Astrocytes, brain aging, and neurodegeneration

Neurobiologyof Aging,Vol. 17, No. 3, pp. 467-480, 1996 Copyright© 1996ElsevierScienceInc. Printedin the USA.All rightsreserved 0197-4580/96$15.00+ .00 ELSEVIER

S0197-4580(96)00014-0

PEER COMMENTARY ASTROCYTES, BRAIN AGING, AND NEURODEGENERATION H Y M A N M. SCHIPPER

Department of Neurology and Neurosurgery, Department of Medicine (Geriatrics), and Centre for Studies in Aging, McGill University; and Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada A subpopulation of astrocytes in striatum, hippocampus, and periventricular brain regions accumulates unique cytoplasmic inclusions with advancing age that are histochemically and morphologically distinct from lipofuscin. The gliosomes exhibit an affinity for Gomori stains, orange-red autofluorescence, and intense, nonenzymatic peroxidase activity likely mediated by ferrous iron. In the hypothalamic arcuate nucleus of adult rats and mice, chronic estrogen exposure induces a marked proliferation of peroxidasepositive astrocytic inclusions in close proximity to degenerating neuritic processes. We hypothesized that the glial peroxidase activity promotes lipid peroxidation of adjacent neuropil constituents in this brain region by oxidizing catecholestrogens and catecholamines to potentially neurotoxic free radical (ortho-semiquinone) derivatives. In support of t~is hypothesis, we demonstrated that dietary supplementation with potent antioxidants (a-tocopherol, 21-aminosteroids) significantly attenuates estradiol-related hypothalamic damage. The sulfhydryl agent, cysteamine (CSH), greatly accelerates the accumulation of peroxidase-positive astrocytic inclusions in situ and in primary brain cell cultures. In the latter, electron microprobe analysis in conjunction with diaminobenzidine cytochemistry confirmed that redox-active (likely ferrous) iron mediates nonenzymatic peroxidase reactions in these ceils. Using transmission electron and immunofluorescenceconfocal microscopy, we determined that the redox-active inclusions are derived from abnormal mitochondria engaged in an autophagic process both in CSH-treated g,tial cultures and in the aging periventricular brain. Furthermore, the transformation of normal mitochondria to mature, Gc,mori-positive inclusions occurs in the context of a cellular stress (heat-shock) response. Evidence implicating intracellular oxidative stress as a final common pathway leading to the formation of these astrocytic inclusions is discussed. Using electron spin resonance spectroscopy, we demonstrated that CSH-induced astrocyte peroxidase activity catalyzes the robust oxidation of 2-hydroxyestradiol to its ortho-semiquinone radical lending further credence to our Free Radical Hypothesis of estradiol neurotoxicity. Moreover, the peroxidase activity in these cells significantlyaugments the bioactivation of dopamine to dopamineo-semiquinone and may thereby render the senescent nervous system particularly prone to parkinsonism and other free radicalrelated neurodegenerations. A model of astrocyte senescence is presented in accord with the prevailing Mitochondrial Hypothesis of Aging. Astrocytes comprise a major class of neuroglia and perform a wide range of adaptive functions in the mammalian nervous sys-

tern. These cells play important roles in maintenance of the bloodbrain barrier and ion homeostasis, the elaboration of a scaffolding for neuronal migration during embryogenesis, sequestration, and metabolism of various neurotransmitters, and production of proinflammatory and immunomodulatory cytokines and neuropeptides [reviewed in (30,106)]. Alternatively, astrocytes may, under certain circumstances, mediate dystrophic effects within the CNS and thereby contribute to a decline in neurologic function. Examples of the latter include the accumulation and release of excitotoxic amino acids following tissue hypoxia, oxidative stress and metal exposure, formation of epileptogenic scar tissue in response to CNS injury, neoplastic transformation and malignant behavior, and metabolism of protoxins (such as MPTP) to potent neurotoxins (MPP+) (2,15,31,50). Astrocyte hypertrophy, accumulation of glial fibrillary acidic protein (GFAP)-positive intermediate filaments, and possibly hyperplasia (reactive gliosis) are characteristic pathological features of the major aging-related neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, and amyotrophic lateral sclerosis (4, 26,49,87,103). Gliosis also occurs, to a lesser extent, in the course of normal brain aging (52,71,92). Increases in monoamine oxidase B (MAO-B) activity accompany reactive gliosis in the aging mammalian brain, in AD, and in experimental models of PD. Under these circumstances, neuronal injury may be fostered by excessive H20 2 derived from the accelerated oxidative deamination of dopamine and other monoamines (77,104). The astrocytic compartment may also contribute to the pathogenesis of AD by serving as an important locus for cathepsin-mediated proteolysis, [3-amyloid metabolism, cytoskeletal protein hyperphosphorylation, and the expression of clusterin and apolipoprotein E (28,63,65,69). On occasion, astrocytes may also exhibit specific cytopathological changes, suggesting that they may be the primary targets of the disease process, as in the case of hepatic encephalopathy and certain rare neurodegenerative conditions (66,93). In this article, astrocytes exhibiting a unique senescent phenotype are described with emphasis on the role(s) these cells may play in normal brain aging and in senescence-dependent neurodegenerative conditions. THE GOMORI-POSITIVE,PERIVENTRICULARGLIALSYSTEM A subpopulation of granule-laden neuroglia has been described in limbic and periventricular brain regions of all vertebrates examined to date (21,23,98,100,107), including humans (78,81,102). These cells were initially identified as astrocytes by electron microscopy on the basis of their attenuated cytoplasm, bundles of

