Lack of zinc finger protein 521 upregulates dopamine ß-hydroxylase expression in the mouse brain, leading to abnormal behavior.

AIM: Previously, we reported that mice deficient in most of the Zfp521 coding region (Zfp521Δ/Δ mice) displayed abnormal behaviors, including hyperlocomotion and lower anxiety. In this study, we aimed to elucidate the involvement and mechanisms of monoamine variation. MAIN METHODS: First, we compared the levels of dopamine (DA), noradrenaline (NA), and serotonin in the brains of Zfp521Δ/Δ and Zfp521+/+ mice using enzyme-linked immunosorbent assay. Next, we elucidated the mechanisms using quantitative PCR and Western Blotting. Additionally, we administered inhibitory drug to the mice and performed behavioral tests. KEY FINDINGS: Our results showed that the DA level decreased and the NA level increased in Zfp521Δ/Δ mice. We found that ZFP521 suppresses the expression of dopamine ß-hydroxylase (DBH), which converts DA into NA. We also demonstrated that paired homeodomain transcription factor 2 and early growth response protein-1, which are the transcription factors for Dbh, were involved in the upregulation of Dbh by ZFP521. The administration of nepicastat, a specific inhibitor of DBH, attenuated the abnormal behaviors of Zfp521Δ/Δ mice. SIGNIFICANCE: These results suggest that the lack of ZFP521 upregulates the expression of DBH, which leads to a decrease in the DA level and an increase in the NA level in the brain, resulting in abnormal behaviors.

Transferrin receptor 1 is required for enucleation of mouse erythroblasts during terminal differentiation.

Enucleation is the process whereby the nucleus is extruded from the erythroblast during late stage mammalian erythropoiesis. However, the specific signaling pathways involved in this process remain unclear. To better understand the mechanisms underlying erythroblast enucleation, we investigated erythroblast enucleation using both the spleens of adult mice with phenylhydrazine-induced anemia and mouse fetal livers. Our results indicated that both iron-bound transferrin (holo-Tf) and the small-molecule iron transporter hinokitiol with iron ions (hinokitiol plus iron) promote hemoglobin synthesis and the enucleation of mouse spleen-derived erythroblasts. Although an antitransferrin receptor 1 (TfR1) monoclonal antibody inhibited both enucleation and hemoglobin synthesis promoted by holo-Tf, it inhibited only enucleation, but not hemoglobin synthesis, promoted by hinokitiol plus iron. Furthermore, siRNA against mouse TfR1 were found to suppress the enucleation of mouse fetal liver-derived erythroblasts, and the endocytosis inhibitor MitMAB inhibited enucleation, hemoglobin synthesis, and the internalization of TfR1 promoted by both types of stimuli. Collectively, our results suggest that TfR1, iron ions, and endocytosis play important roles in mouse erythroblast enucleation.

Abnormal behaviors and developmental disorder of hippocampus in zinc finger protein 521 (ZFP521) mutant mice.

Zinc finger protein 521 (ZFP521) regulates a number of cellular processes in a wide range of tissues, such as osteoblast formation and adipose commitment and differentiation. In the field of neurobiology, it is reported to be an essential factor for transition of epiblast stem cells into neural progenitors in vitro. However, the role of ZFP521 in the brain in vivo still remains elusive. To elucidate the role of ZFP521 in the mouse brain, we generated mice lacking exon 4 of the ZFP521 gene. The birth ratio of our ZFP521Δ/Δ mice was consistent with Mendel's laws. Although ZFP521Δ/Δ pups had no apparent defect in the body and were indistinguishable from ZFP521+/+ and ZFP521+/Δ littermates at the time of birth, ZFP521Δ/Δ mice displayed significant weight reduction as they grew, and most of them died before 10 weeks of age. They displayed abnormal behavior, such as hyper-locomotion, lower anxiety and impaired learning, which correspond to the symptoms of schizophrenia. The border of the granular cell layer of the dentate gyrus in the hippocampus of the mice was indistinct and granular neurons were reduced in number. Furthermore, Sox1-positive neural progenitor cells in the dentate gyrus and cerebellum were significantly reduced in number. Taken together, these findings indicate that ZFP521 directly or indirectly affects the formation of the neuronal cell layers of the dentate gyrus in the hippocampus, and thus ZFP521Δ/Δ mice displayed schizophrenia-relevant symptoms. ZFP521Δ/Δ mice may be a useful research tool as an animal model of schizophrenia.

