Activation of TNFR1 and TLR4 following oxygen glucose deprivation promotes mitochondrial fission in C6 astroglial cells.

Astrocytes have emerged as active players in the innate immune response triggered by various types of insults. Recent literature suggests that mitochondria are key participants in innate immunity. The present study investigates the role of ischemia-induced innate immune response on p65/PGC-1α mediated mitochondrial dynamics in C6 astroglial cells. OGD conditions induced astroglial differentiation in C6 cells and increased the expression of hypoxia markers; HIF-1α, HO-1 and Cox4i2. OGD conditions resulted in induction of innate immune response in terms of expression of TNFR1 and TLR4 along with increase in IL-6 and TNF-α levels. OGD conditions resulted in decreased expression of I-κB with a concomitant increase in phos-p65 levels. The expression of PGC-1α, a key regulator of mitochondrial biogenesis, was also increased. Immunochemical staining suggested that phos-p65 and PGC-1α was co-localized. Studies on mitochondrial fusion (Mfn-1) and fission (DRP1) markers revealed shift toward fission. In addition, mitochondrial membrane potential decreased with increased DNA degradation and apoptosis confirming mitochondrial fission under OGD conditions. However, inhibition of phos-p65 by MG132 reduced the co-localization of phos-p65/ PGC-1α and significantly increased the Mfn-1 expression. The findings demonstrate the involvement of TNFR1 and TLR4 mediated immune response followed by interaction between phos-p65 and PGC-1α in promoting fission in C6 cells under hypoxic condition.

Systemic inflammation without gliosis mediates cognitive deficits through impaired BDNF expression in bile duct ligation model of hepatic encephalopathy.

Chronic liver disease per se induces neuroinflammation that contributes to cognitive deficits in hepatic encephalopathy (HE). However, the processes by which pro-inflammatory molecules result in cognitive impairment still remains unclear. In the present study, a significant increase in the activity of liver function enzymes viz. alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) was observed along with increase in plasma ammonia levels after four weeks of bile duct ligation (BDL) in rats suggesting hepatocellular damage. A significant increase was observed in mRNA expression of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) in brain regions and liver of BDL rats. Concomitantly, IL-6, TNF-α and MCP-1 protein levels were also increased in brain regions, liver and serum of BDL rats suggesting the involvement of blood-brain-axis in inflammatory response. However, a significant decrease was observed in glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule-1 (Iba-1) expression at transcriptional and translation level in brain of BDL rats. Immunohistochemical and flowcytometric analysis revealed reduced number of GFAP-immunopositive astrocytes and Iba1-immunopositive microglia in the brain regions of BDL rats. Further, a significant decline was observed in cognitive functions in BDL rats assessed using Morris water maze and novel object recognition tests. Expression of pro and mature form of brain derived neurotrophic factor (BDNF) and its upstream transcription element showed significant reduction in brain of BDL rats. Taken together, the results of the present study suggest that systemic inflammation and reduced expression of BDNF and its upstream transcription factor plays a key role in cognitive decline in HE.

Mitochondrial dysfunctions contribute to energy deficits in rodent model of hepatic encephalopathy.

Perturbations in the cerebral energy metabolism are anticipated to be an important factor by which ammonia may exert its toxic effects on the central nervous system. The present study was designed to investigate the role of impaired mitochondrial functions and cerebral energy metabolism in the development hepatic encephalopathy (HE) induced by of bile duct ligation (BDL). After four weeks of BDL, a significant increase in hepatic hydroxyproline and collagen content was observed which confirmed biliary fibrosis. Brain regions viz. cortex, hippocampus, striatum and cerebellum of BDL rats had impaired activity of mitochondrial respiratory chain enzymes. This was accompanied by increase in mitochondrial reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl levels in the brain. Mitochondrial redox ratio was significantly reduced in the brain of BDL rats. In addition, mitochondria from brain of BDL rats were depolarized and swollen compared to the sham controls. Ultrastructure analysis of mitochondria from cortex and hippocampus of BDL animals revealed aberrant cristae, ruptured membranes and non-dense matrix. Further, a significant decrease was observed in creatine kinase activity, glucose uptake and CO2 production in the brain regions of BDL rats. ATP/ADP ratio, a critical parameter of cellular energy status, was also significantly reduced in brain regions of rats with HE. Overall, the findings clearly demonstrate that BDL induced HE involves mitochondrial respiratory chain dysfunctions, mitochondrial depolarization and swelling that accentuates oxidative stress which in turn leads to compromise in cerebral energy metabolism thereby contributing to the pathophysiology of chronic HE.

Sulforaphane attenuates postnatal proteasome inhibition and improves spatial learning in adult mice.

Proteasomes are known to degrade proteins involved in various processes like metabolism, signal transduction, cell-cycle regulation, inflammation, and apoptosis. Evidence showed that protein degradation has a strong influence on developing neurons as well as synaptic plasticity. Here, we have shown that sulforaphane (SFN) could prevent the deleterious effects of postnatal proteasomal inhibition on spatial reference and working memory of adult mice. One day old Balb/c mice received intracerebroventricular injections of MG132 and SFN. Sham received an equal volume of aCSF. We observed that SFN pre-administration could attenuate MG132 mediated decrease in proteasome and calpain activities. In vitro findings revealed that SFN could induce proteasomal activity by enhancing the expression of catalytic subunit-ß5. SFN pre-administration prevented the hippocampus based spatial memory impairments during adulthood, mediated by postnatal MG132 exposure. Histological examination showed deleterious effects of MG132 on pyramidal neurons and granule cell neurons in DG and CA3 sub-regions respectively. Furthermore, SFN pre-administration has shown to attenuate the effect of MG132 on proteasome subunit-ß5 expression and also induce the Nrf2 nuclear translocation. In addition, SFN pre-administered mice have also shown to induce expression of pCaMKII, pCreb, and mature/pro-Bdnf, molecules which play a crucial role in spatial learning and memory consolidation. Our findings have shown that proteasomes play an important role in hippocampal synaptic plasticity during the early postnatal period and SFN pre-administration could enhance the proteasomal activity as well as improve spatial learning and memory consolidation.