1Requests for reprints should be addressed to Dr. H. M. Schipper, Lady Davis Institute, Jewish General Hospital, 3755 Cote Ste. Catherine Road, Montreal, Quebec H3T IE2, Canada. 467

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intermediate filaments, and ellipsoidal, euchromatic nuclei (7,12, 14,99). The cytoplasmic granules that distinguish these cells are round to angular in shape, of varying dimensions, and intensely osmiophilic (Fig. 1). The inclusions are often invested with limiting membranes and occasionally appear contiguous with short, tubular elements filled with material of similar electron density (12,13,99). At the light microscopic level, this glial subpopulation is readily delineated by dual labeling for endogenous peroxidase activity (see below) and the astrocyte marker, GFAP (Fig. 2). These glia may also be distinguished on the basis of their affinity for the Gomori stains, aldehyde fuchsin, and chrome alum hematoxylin, and by the metachromasia of their cytoplasmic inclusions in Toluidine blue-stained sections (21,78). Although the cytoplasmic inclusions progressively increase with aging (see below), the absence of lipids (negative Sudan Black and Sudan III reactions) and visible pigment under light microscopy, and the presence of atypical, orange-red autofluorescence exclude lipofuscin as a component of these astrocytic granules (32,33,78). Most significantly, the Gomori-positive gliosomes are rich in iron (37,55,101), may contain other transition metals such as copper (13) and chromium (11,13), and express the metal-binding protein, metallothionein (109). The gliosomes stain intensely with diaminobenzidine (DAB), a marker of endogenous peroxidase activity (44,86,97). In these cells, DAB staining persists after tissue preheating, at extremes of pH, and in the presence of the catalase inhibitor, aminotriazole (48,81,86). The peroxidase activity is, therefore, nonenzymatic in nature (pseudoperoxidase) and is most likely mediated by ferrous iron (34). TOPOGRAPHY OF THE GOMORI-POSITIVE GLIA

Gomori-positive glia were initially described in subependymal brain regions and circumventricular organs (21,32,44,98,107). In the dorsal hippocampus, numerous DAB-positive cells are consistently present in the hilus of the dentate gyrus, the lacunosum molecular layer, and the stratum oriens of regions CA1 and CA2

adjacent to the corpus callosum. Other hippocampal regions, such as the granule cell and inner molecular layers of the dentate gyrus and the pyramidal cell layer and stratum radiatum of CA1 and CA2, contain GFAP-positive astrocytes that are largely devoid of endogenous peroxidase activity (88). In the basal ganglia, Gomoripositive glia are found in the caudate nucleus adjacent to the lateral ventricle and throughout the globus pallidus (88). In the diencephalon, these cells are most numerous throughout the entire rostrocaudal extent of the arcuate nucleus and ventral premammillary area. In the mesencephalon, they are readily detectable in the periaqueductal gray, dorsal to the raph6 nuclear complex, and in superficial layers of the superior colliculi (44). Neuroanatomical maps depicting the distribution of Gomori-positive glia in rat brain and spinal cord have previously been published (44), and further details concerning the topography of these cells within the rat neuraxis are presented in references (78,88). ROLE OF PEROXIDASE-POSITIVE ASTROCYTES IN ESTRADIOL-RELATED HYPOTHALAMIC DAMAGE

In rats and mice, there is a progressive increase in numbers of Gomori-positive astrocyte granules in the hypothalamic arcuate nucleus between 6 and 14 months of age (80). In female rodents, early gonadectomy abolishes the age-related accumulation of these astrocytic inclusions within the arcuate nucleus [(80); Fig. 3A]. Conversely, chronic estrogenization markedly increases numbers of these glial inclusions in this estrogen receptor-rich brain region (7,9,10,14,86,90). In addition, treatment with estradiol valerate results in the progressive degeneration of axo-dendritic profiles with formation of abundant myelin figures, synaptic loss and remodeling, depletion of hypothalamic [3-endorphin, and increased numbers of reactive microglial cells containing phagocytosed debris [reviewed in (6,79)]. The degenerating neural processes were often seen apposed or in close proximity to hypertrophic, Gomoripositive astrocytes exhibiting a massive proliferation of their electron-dense cytoplasmic inclusions (Fig. 1B). Peroxidase reactions

FIG. 1. (A) Ultrastructure of Gomofi-positive astrocyte in the hypothalamic arcuate nucleus of a normal adult female rat. Osmiophilic, Gomori-positive gliosomes (g) and a bundle of intermediate filaments (arrows) are depicted, x17,220, magnification. (B) Gomori-positive astrocyte in the hypothalamic arcuate nucleus of an adult female rat rendered anovulatory with estradiol valerate. This treatment induces a marked accumulation of Gomori-positive cytoplasmic inclusions (g) in close proximity to degenerating dendritic profiles (arrow). N, astrocyte nucleus; F, intermediate filaments: x17,220. [From (7), with permission.]

