Formation of Neurodegenerative Aggresome and Death-Inducing Signaling Complex in Maternal Diabetes-Induced Neural Tube Defects

Formation of neurodegenerative aggresome and death-inducing signaling complex in maternal diabetes-induced neural tube defects

Zhiyong Zhaoa,b,1,2, Lixue Caoa,1, and E. Albert Reecea,b

aDepartment of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Baltimore, MD 21201; and bDepartment of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201

Edited by Ted M. Dawson, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, and accepted by Editorial Board Member Gregg L. Semenza March 13, 2017 (received for review September 27, 2016) Diabetes mellitus in early pregnancy increases the risk in infants of (ROS), which results in oxidative stress (15, 16) and disrupts in- birth defects, such as neural tube defects (NTDs), known as diabetic tracellular signaling, thus altering cellular activities (17). embryopathy. NTDs are associated with hyperglycemia-induced Misfolded proteins are prone to forming aggregates that are protein misfolding and Caspase-8–inducedprogrammedcelldeath. resistant to UPS degradation (18). Aggregation of α-Synuclein The present study shows that misfolded proteins are ubiquitinylated, (α-Syn), Parkin, and Huntingtin (Htt) has been found to be in- suggesting that ubiquitin-proteasomal degradation is impaired. volved in the pathogenesis of Alzheimer’s, Parkinson’s, and Misfolded proteins form aggregates containing ubiquitin-binding Huntington’s diseases (19–22). Protein aggregates can be elimi- protein p62, suggesting that autophagic-lysosomal clearance is nated by the autophagic-lysosomal system (ALS) (23, 24). The insufficient. Additionally, these aggregates contain the neurodegen- transfer of ubiquitinylated proteins to the ALS is mediated by a erative disease-associated proteins α-Synuclein, Parkin, and Huntingtin ubiquitin-binding protein, p62, also known as Sequestosome-1 (25, (Htt). Aggregation of Htt may lead to formation of a death-inducing 26). Protein aggregates containing p62 are commonly seen in signaling complex of Hip1, Hippi, and Caspase-8. Treatment with neurodegenerative diseases, indicating deficiency of the ALS in chemical chaperones, such as sodium 4-phenylbutyrate (PBA), re- the pathogenesis of these diseases (27). These neural degenerative duces protein aggregation in neural stem cells in vitro and in embryos disease-associated proteins are expressed in the developing neural in vivo. Furthermore, treatment with PBA in vivo decreases NTD rate tube (28–30); however, it is unknown whether they are involved in in the embryos of diabetic mice, as well as Caspase-8 activation and hyperglycemia-induced NTDs. cell death. Enhancing protein folding could be a potential interven- In the present study, we aimed to investigate the fate of these tional approach to preventing embryonic malformations in diabetic misfolded proteins and their potential effects on apoptotic regu- pregnancies. lation and NTD formation. We also explored an approach to alleviate protein aggregation by enhancing protein folding using diabetic embryopathy | protein folding | protein aggregation | Caspase-8 | chemical chaperones to provide insights into the mechanisms of chemical chaperone diabetic embryopathy, and to help identify potential strategies to prevent diabetes-induced birth defects. regnancies complicated by diabetes mellitus have a higher Prisk of resulting in infants with congenital birth defects, a Results complication known as diabetic embryopathy (1). The defects in Protein Ubiquitinylation and Aggregation. Misfolded proteins are the central nervous system are the result of perturbed neural tube usually ubiquitinylated and degraded by the UPS (13). However, formation (neurulation) during early embryogenesis and are thus when the UPS is impaired, ubiquitinylated proteins can accu- mulate in the cell. Because we hypothesized that UPS dysfunction referred to as neural tube defects (NTDs) (2). Neurulation is a MEDICAL SCIENCES process that begins with development of the neural folds at about week 2 of gestation in humans and embryonic day 8 (E8) in the Significance mouse, and concludes with fusion of the neural tissues at the dorsal midline, to form the neural tube, by about week 4 in humans Neural tube defects in infants of women with diabetes are as- and E11.5 in the mouse (3, 4). The failure of neural tube closure in sociated with disrupted protein folding and increased pro- diabetic embryopathy is associated with excessive programmed cell grammed cell death in embryonic cells. Misfolded proteins are death (apoptosis) in the neural epithelium (1). Diabetes-induced ubiquitinylated and form aggregates that contain the neurode- apoptosis is mediated by Caspase-8 (5), which is activated by a generative disease-associated proteins α-Synuclein, Parkin, and death-inducing signaling complex (DISC) (6, 7). Huntingtin (Htt). Aggregation of Htt leads to formation of a Maternal hyperglycemia, the major teratogen of diabetes- Hip1–Hippi–Caspase-8 complex to activate Caspase-8. Treatment induced embryonic malformations, exerts profound effects on with chemical chaperones alleviates protein aggregation and intracellular metabolic homeostasis and organelle function (1). decreases neural tube defects in the embryos of diabetic mice. Perturbation of rough endoplasmic reticulum (ER) function has Targeting misfolded proteins could be a potential approach to been implicated in diabetic embryopathy by the observations that preventing birth defects in diabetic pregnancies. factors involved in the unfolded protein response are up-regulated Author contributions: Z.Z. and E.A.R. designed research; Z.Z. and L.C. performed research; (8, 9), due to accumulation of improperly folded proteins in the L.C. analyzed data; and Z.Z. wrote the paper. ER lumen (10). Misfolded proteins are usually degraded through the The authors declare no conflict of interest. ubiquitin-proteasome system (UPS), including the ER-associated This article is a PNAS Direct Submission. T.M.D. is a Guest Editor invited by the Editorial protein degradation pathway (11–13).However,iftheUPSisim- Board. paired, misfolded proteins escape degradation and are released 1Z.Z. and L.C. contributed equally to this work. into the cytosol (14). Accumulation of misfolded cytotoxic proteins 2To whom correspondence should be addressed. Email: [email protected]. within a cell perturbs organelle functions, such as inducing the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mitochondria to generate an abundance of reactive oxygen species 1073/pnas.1616119114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1616119114 PNAS | April 25, 2017 | vol. 114 | no. 17 | 4489–4494 Downloaded by guest on September 25, 2021 ABND DM validated to specifically detect protein aggregates and aggresome- 8 like inclusion bodies in cells and cell lysates (31). In the tissue sections of the dorsal brain (SI Appendix, Fig. S1), 6 more intense Proteostat signals were observed in the DM group

