Formation of neurodegenerative aggresome anddeath-inducing signaling complex in maternaldiabetes-induced neural tube defectsZhiyong 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 ofBiochemistry 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 BoardMember Gregg L. Semenza March 13, 2017 (received for review September 27, 2016)

Diabetes mellitus in early pregnancy increases the risk in infants ofbirth defects, such as neural tube defects (NTDs), known as diabeticembryopathy. NTDs are associated with hyperglycemia-inducedprotein misfolding and Caspase-8–induced programmed cell death.The present study shows that misfolded proteins are ubiquitinylated,suggesting that ubiquitin-proteasomal degradation is impaired.Misfolded proteins form aggregates containing ubiquitin-bindingprotein p62, suggesting that autophagic-lysosomal clearance isinsufficient. Additionally, these aggregates contain the neurodegen-erative disease-associated proteins α-Synuclein, Parkin, and Huntingtin(Htt). Aggregation of Htt may lead to formation of a death-inducingsignaling complex of Hip1, Hippi, and Caspase-8. Treatment withchemical chaperones, such as sodium 4-phenylbutyrate (PBA), re-duces protein aggregation in neural stem cells in vitro and in embryosin vivo. Furthermore, treatment with PBA in vivo decreases NTD ratein the embryos of diabetic mice, as well as Caspase-8 activation andcell death. Enhancing protein folding could be a potential interven-tional approach to preventing embryonic malformations in diabeticpregnancies.

diabetic embryopathy | protein folding | protein aggregation | Caspase-8 |chemical chaperone

Pregnancies complicated by diabetes mellitus have a higherrisk of resulting in infants with congenital birth defects, a

complication known as diabetic embryopathy (1). The defects inthe central nervous system are the result of perturbed neural tubeformation (neurulation) during early embryogenesis and are thusreferred to as neural tube defects (NTDs) (2). Neurulation is aprocess that begins with development of the neural folds at aboutweek 2 of gestation in humans and embryonic day 8 (E8) in themouse, and concludes with fusion of the neural tissues at thedorsal midline, to form the neural tube, by about week 4 in humansand E11.5 in the mouse (3, 4). The failure of neural tube closure indiabetic embryopathy is associated with excessive programmed celldeath (apoptosis) in the neural epithelium (1). Diabetes-inducedapoptosis is mediated by Caspase-8 (5), which is activated by adeath-inducing signaling complex (DISC) (6, 7).Maternal hyperglycemia, the major teratogen of diabetes-

induced embryonic malformations, exerts profound effects onintracellular metabolic homeostasis and organelle function (1).Perturbation of rough endoplasmic reticulum (ER) function hasbeen implicated in diabetic embryopathy by the observations thatfactors involved in the unfolded protein response are up-regulated(8, 9), due to accumulation of improperly folded proteins in theER lumen (10). Misfolded proteins are usually degraded through theubiquitin-proteasome system (UPS), including the ER-associatedprotein degradation pathway (11–13). However, if the UPS is im-paired, misfolded proteins escape degradation and are releasedinto the cytosol (14). Accumulation of misfolded cytotoxic proteinswithin a cell perturbs organelle functions, such as inducing themitochondria to generate an abundance of reactive oxygen species

(ROS), which results in oxidative stress (15, 16) and disrupts in-tracellular signaling, thus altering cellular activities (17).Misfolded proteins are prone to forming aggregates that are

resistant to UPS degradation (18). Aggregation of α-Synuclein(α-Syn), Parkin, and Huntingtin (Htt) has been found to be in-volved in the pathogenesis of Alzheimer’s, Parkinson’s, andHuntington’s diseases (19–22). Protein aggregates can be elimi-nated by the autophagic-lysosomal system (ALS) (23, 24). Thetransfer of ubiquitinylated proteins to the ALS is mediated by aubiquitin-binding protein, p62, also known as Sequestosome-1 (25,26). Protein aggregates containing p62 are commonly seen inneurodegenerative diseases, indicating deficiency of the ALS inthe pathogenesis of these diseases (27). These neural degenerativedisease-associated proteins are expressed in the developing neuraltube (28–30); however, it is unknown whether they are involved inhyperglycemia-induced NTDs.In the present study, we aimed to investigate the fate of these

misfolded proteins and their potential effects on apoptotic regu-lation and NTD formation. We also explored an approach to al-leviate protein aggregation by enhancing protein folding usingchemical chaperones to provide insights into the mechanisms ofdiabetic embryopathy, and to help identify potential strategies toprevent diabetes-induced birth defects.

