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. 2007 Mar 28;2(3):e334.
doi: 10.1371/journal.pone.0000334.

Mechanisms of copper ion mediated Huntington's disease progression

Jonathan H Fox  1 , Jibrin A Kama, Gregory Lieberman, Raman Chopra, Kate Dorsey, Vanita Chopra, Irene Volitakis, Robert A Cherny, Ashley I Bush, Steven Hersch
Affiliations

Mechanisms of copper ion mediated Huntington's disease progression

Jonathan H Fox et al. PLoS One. .

Abstract

Huntington's disease (HD) is caused by a dominant polyglutamine expansion within the N-terminus of huntingtin protein and results in oxidative stress, energetic insufficiency and striatal degeneration. Copper and iron are increased in the striata of HD patients, but the role of these metals in HD pathogenesis is unknown. We found, using inductively-coupled-plasma mass spectroscopy, that elevations of copper and iron found in human HD brain are reiterated in the brains of affected HD transgenic mice. Increased brain copper correlated with decreased levels of the copper export protein, amyloid precursor protein. We hypothesized that increased amounts of copper bound to low affinity sites could contribute to pro-oxidant activities and neurodegeneration. We focused on two proteins: huntingtin, because of its centrality to HD, and lactate dehydrogenase (LDH), because of its documented sensitivity to copper, necessity for normoxic brain energy metabolism and evidence for altered lactate metabolism in HD brain. The first 171 amino acids of wild-type huntingtin, and its glutamine expanded mutant form, interacted with copper, but not iron. N171 reduced Cu(2+)in vitro in a 1:1 copper:protein stoichiometry indicating that this fragment is very redox active. Further, copper promoted and metal chelation inhibited aggregation of cell-free huntingtin. We found decreased LDH activity, but not protein, and increased lactate levels in HD transgenic mouse brain. The LDH inhibitor oxamate resulted in neurodegeneration when delivered intra-striatially to healthy mice, indicating that LDH inhibition is relevant to neurodegeneration in HD. Our findings support a role of pro-oxidant copper-protein interactions in HD progression and offer a novel _target for pharmacotherapeutics.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1
Full-length and N-terminal fragments of human huntingtin interact with copper (II) as measured by immobilized-metal affinity chromatography (IMAC). A. Full-length (FL) normal and mutant huntingtin from human motor cortex interact with copper and elute with peak centered at 128 mM imidazole. FL huntingtin has higher affinity for copper (II) than the copper-binding domain of human soluble APLP2. FL protein migrates at ∼350 kDa. B. N-171 fragments of human huntingtin with 17 or 68 glutamines interact with copper (II) and have identical elution profiles. Proteins with C-terminal FLAG tag elute as a single peak at 32 mM imidazole. Non-FLAG protein elutes primarily at 32 mM imidazole, but also at higher concentrations. NSB = non-specific band. C. N-171-flag fragments of wild-type and mutant huntingtin do not interact with iron (III). For iron-IMAC, iron (III) was loaded onto column at pH 3, adjusted to pH 7, then the IMAC procedure performed immediately. D. N-171-17Q fragment (wild-type) was expressed using the pGEX vector and purified (see methods). Coomassie gel analysis reveals high purity. E. Purified N-171-17Q-flag fragment of huntingtin interacts directly with copper (II) and elutes identically to the in-vitro transcription-translation expressed protein (Figure 1B).
Figure 2
Figure 2
Interaction of N-171 huntingtin with copper (II) by IMAC involves histidine 82 and 98. A. N171-17Q huntingtin contains several potential copper (II) coordinating residues, two histidines (red) and four cysteines (blue). The vertical line represents the exon-1-2 boundary. Flag tag sequence (underlined) partially overlaps with huntingtin sequence (upper case). B. Purified N-171-17Q huntingtin fragment elutes from copper column at pH 4.5–3.5 consistent with interaction with histidine residue(s). Elution solutions were prepared in 20 mM citrate-buffer. C. Modification of histidine 82 and/or 98 to phenylalanine results in elution of protein in washes indicating that both histidines are necessary for interaction with copper (II). D. Huntingtin exon-1-17Q fragment is sufficient for copper (II) interaction.
Figure 3
Figure 3
Copper (II) is reduced by N171-17Q huntingtin and promotes aggregation of cell-free full-length mouse huntingtin. A. N-terminal huntingtin (N-171-17Q) reduces copper (II) as measured by the bathocuproine assay. B. Iron (III) is reduced by N171-17Q to a lesser extent than copper as measured by bathophenanthroline assay. Met = methionine, Phe = phenylalanine, His = histidine, Ins = insulin, VitC = ascorbate. Purified N171-17Q proteins were incubated for 1 hour at 37°C in the presence of 20 µM of copper (II) or iron (III) and 360 µM bathocuproine or bathophenanthroline for copper (II) and iron (III) reduction assays, respectively. n = 4. C. Full-length wild-type mouse huntingtin aggregates following incubation at 37°C. Aggregation is promoted by 10 µM copper (II) and inhibited by EDTA and the brain permeable chelator, clioquinol. Aggregation is not inhibited by co-incubation with catalase.
