Tetsuya Mizuno

Tumor Necrosis Factor-α Induces Neurotoxicity via Glutamate Release from Hemichannels of Activated Microglia in an Autocrine Manner

Glutamate released by activated microglia induces excitoneurotoxicity and may contribute to neuronal damage in neurodegenerative diseases, including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and multiple sclerosis. In addition, tumor necrosis factor-α (TNF-α) secreted from activated microglia may elicit neurodegeneration through caspase-dependent cascades and silencing cell survival signals. However, direct neurotoxicity of TNF-α is relatively weak, because TNF-α also increases production of neuroprotective factors. Accordingly, it is still controversial how TNF-α exerts neurotoxicity in neurodegenerative diseases. Here we have shown that TNF-α is the key cytokine that stimulates extensive microglial glutamate release in an autocrine manner by up-regulating glutaminase to cause excitoneurotoxicity. Further, we have demonstrated that the connexin 32 hemichannel of the gap junction is another main source of glutamate release from microglia besides glutamate transporters. Although pharmacological blockade of glutamate receptors is a promising therapeutic candidate for neurodegenerative diseases, the associated perturbation of physiological glutamate signals has severe adverse side effects. The unique mechanism of microglial glutamate release that we describe here is another potential therapeutic target. We rescued neuronal cell death in vitro by using a glutaminase inhibitor or hemichannel blockers to diminish microglial glutamate release without perturbing the physiological glutamate level. These drugs may give us a new therapeutic strategy against neurodegenerative diseases with minimum adverse side effects.

Production and neuroprotective functions of fractalkine in the central nervous system.

The CX3C-chemokine, fractalkine is reportedly to be expressed in the central nervous system, and up-regulated in certain pathological conditions, such as HIV encephalopathy and multiple sclerosis. In the present study, we examined the production of fractalkine and the expression of its receptor, CX3CR1 in murine glial and neuronal cell in vitro, and investigated its neuroprotective functions. Both fractalkine and CX3CR1 were expressed constitutively in neurons, microglia, and astrocytes. Neither the production of fractalkine nor its receptor expression was up-regulated by lipopolysaccharide (LPS), as measured by mRNA expression and protein synthesis. Fractalkine dose-dependently suppressed the production of nitric oxide (NO), interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha with activated microglia. It also significantly suppressed neuronal cell death induced by microglia activated with LPS and interferon-gamma, in a dose-dependent manner. These results suggest the possible functions of fractalkine as an intrinsic inhibitor against neurotoxicity by activated microglia.

Neuritic beading induced by activated microglia is an early feature of neuronal dysfunction toward neuronal death by inhibition of mitochondrial respiration and axonal transport.

Recent studies suggest that excitotoxicity may contribute to neuronal damage in neurodegenerative diseases including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and multiple sclerosis. Activated microglia have been observed around degenerative neurons in these diseases, and they are thought to act as effector cells in the degeneration of neural cells in the central nervous system. Neuritic beading, focal bead-like swellings in the dendrites and axons, is a neuropathological sign in epilepsy, trauma, ischemia, aging, and neurodegenerative diseases. Previous reports showed that neuritic beading is induced by various stimuli including glutamate or nitric oxide and is a neuronal response to harmful stimuli. However, the precise physiologic significance of neuritic beading is unclear. We provide evidence that neuritic beading induced by activated microglia is a feature of neuronal cell dysfunction toward neuronal death, and the neurotoxicity of activated microglia is mediated through N-methyl-d-aspartate (NMDA) receptor signaling. Neuritic beading occurred concordant with a rapid drop in intracellular ATP levels and preceded neuronal death. The actual neurite beads consisted of collapsed cytoskeletal proteins and motor proteins arising from impaired neuronal transport secondary to cellular energy loss. The drop in intracellular ATP levels was because of the inhibition of mitochondrial respiratory chain complex IV activity downstream of NMDA receptor signaling. Blockage of NMDA receptors nearly completely abrogated mitochondrial dysfunction and neurotoxicity. Thus, neuritic beading induced by activated microglia occurs through NMDA receptor signaling and represents neuronal cell dysfunction preceding neuronal death. Blockage of NMDA receptors may be an effective therapeutic approach for neurodegenerative diseases.