ResultsProtein Ubiquitinylation and Aggregation. Misfolded proteins areusually ubiquitinylated and degraded by the UPS (13). However,when the UPS is impaired, ubiquitinylated proteins can accu-mulate in the cell. Because we hypothesized that UPS dysfunction

Significance

Neural tube defects in infants of women with diabetes are as-sociated with disrupted protein folding and increased pro-grammed cell death in embryonic cells. Misfolded proteins areubiquitinylated and form aggregates that contain the neurode-generative disease-associated proteins α-Synuclein, Parkin, andHuntingtin (Htt). Aggregation of Htt leads to formation of aHip1–Hippi–Caspase-8 complex to activate Caspase-8. Treatmentwith chemical chaperones alleviates protein aggregation anddecreases neural tube defects in the embryos of diabetic mice.Targeting misfolded proteins could be a potential approach topreventing birth defects in diabetic pregnancies.

Author contributions: Z.Z. and E.A.R. designed research; Z.Z. and L.C. performed research;L.C. analyzed data; and Z.Z. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. T.M.D. is a Guest Editor invited by the EditorialBoard.1Z.Z. and L.C. contributed equally to this work.2To whom correspondence should be addressed. Email: zzhao@fpi.umaryland.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1616119114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1616119114 PNAS | April 25, 2017 | vol. 114 | no. 17 | 4489–4494

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plays a role in diabetes-induced NTDs, we generated diabeticpregnant mice (10 wk old) and collected neural tissues of em-bryos at E9.5, an important stage in murine neurulation (3, 4), toquantify changes in levels of ubiquitinylated proteins. In thedorsal brain regions (SI Appendix, Fig. S1), where cranial NTDsoccur, the levels of global protein ubiquitinylation were signifi-cantly higher in the diabetes mellitus (DM) group, comparedwith the nondiabetic (ND) group (Fig. 1 A and B).Accumulation of misfolded proteins has been implicated in

diabetic embryopathy (8, 9), but the fate of these misfolded pro-teins, which can aggregate and become cytotoxic (18), has notbeen previously explored. We used a Proteostat system to examineprotein aggregates in the neural tube of embryos at E9.5 andE10.5. Proteostat is a rotor molecule that emits fluorescence whenit binds to the tertiary structure of aggregated proteins. It has been

validated to specifically detect protein aggregates and aggresome-like inclusion bodies in cells and cell lysates (31).In the tissue sections of the dorsal brain (SI Appendix, Fig. S1),

more intense Proteostat signals were observed in the DM groupthan in the ND group (Fig. 1 C and F). Quantification of thelevels of protein aggregation showed significant increases in theDM group, compared with those in the ND group (Fig. 1I).Aggregates of ubiquitinylated proteins are subject to ALS clear-

ance, mediated by ubiquitin-binding protein p62 (25, 26). Double-labeling with Proteostat to detect protein aggregates and an anti-body to detect p62 revealed colocalization of protein aggregates andp62 (Fig. 1 F–H).To determine whether high glucose induces the protein aggre-

gation observed in embryos of diabetic dams, we used neural stemcells, 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 [normalglucose (NG)] and assessed for protein aggregation. Significantincreases in protein aggregation and colocalization with p62 wereobserved in the cells of the HG group, compared with those in thecells of the NG group (Fig. 1 J–P).

Aggregation of Neurodegenerative Disease-Associated Proteins.Aggregation of α-Syn, Parkin, and Htt is seen in Alzheimer’s,Parkinson’s, and Huntington’s diseases (19). Because these pro-teins are also expressed in the neural epithelium of the developingembryo (SI Appendix, Fig. S2) (28–30), we sought to determinewhether these neurodegenerative disease-associated proteins alsoaggregate in the neural tube of embryos from diabetic mice.We used a proximity ligation assay (PLA) to examine interactions

between p62 and α-Syn, Parkin, and Htt in situ on tissue sections.When two proteins physically interact, the antibodies that recognizethem are in a close proximity so that conjugated oligonucleotides canserve as guides to ligate additional oligonucleotides to form a cir-cular template for DNA synthesis. The DNA amplified from thetemplate is then detected using a fluorescent dye-labeled probe (33).PLA signals in the dorsal region of the neural tube at E9.5 in

the DM group were significantly higher than those in the NDgroup (Fig. 2 A–I), as well as in the neural stem cells cultured inhigh glucose (SI Appendix, Fig. S3). Such protein–protein inter-actions were also detected in high glucose-treated neural stemcells, using a coimmunoprecipitation assay (Fig. 2 J and K).

