Diminished hippocalcin expression in Huntington’s disease brain does not account for increased...

,

*Brain Mind Institute, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne, Switzerland

�MassGeneral Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Charlestown, Massachusetts, USA

�Atomic Energy Commission (CEA), Molecular Imaging Research Center (MIRCen), Fontenay-aux-Roses, France

§CNRS/CEA URA2210, Fontenay-aux-Roses, France

Huntington’s disease is a hereditary neurodegenerativedisorder characterized by both psychiatric and motormanifestations (Young 2003; Luthi-Carter 2007). Neuropa-thologically, the most prominent Huntington’s disease (HD)-related degeneration involves the medium spiny GABAergicoutput neurons of the striatum. HD results from a cytosine-adenine-guanine (CAG) trinucleotide repeat expansion muta-tion encoding an extended polyglutamine stretch within thehuntingtin protein. Mutant huntingtin interacts with amultitude of other cellular proteins (Faber et al. 1998;Goehler et al. 2004; Kaltenbach et al. 2007), and thus itsfunctional sequellae are multipartite. Mutant huntingtin’sassociation with mitochondria and the plasma membrane hasbeen reported to result in metabolic and pro-excitotoxiceffects in HD (Zeron et al. 2002; Benchoua et al. 2006;Fernandes et al. 2007; Bossy-Wetzel et al. 2008). Nucleareffects have been attributed to the accumulation of N-terminal fragments of mutant huntingtin and abnormal

interactions of mutant huntingtin with transcriptionally activeproteins (Luthi-Carter and Cha 2003; Landles and Bates2004). Effects on gene expression include a progressive

Received May 23, 2009; revised manuscript received August 3, 2009;accepted August 4, 2009.Address correspondence and reprint requests to Ruth Luthi-Carter,

Brain Mind Institute, Ecole Polytechnique Federale de Lausanne(EPFL), Station 15, CH1015 Lausanne, Switzerland.E-mail: [email protected] used: 3-NP, 3-nitropropionic acid; BIR, baculovirus

inhibitors of apoptosis repeat; DIV, day in vitro; ECFP, enhanced cyanfluorescent protein; EYFP, enhanced yellow fluorescent protein; HD,Huntington’s disease; HEK, human embryonic kidney; NAIP, neuronalapoptosis inhibitory protein; NeuN, Neuronal Nuclei; NCS, neuronalcalcium sensor; NGS, normal goat serum; NVP-3, neuronal visinin-likeprotein-3; PBS, phosphate-buffered saline; PGK, phosphoglyceratekinase 1; SIN, self-inactivating lentiviral plasmid vector; SIN-W, SINwith a woodchuck regulatory element; TGN, trans-Golgi network; TRE,tetracycline responsive element; WB, western blot.

Abstract

Hippocalcin is a neuronal calcium sensor protein previously

implicated in regulating neuronal viability and plasticity.

Hippocalcin is the most highly expressed neuronal calcium

sensor in the medium spiny striatal output neurons that

degenerate selectively in Huntington’s disease (HD). We have

previously shown that decreased hippocalcin expression oc-

curs in parallel with the onset of disease phenotype in mouse

models of HD. Here we show by in situ hybridization histo-

chemistry that hippocalcin RNA is also diminished by 63% in

human HD brain. These findings lead us to hypothesize that

diminished hippocalcin expression might contribute to striatal

neurodegeneration in HD. We tested this hypothesis by

assessing whether restoration of hippocalcin expression

would decrease striatal neurodegeneration in cellular models

of HD comprising primary striatal neurons exposed to mutant

huntingtin, the mitochondrial toxin 3-nitropropionic acid or an

excitotoxic concentration of glutamate. Counter to our

hypothesis, hippocalcin expression did not improve the sur-

vival of striatal neurons under these conditions. Likewise,

expression of hippocalcin together with interactor proteins

including the neuronal apoptosis inhibitory protein did not in-

crease the survival of striatal cells in cellular models of HD.

These results indicate that diminished hippocalcin expression

does not contribute to HD-related neurodegeneration.

Keywords: 3-nitropropionic acid, calcium, excitotoxicity,

hippocalcin, huntingtin, Huntington’s disease.

