(a) Schematic of sciatic nerve crush (SNC) and optic nerve crush (ONC). Mouse lumbar level 3 and 4 dorsal root ganglia (DRG) and retina were isolated to assess the neuronal stress response after SNC and ONC, respectively. (b) Microarray cross-comparison of injury-regulated mRNAs following SNC or ONC (n = 5 per condition) identifies multiple ISR-associated genes (blue), including Eif4ebp1, Atf4, Chac1 and Eif2ak3 (PERK), upregulated by both insults. (c–d) mRNAs within the ‘ISR-related’ gene set (see Materials and Methods) are observed more frequently amongst upregulated mRNAs than expected by the overall distribution of mRNA expression changes assessed in each microarray study (‘complete gene set’) following SNC (p=2.4 × 10−5, (c)) or ONC (p=9.9 × 10−7, (d)), suggesting the selective activation of the ISR. (e–f) Immunoblots reveal upregulation of the ISR (p-PERK, p-eIF2α, and ATF4), in addition to the JNK-pathway (p-c-Jun) in L3/L4 DRG lysates after SNC (e), and in retina lysates after ONC (f). The time post-injury is indicated in hours. (g) Primary e12.5 mouse DRG cultures deprived of NGF (3 h) or treated with the ER stress inducer thapsigargin (Tgn) in the vehicle DMSO. NGF deprivation engages phosphorylation of PERK (p-PERK/PERK), p-eIF2α and ATF4. Protein levels were normalized to GAPDH and non-NGF deprived vehicle control (n = 6–7/condition, four independent experiments). (h) siRNA targeting each of the four eIF2α kinases differentially impacts ISR activation at 3 h after NGF withdrawal from embryonic DRG neuronal cultures, with only siRNA targeting Eif2ak3 consistently reducing ATF4 protein levels. (i) Representative TUJ-1 immunostainings 42 hr after isolation and siRNA-transfection of adult DRG neurons. (j) OnTarget Plus siRNA-mediated knockdown of Atf4 or Eik2ak3, but not other eIF2α kinases, enhances adult sensory axon regrowth in vitro (n ≥ 8 wells/condition). Molecular weight indicated in kilodaltons (kDa). Data are represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with post-hoc Bonferroni test.