Role of Retinoic Acid and Fibroblast Growth Factor 2 in Neural Differentiation from Cynomolgus Monkey (Macaca fascicularis) Embryonic Stem Cells

Retinoic acid is a widely used factor in both mouse and human embryonic stem cells. It suppresses differentiation to mesoderm and enhances differentiation to ectoderm. Fibroblast growth factor 2 (FGF2) is widely used to induce differentiation to neurons in mice, yet in primates, including humans, it maintains embryonic stem cells in the undifferentiated state. In this study, we established an FGF2 low-dose-dependent embryonic stem cell line from cynomolgus monkeys and then analyzed neural differentiation in cultures supplemented with retinoic acid and FGF2. When only retinoic acid was added to culture, neurons differentiated from FGF2 low-dose-dependent embryonic stem cells. When both retinoic acid and FGF2 were added, neurons and astrocytes differentiated from the same embryonic stem cell line. Thus, retinoic acid promotes the differentiation from embryonic stem cells to neuroectoderm. Although FGF2 seems to promote self-renewal in stem cells, its effects on the differentiation of stem cells are influenced by the presence or absence of supplemental retinoic acid.Abbreviations: EB, embryoid body; ES, embryonic stem; ESM, embryonic stem cell medium; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; LIF, leukemia inhibitory factor; MBP, myelin basic protein; RA, retinoic acid; SSEA, stage-specific embryonic antigen; TRA, tumor-related antigenPluripotent stem cells are potential sources of material for cell replacement therapy and are useful experimental tools for in vitro models of human disease and drug screening. Embryonic stem (ES) cells are capable of extensive proliferation and multilineage differentiation, and thus ES-derived cells are suitable for use in cell-replacement therapies.^18,23 Reported ES cell characteristics including tumorigenic potential, DNA methylation status, expression of imprinted genes, and chromatin structure were elucidated by using induced pluripotent stem cells.^2,11,17 Because the social expectations of regeneration medicine are growing, we must perform basic research with ES cells, which differ from induced pluripotent stem cells in terms of origin, differentiation ability, and epigenetic status.^2,8Several advances in research have been made by using mouse ES cells. Furthermore, primate ES cell lines have been established from rhesus monkeys (Macaca mulatta),²⁴ common marmosets (Callithrix jacchus),²⁵ cynomolgus monkeys (M. fascicularis),²⁰ and African green monkeys (Chlorocebus aethiops).¹⁹ Mouse and other mammalian ES cells differ markedly in their responses to the signaling pathways that support self-renewal.^8,28 Mouse ES cells require leukemia inhibitory factor (LIF)–STAT3 signaling.¹⁴ In contrast, primate ES cells do not respond to LIF. Fibroblast growth factor 2 (FGF2) appears to be the most upstream self-renewal factor in primate ES cells. FGF2 also exerts its effects through indirect mechanisms, such as the TGFβ–Activin–Nodal signaling pathway, in primate ES cells.²¹ In addition to the biologic similarities between monkeys and humans, ES cells derived from cynomolgus monkeys or human blastocysts have extensive similarities that are not apparent in mouse ES cells.^8,14,21,28 Numerous monkey ES cell lines are now available, and cynomolgus monkeys are an efficient model for developing strategies to investigate the efficacy of ES-cell–based medical treatments in humans.Several growth factors and chemical compounds, including retinoic acid (RA),^4,9,13,22,26 FGF2,^9,10,16,22 epidermal growth factor,^9,22 SB431542,^1,4,10 dorsomorphin,^10,27 sonic hedgehog,^{12,13,16,27,29} and noggin,^1,4,9,27 are essential for the differentiation and proliferation or maintenance of neural stem cells derived from primate ES cells. Of these factors, active RA signaling suppresses a mesodermal fate by inhibiting Wnt and Nodal signaling pathways during in vitro culture and leads to neuroectoderm differentiation in ES cells.^4,13,26 RA is an indispensable factor for the specialization to neural cells. FGF2 is important during nervous system development,¹² and FGF2 and RA both are believed to influence the differentiation to neural cells. The current study was done to clarify the mechanism of RA and FGF2 in the induction of differentiation along the neural lineage.We recently established a monkey ES cell line that does not need FGF2 supplementation for maintenance of the undifferentiated state. This ES cell line allowed us to study the role of differentiation to neural cells with RA and enabled us to compare ES cell differentiation in the context of supplementation with RA or FGF2 in culture. To this end, we established a novel cynomolgus monkey cell line derived from ES cells and maintained it in an undifferentiated state in the absence of FGF2 supplementation.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

Neuritin can normalize neural deficits of Alzheimer's disease

