Current Neurovascular Research, Vol. 2, No. 3, 2005
Contents
From the Editor's Perspective: Inflammatory Glial Cells of
the Nervous System: Assistants or Assassins? Pp.187-188
Kenneth Maiese
Original Articles
Increasing Expression of Heme Oxygenase-1 by Proteasome
Inhibition Protects Astrocytes from Heme-mediated Oxidative Injury
Pp.189-196
Jing Chen and Raymond F. Regan
mGluRI Targets Microglial Activation and Selectively
Prevents Neuronal Cell Engulfment Through Akt and Caspase Dependent Pathways
Pp.197-211
Zhao Zhong Chong, Jingqiong Kang, Faqi Li and Kenneth
Maiese
Review Articles
Neuronal and Glial Responses to Polyamines in the
Ischemic Brain Pp.213-223
Katsura Takano, Masato Ogura, Yoichi Nakamura and Yukio
Yoneda
Relaxin in Vascular Physiology and Pathophysiology: Possible
Implications in Ischemic Brain Disease Pp.225-233
Silvia Nistri and Daniele Bani
Recent Progress in Cerebrovascular Gene Therapy
Pp.235-247
Naoyuki Sato, Munehisa Shimamura and Ryuichi Morishita
Role of Kynurenines in the Central and Peripherial
Nervous Systems Pp.249-260
Hajnalka Nemeth, Jozsef Toldi and Laszlo Vecsei
Ferric Cycle Activity and Alzheimer Disease Pp.261-267
Barney E. Dwyer, Atsushi Takeda, Xiongwei Zhu, George
Perry and Mark A. Smith
Abstracts
[Back to top]
From the Editor's Perspective: Inflammatory Glial Cells of the Nervous
System: Assistants or Assassins?
Kenneth Maiese
The maintenance of extracellular homeostasis can be as critical to cellular survival as the protection or repair of intracellular nuclear DNA. Glial cells, such as astrocytes and microglia, appear to have a significant dual role in not only maintaining the extracellular environment, but also potentially leading to cellular injury in the nervous system.
As an example, microglial cells provide an interesting perspective of the "double life" glial cells can lead in the central nervous system. Microglia enter the central nervous system during embryonic development and function similar to peripheral macrophages for the phagocytic removal of apoptotic cells. Although several factors most likely play a role to foster the removal of injured cells by microglia, the early apoptotic translocation of membrane phosphatidylserine (PS) residues from the inner cellular membrane to the outer surface appears to be one of the principal mechanisms for the removal of injured cells by microglia. The phospholipids of the plasma membrane are normally in an asymmetric pattern with the outer leaflet of the plasma membrane consisting primarily of choline-containing lipids, such as phosphatidylcholine and sphingomyelin, and the inner leaflets consisting of aminophospholipids that include phosphatidylethanolamine and PS. The loss of membrane phospholipid asymmetry leads to the externalization of membrane PS residues and serves to identify cells for phagocytosis.
Yet, several additional studies have demonstrated that in addition to the early exposure of PS in apoptotic cells, the increased expression of the phosphatidylserine receptor in microglia also is necessary for microglial recognition of apoptotic cells in the nervous system. Furthermore, recognition of apoptotic cells by microglia also may require secreted factors, such as milk fat globule-EGF-factor 8, fractalkine, Gas6, and lipid lysophosphosphatidylcholine, that can assist microglia with the removal of injured cells.
It is important to note that glial cells, especially astrocytes and microglia, also can have a "dark side" in regards to cell injury and may promote rather than prevent cellular inflammation and injury in the nervous system. For example, if one examines the cellular processes modulated by microglia during Alzheimer's disease, it becomes evident that microglia may play an important part in the development of neurodegenerative diseases. Expression of markers that are indicative of microglial activation have been demonstrated to be significantly increased in patients with Alzheimer's disease. Even during early onset or mild progression of Alzheimer's disease, microglial activation has been shown to exist in regions of the entorhinal, parietal, and cingulate cortex. One of the major pathogens of Alzheimer's disease, namely b-amyloid (Ab), also has been shown to lead to inflammatory cell injury that involves microglial activation. Ab can precipitate a significant inflammatory response with microglial activation and result in the secretion of tumor necrosis factor-a (TNF-a). Microglial cells also have been demonstrated to co-localize with the perivascular deposits of Ab and lead to the progression of amyloid plaques.