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FIG. 3. Effects of aging and long-term gonadectomy on numbers of Gomori-positive astrocytic granules in the arcuate nucleus of male and female rats, (A) Females: numbers of astrocytic granules increase significantly with advancing age (fine crosshatching). Early ovariectomy markedly attenuates this aging effect (heavy crosshatching). (B) Male rats: the age-related increase in numbers of astrocytic granules in male rats is less robust than in females and early castration in the former does not significantly suppress this aging phenomenon. [From (80), with permission.] within arcuate neuroglia may play a pivotal role in the development of estradiol-related neuronal damage in this brain region. In the hypothalamus, 2-hydroxylases and peroxidases convert estradiol to 2-hydroxyestradiol or catecholestrogen (3,53,58). Catecholestrogens may, in turn, be transformed to reactive semiquinone radicals via a peroxidase/H2Oe-catalyzed reaction or by spontaneous autoxidation. The latter pathway also generates potentially neurotoxic reactive oxygen species including H202 and superoxide anion [(42,43); Fig. 4]. We, therefore, hypothesized that the collapse of axo-dendritic profiles and the formation of myelin figures in the arcuate nuclei of hyperestrogenized rats may reflect lipid peroxidation and membrane destruction initiated by estradiolderived radical species (79,86). In support of this hypothesis, we demonstrated that dietary supplementation with potent antioxidants such as a-tocopherol (22) or the 21-aminosteroid U74389F (84), blocks the depletion of hypothalamic [3-endorphin in estradiol valerate-treated rats. Thus, by facilitating the conversion of catechols to their ortho-semiquinone derivatives, peroxidasepositive astrocytes may render the senescent nervous system particularly prone to free radical-related injury. As discussed below, our ability to generate primary brain cell cultures highly enriched for peroxidase-positive astroglia has significantly advanced our knowledge concerning the origin of the peroxidase-positive inclusions, the mechanism(s) responsible for their biogenesis, and the role these cells may play in brain aging and neurodegenerative disease. PEROXIDASE-POSITIVEASTROCYTESIN PRIMARYCULTURE:EFFECTS OF CYSTEAMINE Gomori-positive astrocytes have been detected in fetal rat and human diencephalic explants (101,102) and in dissociated rat brain

cell cultures (91). In the latter, we observed a progressive accumulation of these cells and their granule content between days 10 and 50 in vitro. We demonstrated that exposure to the sulfhydryl agent, 2-mercaptoethylamine or cysteamine (CSH; 88-880 ~M in culture medium administered twice weekly from in vitro days 6-18) induces a massive accumulation of Gomori-positive astrocytes in primary culture. The CSH-treated astrocytes exhibit orange-red autofluorescent granules and nonenzymatic peroxidase activity identical to that of Gomori astrocytes in unstimulated, older cultures and in senescent periventricular brain regions in situ [(18,88,89); Fig. 5]. At the fine structural level, the CSH-induced astrocytic inclusions are membrane bound, variable in size, round or ovoid in shape, and exhibit an intensely electron-dense granular matrix akin to periventricular astrocyte granules in situ [(7,12,13, 55); Fig. 6]. In nonosmicated preparations, DAB reaction product appears diffusely distributed throughout the granule matrix or is restricted to specific intraorganellar compartments. Moreover, elemental iron is detected in the inclusions by electron microprobe analysis, and the presence and concentration of the metal correlates closely with the presence and intensity of DAB staining [(55); Fig. 7]. These observations substantiate the notion that redox-active (likely ferrous) iron mediates nonenzymatic peroxidase reactions in these cells. We determined that within 24--72 h of CSH exposure, many astroglial mitochondria exhibit progressive swelling, rearrangement or dissolution of cristae, subcompartmental sequestration of redox-active iron, and, in some cases, fusion with acid phosphatase-positive lysosomes [(8); Fig. 8]. Using a panel of FITC-labeled antibodies directed against organelle-specific proteins and laser scanning confocal microscopy, we confirmed partial colocalization of lysosomes, and to a lesser extent early endosomes and rough endoplasmic reticulum, to the