Reductions in hippocampal neurite complexity and synaptic plasticity are believed to contribute to the progressive impairment in episodic memory and the mild cognitive decline that occur particularly in the early stages of Alzheimer''s disease (AD). Despite the functional and therapeutic importance for patients with AD, intervention to rescue or normalize dendritic elaboration and synaptic plasticity is scarcely provided. Here we show that overexpression of neuritin, an activity-dependent protein, promoted neurite outgrowth and maturation of synapses in parallel with enhanced basal synaptic transmission in cultured hippocampal neurons. Importantly, exogenous application of recombinant neuritin fully restored dendritic complexity as well as spine density in hippocampal neurons prepared from Tg2576 mice, whereas it did not affect neurite branching of neurons from their wild-type littermates. We also showed that soluble recombinant neuritin, when chronically infused into the brains of Tg2576 mice, normalized synaptic plasticity in acute hippocampal slices, leading to intact long-term potentiation. By revealing the protective actions of soluble neuritin against AD-related neural defects, we provide a potential therapeutic approach for patients with AD.Efficient neuronal communications through synapses are crucial for normal brain functions, whereas alterations in synapse numbers, dendritic spine morphology, and dendritic complexity are thought to be reflected by different forms of synaptic plasticity and are also causally associated with a variety of neurological disorders.^{1, 2, 3, 4, 5} For example, synapse loss and neurite atrophy are the major neurobiological substrates underlying memory impairment in neurodegenerative diseases such as Alzheimer''s disease (AD).^{6, 7} The increased dendritic mislocalization of hyperphosphorylated tau protein, a microtubule-associated protein enriched at axons of mature neurons,⁸ and abundance of soluble oligomeric forms of β-amyloid (Aβ) appear to cause the synaptic defects and disruption of synaptic plasticity involving the progression of AD pathology.^{6, 9, 10} The apparent decreases in neurotrophic factors observed in brains of patients with AD¹¹ have prompted several trials for administration of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), to attenuate and possibly reverse synaptic defects.^{11, 12, 13} However, the truncation or decreased expression of its cognate receptors in AD brains have limited their potential usage as AD therapeutics.^{12, 14, 15}Neuritin, also known as the candidate plasticity gene 15, was originally identified in a screening study for activity-regulated genes and was subsequently found to be one of the signaling molecules downstream to BDNF and its receptor tropomyosin-related kinase receptor type B.^{16, 17} Ensuing studies indicated that neuritin could also be induced by experimental seizure or by normal life experiences, such as sensory stimulation and exercise.^{17, 18, 19, 20, 21, 22} Located in the 6p24-p25 interval on chromosome 6,²³ the neuritin gene encodes a small, highly conserved protein containing a secretory signal sequence at the N-terminus and a consensus sequence for glycosylphosphatidylinositol (GPI) at the C-terminus.¹⁶ This GPI linkage enables neuritin to anchor at cell surfaces, and upon cleavage of GPI by phospholipase the resultant soluble neuritin is released into the extracellular space.^{16, 20, 24, 25, 26}During embryonic neural development, neuritin is mainly expressed in brain regions that undergo a rapid proliferation of neuronal progenitor pools, suggesting a protective role of neuritin for differentiated neurons.^{26, 27} Interestingly, the expression level of neuritin remains elevated after birth or even increases, especially in brain regions presumably exhibiting high neural activity and synaptic plasticity, such as the hippocampus, visual cortex, and external granular layer of the cerebellum.^{16, 19, 20, 26} In addition, neuritin promotes neuritic arbor growth and synaptic formation.^{16, 20, 24, 25, 28, 29, 30, 31} Although various studies have suggested these potent neuritogenetic activities of neuritin, the contribution of neuritin expression to or its effectiveness against neurodegenerative diseases that display neurite atrophy and synapse loss has been largely unexplored.Here we determined that neuritin expression increased neurite complexity and promoted the maturation of individual spines in cultured hippocampal neurons. Consistent with these findings, basal synaptic transmission was enhanced by transient expression of neuritin. Importantly, when exogenously applied, the soluble neuritin peptide rescued the dendrite complexity of neurons prepared from Tg2576 mice, a transgenic mouse model of AD, such that the complexity was comparable to that in wild-type (WT) mice and also normalized synaptic plasticity in the hippocampus of the Tg2576 mice. Taken together, these results suggest that neuritin, particularly a soluble form of neuritin, reverses synaptic defects manifest in Tg2576 mice and that manipulations to increase neuritin levels may be beneficial therapeutic approaches in AD.     

