Jianfeng Liang

Blockade of Gap Junction Hemichannel Suppresses Disease Progression in Mouse Models of Amyotrophic Lateral Sclerosis and Alzheimer's Disease

BACKGROUND Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimers disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases. METHODS AND FINDINGS In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimers disease mouse model. CONCLUSIONS Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases.

Fractalkine Attenuates Excito-neurotoxicity via Microglial Clearance of Damaged Neurons and Antioxidant Enzyme Heme Oxygenase-1 Expression

Glutamate-induced excito-neurotoxicity likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases. Microglial clearance of dying neurons and associated debris is essential to maintain healthy neural networks in the central nervous system. In fact, the functions of microglia are regulated by various signaling molecules that are produced as neurons degenerate. Here, we show that the soluble CX3C chemokine fractalkine (sFKN), which is secreted from neurons that have been damaged by glutamate, promotes microglial phagocytosis of neuronal debris through release of milk fat globule-EGF factor 8, a mediator of apoptotic cell clearance. In addition, sFKN induces the expression of the antioxidant enzyme heme oxygenase-1 (HO-1) in microglia in the absence of neurotoxic molecule production, including NO, TNF, and glutamate. sFKN treatment of primary neuron-microglia co-cultures significantly attenuated glutamate-induced neuronal cell death. Using several specific MAPK inhibitors, we found that sFKN-induced heme oxygenase-1 expression was primarily mediated by activation of JNK and nuclear factor erythroid 2-related factor 2. These results suggest that sFKN secreted from glutamate-damaged neurons provides both phagocytotic and neuroprotective signals.

Excitatory amino acid transporter expression by astrocytes is neuroprotective against microglial excitotoxicity

Glutamate-induced excitotoxicity is considered as a major cause of neurodegenerative disease. Excitatory amino acid transporters (EAATs) on glial cells are responsible for the homeostasis of extracellular glutamate in the central nervous system which may contribute to the prevention of excitotoxic neurodegeneration. However, the differential EAAT expression in astrocytes and microglia is not fully understood. In this study, we compared the expression of EAATs in astrocytes and microglia, and we assessed the neuroprotective and neurotoxic function of astrocytes and microglia by a co-culture system. RT-PCR analyses detected that astrocytes expressed each EAAT (EAAT1-5) whereas microglia did not express EAAT4. Western blot analyses demonstrated that astrocytes express a much larger amount of membrane-localized EAATs than microglia. Astrocytes prevented excito-neurotoxicity by the reduction of exogenous glutamate whereas microglia did not. Conversely, activated microglia released an excess of glutamate that induced excitotoxic neuronal death. Astrocytes rescued neurons from microglial glutamate-induced death in a ratio-dependent manner. Inhibition of EAATs abolished glutamate uptake and the neuroprotective effect of astrocytes, but it did not alter any microglial neurotoxic or neuroprotective effects. These results revealed that astrocytic EAATs can counteract microglial glutamate-induced neuronal death whereas microglial EAATs are inconsequential to neurotoxicity and neuroprotection.

Blockade of microglial glutamate release protects against ischemic brain injury

Glutamate released by activated microglia induces excito-neurotoxicity and may contribute to neurodegeneration in numerous neurological diseases including ischemia, inflammation, epilepsy, and neurodegenerative diseases. We observed that the gap junction blocker carbenoxolone (CBX) or the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON) decreased glutamate release from activated microglia and rescued neuronal death in a dose-dependent manner in vitro. In gerbils, treatment with CBX or DON also prevented the delayed death of hippocampal neurons following transient global ischemia. Thus, blockade of microglial glutamate release may be an effective therapeutic strategy against neurodegeneration after ischemic injury.

Macrophage-induced neurotoxicity is mediated by glutamate and attenuated by glutaminase inhibitors and gap junction inhibitors

