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.     

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.     

Effects of Route of Inoculation and Viral Genetic Variation on Antibody Responses to Polyomavirus SV40 in Syrian Golden Hamsters

Genetic variants of polyomavirus SV40 are powerful agents with which to define viral effects on cells and carcinogenesis pathways. We hypothesized that differences in biologic variation among viral strains affect the process of viral infection and are reflected in antibody responses to the viral nonstructural large T-antigen (TAg) protein but not in neutralizing antibody responses against the inoculated viral particles. We analyzed the production of TAg antibody and neutralizing antibody in Syrian golden hamsters that were inoculated with SV40 viral strains by intracardiac, intravenous, or intraperitoneal routes and remained tumor free. Compared with the intraperitoneal route, intravascular (that is, intravenous, intracardiac) inoculation resulted in increased frequency of responsiveness to TAg but not in higher TAg antibody titers. The intravascular route was superior both for eliciting neutralizing antibody responses and for higher titers of those responses. Viruses with complex regulatory regions induced TAg antibody more often than did viruses with simple regulatory regions after intraperitoneal but not intravascular injections, with no differences in antibody titers. This viral genetic variation had no effect on neutralizing antibody production after intraperitoneal or intravascular inoculations or on neutralizing antibody titers achieved. These findings confirm that SV40 variants differ in their biologic properties. Route of inoculation combined with viral genetic variation significantly influence the development of serum antibodies to SV40 TAg in tumor-free hamsters. Route of inoculation—but not viral genetic variation—is an important factor in production of neutralizing antibody to SV40.Abbreviations: TAg, large T antigenPolyomavirus SV40 was discovered as an unrecognized contaminant of early poliovaccines³⁸ and was shown promptly to be an oncogenic virus.^9,15,17,18 Syrian golden hamsters are the small animal model that is susceptible to SV40 tumorigenicity.^{7,9-13,18,27,35} Since its discovery, SV40 has proven to be an outstanding tool for the discovery of mechanisms underlying carcinogenesis and for viral influences on cellular processes.^{1,3,5,19,23,30}Genetic variants of SV40 exist.^{16,20,26,28,33,34,36,37,41} This variation typically occurs in the viral regulatory region, in which some strains have duplications or rearrangements (or both) in the enhancer region, and in the C-terminal region of the large T-antigen (TAg) gene, in which nucleic acid variations may result in amino acid changes in the protein. TAg is an essential viral replication protein and the major viral oncoprotein. An important question is whether SV40 viral variants differ in their biologic properties, including in host responses to infections, as this could affect the spectrum of viral disease pathogenesis. We previously have shown that the viral regulatory region influences SV40 tumor induction in hamsters^35,40 and vertical transmission of the virus in hamsters²⁹ and that the route of inoculation influences SV40-mediated carcinogenesis.²⁷ Because TAg is not a component of the virus particle but instead is synthesized in virus-infected cells, we hypothesized that differences in antibody responses to TAg reflect biologic variation among viral strains with respect to the process of viral infection. In contrast, we expected that neutralizing antibody responses arise primarily against the injected viral particles, which represent a single serotype, and therefore are less informative about viral variation. We report here that an analysis revealed that the route of inoculation—in combination with viral genetic variation—significantly influences the development of serum antibodies to SV40 large TAg but not the titer of those antibodies in virus-exposed, tumor-free hamsters. The route of inoculation—but not viral genetic variation—influenced both the frequency of development and the titers of virus-neutralizing antibodies in the same animals.     

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.     

Distinct roles of RIP1–RIP3 hetero- and RIP3–RIP3 homo-interaction in mediating necroptosis

