Journal: Molecular Neurobiology

Article Title: Gene Co-expression Analysis of the Human Substantia Nigra Identifies ZNHIT1 as an SNCA Co-expressed Gene that Protects Against α-Synuclein-Induced Impairments in Neurite Growth and Mitochondrial Dysfunction in SH-SY5Y Cells

doi: 10.1007/s12035-022-02768-9

Figure Lengend Snippet: Identification of SNCA co-expressed genes in the human SN reveals enrichment of genes involved in histone deacetylation, and altered ZNHIT1-SNCA co-expression in the SN in PD. A Venn diagram showing the overlap between genes with positive significant co-expression with SNCA in the SN (green), hippocampus (red) and occipital cortex (blue), after Bonferroni correction. This analysis identified n = 303 SNCA co-expressed (SNCA_coEx) genes that were unique to the SN. B Venn diagram showing the overlap between SNCA_coEx genes (green) and genes co-expressed with the dopaminergic markers TH (red) and ALDH1A1 (blue) in the SN. This analysis identified n = 223 SNCA co-expressed (SNCA_coEx) genes that overlapped with TH and ALDH1A1 co-expressed genes. In A and B the total number of genes in each case is shown in parentheses. Raw data was derived from GSE60863. C The list of n = 223 genes were classified using the Panther classification tool ( http://pantherdb.org ), and the table shows the list of functional and/or structural gene classes that were removed from the n = 223 genes to generate a final list of n = 125 SNCA_coEx genes in the SN. D Results of the gene ontology (GO) enrichment analysis of the n = 125 genes, which was performed using STRING ( https://string-db.org ). The p value was corrected for multiple testing using the Benjamini–Hochberg procedure. E Table showing the changes in the co-expression pattern ( r2 – r 1) of SNCA with ZNHIT1 , HDAC5 , HDAC6 , SAP18 and MORF4L1 in control ( n = 8) and PD ( n = 15) SN samples. F Linear regression analysis showing the correlations between ZNHIT1 and SNCA in control ( n = 8), Braak stage 1/2 ( n = 5), Braak stage 3/4 ( n = 7) and Braak stage 5/6 ( n = 8) SN samples. The r values are shown on each linear regression graph. Raw data was derived from GSE49036. All analyses were performed using the R2 genomics analysis and visualisation platform ( https://hgserver1.amc.nl/cgi-bin/r2/main.cgi )

Article Snippet: A Oxygen consumption rate (OCR) in the Seahorse XF Mito Stress Test in SH-SY5Y cells stably expressing GFP or α-Syn and transfected for 72 h with 500 ng of a plasmid expressing either FLAG (Addgene #31,385) or FLAG-tagged ZNHIT1 (Addgene #15,332).

Techniques: Expressing, Derivative Assay, Functional Assay

Journal: Molecular Neurobiology

Article Title: Gene Co-expression Analysis of the Human Substantia Nigra Identifies ZNHIT1 as an SNCA Co-expressed Gene that Protects Against α-Synuclein-Induced Impairments in Neurite Growth and Mitochondrial Dysfunction in SH-SY5Y Cells

doi: 10.1007/s12035-022-02768-9

Figure Lengend Snippet: ZNHIT1 increases incorporation of the histone variant H2A.Z in SH-SY5Y cells and this is unaffected by α -synuclein overexpression. A – F SH-SY5Y cells were transfected with 500 ng of plasmid expressing either FLAG (Addgene #31,385) or FLAG-tagged ZNHIT1 (Addgene #15,332), together with a plasmid expressing either GFP or GFP-tagged wild-type α-synuclein (αSyn) (Addgene #40,824). All analyses were performed at 72 h post-transfection. A – F Graphs and representative photomicrographs of A , B H2A.Z, C , D H3 and E , F acetylated (ac)H3 levels in transfected (GFP +) cells. All data are mean ± SEM expressed as a percentage of the control (FLAG + GFP) from n = 3–6 independent experiments. Two-way ANOVA with post hoc Fishers LSD test (** p < 0.01, *** p < 0.001 vs. FLAG + GFP)

Article Snippet: A Oxygen consumption rate (OCR) in the Seahorse XF Mito Stress Test in SH-SY5Y cells stably expressing GFP or α-Syn and transfected for 72 h with 500 ng of a plasmid expressing either FLAG (Addgene #31,385) or FLAG-tagged ZNHIT1 (Addgene #15,332).