-actin than in the ND group (Fig. 1 C and F). Quantification of the β 4 levels of protein aggregation showed significant increases in the DM group, compared with those in the ND group (Fig. 1I). Ub-proteins 2 Aggregates of ubiquitinylated proteins are subject to ALS clear- Ub-protein/ ance, mediated by ubiquitin-binding protein p62 (25, 26). Double- 0 labeling with Proteostat to detect protein aggregates and an anti- ND DM β-actin body to detect p62 revealed colocalization of protein aggregates and F–H C D E I 150 p62 (Fig. 1 ). To determine whether high glucose induces the protein aggregation observed in embryos of diabetic dams, we used neural stem 100 cells, derived from E9 mouse neural tubes, as a model system (32). The cells were cultured in a concentration of 27.8 mM (500 mg/dL) glucose [high glucose (HG)] or 6 mM (100 mg/dL) glucose [normal F G H 50 glucose (NG)] and assessed for protein aggregation. Significant increases in protein aggregation and colocalization with p62 were observed in the cells of the HG group, compared with those in the cells of the NG group (Fig. 1 J–P).

% (Aggregates/total protein) 0 ND DM Aggregation of Neurodegenerative Disease-Associated Proteins. Aggregation of α-Syn, Parkin, and Htt is seen in Alzheimer’s, 40 J K L P Parkinson’s, and Huntington’s diseases (19). Because these proteins are also expressed in the neural epithelium of the developing 30 embryo (SI Appendix,Fig.S2)(28–30), we sought to determine whether these neurodegenerative disease-associated proteins also 20 aggregate in the neural tube of embryos from diabetic mice. M N O We used a proximity ligation assay (PLA) to examine interactions 10 between p62 and α-Syn, Parkin, and Htt in situ on tissue sections. When two proteins physically interact, the antibodies that recognize % (Positive/total cells) 0 them are in a close proximity so that conjugated oligonucleotides can NG HG serve as guides to ligate additional oligonucleotides to form a cir- cular template for DNA synthesis. The DNA amplified from the Fig. 1. Protein ubiquitinylation and aggregation in cells of the embryonic template is then detected using a fluorescent dye-labeled probe (33). neural tube of diabetic mice and cultured neural stem cells treated with high glucose. (A) Western blot of ubiquitinylated (Ub) proteins in the dorsal region PLA signals in the dorsal region of the neural tube at E9.5 in of the brain of E9.5 embryos, using an anti–Ub-protein antibody. (B)Quanti- the DM group were significantly higher than those in the ND fication of the Western blot bands of the whole lane. β-Actin, loading control. group (Fig. 2 A–I), as well as in the neural stem cells cultured in Data are presented as mean ± SD, *P = 0.005, n = 3embryos.(C, F, J,andM) high glucose (SI Appendix, Fig. S3). Such protein–protein inter- Proteostat detection (red). (D, G, K,andN) p62 immunofluorescence (green). actions were also detected in high glucose-treated neural stem (E, H, L,andO) Merged images of Proteostat, p62, and DAPI. (C–H)Dorsalbrain cells, using a coimmunoprecipitation assay (Fig. 2 J and K). regionsofE9.5embryos.(Inset in C) Diagrammatic representation of a cross- – – section of the neural tube. The box marks the proximal area shown in C H.(C E) Formation of Death-Inducing Signaling Complex. High levels of ap- – Nondiabetic (ND). (F H) Diabetes mellitus (DM). Arrows indicate colocalization optosis in NTDs in diabetic embryopathy are mediated by Caspase-8 of Proteostat and p62. (I) Levels of protein aggregation in neural tissues, assessed by measuring Proteostat fluorescence intensity. Data are presented as (34). Caspase-8 can be activated by a DISC. One such DISC mean ± SD, *P = 0.003, n = 6 embryos (three repeats). (J–O) Neural stem cells. contains Htt-interacting protein 1 (Hip1) and Hip1 protein inter- (J, K,andL)Normalglucose(NG).(M–O) High glucose (HG). (P) Quantification of actor (Hippi), which forms when Htt aggregates to release Hip1 (35, neural stem cells with protein aggregates. Data are presented as mean ± SD, 36). We investigated if Hip1, Hippi, and Caspase-8 form this unique *P = 0.003, n = 4 repeats. (Scale bar = 10 μmforallimages.) DISC in embryos from diabetic dams. Interactions between these proteins were detected, using PLA, in the dorsal region of the neural tube in E9.5 embryos. The levels of these protein complexes were plays a role in diabetes-induced NTDs, we generated diabetic significantly higher in the DM group than in the ND group (Fig. 3). pregnant mice (10 wk old) and collected neural tissues of embryos at E9.5, an important stage in murine neurulation (3, 4), to Effects of Chemical Chaperones on Protein Aggregation in Vitro and quantify changes in levels of ubiquitinylated proteins. In the in Vivo. To examine if protein aggregation in diabetic embryopathy dorsal brain regions (SI Appendix, Fig. S1), where cranial NTDs is the result of aberrant protein folding, we treated embryonic occur, the levels of global protein ubiquitinylation were signifi- neural stem cells with chemical chaperones, including sodium cantly higher in the diabetes mellitus (DM) group, compared 4-phenylbutyrate (PBA), sodium taurochenodeoxycholate (TUDCA), and trimethylamine N-oxide (TMAO). The treatments significantly with the nondiabetic (ND) group (Fig. 1 A and B). decreased protein aggregation in neural stem cells cultured in high Accumulation of misfolded proteins has been implicated in glucose (HG+PBA: 25 μMand50μM; HG+TUDCA: 25 μMand diabetic embryopathy (8, 9), but the fate of these misfolded pro- 50 μM; HG+TMAO: 100 μMand250μM),comparedwiththe teins, which can aggregate and become cytotoxic (18), has not HG+VEH group (Fig. 4 A–C). been previously explored. We used a Proteostat system to examine To investigate whether chemical chaperones can alleviate the protein aggregates in the neural tube of embryos at E9.5 and hyperglycemia-induced protein-folding crisis, and can reduce E10.5. Proteostat is a rotor molecule that emits fluorescence when diabetes-induced embryonic malformations in vivo, we used PBA it binds to the tertiary structure of aggregated proteins. It has been to treat diabetic pregnant mice (100 mg/kg, daily) from E6.5 to

