There is strong evidence that iron homeostasis is impaired in the aging and Alzheimer’s disease (AD) brain and that this contributes to neurodegeneration. Apolipoprotein E (APOE) has been identified as the strongest genetic risk factor for AD. However, the interaction between the two has yet to be fully explored.
Objective:
This study aimed to investigate the relationship between exogenous iron levels and ApoE in neurons and astrocytes.
Methods:
Our study used primary cultured cortical neurons and astrocytes to investigate the changes in ApoE caused by iron. Western blot and RT-PCR were used to measure ApoE.
Results:
We observed that iron upregulated intracellular ApoE levels in both neurons and astrocytes at the post-transcriptional and transcriptional level, respectively. However, there was less full-length ApoE secreted by neurons and astrocytes after iron treatment. We speculate that this might impair brain lipid metabolism and amyloid-β clearance. In terms of ApoE receptors, we observed that neuronal LRP-1 levels were increased by the addition of exogenous iron, which could contribute to AβPP endocytosis in neurons. However, there were no significant changes in neuronal LDLR, astrocyte LDLR, or astrocyte LRP-1.
Conclusion:
Our study reveals that iron may contribute to the pathogenesis of AD by affecting ApoE and its receptors and supports the notion that iron chelation should be investigated as a therapeutic strategy for AD.
SinghN, HaldarS, TripathiAK, HorbackK, WongJ, SharmaD, BeserraA, SudaS, AnbalaganC, DevS, MukhopadhyayCK, SinghA (2014) Brain iron homeostasis: From molecular mechanisms to clinical significance and therapeutic opportunities. Antioxid Redox Signal20, 1324–1363.
2.
SipeJC, LeeP, BeutlerE (2002) Brain iron metabolism and neurodegenerative disorders. Dev Neurosci24, 188–196.
3.
AytonS, LeiP, AdlardPA, VolitakisI, ChernyRA, BushAI, FinkelsteinDI (2014) Iron accumulation confers neurotoxicity to a vulnerable population of nigral neurons: Implications for Parkinson’s disease. Mol Neurodegener9, 27.
4.
BilgicB, PfefferbaumA, RohlfingT, SullivanEV, AdalsteinssonE (2012) MRI estimates of brain iron concentration in normal aging using quantitative susceptibility mapping. Neuroimage59, 2625–2635.
5.
LangkammerC, RopeleS, PirpamerL, FazekasF, SchmidtR (2014) MRI for iron mapping in Alzheimer’s disease. Neurodegener Dis13, 189–191.
6.
PetersDG, ConnorJR, MeadowcroftMD (2015) The relationship between iron dyshomeostasis and amyloidogenesis in Alzheimer’s disease: Two sides of the same coin. Neurobiol Dis81, 49–65.
7.
LeeJY, ChoE, KimTY, KimDK, PalmiterRD, VolitakisI, KimJS, BushAI, KohJY (2010) Apolipoprotein E ablation decreases synaptic vesicular zinc in the brain. Biometals23, 1085–1095.
8.
BushAI (2013) The metal theory of Alzheimer’s disease. J Alzheimers Dis33(Suppl 1), S277–S281.
9.
GreenoughMA, CamakarisJ, BushAI (2013) Metal dyshomeostasis and oxidative stress in Alzheimer’s disease. Neurochem Int62, 540–555.
10.
BertramL, TanziRE (2009) Genome-wide association studies in Alzheimer’s disease. Hum Mol Genet18, R137–R145.
11.
LahiriDK, SambamurtiK, BennettDA (2004) Apolipoprotein gene and its interaction with the environmentally driven risk factors: Molecular, genetic and epidemiological studies of Alzheimer’s disease. Neurobiol Aging25, 651–660.
12.
BuG (2009) Apolipoprotein E and its receptors in Alzheimer’s disease: Pathways, pathogenesis and therapy. Nat Rev Neurosci10, 333–344.
13.
