Huntingtin: an iron-regulated protein essential for normal nuclear

© 2000 Oxford University Press Human Molecular Genetics, 2000, Vol. 9, No. 19 2789–2797

ARTICLE

Huntingtin: an iron-regulated protein essential fornormal nuclear and perinuclear organellesPaige Hilditch-Maguire, Flavia Trettel, Lucius A. Passani, Anna Auerbach1,Francesca Persichetti and Marcy E. MacDonald+

Molecular Neurogenetics Unit, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129,USA and 1Howard Hughes Medical Institute and Skirball Institute for Biomolecular Medicine, Department of CellBiology, New York University School of Medicine, New York, NY 10016, USA

Received 7 July 2000; Revised and Accepted 25 September 2000

Huntington’s disease (HD), with its selective neuronal cell loss, is caused by an elongated glutamine tract inthe huntingtin protein. To discover the pathways that are candidates for the protein’s normal and/or abnormalfunction, we surveyed 19 classes of organelle in Hdhex4/5/Hdhex4/5 knock-out compared with wild-type embryo-nic stem cells to identify any that might be affected by huntingtin deficiency. Although the majority did notdiffer, dramatic changes in six classes revealed that huntingtin’s function is essential for the normal nuclear(nucleoli, transcription factor-speckles) and perinuclear membrane (mitochondria, endoplasmic reticulum,Golgi and recycling endosomes) organelles and for proper regulation of the iron pathway. Moreover, upmodu-lation by deferoxamine mesylate implicates huntingtin as an iron-response protein. However, excess huntingtinproduced abnormal organelles that resemble the deficiency phenotype, suggesting the importance of hunt-ingtin level to the protein’s normal pathway. Thus, organelles that require huntingtin to function suggest rolesfor the protein in RNA biogenesis, trafficking and iron homeostasis to be explored in HD pathogenesis.

INTRODUCTION

Huntingtin is a novel protein which was discovered because ofthe elongation of an N-terminal glutamine tract of >37 residueswhich triggers the loss of striatal neurons in Huntington’sdisease (HD), a dominantly inherited disorder (1,2). Theexpansion confers on the mutant protein a novel attribute (3,4)that may initiate disease by changing an activity of huntingtinor an interacting protein, assuming that these are critical to thetargeted neurons. Alternatively, it may act independently of theprotein’s normal activity, perhaps by disrupting the function ofa cellular constituent that is not a normal interactor.

Conservation in evolution (5) suggests an essential functionfor the ∼350 kDa protein, although this is not evident from itsnovel sequence which features only multiple HEAT proteininteraction domains (6). A broad subcellular distribution,however, implies a function that may involve multiple intra-cellular sites. The bulk of the protein resides in the cytoplasm (7–12), where some is loosely associated with the membrane (7), buta fraction is also found in the nucleus (11,12). Antibodies havedistinguished alternate versions of the protein that are detected indistinct subsets of nuclear and cytoplasmic organelles (13,14),each consistent with a different subset of huntingtin’s binding

partners that have implied roles for huntingtin in RNAbiogenesis (15–18) and in vesicle trafficking (15,19–21).

Homozygous inactivation of the mouse HD gene, Hdh (22–26) has demonstrated that huntingtin’s function is required fornormal embryonic development, during gastrulation (22–25),for extra-embryonic tissue (25) and in neurogenesis (26). Incontrast, the protein appears to be dispensable for the growth,viability (22,23,25) and the neuronal differentiation (27) ofcultured ‘double knock-out’ embryonic stem (ES) cells, butintriguingly is needed for the production of hematopoieticprogenitor cells (28). Although revealing the protein’s essen-tial nature, these analyses of the consequences of huntingtindeficiency at the whole animal and cellular levels have notyielded specific candidate pathways for huntingtin function.

Consequently, we have sought clues to huntingtin’s activityby the identification of organelles that may require the protein.We have conducted a comprehensive survey of the conse-quences of huntingtin deficiency at the subcellular level,comparing Hdhex4/5/Hdhex4/5 knock-out and wild-type ES cells.We have assessed 19 classes of organelle and have found 6 thatrequire huntingtin for normal morphology and function. Theseorganelles reveal a role for huntingtin in the response tohypoxia and also implicate huntingtin function in specificcellular processes that can be investigated in HD pathogenesis.

