Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma

Glioblastoma (GBM), the most prevalent type of primary intrinsic brain cancer in adults, remains universally fatal despite maximal therapy, including radiotherapy and chemotherapy. Cytotoxic therapy generates double-stranded DNA breaks (DSBs), most commonly repaired by homologous recombination (HR). We hypothesized that cancer cells coopt meiotic repair machinery as DSBs are generated during meiosis and repaired by molecular complexes distinct from genotoxic responses in somatic tissues. Indeed, we found that gliomas express meiotic repair genes and their expression informed poor prognosis. We interrogated the function of disrupted meiotic cDNA1 (DMC1), a homolog of RAD51, the primary recombinase used in mitotic cells to search and recombine with the homologous DNA template. DMC1, whose only known function is as an HR recombinase, was expressed by GBM cells and induced by radiation. Although targeting DMC1 in non-neoplastic cells minimally altered cell growth, DMC1 depletion in GBM cells decreased proliferation, induced activation of CHK1 and expression of p21^CIP1/WAF1, and increased RPA foci, suggesting increased replication stress. Combining loss of DMC1 with ionizing radiation inhibited activation of DNA damage responses and increased radiosensitivity. Furthermore, loss of DMC1 reduced tumor growth and prolonged survival in vivo. Our results suggest that cancers coopt meiotic genes to augment survival under genotoxic stress, offering molecular targets with high therapeutic indices.Glioblastomas (GBMs) rank among the deadliest of all human cancers, with only modest improvement in patient survival over recent decades. More than 12 000 GBM patients are diagnosed annually in the United States.^{1, 2} Despite aggressive treatment consisting of maximal safe surgical resection, concurrent radiotherapy and chemotherapy, and adjuvant chemotherapy, median survival remains dismal at 12–15 months.^{3, 4} Although numerous molecular targets have been identified in GBM, no molecularly targeted therapy has demonstrated a survival benefit. Radiotherapy remains the cornerstone of post-surgical GBM therapy with modest additional benefit offered by concurrent administration of the oral methylator, temozolomide. However, radioresistance and tumor recurrence is universal in GBM.^{4, 5, 6} Radiation also damages non-neoplastic brain tissue, resulting in cognitive impairment and decreased quality-of-life.⁷ Focal high-dose radiation reduces toxicity to non-neoplastic tissue, but tumor invasion into normal brain regions limits the survival benefit of highly focused radiotherapy techniques, like gamma knife and proton beam, establishing a need for improved combinatorial treatments, such as radiosensitizers.^{8, 9} To date, no radiosensitizer has successfully increased survival with acceptable toxicity in a clinical trial. Based on this background, we sought novel molecular targets that mediate responses to genotoxic stress and have limited function in normal cells.During mitosis, cells inspect the integrity of their DNA and repair replication errors through cell-state and error-specific mechanisms.¹⁰ Unrepaired or large regions of DNA damage overwhelm replication mechanisms to induce cell death.