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Fig. 3 | Molecular Neurodegeneration

Fig. 3

From: 17q21.31 sub-haplotypes underlying H1-associated risk for Parkinson’s disease are associated with LRRC37A/2 expression in astrocytes

Fig. 3

LRRC37A/2 expression is associated with increased cellular migration, chemotaxis and inflammation. A-B. Significantly enriched A. Migration of cells and B. Chemotaxis pathways associated with LRRC37A/2 overexpression, derived from Ingenuity pathway analysis. Red genes indicate upregulation, green indicates downregulation C. qRT-PCR for LRRC37A expression in H1 and H2 homozygote iPSC-derived neurons and astrocytes. N = 3 in duplicate. ns = not significant, *p < 0.05. D. Western blots for LRRC37A/2 in cytosolic (C) and membrane (M) fractions from (top) iPSC-neurons and (bottom) iPSC-astrocytes. Cytosolic fractions were confirmed by labeling with an anti-HSP90 antibody, and membrane fractions were confirmed by labeling with an anti-Pan-Cadherin antibody, N = 6. E. Proportion of LRRC37A2-expressing astrocytes by 17q21.31 haplotype from human prefrontal cortex snuc-seq data [33]. Dot size = proportion of cells expressing LRRC37A2, depth of color = LRRC37A2 expression level. F. Representative immunofluorescence images of iPSC-derived astrocytes expressing either scrambled control or LRRC37A2 shRNA lentivirus constructs, labeled with antibodies against either IL-16 or IL-32. Scale bar = 10 µm. G. Quantification of mean grey intensity of astrocytes in immunofluorescence images represented in F. Each point represents the average of 12–16 individual images from an individual cell line. Four cell lines were analyzed. Points from the same iPSC line are connected between scrambled and LRRC37A2 shRNA conditions. Overall p-value indicates paired t-test of cell line image means. Asterisks denote significant between scrambled and LRRC37A2 conditions for each paired line. *p < 0.05, ***p < 0.001, ns = not significant. H. Wound confluence (%) of scratch wound repair over 50 h in scrambled control (dashed lines) and LRRC37A2 shRNA (solid lines) treated iPSC-derived astrocytes treated with either 200 ng/ml α-synuclein monomers (orange) or PBS (green). Statistical difference between groups over time was determined by ANCOVA and post-hoc Tukey tests. *p < 0.05, ***p < 0.001. N = 4. I. Representative images with quantification masks of iPSC-astrocytes at 0 h,16 h and 24 h following induction of a scratch wound in scrambled control (top) and LRRC37A2 shRNA (bottom) treated cells, with (right panels) and without (left panels) incubation with 200 ng/ml α-synuclein. Yellow masks indicate cell confluence, blue indicates the scratch wound area, and grey indicates cellular migration into the original wound area. Scale bar = 600 µm

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