Inhibitory effect of 2-(piperidinoethoxyphenyl)-3-(4-hydroxyphenyl)-2H-benzo(b)pyran (K-1) on human primary endometrial hyperplasial cells mediated via combined suppression of Wnt/β-catenin signaling and PI3K/Akt survival pathway

Endometrial hyperplasia is a precursor to the most common gynecologic cancer diagnosed in women. Apart from estrogenic induction, aberrant activation of the Wnt/β-catenin signal is well known to correlate with endometrial hyperplasia and its carcinoma. The benzopyran compound 2-(piperidinoethoxyphenyl)-3-(4-hydroxyphenyl)-2H-benzo (b) pyran(K-1), a potent antiestrogenic agent, has been shown to have apoptosis-inducing activity in rat uterine hyperplasia. The current study was undertaken to explore the effect of the benzopyran compound K-1 on growth and Wnt signaling in human endometrial hyperplasial cells. Primary culture of atypical endometrial hyperplasial cells was characterized by the epithelial cell marker cytokeratin-7. Results revealed that compound K-1 reduced the viability of primary endometrial hyperplasial cells and expression of ERα, PR, PCNA, Wnt7a, FZD6, pGsk3β and β-catenin without affecting the growth of the primary culture of normal endometrial cells. The β-catenin target genes CyclinD1 and c-myc were also found to be reduced, whereas the expression of axin2 and Wnt/β-catenin signaling inhibitor Dkk-1 was found to be upregulated, which caused the reduced interaction of Wnt7a and FZD6. Nuclear accumulation of β-catenin was found to be decreased by compound K-1. K-1 also suppressed the pPI3K/pAkt survival pathway and induced the cleavage of caspases and PARP, thus subsequently causing the apoptosis of endometrial hyperplasial cells. In conclusion, compound K-1 suppressed the growth of human primary endometrial hyperplasial cells through discontinued Wnt/β-catenin signaling and induced apoptosis via inhibiting the PI3K/Akt survival pathway.Wnt/β-catenin signaling is known to have a prominent role in a number of developmental processes,¹ differentiation and proliferation,² survival and adhesion³ as well as the regulation of menstrual cycle.⁴ In the endometrium, Wnt/β-catenin signaling is under the control of finely tuned hormonal equilibrium, for example, estrogen induces the nuclear accumulation of β-catenin during the proliferative phase, which is later decreased during the secretary phase by progesterone, in the menstrual cycle.^{5, 6} Aberrant regulation of the Wnt signaling pathway results in neoplastic transformation and thus lays at the root of initiation and progression of many malignancies.^{7, 8} Moreover, nuclear β-catenin staining has been found to be prominent in the early onset of endometrial cancer, endometrial hyperplasia and well-differentiated endometriod carcinomas.^{9, 10}Unopposed estrogen action induces proliferative disorders and changes in the tissue structure of uterus, culminating into atypical hyperplasia and subsequently, the endometrial carcinogenesis.^{11, 12} The regression of hyperplastic to normal endometrium is the main purpose of any conservative treatment in order to prevent the development of adenocarcinoma. Currently, as a mode of treatment for hyperplasia, cyclical progestin therapy is recommended, whereas in patients with cellular atypia, hysterectomy is recommended. However, neither progestin treatment, the major hormonal therapy for endometrial cancer, nor cytotoxic chemotherapy showed substantial benefits for the treatment of endometrial hyperplasia. Therefore, future research activities must evaluate new compounds and treatment strategies.In our prior studies, it has been demonstrated that benzopyrans are the class of potent antiestrogenic compounds^{13, 14} that showed antiproliferative and apoptotic activity in rat endometrial hyperplasia¹⁵ and in human endometrial cancer cells.^{16, 17} However, the complete mechanism of action of 2-(piperidinoethoxyphenyl)-3-(4-hydroxyphenyl)-2H-benzo(b)pyran (K-1), responsible for inhibition of endometrial hyperplasia, remains to be explored in human endometrial hyperplasial cells. Because estrogen is one of the factor responsible for inducing Wnt/β-catenin signaling that may be responsible for inducing proliferation and hyperplasia formation in endometrium,^{4, 5} we further analyzed whether the benzopyran compound (K-1) modulates Wnt/β-catenin signaling in human endometrial hyperplasial cells from proliferation (Wnt-On) to differentiation (Wnt-Off). Accordingly, in the current study, we explored the antiproliferative efficacy of K-1 on primary culture cells derived from human endometrial hyperplasial tissue and investigated the Wnt/β-catenin pathway and its downstream signaling. We have also studied the effect of K-1 on cell survival pathway to substantiate the above conjecture.     