4490 | www.pnas.org/cgi/doi/10.1073/pnas.1616119114 Zhao et al. Downloaded by guest on September 25, 2021 ND DM A B C 5 α -Syn Input 4 J (5%) IgG 3 70 kDa p62 2

Dots/cell 1 50 kDa p62- α -Syn 0 ND DM D E F 1.0 0.8

0.6

0.4 Dots/cell K Htt p62-Parkin 0.2

0.0 70 kDa ND DM p62 I 2.5 G H 50 kDa 2.0

1.5

1.0 Dots/cell p62-Htt 0.5

0.0 ND DM

Fig. 2. Interactions of p62 with neurodegenerative proteins. (A–I) PLA to detect interaction between p62 and α-Syn (A and B), Parkin (D and E), or Htt (G and H) in the dorsal brain regions of E9.5 embryos. PLA signals are red dots. DAPI counterstaining is blue. (Inset in A) Diagrammatic representation of a cross-section of the neural tube. The box marks the proximal area shown in the figures. (C, D,andI) Quantification of PLA signals. Data are presented as mean ± SD, *P = 0.002, n = 3embryos(C); *P = 0.0009, n = 4 embryos (F); *P = 0.0003, n = 3embryos(I). (J and K) Coimmunoprecipitation assay of neural stem cells treated with a high concentration of glucose (27.8 mM or 500 mg/dL) for 18 h. Pull-down with antibodies recognizing α-Syn (J)andHtt(K); Western blot detection of p62 with an anti- p62 antibody. Arrows and open stars indicate full-length and degraded products of p62, respectively. Arrowheads indicate molecular weight markers (70 kDa and 50 kDa). n = 3 repeats. ND, nondiabetic; DM, diabetes mellitus. (Scale bar = 20 μminA–H.)

E9.5, the period of early neurulation. After treatment, neural (p-eIF2α). Maternal diabetes also compromises cell survival tissues were collected at E9.5 and E10.5 for examination of protein regulation, indicated by decreased expression of protein kinase modification and aggregation. In the embryos of the DM+PBA Bα (p-Pkbα) and mechanistic target of rapamycin (p-mTOR) (8, group, the levels of global protein ubiquitinylation and aggregation 15). In the present study, PBA treatment significantly decreased were significantly decreased, compared with those in the DM+VEH the levels of Atf6 (Fig. 6A), p-Perk (Fig. 6B), and p-eIF2α (Fig. group (Fig. 4 D and E). The involvement of α-Syn, Parkin, and Htt 6C) and increased the activation of p-Pkbα (Fig. 6D) and in the p62-containing inclusion bodies was also significantly de- p-mTOR (Fig. 6E), compared with the levels of the proteins in MEDICAL SCIENCES creased, compared with that in the DM+VEH group (Fig. 4 F–H). the DM+VEH group, at E9.5 and E10.5.