Accelerated destruction of erythrocytes in Tie2 promoter-driven STAT3 conditional knockout mice.

AIMS: STAT3 is a key modulator of activation and differentiation of macrophages. But it is still unknown if deficiency of STAT3 activates macrophages to destroy erythrocytes by phagocytosis. We generated STAT3 conditional knockout mice by crossing floxed STAT3 mice with Tie2 promoter-driven Cre-recombinase transgenic mice and clarified that Stat3 plays a critical role in the formation and activation of macrophages. MAIN METHODS: Blood cell count, reticulocyte count, serum lactate dehydrogenase, erythropoietin, iron and ferritin concentration, and life span of the erythrocytes in Tie2 promoter-driven STAT3 conditional knockout mice were analyzed. To explore the erythropoietic function of the mice, we subjected them to brief hemolytic anemia by injecting them intraperitoneally with phenylhydrazine. The fragility of erythrocytes was examined by scanning electron microscopy and osmotic tolerance test. KEY FINDINGS: The conditional knockout mice had mild normocytic anemia. They also displayed higher lactate dehydrogenase, ferritin and erythropoietin concentration, higher reticulocyte count, and a shorter lifespan of erythrocytes compared with wild-type controls. These data suggest that destruction of erythrocytes and secondary blood formation were accelerated in the STAT3 conditional knockout mice. It didn't appear due to the fragility of erythrocytes. A few of the conditional knockout mice suddenly developed acute severe anemia, high body temperature and massive splenomegaly, and died within 2weeks after the onset of anemia. SIGNIFICANCE: This study provided evidence that STAT3 have a critical role in the destruction of erythrocytes by resident macrophages in the spleen.

Essential role of p38 MAPK in caspase-independent, iPLA(2)-dependent cell death under hypoxia/low glucose conditions.

The mechanisms of cell death induced by hypoxia or ischemia are not yet fully understood. We have previously demonstrated that cell death induced by hypoxia occurs independently of caspases, and is mediated by phospholipase A(2) (PLA(2)). Here, we show that p38 mitogen-activated protein kinase is activated under hypoxia. A selective inhibitor of p38 or decrease in the p38alpha protein level prevents hypoxia-induced cell death. The p38 inhibitor abolishes PLA(2) activation by hypoxia, indicating that p38 acts upstream of PLA(2). The antioxidant N-acetyl-cysteine inhibits activation of p38 and cell death induced by hypoxia, indicating that reactive oxygen species (ROS) are responsible for p38 activation. These results demonstrate that the ROS/p38/PLA(2) signaling axis has a crucial role in caspase-independent cell death induced by hypoxia.

Expression of Disabled 1 suppresses astroglial differentiation in neural stem cells.

Disabled 1 (Dab1), a cytoplasmic adaptor protein expressed predominantly in the CNS, transduces a Reelin-initiated signaling that controls neuronal migration and positioning during brain development. To determine the role of Dab1 in neural stem cell (NSC) differentiation, we established a culture of neurospheres derived from the embryonic forebrain of the Dab1(-/-) mice, yotari. Differentiating Dab1(-/-) neurospheres exhibited a higher expression of GFAP, an astrocytic marker, at the expense of neuronal markers. Under Dab1-deficient condition, the expression of NeuroD, a transcription factor for neuronal differentiation, was decreased and the JAK-STAT pathway was evidently increased during differentiation of NSC, suggesting the possible involvement of Dab1 in astrocyte differentiation via JAK-STAT pathway. Notably, expression of neural and glial markers and the level of JAK-STAT signaling molecules were not changed in differentiating NSC by Reelin treatment, indicating that differentiation of NSC is Reelin-independent. Immunohistochemical analyses showed a decrease in the number of neurons and an increase in the number of GFAP-positive cells in developing yotari brains. Our results suggest that Dab1 participates in the differentiation of NSCs into a specific cell lineage, thereby maintaining a balance between neurogenesis and gliogenesis.

Protective effect of ginsenosides Rg(2) and Rh(1) on oxidation-induced impairment of erythrocyte membrane properties.