+To whom correspondence should be addressed. Tel: +1 617 726 5089; Fax: +1 617 726 5735; Email: [email protected]

2790 Human Molecular Genetics, 2000, Vol. 9, No. 19

RESULTS

Select nuclear and perinuclear organelles are abnormal inHdhex4/5/Hdhex4/5 ES cells

To probe huntingtin function we have investigated whethercomplete deficiency for this novel protein would perturborganelles that may require its activity. Consequently, wecompared parental ES cells that express huntingtin byimmunoblot analysis (22) and targeted Hdhex4/5/Hdhex4/5 knock-out ES cells which lack the protein (22), using confocal anti-body or lectin staining with a total of 24 markers that detect 19different classes of organelle. The results of this survey aresummarized in Table 1.

For the majority of the markers, Hdhex4/5/Hdhex4/5 and wild-type ES cells exhibited similar staining patterns. The plasmaand nuclear membranes, nuclear coil bodies or splicing-speckles detected by two huntingtin partners, HYPI/symplekinand HYPA/FBP-11, the cytoskeleton, centrosome and distinctendosomes (early, sorting, late) or lysosomes all appeared rela-tively unaffected by the absence of huntingtin. In contrast, 10of the markers that probed six kinds of organelle exhibiteddramatically different staining patterns. Two of these were

nuclear: nucleoli and transcription-speckles; and four otherswere perinuclear: mitochondrial clusters, the endoplasmicreticulum (ER), Golgi complex and recycling vesicles.

Abnormal nuclear organelles involved in RNA biogenesis

The abnormal marker staining patterns reflected aberrantorganelle morphology, typically a reduced size or an alteredintracellular distribution. This is illustrated for the affectednuclear organelles in Figure 1. The nucleoli were stained onlyweakly for fibrillarin and were collapsed necklaces rather thanrobust clusters. These were located within the nuclei boundedby the lamin A-reactive nuclear envelope. However, transcrip-tion factor-speckles defined by huntingtin partners HYPB andN-CoR, in each case, were abnormally localized to the cyto-plasm, indicating that huntingtin is needed for the normalnuclear localization of these complexes.

As these organelles are involved in rRNA and mRNAbiosynthesis, we tested cellular attributes that are determinedby normal gene expression and protein synthesis. Consistentwith the normal growth properties of the knock-out cells, flow

Table 1. Summary of wild-type and Hdhex4/5/Hdhex4/5 ES cell organellesurvey results

Marker protein/lectin Organelles with staining pattern in wild-typeand Hdhex4/5/Hdhex4/5 ES cells that are:

Ref.

Similar Different

Fibrillarin Nucleoli 40

HYPA/FBP-11 Nuclear splicing factor-speckles

15,16

HYPB Nuclear transcriptionfactor-speckles

16

HYPI/symplekin Nuclear coil bodies 15,18

N-CoR Nuclear transcriptionfactor-speckles

17

NuMA Nuclear matrix 40

Lamin A, lamin B,syntaxin 1A

Nuclear membrane 40

γ-tubulin Centrosome 40

α-tubulin, dynein Microtubulecytoskeleton

40

Actin Actin cytoskleton 40

Calveolin Plasma membrane, non-clathrin vesicles

40

LDL receptor Early, sorting, lateendosomes lysosomes

40

Rab5a Early endosomes 40

β-COP, GM130,VVL, Arf1

Golgi apparatus 40

Transferrin receptor Early, sortingendosomes

Perinuclear recyclingendosomes

40

ConA Endoplasmic reticulum 40

Figure 1. Nuclear defects in Hdhex4/5/Hdhex4/5 ES cells: collapsed nucleoli andmislocalization. Wild-type (WT) and Hdhex4/5/Hdhex4/5 (dKO) ES cells stainedwith antibodies to fibrillarin (green) and nuclear envelope protein, lamin A(red), reveal compact nucleoli in parental cells but collapsed necklaces in thedKO cells (top). HYPB (BF-1) and NCoR antibodies (white) detect nuclearspeckles in WT and dKO cells, respectively, plus cytoplasmic puncta in dKOcells only (arrow), denoting mislocalization of huntingtin partners (middle andbottom). Data were collected and analyzed identically for WT and dKO EScells.

Human Molecular Genetics, 2000, Vol. 9, No. 19 2791

cytometry indicated that huntingtin deficiency did not altereither DNA content or cell size, although mini-nuclei werefound in rare Hdhex4/5/Hdhex4/5 but not wild-type ES cells (datanot shown). Thus, although the protein is essential for normalnucleoli and transcription factor-speckles, huntingtin deficiencyappears not to globally disrupt nuclear function.