Formation of Death-Inducing Signaling Complex. High levels of ap-optosis in NTDs in diabetic embryopathy are mediated by Caspase-8(34). Caspase-8 can be activated by a DISC. One such DISCcontains Htt-interacting protein 1 (Hip1) and Hip1 protein inter-actor (Hippi), which forms when Htt aggregates to release Hip1 (35,36). We investigated if Hip1, Hippi, and Caspase-8 form this uniqueDISC in embryos from diabetic dams. Interactions between theseproteins were detected, using PLA, in the dorsal region of the neuraltube in E9.5 embryos. The levels of these protein complexes weresignificantly higher in the DM group than in the ND group (Fig. 3).

Effects of Chemical Chaperones on Protein Aggregation in Vitro andin Vivo. To examine if protein aggregation in diabetic embryopathyis the result of aberrant protein folding, we treated embryonicneural stem cells with chemical chaperones, including sodium4-phenylbutyrate (PBA), sodium taurochenodeoxycholate (TUDCA),and trimethylamine N-oxide (TMAO). The treatments significantlydecreased protein aggregation in neural stem cells cultured in highglucose (HG+PBA: 25 μM and 50 μM; HG+TUDCA: 25 μM and50 μM; HG+TMAO: 100 μM and 250 μM), compared with theHG+VEH group (Fig. 4 A–C).To investigate whether chemical chaperones can alleviate the

hyperglycemia-induced protein-folding crisis, and can reducediabetes-induced embryonic malformations in vivo, we used PBAto treat diabetic pregnant mice (100 mg/kg, daily) from E6.5 to

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Fig. 1. Protein ubiquitinylation and aggregation in cells of the embryonicneural tube of diabetic mice and cultured neural stem cells treated with highglucose. (A) Western blot of ubiquitinylated (Ub) proteins in the dorsal regionof the brain of E9.5 embryos, using an anti–Ub-protein antibody. (B) Quanti-fication of the Western blot bands of the whole lane. β-Actin, loading control.Data are presented as mean ± SD, *P = 0.005, n = 3 embryos. (C, F, J, and M)Proteostat detection (red). (D, G, K, and N) p62 immunofluorescence (green).(E, H, L, andO) Merged images of Proteostat, p62, and DAPI. (C–H) Dorsal brainregions of E9.5 embryos. (Inset in C) Diagrammatic representation of a cross-section of the neural tube. The boxmarks the proximal area shown in C–H. (C–E)Nondiabetic (ND). (F–H) Diabetes mellitus (DM). Arrows indicate colocalizationof Proteostat and p62. (I) Levels of protein aggregation in neural tissues,assessed by measuring Proteostat fluorescence intensity. Data are presented asmean ± SD, *P = 0.003, n = 6 embryos (three repeats). (J–O) Neural stem cells.(J, K, and L) Normal glucose (NG). (M–O) High glucose (HG). (P) Quantification ofneural stem cells with protein aggregates. Data are presented as mean ± SD,*P = 0.003, n = 4 repeats. (Scale bar = 10 μm for all images.)

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E9.5, the period of early neurulation. After treatment, neuraltissues were collected at E9.5 and E10.5 for examination of proteinmodification and aggregation. In the embryos of the DM+PBAgroup, the levels of global protein ubiquitinylation and aggregationwere significantly decreased, compared with those in the DM+VEHgroup (Fig. 4 D and E). The involvement of α-Syn, Parkin, and Httin the p62-containing inclusion bodies was also significantly de-creased, compared with that in the DM+VEH group (Fig. 4 F–H).

Effects of PBA on NTDs, Apoptosis, and Caspase-8 Activation. In hu-man diabetic pregnancies, NTDs in offspring are present in thebrain and spinal cord, and are frequently manifested as exence-phaly, anencephaly, and spina bifida (37). Our mouse model sys-tem recapitulates these anomalies. Embryos from the DM+VEHgroup displayed NTDs in the forebrain (presumptive anenceph-aly), midbrain and/or hindbrain (presumptive exencephaly), andspinal cord (spina bifida) (Fig. 5B) (38).The NTD rate, examined at E10.5, in the DM+VEH group was

significantly higher than that in the ND group (Table 1). Whendiabetic pregnant mice were treated with PBA, the NTD rate wassignificantly lower than that in the DM+VEH group, but similar tothat in the ND group (Table 1 and Fig. 5 A–C). PBA treatmentalso significantly reduced the levels of apoptosis in the dorsal re-gion of the neural tube at E9.5 and E10.5 (Fig. 5 D–F) and alsoreduced the levels of Caspase-8 activation (Fig. 5 G and H).

