Chromosome 22q11.2 deletion causes PERK-dependent vulnerability in dopaminergic neurons

Abstract

Background: The chromosome 22q11.2 deletion is an extremely high risk genetic factor for various neuropsychiatric disorders; however, the 22q11.2 deletion-related brain pathology in humans at the cellular and molecular levels remains unclear.

Methods: We generated iPS cells from healthy controls (control group) and patients with 22q11.2 deletion (22DS group), and differentiated them into dopaminergic neurons. Semiquantitative proteomic analysis was performed to compare the two groups. Next, we conducted molecular, cell biological and pharmacological assays.

Findings: Semiquantitative proteomic analysis identified 'protein processing in the endoplasmic reticulum (ER)' as the most altered pathway in the 22DS group. In particular, we found a severe defect in protein kinase R-like endoplasmic reticulum kinase (PERK) expression and its activity in the 22DS group. The decreased PERK expression was also shown in the midbrain of a 22q11.2 deletion mouse model. The 22DS group showed characteristic phenotypes, including poor tolerance to ER stress, abnormal F-actin dynamics, and decrease in protein synthesis. Some of phenotypes were rescued by the pharmacological manipulation of PERK activity and phenocopied in PERK-deficient dopaminergic neurons. We lastly showed that DGCR14 was associated with reduction in PERK expression.

Interpretation: Our findings led us to conclude that the 22q11.2 deletion causes various vulnerabilities in dopaminergic neurons, dependent on PERK dysfunction.

Funding: This study was supported by the AMED under grant nos JP20dm0107087, JP20dm0207075, JP20ak0101113, JP20dk0307081, and JP18dm0207004h0005; the MEXT KAKENHI under grant nos. 16K19760, 19K08015, 18H04040, and 18K19511; the Uehara Memorial Foundation under grant no. 201810122; and 2019 iPS Academia Japan Grant.

Keywords: 22q11.2 deletion; Dopaminergic neurons; Neuropsychiatric disorders; PERK; iPS cells.

Fig. 1

Characterisation of 22q11.2DS iPSC-derived dopaminergic neurons. (a) Schematic illustration of the differentiation of dopaminergic neurons. (b) Representative image of dopaminergic neurons (Day 24) immunostained for TH and βIII-tubulin. The yellow scale bar in the images represents 50 μm. White arrows indicate TH-negative, βIII-tubulin-positive (+) neurons. (c) Analysis of dopaminergic neuron differentiation efficiency by quantifying the ratio of TH+ to βIII-tubulin+ cells at Day 24. The bars represent means ± SEs. Five fields were analyzed. (d) Schematic illustration of the proteomic analysis. DA neuron = dopaminergic neurons. (e) KEGG pathway analysis. Proteins used showed a fold change (FC) > 10 and p < 0.05 (Control vs. 22DS). (f) Cell viability in the presence of ER stress. Control: n = 12 (Control1, n = 4; Control2, n = 4; Control3, n = 4); 22DS: n = 30 (22DS1_1, n = 4; 22DS1_2, n = 4; 22DS2_1, n = 4; 22DS2_9, n = 4; 22DS3_1, n = 7; 22DS3_5, n = 7). The plots represent the means ± SEs. *p < 0.05, **p < 0.01 vs. without TCM in the control group, †††p < 0.001 vs. without TCM in the 22DS group. (g) Representative images of immunostained cleaved-caspase3+ cells. White arrows indicate cleaved-caspase3+ cells. The yellow scale bar in the image represents 50 μm. (h) Quantification of the celaved-caspase 3+ cell ratio. Control: n = 12 (each, n = 4 fields); 22DS: n = 24 (each, n = 4 fields). Bars represent means ± SEs. **p < 0.01. Each plot represents the value of each line.

