Helicobacter typhlonius and Helicobacter rodentium Differentially Affect the Severity of Colon Inflammation and Inflammation-Associated Neoplasia in IL10-Deficient Mice

Infection with Helicobacter species is endemic in many animal facilities and may alter the penetrance of inflammatory bowel disease (IBD) phenotypes. However, little is known about the relative pathogenicity of H. typhlonius, H. rodentium, and combined infection in IBD models. We infected adult and neonatal IL10^−/− mice with H. typhlonius, H. rodentium, or both bacteria. The severity of IBD and incidence of inflammation-associated colonic neoplasia were assessed in the presence and absence of antiHelicobacter therapy. Infected IL10^−/− mice developed IBD with severity of noninfected (minimal to no inflammation) < H. rodentium < H. typhlonius < mixed H. rodentium + H. typhlonius (severe inflammation). Inflammation-associated colonic neoplasia was common in infected mice and its incidence correlated with IBD severity. Combined treatment with amoxicillin, clarithromycin, metronidazole, and omeprazole eradicated Helicobacter in infected mice and ameliorated established IBD in both infected and noninfected mice. Infection of IL10^−/− mice with H. rodentium, H. typhlonius, or both organisms can trigger development of severe IBD that eventually leads to colonic neoplasia. The high incidence and multiplicity of neoplastic lesions in infected mice make this model well-suited for future research related to the development and chemoprevention of inflammation-associated colon cancer. The similar antiinflammatory effect of antibiotic therapy in Helicobacter-infected and -noninfected IL10^−/− mice with colitis indicates that unidentified microbiota in addition to Helicobacter drive the inflammatory process in this model. This finding suggests a complex role for both Helicobacter and other intestinal microbiota in the onset and perpetuation of IBD in these susceptible hosts.Abbreviations: IBD, Inflammatory bowel diseaseInflammatory bowel disease (IBD) is hypothesized to develop due to aberrant immune responses induced by gut microbes.⁵ IBD does not occur in germ-free IL10^−/− mice,^2,15 indicating the importance of microorganisms as environmental triggers of intestinal inflammation. However, conventionally colonized or specific pathogen-free IL10^−/− mice may develop colitis spontaneously^2,32 or in response to specific triggers such as nonsteroidal antiinflammatory drugs^3,14 or infections with certain bacteria.^6,16,18 The normal lack of ongoing immune responses against bacteria in subjects without IBD has been attributed to the immunologic tolerance that specifically downregulates immune responses against antigens derived from these bacteria. Nevertheless, despite a large number of studies, no single bacterial type has fulfilled Koch postulates and been confirmed as a cause of IBD in animals or humans.Previous studies used fluorescence in situ hybridization with probes specific for bacterial 16S rRNA combined with conventional histologic techniques to study the relationships between various species of intestinal bacteria and the mucosa in mice and humans with IBD.^33,34 Those studies showed that in normal mice, most bacterial groups are separated from the mucosal surface by either a mucus layer that excludes bacteria or, in the cecum and proximal colon, by an ‘interlaced’ layer that is composed of tightly packed bacteria. The interlaced or mucus layer thus limits the contact of the bulk of the enteric bacteria with the mucosal epithelium. In contrast, complex biofilms composed of multiple species of bacteria that were firmly adherent to the mucosal surface were identified in the majority of colon tissue samples collected from humans and mice with IBD.^33,34 The presence of a biofilm abrogates the protective effects of the normal layer of mucus and can allow luminal bacterial antigens and toxins to reach the unshielded epithelial surface, where they can trigger cascades of host inflammatory responses. Situations that cause defects in the epithelial surface or degrade the protective qualities of mucus or the interlaced layer (or both) may allow contact of bacterial antigens and adjuvants with immune cells located in the lamina propria and lead to the generation of immune responses that result in IBD.³⁴Helicobacter species are used frequently to model microbial triggers of colon inflammation, because they have previously been linked to the development of both IBD- and inflammation-associated neoplasia.^11,21,29 Most studies have been performed by using Helicobacter hepaticus or H. bilis.²⁰ However, H. typhlonius, H. rodentium, H. muridarum, H. ganmani, H. trogontum and other species^{8,12,17,29,35} can also be endemic in research animal facilities. The pathophysiologic effects of these less-common Helicobacter species are, for the most part, poorly investigated.Most rodent Helicobacter species are urease-negative and therefore preferentially colonize the intestine, but some species produce urease enzyme and can translocate to the liver or colonize the biliary system.¹³ H. typhlonius was shown to cause an enteric disease characterized by mucosal hyperplasia and associated inflammation in the cecum and colon in immunodeficient mice^11,23 and IL10^−/− mice.¹⁸ H. typhlonius is genetically related most closely to H. hepaticus, having only 2.36% difference in the 16S rRNA gene sequence, but H. typhlonius has a unique intervening sequence in this gene that makes it easily recognizable by PCR.^9,12 Molecular detection of this pathogen with PCR is rapid, sensitive and allows the detection of the early phases of infection; further enhanced sensitivity is achieved with nested primers.²² One of the most important features of PCR is that it can be performed noninvasively on fecal pellets. Data regarding the pathogenetic mechanisms of H. rodentium are scarce.^35,36 H. rodentium alone apparently does not cause hepatitis or enteritis in A/JCr or C.B-17/IcrCrl-scidBr mice; however, coinfection with H. hepaticus and H. rodentium was associated with augmented cecal gene expression and clinical diarrheal disease in immunodeficient mice compared with mice infected with H. hepaticus alone.²³Previous reports demonstrated that H. typhlonius was capable of initiating colitis in adult IL10^−/− mice.^10,11 In those studies, colitis was relatively mild, with no development of inflammation-associated neoplasia. H. rodentium has been described to be nonpathogenic in adult wild-type mice but did enhance cytokine production in the cecum of mice also infected with H. hepaticus.²³ We recently observed rapid onset of severe IBD and a high incidence of inflammation-associated neoplasia in IL10^−/− mice that were coinfected with both H. typhlonius and H. rodentium as pups.¹⁶ The current study was undertaken to determine the relative roles of H. rodentium and H. typhlonius, individually and in combination, and age at infection in the development of colon inflammation and inflammation-associated neoplasia in IL10^−/− mice. Novel features of our model include controlled infection of the combination of H. typhlonius and H. rodentium⁹ and infection of IL10^−/− mice during the neonatal period.     

