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Heme Deficiency May Be a Factor in the Mitochondrial and Neuronal Decay of Aging

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Heme Deficiency May Be a Factor in the Mitochondrial and Neuronal Decay of Aging Heme deficiency may be a factor in the mitochondrial and neuronal decay of aging Hani Atamna*, David W. Killilea, Alison Nisbet Killilea, and Bruce N. Ames* Children’s Hospital Oakland Research Institute, Oakland, CA 94609 Contributed by Bruce N. Ames, September 26, 2002 Heme, a major functional form of iron in the cell, is synthesized in other environmental toxins (15, 16). Ferrochelatase is inacti- the mitochondria by ferrochelatase inserting ferrous iron into vated when its iron-sulfur cluster disassembles because intracel- protoporphyrin IX. Heme deficiency was induced with N-methyl- lular iron levels are low (17) or nitric oxide (NO) is present (18). protoporphyrin IX, a selective inhibitor of ferrochelatase, in two Iron deficiency is by far the most studied; it impairs cognitive human brain cell lines, SHSY5Y (neuroblastoma) and U373 (astro- function in children, impacts the morphology and the physiology cytoma), as well as in rat primary hippocampal neurons. Heme of brain even before changes in hemoglobin appear (19), dam- deficiency in brain cells decreases mitochondrial complex IV, acti- ages mitochondria (20), and causes oxidative stress (20). Iron vates nitric oxide synthase, alters amyloid precursor protein, and deficiency in children is associated with difficulties in performing corrupts iron and zinc homeostasis. The metabolic consequences cognitive tasks and retardation in the development of the CNS resulting from heme deficiency seem similar to dysfunctional (21, 22). Prenatal iron deficiency causes loss of cytochrome c neurons in patients with Alzheimer’s disease. Heme-deficient oxidase (complex IV) in selected regions in the brain of rats (23) SHSY5Y or U373 cells die when induced to differentiate or to and causes a decrease in ferrochelatase (17). Iron deficiency also proliferate, respectively. The role of heme in these observations occurs in the elderly (24), but surprisingly little work has been could result from its interaction with heme regulatory motifs in reported concerning heme and iron deficiency in brain dysfunc- specific proteins or secondary to the compromised mitochondria. tion with age. The increase in the levels of iron with age could Common causes of heme deficiency include aging, deficiency of occur as a result of the inefficient synthesis of heme, suggesting iron and vitamin B6, and exposure to toxic metals such as alumi- a functional deficiency of iron in advanced age (8). num. Iron and B6 deficiencies are especially important because In this study we tested the hypothesis of a possible role of heme they are widespread, but they are also preventable with supple- deficiency in neuronal decay. We found that heme deficiency mentation. Thus, heme deficiency or dysregulation may be an changes mitochondrial complex IV, APP, NO synthase (NOS), important and preventable component of the neurodegenerative and zinc and iron homeostasis. Many of the phenotypic changes process. seen in heme-deficient cells are also seen in the aging brain and are even more pronounced in neurodegenerative diseases, such iron ͉ complex IV ͉ amyloid precursor protein (APP) ͉ nitric oxide as AD. In addition, brain cells that were heme-deficient failed to synthase ͉ oxidative stress differentiate or to complete a successful cell cycle, which sug- gests a unique function of heme that is beyond the classic he causes that underlie the neurodegeneration accompany- perception of heme in cell biology. ing Alzheimer’s disease (AD) are unknown. Normal aging of T Materials and Methods the brain and the pathology of the neurodegeneration caused by AD share: the decline in complex IV (1), mitochondrial dys- Materials. N-methylprotoporphyrin (NMP) was from Porphyrin function (2), hypometabolism (3, 4), oxidative stress (5), loss of Products (Logan, UT). Anti-human cytochrome c oxidase sub- iron homeostasis (6), and dystrophic neurons. Several mutations unit II mouse monoclonal antibody (12c4-f12) was from Molec- causing altered proteins amyloid precursor protein (APP), PS1, ular Probes; anti-human APP (preA4695) mouse monoclonal and PS2 have been identified in familial cases of AD. Aging is antibody against the N terminus (MAB348 clone 22C11) was a risk factor even in these familial cases, which represent a small from Chemicon; anti-neuronal NOS (NOS1) rabbit-polyclonal portion of the total cases of AD. antibody against the C terminus (R-20) was from Santa Cruz Heme synthesis declines with age (7). This decline could Biotechnology. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetra- explain the age-associated loss of iron homeostasis, because zolium bromide, thymidine, laminin, and poly-D-lysine were insufficient levels of heme would compromise iron regulation from Sigma. Cell media (DMEM), nerve growth factor, nones- sential amino acids, and sodium pyruvate were from Invitrogen. and vice versa (8). Furthermore, we demonstrated that heme CELL BIOLOGY ͞ deficiency shows a selective decrease in mitochondrial complex The reagents for SDS PAGE and Western blotting were of the IV that leads to oxidative stress and corruption of Ca2ϩ ho- highest grade available. meostasis (9). Heme functions in hemoglobin and enzymes, and it regulates the activity of proteins and gene expression through Tissue Cultures. Human neuroblastoma (SHSY5Y) and astrocy- the heme regulatory motifs (HRMs) in several proteins (10–12). toma (U373) were purchased from the American Type Culture Also, a role of heme in promoting neurite outgrowth and Collection and maintained as described by the supplier. In brief, neurogenesis has been known for some time (13). Thus, dys- the cells were split two times every week (except SHSY5Y were ͞ functional heme metabolism could alter gene expression in brain split on a weekly basis) by trypsin EDTA and seeded as 1:10 cells, perhaps even preceding altered cytochrome activity. (U373) or 1:5 (SHSY5Y) dilutions. The cells were maintained in ͞ ͞ Heme synthetic pathway depends on essential micronutrients, DMEM 1% non-essential amino acids 1% sodium pyruvate at ͞ such as vitamin B6, lipoic acid, iron, copper, and zinc (8). About 37°C in a 5% CO2 95% air-equilibrated incubator. 10% of the U.S. population ingest Ͻ50% of the recommended daily allowance (RDA) for B6 (14); Ϸ25% of menstruating Ͻ Abbreviations: NO, nitric oxide; NOS, NO synthase; NOS1, neuronal NOS; APP, amyloid women in the U.S. ingest 50% of the RDA for iron. Iron precursor protein; NMP, N-methylprotoporphyrin IX; AD, Alzheimer’s disease; HRM, heme deficiency is the most common micronutrient deficiency in the regulatory motif. world in menstruating women and in children (14). In addition, *To whom correspondence may be addressed. E-mail: [email protected] or bames@ heme synthesis is susceptible to exogenous lead, aluminum, and chori.org. www.pnas.org͞cgi͞doi͞10.1073͞pnas.192585799 PNAS ͉ November 12, 2002 ͉ vol. 99 ͉ no. 23 ͉ 14807–14812 Downloaded by guest on September 24, 2021 Preparation of Hippocampal Neurons. Children’s Hospital’s Animal Care and Use Committee approved the animal experimentation performed in this study. Hippocampal primary neurons were prepared by using 12-h-old neonatal Fischer 344 rats according to the published protocol (25). Fig. 1. Heme deficiency selectively prevents assembly of cytochrome c Inhibition of Heme Synthesis. N-methylprotoporphyrin IX (NMP) oxidase. Human neuroblastoma (SHSY5Y) and astrocytoma (U373) cells were Ϯ ␮ mimics protoporphyrin IX, the substrate for ferrochelatase, maintained for 6 days with a medium 10 M NMP. After that the proteins were separated by SDS͞PAGE (12%), Western blotted, and analyzed by mono- except that a methyl group is added to one of its nitrogens. NMP clonal antibodies for subunit II of complex IV. The data presented are from one binds ferrochelatase with affinity similar to protoporphyrin IX, representative experiment of at least three independent experiments. Con, but the methyl group prevents iron from being inserted into control; COXII, subunit II of complex IV. NMP (26); thus, it is a selective and specific inhibitor for ferrochelatase (27). NMP has been used previously in several studies to inhibit heme synthesis (9, 28). Cells treated with NMP Heme Deficiency Alters Endogenous APP. We examined the effect of are still able to synthesize heme up to 40% of the controls (29) heme deficiency on APP. Monomeric APP decreases in brain Ϸ by up-regulating ferrochelatase (9). To induce heme deficiency, cells after 5 days of heme deficiency to 50% of the controls, Ն NMP was applied at concentrations and intervals as stated in whereas a dimeric and higher molecular mass species ( 200 figure legends. kDa) appears as detected by antibody specific for APP (Fig. 2A). Immuno (Western) blotting with SDS͞10–12% polyacryl- APP in hippocampal primary neurons was similarly altered by amide gel, poly(vinylidene difluoride) membranes, and specific heme deficiency (Fig. 2B). antibodies against the selected proteins was applied to evaluate the effect of NMP on complex IV, NOS, and APP. NOS Is Up-Regulated in Heme-Deficient Cells. Heme deficiency is associated with an increase in the activity of NOS in SHSY5Y Production of Nitrate͞Nitrite as a Measure for NOS Activity. The level (Fig. 3A) and U373 (Fig. 3B). The activity of NOS was deter- of nitrate͞nitrite produced by the cells was measured in the mined by measuring the levels of nitrite and nitrate that were medium by Griess Reagents by using a nitrate͞nitrite colori- released to the medium. The level of nitrite and nitrate are metric assay kit from Alexis Biochemicals (Carlsbad, CA). normalized to the ability of the cells to reduce 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Quantification of Cellular Metal Content. Iron and zinc content was A similar increase in NOS activity was observed when the results determined by inductively coupled plasma spectrometry (IRIS were normalized to protein (data not shown). Heme deficiency 5900, Thermo Elemental, Franklin, MA) by using the protocol also induced a similar increase in NOS activity in primary rat from a previous report (30). Metal content was expressed as hippocampal neurons (data not shown).
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