Activation of HIPK2 Promotes ER Stress-Mediated Neurodegeneration in Amyotrophic Lateral Sclerosis

Abstract

Persistent accumulation of misfolded proteins causes endoplasmic reticulum (ER) stress, a prominent feature in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here we report the identification of homeodomain interacting protein kinase 2 (HIPK2) as the essential link that promotes ER-stress-induced cell death via the IRE1α-ASK1-JNK pathway. ER stress, induced by tunicamycin or SOD1(G93A), activates HIPK2 by phosphorylating highly conserved serine and threonine residues (S359/T360) within the activation loop of the HIPK2 kinase domain. In SOD1(G93A) mice, loss of HIPK2 delays disease onset, reduces cell death in spinal motor neurons, mitigates glial pathology, and improves survival. Remarkably, HIPK2 activation positively correlates with TDP-43 proteinopathy in NEFH-tTA/tetO-hTDP-43ΔNLS mice, sporadic ALS and C9ORF72 ALS, and blocking HIPK2 kinase activity protects motor neurons from TDP-43 cytotoxicity. These results reveal a previously unrecognized role of HIPK2 activation in ER-stress-mediated neurodegeneration and its potential role as a biomarker and therapeutic target for ALS. VIDEO ABSTRACT.

Published by Elsevier Inc.

Figures

Figure 1. Cortical and spinal motor neurons in Hipk2−/− mice are more resistant to tunicamycin-induced neurodegeneration

(A–L) Three-month-old wild type and Hipk2−/− mice treated with tunicamycin show TUNEL+ apoptotic neurons in sensorimotor cortex. (M–N) Number of TUNEL+ and NeuN+ cortical neurons in the sensorimotor cortex of wild type and Hipk2−/− mutants. * p < 0.05, two-tailed unpaired Student’s t test. (O–V) Wild type and Hipk2−/− mice injected with tunicamycin show TUNEL+ motor neurons in spinal cord. (W–X) Number of TUNEL+ motor neurons in the cervical spinal cord of wild type and Hipk2−/− mice. * p < 0.05, two-tailed unpaired Student’s t test. (Y) Protein lysates and RNA samples were prepared from the cerebral cortex of wild type and Hipk2−/− mice at 1, 2, 3 or 4 days after tunicamycin injection to characterize IRE1α-ASK1-JNK activation and Xbp1 splicing. (Z) Quantification of the time-dependent p-JNK levels induced by tunicamycin. **** p < 0.0001, two-way ANOVA.

Figure 2. ER stress promotes sequential activation of the ASK1-HIPK2-JNK pathway by phosphorylating HIPK2 at S359/T360

(A) Experimental procedures using in vitro kinase assays and liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize HIPK2 phosphorylation. (B–C) Protein lysates from HEK293 cells treated with tunicamycin were used for in vitro kinase assays to detect HIPK2 activation measured by incorporation of γ-P32-ATP, and to detect p-ASK1 and p-JNK. (D) (Top) Representative LC-MS/MS spectrum of a singly phosphorylated HIPK2 tryptic peptide spanning the S359, T360 and Y361 residues shows overlapping ion series corresponding to phosphorylation at T360 and Y361, highlighted in blue and green, respectively. All assigned ion peaks are marked in red. (bottom) Relative phosphorylation of HIPK2 at the two sites with and without tunicamycin treatment was compared by calculating peak areas of extracted ion chromatographs. (E) Protein lysates from HEK293 cells expressing HIPK2-WT, HIPK2-S359A, HIPK2-T360A or HIPK2-Y361F were used in in vitro kinase assays to detect γ-P32-ATP incorporation in HIPK2 after tunicamycin treatment. (F) HEK293 cells expressing HIPK2-WT, HIPK2-S359A, HIPK2-T360A and HIPK2 kinase-dead HIPK2-K221A were treated with tunicamycin. Cell viability was measured using MTT assay. Two-way ANOVA, * p < 0.05. (G) Western blot using epitope-specific p-HIPK2 [S359/T360] Ab detects phosphorylated HIPK2 in tunicamycin-treated HEK293 cells. (H) HEK293 cells were treated with scramble siRNA or ASK1 siRNA and tunicamycin to determine the effect on JNK phosphorylation. (I) HEK293 cells co-expressing ASK1 and HIPK2-WT, HIPK2-S359A or kinase-dead HIPK2 (HIPK2-K221A) to determine the effect of ASK1 on HIPK2 phosphorylation. (J) HEK293 cells expressing GFP-HIPK2 and HA-JNK2 treated with tunicamycin were used in co-immunoprecipitation assays to demonstrate HIPK2-JNK protein complex formation. “U” for untransfected HEK293 cells. (K) HEK293 cells expressing different HIPK2 constructs were treated with tunicamycin to characterize endogenous JNK activation under ER stress. (L) HEK293 cells were treated with control or Hipk2 siRNA and subsequently treated with tunicamycin to determine the effect on JNK phosphorylation. (M) Lysates from HEK293 cells expressing HIPK2-WT, HIPK2-S359A, HIPK2-T360A, or HIPK2-Y361F and treated with tunicamycin were used to characterize the effect of HIPK2 phosphorylation on S359 and T360 in JNK phosphorylation. (N) Diagram showing ER stress promotes a cascade of protein kinase activation, including (1) ASK1 activation, (2) ASK1 interaction with HIPK2 and ASK1-mediated site-specific phosphorylation of HIPK2, (3) HIPK2 kinase activation, and (4) HIPK2-JNK protein complex formation and JNK activation.

