Albert Quintana

Interleukin-6, a major cytokine in the central nervous system.

Interleukin-6 (IL-6) is a cytokine originally identified almost 30 years ago as a B-cell differentiation factor, capable of inducing the maturation of B cells into antibody-producing cells. As with many other cytokines, it was soon realized that IL-6 was not a factor only involved in the immune response, but with many critical roles in major physiological systems including the nervous system. IL-6 is now known to participate in neurogenesis (influencing both neurons and glial cells), and in the response of mature neurons and glial cells in normal conditions and following a wide arrange of injury models. In many respects, IL-6 behaves in a neurotrophin-like fashion, and seemingly makes understandable why the cytokine family that it belongs to is known as neuropoietins. Its expression is affected in several of the main brain diseases, and animal models strongly suggest that IL-6 could have a role in the observed neuropathology and that therefore it is a clear target of strategic therapies.

mTOR Inhibition Alleviates Mitochondrial Disease in a Mouse Model of Leigh Syndrome

More from mTOR Leigh syndrome is a rare, untreatable, inherited neurodegenerative disease in children that is caused by functional disruption of mitochondria, the cells energy-producing organelles. Johnson et al. (p. 1524, published online 14 November; see Perspective by Vafai and Mootha) show that rapamycin, a drug used clinically as an immunosuppressant and for treatment of certain cancers, delayed the onset and progression of neurological symptoms in a mouse model of Leigh syndrome and significantly extended survival of the animals. Rapamycin inhibits the so-called “mTOR” signaling pathway, which is currently under intense study because it plays a contributory role in many common diseases. A drug in clinical use for other disorders delays progression of an untreatable mitochondrial disease in knockout mice. [Also see Perspective by Vafai and Mootha] Mitochondrial dysfunction contributes to numerous health problems, including neurological and muscular degeneration, cardiomyopathies, cancer, diabetes, and pathologies of aging. Severe mitochondrial defects can result in childhood disorders such as Leigh syndrome, for which there are no effective therapies. We found that rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, robustly enhances survival and attenuates disease progression in a mouse model of Leigh syndrome. Administration of rapamycin to these mice, which are deficient in the mitochondrial respiratory chain subunit Ndufs4 [NADH dehydrogenase (ubiquinone) Fe-S protein 4], delays onset of neurological symptoms, reduces neuroinflammation, and prevents brain lesions. Although the precise mechanism of rescue remains to be determined, rapamycin induces a metabolic shift toward amino acid catabolism and away from glycolysis, alleviating the buildup of glycolytic intermediates. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.

Complex I deficiency due to loss of Ndufs4 in the brain results in progressive encephalopathy resembling Leigh syndrome

To explore the lethal, ataxic phenotype of complex I deficiency in Ndufs4 knockout (KO) mice, we inactivated Ndufs4 selectively in neurons and glia (NesKO mice). NesKO mice manifested the same symptoms as KO mice including retarded growth, loss of motor ability, breathing abnormalities, and death by ~7 wk. Progressive neuronal deterioration and gliosis in specific brain areas corresponded to behavioral changes as the disease advanced, with early involvement of the olfactory bulb, cerebellum, and vestibular nuclei. Neurons, particularly in these brain regions, had aberrant mitochondrial morphology. Activation of caspase 8, but not caspase 9, in affected brain regions implicate the initiation of the extrinsic apoptotic pathway. Limited caspase 3 activation and the predominance of ultrastructural features of necrotic cell death suggest a switch from apoptosis to necrosis in affected neurons. These data suggest that dysfunctional complex I in specific brain regions results in progressive glial activation that promotes neuronal death that ultimately results in mortality.

Metallothionein reduces central nervous system inflammation, neurodegeneration, and cell death following kainic acid-induced epileptic seizures

We examined metallothionein (MT)‐induced neuroprotection during kainic acid (KA)‐induced excitotoxicity by studying transgenic mice with MT‐I overexpression (TgMT mice). KA induces epileptic seizures and hippocampal excitotoxicity, followed by inflammation and delayed brain damage. We show for the first time that even though TgMT mice were more susceptible to KA, the cerebral MT‐I overexpression decreases the hippocampal inflammation and delayed neuronal degeneration and cell death as measured 3 days after KA administration. Hence, the proinflammatory responses of microglia/macrophages and lymphocytes and their expression of interleukin (IL)‐1, IL‐6, IL‐12, tumor necrosis factor‐α and matrix metalloproteinases (MMP‐3, MMP‐9) were significantly reduced in hippocampi of TgMT mice relative to wild‐type mice. Also by 3 days after KA, the TgMT mice showed significantly less delayed damage, such as oxidative stress (formation of nitrotyrosine, malondialdehyde, and 8‐oxoguanine), neurodegeneration (neuronal accumulation of abnormal proteins), and apoptotic cell death (judged by TUNEL and activated caspase‐3). This reduced bystander damage in TgMT mice could be due to antiinflammatory and antioxidant actions of MT‐I but also to direct MT‐I effects on the neurons, in that significant extracellular MT presence was detected. Furthermore, MT‐I overexpression stimulated astroglia and increased immunostaining of antiinflammatory IL‐10, growth factors, and neurotrophins (basic fibroblastic growth factor, transforming growth factor‐β, nerve growth factor, brain‐derived neurotrophic factor, glial‐derived neurotrophic factor) in hippocampus. Accordingly, MT‐I has different functions that likely contribute to the increased neuron survival and improved CNS condition of TgMT mice. The data presented here add new insight into MT‐induced neuroprotection and indicate that MT‐I therapy could be used against neurological disorders.

