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.     

Dysferlin regulates cell membrane repair by facilitating injury-triggered acid sphingomyelinase secretion

Dysferlin deficiency compromises the repair of injured muscle, but the underlying cellular mechanism remains elusive. To study this phenomenon, we have developed mouse and human myoblast models for dysferlinopathy. These dysferlinopathic myoblasts undergo normal differentiation but have a deficit in their ability to repair focal injury to their cell membrane. Imaging cells undergoing repair showed that dysferlin-deficit decreased the number of lysosomes present at the cell membrane, resulting in a delay and reduction in injury-triggered lysosomal exocytosis. We find repair of injured cells does not involve formation of intracellular membrane patch through lysosome–lysosome fusion; instead, individual lysosomes fuse with the injured cell membrane, releasing acid sphingomyelinase (ASM). ASM secretion was reduced in injured dysferlinopathic cells, and acute treatment with sphingomyelinase restored the repair ability of dysferlinopathic myoblasts and myofibers. Our results provide the mechanism for dysferlin-mediated repair of skeletal muscle sarcolemma and identify ASM as a potential therapy for dysferlinopathy.Dysferlinopathy is a progressive muscle wasting disease, which is classified as limb-girdle muscular dystrophy type 2B (LGMD2B) or Miyoshi muscular dystrophy 1, based on its muscle involvement.^{1, 2} Dysferlin deficit leads to altered vesicle formation and trafficking,^{3, 4} poor repair of injured cell membranes,^{5, 6} and increased muscle inflammation.^{7, 8} Dysferlin contains C2 domains that are found in Ca²⁺-dependent membrane fusion proteins such as synaptotagmins.⁹ Thus, dysferlin is thought to regulate muscle function by regulating vesicle trafficking and fusion.^{10, 11, 12, 13} Dysferlin deficiency has also been implicated in conflicting reports regarding the fusion ability of dysferlinopathic myoblasts.^{4, 14, 15, 16} With such diverse roles for dysferlin, the mechanism through which dysferlin deficiency results in muscle pathology is unresolved. As skeletal muscle-specific re-expression of dysferlin rescues all dysferlinopathic pathologies,^{17, 18} myofiber repair has been suggested to be the unifying deficit underlying muscle pathology in dysferlinopathy.¹⁹ Repair of injured cell membranes requires subcellular compartments, which in mammalian cells include lysosomes,¹¹ enlargeosomes,²⁰ caveolae,²¹ dysferlin-containing vesicles,⁵ and mitochondria.²²Cells from muscular dystrophy patients that have normal dysferlin expression exhibit normal lysosome and enlargeosome exocytosis.²³ However, dysferlinopathic muscle cells exhibit enlarged LAMP2-positive lysosomes, reduced fusion of early endosomes, altered expression of proteins regulating late endosome/lysosome fusion, and reduced injury-triggered cell-surface levels of LAMP1.^{4, 11, 12} In non-muscle cells, lack of dysferlin reduces lysosomal exocytosis.²⁴ These findings implicate lysosomes in dysferlin-mediated muscle cell membrane repair. In one model for lysosome-mediated cell membrane repair, Ca²⁺ triggers vesicle–vesicle fusion near the site of injury, forming ‘membrane patch'', which fuses to repair the wounded cell membrane.^{25, 26, 27, 28} In another model, lysosome exocytosis following cell membrane injury by pore-forming toxins leads to secretion of the lysosomal enzyme acid sphingomyelinase (ASM), which causes endocytosis of pores in the damaged cell membranes.^{21, 29, 30} Both these models have been suggested to be involved in the repair of injured muscle cells.^{21, 28}To examine the muscle cell pathology in dysferlinopathy, we have developed dysferlinopathic mouse and human models. Use of these models shows that a lack of dysferlin does not alter myogenic differentiation but causes poor repair of even undifferentiated muscle cells. We show that dysferlin is required for tethering lysosomes to the cell membrane. Fewer lysosomes at the cell membrane in dysferlinopathic cells results in slow and reduced lysosome exocytosis following injury. This reduction in exocytosis reduces injury-triggered ASM secretion, which is responsible for the poor repair of dysferlinopathic muscle cells. Extracellular sphingomyelinase (SM) fully rescues the repair deficit in dysferlinopathic cells and mouse myofibers, offering a potential drug-based therapy for dysferlinopathy.     

