Macrophage Migration Inhibitory Factor Deficiency Ameliorates High-Fat Diet Induced Insulin Resistance in Mice with Reduced Adipose Inflammation and Hepatic Steatosis

Macrophage infiltration is a critical determinant of high-fat diet induced adipose tissue inflammation and insulin resistance. The precise mechanisms underpinning the initiation of macrophage recruitment and activation are unclear. Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, displays chemokine-like properties. Circulating MIF levels are elevated during obesity however its role in high-fat diet induced adipose inflammation and insulin resistance remains elusive. Wildtype and MIF^−/− C57Bl\6J mice were fed chow or high-fat diet. Body weight and food intake was assessed. Glucose homeostasis was monitored by glucose and insulin tolerance tests. Adipose tissue macrophage recruitment and adipose tissue insulin sensitivity was evaluated. Cytokine secretion from stromal vascular fraction, adipose explants and bone marrow macrophages was measured. Inflammatory signature and insulin sensitivity of 3T3-L1-adipocytes co-cultured with wildtype and MIF^−/− macrophage was quantified. Hepatic triacylglyceride levels were assessed. MIF^−/− exhibited reduced weight gain. Age and weight-matched obese MIF^−/− mice exhibited improved glucose homeostasis coincident with reduced adipose tissue M1 macrophage infiltration. Obese MIF^−/− stromal vascular fraction secreted less TNFα and greater IL-10 compared to wildtype. Activation of JNK was impaired in obese MIF^−/−adipose, concomitant with pAKT expression. 3T3-L1-adipocytes cultured with MIF^−/− macrophages had reduced pro-inflammatory cytokine secretion and improved insulin sensitivity, effects which were also attained with MIF inhibitor ISO-1. MIF^−/− liver exhibited reduced hepatic triacyglyceride accumulation, enhanced pAKT expression and reduced NFκB activation. MIF deficiency partially protects from high-fat diet induced insulin resistance by attenuating macrophage infiltration, ameliorating adipose inflammation, which improved adipocyte insulin resistance ex vivo. MIF represents a potential therapeutic target for treatment of high-fat diet induced insulin resistance.     

MIF synergizes with Trypanosoma cruzi antigens to promote efficient dendritic cell maturation and IL-12 production via p38 MAPK

Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of the MIF-dependent responses to Trypanosoma cruzi, we investigated host resistance in MIF-/- mice (on the BALB/c background) during an intraperitoneal infection. We focused on the potential involvement of MIF in dendritic cell (DC) maturation and cytokine production. Following a challenge with 5 x 10³ T. cruzi parasites, wild type (WT) mice developed a strong IL-12 response and adequate maturation of the draining mesenteric lymph node DCs and were resistant to infection. In contrast, similarly infected MIF^-/- mice mounted a weak IL-12 response, displayed immature DCs in the early phases of infection and rapidly succumbed to T. cruzi infection. The lack of maturation and IL-12 production by the DCs in response to total T. cruzi antigen (TcAg) was confirmed by in vitro studies. These effects were reversed following treatment with recombinant MIF. Interestingly, TcAg-stimulated bone marrow-derived DCs from both WT and MIF^-/- mice had increased ERK1/2 MAPK phosphorylation. In contrast, p38 phosphorylation was only upregulated in WT DCs. Reconstitution of MIF to MIF^-/- DCs upregulated p38 phosphorylation. The MIF-p38 pathway affected MHC-II and CD86 expression as well as IL-12 production. These findings demonstrate that the MIF-induced early DC maturation and IL-12 production mediates resistance to T. cruzi infection, probably by activating the p38 pathway.     

Behavioral Phenotyping of Parkin-Deficient Mice: Looking for Early Preclinical Features of Parkinson's Disease