J. Neurochem. (2009) 111, 460–472.

JOURNAL OF NEUROCHEMISTRY | 2009 | 111 | 460–472 doi: 10.1111/j.1471-4159.2009.06344.x

460 Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 460–472� 2009 The Authors

decrease in the expression of neurotransmitter signaling- andcalcium-related genes, which have been observed in multipleHD model systems (Luthi-Carter et al. 2000; Desplats et al.2006; Kuhn et al. 2007; Runne et al. 2008).

A prominent subclass of calcium-related genes whoseexpression is diminished in mouse models of HD are theneuronal calcium sensors (NCSs). NCSs comprise a familyof EF hand-containing calcium binding proteins (Zozulyaand Stryer 1992) that have been implicated in many centralnervous system functions including phototransduction, excit-atory neurotransmission, neuronal plasticity, and neuronalsurvival (reviewed in Burgoyne et al. 2004; Burgoyne 2007).NCSs possess no inherent enzymatic properties, but exerttheir calcium-dependent functions through interactions withother proteins. Thus, the identification of calcium-dependentNCS interactions is important for the elucidation of specificNCS activities.

The distinct distributions of different NCSs in themammalian CNS suggests that their functions are region-and cell-type specific. The NCS protein hippocalcin isenriched in hippocampal pyramidal neurons and in mediumspiny neurons of the striatum (Mercer et al. 2000; Paterliniet al. 2000). The molecular and cellular functions ofhippocalcin remain incompletely understood. Mice lackinghippocalcin have no obvious brain structural abnormalitiesbut show mild deficits in spatial and associative memory(Kobayashi et al. 2005). Suggested roles for hippocalcininclude the regulation of slow afterhyperpolarization(Tzingounis et al. 2007), gene transcription (Kobayashiet al. 2005) and neurite outgrowth (Oh et al. 2008).

Another function previously ascribed to hippocalcin is itsneuroprotective activity. An interaction between hippocalcinand the baculovirus inhibitors of apoptosis repeat 3 (BIR3)domain of the neuronal anti-apoptosis protein (NAIP) wasshown to synergistically inhibit caspase 3-activated, caspase12-induced and age-dependent neurodegeneration (Merceret al. 2000; Lindholm et al. 2002; Korhonen et al. 2005). Inaddition, hippocampal neurons from hippocalcin-deficientmice have been shown to be more vulnerable to degenerationinduced by thapsigargin, a SERCA pump inhibitor, as well asto excitotoxicity caused by the glutamate receptor agonistskainic acid and quinolinic acid (Korhonen et al. 2005;Masuo et al. 2007).

Our group has previously shown that hippocalcin is one ofthe most strongly down-regulated genes in the striata of HDmice (Luthi-Carter et al. 2000) and that this change inexpression coincides temporally with behavioral signs ofdisease in these animals (Luthi-Carter et al. 2002). In thepresent study, we further demonstrate that hippocalcinexpression is also significantly diminished in human HDbrain. These findings, together with previous reports ofhippocalcin’s anti-apoptotic effects in other neuronal sub-types, led us to hypothesize that hippocalcin might protectmedium spiny neurons from HD-related degeneration.

Therefore, we tested whether heterologous expression ofhippocalcin could mitigate striatal neurotoxicities associatedwith HD. These studies assessed hippocalcin’s effects onthree major mechanisms of HD-related neurodegneration,namely, the effects of mutant huntingtin fragment accumu-lation, mitochondrial metabolism abnormalities and gluta-mate-receptor-mediated excitotoxicity.

Materials and methods

Human brain tissueHuman brain tissues were obtained from the Harvard Brain Tissue

Resource Center (Belmont, MA, USA) and Dr. Anne B. Young

(Massachusetts General Hospital, Charlestown, MA, USA). HD

brain samples included tissue from symptomatic HD gene-positive

patients (n = 12, nine male, three female; mean age 57.1; pathologic

grades: 1 HD0, 2 HD1, 5 HD2, 4 HD3) and age- and gender-

matched controls (n = 18; 12 male, 5 female; mean age 57.8).

Tissue blocks were tested for RNA integrity by capillary electro-

phoresis of RNA samples on a Agilent 2100 Bioanalyzer, as

described previously (Hodges et al. 2006). Brain sections (12 lm)

were mounted on Superfrost Plus slides. All tissue was stored at

)80�C before and after sectioning.

