Genome-wide microarray analysis of brain gene expression in mice on a short-term high iron diet

doi:10.1016/j.neuint.2010.03.015

Neurochemistry International

Volume 56, Issues 6–7, May–June 2010, Pages 856-863

https://doi.org/10.1016/j.neuint.2010.03.015 Get rights and content

Abstract

The effects of systemic iron overload on the brain are unclear. Microarray analysis of brain gene expression in mice following short-term iron supplementation revealed altered expression of 287 genes, although most changes were small. Transcripts for the iron storage protein ferritin light chain increased 20% (p = 0.002) and transcripts for iron regulatory protein 1, which negatively regulates ferritin translation, decreased 28% (p = 0.048). There were expression changes for genes involved in important brain functions such as neurotransmission and nitric oxide signaling, which is dependent on iron. Few changes related to reactive oxygen species, inflammation or apoptosis, however expression changes were observed for genes causatively linked to neurological disorders, including Charcot-Marie-Tooth disease, neuronal ceroid lipofuscinosis and mucolipidosis. The latter involve intralysosomal lipofuscin build-up that may reflect lysosomal iron accumulation. The findings suggest that high iron intake may cause subtle brain effects of clinical relevance in some circumstances.

Introduction

Iron is necessary for neurogenesis and brain development, and for essential brain functions such as neurotransmission, but too much iron can be toxic (Rouault and Cooperman, 2006, Madsen and Gitlin, 2007). Genetic mutations affecting brain iron homeostasis can cause fatal neurodegenerative diseases involving impaired movement or behavior. Examples include neurodegeneration with brain iron accumulation type 1 and neuroferritinopathy (Burn and Chinnery, 2006, Gregory et al., 2009). While these severe neurogenetic conditions are rare, as many as 10% of people in Western countries have abnormally high body iron stores for genetic, dietary or other reasons (Olynyk et al., 1999, Hovell et al., 2001, Adams et al., 2005). How this affects brain function is not well understood.

The blood–brain barrier limits iron entry into the brain (Moos and Morgan, 2000, Moos and Morgan, 2002, Deane et al., 2004) but high body iron status may nevertheless lead to increased iron in specific brain regions in rats (Pinero et al., 2000) and humans (Golub et al., 2005). Animal models have been used to investigate the effects of high body iron status on the brain and the molecular mechanisms involved. However, to date, such studies have only investigated changes in the levels of specific iron-related transcripts or proteins and have not looked at genome-wide changes (Moos et al., 1999, Pinero et al., 2000, Burdo et al., 2004, Chang et al., 2005, Ke et al., 2005, Qian et al., 2007).

One study has investigated genome-wide changes in brain gene expression in a rat model of iron deficiency (Clardy et al., 2006). In this study, brain gene expression was assessed in 21 day-old rat pups from mothers fed an iron deficient diet through gestation and lactation. A total of 334 genes were identified as having altered expression in the brains of iron-deficient rats, including a number of myelin-related genes. This suggests that systemic iron status can influence brain gene expression in some circumstances but so far there have been no reported studies of genome-wide brain gene expression in models of iron overload. In this exploratory study we have used microarray analysis to examine the effects of short-term increases in dietary iron intake on gene expression in the mouse brain.

Section snippets

Animals

Brain tissue from male AKR strain mice maintained at the University of Western Australia was kindly provided by Dr. D. Trinder and Dr. R.M. Graham. All protocols were approved by the Animal Ethics Committee of the University of Western Australia. After weaning, mice were maintained on normal chow (‘control’) or a high-iron diet (‘iron-supplemented’ - normal chow until 7 weeks of age then normal chow supplemented with 2% carbonyl iron for 3 weeks). The high iron diet causes increased serum iron

Results

The iron supplementation period of three weeks used in this study was chosen because it causes liver iron loading comparable to that in mild human hemochromatosis but no gross damage to the liver or other tissues (Drake et al., 2007), providing insights into the early changes occurring in response to iron loading, uncomplicated by subsequent secondary effects. Measurement of liver non-heme iron confirmed liver iron loading in the iron-supplemented mice (19.5 ± 1.4 μmol Fe/g wet wt liver) compared