There is considerable evidence showing that the neurodegenerative processes that lead to sporadic Parkinson''s disease (PD) begin many years before the appearance of the characteristic motor symptoms. Neuropsychiatric, sensorial and cognitive deficits are recognized as early non-motor manifestations of PD, and are not attenuated by the current anti-parkinsonian therapy. Although loss-of-function mutations in the parkin gene cause early-onset familial PD, Parkin-deficient mice do not display spontaneous degeneration of the nigrostriatal pathway or enhanced vulnerability to dopaminergic neurotoxins such as 6-OHDA and MPTP. Here, we employed adult homozygous C57BL/6 mice with parkin gene deletion on exon 3 (parkin ^−/−) to further investigate the relevance of Parkin in the regulation of non-motor features, namely olfactory, emotional, cognitive and hippocampal synaptic plasticity. Parkin ^−/− mice displayed normal performance on behavioral tests evaluating olfaction (olfactory discrimination), anxiety (elevated plus-maze), depressive-like behavior (forced swimming and tail suspension) and motor function (rotarod, grasping strength and pole). However, parkin ^−/− mice displayed a poor performance in the open field habituation, object location and modified Y-maze tasks suggestive of procedural and short-term spatial memory deficits. These behavioral impairments were accompanied by impaired hippocampal long-term potentiation (LTP). These findings indicate that the genetic deletion of parkin causes deficiencies in hippocampal synaptic plasticity, resulting in memory deficits with no major olfactory, emotional or motor impairments. Therefore, parkin ^−/− mice may represent a promising animal model to study the early stages of PD and for testing new therapeutic strategies to restore learning and memory and synaptic plasticity impairments in PD.     

MIF induces osteoclast differentiation and contributes to progression of periodontal disease in mice

Periodontal disease (PD) is a chronic inflammatory and alveolar bone destructive disease triggered by microorganisms from the oral biofilm. Oral inoculation of mice with the periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) induces marked alveolar bone loss and local production of inflammatory mediators, including Macrophage Migration Inhibitory Factor (MIF). The role of MIF for alveolar bone resorption during PD is not known. In the present study, experimental PD was induced in BALB/c wild-type mice (WT) and MIF knockout mice (MIF^?/?) through oral inoculation of Aa. Despite enhanced number of bacteria, MIF^?/? mice had reduced infiltration of TRAP-positive cells and reduced alveolar bone loss. This was associated with decreased neutrophil accumulation and increased levels of IL-10 in periodontal tissues. TNF-α production was similar in both groups. In vitro, LPS from Aa enhanced osteoclastic activity in a MIF-dependent manner. In conclusion, MIF has role in controlling bacterial growth in the context of PD but contributes more significantly to the progression of bone loss during PD by directly affecting differentiation and activity of osteoclasts.     

Serpina3c protects against high-fat diet-induced pancreatic dysfunction through the JNK-related pathway

BackgroundSerpina3 is a member of the serine protease inhibitor family and is involved in the inflammatory response. In this study, we investigated the effect of Serpina3c on pancreatic function in hypercholesterolemic mice.MethodsTo investigate the role of Serpina3c in hyperlipidaemia, Serpina3c knockout mice were bred with Apoe-knockout mice (on a C57BL/6 background) to generate heterozygous Serpina3c-Apoe double knockout (Serpina3c^+/−/Apoe^+/−) mice and were then bred to obtain homozygotes. C57BL/6, Serpina3c^−/−, Apoe^−/−, and Apoe^−/-Serpina3c^−/− mice were fed normal chow, and Apoe^−/− and Apoe^−/-Serpina3c^−/− mice were fed a high-fat diet (HFD). After feeding for 3 months, the mice were monitored for body weight, blood glucose, glucose tolerance, and insulin tolerance test (ITT). ELISA and immunohistochemistry were used to detect insulin levels and glucagon expression. Immunohistochemical staining for macrophages in the pancreas was also performed. Western blot analysis was performed on pancreatic tissues to detect the protein levels of insulin-associated molecules, the metalloproteinase MMP2, the tissue inhibitor TIMP2 and components of the JNK-related pathway.ResultsBlood glucose levels, glucose tolerance, and ITT were not significantly different among the groups. Serpina3c knockout resulted in blood lipid abnormalities in mice under HFD conditions. Insulin secretion was decreased in Apoe^−/-Serpina3c^−/− mice compared with Apoe^−/− mice under normal chow conditions. In addition, Apoe^−/-Serpina3c^−/− mice exhibited increased insulin and glucagon secretion and expression after three months of HFD feeding, but insulin secretion was decreased in Apoe^−/-Serpina3c^−/− mice compared with Apoe^−/− mice after the fifth month of HFD feeding. Serpina3c knockout increased MMP2 protein levels, whereas TIMP2 levels in the pancreas were decreased. Furthermore, Serpina3c knockout significantly upregulated the number of macrophages in the pancreas under HFD conditions. The JNK/AKT/FOXO1/PDX-1 axis was found to be involved in Serpina3c-regulated insulin secretion.ConclusionThese novel findings show that Serpina3c could play a protective role in insulin secretion partly through the JNK-related pathway under HFD conditions.     