Necroptosis is mediated by a signaling complex called necrosome, containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL). It is known that RIP1 and RIP3 form heterodimeric filamentous scaffold in necrosomes through their RIP homotypic interaction motif (RHIM) domain-mediated oligomerization, but the signaling events based on this scaffold has not been fully addressed. By using inducible dimer systems we found that RIP1–RIP1 interaction is dispensable for necroptosis; RIP1–RIP3 interaction is required for necroptosis signaling, but there is no necroptosis if no additional RIP3 protein is recruited to the RIP1–RIP3 heterodimer, and the interaction with RIP1 promotes the RIP3 to recruit other RIP3; RIP3–RIP3 interaction is required for necroptosis and RIP3–RIP3 dimerization is sufficient to induce necroptosis; and RIP3 dimer-induced necroptosis requires MLKL. We further show that RIP3 oligomer is not more potent than RIP3 dimer in triggering necroptosis, suggesting that RIP3 homo-interaction in the complex, rather than whether RIP3 has formed homo polymer, is important for necroptosis. RIP3 dimerization leads to RIP3 intramolecule autophosphorylation, which is required for the recruitment of MLKL. Interestingly, phosphorylation of one of RIP3 in the dimer is sufficient to induce necroptosis. As RIP1–RIP3 heterodimer itself cannot induce necroptosis, the RIP1–RIP3 heterodimeric amyloid fibril is unlikely to directly propagate necroptosis. We propose that the signaling events after the RIP1–RIP3 amyloid complex assembly are the recruitment of free RIP3 by the RIP3 in the amyloid scaffold followed by autophosphorylation of RIP3 and subsequent recruitment of MLKL by RIP3 to execute necroptosis.Necroptosis is a type of programmed necrosis characterized by necrotic morphological changes, including cellular organelle swelling, cell membrane rupture,^{1, 2, 3} and dependence of receptor-interacting protein (RIP)1⁴ and RIP3.^{5, 6, 7} Physiological function of necroptosis has been illustrated in host defense,^{8, 9, 10, 11} inflammation,^{12, 13, 14, 15, 16} tissue injury,^{10, 17, 18} and development.^{19, 20, 21}Necroptosis can be induced by a number of different extracellular stimuli such as tumor necrosis factor (TNF). TNF stimulation leads to formation of TNF receptor 1 (TNFR1) signaling complex (named complex I), and complex II containing RIP1, TRADD, FAS-associated protein with a death domain (FADD), and caspase-8, of which the activation initiates apoptosis. If cells have high level of RIP3, RIP1 recruits RIP3 to form necrosome containing FADD,^{22, 23, 24} caspase-8, RIP1, and RIP3, and the cells undergo necroptosis.^{25, 26} Caspase-8 and FADD negatively regulates necroptosis,^{27, 28, 29, 30} because RIP1, RIP3, and CYLD are potential substrates of caspase-8.^{31, 32, 33, 34} Necrosome also suppresses apoptosis but the underlying mechanism has not been described yet. Mixed-lineage kinase domain-like (MLKL) is downstream of RIP3,^{35, 36} and phosphorylation of MLKL is required for necroptosis.^{37, 38, 39, 40, 41, 42}Apoptosis inducing complex (complex II) and necrosome are both supramolecular complexes.^{43, 44, 45} A recent study showed that RIP1 and RIP3 form amyloidal fibrils through their RIP homotypic interaction motif⁴⁶ (RHIM)-mediated polymerization, and suggested that amyloidal structure is essential for necroptosis signaling.⁴⁷ The RIP1–RIP3 heterodimeric amyloid complex is believed to function as a scaffold that brings signaling proteins into proximity to permit their activation. However, RIP1 and RIP3 also can each form fibrils on their own RHIM domains in vitro. It is unclear how the homo- and hetero-interactions are coordinated and organized on the amyloid scaffold to execute their functions in necroptosis. Here, we used inducible dimerization systems to study the roles of RIP1–RIP1, RIP1–RIP3, and RIP3–RIP3 interactions in necroptosis signaling. Our data suggested that it is the RIP1–RIP3 interaction in the RIP1–RIP3 heterodimeric amyloid complex that empowers to recruit other free RIP3; homodimerization of RIP3 triggers its autophosphorylation and only the phosphorylated RIP3 can recruit MLKL to execute necroptosis.     

Epigallocatechin-3-gallate opposes HBV-induced incomplete autophagy by enhancing lysosomal acidification,which is unfavorable for HBV replication

Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, exhibits diverse beneficial properties, including antiviral activity. Autophagy is a cellular process that is involved in the degradation of long-lived proteins and damaged organelles. Recent evidence indicates that modulation of autophagy is a potential therapeutic strategy for various viral diseases. In the present study, we investigated the effect of EGCG on hepatitis B virus (HBV) replication and the possible involvement of autophagy in this process. Our results showed that HBV induced autophagosome formation, which was required for replication of itself. However, although EGCG efficiently inhibited HBV replication, it enhanced, but not inhibited, autophagosome formation in hepatoma cells. Further study showed that HBV induced an incomplete autophagy, while EGCG, similar to starvation, was able to induce a complete autophagic process, which appeared to be unfavorable for HBV replication. Furthermore, it was found that HBV induced an incomplete autophagy by impairing lysosomal acidification, while it lost this ability in the presence of EGCG. Taken together, these data demonstrated that EGCG treatment opposed HBV-induced incomplete autophagy via enhancing lysosomal acidification, which was unfavorable for HBV replication.Macroautophagy (hereafter autophagy) is a conserved cellular process through which cytoplasmic materials are sequestered into double-membrane vacuole called autophagosomes and destined for degradation through fusion with lysosomes.^{1, 2, 3} Accumulating evidence indicates that autophagy is involved in diverse pathophysiological processes, including cancer, neurodegenerative disorders, and cardiovascular diseases.^{4, 5, 6, 7} Recent studies show that autophagy has an important role in regulating the replication of many viruses, including dengue virus, coxsackievirus B3 virus (CVB3), hepatitis C virus (HCV), and influenza virus A.^{8, 9, 10, 11, 12} Several investigations also indicate that autophagy has an important role in hepatitis B virus (HBV) replication: autophagy is induced by HBV and is required for HBV replication; however, the underlying mechanisms remains still unclear.^{13, 14, 15, 16}Green tea is the most commonly consumed beverage worldwide. In traditional Chinese medicine, green tea is considered to have beneficial properties for human health, including antitumorigenic, antioxidant, and anti-inflammatory activities.^{17, 18, 19} Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea and appears to be the primary active ingredient accounting for the latter''s biological effects. In recent years, EGCG is revealed to display inhibitory effect on diverse viruses, such as human immunodeficiency virus type-1, Epstein–Barr virus (EBV), and HCV.^{20, 21, 22, 23, 24, 25} Of interest, EGCG is also found to regulate autophagy formation, although it seems to be cell-type specific.^{26, 27, 28, 29, 30} Given the potential therapeutic effect of EGCG on viral infection and its role in autophagy regulation, we investigated the effect of EGCG on HBV replication and the possible involvement of autophagy in this process.Here we showed that HBV induced an incomplete autophagy that was required for HBV replication; however, a complete autophagic process induced by EGCG appeared to be unfavorable for HBV replication. Further study showed that HBV hampered the autophagic flux by impairing lysosomal acidification, which could be opposed by the treatment of EGCG.     

