PMC

Hypothermic UPR-mediated preconditioning of the ER protects hCNs against oxidative stress.
(A) Oxidative stress-mediated injury is increased by PERK inhibitor (PI) (N = 3; n = 3; n_HES1 = 1, n_HES2 = 1, n_IPS1 = 1; 28 °C, P < 0.0005; 37 °C, P = 0.016). Hypothermia remained protective only at 200 μM H₂O₂ (P = 0.023).
(B) H₂O₂ injury is increased by Tm (N = 3; n = 7; n_HES1 = 5, n_HES2 = 1, n_IPS1 = 1; 28 °C, P < 0.05; 37 °C, P < 0.0005). Hypothermia reduced the toxic effect of Tm (P = 0.062). Note that log scale was required to accommodate magnitude of injury changes across H₂O₂ concentrations in Fig. 3A and B.
(C) Proposed mechanism of ER-hormesis in cooled hCNs. UPR pathways are depicted, together with known regulatory feedback pathways. Filled boxes denote components induced at transcript and/or protein level in hCNs with cooling. Phospho-IRE1α and phospho-PERK were not assessed. Orange arrows indicate hormetic elements that resolve the UPR and increase ER resilience to stress.
See also Fig. S3.

Hypothermia induces mild ER stress in hCNs with full activation of the UPR.
(A) BiP transcripts after cooling (left, N = 3; n = 22; n_HES1 = 11, n_HES2 = 6, n_IPS1 = 5; 32 °C, P = 0.006; 28 °C, P < 0.0005) or Tm treatment (right, N = 3; n = 8; n_HES1 = 4, n_HES2 = 2, n_IPS1 = 2, P = 0.004).
(B) Total BiP protein expression (N = 2; n = 3; n_HES1 = 2; n_HES2 = 1; 28 °C, P = 0.051).
(C) IRE1α transcripts (P < 0.01, N = 3; n = 14; n_HES1 = 7, n_HES2 = 4, n_IPS1 = 3).
(D) Immunoblots of fractionated lysates (C = cytoplasmic, H = high-detergent) from hCNs. Note increased BiP, full length (fATF6), and cleaved (cATF6) sitting in the high detergent fraction at 28 °C. This is consistent with nuclear translocation of cATF6 and upregulation of its target transcripts (BiP and unspliced XBP1 — as shown in Figs. 2A, E, and S2B).
(E) Gel images of RT-PCR products. Faint bands at 263 bp confirm mild splicing of XBP1 in hypothermic hCNs relative to negative (37 °C) and positive (Tm-treated) controls. GAPDH = reference target.
(F) qRT-PCR analysis of XBP1s transcript after cooling (left, N = 3; n = 22; n_HES1 = 11, n_HES2 = 6, n_IPS1 = 5; 28 °C, P = 0.003) or Tm-treatment (right, N = 3; n = 8; n_HES1 = 4, n_HES2 = 2, n_IPS1 = 2, P < 0.0005).
(G) CHOP transcripts after cooling (left, N = 3; n = 22; n_HES1 = 11, n_HES2 = 6, n_IPS1 = 5; 32 °C, P = 0.011; 28 °C, P = 0.001) or Tm treatment (right, N = 3; n = 8; n_HES1 = 4, n_HES2 = 2, n_IPS1 = 2, P < 0.0005).
(H) GADD34 transcripts (N = 3; n = 7; n_HES1 = 3, n_HES2 = 2, n_IPS1 = 2, 32 °C, P < 0.0005; 28 °C, P < 0.0005; Tm, P < 0.0005).
See also Fig. S2.

Mild-to-moderate hypothermia elicits a cold-shock response in hCNs.
(A) RBM3 transcripts (left, N = 3; n = 14; n_HESI = 7, n_HES2 = 4, n_IPS1 = 3) with significant increases after 3 h (32 °C P = 0.011, 28 °C P = 0.003) and 24 h (P < 0.0005). Cell counts for RBM3-positive nuclei (right, N = 3; n = 6; n_HES1 = 4, n_HES2 = 1, n_IPS1 = 1, mean 37.2% (total 902 out of 2096 cells) at 28 °C, P = 0.039; mean 46.0% (total 1050 out of 2377 cells) at 32 °C, P = 0.001; mean 20.8% (total 531 out 2062 cells) at 37 °C). For each independent hCN batch and temperature condition, a minimum of 10 fields of view at 63 × were counted (pooled from two replicate coverslips). Counts are presented as mean % + standard error of the mean (SEM).
(B) CIRBP transcripts (left, N = 3; n = 14; n_HES1 = 7, n_HES2 = 4, n_IPS1 = 3, P < 0.0005 at 24 h) and cell counts (right, N = 3; n = 6; n_HES1 = 4, n_HES2 = 1, n_IPS1 = 1, mean 48.9% (total 719 out of 1416 cells) at 28 °C; mean 52.3% (total 985 out of 1882 cells) at 32 °C; mean 13.1% (total 252 out of 1850 cells) at 37 °C, P < 0.0005).
(C and D) Fluorescent micrographs of hCNs co-stained for neuronal and cold-shock markers, scale bar = 10 μm.
(E) Subcellular expression of RBM3 and CIRBP by immunoblot, alongside human foetal and adult cortex. GAPDH and hnRNP A1 are loading controls. The stability of hnRNP A1 expression under mild hypothermic conditions in human cells has reported elsewhere ().
(F) Quantitative Western analysis of RBM3 and CIRBP (N = 3; n ≥ 4; n_HES1 ≥ 2; n_HES2 = 1, n_IPS1 = 1). RBM3 expression was greatest at 28 °C (P = 0.002); CIRBP expression peaked at 32 °C (P < 0.0005).
(G) Correlation of RBM3 and CIRBP transcripts (37 °C P = 0.001, 32 °C P = 0.012, 28 °C P < 0.0005).
See also Fig. S1.