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.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Cerium oxide nanoparticles protect against Aβ-induced mitochondrial fragmentation and neuronal cell death

Evidence indicates that nitrosative stress and mitochondrial dysfunction participate in the pathogenesis of Alzheimer''s disease (AD). Amyloid beta (Aβ) and peroxynitrite induce mitochondrial fragmentation and neuronal cell death by abnormal activation of dynamin-related protein 1 (DRP1), a large GTPase that regulates mitochondrial fission. The exact mechanisms of mitochondrial fragmentation and DRP1 overactivation in AD remain unknown; however, DRP1 serine 616 (S616) phosphorylation is likely involved. Although it is clear that nitrosative stress caused by peroxynitrite has a role in AD, effective antioxidant therapies are lacking. Cerium oxide nanoparticles, or nanoceria, switch between their Ce³⁺ and Ce⁴⁺ states and are able to scavenge superoxide anions, hydrogen peroxide and peroxynitrite. Therefore, nanoceria might protect against neurodegeneration. Here we report that nanoceria are internalized by neurons and accumulate at the mitochondrial outer membrane and plasma membrane. Furthermore, nanoceria reduce levels of reactive nitrogen species and protein tyrosine nitration in neurons exposed to peroxynitrite. Importantly, nanoceria reduce endogenous peroxynitrite and Aβ-induced mitochondrial fragmentation, DRP1 S616 hyperphosphorylation and neuronal cell death.Nitric oxide (NO) is a neurotransmitter and neuromodulator required for learning and memory.¹ NO is generated by NO synthases, a group of enzymes that produce NO from L-arginine. In addition to its normal role in physiology, NO is implicated in pathophysiology. When overproduced, NO combines with superoxide anions (O₂·⁻), byproducts of aerobic metabolism and mitochondrial oxidative phosphorylation, to form peroxynitrite anions (ONOO⁻) that are highly reactive and neurotoxic. Accumulation of these reactive oxygen species (ROS) and reactive nitrogen species (RNS), known as oxidative and nitrosative stress, respectively, is a common feature of aging, neurodegeneration and Alzheimer''s disease (AD).¹Nitrosative stress caused by peroxynitrite has a critical role in the etiology and pathogenesis of AD.^{2, 3, 4, 5, 6, 7} Peroxynitrite is implicated in the formation of the two hallmarks of AD, Aβ aggregates and neurofibrillary tangles containing hyperphosphorylated Tau protein.^{1, 4, 7} In addition, peroxynitrite promotes the nitrotyrosination of presenilin 1, the catalytic subunit of the γ-secretase complex, which shifts production of Aβ to amyloid beta (Aβ)42 and increases the Aβ42/Aβ40 ratio, ultimately resulting in an increased propensity for aggregation and neurotoxicity.⁵ Furthermore, nitration of Aβ tyrosine 10 enhances its aggregation.⁶ Peroxynitrite can also modify enzymes, such as triosephosphate isomerase,⁴ and activate kinases, including Jun amino-terminal kinase and p38 mitogen-activated protein kinase, which enhance neuronal cell death.^{8, 9} Moreover, peroxynitrite can trigger the release of free metals such as Zn²⁺ from intracellular stores with consequent inhibition of mitochondrial function and enhancement of neuronal cell death.^{10, 11, 12} Finally, peroxynitrite can irreversibly inhibit complexes I and IV of the mitochondrial respiratory chain.^{11, 13}Because mitochondria have a critical role in neurons as energy producers to fuel vital processes such as synaptic transmission and axonal transport,¹⁴ and mitochondrial dysfunction is a well-documented and early event in AD,¹⁵ it is important to consider how peroxynitrite and nitrosative stress affect mitochondria. Although the ultimate cause of mitochondrial dysfunction in AD remains unclear, an imbalance in mitochondrial fission and fusion is one possibility.^{1, 14, 16, 17, 18} Notably, peroxynitrite, N-methyl D-aspartate (NMDA) receptor activation and Aβ can induce mitochondrial fragmentation by activating mitochondrial fission and/or inhibiting fusion.¹⁶ Mitochondrial fission and fusion is regulated by large GTPases of the dynamin family, including dynamin-related protein 1 (DRP1) that is required for mitochondrial division,¹⁹ and inhibition of mitochondrial division by overexpression of the GTPase-defective DRP1^K38A mutant provides protection against peroxynitrite-, NMDA- and Aβ-induced mitochondrial fragmentation and neuronal cell death.