^{10, 11} DNA double-strand breaks (DSBs) are detrimental as they cause large-scale chromosomal rearrangements.¹⁰ The homologous recombination (HR) pathway is primarily used to repair DSBs during S- and G₂-phases, providing access to both sister and homologous chromosomes as repair templates.^{7, 12} RADiation sensitive 51 (RAD51) is a key recombinase important in HR and replication fork maintenance, functioning in both mitotic and meiotic cells.^{7, 12, 13, 14, 15} Phosphorylated RAD51 replaces replication protein A (RPA) upon DNA loading.¹⁶ Recombination mediated by RAD51 with the intact DNA template strand results in a relatively error-free repair.¹²In contrast to mitosis, germ cells undergoing meiosis actively generate genetic diversity through induction of programmed DSBs, which are repaired through HR.^{17, 18, 19} In meiotic HR, RAD51 functions in conjunction with the meiosis-specific recombinase, disrupted meiotic cDNA1 (DMC1). RAD51 and DMC1 are loaded onto DNA by a meiosis-specific accessory protein complex, homologous-pairing protein 2 (HOP2)–meiotic nuclear divisions 1 (MND1), to promote homologous strand invasion and dissociation-loop (D-loop) formation.^{20, 21} D-loops formed using the DMC1–RAD51 complex are more resistant to dissociation as opposed to D-loops formed by RAD51 alone, increasing the likelihood of DNA crossover events.²⁰ In addition, DMC1-directed crossovers preferentially utilize the homologous chromosome further increasing genetic variation.²²GBM cells commonly harbor genetic lesions that promote unrestrained proliferation but also stimulate genotoxic stress responses. Neoplastic cells do not require perfect fidelity of repair. In fact, dysfunctional repair accelerates genetic evolution of clones, but cancer cells must acquire mechanisms to bypass cell death or senescence in response to exogenous stressors.^{11, 23} Radiotherapy targets proliferating cancer cells by production of reactive oxygen species, leading to generation of DSBs and activation of the DNA damage response (DDR) pathway.^{11, 24} DSBs generated as a result of ionizing radiation (IR) are repaired through HR or non-homologous end joining (NHEJ).^{7, 12, 25, 26} Terminally differentiated neurons are post-mitotic and rely on NHEJ as a means to repair DNA DSBs. Therefore, inhibition of the NHEJ pathway may result in unfavorable normal neural cell toxicity.²⁶The HR pathway is an attractive target as it is linked to increased genetic variation and loss of heterozygosity (LOH).^{12, 27} Multiple HR checkpoints have been proposed as potential therapeutic targets for GBM.^{28, 29, 30, 31} Although the prognostic value of RAD51 expression in GBM is unresolved,^{29, 32, 33} RAD51 is consistently elevated in GBM compared with normal brain.³³ Reducing RAD51 expression radiosensitizes GBM cells,²⁹ but may have a limited therapeutic index because of the potentially toxic effects on non-neoplastic cells. In this study, we investigated the aberrant activity of meiotic HR regulators in glioma, focusing on the meiosis-specific DMC1. Activation of meiotic repair genes in neoplastic cells selectively provides tumor cells with a repair mechanism to evade cell death caused by DNA damage, yet increase genetic diversity to drive clonal evolution.     