Live imaging the phagocytic activity of inner ear supporting cells in response to hair cell death

Hearing loss and balance disorders affect millions of people worldwide. Sensory transduction in the inner ear requires both mechanosensory hair cells (HCs) and surrounding glia-like supporting cells (SCs). HCs are susceptible to death from aging, noise overexposure, and treatment with therapeutic drugs that have ototoxic side effects; these ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic drug cisplatin. Although both classes of drugs are known to kill HCs, their effects on SCs are less well understood. Recent data indicate that SCs sense and respond to HC stress, and that their responses can influence HC death, survival, and phagocytosis. These responses to HC stress and death are critical to the health of the inner ear. Here we have used live confocal imaging of the adult mouse utricle, to examine the SC responses to HC death caused by aminoglycosides or cisplatin. Our data indicate that when HCs are killed by aminoglycosides, SCs efficiently remove HC corpses from the sensory epithelium in a process that includes constricting the apical portion of the HC after loss of membrane integrity. SCs then form a phagosome, which can completely engulf the remaining HC body, a phenomenon not previously reported in mammals. In contrast, cisplatin treatment results in accumulation of dead HCs in the sensory epithelium, accompanied by an increase in SC death. The surviving SCs constrict fewer HCs and display impaired phagocytosis. These data are supported by in vivo experiments, in which cochlear SCs show reduced capacity for scar formation in cisplatin-treated mice compared with those treated with aminoglycosides. Together, these data point to a broader defect in the ability of the cisplatin-treated SCs, to preserve tissue health in the mature mammalian inner ear.Hearing loss affects more than 360 million people worldwide and is often irreversible.¹ Mechanosensory hair cells (HCs), the receptor cells of hearing and balance, are not regenerated in the adult mammal and their death results in permanent hearing loss.^{2, 3} HCs are surrounded by glia-like supporting cells (SCs) that are necessary for HC survival and function (reviewed in Monzack et al.).⁴ SCs perform many functions, including providing critical trophic factors, preventing excitotoxicity, and mediating regeneration in those systems (non-mammalian vertebrates) capable of replacing lost HCs.^{5, 6, 7, 8, 9, 10, 11} When HCs die, SCs also preserve the integrity and function of the remaining tissue by forming scars and clearing dead HCs.^{2, 12, 13, 14, 15, 16, 17} Maintaining a fluid barrier at the surface of the sensory epithelium after damage is necessary to preserve the electro-chemical gradient that drives HC depolarization and therefore sensory transduction after the onset of hearing (reviewed in Wangemann).¹⁸Several major stressors cause HC death,^{19, 20, 21, 22} including aging, noise trauma, and exposure to therapeutic drugs with ototoxic side effects. When a HC is killed by noise or aminoglycoside antibiotics, surrounding SCs form a filamentous actin (F-actin) cable that constricts the HC at its apex.^{2, 12, 13, 14, 15, 16, 17} This process separates the apical portion of the cell, including the stereocilia bundle, from the HC body and preserves a sealed reticular lamina.²³ In the chick utricle, following the apical constriction of dead HCs, the SCs engulf and phagocytose the remaining HC corpse.¹⁵ Additional data from the chick indicate that the ototoxic drug cisplatin impairs some SC functions, including regeneration of HCs or clearance of HC debris.²⁴ We hypothesized that SCs would have significant phagocytic activity in the mature mammalian inner ear, and that cisplatin would impair this activity. To examine these dynamic processes, we live-imaged SC phagocytic activity in the adult mouse utricle and compared the SC responses with HC stress and death caused by aminoglycosides versus cisplatin.     

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.     