Clinicopathologic Characteristics,Prevalence, and Risk Factors of Spontaneous Diabetes in Sooty Mangabeys (Cercocebus atys)

In 2008, clinical observations in our colony of sooty mangabeys (Cercocebus atys) suggested a high frequency of type 2 diabetes. Postmortem studies of diabetic animals revealed dense amyloid deposits in pancreatic islets. To investigate these findings, we screened our colony (97 male mangabeys; 99 female mangabeys) for the disease from 2008 to 2012. The overall prevalence of diabetes was 11% and of prediabetes was 7%, which is nearly double that reported for other primate species (less than 6%). Fructosamine and triglyceride levels were the best indicators of diabetes; total cholesterol and glycated hemoglobin were not associated with disease. Increasing age was a significant risk factor: prevalence increased from 0% in infants, juveniles, and young adults to 11% in adults and 19% in geriatric mangabeys. Sex, medroxyprogesterone acetate exposure, and SIV status were unrelated to disease. Weight was marginally higher in prediabetics, but body condition did not indicate obesity. Of the 49 mangabeys that were necropsied after clinical euthanasia or death from natural causes, 22 were diabetic; all 22 animals demonstrated pancreatic amyloid, and most had more than 75% of islets replaced with amyloid. We conclude that type 2 diabetes is more common in mangabeys than in other primate species. Diabetes in mangabeys has some unusual pathologic characteristics, including the absence of altered cholesterol levels and glycated hemoglobin but a robust association of pancreatic insular amyloidosis with clinical diabetes. Future research will examine the genetic basis of mangabey diabetes and evaluate additional diagnostic tools using imaging and serum markers.Abbreviations: HbA1c, glycated hemoglobin; MPA, medroxyprogesterone acetate; YNPRC, Yerkes National Primate Research CenterSooty mangabeys (Cercocebus atys) are Old World NHP that are native to West Africa. Historically their use in research has been limited to infectious disease studies, leprosy studies, and behavioral research.^14,25 Over the past 20 to 30 y, they have been used in HIV–AIDS research. Mangabeys are natural hosts of SIV_smm, which is recognized as the origin of HIV2 infection in humans.^7,8,30,36,42 SIV typically is nonpathogenic in mangabeys despite high levels of virus replication, which makes this species a unique and invaluable model in AIDS research.^7,30,36,42 Our facility maintains a colony of approximately 200 sooty mangabeys. In 2008 clinical observations of relative hyperglycemia, glucosuria, and weight loss in our colony suggested that type 2 diabetes mellitus occurred at a relatively high frequency in this population. Spontaneous diabetes was found in 10% of the colony, and 5% of animals were prediabetic; this incidence is higher than that typically reported for other NHP species, such as cynomolgus macaques (less than 1% to 2%)²² and chimpanzees (less than 1%).³⁷ The prevalence of spontaneous diabetes in humans is typically 8.3%.^2,6,22,37 In addition, necropsies revealed that many affected animals had dense amyloid deposits in pancreatic islet cells. Insular amyloidosis was seen on histology, with a total replacement of islets by amyloid deposition in advanced diabetes. Advanced diabetes was determined by increased weight loss and severity of relative hyperglycemia. The increased clinical prevalence of diabetes in our mangabey colony prompted additional characterization of the clinicopathologic profile, risk factors, and prevalence of diabetes in our mangabey colony.The form of diabetes in this mangabey colony is characterized as type 2 diabetes mellitus, as they have hyperglycemia, hypertriglyceridemia, and islet amyloidosis. Type 2 diabetes mellitus is the most common of the 3 forms of diabetes, and has been documented in humans and NHP,^22,31,37,55 including rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis), Celebes crested macaques (Macaca nigra), bonnet macaques (Macaca radiate), pigtailed macaques (Macaca nemestrina), vervet monkeys (Chlorocebus pygerythrus), squirrel monkeys (Saimiri sciureus), chimpanzees (Pan troglodytes), and woolly monkeys (Lagothrix spp.).^{1,24,31,52,55} Type 2 diabetes is a chronic metabolic disorder in which insulin resistance occurs in liver, muscle, and adipose tissue. As type 2 diabetes progresses, it also can be characterized as a relative insulin deficiency.^{1,6,15,22,29,31,37,55} The initial clinical presentation of diabetes in humans and NHP includes polydipsia, polyuria, polyphagia, weight loss, and lethargy.