Other neurodegenerative diseases that may be acute or chronic in nature also point to a critical association of glial activation with cellular injury. In Huntington's disease and amyotrophic lateral sclerosis, significant microglial activation has been reported in regions of the nervous system that are specific for these disease entities. Furthermore, microglial activation appears also to have a prominent role during acute injury of the nervous system. Following the induction of ischemic injury to cells, activation of microglia can parallel the induction of cellular apoptosis and correlate directly with the severity of the ischemic insult. These studies suggest that in cases of microglial activation, glial cells may be precipitate the early stages of several disease entities of the nervous system.
Although it is sometimes unclear whether glial cells mediate cell injury or become activated as a result of cell dysfunction, glial cells, such as microglia, have been shown to cause cellular damage either through the generation of reactive oxygen species or through the production of cytokines. For example, microglia stimulated by phorbol myristate acetate can release superoxide radicals. In addition, administration of scavenger agents for reactive oxygen species in the presence of activated microglia can prevent cellular injury, suggesting that oxidative stress generated by microglia can result in the demise of cells. Additional work has shown that microglia promote the production of pro-inflammatory cytokines, such as TNF-a and interleukin-1ß, as well as fatty acid metabolites, such as eicosanoids, that can precipitate cell death. TNF-a production by microglia also may be linked to neurodegeneration by increasing the sensitivity of neurons to free radical exposure.
In this issue of Current Neurovascular Research, new insights into the potential role of inflammatory cells are presented in both original articles and timely reviews that capture some of the unique qualities of astrocytes and microglia as essential mediators for both cellular survival and cellular disposal. In our initial article, Chen and Regan provide exciting and novel evidence for the role of the stress responsive transcription factor, Nrf2, as well as heme oxygenase-1 (HO-1) to offer cytoprotection to astrocytes. Under certain conditions, astrocytes can be supportive of neuronal function. The authors show that increased expression of Nrf2 in combination with HO-1 expression is associated with enhanced cortical astrocyte survival during insults such as acute cerebral hemorrhage that involve heme-mediated oxidative stress injury. In addition, a complimentary reduction in astrocytic protein oxidation during heme-mediated cell toxicity occurs during Nrf2 and HO-1 expression. Taken together, this work suggests that targeting overexpression of either Nrf2 or HO-1 may offer fertile ground for the development of strategies to protect the cerebral cortex from the devastating effects of acute cerebral hemorrhage.
Chong et al. provide an alternative, but compelling view to suppress rather than support glial cell function by illustrating the necessity to prevent acute inflammatory microglial activation during oxidative stress-induced neuronal injury. Targeting the metabotropic glutamate system, a family of GTP-binding proteins, they show that activation of group I metabotropic glutamate receptors (mGluRIs) can prevent the degradation of nuclear DNA during free radical exposure in primary hippocampal neurons. Yet, potentially more critical is the demonstration that mGluRI activation prevents neuronal PS membrane exposure and blocks the highly specific engulfment of individual living neurons by activated microglia. Furthermore, mGluRI activation employs a central cytoprotective pathway that involves protein kinase B (Akt1) to control microglia activity through pathways that involve mitochondrial permeability transition pore complex formation and caspase 9-like activity.
Our review articles for this issue extend the novel findings of these original articles and provide further insights into the complicated role of neuronal and glial interactions in the brain. The work by Takano et al. outlines the role of polyamines in the pathogenesis of ischemic cerebral injury and the intimate link of polyamines to excitotoxicity, reactive oxygen species, and cellular structural damage. In particular, these authors highlight the different susceptibility between neurons and glial cells to polyamine induced injury and suggest that mechanisms not seen in neurons can determine glial cell injury, highlighting the potential for the development of specific and independent cell directed therapies for disorders such as stroke. Nistri and Bani focus on the novel role of the peptide hormone relaxin and its ability to protect cells of either the cardiovascular or the nervous system. They discuss the broad function of relaxin via autocrine and paracrine routes in a variety of cell populations that lead to vasodilatation, angiogenesis, modulation of nitric oxide, and the control of inflammatory cell migration. They further describe several treatment options using relaxin mediated pathways in reference to ischemia and reperfusion injury.