Cell cycle-dependent Cdc25C phosphatase determines cell survival by regulating apoptosis signal-regulating kinase 1

Cdc25C (cell division cycle 25C) phosphatase triggers entry into mitosis in the cell cycle by dephosphorylating cyclin B-Cdk1. Cdc25C exhibits basal phosphatase activity during interphase and then becomes activated at the G₂/M transition after hyperphosphorylation on multiple sites and dissociation from 14-3-3. Although the role of Cdc25C in mitosis has been extensively studied, its function in interphase remains elusive. Here, we show that during interphase Cdc25C suppresses apoptosis signal-regulating kinase 1 (ASK1), a member of mitogen-activated protein (MAP) kinase kinase kinase family that mediates apoptosis. Cdc25C phosphatase dephosphorylates phospho-Thr-838 in the activation loop of ASK1 in vitro and in interphase cells. In addition, knockdown of Cdc25C increases the activity of ASK1 and ASK1 downstream targets in interphase cells, and overexpression of Cdc25C inhibits ASK1-mediated apoptosis, suggesting that Cdc25C binds to and negatively regulates ASK1. Furthermore, we showed that ASK1 kinase activity correlated with Cdc25C activation during mitotic arrest and enhanced ASK1 activity in the presence of activated Cdc25C resulted from the weak association between ASK1 and Cdc25C. In cells synchronized in mitosis following nocodazole treatment, phosphorylation of Thr-838 in the activation loop of ASK1 increased. Compared with hypophosphorylated Cdc25C, which exhibited basal phosphatase activity in interphase, hyperphosphorylated Cdc25C exhibited enhanced phosphatase activity during mitotic arrest, but had significantly reduced affinity to ASK1, suggesting that enhanced ASK1 activity in mitosis was due to reduced binding of hyperphosphorylated Cdc25C to ASK1. These findings suggest that Cdc25C negatively regulates proapoptotic ASK1 in a cell cycle-dependent manner and may play a role in G₂/M checkpoint-mediated apoptosis.Cell division cycle 25 (Cdc25) phosphatases are dual-specificity phosphatases involved in cell cycle regulation. By removing inhibitory phosphate groups from phospho-Thr and phospho-Tyr residues of cyclin-dependent kinases (CDKs),¹ Cdc25 proteins regulate cell cycle progression in S phase and mitosis. In mammals, three isoforms of Cdc25 phosphatases have been reported: Cdc25A, which controls the G₁/S transition;^{2, 3} Cdc25B, which is a mitotic starter;⁴ and Cdc25C, which controls the G₂/M phase.⁵ Overexpression of Cdc25 phosphatases is frequently associated with various cancers.⁶ Upon exposure to DNA-damaging reagents like UV radiation or free oxygen radicals, Cdc25 phosphatases are key targets of the checkpoint machinery, resulting in cell cycle arrest and apoptosis. The 14-3-3 proteins bind to phosphorylated Ser-216 of Cdc25C and induce Cdc25C export from the nucleus during interphase in response to DNA damage,^{7, 8} but they have no apparent effect on Cdc25C phosphatase activity.^{9, 10} In addition, hyperphosphorylation of Cdc25C correlates to its enhanced phosphatase activity.¹¹ Most studies with Cdc25C have focused on its role in mitotic progression. However, the role of Cdc25C is not clear when it is sequestered in the cytoplasm by binding to 14-3-3.Apoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAPKKK5), is a ubiquitously expressed enzyme with a molecular weight of 170 kDa. The kinase activity of ASK1 is stimulated by various cellular stresses, such as H₂O₂,^{12, 13} tumor necrosis factor-α (TNF-α),¹⁴ Fas ligand,¹⁵ serum withdrawal,¹³ and ER stress.¹⁶ Stimulated ASK1 phosphorylates and activates downstream MAP kinase kinases (MKKs) involved in c-Jun N-terminal kinase (JNK) and p38 pathways.^{17, 18, 19} Phosphorylation and activation of ASK1 can induce apoptosis, differentiation, or other cellular responses, depending on the cell type. ASK1 is regulated either positively or negatively depending on its binding proteins.^{12, 13, 15, 18, 19, 20, 21, 22, 23, 24, 25}ASK1 is regulated by phosphorylation at several Ser/Thr/Tyr residues. Phosphorylation at Thr-838 leads to activation of ASK1, whereas phosphorylation at Ser-83, Ser-967, or Ser-1034 inactivates ASK1.^{24, 26, 27, 28} ASK1 is basally phosphorylated at Ser-967 by an unidentified kinase, and 14-3-3 binds to this site to inhibit ASK1.²⁴ Phosphorylation at Ser-83 is known to be catalyzed by Akt or PIM1.^{27, 29} Oligomerization-dependent autophosphorylation at Thr-838, which is located in the activation loop of the kinase domain, is essential for ASK1 activation.^{14, 18, 30} Phosphorylation at Tyr-718 by JAK2 induces ASK1 degradation.³¹ Several phosphatases that dephosphorylate some of these sites have been identified. Serine/threonine protein phosphatase type 5 (PP5) and PP2C dephosphorylate phosphorylated (p)-Thr-838,^{28, 32} whereas PP2A and SHP2 dephosphorylate p-Ser-967 and p-Tyr-718, respectively.^{31, 33} Little is known about the kinase or phosphatase that regulates phosphorylation at Ser-1034. Although ASK1 phosphorylation is known to be involved in the regulation of apoptosis, only a few reports show that ASK1 phosphorylation or activity is dependent on the cell cycle.^{21, 34}In this study, we examined the functional relationship between Cdc25C and ASK1 and identified a novel function of Cdc25C phosphatase that can dephosphorylate and inhibit ASK1 in interphase but not in mitosis. Furthermore, we demonstrated that Cdc25C phosphorylation status plays a critical role in the interaction with and the activity of ASK1. These results reveal a novel regulatory function of Cdc25C in the ASK1-mediated apoptosis signaling pathway.     