FIG. 2. Identification of Gomori-positive astrocytes by DAB-GFAP double label immunohistochemistry. Hypothalamic arcuate nucleus. Long arrow: brown reaction product (endogenous peroxidase activity) within pink (GFAP-positive) astrocyte. Short arrow: astrocytic process replete with endogenous peroxidase activity. Arrowhead: astrocyte with several tiny DAB-positive inclusions (40 micron section; methyl green counterstain), x529.2. [From (86), with permission.] FIG. 10. Laser scanning confocal micrograph of adult rat arcuate nucleus stained with anti-CLSO. Consistent colocalization of the mitochondrial marker (green) to the autofluorescent glial granules (red) produces yellow fluorescence. Bar = 10 IxM. [From (13), with permission.] FIG. 12. Immunolocalization of stress proteins to autofluorescent astrocyte granules in rat brain sections and CSH-treated glial cultures. HSP27 shows intense colocalization (yellow fluorescence) to astrocyte granules in situ (A; empty arrows) and in culture (B; empty arrows). Solid arrows indicate smaller granules devoid of HSP27-immunoreactivity (bars = 100 I~m for A; 10 Ixm for B). Within the subependymal zone of the third ventricle (V), ubiquitin exhibits strong colocalization (yellow fluorescence) to the autofluorescent granules (C; arrows). (Bar = 25 txm). In vitro, the larger autofluorescent granules are ubiquitinated (D; empty arrows) whereas many smaller granules are not ubiquitin-immunoreactive (solid arrow). Occasional ubiquitin staining of granule-free cytoplasm is shown in D (arrowhead) (bar = l0 ~m). In brain sections (E), strong aB-crystallin staining can be seen in cells along the third ventricular wall (V). Both in situ (E) and in vitro (F), c~B-crystallinmanifests strong immunolabeling of astroglia but no colocalization to the autoflnorescent inclusions (arrows) (bars = 10 Ixm for E and F). [From (62), with permission.]

ASTROCYTES, BRAIN AGING, AND NEURODEGENERATION

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FIG. 5. Embryonic day 17 rat brain cell cultures (18 days in vitro). (A) Untreated control. DAB stain for endogenous peroxidase activity. Astrocytes devoid of DAB-positive granules are observed. Methyl green counterstain, x292. B: Effects of cysteamine (880 IxM twice weekly in medium from day 6). DAB stain. Astrocytes exhibit a massive accumulation of cytoplasmic peroxidase-positive inclusions. Methyl green counterstain, x292. (C) Untreated embryonic day 17 rat brain cell culture photographed for autofluorescence. Astrocytes exhibit faint or no orange-red autofluorescence. ×292. (D) Cysteamine-treated brain cell culture. Cysteamine-induced astrocyte granules and cytoplasm emit intense orange-red autofluorescence, x292. [From (91), with permission.]

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FIG. 6. (A) Transmission electron microscopy of astrocytes from control culture (unexposed to cysteamine). Segments of four cells are visible. Polysomes, short cisternae of rough endoplasmic reticulum and bundles of intermediate filaments are scattered throughout the cytoplasm. A small Golgi apparatus (arrowheads) and a dense inclusion body (arrow) are depicted. Bar = 0.83 ~M. (B) Astrocyte from cysteamine-treated culture. The cytoplasm is replete with large osmiophilic inclusions. Many of the gliosomes exhibit concentric stacks of membrane along segments of their periphery (arrows). Bar = 0.45 p,M. (Reproduced with permission, McLaren, J.; Brawer, J. R.; Schipper, H. M. J. Histochem. Cytochem. 40:1887-1897; 1992).

red autofluorescent granules induced in cultured astroglia by CSH exposure (83). In young adult rats, subcutaneous CSH injections (150-300 mg/kg twice weekly for 3 weeks) induce two- to threefold increases in numbers of peroxidase-positive astrocyte granules in striatum, hippocampus, and other brain regions [(89); Fig. 9). As in the case of the CSH-treated cultures, peroxidase-positive glial granules in the intact rat and human brain invariably exhibit mitochondrial epitopes in immunohistochemical preparations [(! 3, 81); Fig. 10). Taken together, these observations indicate that (a) the peroxidase-positive astrocyte granules are derived from abnormal mitochondria engaged in a complex macroautophagic process, and (b) CSH accelerates the appearance of a senescent phenotype in these cells. THE BIOGENESISOF ASTROCYTICINCLUSIONS:A CELLULARSTRESS RESPONSE? Recent evidence from our laboratory suggests that activation of the cellular heat-shock (stress) response plays an important role in the biogenesis of peroxidase-positive astrocytic inclusions. Within 6 h of CSH exposure, cultured rat astroglia exhibit robust increases in the expression of heat-shock protein (HSP)27, HSP90, heme oxygenase-1 (HO-1), and ubiquitin as determined by Western blotting and immunofluorescence microscopy (17,61,62). In contrast, CSH treatment only moderately enhanced HSP72 immunoreactivity and had no appreciable effect on glucose regulated protein (GRP)94, which was constitutively present in our glial cultures (17,61). In the case of HSP27, HSP90, and HO-1, upregulation by CSH probably occurs at the transcriptional level as determined by slot blot hybridization and Northern analyses [(17); Fig. 11). In spite of the mitochondrial injury incurred by CSH exposure (see above), CSH-pretreated astroglia exhibit enhanced resistance to H202 toxicity and mechanoenzymatic stress (trypsinization) relative to nonpretreated controls providing physiological evidence of an antecedent cellular stress response (61). We also determined that systemic administration of CSH to young adult rats induces significant increases in concentrations of peroxidase-positive astrocyte granules and numbers of GFAP-positive astrocytes expressing HSP27, 72, and 90 in dorsal hippocampus, striatum, cor-

