Figure 4

Acute inhibition of USP14 causes synaptic transmission deficits at the adult NMJ. (A) Example traces of MEPCs recorded from diaphragm muscle fibers isolated from 4- to 6-week-old wild type (n = 10 endplates, 4 mice) and TgUsp14CA (n = 7 endplates, 3 mice) muscles in the presence of vehicle or the USP14 inhibitor IU1. Quantitation of (B) MEPC frequency, (C) amplitude, and (D) decay constants in muscles from wild type and TgUsp14CA muscles before (-) and after (+) 5 μM IU1 application. Quantitation of (E) EPC amplitude and (F) quantal content in wild type muscles (n = 10 endplates, 4 mice) before (-) and after (+) addition of 5 μM IU1. In (B)-(F), symbols represent Wilcoxon matched-pairs signed rank tests, where *p < 0.05 (G) Schematic of experimental design, where adult wild type mice (12- to 14-weeks-old) were given intramuscular injections of 100 μL of 100 μM IU1 or vehicle every other day for 1 week, and (H) the abundance of AChR subunit mRNA in gastrocnemius muscles was compared using qPCR. Data are shown as mean ± SEM and were normalized to vehicle treatment. n = 3 muscles per condition, run in triplicate. (I) Whole mount immunostaining of gastrocnemius muscles from wild type mice given intramuscular injections of IU1 or vehicle as described above. Endplates were labeled with rhodamine-conjugated α-BTX, and motor neurons were visualized via expression of YFP under the Thy1.2 promoter. Scale bars = 50 μm.