Discussion

The findings suggest that short-term iron supplementation in adulthood can influence brain gene expression, even in a mild model with at most restricted brain iron loading. The observed gene expression changes are likely to reflect early primary effects, since the lack of tissue damage in this short-term model will limit compensatory effects or repair responses secondary to damage. The expression changes were generally small and remain to be confirmed but exhibited considerable biological

Acknowledgements

We gratefully acknowledge Dr Deborah Trinder and Dr Ross Graham for providing brain tissue samples and performing non-heme iron assays for this study. This work was funded by the University of Newcastle. The authors have no conflicts of interest to declare.

References (85)

G. Bach
Mucolipidosis type IV
Mol Genet Metab
(2001)
R. Bender et al.
Adjusting for multiple testing—When and how?
J Clin Epidemiol
(2001)
A. Bomford et al.
Adaptive responses of rat tissue isoferritins to iron administration. Changes in subunit synthesis, isoferritin abundance, and capacity for iron storage
J Biol Chem
(1981)
M.O. Bostanci et al.
Nitric oxide synthesis inhibition attenuates iron-induced neurotoxicity: a stereological study
Neurotoxicology
(2008)
U.T. Brunk et al.
Lipofuscin: mechanisms of age-related accumulation and influence on cell function
Free Radic Biol Med
(2002)
J. Burn et al.
Neuroferritinopathy
Semin Pediatr Neurol
(2006)
A.H. Crosby et al.
Is the transportation highway the right road for hereditary spastic paraplegia?
Am J Hum Genet
(2002)
O. Kakhlon et al.
Repression of ferritin expression increases the labile iron pool, oxidative stress, and short-term growth of human erythroleukemia cells
Blood
(2001)
Y. Ke et al.
Age-dependent and iron-independent expression of two mRNA isoforms of divalent metal transporter 1 in rat brain
Neurobiol Aging
(2005)
T. Moos et al.
Iron-independent neuronal expression of transferrin receptor mRNA in the rat
Brain Res Mol Brain Res
(1999)

D. Nikitovic et al.

S-nitrosoglutathione is cleaved by the thioredoxin system with liberation of glutathione and redox regulating nitric oxide

J Biol Chem

(1996)

A.J. Okumura et al.

Involvement of a novel Q-SNARE, D12, in quality control of the endomembrane system

J Biol Chem

(2006)

D. Pacheco-Alvarez et al.

Biotin availability regulates expression of the sodium-dependent multivitamin transporter and the rate of biotin uptake in HepG2 cells

Mol Genet Metab

(2005)

D.J. Pinero et al.

Variations in dietary iron alter brain iron metabolism in developing rats

J Nutr

(2000)

P. Ponka

Rare causes of hereditary iron overload

Semin Hematol

(2002)

T.A. Rouault et al.

Brain iron metabolism

Semin Pediatr Neurol

(2006)

T. Sardon et al.

Nitric oxide-dependent and independent down-regulation of NO-sensitive guanylyl cyclase in neural cells

Toxicol Lett

(2004)

S. Sastry et al.

Time-dependent changes in iron levels and associated neuronal loss within the substantia nigra following lesions within the neostriatum/globus pallidus complex

Neuroscience

(1995)

H.H. Schmidt et al.

NO at work

Cell

(1994)

E. Siintola et al.

The novel neuronal ceroid lipofuscinosis gene MFSD8 encodes a putative lysosomal transporter

Am J Hum Genet

(2007)

T.J. Sobotka et al.

Neurobehavioral dysfunctions associated with dietary iron overload

Physiol Behav

(1996)

J.M. Tepper et al.

GABAergic control of substantia nigra dopaminergic neurons

Prog Brain Res

(2007)

A. Terman et al.

Lipofuscin

Int J Biochem Cell Biol

(2004)

P.C. Adams et al.