The effect of PTZ-induced epileptic seizures on hippocampal expression of PSA-NCAM in offspring born to kindled rats

Background

Maternal epileptic seizures during pregnancy can affect the hippocampal neurons in the offspring. The polysialylated neural cell adhesion molecule (PSA-NCAM), which is expressed in the developing central nervous system, may play important roles in neuronal migration, synaptogenesis, and axonal outgrowth. This study was designed to assess the effects of kindling either with or without maternal seizures on hippocampal PSA-NCAM expression in rat offspring.

Methods

Forty timed-pregnant Wistar rats were divided into four groups: A) Kind⁺/Seiz⁺, pregnant kindled (induced two weeks prior to pregnancy) rats that received repeated intraperitoneal (i.p.) pentylenetetrazol, PTZ injections on gestational days (GD) 14-19; B) Kind^-/Seiz⁺, pregnant non-kindled rats that received PTZ injections on GD14-GD19; C) Kind⁺/Seiz^-, pregnant kindled rats that did not receive any PTZ injections; and D) Kind^-/Seiz^-, the sham controls. Following birth, the pups were sacrificed on PD1 and PD14, and PSA-NCAM expression and localization in neonates’ hippocampi were analyzed by Western blots and immunohistochemistry.

Results

Our data show a significant down regulation of hippocampal PSA-NCAM expression in the offspring of Kind⁺/Seiz⁺ (p = 0.001) and Kind^-/Seiz⁺ (p = 0.001) groups compared to the sham control group. The PSA-NCAM immunoreactivity was markedly decreased in all parts of the hippocampus, especially in the CA3 region, in Kind⁺/Seiz⁺ (p = 0.007) and Kind^-/Seiz⁺ (p = 0.007) group’s newborns on both PD1 and 14.

Conclusion

Our findings demonstrate that maternal seizures but not kindling influence the expression of PSA-NCAM in the offspring’s hippocampi, which may be considered as a factor for learning/memory and cognitive impairments reported in children born to epileptic mothers.     

Dissociated Fear and Spatial Learning in Mice with Deficiency of Ataxin-2

Mouse models with physiological and behavioral differences attributable to differential plasticity of hippocampal and amygdalar neuronal networks are rare. We previously generated ataxin-2 (Atxn2) knockout mice and demonstrated that these animals lacked obvious anatomical abnormalities of the CNS, but showed marked obesity and reduced fertility. We now report on behavioral changes as a consequence of Atxn2-deficiency. Atxn2-deficiency was associated with impaired long-term potentiation (LTP) in the amygdala, but normal LTP in the hippocampus. Intact hippocampal plasticity was associated behaviorally with normal Morris Water maze testing. Impaired amygdala plasticity was associated with reduced cued and contextual fear conditioning. Conditioned taste aversion, however, was normal. In addition, knockout mice showed decreased innate fear in several tests and motor hyperactivity in open cage testing. Our results suggest that Atxn2-deficiency results in a specific set of behavioral and cellular disturbances that include motor hyperactivity and abnormal fear-related behaviors, but intact hippocampal function. This animal model may be useful for the study of anxiety disorders and should encourage studies of anxiety in patients with spinocerebellar ataxia type 2 (SCA2).     