Disruption of the temporally regulated cloaca endodermal β-catenin signaling causes anorectal malformations

The cloaca is temporally formed and eventually divided by the urorectal septum (URS) during urogenital and anorectal organ development. Although congenital malformations, such as anorectal malformations (ARMs), are frequently observed during this process, the underlying pathogenic mechanisms remain unclear. β-Catenin is a critical component of canonical Wnt signaling and is essential for the regulation of cell differentiation and morphogenesis during embryogenesis. The expression of β-catenin is observed in endodermal epithelia, including URS epithelia. We modulated the β-catenin gene conditionally in endodermal epithelia by utilizing tamoxifen-inducible Cre driver line (Shh^CreERT2). Both β-catenin loss- and gain-of-function (LOF and GOF) mutants displayed abnormal clefts in the perineal region and hypoplastic elongation of the URS. The mutants also displayed reduced cell proliferation in the URS mesenchyme. In addition, the β-catenin GOF mutants displayed reduced apoptosis and subsequently increased apoptosis in the URS epithelium. This instability possibly resulted in reduced expression levels of differentiation markers, such as keratin 1 and filaggrin, in the perineal epithelia. The expression of bone morphogenetic protein (Bmp) genes, such as Bmp4 and Bmp7, was also ectopically induced in the epithelia of the URS in the β-catenin GOF mutants. The expression of the Msx2 gene and phosphorylated-Smad1/5/8, possible readouts of Bmp signaling, was also increased in the mutants. Moreover, we introduced an additional mutation for a Bmp receptor gene: BmprIA. The Shh^CreERT2/+; β-catenin^flox(ex3)/+; BmprIA^flox/− mutants displayed partial restoration of URS elongation compared with the β-catenin GOF mutants. These results indicate that some ARM phenotypes in the β-catenin GOF mutants were caused by abnormal Bmp signaling. The current analysis revealed the close relation of endodermal β-catenin signaling to the ARM phenotypes. These results are considered to shed light on the pathogenic mechanisms of human ARMs.During embryonic development, the cloaca is a temporal structure that is subsequently divided into the urogenital sinus and rectum by the urorectal septum (hereafter referred to as the URS).^{1, 2, 3} The URS develops from the proximal umbilical mesenchyme (the rostral portion of the cloaca) around embryonic day 10.5 (E10.5) (Figure 1a, asterisk)^{3, 4, 5} and subsequently extends caudally along the cloaca reaching the cloacal membrane. Part of the cloacal membrane degrades with the approximation of the URS tip. As a result, the cloaca is divided into the urogenital sinus and rectum, and the tip of the URS (endodermal epithelia) contributes to forming the ectodermal epithelia of the perineum and external genitalia.⁶ The contribution of these cells and growth factors expressed in the endodermal epithelia are essential for the proper morphogenesis of the perineal region, based on the phenotypes of several mutants. For instance, Sonic hedgehog (Shh) is expressed in the endodermal epithelia and has an essential role in both URS formation and GT protrusion by affecting neighboring mesenchymal cells.^{7, 8, 9, 10, 11} Apoptotic cells are observed in the urogenital tract during URS formation, being distributed primarily in the epithelial layers of the URS, cloacal membrane and mesenchyme of the dorsal surface of the caudal hindgut at E11.5 and E11.75.^{5, 12} From E12.5, apoptotic cells are also observed in the URS mesenchyme and are thought to be involved in the transformation of the URS and disintegration of the cloacal membrane.⁵Open in a separate windowFigure 1Temporally labeled Shh-expressing URS endodermal cells contribute to the ectodermal epithelia. The expression of the Shh gene in sagittal sections of wild-type embryos at E10.5, E11.5, E12.5 and E13.5 (a–d). The asterisk in (a) indicates future URS derived from the proximal umbilical mesenchyme. Ventral view of the genital tubercle (e–j). Tissue labeling experiments of Shh-expressing cells were performed at E15.5. Shh^CreERT2/+; R26^LacZ/+ embryos subsequent to TM administration at E8.5, E9.5, E10.5, E11.5, E12.5 or E13.5 (e–j). The red arrows indicate the LacZ-positive ectodermal cells. β-Catenin was expressed in the endoderm, including the URS epithelia, at E11.5 in the wild-type embryos (k). b, Bladder; c, cloaca; gt, genital tubercle; hl, hindlimb; r, rectum; t, tail; uc, umbilical cord; urs, urorectal septumSeveral congenital anomalies are frequently observed during urogenital organ development. These abnormalities are usually accompanied by deficient excretory and copulatory functions, influencing the quality of life of the patient. In particular, the incidence of anorectal malformations (ARMs) is approximately 1 in 5000 human births;¹³ however, the underlying pathogenic mechanisms of this condition are currently unknown. ARM phenotypes are observed in several diseases, including Currarino syndrome, Townes Brocks syndrome and VACTERL complex.^{14, 15, 16, 17} Affected patients often display other malformations, such as anal fistulas, sacral malformations and renal malformations. Human and mouse genetic analyses have shed light on the possible genetic causes of some of these abnormalities.Several mouse mutants for hedgehog signaling genes, ephrin-Eph signaling genes, fibroblast growth factor (Fgf) signaling genes and Wnt signaling genes are reported to display ARM phenotypes.^{18, 19, 20, 21, 22} Another causative factor for the development of ARM is all-trans retinoic acid (RA), a teratogen and active form of vitamin A. RA treatment in pregnant mice results in imperforate anus in embryos.^{23, 24} Although these reports have identified causative factors for ARM phenotypes, the pathogenic mechanisms underlying the development of ARM and URS remain elusive.The Wnt signaling pathway is essential for embryonic development, and its dysregulation has been implicated in developmental disorders and human diseases. Wnt signaling is transmitted primarily via three divergent pathways: the canonical Wnt/β-catenin pathway, the planner cell polarity pathway and the Wnt/Ca²⁺ pathway.^{25, 26} β-Catenin is a key factor for the canonical Wnt pathway and also acts as a subunit of the cadherin protein complex, which controls cell–cell adhesion. Owing to the early lethality of β-catenin-deficient mice,²⁷ analyses of the function of β-catenin in organogenesis have been performed using conditional mutants. Previous reports have revealed that β-catenin induces the differentiation of hair follicles during hair/skin development according to loss- and gain-of-function (LOF and GOF) approaches.^{28, 29} The ectopic activation of canonical Wnt signaling results in the ectopic induction of bone morphogenetic protein (Bmp) signaling, which is essential for hair follicle formation.^{30, 31} With respect to genital tubercle (primordia of external genitalia: GT) development, region-specific (ectodermal, endodermal and mesenchymal) modulation of the β-catenin gene has revealed essential functions, such as regulation of cell proliferation, epithelial integrity and protrusion/elongation of GT.^{8, 32, 33} However, the regulatory functions of the β-catenin gene during the development of the URS have not been investigated.The current study aimed to investigate the function of the β-catenin gene in URS development. We modulated the β-catenin activity in the endodermal epithelia and studied the phenotypic consequences of dysregulated endodermal β-catenin signaling for urorectal development. Both β-catenin LOF and GOF mutations resulted in ARM phenotypes. The β-catenin GOF mutation led to the ectopic induction of Bmp signaling. Moreover, the ARM phenotypes in the β-catenin GOF mutants were restored by additionally introducing the BmprIA gene mutation. These results suggest that adequately controlled β-catenin signaling and its downstream growth factor signaling are essential for proper URS formation.     

