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. 2002 Nov 12;99(23):14807-12.
doi: 10.1073/pnas.192585799. Epub 2002 Nov 4.

Heme deficiency may be a factor in the mitochondrial and neuronal decay of aging

Hani Atamna  1 , David W Killilea, Alison Nisbet Killilea, Bruce N Ames
Affiliations

Heme deficiency may be a factor in the mitochondrial and neuronal decay of aging

Hani Atamna et al. Proc Natl Acad Sci U S A. .

Abstract

Heme, a major functional form of iron in the cell, is synthesized in the mitochondria by ferrochelatase inserting ferrous iron into protoporphyrin IX. Heme deficiency was induced with N-methylprotoporphyrin IX, a selective inhibitor of ferrochelatase, in two human brain cell lines, SHSY5Y (neuroblastoma) and U373 (astrocytoma), as well as in rat primary hippocampal neurons. Heme deficiency in brain cells decreases mitochondrial complex IV, activates nitric oxide synthase, alters amyloid precursor protein, and corrupts iron and zinc homeostasis. The metabolic consequences resulting from heme deficiency seem similar to dysfunctional neurons in patients with Alzheimer's disease. Heme-deficient SHSY5Y or U373 cells die when induced to differentiate or to proliferate, respectively. The role of heme in these observations could result from its interaction with heme regulatory motifs in specific proteins or secondary to the compromised mitochondria. Common causes of heme deficiency include aging, deficiency of iron and vitamin B6, and exposure to toxic metals such as aluminum. Iron and B6 deficiencies are especially important because they are widespread, but they are also preventable with supplementation. Thus, heme deficiency or dysregulation may be an important and preventable component of the neurodegenerative process.

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Figures

Fig 1.
Fig 1.
Heme deficiency selectively prevents assembly of cytochrome c oxidase. Human neuroblastoma (SHSY5Y) and astrocytoma (U373) cells were maintained for 6 days with a medium ± 10 μM NMP. After that the proteins were separated by SDS/PAGE (12%), Western blotted, and analyzed by monoclonal antibodies for subunit II of complex IV. The data presented are from one representative experiment of at least three independent experiments. Con, control; COXII, subunit II of complex IV.
Fig 2.
Fig 2.
Heme deficiency induces dimers and aggregates of APP in human brain cells and in rat hippocampal primary neurons. (A) Human brain cells (SHSY5Y and U373). (B) Rat hippocampal primary neurons. After induction of heme deficiency (A, 10 μM NMP for 6 days or B, different concentrations of NMP) the cells were harvested, and proteins were separated on SDS/PAGE, Western blotted, and analyzed by specific antibody for APP. The bars label APP aggregates. The data are from one of six independent experiments (human brain cells) and three experiments of hippocampal primary neurons. MWM, molecular weight markers.
Fig 3.
Fig 3.
Heme deficiency induces NOS1 in human brain cells. Heme deficiency was induced by incubation with 10 μM NMP for 6 days. The medium of heme-deficient and control cells was tested for the level of nitrite/nitrate as a measure for NOS1 activity. The data of NOS activity were normalized to 1 OD at 585 nm produced by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. (A) SHSY5Y. (B) U373. The data are a mean ± SD of one experiment of four. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 4.
Fig 4.
Complex IV is the first to decrease in heme-deficient human brain cells. Heme deficiency was induced by incubation with 10 μM NMP. The cells were harvested and prepared for analysis at different intervals. The level of COXII (subunit II of complex IV), NOS, and APP were evaluated by SDS/PAGE followed by Western blotting. The data are from one of three independent experiments with U373 cells.
Fig 5.
Fig 5.
Heme deficiency increases iron and decreases zinc in human astrocytoma (U373). The level of intracellular iron (A) and zinc (B) in heme-deficient cells was evaluated by inductively coupled plasma spectrometry and compared with heme-sufficient (control) cells maintained for 7 days in culture conditions. The data are mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01. HS, heme-sufficient (control); d, days.
Fig 6.
Fig 6.
Heme deficiency compromises differentiation in human neuroblastoma. Heme deficiency was induced by incubation with 10 μM NMP for 5 days. The differentiation of SHSY5Y was induced by DMEM + 50 ng/ml nerve growth factor. Heme-sufficient (a) and heme-deficient (c) cells seemed completely normal before the differentiation. Heme-sufficient cells differentiated to neurons (b) as expected within 3 h, whereas heme-deficient cells failed to complete the differentiation, lost their axons, and died within 3–4 h after differentiation (d). Shown is one representative experiment of four.
Fig 7.
Fig 7.
Heme deficiency compromises proliferation in human astrocytoma. Heme deficiency was induced as described in Fig. 6 in U373 cells. The cells were induced to proliferate by replacing the week-old medium with fresh complete medium. Heme-sufficient (a) and heme-deficient (c) cells seemed completely normal before the old medium was replaced by the fresh medium. Heme-sufficient cells were not affected by the fresh medium (b), whereas heme deficiency impaired cellular response to serum in heme-deficient cells (d) and the cells died within 7 h. Shown is one representative experiment of four.
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