Anxiety and increased 5‐HT1A receptor response in NCAM null mutant mice

Mice deficient in the neural cell adhesion molecule (NCAM) show behavioral abnormalities as adults, including altered exploratory behavior, deficits in spatial learning, and increased intermale aggression. Here, we report increased anxiety‐like behavior of homozygous (NCAM^−/−) and heterozygous (NCAM^+/−) mutant mice in a light/dark avoidance test, independent of genetic background and gender. Anxiety‐like behavior was reduced in both NCAM^+/+ and NCAM^−/− mice by systemic administration of the benzodiazepine agonist diazepam and the 5‐HT_1A receptor agonists buspirone and 8‐OH‐DPAT. However, NCAM^−/− mice showed anxiolytic‐like effects at lower doses of buspirone and 8‐OH‐DPAT than NCAM^+/+ mice. Such increased response to 5‐HT_1A receptor stimulation suggests a functional change in the serotonergic system of NCAM^−/− mice, likely involved in the control of anxiety and aggression. However, 5‐HT_1A receptor binding and tissue content of serotonin and its metabolite 5‐hydroxyindolacetic acid were found unaltered in every brain area of NCAM^−/− mice investigated, indicating that expression of 5‐HT_1A receptors as well as synthesis and release of serotonin are largely unchanged in NCAM^−/− mice. We hypothesize a critical involvement of endogenous NCAM in serotonergic transmission via 5‐HT_1A receptors and inwardly rectifying K⁺ channels as the respective effector systems. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 343–355, 1999     

Adipose monocyte chemotactic protein-1 deficiency reduces high-fat diet-enhanced mammary tumorigenesis in MMTV-PyMT mice

Monocyte chemotactic protein-1 (MCP-1) is an adipokine with demonstrated carcinogenic potential. However, there is a lack of evidence whether adipose-produced MCP-1 contributes to breast cancer. We tested the hypothesis that adipose-produced MCP-1 contributes to mammary tumorigenesis in this study. In a breast cancer model of mouse mammary tumor virus-polyomavirus middle T-antigen (MMTV-PyMT), mice with or without adipose MCP-1 knockout [designated as Mcp-1^−/− or wild-type (WT)] were fed the standard AIN93G diet (16% of energy from soybean oil) or a high-fat diet (HFD, 45% of energy from soybean oil). Adipose MCP-1 knockout reduced Mcp-1 expression in adipose tissue and concentrations of MCP-1 in plasma. Mcp-1^−/− mice fed the HFD had less body fat than their WT counterparts. Adipose MCP-1 knockout attenuated HFD-enhanced mammary tumorigenesis, evidenced by lower mammary tumor volume. Furthermore, Mcp-1^−/− mice, regardless of diet, had a longer tumor latency and less tumor weight than WT mice. When fed the HFD, Mcp-1^−/− mice, compared to WT mice, exhibited lower concentrations of insulin, leptin, resistin, vascular endothelial growth factor and hepatic growth factor in plasma. In summary, adipose MCP-1 deficiency attenuated HFD-enhanced MMTV-PyMT mammary tumorigenesis. This attenuation can be attributed to less body adiposity, improvement in insulin sensitivity and down-regulation in protumorigenic inflammation cytokines and angiogenic factors in Mcp-1^−/− mice. It concludes that adipose-produced MCP-1 contributes to mammary tumorigenesis in the MMTV-PyMT mouse model.     

Macrophage migration inhibitory factor (MIF): Genetic evidence for participation in early onset and early stage rheumatoid arthritis

Macrophage migration inhibitory factor (MIF) is an upstream pro-inflammatory cytokine that is associated with the pathogenesis of autoimmune inflammatory diseases including rheumatoid arthritis (RA). Two polymorphisms in the upstream region exist in the MIF gene and are associated with RA susceptibility or severity in different populations. In this case-control study, we investigated whether MIF polymorphisms are associated with RA susceptibility or activity in a western Mexican population .The relationship of MIF levels with clinical features of disease also was assessed. Genotyping of the ?794 CATT_5–8 (rs5844572) and the ?173 G > C (rs755622) polymorphisms was performed by PCR and PCR-RFLP respectively on 226 RA patients and 210 healthy subjects. Serum MIF levels were determined by ELISA. We found a significant association between the ?794 CATT_5–8 6,7 MIF genotype with RA. Moreover, we detected an association between the ?794 CATT₇ allele with early onset RA. The ?794 CATT₇ and ?173^*C alleles, which are in linkage disequilibrium, were associated with high disease activity on RA patients. A positive correlation between circulating MIF levels and C-reactive protein, erythrocyte sedimentation rate, rheumatoid factor, anti-citrullinated protein/peptides antibodies and TNFα was detected. MIF levels appear to be associated with disease progression rather than disease activity, which is distinct from the established relationship between disease activity and TNFα levels. In conclusion, the MIF gene and protein are associated with RA in a western Mexican population, with a main contribution onto early onset and early stages of disease.     