Modulation of A1 and A2B adenosine receptor activity: a new strategy to sensitise glioblastoma stem cells to chemotherapy

Therapies that target the signal transduction and biological characteristics of cancer stem cells (CSCs) are innovative strategies that are used in combination with conventional chemotherapy and radiotherapy to effectively reduce the recurrence and significantly improve the treatment of glioblastoma multiforme (GBM). The two main strategies that are currently being exploited to eradicate CSCs are (a) chemotherapeutic regimens that specifically drive CSCs toward cell death and (b) those that promote the differentiation of CSCs, thereby depleting the tumour reservoir. Extracellular purines, particularly adenosine triphosphate, have been implicated in the regulation of CSC formation, but currently, no data on the role of adenosine and its receptors in the biological processes of CSCs are available. In this study, we investigated the role of adenosine receptor (AR) subtypes in the survival and differentiation of CSCs isolated from human GBM cells. Stimulation of A₁AR and A_2BAR had a prominent anti-proliferative/pro-apoptotic effect on the CSCs. Notably, an A₁AR agonist also promoted the differentiation of CSCs toward a glial phenotype. The differential effects of the two AR agonists on the survival and/or differentiation of CSCs may be ascribed to their distinct regulation of the kinetics of ERK/AKT phosphorylation and the expression of hypoxia-inducible factors. Most importantly, the AR agonists sensitised CSCs to the genotoxic activity of temozolomide (TMZ) and prolonged its effects, most likely through different mechanisms, are as follows: (i) by A_2BAR potentiating the pro-apoptotic effects of TMZ and (ii) by A₁AR driving cells toward a differentiated phenotype that is more sensitive to TMZ. Taken together, the results of this study suggested that the purinergic system is a novel target for a stem cell-oriented therapy that could reduce the recurrence of GBM and improve the survival rate of GBM patients.Glioblastoma multiforme (GBM), classified as grade IV on the World Health Organization scale,¹ is the most common type of primary malignant brain tumour.² The current therapeutic strategy includes surgery followed by radiation and chemotherapy using temozolomide (TMZ). This therapeutic approach slightly improves the survival rate of GBM patients, but their prognosis remains poor and most patients die of tumour recurrence.³ The causes of the recurrence of GBM are complex and include the high proliferative index of the tumour cells and their resistance to chemotherapy and radiotherapy, particularly in the case of the cancer stem cells (CSCs). These cells have been proposed to not only initiate the genesis of GBM and contribute to its highly proliferative nature, but to also be the basis for its recurrences following treatment. Moreover, it has been reported that the most aggressive or refractory cancers contain the highest number of CSCs.^{4, 5, 6}These findings suggest that innovative stem cell-orientated therapy may be an effective strategy to reduce tumour recurrence and significantly improve GBM treatment outcomes.^{7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18} This type of therapy may not be easy to implement because CSCs have been shown to have a low level of reactive oxygen species¹⁹ and to be more resistant to ionising radiation,²⁰ vincristine,²¹ hypoxia and other chemotherapeutics²² compared with non-CSCs. In contrast, the preferential elimination of the CSC population may contribute to the effectiveness of TMZ, which is the most effective pharmacologic agent used in glioma treatment;²³ however, the activity of TMZ appears to be short lived because the drug causes the reversible blockage of the cell cycle of CSCs.²⁴ Moreover, long-term TMZ therapy results in the occurrence of drug-resistant GBM cells,²⁵ indicating the need to develop distinct strategies to overcome this resistance.Extracellular purines have been implicated in several aspects of GBM biology, such as proliferation,²⁶ migration,²⁷ invasion²⁸ and death.²⁹ The concentration of adenosine in the extracellular fluid of glioma tissue was reported to be in the low micromolar range,³⁰ which is sufficiently high to stimulate all the four of the adenosine receptor (AR) subtypes (A₁, A_2A, A_2B and A₃).