^{1,6,22,27,31,37,55} Similar presentation was observed in our colony of diabetic mangabeys.Diagnostic criteria of diabetes in NHP species is similar to that for humans and is based on clinical symptoms and routine lab tests, including serum chemistry panel to evaluate persistent fasting hyperglycemia, hypertriglyceridemia, and hypercholesterolemia.^{2,6,11,16-18,21,22,29,31,37,48-50,52,55} Hypertriglyceridemia and hypercholesterolemia frequently are elevated due to diabetes and therefore are used as supportive diagnostic markers. In addition, the disease is characterized by transient hyperinsulinemia followed by insulin deficiency subsequent to glucose challenge. Urinalysis is used to evaluate glucosuria and ketonuria. These tests are not exclusive for diagnosing diabetes and can be inconsistent between species, thus making conclusive diagnosis challenging. For example, hyperglycemia can be a transient finding associated with recent food intake or stress associated with restraint for blood sample collection or anesthetic access, whereas hypertriglyceridemia can be seen in obese animals and those with other metabolic diseases such as pancreatitis and hypothyroidism.^1,22,37,55The typical clinical approach to the diagnosis of diabetes in NHP and other veterinary patients includes evaluation of fructosamine and glycated hemoglobin (HbA1c) levels and glucose tolerance testing. These tests are indices of glycemic control and are used in clinical settings primarily to assess prognosis and response to treatment; they are also useful for the initial diagnosis of diabetes when used in parallel with serum chemistry markers. Fructosamine and HbA1c can both provide information on long-term glycemic control, because fructosamine reflects average blood glucose levels over 2 to 3 wk whereas HbA1c reflects average blood glucose over 2 to 3 mo preceding blood collection. HbA1c is the primary test for diabetes in human medicine,^6,31,35,37 whereas fructosamine is commonly used in veterinary medicine. Glucose tolerance testing provides an indirect measure of insulin sensitivity, but it is not frequently used clinically in NHP because of the requirement for prolonged physical restraint or sedation.^{1,21,22,26,27,34,37,55}Prevention and management of diabetes in NHP and humans can be achieved by identifying potential risk factors, including age, weight, sex, genetics, hormone drug exposure, and viral status.^{1,6,15,22,29,31,37,42,55} Advanced age, obesity, sex, and genetics are associated with diabetes in some species of NHP and humans.^{1,6,15,22,29,31,37,55} In addition, exposure to drugs such as medroxyprogesterone acetate (MPA) is suspected to be linked to diabetes due to the hormonal effects of progesterone impacting glucoregulatory function.^{1,6,10,22,23,31,34,55} MPA exposure is of interest, because it is used regularly in our mangabey colony as both a contraceptive and as therapy for endometriosis. In addition, SIV status is being evaluated as a risk factor, because a portion of our colony is SIV positive. Although HIV is not thought to be associated with diabetes in people, SIV pathogenesis in mangabeys differs; therefore it was of interest to explore the possible association of SIV and diabetes in mangabeys.^7,30,36,42 Pancreatic insular amyloidosis has been documented to be associated with type 2 diabetes in several species. Amyloidosis is a group of disorders that are caused by extracellular deposition of misfolded proteins that can result in impaired function of any organ.^{15,20,23,28,32,43,45,48,49} Because a high incidence of pancreatic insular amyloid was noted at necropsy, we sought to document the relationship with clinical diabetes in mangabeys.Spontaneous type 2 diabetes mellitus has been well documented in several species of NHP. Because the literature contains little information regarding the clinicopathologic features (the ‘profile’), risk factors, and prevalence of spontaneous diabetes mellitus in sooty mangabeys, the primary aims of the current study were 1) to determine whether elevated levels of fasting blood glucose, fructosamine, HbA1c, triglycerides, and total cholesterol levels are reliable diagnostic markers of type 2 diabetes mellitus in this NHP species; 2) to determine whether age, sex, MPA exposure, and SIV status influence the risk of diabetes; 3) to determine whether body weight influences diabetic status; 4) to evaluate the relationship between pancreatic amyloidosis and diabetes mellitus; and 5) to characterize the prevalence of diabetes mellitus in the mangabey population at our institution. To our knowledge, this report is the first to describe the natural occurrence of type 2 diabetes mellitus within a captive colony of sooty mangabeys. We hypothesized that blood glucose, fructosamine, HbA1c, triglyceride, and total cholesterol would be reliable diagnostic markers and that age, sex, and MPA exposure would influence the risk of diabetes in this species.     