Fig. 2

Downregulation of PERK expression and activity in 22q11.2DS iPSC-derived dopaminergic neurons. (a) Immunoblotting for ER stress sensors and their related proteins using dopaminergic neurons (Day 24). P-PERK: phosphorylated PERK; P-eIF2α: phosphorylated eIF2α; P-IRE1: phosphorylated IRE1. (b–i) Quantification of the PERK/β-actin, P-eIF2α/eIF2α, ATF4/β-actin, GADD34/β-actin, ATF6/β-actin, GRP78/β-actin, IRE1/β-actin, P-IRE1/IRE1 ratios. Immunoblots were independently performed four (b and d; control [n = 12], 22DS [n = 24]), three (f and h; control [n = 9], 22DS [n = 18]) or two times (c, e, g and i; control [n = 6], 22DS [n = 12]). The value of Control1 was set to 1. Each plot represents the value of each line. Bars represent means ± SEs. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3

Examination of PERK expression in the brain of 22q11.2DS model mouse and iPSCs. (a) Schematic diagram of preparing brain slices from adult WT and Del(3.0Mb)/+ mice. SN = substantial nigra. (b–e) Immunoblot analysis using area A and area B. Yellow arrows indicate the section used for quantification. b and c: PERK and TH were simultaneously detected, with GAPDH as the internal standard. d: Double detection of PERK and TH. e: Decreased expression of SNAP29, one of the factors in the 22q11.2 deletion region. (f–h) Quantification of the TH/GAPDH, PERK/GAPDH and PERK/TH ratios in the area A of section no. 3. f, g and h correspond to b, c and d, respectively. Quantitative detection by immunoblot was performed by Odyssey. Immunoblots were independently performed four (f and g) or six (h) times. The numbers indicated in the bars represent relative values (WT = 100). The bars represent means ± SEs. ***p < 0.001. (i) Immunoblotting for PERK, ATF6, IRE1 and β-actin using proteins extracted from iPSCs. (j) Quantification of the PERK/β-actin, ATF6/β-actin and IRE1/β-actin ratios. Two independent experiments were performed (in summary, Control: n = 6; 22DS: n = 12). The value of Control1 is 1. Each plot represents the value of each line. The bars represent means ± SEs.

Fig. 4

Generation of PERK-deficient iPSCs. (a) Cell viability in the presence of TCM with or without salubrinal (Sal). n = 24 (22DS1_1, n = 6; 22DS1_2, n = 6; 22DS2_1, n = 6; 22DS2_9, n = 6). Each plot represents the value of each line. The bars represent means ± SEs. **p < 0.01, ***p < 0.001. (b) Quantification of the caspase 3-positive (+) cell ratio using the 22DS group. TCM treatment (0.4 μg/ml) with or without Sal. Twenty fields were used (each, n = 5). The plots represent means ± SEs. ** p < 0.01. (c) The target site of the CRISPR/Cas9 system used in this study. (d) Indel patterns of the PERK-deficient isogenic lines. (e) Representative images of dopaminergic neurons (Day 24) immunostained for TH and βIII-tubulin. The scale bar represents 50 μm. (f) Analysis of dopaminergic neuron differentiation efficiency via the quantification of the ratio of TH+ to βIII-tubulin+ cells at Day 24. The bars represent means ± SEs. Five fields were used. (g) Immunoblotting of PERK and β-actin using DA neurons at Day 24. (h) Measurement of neurite length. The numbers indicated in the bars represent the number of counted cells. The bars represent means ± SEs. (i) Cell viability in the presence of ER stress (n = 7). The plots represent the means ± SEs. *** p < 0.001, ††† p < 0.001 vs. without TCM. (j) Representative images of immunostaining for cleaved-Caspase3 using dopaminergic neurons. White arrows indicate Caspase3+ cells. The yellow scale bar in the image represents 50 μm. (k) Quantification of the cleaved-caspase 3+ cell ratio (n = 14). The bars represent means ± SEs. * p < 0.05, ** p < 0.01. Each plot represents the value of each line.

