Fig. 7.

mTORC1 activity regulates PARK7 protein expression in PSD and protein synthesis in dendrites. A, DAVID clustering results for PARK7 terminal hub proteins. PARK7 associates with proteins that are involved in translation, ribonucleoprotein (RNP) complex biogenesis, and ribosome assembly. The abscissa accounts the number of proteins that have been shown to associate with PARK7 for each of the GO biological process (ordinate) (supplemental Table S10). B, Heatmap for PARK7 in PSD (P) and soluble (S) fractions. Heatmap is shown as log₂ fold-change. In PSD, PARK7 is an out-of-range protein with fold-change of 0/Control (red textured). C, mTORC1 inhibition for 1h by intraperitoneal injection of rapamycin reduces PARK7 levels in PSD-enriched fraction of rat cortices, similar to MS/MS data in B. Top: representative Western blots. Bottom: densitometric analysis of Western blots. (DMSO = 1.00 ± 0.06; Rapamycin, RAPA, = 0.24 ± 0.10; n = 3 animals per condition; **, p < 0.002). D, Representative images of cultured neurons treated with carrier (DMSO, left) and rapamycin (200 nm, right) showing differential expression of PARK7 (red), PSD-95 (green), and MAP2 (blue). Scale bar 20 μm. E, Blown up dendrites indicated by dotted box in D, of PARK7 (top), PSD-95 (middle) and merged image showing colocalization (bottom). White arrows indicate PARK7 that colocalize with the postsynaptic protein PSD-95. Blue arrows indicate PARK7 that colocalize with the presynaptic protein synapsin. F, Same dendrite as in D, representative staining of synapsin (top) colocalized with PARK7 (bottom, merged). Scale bar = 5 μm. G, Normalized PCC averaged over many dendrites for PARK7 and PSD-95 or PARK7 and synapsin. All raw PCC values were normalized to the average PCC between PARK7 and synapsin in control condition (DMSO, D). Dotted line indicates the average normalized PCC of PARK7 and synapsin in DMSO. Note, PCC of PARK7 with PSD-95 (DMSO, D, = 1.47 ± 0.08, n = 24 dendrites; Rapamycin, R, = 1.17 ± 0.05, n = 24 dendrites) and synapsin (DMSO = 1.00 ± 0.08, n = 24 dendrites; Rapamycin = 0.67 ± 0.06, n = 24 dendrites) decreased at 1 hour after mTORC1 inhibition. Statistics: * and **, significantly different by one-way ANOVA, Tukey's multiple comparison; ^#, significantly different by single t test from PCC of PARK7 with synapsin in DMSO. H, Representative images of newly synthesized PARK7 protein, as determined by BONCAT-PLA, when mTORC1 is active (left, top) or repressed (right, top) in or near MAP2-positive dendrites (middle panels). Arrows indicate new PARK7 protein. Overlay images of new PARK7 proteins and MAP2 (bottom panels). Scale = 5 μm. See supplemental Fig. S7. I, Rapamycin reduces new PARK7 (DMSO = 1.00 ± 0.11, n = 26 dendrites; RAPA = 0.76 ± 0.05, n = 36 dendrites). Statistics: *, p < 0.05; Student's t test. Chicken anti-MAP2 was imaged with 405 nm laser (see “Experimental Procedures”).