In situ hybridization histochemistryAn anti-sense hippocalcin probe and sense control probe were

generated using I.M.A.G.E. clone 3355354 as a template for in vitrotranscription of 35S-labeled riboprobes in Riboprobe System

(Promega, Madison, WI, USA). In situ hybridization was performed

according to (Landwehrmeyer et al. 1995). Slides were apposed to

BioMax film (Kodak, Rochester, NY, USA) and exposed for 2–

4 days. Developed films were analyzed using an M1 computer-

based image analysis system (Imaging Research, St. Catherine’s,

Canada) to measure the optical densities of the autoradiograms of

each section. With the experimenter blinded to condition, a

standardized area of the caudate autoradiogram of approx.

30 mm2 was sampled so as to record a representative Optical

Density for each brain.

Plasmid expression vectorsHuman cDNAs encoding either full-length hippocalcin (GenBank:

BC001777, nucleotides 41–622) or amino acids 2–72 of hippocalcin

(GenBank: BC001777, nucleotides 41–43 + 47–256) were ampli-

fied using the polymerase chain reaction (PCR) and cloned into the

shuttle plasmid pENTR/D-TOPO (Invitrogen, Carlsbad, CA, USA)

using the pENTR Directional TOPO� Cloning Kit (Invitrogen). C-

terminally 6· histidine-tagged constructs were created by modifying

the reverse PCR primer. The cDNAs were then transferred from the

shuttle vectors into a self-inactivating lentiviral plasmid vector with

a woodchuck regulatory element (SIN-W) under the control of the

mouse phosphoglycerate kinase 1 (PGK) promoter using Invitro-

gen’s Gateway� recombination technology. The same procedure

was used to clone the cDNA encoding enhanced cyan fluorescent

protein (ECFP), which was amplified from the pcDNA3.1-ECFP

plasmid (kindly provided by Dr. Harald Hirling, Brain Mind

Institute, EPFL). To create a hippocalcin-ECFP fusion construct,

EcoRI and XhoI restriction sites were added to 5¢- and 3¢-ends

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 460–472

Decreased hippocalcin in Huntington’s disease | 461

correspondingly of human hippocalcin cDNA using PCR, and the

resulting product was inserted upstream of and in frame with the

ECFP gene in the pcDNA3.1-ECFP plasmid. The obtained

pcDNA3.1-HPCA-ECFP plasmid was used as a substrate for the

PCR-amplification of the hippocalcin-ECFP cDNA. This cDNAwas

then cloned into SIN-W-PGK lentiviral vector using the pENTR�and Gateway� procedures described above.

cDNAs encoding potential hippocalcin interaction partners with

C-terminal c-myc-tags were cloned into the SIN-W-PGK lentiviral

vector using the same pENTR� and Gateway� strategies. These

cDNAs included human GABAA receptor-associated-like protein

2 (GABARAPL2, GenBank: AF087848, nucleotides 108–461),

human kinesin light chain (KNS2, GenBank: NM_005552,

nucleotides 309–1991), human tyro1/ephrin EA4 receptor

(EphA4, GenBank: NM_004438, nucleotides 43–3003), human

glutathione peroxidase 4 (GPX4, GenBank: BC022071, nucleo-

tides 56–649), rat ATP synthase a (GenBank: NM_023093,

nucleotides 50–1711), rat ATP synthase b (GenBank:

NM_134364, nucleotides 23–1612), rat 3-hydroxyacyl-CoA dehy-

drogenase subunit A (ECHA, GenBank: NM_130826, nucleotides

17–2308), rat 3-hydroxyacyl-CoA dehydrogenase subunit B

(ECHB, GenBank: NM_133618, nucleotides 1–1428), rat pro-

hibitin (PHB, GenBank: NM_031851, nucleotides 59–877).

cDNA encoding BIR domains 1–3 of human NAIP (GenBank

NM_004536, nucleotides 896–1753) was cloned in-frame with N-

terminal hemagglutinin- (HA-) tag into the SIN-W-PGK lentiviral

vector using the same protocol. cDNA encoding human GABAA

receptor-associated-like protein 1 (GABARAPL1, GenBank:

NM_031412, nucleotides 241–594) was cloned in-frame with

C-terminal enhanced yellow fluorescent protein (EYFP)-tag into

the pcDNA3.1-EYFP plasmid using the same procedure as for the

hippocalcin-ECFP fusion construct.

