Fig. 7. Metallothioneins are implicated in GWA studies and promote neuronal viability.

A Genome-wide Association study (GWAS) of residual age of motor onset in HD. This study combines data from the Venezuelan Kindreds and GeM-HD consortium of patients. A LocusZoom plot of the metallothionein (MT) gene cluster (box) on chromosome 16 is shown, depicting the SNPs in the association analysis on chr16 and the log-transformed associated Wald’s p-value (y-axis) measured using a mixed linear model. SNPs are color-coded by the r2 of linkage disequilibrium (LD) with the representative SNP rs74611520 (diamond). Two neighboring SNPs (rs2518054, rs3812963) in high linkage disequilibrium are indicated by red triangles and outlined by dotted ellipse. B Glutamate levels in media conditioned by MT3 astrocytes normalized by levels in control human astrocytes (HA). Paired one-tailed t-test with N = 3 experiments. p = 0.0011. C A cartoon depiction of the design of the astrocyte-neuron co-culture viability experiment. Neurons (GFP−) and astrocytes (GFP+) were separated using flow cytometry-activated sorting (FACS). Cd: Cadmium. D Bar plots of the viability of FACS sorted MT3 overexpressing astrocytes normalized by viability of control GFP astrocytes under the indicated conditions. n = 3–4 independent biological replicates as indicated. Paired one-tailed t-tests. E Bar plots of FACS sorted murine neuronal viability when co-cultured with MT3 overexpressing astrocytes normalized by neuronal viability of sorted neurons co-cultured with control GFP+ astrocytes. Treatment conditions as per D. n = 3–4 independent biological replicates as indicated. Paired one-tailed t-tests. P values are indicated. F Expression of Annexin V and Caspase 3/7 in HD-derived directly reprogrammed SPNs co-cultured with control (GFP) versus MT3 astrocytes at 30 and 32 days in co-culture. The values are expressed as fold change from control. N = 3 biological replicates. The p values are indicated. One-tailed one-sample t-test. G Example of Annexin V signal in control versus HD derived directly reprogrammed SPNs co-cultured with control astrocytes at day 30 demonstrating significant neurodegeneration in HD co-cultures evidenced by the increase in Annexin V signal. N = 4 and 6 technical replicates for control and HD, respectively, two-tailed unpaired t-test, the p values are indicated. For B, D–G, the data are shown as mean ± SEM. H Summary of our understanding of astrocytic states and regional heterogeneity in HD as it relates to neuroprotection. Panels C and H were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.