Hemochromatosis and iron-overload screening in a racially diverse population

N Engl J Med

(2005)

F. Antunes et al.

Apoptosis induced by exposure to a low steady-state concentration of H₂O₂ is a consequence of lysosomal rupture

Biochem J

(2001)

P. Arosio et al.

Structural and functional studies of human ferritin H and L chains

Curr Stud Hematol Blood Transfus

(1991)

R. Bargal et al.

Identification of the gene causing mucolipidosis type IV

Nat Genet

(2000)

R.V. Baxter et al.

Ganglioside-induced differentiation-associated protein-1 is mutant in Charcot-Marie-Tooth disease type 4A/8q21

Nat Genet

(2002)

M. Benhar et al.

Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins

Science

(2008)

N.G. Bowery et al.

GABA and glycine as neurotransmitters: a brief history

Br J Pharmacol

(2006)

J.R. Burdo et al.

Regulation of the profile of iron-management proteins in brain microvasculature

J Cereb Blood Flow Metab

(2004)

B.J. Burri et al.

Heterogeneity of holocarboxylase synthetase in patients with biotin-responsive multiple carboxylase deficiency

Am J Hum Genet

(1985)

Y.Z. Chang et al.

Effects of development and iron status on ceruloplasmin expression in rat brain

J Cell Physiol

(2005)

S.L. Clardy et al.

Acute and chronic effects of developmental iron deficiency on mRNA expression patterns in the brain

J Neural Transm Suppl

(2006)

J.D. Cooper

Progress towards understanding the neurobiology of Batten disease or neuronal ceroid lipofuscinosis

Curr Opin Neurol

(2003)

A. Cuesta et al.

The gene encoding ganglioside-induced differentiation-associated protein 1 is mutated in axonal Charcot-Marie-Tooth type 4A disease

Nat Genet

(2002)

R. Deane et al.

Brain capillary endothelium and choroid plexus epithelium regulate transport of transferrin-bound and free iron into the rat brain

J Neurochem

(2004)

X.P. Dong et al.

The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel

Nature

(2008)

A.S. Dornelles et al.

mRNA expression of proteins involved in iron homeostasis in brain regions is altered by age and by iron overloading in the neonatal period

Neurochem Res

(2010)

S.F. Drake et al.

Iron absorption and hepatic iron uptake are increased in a transferrin receptor 2 (Y245X) mutant mouse model of hemochromatosis type 3

Am J Physiol Gastrointest Liver Physiol

(2007)

F. Dupic et al.

Duodenal mRNA expression of iron related genes in response to iron loading and iron deficiency in four strains of mice

Gut

(2002)

S.C. Fagan et al.

Targets for vascular protection after acute ischemic stroke

Stroke

(2004)

Cited by (24)

