Juan Hidalgo

Roles of the metallothionein family of proteins in the central nervous system.

Metallothioneins (MTs) constitute a family of proteins characterized by a high heavy metal [Zn(II), Cu(I)] content and also by an unusual cysteine abundance. Mammalian MTs are comprised of four major isoforms designated MT-1 trough MT-4. MT-1 and MT-2 are expressed in most tissues including the brain, whereas MT-3 (also called growth inhibitory factor) and MT-4 are expressed predominantly in the central nervous system and in keratinizing epithelia, respectively. All MT isoforms have been implicated in disparate physiological functions, such as zinc and copper metabolism, protection against reactive oxygen species, or adaptation to stress. In the case of MT-3, an additional involvement of this isoform in neuromodulatory events and in the pathogenesis of Alzheimers disease has also been suggested. It is essential to gain insight into how MTs are regulated in the brain in order to characterize MT functions, both in normal brain physiology, as well as in pathophysiological states. The focus of this review concerns the biology of the MT family in the context of their expression and functional roles in the central nervous system.

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.

Strongly compromised inflammatory response to brain injury in interleukin‐6‐deficient mice

Injury to the central nervous system (CNS) elicits an inflammatory response involving activation of microglia, brain macrophages, and astrocytes, processes likely mediated by the release of proinflammatory cytokines. In order to determine the role of interleukin‐6 (IL‐6) during the inflammatory response in the brain following disruption of the blood–brain barrier (BBB), we examined the effects of a focal cryo injury to the fronto‐parietal cortex in interleukin‐6‐deficient (IL‐6−/−) and normal (IL‐6+/+) mice. In IL‐6+/+ mice, brain injury resulted in the appearance of brain macrophages and reactive astrocytes surrounding the lesion site. In addition, expression of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and metallothionein‐I+II (MT‐I+II) were increased in these cells, while the brain‐specific MT‐III was only moderately upregulated. In IL‐6−/− mice, however, the response of brain macrophages and reactive astrocytes was markedly depressed and the number of NSE positive neurons was reduced. Brain damage‐induced GM‐CSF and MT‐I+II expression were also markedly depressed compared to IL‐6+/+ mice. In contrast, MT‐III immunoreactivity was markedly increased in brain macrophages and astrocytes. In situ hybridization analysis indicates that MT‐I+II but not MT‐III immunoreactivity reflect changes in the messenger levels. The number of cell divisions was similar in IL‐6+/+ and IL‐6−/− mice. The present results demonstrate that IL‐6 is crucial for the recruitment of myelo‐monocytes and activation of glial cells following brain injury with disrupted BBB. Furthermore, our results suggest IL‐6 is important for neuroprotection and the induction of GM‐CSF and MT expression. The opposing effect of IL‐6 on MT‐I+II and MT‐III levels in the damaged brain suggests MT isoform‐specific functions. GLIA 25:343–357, 1999.

MHC class II-dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies

Antigen presentation, but not antibody secretion, by B cells drives CNS autoimmunity induced by immunization with human MOG.

Metallothionein in the central nervous system: Roles in protection, regeneration and cognition.

Metallothionein (MT) is an enigmatic protein, and its physiological role remains a matter of intense study and debate 50 years after its discovery. This is particularly true of its function in the central nervous system (CNS), where the challenge remains to link its known biochemical properties of metal binding and free radical scavenging to the intricate workings of brain. In this compilation of four reports, first delivered at the 11th International Neurotoxicology Association (INA-11) Meeting, June 2007, the authors present the work of their laboratories, each of which gives an important insight into the actions of MT in the brain. What emerges is that MT has the potential to contribute to a variety of processes, including neuroprotection, regeneration, and even cognitive functions. In this article, the properties and CNS expression of MT are briefly reviewed before Dr Hidalgo describes his pioneering work using transgenic models of MT expression to demonstrate how this protein plays a major role in the defence of the CNS against neurodegenerative disorders and other CNS injuries. His groups work leads to two further questions, what are the mechanisms at the cellular level by which MT acts, and does this protein influence higher order issues of architecture and cognition? These topics are addressed in the second and third sections of this review by Dr West, and Dr Levin and Dr Eddins, respectively. Finally, Dr Aschner examines the ability of MT to protect against a specific toxicant, methylmercury, in the CNS.