Effects of Helicobacter Infection on Research: The Case for Eradication of Helicobacter from Rodent Research Colonies

Infection of mouse colonies with Helicobacter spp. has become an increasing concern for the research community. Although Helicobacter infection may cause clinical disease, investigators may be unaware that their laboratory mice are infected because the pathology of Helicobacter species is host-dependent and may not be recognized clinically. The effects of Helicobacter infections are not limited to the gastrointestinal system and can affect reproduction, the development of cancers in gastrointestinal organs and remote organs such as the breast, responses to vaccines, and other areas of research. The data we present in this review show clearly that unintentional Helicobacter infection has the potential to significantly interfere with the reliability of research studies based on murine models. Therefore, frequent screening of rodent research colonies for Helicobacter spp. and the eradication of these pathogens should be key goals of the research community.The reliability of an experiment that uses an in vivo model system depends on understanding and controlling all variables that can influence the experimental outcome. Infections of mouse colonies are important to the scientific community because they can introduce such harmful variables. Therefore, the ultimate goal of laboratory animal facilities is to maintain disease-free animals, to eliminate those unwanted variables.Numerous pathogenic microbes can interfere with animal research (reviewed in reference 57), and colonization of mouse colonies with members of the family Helicobacteriaceae is an increasing concern for the research community. Naturally acquired Helicobacter infections have been reported in all commonly used laboratory rodent species.^{3,10,36,44,45,49,82,124} A study of mice derived from 34 commercial and academic institutions in Canada, Europe, Asia, Australia, and the United States showed that 88% of these institutions had mouse colonies infected with 1 or more Helicobacter spp.¹⁰⁹ Approximately 59% of these mice were infected with Helicobacter hepaticus ; however monoinfections with other species also were encountered. In another study, at least 1 of 5 Helicobacter spp. was detected in 88% of the 40 mouse strains tested.⁴Surveys such as these have established that a broad range of Helicobacter spp. may be present in mouse research colonies. Several of those Helicobacter species cause disease in laboratory mice. H. hepaticus first was identified as a pathogen when it was discovered to be the cause of chronic hepatitis and hepatocellular carcinoma in mice,^26,31,116 either alone or in combination with other Helicobacter spp.⁷⁸ In addition, H. typhlonius causes intestinal inflammation in mice with immunodeficiency or defects in immune regulation;^28,37 H. muridarum has been associated with gastritis,⁸⁶ and H. bilis has been associated with hepatitis^35,38 and colitis.^60,61 Although, H. rodentium appears to be relatively nonpathogenic in wild-type and SCID mice,⁷⁸ combined infection with H. rodentium and H. typhlonius results in a high incidence of inflammation-associated neoplasia in IL10^−/− mice.^9,46 Further, it is becoming increasingly clear that the effects of Helicobacter infections are not limited to the gastrointestinal system. Helicobacter infections have been documented to directly or indirectly affect responses as diverse as reproduction, development of breast cancer, and altered immune responses to vaccines.^65,95,99 In addition to effects on rodents, Helicobacter spp. can infect other laboratory animals^{2,5,27,29,33,36,107} and can colonize different anatomic regions of the gastrointestinal system.³⁵ This review focuses on the potential effect of these organisms on in vivo experiments and biomedical research. The results summarized here emphasize the importance of knowledge of colony infection status and prevention of unintentional infections to achieve the goal of providing a consistent and reliable environment for research studies.     