Exportin 1-dependent export of nuclear versions ofhuntingtin to the cytoplasm

To characterize the nuclear versions of huntingtin that wereimplicated by the abnormal nuclear organelles, we testedwhether the export to the cytoplasm might involve exportin1 (crm1) by treating cells with the inhibitor leptomycin B(29,30). To assess the nuclear amino-terminal-accessibleversion of the protein (14), we first stained wild-type ES cellswith reagent AP229. However, the low level of signal in thesecells was not suited to the confocal format. Consequently, weexamined STHdh+/Hdh+ mouse striatal cells, which exhibitreadily detectable AP229-reactive N-terminal-accessibleprotein in splicing-speckles (14). The results indicated thatleptomycin B prevented the cytoplasmic AP229-reactivespeckles that were evident in the untreated cells, indicatingexportin 1-dependent nuclear export (Fig. 2). This impliesnuclear export signals (NES) in huntingtin that are involved inthe nuclear–cytoplasmic localization of the 350 kDa protein.

Abnormal perinuclear mitochondrial clusters

In the cytoplasm our survey detected abnormally distributedmitochondria in the absence of huntingtin. The results ofstaining for Grp75, a mitochondrial matrix protein, are shown

in Figure 3. Perinuclear clusters, that are associated withreplication and coordinate transcription of mitochondrial andnuclear genes involved in energy biogenesis (31), were evidentin wild-type cells. In contrast, Hdhex4/5/Hdhex4/5 ES cells did notexhibit clusters but instead displayed linear mitochondrialarrays. These arrays were abundant in all cells, however,suggesting normal segregation of the mitochondria after celldivision. Consistent with this possibility, staining for α-tubulindemonstrated that the cytoskeleton and microtubule organizingcenter, which are involved in both segregation and perinuclearcluster formation, were not noticeably altered by huntingtindeficiency (Fig. 3). This finding implies that the normalassembly of mitochondria around the nucleus has somespecific requirement for huntingtin.

Abnormal ER and Golgi in the absence of huntingtin

The size of each perinuclear component of the secretoryapparatus was reduced by huntingtin deficiency. Figure 4aillustrates the hearty perinuclear Golgi clusters detected byGM130 and by vicia villosa lectin (VVL) in the wild-typecells. In contrast, the knock-out cells exhibited weak, disperseGolgi membrane (cis and trans) that were, however, locatednear the lamin B-stained nuclear membrane. Perinuclearsignals for the Golgi membrane fusion proteins, β-COPcoatmer protein and Arf1 ADP-ribosylation factor, were alsoreduced, suggesting impaired trafficking (data not shown).Consistent with this possibility, the Concanavalin A (ConA)-‘stained’ rough ER (Fig. 4b) appeared to be diminished and didnot properly extend toward the edges of the cell. To directlytest ER–Golgi membrane trafficking, we co-stained cells that

Figure 2. Leptomycin B blocks nuclear export of AP229-positive huntingtin.Confocal images of STHdh+/Hdh+ cells stained with AP229 (white) before(top) or following (bottom) leptomycin B (LMB) treatment. Treatment resultsin abrogation of cytoplasmic staining (arrow) which can be seen at low laserpower but is more evident using high laser power.

Figure 3. Abnormal distribution of mitochondria in Hdhex4/5/Hdhex4/5 ES cells.Confocal images of wild-type (WT) and Hdhex4/5/Hdhex4/5 (dKO) ES cellsstained for mitochondrial proteins, Grp75 (white) and α-tubulin (white). Peri-nuclear clustering of mitochondria in dKO cells is absent (top) despite compa-rable microtubule distribution in WT and dKO ES cells (bottom). Data werecollected and analyzed identically for WT and dKO ES cells.



had been treated with Brefeldin A, which is an inhibitor of Arfactivation (32). The results (Fig. 4b) revealed the expectedintermixing of Golgi and ER vesicles in wild-type cells. Incontrast, Hdhex4/5/Hdhex4/5 cells exhibited engorged ConA-filledER balloons, ringed by GM130-reactive dots, that indicatedabnormal ER–Golgi membrane fusion.