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore.Mitochondrial permeabilization during apoptosis is regulated by the Bcl-2 family of proteins.^{1, 2, 3} Although the Bcl-2 homology 3 (BH3)-only members such as Bid and Bim trigger apoptosis by binding to other family members, the prosurvival members block apoptosis by sequestering their pro-apoptotic relatives. Two remaining members, Bak and Bax, form the apoptotic pore within the mitochondrial outer membrane (MOM).Bak and Bax are globular proteins comprising nine α-helices.^{4, 5} They are activated by BH3-only proteins binding to the α2–α5 surface groove,^{6, 7, 8, 9, 10, 11, 12} or for Bax, to the α1/α6 ‘rear pocket''.¹³ Binding triggers dissociation of the latch domain (α6–α8) from the core domain (α2–α5), together with exposure of N-terminal epitopes and the BH3 domain.^{6, 7, 14, 15, 16} The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers.^{6, 7, 14, 17, 18} In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer.¹⁹ In Bax, the α5 and α6 helices may insert into the MOM,²⁰ although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface.^{7, 21, 22, 23} The dimers can be linked via cysteine residues placed in α6,^{18, 24, 25} and more recently via cysteine residues in either α3 or α5,^{6, 21} allowing detection of the higher-order oligomers associated with pore formation.^{26, 27} However, whether these interactions are required for high-order oligomers and pore formation remains unclear.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the MOM in healthy cells,²⁸ whereas the Bax C terminus is either exposed²⁹ or sequestered within the hydrophobic groove until apoptotic signals trigger Bax translocation.^{5, 30, 31} The hydrophobic stretch is important, as substituting polar or charged residues decreased targeting of Bak and Bax.^{10, 32} Mitochondrial targeting is also controlled by basic residues at the far C termini,^{32, 33, 34} and by interaction with VDAC2^{35, 36} via the Bak and Bax C termini.^{37, 38} Retrotranslocation of Bak and Bax was also altered by swapping the C termini.³⁹The membrane topology of the Bak and Bax C termini before and after apoptosis has not been examined directly, due in part to difficulty in reconstituting oligomers of full-length Bak in artificial membranes. Nor is it known whether the C termini contribute to pore formation by promoting oligomerization or disturbing the membrane. To address these questions synthetic peptides based on the Bak and Bax C termini have been studied in model membranes. The peptides adopt a predominantly α-helical secondary structure,^{40, 41, 42, 43} with orientation affected by lipid composition.^{42, 44, 45} The peptides could also permeabilize lipid vesicles,^{41, 43, 46, 47} suggesting that the C termini in full-length Bak and Bax may contribute to pore formation.Here we examined the membrane topology of the C termini within full-length Bak and Bax in the MOM, both before and after apoptotic pore formation. After pore formation the α9 helices of Bak (and of Bax) became juxtaposed but did not line the surface of a pore. The α9:α9 interaction occurred after Bak activation and conformation change, but was promoted by formation of BH3:groove dimers. Combining linkage at more than one interface indicated that the Bak α9:α9 interface can link BH3:groove dimers to high-order oligomers, and moreover, that the α6–α9 region is flexible in oligomerized Bak.     