Applying microarray-based technique to study and analyze silkworm (Bombyx mori) transcriptomic response to long-term high iron diet
2019, Genomics
To investigate the biological processes affected by long-term iron supplementation, newly hatched silkworms were exposed to high iron mulberry diet (10 and 100 ppm) and its effect on silkworm transcriptom was determined. The results showed that the silkworm was responsive to iron by increasing iron concentration and ferritin levels in the hemolymph and by regulating the expression of many other genes. A total of 523 and 326 differentially expressed genes were identified in 10 and 100 ppm Fe group compared to the control, respectively. Of these genes, 249 were shared between in both the 10 ppm and 100 ppm Fe group, including 152 up-regulated and 97 down-regulated genes. These shared genes included 19 known Fe regulated, 24 immune-related, 12 serine proteases and serine proteases homologs, 41 cuticular and cuticle genes. Ten genes (carboxypeptidases A, serine protease homologs 85, fibrohexamerin/P25, transferrin, sex-specific storage-protein 2, fungal protease inhibitor F, insect intestinal mucin, peptidoglycan recognition protein B, cuticle protein CPH45, unknown gene) were involved in the regulation of iron overload responses.
Associations between changes in ferritin levels and susceptibility-weighted imaging filtered phase in patients with relapsing-remitting multiple sclerosis over 24weeks of therapy with subcutaneous interferon beta-1a three times weekly
2015, Journal of Neuroimmunology
Citation Excerpt :
For example, in elderly healthy volunteers, a relationship was shown between serum iron and deep gray matter iron, but there was no corresponding relationship with serum ferritin (House et al., 2010). However, in mice fed a short-term iron-overload diet, serum iron and liver iron were significantly increased, but there was no change in brain iron level (Johnstone and Milward, 2010). In this study, we had a unique opportunity to combine blood assays and MRI analysis to study both serum changes and iron-sensitive MRI phase data.
Subanalysis of a pilot study (NCT01085318) assessed correlations between serum ferritin and imaging assessments in relapsing–remitting multiple sclerosis patients (n = 23) receiving 44 μg interferon beta-1a subcutaneously three times weekly. At baseline, 12, and 24 weeks, mean ferritin was 75, 127 (p < 0.001 vs baseline), and 101 (p = 0.020 vs baseline) ng/mL. No relationship between ferritin and susceptibility-weighted imaging (SWI)-filtered phase of subcortical deep gray matter was found. Increasing ferritin correlated with decreasing lesion numbers on both fluid attenuated inversion recovery and SWI phase at 12 weeks (r = − 0.62; p = 0.003; n = 21), and with decreasing gadolinium-enhancing lesion volume at 24 weeks (r = − 0.71; p = 0.050; n = 8).
Cell biology and function of neuronal ceroid lipofuscinosis-related proteins
2013, Biochimica et Biophysica Acta - Molecular Basis of Disease
Neuronal ceroid lipofuscinoses (NCL) comprise a group of inherited lysosomal disorders with variable age of onset, characterized by lysosomal accumulation of autofluorescent ceroid lipopigments, neuroinflammation, photoreceptor- and neurodegeneration. Most of the NCL-related genes encode soluble and transmembrane proteins which localize to the endoplasmic reticulum or to the endosomal/lysosomal compartment and directly or indirectly regulate lysosomal function. Recently, exome sequencing led to the identification of four novel gene defects in NCL patients and a new NCL nomenclature currently comprising CLN1 through CLN14. Although the precise function of most of the NCL proteins remains elusive, comprehensive analyses of model organisms, particularly mouse models, provided new insight into pathogenic mechanisms of NCL diseases and roles of mutant NCL proteins in cellular/subcellular protein and lipid homeostasis, as well as their adaptive/compensatorial regulation at the transcriptional level. This review summarizes the current knowledge on the expression, function and regulation of NCL proteins and their impact on lysosomal integrity. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.
Brain transcriptome perturbations in the transferrin receptor 2 mutant mouse support the case for brain changes in iron loading disorders, including effects relating to long-term depression and long-term potentiation
2013, Neuroscience
Citation Excerpt :
Brains were removed, snap-frozen in liquid nitrogen and stored at −80 °C. Non-heme iron levels in brain and liver homogenates were assessed using the method of Kaldor (1954), as described previously (Johnstone and Milward, 2010a; Johnstone et al., 2012a). Total brain iron levels were assessed by inductively coupled plasma atomic emission spectroscopy (ICP-AES).
Iron abnormalities within the brain are associated with several rare but severe neurodegenerative conditions. There is growing evidence that more common systemic iron loading disorders such as hemochromatosis can also have important effects on the brain. To identify features that are common across different forms of hemochromatosis, we used microarray and real-time reverse transcription polymerase chain reaction (RT-PCR) to assess brain transcriptome profiles of transferrin receptor 2 mutant mice (Tfr2^mut), a model of a rare type of hereditary hemochromatosis, relative to wildtype control mice. The results were compared with our previous findings in dietary iron-supplemented wildtype mice and Hfe^−/− mice, a model of a common type of hereditary hemochromatosis. For transcripts showing significant changes relative to controls across all three