³¹ Each of the ARs have a pivotal role in the control of tumour growth and invasiveness^{32, 33, 34} but to date, no data on their role in CSC biology are available. Recently, it was demonstrated that treatment with adenosine triphosphate reduced the rate of sphere formation by glioma cells and that purinergic receptors are differentially expressed in spheres of tumour cells and adherent cells.³³ In this study, we investigated the role of AR subtypes in the survival and differentiation of CSCs. Globally, our data clarified the role of each AR subtype in CSC functionality and suggested that the purinergic system is a novel pharmacological target for the development of new anti-CSC therapies, particularly those aimed at the treatment of GBM recurrences.     

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.     

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.     

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.     

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.     

Self-Injurious Behavior Secondary to Cytomegalovirus-Induced Neuropathy in an SIV-Infected Rhesus Macaque (Macaca mulatta)

A 3.5-y-old, female rhesus macaque (Macaca mulatta) inoculated with SIVmac239 presented 8 mo later for inappetence and facial bruising. Physical examination revealed a superficial skin abrasion below the left eye, bruising below the left brow, and epistaxis of the left nostril. There were no significant findings on CBC, serum chemistry, urinalysis, or radiographs. Differential diagnoses included infectious etiologies, self-injurious behavior, immune-mediated dermatitis, and neoplasia. Lack of response to antibiotic and analgesic therapy and observations of the macaque made it apparent that the skin lesions were self-inflicted. The excoriations rapidly progressed to extend over the nose, and the left palpebrae became edematous. Euthanasia was elected because the macaque appeared to be experiencing continued discomfort despite analgesic therapy. Histopathologic examination revealed systemic cytomegalovirus (CMV) infection involving the facial nerves, periocular nerves, meninges, and perimesenteric lymph nodes. CMV is a common infection in macaques, with adult seroprevalence close to 100% in most colonies. Infection in immunocompetent animals is usually asymptomatic but can cause significant clinical disease in immunodeficient hosts. CMV is associated with a painful peripheral neuropathy in human AIDS patients, and analgesic treatment is often unsatisfactory. Peripheral neuropathy secondary to CMV should be considered as an underlying cause of self-injurious behavior in SIV-infected macaques. Macaques affected by other diseases and disorders may also be at risk for development of painful peripheral neuropathies.Abbreviations: CMV, cytomegalovirus; HCMV, human CMV; RhCMV, rhesus CMV; SIB, self-injurious behaviorRhesus macaques (Macaca mulatta) are one of the most commonly used NHP species in biomedical research.^9,25 They generally adapt well to captivity, but some develop abnormal behaviors such as stereotypies and self-injurious behavior (SIB).³² Examples of SIB include excessive hair-plucking, head banging, and self-biting that causes wounds in some cases.³² Self-inflicted wounding has been reported in 11% to 14% of individually housed rhesus macaques and can be difficult to manage.^26,32,39 The most commonly discussed risk factors for the development of SIB are related to housing and management, and little is known about potential physiologic causes, such as neuropathic pain.^18,26,32 Compulsive SIB directed toward a specific body part due to neuropathic pain or pruritus occurs in humans.^12,28Rhesus cytomegalovirus (RhCMV) is enzootic in rhesus macaques with close to 100% seroprevalence by 1 y of age in both wild and captive populations.^22,42 As with human cytomegalovirus (HCMV), RhCMV infection is generally subclinical in immunocompetent animals but can cause serious disease in immunodeficient macaques.⁸ RhCMV can be highly pathogenic in SIV-infected animals, and HCMV is the most common viral opportunistic infection in humans with AIDS.^8,37 HCMV infection has been associated with painful peripheral neuropathies in AIDS patients.^17,36,37 We here report a case of SIB associated with RhCMV-induced peripheral neuropathy in an immunocompromised macaque.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.     