Inhibition of phosphodiesterase 5 reduces bone mass by suppression of canonical Wnt signaling

Inhibitors of phosphodiesterase 5 (PDE5) are widely used to treat erectile dysfunction and pulmonary hypertension in clinics. PDE5, cyclic guanosine monophosphate (cGMP), and protein kinase G (PKG) are important components of the non-canonical Wnt signaling. This study aimed to investigate the effect of PDE5 inhibition on canonical Wnt signaling and osteoblastogenesis, using both in vitro cell culture and in vivo animal models. In the in vitro experiments, PDE5 inhibition resulted in activation of cGMP-dependent protein kinase 2 and consequent inhibition of glycogen synthase kinase 3β phosphorylation, destabilization of cytosolic β-catenin and the ultimate suppression of canonical Wnt signaling and reduced osteoblastic differentiation in HEK293T and C3H10T1/2 cells. In animal experiments, systemic inhibition of PDE5 suppressed the activity of canonical Wnt signaling and osteoblastogenesis in bone marrow-derived stromal cells, resulting in the reduction of bone mass in wild-type adult C57B/6 mice, significantly attenuated secreted Frizzled-related protein-1 (SFRP1) deletion-induced activation of canonical Wnt signaling and excessive bone growth in adult SFRP1^−/− mice. Together, these results uncover a hitherto uncharacterized role of PDE5/cGMP/PKG signaling in bone homeostasis and provide the evidence that long-term treatment with PDE5 inhibitors at a high dosage may potentially cause bone catabolism.In the canonical Wnt (Wnt/β-catenin (β-cat)) signaling cascade, Wnt binds to Frizzled (Frz) receptors and the low-density lipoprotein receptor-related protein (LRP) 5 or 6, thereby activating dishevelled, suppressing the glycogen synthase kinase 3β (GSK3β) activity and inhibiting phosphorylation of β-cat at Thr41, Ser37, and Ser33 sites. The stabilized cytosolic β-cat enters the nucleus and consequently activates its downstream target genes via lymphoid enhancer-binding factor-1 (Lef-1) and T-cell factors.^{1, 2} This signaling is fine-tuned in part via a negative feedback mechanism involving secreted and transmembrane Wnt inhibitors and activators, secreted Frz-related proteins (SFRPs), and Dickkopf-1 (Dkk1).^{3, 4}Canonical Wnt signaling is critical not only to bone development in embryogenesis but also to the maintenance of bone mass during adult life.⁵ The initial evidence came from the discoveries that in humans loss- or gain-of-function mutations in LRP5 were linked with the osteoporosis-pseudoglioma syndrome and a high-bone-density syndrome, respectively.^{6, 7, 8} Subsequent studies in mice showed that Wnt signaling might promote ossification by inducing the differentiation of bone-forming osteoblasts, suppressing the development of bone-resorbing osteoclasts, and driving the differentiation of multi-potent stem cells toward an osteoblast cell fate.⁹Non-canonical Wnt signaling is β-cat independent and consists of two main pathways: the Rho small GTPases-mediated planar cell polarity pathway and the Wnt/Ca²⁺ pathway,¹⁰ involved in various aspects of cell fate differentiation and cell movement. Non-canonical Wnt signaling has profound effects on tissue morphogenesis in a variety of vertebrate species.¹⁰ The potential role for non-canonical Wnt signaling in bone formation has been investigated recently in limited studies, which have shown that the non-canonical Wnt-Gαq/11-PKC pathway operates in mammalian osteoprogenitors to promote osteoblast development, and that Wnt16 exhibits a stimulatory effect on bone metabolism.^{11, 12, 13} Nevertheless, the molecular events in the non-canonical Wnt signaling regulation of bone development and homeostasis have yet to be further elucidated.Phosphodiesterases (PDEs) are a large family of enzymes that cleave cyclic nucleotides. To date, 11 PDE subtypes have been identified, among which PDE5 has been most extensively studied. PDE5, cyclic guanosine monophosphate (cGMP), and cGMP-dependent protein kinase (PKG) are among the major components of the non-canonical Wnt signaling pathway and are involved in the regulation of intracellular Ca²⁺ concentration.^{14, 15} It is now well established that PDE5 degrades 3''-5′- cGMP and its inhibition leads to an increase in intracellular cGMP levels and activation of protein kinase G (PKG), resulting in a decrease in Ca²⁺ influx and consequent relaxation of smooth muscles, which produces the therapeutic effects in clinical erectile dysfunction (ED) and pulmonary hypertension (PH).¹⁴ Currently, little is known regarding the involvement of PDE5 in Wnt signaling regulation of bone formation and homeostasis. The objective of this study was to determine the effect of PDE5 inhibition on canonical Wnt signaling and bone mass.     

