Laminin is induced in astrocytes of adult brain by injury.

Laminin is a high mol. wt. non-collagenous matrix glycoprotein, confined in adult tissues to basement membranes. In normal rat brain we found laminin mainly in vessel walls but, after injury, induced by stereotaxic injection of a neurotoxin, laminin immunoreactivity appeared also in reactive astrocytes, which are characteristically positive for the glial fibrillary acidic protein (GFAP). Laminin was first detected in GFAP-immunoreactive glial cells 24 h after injury. Four days later the majority of reactive astrocytes in the gray matter were positive for laminin and the laminin immunoreactivity, but not that of GFAP, gradually subsided within a month. Fibronectin, the other major matrix glycoprotein, was found only in capillary structures both in normal and lesioned brain tissue. The results indicate that mature astrocytes have the potential to produce laminin and suggest a role for this glycoprotein in brain regeneration.     

Glial responses to steroids as markers of brain aging.

Glia mediate neuroendocrine and neuroimmune functions that are altered during the process of normal aging. The biological functions of glia are also important in synaptic remodeling and the loss of synaptic connections that occur during aging. These functions are carried out by changes in glia, including changes in shape, interactions with neurons and other glia, and gene expression. The predominant change that occurs in glia during aging is glial activation, which can progress to reactive gliosis in response to neurodegeneration. More markers are needed to distinguish normal and reactive glia. During aging, astrocytes hypertrophy and exhibit signs of metabolic activation, and astrocytic processes surround neurons. Microglia also become activated and subsets of activated microglial increase in number and may enter the phagocytic or reactive stage. Glial markers of brain aging and glial activation include glial fibrillary acidic protein (GFAP) and transforming growth factor (TGF)-beta1, which are increased in astrocytes and microglia, respectively. Steroids regulate the interactions between glia and neurons and glial gene expression, including GFAP and TGF-beta1. Therefore, changes in these parameters during aging may be due to altered steroid regulation. In general, the effects of steroids oppose the effects of aging. Recent data indicate that steroid treatment can decrease the expression of GFAP in the aged brain, yet GFAP is resistant to down-regulation by endogenous glucocorticoids. Cellular and molecular markers of glial activation are being used to determine how changes in neuroendocrine and neuroimmune regulation contribute to repair and functional recovery that may reverse synaptic loss and cognitive impairment during aging.     

Apolipoprotein D expression in substantia nigra of Parkinson disease

Apolipoprotein D (apo D), a lipocalin transporter of small hydrophobic molecules could play an important role in several neurodegenerative diseases. However, its role in those diseases remains unclear. Increments of apo D have been reported in relation with injury and degeneration in the nervous system. Recently increases of apo D level have been reported in schizophrenia, a neuropathologic disease where the oxidative stress and lipid abnormalities may be involved. Apo D could act as a sequestering molecule binding excess of arachidonic acid in cells. In order to determine the relationship between apo D expression and other neurodegenerative pathologies related to oxidative damage, we studied the presence of apo D in the substantia nigra of control and Parkinson disease (PD) subjects. We found dopaminergic neurons were not immunoreactive for apo D, control or PD subjects. However, surrounding glial cells showed immunostaining for apo D and signal increases in PD cases. These findings support the role of apolipoprotein D in neuroprotection and the importance of glia in the amount of this protein in the central nervous system.     

Role of the lesion scar in the response to damage and repair of the central nervous system

Traumatic damage to the central nervous system (CNS) destroys the blood-brain barrier (BBB) and provokes the invasion of hematogenous cells into the neural tissue. Invading leukocytes, macrophages and lymphocytes secrete various cytokines that induce an inflammatory reaction in the injured CNS and result in local neural degeneration, formation of a cystic cavity and activation of glial cells around the lesion site. As a consequence of these processes, two types of scarring tissue are formed in the lesion site. One is a glial scar that consists in reactive astrocytes, reactive microglia and glial precursor cells. The other is a fibrotic scar formed by fibroblasts, which have invaded the lesion site from adjacent meningeal and perivascular cells. At the interface, the reactive astrocytes and the fibroblasts interact to form an organized tissue, the glia limitans. The astrocytic reaction has a protective role by reconstituting the BBB, preventing neuronal degeneration and limiting the spread of damage. While much attention has been paid to the inhibitory effects of the astrocytic component of the scars on axon regeneration, this review will cover a number of recent studies in which manipulations of the fibroblastic component of the scar by reagents, such as blockers of collagen synthesis have been found to be beneficial for axon regeneration. To what extent these changes in the fibroblasts act via subsequent downstream actions on the astrocytes remains for future investigation.     