Comparison of Biomarkers of Oxidative Stress and Cardiovascular Disease in Humans and Chimpanzees (Pan troglodytes)

In the oxidative stress hypothesis of aging, the aging process is the result of cumulative damage by reactive oxygen species. Humans and chimpanzees are remarkably similar; but humans live twice as long as chimpanzees and therefore are believed to age at a slower rate. The purpose of this study was to compare biomarkers for cardiovascular disease, oxidative stress, and aging between male chimpanzees and humans. Compared with men, male chimpanzees were at increased risk for cardiovascular disease because of their significantly higher levels of fibrinogen, IGF1, insulin, lipoprotein a, and large high-density lipoproteins. Chimpanzees showed increased oxidative stress, measured as significantly higher levels of 5-hydroxymethyl-2-deoxyuridine and 8-iso-prostaglandin F_2α, a higher peroxidizability index, and higher levels of the prooxidants ceruloplasmin and copper. In addition, chimpanzees had decreased levels of antioxidants, including α- and β-carotene, β-cryptoxanthin, lycopene, and tocopherols, as well as decreased levels of the cardiovascular protection factors albumin and bilirubin. As predicted by the oxidative stress hypothesis of aging, male chimpanzees exhibit higher levels of oxidative stress and a much higher risk for cardiovascular disease, particularly cardiomyopathy, compared with men of equivalent age. Given these results, we hypothesize that the longer lifespan of humans is at least in part the result of greater antioxidant capacity and lower risk of cardiovascular disease associated with lower oxidative stress.Abbreviations: 5OHmU, 5-hydroxymethyl-2-deoxyuridine; 8isoPGF_2α, 8-iso-prostaglandin F_2α; HDL, high-density lipoprotein; IGF1, insulin-like growth factor 1; LDL, low-density lipoprotein; ROS, reactive oxygen speciesAging is characterized as a progressive reduction in the capacity to withstand the stresses of everyday life and a corresponding increase in risk of mortality. According to the oxidative stress hypothesis of aging, much of the aging process can be accounted for as the result of cumulative damage produced by reactive oxygen species (ROS).^{6,21,28,41,97} Endogenous oxygen radicals (that is, ROS) are generated as a byproduct of normal metabolic reactions in the body and subsequently can cause extensive damage to proteins, lipids, and DNA.^6,41 Various prooxidant elements, in particular free transition metals, can catalyze these destructive reactions.⁶ The damage caused by ROS can be counteracted by antioxidant defense systems, but the imbalance between production of ROS and antioxidant defenses, over time, leads to oxidative stress and may contribute to the rate of aging.^28,97Oxidative stress has been linked to several age-related diseases including neurodegenerative diseases, ophthalmologic diseases, cancer, and cardiovascular disease.^21,28,97 Of these, cardiovascular disease remains the leading cause of adult death in the United States and Europe.⁷¹ In terms of cardiovascular disease, oxidative stress has been linked to atherosclerosis, hypertension, cardiomyopathy, and chronic heart failure in humans.^55,78,84 Increases in oxidant catalysts (prooxidants)—such as copper, iron, and cadmium—have been associated with hypertension, coronary artery disease, atherosclerosis, and sudden cardiac death.^98,102,106 Finally, both endogenous and exogenous antioxidants have been linked to decreased risk of cardiovascular disease, although the mechanisms behind this relationship are unclear.^11,52,53 However, the oxidative stress hypothesis of aging aims to explain not only the mechanism of aging and age-related diseases (such as cardiovascular disease) in humans but also the differences between aging rates and the manifestations of age-related diseases across species.The differences in antioxidant and ROS levels between animals and humans offer promise for increasing our understanding of human aging. Additional evidence supporting the oxidative stress hypothesis of aging has come from comparative studies linking differences in aging rates across taxa with both antioxidant and ROS levels.^{4,17-21,58,71,86,105} In mammals, maximum lifespan potential is positively correlated with both serum and tissue antioxidant levels.^{17,18,21,71,105} Research has consistently demonstrated that the rate of oxidative damage varies across species and is negatively correlated with maximum lifespan potential.^{4,19,20,58,71,86} However, few studies involved detailed comparisons of hypothesized biochemical indicators of aging and oxidative stress between humans and animals.⁶ This type of interspecies comparison has great potential for directly testing the oxidative stress hypothesis of aging.Much evolutionary and genetic evidence supports remarkable similarity between humans and chimpanzees.^95,100 Despite this similarity, humans have a lifespan of almost twice that of chimpanzees.^3,16,47 Most comparative primate aging research has focused on the use of a macaque model,^62,81,88 and several biochemical markers of age-related diseases have been identified in both humans and macaque monkeys.^{9,22,28,81,93,97} Several other species of monkeys have also been used in research addressing oxidative stress, antioxidant defenses, and maximum lifespan potential.^18,21,58,105 However, no study to date has examined biochemical indicators of oxidative stress and aging in chimpanzees and humans as a test of the oxidative stress hypothesis for aging. The purpose of this study is to compare biochemical markers for cardiovascular disease, oxidative stress, and aging directly between male chimpanzees and humans. Given the oxidative stress hypothesis for aging and the known role of oxidative stress in cardiovascular disease, we predict that chimpanzees will show higher levels of cardiovascular risk and oxidative stress than humans.     