The extract from Panax ginseng has been reported to improve the microcirculation in various organs. However, the mechanisms underlying this phenomenon are still poorly understood. In the present study, using the rheological properties of erythrocytes as an index, we have screened the components of Panax ginseng extract and identified Rg(2) and Rh(1) as the active ingredients. These two ginsenosides prevented the oxidative stress-induced elevation of erythrocyte suspension viscosity and the impairment of erythrocyte elongation in response to shear stress. Rg(2) and Rh(1) ginsenosides did not have antioxidant activity in an aqueous phase and did not inhibit the peroxidation of membrane lipids, either. However, they inhibited the oxidation-induced decrease of SH-groups in band 3 (anion exchanger-1), one of the important structural proteins of the erythrocyte membrane, but not in other structural proteins: bands 1 and 2 (spectrins), band 4.2 or band 5 (actin). These results suggest that ginsenosides Rg(2) and Rh(1) protect the rheological functions of erythrocytes against oxidative stress by preventing the oxidation of SH-groups in band 3 protein.

Participation of caspase-3-like protease in oxidation-induced impairment of erythrocyte membrane properties.

Erythrocytes are very susceptible to oxidative stress, having a high content of intracellular oxygen and hemoglobin. In the present study, exposure to oxidative stress resulted in a significant impairment of erythrocyte membrane functions, such as deformability and anion exchange. Band 3 protein, also known as anion exchanger-1, plays an important role in these two functions. We show that oxidative stress activated caspase-3 inside the erythrocytes, which resulted in band 3 protein cleavage. Interestingly, inhibition of the caspase-3 with its specific inhibitor not only suppressed the digestion of band 3 protein, but also blunted the functional damage to erythrocytes, such as deformability and anion exchange, without changing the level of peroxidation of membrane lipids. These results provide experimental evidence that activation of caspase-3 plays an important role in the oxidative stress-induced impairment of membrane functions of erythrocytes.

Reelin signals survival through Src-family kinases that inactivate BAD activity.

Reelin plays an important role in the migration of embryonic neurons, but its continuing presence suggests additional functions in the brain. We now report a novel function where reelin protects P19 embryonal cells from apoptosis during retinoic acid-induced neuronal differentiation. This increased survival is associated with reelin activation of the phosphatidyl-inositol-3-kinase (PI3 K)/Akt pathway. When PI3 K was inhibited with LY294002, reelin failed to protect against this retinoic acid-induced apoptosis. The protective effect of reelin includes activating the Src-family kinases/PI3 K/Akt pathway which then led to selective phosphorylation of Bcl-2/Bcl-XL associated death promoter (BAD) at serine-136, while the phosphorylation-incompetent mutation of BAD (S136A) suppressed this protection. These and additional studies define a novel pathway where reelin binds apoE receptors, significantly activates the PI3 K/Akt pathway causing phosphorylation of BAD which helps to protect cells from apoptosing, thus serving an important role in promoting the survival of maturing neurons in the brain.

Intravenous infusion of dihydroginsenoside Rb1 prevents compressive spinal cord injury and ischemic brain damage through upregulation of VEGF and Bcl-XL.

Red ginseng root (Panax Ginseng CA Meyer) has been used clinically by many Asian people for thousands of years without any detrimental effects. One of the major components of Red ginseng root is ginsenoside Rb(1) (gRb1). Previously, we showed that intravenous infusion of gRb1 ameliorated ischemic brain damage through upregulation of an anti-apoptotic factor, Bcl-x(L) and that topical application of gRb1 to burn wound lesion facilitated wound healing through upregulation of vascular endothelial growth factor (VEGF). In the present study, we produced dihydroginsenoside Rb1 (dgRb1), a stable chemical derivative of gRb1, and showed that intravenous infusion of dgRb1 improved spinal cord injury (SCI) as well as ischemic brain damage. As we expected, the effective dose of dgRb1 was ten times lower than that of gRb1. Intravenous infusion of dgRb1 at this effective dose did not affect brain temperature, blood pressure or cerebral blood flow, suggesting that dgRb1 rescued damaged neurons without affecting systemic parameters. In subsequent in vitro studies that focused on dgRb1-induced expression of gene products responsible for neuronal death or survival, we showed that dgRb1 could upregulate the expression of not only Bcl-x(L), but also a potent angiogenic and neurotrophic factor, VEGF. We also showed that dgRb1-induced expression of bcl-x(L) and VEGF mRNA was HRE (hypoxia response element) and STRE (signal transducers and activators of transcription 5 (Stat5) response element) dependent, respectively.

