Figure 3.

ARHGAP24 isoform 1 is an important regulator of neuronal morphology. (a) Schematic diagrams of ARHGAP24 isoforms (upper panels). Magnified area indicates the position of uORF (upstream open-reading frame) in isoform 1. Lower panel displays aligned RNA-SEQ reads (from Control 1 and Patient 1 only, shown as representative) to ARHGAP24 (snap shot from UCSC Genome Browser). (b) ARHGAP24 isoform 1 is a target of NMD. ARHGAP24 isoform 1 was significantly upregulated in HeLa cells depleted of UPF1, UPF2 and SMG1, relative to HeLa cells depleted of Luciferase (negative control). Expression value was measured by reverse-transcriptase quantitative PCR (RT-qPCR) using relative standard-curve method, normalized against ACTB in the same sample, and converted to fold change. *P < 0.05, **P < 0.01 by Student’s two-tailed t-test. (c) ARHGAP24 isoform 1 was significantly upregulated in normal control LCLs (n = 6) after treatment with Cycloheximide (CHX) for 6h. Expression value was measured by RT-qPCR using relative standard-curve method, normalized to ACTB level in the same sample, and converted into fold-change. Result is shown as representative of two independent experiments. *P < 0.05 by Student’s one-tailed t-test. (d) Representative images of day 4 cultures of primary hippocampal neurons exogenously expressing green fluorescent protein (GFP) only (Control) or together with ARHGAP24 isoform1. Upper panels show GFP expression within transfected cells. Bottom panels show same cells stained with MAP2 (red) and Tau1 (cyan) to reveal dendritic and axonal domains, respectively. (e) Ectopic expression of ARHGAP24 isoform 1 inhibits axonal outgrowth. Mean primary axonal length compared at day 4 and 8 of culture. (f) Ectopical expression of ARHGAP24 isoform 1 inhibits neuronal arborization. Total, axonal and dendritic neurite termini compared at day 4 and 8 of culture. Experiments in panels e and f were done in triplicate with at least 25 neurons scored per repeat. *P < 0.05 by Student’s paired two-tailed t-test.