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.     

Platelet-activating factor induces cell cycle arrest and disrupts the DNA damage response in mast cells

Platelet-activating factor (PAF) is a potent phospholipid modulator of inflammation that has diverse physiological and pathological functions. Previously, we demonstrated that PAF has an essential role in ultraviolet (UV)-induced immunosuppression and reduces the repair of damaged DNA, suggesting that UV-induced PAF is contributing to skin cancer initiation by inducing immune suppression and also affecting a proper DNA damage response. The exact role of PAF in modulating cell proliferation, differentiation or transformation is unclear. Here, we investigated the mechanism(s) by which PAF affects the cell cycle and impairs early DNA damage response. PAF arrests proliferation in transformed and nontransformed human mast cells by reducing the expression of cyclin-B1 and promoting the expression of p21. PAF-treated cells show a dose-dependent cell cycle arrest mainly at G2–M, and a decrease in the DNA damage response elements MCPH1/BRIT-1 and ataxia telangiectasia and rad related (ATR). In addition, PAF disrupts the localization of p-ataxia telangiectasia mutated (p-ATM), and phosphorylated-ataxia telangiectasia and rad related (p-ATR) at the site of DNA damage. Whereas the potent effect on cell cycle arrest may imply a tumor suppressor activity for PAF, the impairment of proper DNA damage response might implicate PAF as a tumor promoter. The outcome of these diverse effects may be dependent on specific cues in the microenvironment.Ultraviolet (UV)-mediated immunosuppression poses a major risk for skin cancer induction,^{1, 2} and many have reported that an essential mediator in this process is UV-induced platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine).^{3, 4, 5} PAF is a phospholipid, first discovered as a secreted component by activated innate immune cells,^{6, 7} that mediates its activity by binding to a G-protein-coupled receptor.⁸ It is involved in a variety of mechanisms including the release of histamine in activated leukocytes,^{9, 10, 11} anaphylaxis, and phagocytosis.¹²Exposure to low doses of UV radiation activates PAF release by keratinocytes,^{13, 14} so it is likely that most of the population is regularly exposed to keratinocyte-derived PAF. In previous studies we showed that PAF upregulates both CXCR4 on mast cells and its ligand (CXCL12) on draining lymph node cells, promoting the migration of dermal mast cells from inflamed skin to the lymph nodes.¹⁵ Mast cells that reach the draining lymph nodes activate immune suppression by releasing interleukin 10.¹⁶ Blocking mast cell migration by using a CXCR4 antagonist, AMD3100, blocks UV-induced immune suppression and the induction of skin cancer.^{15, 17} No immune suppression is noted when PAF receptor-deficient mice (PAFR^-/-) are exposed to UV radiation,^{4, 5} nor can one reconstitute immune suppression when PAFR^-/- mast cells are used to reconstitute mast cell-deficient mice.¹⁸ PAF also has a critical role in skin cancer induction and progression,^{19, 20} and this may reflect its capacity to both induce immune suppression and hamper DNA repair.²¹Hanahan and Weinberg recognized the important roles inflammation and immune evasion play in the initiation of cancer.²² UV-induced PAF by activating immune suppression, retarding DNA repair and activating inflammation clearly constitutes an important hallmark for cancer induction. Supporting this idea is the observation that PAF is involved in a variety of other cancers besides skin cancer.^{23, 24, 25, 26, 27} Although we previously demonstrated that PAF suppresses the rate of DNA repair in vivo,²¹ little is known regarding the mechanisms involved. In this study we performed a series of experiments to determine how PAF affects DNA repair by examining important checkpoints that regulate DNA repair and cell cycle progression. We primarily used mast cells because of the critical role these cells have in UV-induced immune suppression and skin cancer induction,^{15, 28} and also because the dermis where they reside is targeted by UV-induced PAF.¹⁸     