Lentiviral vector productionSIN-W-PGK and SIN-W-tetracycline responsive element (TRE)

expression vectors were used for the production of lentiviruses in

human cells (HEK293T) using a four-plasmid system as described

previously (Hottinger et al. 2000). Viral pellets were resuspended

in phosphate-buffered saline (PBS) with 0.5% bovine serum

albumin and stored at )80�C. Prior to utilization, viral stocks

were diluted with cell culture medium to a concentration of

1500 ng p24 antigen/mL as measured by ELISA (RETROtek,

Gentaur, Kampenhout, Belgium), and applied to the cell cultures at

the concentration of 50 ng p24/mL of culture medium for each

virus.

Primary striatal neuron culturesPrimary cultures of medium spiny neurons were prepared according

to (Zala et al. 2005). Briefly, ganglionic eminences of rat day 16

embryos were isolated and dissociated by repeated pipetting. Cells

were pelleted by centrifugation, resuspended in the culture medium

[Neurobasal medium (Invitrogen) complemented with 1% B27

(Invitrogen), Pen-Strep, L-Glutamine and KCl], plated in poly-L-

lysine-coated multi-well culture dishes at a density of 150 000 cells/

cm2 and cultured at 37�C in 5% CO2/air atmosphere. For lentiviral-

mediated protein expression, cultures were infected on day in vitro(DIV) 1, half the medium was replaced on DIV 4, and half of the

medium was replaced weekly thereafter.

In vitro model of Huntington’s diseaseAn in vitro model of Huntington’s disease was implemented using

lentiviral vectors described in (Regulier et al. 2003). This model

involves the lentiviral-mediated over-expression of N-terminal 171

amino acid fragments of wild-type huntingtin (with 18 glutamine

repeats) or mutant huntingtin (with 82 glutamine repeats) in striatal

neuron (ganglionic eminence) cultures under the control of TRE

promoter (SIN-W-TRE). SIN-W-TRE-htt171-82Q- but not SIN-W-

TRE-htt171-18Q-exposed cells exhibit a rapid pathology that leads

to intracellular huntingtin inclusions at 1–2 weeks and decreased

expression of the neuronal marker Neuronal Nuclei (NeuN) as early

as at 2 weeks after infection. To estimate the effects of heterologous

expression of hippocalcin, its interactors or their combination,

cultured neurons were co-infected with SIN-W-TRE-htt171-18/82Q

(25 ng p24/mL of culture medium), SIN-W-PGK-tTA (tetracycline

transactivator, 25 ng p24/mL of culture medium) and the SIN-W-

PGK vector with the gene of interest (25 ng p24/mL of culture

medium) on DIV 1. Half the medium was replaced on DIV 4, and

half of the medium was replaced weekly thereafter. Neurons were

fixed at DIV 21 and their survival assessed by NeuN-positive cell

counting.

Glutamate stimulation of cultured neuronsAt DIV 11–13, striatal neurons were exposed for 15 min to 10 lMglutamate in culture medium supplemented with 50 lM glycine and

1.35 mM CaCl2. Control (mock) treatment medium contained no

glutamate, only glycine and CaCl2. Following the treatment, cells

were washed once with fresh culture medium and placed in

conditioned culture medium for 48 h, after which they were fixed

and their survival assessed by NeuN-positive cell counting.

3-Nitropropionic acid treatmentAt DIV 11–13, 300 lM 3-nitropropionic acid (3-NP) was added to

the striatal neuron culture medium. Forty-eight hours later cells

were fixed and their survival assessed by NeuN-positive cell

counting.

Neuronal survival assessment by NeuN-positive cell countingPrimary neurons were washed with PBS and fixed with 4%

paraformaldehyde in PBS (Fluka, Buchs, Switzerland) for 15 min

at 4�C. After fixation, cells were washed three times with ice-cold

PBS and blocked with 10% normal goat serum (NGS, Invitrogen)

in PBS supplemented with 0.1% Triton X-100 (Sigma, St Louis,

MO, USA). An anti-NeuN monoclonal antibody (Chemicon,

Temecula, CA, USA) was diluted 1 : 500 in PBS with 5% NGS

and 0.1% Triton X-100, and incubated with the cells overnight at

4�C. Cells were washed three times with PBS and incubated with

goat-anti-mouse Cy3-conjugated antibody (1 : 1000 dilution,

Invitrogen) in PBS with 1% NGS, followed by three washes

with PBS. Images were taken on an automated high-throughoutput

imaging system BD Pathway 855 Bioimager (4· objective,

Becton-Dickinson, Franklin Lakes, NJ, USA). Image processing

and cell counting was performed using the public domain software

ImageJ.