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

Transforming growth factor-β₁ (TGF-β₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β₁ promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β₁-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),¹ diabetic cardiomyopathy² and hypertension.³ Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β₁.⁴ TGF-β₁ signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.^{5, 6} Elevated TGF-β₁ is observed in post-MI rat heart⁷ and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.⁸ The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.^{9, 10}Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.^{11, 12, 13, 14} Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.^{15, 16} Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.^{17, 18, 19, 20} The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.^{11, 12, 13, 14, 15, 16, 17, 18, 21, 22}Recent studies have demonstrated that TGF-β₁ may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells^{15, 23, 24} but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.²⁵ Moreover, it has recently been reported that autophagy can negatively¹⁵ and positively^{25, 26, 27} regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β₁-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart.     

Alzheimer's disease-related amyloid-β induces synaptotoxicity in human iPS cell-derived neurons

Human induced pluripotent stem cell (iPSC)-derived neurons have been proposed to be a highly valuable cellular model for studying the pathomechanisms of Alzheimer''s disease (AD). Studies employing patient-specific human iPSCs as models of familial and sporadic forms of AD described elevated levels of AD-related amyloid-β (Aβ). However, none of the present AD iPSC studies could recapitulate the synaptotoxic actions of Aβ, which are crucial early events in a cascade that eventually leads to vast brain degeneration. Here we established highly reproducible, human iPSC-derived cortical cultures as a cellular model to study the synaptotoxic effects of Aβ. We developed a highly efficient immunopurification procedure yielding immature neurons that express markers of deep layer cortical pyramidal neurons and GABAergic interneurons. Upon long-term cultivation, purified cells differentiated into mature neurons exhibiting the generation of action potentials and excitatory glutamatergic and inhibitory GABAergic synapses. Most interestingly, these iPSC-derived human neurons were strongly susceptible to the synaptotoxic actions of Aβ. Application of Aβ for 8 days led to a reduction in the overall FM4–64 and vGlut1 staining of vesicles in neurites, indicating a loss of vesicle clusters. A selective analysis of presynaptic vesicle clusters on dendrites did not reveal a significant change, thus suggesting that Aβ impaired axonal vesicle clusters. In addition, electrophysiological patch-clamp recordings of AMPA receptor-mediated miniature EPSCs revealed an Aβ-induced reduction in amplitudes, indicating an impairment of postsynaptic AMPA receptors. A loss of postsynaptic AMPA receptor clusters was confirmed by immunocytochemical stainings for GluA1. Incubation with Aβ for 8 days did not result in a significant loss of neurites or cell death. In summary, we describe a highly reproducible cellular AD model based on human iPSC-derived cortical neurons that enables the mechanistic analysis of Aβ-induced synaptic pathomechanisms and the development of novel therapeutic approaches.In Alzheimer''s disease (AD), synapse damage and synapse loss are thought to underlie cognitive deficits.¹ Oligomers of the amyloid-β (Aβ) peptide appear to induce synaptic failure as an early event in the etiology of AD.^{2, 3, 4} However, despite its well-established synapse-impairing effects in rodent models,^{5, 6, 7} the synaptotoxic actions of Aβ most relevant for the human disease have not been identified in a human model system. Several studies have investigated the synaptotoxic effects of Aβ in cultured rodent neurons and in transgenic mouse models revealing a multitude of potential mechanisms affecting synapses. Postsynaptic Aβ actions result in the loss of functional (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type) glutamate receptors,^{8, 9, 10} involve long-term depression-like mechanisms,^{9, 11, 12} and lead to the degradation of the entire postsynapse (dendritic spines).^{9, 11, 13} In addition, several distinct presynaptic Aβ actions on the synaptic vesicle cycle have been described.^{10, 14} Furthermore, Aβ-induced impairments of axonal transport regulation and Aβ-induced axon degeneration have been found in rodent neurons.^{15, 16, 17} This puzzling diversity of Aβ-induced synapse-related defects raises the question whether all of them are involved in the early pathomechanisms of human AD.In addition to well-established animal systems, the modelling of human neurological disease pathologies by human induced pluripotent stem cell (hiPSC) technology¹⁸ has been proposed as an innovative approach.^{19, 20, 21} The in vitro differentiation of hiPSCs to excitable neurons has been reported using a variety of protocols.^{22, 23, 24} However, quantitative analysis of both functional glutamatergic and GABAergic synapses has been difficult to achieve.^{19, 25, 26} In addition to studying the functional properties of iPSC-derived human neurons from healthy individuals, the in vitro differentiation of patient-derived iPSCs has been used to model complex neurodevelopmental and neurodegenerative diseases.^{19, 27, 28} Recently, iPSCs derived from AD patients have been reported to exhibit increased secretion of Aβ upon in vitro neuronal differentiation; however, neither a loss of synapses nor an impairment of synapse function was detected.^{21, 29, 30, 31, 32, 33} Here we describe a hiPSC-based, carefully optimized in vitro differentiation protocol, including a novel immunopanning step, which enabled us to study the deleterious effects of application of Aβ on human cortical neurons and on human synapses.     