Site-Specific Production of IL-6 in the Central Nervous System Retargets and Enhances the Inflammatory Response in Experimental Autoimmune Encephalomyelitis

IL-6 is crucial for the induction of many murine models of autoimmunity including experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. To establish the role of site-specific production of IL-6 in autoimmunity, we examined myelin oligodendrocyte glycoprotein immunization-induced EAE in transgenic mice (GFAP-IL6) with IL-6 production restricted to the cerebellum. Myelin oligodendrocyte glycoprotein-immunized (Mi-) GFAP-IL6 mice developed severe ataxia but no physical signs of spinal cord involvement, which was in sharp contrast to Mi-wild type (WT) animals that developed classical EAE with ascending paralysis. Immune pathology and demyelination were nearly absent from the spinal cord, but significantly increased in the cerebellum of Mi-GFAP-IL6 mice. Tissue damage in the cerebellum in the Mi-GFAP-IL6 mice was accompanied by increased total numbers of infiltrating leukocytes and increased proportions of both neutrophils and B-cells. With the exception of IL-17 mRNA, which was elevated in both control immunized and Mi-GFAP-IL6 cerebellum, the level of other cytokine and chemokine mRNAs were comparable with Mi-WT cerebellum whereas significantly higher levels of IFN-γ and TNF-α mRNA were found in Mi-WT spinal cord. Thus, site-specific production of IL-6 in the cerebellum redirects trafficking away from the normally preferred antigenic site the spinal cord and acts as a leukocyte “sink” that markedly enhances the inflammatory cell accumulation and disease. The mechanisms underlying this process likely include the induction of specific chemokines, activation of microglia, and activation and loss of integrity of the blood-brain barrier present in the cerebellum of the GFAP-IL6 mice before the induction of EAE.

Astrocyte-targeted expression of interleukin-6 protects the central nervous system during neuroglial degeneration induced by 6-aminonicotinamide

6‐Aminonicotinamide (6‐AN) is a niacin antagonist, which leads to degeneration of gray matter astrocytes mainly in the brainstem. We have examined the role of interleukin‐6 (IL‐6) in this degenerative process by using transgenic mice with astrocyte‐targeted IL‐6 expression (GFAP‐IL6 mice). This study demonstrates that transgenic IL‐6 expression significantly increases the 6‐AN‐induced inflammatory response of reactive astrocytes, microglia/macrophages, and lymphocytes in the brainstem. Also, IL‐6 induced significant increases in proinflammatory cytokines IL‐1, IL‐12, and tumor necrosis factor‐α as well as growth factors basic fibroblast growth factor (bFGF), transforming growth factor‐β, neurotrophin‐3, angiopoietin, vascular endothelial growth factor, and the receptor for bFGF. In accordance, angiogenesis was increased in GFAP‐IL6 mice relative to controls after 6‐AN. Moreover, oxidative stress and apoptotic cell death were significantly reduced by transgenic IL‐6 expression. IL‐6 is also a major inducer in the CNS of metallothionein I and II (MT‐I+II), which were significantly increased in the GFAP‐IL6 mice. MT‐I+II are antioxidants and neuroregenerative factors in the CNS, so increased MT‐I+II levels in GFAP‐IL6 mice could contribute to the reduction of oxidative stress and cell death in these mice.

Differential role of tumor necrosis factor receptors in mouse brain inflammatory responses in cryolesion brain injury