Characterization of NO and cytokine production in immune-activated microglia and peritoneal macrophages derived from a mouse model expressing the human NOS2 gene on a mouse NOS2 knockout background.

Significant differences exist in the production and release of nitric oxide (NO) from human macrophages versus macrophages of mouse origin. Human macrophages have been shown to respond poorly to stimuli that provoke strong inflammatory reactions from mouse macrophages. To address the differences in macrophage function in an animal model, a transgenic mouse was created that contained the entire human NOS2 gene, including the human promoter and all of its exons and introns. The huNOS2 transgenic mouse was then mated to mice lacking a functional NOS2 gene (muNOS2(/) or NOS2 knockout mice) to generate a double transgenic mouse (huNOS2(+/0)/muNOS2(/)) that expresses a functional human NOS2 gene in place of the mouse NOS2 gene. These double transgenic mice were found to express only human NOS2 mRNA and human iNOS proteins in response to immune stimulation. The production and release of nitric oxide from isolated macrophages from the doubly transgenic mouse also more closely paralleled human responses rather than mouse. Peritoneal macrophages from double transgenic mice generated nanomolar levels of nitrite in response to inflammatory stimuli, while peritoneal macrophages from wild-type mice generated micromolar levels of nitrite in response to the same inflammatory stimuli. Similarly, microglia from the huNOS2(+/0)/muNOS2(/) mice accumulated nanomolar levels of nitrite following inflammatory stimulation. Reduced nitrite release persisted in spite of normal responsiveness to inflammatory stimulation as measured by tumor necrosis factor alpha and interleukin-6 production and release. These data suggest that the human-specific release of nanomolar levels of nitrite may largely result from differences between the human and mouse NOS2 genes, which may program different degrees of nitric oxide responses to inflammatory signals in humans than in mice.

Prevention of ischemic neuronal death by intravenous infusion of a ginseng saponin, ginsenoside Rb(1), that upregulates Bcl-x(L) expression.

Almost all agents that exhibit neuroprotection when administered into the cerebral ventricles are ineffective or much less effective in rescuing damaged neurons when infused into the blood stream. Search for an intravenously infusible drug with a potent neuroprotective action is essential for the treatment of millions of patients suffering from acute brain diseases. Here, we report that postischemic intravenous infusion of a ginseng saponin, ginsenoside Rb(1) (gRb(1)) (C(54)H(92)O(23), molecular weight 1109.46) to stroke-prone spontaneously hypertensive rats with permanent occlusion of the middle cerebral artery distal to the striate branches significantly ameliorated ischemia-induced place navigation disability and caused an approximately 50% decrease in the volume of the cortical infarct lesion in comparison with vehicle-infused ischemic controls. In subsequent studies that focused on gRb(1)-induced expression of gene products responsible for neuronal death or survival, we showed that gRb(1) stimulated the expression of the mitochondrion-associated antiapoptotic factor Bcl-x(L) in vitro and in vivo. Moreover, we revealed that a Stat5 responsive element in the bcl-x promoter became active in response to gRb(1) treatment. Ginsenoside Rb(1) appears to be a promising agent not only for the treatment of cerebral stroke, but also for the treatment of other diseases involving activation of mitochondrial cell death signaling.

A novel alternative splice variant of nicastrin and its implication in Alzheimer disease.

Nicastrin interacts with gamma-secretase complex components predominantly via the N-terminal third of the transmembrane domain. The authentic transmembrane domain is critically required for the interaction with gamma-secretase complex components and for formation of an active gamma-secretase complex. In this study, we have identified a novel alternatively spliced transcript of nicastrin in human brain tissue. This transcript (NCSTN-DeltaE16) lacks exon 16 of nicastrin mRNA, which leads to deletion of 71 amino acids just upstream of its transmembrane domain. Its expression pattern was analyzed in the hippocampus of patients with pathologically diagnosed Alzheimer disease (cases) and non-Alzheimer dementia (controls). In patients with the APOE-epsilon4 allele, the frequency of Alzheimer disease appeared to be increased in the NCSTN-DeltaE16-positive group, but the association was not statistically significant. In conclusion, the expression of NCSTN-DeltaE16 transcript may confer some additional risk for developing Alzheimer disease beyond the risk due to ApoE-epsilon4 allele. Further investigation in larger scale population would be necessary to address its potential implication in Alzheimer disease.