Sato et al. compliments the analysis of targeted therapy for diseases of the nervous system by providing us with insight into the applications of gene therapy for cerebrovascular disease. They examine the utility of several viral vectors, such as adenovirus, adeno-associated virus, or herpes-simplex virus as well non-viral gene transfer. The authors also identify specific routes of delivery using both neuronal and glial cell derived systems to express trophic and angiogenic factors for cell protection and potential progenitor cell development. In the next review, Németh et al. provide us with additional insight into the complex parameters that can govern cell survival in the nervous system in regards to kynurenine and the metabolites of this agent. Multiple neurodegenerative disorders as well as systemic disorders are discussed, providing increased understanding for the extremely broad role that kynurenines possess throughout the body. This work focuses upon the vital link kynurenines have in the nervous system to a host of disorders, such as the association of kynurenine metabolites in glial cells with chronic epilepsy. As an alternative hypothesis on potential mechanisms of cell injury during oxidative stress, Smith and Dwyer complete this issue with their thought provoking discussion on the potential of heme deficiency to lead to some of the pathological consequences associated with Alzheimer's disease.
Given the varied nature of diseases of the nervous system and the multiple cellular pathways that must function together to lead to a specific disease pathology, it should come as no surprise that the complexity that occurs inside a cell will also define the varied relationships that can result between cells. For example, activated microglia may assist during the recovery phase in the brain following an injury, such as with the removal of injured cells and debri following cerebral hemorrhage. Yet, under different conditions, these cellular scavengers of the brain also may be the principal source for escalating tissue inflammation and promoting apoptotic cell injury in otherwise functional and intact neighboring cells of the brain. In light of the significant and multifaceted role inflammatory glial cells may play during a variety of disorders of the nervous system, investigations that seek to elucidate the cellular pathways controlled by these cells may greatly further not only our understanding of disease mechanisms, but also our development of targeted treatments for acute and chronic diseases of the nervous system. Continued progression of innovative investigations and especially those that examine previously unexplored pathways of glial cell biology, such as those presented in this issue of Current Neurovascular Research, should bring us closer to determining whether we have an "assistant" or an "assassin" at hand.
[Back to top]
Increasing Expression of Heme Oxygenase-1 by Proteasome Inhibition Protects
Astrocytes from Heme-mediated Oxidative Injury
Jing Chen and Raymond F. Regan
Hemin is released from hemoglobin after CNS hemorrhage, and may contribute to cell loss in surrounding tissue. Heme oxygenase-1 (HO-1) is induced by these injuries, and may have an effect on cell viability. In a prior study, we reported that increasing HO-1 expression by adenoviral gene transfer prior to hemin exposure attenuated oxidative stress and cell death in astrocytes. However, rapid gene transfer to the CNS may not be feasible. HO-1 expression is controlled by a stress-responsive transcription factor, Nrf2, which is a labile protein that is subject to proteasomal degradation. In this study, we hypothesized that preventing degradation of Nrf2 with a lipid-soluble proteasome inhibitor would increase HO-1 expression and protect astrocytes from hemin. Treatment of cortical astrocyte cultures with 1 mM MG-132 resulted in a rapid increase in Nrf2, to a level that was five-fold that of vehicle-treated cultures by 2 h. This was followed by a three to six-fold increase in HO-1 expression that persisted through the 16 h observation period. Exposure of cultures to 30 mM or 60 mM hemin for 8 h resulted in death, as measured by LDH release, of 39±3.0 or 67.5±5.9% of astrocytes. Pre-treatment with MG-132 prevented approximately half of this injury. Cytoprotection persisted at 24 h, and was also observed when injury was assessed via the MTT assay. Astrocyte protein oxidation produced by hemin was also significantly attenuated by MG-132 pre-treatment. These results suggest that increasing HO-1 expression with a proteasome inhibitor protects astrocytes from heme-mediated oxidative injury. This pharmacological approach may provide a mechanism for rapidly upregulating HO-1 in astrocytes after CNS hemorrhage.
[Back to top] mGluRI
Targets Microglial Activation and Selectively Prevents Neuronal Cell Engulfment
Through Akt and Caspase Dependent Pathways
Zhao Zhong Chong, Jingqiong Kang, Faqi Li and Kenneth
Maiese
Metabotropic glutamate receptors (mGluRs) are expressed throughout the mammalian central nervous system and integrate a host of signal transduction pathways that determine cellular function, plasticity and injury. Yet, one of the more unique regulatory functions of this family of GTP-binding proteins involves cytoprotection in the nervous system. Here, we demonstrate that activation of group I metabotropic glutamate receptors (mGluRIs) in primary hippocampal neurons not only provides intrinsic cellular protection for the maintenance of genomic DNA integrity, but also prevents inflammatory microglial activation and specific neuronal cell engulfment during free radical oxidative stress. Loss of cellular membrane asymmetry and exposure of membrane phosphatidylserine (PS) residues were necessary and sufficient to result in microglial activation and proliferation, since administration of an antibody to the PS receptor could block microglial activity. Through the continuous assessment of individual neurons in real time, activation of mGluRIs was documented to block neuronal PS exposure and prevented subsequent neuronal cell engulfment by microglia seeking "PS tagged" neurons. Furthermore, regulation of both cellular integrity and microglial activity by mGluRI activation was dependent upon the activation and phosphorylation of protein kinase B (Akt1), prevention of mitochondrial membrane depolarization with associated permeability transition pore complex formation, and the down regulation of caspase 9-like activity. Our work defines a significant role of mGluRIs for the modulation of cellular survival and inflammation in the nervous system during oxidative stress.