We have shown previously, that the most neurotoxic factor from activated microglia is glutamate that is produced by glutaminase utilizing extracellular glutamine as a substrate. Drugs that inhibit glutaminase or gap junction through which the glutamate is released were effective in reducing neurotoxic activity of microglia. In this study, to elucidate whether or not a similar mechanism is operating in macrophages infiltrating into the central nervous system during inflammatory, demyelinating, and ischemic brain diseases, we examined the neurotoxicity induced by macrophages, in comparison with microglia in vitro. LPS- or TNF-alpha-stimulated macrophage-conditioned media induced robust neurotoxicity, which was completely inhibited by the NMDA receptor antagonist MK801. Both the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine (DON), and the gap junction inhibitor carbenoxolone (CBX), effectively suppressed glutamate production and subsequent neurotoxicity by activated macrophages. These results revealed that macrophages produce glutamate via glutaminase from extracelluar glutamine, and release it through gap junctions. This study demonstrated that a similar machinery is operating in macrophages as well, and DON and CBX that prevent microglia-mediated neurotoxicity should be effective for preventing macrophage-mediated neurotoxicity. Thus, these drugs may be effective therapeutic reagents for inflammatory, demyelinating, and ischemic brain diseases.

Glutamate induces neurotrophic factor production from microglia via protein kinase C pathway

Microglia are intrinsic immune cells in the central nervous system and play key roles in the pathogenesis of various central nervous system disorders. Microglia have been shown to attack damaged neurons by secreting a variety of neurotoxic factors including inflammatory cytokines, reactive oxygen species and glutamate. On the other hand, they can produce neurotrophic factors (NTFs) which support neuronal survival and growth. However, the precise mechanism that regulates microglial NTF production is not fully understood, and the relation between glutamate and NTFs remains unclear. In the present study, we show that glutamate significantly induces microglial NTF production by the activation of N-methyl-d-aspartate (NMDA) receptors, group III metabotropic glutamate receptors, and glutamate transporters. Activation of NMDA receptors and group III metabotropic glutamate receptors induces intracellular Ca(2+) release from the endoplasmic reticulum. Further, stimulation of glutamate transporters leads to influx of extracellular Ca(2+) in a Na(+)-dependent manner. This intracellular Ca(2+) elevation activates the protein kinase C pathway which induces microglial NTF expression and production. These results suggest that microglia play a neuroprotective role during the excitotoxic state in neurodegenerative diseases.

Tumor necrosis factor-α promotes granulocyte-macrophage colony-stimulating factor-stimulated microglia to differentiate into competent dendritic cell-like antigen-presenting cells

Objective:  To examine whether microglia differentiate to dendritic cells (DC) and those DCs can acquire the full characteristic phenotype of mature stimulatory DCs to participate in functional responses.

Comparison research on excitatory amino acid transporters between astrocyte and microglia

The association between protein aggregation and the pathogenesis of Parkinson’s disease has been widely documented. In order to understand how Lewy bodies are formed in dopaminergic neurons, we investigated the effect of dopamine and l-dopa on the aggregate formation using neural-differentiated PC12 cells and Tet-off conditioned alpha-synuclein expressing cells. The tyrosine hydroxylase inhibitor, alpha-methyl tyrosine dramatically reduced the formation of aggregates under the proteasome-inhibited condition and upregulated proteasome activity. An in vitro aggregation assay for ubiquitin and alpha-synuclein demonstrated that dopamine and l-dopa enhanced the oligomerization of ubiquitin and alpha-synuclein, respectively. Moreover, a scavenger of quinone body cystein dramatically decreased oligomerization. These data suggest that dopamine and l-dopa enhance the formation of aggregates via quinone formation in dopaminergic neurons.

The Direct and Indirect Effects of Serofendic Acid on Neuroprotection

Abstract:  Serofendic acid is a novel neuroprotective factor isolated from fetal calf serum. To elucidate the mechanisms how serofendic acid exerts neuroprotection, we examined its effects on glutamate‐induced excito‐toxicity in mouse cortical neurons. The effects of serofendic acid on inflammatory cytokine and neurotrophin production by glial cells were also examined to evaluate the indirect neuroprotection. Serofendic acid significantly and dose dependently increased survival of mouse cortical neurons after 10 μM N‐methyl‐D‐asparate (NMDA) exposure. However, it did not affect production of inflammatory cytokines and neurotrophins by microglia as assessed by reverse transciption polymerase chain reaction (RT‐PCR) for mRNA expression and ELISA for protein levels, though it suppressed tumor necrosis factor (TNF)‐α production by astrocytes. Thus, serofendic acid works directly on neurons to protect against glutamate toxicity. Suppression of TNF‐α production by astoryctes may also synergistically exert neuroprotective functions of serofendic acid. Serofendic acid may be of use for the future therapeutic strategy against ischemic and degenerative neurological disorders.