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.     

A Host RNA Helicase-Like Protein,AtRH8, Interacts with the Potyviral Genome-Linked Protein,VPg, Associates with the Virus Accumulation Complex,and Is Essential for Infection

The viral genome-linked protein, VPg, of potyviruses is a multifunctional protein involved in viral genome translation and replication. Previous studies have shown that both eukaryotic translation initiation factor 4E (eIF4E) and eIF4G or their respective isoforms from the eIF4F complex, which modulates the initiation of protein translation, selectively interact with VPg and are required for potyvirus infection. Here, we report the identification of two DEAD-box RNA helicase-like proteins, PpDDXL and AtRH8 from peach (Prunus persica) and Arabidopsis (Arabidopsis thaliana), respectively, both interacting with VPg. We show that AtRH8 is dispensable for plant growth and development but necessary for potyvirus infection. In potyvirus-infected Nicotiana benthamiana leaf tissues, AtRH8 colocalizes with the chloroplast-bound virus accumulation vesicles, suggesting a possible role of AtRH8 in viral genome translation and replication. Deletion analyses of AtRH8 have identified the VPg-binding region. Comparison of this region and the corresponding region of PpDDXL suggests that they are highly conserved and share the same secondary structure. Moreover, overexpression of the VPg-binding region from either AtRH8 or PpDDXL suppresses potyvirus accumulation in infected N. benthamiana leaf tissues. Taken together, these data demonstrate that AtRH8, interacting with VPg, is a host factor required for the potyvirus infection process and that both AtRH8 and PpDDXL may be manipulated for the development of genetic resistance against potyvirus infections.Plant viruses are obligate intracellular parasites that infect many agriculturally important crops and cause severe losses each year. One of the common characteristics of plant viruses is their relatively small genome that encodes a limited number of viral proteins, making them dependent on host factors to fulfill their infection cycles (Maule et al., 2002; Whitham and Wang, 2004; Nelson and Citovsky, 2005; Decroocq et al., 2006). In order to establish a successful infection, the invading virus must recruit an array of host proteins (host factors) to translate and replicate its genome and to move locally from cell to cell via the plasmodesmata and systemically via the vascular system. It has been suggested that down-regulation or mutation of some of the required host factors may result in recessively inherited resistance to viruses (Kang et al., 2005b).Potyviruses, belonging to the genus Potyvirus in the family Potyviradae, constitute the largest group of plant viruses (Rajamäki et al., 2004). Potyviruses have a single positive-strand RNA genome approximately 10 kb in length, with a viral genome-linked protein (VPg) covalently attached to the 5′ end and a poly(A) tail at the 3′ end (Urcuqui-Inchima et al., 2001; Rajamäki et al., 2004). The viral genome contains a single open reading frame (ORF) that translates into a polypeptide with a molecular mass of approximately 350 kD, which is cleaved into 10 mature proteins by viral proteases (Urcuqui-Inchima et al., 2001). Recently, a novel viral protein resulting from a frameshift in the P3 cistron has been reported (Chung et al., 2008). Of the 11 viral proteins, VPg is a multifunctional protein and the only other viral protein present in the viral particles (virions) besides the coat protein and the cylindrical inclusion protein (CI; Oruetxebarria et al., 2001; Puustinen et al., 2002; Gabrenaite-Verkhovskaya et al., 2008). The nonstructural protein is linked to the viral RNA by a phosphodiester bond between the 5′ terminal uridine residue of the RNA and the O⁴-hydroxyl group of amino acid Tyr (Murphy et al., 1996; Oruetxebarria et al., 2001; Puustinen et al., 2002). Mutation of the Tyr residue that links VPg to the viral RNA abolishes virus infectivity completely (Murphy et al., 1996). In infected cells, VPg and its precursor NIa are present in the nucleus and in the membrane-associated virus replication vesicles in the cytoplasm (Carrington et al., 1993; Rajamäki and Valkonen, 2003; Cotton et al., 2009). As a component of the replication complex, VPg may serve as a primer for viral RNA replication (Puustinen and Mäkinen, 2004) and as an analog of the m⁷G cap of mRNAs for the viral genome to recruit the translation complex for translation (Michon et al., 2006; Beauchemin et al., 2007; Khan et al., 2008). Furthermore, VPg has been suggested to be an avirulence factor for recessive resistance genes in diverse plant species (Moury et al., 2004; Kang et al., 2005b; Bruun-Rasmussen et al., 2007). Thus, VPg plays a pivotal role in the virus infection process. The molecular identification of VPg-interacting host proteins and the subsequent functional characterization of such interactions may advance knowledge of the intricate virus replication mechanisms and help develop novel antiviral strategies.Previous studies have shown that VPg and its precursor NIa interact with several host proteins, including three essential components of the host protein translation apparatus (Thivierge et al., 2008). The first protein is the cellular translation initiation factor eIF4E or its isoform eIF(iso)4E, identified through a yeast two-hybrid screen using VPg as a bait (Wittmann et al., 1997; Schaad et al., 2000). The protein complex of VPg and eIF4E is an essential component for virus infectivity (Robaglia and Caranta, 2006). Mutations and knockout of eIF4E or eIF(iso)4E confer resistance to infection (Lellis et al., 2002; Ruffel et al., 2002; Nicaise et al., 2003; Gao et al., 2004; Kang et al., 2005a; Ruffel et al., 2005; Decroocq et al., 2006; Bruun-Rasmussen et al., 2007). It is well known that potyviruses recruit selectively one of the eIF4E isoforms, depending on specific virus-host combinations (German-Retana et al., 2008). For instance, in Arabidopsis (Arabidopsis thaliana), eIF(iso)4E is required for infection by Turnip mosaic virus (TuMV), Plum pox virus (PPV), and Lettuce mosaic virus, while eIF4E is indispensable for infection by Clover yellow vein virus (Duprat et al., 2002; Lellis et al., 2002; Sato et al., 2005; Decroocq et al., 2006). The second cellular protein interacting with VPg is another translation initiation factor, eIF4G. Analysis of Arabidopsis knockout mutants for eIF4G or its isomers eIF(iso)4G1 and eIF(iso)4G2 has yielded results supporting the idea that the recruitment of eIF4G for potyvirus infection is also isoform dependent (Nicaise et al., 2007). Recently, poly(A)-binding protein (PABP), the translation initiation factor that bridges the 5′ and 3′ termini of the mRNA into proximity, has been proposed to be essential for efficient multiplication of TuMV (Dufresne et al., 2008). PABP was previously documented to interact with NIa, a VPg precursor containing both VPg and the proteinase NIa-Pro (Léonard et al., 2004). As the translation factors eIF(iso)4E and PABP have been found to be internalized in virus-induced vesicles, it has been suggested that the interactions between VPg and these translation factors are crucial for viral RNA translation and/or replication (Beauchemin and Laliberté, 2007; Beauchemin et al., 2007; Cotton et al., 2009). Besides these three translation factors, a Cys-rich plant protein, potyvirus VPg-interaction protein, was also found to associate with VPg (Dunoyer et al., 2004). This plant-specific VPg-interacting host protein contains a PHD finger domain and acts as an ancillary factor to support potyvirus infection and movement (Dunoyer et al., 2004).In this study, we describe the identification of an Arabidopsis DEAD-box RNA helicase (DDX), AtRH8, and a peach (Prunus persica) DDX-like protein, PpDDXL, both interacting with the potyviral VPg protein. Using the atrh8 mutant, we demonstrate that AtRH8 is not required for plant growth and development in Arabidopsis but is necessary for infection by two plant potyviruses, PPV and TuMV. Furthermore, we present evidence that AtRH8 colocalizes with the virus accumulation complex in potyvirus-infected leaf tissues, which reveals a possible role of AtRH8 in virus infection. Finally, we have identified the VPg-binding region (VPg-BR) of AtRH8 and PpDDX and show that overexpression of the VPg-BR either from AtRH8 or PpDDXL suppresses virus accumulation.     