Production and functions of IL-17 in microglia

Interleukin (IL)-17-producing helper T cells may play a pivotal role in the pathogenesis of multiple sclerosis. Here, we examined the effects of IL-17 on microglia, which are known to be critically involved in multiple sclerosis. Treatment with IL-17 upregulated the microglial production of IL-6, macrophage inflammatory protein-2, nitric oxide, adhesion molecules, and neurotrophic factors. We also found that IL-17 was produced by microglia in response to IL-23 or IL-1beta. Because microglia produce IL-1beta and IL-23, these cytokines may act in an autocrine manner to induce IL-17 expression in microglia, and thereby contribute to autoimmune diseases, such as MS, in the central nervous system.

Neuroprotective role of phosphodiesterase inhibitor ibudilast on neuronal cell death induced by activated microglia

The phosphodiesterase inhibitor, ibudilast, has many effects on lymphocytes, endothelial cells, and glial cells. We examined the neuroprotective role of ibudilast in neuron and microglia co-cultures. Ibudilast significantly suppressed neuronal cell death induced by the activation of microglia with lipopolysaccharide (LPS) and interferon (IFN)-gamma. To examine the mechanisms by which ibudilast exerts a neuroprotective role against the activation of microglia, we examined the production of inflammatory and anti-inflammatory mediators and trophic factors following ibudilast treatment. In a dose-dependent manner, ibudilast suppressed the production of nitric oxide (NO), reactive oxygen species, interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha and enhanced the production of the inhibitory cytokine, IL-10, and additional neurotrophic factors, including nerve growth factor (NGF), glia-derived neurotrophic factor (GDNF), and neurotrophin (NT)-4 in activated microglia. Thus, ibudilast-mediated neuroprotection was primarily due to the inhibition of inflammatory mediators and the upregulation of neurotrophic factor. In the CA1 region of hippocampal slices, long-term potentiation (LTP) induced by high frequency stimulation (HFS) could be inhibited with LPS and interferon-gamma stimulation. Ibudilast returned this LTP inhibition to the levels observed in controls. These results suggest that ibudilast may be a useful neuroprotective and anti-dementia agent counteracting neurotoxicity in activated microglia.

LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma

Malignant mesothelioma (MM) is an aggressive neoplasm associated with asbestos exposure. We carried out genome-wide array-based comparative genomic hybridization analysis with 14 MM cell lines. Three cell lines showed overlapping homozygous deletion at chromosome 13q12, which harbored the LATS2 (large tumor suppressor homolog 2) gene. With 6 other MM cell lines and 25 MM tumors, we found 10 inactivating homozygous deletions or mutations of LATS2 among 45 MMs. LATS2 encodes a serine/threonine kinase, a component of the Hippo tumor-suppressive signaling pathway, and we transduced LATS2 in MM cells with its mutation. Transduction of LATS2 inactivated oncoprotein YAP, a transcriptional coactivator, via phosphorylation, and inhibited MM cell growth. We also analyzed LATS2 immunohistochemically and found that 13 of 45 MM tumors had low expression of LATS2. Because NF2 is genetically mutated in 40% to 50% of MM, our data indicate that Hippo pathway dysregulation is frequent in MM cells with inactivation of LATS2 or an upstream regulator of this pathway, Merlin, which is encoded by NF2. Thus, our results suggest that the inactivation of LATS2 is one of the key mechanisms for constitutive activation of YAP, which induces deregulation of MM cell proliferation.

Pleomorphic Carcinoma of the Lung: Clinicopathologic Characteristics of 70 Cases

Pleomorphic carcinoma (PC) of the lung is rare, and it is classified as a subtype of sarcomatoid carcinoma of the lung in the World Health Organization histologic classification of lung tumors. In this study, 70 cases of PC surgically resected were reviewed to identify its clinicopathologic characteristics. There were 57 men and 13 women, and their mean age was 66 years (range: 29 to 80 y). Sixty-eight tumors contained identifiable epithelial components, and the other 2 consisted of spindle cells and giant cells alone. An adenocarcinoma component was found in 34 cases, a squamous cell carcinoma component in 13, and a large cell carcinoma component in 40. The overall survival rate and disease-free survival rate were 36.6% and 40.7%, respectively, and both rates were significantly lower than for other nonsmall cell lung carcinomas. When the PC patients were divided into 3 groups according to the predominant epithelial component, an adenocarcinoma group, squamous cell carcinoma group, and large cell carcinoma group, there were no significant differences in the overall survival rate and median survival time between the 3 groups. Univariate analysis revealed that advanced stage (stage III), mediastinal lymph node metastasis, lymphatic permeation, and histologically diagnosed massive coagulation necrosis (>25% of the tumor) predicted poorer disease-free survival. Multivariate analysis showed that massive necrosis alone was an independent prognostic factor. We concluded that PC should be considered as an aggressive disease and massive necrosis should be routinely reported and used as a factor in clinical assessments.

