Poonlarp Cheepsunthorn

Characterization of a novel brain‐derived microglial cell line isolated from neonatal rat brain

We observed highly aggressively proliferating immortalized (HAPI) cells growing in cultures that had been enriched for microglia. The cells were initially obtained from mixed glial cultures prepared from 3‐day‐old rat brains. HAPI cells are typically round with few or no processes when cultured in 10% serum containing medium. As the percentage of serum in the medium is decreased, the HAPI cells have more processes. HAPI cells stain for the isolectin B4, OX‐42, and GLUT5, which are markers for microglial cells, but the cells do not immunolabel with A2B5, a marker of cells in the oligodendroglial cell lineage, or with the astrocyte‐specific marker, glial fibrillary aciidic protein (GFAP). In addition, HAPI cells are capable of phagocytosis. We conclude that HAPI cells are of microglia/macrophage lineage. Exposing HAPI cells to lipopolysaccharide (LPS) induces the mRNAs for tumor necrosis factor‐α (TNF‐α) and inducible nitric oxide synthase (iNOS). LPS exposure also induces secretion of TNF‐α and production of nitric oxide (NO) in HAPI cells. Because activation of microglia is associated with an increase in iron accumulation and ferritin expression, we tested the hypothesis that iron status affects the production of TNF‐α and NO. Our studies demonstrate that both iron chelation and iron loading diminished the LPS‐induced effect of TNF‐α and NO. The results of this study indicate that HAPI cells possess the characteristics of microglia/brain macrophages, providing an alternative cell culture model for the study of microglia. In addition, we demonstrate that the activation of microglial cells could be modified by iron. GLIA 35:53–62, 2001.

Cellular distribution of ferritin subunits in postnatal rat brain

The normal development of the brain requires finely coordinated events, many of which require iron. Consequently, iron must be available to the brain in a timely manner and in a bioavailable form. However, the brain also requires stringent mechanisms to protect itself from iron‐induced oxidative damage. The protein that is best suited to making iron available but also adequately protecting the cell is the intracellular iron storage protein ferritin. Typically, ferritin is composed of 24 subunits of H and L chains, which are functionally distinct. This study was undertaken to determine the expression of ferritin subunits during normal development of the postnatal rat brain. There is a shift in ferritin‐containing cell types during development from predominantly microglia at postnatal day 5 (PND 5) to predominantly oligodendrocytes by PND 30. At PND 5, microglia are found throughout gray and white matter areas of the brain, but only amoeboid microglia in discrete foci in the subcortical white matter are ferritin positive. At PND 15, some oligodendrocytes in the subcortical white matter express ferritin, but the majority of ferritin‐containing cells within white matter are still microglia. By PND 30, the predominant ferritin‐containing cell type within white matter are oligodendrocytes. Generally, the cellular distribution of both ferritin subunits were identical with one major exception; H‐ferritin, but not L‐ferritin, was present in neuronal nuclei in the cortex. These data suggest that microglia play a role in brain iron homeostasis during normal postnatal development and may influence myelination by competing with oligodendrocytes for iron. J. Comp. Neurol. 400:73–86, 1998.

Characterization of putative Japanese encephalitis virus receptor molecules on microglial cells.

Japanese encephalitis virus (JEV) a mosquito‐borne flavivirus is a major cause of viral encephalitis in Asia. While the principle target cells for JEV in the central nervous system are believed to be neurons, microglia are activated in response to JEV and have been proposed to act as a long lasting virus reservoir. Viral attachment to a host cell is the first step of the viral entry process and is a critical mediator of tissue tropism. This study sought to identify molecules associated with JEV entry to microglial cells. Virus overlay protein‐binding assay (VOPBA) and liquid chromatography–mass spectrometry (LC/MS/MS) identified the 37/67 kDa high‐affinity laminin receptor protein and nucleolin as a potential JEV‐binding proteins. These proteins were subsequently investigated for a contribution to JEV entry to mouse microglial BV‐2 cells together with other possible candidate receptor molecules including Hsp70, Hsp90, GRP78, CD14, and CD4. In antibody mediated inhibition of infection experiments, both anti‐laminin receptor and anti‐CD4 antibodies significantly reduced virus entry while anti‐Hsp70 and 90 antibodies produced a slight reduction. Significant inhibition of virus entry (up to 80%) was observed in the presence of lipopolysaccharide (LPS) which resulted in a complete down‐regulation of CD4 and moderate down‐regulation of CD14. These results suggest that multiple receptor proteins may mediate the entry of JEV to microglial cells, with CD4 playing a major role. J. Med. Virol. 84:615–623, 2012.

Highly permissive infection of microglial cells by Japanese encephalitis virus: a possible role as a viral reservoir.

Japanese encephalitis virus (JEV), a mosquito-borne Flavivirus, is a major cause of acute encephalitis, and neurons have been proposed to be the principle JEV target cells in the central nervous system. However, clinically, infection with JEV leads to increased levels of cytokines and chemokines in the serum and cerebrospinal fluid (CSF) the levels of which correlate with the mortality rate of patients. This research aimed to study the role of microglial cells in JEV infection. Mouse microglial cells (BV-2) supported the replication of JEV with extracellular production of virus by 10h post-infection, and virus titer reached a maximum (2.55x10(10)pfu/ml) by day 3 post-infection. While apoptosis was induced in response to virus infection, no alteration in nitric oxide production was observed. Microglial cells remained productively infected with JEV for up to 16 weeks without significant morphological alterations, and the released virions were infectious to mouse neuroblastoma (NA) cells. The high virus production and long persistence of JEV in microglial cells suggests that these cells may serve as viral reservoirs for the infection of neurons in the CNS.