models, there was perfect (100%) directional concordance (i.e. transcripts were increased in all models or decreased in all models). Comparison of the two models of hereditary hemochromatosis, which showed more pronounced changes than the dietary iron-supplemented mice, revealed numerous common molecular effects. Pathway analyses highlighted changes for genes relating to long-term depression (6.8-fold enrichment, p = 5.4 × 10⁻⁷) and, to a lesser extent, long-term potentiation (3.7-fold enrichment, p = 0.01), with generalized reductions in transcription of key genes from these pathways, which are involved in modulating synaptic strength and efficacy and are essential for memory and learning. The agreement across the models suggests the findings are robust and strengthens previous evidence that iron loading disorders affect the brain. Perturbations of brain phenomena such as long-term depression and long-term potentiation might partly explain neurologic symptoms reported for some hemochromatosis patients.
Metals, oxidative stress and neurodegeneration: A focus on iron, manganese and mercury
2013, Neurochemistry International
Citation Excerpt :
One important (but not fully explored) aspect on Fe homeostasis is how dietary or genetic Fe loading can modify the metabolism of proteins involved in Fe absorption, trafficking and storage in brain tissues. Clarifying such aspects would contribute on understanding how Fe participates in neurodegenerative processes; such knowledge may improve treatment options in a range of neurodegenerative disorders (Johnstone and Milward, 2010a,b). As discussed above, free cationic Fe can be extremely toxic via disruption of mitochondrial function, and theoretically, Fe2+ ↔ Fe3+ redox changes can be coupled with formation of extremely reactive species, such as hydroxyl radical (OH).
Essential metals are crucial for the maintenance of cell homeostasis. Among the 23 elements that have known physiological functions in humans, 12 are metals, including iron (Fe) and manganese (Mn). Nevertheless, excessive exposure to these metals may lead to pathological conditions, including neurodegeneration. Similarly, exposure to metals that do not have known biological functions, such as mercury (Hg), also present great health concerns. This review focuses on the neurodegenerative mechanisms and effects of Fe, Mn and Hg. Oxidative stress (OS), particularly in mitochondria, is a common feature of Fe, Mn and Hg toxicity. However, the primary molecular targets triggering OS are distinct. Free cationic iron is a potent pro-oxidant and can initiate a set of reactions that form extremely reactive products, such as OH. Mn can oxidize dopamine (DA), generating reactive species and also affect mitochondrial function, leading to accumulation of metabolites and culminating with OS. Cationic Hg forms have strong affinity for nucleophiles, such as –SH and –SeH. Therefore, they target critical thiol- and selenol-molecules with antioxidant properties. Finally, we address the main sources of exposure to these metals, their transport mechanisms into the brain, and therapeutic modalities to mitigate their neurotoxic effects.
Brain transcriptome perturbations in the Hfe <sup>-/-</sup> mouse model of genetic iron loading
2012, Brain Research
Citation Excerpt :
The mechanisms underlying these phenotypic changes, whether due to brain iron dyshomeostasis, disruption of some other important brain mechanism involving HFE or indirect effects of peripheral systemic iron overload, remain unclear. We have previously investigated brain transcript levels in 10 week-old wildtype mice fed an iron-supplemented diet (2% carbonyl iron) for three weeks (Johnstone and Milward, 2010b). These mice showed significant changes in transcript levels for a number of important genes, however these changes were generally small in magnitude, possibly due to the acute nature of the iron loading.
Severe disruption of brain iron homeostasis can cause fatal neurodegenerative disease, however debate surrounds the neurologic effects of milder, more common iron loading disorders such as hereditary hemochromatosis, which is usually caused by loss-of-function polymorphisms in the HFE gene. There is evidence from both human and animal studies that HFE gene variants may affect brain function and modify risks of brain disease. To investigate how disruption of HFE influences brain transcript levels, we used microarray and real-time reverse transcription polymerase chain reaction to assess the brain transcriptome in Hfe^−/− mice relative to wildtype AKR controls (age 10 weeks, n ≥ 4/group). The Hfe^−/− mouse brain showed numerous significant changes in transcript levels (p < 0.05) although few of these related to proteins directly involved in iron homeostasis. There were robust changes of at least 2-fold in levels of transcripts for prominent genes relating to transcriptional regulation (FBJ osteosarcoma oncogene Fos, early growth response genes), neurotransmission (glutamate NMDA receptor Grin1, GABA receptor Gabbr1) and synaptic plasticity and memory (calcium/calmodulin-dependent protein kinase IIα Camk2a). As previously reported for dietary iron-supplemented mice, there were altered levels of transcripts for genes linked to neuronal ceroid lipofuscinosis, a disease characterized by excessive lipofuscin deposition. Labile iron is known to enhance lipofuscin generation which may accelerate brain aging. The findings provide evidence that iron loading disorders can considerably perturb levels of transcripts for genes essential for normal brain function and may help explain some of the neurologic signs and symptoms reported in hemochromatosis patients.