BrechtWJ, HarrisFM, ChangS, TesseurI, YuGQ, XuQ, Dee FishJ, Wyss-CorayT, ButtiniM, MuckeL, MahleyRW, HuangY (2004) Neuron-specific apolipoprotein e4 proteolysis is associated with increased tau phosphorylation in brains of transgenic mice. J Neurosci24, 2527–2534.
PfriegerFW (2003) Cholesterol homeostasis and function in neurons of the central nervous system. Cell Mol Life Sci60, 1158–1171.
16.
HanSH, KimJS, LeeY, ChoiH, KimJW, NaDL, YangEG, YuMH, HwangD, LeeC, Mook-JungI (2014) Both targeted mass spectrometry and flow sorting analysis methods detected the decreased serum apolipoprotein E level in Alzheimer’s disease patients. Mol Cell Proteomics13, 407–419.
17.
GuptaVB, LawsSM, VillemagneVL, AmesD, BushAI, EllisKA, LuiJK, MastersC, RoweCC, SzoekeC, TaddeiK, MartinsRN (2011) Plasma apolipoprotein E and Alzheimer disease risk: The AIBL study of aging. Neurology76, 1091–1098.
18.
BalesKR (2010) Brain lipid metabolism, apolipoprotein E and the pathophysiology of Alzheimer’s disease. Neuropharmacology59, 295–302.
19.
NambaY, TomonagaM, KawasakiH, OtomoE, IkedaK (1991) Apolipoprotein E immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in Alzheimer’s disease and kuru plaque amyloid in Creutzfeldt-Jakob disease. Brain Res541, 163–166.
20.
VerghesePB, CastellanoJM, GaraiK, WangY, JiangH, ShahA, BuG, FriedenC, HoltzmanDM (2013) ApoE influences amyloid-beta (Abeta) clearance despite minimal apoE/Abeta association in physiological conditions. Proc Natl Acad Sci U S A110, E1807–E1816.
21.
HoltzmanDM, HerzJ, BuG (2012) Apolipoprotein E and apolipoprotein E receptors: Normal biology and roles in Alzheimer disease. Cold Spring Harb Perspect Med2, a006312.
22.
RebeckGW, LaDuMJ, EstusS, BuG, WeeberEJ (2006) The generation and function of soluble apoE receptors in the CNS. Mol Neurodegener1, 15.
23.
HerzJ, StricklandDK (2001) LRP: A multifunctional scavenger and signaling receptor. J Clin Invest108, 779–784.
24.
HerzJ, KowalRC, GoldsteinJL, BrownMS (1990) Proteolytic processing of the 600kd low density lipoprotein receptor-related protein (LRP) occurs in a trans-Golgi compartment. EMBO J9, 1769–1776.
25.
Vazquez-HigueraJL, MateoI, Sanchez-JuanP, Rodriguez-RodriguezE, PozuetaA, InfanteJ, BercianoJ, CombarrosO (2009) Genetic interaction between tau and the apolipoprotein E receptor LRP1 Increases Alzheimer’s disease risk. Dement Geriatr Cogn Disord28, 116–120.
26.
RodriguezE, MateoI, LlorcaJ, Sanchez-QuintanaC, InfanteJ, BercianoJ, CombarrosO (2006) Genetic interaction between two apolipoprotein E receptors increases Alzheimer’s disease risk. J Neurol253, 801–803.
27.
AkramA, SchmeidlerJ, KatselP, HofPR, HaroutunianV (2012) Association of ApoE and LRP mRNA levels with dementia and AD neuropathology. Neurobiol Aging33, 628e621–628 e614.
AdlardPA, BicaL, WhiteAR, NurjonoM, FilizG, CrouchPJ, DonnellyPS, CappaiR, FinkelsteinDI, BushAI (2011) Metal ionophore treatment restores dendritic spine density and synaptic protein levels in a mouse model of Alzheimer’s disease. PLoS One6, e17669.