A re-orientation assay (33) demonstrated impaired perinu-clear translocation of the Golgi apparatus in the absence ofhuntingtin. The VVL signals in the wild-type ES cellsbordering a scrape in the monolayer are aligned, reflecting arepositioning of the Golgi to a perinuclear location that isnearest the extending edge of the cell (Fig. 4c). In contrast, theknock-out ES cells were unable to rapidly shift their weakGolgi, indicating that perinuclear membrane trafficking wasimpaired.

Abnormal perinuclear recycling endosomes

Perinuclear recycling endosomes were detected by the trans-ferrin receptor, and were also reduced in the cells that lackhuntingtin (Fig. 5a), although an over-abundance of signal wasfound throughout the cytoplasm. Brefeldin A treatment toinhibit membrane fusion revealed diminished perinuclearmembrane in the knock-out cells compared with the wild-typecells.

To determine whether this deficit was restricted to theperinuclear recycling endosomes, we tested the uptake andtransport of extracellular FITC-tagged transferrin by ES cellsthat had been stimulated by the iron chelator, deferoxaminemesylate (34). The results (Fig. 5b) confirmed trafficking ofFITC–transferrin to both the early and sorting endosomes inthe knock-out ES cells, although a weak perinuclear ligandsignal indicated impaired transport to perinuclear recyclingendosomes compared with the wild-type cells. Furthermore,consistent with the normal low density lipoprotein (LDL)receptor staining found in the marker survey, the receptor-mediated uptake and transport of extracellular Dil-tagged LDLto the lysosomes via the early–late and sorting endosomes (34)was indistinguishable in knock-out and wild-type ES cells. Ofthe endosomal compartments, the absence of huntingtin affectsprimarily the perinuclear recycling endosomes. Thus, hunt-ingtin function is implicated in a perinuclear process that isessential both for the normal trafficking of secretorymembrane and for the assembly of mitochondria near thenucleus.

Huntingtin and the cellular iron pathway

The abnormalities in perinuclear transferrin receptor traf-ficking and mitochondrial cluster-tethering also suggested thatiron metabolism might be abnormal in the absence of hunt-ingtin. Therefore, we tested the levels of transferrin receptor innaïve and deferoxamine treated ES cells by immunoblot

analyses. Typical results are shown in Figure 6a; theserevealed an expected ∼3.9-fold increase (n = 3) in transferrinreceptor levels in the ‘treated’ compared with the ‘untreated’wild-type lysate. In contrast, the naïve knock-out cell extractexhibited a strong band that was ∼4.2 fold (n = 3) increasedcompared with the naïve wild-type lysate. Furthermore, thisabnormally increased level was only marginally elevated(∼1.3-fold; n = 3) by deferoxamine mesylate, implicating hunt-ingtin in the normal regulation of the iron pathway.

Consequently, we assessed the iron modulation of huntingtinitself by probing the immunoblots in Figure 6a with huntingtinreagent monoclonal antibody (mAb) 2166 (Fig. 6). The ∼350kDa protein was not detected in the Hdhex4/5/Hdhex4/5 ES cellextract as expected (22). However, the huntingtin banddetected in the naïve wild-type proteins was increased withdeferoxamine mesylate treatment by ∼4.5-fold (n = 3), indi-cating that huntingtin was upregulated by stimulation of theiron pathway.

We then searched the 5′ and 3′ non-coding regions of themouse, rat and human HD genes for canonical CAGUGXmotifs (35) but we failed to find any that were likely to formthe ‘hair-pin’ iron-responsive element (IRE) implicated in themRNA stabilization of iron response proteins (35). However,searches of the MatInspector matrices (http://www.gsf.de/cgi-bin/matsearch ) with the promoter region sequences (36) iden-tified a core binding site for the HIF-1 hypoxia-inducible trans-cription factor (AHRARNT). As shown in Figure 6b, thissequence is conserved in the mouse, the rat and the human HDhomologs, suggesting a hypoxia response element (HRE). Thiselement is also found in HIF-1 target genes such as thoseencoding the glucose transporter and transferrin receptor (37),suggesting that this hypoxia transcription factor may also coor-dinately regulate huntingtin levels.