[Back
to top] Neuronal and Glial
Responses to Polyamines in the Ischemic Brain
Katsura Takano, Masato Ogura, Yoichi Nakamura and Yukio
Yoneda
The polyamines, putrescine, spermidine and spermine are present in most living cells, with the essentiality for normal cell function, cellular growth and differentiation. In the mammalian brain, polyamines are also present at relatively high concentrations with different regional distribution profiles. Cerebral ischemia is a leading cause of disability and mortality in humans, and believed to yield a cascade of cytotoxic molecules responsible for the death of viable cells in the brain. Polyamines have been implicated in the pathogenesis of ischemic brain damage. For example, polyamine biosynthesis is increased after the onset of cerebral ischemia through an induction of ornithine decarboxylase, a key enzyme in the polyamine biosynthetic pathway. The administration of a drug that inhibits ornithine decarboxylase activity prevents the development of ischemic brain damage, suggesting a critical role of the accumulation of polyamines in the ischemic brain in the pathogenesis of stroke. Both spermine and spermidine are linked to the development of glutamate-mediated neurotoxicity, for they can bind to the N-methyl-D-aspartate (NMDA)-sensitive subtype of glutamate receptors to potentiate cellular responses to glutamate. Moreover, polyamines are metabolized by polyamine oxidases after acetylation to produce different cytotoxic aldehydes and reactive oxygen species such as hydrogen peroxide, which possibly damage proteins, DNA and lipids. Polyamines have been extensively studied in the ischemic brain, particularly with respect to neuronal responses such as NMDA receptor-mediated excitotoxicity. However, little is known about glial responses to polyamines in the ischemic brain to date. In this review, we would summarize previous studies related to neuronal and glial responses to polyamines in the ischemic brain.
[Back to top]
Relaxin in Vascular Physiology and Pathophysiology: Possible Implications in
Ischemic Brain Disease
Silvia Nistri and Daniele Bani
The hormone relaxin, known for its action on the female reproductive tract, is also able to act on organs and systems different from the reproductive ones, including the blood vessels, the heart and the brain. Relaxin causes vasodilation in several organs stimulating the biosynthetic pathway of nitric oxide (NO), a potent vasodilator. Relaxin also has a cardioprotective action: it reduces the inflammatory activation of neutrophils and their adhesion to the endothelium, and protects against myocardial injury caused by ischemia and reperfusion (I-R) in experimental animal models of myocardial infarction. Its mechanisms of action chiefly depend on the hormone’s vasodilatory and anti-inflammatory properties. Recently, an additional form of relaxin has been discovered in the brain, where it has been postulated to act locally as a neurotransmitter. Relaxin, acting mainly on circumventricular organs, stimulates water drinking and vasopressin release and appears to be involved in the regulation of behavioural processes. Based on its properties on the cardiovascular system, it is possible to hypothesise that relaxin could regulate the vascular tone in the central nervous system and, going a step further, could protect the brain from IR-induced damage, possibly by an NO-mediated mechanism. This latter possibility is supported by the observation that relaxin is able to up regulate the endogenous production of NO in several target cells, as NO, at appropriate levels, is known to be involved in the protection against neural pathophysiological processes such as I-R-induced injury.