Fig. 5

Impairment of F-actin dynamics and PERK dysfunction. (a) Schematic illustration of experimental timing. (b) Representative images of Control1 and 22DS1_1 stained with phalloidin at Day 22. The yellow scale bar in the images represents 50 μm. The magenta box represents filopodia. The yellow arrow represents lamellipodia. (c) Ratio of cells exhibiting typical lamellipodia. The numbers over the bars represent the number of counted cells. ***p < 0.001. (d) Measurement of filopodium length. The numbers indicated in the bars represent the number of counted filopodia. The bars represent means ± SEs. ***p < 0.001. (e) Representative results for the automatic detection system. A skeletonised region (Upper), except for the region detected as a cell body (Middle), is recognised as a filopodia-like protuberance (Lower). (f) Representative images of cells stained with phalloidin at Day 22 with or without Sal (40 μM) in the 22DS group. The yellow scale bar represents 50 μm. (g) Measurement of protuberance length using an automated detection system. The numbers indicated in bars represent the number of counted fields. Each plot represents the value of each line. The bars represent means ± SEs. *** p < 0.001. (h) Representative images of cells stained with phalloidin at Day 22 with or without GSK2656157 (GSK, 0.4 μM) or Sal (40 μM) in the control group. The yellow scale bar represents 50 μm. (i) Measurement of protuberance length using an automated detection system. The numbers indicated in bars represent the number of counted fields. Each plot represents the value of each line. The bars represent means ± SEs. *** p < 0.001. (j) Summary of the effects of pharmacological manipulation (g and i) for PERK activity. The average length of the control group was set as 100. (k) Representative images of Control4 and KO#4 stained with phalloidin at Day 22. The yellow scale bar represents 50 μm. (l) Measurement of filopodium length. The numbers indicated in the bars represent the number of counted filopodia. The bars represent means ± SEs. *** p < 0.001.

Fig. 6

Global protein synthesis and PERK dysfunction. (a) Immunoblotting for puromycin and β-actin in the control and 22DS groups at Day 24. Treatment with puromycin lasted for 30 min. (b) Quantification of puromycilated protein signal intensity in a. The value of Control1 was set to 1. Two independent experiments were performed (in summary, Control, n = 6; 22DS, n = 8). Each plot represents the value of each line. The bars represent means ± SEs. ** p < 0.01. (c) Immunoblotting for puromycin and β-actin using the 22DS group at Day 24 with or without Sal (40 μM). Treatment with puromycin lasted for 30 min. (d) Immunoblotting for puromycin and β-actin using Control4, KO#4 and KO#10 DA neurons at Day 24. Treatment with puromycin lasted for 30 min. (e) Quantification of puromycilated protein signal intensity in d. The value of Control4 was set to 1. Three independent experiments were performed. Each plot represents the value of each line. The bars represent means ± SEs.

Fig. 7

ER-mitochondria contacts in the 22DS and PERK-deficient groups. (a) Representative images of dopaminergic neurons from Control4 and KO#4 (Day 24) immunostained for calreticulin (for ER) and TOMM20 (for mitochondria). The yellow scale bar in the image represents 10 μm. (b) Left panel: Representative results of the co-localisation analysis that was performed using Mander's method. Right panel: Mander's co-localisation analysis. The numbers indicated in the bars represent the number of counted fields. The bars represent means ± SEs. *** p < 0.001. (c) Representative images of dopaminergic neurons from Control1 and 22DS1_1 (Day 24) immunostained for calreticulin and TOMM20. (d) Left panel: Representative results of a co-localisation analysis that was performed using Mander's method. The yellow scale bar in the image represents 10 μm. Right panel: Mander's co-localisation analysis. The numbers indicated in the bars represent the number of counted fields. The bars represent means ± SEs. *** p < 0.001.

Fig. 8

DGCR14 is associated with the downregulation of PERK expression. (a) Relative expression levels of DGCR14 in DGCR14 knockdown cells. *** p < 0.001. (b) Immunoblotting for PERK and β-actin using the negative target and DGCR14 knockdown cells. ‘Negative target’ means the use of the negative control RNAi. (c) Quantification of the PERK/β-actin ratio. Three independent experiments were performed. The bars represent means ± SEs. * p < 0.05, ** p < 0.01. The value of N was set to 1. (d) Quantification of the mRNA expression level of DGCR14 in iPSCs and dopaminergic neurons (Day 24) derived from healthy controls and 22q11.2DS patients. The bars represent means ± SEs. 22DS1_2 and 22DS2_1 in dopaminergic neurons: n = 4; others: n = 3. *** p < 0.001. (e) Immunoblotting for puromycin and β-actin in DGCR14 knockdown cells. (f) Quantification of puromycilated protein signal intensity in e. The value of N was set to 1. The bars represent means ± SEs. Each n = 4. ** p < 0.01, *** p < 0.001 vs N. (g) Immunoblotting for puromycin and β-actin in dopaminergic neurons treated with cycloheximide (CHX). (h) Representative images of immunostaining for βIII-tubulin using CHX-treated dopaminergic neurons. The yellow bar in the image was 100 μm. (i) Measurement of neurite length. The numbers indicated in/over the bars represent the number of counted cells. The bars represent means ± SEs. *** p < 0.001. (j) Graphic abstract.