TRAF2 is a biologically important necroptosis suppressor

Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor-interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)—driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)—previously implicated in apoptosis suppression—also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα–driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.Apoptotic cell death is mediated by caspases and has distinct morphological features, including membrane blebbing, cell shrinkage and nuclear fragmentation.^{1, 2, 3, 4} In contrast, necroptotic cell death is caspase-independent and is characterized by loss of membrane integrity, cell swelling and implosion.^{1, 2, 5} Nevertheless, necroptosis is a highly regulated process, requiring activation of RIPK1 and RIPK3, which form the core necrosome complex.^{1, 2, 5} Necrosome assembly can be induced via specific death receptors or toll-like receptors, among other modules.^{6, 7, 8, 9} The activated necrosome engages MLKL by RIPK3-mediated phosphorylation.^{6, 10, 11} MLKL then oligomerizes and binds to membrane phospholipids, forming pores that cause necroptotic cell death.^{10, 12, 13, 14, 15} Unchecked necroptosis disrupts embryonic development in mice and contributes to several human diseases.^{7, 8, 16, 17, 18, 19, 20, 21, 22}The apoptotic mediators FADD, caspase-8 and cFLIP suppress necroptosis.^{19, 20, 21, 23, 24} Elimination of any of these genes in mice causes embryonic lethality, subverted by additional deletion of RIPK3 or MLKL.^{19, 20, 21, 25} Necroptosis is also regulated at the level of RIPK1. Whereas TNFα engagement of TNFR1 leads to K63-linked ubiquitination of RIPK1 by cellular inhibitor of apoptosis proteins (cIAPs) to promote nuclear factor (NF)-κB activation,²⁶ necroptosis requires suppression or reversal of this modification to allow RIPK1 autophosphorylation and consequent RIPK3 activation.^{2, 23, 27, 28} CYLD promotes necroptotic signaling by deubiquitinating RIPK1, augmenting its interaction with RIPK3.²⁹ Conversely, caspase-8-mediated CYLD cleavage inhibits necroptosis.²⁴TRAF2 recruits cIAPs to the TNFα-TNFR1 signaling complex, facilitating NF-κB activation.^{30, 31, 32, 33} TRAF2 also supports K48-linked ubiquitination and proteasomal degradation of death-receptor-activated caspase-8, curbing apoptosis.³⁴ TRAF2 KO mice display embryonic lethality; some survive through birth but have severe developmental and immune deficiencies and die prematurely.^{35, 36} Conditional TRAF2 KO leads to rapid intestinal inflammation and mortality.³⁷ Furthermore, hepatic TRAF2 depletion augments apoptosis activation via Fas/CD95.³⁴ TRAF2 attenuates necroptosis induction in vitro by the death ligands Apo2L/TRAIL and Fas/CD95L.³⁸ However, it remains unclear whether TRAF2 regulates TNFα-induced necroptosis—and if so—how. Our present findings reveal that TRAF2 inhibits TNFα necroptotic signaling. Furthermore, our results establish TRAF2 as a biologically important necroptosis suppressor in vitro and in vivo and provide initial insight into the mechanisms underlying this function.     

Inhibition of the MAP3 kinase Tpl2 protects rodent and human β-cells from apoptosis and dysfunction induced by cytokines and enhances anti-inflammatory actions of exendin-4

