB360, Millipore), Iba-1 (019-19741, Wako), NeuN (MAB377, Millipore), APP (512700, Invitrogen), PLP (MAB388, Millipore), MBP (sc-13914, Santa Cruz Biotechnology; MAB384, Millipore), MAG (34-6200, Invitrogen), MOG (sc-73330, Santa Cruz Biotechnology), CNPase (NE1020, Millipore), β-actin (MA515739, Pierce), GAPDH (MA515738, Pierce), acetylated histone 3 (Millipore 06-599, 1:200); acetylated histone 4 (Millipore 06-866, 1:200), tri-methyl K9 histone 3 (Abcam ab8898, 1:400); tri-methyl K27 histone 3 (Millipore 07-449, 1:200), CC1 (APC, Calbiochem OP-80, 1:50); HMGCR (Abcam, ab174830), and FASN (Abcam, ab22759).

Techniques: Expressing, Western Blot, Quantitation Assay, Over Expression, Staining, Marker, Activity Assay

Journal: The Journal of Neuroscience

Article Title: Defects of Lipid Synthesis Are Linked to the Age-Dependent Demyelination Caused by Lamin B1 Overexpression

doi: 10.1523/JNEUROSCI.1668-15.2015

Figure Lengend Snippet: Altered age-dependent oligodendrocyte-specific chromatin modifications in Plp-FLAG-LMNB1 TG mice. A–D, Confocal images of spinal cord sections from WT and TG mice at 3 months (3 mo) and 13 months (13 mo) stained with antibodies against different histone modifications in oligodendrocytes. In all cases, the histone antibodies are in green, the oligodendrocyte marker CC1 in red, and DAPI in blue. For each figure, bottom shows a quantitative plot of the immunoreactivity at different time points (data plotted are mean ± SEM). H3K9me3 staining (A) and HeK27me3 staining (B). Note that both these repressive marks show significant increases in TG samples at both the 13 and 3 month time points relative to WT. AcH3 staining (C) and AcH4 staining (D). Note that both these activating histone marks show a reduction in TG samples relative to WT at the 13 month time point. For all graphs n = 3–4 animals per genotype at each time point. Data plotted are mean ± SEM; ***p < 0.001.

Article Snippet: The following antibodies were used in this study: FLAG (F7425, F4042 both from Sigma-Aldrich), Lamin B1 (ab16048, Abcam; 33-2000, Invitrogen; sc-6217, Santa Cruz Biotechnology), APC/CC-1 (OP 80, Millipore), GFAP (MAB360, Millipore), Iba-1 (019-19741, Wako), NeuN (MAB377, Millipore), APP (512700, Invitrogen), PLP (MAB388, Millipore), MBP (sc-13914, Santa Cruz Biotechnology; MAB384, Millipore), MAG (34-6200, Invitrogen), MOG (sc-73330, Santa Cruz Biotechnology), CNPase (NE1020, Millipore), β-actin (MA515739, Pierce), GAPDH (MA515738, Pierce), acetylated histone 3 (Millipore 06-599, 1:200); acetylated histone 4 (Millipore 06-866, 1:200), tri-methyl K9 histone 3 (Abcam ab8898, 1:400); tri-methyl K27 histone 3 (Millipore 07-449, 1:200), CC1 (APC, Calbiochem OP-80, 1:50); HMGCR (Abcam, ab174830), and FASN (Abcam, ab22759).

Techniques: Staining, Marker

Journal: The Journal of Neuroscience

Article Title: Defects of Lipid Synthesis Are Linked to the Age-Dependent Demyelination Caused by Lamin B1 Overexpression

doi: 10.1523/JNEUROSCI.1668-15.2015

Figure Lengend Snippet: Plp-FLAG-LMNB1 TG mice do not show significant alterations of myelin-specific proteins. A, Immunoblots of spinal cord protein lysates from 13 month WT and TG animals probed with antibodies against the myelin-specific proteins CNPase, MBP, MOG, MAG, and PLP1. B, Quantitation of immunoblot from A show no significant differences in the levels of these proteins between WT and TG samples. Data plotted are mean band intensities ± SEM, first normalized to loading control and then to WT samples (n = 8 for TG; n = 4 for WT). C, Quantitation of real time PCR analysis of the expression levels of these genes also shows no significant differences between TG and WT samples. Data plotted are mean expression levels ± SEM, first normalized to internal control and then to WT samples (n = 3–4 animals for each genotype).

Article Snippet: The following antibodies were used in this study: FLAG (F7425, F4042 both from Sigma-Aldrich), Lamin B1 (ab16048, Abcam; 33-2000, Invitrogen; sc-6217, Santa Cruz Biotechnology), APC/CC-1 (OP 80, Millipore), GFAP (MAB360, Millipore), Iba-1 (019-19741, Wako), NeuN (MAB377, Millipore), APP (512700, Invitrogen), PLP (MAB388, Millipore), MBP (sc-13914, Santa Cruz Biotechnology; MAB384, Millipore), MAG (34-6200, Invitrogen), MOG (sc-73330, Santa Cruz Biotechnology), CNPase (NE1020, Millipore), β-actin (MA515739, Pierce), GAPDH (MA515738, Pierce), acetylated histone 3 (Millipore 06-599, 1:200); acetylated histone 4 (Millipore 06-866, 1:200), tri-methyl K9 histone 3 (Abcam ab8898, 1:400); tri-methyl K27 histone 3 (Millipore 07-449, 1:200), CC1 (APC, Calbiochem OP-80, 1:50); HMGCR (Abcam, ab174830), and FASN (Abcam, ab22759).