Techniques: Variant Assay, Over Expression, Transfection, Plasmid Preparation, Expressing

Journal: Molecular Neurobiology

Article Title: Gene Co-expression Analysis of the Human Substantia Nigra Identifies ZNHIT1 as an SNCA Co-expressed Gene that Protects Against α-Synuclein-Induced Impairments in Neurite Growth and Mitochondrial Dysfunction in SH-SY5Y Cells

doi: 10.1007/s12035-022-02768-9

Figure Lengend Snippet: ZNHIT1 promotes neurite growth and protects against α -synuclein-induced reductions in neurite growth and cell viability in SH-SY5Y cells. A Representative photomicrographs, B graph of neurite length and C graph of cell viability of SH-SY5Y cells transfected with 500 ng of a plasmid expressing either FLAG (Addgene #31,385) or FLAG-tagged ZNHIT1 (Addgene #15,332), together with a plasmid expressing either GFP or GFP-tagged wild-type α-synuclein (αSyn) (Addgene #40,824). All analyses were performed at 72 h post-transfection. D , E Graph and representative photomicrographs showing the relative levels of ZNHIT1 (red) in transfected GFP + cells (green). F , G Graph and representative photomicrographs showing the relative levels of αSyn (red) in transfected GFP + cells (green). All data are mean ± SEM expressed as a percentage of the control (FLAG + GFP) from n = 3–6 independent experiments. Two-way ANOVA with post hoc Fishers LSD test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. FLAG + GFP or as indicated)

Article Snippet: A Oxygen consumption rate (OCR) in the Seahorse XF Mito Stress Test in SH-SY5Y cells stably expressing GFP or α-Syn and transfected for 72 h with 500 ng of a plasmid expressing either FLAG (Addgene #31,385) or FLAG-tagged ZNHIT1 (Addgene #15,332).

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: Molecular Neurobiology

Article Title: Gene Co-expression Analysis of the Human Substantia Nigra Identifies ZNHIT1 as an SNCA Co-expressed Gene that Protects Against α-Synuclein-Induced Impairments in Neurite Growth and Mitochondrial Dysfunction in SH-SY5Y Cells

doi: 10.1007/s12035-022-02768-9

Figure Lengend Snippet: Bioinformatics analysis implicates ZNHIT1 co-expressed genes in the regulation of mitochondrial function. A Protein–protein interaction (PPI) network generated from the list of genes that were strongly co-expressed ( r > 0.7) with ZNHIT1 in the human SN. B Table showing the significantly enriched gene ontology (GO) biological processes (bp) categories resulting from a gene set enrichment analysis of ZHNIT1 co-expressed genes. Raw data was derived from GSE:60,863. Gene co-expression analysis was performed using the R2 genomics analysis and visualisation platform ( https://hgserver1.amc.nl/cgi-bin/r2/main.cgi ). The PPI network and GO analysis were performed using STRING ( https://string-db.org )

Article Snippet: A Oxygen consumption rate (OCR) in the Seahorse XF Mito Stress Test in SH-SY5Y cells stably expressing GFP or α-Syn and transfected for 72 h with 500 ng of a plasmid expressing either FLAG (Addgene #31,385) or FLAG-tagged ZNHIT1 (Addgene #15,332).

Techniques: Generated, Derivative Assay, Expressing

Journal: Molecular Neurobiology

Article Title: Gene Co-expression Analysis of the Human Substantia Nigra Identifies ZNHIT1 as an SNCA Co-expressed Gene that Protects Against α-Synuclein-Induced Impairments in Neurite Growth and Mitochondrial Dysfunction in SH-SY5Y Cells

doi: 10.1007/s12035-022-02768-9

Figure Lengend Snippet: Bioenergetic state analysis shows that overexpression of ZNHIT1 prevents α-synuclein-induced impairments in mitochondrial function. A Oxygen consumption rate (OCR) in the Seahorse XF Mito Stress Test in SH-SY5Y cells stably expressing GFP or α-Syn and transfected for 72 h with 500 ng of a plasmid expressing either FLAG (Addgene #31,385) or FLAG-tagged ZNHIT1 (Addgene #15,332). B – G Graphs of individual parameters of respiration showing B basal respiration, C proton leak, D maximal respiration, E non-mitochondrial respiration, F ATP production, G spare respiratory capacity and H coupling efficiency. Data are mean ± SEM of OCR values normalised to protein content per well from n = 4 independent replicates. Two-way ANOVA or mixed effects model and post hoc Fisher’s LSD test (* p < 0.05, ** p < 0.01 vs FLAG + GFP)

Article Snippet: A Oxygen consumption rate (OCR) in the Seahorse XF Mito Stress Test in SH-SY5Y cells stably expressing GFP or α-Syn and transfected for 72 h with 500 ng of a plasmid expressing either FLAG (Addgene #31,385) or FLAG-tagged ZNHIT1 (Addgene #15,332).

Techniques: Over Expression, Stable Transfection, Expressing, Transfection, Plasmid Preparation