FABP7 expression in normal and stab-injured brain cortex and its role in astrocyte proliferation

Reactive gliosis, in which astrocytes as well as other types of glial cells undergo massive proliferation, is a common hallmark of all brain pathologies. Brain-type fatty acid-binding protein (FABP7) is abundantly expressed in neural stem cells and astrocytes of developing brain, suggesting its role in differentiation and/or proliferation of glial cells through regulation of lipid metabolism and/or signaling. However, the role of FABP7 in proliferation of glial cells during reactive gliosis is unknown. In this study, we examined the expression of FABP7 in mouse cortical stab injury model and also the phenotype of FABP7-KO mice in glial cell proliferation. Western blotting showed that FABP7 expression was increased significantly in the injured cortex compared with the contralateral side. By immunohistochemistry, FABP7 was localized to GFAP(+) astrocytes (21% of FABP7(+) cells) and NG2(+) oligodendrocyte progenitor cells (62%) in the normal cortex. In the injured cortex there was no change in the population of FABP7(+)/NG2(+) cells, while there was a significant increase in FABP7(+)/GFAP(+) cells. In the stab-injured cortex of FABP7-KO mice there was decrease in the total number of reactive astrocytes and in the number of BrdU(+) astrocytes compared with wild-type mice. Primary cultured astrocytes from FABP7-KO mice also showed a significant decrease in proliferation and omega-3 fatty acid incorporation compared with wild-type astrocytes. Overall, these data suggest that FABP7 is involved in the proliferation of astrocytes by controlling cellular fatty acid homeostasis.     

Connecting Malfunctioning Glial Cells and Brain Degenerative Disorders

The DNA damage response(DDR) is a complex biological system activated by different types of DNA damage.Mutations in certain components of the DDR machinery can lead to genomic instability disorders that culminate in tissue degeneration,premature aging,and various types of cancers.Intriguingly,malfunctioning DDR plays a role in the etiology of late onset brain degenerative disorders such as Parkinson's,Alzheimer's,and Huntington's diseases.For many years,brain degenerative disorders were thought to result from aberrant neural death.Here we discuss the evidence that supports our novel hypothesis that brain degenerative diseases involve dysfunction of glial cells(astrocytes,microglia,and oligodendrocytes).Impairment in the functionality of glial cells results in pathological neuro-glial interactions that,in turn,generate a ‘‘hostile" environment that impairs the functionality of neuronal cells.These events can lead to systematic neural demise on a scale that appears to be proportional to the severity of the neurological deficit.     

The role of tachykinins via NK1 receptors in progression of human gliomas

In recent years, it has become evident that astrocytes harbor functional receptors to many neurotransmitters. including substance P (SP), an undecapeptide belonging to the tachykinin family of neuropeptides. SP is an important stimulus for reactive astrocytes in CNS development, infection and injury, and provides a link for bi-directional interactions between glial cells and neurons. In brain tumors, malignant glial cells originating from astrocytes, via NK1 receptors, are triggered by tachykinins, SP and neurokinin A (NKA), to release soluble mediators, in particular cytokines, and increase their proliferative rate. In this paper, we review the results obtained in in vitro and in vivo studies on the role of SP as an inducer of human glioma responses that may be relevant for tumor progression. In addition, the presence of SP and the expression of NK1 receptors in glioma explants have been examined. We discuss the possible use of selective NK1 receptor antagonists as a therapeutic approach to treat malignant gliomas.     

Analysis of glial acidic fibrillary protein in the human entorhinal cortex during aging and in Alzheimer's disease

Glial fibrillary acidic protein, GFAP, is a major intermediate filament protein of glial cells and major cytoskeletal structure in astrocytes. The entorhinal cortex has a key role in memory function and is one of the first brain areas to reveal hallmark structures of Alzheimer's disease and therefore provides an ideal tissue to investigate incipient neurodegenerative changes. Here we have analyzed age- and disease-related occurrence and composition of GFAP in the human entorhinal cortex by using one- and two-dimensional electrophoresis, Western blots and immunocytochemistry combined with confocal microscopy. A novel monoclonal antibody, GF-02, was characterized that mainly reacted with intact GFAP molecules and indicated that more acidic and soluble GFAP forms were also more susceptible to degradation. GFAP and vimentin increased with aging and in Alzheimer's disease (AD). Two-dimensional electrophoresis and Western blots revealed a complex GFAP pattern, both in aging and AD with different modification and degradation forms. Immunohistochemistry indicated that reactive astrocytes mainly accumulated in relation to neurofibrillary tangles and senile plaques in deeper entorhinal cortex layers. GFAP may be used as an additional but not exclusive diagnostic tool in the evaluation of neurodegenerative diseases because its levels change with age and respond to senile plaque and tangle formation.     