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.     

A sphingosine kinase inhibitor combined with temozolomide induces glioblastoma cell death through accumulation of dihydrosphingosine and dihydroceramide,endoplasmic reticulum stress and autophagy

Glioblastomas (GBMs) are very aggressive tumors with low chemosensitivity. The DNA-alkylating agent temozolomide (TMZ) is currently the most efficient chemotoxic drug for GBM therapy; however, many patients develop resistance to TMZ. Combining TMZ with another agent could present an improved treatment option if it could overcome TMZ resistance and avoid side effects. Sphingosine kinase inhibitors (SKIs) have emerged as anticancer agents. Sphingosine kinases are often overexpressed in tumors where their activity of phosphorylating sphingosine (Sph) contributes to tumor growth and migration. They control the levels of the pro-apoptotic ceramide (Cer) and Sph and of the pro-survival sphingosine-1 phosphate. In the present work, TMZ was combined with a specific SKI, and the cytotoxic effect of each drug alone or in combination was tested on GBM cell lines. The combination of sublethal doses of both agents resulted in the cell death potentiation of GBM cell lines without affecting astrocyte viability. It triggered a caspase-3-dependent cell death that was preceded by accumulation of dihydrosphingosine (dhSph) and dihydroceramide (dhCer), oxidative stress, endoplasmic reticulum stress, and autophagy. Autophagy was identified as the crucial switch that facilitated induction of this cell death potentiation. The sublethal dose of the inhibitor induced these stress events, whereas that of TMZ induced the destructive autophagy switch. Remarkably, neither Cer nor Sph, but rather the Cer intermediates, dhSph and dhCer, was involved in the cytotoxicity from the combination. Cell lines sensitive to the combination expressed low levels of the antioxidant enzyme glutathione peroxidase-1, indicating this enzyme as a potential marker of sensitivity to such treatment. This work shows for the first time a strong interaction between a SKI and TMZ, leading to a tumor cell-specific death induction. It further demonstrates the biological relevance of dihydrosphingolipids in cell death mechanisms and emphasizes the potential of drugs that affect sphingolipid metabolism for cancer therapy.Glioblastoma (GBM) is a devastating cancer with poor prognosis. The DNA-alkylating agent temozolomide (TMZ) is currently the most efficient drug in GBM therapy; however, not all patients benefit from TMZ and those who initially do benefit become resistant to TMZ over time, pointing out the urgent need for novel therapies.^1,2 Modulating the metabolism of bioactive sphingolipids has been shown to have a potential in treating malignancies.³ Particularly, inhibitors of the sphingosine kinases (SK) emerge as interesting anticancer agents.⁴ SK exist as two isoforms, SK1 mainly found in the cytoplasm and SK2 found in the nucleus. Pro-survival as well as pro-apoptotic effects have been reported for both isoforms.⁵ These enzymes have a central role in the so-called ‘sphingolipid rheostat'' as they control the balance between the levels of the sphingolipids ceramide (Cer), sphingosine (Sph), and sphingosine-1 phosphate (S1P). As such, they control cell fate by regulating the relative amounts of pro-apoptotic Cer and Sph to pro-survival S1P.⁶ S1P acts extracellularly as a ligand to S1P receptors, leading to increased tumor cell migration and proliferation.^7,8 Thus, blocking SK with a specific inhibitor would not only decrease the levels of S1P and hence tumor migration, but also lead to an increase in Cer and Sph, thereby inducing cell death. In various studies (reviewed in Heffernan-Stroud and Obeid⁹), pharmacological SK inhibitors were reported to sensitize cells towards chemotoxic drugs such as doxorubicin and etoposide, to decrease viability and to reduce migration in different tumor cell lines, including TMZ-resistant GBM cell lines.¹⁰ We have previously shown that the sphingosine kinase inhibitor (SKI)-II,¹¹ which inhibits both SK1 and SK2,⁴ induced death in murine and human GBM cells but not in normal and non-transformed astrocytes.¹²On the basis of these observations, we hypothesize that a combination of low doses of TMZ and SKI-II may overcome TMZ resistance and lead to a tumor-specific cell death. In GBM cells, TMZ was reported to induce a late apoptosis triggered by O⁶-methylguanine lesion,^13,14 mitotic catastrophe,¹⁵ and autophagy.¹⁶ The death mechanisms triggered by SKI have not been characterized in detail, except for the role of pro-apoptotic Cer,¹⁷ of which the concentration is expected to rise after SK inhibition. Interference with sphingolipid metabolism is expected to induce cellular stress at the various organelles where sphingolipids are generated or metabolized (endoplasmic reticulum (ER), mitochondria, lysosome).¹⁸ We reported that SKI-II induces lysosome stress in GBM cells, as indicated by lysosome enlargement and subsequent cell death.¹²In this report, we show that a combination of sublethal doses of SKI-II and TMZ triggers a significant increase in death of human GBM cells but not of human astrocytes. We identify the steps induced by SKI-II, TMZ, and both combined thatlead to this specific cell death.     

Spontaneous Osteoblastic Osteosarcoma in a Mongolian Gerbil (Meriones unguiculatus)