Interleukin-6 Regulation of AMP-Activated Protein Kinase: Potential Role in the Systemic Response to Exercise and Prevention of the Metabolic Syndrome

Interleukin (IL)-6 is a pleiotropic hormone that has both proinflammatory and anti-inflammatory actions. AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that among its other actions responds to decreases in cellular energy state by enhancing processes that generate ATP and inhibiting others that consume ATP but are not acutely necessary for survival. IL-6 is synthesized and released from skeletal muscle in large amounts during exercise, and in rodents, the resultant increase in its concentration correlates temporally with increases in AMPK activity in multiple tissues. That IL-6 may be responsible in great measure for these increases in AMPK is suggested by the fact it increases AMPK activity both in muscle and adipose tissue in vivo and in incubated muscles and cultured adipocytes. In addition, we have found that AMPK activity is diminished in muscle and adipose tissue of 3-month-old IL-6 knockout (KO) mice at rest and that the absolute increases in AMPK activity in these tissues caused by exercise is diminished compared with control mice. Except for an impaired ability to exercise and to oxidize fatty acids, the IL-6 KO mouse appears normal at 3 months of age. On the other hand, by age 9 months, it manifests many of the abnormalities of the metabolic syndrome including obesity, dyslipidemia, and impaired glucose tolerance. This, plus the association of decreased AMPK activity with similar abnormalities in a number of other rodents, suggests that a decrease in AMPK activity may be a causal factor. Whether increases in IL-6, by virtue of their effects on AMPK, contribute to the reported ability of exercise to diminish the prevalence of type 2 diabetes, coronary heart disease, and other disorders associated with the metabolic syndrome remains to be determined.

Impaired inflammatory response and increased oxidative stress and neurodegeneration after brain injury in interleukin-6-deficient mice.

In order to determine the role of the neuropoietic cytokine interleukin‐6 (IL‐6) during the first 3 weeks after a focal brain injury, we examined the inflammatory response, oxidative stress and neuronal survival in normal and interleukin‐6‐deficient (knockout, IL‐6KO) mice subjected to a cortical freeze lesion. In normal mice, the brain injury was followed by reactive astrogliosis and recruitment of macrophages from 1 day postlesion (dpl), peaking at 3–10 dpl, and by 20 dpl the transient immunoreactions were decreased, and a glial scar was present. In IL‐6KO mice, the reactive astrogliosis and recruitment of macrophages were decreased throughout the experimental period. The expression of the antioxidant and anti‐apoptotic factors metallothionein I+II (MT‐I+II) was increased prominently by the freeze lesion, but this response was significantly reduced in the IL‐6 KO mice. By contrast, the expression of the antioxidants Cu/Zn‐superoxide dismutase (Cu/Zn‐SOD), Mn‐SOD, and catalase remained unaffected by the IL‐6 deficiency. The lesioned mice showed increased oxidative stress, as judged by malondialdehyde (MDA) and nitrotyrosine (NITT) levels and by formation of inducible nitric oxide synthase (iNOS). IL‐6KO mice showed higher levels of MDA, NITT, and iNOS than did normal mice. Concomitantly, in IL‐6KO mice the number of apoptotic neurons was significantly increased as judged by TUNEL staining, and regeneration of the tissue was delayed relative to normal mice. The changes in neuronal tissue damage and in brain regeneration observed in IL‐6KO mice are likely caused by the IL‐6‐dependent decrease in MT‐I+II expression, indicating IL‐6 and MT‐I+II as neuroprotective factors during brain injury. GLIA 32:271–285, 2000.