30.
ChenSH, LinJK, LiuSH, LiangYC, Lin-ShiauSY (2008) Apoptosis of cultured astrocytes induced by the copper and neocuproine complex through oxidative stress and JNK activation. Toxicol Sci102, 138–149.
31.
WangXF, CynaderMS (2001) Pyruvate released by astrocytes protects neurons from copper-catalyzed cysteine neurotoxicity. J Neurosci21, 3322–3331.
32.
WhiteAR, HuangX, JoblingMF, BarrowCJ, BeyreutherK, MastersCL, BushAI, CappaiR (2001) Homocysteine potentiates copper- and amyloid beta peptide-mediated toxicity in primary neuronal cultures: Possible risk factors in the Alzheimer’s-type neurodegenerative pathways. J Neurochem76, 1509–1520.
33.
AimoL, OteizaPI (2006) Zinc deficiency increases the susceptibility of human neuroblastoma cells to lead-induced activator protein-1 activation. Toxicol Sci91, 184–191.
34.
LeiP, AytonS, AppukuttanAT, VolitakisI, AdlardPA, FinkelsteinDI, BushAI (2015) Clioquinol rescues Parkinsonism and dementia phenotypes of the tau knockout mouse. Neurobiol Dis81, 168–175.
35.
GhoshMC, ZhangL, RouaultTA (2015) Iron misregulation and neurodegenerative disease in mouse models that lack iron regulatory proteins. Neurobiol Dis81, 66–75.
36.
HareDJ, GrubmanA, RyanTM, LothianA, LiddellJR, GrimmR, MatsudaT, DoblePA, ChernyRA, BushAI, WhiteAR, MastersCL, RobertsBR (2013) Profiling the iron, copper and zinc content in primary neuron and astrocyte cultures by rapid online quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry. Metallomics5, 1656–1662.
37.
WardRJ, ZuccaFA, DuynJH, CrichtonRR, ZeccaL (2014) The role of iron in brain ageing and neurodegenerative disorders. Lancet Neurol13, 1045–1060.
38.
MiyataM, SmithJD (1996) Apolipoprotein E allele-specific antioxidant activity and effects on cytotoxicity by oxidative insults and beta-amyloid peptides. Nat Genet14, 55–61.
39.
DoreyE, ChangN, LiuQY, YangZ, ZhangW (2014) Apolipoprotein E, amyloid-beta, and neuroinflammation in Alzheimer’s disease. Neurosci Bull30, 317–330.
40.
WardRJ, ZuccaFA, DuynJH, CrichtonRR, ZeccaL (2014) The role of iron in brain ageing and neurodegenerative disorders. Lancet Neurol13, 1045–1060.
41.
PoyntonC, JenkinsonM, AdalsteinssonE, SullivanE, PfefferbaumA, WellsW (2015) Quantitative susceptibility mapping by inversion of a perturbation field model: Correlation with brain iron in normal aging. IEEE Trans Med Imaging34, 339–353.
42.
RamosP, SantosA, PintoNR, MendesR, MagalhaesT, AlmeidaA (2014) Iron levels in the human brain: A post-mortem study of anatomical region differences and age-related changes. J Trace Elem Med Biol28, 13–17.
43.
BushAI (2003) The metallobiology of Alzheimer’s disease. Trends Neurosci26, 207–214.
44.
LeVineSM, WulserMJ, LynchSG (1998) Iron quantification in cerebrospinal fluid. Anal Biochem265, 74–78.
45.
BishopGM, RobinsonSR, LiuQ, PerryG, AtwoodCS, SmithMA (2002) Iron: A pathological mediator of Alzheimer disease?. Dev Neurosci24, 184–187.
46.
JiangD, LiX, WilliamsR, PatelS, MenL, WangY, ZhouF (2009) Ternary complexes of iron, amyloid-beta, and nitrilotriacetic acid: Binding affinities, redox properties, and relevance to iron-induced oxidative stress in Alzheimer’s disease. Biochemistry48, 7939–7947.