Tumor necrosis factor‐α (TNF‐α) is one of the mediators dramatically increased after traumatic brain injury that leads to the activation, proliferation, and hypertrophy of mononuclear, phagocytic cells and gliosis. Eventually, TNF‐α can induce both apoptosis and necrosis via intracellular signaling. This cytokine exerts its functions via interaction with two receptors: type‐1 receptor (TNFR1) and type‐2 receptor (TNFR2). In this work, the inflammatory response after a freeze injury (cryolesion) in the cortex was studied in wild‐type (WT) animals and in mice lacking TNFR1 (TNFR1 KO) or TNFR2 (TNFR2 KO). Lack of TNFR1, but not of TNFR2, significantly decreased the inflammatory response and tissue damage elicited by the cryolesion at both 3 and 7 days postlesion, with decreased gliosis, lower IL‐1β immunostaining, and a reduction of apoptosis markers. Cryolesion produced a clear induction of the proinflammatory cytokines interleukin (IL)‐1α, IL‐1β, IL‐6, and TNF‐α; this induction was significantly lower in the TNFR1 KO mice. Host response genes (ICAM‐1, A20, EB22/5, and GFAP) were also induced by the cryolesion, but to a lesser extent in TNFR1 KO mice. Lack of TNFR1 signaling also affected the expression of apoptosis/cell death‐related genes (Fas, Rip, p53), matrix metalloproteinases (MMP3, MMP9, MMP12), and their inhibitors (TIMP1), suggesting a role of TNFR1 in extracellular matrix remodeling after injury. However, GDNF, NGF, and BDNF expression were not affected by TNFR1 deficiency. Overall, these results suggest that TNFR1 is involved in the early establishment of the inflammatory response and that its deficiency causes a decreased inflammatory response and tissue damage following brain injury.

Lack of GPR88 enhances medium spiny neuron activity and alters motor- and cue-dependent behaviors.

The striatum regulates motor control, reward and learning. Abnormal function of striatal GABAergic medium spiny neurons (MSNs) is believed to contribute to the deficits in these processes that are observed in many neuropsychiatric diseases. The orphan G protein–coupled receptor GPR88 is robustly expressed in MSNs and is regulated by neuropharmacological drugs, but its contribution to MSN physiology and behavior is unclear. We found that, in the absence of GPR88, MSNs showed increased glutamatergic excitation and reduced GABAergic inhibition, which promoted enhanced firing rates in vivo, resulting in hyperactivity, poor motor coordination and impaired cue-based learning in mice. Targeted viral expression of GPR88 in MSNs rescued the molecular and electrophysiological properties and normalized behavior, suggesting that aberrant MSN activation in the absence of GPR88 underlies behavioral deficits and its dysfunction may contribute to behaviors observed in neuropsychiatric disease.

Brain response to traumatic brain injury in wild-type and interleukin-6 knockout mice: a microarray analysis

Traumatic injury to the brain is one of the leading causes of injury‐related death or disability. Brain response to injury is orchestrated by cytokines, such as interleukin (IL)‐6, but the full repertoire of responses involved is not well known. We here report the results obtained with microarrays in wild‐type and IL‐6 knockout mice subjected to a cryolesion of the somatosensorial cortex and killed at 0, 1, 4, 8 and 16 days post‐lesion. Overall gene expression was analyzed by using Affymetrix genechips/oligonucleotide arrays with ∼12 400 probe sets corresponding to ∼10 000 different murine genes (MG_U74Av2). A robust, conventional statistical method (two‐way anova) was employed to select the genes significantly affected. An orderly pattern of gene responses was clearly detected, with genes being up‐ or down‐regulated at specific timings consistent with the processes involved in the initial tissue injury and later regeneration of the parenchyma. IL‐6 deficiency showed a dramatic effect in the expression of many genes, especially in the 1 day post‐lesion timing, which presumably underlies the poor capacity of IL‐6 knockout mice to cope with brain damage. The results highlight the importance of IL‐6 controlling the response of the brain to injury as well as the suitability of microarrays for identifying specific targets worthy of further study.

RiboTag Analysis of Actively Translated mRNAs in Sertoli and Leydig Cells In Vivo

Male spermatogenesis is a complex biological process that is regulated by hormonal signals from the hypothalamus (GnRH), the pituitary gonadotropins (LH and FSH) and the testis (androgens, inhibin). The two key somatic cell types of the testis, Leydig and Sertoli cells, respond to gonadotropins and androgens and regulate the development and maturation of fertilization competent spermatozoa. Although progress has been made in the identification of specific transcripts that are translated in Sertoli and Leydig cells and their response to hormones, efforts to expand these studies have been restricted by technical hurdles. In order to address this problem we have applied an in vivo ribosome tagging strategy (RiboTag) that allows a detailed and physiologically relevant characterization of the “translatome” (polysome-associated mRNAs) of Leydig or Sertoli cells in vivo. Our analysis identified all previously characterized Leydig and Sertoli cell-specific markers and identified in a comprehensive manner novel markers of Leydig and Sertoli cells; the translational response of these two cell types to gonadotropins or testosterone was also investigated. Modulation of a small subset of Sertoli cell genes occurred after FSH and testosterone stimulation. However, Leydig cells responded robustly to gonadotropin deprivation and LH restoration with acute changes in polysome-associated mRNAs. These studies identified the transcription factors that are induced by LH stimulation, uncovered novel potential regulators of LH signaling and steroidogenesis, and demonstrate the effects of LH on the translational machinery in vivo in the Leydig cell.