Isolation of Helicobacter spp. from Mice with Rectal Prolapses

Enterohepatic Helicobacter species (EHS) often are associated with typhlocolitis and rectal prolapse in mice. We sought to describe rectal prolapses histologically, relate lesions to mouse genotype and EHS infection status, and characterize EHS pathogens on our campus. Our mouse population was housed among 6 facilities on our main campus and a seventh, nearby facility. We investigated cases of rectal prolapse over 1 y and included 76 mice, which were broadly categorized according to genotype. Microscopically, lesions ranged from mild to severe typhlocolitis, often with hyperplastic and dysplastic foci. Neoplastic foci tended to occur at the ileocecal–colic junction. Lesions were most severe in strains that had lower-bowel inflammatory disease, notably IL10, Rag1, and Rag2 knockout strains; prolapses occurred in these strains when housed both in areas with endemic EHS and in our Helicobacter-free barrier facility. Most mice with rectal prolapses were immunocompromised genetically modified mice; however, the most frequently sampled strain, the lamellipodin knockout, was noteworthy for its high incidence of rectal prolapse, localized distal colonic and rectal lesions, and lack of known immunodeficiency. This strain is being explored as a model of rectal carcinoma. Most of the colons examined tested PCR-positive for EHS, often with coinfections. Although H. bilis is prevalent on our campus, we did not find this organism in any mice exhibiting clinical signs of rectal prolapse. Identification of H. apodemus in 22% of cases has fueled increased surveillance on our campus to characterize this organism and differentiate it from the closely related H. rodentium.Abbreviations: EHS, enterohepatic Helicobacter species; IBD, inflammatory bowel disease; RFLP, restriction-fragment–length polymorphism; RP, rectal prolapseRectal prolapse (RP) occurs commonly in laboratory mice and is often associated with lower-bowel inflammation. Mice have a relatively short and poorly supported distal colon, which lacks a serosal covering.³⁰ This anatomic weakness, coupled with a microbial insult, toxic injury, or space-occupying neoplastic masses within the gastrointestinal tract, are the predisposing factors for tenesmus and RP (Figure 1). In the context of microbial insults, the pathogenesis involves diffuse or multifocal inflammation in the more proximal segments of colon or distal colon, which can result in thickened edematous tissue and tenesmus, triggering a prolapse.^6,30,40 Bacteria most often associated with this condition are the enterohepatic Helicobacter species (EHS) and Citrobacter rodentium; although in theory any pathogenic bacteria causing colitis may predispose mice to RP.^1,11,13,38Open in a separate windowFigure 1.Mouse rectal prolapse. An example of the clinical presentation of rectal prolapse in laboratory mice. Note the attachment of bedding and nesting material in the film of mucous that frequently is seen covering the exposed rectal tissue. Generally the tissue becomes severely erythematous, as can be appreciated in this photograph.Although the clinical presentation of RP may occur in immunocompetent mice, it is most often associated with mice that have a spontaneous or transgenic mutation causing immunodeficiency.^11,13,38 Indeed, these naturally occurring murine pathogens are used to model inflammatory bowel disease in strains that are highly susceptible to typhlocolitis with EHS infection; examples include Il10^−/− and Rag-deficient mice.^{3,5,8,9,13,16,19,20,22,40} In addition, H. hepaticus and other EHS including H. typhlonius, H. rodentium, and H. bilis, which are known to persistently colonize the intestinal crypt of the lower bowel, have been shown to induce colitis-associated cancer in susceptible immunodeficient strains of mice.^{4,7,9,23,24,27,29,31}In 1999, our institution introduced a rodent importation policy to reduce the introduction of murine pathogens. As part of this program, all approved commercial vendors were screened to ensure animals were SPF for EHS. Any random-source mice (typically imported from other academic institutions for collaborative projects) were required to be rederived by embryo transfer. In comparing PCR data between 1999 (prior to implementing the ET policy) and 2009, we found that after more than a decade of strict rederivation and husbandry practices that reduce fecal–oral transmission, EHS prevalence was markedly reduced.²¹ Despite this success, these practices did not completely eradicate rodent EHS. Of particular note, 2 facilities on campus house well-established long-term breeding colonies, many of which are unique transgenic lines with various immunodeficiencies, that are used primarily for immunology and cancer research. Rederivation of each of these strains was considered to be cost-prohibitive; thus EHS has remained endemic in these breeding colonies for more than a decade, as evident by our recent surveillance for EHS prevalence.²¹ The species known to be prevalent on our campus prior to the current study included H. hepaticus, H. rodentium, H. typhlonius, and H. bilis; in a few isolated areas, H. mastomyrinus was identified also.²¹Although EHS infections often are subclinical, we sought to correlate the presence of EHS-endemic areas with clinical lower-bowel inflammation (evident by rectal prolapse). In this survey of laboratory mice at our institution, we identified patterns in mouse strain susceptibility to RP, RP association with EHS, and histopathologic findings and correlated specific EHS species with clinical disease. Because we sought to study spontaneous infections, we excluded any mice on study with experimentally induced inflammatory bowel disease (IBD), including Helicobacter-induced IBD and chemically induced colitis models.From July 2011 to July 2012, a total of 63 mice with RP from these 6 facilities at our institution were necropsied as part of this investigation. In addition, 13 mice with RP were identified at a nearby research institute housing mice known to have endemic EHS.     

The Effect of Helicobacter hepaticus Infection on Immune Responses Specific to Herpes Simplex Virus Type 1 and Characteristics of Dendritic Cells

Infection of mice with Helicobacter hepaticus is common in research colonies, yet little is known about how this persistent infection affects immunologic research. The goal of this study was to determine whether H. hepaticus infection status can modulate immune responses specific to herpes simplex virus type 1 (HSV1) and the phenotypic and functional characteristics of dendritic cells (DC) of mice. We compared virus-specific antibody and T cell-mediated responses in H. hepaticus-infected and noninfected mice that were inoculated intranasally with HSV1. The effect of H. hepaticus on the HSV1-specific antibody and T cell-mediated immune responses in superficial cervical and tracheobronchal lymph nodes (LN) did not reach statistical significance. Surface expression of the maturation-associated markers CD40, CD80, CD86, and MHC II and percentages of IL12p40- and TNFα-producing DC from spleen and colic LN in H. hepaticus-infected mice and noninfected mice were measured in separate experiments. Expression of CD40, CD86, and MHC II and percentages of IL12p40- and TNFα-producing DC from colic LN were decreased in H. hepaticus-infected mice. In contrast, H. hepaticus infection did not reduce the expression of these molecules by splenic DC. Expression of CD40, CD80, CD86, and MHC II on splenic DC from H. hepaticus-infected mice was increased after in vitro lipopolysaccharide stimulation. These results indicate that H. hepaticus infection can influence the results of immunologic assays in mice and support the use of H. hepaticus-free mice in immunologic research.Abbreviations: DC, dendritic cells; HSV1, herpes simplex virus type 1; LN, lymph nodes; MHC II, major histocompatibility complex class II; MHV, mouse hepatitis virus; OVA, ovalbumin peptide SIINFEKL; PE, phycoerythrinHelicobacter hepaticus is a gram-negative, microaerophilic, curved to spiral-shaped bacterium with bipolar, sheathed flagella. H. hepaticus was described for the first time in 1994 as the cause of chronic active hepatitis associated with a high incidence of hepatocellular neoplasms in mice on a long-term toxicology study.³⁹ Since then, H. hepaticus has been identified as a common contaminant of mouse colonies at a variety of research institutions. Although commercial breeders produce H. hepaticus-free animals, many mouse colonies at public and private research institutions still harbor H. hepaticus. A recent survey found H. hepaticus-infected mice in 59% of commercial and academic institutions in Canada, Europe, Asia, Australia, and the United States.³⁵H. hepaticus persistently colonizes the hepatic bile canaliculi and the cecal and colonic mucosa of mice.^9,39 Infection can cause chronic active hepatitis, hepatocellular neoplasms, and typhlocolitis, which vary in severity depending on the strain, age, gender, and immune status of the mouse.^5,9,11,39 In adult immunocompetent mice, H. hepaticus infection is usually asymptomatic. However, immune-dysregulated mice can develop inflammatory bowel disease, which may present as rectal prolapse or diarrhea.¹⁶Mice initiate immune responses against H. hepaticus primarily through its interaction with Toll-like receptor 2 on antigen-presenting cells.²¹ Both systemic and local (at the site of infection) H. hepaticus-specific Th1-type immune responses are induced in immunocompetent mice.^26,40 Systemic antibody and cell-mediated immunity against the bacteria persist for at least 46 wk after experimental inoculation.⁴⁰ Gene expression profiles of cecal tissue of H. hepaticus-infected mice have shown that inflammatory responses differ depending on the mouse strain. For example, A/JCr mice had significant and prolonged expression of the Th1-type cytokines IFNγ and IL12p40 in cecal mucosa, and these expression levels persisted for at least 3 mo after H. hepaticus infection. However, C57BL/6 mice had a lesser elevation of IFNγ gene expression without an effect on IL12p40. IFN γ expression waned by 1 mo after inoculation in C57BL/6 mice.²⁵ In addition, H. hepaticus-specific secretory IgA antibodies are persistently detected in the feces of mice.⁴⁰ How these immune responses in H. hepaticus-infected mice might affect immunologic research is unknown.The goal of this study was to determine whether immune responses to herpes simplex virus type 1 (HSV1) and the phenotypic and functional characteristics of dendritic cells (DC) are altered in H. hepaticus-infected mice. The intranasal HSV1 infection model is used widely to study immune mechanisms in mice. Immunity to HSV1 consists of virus-neutralizing antibodies in the serum and virus-specific T cells in the draining LN. Superficial cervical and mediastinal LN have been described as draining LN for intranasal HSV1 infection.² The response to HSV1 infection peaks at 7 d after infection and leads to clearance of the viral load.² In this study, we compared levels of HSV1-specific antibody and T cell-mediated immune responses between H. hepaticus-infected and noninfected mice.Dendritic cells are important components of the immune system that play a role in antigen processing and presentation. On exposure to foreign antigen, DC mature and express increased levels of major histocompatibility complex class II proteins (MHC II), CD40, CD80, and CD86 on the cell surface. These maturation-associated cell surface markers interact with naive T and B cells to initiate antibody- and cell-mediated immune responses against foreign antigens.²⁷ In addition, mature DC secrete proinflammatory cytokines, including TNFα and IL12p40. These cytokines lead to increased vascular permeability, complement activity, lymphocyte activation, lymphocyte proliferation, and increased antibody production.²⁷ To determine whether infection with H. hepaticus affects characteristics of DC, we measured the expression of the maturation-associated cell surface markers CD40, CD80, CD86, and MHC II and proinflammatory cytokines IL12p40 and TNFα by DC derived from the spleen and colic LN of H. hepaticus-infected and noninfected mice. Our findings indicate that H. hepaticus infection can influence the various aspects of immune responsiveness and, therefore, must be considered as a potential variable in studies in which immune function is a measurable outcome.     