BDNF produced by cerebral microglia promotes cortical plasticity and pain hypersensitivity after peripheral nerve injury

Peripheral nerve injury–induced mechanical allodynia is often accompanied by abnormalities in the higher cortical regions, yet the mechanisms underlying such maladaptive cortical plasticity remain unclear. Here, we show that in male mice, structural and functional changes in the primary somatosensory cortex (S1) caused by peripheral nerve injury require neuron-microglial signaling within the local circuit. Following peripheral nerve injury, microglia in the S1 maintain ramified morphology and normal density but up-regulate the mRNA expression of brain-derived neurotrophic factor (BDNF). Using in vivo two-photon imaging and Cx3cr1^CreER;Bdnf^flox mice, we show that conditional knockout of BDNF from microglia prevents nerve injury–induced synaptic remodeling and pyramidal neuron hyperactivity in the S1, as well as pain hypersensitivity in mice. Importantly, S1-targeted removal of microglial BDNF largely recapitulates the beneficial effects of systemic BDNF depletion on cortical plasticity and allodynia. Together, these findings reveal a pivotal role of cerebral microglial BDNF in somatosensory cortical plasticity and pain hypersensitivity.

This study reveals that brain-derived neurotrophic factor (BDNF) from cerebral microglia contributes to nerve injury-induced synaptic remodeling and neuronal hyperactivity, and ultimately contributes to pain sensitivity in mice; removal of microglial BDNF has beneficial effects on cortical plasticity and pain.     

Regulation of glucose homeostasis by KSR1 and MARK2

Protein scaffolds control the intensity and duration of signaling and dictate the specificity of signaling through MAP kinase pathways. KSR1 is a molecular scaffold of the Raf/MEK/ERK MAP kinase cascade that regulates the intensity and duration of ERK activation. Relative to wild-type mice, ksr1^-/- mice are modestly glucose intolerant, but show a normal response to exogenous insulin. However, ksr1^-/- mice also demonstrate a three-fold increase in serum insulin levels in response to a glucose challenge, suggesting a role for KSR1 in insulin secretion. The kinase MARK2 is closely related to C-TAK1, a known regulator of KSR1. Mice lacking MARK2 have an increased rate of glucose disposal in response to exogenous insulin, increased glucose tolerance, and are resistant to diet-induced obesity. mark2^-/-ksr1^-/- (DKO) mice were compared to wild type, mark2^-/-, and ksr1^-/- mice for their ability to regulate glucose homeostasis. Here we show that disruption of KSR1 in mark2^-/- mice reverses the increased sensitivity to exogenous insulin resulting from MARK2 deletion. DKO mice respond to exogenous insulin similarly to wild type and ksr1^-/- mice. These data suggest a model whereby MARK2 negatively regulates insulin sensitivity in peripheral tissue through inhibition of KSR1. Consistent with this model, we found that MARK2 binds and phosphorylates KSR1 on Ser392. Phosphorylation of Ser392 is a critical regulator of KSR1 stability, subcellular location, and ERK activation. These data reveal an unexpected role for the molecular scaffold KSR1 in insulin-regulated glucose metabolism.     

Polμ deficiency induces moderate shortening of P53−/− mouse lifespan and modifies tumor spectrum

Non-homologous end joining (NHEJ) is the main mechanism for double strand break (DSB) DNA repair. The error-prone DNA polymerase mu (Polμ) is involved in immunoglobulin variable region rearrangement and in general, NHEJ in non-lymphoid cells. Deletion of NHEJ factors in P53^−/− mice, which are highly prone to development of T cell lymphoma, generally increases cancer incidence and shifts the tumor spectrum towards aggressive pro-B lymphoma. In contrast, Polμ deletion increased sarcoma incidence, proportionally reducing pro-B lymphoma development on the P53-deficient background. Array comparative genomic hybridization (aCGH) analyses showed DNA copy number alterations in both P53^−/− and Polμ^−/−P53^−/− lymphomas. Our results also indicate that the increase in sarcoma incidence in Polμ^−/−P53^−/− mice could be associated with Cdk4 and Kub3 amplification and overexpression. These results identify a role for Polμ in the prevention of sarcomagenesis on a murine P53-deficient background, in contrast to most other NHEJ factors.     