Increased cellular iron levels affect matrix metalloproteinase expression and phagocytosis in activated microglia

Activation of microglia could be beneficial and yet simultaneously harmful depending upon nature of pathological milieu. Regardless of disease-specific etiology, iron accumulation, particularly in activated microglia, is a notable feature associated with a series of neuropathologies, including Alzheimers diseases. Although mounting evidence supports the role of iron in oxidative brain injury, knowledge on its regulatory role in neuroinflammation is still scarce. Here, we hypothesize that cellular iron status may be involved in determining the roles of activated microglia in neuroinflammatory processes. In this study, we examined effects of iron on expression of MMPs known to be involved in nervous system inflammation and degeneration using rat microglial cell line (HAPI). Stimulation experiments were performed using lipopolysaccharide (LPS). We demonstrated by RT-PCR that increased cellular iron levels enhanced the expression of MMP-9 in activated microglia, but had no effect on MMP-1. Studies using western blot and gelatin zymography analyses demonstrated that increased cellular iron levels in activated microglia enhanced the secretion of MMP-9 and MMP-1. Taken together, these results demonstrated regulatory roles of iron in the expression of MMPs by activated microglia at the transcription and translation levels. Using a colorimetric NBT reduction assay, we showed that increased cellular iron levels impaired zymosan phagocytic activity in activated microglia. Thus, these findings further our understanding toward the consequences of iron accumulation by activated microglia in neurodegeneration and suggest a possible link between iron metabolism in activated microglia and neuroinflammation.

The H63D HFE gene variant promotes activation of the intrinsic apoptotic pathway via mitochondria dysfunction following β-amyloid peptide exposure.

Numerous epidemiological studies suggest that the expression of the HFE allelic variant H63D may be a risk factor or genetic modifier for Alzheimers disease (AD). The H63D variant alters cellular iron homeostasis and increases baseline oxidative stress. The elevated cellular stress milieu, we have proposed, may alter cellular responses to genetic and environmental determinants of AD. Accumulation of β‐amyloid peptides (Aβ) is one of the most prominent pathogenic characteristics of AD. Several studies have demonstrated that Aβ can induce neuronal cell death through apoptosis. In this study, we provide evidence that an Aβ25–35 fragment, which contains the cytotoxic sequence of the amyloid peptide, activates the intrinsic apoptotic pathway in SH‐SY5Y human neuroblastoma cells expressing the HFE allelic variant H63D to a greater extent than in cells with wild‐type (WT) HFE. Specifically, Aβ25–35 peptide exposure significantly induced Bax translocation from the cytosol to the mitochondria in H63D‐expressing cells compared with WT cells. This translocation was associated with increased cytochrome c release from mitochondria and an increase in active caspase‐9 and caspase‐3 activity in H63D cells. Consequently, there is increased apoptosis in cells expressing the H63D variant as opposed to cells expressing WT HFE. We also found increased amyloid precursor protein (APP) and Aβ1–42 peptide in the mitochondrial compartment as well as increased mitochondrial stress in H63D‐expressing cells compared with WT. These findings support our hypothesis that the presence of the HFE H63D allele enables factors that trigger neurodegenerative processes associated with AD and predisposes cells to cytotoxcity.

The effects of okra (Abelmoschus esculentus Linn.) on the cellular events associated with Alzheimer's disease in a stably expressed HFE neuroblastoma SH-SY5Y cell line.

It has been reported that persons carrying the H63D variant in their hemochromatosis (HFE) gene are at increased risk of Alzheimers disease (AD). We investigated the possibility that okra (Abelmoschus esculentus) and quercetin could mitigate this risk factor by examining its effect on AD-associated cellular events in HFE stably expressing SH-SY5Y cells. Treatment of H63D HFE cells either with okra or quercetin significantly decreased reactive oxygen species (ROS), hydrogen peroxide (H2O2), and protein oxidation compared to untreated cells. The levels of tau phosphorylation at serine-199, serine-202, and serine-396 sites were also significantly decreased when cells were treated with okra. Exposure of the H63D and wild type (WT) cells to iron increased tau phosphorylation, but this response was decreased significantly when cells were treated with okra. The mechanism responsible for these changes appears to be related to decreased glycogen synthase kinase (GSK)-3β activity, an upstream signaling kinase of tau phosphorylation. We also established that okra treatment dramatically decreases intracellular iron levels in H63D cells compared to untreated cells. Our results provide important in vitro data on the effects of okra on various AD-associated cellular processes in H63D variant HFE cells. These results suggest okra may be beneficial in people expressing the H63D variant to reduce the risk of AD and other neurodegenerative diseases related to oxidative stress. Further in vivo studies would help confirm this.

Valproic acid attenuates nitric oxide and interleukin‑1β production in lipopolysaccharide‑stimulated iron‑rich microglia

Iron accumulation in activated microglia has been consistently reported in neurodegenerative diseases. Previous results suggest that these cells facilitate neuroinflammation leading to neuronal cell death. Therefore, chemical compounds that alleviate the activation of iron-rich microglia may result in neuroprotection. In the present study, the effect of valproic acid (VPA) on microglial activation under iron-rich conditions was investigated. BV-2 microglial cells were exposed to lipopolysaccharide (LPS; 1 µg/ml) and iron (300 µg/ml) with or without VPA (1.6 mM). The results demonstrated that VPA attenuated the activation of iron-rich BV2 cells induced by LPS by down-regulating the mRNA expression of inducible nitric oxide (NO) synthase and interleukin 1β (IL-1β; P<0.01), to ultimately reduce the production of NO and IL-1β (P<0.01). These events were accompanied by an attenuation in the nuclear translocation of nuclear factor-κB p65 subunit (P<0.01). These findings suggest that VPA may be therapeutically useful for attenuating the activation of iron-rich microglia.