Overexpressed protein produces a phenotype thatresembles Hdh deficiency

To explore whether the level of huntingtin is important for itscellular pathway in ES cells and in striatal cells that aretargeted in HD, we assessed the impact of excess protein onorganelles that were found to require its function. The wild-type and the double knock-out ES cells and the STHdh+/Hdh+

striatal cells were transiently transfected with HD1-3144Q23,which drives expression of full-length normal huntingtin (37).The results of co-staining of huntingtin with HF1 and eitherfibrillarin or GM130 reagents to detect nucleoli and Golgimembrane, respectively, are shown in Figure 7. These imagesdemonstrated that both the wild-type ES cells and the striatalcells that overexpressed huntingtin exhibited collapsednucleoli and abnormal fragmented Golgi compared with theiruntransfected neighbors. In addition, the overexpressed hunt-ingtin also worsened the abnormal organelles that character-

Figure 4. Abnormal Hdhex4/5/Hdhex4/5 ER–Golgi complex reveals aberrant membranes. (a) Fragmented Hdhex4/5/Hdhex4/5 (dKO) membranes are revealed in confocalimages of perinuclear Golgi complexes in wild-type (WT) and dKO cells stained with GM130 (green) and lamin B for nuclear envelope (red) (top), and VVL (red)(bottom). (b) Abnormal ConA-stained ER (green) in dKO cells fails to extend to the periphery, as in WT cells. Brefeldin A (BFA) treatment in dKO cells inducesaberrant ConA balls (red) ringed with green non-colocalizing GM130-positive membranes (merge). ConA-reactive and GM130-positive membranes in BFA-treated WT cells partially overlap (merge). (c) Wound-healing, VVL-reactive Golgi membranes (red) re-polarize toward the leading edge in WT cells but remaindisorganized in dKO cells. Data were collected and analyzed identically for WT and dKO ES cells.


ized the knock-out ES cells. In these experiments staining forperinuclear transferrin receptor also demonstrated a reductionin the recycling endosome compartment in all the cell typesoverexpressing huntingtin (data not shown). Thus, over-expressed huntingtin produced a set of nuclear and perinuclearabnormalities that mirrored huntingtin deficiency, stronglysuggesting a dominant-negative impact on huntingtin’spathway that may reflect the overwhelming of a criticallimiting component.

DISCUSSION

Huntingtin’s novel sequence does not predict the protein’sphysiological role or reveal the mechanism by which theexpanded polyglutamine segment in the mutant proteintriggers the selective degeneration of striatal neurons. Touncover these processes we have conducted a survey to deter-mine which cellular organelles are chiefly affected by the hunt-ingtin deficiency. Our findings indicate that huntingtin is aniron-regulated protein that is essential for normal nuclear andperinuclear organelles that implicate the protein in ironhomeostasis, RNA biogenesis and trafficking, providing avariety of candidates for the protein’s normal and/or abnormalpathway.

Although abnormal columnar epithelial cells in huntingtin-deficient embryos (25) and impaired erythroid progenitorsfrom knock-out ES cells (28) have previously implied aconnection, our findings demonstrate an essential role for hunt-

Figure 5. Transferrin receptor recycling is compromised in Hdhex4/5/Hdhex4/5

cells. (a) Perinuclear recycling compartment in untreated cells (–BFA),revealed by antibody stain of endogenous transferrin receptor (Tfn R) (green),is robust in wild-type (WT) and diminished in Hdhex4/5/Hdhex4/5 (dKO) cells.Brefeldin A (+BFA) swollen recycling compartment is reduced in dKO cellscompared with WT cells. (b) Functional tracking of early, late and recyclingendosomes in ES cells of Tfn R via FITC-tagged ligand (green) reveals peri-nuclear foci and cytoplasmic dots in WT cells but only sparse puncta in theperiphery of dKO cells. Trafficking of lysosomal-fated Dil-LDL (red) in WTand dKO cells is similar, with numerous cytoplasmic puncta. Data were col-lected and analyzed identically for WT and dKO ES cells.

Figure 6. Huntingtin modulates Tfn R and is itself upregulated by iron deple-tion. (a) Immunoblot analysis of extracts of wild-type (WT) and Hdhex4/5/Hdhex4/5 (dKO) ES cells untreated (–) or treated (+) with deferoxaminemesylate (DM). The blot was probed for transferrin receptor (Tfn R), revealingupregulated levels in DM-treated wild-type extract. In naïve dKO extract basallevels were abnormally high and only modestly increased by DM. Staining thesame blot for huntingtin (Httn) with mAb 2166 reveals the ∼350 kDa band inproteins from untreated WT, but not dKO, cells. The Httn band is dramaticallyaugmented in extracts from DM-treated WT cells. Equal loading of proteins isshown by detection of fodrin (Spectrin). (b) Location of a conserved HIF-1transcription factor binding site (HBS) in the HD promotor region. Shown isthe core HBS (underlined) and preferred flanking DNA sequence identified byMatInspector version 2.2 in the promoter region (36) upstream of the ATG startsite (+1) in the human (HD) (GenBank accession no. L12392), mouse (Hdh)(GenBank accession no. L34008) and rat (rhd) (GenBank accession no.AJ224197) HD genes. Functional HBS sites in the mouse glucose transporter-1 gene (GLUT-1) and human and mouse transferrin receptor (TfR) genes fromLok and Ponka (37) are given below.