Suppression of Stat3 promotes neurogenesis in cultured neural stem cells.

To investigate the effects of signal transducer and activator of transcription 3 (Stat3) on neural stem cell fate, stem cells were inoculated with an adenovirus vector expressing dominant negative form of Stat3 (Stat3F). One day later, a promoter assay revealed significant reduction of the transcriptional level in the transfected cells. Three days later, Western blot analysis and immunocytochemical analysis revealed that the protein level of microtubule-associated protein (MAP)2 and the number of MAP2-positive cells were increased significantly in the transfected cells whereas the protein level of glial fibrillary acidic protein (GFAP) and the number of GFAP-positive cells were decreased significantly. In addition, mRNA levels of Notch family members (Notch1, 2, and 3) and of inhibitory basic helix-loop-helix (bHLH) factors (Hes5, Id2, and Id3) were significantly downregulated at 3 days after viral inoculation with Stat3F; however, mRNA levels of bHLH determination factors (Math1 and Neurogenin3) and bHLH differentiation factors (NeuroD1 and NeuroD2) were significantly upregulated. These data indicated that suppression of Stat3 directly induced neurogenesis and inhibited astrogliogenesis in neural stem cells.

L-serine-mediated release of apolipoprotein E and lipids from microglial cells.

Apolipoprotein E (ApoE), one of the genetic risk factors for Alzheimer's disease, is considered to have a critical role in transporting lipids in the brain. In the present study, we investigated ApoE release in primary rat microglial cultures. Microglial cells released ApoE in response to L-Ser in culture medium, and ApoE-immunoreactivity was detected in granules in the cell periphery and in perinuclear structures. Immunocytochemical studies, immunoblotting, and reverse transcription-polymerase chain reaction (RT-PCR) results all supported the notion that microglial cells are the potential source of ApoE in the brain. L-Ser enhanced ApoE release in a concentration-dependent manner without upregulating ApoE mRNA expression. Astrocytes presumably enhanced production and release of ApoE by microglial cells through secretion of L-Ser. As revealed by gel chromatography, ApoE was secreted as a component of lipoproteins, and L-Ser enhanced release of cholesterol and triglycerides together with ApoE. Activation of microglial cells by lipopolysaccharides and serum resulted in an overall decrease of the ApoE release. These findings suggest that microglial cells are a significant source of lipoproteins containing ApoE in the brain under physiological conditions, and that L-Ser is an important mediator of the neuron-astrocyte-microglia network in the brain.

NFkappaB regulates plasma apolipoprotein A-I and high density lipoprotein cholesterol through inhibition of peroxisome proliferator-activated receptor alpha.

The levels of plasma HDL cholesterol and apoA-I in NFkappaB p50 subunit-deficient mice were significantly higher than those in wild-type mice under regular and high fat diets, without any significant difference in the level of total cholesterol. To examine the role of NFkappaBin lipid metabolism, we studied its effect on the regulation of apoA-I secretion from human hepatoma HepG2 cells. Lipopolysaccharide-induced activation of NFkappaB reduced the expression of apoA-I mRNA and protein, whereas adenovirus-mediated expression of IkappaBalpha super-repressor ameliorated the reduction. This IkappaBalpha-induced apoA-I increase was blocked by preincubation with MK886, a selective inhibitor of peroxisome proliferator-activated receptor alpha (PPARalpha), suggesting that NFkappaB inactivation induces apoA-I through activation of PPARalpha. To further support this idea, the expression of IkappaBalpha increased apoA-I promoter activity, and this increase was blocked by preincubation with MK886. Mutations in the putative PPARalpha-binding site in the apoA-I promoter or lack of the site abrogated these changes. Taking these results together, inhibition of NFkappaB increases apoA-I and HDL cholesterol through activation of PPARalpha in vivo and in vitro. Our data suggest a new aspect of lipid metabolism and may lead to a new paradigm for prevention and treatment of atherosclerotic disease.