Effects of PBA on NTDs, Apoptosis, and Caspase-8 Activation. In hu- Discussion man diabetic pregnancies, NTDs in offspring are present in the Maternal diabetes mellitus perturbs protein folding in cells of the brain and spinal cord, and are frequently manifested as exence- embryo, leading to accumulation of improperly folded proteins (8, phaly, anencephaly, and spina bifida (37). Our mouse model sys- 9). However, the fate of these abnormally folded proteins, which tem recapitulates these anomalies. Embryos from the DM+VEH likely interfere with normal intracellular signaling and cellular group displayed NTDs in the forebrain (presumptive anenceph- activities, has not been explored. Here, we show that misfolded aly), midbrain and/or hindbrain (presumptive exencephaly), and proteins form aggregates and are resistant to degradation via the spinal cord (spina bifida) (Fig. 5B) (38). UPS and ALS. In addition, the protein aggregates contain α-Syn, The NTD rate, examined at E10.5, in the DM+VEH group was Parkin, and Htt, which are also involved in the pathogenesis of significantly higher than that in the ND group (Table 1). When neurodegenerative diseases, including Alzheimer’s, Parkinson’s, diabetic pregnant mice were treated with PBA, the NTD rate was and Huntington’sdiseases(19–22). The presence of these aggre- significantly lower than that in the DM+VEH group, but similar to gates in maternal diabetes-induced embryonic NTDs suggests that that in the ND group (Table 1 and Fig. 5 A–C). PBA treatment similar molecular processes may be occurring in diabetic embry- also significantly reduced the levels of apoptosis in the dorsal re- opathy and neurodegenerative diseases. gion of the neural tube at E9.5 and E10.5 (Fig. 5 D–F)andalso To control the quality of proteins, abnormally folded proteins reduced the levels of Caspase-8 activation (Fig. 5 G and H). are usually flagged with ubiquitin and degraded by the UPS (12). In the neural tube of embryos of diabetic mice, global protein Effects of PBA on Cellular Stress and Cell Survival Signaling. It was ubiquitinylation is increased, indicating a significant accumulation previously shown that maternal diabetes exacerbates ER stress, of misfolded proteins. Abnormally folded proteins can aggregate indicated by an increased expression of activating transcription and form inclusion bodies in the cell, thereby perturbing intracel- factor 6 (Atf6), phosphorylated (p) protein kinase RNA-like ER lular signaling and cell activity. Although these aggregates can be kinase (PERK), and α-subunit of eukaryotic initiation factor 2 eliminated by p62-mediated ALS (25, 26), the accumulation of

Zhao et al. PNAS | April 25, 2017 | vol. 114 | no. 17 | 4491 Downloaded by guest on September 25, 2021 ND DM is to regulate mitochondrial morphogenesis and remove damaged A B C 5 mitochondria through a form of ALS (50, 51). Mutations in 4 PINK1 are associated with mitochondrial dysmorphogenesis and ’ 3 dysfunction, which are also characteristics of Parkinson s disease 2 (52, 53). In diabetic embryopathy, the aggregation of Parkin may perturb mitochondrial dynamics and protein quality control,

Dots/cell 1 Hip1-Hippi thereby leading to embryonic malformations (15). 0 ND DM Htt aggregation causes neuronal death via multiple mechanisms, including activating Caspase-8 (54). Caspase-8–dependent apo- D E F 3 ptosis has been observed in diabetes-induced NTDs (5). The DISC, which activates Caspase-8 in the tumor necrosis factor re- 2 ceptor (TNFR) and Fas receptor signaling systems, consists of Fas- associated death domain-containing protein, TNFR-associated 1 Dots/cell factor 2, and TNFR-associated death domain protein (6, 7).

Hip1-Casp8 However, in Huntington’s disease, Caspase-8 can be activated by a 0 ND DM unique DISC, consisting of HIP1 and HIPPI (55). HTT aggrega- 6 tion releases HIP1 from the cell membrane to make it available for G H I DISC formation (36). In this study, we observed that Hip1, Hippi, 4 and Caspase-8 form a complex in the neural tissues of embryos from diabetic dams, suggesting that Htt aggregation serves as one 2 mechanism of Caspase-8 activation in diabetic embryopathy.

Dots/cell The pathway that begins with disrupted protein folding and Hippi-Casp8 0 ends with protein aggregation in embryonic cells subjected to ND DM

Fig. 3. Protein interactions in the embryonic neural tube of diabetic mice. PLA assay to detect interaction between Hip1 and Hippi (A and B), Hip1 and A BC Caspase-8 (D and E), and Hippi and Caspase-8 (G and H) in the dorsal brain of 40 40 E9.5 embryos. PLA signals are red dots. DAPI counterstaining is blue. (Inset in 40 30 A) Diagrammatic representation of a cross-section of the neural tube. The 30 30 box marks the proximal area shown in the figures. (A, D,andG) Nondiabetic 20 (ND). (B, E, and H) Diabetes mellitus (DM). (Scale bar = 20 μm.) (C, F, and I) 20 20 ± = = Quantification of PLA signals. Data are presented as mean SD, *P 0.0004, n 10 10 10 3embryos(C); *P = 0.0007, n = 3embryos(F); *P = 0.0001, n = 3embryos(I). % (Positive/total cells) % (Positive/total cells) 0 0 % (Positive/total cells) 0

NG NG NG 25 µM 50 µM 25 µM 50 µM HG+VEH HG+VEH 100 µM 250 µM p62-containing aggregates indicates an impairment of the ALS in HG+PBA HG+TUDCA HG+VEH HG+TMAO embryonic cells of diabetic mice. D E Three proteins associated with neurodegenerative diseases, 150 α-Syn, Parkin, and Htt, also form aggregates in the embryos of 8 diabetic mice. These proteins are expressed in the neural epithe- 6 100 – β -actin lium during neurulation (28 30). Aggregation of these proteins 4 50 may abolish their normal functions and also disturb intracellular 2 signaling, as seen in neurodegenerative diseases (19–22).