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.     

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.     

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.     

Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling

Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.The sphingomyelin pathway is initiated by the hydrolysis of sphingomyelin to generate the second messenger ceramide.¹ Sphingomyelin hydrolysis is a major pathway for stress-induced ceramide generation. Neutral sphingomyelinase (nSMase) is activated by a variety of environmental stress conditions, such as heat shock,^{1, 2, 3} oxidative stress (hydrogen peroxide (H₂O₂), oxidized lipoproteins),¹ ultraviolet (UV) radiation,¹ chemotherapeutic agents,⁴ and β-amyloid peptides.^{5, 6} Cytokines, including tumor necrosis factor (TNF)-α,^{7, 8, 9} interleukin (IL)-1β,¹⁰ Fas ligand,¹¹ and their associated proteins, also trigger the activation of nSMase.¹² Membrane-bound Mg²⁺-dependent nSMase is considered to be a strong candidate for mediating the effects of stress and inflammatory cytokines on ceramide.³Among the four vertebrate nSMases, nSMase1 (SMPD2) was the first to be cloned and is localized in the endoplasmic reticulum (ER) and Golgi apparatus.¹³ Several studies have focused on the potential signaling roles of nSMase1, and some reports have suggested that nSMase1 is important for ceramide generation in response to stress.^{5, 6, 14, 15} In addition, nSMase1 is responsible for heat-induced apoptosis in zebrafish embryonic cultured (ZE) cells, and a loss-of-function study showed a reduction in ceramide generation, caspase-3 activation, and apoptosis in zebrafish embryos.¹⁶ However, nSMase1-knockout mice showed no lipid storage diseases or abnormalities in sphingomyelin metabolism.¹⁷ Therefore, the molecular mechanisms by which nSMase1 is activated have yet to be elucidated.Environmental stress and inflammatory cytokines^{1, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27} stimulate stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) signaling, which involves the sequential activation of members of the mitogen-activated protein kinase (MAPK) family, including MAPK/ERK kinase kinase (MEKK)1/MAPK kinase (MKK)4, and/or SAPK/ERK kinase (SEK)1/MKK7, JNK, and c-jun. Both the JNK and sphingomyelin signaling pathways coordinately mediate the induction of apoptosis.¹ However, possible crosstalk between the JNK and sphingomyelin signaling pathways has not yet been characterized. Previously, we used SDS-PAGE to determine that nSMase1 polypeptides migrated at higher molecular masses,¹⁶ suggesting that the sphingomyelin signaling pathway might cause the production of a chemically modified phosphorylated nSMase1, which is stimulated under stressed conditions in ZE cells.¹⁶ Here, we demonstrate that JNK signaling results in the phosphorylation of Ser-270 of nSMase1, which initiates ceramide generation and apoptosis. We also provide evidence for a signaling mechanism that integrates cytokine- and stress-activated apoptosis in vertebrate cells. We studied stress-induced ceramide generation in two cell types: ZE cells and human leukemia Jurkat T-lymphoid cells. Stress-induced apoptosis has been investigated in these systems previously.^{16, 28}     

Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity.Obesity, a medical condition predisposing to diabetes, cardiovascular diseases, cancer, and complicating other life-threatening diseases, is becoming an increasingly important social problem.^{1, 2, 3} Development of pharmacological approaches to reduction of body fat has remained a daunting task.⁴ Approved obesity treatments typically produce only moderate and temporary effects.^2,5 White adipocytes are the differentiated cells of white adipose tissue (WAT) that store triglycerides in lipid droplets.^6,7 In contrast, adipocytes of brown adipose tissue (BAT) dissipate excess energy through adaptive thermogenesis. Under certain conditions, white adipocytes can become partially replaced with brown-like ‘beige'' (‘brite'') adipocytes that simulate the thermogenic function of BAT adipocytes.^7,8 Obesity develops in the context of positive energy balance as a result of hypertrophy and hyperplasia of white adipocytes.⁹Expansion and renewal of the white adipocyte pool in WAT continues in adulthood.^10,11 This process is believed to rely on proliferation and self-renewal of mesenchymal precursor cells¹² that we term white adipocyte progenitors (WAPs). WAPs reside within the population of adipose stromal cells (ASCs)¹³ and are functionally similar to bone marrow mesenchymal stem cells (MSCs).^{14, 15, 16} ASCs can be isolated from the stromal/vascular fraction (SVF) of WAT based on negativity for hematopoietic (CD45) and endothelial (CD31) markers.^17,18 ASCs support vascularization as mural/adventitial cells secreting angiogenic factors^5,19 and, unlike bone marrow MSCs, express CD34.^19,20 WAPs have been identified within the ASC population based on expression of mesenchymal markers, such as platelet-derived growth factor receptor-β (PDGFRβ, aka CD140b) and pericyte markers.^17,18 Recently, a distinct ASC progenitor population capable of differentiating into both white and brown adipocytes has been identified in WAT based on PDGFRα (CD140a) expression and lack of PDGFRβ expression.^21,22 The physiological relevance of the two precursor populations residing in WAT has not been explored.We have previously established an approach to isolate peptide ligands binding to receptors selectively expressed on the surface of cell populations of interest.^{23, 24, 25, 26, 27} Such cell-targeted peptides can be used for targeted delivery of experimental therapeutic agents in vivo. A number of ‘hunter-killer'' peptides²⁸ composed of a cell-homing domain binding to a surface marker and of KLAKLAK₂ (sequence KLAKLAKKLAKLAK), a moiety inducing apoptosis upon receptor-mediated internalization, has been described by our group.^26,29 Such bimodal peptides have been used for depletion of malignant cells and organ-specific endothelial cells in preclinical animal models.^26,30,31 Recently, we isolated a cyclic peptide WAT7 (amino acid sequence CSWKYWFGEC) based on its specific binding to ASCs.²⁰ We identified Δ-decorin (ΔDCN), a proteolytic cleavage fragment of decorin, as the WAT7 receptor specifically expressed on the surface of CD34+PDGFRβ+CD31-CD45- WAPs and absent on MSCs in other organs.²⁰Here, we investigated whether WAPs are required for obesity development in adulthood. By designing a new hunter-killer peptide that directs KLAKLAK₂ to WAPs through WAT7/ΔDCN interaction, we depleted WAP in the mouse diet-induced obesity model. We demonstrate that WAP depletion suppresses WAT growth. We show that, in response to WAP deficiency, WAT becomes populated with beige adipocytes. Consistent with the reported thermogenic function of beige adipocytes,^32,33 the observed WAT remodeling is associated with increased energy expenditure. We identify a population of PDGFRα-positive, PDGFRβ-negative ASCs reported recently²² as a population surviving WAP depletion and responsible for WAT browning.     

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.     

Restoration of miR-101 suppresses lung tumorigenesis through inhibition of DNMT3a-dependent DNA methylation

The deregulation of miR-101 and DNMT3a has been implicated in the pathogenesis of multiple tumor types, but whether and how miR-101 silencing and DNMT3a overexpression contribute to lung tumorigenesis remain elusive. Here we show that miR-101 downregulation associates with DNMT3a overexpression in lung cancer cell lines and patient tissues. Ectopic miR-101 expression remarkably abrogated the DNMT3a 3′-UTR luciferase activity corresponding to the miR-101 binding site and caused an attenuated expression of endogenous DNMT3a, which led to a reduction of global DNA methylation and the re-expression of tumor suppressor CDH1 via its promoter DNA hypomethylation. Functionally, restoration of miR-101 expression suppressed lung cancer cell clonability and migration, which recapitulated the DNMT3a knockdown effects. Interestingly, miR-101 synergized with decitabine to downregulate DNMT3a and to reduce DNA methylation. Importantly, ectopic miR-101 expression was sufficient to trigger in vivo lung tumor regression and the blockage of metastasis. Consistent with these phenotypes, examination of xenograft tumors disclosed an increase of miR-101, a decrease of DNMT3a and the subsequent DNA demethylation. These findings support that the loss or suppression of miR-101 function accelerates lung tumorigenesis through DNMT3a-dependent DNA methylation, and suggest that miR-101-DNMT3a axis may have therapeutic value in treating refractory lung cancer.Owing to a high propensity for recurrence and a high rate of metastasis at the advanced stages,^{1, 2, 3} lung cancer remains the leading cause of cancer-related mortality. DNA methylation is a major epigenetic rule controlling chromosomal stability and gene expression.^{4, 5} It is under control of DNA methyltransferases (DNMTs), whose overexpression in lung cancer cells predicts worse outcomes.^{6, 7} It is postulated that DNMT overexpression induces DNA hypermethylation and silencing of tumor suppressor genes (TSGs), leading to an aggressive lung cancer. Indeed, enforced expression of DNMT1 or DNMT3a increases DNA methylation, while the abolition of DNMT expression by genetic depletion, microRNAs (miRs) or small molecules reduces genome-wide and gene-specific DNA methylation and restores TSG expression.^{8, 9, 10, 11, 12, 13} As TSGs are the master controllers for cell multiplicity and their silencing predicts poor prognosis,^{14, 15} TSG re-expression via promoter DNA hypomethylation inhibits cell proliferation and induces cell differentiation.^{13, 16} Thus, DNMT gene abundance could serve as a target for anticancer therapy, but how DNMT upregulation occurs in lung cancer is incompletely understood.MiRs are small non-coding RNAs that crucially regulate target gene expression. Up to 30% of all protein-coding genes are predicted to be targeted by miRs,^{17, 18} supporting the key roles of miRs in controlling cell fate.^{19, 20, 21, 22} Research is showing that certain miRs are frequently dysregulated in cancers, including lung cancer.^{7, 23, 24} As miR targets can promote or inhibit cancer cell expansion, miRs have huge potential for acting as bona fide oncogenes (i.e., miR-21) or TSGs (i.e., miR-29b).^{7, 25} We and others demonstrated that the levels of DNMT1 or DNMT3a or DNMT3b are regulated by miR-29b, miR-148, miR-152 or miR-30c,^{7, 13, 26, 27} and overexpression of these miRs results in DNA hypomethylation and TSG reactivation with the concurrent blockage of cancer cell proliferation.^{7, 13} These findings underscore the importance of miRs as epigenetic modulators and highlight their therapeutic applications.MiR-101 is frequently silenced in human cancers^{28, 29, 30, 31} and, importantly, exhibits antitumorigenic properties when overexpressed. Mechanistically, miR-101 inactivation by genomic loss causes the overexpression of EZH2, a histone methyltransferase, via 3′-UTR targeting, which is followed by histone hypermethylation and aggressive tumorigenesis.^{29, 30, 32} However, whether and how miR-101 silencing contributes to DNA hypermethylation patterning in lung cancer is unclear. In this study, we explore the role of miR-101 in regulating DNMT3a expression and the impacts of miR-101-DNMT3a nexus on lung cancer pathogenesis. We showed that the expression of miR-101 and DNMT3a was negatively correlated in lung cancer. We presented evidence that ectopic miR-101 expression decreased DNMT3a levels, reduced global DNA methylation and upregulated CDH1 via its promoter DNA demethylation. The biological significance of miR-101-mediated DNA hypomethylation and CDH1 re-expression was evident by its inhibition of lung tumor cell growth in vitro and in vivo. Thus, our findings mechanistically and functionally link miR-101 silencing to DNA hypermethylation in lung cancer cells.     