Proinflammatory cytokines exert cytotoxic effects on β-cells, and are involved in the pathogenesis of type I and type II diabetes and in the drastic loss of β-cells following islet transplantation. Cytokines induce apoptosis and alter the function of differentiated β-cells. Although the MAP3 kinase tumor progression locus 2 (Tpl2) is known to integrate signals from inflammatory stimuli in macrophages, fibroblasts and adipocytes, its role in β-cells is unknown. We demonstrate that Tpl2 is expressed in INS-1E β-cells, mouse and human islets, is activated and upregulated by cytokines and mediates ERK1/2, JNK and p38 activation. Tpl2 inhibition protects β-cells, mouse and human islets from cytokine-induced apoptosis and preserves glucose-induced insulin secretion in mouse and human islets exposed to cytokines. Moreover, Tpl2 inhibition does not affect survival or positive effects of glucose (i.e., ERK1/2 phosphorylation and basal insulin secretion). The protection against cytokine-induced β-cell apoptosis is strengthened when Tpl2 inhibition is combined with the glucagon-like peptide-1 (GLP-1) analog exendin-4 in INS-1E cells. Furthermore, when combined with exendin-4, Tpl2 inhibition prevents cytokine-induced death and dysfunction of human islets. This study proposes that Tpl2 inhibitors, used either alone or combined with a GLP-1 analog, represent potential novel and effective therapeutic strategies to protect diabetic β-cells.It is now clear that chronic inflammation is a hallmark of type I and type II diabetes, affecting both β-cell mass and insulin secretion.¹ Type I diabetes is characterized by drastic decreases in β-cell mass and insulin secretion, in part mediated by proinflammatory cytokines produced following autoimmune activation.¹ Proinflammatory cytokines, particularly interleukin-1β (IL-1β), in combination with interferon-γ (IFN-γ) and/or tumor necrosis factor-α (TNF-α), promote death by apoptosis and decrease function of differentiated β-cells, leading to β-cell destruction.¹ Pancreatic islet transplantation is a promising alternative therapy for some type I diabetic patients.² However, clinical outcome is not always achieved because of significant loss of islet mass during and after transplantation.³ Up to 80% of transplanted islets can die during the post-transplantation period as a result of apoptosis because of several mechanisms, notably the instant blood-mediated inflammatory response (IBMIR) and the release of a mix of cytokines including IL-1β, TNF-α and IFN-γ.⁴Immune-modulatory strategies for type I diabetes therapy and improvement of islet transplantation outcomes have emerged, targeting a single specific cytokine, such as IL-1β or TNF-α.^{2, 5} However, these strategies may only target inflammation partially.² Indeed, multiple cytokines, originating from surrounding immune cells and/or β-cells themselves, are more likely to be present simultaneously^{4, 6} and act synergistically to induce β-cell death and dysfunction.^{7, 8, 9} Preclinical and clinical studies demonstrated that glucagon-like peptide-1 (GLP-1) analogs, in addition to regulating glucose homeostasis in vivo, contribute to the restoration of normoglycemia after islet transplantation.^{10, 11, 12, 13} GLP-1 receptor (GLP-1R) analogs protect β-cell survival and function from proinflammatory cytokine attack.^{12, 14, 15} However, some studies have shown only modest and short-term anti-inflammatory effects of GLP-1 analogs when used alone.^{11, 13, 16}Mitogen-activated protein kinases (MAPKs) (i.e., extracellular-regulated kinase-1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK) play important roles in cytokine-induced β-cell dysfunction and death.¹ Conversely, ERK1/2 are involved in the beneficial effects of glucose and GLP-1 analogs.^{17, 18, 19} In this context, upstream protein kinases that specifically control the activation of MAPK in response to a combination of inflammatory cytokines (IL-1β, TNF-α and IFN-γ), rather than a single cytokine, may be useful targets for therapeutic interventions against pancreatic β-cell failure.The serine/threonine kinase tumor progression locus 2 (Tpl2) (also known as COT (Cancer Osaka Thyroid) in humans) is a member of the MAP3K family (the MAP3K8) whose activation stimulates primarily the ERK1/2 pathway, but also JNK and/or p38 MAPK in some cell types, specifically in response to various inflammatory stimuli.^{20, 21, 22} Dysregulation of Tpl2 expression and signaling is associated with acute and chronic inflammatory diseases,^{20, 21, 22} and several studies highlight a critical function of Tpl2 in the control of inflammatory responses and survival in adipocytes, fibroblasts and immune and epithelial cells.^{21, 22, 23, 24}However, there is currently nothing known about the effects of Tpl2 in β-cells. The aim of this study was to determine whether Tpl2 may be a new key inflammatory regulator in β-cells or islets. We demonstrate that Tpl2 contributes to cytokine-induced β-cell apoptosis and dysfunction, and suggest that Tpl2 inhibition, either alone or combined with a GLP-1 receptor agonist, represents a potential new therapeutic strategy for the treatment of diabetes.     