pus callosum, and, to a much lesser extent, cerebral and cerebellar cortices (89). Along similar lines, estradiol valerate elicits both a heat-shock response and subsequent granulation in astrocytes residing in estradiol receptor-rich brain regions including the arcuate nucleus and the third ventricular subependymal zone (60). Finally, confocal microscopy revealed consistent colocalization of HSP27, GRP94, ubiquitin, and to a lesser extent, HSP72 (but not HSP90 or ctB-crystallin) to the red autofluorescent astrocyte granules in CSH-treated cultures and in the third ventricular subependymal zone of adult male rats [(62); Fig. 12). These latter observations greatly extend our previous histochemical studies underscoring the identical origin of the CSH-induced astroglial inclusions and those which spontaneously accumulate in the aging periventricular brain. Moreover, the data suggest that chronic or repeated induction of the cellular stress response plays an important role in the biogenesis of peroxidase-positive "stress granules" in astrocytes of the aging periventricular brain akin to the formation of "heat shock granules" observed in other cells following sustained stress (45, 67,70). A considerable body of evidence suggests that intracellular oxidative stress may be the "final common pathway" responsible for the transformation of normal astrocyte mitochondria to peroxidase-positive inclusions both in vitro and in the intact aging brain: (a) prior to inducing astrocyte granulation, both CSH and estradiol valerate upregulate stress proteins that typically respond to oxidative stress (e.g., HSP27, 90) but have little or no effect on redoxinsensitive proteins such as GRP94 (60,61). (b) In the presence of transition metals, CSH undergoes redox cycling with the generation of pro-oxidant species including thiyl radicals, superoxide, H202, and the hydroxyl radical (59). In isolated astroglial mitochondria, CSH induces lipid peroxidation, which can be blocked by catalase coincubation implicating CSH-derived H202 in the biogenesis of the astrocytic inclusions (54). H202 induces HSP and HO-1 expression in rat astrocytes (27) and stimulates the accumulation of peroxidase-positive astrocyte granules in primary culture following prolonged treatment (61). (c) Ionizing radiation, a known generator of intracellular pro-oxidant intermediates, increases numbers of peroxidase-positive glial granules in the rat

ASTROCYTES, BRAIN AGING, AND NEURODEGENERATION

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FIG. 7. (a) Peroxidase activity in astrocyte from cysteamine-treated culture. Inclusions within this nonosmicated cell exhibit varying degrees of peroxidase activity indicated by the granular DAB reaction product. Inclusions D and E show little DAB reaction product, whereas inclusions C and F are strongly positive. The DAB precipitate :in the latter appears localized to specific intra-organellar compartments. Bar = 0.4 IxM. (b) X-ray emission spectra derived from the cell depicted in A. Emission peaks indicating the various elemental constituents are labeled. The concentration of a given element is proportional to the area under the peak(s) fiar that element. The large peak at the right of each histogram indicates copper resulting from the use of copper grids. (A) This emission spectrum was generated by a region of clear cytoplasm. Note the absence of a peak for iron. (B) This spectrum was generated by a euchromatic region of nucleus Note the absence of a peak for iron. (C) This spectrum was generated by inclusion C in A. The two iron peaks (arrows) are indicative of a high iron concentration within this inclusion. (D) Spectrum for inclusion D in A. There is only a single small iron peak (arrow) indicative of a relatively low concentrati,an of iron. (E) Spectrum for inclusion E in A. The single small iron peak (arrow) indicates a low iron concentration. (F) Spectrum for inclusion F in A. The twin peaks (arrows) indicate a high concentration of iron. [From (55), with permission.]

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FIG. 8. Cysteamine induced mitochondrial pathology in cultured astroglia. (A) Mitochondrial swelling and dissolution of cristae after 24 h of CSH exposure. Bar = 300 nm. (B) Mitochondrial macroautophagy after 12 days of CSH exposure. Bar = 500 nm. [From (8), with permission.]

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FIG. 9. Effects of subcuteneous CSH administration (see text for treatment regimen) on numbers of DAB-positive astrocyte and ependymal granules in various brain regions. Columns and vertical bars represent means and standard errors of the means, respectively. Asterisks denote statistically significant increases in granule numbers relative to untreated controls (p < 0.05). From ref. 89, reproduced with permission from the Journal of Neuropathology and Experimental Neurology.