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.     

Neuritin 1 promotes retinal ganglion cell survival and axonal regeneration following optic nerve crush

Neuritin 1 (Nrn1) is an extracellular glycophosphatidylinositol-linked protein that stimulates axonal plasticity, dendritic arborization and synapse maturation in the central nervous system (CNS). The purpose of this study was to evaluate the neuroprotective and axogenic properties of Nrn1 on axotomized retinal ganglion cells (RGCs) in vitro and on the in vivo optic nerve crush (ONC) mouse model. Axotomized cultured RGCs treated with recombinant hNRN1 significantly increased survival of RGCs by 21% (n=6–7, P<0.01) and neurite outgrowth in RGCs by 141% compared to controls (n=15, P<0.05). RGC transduction with AAV2-CAG–hNRN1 prior to ONC promoted RGC survival (450%, n=3–7, P<0.05) and significantly preserved RGC function by 70% until 28 days post crush (dpc) (n=6, P<0.05) compared with the control AAV2-CAG–green fluorescent protein transduction group. Significantly elevated levels of RGC marker, RNA binding protein with multiple splicing (Rbpms; 73%, n=5–8, P<0.001) and growth cone marker, growth-associated protein 43 (Gap43; 36%, n=3, P<0.01) were observed 28 dpc in the retinas of the treatment group compared with the control group. Significant increase in Gap43 (100%, n=5–6, P<0.05) expression was observed within the optic nerves of the AAV2–hNRN1 group compared to controls. In conclusion, Nrn1 exhibited neuroprotective, regenerative effects and preserved RGC function on axotomized RGCs in vitro and after axonal injury in vivo. Nrn1 is a potential therapeutic target for CNS neurodegenerative diseases.Central nervous system (CNS) trauma and neurodegenerative disorders trigger a cascade of intrinsic and extrinsic cellular events resulting in regenerative failure and subsequent damage to neurons.^{1, 2, 3, 4, 5} The intrinsic factors include deregulation in growth-promoting factors, apoptotic factors, intracellular signaling molecules and trophic factors.⁶ Similarly, the extrinsic factors correlate to growth inhibition due to inhibitory cues^{3, 7, 8, 9, 10, 11, 12, 13} that include myelin and myelin associated inhibitors, glial scarring,^{5, 14} slow clearance of axonal debris,⁷ incorrect development of neuronal projections⁶ and CNS inflammation.^{15, 16} Progressive degeneration of mature retinal ganglion cells (RGCs) has been associated with loss of trophic support,^{8, 9} detrimental inflammatory processes/immune regulation^{10, 11} and apoptotic effectors.^{9, 12, 13, 15, 17}After injury, mammalian RGC axons show only a short-lived sprouting response but no long-distance regeneration through the optic nerve (ON).¹⁶ Glial responses around the affected area are initiated by injured CNS axons.¹⁸ Axons undergoing Wallerian degeneration are surrounded by astrocytes that upregulate glial fibrillary acidic protein (Gfap) expression and these reactive astrocytes contribute to trauma-induced neurodegeneration.¹⁹ Glial scarring inhibits axonal transport after ON crush (ONC)^{5, 14} decreasing transport of proteins involved in neuroprotection and synaptic plasticity. Regenerative failure is a critical endpoint of these destructive triggers culminating in neuronal apoptosis^{3, 20, 21} and inhibition of functional recovery. Intrinsic factors affecting axonal regeneration after CNS injury are crucial for recovery and thus, dysregulation of genes involved in axonal plasticity and outgrowth can prove detrimental to the neuronal recovery.^{22, 23, 24}Current neuroprotection approaches include promoting survival of RGCs by intraocular injections of recombinant factors like ciliary neurotrophic factor (CNTF) and peripheral nerve (PN) transplantations in vitro²⁵ and in vivo after injury.²⁶ Studies performed with glial cell-line-derived neurotrophic factor and neurturin protect RGCs from axotomy-induced apoptosis.²⁷ Further, in the ON injury model, RGC survival was promoted after deletion of CCAAT/enhancer binding protein homologous protein²⁸ and enhanced regeneration observed with co-deletion of kruppel-like factor 4 (Klf4) and suppressor of cytokine signaling 3 (Socs3).²⁹ Intraocular administration of neurotrophin-4 (NT-4) and brain-derived neurotrophic factor (BDNF) after ON transection has also exerted neuroprotective effects on axotomized RGCs. In addition, PNs transplanted adjacent to ONs, ex vivo PN grafts with lenti-viral transduced Schwann cells, and stimulation of inflammatory processes have strong pro-regenerative effects on injured RGCs.^{26, 30, 31, 32, 33}In addition, using adeno-associated-virus (AAV) therapy, AAV mediated expression of CNTF in bcl2 overexpressing transgenic mice increases cell viability and axonal regeneration,³⁴ whereas BDNF promotes survival of RGCs.³⁵ Likewise, experiments with AAV–BDNF, –CNTF and –growth-associated protein 43 (GAP43) have shown that AAV–CNTF was the most crucial for promoting both long-term survival and regeneration.³⁶ The positive effects of CNTF are observed mainly through simultaneous deletion of both PTEN and SOCS3³⁷ and the concurrent activation of mTOR and STAT3 pathways.³⁸ Although CNTF shows robust increase and sustained axon regeneration in injured ONs of rodents, it causes axonal misguidance and aberrant growth.³⁹ Furthermore, it has been shown that CNTF acts as a chemoattractant. CNTF administration onto autologous PN grafts transplanted within transected ON increased regeneration, but these effects were significantly reduced after removal of macrophages from this site.⁴⁰ In addition, the effects of CNTF using PN grafts at ON transection sites are further subject to debate, as previously it has been shown that Ad-CNTF injections preserved RGC axons but did not induce regeneration of axotomized RGCs.⁴¹ Thus, other studies have addressed RGC survivability and axonal regeneration with CNTF and other growth factors,^{35, 36} but most trophic factors affect neuronal survival and regeneration differentially.Previous studies targeting neuronal apoptosis by overexpressing intrinsic growth factors, inhibiting apoptosis and enhancing regeneration in CNS trauma models have established that a multifactorial approach is required for successful and long-lasting therapeutic outcomes.^{6, 36} Current gaps still exist for a key gene that could effectively target neuroprotection, enhance neuron regeneration and sustain neuronal function.One key gene implicated in neuronal plasticity is Neuritin 1 (Nrn1), also known as candidate plasticity gene 15. It has multiple functions and was first identified and characterized when screening for candidate plasticity genes in the rat hippocampal dentate gyrus activated by kainate.^{42, 43, 44} Nrn1 is highly conserved across species⁴⁵ and translates to an extracellular, glycophosphatidylinositol-linked protein (GPI-linked protein), which can be secreted as a soluble form. Nrn1 stimulates axonal plasticity, dendritic arborization and synapse maturation in the CNS.⁴⁶ During early embryonic development, Nrn1 promotes the survival of neural progenitors and differentiated neurons,⁴⁷ while later in development it promotes axonal and dendritic growth and stabilization, allowing maturation and formation of synapses.^{43, 46, 48} In the adult brain, Nrn1 has been correlated with activity-dependent functional plasticity^{45, 49} and is expressed in post mitotic neurons.Nrn1 may be a crucial gene for neuroprotection and regeneration because growth factors such as nerve growth factor (NGF), BDNF and NT-3 as well as neuronal activity can potentiate the expression of Nrn1.^{44, 50} In addition, we reported that Nrn1 mRNA expression appears to be biphasic after ON axonal trauma, indicating a transient attempt by RGCs at neuroprotection/neuroregeneration in response to ONC injury.⁵¹ The dynamic regulation of Nrn1 coupled with neurotrophic effects may promote axonal regeneration in the CNS. To overcome CNS trauma, a new therapy geared towards neuroprotection and effective axonal regeneration is required to enhance a future multifactorial approach. The purpose of this study is to evaluate the therapeutic effects of Nrn1 in mouse RGC cultures as well as in the mouse ONC model. We have identified a distinct neuroprotective and regenerative strategy that prevents neurodegeneration after ON injury. AAV2–hNRN1 expression vectors partially rescued RGCs from apoptosis, maintained RGC function, and initiated regeneration of injured axons.     