Figure 4
Figure 4
Brain copper and iron levels in pre-clinical and impaired Huntington's disease (HD) mice. A–B. Twelve-week-old R6/2 and 12-month-old CAG140 mice represent late stage and pre-clinical HD, respectively. A. Open-field activity of R6/2 transgenic and CAG140 knock-in mice. R6/2 mice at 12-weeks have significantly decreased activity. CAG140 mice at 8–18 months are the same as wild-type littermates consistent with pre-clinical disease. B. Brain weights of 12-week-old R6/2 mice are decreased 12% consistent with severe brain atrophy and late-stage HD. Twelve-month-old CAG140 mouse brain weights are normal (p = 0.2367). C–D. Copper and iron levels in brains of 12-month-old CAG140 HD knock-in mice (pre-clinical HD) as measured by inductively-coupled-plasma (ICP) mass spectroscopy. C. Copper levels are unaltered. D. Iron levels are increased 15% in cortex. E–F. Copper and iron levels in brains of 12-week-old R6/2 HD mice (late-stage HD) as measured by ICP spectroscopy. E. Copper levels are significantly increased in striatum and cortex. F. Striatal and cortical iron levels are increased, but not significantly (p-values are 0.2897 and 0.0782, respectively). n = 10–14. Values are shown as mean±SEM. Bars: white = CAG140; cross-hatched = R6/2; gray = wild-type litter mates of R6/2; black = wild-type litter mates of CAG140. p-values: *<0.05, **<0.01, *** = p<0.0001
Figure 5
Figure 5
Mutant huntingtin, copper and iron distribution in biochemical fractions of 12-week-old HD mouse cerebral cortex. A. Copper is increased in soluble and membrane fractions. B. Iron is increased in soluble, membrane and pellet fractions. Metals were measured by ICP-MS. n = 10–11. p-values after Dunn-Šidák correction for multiple testing *<0.05, **<0.01, ***<0.001 C. Distribution of mutant huntingtin in pellet (P), soluble (S) and membrane (M) fractions as determined by Western blotting using a polyglutamine specific antibody (IC2-Chemicon). Pellets were treated with 90% formic acid for 1 hour at 37°C, lyophilized then resuspended in SDS-PAGE buffer. Mutant huntingtin is identified by the presence of a band in transgenic mice (Tg) but not wild-type (Wt) mice (red arrow for soluble fraction). WetWtEq = wet weight equivalents.
Figure 6
Figure 6
Decreased L-lactate dehydrogenase (LDH) activity in R6/2 HD mice is relevant to neurodegeneration. For A and C, black = wild-type, cross-hatched = transgenic. A. Activity of the copper-sensitive enzyme LDH is decreased in forebrains of R6/2 HD mice from 8-weeks. Time points were determined on consecutive generations of mice, thus only within time point comparisons are valid. n = 10. B. Total actin-normalized LDH monomer levels are unaltered (n = 4) but all five LDH isoenzyme activities are decreased at 12-weeks (n = 4, p = 0.0421). C. Lactate levels are increased in striatum and cortex of 12-week-old R6/2 HD mice. D. LDH is exceptionally sensitive to copper, but not iron or manganese, mediated inactivation. Five µg/ml purified LDH (Roche) was incubated with copper (II), iron (III), manganese (II) chloride in chelex-treated PBS for 1 hour at 37°C before LDH analysis. n = 8 Symbols: black circles = copper, white circles = iron, triangles = manganese. P-values: *** = p<0.0001, ** = p<0.001, E. The LDH inhibitor oxamate results in acute degeneration in wild-type mice when delivered intra-striatially, as compared to control treated mice.
Figure 7
Figure 7
Amyloid precursor protein (APP), a copper exporting protein, is decreased at the protein but not transcript level in the brains of 12-week-old HD transgenic mice. A–D. Soluble and membrane forms of APP are significantly decreased in striatum and cortex of R6/2 HD transgenic mice. APP-like protein 2 (APLP2) is decreased in striatal soluble fraction only. APLP1 levels are unaffected. β-actin is loading control for soluble fraction. C–D. Expression levels for transgenic mice are shown relative to respective wild-types (normalized to 100%). Black bars = membrane fraction, cross-hatched bars = soluble fraction. n = 11, a = p<0.0001 E–G. Transcripts coding for APP, APLP2 and APLP1 are unaltered in striatum and cortex. n = 10 Gray bars = wild-type, white = R6/2 transgenic. H. Analysis of copper-transporting ATPase expression in HD mice. Menke's-disease protein (ATP7A) expression is unaltered. Wilson's disease protein (ATP7B) is increased in cortex only (p = 0.0174). n = 7–10, WT = wild-type, TG = transgenic.
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