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ASTROCYTES, BRAIN AGING, AND NEURODEGENERATION

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FIG. 13. ESR spectra of metal-complexedsemiquinonesfrom the peroxidase-H202 oxidation of 2-hydroxy(catechol)estradiol. (A) Top: autoxidation of 2-hydroxyestradiolin serum-freemedium in the absence of cells following alkalinizationto pH 10.0 with NaOH. Mediumcontained2-hydroxyestradiol (10-2 M), MgCI2 (0.5 M), and NaOH in DMEM. The characteristic o-semiquinonespeclrum is identical to that observed by Kalyanaramanet al. (1984). Bottom: computer-simulatedspectrum of the 2-hydroxyestradiol o-semiquinonederived from measured hyperfinecoupling constants. (B) Incubationof 2-hydroxyestradiol(10-2 M), MgC12(0.5 M), NADPH (0.3 M), and H202 (0.1 raM) with tissue homogenate derived from untreated (control) brain cell culture (pH 7.0). The gain settings in B and C are identical,permittingdirect amplitudecomparisons.(C) Incubation as in B with tissue homogenate derived from cysteaminepretreated (peroxidase-enriched) brain cell culture. An intense o-semiquinonesignal is observedwith hyperfinestructure identicalto the pattern obtainedin the cell-free 2-hydroxyestradiolautoxidationexperiment(A). The peroxidase activityinducedin astrocytesby cysteaminecatalyzescatechol oxidationto o-semiquinoneradicals. [From (85), with permission.] hypothalamus in a dose-de,pendent manner (97). If a causal relationship to intracellular oxidative stress is confirmed, determination of the topography and intensity of endogenous glial peroxidase activity may permit accurate "mapping" of CNS regions particularly prone to chronic oxidative stress during normal aging and under pathologic conditions.

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tem to oxidative injury by promoting the conversion of neutral catechols to potentially neurotoxic semiquinone derivatives (Fig. 4). Electron spin resonance spectroscopy (ESR) with magnesium spin stabilization was used to determine whether CSH-induced peroxidase activity in cultured astroglia is capable of oxidizing catecholestrogens and catecholamines to their respective orthosemiquinone radicals (85). Incubation of 2-hydroxyestradiol (10-4-10-2 M) with homogenates derived from untreated (control) astroglial monolayers in the presence of n202 and NADPH (pH 7.0) yielded no or barely detectable o-semiquinone spectra (Fig. 13). In contrast, intense o-semiquinone spectra indicative of robust catechol oxidation were consistently observed following incubation of equimolar concentrations of 2-hydroxyestradiol with homogenates obtained from CSH-pretreated (peroxidase-enriched) astrocyte monolayers in the presence of appropriate cofactors (Fig. 13). In the absence of H202 substrate, there was a marked reduction in signal amplitude attesting to the important role of glial peroxidase activity in the augmentation of catecholestrogen metabolism in our system (85). On the other hand, NADPH enhanced, but was not an absolute prerequisite for, glial peroxidase-catalyzed oxidation of catecholestrogen. Although NADPH is an important cofactor for peroxidase-driven oxidations in cell-free systems (42, 43), the high sulfhydryl content of Gomori-positive astrocytes (32, 97), or equivalent reducing substances appear capable of sustaining peroxidase-mediated reactions in these brain cell preparations in the absence of exogenous NADPH. The results of the ESR experiments, in conjunction with the aforementioned protective effects of (x-tocopherol and 21-aminosteroids on estradiol-induced depletion of hypothalamic [3-endorphin, further support our contention that free radical generation by peroxidase-positive astrocytes may mediate, at least in part, the dystrophic effects of estradiol in this brain region. The high thiol and stress protein content of Gomori astrocytes (see previous sections) could serve to protect against oxidative injury within the glia themselves. However, H202 is lipid soluble and can easily traverse plasma membranes to reach the intercellular space while superoxide can be extruded from cells via anion channels (47). Thus, leakage of free radicals from peroxidase-positive astrocytes into the surrounding neuropil may promote lipid peroxidation and degeneration of nearby dendrites and other vulnerable neuronal constituents. As discussed above, the latter may account for the neuropathological profile depicted in Fig. lB. As in the case of 2-hydroxyestradiol, we demonstrated that, in the presence of H202, CSH-induced peroxidase activity in cultured astroglia significantly enhances the oxidation of the catecholamine, dopamine, to its dopamine-o-semiquinonederivative (85). This observation is consistent with previous reports that dopamine and norepinephrine are readily oxidized to semiquinones with proven neurotoxic activity in vitro via peroxidase-mediated reactions (56). Furthermore, redox-active glial iron may facilitate the nonenzymatic oxidation of (a) the protoxin, MPTP, to the dopaminergic toxin, MPP+, in the presence of MAO inhibitors (24); and (b) the dopamine precursor, DOPA, to 2,4,5-trihydroxyphenylalanine (TOPA) and the non-NMDA excitotoxin, TOPAquinone (64). Thus, by promoting the bioactivation of protoxins, the age-dependent increases in numbers of peroxidase-positive astrocytes that have been documented in rodent and human brain may render the senescent nervous system particularly vulnerable to parkinsonism and other free radical-related neurodegenerations.