Figure 3. Loss of HIPK2 blocks JNK activation and neuronal cell death in SOD1G93A model of ALS

(A–H) Cervical spinal cord from P90 wild type and SOD1G93A were stained using C4F6 and HIPK2 antibodies. (I) HIPK2 signal intensity in the spinal motor neurons of SOD1G93A mice at P60 and P90 is plotted as a function of the signal intensity for misfolded SOD1G93A proteins (C4F6 intensity). Pearson correlation coefficients, with 95% confidence and the p values. (J) Spinal cord tissues from wild type and SOD1G93A mice at 1-, 2- and 3-month-old were used in immunoprecipitation-in vitro kinase assays for HIPK2 activation, and to detect the presence of p-IRE1α, p-ASK1, and p-JNK and Xbp1 splicing. (K) Quantification of HIPK2 [P32] and p-JNK in wild type and SOD1G93A mice. (L) Lysates from the cervical spinal cord of 3 SOD1-ALS patients and 3 age-matched controls were analyzed in western blots to detect the presence of IRE1α, HIPK2 and JNK phosphorylation. (M) Relative signal intensity of p-HIPK2 [S359/T360], p-IRE1α and p-JNK. Two-tailed unpaired Student’s t test, * p < 0.05 and ** p < 0.01. (N–R) Two-color immunohistochemistry for misfolded SOD1 proteins (detected by C4F6, brown color) and HIPK2 (blue color) was performed in spinal motor neurons of control and ALS patients with SOD1G93A or SOD1I133T mutation. Arrowheads in L–O indicate spinal motor neurons, whereas arrows indicate glial cells. The intensity of HIPK2 in motor neurons was quantified using NIH ImageJ. (S) Spinal cord tissues from P90 wild type, SOD1G93A, SOD1G93A;Hipk2−/− and SOD1G93A;Hipk2−/− mice were used to detect activation of ER stress markers p-IRE1α, p-ASK1 and Xbp1 mRNA splicing. (T) Quantification of p-JNK and p-c-Jun signal intensity. Two-tailed unpaired Student’s t test, * p < 0.05, ** p < 0.01. (U–W) Confocal images of p-c-Jun in ChAT+ motor neurons in the cervical spinal cord of P120 wild type, SOD1G93A and SOD1G93A;Hipk2−/− mice. (X–Z) Confocal images of activated caspase 3+ motor neurons in the cervical spinal cord of P120 wild type, SOD1G93A and SOD1G93A;Hipk2−/− mice. (Z′) Quantification of p-c-Jun+ motor neurons and caspase 3+ neurons. Student’s t test, *** p < 0.001.

Figure 4. HIPK2 deletion in SOD1G93A mice attenuates neurodegeneration, delays disease onset, prolongs survival and improves motor functions

(A–F) Immunostaining of ChAT+ neurons in the cervical spinal cord of wild type, SOD1G93A and SOD1G93A;Hipk2−/− mice at P90 and P120. (G–L) Immunostaining for GFAP+ astrocytes and Iba1+ microglia in wild type, SOD1G93A and SOD1G93A;Hipk2−/− mice at P120. (M–N) Number of ChAT+ motor neurons, GFAP+ astrocytes and Iba1+ microglia in wild type, SOD1G93A and SOD1G93A;Hipk2−/− mice at P90 and P120. Two-tailed unpaired Student’s t test. *** p < 0.005, ** p < 0.01, and * p < 0.05. (O) Kaplan–Meier analyses of disease onset and survival in wild type, SOD1G93A mice (B6SJL), SOD1G93A (B6SJL × B6;129) and SOD1G93A;Hipk2−/− mice. (P) Rotarod testing in P120 wild type, Hipk2−/−, SOD1G93A and SOD1G93A;Hipk2−/− mice. Two-way ANOVA, * p = 0.04, ** p = 0.0082, *** p = 0.002, ns, not significant.