¹⁶The exact mechanism of peroxynitrite-induced mitochondrial fragmentation remains unclear. A recent report suggested that S-nitrosylation of DRP1 at cysteine 644 increases DRP1 activity and is the cause of peroxynitrite-induced mitochondrial fragmentation in AD;²⁰ however, the work remains controversial, suggesting that alternative pathways might be involved.²¹ For example, peroxynitrite also causes rapid DRP1 S616 phosphorylation that promotes its translocation to mitochondria and organelle division.^{21, 22} In mitotic cells, DRP1 S616 phosphorylation is mediated by Cdk1/cyclinB1 and synchronizes mitochondrial division with cell division.²³ Interestingly, DRP1 is S616 hyperphosphorylated in AD brains, suggesting that this event might contribute to mitochondrial fragmentation in the disease.^{21, 22} A recent report indicates that Cdk5/p35 is responsible for DRP1 S616 phosphorylation,²⁴ and notably aberrant Cdk5/p35/p25 signaling is associated with AD pathogenesis.²⁵ Thus, we explored here the possible role of DRP1 S616 hyperphosphorylation in Aβ- and peroxynitrite-mediated mitochondrial fragmentation.Under normal conditions, accumulated mitochondrial superoxide anions and hydrogen peroxide (H₂O₂) can be neutralized by superoxide dismutase (SOD) and catalase. Nitrosative stress in aging and AD might be explained by a loss of antioxidant enzymes. Previous studies suggest that expression of SOD subtypes is decreased in the human AD brain.^{26, 27} Furthermore, SOD1 deletion in a mouse model of AD increased the burden of amyloid plaques.²⁶ By contrast, overexpression of SOD2 in a mouse model of AD decreased the Aβ42/Aβ40 ratio and alleviated memory deficits.^{28, 29} There is currently a lack of antioxidants that can effectively quench superoxide anions, H₂O₂ or peroxynitrite and provide lasting effects. Cerium is a rare earth element and cerium oxide (CeO₂) nanoparticles, or nanoceria, shuttle between their 3+ or 4+ states. Oxidation of Ce⁴⁺ to Ce³⁺ causes oxygen vacancies and defects on the surface of the crystalline lattice structure of the nanoparticles, generating a cage for redox reactions to occur.³⁰ Accordingly, nanoceria mimic the catalytic activities of antioxidant enzymes, such as SOD^{31, 32} and catalase,³³ and are able to neutralize peroxynitrite.³⁴ Because of these antioxidant properties, we hypothesized that nanoceria could detoxify peroxynitrite and protect against Aβ-induced DRP1 S616 hyperphosphorylation, mitochondrial fragmentation and neuronal cell death.     

In a three-dimensional reconstructed human epidermis filaggrin-2 is essential for proper cornification

Atopic dermatitis is a chronic inflammatory skin disease with defects in the epidermal barrier. In a cohort of African-American children, a FLG2 nonsense mutation has been associated with the disease. In the epidermis of European patients, the expression of filaggrin-2, the filaggrin-related protein encoded by FLG2, is decreased. To describe the function of filaggrin-2 and evaluate the impact of its deficiency, its expression was downregulated using lentivirus-mediated shRNA interference in a three-dimensional reconstructed human epidermis (RHE) model. This resulted in parakeratosis and a compact stratum corneum, presence of abnormal vesicles inside the corneocytes, increased pH and reduced amounts of free amino acids at the RHE surface, leading to increased sensitivity to UVB radiations. The expression of differentiation markers was slightly modified. However, we observed reduced proteolytic processing of corneodesmosin, hornerin and filaggrin in parallel with reduced amounts of caspase-14 and bleomycin hydrolase. Our data demonstrated that filaggrin-2 is important for a proper cornification and a functional stratum corneum. Its downregulation in atopic patients may be involved in the disease-associated epidermis impairment.Atopic dermatitis (AD; OMIM #603165), also known as atopic eczema, is a very common inflammatory skin disease.^{1, 2} It is the result of complex interactions between genetic and environmental factors. The most robust and widely replicated genetic risk factor for the disease corresponds to nonsense mutations of the gene FLG.^{3, 4} This gene encodes filaggrin, an S100 fused-type protein essential for the epidermal barrier functions.^{3, 4, 5} Filaggrin deficiency is responsible for decreased amounts of free amino acids in the stratum corneum (SC),^{6, 7, 8, 9} abnormal keratinocyte differentiation,^{6, 10} epidermal barrier defects and enhanced percutaneous sensitization,^{6, 7, 9} all characteristics of the atopic skin. However, a significant number of Asian and European patients with AD do not display any of the numerous known FLG mutations,^{3, 4} in particular in the South of Europe.