PROTOXINBIOACTIVATIONBY PEROXIDASE-POSITIVEASTROCYTES IN PRIMARYCULTURE

GLIALIRONSEQUESTRATIONAND PARKINSON'SDISEASE

As discussed above, the stress-related accumulation of peroxidase-positive glial granules may sensitize the aging nervous sys-

Idiopathic Parkinson's Disease (PD) is a movement disorder of uncertain etiology characterized by the accelerated loss of dopa-

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minergic neurons in the pars compacta of the substantia nigra (38). Mounting evidence indicates that the excessive production of neurotoxic free radicals may play an important role in the pathogenesis of this disorder (19,29,41). Abnormally high levels of tissue iron reported in the substantia nigra and basal ganglia of parkinsonian subjects have been implicated as a major generator of reactive oxygen species in this condition (76,108). Redox-active iron may promote oxidative stress within the degenerating basal ganglia by several mechanisms. For example, the oxidation of dopamine via MAO in neurons and astroglia generates H202, which in turn, may be reduced to the highly cytotoxic hydroxyl radical in the presence of ferrous iron. As discussed above, the latter may also behave as a nonenzymatic peroxidase activity capable of converting catecholamines to potentially neurotoxic quinodal metabolites (34,35,85). Attempts to ameliorate iron-mediated neuronal injury in PD presupposes some understanding of the regulatory mechanisms subserving iron metabolism and sequestration in the aging and degenerating CNS. Several important, but as yet unanswered questions in this regard include the following: (a) What is the role of heme vs. nonheme iron in PD and other aging-related neurodegenerative conditions? (b) Which cell type(s) and subcellular compartments are responsible for the abnormal sequestration of brain iron in these degenerative disorders? (c) Does induction of a cellular stress (heat-shock) response facilitate trapping of redoxactive iron in neural tissues? We have begun addressing some of these issues by focusing on the mechanisms responsible for the accumulation of the iron-rich cytoplasmic inclusions in aging peri-

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ventricular astrocytes and in CSH-exposed astroglial cultures. Several laboratories including our own (33,78) have previously concluded on the basis of histochemical and spectrofluorometric data that porphyrins and heine ferrous iron are responsible, respectively, for the orange-red autofluorescence and nonenzymatic peroxidase activity in these glial inclusions. However, we recently determined that CSH suppresses the incorporation of the heme precursors, A-amino[laC]-levulinicacid and [14C]glycine into astroglial porphyrin and heme in primary culture prior to and during the time when increased iron content is detectable in swollen astrocyte mitochondria by microprobe analysis [(8,105); Fig. 14). Thus, contrary to hypothesis, de novo biosynthesis of porphyrins and heme is not responsible for the increased mitochondrial iron content, autofluorescence, and peroxidase activity observed in cultured astroglia following CSH exposure. Because the CSHinduced astroglial inclusions are morphologically and histochemically identical to the iron-laden astrocyte granules that normally accumulate in the aging periventricular brain, it would seem highly unlikely that augmentation of porphyrin-heme biosynthesis is responsible for the biogenesis of the latter as well. Oxidized mitochondrial flavoproteins exhibit fluorescence emission spectra that may be difficult to distinguish from porphyrins (25,46) and are likely mediators of orange-red autofluorescence in these astrocytic inclusions. We also showed that following inhibition of porphyrinheme biosynthesis, CSH significantly augmented the incorporation of 59Fe into astroglial mitochondria without affecting transfer of the metal into whole-cell and lysosomal compartments [(105); Fig.

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FIG. 14. Incorporation of I~4C]ALA into (A) uroporphyrin, (B) coproporphyrin, (C) protoporphyrin, and (D) hemin in control untreated (O) and CSH-treated (0) astrocytes. Data are presentedas mean_+SD (bars) of three to six observations.*p < 0.05, **p < 0.01 for significanceof differencerelative to untreated controls. CSH suppresses porphyrin-hemebiosynthesisin cultured astroglia. [From (105), with permission.]

ASTROCYTES, BRAIN AGING, AND NEURODEGENERATION

15). This CSH effect was clearly demonstrable when inorganic 59FeC13, but not 59Fe-diferric transferrin, served as the metal donor. These findings are consistent with previous reports that intracellular transport of low molecular weight, inorganic iron may be 5- to 10-fold more efficient than that of transferrin-bound iron in various tissues, including melanoma cells (73-75), Chinese hamster ovary cells (16), and K562 ceils (39). Our observations support the conclusion of Adam s and co-workers (1) that inhibition of heme biosynthesis stimulates the selective transport of low molecular weight iron from the cytoplasm to the mitochondrial compartment. Our observations on CSH-stressed astroglia suggest a model for inclusion formation, iron sequestration, and the perpetuation of oxidative injury in the aging and degenerating nervous system: (a) in senescent periventricular and limbic brain regions, unidentified