Alpha-Synuclein affects neurite morphology,autophagy, vesicle transport and axonal degeneration in CNS neurons

Many neuropathological and experimental studies suggest that the degeneration of dopaminergic terminals and axons precedes the demise of dopaminergic neurons in the substantia nigra, which finally results in the clinical symptoms of Parkinson disease (PD). The mechanisms underlying this early axonal degeneration are, however, still poorly understood. Here, we examined the effects of overexpression of human wildtype alpha-synuclein (αSyn-WT), a protein associated with PD, and its mutant variants αSyn-A30P and -A53T on neurite morphology and functional parameters in rat primary midbrain neurons (PMN). Moreover, axonal degeneration after overexpression of αSyn-WT and -A30P was analyzed by live imaging in the rat optic nerve in vivo. We found that overexpression of αSyn-WT and of its mutants A30P and A53T impaired neurite outgrowth of PMN and affected neurite branching assessed by Sholl analysis in a variant-dependent manner. Surprisingly, the number of primary neurites per neuron was increased in neurons transfected with αSyn. Axonal vesicle transport was examined by live imaging of PMN co-transfected with EGFP-labeled synaptophysin. Overexpression of all αSyn variants significantly decreased the number of motile vesicles and decelerated vesicle transport compared with control. Macroautophagic flux in PMN was enhanced by αSyn-WT and -A53T but not by αSyn-A30P. Correspondingly, colocalization of αSyn and the autophagy marker LC3 was reduced for αSyn-A30P compared with the other αSyn variants. The number of mitochondria colocalizing with LC3 as a marker for mitophagy did not differ among the groups. In the rat optic nerve, both αSyn-WT and -A30P accelerated kinetics of acute axonal degeneration following crush lesion as analyzed by in vivo live imaging. We conclude that αSyn overexpression impairs neurite outgrowth and augments axonal degeneration, whereas axonal vesicle transport and autophagy are severely altered.Growing evidence suggests that Parkinson''s disease (PD) pathology starts at the presynaptic terminals and the distal axons and is then propagated back to the soma in a ''dying back'' pattern.^{1, 2} Accordingly, at the time of clinical onset, there is only a 30% loss of total substantia nigra pars compacta neurons but a far more severe loss of striatal dopaminergic markers (70–80%), suggesting that axonal terminals of the nigrostriatal pathway are affected earlier.¹ It is thus essential to understand the pathomechanisms specifically affecting the axon in PD in order to interfere with early disease progression.Neurodegeneration in PD is accompanied by the appearance of intraneuronal protein aggregates, denoted Lewy bodies (LBs).³ Interestingly, also LB pathology is initially found in the distal axons before becoming evident in the neuronal somata, and dystrophic neurites, so called ''Lewy neurites'', outnumber LBs in the early stages of PD.^{2, 4, 5} A main component of LBs is the protein alpha-synuclein (αSyn) that is not only widely used as a histopathological marker for PD but is also believed to have a major role in PD pathogenesis.^{6, 7} The importance of αSyn is further underlined by the discovery of αSyn point mutations (e.g. Ala53Thr (A53T), Ala30Pro (A30P)) and multiplications of the αSyn gene, all of which cause autosomal dominant forms of PD.^{8, 9, 10} However, neither the physiological functions nor the pathogenetic mechanisms of αSyn are well understood.⁷The biological effects of αSyn expression strongly depend on the model system. Wild-type (WT) human αSyn does not lead to major clinical or histological abnormalities when expressed in transgenic mice,^{11, 12} but its overexpression mediated by adeno-associated viral vectors (AAV) results in severe neurodegeneration, suggesting a dose-dependent toxic effect.^{13, 14} Different human αSyn-A30P and -A53T transgenic mouse lines develop severe motor impairments, partly resembling symptoms of human PD, accompanied by a degeneration of the nigrostriatal neuronal system and LB-like pathology.^{11, 12, 15} In line with the pathological findings in human PD, the axonal compartment is affected early and most prominently in these animal models.Different putative pathomechanisms of αSyn toxicity have been explored. For example, the cytoskeleton is an important molecular target of αSyn. Multimeric forms of αSyn were shown to impair the polymerization of tubulin and microtubule formation.^{16, 17} Overexpression of αSyn increased actin instability and induced actin bundling in cultured hippocampal neurons.¹⁸ There are, however, divergent data on the resulting effects of αSyn overexpression on neurite outgrowth and integrity in different model systems.^{19, 20, 21, 22}Moreover, a dysregulation of autophagy has been implicated in PD pathology. Aberrant αSyn is normally degraded by autophagy and only to a negligible degree by the proteasome.²³ Several studies have shown that the inhibition of autophagy results in an accumulation and increased toxicity of αSyn, whereas the activation of autophagy has therapeutic effects in PD models.^{23, 24, 25, 26} However, the direct effects of αSyn and its mutants on autophagy seem to rely strongly on the model system and the published data are highly controversial.^{24, 26, 27, 28, 29, 30, 31, 32}Given the central role of axonal degeneration in PD, it is likely that disturbances of axonal transport are involved.³³ In support of this proposition, the motor protein kinesin was shown to be decreased early and stage-dependently in PD patients, preceding the loss of substantia nigra neurons.³⁴ αSyn itself is actively transported along the axons, mainly by the slow component of axonal transport, but the role of αSyn in axonal vesicle transport is unclear.³⁵Here, we present a comprehensive analysis of the effects of αSyn on neurite morphology and examine important pathomechanisms.