Cholesterol-dependent generation of a seeding amyloid beta-protein in cell culture.

Deposition of aggregated amyloid β-protein (Aβ), a proteolytic cleavage product of the amyloid precursor protein (1), is a critical step in the development of Alzheimer’s disease (2). However, we are far from understanding the molecular mechanisms underlying the initiation of Aβ polymerization in vivo. Here, we report that a seeding Aβ, which catalyzes the fibrillogenesis of soluble Aβ, is generated from the apically missorted amyloid precursor protein in cultured epithelial cells. Furthermore, the generation of this Aβ depends exclusively on the presence of cholesterol in the cells. Taken together with mass spectrometric analysis of this novel Aβ and our recent study (3), it is suggested that a conformationally altered form of Aβ, which acts as a “seed” for amyloid fibril formation, is generated in intracellular cholesterol-rich microdomains.

Production and functions of IL-33 in the central nervous system

Interleukin-33 (IL-33) is a novel multifunctional IL-1 family cytokine. IL-33 signals via a heterodimer composed of IL-1 receptor-related protein ST2 and IL-1 receptor accessory protein (IL-1RAcP). IL-33 has been shown to activate T helper 2 cells (Th2), mast cells and basophils to produce a variety of Th2 cytokines and mediate allergic-type immune responses. Recent studies have revealed that glial cells are induced to express IL-33 mRNA and protein. However, the functions of IL-33 and its producing cells in the central nervous system (CNS) are still uncertain. In this study, we investigated the expression and function of IL-33 in the CNS. IL-33 is produced by endothelial cells and astrocytes but not by microglia or neurons. The IL-33 receptors are expressed mainly in microglia and astrocytes. IL-33 dose-dependently induces the proliferation of microglia and enhances the production of pro-inflammatory cytokines, such as IL-1β and TNFα, as well as the anti-inflammatory cytokine IL-10. It also enhances chemokines and nitric oxide production and phagocytosis by microglia. Thus, IL-33 produced in the CNS activates microglia and may function as a pro-inflammatory mediator in the pathophysiology of the CNS.

YAP induces malignant mesothelioma cell proliferation by upregulating transcription of cell cycle-promoting genes

Malignant mesothelioma (MM) shows frequent inactivation of the neurofibromatosis type 2 (NF2) –tumor-suppressor gene. Recent studies have documented that the Hippo signaling pathway, a downstream cascade of Merlin (a product of NF2), has a key role in organ size control and carcinogenesis by regulating cell proliferation and apoptosis. We previously reported that MMs show overexpression of Yes-associated protein (YAP) transcriptional coactivator, the main downstream effector of the Hippo signaling pathway, which results from the inactivation of NF2, LATS2 and/or SAV1 genes (the latter two encoding core components of the mammalian Hippo pathway) or amplification of YAP itself. However, the detailed roles of YAP remain unclear, especially the target genes of YAP that enhance MM cell growth and survival. Here, we demonstrated that YAP-knockdown inhibited cell motility, invasion and anchorage-independent growth as well as cell proliferation of MM cells in vitro. We analyzed genes commonly regulated by YAP in three MM cell lines with constitutive YAP-activation, and found that the major subsets of YAP-upregulating genes encode cell cycle regulators. Among them, YAP directly induced the transcription of CCND1 and FOXM1, in cooperation with TEAD transcription factor. We also found that knockdown of CCND1 and FOXM1 suppressed MM cell proliferation, although the inhibitory effects were less evident than those of YAP knockdown. These results indicate that constitutive YAP activation in MM cells promotes cell cycle progression giving more aggressive phenotypes to MM cells.