Localization of immunoglobulins G, A and M in glial cells of reactive and neoplastic origin

The localization of immunoglobulins G, A and M in glial cells of neoplastic and reactive origin have been investigated by the use of the PAP (peroxidase-antiperoxidase) method on paraffin embedded tissue previously fixed in calcium formol. It has been found, that some glial cells of astrocyte type showed a very intense staining when oligoclonal antibodies to human immunoglobulins G, A and M specific for gamma, alpha, and mu chains were used. The localization of immunoglobulins was disclosed in astrocytes of various morphology; astrocytes with well developed processes, gemistocyte type cells without or only with short and thick cell processes and in small cells with scanty cytoplasm. The number of cells with immunoglobulins localized is very small. No positive results have been noted if the normal brain tissue is concerned. The specificity of the method is discussed.     

Intermediate filaments regulate astrocyte motility.

Intermediate filaments (IFs) compose, together with actin filaments and microtubules, the cytoskeleton and they exhibit a remarkable but still enigmatic cell-type specificity. In a number of cell types, IFs seem to be instrumental in the maintenance of the mechanical integrity of cells and tissues. The function of IFs in astrocytes has so far remained elusive. We have recently reported that glial scar formation following brain or spinal cord injury is impaired in mice deficient in glial fibrillary acidic protein and vimentin. These mice lack IFs in reactive astrocytes that are normally pivotal in the wound repair process. Here we show that reactive astrocytes devoid of IFs exhibit clear morphological changes and profound defects in cell motility thereby revealing a novel function for IFs.     

Molecular mechanisms of neuroprotective action of immunosuppressants--facts and hypotheses

Cyclosporin A (CsA) and FK506 (Tacrolimus) are short polypeptides which block the activation of lymphocytes and other immune system cells. Immunosuppressants exert neuroprotective and neurotrophic action in traumatic brain injury, sciatic nerve injury, focal and global ischemia in animals. Their neuroprotective actions are not understood and many hypotheses have been formed to explain such effects. We discuss a role of drug target - calcineurin in neuroprotective action of immunosuppressants. Protein dephosphorylation by calcineurin plays an important role in neuronal signal transduction due to its ability to regulate the activity of ion channels, glutamate release, and synaptic plasticity. In vitro FK506 protects cortex neurons from NMDA-induced death, augments NOS phosphorylation inhibiting its activity and NO synthesis. However, in vivo experiments demonstrated that FK506 in neuroprotective doses did not block excitotoxic cell death nor did it alter NO production during ischemia/reperfusion. Tissue damage in ischemia is the result of a complex pathophysiological cascade, which comprises a variety of distinct pathological events. Resident non-neuronal brain cells respond rapidly to neuronal cell death and may have both deleterious and useful role in neuronal damage. There is increasing evidence that reactive gliosis and post-ischemic inflammation involving microglia contribute to ischemic damage. We have demonstrated that FK506 modulates hypertrophic/proliferative responses and proinflammatory cytokine expression in astrocytes and microglia in vitro and in focal transient brain ischemia. Our findings suggest that astrocytes and microglia are direct targets of FK506 and modulation of glial response and inflammation is a possible mechanism of FK506-mediated neuroprotection in ischemia.     

Astrocyte,the star <Emphasis Type="Italic">avatar</Emphasis>: redefined

Until recently, the neuroscience community held the belief that glial cells such as astrocytes and oligodendrocytes functioned solely as “support” cells of the brain. In this role, glial cells simply provide physical support and housekeeping functions for the more important cells of the brain, the neurons. However, this view has changed radically in recent years with the discovery of previously unrecognized and surprising functions for this underappreciated cell type. In the past decade or so, emerging evidence has provided new insights into novel glial cell activities such as control of synapse formation and function, communication, cerebrovascular tone regulation, immune regulation and adult neurogenesis. Such advances in knowledge have effectively elevated the role of the astrocyte to one that is more important than previously realized. This review summarizes the past and present knowledge of glial cell functions that has evolved over the years, and has resulted in a new appreciation of astrocytes and their value in studying the neurobiology of human brain cells and their functions. In this review, we highlight recent advances in the role of glial cells in physiology, pathophysiology and, most importantly, in adult neurogenesis and “stemness”, with special emphasis on astrocytes.     