Synaptic dysfunction,memory deficits and hippocampal atrophy due to ablation of mitochondrial fission in adult forebrain neurons

Well-balanced mitochondrial fission and fusion processes are essential for nervous system development. Loss of function of the main mitochondrial fission mediator, dynamin-related protein 1 (Drp1), is lethal early during embryonic development or around birth, but the role of mitochondrial fission in adult neurons remains unclear. Here we show that inducible Drp1 ablation in neurons of the adult mouse forebrain results in progressive, neuronal subtype-specific alterations of mitochondrial morphology in the hippocampus that are marginally responsive to antioxidant treatment. Furthermore, DRP1 loss affects synaptic transmission and memory function. Although these changes culminate in hippocampal atrophy, they are not sufficient to cause neuronal cell death within 10 weeks of genetic Drp1 ablation. Collectively, our in vivo observations clarify the role of mitochondrial fission in neurons, demonstrating that Drp1 ablation in adult forebrain neurons compromises critical neuronal functions without causing overt neurodegeneration.In addition to their crucial importance in energy conversion, mitochondria serve many other housekeeping functions, including calcium buffering, amino-acid and steroid biosynthesis as well as fatty acids beta-oxidation and regulation of cell death. During the past decade, it has become increasingly clear that processes regulating mitochondrial morphology and ultrastructure are influenced by specific cellular requirements upon which mitochondria, in a precisely regulated manner, undergo fusion and division events.¹ Maintaining this balance is especially important for highly energy-consuming, polarized cells such as neurons, where single organellar units sprouting from the mitochondrial network are transported along the cytoskeleton into dendrites and spines to meet local energy requirements.² In addition, elaborate quality-control mechanisms also rely on mitochondrial dynamics: whereas defective organelles are sequestered by fission, enabling their removal from the mitochondrial network,^{3, 4} fusion supports qualitative homogeneity of the syncytium through complementation.⁵Mitochondrial fusion and fission are mediated by large GTPases of the dynamin superfamily.⁶ The outer mitochondrial membrane mitofusins 1 (MFN1) and 2 (MFN2) tether mitochondrial membranes by homodimer or heterodimer formation,⁷ thereby initiating fusion of the organelles, a process that also involves the inner mitochondrial membrane-associated GTPase Optic Atrophy 1.⁸ In addition, MFN2 also mediates contacts between mitochondria and endoplasmic reticulum.⁹ The only known mammalian mitochondrial fission protein, Dynamin-Related Protein 1 (Drp1), translocates upon dephosphorylation by calcineurin¹⁰ to fission sites where it binds to mitochondrial fission factor.¹¹ Drp1 translocation is preceded by ER membranes wrapping around mitochondria to constrict the organelles,¹² thereby facilitating the formation of multimeric Drp1 complexes that, upon GTP hydrolysis, further tighten to complete the process of mitochondrial fission.¹³Genetic evidence in mice and humans indicates that mitochondrial dynamics are crucially important in neurons: in humans, a sporadic dominant-negative DRP1 mutation caused a lethal syndromic defect with abnormal brain development;¹⁴ similarly, constitutive Drp1 knockout in the mouse brain leads to lethal neurodevelopmental defects.^{15, 16} Although the crucial role of Drp1 during brain development is undisputed, studies on Drp1 function in postmitotic (adult) neurons are scarce; likewise, Drp1 ablation studies in primary cultures have so far failed to yield a conclusive picture. In vitro, Drp1 ablation is reported to lead to a super-elongated neuroprotective^{17, 18, 19, 20, 21, 22, 23, 24} or an aggregated mitochondrial phenotype associated with neurodegeneration.^{15, 16, 25, 26, 27} These discrepancies are probably due to different experimental conditions: neuronal health is indeed influenced by the onset and duration of Drp1 inhibition, which varies considerably among the cited reports,²⁸ and different types of neuronal cultures studied display different sensitivity to Drp1 inhibition. In vivo, Drp1 ablation in Purkinje cells results in oxidative stress and neurodegeneration,²⁹ demonstrating that Drp1 is essential for postmitotic neurons'' health. In contrast, transient pharmacological Drp1 inhibition is neuroprotective in several mouse ischemia models, indicating that temporarily blocking mitochondrial fission holds therapeutic potential.^{30, 31, 32}To elucidate the consequences of blocked mitochondrial fission in the central nervous system in vivo, we bypassed the critical role of Drp1 during brain development by generating Drp1^flx/flx mice¹⁵ expressing tamoxifen-inducible Cre recombinase under the control of the CaMKIIα promoter.³³ Upon induced Drp1 deletion in postmitotic adult mouse forebrain neurons, mice develop progressive, neuronal subtype-specific alterations in mitochondrial shape and distribution in the absence of overt neurodegeneration. In addition, respiratory capacity, ATP content, synaptic reserve pool vesicle recruitment as well as spatial working memory are impaired, demonstrating that severely dysregulated mitochondrial dynamics can compromise critical neuronal functions in vivo without causing neuronal cell death.     