Apolipoprotein E and Reelin ligands modulate tau phosphorylation through an apolipoprotein E receptor/disabled-1/glycogen synthase kinase-3beta cascade.

Neurofibrillary tangles comprised of highly phosphorylated tau proteins are a key component of Alzheimer's disease pathology. Mice lacking Reelin (Reln), double-knockouts lacking the VLDL receptor (VLDLR) and ApoE receptor2 (ApoER2), and mice lacking disabled-1 (Dab1) display increased levels of phosphorylated tau. Because Reln binds to recombinant ApoE receptors, assembly of a Reln/ApoE-receptor/Dab1 (RAD) complex may initiate a signal transduction cascade that controls tau phosphorylation. Conversely, disruption of this RAD complex may increase tau phosphorylation and lead to neurodegeneration. To substantiate this concept, we mated Reln-deficient mice to ApoE-deficient mice and found that in the absence of Reln, tau phosphorylation increased as the amount of ApoE decreased. Paralleling the change in tau phosphorylation levels, we found that GSK-3beta activity increased in Reln-deficient mice and further increased in mice lacking both Reln and ApoE. CDK-5 activity was similar in mice lacking Reln, ApoE, or both. GSK-3beta and CDK-5 activity increased in Dab1-deficient mice, independent of ApoE levels. Further supporting the idea that increased tau phosphorylation results primarily from increased kinase activity, the activity of two phosphatases was similar in all conditions tested. These data support a novel, ligand-mediated signal transduction cascade--initiated by the assembly of a RAD complex that suppresses kinase activity and controls tau phosphorylation.

The 4,752 C/T polymorphism in the presenilin 1 gene increases the risk of Alzheimer's disease in apolipoprotein E4 carriers.

OBJECTIVE: We sought to establish an association between sporadic Alzheimer's disease (AD) and presenilin 1 (PSEN1) gene polymorphisms in the Japanese population. METHODS: A 5 kb fragment containing the putative promoter of the PSEN1 gene for randomly selected control subjects was subcloned into plasmid and sequenced to screen novel polymorphisms in this region. Patients and controls were genotyped for five polymorphic markers in the PSEN1 region. We then constructed haplotypes using the computer program HAPLO and compared the frequencies between cases and controls. SUBJECTS: A total of 189 AD cases (NINCDS-ADRDA criteria) and 240 controls were studied. RESULTS: We discovered a novel polymorphism with high heterozygosity on -4,752 of the PSEN1 promoter region. A significant association was observed between the -4,752 C/T polymorphism and late-onset AD. The odds ratio for AD associated with the CC vs non-CC genotype was 1.59 (95% CI = 1.01-2.51), while that of epsilon 4 vs non-epsilon 4 in APOE gene was 4.41 (95% CI = 2.72-7.16). The C allele was associated with a further increase in the risk of AD in APOE epsilon 4 carriers. We found 12 major haplotypes using five polymorphisms. The distribution pattern was significantly different between cases and controls. CONCLUSION: The PSEN1 gene -4,752 C/T polymorphism modifies the risk for AD.

N141I mutant presenilin-2 gene enhances neuronal cell death and decreases bcl-2 expression.

A missense mutation (N1411) in Presenilin-2 (PS-2) gene is associated with early-onset familial Alzheimer's disease. In this study, SK-N-SH human neuroblastoma cells were transfected with wild-type and mutant PS-2 gene to examine presenilin-2 effects on apoptosis. Serum deprivation resulted in enhanced apoptosis in mutant PS-2 comparing with wild-type PS-2. Similarly, mutant PS-2 induced lactate dehydrogenase release to greater extent than wild-type PS-2. Time course experiment demonstrated that the increase in caspase-3-like activity was more pronounced and accelerated in mutant PS-2, compared to wild-type PS-2. While a significant decrease in bcl-2, an anti-apoptotic molecule, occurred in the cells overexpressing mutant PS-2, no significant change was observed in bax, a pro-apoptotic molecule, as compared with the cells overexpressing wild-type PS-2. Our study demonstrated that mutant PS-2 induces apoptosis accompanied by increased caspase-3-like activity and decreased bcl-2 expression in neuronal cells after serum-deprivation.