Inorganic mercury in human astrocytes,oligodendrocytes, corticomotoneurons and the locus ceruleus: implications for multiple sclerosis,neurodegenerative disorders and gliomas

Neurotoxic metals have been implicated in the pathogenesis of multiple sclerosis, neurodegenerative disorders and brain tumours but studies of the location of heavy metals in human brains are rare. In a man who injected himself with metallic mercury the cellular location of mercury in his brain was studied after 5 months of continuous exposure to inorganic mercury arising from metallic mercury deposits in his organs. Paraffin sections from the primary motor and sensory cortices and the locus ceruleus in the pons were stained with autometallography to detect inorganic mercury and combined with glial fibrillary acidic protein immunohistochemistry to identify astrocytes. Inorganic mercury was found in grey matter subpial, interlaminar, protoplasmic and varicose astrocytes, white matter fibrous astrocytes, grey but not white matter oligodendrocytes, corticomotoneurons and some locus ceruleus neurons. In summary, inorganic mercury is taken up by five types of human brain astrocytes, as well as by cortical oligodendrocytes, corticomotoneurons and locus ceruleus neurons. Mercury can induce oxidative stress, stimulate autoimmunity and damage DNA, mitochondria and lipid membranes, so its location in these CNS cells suggests it could play a role in the pathogenesis of multiple sclerosis, neurodegenerative conditions such as Alzheimer’s disease and amyotrophic lateral sclerosis, and glial tumours.     

A combined staining method for argyrophilic nucleolar organizer regions and for glial fibrillary acidic protein in astrocytes of human brain

Summary Different protocols are described for the combined staining method by which argyrophilic nucleolar organizer region sites can be evaluated in human astrocytes that are immunoreactive for glial fibrillary acidic protein. Among the four protocols studied, the following method was superior to others in terms of unambiguous visualization of the regions in glial fibrillary acidic protein-positive astrocytes; the first step was immunostaining for the protein with a blue colour reaction of alkaline phosphatase, followed by sequential colloidal silver staining for the regions. By this double staining method, we have demonstrated that the reactive astrocytes found in white matter around the metastatic lesion of carcinoma and the infarction, contain more argyrophilic nucleolar organizer regions in terms of the count as well as the area than glial fibrillary acidic protein-positive astrocytes present in the white matter of the normal brain. In conclusion, the double staining may provide valuable information on the cellular activity of astroglia when performed on routine formalin-fixed paraffin sections of the human brain.     

Downregulation of FIP200 induces apoptosis of glioblastoma cells and microvascular endothelial cells by enhancing Pyk2 activity

The expression of focal adhesion kinase family interacting protein of 200-kDa (FIP200) in normal brain is limited to some neurons and glial cells. On immunohistochemical analysis of biopsies of glioblastoma tumors, we detected FIP200 in the tumor cells, tumor-associated endothelial cells, and occasional glial cells. Human glioblastoma tumor cell lines and immortalized human astrocytes cultured in complete media also expressed FIP200 as did primary human brain microvessel endothelial cells (MvEC), which proliferate in culture and resemble reactive endothelial cells. Downregulation of endogenous expression of FIP200 using small interfering RNA resulted in induction of apoptosis in the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC. It has been shown by other investigators using cells from other tissues that FIP200 can interact directly with, and inhibit, proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK). In the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC, we found that downregulation of FIP200 increased the activity of Pyk2 without increasing its expression, but did not affect the activity or expression of FAK. Coimmunoprecipitation and colocalization studies indicated that the endogenous FIP200 was largely associated with Pyk2, rather than FAK, in the glioblastoma tumor cells and brain MvEC. Moreover, the pro-apoptotic effect of FIP200 downregulation was inhibited significantly by a TAT-Pyk2-fusion protein containing the Pyk2 autophosphorylation site in these cells. In summary, downregulation of endogenous FIP200 protein in glioblastoma tumor cells, astrocytes, and brain MvECs promotes apoptosis, most likely due to the removal of a direct interaction of FIP200 with Pyk2 that inhibits Pyk2 activation, suggesting that FIP200 expression may be required for the survival of all three cell types found in glioblastoma tumors.     

