Invited Symposium: Oxidative Stress and the CNS
Iron Deposition and Neurodegeneration
The pathological deposition of redox-active brain iron has been implicated as a major generator of reactive oxygen species (ROS) in PD, AD, and other aging-related neurodegenerative conditions. Yet, the mechanisms responsible for the abnormal patterns of neural iron sequestration in these disorders remain incompletely understood. In this paper, data germane to this issue are summarized and a model of aberrant brain iron deposition based on the over-expression of heme oxygenase-1 (HO-1) in astroglia is presented.
Abnormally high levels of tissue iron have been consistently documented in the substantia nigra and basal ganglia of PD subjects (14,15,33,43). In PD, the excessive iron deposition primarily affects the zona compacta of the substantia nigra and correlates with the disappearance of dopaminergic neurons in this brain region (13,43,44). The excessive nigral iron appears to be predominantly sequestered within astrocytes, microglia, macrophages and microvessels in areas depleted of neuromelanin-containing (dopaminergic) neurons.
Histochemical evidence for substantial iron deposition in PD-affected nigral or striatal neurons, on the other hand, is scant or non-existent (12,24,43). In glia and other non-neuronal cells, augmented tissue iron levels are accompanied by changes in the expression of several important iron-binding proteins and their receptors. In general, increased expression of tissue ferritin, the major intracellular sequester of ferric iron, parallels the distribution of the excess iron. The iron-binding protein, transferrin, is responsible for the extracellular transport of ferric iron and its delivery to most mammalian tissues. To maintain tissue iron homeostasis, plasma membrane transferrin receptor densities and intracellular ferritin concentrations are tightly regulated (at transcriptional and post-transcriptional levels) by iron bio-availability and intracellular iron stores (19,32,41).
In contrast to the ferritin data, the concentration of transferrin binding sites remains unchanged or varies inversely with elevated iron stores in the substantia nigra and striatum of PD subjects (10,12,16,22). Thus, transferrin and its receptor may play a limited role, if any, in the pathological deposition of iron in aging and degenerating CNS tissues (10,12,16,22). The potential role of alternative iron transport mechanisms, such as that mediated by lactoferrin and the lactoferrin receptor, is reviewed elsewhere (35). By participating in Fenton reactions, the excesss iron may generate the highly cytotoxic hydroxyl radical. In PD brain, ferrous iron may also behave as a non-enzymatic peroxidase activity capable of oxidizing catecholamines, such as dopamine, to neurotoxic semiquinone radicals in the presence of H2O2 (23,38). Because aging subcortical astrocytes may exhibit both enhanced MAO-B activity and abundant mitochondrial iron (5), H2O2 produced by MAO-B oxidation of dopamine could conceivably serve as a co-factor for further dopamine oxidation (to potentially neurotoxic ortho-semiquinones) by peroxidase-mediated reactions. In addition to dopamine, redox-active glial iron may also facilitate the non-enzymatic oxidation of: a) the pro-toxin, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), to the dopaminergic toxin, MPP+, in the presence of MAO inhibitors (6), and b) the dopamine precursor, DOPA, to 2,4,5-trihydroxyphenylalanine (TOPA) and the non-NMDA excitotoxin, TOPA-quinone (29).
As in the case of PD, derangements of iron homeostasis and excessive deposition of this transition metal are characteristic of Alzheimer-diseased brain tissues. In the AD hippocampus, increased deposition of non-heme iron has been shown to occur in NFT-bearing neurons, astrocytes, microglia, and in the vicinity of neuritic plaques (4,12,14,25). There appears to be an overall decrease in levels of immunoreactive transferrin and transferrin receptor concentrations in AD-affected cortical and subcortical brain tissue in comparison with those of age-matched, non-demented controls (4,16). This apparent mis-match of brain iron and transferrin/transferrin receptor is analogous to that observed in the substantia nigra of PD subjects and further suggests that the transferrin pathway of iron mobilization may contribute little to the pathological deposition of brain iron observed in these aging-related neurodegenerative conditions. In the AD brain, the pathological iron stores may promote the generation of neurotoxic ROS as described above for PD-affected tissues. Furthermore, the amyloid precursor protein gene contains iron response element-like consensus sequences suggesting that brain amyloid deposition in AD may be iron-sensitive (12).