Trophoblast Deportation to the Lungs of Cotton Rats (Sigmodon hispidus)

Cotton rats (Sigmodon hispidus) have been used to study a variety of infectious agents, particularly human respiratory viral pathogens. During the course of comprehensive pathologic evaluations of aging breeders from our breeding colony, 6 of 22 (27%) female cotton rats had histologic evidence, limited to the lungs, of embolized cells that were confirmed to be trophoblastic in origin by HSD3B1 immunoreactivity. When pulmonary trophoblast emboli were numerous, they usually were associated with additional histologic findings in the lungs, including pulmonary edema and hemorrhage, endothelial hypertrophy, fibrinoid vascular necrosis, and abundant alveolar macrophages containing fresh fibrin and hemolyzing erythrocytes. Of the 6 cotton rats with pulmonary trophoblast emboli, 5 (83%) were at 8 to 18 d of the 27-d gestation period, with the greatest number of emboli per lung present between days 10 through 14. The remaining cotton rat had a focal pulmonary trophoblast embolus and was not pregnant but had delivered a litter 3 mo previously. Three other cotton rats in either the early or late stages of gestation showed no histologic evidence of pulmonary trophoblast deportation. This report is the first to document pulmonary trophoblast emboli in cotton rats. This finding suggests that cotton rats may be an alternative animal model for the study of normal and aberrant trophoblast deportation in routine pregnancies and gestational pathologic conditions in women.Abbreviations: HSD3B1, hydroxyl-C-5-steroid dehydrogenaseCotton rats (Sigmodon hispidus) are a relevant animal model for the study of human respiratory²³ viral pathogens, with increasing usage by academic and industrial institutions. The hemochorial placentation in Sigmodontinae²² is similar to that of humans and several laboratory animal species including mice, rats, hamsters, rabbits, guinea pigs, chinchillas, and nonhuman primates.^{10,20,40,42,44,49} In these species, one or more layers of analogous trophoblast types comprise the interhemal barrier between maternal and fetal blood supplies. Placental trophoblasts perform a number of critical functions during gestation, including mediation of uterine implantation and invasion, nutrient exchange, regulation of maternal blood flow, and hormone production.^{1,19,26-28,35,38,46,47}As a consequence of their inherent invasiveness, placental trophoblasts migrate into maternal uterine blood vessels, after which syncytiotrophoblasts (syncytial knots) are normally deported daily to the lungs in humans.^2,3,17 Deportation increases with frequency as gestation progresses,^3,4 with gestational pathologic conditions such as preeclampsia and eclampsia,^2,3,18,36 and after cesarean sections⁵⁰ and abortions.⁴⁸ The current thinking is that these syncytial knots undergo programmed cell death and apoptotic shedding during routine pregnancy, in contrast to conditions like preeclampsia and eclampsia, during which aberrant intervillous hemodynamics resulting in hypoxia favor necrosis and associated inflammation.^18,25,29,30 In addition, spontaneous trophoblast emboli have been documented in the lungs and a few other tissues, including uterus, adrenal gland, spleen, and liver of chinchillas,^6,11,52 hamsters,^7,41 and porcupines.²⁴ Experimentally, trophoblast invasion has been further studied in mice^8,9 and hamsters.⁵ To our knowledge, pulmonary trophoblast emboli in cotton rats have not previously been reported.Pairs of cotton rat breeders were maintained for the production of animals to be used in various studies investigating human respiratory viruses, including measles, respiratory syncytial, and parainfluenza viruses. During the course of comprehensive pathologic evaluations of aging breeders, 6 female cotton rats were incidentally found to have pulmonary trophoblast emboli. The purposes of the present case series were to characterize the embolized trophoblasts and associated pulmonary histopathology in these cotton rats and to correlate the incidence with gestational stage.     

HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death

Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90''s previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3.Necroptosis is an inflammatory, caspase-independent form of regulated cell death characterised by loss of cellular membrane integrity and release of cytoplasmic contents.¹ It is believed to have evolved as a defence mechanism against viruses;^{2, 3} however, there is increasing evidence that deregulated necroptosis has a role in the pathogenesis of a range of inflammatory, autoimmune and neurodegenerative diseases.^{4, 5, 6, 7, 8} Reduced capacity to undergo necroptosis has been correlated to increased aggressiveness of cancers;^{9, 10} and therapeutic initiation of necroptosis is currently being investigated as a cancer therapy.^{11, 12} Additionally, there is emerging evidence that the necroptotic signalling pathway has a general role in the modulation of inflammation.^{13, 14, 15, 16, 17} As such, unravelling the molecular events governing necroptosis, and potential avenues for therapeutic intervention, is of enormous interest.Necroptosis is initiated through activation of death receptors, such as Tumour Necrosis Factor Receptor 1 (TNFR1), or through microbial activation of pattern recognition receptors, such as Toll-like receptors or intracellular viral DNA sensors.^{3, 18, 19, 20} Receptor ligation initiates a signalling cascade, whereby Receptor Interacting Protein Kinase (RIPK)-3 oligomerises and is phosphorylated, a process known to be regulated by association with other effectors, such as the protein kinase RIPK1, TIR-domain-containing adapter-inducing IFN-β (TRIF), or DNA-dependent activator of IFN regulatory factors (DAI), via their RIP Homotypic Interaction Motifs (RHIMs).^{2, 21, 22} Once activated, RIPK3 phosphorylates the pseudokinase domain of Mixed Lineage Kinase domain-Like (MLKL), the most downstream known obligate effector of the necroptotic signalling pathway, to induce its activation.^{23, 24} MLKL phosphorylation is thought to trigger a molecular switch,^{25, 26, 27} leading to the unleashing of the N-terminal executioner four-helix bundle (4HB) domain,²⁸ MLKL oligomerisation and translocation to cellular membranes where cell death occurs via an incompletely-understood mechanism.^{28, 29, 30}Molecular chaperones have an integral role in modulating both the structure and function of proteins. One such chaperone is heat-shock protein 90 (HSP90), which interacts with a diverse group of protein ‘clients'', the largest group comprising the kinases and pseudokinases, with 50% of the human kinome estimated to interact with HSP90.³¹ These interactions are dependent on the recognition of the kinase or pseudokinase domain by the HSP90 co-chaperone Cdc37, which enables HSP90 to confer protein stabilisation, assist in late-stage folding and conformational modifications, and mediate intracellular transport.^{32, 33, 34, 35}It has already been demonstrated that the necroptotic pathway is subject to modulation by HSP90. RIPK1 is well established as an HSP90 client protein, with a number of studies finding HSP90 inhibition affects both the stability and function of RIPK1 and promotes an apoptotic phenotype.^{36, 37, 38, 39, 40, 41} More recently, RIPK3 was also identified as an HSP90 client.^{2, 42, 43} Surprisingly, HSP90 inhibition did not markedly impact RIPK3 abundance or stability, but rather was essential for RIPK3''s necroptotic functions, such as phosphorylation of MLKL.⁴² However, whether MLKL itself is a client of HSP90 has not been investigated.In this study, using a phenotypic screen for small-molecule inhibitors of MLKL-driven cell death, we identified HSP90 as a modulator of necroptosis that functions on, or downstream of, the terminal effector, MLKL. HSP90 inhibition did not markedly reduce levels of MLKL in human U937 or mouse dermal fibroblasts, suggesting instead that HSP90 has an active role in governing MLKL-mediated cell death. This idea is supported by our finding that cell death driven by the S345D activated mutant of MLKL in Ripk3-deficient fibroblasts in the absence of necroptotic stimuli was suppressed by three distinct chemical classes of HSP90 inhibitor, but MLKL abundance was not impacted by HSP90 inhibition. Although our data indicate that MLKL binds HSP90 weakly or transiently, HSP90 activity was essential for the assembly of MLKL into high molecular weight complexes and the membrane translocation known to precede cell death. These findings suggest an expanded role for HSP90 in regulating necroptosis, and further our understanding of the mechanisms controlling MLKL-mediated cell death.