Targeting microglial KATP channels to treat neurodegenerative diseases: a mitochondrial issue

Neurodegeneration is a complex process involving different cell types and neurotransmitters. A common characteristic of neurodegenerative disorders is the occurrence of a neuroinflammatory reaction in which cellular processes involving glial cells, mainly microglia and astrocytes, are activated in response to neuronal death. Microglia do not constitute a unique cell population but rather present a range of phenotypes closely related to the evolution of neurodegeneration. In a dynamic equilibrium with the lesion microenvironment, microglia phenotypes cover from a proinflammatory activation state to a neurotrophic one directly involved in cell repair and extracellular matrix remodeling. At each moment, the microglial phenotype is likely to depend on the diversity of signals from the environment and of its response capacity. As a consequence, microglia present a high energy demand, for which the mitochondria activity determines the microglia participation in the neurodegenerative process. As such, modulation of microglia activity by controlling microglia mitochondrial activity constitutes an innovative approach to interfere in the neurodegenerative process. In this review, we discuss the mitochondrial KATP channel as a new target to control microglia activity, avoid its toxic phenotype, and facilitate a positive disease outcome.

Targeting microglial KATP channels to treat neurodegenerative diseases: a mitochondrial issue

Neurodegeneration is a complex process involving different cell types and neurotransmitters. A common characteristic of neurodegenerative disorders is the occurrence of a neuroinflammatory reaction in which cellular processes involving glial cells, mainly microglia and astrocytes, are activated in response to neuronal death. Microglia do not constitute a unique cell population but rather present a range of phenotypes closely related to the evolution of neurodegeneration. In a dynamic equilibrium with the lesion microenvironment, microglia phenotypes cover from a proinflammatory activation state to a neurotrophic one directly involved in cell repair and extracellular matrix remodeling. At each moment, the microglial phenotype is likely to depend on the diversity of signals from the environment and of its response capacity. As a consequence, microglia present a high energy demand, for which the mitochondria activity determines the microglia participation in the neurodegenerative process. As such, modulation of microglia activity by controlling microglia mitochondrial activity constitutes an innovative approach to interfere in the neurodegenerative process. In this review, we discuss the mitochondrial KATP channel as a new target to control microglia activity, avoid its toxic phenotype, and facilitate a positive disease outcome.

Microglial cells in astroglial cultures: a cautionary note

Primary rodent astroglial-enriched cultures are the most popular model to study astroglial biology in vitro. From the original methods described in the 1970's a great number of minor modifications have been incorporated into these protocols by different laboratories. These protocols result in cultures in which the astrocyte is the predominant cell type, but astrocytes are never 100% of cells in these preparations. The aim of this review is to bring attention to the presence of microglia in astroglial cultures because, in my opinion, the proportion of and the role that microglial cells play in astroglial cultures are often underestimated. The main problem with ignoring microglia in these cultures is that relatively minor amounts of microglia can be responsible for effects observed on cultures in which the astrocyte is the most abundant cell type. If the relative contributions of astrocytes and microglia are not properly assessed an observed effect can be erroneously attributed to the astrocytes. In order to illustrate this point the case of NO production in activated astroglial-enriched cultures is examined. Lipopolysaccharide (LPS) induces nitric oxide (NO) production in astroglial-enriched cultures and this effect is very often attributed to astrocytes. However, a careful review of the published data suggests that LPS-induced NO production in rodent astroglial-enriched cultures is likely to be mainly microglial in origin. This review considers cell culture protocol factors that can affect the proportion of microglial cells in astroglial cultures, strategies to minimize the proportion of microglia in these cultures, and specific markers that allow the determination of such microglial proportions.

The L-type voltage-gated calcium channel modulates microglial pro-inflammatory activity

Under pathological conditions, microglia, the resident CNS immune cells, become reactive and release pro-inflammatory cytokines and neurotoxic factors. We investigated whether this phenotypic switch includes changes in the expression of the L-type voltage-gated calcium channel (VGCC) in a rat model of N-methyl-d-aspartate-induced hippocampal neurodegeneration. Double immunohistochemistry and confocal microscopy evidenced that activated microglia express the L-type VGCC. We then analyzed whether BV2 microglia express functional L-type VGCC, and investigated the latter's role in microglial cytokine release and phagocytic capacity. Activated BV2 microglia express the CaV1.2 and CaV1.3 subunits of the L-type VGCC determined by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry. Depolarization with KCl induced a Ca2+ entry facilitated by Bay k8644 and partially blocked with nifedipine, which also reduced TNF-α and NO release by 40%. However, no nifedipine effect on BV2 microglia viability or phagocytic capacity was observed. Our results suggest that in CNS inflammatory processes, the L-type VGCC plays a specific role in the control of microglial secretory activity.