Iron Sequestration in Aging Astroglia: A Model
For a number of years, our laboratory has focused on the mechanisms responsible for the accumulation of iron-rich cytoplasmic inclusions in aging subcortical astrocytes and in astroglial cultures subjected to oxidative stress. We determined that the sulfhydryl agent, cysteamine (CSH), accelerates the aging-related accrual of iron-rich cytoplasmic inclusions in hippocampal, striatal and other subcortical astroglia in situ and in primary neuroglial cultures. We showed that these iron-containing glial granules are derived from oxidatively-damaged mitochondria in the context of a cellular stress (heat shock) response. Comprehensive reference lists and figures depicting the morphology and histochemistry of this senescent glial phenotype are provided in several published reviews (26,28,34). We subsequently demonstrated that CSH suppresses the incorporation of the heme precursors, d-amino[14C]-levulinic acid and [14C]glycine into astroglial porphyrin and heme in primary culture prior to and during the time when increased iron content is noted in swollen astrocyte mitochondria by microprobe analysis (34,42). Thus, de novo biosynthesis of porphyrins and heme is not responsible for the increased mitochondrial iron content and peroxidase activity observed in cultured astroglia following CSH exposure.
Following suppression of porphyrin-heme biosynthesis, CSH enhances the uptake of 59Fe (or 55Fe) into astroglial mitochondria without significantly affecting transfer of the metal into whole-cell and lysosomal compartments (42). This CSH effect was clearly demonstrable when inorganic 59FeCl3, but not 59Fe-diferric transferrin, served as the metal donor. The latter observation is consistent with the aforementioned findings that transferrin and its receptor are relatively deficient in PD-affected neural tissues exhibiting iron overload. Akin to the effects of CSH, dopamine (1 ÁM) stimulates the sequestration of non-transferrin-bound 55Fe in the mitochondrial compartment of cultured astroglia without affecting the disposition of transferrin-derived 55Fe. L-DOPA (25 ÁM) weakly recapitulated the effects of dopamine on glial iron sequestration whereas equimolar concentrations of norepinephrine were entirely inert in this regard (36). The effects of dopamine on glial iron trapping were abrogated by co-administration of ascorbate (200 ÁM), but not by the D1 antagonist, SCH23390, or the D2 blocker, sulpiride, suggesting that dopamine-derived free radicals promote the sequestration of non-transferrin derived iron within astroglial mitochondria.
Role of HO-1 in Brain Iron Deposition
HO-1 and Glial Iron Sequestration
Heme oxygenase-1 (HO-1) is a 32-kd member of the stress protein superfamily that mediates the rapid catabolism of heme to biliverdin in brain and other tissues (8,9,20,31,40). The HO-1 gene has a heat-shock consensus sequence in its promoter region and is strongly induced by oxidative stress, metal ions, sulfhydryl compounds, and hyperthermia (1,7,17,18,30). In response to oxidative stress, induction of HO-1 may protect cells by degrading prooxidant metalloprophyrins, such as heme, to bile pigments (biliverdin, bilirubin) with free radical-scavenging capabilities (1). Alternatively, free ferrous iron and carbon monoxide released during HO-1-catalyzed heme degradation may exacerbate oxidative stress by damaging mitochondria and stimulating ROS generation within this subcellular compartment (45).