[Back to top] Recent
Progress in Cerebrovascular Gene Therapy
Naoyuki Sato, Munehisa Shimamura and Ryuichi Morishita
Gene therapy provides a potential strategy for the treatment of cardiovascular disease such as peripheral arterial disease, myocardial infarction, restenosis after angioplasty, and vascular bypass graft occlusion. Currently, more than 20 clinical studies of gene therapy for cardiovascular disease are in progress. Although cerebrovascular gene therapy has not proceeded to clinical trials, in contrast to cardiovascular gene therapy, there have been several trials in experimental models. Three major potential targets for cerebrovascular gene therapy are vasospasm after subarachnoid hemorrhage (SAH), ischemic cerebrovascular disease, and restenosis after angioplasty, for which current therapy is often inadequate. In experimental SAH models, strategies using genes encoding a vasodilating protein or decoy oligodeoxynucleotides have been reported to be effective against vasospasm after SAH. In experimental ischemic cerebrovascular disease, gene therapy using growth factors, such as Brain-derived neurotrophic factor (BDNF), Fibroblast growth factor-2 (FGF-2), or Hepatocyte growth factor (HGF), has been reported to be effective for neuroprotection and angiogenesis. Nevertheless, cerebrovascular gene therapy for clinical human treatment still has some problems, such as transfection efficiency and the safety of vectors. Development of an effective and safe delivery system for a target gene will make human cerebrovascular gene therapy possible.
[Back to top] Role
of Kynurenines in the Central and Peripherial Nervous Systems
Hajnalka Nemeth, Jozsef Toldi and Laszlo Vecsei
Kynurenine (KYN) is an intermediate in the pathway of the metabolism of tryptophan to nicotinic acid. KYN is formed in the mammalian brain (40%) and is taken up from the periphery (60%), indicating that it can be transported across the blood-brain barrier (BBB). In the brain, KYN can be converted to two other components of the pathway: the neurotoxic quinolinic acid (QUIN) and the neuroprotective kynurenic acid (KYNA). QUIN is probably the most widely studied metabolite of KYN, because it may cause excitotoxic neuronal cell loss and convulsions by interacting with the N-methyl-D-aspartate (NMDA) receptor complex, a type of glutamate receptor. KYNA is another metabolite of KYN; its synthesis is catalysed by KYN aminotransferases. This is the only known endogenous NMDA receptor inhibitor, which can act at the glycine site on the receptor complex. Furthermore, KYNA non-competitively inhibits a7 nicotinic acetylcholine presynaptic receptors (nAChRs), inhibiting glutamate release, and regulates the expression of a4b2 nAChR. It is well-known that the activation of excitatory amino acid (EAA) receptors can play a role in a number of neurodegenerative disorders, such as Parkinson’s disease, Alzheimer’s disease, stroke and epilepsy. Various studies have been made of whether the EAA receptor antagonist KYNA can exert a therapeutic effect in these neurological disorders. It has been established that KYNA has only a very limited ability to cross the BBB. Other KYNA derivatives have been synthesised (e.g. glucosamine-KYNA, 4-chloro-KYNA and 7-chloro-KYNA), which are well transported across the BBB and act on the glutamate receptors. Moreover, it has been demonstrated that probenecid, a known inhibitor of the transport of organic acids (e.g. KYNA), increases the cerebral concentration of KYNA. There is another new perspective to the maintenance of a high level of KYNA in the brain: the use of enzyme inhibitors, which can block the synthesis of the neurotoxic QUIN. These are some of the most promising possibilities as novel therapeutic strategies for the treatment of neurodegenerative diseases, in which the hyperactivation of amino acid receptors could be involved. The presence and importance of KYN derivatives in the periphery are also discussed in the light of recent publications.
[Back to top] Ferric Cycle
Activity and Alzheimer Disease
Barney E. Dwyer, Atsushi Takeda, Xiongwei Zhu, George
Perry and Mark A. Smith
Elevated plasma homocysteine is an independent risk factor for the development of Alzheimer disease, however, the precise mechanisms underlying this are unclear. In this article, we expound on a novel hypothesis depicting the involvement of homocysteine in a vicious circle involving iron dysregulation and oxidative stress designated as the ferric cycle (Dwyer et al., 2004). Moreover, we suspect that the development of a critical heme deficiency in vulnerable neurons is an additional consequence of ferric cycle activity. Oxidative stress and heme deficiency are consistent with many pathological changes found in Alzheimer disease including mitochondrial abnormalities and impaired energy metabolism, cell cycle and cell signaling abnormalities, neuritic pathology, and other features of the disease involving alterations in iron homeostasis such as the abnormal expression of heme oxygenase-1 and iron response protein 2. Based on the ferric cycle concept, we have developed a model of Alzheimer disease development and progression, which offers an explanation for why sporadic Alzheimer disease is different than normal aging and why familial Alzheimer disease and sporadic Alzheimer disease could have different etiologies but a common end-stage.