Figure 5. Activation of IRE1α-HIPK2-JNK in SOD1G93A and NEFH-tTA/tetO-hTDP-43ΔNLS, but not FUS-R521C, ALS models

(A–B) Spinal cord lysates from SOD1G93A, FUS-R521C and wild type littermates were used in western blots to compare the levels of p-IRE1α, p-HIPK2 [S359/T360] and p-JNK. Student’s t test, * p < 0.05 and ** p < 0.01. (C–D) RIPA soluble lysates are prepared from the cortex non-transgenic control (nTg), NEFH-tTA mice (tTA), and NEFH-tTA/tetO-hTDP-43ΔNLS mice that are “off doxycycline” for 1, 2, 4, 6, 7, 11 and 15 weeks to detect activated and total IRE1α, HIPK2 and JNK. Two-tailed unpaired Student’s t test, * p < 0.05, ** p < 0.01. (E–K) Confocal images of HIPK2 in ChAT+ motor neurons in NEFH-tTA mice and NEFH-tTA/tetO-hTDP-43ΔNLS (rNLS8) mice that are “off doxycycline” for 10 weeks. Arrows indicate ChAT+ neurons, and arrowheads ChAT− neurons. Relative signal intensity of HIPK2 was quantified by NIH ImageJ. Two-tailed unpaired Student’s t test, *** p < 0.001.

Figure 6. HIPK2-JNK activation positively correlates with TDP-43 proteinopathy in sporadic ALS and familial ALS with C9ORF72 mutations

(A–B) Spinal cord tissues from SALS and C9-ALS cases were used in western blots to detect p-HIPK2 [S359/T360], ubiquinated TDP-43, p-TDP-43 [S409/410], and p-JNK. (C–F) Relative abundance of p-HIPK2 [S359/T360], p-TDP-43 [S409/410], ubiquitinated TDP-43 and p-JNK. Two-tailed Student’s t test. (G–L) Relative abundance of p-HIPK2 [S359/T360] is plotted as a function of the signal intensity of p-TDP-43 [S409/410], ubiquitinated TDP-43 or p-JNK in SALS cases (G–I) and C9-ALS (J–L). Pearson correlation coefficients and p values are indicated. Dash lines represent 95% confidence. (M–P) Spinal cord tissues from control, SALS and C9-ALS cases were stained with HIPK2 antibody, and the intensity of HIPK2 quantified using NIH ImageJ.

Figure 7. HIPK2 kinase inhibitors block S359/T360 phosphorylation and attenuate ER stress-induced cell death

(A) HIPK2 kinase inhibitors (1 μM each) were tested in HEK293 cells for their ability to block HIPK2 phosphorylation and JNK phosphorylation. (B) Quantification of p-HIPK2 [S359/T360] and p-JNK. Two-tailed unpaired Student’s t test; * p < 0.05, ** p < 0.01, *** p < 0.005 and **** p < 0.0001. (C) HEK293 cells were treated with tunicamycin (1μg/ml) and HIPK2 inhibitors at the designated concentrations. Cell viability is determined using MTT assays. 2 way ANOVA; ** p < 0.01 and *** p < 0.005. (D) Motor neurons were treated with DMSO, tunicamycin (1 μg/ml) or tunicamycin and HIPK2 inhibitor A64 (1μM). Protein lysates from these neurons were used to characterize HIPK2 and JNK phosphorylation. (E–J) Cortical neurons were treated with DMSO, tunicamycin (1 μg/ml) or tunicamycin and HIPK2 inhibitor A64 (1 μM). Cell death was analyzed using antibodies for activated caspase 3. (K) Quantificaiton of caspase 3+ neurons. Data represents mean ± SEM. Two-tailed unpaired Student’s t test; * p < 0.05. (L) Motor neurons transfected with mutant SOD1G93A were treated with HIPK2 inhibitor A64, and cell death quantified using activated caspase 3 antibody.

Figure 8. HIPK2 inhibitor A64 attenuates TDP-43 proteinopathy-induced cell death in HEK293 cells and spinal motor neurons

(A) Lysates from HEK293 cells expressing wild type or mutant TDP43 were used in western blots to determine TDP-43 ubiquitination, phosphorylation on TDP-43, IRE1α and HIPK2, and Xbp1 splicing. (B) Quantification of p-HIPK2 [S359/T360] and ubiquitinated TDP-43. * p < 0.05, unpaired two-tailed Student’s t test. (C) HEK293 cells were co-transfected with TDP-43 constructs, together with HIPK2-WT, HIPK2-S359A or HIPK2-WT treated with HIPK2 inhibitor A64 (1 μM). Two days after transfection, cells were harvested for MTT assays. Two way ANOVA, *** p < 0.001 and **** p < 0.0001. (D–H) Activated caspase 3 in rat spinal motor neurons two days after transfection with GFP, Myc-hTDP-43-WT or TDP-43G348C-Myc-His, and treated with DMSO or HIPK2 inhibitor A64 (1 μM). (I) Qunatification of apoptotic motor neurons. * p < 0.05, two-tailed Student’s t test.