47.
AytonS, FauxNG, BushAI (2015) Ferritin levels in the cerebrospinal fluid predict Alzheimer’s disease outcomes and are regulated by APOE. Nat Commun6, 6760.
48.
PoirierJ, MironJ, PicardC, GormleyP, TherouxL, BreitnerJ, DeaD (2014) Apolipoprotein E and lipid homeostasis in the etiology and treatment of sporadic Alzheimer’s disease. Neurobiol Aging35(Suppl 2), S3–S10.
49.
FryerJD, DemattosRB, McCormickLM, O’DellMA, SpinnerML, BalesKR, PaulSM, SullivanPM, ParsadanianM, BuG, HoltzmanDM (2005) The low density lipoprotein receptor regulates the level of central nervous system human and murine apolipoprotein E but does not modify amyloid plaque pathology in PDAPP mice. J Biol Chem280, 25754–25759.
50.
WahrleSE, JiangH, ParsadanianM, LegleiterJ, HanX, FryerJD, KowalewskiT, HoltzmanDM (2004) ABCA1 is required for normal central nervous system ApoE levels and for lipidation of astrocyte-secreted apoE. J Biol Chem279, 40987–40993.
51.
Hirsch-ReinshagenV, ZhouS, BurgessBL, BernierL, McIsaacSA, ChanJY, TansleyGH, CohnJS, HaydenMR, WellingtonCL (2004) Deficiency of ABCA1 impairs apolipoprotein E metabolism in brain. J Biol Chem279, 41197–41207.
52.
WahrleSE, JiangH, ParsadanianM, KimJ, LiA, KnotenA, JainS, Hirsch-ReinshagenV, WellingtonCL, BalesKR, PaulSM, HoltzmanDM (2008) Overexpression of ABCA1 reduces amyloid deposition in the PDAPP mouse model of Alzheimer disease. J Clin Invest118, 671–682.
53.
LiuQ, ZerbinattiCV, ZhangJ, HoeHS, WangB, ColeSL, HerzJ, MugliaL, BuG (2007) Amyloid precursor protein regulates brain apolipoprotein E and cholesterol metabolism through lipoprotein receptor LRP1. Neuron56, 66–78.
54.
ZarowC, VictoroffJ (1998) Increased apolipoprotein E mRNA in the hippocampus in Alzheimer disease and in rats after entorhinal cortex lesioning. Exp Neurol149, 79–86.
55.
HuangY, MahleyRW (2014) Apolipoprotein E: Structure and function in lipid metabolism, neurobiology, and Alzheimer’s diseases. Neurobiol Dis 72 Pt A3–12.
56.
HuangY, LiuXQ, Wyss-CorayT, BrechtWJ, SananDA, MahleyRW (2001) Apolipoprotein E fragments present in Alzheimer’s disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons. Proc Natl Acad Sci U S A98, 8838–8843.
57.
JonesPB, AdamsKW, RozkalneA, Spires-JonesTL, HshiehTT, HashimotoT, von ArminCA, MielkeM, BacskaiBJ, HymanBT (2011) Apolipoprotein E: Isoform specific differences in tertiary structure and interaction with amyloid-beta in human Alzheimer brain. PLoS One6, e14586.
58.
RebeckGW, ReiterJS, StricklandDK, HymanBT (1993) Apolipoprotein E in sporadic Alzheimer’s disease: Allelic variation and receptor interactions. Neuron11, 575–580.
59.
RappA, GmeinerB, HuttingerM (2006) Implication of apoE isoforms in cholesterol metabolism by primary rat hippocampal neurons and astrocytes. Biochimie88, 473–483.
60.
ArelinK, KinoshitaA, WhelanCM, IrizarryMC, RebeckGW, StricklandDK, HymanBT (2002) LRP and senile plaques in Alzheimer’s disease: Colocalization with apolipoprotein E and with activated astrocytes. Brain Res Mol Brain Res104, 38–46.