^{11, 12} In addition, FLG nonsense mutations have not been detected in Ethiopian and South African populations,^{13, 14} and are not associated with AD in African-American patients.^{15, 16} Hence, the defect in epidermal barrier functions of patients without FLG mutations, including those of African ancestry, may be associated with other inherited/acquired abnormalities that compromise keratinocyte differentiation.¹⁷Recently, mutations in the FLG2 gene, in particular a nonsense mutation, were shown to be associated with persistent AD in a cohort of 60 US patients of African ancestry.¹⁸ FLG2 encodes filaggrin-2, another S100 fused-type protein.¹⁹ Filaggrin-2 is very similar to filaggrin in terms of protein structure, amino-acid composition, pattern of expression and of biochemical properties. It is synthetized by granular keratinocytes as a large precursor consisting of 23 homologous repeats and a S100-homologous N-terminal domain. Filaggrin-2 and filaggrin are colocalized in keratohyalin granules in granular keratinocytes, and in the cytoplasmic matrix of the lower corneocytes.^{20, 21} In the upper SC, filaggrin-2 is deiminated and degraded by calpain 1.²⁰ The role of filaggrin-2 in the SC remains to be discovered, but it may be similar to that of filaggrin.²² In addition, FLG2 is one of the genes that are the most downregulated after cholesterol depletion of keratinocytes, an experimental model of AD.²³ Importantly, we and others have shown that filaggrin-2 expression is reduced, probably by pro-inflammatory cytokines, in the epidermis of European patients.^{12, 24}To analyze the function of filaggrin-2 and understand the effect of its deficiency in AD in an immunological cell-free context, we downregulated its expression with shRNA technology in reconstructed human epidermis (RHE). This technology has previously been used with success to demonstrate the importance of filaggrin in the human epidermis.⁶ The research focused particularly on epidermal differentiation, SC properties and permeability barrier.     

Neuronal NLRP1 inflammasome activation of Caspase-1 coordinately regulates inflammatory interleukin-1-beta production and axonal degeneration-associated Caspase-6 activation

Neuronal active Caspase-6 (Casp6) is associated with Alzheimer disease (AD), cognitive impairment, and axonal degeneration. Caspase-1 (Casp1) can activate Casp6 but the expression and functionality of Casp1-activating inflammasomes has not been well-defined in human neurons. Here, we show that primary cultures of human CNS neurons expressed functional Nod-like receptor protein 1 (NLRP1), absent in melanoma 2, and ICE protease activating factor, but not the NLRP3, inflammasome receptor components. NLRP1 neutralizing antibodies in a cell-free system, and NLRP1 siRNAs in neurons hampered stress-induced Casp1 activation. NLRP1 and Casp1 siRNAs also abolished stress-induced Casp6 activation in neurons. The functionality of the NLRP1 inflammasome in serum-deprived neurons was also demonstrated by NLRP1 siRNA-mediated inhibition of speck formation of the apoptosis-associated speck-like protein containing a caspase recruitment domain conjugated to green fluorescent protein. These results indicated a novel stress-induced intraneuronal NLRP1/Casp1/Casp6 pathway. Lipopolysaccharide induced Casp1 and Casp6 activation in wild-type mice brain cortex, but not in that of Nlrp1^−/− and Casp1^−/− mice. NLRP1 immunopositive neurons were increased 25- to 30-fold in AD brains compared with non-AD brains. NLRP1 immunoreactivity in these neurons co-localized with Casp6 activity. Furthermore, the NLRP1/Casp1/Casp6 pathway increased amyloid beta peptide 42 ratio in serum-deprived neurons. Therefore, CNS human neurons express functional NLRP1 inflammasomes, which activate Casp1 and subsequently Casp6, thus revealing a fundamental mechanism linking intraneuronal inflammasome activation to Casp1-generated interleukin-1-β-mediated neuroinflammation and Casp6-mediated axonal degeneration.The lack of efficient treatment for Alzheimer disease (AD) is of high social and economical cost and a growing concern with the aging of the world''s population.