Effects of PBA on Cellular Stress and Cell Survival Signaling. It waspreviously shown that maternal diabetes exacerbates ER stress,indicated by an increased expression of activating transcriptionfactor 6 (Atf6), phosphorylated (p) protein kinase RNA-like ERkinase (PERK), and α-subunit of eukaryotic initiation factor 2

(p-eIF2α). Maternal diabetes also compromises cell survivalregulation, indicated by decreased expression of protein kinaseBα (p-Pkbα) and mechanistic target of rapamycin (p-mTOR) (8,15). In the present study, PBA treatment significantly decreasedthe levels of Atf6 (Fig. 6A), p-Perk (Fig. 6B), and p-eIF2α (Fig.6C) and increased the activation of p-Pkbα (Fig. 6D) andp-mTOR (Fig. 6E), compared with the levels of the proteins inthe DM+VEH group, at E9.5 and E10.5.

DiscussionMaternal diabetes mellitus perturbs protein folding in cells of theembryo, leading to accumulation of improperly folded proteins (8,9). However, the fate of these abnormally folded proteins, whichlikely interfere with normal intracellular signaling and cellularactivities, has not been explored. Here, we show that misfoldedproteins form aggregates and are resistant to degradation via theUPS and ALS. In addition, the protein aggregates contain α-Syn,Parkin, and Htt, which are also involved in the pathogenesis ofneurodegenerative diseases, including Alzheimer’s, Parkinson’s,and Huntington’s diseases (19–22). The presence of these aggre-gates in maternal diabetes-induced embryonic NTDs suggests thatsimilar molecular processes may be occurring in diabetic embry-opathy and neurodegenerative diseases.To control the quality of proteins, abnormally folded proteins

are usually flagged with ubiquitin and degraded by the UPS (12).In the neural tube of embryos of diabetic mice, global proteinubiquitinylation is increased, indicating a significant accumulationof misfolded proteins. Abnormally folded proteins can aggregateand form inclusion bodies in the cell, thereby perturbing intracel-lular signaling and cell activity. Although these aggregates can beeliminated by p62-mediated ALS (25, 26), the accumulation of

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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 ofthe neural tube. The box marks the proximal area shown in the figures. (C, D, and I) Quantification of PLA signals. Data are presented as mean ± SD, *P = 0.002,n = 3 embryos (C); *P = 0.0009, n = 4 embryos (F); *P = 0.0003, n = 3 embryos (I). (J and K) Coimmunoprecipitation assay of neural stem cells treated with a highconcentration of glucose (27.8 mMor 500 mg/dL) for 18 h. Pull-downwith antibodies recognizing α-Syn (J) and Htt (K); Western blot detection of p62with an anti-p62 antibody. Arrows and open stars indicate full-length and degraded products of p62, respectively. Arrowheads indicate molecular weight markers (70 kDa and50 kDa). n = 3 repeats. ND, nondiabetic; DM, diabetes mellitus. (Scale bar = 20 μm in A–H.)

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p62-containing aggregates indicates an impairment of the ALS inembryonic cells of diabetic mice.Three proteins associated with neurodegenerative diseases,

α-Syn, Parkin, and Htt, also form aggregates in the embryos ofdiabetic mice. These proteins are expressed in the neural epithe-lium during neurulation (28–30). Aggregation of these proteinsmay abolish their normal functions and also disturb intracellularsignaling, as seen in neurodegenerative diseases (19–22).Protein aggregates containing α-Syn, known as Lewy bodies