View all citing articles on Scopus

View full text

Genome-wide microarray analysis of brain gene expression in mice on a short-term high iron diet

Abstract

Introduction

Section snippets

Animals

Results

Discussion

Acknowledgements

Mol Genet Metab

J Clin Epidemiol

J Biol Chem

Neurotoxicology

Free Radic Biol Med

Semin Pediatr Neurol

Am J Hum Genet

Blood

Neurobiol Aging

Brain Res Mol Brain Res

J Biol Chem

J Biol Chem

Mol Genet Metab

J Nutr

Semin Hematol

Semin Pediatr Neurol

Toxicol Lett

Neuroscience

Cell

Am J Hum Genet

Physiol Behav

Prog Brain Res

Int J Biochem Cell Biol

Hemochromatosis and iron-overload screening in a racially diverse population

N Engl J Med

Apoptosis induced by exposure to a low steady-state concentration of H2O2 is a consequence of lysosomal rupture

Biochem J

Structural and functional studies of human ferritin H and L chains

Curr Stud Hematol Blood Transfus

Identification of the gene causing mucolipidosis type IV

Nat Genet

Ganglioside-induced differentiation-associated protein-1 is mutant in Charcot-Marie-Tooth disease type 4A/8q21

Nat Genet

Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins

Science

GABA and glycine as neurotransmitters: a brief history

Br J Pharmacol

Regulation of the profile of iron-management proteins in brain microvasculature

J Cereb Blood Flow Metab

Heterogeneity of holocarboxylase synthetase in patients with biotin-responsive multiple carboxylase deficiency

Am J Hum Genet

Effects of development and iron status on ceruloplasmin expression in rat brain

J Cell Physiol

Acute and chronic effects of developmental iron deficiency on mRNA expression patterns in the brain

J Neural Transm Suppl

Progress towards understanding the neurobiology of Batten disease or neuronal ceroid lipofuscinosis

Curr Opin Neurol

The gene encoding ganglioside-induced differentiation-associated protein 1 is mutated in axonal Charcot-Marie-Tooth type 4A disease

Nat Genet

Brain capillary endothelium and choroid plexus epithelium regulate transport of transferrin-bound and free iron into the rat brain

J Neurochem

The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel

Nature

mRNA expression of proteins involved in iron homeostasis in brain regions is altered by age and by iron overloading in the neonatal period

Neurochem Res

Iron absorption and hepatic iron uptake are increased in a transferrin receptor 2 (Y245X) mutant mouse model of hemochromatosis type 3

Am J Physiol Gastrointest Liver Physiol

Duodenal mRNA expression of iron related genes in response to iron loading and iron deficiency in four strains of mice

Gut

Targets for vascular protection after acute ischemic stroke

Stroke

Apoptosis induced by exposure to a low steady-state concentration of H₂O₂ is a consequence of lysosomal rupture