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ANA_22337_sm_suppinfofigures.doc695KFigure S1: Microglial morphology in control and demyelinated MS hippocampus. Modest microglial activation was seen in control (a) and demyelinated MS hippocampus (b) when stained with Iba1 antibody. Figure S2: mRNA encoding myelin proteins are significantly decreased in demyelinated MS Hippocampus. mRNA encoding proteolipid protein (PLP), myelin basic protein (MBP), 2′,3′-cyclic nucleotide 3′ phosphodiesterase (CNP), myelin oligodendrocyte protein (MOG), oligodendrocyte myelin glycoprotein (OMGP), and myelin-associated oligodendrocyte basic protein (MOBP) are significantly decreased in MS demyelinated hippocampus (n=4) compared to myelinated hippocampus (n=4). White bars: myelinated MS; black bars: demyelinated MS. Error: ±S.E.M; *p<0.05, ** P<0.005, ***p<0.0005, ****p<0.00005 Figure S3: Neuronal housekeeping genes are not altered in demyelinated hippocampi. (a) Normalized intensity of mRNAs encoding TAU, neurofilament-heavy chain (NF-H), neurofilament-medium chain (NF-M), neurofilament-light chain (NF-L) and MAP2 were similar between MS myelinated (n=4) and MS demyelinated (n=4) hippocampus. (b) Protein levels of the neuronal markers, HuR and NF-L, were also similar between control, myelinated MS (Mye) and demyelinated MS (Demye) hippocampi. (c) Significant decrease in mRNA's encoding NF-H, NF-M and NF-L were reported in severe AD samples compared to moderate AD samples. Data retrieved from Gene Expression Omnibus (GEO) using accession number GDS810 (18). Error: ±S.E.M; *p<0.05 Figure S4: kinesin-mediated transport mRNAs were similar in MS demyelinated motor cortex. Intensity values of mRNAs encoding Kinesin 1A (KIF1A), Kinesin 3A (KIF3A), Kinesin 5B (KIF5B), Kinesin 5C (KIF5C), Kinesin 15 (KIF15) and Kinectin 1 (KTN1) were similar between control (white bars), myelinated MS (gray bars) and demyelinated MS (black bars) motor cortex samples (n=6). Error bars indicate S.E.M. Figure S5: mRNA encoding synaptic proteins were significantly decreased in MS emyelinated hippocampus. RT-PCR analysis detected decreased levels of mRNAs encoding post synaptic density protein 95 (PSD95), calmodulin-associated serine/threonin kinase (CASK), Neurexin 1 (NRXN1) and Neuroligin 1 (NLG1) in demyelinated MS (n=5, black bars) hippocampus compared to myelinated MS (n=5, gray bars) and control (n=5, white) hippocampus. Ratio of threshold cycles (Ct) for individual genes normalized to GAPDH (delta Ct) are plotted. Error Bars: ±S.E.M; *p<0.05, ** P<0.005, ***p<0.0005. Figure S5: mRNA encoding synaptic proteins were significantly decreased in MS demyelinated hippocampus. RT-PCR analysis detected decreased levels of mRNAs encoding post synaptic density protein 95 (PSD95), calmodulin-associated serine/threonin kinase (CASK), Neurexin 1 (NRXN1) and Neuroligin 1 (NLG1) in demyelinated MS (n=5, black bars) hippocampus compared to myelinated MS (n=5, gray bars) and control (n=5, white) hippocampus. Ratio of threshold cycles (Ct) for individual genes normalized to GAPDH (delta Ct) are plotted. Error Bars: ±S.E.M; *p<0.05, ** P<0.005, ***p<0.0005. Figure S7: GABA receptors were increased in MS demyelinated hippocampi (a) mRNAs encoding GABA receptors GABAβ3, GABAγ2 GABAα5 and GABA synthesizing enzyme, GAD67, were significantly increased in microarrays and by quantitative RT-PCR. mRNA levels of GABAα1 were however decreased in MS demyelinated hippocampus. (b) Protein levels of GABAα5 was also increased in MS demyelinated hippocampus. Error Bars: ±S.E.M; *p<0.05
ANA_22337_sm_suppinfotables.doc148KSupplemental Table S1: Demographic of MS and Control Patients Supplemental Table S2: Primer Sequences Supplemental Table S3: List of Antibodies

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