Within 6 h of CSH exposure, we observed 4-10 fold increases in HO-1 mRNA and protein levels, robust HO-1 immunofluorescent staining, and a 3-fold increase in HO enzymatic activity in cultured rat astroglia (3,21,27). As in the case of CSH, H2O2, menadione and dopamine (but not norepinephrine) consistently augment HO-1 expression in cultured astroglia prior to promoting the trapping of non-transferrin bound 55Fe by the mitochondrial compartment (2,36). The effects of dopamine on HO-1 expression were blocked by ascorbate implicating a free radical mechanism of action. Dopamine-induced mitochondrial iron sequestration was abrogated by administration of the heme oxygenase inhibitors, tin mesoporphyrin or dexamethasone indicating that upregulation of HO-1 is necessary for subsequent mitochondrial iron deposition in these cells. Moreover, overexpression of the human HO-1 gene in cultured rat astroglia by transient transfection stimulated mitochondrial 55Fe deposition, an effect that was again preventible by tin mesoporphyrin or dexamethasone administration (11). We hypothesize that free ferrous iron and carbon monoxide generated by HO-1-mediated heme degradation promote mitochondrial membrane injury and the deposition of redox-active iron within this organelle.
HO-1 Expression in AD and PD Brain
Using immunolabeling techniques and laser scanning confocal microscopy, we observed intense HO-1 immunoreactivity of HO-1 in neurons of the hippocampus and temporal cortex of Alzheimer-diseased brain relative to age-matched control specimens (37). We demonstrated consistent colocalization of HO-1 to GFAP-positive astrocytes, neurofibrillary tangles and senile plaques in the AD tissues. In AD hippocampus, approximately 86% of GFAP-positive astrocytes expressed HO-1 whereas only 6.8% of hippocampal astrocytes in normal senescent control tissues were immunopositive for this enzyme (p < 0.0001). In the substantia nigra, a region exhibiting little or no Alzheimer pathology, the proportion of astrocytes expressing HO-1 in the AD group (12.8%) was not significantly different from the controls (6.4%). Robust 32 kDa bands corresponding to HO-1 were observed by Western blotting of protein extracts derived from AD temporal cortex and hippocampus after SDS-PAGE, whereas HO-1 bands were very faint or absent in protein extracts prepared from control specimens (37). Our findings indicate that HO-1 is significantly overexpressed in neurons and astrocytes of Alzheimer-diseased hippocampus and cerebral cortex relative to control brains.
In a second study (39), immunohistochemistry was used to assess HO-1 expression in post-mortem human brain specimens derived from PD and control subjects. In the substantia nigra of both PD and control specimens, moderate HO-1 immunoreactivity was consistently observed in neuromelanin-containing (dopaminergic) neurons. Lewy bodies, present only in nigral neurons of the PD subjects, exhibited intense HO-1 immunostaining in their peripheries. In both PD and control specimens, neuronal HO-1 staining was faint or non-detectable in the other brain regions examined. The proportion of GFAP-positive astroglia expressing HO-1 in PD substantia nigra (77.1%) was significantly greater than that observed in the substantia nigra of control subjects (18.7%). In the other regions surveyed, including the caudate, putamen and globus pallidus, percentages of GFAP-positive astroglia co-expressing HO-1 were relatively low and did not differ significantly between control and PD specimens.
Up-regulation of HO-1 in AD- and PD-affected neural tissues support the contention that the latter are experiencing chronic oxidative stress. The physiological significance of this HO-1 response, however, remains open to interpretaiton. It may be argued that intracellular degradation of pro-oxidant heme to bile pigments with anti-oxidant properties (biliverdin, bilirubin) may serve as an attempt to restore the redox microenvironment in the face of unremitting oxidative stress (1). On the other hand, as described above, HO-1-catalyzed heme degradation liberates free iron and CO which may exacerbate intracellular oxidative stress by stimulating free radical production within the mitochondrial compartment (45). In this paper, we reviewed evidence from our laboratory that HO-1 induction in cultured rat astroglia promotes the sequestration of non-transferrin-derived iron by the mitochondrial compartment. Taken together, our findings indicate that stress-induced up-regulation of HO-1 in astroglia may contribute to the abnormal patterns of iron deposition and mitochondrial insufficiency documented in the brains of AD and PD subjects.
|Schipper, H.M.; (1998). Glial HO-1 Expression, Iron Deposition and Oxidative Stress in Neurodegenerative Diseases. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/juurlink/schipper0139/index.html|
|© 1998 Author(s) Hold Copyright|