Papel de la Interleucina 6 (IL-6) en la regulación de la expresión de Osteopontina (OPN) y CD44 tras axotomía del nervio facial

Investigación producida a partir de una estancia en la University of Sidney, Australia, entre octubre del 2008 y enero del 2009. Se ha desarrollado el proyecto titulado "Papel de la interleucina 6 (IL6) en la regulación de la expresión de Osteopontina (OPN) y de CD44 tras axotomía del nervio facial". Tras efectuar una transección del nervio facial, se indujo una reactividad glial en el núcleo facial (NF) localizado en el tronco cerebral, utilizando ratones transgénicos que sobrexpresan IL6 bajo promotor GFAP (tg GFAP-IL6), es decir selectivamente en astrocitos. Se han utilizado técnicas histoquímicas e inmunohistoquímicas, así como también se ha completado el estudio utilizando análisis de RPA, western blotting y citometría de flujo para la identificación de poblaciones celulares. Los resultados obtenidos indican que la OPN se expresa constitutivamente en las neuronas del NF. Tras axotomía del nervio facial, la expresión de OPN y CD44 incrementa en los ratones WT, mientras que en los tg GFAP-IL6 disminuye significativamente, sugiriendo que la IL6 podría estar involucrada en la modulación de la expresión de ambas moléculas. Sin embargo, no se ha visto diferencias en otros receptores de OPN como la integrina Alpha-5. La ctometría de flujo corroboró algunos de los resultados histológicos sobre la reactividad microglial y permitió concluir que la proporción de microglía activada (CD11b+/CD45+mid) y macrófagos (CD11b+/CD45+high) que expresan CD44 incrementa en in los tg GFAP-IL6 versus WT donde la mayor parte de microglia activada mostraba un perfil CD11b+/CD45+low.

Pro-inflammatory gene expression and neurotoxic effects of activated microglia are attenuated by absence of CCAAT/enhancer binding protein beta

Background. Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression. This response, known as glial activation or neuroinflammation, can be detrimental to the surrounding tissue. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) is an important regulator of gene expression in inflammation but little is known about its involvement in glial activation. To explore the functional role of C/EBPß in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBPß-null glial cultures. Methods. Due to fertility and mortality problems associated with the C/EBPß-null genotype we developed a protocol to prepare mixed glial cultures from cerebral cortex of a single mouse embryo with high yield. Wild-type and C/EBPß-null glial cultures were compared in terms of total cell density by Hoechst-33258 staining; microglial content by CD11b immunocytochemistry; astroglial content by GFAP western blot; gene expression by quantitative real-time PCR, western blot, immunocytochemistry and Griess reaction; and microglial neurotoxicity by estimating MAP2 content in neuronal/microglial cocultures. C/EBPß DNA binding activity was evaluated by electrophoretic mobility shift assay and quantitative chromatin immunoprecipitation. Results. C/EBPß mRNA and protein levels, as well as DNA binding, were increased in glial cultures by treatment with lipopolysaccharide (LPS) or LPS + interferon ¿ (IFN¿). Quantitative chromatin immunoprecipitation showed binding of C/EBPß to pro-inflammatory gene promoters in glial activation in a stimulus- and gene-dependent manner. In agreement with these results, LPS and LPS+IFN¿ induced different transcriptional patterns between pro-inflammatory cytokines and NO synthase-2 genes. Furthermore, the expressions of IL-1ß and NO synthase-2, and consequent NO production, were reduced in the absence of C/EBPß. In addition, neurotoxicity elicited by LPS+IFN¿-treated microglia co-cultured with neurons was completely abolished by the absence of C/EBPß in microglia.

Oral administration of the KATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis

Background Multiple Sclerosis (MS) is an acquired inflammatory demyelinating disorder of the central nervous system (CNS) and is the leading cause of nontraumatic disability among young adults. Activated microglial cells are important effectors of demyelination and neurodegeneration, by secreting cytokines and others neurotoxic agents. Previous studies have demonstrated that microglia expresses ATP-sensitive potassium (KATP) channels and its pharmacological activation can provide neuroprotective and anti-inflammatory effects. In this study, we have examined the effect of oral administration of KATP channel opener diazoxide on induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Methods Anti-inflammatory effects of diazoxide were studied on lipopolysaccharide (LPS) and interferon gamma (IFNy)-activated microglial cells. EAE was induced in C57BL/6J mice by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Mice were orally treated daily with diazoxide or vehicle for 15 days from the day of EAE symptom onset. Treatment starting at the same time as immunization was also assayed. Clinical signs of EAE were monitored and histological studies were performed to analyze tissue damage, demyelination, glial reactivity, axonal loss, neuronal preservation and lymphocyte infiltration. Results Diazoxide inhibited in vitro nitric oxide (NO), tumor necrosis factor alpha (TNF-¿) and interleukin-6 (IL-6) production and inducible nitric oxide synthase (iNOS) expression by activated microglia without affecting cyclooxygenase-2 (COX-2) expression and phagocytosis. Oral treatment of mice with diazoxide ameliorated EAE clinical signs but did not prevent disease. Histological analysis demonstrated that diazoxide elicited a significant reduction in myelin and axonal loss accompanied by a decrease in glial activation and neuronal damage. Diazoxide did not affect the number of infiltrating lymphocytes positive for CD3 and CD20 in the spinal cord. Conclusion Taken together, these results demonstrate novel actions of diazoxide as an anti-inflammatory agent, which might contribute to its beneficial effects on EAE through neuroprotection. Treatment with this widely used and well-tolerated drug may be a useful therapeutic intervention in ameliorating MS disease.