Ub-proteins/ 0 0

Protein aggregates containing α-Syn, known as Lewy bodies % (Aggregates/total protein) (LBs), are seen in many diseases, including neurodegenerative ND ND DM+PBA diseases, dementia with LBs, and multiple system atrophy, and thus DM+VEH DM+VEHDM+PBA are referred to as syncleinopathies (39, 40). The role of α-Syn in- FG5 1.0 H2.5 clusion bodies in the pathogenesis of neurodegenerative diseases is 4 0.8 2.0 not completely understood; however, α-Syn interacts with phos- 3 0.6 1.5 2 0.4 1.0 Dots/cell pholipids on plasma membranes and may be involved in altered Dots/cell Dots/cell phospholipid metabolism (41). Aberrant phospholipid metabolism 1 0.2 0.5 has also been suggested as an important process in diabetic 0 0.0 0.0 ND ND embryopathy (15). α-Syn is involved in regulation of mitochondrial ND DM+VEHDM+PBA DM+VEHDM+PBA dynamics, such as fusion and fission (42), and LBs localized in the DM+VEH DM+PBA mitochondria can influence electron transport to generate ROS Fig. 4. Effects of chemical chaperones on protein aggregation in neural (43, 44). Both perturbed mitochondrial dynamics and ROS over- stem cells and embryonic neural tube of diabetic mice. (A–C) Neural stem production are associated with hyperglycemia-induced embryonic cells cultured in high glucose and treated with chemical chaperones for 18 h. malformations in diabetic embryopathy (1, 15); therefore, the role Data are presented as mean ± SD. (A) PBA, 25 μM and 50 μM. *P < 0.0001, of LBs in diabetic embryopathy deserves further investigation. n = 4 repeats. (B) TUDCA, 25 μM and 50 μM. *P < 0.0001, n = 4 repeats. (C) Parkin is an E3 ubiquitin ligase, which plays a role in protein TMAO, 100 μM and 250 μM. *P < 0.0001, n = 4 repeats. (D–H) Embryos from degradation via the UPS (45, 46). Mutations in the PARK2 gene diabetic mice treated with PBA (100 mg/kg) from E6.5 to E9.5. Data are pre- cause PARKIN aggregation and loss of function in neurons, which sented as mean ± SD. (D) Levels of protein ubiquitinylation in the brain tissues of = = are hallmarks of Parkinson’s disease (47). In the present study, we E9.5 embryos, assessed using Western blot assay. *P 0.005, n 3embryos.(E) observed Parkin aggregation in the neural cells of embryos from Levels of protein aggregation in brain tissues of E9.5 embryos, assessed by measuring Proteostat fluorescence intensity. *P = 0.0002, n = 6embryos diabetic dams, suggesting that an impaired Parkin-mediated UPS (three repeats). (F–H) PLA assay of interaction of p62 with α-Syn (F:*P = 0.005, may be involved in diabetic embryopathy. PARKIN interacts with n = 3 embryos), Parkin (G:*P = 0.0001, n = 3 embryos), and Htt (H:*P = 0.0001, PTEN-induced putative kinase 1 (PINK1) on the mitochondrial n = 3 embryos) in the dorsal region of neural tube at E9.5. DM, diabetes mellitus; membrane (48, 49). The action of the PARKIN–PINK1 complex HG, high glucose; ND, nondiabetic; NG, normal glucose; VEH, vehicle.

4492 | www.pnas.org/cgi/doi/10.1073/pnas.1616119114 Zhao et al. Downloaded by guest on September 25, 2021 123 ABC G 50 kDa

30 kDa

18 kDa -actin DEF H 0.8 0.6

0.4

0.2

Casp8/ -actin0.0 12 3

Fig. 5. Effect of PBA on NTDs, apoptosis, and Caspase-8 activation in the embryos of diabetic mice. Diabetic pregnant dams were treated with PBA (100 mg/kg) from E6.5 to E9.5. (A–C) Embryos at E10.5. Arrow indicates open neural tube. (D–F) TUNEL assay of brain region of the neural tube of E9.5 embryos. (A and D) ND. (B and E)DM+VEH. (C and F)DM+PBA. (G) Western blot of Caspase-8 cleavage in the brain region of the neural tube. Arrowheads indicate cleaved products of Caspase-8 (43 kDa and 18 kDa). 1, ND; 2, DM+VEH; 3, DM+PBA; β-actin, loading control. (H) Quantification of the 18-kDa bands. Data are presented as mean ± SD, *P = 0.0003, n = 4 embryos. (Scale bar = 0.5 mm in A–C;10μminD–F.)