Identification of Guanosine 5'-diphosphate as Potential Iron Mobilizer: Preventing the Hepcidin-Ferroportin Interaction and Modulating the Interleukin-6/Stat-3 Pathway.

Hepcidin, a peptide hormone, is a key regulator in mammalian iron homeostasis. Increased level of hepcidin due to inflammatory conditions stimulates the ferroportin (FPN) transporter internalization, impairing the iron absorption; clinically manifested as anemia of inflammation (AI). Inhibiting hepcidin-mediated FPN degradation is proposed as an important strategy to combat AI. A systematic approach involving in silico, in vitro, ex vivo and in vivo studies is employed to identify hepcidin-binding agents. The virtual screening of 68,752 natural compounds via molecular docking resulted into identification of guanosine 5'-diphosphate (GDP) as a promising hepcidin-binding agent. The molecular dynamics simulations helped to identify the important hepcidin residues involved in stabilization of hepcidin-GDP complex. The results gave a preliminary indication that GDP may possibly inhibit the hepcidin-FPN interactions. The in vitro studies revealed that GDP caused FPN stabilization (FPN-GFP cell lines) and increased the FPN-mediated cellular iron efflux (HepG2 and Caco-2 cells). Interestingly, the co-administration of GDP and ferrous sulphate (FeSO4) ameliorated the turpentine-induced AI in mice (indicated by increased haemoglobin level, serum iron, FPN expression and decreased ferritin level). These results suggest that GDP a promising natural small-molecule inhibitor that targets Hepcidin-FPN complex may be incorporated with iron supplement regimens to ameliorate AI.

Mitochondria as a centrally positioned hub in the innate immune response.

Mitochondria are vital organelles involved in numerous cellular functions ranging from energy metabolism to cell survival. Emerging evidence suggests that mitochondria provide a platform for signaling pathways involved in innate immune response. Mitochondrial ROS (mtROS) production, mitochondrial DNA (mtDNA) release, mitochondrial antiviral signaling protein (MAVS) are key triggers in the activation of innate immune response following variety of stress signals that include infection, tissue damage and metabolic dysregulation. The process is mediated through pattern recognition receptors (PRRs) that consist of retinoic acid inducible gene like receptors (RLRs), c-type lectin receptors (CLRs), toll type receptors (TLRs) and nuclear oligomerization-domain like receptors (NLRs). These signals converge to form a multiprotein complex called inflammasome that leads to caspase-1 activation to promote processing of precursor cytokines (pro-IL1ß and pro-IL-18) to active cytokines (IL-1ß and IL-18). It appears that mitochondria induced inflammasome activation contributes to inflammatory process in many diverse disorders. Therefore, strategies aimed at modulating mitochondria mediated inflammasome activation might be beneficial in many pathophysiological conditions. This article is part of a Special Issue entitled: Oxidative Stress and Mitochondrial Quality in Diabetes/Obesity and Critical Illness Spectrum of Diseases - edited by P. Hemachandra Reddy.

Migration and Phagocytic Ability of Activated Microglia During Post-natal Development is Mediated by Calcium-Dependent Purinergic Signalling.

Microglia play an important role in synaptic pruning and controlled phagocytosis of neuronal cells during developmental stages. However, the mechanisms that regulate these functions are not completely understood. The present study was designed to investigate the role of purinergic signalling in microglial migration and phagocytic activity during post-natal brain development. One-day-old BALB/c mice received lipopolysaccharide (LPS) and/or a purinergic analogue (2-methylthioladenosine-5'-diphosphate; 2MeSADP), intracerebroventrically (i.c.v.). Combined administration of LPS and 2MeSADP resulted in activation of microglia as evident from increased expression of ionised calcium-binding adapter molecule 1 (Iba1). Activated microglia showed increased expression of purinergic receptors (P2Y2, P2Y6 and P2Y12). LPS either alone or in combination with 2MeSADP induced the expression of Na(+)/Ca(2+) exchanger (NCX-1) and P/Q-type Ca(2+) channels along with MARCKS-related protein (MRP), which is an integral component of cell migration machinery. In addition, LPS and 2MeSADP administration induced the expression of microglial CD11b and DAP12 (DNAX-activation protein 12), which are known to be involved in phagocytosis of neurons during development. Interestingly, administration of thapsigargin (TG), a specific Ca(2+)-ATPase inhibitor of endoplasmic reticulum, prevented the LPS/2MeSADP-induced microglial activation and migration by down-regulating the expression of Iba1 and MRP, respectively. Moreover, TG also reduced the LPS/2MeSADP-induced expression of CD11b/DAP12. Taken together, the findings reveal for the first time that Ca(2+)-mediated purinergic receptors regulate the migration and phagocytic ability of microglia during post-natal brain development.

Specific noninvasive detection of Leishmania donovani in desquamated buccal cell swab samples from human visceral Leishmaniasis-HIV coinfected patients.

Diagnosis of human immunodeficiency virus (HIV) infection with visceral leishmaniasis (VL) coinfection is challenging. Specific diagnosis of VL in HIV-coinfected patients was evaluated by molecular methods in desquamated buccal swab samples, demonstrating 86.3% sensitivity and 98.3% specificity in controls. This test holds significant potential for development as a noninvasive diagnostic tool for VL in HIV-coinfected patients.