Techniques: Western Blot, Quantitation Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: The Journal of Neuroscience

Article Title: Defects of Lipid Synthesis Are Linked to the Age-Dependent Demyelination Caused by Lamin B1 Overexpression

doi: 10.1523/JNEUROSCI.1668-15.2015

Figure Lengend Snippet: Age-dependent reduction of lipid gene expression in Plp-FLAG-LMNB1 TG mice. A, QT-PCR validation of RNA-Seq data showed that all nine lipid synthesis genes common between 3 and 13 month time points were consistently downregulated in TG samples and showed significant age-dependent decline comparative to age matched WT controls. Expression levels of ApoE and Fasn are also shown. In addition, Srebf2 showed significant age-dependent reductions in expression in TG samples, whereas Srebf1a and 1c show reduction in expression only at earlier time points. Expression levels of TG samples are first normalized to internal control and then to WT controls for each time point. Data plotted are mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; n = 3–4 animals per genotype for each time point. B, Protein levels of HMGCR are significantly reduced in 13 month TG spinal cords, data plotted are mean ± SEM; **p < 0.01; n = 3. C, Oligodendrocyte-specific reductions of FASN expression in 13 month TG spinal cord sections. Arrows point to oligodendrocytes stained with antibodies against CC1 and FASN. Scale bar, 25 μm.

Article Snippet: The following antibodies were used in this study: FLAG (F7425, F4042 both from Sigma-Aldrich), Lamin B1 (ab16048, Abcam; 33-2000, Invitrogen; sc-6217, Santa Cruz Biotechnology), APC/CC-1 (OP 80, Millipore), GFAP (MAB360, Millipore), Iba-1 (019-19741, Wako), NeuN (MAB377, Millipore), APP (512700, Invitrogen), PLP (MAB388, Millipore), MBP (sc-13914, Santa Cruz Biotechnology; MAB384, Millipore), MAG (34-6200, Invitrogen), MOG (sc-73330, Santa Cruz Biotechnology), CNPase (NE1020, Millipore), β-actin (MA515739, Pierce), GAPDH (MA515738, Pierce), acetylated histone 3 (Millipore 06-599, 1:200); acetylated histone 4 (Millipore 06-866, 1:200), tri-methyl K9 histone 3 (Abcam ab8898, 1:400); tri-methyl K27 histone 3 (Millipore 07-449, 1:200), CC1 (APC, Calbiochem OP-80, 1:50); HMGCR (Abcam, ab174830), and FASN (Abcam, ab22759).

Techniques: Expressing, RNA Sequencing Assay, Staining

Journal: The Journal of Neuroscience

Article Title: Defects of Lipid Synthesis Are Linked to the Age-Dependent Demyelination Caused by Lamin B1 Overexpression

doi: 10.1523/JNEUROSCI.1668-15.2015

Figure Lengend Snippet: MALDI imaging of spinal cord and model for disease mechanism in Plp-FLAG-LMNB1 TG mice. A, MALDI lipid imaging of spinal cord sections from 3-month-old TG and WT mice for individual PE and PC molecular species (listed below each image). Images are pseudocolored and the TG intensities have been normalized to maximal WT intensity to allow direct comparison across TG and WT images. Intensities of these lipid species showed reduction in white matter regions of TG spinal cord. For PE images, p16:0/18:1 and p18:0/18:1 refer to the plasmalogen type of fatty acid side chains. In the case of PC (18:1/20:0), (18:0/20:1), both these species are isobaric and cannot be differentiated by MALDI imaging. Scale bar, 500 μm. B, Mechanistic model linking lamin B1 overexpression, chromatin modification, lipid synthesis, and demyelination. This model provides a rationale for the age dependence and white matter involvement in ADLD.

Article Snippet: The following antibodies were used in this study: FLAG (F7425, F4042 both from Sigma-Aldrich), Lamin B1 (ab16048, Abcam; 33-2000, Invitrogen; sc-6217, Santa Cruz Biotechnology), APC/CC-1 (OP 80, Millipore), GFAP (MAB360, Millipore), Iba-1 (019-19741, Wako), NeuN (MAB377, Millipore), APP (512700, Invitrogen), PLP (MAB388, Millipore), MBP (sc-13914, Santa Cruz Biotechnology; MAB384, Millipore), MAG (34-6200, Invitrogen), MOG (sc-73330, Santa Cruz Biotechnology), CNPase (NE1020, Millipore), β-actin (MA515739, Pierce), GAPDH (MA515738, Pierce), acetylated histone 3 (Millipore 06-599, 1:200); acetylated histone 4 (Millipore 06-866, 1:200), tri-methyl K9 histone 3 (Abcam ab8898, 1:400); tri-methyl K27 histone 3 (Millipore 07-449, 1:200), CC1 (APC, Calbiochem OP-80, 1:50); HMGCR (Abcam, ab174830), and FASN (Abcam, ab22759).

Techniques: Imaging, Over Expression, Modification