(LBs), are seen in many diseases, including neurodegenerativediseases, dementia with LBs, and multiple system atrophy, and thusare referred to as syncleinopathies (39, 40). The role of α-Syn in-clusion bodies in the pathogenesis of neurodegenerative diseases isnot completely understood; however, α-Syn interacts with phos-pholipids on plasma membranes and may be involved in alteredphospholipid metabolism (41). Aberrant phospholipid metabolismhas also been suggested as an important process in diabeticembryopathy (15). α-Syn is involved in regulation of mitochondrialdynamics, such as fusion and fission (42), and LBs localized in themitochondria can influence electron transport to generate ROS(43, 44). Both perturbed mitochondrial dynamics and ROS over-production are associated with hyperglycemia-induced embryonicmalformations in diabetic embryopathy (1, 15); therefore, the roleof LBs in diabetic embryopathy deserves further investigation.Parkin is an E3 ubiquitin ligase, which plays a role in protein

degradation via the UPS (45, 46). Mutations in the PARK2 genecause PARKIN aggregation and loss of function in neurons, whichare hallmarks of Parkinson’s disease (47). In the present study, weobserved Parkin aggregation in the neural cells of embryos fromdiabetic dams, suggesting that an impaired Parkin-mediated UPSmay be involved in diabetic embryopathy. PARKIN interacts withPTEN-induced putative kinase 1 (PINK1) on the mitochondrialmembrane (48, 49). The action of the PARKIN–PINK1 complex

is to regulate mitochondrial morphogenesis and remove damagedmitochondria through a form of ALS (50, 51). Mutations inPINK1 are associated with mitochondrial dysmorphogenesis anddysfunction, which are also characteristics of Parkinson’s disease(52, 53). In diabetic embryopathy, the aggregation of Parkin mayperturb mitochondrial dynamics and protein quality control,thereby leading to embryonic malformations (15).Htt aggregation causes neuronal death via multiple mechanisms,

including activating Caspase-8 (54). Caspase-8–dependent apo-ptosis has been observed in diabetes-induced NTDs (5). TheDISC, which activates Caspase-8 in the tumor necrosis factor re-ceptor (TNFR) and Fas receptor signaling systems, consists of Fas-associated death domain-containing protein, TNFR-associatedfactor 2, and TNFR-associated death domain protein (6, 7).However, in Huntington’s disease, Caspase-8 can be activated by aunique DISC, consisting of HIP1 and HIPPI (55). HTT aggrega-tion releases HIP1 from the cell membrane to make it available forDISC formation (36). In this study, we observed that Hip1, Hippi,and Caspase-8 form a complex in the neural tissues of embryosfrom diabetic dams, suggesting that Htt aggregation serves as onemechanism of Caspase-8 activation in diabetic embryopathy.The pathway that begins with disrupted protein folding and

ends with protein aggregation in embryonic cells subjected to

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Fig. 4. Effects of chemical chaperones on protein aggregation in neuralstem cells and embryonic neural tube of diabetic mice. (A–C) Neural stemcells cultured in high glucose and treated with chemical chaperones for 18 h.Data are presented as mean ± SD. (A) PBA, 25 μM and 50 μM. *P < 0.0001,n = 4 repeats. (B) TUDCA, 25 μM and 50 μM. *P < 0.0001, n = 4 repeats. (C)TMAO, 100 μM and 250 μM. *P < 0.0001, n = 4 repeats. (D–H) Embryos fromdiabetic mice treated with PBA (100 mg/kg) from E6.5 to E9.5. Data are pre-sented as mean ± SD. (D) Levels of protein ubiquitinylation in the brain tissues ofE9.5 embryos, assessed using Western blot assay. *P = 0.005, n = 3 embryos. (E)Levels of protein aggregation in brain tissues of E9.5 embryos, assessed bymeasuring Proteostat fluorescence intensity. *P = 0.0002, n = 6 embryos(three repeats). (F–H) PLA assay of interaction of p62 with α-Syn (F: *P = 0.005,n = 3 embryos), Parkin (G: *P = 0.0001, n = 3 embryos), and Htt (H: *P = 0.0001,n = 3 embryos) in the dorsal region of neural tube at E9.5. DM, diabetes mellitus;HG, high glucose; ND, nondiabetic; NG, normal glucose; VEH, vehicle.

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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 andCaspase-8 (D and E), and Hippi and Caspase-8 (G and H) in the dorsal brain ofE9.5 embryos. PLA signals are red dots. DAPI counterstaining is blue. (Inset inA) Diagrammatic representation of a cross-section of the neural tube. Thebox marks the proximal area shown in the figures. (A, D, and G) Nondiabetic(ND). (B, E, and H) Diabetes mellitus (DM). (Scale bar = 20 μm.) (C, F, and I)Quantification of PLA signals. Data are presented as mean± SD, *P = 0.0004, n =3 embryos (C); *P = 0.0007, n = 3 embryos (F); *P = 0.0001, n = 3 embryos (I).