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.     

Thrombospondin-1 signaling through CD47 inhibits cell cycle progression and induces senescence in endothelial cells

CD47 signaling in endothelial cells has been shown to suppress angiogenesis, but little is known about the link between CD47 and endothelial senescence. Herein, we demonstrate that the thrombospondin-1 (TSP1)-CD47 signaling pathway is a major mechanism for driving endothelial cell senescence. CD47 deficiency in endothelial cells significantly improved their angiogenic function and attenuated their replicative senescence. Lack of CD47 also suppresses activation of cell cycle inhibitors and upregulates the expression of cell cycle promoters, leading to increased cell cycle progression. Furthermore, TSP1 significantly accelerates replicative senescence and associated cell cycle arrest in a CD47-dependent manner. These findings demonstrate that TSP1-CD47 signaling is an important mechanism driving endothelial cell senescence. Thus, TSP1 and CD47 provide attractive molecular targets for treatment of aging-associated cardiovascular dysfunction and diseases involving endothelial dysregulation.Endothelial cell (EC) senescence is accompanied with vascular dysfunction, including arterial stiffening and remodeling,¹ impaired angiogenesis,^{2, 3} reduced endothelial repair capability and increased incidence of cardiovascular disease.^{4, 5, 6} Cellular senescence can occur in vivo or in vitro in response to various stressors,^{7, 8, 9, 10} leading to suppression of cell proliferation. EC senescence has been reported to contribute to the pathogenesis of age-associated vascular diseases, such as atherosclerosis.¹¹ Thus, further understanding the mechanisms of EC senescence may help to identify effective targets for antisenescence therapy and treatment aging-associated cardiovascular disorders.Previous studies have shown that the secreted matricellular protein thrombospondin-1 (TSP1) is as potent inhibitor of angiogenesis¹² and its antiangiogenic activity is mediated by its receptors, CD36^{13, 14} and CD47.^{15, 16} CD47 is a ubiquitously expressed transmembrane protein that serves as a ligand for signal regulatory protein-α and is a signaling receptor of TSP1. The TSP1-CD47 pathway has an important role in several fundamental cellular functions, including proliferation, apoptosis, inflammation and atherosclerotic response.¹⁷ Ligation of CD47 by TSP1 has been shown to inhibit nitric oxide (NO)/cGMP signaling in vascular cells, leading to suppression of angiogenic responses.¹⁶ Recently, it was reported that lack of CD47 expression in ECs may enable these cells to spontaneously gain characteristics of embryonic stem cells.¹⁸ However, the potential role of CD47 in regulation of EC senescence has not been well explored. The present study was initiated to determine the role and mechanisms of TSP1-CD47 signaling pathway in regulating cell cycle progression and replicative senescence of ECs.     

Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells

Billions of inflammatory leukocytes die and are phagocytically cleared each day. This regular renewal facilitates the normal termination of inflammatory responses, suppressing pro-inflammatory mediators and inducing their anti-inflammatory counterparts. Here we investigate the role of the receptor tyrosine kinase (RTK) Mer and its ligands Protein S and Gas6 in the initial recognition and capture of apoptotic cells (ACs) by macrophages. We demonstrate extremely rapid binding kinetics of both ligands to phosphatidylserine (PtdSer)-displaying ACs, and show that ACs can be co-opsonized with multiple PtdSer opsonins. We further show that macrophage phagocytosis of ACs opsonized with Mer ligands can occur independently of a requirement for αV integrins. Finally, we demonstrate a novel role for Mer in the tethering of ACs to the macrophage surface, and show that Mer-mediated tethering and subsequent AC engulfment can be distinguished by their requirement for Mer kinase activity. Our results identify Mer as a receptor uniquely capable of both tethering ACs to the macrophage surface and driving their subsequent internalization.Many diseases, including rheumatoid arthritis, pulmonary fibrosis, adult respiratory distress syndrome, and inflammatory bowel disease,^{1, 2, 3, 4} are commonly marked by impaired resolution of inflammation that is linked to defects in the phagocytic clearance of apoptotic cells.^{5, 6, 7} Apoptotic cell (AC) clearance normally eliminates a plethora of pro-inflammatory stimuli,^{8, 9} and the recognition of ACs by phagocytes¹⁰ limits progression to necrosis,¹¹ suppresses pro-inflammatory mediator production, and induces IL-10 and TGF-β release.^{12, 13} As defective clearance of ACs is associated with the development of inflammatory disease and autoimmunity,^{14, 15} new therapeutic approaches designed to increase the capacity of phagocytes to remove ACs could effectively promote the resolution of inflammation.Phagocytosis of ACs can be regulated by soluble mediators, including cytokines,^{16, 17} prostaglandins and lipoxins,^{17, 18, 19} serum proteins,²⁰ agonists of Liver X receptors (LXRs),^{17, 21} and glucocorticoids (GC).^{17, 22} In particular, LXR agonists and GCs promote phagocytosis of ACs predominantly via a Tyro3/Axl/Mer (TAM) receptor tyrosine kinase (RTK)-dependent pathway.^{17, 21, 23} There are two established ligands for the TAM RTKs, Protein S (gene name Pros1), which activates Tyro3 and Mer, and Gas6, which activates all three TAMs,^{24, 25} although other ligands have been suggested.^{26, 27} The amino terminal Gla domains of Protein S and Gas6 bind to phosphatidylserine (PtdSer) on the plasma membrane of ACs,²⁸ a potent ‘eat-me'' signal by which ACs are recognized by phagocytes.²⁹ TAM receptors bind to the carboxy terminal domains of Protein S and Gas6, which effectively act as molecular ‘bridges'' between PtdSer on the AC and TAM receptors on the phagocyte.^{17, 30, 31} TAM receptor- and ligand-deficient mice exhibit defective phagocytic pruning of photoreceptor outer segments by retinal pigment epithelial (RPE) cells of the eye,^{32, 33, 34} defective clearance of apoptotic germ cells by Sertoli cells of the testis,³⁵ and defective clearance of ACs by macrophages/dendritic cells in lymphoid organs.³⁶ These phenotypes are also detectable in Mer (gene name Mertk) single knockouts.³⁷ In addition to phagocytic clearance, TAM signaling also has a pivotal role in controlling the innate immune response to pathogenic stimuli.^{13, 17, 38}Although the importance of Mer in the internalization of ACs by macrophages is now well-established, this receptor has been thought not to have a significant role in the initial ‘tethering'' of ACs to the macrophage surface.^{36, 39} In their studies, Scott et al.³⁶ used peritoneal macrophages for which tethering of ACs has now been shown to be mediated by T-cell immunoglobulin and mucin domain-containing molecule 4 (TIM4).³⁹ Subsequent internalization of tethered ACs is then mediated by either integrin αvβ3- or Mer-mediated signaling.^{39, 40} Similarly, for RPE cells, the initial capture of photoreceptor outer segments by RPE cells required the integrin αvβ5,⁴¹ with Mer-dependent signaling necessary for subsequent internalization. To further probe the mechanistic role of Mer in AC recognition and engulfment, we have now examined macrophages that predominantly use a Mer-dependent AC phagocytosis mechanism.^{17, 23} We show that in these cells, which do not express TIM4, Mer has the capacity to serve a unique dual role in mediating both tethering of ACs to the macrophage surface as well as subsequent AC engulfment.     

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.