hyperglycemic conditions is demonstrated by the treatment with models by restoring protein folding, thereby reducing cellular three different chemical chaperones (PBA, TUDCA, and TMAO). stress (61, 62). Chemical chaperones have also been tested in The involvement of protein folding and aggregation in the humans to treat protein-folding–related diseases (57, 63, 64). The pathogenesis of diabetic embryopathy is further demonstrated by observation that PBA reduces embryonic malformations in di- in vivo treatment with PBA. More importantly, PBA treatment abetic mice suggests that targeting aberrant protein folding could significantly reduces NTDs in embryos of diabetic dams. Such a be a means to prevent birth defects in diabetic pregnancies. significant effect is associated with reductions of Caspase-8 acti- In addition to enhancing protein folding, targeting protein ag- vation and apoptosis, alleviation of ER stress, and restoration of gregation could mitigate the cytotoxicity of protein inclusion cell survival regulation. PBA, a Food and Drug Administration- bodies (65). For example, efforts have been taken to block or approved drug, has been shown to protect neuronal death by disrupt α-Syn oligomization in Parkinson’s disease with promising enhancing proper protein folding (56) and is used to ameliorate protein folding-associated diseases (57). These observations outcomes (66). The delineation of the protein-folding crisis and suggest that targeting the protein-folding crisis is a potential aggregation in hyperglycemia-induced embryonic malformations interventional approach to prevent diabetes-associated birth provides opportunities for developing interventions at multiple defects. levels to prevent birth defects in diabetic pregnancies. Previous efforts to reduce embryonic malformations in diabetic MEDICAL SCIENCES embryopathy have focused on amelioration of oxidative stress using antioxidants (1). However, the effectiveness of antioxidants in humans remains questionable, as multiple large-scale human trials that have used antioxidants (i.e., vitamin C and vitamin E) to treat other diseases (i.e., preeclampsia, cardiovascular disease) have revealed unsatisfactory results (58–60). Although the reasons for this failure have not been identified, it is speculated that other cellular stress conditions (i.e., ER stress), which may not be af- fected by antioxidant therapies, may be involved (60). Chemical chaperones have been shown to improve diabetes in animal

Table 1. NTD rate in the embryos of diabetic mice treated with PBA Total no. of Blood embryos Embryos glucose, mg/dL; Groups (litters) with NTDs NTD rate, % mean ± SD

ND 85 (11) 1 1.18 121.7 ± 16.8 Fig. 6. Changes in ER stress and cell survival factors by PBA treatment in the DM+VEH 96 (13) 23 23.96 311.5 ± 7.7 embryonic neural tube of diabetic mice. Data are presented as mean ± SD. – = = = = DM+PBA 123 (17) 5 4.06 291.7 ± 9.7 (A C) ER stress factors Atf6 (*P 0.045, n 3 embryos), p-Perk (*P 0.04, n 3 embryos), and p-eIF2α (*P = 0.042, n = 5 embryos). (D and E) Cell survival DM, diabetes mellitus; ND, nondiabetic; NTDs: open forebrain, midbrain, factors p-Pkbα (*P = 0.0013, n = 4 embryos) and p-mTOR (*P = 0.032, n = hindbrain, and/or spinal cord. PBA, phenylbutyrate; VEH, vehicle. P = 0.009 4 embryos). β-Actin, loading control; DM, diabetes mellitus; ND, nondiabetic; (DM+PBA vs. DM+VEH); P = 0.0016 (DM+VEH vs. ND); P = 0.17 (DM+PBA vs. ND). PBA, phenylbutyrate; VEH, vehicle.

Zhao et al. PNAS | April 25, 2017 | vol. 114 | no. 17 | 4493 Downloaded by guest on September 25, 2021 Materials and Methods and Western blot assay, as well as statistical analyses, can be found in the SI Appendix, Supplementary Materials and Methods. Diabetes mellitus was induced in female mice (C57BL/6J) by i.v. injection of streptozotocin. The use of animals was approved by the Institutional Animal ACKNOWLEDGMENTS. We thank Hua Li for technical assistance, Dr. Min Care and Use Committee of University of Maryland, Baltimore. Detailed Zhan for statistical analyses, and Dr. Julie Rosen for critical reading and procedures about the generation of diabetic mice, cell culture, protein ag- editing. The work was supported by the National Institutes of Health under gregation assay, TUNEL, proximity ligation assay, coimmunoprecipitation, Award Numbers HD076245 (to Z.Z.) and HD075995 (to Z.Z.).

