Journal: bioRxiv

Article Title: Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS

doi: 10.1101/2023.12.13.571549

Figure Lengend Snippet: Effects of HDAC inhibitors, arimoclomol and combined treatments on HSPA1A in motor neurons expressing TDP-43 G348C . A Phase contrast image of spinal cord – DRG culture. Arrows point to motor neurons. B Small but significant increase in the percentage of motor neurons with HSPA1A immunoreactivity three days following microinjection of plasmid encoding TDP-43 G348C , compared to absence of labeling in neurons injected with mCherry (control). C-F Percentage of HSPA1A immunopositive motor neurons after three days of treatment with vehicle, the HDAC inhibitors C SAHA, D RGFP109, E RGFP963 or F Tubastatin A, or with arimoclomol alone or in combination with the respective HDAC inhibitor. HDAC inhibitors with class I activity ( C-E ) significantly increased the percentage of neurons expressing HSPA1A. Arimoclomol and the HDAC6 inhibitor Tubastatin A were ineffective. Data presented as mean ± S.D., n = 9 cultures. Statistical significance was evaluated by one-way ANOVA followed by Bonferroni post hoc analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bar = 20μm.

Article Snippet: Primary antibodies were: rabbit anti-TDP-43 (EPR18554 1:500 Abcam, Cambridge, UK), rabbit anti-FUS (11570-1-AP 1:300, Proteintech, Rosemont, IL, USA); mouse antibody specific for human SOD1 (SD-G6 1:100, Millipore Sigma), Mouse anti-human HSP70 specific for stress-inducible HSPA1A (SMC-100B 1:100, Stressmarq Biosciences Inc., Victoria, BC, Canada), mouse anti-HSPA8/HSC71/Hsc70 (13D3) antibody (NB120-2788 1:300, Novus Biologicals, Colorado, USA), mouse anti-FLAG M2 (F1804 1:400, Millipore Sigma), rabbit anti-acetylated H3K9/K14 (9677 1:400, Cell Signaling, Danvers, MA, USA), rabbit anti-Brg1 (21634-1-AP 1:600, Proteintech), rabbit anti-MAP2 (AB5622 1:500, Millipore Sigma), chicken anti-GFP (GFP1010 1:500 AVES, CA, USA).

Techniques: Expressing, Microinjection, Plasmid Preparation, Labeling, Injection, Control, Activity Assay

Journal: bioRxiv

Article Title: Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS

doi: 10.1101/2023.12.13.571549

Figure Lengend Snippet: Hspa1a mRNA in ALS culture models using single molecule fluorescence in situ hybridization (smFISH). Cultures containing motor neurons expressing TDP-43 G348C , FUS R521G or SOD1 G93A were treated with vehicle (DMSO), 4 µM arimoclomol, 1µM RGFP963 or the combination of arimoclomol and RGFP963 (Combo). smFISH was conducted on day three. A The highly expressed Hspa8 mRNA is presented as a reference control for comparison to the minimal labeling of Hspa1a mRNA in B,C,D . B No significant impact of TDP-43 G348C or drug treatments on the number of Hspa1a mRNA spots. C Scarcity of Hspa1a mRNA spots in neurons expressing FUS R521G ; no significant effect of drug treatments. D Low numbers of Hspa1a mRNA spots in neurons expressing SOD1 G93A ; a small but significant increase in a subset of neurons by arimoclomol and RGFP963 treatments. Data are presented as mean ± S.D. n = 9-24 neurons per group. E-G Number of transcription sites by smFISH. E Example of H spa1a transcription site labeling. F,G Effect of ALS variants and combination drug treatment on the number of Hspa1a transcription sites on F day one and G day three following microinjection of expression vectors. n = 6-30 neurons per group. Statistical significance was evaluated through one-way ANOVA followed by Bonferroni post hoc analysis. *p<0.05. Scale bar = 15μm.

Article Snippet: Primary antibodies were: rabbit anti-TDP-43 (EPR18554 1:500 Abcam, Cambridge, UK), rabbit anti-FUS (11570-1-AP 1:300, Proteintech, Rosemont, IL, USA); mouse antibody specific for human SOD1 (SD-G6 1:100, Millipore Sigma), Mouse anti-human HSP70 specific for stress-inducible HSPA1A (SMC-100B 1:100, Stressmarq Biosciences Inc., Victoria, BC, Canada), mouse anti-HSPA8/HSC71/Hsc70 (13D3) antibody (NB120-2788 1:300, Novus Biologicals, Colorado, USA), mouse anti-FLAG M2 (F1804 1:400, Millipore Sigma), rabbit anti-acetylated H3K9/K14 (9677 1:400, Cell Signaling, Danvers, MA, USA), rabbit anti-Brg1 (21634-1-AP 1:600, Proteintech), rabbit anti-MAP2 (AB5622 1:500, Millipore Sigma), chicken anti-GFP (GFP1010 1:500 AVES, CA, USA).

Techniques: Fluorescence, In Situ Hybridization, Expressing, Control, Comparison, Labeling, Microinjection