Microglia, Calcification and Neurodegenerative Diseases

Neurodegeneration is a complex process involving different cell types andneurotransmitters. A common characteristic of neurodegenerative disorders such asAlzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis, Huntington’s disease (HD) and Amyotrophic Lateral Sclerosis (ALS) is the occurrence of a neuroinflammatoryreaction in which cellular processes involving glial cells (mainly microglia and astrocytes) and T cells are activated in response to neuronal death. This inflammatory reaction has recently received attention as an unexpected potential target for the treatment of these diseases.Microglial cells have a mesenchymal origin, invade the central nervous system (CNS)prenatally (Chan et al., 2007b) and are the resident macrophages in the CNS (Ransohoff &Perry, 2009). They comprise approximately 10-20% of adult glia and serve as the CNS innateimmune system. In neurodegenerative diseases, microglia is activated by misfoldedproteins. In the case of AD, amyloid- (A ) peptides accumulate extracellularly and activate the microglia locally. In the case of PD, ALS and HD, the misfolded proteins accumulate intracellularly but are still associated with activation of the microglia (Perry et al., 2010). Reactive microglia in the substantia nigra and striatum of PD brains have been described, and increased levels of proinflammatory cytokines and inducible nitric oxide synthase havebeen detected in these brain regions, providing evidence of a local inflammatory reaction (Hirsch & Hunot, 2009). The injection of lipopolysaccharide (a potent microglia activator) into the substantia nigra produces microglial activation and the death of dopaminergic cells. These findings support the hypothesis that microglial activation and neuroinflammationcontribute to PD pathogenesis (Herrera et al., 2000)...

Crucial role of CB(2) cannabinoid receptor in the regulation of central immune responses during neuropathic pain

Neuropathic pain is a clinical manifestation of nerve injury difficult to treat even with potent analgesic compounds. Here, we used different lines of genetically modified mice to clarify the role played by CB2 cannabinoid receptors in the regulation of the central immune responses leading to the development of neuropathic pain. CB2 knock-out mice and wild-type littermates were exposed to sciatic nerve injury, and both genotypes developed a similar hyperalgesia and allodynia in the ipsilateral paw. Most strikingly, knock-outs also developed a contralateral mirror image pain, associated with an enhanced microglial and astrocytic expression in the contralateral spinal horn. In agreement, hyperalgesia, allodynia, and microglial and astrocytic activation induced by sciatic nerve injury were attenuated in transgenic mice overexpressing CB2 receptors. These results demonstrate the crucial role of CB2 cannabinoid receptor in modulating glial activation in response to nerve injury. The enhanced manifestations of neuropathic pain were replicated in irradiated wild-type mice reconstituted with bone marrow cells from CB2 knock-outs, thus demonstrating the implication of the CB2 receptor expressed in hematopoietic cells in the development of neuropathic pain at the spinal cord.

Pattern of injury with a graded excitotoxic insult and ensuing chronic medial septal damage in the rat brain

Brain damage caused by an acute injury depends on the initial severity of the injury and the time elapsed after the injury. To determine whether these two variables activate common mechanisms, we compared the response of the rat medial septum to insult with a graded series of concentrations of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) with the time-course effects of a low dose of AMPA. For this purpose we conducted a dose-response study at concentrations of AMPA between 0.27 and 10.8 nmol to measure atrophy of the septal area, losses of cholinergic and GABAergic neurons, astroglial and microglial reactions, and calcification. Cholinergic neurons, whose loss paralleled the degree of septal atrophy produced by AMPA, are more sensitive than GABAergic neurons to the injury produced by AMPA. At doses of AMPA above 2.7 nmol, calcification and the degree of microglial reaction increased only in the GABAergic region of the septal area, whereas atrophy and neuronal loss reached a plateau. We chose the 2.7-nmol dose of AMPA to determine how these parameters were modified between 4 days and 6 months after injection. We found that atrophy and neuronal loss increased progressively through the 6-month study period, whereas astrogliosis ceased to be observed after 1 month, and calcium precipitates were never detected. We conclude that septal damage does not increase with the intensity of an excitotoxic insult. Rather, it progresses continuously after the insult. Because these two situations involve different mechanisms, short-term paradigms are inappropriate for interpreting the pathogenic mechanisms responsible for long-term neurodegenerative processes.