Serologic Evaluation of Clinical and Subclinical Secondary Hepatic Amyloidosis in Rhesus Macaques (Macaca mulatta)

Secondary hepatic amyloidosis in nonhuman primates carries a grave prognosis once animals become clinically ill. The purpose of this study was to establish serologic parameters that potentially could be used to identify rhesus macaques undergoing subclinical development of secondary hepatic amyloidosis. A retrospective analysis was completed by using serum biochemical profiles from 26 histologically diagnosed amyloidotic macaques evaluated at 2 stages of disease, clinical and subclinical (3 to 32 mo prior to clinical signs of disease). Standard serum biochemistry values for cases were compared with institutional age- and gender-specific references ranges by construction of 95% confidence intervals for the difference between means. In addition, 19 histologically diagnosed amyloidotic macaques and 19 age-matched controls were assayed for changes in various parameters by using routinely banked, frozen (–80 °C) sera available from clinical and subclinical time points. Clinically amyloidotic animals displayed increased levels of alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, gamma glutamyltranspeptidase, and macrophage colony-stimulating factor and significantly decreased quantities of albumin and total cholesterol. Subclinical amyloidotic animals displayed increased levels of alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, and serum amyloid A and decreased concentrations of albumin and total cholesterol. The serologic parameters studied indicate a temporal relationship of these factors not previously described, show a clear pattern of disease progression, and could be useful in subclinical disease detection.Abbreviations: mCSF, macrophage colony stimulating factor; SAA, serum amyloid AAmyloid is an eosinophilic substance made of insoluble fibrillar protein.³² When deposited extracellularly, amyloid causes displacement of tissue form and disruption of organ function.³² Persistent accretion of amyloid can result in organ failure and ultimately animal death.²² Clinical signs of disease depend on the tissues affected and the degree of involvement.³² Amyloidosis has been well documented in humans, other mammals, birds, and reptiles.³⁸ In humans, amyloidosis plays a key role in many diseases, including Alzheimer disease, type II diabetes, rheumatoid arthritis, and Down syndrome.^15,20,35,38Amyloidosis generally is classified into 3 categories: primary, secondary, and hereditary. Primary amyloidosis consists of the immunoglobulin- and myeloma-associated types. Secondary (reactive) amyloidosis is associated with chronic inflammation.²⁴ Common causes of secondary amyloidosis in humans include rheumatoid arthritis, idiopathic colitis, infectious diseases, such as tuberculosis and leprosy, and malignant tumors, such as mesothelioma and Hodgkins disease.²⁸ Hereditary amyloid syndromes are rare and include Mediterranean fever, Muckle–Wells syndrome, and familial amyloid cardiomyopathy.^32,38Secondary amyloidosis is the most common form of amyloidosis in animals.³⁸ Amyloidosis occurs in many species of nonhuman primates including the common marmoset (Callithrix jacchus),²³ squirrel monkey (Saimiri sciureus),³⁴ rhesus macaque (Macaca mulatta),^9,10 pigtailed macaque (Macaca nemestrina),^18,27 crab-eating macaque (Macaca fascicularis),²⁷ barbary ape (Macaca sylvanus),⁶ baboon (Papio spp.),¹⁷ mandrill (Papio sphinx), and chimpanzee (Pan troglodytes).^16,39 Although a definitive cause of secondary amyloidosis has not been identified in nonhuman primates, this condition has been associated with chronic inflammation due to rheumatoid arthritis,⁶ viral infection,¹⁸ parasitism,¹ respiratory disease,^27,30 trauma,³⁰ and bacterial enterocolitis.^27,30,31 Shigella spp. have received particular attention as a common etiology linking enterocolitis with amyloidosis.^4,7,38Previous research on amyloidosis in nonhuman primates has yielded clinical and serologic profiles in end-stage amyloidotic animals, but little is known about the serologic status in the subclinical stages of disease. Amyloid can accumulate for as long as 3 y before severe organ disruption occurs¹⁴ and clinical signs of amyloidosis become evident.¹⁶ With appropriate analysis, detection of amyloidosis could occur much earlier than typically now achieved, thus allowing for targeted preventative therapy to potentially halt the progression of this insidious disease.     