Formation and function of apolipoprotein E-containing lipoproteins in the nervous system

The strongest known genetic risk factor for the development of late-onset Alzheimer disease is inheritance of the apolipoprotein (apo) E4 (ε4 allele) although the mechanisms underlying this connection are still not entirely clear. In this review, we shall discuss the role of apo E in the brain, particularly in relation to Alzheimer disease. Cholesterol transport and homeostasis in the central nervous system (CNS) are separated from that in the peripheral circulation by the blood–brain barrier. However, the brain operates its own lipoprotein transport system that is mediated by high density lipoprotein-sized, apo E-containing lipoproteins that are synthesized and secreted by glial cells (primarily astrocytes). Several ATP-binding cassette (ABC) transporters are expressed in the brain, including ABCA1 and ABCG1 which play important roles in the transfer of phospholipids and cholesterol to apo E. The astrocyte-derived apo E-containing lipoproteins can bind to, and be internalized by, receptors of the low density lipoprotein receptor superfamily that are located on the surface of neurons. In addition to these receptors serving as endocytosis receptors for lipoproteins, several of these receptors also act as signaling receptors in neurons and activate pathways involved in axonal growth, as well as neuronal survival. These beneficial pathways appear to be enhanced to a greater extent by apo E3 than by apo E4. Apo E has also been implicated in the deposition of amyloid plaques since apo E3, more readily than apo E4, forms a complex with Aß peptides, and mediates the degradation of amyloid deposits.     

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and the PACAP-selective receptor in cultured rat astrocytes, human brain tumors, and in response to acute intracranial injury

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide with diverse activities in the nervous system. In addition to its more classic role as a neurotransmitter, PACAP functions as a neurotrophic factor. PACAP exerts these activities by binding to PACAP-selective (PAC1) or nonselective (VPAC1, VPAC2) receptors (-R). Glial cells also exhibit PACAP binding, which is associated with the increased proliferation of astrocytes. The present report demonstrates a distinct spatiotemporal regulation of PACAP, PAC1-R, VPAC1-R, and VPAC2-R expression in primary cultured rat astrocytes. To determine the role of PACAP and PAC1-R expression on glial proliferation, two in vivo models were examined--human brain tumors of glial origin and the reactive gliosis induced by a penetrating stab wound to the mature rat brain. Relative to normal human brain, PAC1-R expression is significantly upregulated in glioma, particularly oligodendrogliomas. While similar polymerase chain reaction (PCR) analysis does not detect PACAP expression, in situ hybridization studies reveal PACAP expression in a limited number of cells within the tumor. In sharp contrast, neither PACAP nor PAC1-R expression are upregulated consequent to injury. These results suggest a distinct role for PACAP and PAC1-R in glioma development and nervous system response to injury.     

Lipocalin-type prostaglandin D2 synthase protein regulates glial cell migration and morphology through myristoylated alanine-rich C-kinase substrate: prostaglandin D2-independent effects

Prostaglandin D synthase (PGDS) is responsible for the conversion of PGH(2) to PGD(2). Two distinct types of PGDS have been identified: hematopoietic-type PGDS (H-PGDS) and lipocalin-type PGDS (L-PGDS). L-PGDS acts as both a PGD(2)-synthesizing enzyme and as an extracellular transporter of various lipophilic small molecules. Although L-PGDS is one of the most abundant proteins in the cerebrospinal fluid, little is known about the function of L-PGDS in the central nervous system (CNS). To better understand the role of L-PGDS in the CNS, effects of L-PGDS on the migration and morphology of glial cells were investigated. The L-PGDS protein accelerated the migration of cultured glial cells. Expression of the L-pgds gene was detected in glial cells and neurons. L-PGDS protein also induced morphological changes in glia similar to the characteristic phenotypic changes in reactive gliosis. L-PGDS-induced cell migration was associated with augmented formation of actin filaments and focal adhesion, which was accompanied by activation of AKT, RhoA, and JNK pathways. L-PGDS protein injected into the mouse brain promoted migration and accumulation of astrocytes in vivo. Furthermore, the cell migration-promoting effect of L-PGDS on glial cells was independent of the PGD(2) products. The L-PGDS protein interacted with myristoylated alanine-rich protein kinase C substrate (MARCKS) to promote cell migration. These results demonstrate the critical role of L-PGDS as a secreted lipocalin in the regulation of glial cell migration and morphology. The results also indicate that L-PGDS may participate in reactive gliosis in an autocrine or paracrine manner, and may have pathological implications in neuroinflammatory diseases.