Additional file 4 of Elevated ganglioside GM2 activator (GM2A) in human brain tissue reduces neurite integrity and spontaneous neuronal activity
Creators: name: Hsieh, Yi-Chen
name: Negri, Joseph
name: He, Amy
name: Pearse, Richard V.
name: Liu, Lei
name: Duong, Duc M.
name: Chibnik, Lori B.
name: Bennett, David A.
name: Seyfried, Nicholas T.
name: Young-Pearse, Tracy L.
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Neuroscience
subject: Pharmacology
subject: Mental Health

description: Additional file 4: Supplemental Fig. 1. Effects of human brain extracts on spontaneous firing of cultured cortical neurons are heat labile and are strongest in a high molecular weight fraction. (A) Images of primary rat cortical cultures stained for neuronal markers TAU and DAPI the nuclear marker following 4-day treatment with control brain extract (MGH1887) or AD brain extract (ALB01). Scale bars = 100 μm. (B) Titration of AD brain extract (ALB01, red) compared to an LP-NCI brain extract (MGH1887, blue) at 4-days post-treatment in primary rat cortical neurons. (C) Denaturing extracts by boiling rescues effects on spontaneous firing Spontaneous firing activity within rat cortical cultures following 4-day treatment with native (open points) or boiled (shaded points) human brain extracts. Each point represents a single multi-electrode array, bars represent mean  ± SEM. Significant differences between conditions indicated by brackets and associated p-value. Supplemental Fig. 2. Generation and testing of cell-derived GM2A in neurite integrity assay. (A) Western blot probed for GM2A measuring GM2A levels in TBS-soluble brain extracts from 4 individuals: (1) MGH1887, (2) MGH1837, (3) RM304, and (4) RM305. Serial diluted recombinant GM2A ranging from 0.005 to 0.05 μg served as standards. GM2A is estimated to be ~ 1 ng/uL in the brain extracts. (B) Neurite integrity of Day 21 human iNs treated with 2 or 5 ng/uL recombinant GM2A was comparable to that of untreated iNs (0 ng/uL recombinant GM2A). (C) Western blot probed for GM2A measuring GM2A levels in TBS-soluble protein lysates from HEK293 transfected with GM2A or control cDNA construct. Lysates from 2 independent transfections of the GM2A construct were measured. Serial diluted recombinant GM2A ranging from 0.0025 to 0.01 μg served as standards. Total GM2A (endogenous and GFP-tagged) is estimated to be ~ 25 ng/uL in the TBS-soluble protein lysates from GM2A-transfected HEK293 cells. (D-E) Neurite integrity of Day 21 human iNs treated with 0.25 or 0.5 ng/uL HEK293-derived, TBS-soluble GM2A started to show significant reduction 4 hr. after the treatment (E) and GM2A-mediated reduction of neurite integrity is dose-dependent (D&amp;E). N = 10 for each condition. Statistical analyses used unpaired t-test at each time point with Holm-Šídák method. *, p &lt; 0.05; **, p &lt; 0.01; ****, p &lt; 0.0001. (F-G) Neurite integrity of Day 21 human iNs treated with TBS-soluble protein lysates derived from HEK293 cells transfected with GM2A or control cDNA construct. Neurite integrity assessed every 4 hrs (F) and 24-hr post-treatment (G) are presented. N = 15 for each condition. Error bars represent mean  ± SEM. Statistical analyses used unpaired t-test at each time point with Holm-Šídák method. **, p &lt; 0.01. Supplemental Fig. 3. Expression of synaptic proteins in neurons with elevated GM2A. (A-G) Western blot probed for VGLUT2, HOMER1, GM2A, and GAPDH in Day 21 human iNs overexpressing GM2A or control at MOI = 5 (transduced on Day 5), and in Day 21 human iNs treated with AD brain extract (ALB) or control (treated from Day 17). N = 5 for each condition. (H-L) Images of VGLUT2 and HOMER1 immunostains in Day 21 human iNs overexpressing GM2A or control at MOI = 5 (transduced on Day 5), and in Day 21 human iNs treated with AD brain extract (ALB) or control (treated from Day 17). The insets show the quantified, representative neuritic segments (N = 15 for each condition). Scale bars: 20 μm. Statistical analyses used unpaired t-test. *, p &lt; 0.05; **, p &lt; 0.01; ****, p &lt; 0.0001; ns, not significant.