Figure 3. Atf4mRNA is recruited into Aβ_1–42-treated axons, and axonal ATF4 protein is locally synthesized and retrogradely transported.

(A) Log₂ fold change for Atf4 mRNA as determined by real time RT-PCR and DESeq2 (TMM). ***p<0.001.

(B) Hippocampal neurons were cultured in microfluidic chamber for 9–10 DIV, axons were treated with vehicle, Aβ_scrambled or Aβ_1–40 for 18 h, and axonal Atf4 mRNA levels were measured by quantitative FISH. Mean ±SEM of 25–30 optical fields per condition (n=5–6 biological replicates).

(C) Axons were treated with Aβ_1–42 for the indicated times, and axonal Atf4 mRNA levels were measured by quantitative FISH. Mean ±SEM of 25–40 axonal fields per condition (n=5–8 biological replicates per group). The background fluorescence was determined using a non-targeting probe (neg. probe) and set to zero. *p<0.05; **p<0.01; ***p<0.001. Scale bar, 5 µm.

(D) Neurons were cultures and treated as in C. Axonal ATF4 protein levels were measured by quantitative immunofluorescence. Mean ±SEM of 20–40 axonal fields per condition (n=4–8 biological replicates per group). *p<0.05; **p<0.01. Scale bar, 5 µm.

(E) Hippocampal neurons were cultured and treated as in B. 3 h prior to sample processing axons were treated with DMSO, anisomycin or emetine. Axonal ATF4 protein levels were determined by quantitative immunofluorescence. Mean ±SEM of 25–35 axonal fields per condition (n=5–7 biological replicates per group). ***p<0.001: *p<0.05. Scale bar, 5 µm.

(F) Hippocampal neurons were cultured in microfluidic chambers for 8 DIV. Axons were transfected with a control (ctrl.) siRNA or a siRNA targeting Atf4. 24 h after transfection axons were treated with vehicle or Aβ_1–42 for 18 h. ATF4 protein levels were measured by quantitative immunofluorescence. Mean ±SEM of 35–55 axonal fields per condition (n=7–11 biological replicates per group). **p<0.01: *p<0.05.

(G) Axons were treated with vehicle or Aβ_1–42 for 24h, in the presence or absence of ciliobrevin A for 6h. Anisomycin was added to the cell body or the axonal compartment for 3 h. Axons were immunostained for ATF4 protein. Mean ±SEM of 30–40 axonal fields per condition (n=6–8 biological replicates per group). *p<0.05.

(H) Axons were transfected with a control siRNA or siRNAs targeting Atf4 mRNA and treated with Aβ_1–42 and ciliobrevin A as in G. Axons were immunostained for ATF4 protein. Mean ±SEM of 30–40 axonal fields per condition (n=6–8 biological replicates per group). *p<0.05.

(I) Neurons were cultured and treated as in C. eIF2α and p-eIF2α levels were determined by quantitative immunofluorescence. Mean ±SEM of 20–35 axonal fields per condition (n=4–7 biological replicates per group).

(J) Neurons were cultured as in B. Axonal were treated for 18 h with tunicamycin (Tm) or thapsigargin (Tg) and Atf4 mRNA levels were determined by quantitative FISH. Mean ±SEM of 30 optical fields per condition (n=6 biological replicates).

Scale bars, 5 µm. See also Figure S3 and Supplemental Table S1.