Hepatocyte-specific lysosomal acid lipase deficiency protects mice from diet-induced obesity but promotes hepatic inflammation

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CE) and triglycerides (TG) to generate fatty acids (FA) and cholesterol. LAL deficiency (LAL-D) in both humans and mice leads to hepatomegaly, hypercholesterolemia, and shortened life span. Despite its essential role in lysosomal neutral lipid catabolism, the cell type-specific contribution of LAL to disease progression is still elusive. To investigate the role of LAL in the liver in more detail and to exclude the contribution of LAL in macrophages, we generated hepatocyte-specific LAL-deficient mice (Liv-Lipa^−/−) and fed them either chow or high fat/high cholesterol diets (HF/HCD). Comparable to systemic LAL-D, Liv-Lipa^−/− mice were resistant to diet-induced obesity independent of food intake, movement, and energy expenditure. Reduced body weight gain was mainly due to reduced white adipose tissue depots. Furthermore, Liv-Lipa^−/− mice exhibited improved glucose clearance during glucose and insulin tolerance tests compared to control mice. Analysis of hepatic lipid content revealed a massive reduction of TG, whereas CE concentrations were markedly increased, leading to CE crystal formation in the livers of Liv-Lipa^−/− mice. Elevated plasma transaminase activities, increased pro-inflammatory cytokines and chemokines as well as hepatic macrophage infiltration indicated liver inflammation. Our data provide evidence that hepatocyte-specific LAL deficiency is sufficient to alter whole-body lipid and energy homeostasis in mice. We conclude that hepatic LAL plays a pivotal role by preventing liver damage and maintaining lipid and energy homeostasis, especially during high lipid availability.     

Lipodystrophic gene Agpat2 deficiency aggravates hyperlipidemia and atherosclerosis in Ldlr−/− mice

AimsDysfunction of adipose tissue increases the risk of cardiovascular disease. It was well established that obesity aggravates atherosclerosis, but the effect of adipose tissue loss on atherosclerosis has been less studied. AGPAT2 is the first causative gene of congenital generalized lipodystrophy (CGL), but the role of AGPAT2 on atherosclerosis has not been reported. Hypertriglyceridemia is one of the clinical manifestations of CGL patients, but it is usually absent in CGL mouse model on a normal diet. This study will investigate the effect of Agpat2 on hyperlipidemia and atherosclerosis.Methods and resultsIn this study, Agpat2 knockout (Agpat2^−/−) mice were generated using CRISPR/Cas system, which showed severe loss of adipose tissue and fatty liver, consistent with previous reports. Agpat2^−/− mice were then crossed with hypercholesterolemic and atherosclerotic prone LDL receptor knockout (Ldlr^−/−) mice to obtain double knockout mouse model (Agpat2^−/−Ldlr^−/−). Plasma lipid profile, insulin resistance, fatty liver, and atherosclerotic lesions were observed after 12 weeks of the atherogenic high-fat diet (HFD) feeding. We found that compared with Ldlr^−/− mice, Agpat2^−/−Ldlr^−/− mice showed significantly higher plasma total cholesterol and triglycerides after HFD feeding. Agpat2^−/−Ldlr^−/− mice also developed hyperglycemia and hyperinsulinemia, with increased pancreatic islet area. The liver weight of Agpat2^−/−Ldlr^−/− mice was about 4 times higher than that of Ldlr^−/− mice. The liver lipid deposition was severe and Sirius red staining showed liver fibrosis. In addition, in Agpat2^−/−Ldlr^−/− mice, the area of atherosclerotic lesions in aortic arch and aortic root was significantly increased.ConclusionsOur results show that Agpat2 deficiency led to more severe hyperlipidemia, liver fibrosis and aggravation of atherosclerosis in Ldlr^−/− mice. This study provided additional insights into the role of adipose tissue in hyperlipidemia and atherosclerosis.     