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hyperglycemic conditions is demonstrated by the treatment withthree different chemical chaperones (PBA, TUDCA, and TMAO).The involvement of protein folding and aggregation in thepathogenesis of diabetic embryopathy is further demonstrated byin vivo treatment with PBA. More importantly, PBA treatmentsignificantly reduces NTDs in embryos of diabetic dams. Such asignificant effect is associated with reductions of Caspase-8 acti-vation and apoptosis, alleviation of ER stress, and restoration ofcell survival regulation. PBA, a Food and Drug Administration-approved drug, has been shown to protect neuronal death byenhancing proper protein folding (56) and is used to ameliorateprotein folding-associated diseases (57). These observationssuggest that targeting the protein-folding crisis is a potentialinterventional approach to prevent diabetes-associated birthdefects.Previous efforts to reduce embryonic malformations in diabetic

embryopathy have focused on amelioration of oxidative stressusing antioxidants (1). However, the effectiveness of antioxidantsin humans remains questionable, as multiple large-scale humantrials that have used antioxidants (i.e., vitamin C and vitamin E) totreat other diseases (i.e., preeclampsia, cardiovascular disease)have revealed unsatisfactory results (58–60). Although the reasonsfor this failure have not been identified, it is speculated that othercellular stress conditions (i.e., ER stress), which may not be af-fected by antioxidant therapies, may be involved (60). Chemicalchaperones have been shown to improve diabetes in animal

models by restoring protein folding, thereby reducing cellularstress (61, 62). Chemical chaperones have also been tested inhumans to treat protein-folding–related diseases (57, 63, 64). Theobservation that PBA reduces embryonic malformations in di-abetic mice suggests that targeting aberrant protein folding couldbe a means to prevent birth defects in diabetic pregnancies.In addition to enhancing protein folding, targeting protein ag-

gregation could mitigate the cytotoxicity of protein inclusionbodies (65). For example, efforts have been taken to block ordisrupt α-Syn oligomization in Parkinson’s disease with promisingoutcomes (66). The delineation of the protein-folding crisis andaggregation in hyperglycemia-induced embryonic malformationsprovides opportunities for developing interventions at multiplelevels to prevent birth defects in diabetic pregnancies.

Fig. 6. Changes in ER stress and cell survival factors by PBA treatment in theembryonic neural tube of diabetic mice. Data are presented as mean ± SD.(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 survivalfactors p-Pkbα (*P = 0.0013, n = 4 embryos) and p-mTOR (*P = 0.032, n =4 embryos). β-Actin, loading control; DM, diabetes mellitus; ND, nondiabetic;PBA, phenylbutyrate; VEH, vehicle.

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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 cleavedproducts 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 arepresented as mean ± SD, *P = 0.0003, n = 4 embryos. (Scale bar = 0.5 mm in A–C; 10 μm in D–F.)

Table 1. NTD rate in the embryos of diabetic mice treated withPBA

Groups

Total no. ofembryos(litters)

Embryoswith NTDs NTD rate, %

Bloodglucose, mg/dL;

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ND 85 (11) 1 1.18 121.7 ± 16.8DM+VEH 96 (13) 23 23.96 311.5 ± 7.7DM+PBA 123 (17) 5 4.06 291.7 ± 9.7

DM, diabetes mellitus; ND, nondiabetic; NTDs: open forebrain, midbrain,hindbrain, and/or spinal cord. PBA, phenylbutyrate; VEH, vehicle. P = 0.009(DM+PBA vs. DM+VEH); P = 0.0016 (DM+VEH vs. ND); P = 0.17 (DM+PBA vs. ND).

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Materials and MethodsDiabetes mellitus was induced in female mice (C57BL/6J) by i.v. injection ofstreptozotocin. The use of animals was approved by the Institutional AnimalCare and Use Committee of University of Maryland, Baltimore. Detailedprocedures about the generation of diabetic mice, cell culture, protein ag-gregation assay, TUNEL, proximity ligation assay, coimmunoprecipitation,

and Western blot assay, as well as statistical analyses, can be found in the SIAppendix, Supplementary Materials and Methods.

ACKNOWLEDGMENTS. We thank Hua Li for technical assistance, Dr. MinZhan for statistical analyses, and Dr. Julie Rosen for critical reading andediting. The work was supported by the National Institutes of Health underAward Numbers HD076245 (to Z.Z.) and HD075995 (to Z.Z.).

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