Metallothionein-1+2 protect the CNS after a focal brain injury

We have evaluated the physiological relevance of metallothionein-1+2 (MT-1+2) in the CNS following damage caused by a focal cryolesion onto the cortex. In comparison to normal mice, transgenic mice overexpressing the MT-1 isoform (TgMTI* mice) showed a significant decrease of the number of activated microglia/macrophage and of CD3+ T lymphocytes in the area surrounding the lesion, while astrocytosis was increased. The TgMTI* mice showed a diminished peripheral macrophage but not CD3 T cell response to the cryolesion. This altered inflammatory response produced a decreased expression of the proinflammatory cytokines IL-1beta, IL-6, and TNF-alpha and an increased expression of the growth factors bFGF, TGFbeta1, and VEGF in the TgMTI* mice relative to control mice, which might be related to the increased angiogenesis and regeneration of the parenchyma of the former mice. The overexpression of MT-1 dramatically reduced the cryolesion-induced oxidative stress and neuronal apoptosis. Remarkably, these effects were also obtained by the intraperitoneal administration of MT-2 to both normal and MT-1+2 knock-out mice. These results fully support the notion that MT-1+2 are essential in the CNS for coping with focal brain injury and suggest a potential therapeutic use of these proteins.

ER Stress Cooperates with Hypernutrition to Trigger TNF-Dependent Spontaneous HCC Development

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of viral hepatitis, insulin resistance, hepatosteatosis, and nonalcoholic steatohepatitis (NASH), disorders that increase risk of hepatocellular carcinoma (HCC). To determine whether and how ER stress contributes to obesity-driven hepatic tumorigenesis we fed wild-type (WT) and MUP-uPA mice, in which hepatocyte ER stress is induced by plasminogen activator expression, with high-fat diet. Although both strains were equally insulin resistant, the MUP-uPA mice exhibited more liver damage, more immune infiltration, and increased lipogenesis and, as a result, displayed classical NASH signs and developed typical steatohepatitic HCC. Both NASH and HCC development were dependent on TNF produced by inflammatory macrophages that accumulate in the MUP-uPA liver in response to hepatocyte ER stress.

Enhanced seizures and hippocampal neurodegeneration following kainic acid‐induced seizures in metallothionein‐I + II‐deficient mice

Metallothioneins (MTs) are major zinc binding proteins in the CNS that could be involved in the control of zinc metabolism as well as in protection against oxidative stress. Mice lacking MT‐I and MT‐II (MT‐I + II deficient) because of targeted gene inactivation were injected with kainic acid (KA), a potent convulsive agent, to examine the neurobiological importance of these MT isoforms. At 35 mg/kg KA, MT‐I + II deficient male mice showed a higher number of convulsions and a longer convulsion time than control mice. Three days later, KA‐injected mice showed gliosis and neuronal injury in the hippocampus. MT‐I + II deficiency decreased both astrogliosis and microgliosis and potentiated neuronal injury and apoptosis as shown by terminal deoxynucleotidyl transferase‐mediated in situ end labelling (TUNEL), detection of single stranded DNA (ssDNA) and by increased interleukin‐1β‐converting enzyme (ICE) and caspase‐3 levels. Histochemically reactive zinc in the hippocampus was increased by KA to a greater extent in MT‐I + II‐deficient compared with control mice. KA‐induced seizures also caused increased oxidative stress, as suggested by the malondialdehyde (MDA) and protein tyrosine nitration (NITT) levels and by the expression of MT‐I + II, nuclear factor‐κB (NF‐κB), and Cu/Zn‐superoxide dismutase (Cu/Zn‐SOD). MT‐I + II deficiency potentiated the oxidative stress caused by KA. Both KA and MT‐I + II deficiency significantly affected the expression of MT‐III, granulocyte‐macrophage colony stimulating factor (GM‐CSF) and its receptor (GM‐CSFr). The present results indicate MT‐I + II as important for neuron survival during KA‐induced seizures, and suggest that both impaired zinc regulation and compromised antioxidant activity contribute to the observed neuropathology of the MT‐I + II‐deficient mice.