61.
SpuchC, OrtolanoS, NavarroC (2012) LRP-1 and LRP-2 receptors function in the membrane neuron. Trafficking mechanisms and proteolytic processing in Alzheimer’s disease. Front Physiol3, 269.
62.
ZerbinattiCV, WahrleSE, KimH, CamJA, BalesK, PaulSM, HoltzmanDM, BuG (2006) Apolipoprotein E and low density lipoprotein receptor-related protein facilitate intraneuronal Abeta42 accumulation in amyloid model mice. J Biol Chem281, 36180–36186.
63.
KimJ, CastellanoJM, JiangH, BasakJM, ParsadanianM, PhamV, MasonSM, PaulSM, HoltzmanDM (2009) Overexpression of low-density lipoprotein receptor in the brain markedly inhibits amyloid deposition and increases extracellular A beta clearance. Neuron64, 632–644.
64.
BasakJM, VerghesePB, YoonH, KimJ, HoltzmanDM (2012) Low-density lipoprotein receptor represents an apolipoprotein E-independent pathway of Abeta uptake and degradation by astrocytes. J Biol Chem287, 13959–13971.
65.
LiY, MarzoloMP, van KerkhofP, StrousGJ, BuG (2000) The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein. J Biol Chem275, 17187–17194.
66.
RebeckGW, MoirRD, MuiS, StricklandDK, TanziRE, HymanBT (2001) Association of membrane-bound amyloid precursor protein APP with the apolipoprotein E receptor LRP. Brain Res Mol Brain Res87, 238–245.
67.
ZerbinattiCV, WozniakDF, CirritoJ, CamJA, OsakaH, BalesKR, ZhuoM, PaulSM, HoltzmanDM, BuG (2004) Increased soluble amyloid-beta peptide and memory deficits in amyloid model mice overexpressing the low-density lipoprotein receptor-related protein. Proc Natl Acad SciU S A101, 1075–1080.
68.
GuoLY, AlekseevO, LiY, SongY, DunaiefJL (2014) Iron increases APP translation and amyloid-beta production in the retina. Exp Eye Res129, 31–37.
69.
KimCH, YooYM (2013) Altered APP carboxyl-terminal processing under ferrous iron treatment in PC12 cells. Korean J Physiol Pharmacol17, 189–195.
70.
Avramovich-TiroshY, AmitT, Bar-AmO, WeinrebO, YoudimMB (2008) Physiological and pathological aspects of Abeta in iron homeostasis via 5’UTR in the APP mRNA and the therapeutic use of iron-chelators. BMC Neurosci9(Suppl) 2, S2.
71.
BandyopadhyayS, HuangX, ChoH, GreigNH, YoudimMB, RogersJT (2006) Metal specificity of an iron-responsive element in Alzheimer’s APP mRNA 5’untranslated region, tolerance of SH-SY5Y and H4 neural cells to desferrioxamine, clioquinol, VK-28, and a piperazine chelator. J Neural Transm Suppl237–247.
72.
RogersJT, BushAI, ChoHH, SmithDH, ThomsonAM, FriedlichAL, LahiriDK, LeedmanPJ, HuangX, CahillCM (2008) Iron and the translation of the amyloid precursor protein (APP) and ferritin mRNAs: Riboregulation against neural oxidative damage in Alzheimer’s disease. Biochem Soc Trans36, 1282–1287.
73.
HassS, FresserF, KochlS, BeyreutherK, UtermannG, BaierG (1998) Physical interaction of ApoE with amyloid precursor protein independent of the amyloid Abeta region in vitro. J Biol Chem273, 13892–13897.
74.
HuangY (2010) Abeta-independent roles of apolipoprotein E4 in the pathogenesis of Alzheimer’s disease. Trends Mol Med16, 287–294.