¹ Therapies eliminating amyloid beta peptide (Aβ) from AD brains have unfortunately failed to stem progressive cognitive decline. These disappointing results have forced scientists to reconsider treatments against AD; some focusing on targeting Aβ earlier in disease, while others attempting to disaggregate the Tau protein in neurofibrillary tangles (NFT). Recently, the association of several immune responsive genes with increased AD risks^{2, 3, 4} have additionally revived interest in a possible etiological role for inflammation in AD.AD brain inflammation is attributed to activated microglia, which remove Aβ, and secrete neurotoxic molecules that induce neurodegeneration. Interleukin-1-beta (IL-1β), a critical component of brain neuroinflammation, is increased in AD brains⁵ and may contribute to AD pathology by increasing amyloid precursor protein (APP) gene expression, Tau hyperphosphorylation and memory impairment.⁶ However, anti-inflammatory therapies have not provided the expected beneficial effect in AD patients,⁷ suggesting that microglial inflammation may be a consequence of AD. Degenerating neurons are renowned initiators of brain inflammatory responses and the loss of synapses remains the best correlative marker of dementia in AD.⁸ This has incited us to study the response of human neurons to stress and to determine whether specific neuronal molecular events were initiated that link axonal degeneration to an inflammatory response.The active cysteinal Caspase-6 protease (Casp6), associated with axonal degeneration,^{9, 10, 11, 12, 13} is highly abundant in NFT, neuropil threads, and neuritic plaques of AD brains.¹⁴ In some aged non-cognitively impaired individuals, Casp6 activity in the entorhinal cortex and CA1 regions of the hippocampus,¹⁵ two areas initially affected by NFT pathology in AD,¹⁶ correlates significantly with lower cognitive performance.¹⁷ The expression of active Casp6 in CA1 pyramidal neurons of mouse brains is sufficient to induce age-dependent cognitive impairment, in the absence of plaques and tangles, which suggests that active Casp6 in AD brains could be a major contributor to axonal degeneration and cognitive decline.¹⁸Despite substantial evidence implicating Casp6 in AD, the pathways leading to Casp6 activation in neurons are unclear. Caspase-1 (Casp1) activates Casp6 in primary cultures of human CNS neurons.¹⁹ Inflammasome multiprotein complexes, constituted of danger sensing nucleotide-binding oligomerization domain-like receptors or the DNA sensing absent in melanoma 2 (AIM2) component, and the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), recruit and induce Casp1 self-activation.^{20, 21} Functional Nod-like receptor protein 1 (NLRP1), Nod-like receptor protein 3 (NLRP3), AIM2, and ICE protease activating factor (IPAF-1) inflammasomes have been characterized primarily in peripheral macrophages²² and CNS microglia.^{23, 24} Recently, reports have indicated inflammasome receptor expression and activation in rodent neurons. Rat cerebellar granule neurons submitted to oxygen and glucose deprivation or reduced potassium levels increased Nlrp1 mRNA levels.^{25, 26} Nuclear Nlrp1 or functional Nlrp1 inflammasome complexes increased in rat cortical neurons after traumatic brain injury, stroke, and glucose-oxygen deprivation insults.^{27, 28, 29, 30, 31} Neuronal Nlrp1 increased in rats submitted to spinal cord or sciatic nerve injury,^{29, 32} and in aging rat hippocampus or ethanol treated hippocampal slice cultures.^{33, 34} Aim2 induced pyroptosis in rat cortical neuron cultures and traumatic brain injury.³⁵ Nlrp1 has been reported in human brain pyramidal neurons³⁶ and inflammasome receptor mRNAs were observed in human neuron cultures and human Rasmussen''s encephalitis.³⁷Here, we assessed which inflammasome could activate Casp1 and subsequently Casp6 in human primary CNS cultures. We determined which inflammasomes were expressed in naive and stressed neurons and used siRNAs and S-100 cell-free extracts treated with specific inflammasome activators, or antibody blockers, to identify the functional inflammasome. We uncovered that the NLRP1, AIM2, and IPAF-1, but not the NLRP3, inflammasomes were expressed and functional in neurons and that the NLRP1 inflammasome was responsible for Casp1 and subsequently Casp6 activation in serum-deprived and benzylated ATP (BzATP)-stressed neurons. NLRP1 was co-localized with Casp6 activity, immunostained 25- to 30-fold more neurons in AD, and increased Aβ₄₂ in serum-deprived neurons. The NLRP1–Casp1–Casp6 pathway was blocked in lipopolysaccharide (LPS)-treated Nlrp1^−/− and Casp1^−/− mice brains. These results reveal a molecular cascade linking neuronal inflammasome-mediated Casp1 activation to Casp6 activation and provide unexpected novel common neuronal therapeutic targets against neuroinflammation, axonal degeneration, and cognitive impairment in AD.     