ingtin function in iron homeostasis. Huntingtin was requiredfor normal regulation of a key iron protein, transferrinreceptor, and in response to iron need was modulated with it.This may involve an HIF-1 binding site that suggests coordi-nate regulation of huntingtin with diverse hypoxia responseproteins at the transcriptional level. Interestingly, normal peri-nuclear mitochondrial clustering also required huntingtin func-tion, implying a role for perinuclear versions of the protein inproperly localizing mitochondria that are importing nuclearproducts for the linked energy–iron pathways.

Our survey has also revealed a role for huntingtin function innormal membrane trafficking of perinuclear portions of thesecretory apparatus (ER, Golgi, recycling endosomes), thatmay be the same activity that is involved in mitochondrialclustering. This role is consistent with results of antibodylocalization (7,14) and with a subset of huntingtin-interactingproteins that participate in membrane function (15,19–21).Intriguingly, a version of huntingtin with ‘internal-accessible’epitopes that colocalizes with perinuclear membranes alsoresides in the nucleolus (14) and may be important to normalnucleolar morphology that was uncovered in our survey. More-over, these locations imply that this form of huntingtin may beinvolved in a process that is essential to nucleoli and to thearrangement of membrane near the nucleus.

The necessity for the function of huntingtin in the normalnuclear localization of its transcription factor partners (HYPBand N-coR), however, may involve an alternate version of hunt-ingtin with amino-terminal-accessible epitopes (11,12,14). Thisform of the protein colocalizes with nuclear-speckles and thenuclear matrix, consistent with huntingtin’s pre-mRNAsplicing and polyadenylation complex factors (15–17) thathave supported a role for the protein in RNA biogenesis. Ourdata indicate that huntingtin is exported from the nucleus to the

cytoplasm via an exportin 1-dependent pathway. This mayentail conformational properties of huntingtin (14) that may beinvolved in masking/unmasking of NES motifs, determiningthe proper distribution of huntingtin in the nucleus and thecytoplasm.

Essential nuclear and perinuclear organelles that requirehuntingtin function were also disrupted by excess protein. Thisfinding suggests that some critical constituent of huntingtin’spathway is limiting, implying the importance of regulatedhuntingtin levels. Exploration of the protein’s normal andabnormal functions, therefore, may require accurately expressedprotein. Indeed, the organelles that require huntingtin functionimplicate specific pathways involved in essential cellularprocesses, including rRNA and mRNA biogenesis, perinuclearmembrane trafficking and iron metabolism, to be investigatedin HD patient tissue and in model systems.

MATERIALS AND METHODS

Cell culture and cell assays

R1 wild-type and Hdhex4/5/Hdhex4/5 ES cells have been describedpreviously (22) and were maintained on gelatinized dishes in ESculture medium supplemented with 106 U/l leukemia inhibitoryfactor (LIF) (ESGRO; Life Technologies, Gaithersburg, MD).Twenty-four hours before an uptake experiment the medium wasreplaced with fresh ES culture medium containing 4 µM deferox-amine mesylate (Sigma, St Louis, MO). Uptake of fluorescenttransferrin and LDL was performed at steady state levels over30 min as described previously (37). Brefeldin A (Calbiochem,La Jolla, CA) treatment (5 µM) was for 90 min. STHdh+/Hdh+

striatal progenitor cells and their growth at 33°C have beendescribed (14). Leptomycin B treatment was for 2 h (100 ng/ml)