Spontaneous neoplasms in Mongolian gerbils have an incidence of 20% to 26.8%, but osteosarcomas occur at a much lower rate. Here we report a 1-y-old Mongolian gerbil with a spontaneous osteosarcoma at the level of the proximal tibia, with metastases to the pectoral muscles and lungs. Grossly, the tibial mass obliterated the tibia and adjacent muscles, and an axillary mass with a bloody, cavitary center expanded the pectoral muscles. Microscopically, the tibial mass was an infiltrative, osteoblastic mesenchymal neoplasm, and the axillary mass was an anaplastic mesenchymal neoplasm with hemorrhage. The lung contained multiple metastatic foci. Immunohistochemistry for osteonectin was strongly positive in the tibial, axillary, and pulmonary metastases. Although osteosarcoma is the most common primary malignant bone neoplasm that occurs spontaneously in all laboratory and domestic animal species and humans, it arises less frequently than does other neoplasms. The current case of spontaneous osteoblastic osteosarcoma of the proximal tibia and metastases to the pectoral muscles and lung in a Mongolian gerbil is similar in presentation, histology, and predilection site of both osteoblastic and telangiectatic osteosarcomas in humans. In addition, this case is an unusual manifestation of osteosarcoma in the appendicular skeleton of a Mongolian gerbil.Mongolian gerbils are used frequently in biologic research,^{1,2,4,9,10,12-14} particularly in oncogenic studies and filariasis research studying Brugia malayi.² There have been several reports^{1,6,10,11,13-15} of spontaneous neoplasms, particularly in gerbils 2 y of age and older, typically occurring with the highest incidences in the skin, reproductive tract, and adrenal glands; however, neoplasms have also been reported in the thyroid, thymus, liver, kidney, pancreas, and bone.^{1,6,10,11,13-15} The incidence of spontaneous neoplasms occurring in the subfamily Gerbillinae ranges from 20% to 26.8%,^{1,6,10,11,13-15} depending on the study, age, and sex of the animals.With a lower incidence than those reported for other neoplasms, osteosarcomas in gerbils have been described in the ramus of the mandible and as an extraskeletal mass throughout the peritoneum.^10,11 The usual age of onset for osteosarcomas in Mongolian gerbils is approximately 3 y (36 to 39 mo); however, no tumor type has been reported at less than 2 y of age in this species.^10,11 Here we report a spontaneous osteosarcoma that occurred at the level of the proximal tibia, with metastases to the pectoral muscles and lung, in a 1-y-old Mongolian gerbil.     

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.     

Blood Supply to the Chicken Femoral Head

The purpose of this study was to conduct a comprehensive evaluation of the vascular supply to the femoral head, including the vessels that give rise to the terminal perfusing branches. Using a casting agent, we highlighted the anatomy of the external iliac and ischiatic arteries with their associated branches after anatomic dissection of 24 hips from 12 Leghorn chickens. We confirmed published findings regarding perfusion of the femoral head and identified 3 previously undescribed arterial branches to this structure. The first branch (the acetabular branch of the femoralis artery) was supplied by the femoralis artery and directly perfused the acetabulum and femoral head. The second branch (the lateral retinacular artery) was a tributary of the femoralis artery that directly supplied the femoral head. Finally, we found that the middle femoral nutrient artery supplies a previously undescribed ascending intraosseous branch (the ascending branch of the middle femoral nutrient artery) that perfuses the femoral head. Precise understanding of the major vascular branches to the femoral head would allow for complete or selective ligation of its blood supply and enable the creation of a reproducible bipedal model of femoral head osteonecrosis.Like humans, chickens are bipedal animals that rely on the hip joint to absorb the majority of the body''s weight. This anatomy, in concert with their high activity level, makes chickens an attractive model for the study of osteonecrosis of the femoral head in humans. The vast majority of animal research on osteonecrosis of the femoral head has been performed on quadrupedal animals,^{3,4,10,19,25,26,28,29,31,36,37,41,51,52} thus limiting its application to bipedal species because most quadruped models fail to progress to end-stage mechanical collapse similar to that in humans.⁶Avascular necrosis is the death of bone that occurs from ischemia due to disruption of the vascular supply to bone through direct or indirect mechanisms.³⁸ Avascular necrosis should be differentiated from the broader term of osteonecrosis, which refers to bone death in general.³² Causes of femoral head osteonecrosis include direct and indirect disruption of vascular supply (traumatic injury, intravascular coagulation, extrinsic compression) as well as changes in cellular differentiation and cellular apoptosis.^{4,7,12,15,17,18,24,30-32,38,49,50} Accordingly, causes of osteonecrosis are both traumatic and nontraumatic.^16,31,32The arterial anatomy in the chicken hindlimb has been outlined by several authors.^{20,22,27,35,42,44,45} Briefly, the external iliac and ischiatic artery arise from the abdominal aorta to provide blood supply to the chicken hind limb. The external iliac artery has 2 main branches—the femoralis and femoral circumflex arteries—that distribute blood to the chicken hindlimb. The ischiatic artery provides 3 main branches: the trochanteric artery, superior femoral nutrient artery, and middle femoral nutrient artery. Although the terminal vascular supply to the femoral head of Leghorn and Broiler chickens has been described,^46,47 the origin of these terminal arteries with reference to the ischiatic and femoralis arteries and their respective branches has not been addressed. The current study will describe the blood vessels that feed these terminal branches to the chicken femoral head.