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.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

Adiponectin receptor-mediated signaling ameliorates cerebral cell damage and regulates the neurogenesis of neural stem cells at high glucose concentrations: an in vivo and in vitro study

In the central nervous system (CNS), hyperglycemia leads to neuronal damage and cognitive decline. Recent research has focused on revealing alterations in the brain in hyperglycemia and finding therapeutic solutions for alleviating the hyperglycemia-induced cognitive dysfunction. Adiponectin is a protein hormone with a major regulatory role in diabetes and obesity; however, its role in the CNS has not been studied yet. Although the presence of adiponectin receptors has been reported in the CNS, adiponectin receptor-mediated signaling in the CNS has not been investigated. In the present study, we investigated adiponectin receptor (AdipoR)-mediated signaling in vivo using a high-fat diet and in vitro using neural stem cells (NSCs). We showed that AdipoR1 protects cell damage and synaptic dysfunction in the mouse brain in hyperglycemia. At high glucose concentrations in vitro, AdipoR1 regulated the survival of NSCs through the p53/p21 pathway and the proliferation- and differentiation-related factors of NSCs via tailless (TLX). Hence, we suggest that further investigations are necessary to understand the cerebral AdipoR1-mediated signaling in hyperglycemic conditions, because the modulation of AdipoR1 might alleviate hyperglycemia-induced neuropathogenesis.Adiponectin secreted by the adipose tissue^{1, 2} exists in either a full-length or globular form.^{3, 4, 5, 6} Adiponectin can cross the blood–brain barrier, and various forms of adiponectin are found in the cerebrospinal fluid.^{7, 8, 9, 10, 11} Adiponectin exerts its effect by binding to the adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2)^{12, 13} that have different affinities for the various circulating adiponectins.^{12, 14, 15, 16, 17} Several studies reported that both receptor subtypes are expressed in the central nervous system (CNS).^{7, 12, 18} As adiponectin modulates insulin sensitivity and inflammation,¹⁹ its deficiency induces insulin resistance and glucose intolerance in animals fed a high-fat diet (HFD).^{19, 20, 21} In addition, adiponectin can ameliorate the glucose homeostasis and increase insulin sensitivity.^{22, 23, 24} Adiponectin, which is the most well-known adipokine, acts mainly as an anti-inflammatory regulator,^{25, 26} and is associated with the onset of neurological disorders.²⁷ In addition, a recent study reported that adiponectin promotes the proliferation of hippocampal neural stem cells (NSCs).²⁸ Considering that adiponectin acts by binding to the adiponectin receptors, investigation of the adiponectin receptor-mediated signaling in the brain is crucial to understand the cerebral effects of adiponectin and the underlying cellular mechanisms.The prevalence of type II diabetes mellitus (DM2) and Alzheimer''s disease increases with aging.²⁹ According to a cross-sectional study, in people with DM2, the risk of dementia is 2.5 times higher than that in the normal population.^{30, 31} A study performed between 1980 and 2002 suggested that an elevated blood glucose level is associated with a greater risk for dementia in elderly patients with DM2.³² In addition, according to a 9-year-long longitudinal cohort study, the risk of developing Alzheimer''s disease was 65% higher in people with diabetes than in control subjects.³³ A community-based cohort study also reported that higher plasma glucose concentrations are associated with an increased risk for dementia, because the higher glucose level has detrimental effects on the brain.³¹ High blood glucose level causes mitochondria-dependent apoptosis,^{34, 35, 36} and aggravates diverse neurological functions.^{37, 38} Inflammation and oxidative stress, which are commonly observed in people with diabetes, inhibit neurogenesis.^{39, 40, 41} Similarly, neurogenesis is decreased in mice and rats with genetically induced type I diabetes.^{42, 43} In addition, diabetic rodents have a decreased proliferation rate of neural progenitors.^{43, 44} Furthermore, several studies suggested that an HFD leads to neuroinflammation, the impairment of synaptic plasticity, and cognitive decline.^{45, 46}Here, we investigated whether AdipoR1-mediated signaling is associated with cell death in the brain of mice on a HFD, and whether high glucose level modifies the proliferation and differentiation capacity of NSCs in vitro. Our study provides novel findings about the role of AdipoR1-mediated signaling in hyperglycemia-induced neuropathogenesis.     

Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation

Oxygen signaling is critical for stem cell regulation, and oxidative stress-induced stem cell apoptosis decreases the efficiency of stem cell therapy. Hypoxia activates O-linked β-N-acetyl glucosaminylation (O-GlcNAcylation) of stem cells, which contributes to regulation of cellular metabolism, as well as cell fate. Our study investigated the role of O-GlcNAcylation via glucosamine in the protection of hypoxia-induced apoptosis of mouse embryonic stem cells (mESCs). Hypoxia increased mESCs apoptosis in a time-dependent manner. Moreover, hypoxia also slightly increased the O-GlcNAc level. Glucosamine treatment further enhanced the O-GlcNAc level and prevented hypoxia-induced mESC apoptosis, which was suppressed by O-GlcNAc transferase inhibitors. In addition, hypoxia regulated several lipid metabolic enzymes, whereas glucosamine increased expression of glycerol-3-phosphate acyltransferase-1 (GPAT1), a lipid metabolic enzyme producing lysophosphatidic acid (LPA). In addition, glucosamine-increased O-GlcNAcylation of Sp1, which subsequently leads to Sp1 nuclear translocation and GPAT1 expression. Silencing of GPAT1 by gpat1 siRNA transfection reduced glucosamine-mediated anti-apoptosis in mESCs and reduced mammalian target of rapamycin (mTOR) phosphorylation. Indeed, LPA prevented mESCs from undergoing hypoxia-induced apoptosis and increased phosphorylation of mTOR and its substrates (S6K1 and 4EBP1). Moreover, mTOR inactivation by rapamycin (mTOR inhibitor) increased pro-apoptotic proteins expressions and mESC apoptosis. Furthermore, transplantation of non-targeting siRNA and glucosamine-treated mESCs increased cell survival and inhibited flap necrosis in mouse skin flap model. Conversely, silencing of GPAT1 expression reversed those glucosamine effects. In conclusion, enhancing O-GlcNAcylation of Sp1 by glucosamine stimulates GPAT1 expression, which leads to inhibition of hypoxia-induced mESC apoptosis via mTOR activation.Stem cells in the body are exposed to low oxygen pressure owing to the physiological distribution of vessels.¹ This hypoxic niche for stem cells is essential to maintain the metabolic characteristics of stem cells.² Thus, describing the oxygen nature of this stem cell niche is important for elucidating stem cell regulation. Oxygen signaling is a major determinant of cell fate-controlling cellular processes. Control of oxygen signaling in stem cells has the potential to regulate embryonic development, cell cultivation, cell reprogramming, and transplantation in regenerative medicine.^{1, 3, 4, 5, 6} There are many reports showing the effects of hypoxia on various kinds of stem cells, and it has been shown that hypoxia has a paradoxical role in stem cell behaviors and cell fate regulation related to stem cell type, ageing, and oxygen concentration.^{3, 7, 8, 9} Studies of mechanisms by which stem cells function under hypoxia, and how they are regulated, have been undertaken. Several investigators recently reported that hypoxia-mediated stem cell metabolic alteration is associated with stem cell function; as a result, interest in the interaction between hypoxia and stem cell metabolism is growing.^{10, 11} However, which metabolic factors are important for stem cell fate under hypoxia have not been elucidated.O-linked β-N-acetyl glucosaminylation (O-GlcNAcylation) is affected by cellular nutrient status and extra-cellular stresses including hypoxia.^{12, 13, 14} A hypoxia-induced glycolytic switch primarily stimulates hexosamine biosynthetic pathway (HBP) flux, which induces O-GlcNAcylation signaling.¹⁵ O-GlcNAcylation is catalyzed by O-linked N-acetyl glucosamine transferase (OGT) to add N-acetyl glucosamine to the serine or threonine residues of proteins.^{16, 17, 18} O-GlcNAcylation acts as an essential factor for controlling physiological processes including migration, proliferation, and survival in stem cells, and recently it was considered as a potential strategy for use in stem cell therapy.^{19, 20, 21} In addition, as many human metabolic diseases such as diabetes and cancer are attributed to aberrant O-GlcNAcylation, unraveling HBP-mediated O-GlcNAc signaling is important in the development of practical strategies for metabolic diseases treatment. For example, Liu et al.²² showed that glucosamine-mediated O-GlcNAcylation induced resistance to tissue damage resulting from ischemic injury and provided cardio-protection in an animal model. Furthermore, O-GlcNAcylation interacts with other nutrient metabolic pathways such as lipogenesis, gluconeogenesis, and glycogen synthesis.^{12, 23, 24} Among these metabolic pathways, lipid metabolism is reported to have a central role in controlling stem cell fate.^{25, 26} Collectively, these results suggest that O-GlcNAcylation can be a useful tool for use in cellular metabolic regulation, and identification of an O-GlcNAcylation-regulating potential lipid metabolic factor, which is important for stem cell regulation, may suggest potentially useful metabolic approach in stem cell therapy.Embryonic stem cells (ESCs) are distinctive in that they have a self-renewal capacity, exhibit pluripotency to enable differentiation into cellular derivatives of three lineages, and may be used as a representative in vitro model in the study of early embryo development, pluripotent stem cell physiology, and clinical applications.^{27, 28, 29} Despite the clinical limitation associated with ESCs and the possibility of cancer formation, several studies into the therapeutic effects of ESCs in regenerative medicine have been reported. Indeed, administrations of human or mouse ESCs (mESCs) has induced a paracrine effect and improved damaged cell functions.^{30, 31, 32} However, despite the benefit of ESCs in regenerative medicine, ESC apoptosis remains an impediment to ESC applications using hypoxia.^{33, 34, 35} Thus, researchers are investigating ways to minimize ESC apoptosis and control ESC fate under hypoxia. In this study, we used glucosamine to induce O-GlcNAcylation. Therefore, our study investigated the role of O-GlcNAcylation via glucosamine (GlcN) which is recognized as a HBP activator³⁶ in lipid metabolism and in protection of mESC apoptosis under hypoxia.