Figure 7. Overexpression of huntingtin results in dominant negative phenotypes. Merged confocal images of (a) wild-type (WT) and Hdhex4/5/Hdhex4/5 (dKO) EScells and (b) STHdh+/Hdh+cells, transfected with HD1-3144Q23. Typical cells overexpressing HF1-reactive full-length huntingtin with 23 glutamines (red),costained for fibrillarin (green) and GM130 (green) to detect nucleoli and Golgi membranes, respectively. Both WT and STHdh+/Hdh+ transfectants which over-express huntingtin (red) exhibit collapsed fibrillarin-positive nucleoli and fragmented Golgi rather than robust organelles (green) in surrounding untransfectedcells. Overexpression in dKO cells further worsens the aberrant organelle phenotypes. Data were collected and analyzed identically for all WT and dKO ES cellsand striatal cells.


at 33°C. Wounding–healing entailed a toothpick scrape througha 90% confluent monolayer (33), followed by incubation at 37°Cfor 1 h.

Antibodies and fluorescent labels

Ligands used in this study were as follows: fluoroscein-labeledtransferrin, Dil-labeled LDL, Texas Red-conjugated ConA andbiotinylated VVL (Molecular Probes, Eugene, OR). Antibodyreagents used in this study were as follows: GM130, caveolin,NuMA, symplekin (Transduction Laboratories, San Diego,CA); lamin A, lamin B, N-CoR, Rabs 1A, 5A and 6 (SantaCruz Biotechnology, Santa Cruz, CA); AF-1 for HYPA andBF-1 for HYPB (16); fibrillarin, actin, α- and γ-tubulin, dyneinand β-COP (Sigma); anti-syntaxin 1A (StressGen Biotechnol-ogies, Victoria, Canada); huntingtin mAb 2166 (Chemicon,Temecula, CA), HF1 (37) and spectrin (Chemicon); rat mAbtransferrin receptor (Tfn R; Biosource International, Camarillo,CA). Secondary antibodies conjugated to horseradish peroxidaseor fluorescent labels were from Amersham Lifesciences(Piscataway, NJ) and Jackson ImmunoResearch (West Grove,PA), respectively.

Immunofluorescence, fluorescence-activated cell sorting(FACS) and confocal microscopy

ES cells were fixed in 4% paraformaldehyde, permeabilizedfor 5 min in 0.1% Triton X-100 in phosphate-buffered saline(PBS), treated for 10 min with blocking solution (1% bovineserum albumin in PBS) and incubated for 90 min in blockingsolution containing primary antibodies/lectins. After severalwashes in PBS, cells were incubated for a further 1 h inblocking solution containing secondary antibodies and thenrinsed in PBS. Cells were examined with a BioRad (Hercules,CA) MRC-1024 laser confocal microscope using 20× and 40×objective lenses. Digitized images for each field were saved asseparate files for each channel and were merged using AdobePhotoShop.

Transfection

Full-length huntingtin constructs HD1-3144Q23 and HD1-3144Q113 (40) were introduced into ES cells seeded at densitiesof ∼5 × 103 cells per 24-well plate using the SuperFect Transfec-tion kit (Qiagen, Valencia, CA) and immunofluorescence wasexamined 72 h after transfection.

Immunoblot analysis

Soluble proteins were extracted from PBS-washed ES cells byneedle sheering in buffer containing 50 mM Tris–Cl pH 7.5,10% glycerol, 5 mM magnesium acetate, 0.2 mM EDTA andComplete Protease Inhibitors (Roche Diagnostics,Indianapolis, IN), followed by three freeze–thaw cycles andcentrifugation for 2 min at 17 110 g. Supernatants (25 µg) wereboiled for 5 min in Laemmli loading buffer, separated on a 6%SDS–polyacrylamide gel and transferred onto nitrocellulosemembranes. Proteins were detected by chemiluminescence(KPL Laboratories, Gaithersburg, MD) following incubationwith primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Quantitation was bydensitometry of transferrin receptor or huntingtin band (on

non-saturating exposures) and normalization to the spectrin(fodrin) band in the same lanes.

ACKNOWLEDGEMENTS

We thank Drs J.F. Gusella and T. Greenamyre for criticaldiscussion, Drs A. Bernards and B. Terns for the gifts of E-cadherin and fibrillarin antibodies and Dr M. Yoshida forleptomycin B. L.A.P. is supported by a fellowship from theHereditary Disease Foundation. The research was supportedby NIH grants NS32765 and NS16367 (Huntington’s DiseaseCenter Without Walls), a grant from the Foundation for theCare and Cure of Huntington’s Disease and Telethon, Italy.

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Huntingtin: an iron-regulated protein essential for normal nuclear