Genetic deletion of the neuronal glutamate transporter,EAAC1, results in decreased neuronal death after pilocarpine-induced status epilepticus

Excitatory amino acid carrier 1 (EAAC1 also called EAAT3) is a Na⁺-dependent glutamate transporter expressed by both glutamatergic and GABAergic neurons. It provides precursors for the syntheses of glutathione and GABA and contributes to the clearance of synaptically released glutamate. Mice deleted of EAAC1 are more susceptible to neurodegeneration in models of ischemia, Parkinson’s disease, and aging. Antisense knock-down of EAAC1 causes an absence seizure-like phenotype. Additionally, EAAC1 expression increases after chemonvulsant-induced seizures in rodent models and in tissue specimens from patients with refractory epilepsy. The goal of the present study was to determine if the absence of EAAC1 affects the sensitivity of mice to seizure-induced cell death. A chemoconvulsant dose of pilocarpine was administered to EAAC1^−/− mice and to wild-type controls. Although EAAC1^−/− mice experienced increased latency to seizure onset, no significant differences in behavioral seizure severity or mortality were observed. We examined EAAC1 immunofluorescence 24 h after pilocarpine administration and confirmed that pilocarpine causes an increase in EAAC1 protein. Forty-eight hours after induction of seizures, cell death was measured in hippocampus and in cortex using Fluoro-Jade C. Surprisingly, there was ∼2-fold more cell death in area CA1 of wild-type mice than in the corresponding regions of the EAAC1^−/− mice. Together, these studies indicate that absence of EAAC1 results in either a decrease in pilocarpine-induced seizures that is not detectable by behavioral criteria (surprising, since EAAC1 provides glutamate for GABA synthesis), or that the absence of EAAC1 results in less pilocarpine/seizure-induced cell death, possible explanations as discussed.     

Inducible nitric oxide synthase-deficient mice show exacerbated inflammatory process and high production of both Th1 and Th2 cytokines during paracoccidioidomycosis

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by fungus Paracoccidioides brasiliensis. To analyze the influence of inducible nitric oxide synthase (iNOS) in this disease, iNOS-deficient (iNOS^−/−) and wild-type (WT) mice were infected intravenously with P. brasiliensis 18 isolate. We found that, unlike WT mice, iNOS^−/− mice did not control fungal proliferation, and began to succumb to infection by day 50 after inoculation of yeast cells. Typical inflammatory granulomas were found in WT mice, while, iNOS^−/− mice presented incipient granulomas with intense inflammatory process and necrosis. Additionally, splenocytes from iNOS^−/− mice did not produce nitric oxide, however, their proliferative response to Con-A was impaired, just like infected WT mice. Moreover, infected iNOS^−/− mice presented a mixed pattern of immune response, releasing high levels of both Th1 (IL-12, IFN-γ and TNF-α) and Th2 (IL-4 and IL-10) cytokines. These data suggest that the enzyme iNOS is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, by influencing cytokines production, and by appeasing the development of a high inflammatory response and consequently formation of necrosis. However, iNOS-derived nitric oxide seems not being the unique factor responsible for immunosuppression observed in infections caused by P. brasiliensis.     

IGF‐I deficient mice show reduced peripheral nerve conduction velocities and decreased axonal diameters and respond to exogenous IGF‐I treatment

Although insulin‐like growth factor‐I (IGF‐I) can act as a neurotrophic factor for peripheral neurons in vitro and in vivo following injury, the role IGF‐I plays during normal development and functioning of the peripheral nervous system is unclear. Here, we report that transgenic mice with reduced levels (two genotypes: heterozygous Igf1^+/− or homozygous insertional mutant Igf1^m/m) or totally lacking IGF‐I (homozygous Igf1^−/−) show a decrease in motor and sensory nerve conduction velocities in vivo. In addition, A‐fiber responses in isolated peroneal nerves from Igf1^+/− and Igf1^−/− mice are impaired. The nerve function impairment is most profound in Igf1^−/− mice. Histopathology of the peroneal nerves in Igf1^−/− mice demonstrates a shift to smaller axonal diameters but maintains the same total number of myelinated fibers as Igf1^+/+ mice. Comparisons of myelin thickness with axonal diameter indicate that there is no significant reduction in peripheral nerve myelination in IGF‐I–deficient mice. In addition, in Igf1^m/m mice with very low serum levels of IGF‐I, replacement therapy with exogenous recombinant hIGF‐I restores both motor and sensory nerve conduction velocities. These findings demonstrate not only that IGF‐I serves an important role in the growth and development of the peripheral nervous system, but also that systemic IGF‐I treatment can enhance nerve function in IGF‐I–deficient adult mice. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 142–152, 1999