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oxidative or sulfhydryl stressors (simulated by CSH exposure) induce mitochondrial swelling and the accumulation of autofluorescent, oxidized flavoproteins in a subpopulation of astroglia. Eventual fusion of damaged mitochondria with lysosomes (macroautophagy) results in the formation of Gomori-positive, cytoplasmic inclusions in these cells. (b) The latter undergo a cellular stress (heat-shock) response characterized by upregulation of various HSP and HO-1. HSP confer relative cytoprotection against subsequent stressors and HO-1 may serve to normalize the intracellular redox microenvironment by converting pro-oxidant heme to the antioxidant bilirubin (27,82). (c) Enzymes of the porphyrinheme biosynthetic pathway are directly inactivated by the oxidative or sulfhydryl stressor, or their synthesis is suppressed as part of a generalized cellular heat-shock response. (d) Inhibition of porphyrin-heme biosynthesis or direct oxidative damage to mitochondrial membranes promotes the selective transport of low molecular weight, nonheme iron into the mitochondrial compartment. (e) By promoting further oxidative stress, the redox-active mitochondrial iron participates in a vicious cycle of pathologic events whereby damage to glial mitochondria as well as to the surrounding neuropil is perpetuated. In this regard, aging-related neural injury could progress long after dissipation of the initiating neurotoxic insult. This model of astrocyte senescence is consistent with the Mitochondrial Hypothesis of Aging, which states that oxidative damage to mitochondria results in bioenergetic failure, a vicious spiral of augmented mitochondrial free radical generation and injury, and progressive tissue aging (5,51,57,94,95). Our model accounts for the observation that mosaicism for specific mitochondrial DNA mutations in the normal aging human brain is most striking in regions particularly rich in intracellular iron such as the caudate, putamen, and substantia nigra (96). Moreover, our findings raise the possibility that exacerbation of stress-related trapping of nonheme iron by astroglial mitochondfia may be an important mechanism underlying the pathologic accumulation of redox-active iron in the basal ganglia of PD subjects. Such iron could conceivably originate from degenerating neurons, glia, or myelin or from the cerebrospinal fluid (CSF). Micromolar quantities of chelatable, low molecular weight iron is present in normal CSF, and the concentration of this metal in CSF has been shown to increase under neuropathological conditions (36). Consistent with our model are reports that a significant proportion of the excess iron in PD brain may, indeed, be localized to astroglial mitochondria (20,40,68), and that deficiencies of mitochondrial electron transport are prevalent in the brains of PD subjects [re-

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HOURS OF TREATMENT FIG. 15. Iron-59 uptake in cultured control (O) and CSH-treated (0) astrocytes exposed to 59FEC13: (A) total cell, (B) lysosomal, and (C) mitochondrial fractions. Data are presented as mean ± SD (bars) of three to six observations. *p < 0.05 for significance of difference relative to untreated controls. CSH promotes the sequestration of iron within the mitochondrial compartment. [FrorrL(105), with permission.]

Autoxidation FIG. 16. Free radical generation by peroxidase-positive astrocyte granules in the aging brain. Implications for the pathogenesis of Parkinson's disease (please see text for description). P - F e 2 = ferrous iron with pseudoperoxidase activity; MAO-B = monoarnine o×idase B; Q = quinone; SQo = semiquinone.

478

SCHIPPER

viewed in (72)]. Most importantly, by oxidizing dopamine and environmentally derived xenobiotics to neurotoxic intermediates (see previous section), the redox-active glial iron could promote further nigrostriatal degeneration in patients with PD. Since agingrelated increases in both MAO-B activity and redox-active mitochondrial iron occur within the same cell population (astrocytes), it is conceivable that H20 2 produced by MAO-catalyzed oxidation of dopamine serves as a cofactor for further dopamine oxidation (to o-semiquinones) via peroxidase-mediated reactions (Fig. 16). If nonenzymatic bioactivation of protoxins proves to be important in PD and other neurodegenerative disorders, then administration of mitochondrial protectants (such as acetyl-e-carnitine) or centrally active iron chelators in conjunction with M A O inhibitors (Ldeprenyl) may retard the rate of neuronal depletion and clinical decline in these conditions.

CONCLUSION The progressive accumulation of autofluorescent, peroxidasepositive astrocytic granules represents a fundamental and highly consistent biomarker of aging in the vertebrate CNS. Although, these glial inclusions were first identified almost half a century ago on the basis of their affinity for Gomori stains, it is only in recent years that we have begun to elucidate the subcellular origin of these inclusions, the mechanism(s) responsible for their biogenesis, and their potential role in brain aging and neurodegeneration. The current state of our knowledge strongly suggests that these gliosomes are "stress granules," which ultimately derive from ef-

fete, metal-laden mitochondria engaged in a complex autophagic process. The fact that oxidative (or sulfhydryl) stress appears to initiate the formation of astrocytic inclusions that are themselves redox-active has, we believe, some important implications. First, determination of the topography of these glial inclusions may permit " m a p p i n g " of CNS regions at increased risk for chronic oxidative injury during normal aging and under pathological conditions. Second, the ability to experimentally recapitulate the development of this senescent glial phenotype in primary culture provides a powerful model to investigate mechanisms of pathologic brain iron sequestration germane to the pathogenesis of Parkinson's disease and other aging-dependent neurological disorders. Finally, the nonenzymatic peroxidase activity manifest in these cells may promote the bioactivation of catechols and other protoxins to potential neurotoxins and thereby perpetuate "secondary" neural damage long after initiating neurotoxic insults have dissipated. If the latter is true, attempts to pharmacologically inhibit metal sequestration by "stressed" astroglial mitochondria may constitute a rational and effective strategy in the management of Parkinson's disease and other aging-related neurodegenerative afflictions.

ACKNOWLEDGEMENTS The excellent secretarial assistance of Mrs. Rhona Rosenzweig and Ms. Mary Tomaras is greatly appreciated. The author is supported by grants from the Medical Research Council of Canada and the Fonds de la Recherche en Sant6 du Qurbec.

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