Noncanonical Modulation of the eIF2 Pathway Controls an Increase in Local Translation during Neural Wiring
Roberta Cagnetta, Hovy Ho-Wai Wong, Christian K. Frese, Giovanna R. Mallucci, Jeroen Krijgsveld, Christine E. Holt
Molecular Cell 73, 1–16. February 7, 2019 a 2018 The Authors. Published by Elsevier Inc. https://doi.org/10.1016/j.molcel.2018.11.013
- eIF2α phosphorylation underlies Sema3A-induced upregulation of global translation
- pSILAC-SP3 reveals 75 nascent proteins regulated by the Sema3A-p-eIF2α pathway
- eIF2B modulation is the key node between Sema3A and canonical stress responses
- PERK signaling is required for neural wiring
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Local translation is rapidly regulated by extrinsic sig- nals during neural wiring, but its control mechanisms remain elusive. Here we show that the extracellular cue Sema3A induces an initial burst in local transla- tion that precisely controls phosphorylation of the translation initiation factor eIF2a via the unfolded protein response (UPR) kinase PERK. Strikingly, in contrast to canonical UPR signaling, Sema3A- induced eIF2a phosphorylation bypasses global translational repression and underlies an increase in local translation through differential activity of eIF2B mediated by protein phosphatase 1. Ultrasen- sitive proteomics analysis of axons reveals 75 proteins translationally controlled via the Sema3A- p-eIF2a pathway. These include proteostasis- and actin cytoskeleton-related proteins but not canonical stress markers. Finally, we show that PERK signaling is needed for directional axon migration and visual pathway development in vivo. Thus, our findings reveal a noncanonical eIF2 signaling pathway that controls selective changes in axon translation and is required for neural wiring.
Figure 1. eIF2a Phosphorylation Underlies Sema3A-Induced Upregulation of Axonal Protein Synthesis
(A and B) IF representative images (A) and quantification (B) for total-eIF2a and p-eIF2a in growth cones treated with Tg (15 min) or Sema3A (10 min) (unpaired t test).
(C and D) IF representative images (C) and quantification (D) for puromycin in growth cones incubated with puromycin and co-treated with Tg (15 min) or Sema3A (10 min) and ISRIB (one-way ANOVA with Bonferroni’s multiple comparisons test).
Error bars indicate SEM. Scale bars, 5 mm. See also Figure S1.
Figure 2. pSILAC-SP3 Reveals 75 Nascent Proteins Regulated by the Sema3A-p-eIF2a Pathway
(A) Schematic of the pSILAC-SP3 methodology applied to somaless retinal axons.
(B) Subset of NSPs regulated in response to Sema3A by p-eIF2a. Only significant NSP changes are shown (blue, downregulation; red, upregulation; p < 0.10). (C) KEGG pathway analysis (red, upregulated pathway; blue, downregulated pathway; cutoff R 2 proteins per pathway).
Figure 6. PERK Signaling Is Required for Visual Pathway Development In Vivo
(A) Experimental outline to investigate the contribution of axonal PERK in RGCs only and Slit1 in the optic tract pathway substrate. Unilateral MO injection leads to targeted KD in half of the nervous system.
(B) Schematic of axons navigating the optic tract and reaching the tectum. TPB, tectal posterior boundary; TAB, tectal anterior boundary; TPA tectal projection angle; MDT, mid-diencephalic turn; A, anterior; P, posterior; OC, optic chiasm; Tec, tectum; Di, diencephalon; Hy, hypothalamus; Tel, telencephalon.
(C–G) Representative images of DiI-filled stage 41 retinotectal projections in Control MO (C), unilateral KD of PERK in the axons (D), unilateral KD of Slit1 in the optic tract substrate (E), or both (F and G) (Ax, axon; Br, brain).
(H) Cumulative distribution of MDT angle measurements in unilateral KD of PERK in the axons or Slit1 in the optic tract substrate or both (one-way ANOVA with Bonferroni’s multiple comparisons test).
(I) Penetrance for MDT angles of less than 45 (Fisher’s exact test).
(J) Cumulative distribution of TPA measurements in unilateral KD of PERK in the axons or Slit1 in the optic tract substrate or both. Positive values indicate angles pointing toward the TPB, and negative values indicate angles pointing toward the TAB (one-way ANOVA with Bonferroni’s multiple comparisons test).
(K) Penetrance of posterior tectum avoidance, measured as TPA < mean TPA in CoMO (i.e., 8.6) (Fisher’s exact test).
(L) Single RGC axons in the tectum and line drawings of the corresponding trajectories shown by color-coded branch order: white, axon shaft; branches: red, primary; blue, secondary; yellow, tertiary.
(M) Number of axon branches in the various orders and total number of branches in the PERK morphants (two-way ANOVA).
(N) Length of axon branches in the PERK morphants (unpaired t test).
(O) Formulation of axon complexity index (ACI). Color indicates the branch order as in (L).
(P) ACI in the PERK morphants (Fisher’s exact test).
Error bars indicate SEM. Scale bars, 100 mm (C–G) and 20 mm (L). See also Figure S6.
Adapted with permission from Elsevier Inc. on behalf of Molecular Cell: Cagnetta et al. (2018). Molecular Cell 73, 1–16. February 7, 2019 a 2018 The Authors. Published by Elsevier Inc. https://doi.org/10.1016/j.molcel.2018.11.013. Copyright (2018).
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