1. Zhao Z, Reece EA (2013) New concepts in diabetic embryopathy. Clin Lab Med 33: 35. Bates G (2003) Huntingtin aggregation and toxicity in Huntington’s disease. Lancet 207–233. 361:1642–1644. 2. Greene ND, Copp AJ (2014) Neural tube defects. Annu Rev Neurosci 37:221–242. 36. Gervais FG, et al. (2002) Recruitment and activation of caspase-8 by the Huntingtin- 3. O’Rahilly R, Muller F (1994) Neurulation in the normal human embryo. Ciba Found interacting protein Hip-1 and a novel partner Hippi. Nat Cell Biol 4:95–105. Symp 181:70–82. 37. Correa A, et al. (2008) Diabetes mellitus and birth defects. Am J Obstet Gynecol 199: 4. Copp AJ, Greene ND, Murdoch JN (2003) The genetic basis of mammalian neurulation. 237.e1–9. Nat Rev Genet 4:784–793. 38. Zhao Z (2010) Cardiac malformations and alteration of TGFb signaling system in di- 5. Zhao Z, Yang P, Eckert RL, Reece EA (2009) Caspase-8: A key role in the pathogenesis abetic embryopathy. Birth Defects Res B Dev Reprod Toxicol 89:97–105. of diabetic embryopathy. Birth Defects Res B Dev Reprod Toxicol 86:72–77. 39. Spillantini MG, et al. (1997) α-Synuclein in Lewy bodies. Nature 388:839–840. 6. Tibbetts MD, Zheng L, Lenardo MJ (2003) The death effector domain protein family: 40. Goedert M (2001) α-Synuclein and neurodegenerative diseases. Nat Rev Neurosci Regulators of cellular homeostasis. Nat Immunol 4:404–409. 2:492–501. 7. Sheikh MS, Huang Y (2003) Death receptor activation complexes: It takes two to 41. Dikiy I, Eliezer D (2012) Folding and misfolding of α-synuclein on membranes. Biochim activate TNF receptor 1. Cell Cycle 2:550–552. Biophys Acta 1818:1013–1018. 8. Zhao Z, Eckert RL, Reece EA (2012) Reduction in embryonic malformations and alle- 42. Norris KL, et al. (2015) Convergence of Parkin, PINK1, and α-Synuclein on stress- viation of endoplasmic reticulum stress by nitric oxide synthase inhibition in diabetic induced mitochondrial morphological remodeling. J Biol Chem 290:13862–13874. α embryopathy. Reprod Sci 19:823–831. 43. Reeve AK, et al. (2015) Aggregated -synuclein and complex I deficiency: Exploration 9. Zhao Z (2012) Endoplasmic reticulum stress in maternal diabetes-induced cardiac of their relationship in differentiated neurons. Cell Death Dis 6:e1820. malformations during critical cardiogenesis period. Birth Defects Res B Dev Reprod 44. Devi L, Raghavendran V, Prabhu BM, Avadhani NG, Anandatheerthavarada HK (2008) α Toxicol 95:1–6. Mitochondrial import and accumulation of -synuclein impair complex I in human 10. Walter P, Ron D (2011) The unfolded protein response: From stress pathway to ho- dopaminergic neuronal cultures and Parkinson disease brain. J Biol Chem 283: – meostatic regulation. Science 334:1081–1086. 9089 9100. 11. Meusser B, Hirsch C, Jarosch E, Sommer T (2005) ERAD: The long road to destruction. 45. Trempe JF, et al. (2013) Structure of parkin reveals mechanisms for ubiquitin ligase – Nat Cell Biol 7:766–772. activation. Science 340:1451 1455. 12. Glickman MH, Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway: 46. Zhang Y, et al. (2000) Parkin functions as an E2-dependent ubiquitin-protein ligase Destruction for the sake of construction. Physiol Rev 82:373–428. and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1. – 13. Jarosch E, Lenk U, Sommer T (2003) Endoplasmic reticulum-associated protein deg- Proc Natl Acad Sci USA 97:13354 13359. 47. Sriram SR, et al. (2005) Familial-associated mutations differentially disrupt the solu- radation. Int Rev Cytol 223:39–81. bility, localization, binding and ubiquitination properties of parkin. Hum Mol Genet 14. Tsai B, Ye Y, Rapoport TA (2002) Retro-translocation of proteins from the endo- 14:2571–2586. plasmic reticulum into the cytosol. Nat Rev Mol Cell Biol 3:246–255. 48. Kim Y, et al. (2008) PINK1 controls mitochondrial localization of Parkin through direct 15. Cao L, et al. (2016) Identification of novel cell survival regulation in diabetic embry- phosphorylation. Biochem Biophys Res Commun 377:975–980. opathy via phospholipidomic profiling. Biochem Biophys Res Commun 470:599–605. 49. Kawajiri S, et al. (2010) PINK1 is recruited to mitochondria with parkin and associates 16. Yang X, Borg LA, Eriksson UJ (1995) Altered mitochondrial morphology of rat em- with LC3 in mitophagy. FEBS Lett 584:1073–1079. bryos in diabetic pregnancy. Anat Rec 241:255–267. 50. Poole AC, et al. (2008) The PINK1/Parkin pathway regulates mitochondrial morphol- 17. Ron D, Walter P (2007) Signal integration in the endoplasmic reticulum unfolded ogy. Proc Natl Acad Sci USA 105:1638–1643. protein response. Nat Rev Mol Cell Biol 8:519–529. 51. Vives-Bauza C, et al. (2010) PINK1-dependent recruitment of Parkin to mitochondria 18. Moreno-Gonzalez I, Soto C (2011) Misfolded protein aggregates: Mechanisms, in mitophagy. Proc Natl Acad Sci USA 107:378–383. structures and potential for disease transmission. Semin Cell Dev Biol 22:482–487. 