Cellular Compensatory Mechanisms in the CNS of Dysmyelinated Rats

Loss or absolute lack of myelin in the CNS results in remarkable compensation at the cellular level. In this study on the natural progression of neuropathology in the CNS in 2 related but distinct long-lived dysmyelinated rats, total lack of myelin was associated with remarkable glial cell proliferation and ineffective myelinating activity throughout life in Long Evans Bouncer (LE-bo) rats; conversely, in Long Evans Shaker (LES) rats, futile myelinating activity ceased when rats were advanced in age. Progressively severe astrogliosis separates individual axons from each other and coincides with widespread, abundant axonal sprouting throughout the life in both rat strains. Severely dysmyelinated Long Evans rats can serve as excellent models to elucidate the cellular and molecular mechanisms of neuroglial compensation to lack or loss of myelin in vivo and to study axonal plasticity in the adult demyelinated CNS.Abbreviations: ³H-TdR, [³H]-thymidine; LES, Long Evans Shaker; LE-bo, Long Evans BouncerCompensatory cellular responses to loss of myelin in multiple sclerosis, experimental allergic encephalomyelitis or spinal cord injury, and both loss and absolute lack of myelin in dysmyelinated CNS are not well understood. Morphologic studies indicate that recent demyelinated lesions show proliferation of oligodendrocyte-type cells coinciding with repopulation of demyelinated areas and precocious remyelination.^34,36,38 Newly formed myelin sheaths are thin and may form shortened internodes that can be interspersed by demyelinated axonal segments.³⁵ Compensatory remyelination in human patients affected by multiple sclerosis^26,34,41 involves oligodendrocyte progenitor cells that populate the adult CNS and can be active in areas of demyelination.^45,51,52 However, remyelination is incomplete and fails altogether after recurrent bouts of demyelination.¹ Demyelinated plaques in sclerotic lesions can have surviving axons arranged in bundles which separated from each other by hypertrophied astrocyte processes.¹Cellular mechanisms of compensation in response to demyelination can be studied systematically and in detail in animal models of experimental allergic encephalomyelitis^5,39 or viral encephalitis, such as intracerebral inoculation of Theiler virus in CD1 mice.⁴³ Remyelination in areas of chronic demyelination requires increased mitotic activity, resulting in generation of astrocyte and oligodendrocyte progenitors and their migration into demyelinating areas with subsequent myelination and astrogliosis.^8,37,40 After immunoglobulin treatment of Theiler virus-inoculated SJL/J mice, their demyelinated lesions can have proliferation and maturation of oligodendrocyte progenitors followed by spontaneous remyelination.⁴⁴ Glial cell proliferation near lymphocytes and hypertrophied astrocytes suggested a beneficial role of at least some proinflammatory mechanisms in remyelination. Intracerebral inoculation of C57Bl/6N mice with mouse hepatitis virus produced demyelination followed by transient increase in proliferation of glial progenitors, which differentiated into oligodendrocytes and astrocytes with remyelination and astrogliosis.¹⁵ Other experimental methods of demyelination, such as oral administration of cuprizone resulting in demyelination of cerebellar peduncles in CD1 mice²⁵ and intraspinal injection of ethidium bromide in rats,¹⁰ have been used. Consistently demyelination was followed by increased mitotic activity with subsequent generation of astrocytes and oligodendrocytes and spontaneous remyelination and astrogliosis.^10,25,27Paracrine signaling involving oligodendrocytes and axons has been implicated in determination of the number of oligodendrocytes required to myelinate a population of axons in an area of CNS;³ therefore, dysmyelinated rodent models can afford us insight into cellular mechanisms of compensation to hypomyelination.^4,49 The failure of the jimpy (jp) mouse, which carries a mutation in proteolipid protein,⁴⁷ to generate a normal amount of myelin results in severe hypomyelination due to oligodendrocyte dysfunction^19,29 and coincides with increased proliferation of cells of oligodendrocyte lineage that is balanced by increased oligodendrocyte death.^2,9,18,47,53 Vigorous proliferation of glial cell progenitors in the spinal cord and optic nerve of normal mice declines postpartum and is arrested or negligible by the third week of life.⁴⁶ In contrast, immature jimpy glial cells show even more robust proliferation in the neonatal life, as measured by intranuclear internalization of tritiated thymidine ([³H]-TdR); this proliferation declines, as in glial cells of normal mice, but is still remarkable by the week 3.⁴⁶ In the spinal cord of myelin-deficient (md) rats, another severely dysmyelinated mutant strain with abnormal proteolipid protein, the proliferation of glial cells predominantly of oligodendrocyte lineage was increased as in jimpy cells, and inhibition of this proliferation was delayed until the third week postpartum.²⁴ Despite increased proliferation of oligodendrocytes during the postnatal period, the number of oligodendrocytes markedly declined in relation to oligodendrocyte apoptosis in a longer-surviving substrain of myelin-deficient rats¹⁴ suggesting the lack or insufficient proliferation of oligodendrocyte progenitors to counteract cell death. In the optic nerve of the Long Evans Shaker (LES) rat, a severely dysmyelinated but long-surviving rat with a mutation in myelin basic protein,³⁰ proliferation of glial cells is enhanced and their inhibition delayed, resulting in increased numbers of glial cells predominantly of oligodendrocyte morphology.²² Inhibition of proliferation of oligodendrocyte progenitors was delayed in the spinal cord of shiverer (shi) mouse, another rat mutant in myelin basic protein, coinciding with a remarkable increase in oligodendrocyte numbers.⁶ Although the molecular mechanisms regulating increased proliferation of oligodendrocyte progenitors and delayed inhibition of this proliferation in the postnatal period are unknown, a failure of myelination is considered to induce the proliferative response⁴In severely dysmyelinated mutants such as the shiverer mouse and LES rat, oligodendrocytes develop pathology characterized by accumulation of a membraneous material which forms vesicles limited by a pentalamellar membrane where at regular intervals 3 electrodense lines are separated by 2 electrolucent lines.^13,22 Analysis of electron micrographs of degenerating oligodendrocytes in cases of Pelizaeus–Merzbacher disease revealed that vesicles have limiting membrane with multiple dense lines regularly spaced at 5.8 nm ⁵⁰ Similar vesicles in degenerating oligodendrocytes have been observed in jimpy mice and LES and Long Evans Bouncer (LE-bo) rats, in which the dense lines in the vesicle walls are spaced at approximately 5-nm intervals.^21,22 Although the membraneous material has not been analyzed chemically yet, its morphology including the regular periodicity of dense lines and formation of vesicles suggest a lipid-rich material whose hydrophobic properties induce it to form vesicles in the aqueous environment⁴⁸ of the mutated oligodendrocytes. In comparison, the periodicity of dense lines in the CNS myelin sheath averages 15 nm.²³Lack of myelin in the CNS coincides with astrogliosis, resulting in the enveloping of small bundles of naked axons by hypertrophied astrocyte processes, which effectively separates the axonal bundles from each other in jimpy¹⁹ and shiverer mice^19,33,42 and LES²² and old taiep rats (which progressively lose CNS myelin).²⁸ Review of electron micrographs in published literature revealed that small (less than 0.3 μm) structures containing neurotubules and neurofilaments, which are considered to be axonal sprouts, are common in dysmyelinated CNS in jimpy¹⁹ and shiverer^17,33 mice and LES³² and old taiep²⁸ rats. Although the precise mechanisms of astrogliosis in which hypertrophied astrocyte processes separate bundles of naked axons are unknown, this reaction can be considered compensatory for lack of myelin and an attempt to isolate naked axons. Axonal sprouting, indicative of axonal plasticity in a severely dysmyelinated environment, has been demonstrated in the optic nerve of mature LES rats.³² This observation supports the notion of inhibition of axonal plasticity and regeneration in presence of CNS myelin^7,11 but perhaps not in presence of hypertrophied astroglial processes; this finding also indicates the usefulness of dysmyelinated animals in in vivo studies of axonal regeneration relevant to spinal cord injury.The present study was undertaken to analyze the natural progression of neuropathology in the CNS of 2 severely dysmyelinated long-lived mutant rat strains, LES and LE-bo, and to characterize the mechanisms of cellular response to lack of myelin.