52. Lim KL, Ng XH, Grace LG, Yao TP (2012) Mitochondrial dynamics and Parkinson’s 19. Ross CA, Poirier MA (2004) Protein aggregation and neurodegenerative disease. Nat disease: Focus on parkin. Antioxid Redox Signal 16:935–949. Med 10:S10–S17. 53. Bueler H (2009) Impaired mitochondrial dynamics and function in the pathogenesis 20. Selkoe DJ (2011) Alzheimer’s disease. Cold Spring Harb Perspect Biol 3:a004457. of Parkinson’s disease. Exp Neurol 218:235–246. 21. Irwin DJ, Lee VM, Trojanowski JQ (2013) Parkinson’s disease dementia: Convergence 54. Labbadia J, Morimoto RI (2013) Huntington’s disease: Underlying molecular mecha- of α-synuclein, tau and amyloid-beta pathologies. Nat Rev Neurosci 14:626–636. nisms and emerging concepts. Trends Biochem Sci 38:378–385. 22. Ross CA, Tabrizi SJ (2011) Huntington’s disease: From molecular pathogenesis to 55. Mattson MP (2002) Accomplices to neuronal death. Nature 415:377–379. clinical treatment. Lancet Neurol 10:83–98. 56. Mimori S, et al. (2013) 4-Phenylbutyric acid protects against neuronal cell death by 23. Boya P, Reggiori F, Codogno P (2013) Emerging regulation and functions of auto- primarily acting as a chemical chaperone rather than histone deacetylase inhibitor. phagy. Nat Cell Biol 15:713–720. Bioorg Med Chem Lett 23:6015–6018. 24. Mizushima N, Komatsu M (2011) Autophagy: Renovation of cells and tissues. Cell 147: 57. Rubenstein RC, Zeitlin PL (1998) A pilot clinical trial of oral sodium 4-phenylbutyrate – 728 741. (Buphenyl) in ΔF508-homozygous cystic fibrosis patients: Partial restoration of nasal 25. Rogov V, Dotsch V, Johansen T, Kirkin V (2014) Interactions between autophagy re- epithelial CFTR function. Am J Respir Crit Care Med 157:484–490. ceptors and ubiquitin-like proteins form the molecular basis for selective autophagy. 58. Villar J, et al. (2009) World Health Organisation multicentre randomised trial of – Mol Cell 53:167 178. supplementation with vitamins C and E among pregnant women at high risk for pre- 26. Katsuragi Y, Ichimura Y, Komatsu M (2015) p62/SQSTM1 functions as a signaling hub eclampsia in populations of low nutritional status from developing countries. Br J – and an autophagy adaptor. FEBS J 282:4672 4678. Obstet Gynaecol 116:780–788. 27. Zatloukal K, et al. (2002) p62 is a common component of cytoplasmic inclusions in 59. Briasoulis A, Tousoulis D, Antoniades C, Stefanadis C (2009) The oxidative stress – protein aggregation diseases. Am J Pathol 160:255 263. menace to coronary vasculature: Any place for antioxidants? Curr Pharm Des 15: 28. Dragatsis I, Efstratiadis A, Zeitlin S (1998) Mouse mutant embryos lacking huntingtin 3078–3090. are rescued from lethality by wild-type extraembryonic tissues. Development 125: 60. Steinhubl SR (2008) Why have antioxidants failed in clinical trials? Am J Cardiol 101: 1529–1539. 14D–19D. 29. Kuhn K, Zhu XR, Lubbert H, Stichel CC (2004) Parkin expression in the developing 61. Ozcan U, et al. (2006) Chemical chaperones reduce ER stress and restore glucose mouse. Brain Res Dev Brain Res 149:131–142. homeostasis in a mouse model of type 2 diabetes. Science 313:1137–1140. 30. Zhong SC, et al. (2010) Expression and subcellular location of α-synuclein during 62. Engin F, et al. (2013) Restoration of the unfolded protein response in pancreatic beta mouse-embryonic development. Cell Mol Neurobiol 30:469–482. cells protects mice against type 1 diabetes. Sci Transl Med 5:211ra156. 31. Shen D, et al. (2011) Novel cell- and tissue-based assays for detecting misfolded and 63. Parenti G, Andria G, Valenzano KJ (2015) Pharmacological chaperone therapy: Pre- aggregated protein accumulation within aggresomes and inclusion bodies. Cell clinical development, clinical translation, and prospects for the treatment of lyso- Biochem Biophys 60:173–185. somal storage disorders. Mol Ther 23:1138–1148. 32. Schlett K, Madarasz E (1997) Retinoic acid induced neural differentiation in a neu- 64. Ebrahimi-Fakhari D, Wahlster L, McLean PJ (2011) Molecular chaperones in Parkinson’s roectodermal cell line immortalized by p53 deficiency. J Neurosci Res 47:405–415. disease: Present and future. J Parkinsons Dis 1:299–320. 33. Soderberg O, et al. (2008) Characterizing proteins and their interactions in cells and 65. Ignatova Z, Gierasch LM (2006) Inhibition of protein aggregation in vitro and in vivo tissues using the in situ proximity ligation assay. Methods 45:227–232. by a natural osmoprotectant. Proc Natl Acad Sci USA 103:13357–13361. 34. Cao L, et al. (2016) Amelioration of intracellular stress and reduction of neural tube 66. Dehay B, et al. (2015) Targeting α-synuclein for treatment of Parkinson’s disease: defects in embryos of diabetic mice by phytochemical quercetin. Sci Rep 6:21491. Mechanistic and therapeutic considerations. Lancet Neurol 14:855–866.

4494 | www.pnas.org/cgi/doi/10.1073/pnas.1616119114 Zhao et al. Downloaded by guest on September 25, 2021