Human embryonic kidney 293T cell culture and protein expressionHuman embryonic kidney (HEK) 293T cells were cultured in

Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 460–472� 2009 The Authors

462 | N. Rudinskiy et al.

and Pen-Strep at 37�C in a 5% CO2/air atmosphere. Cells were

plated in 10 cm culture dishes at a density of 3.106 cells/dish.

A calcium phosphate method was used to transfect the cells with

10–15 lg of SIN-W-PGK or pcDNA3.1 mammalian expression

vectors. The culture medium was changed 6 h post-transfection and

protein was harvested 48–72 h later.

ImmunoblottingCells were washed twice with PBS and harvested in lysis buffer

comprised of 20 mM HEPES, pH 7.4, 1% Triton X-100, 100 mM

KCl, 0.1 mM dithiothreitol, 2 mM EDTA, 2 mM EGTA (or 2 mM

CaCl2 instead of EDTA and EGTA where indicated) and protease

inhibitor cocktail (Sigma-Aldrich, Cat# P8340). Lysates were

incubated on ice for 30 min and vortexed every 10 min. Protein

extracts were cleared of cell debris by centrifugation at 20 000 g for

15 min at 4�C, separated by sodium dodecyl sulfate–polyacrylamide

gel electrophoresis (SDS–PAGE) on a 12% polyacrylamide gel, and

analyzed by immunoblotting.

A novel antiserum was raised against a C-terminal hippocalcin

peptide (H2N-VRLLQCDPSSASQF-COOH). In western blot

(WB) tests, this antiserum (HPCA2005) detected endogenous

hippocalcin in rat brain protein extracts and heterologously

expressed hippocalcin in transfected HEK293T cells (Fig. S1a).

Neuronal visinin-like protein 3 (NVP-3) shares high C-terminal

homology with hippocalcin, and thus heterologously expressed

human NVP-3 is also detected by WB with HPCA2005

(Fig. S1a). Co-incubation of antisera with the immunization

peptide during WB blocked its binding to hippocalcin and

NVP-3 (Fig. S1b). Though HPCA2005 also has the potential to

interact with neurocalcin delta, the liquid-chromatography mass-

spectrometric analysis (LC-MS) of proteins immunoprecipitated

from rat striatal lysates using HPCA2005 identified hippocalcin as

the only member of the NCS family immunoprecipitated with this

antibody.

Mouse anti-c-myc monoclonal antibody (EPFL Protein Produc-

tion Core Facility), rabbit 6·-histidine tag polyclonal antibody (PA1

23022) from Affinity BioReagents (Rockford, IL, USA), or rabbit

anti-green fluorescent protein polyclonal antibody (ab290) from

Abcam (Cambridge, MA, USA), were used to detect fusion proteins,

mouse anti-a-tubulin monoclonal antibody (T5168) from Sigma was

used to detect tubulin, which served as a loading control. Other

primary antibodies used were ATP synthase a (612516) from BD

Transduction Laboratories (Lexington, KY, USA) and prohibitin

(MS-261) from Lab Vision (Fremont, CA, USA). Secondary

antibodies consisted of Alexa Fluor 680-conjugated polyclonal goat

anti-mouse or goat anti-rabbit IgG or rabbit anti-goat (Invitrogen) or

IRDye 800CW-conjugated donkey anti-mouse IgG (LI-COR).

Protein bands were visualized using the Odyssey Infrared Imaging

System (LI-COR).

Living cell confocal microscopy and immunocytochemistry foranalysis of hippocalcin subcellular localizationFor living cell microscopy, cultured primary striatal neurons were

grown on poly-L-lysine-coated glass-bottomed 35-mm culture

dishes (FluoroDish-35, World Precision Instruments, Boulevard

Sarasota, FL, USA). Neurons were infected with ECFP or

hippocalcin-ECFP lentiviral expression vectors on DIV 1 and

were used for experiments on DIV 11–13. Culture dishes were

placed in the imaging chamber of an inverted confocal laser-

scanning TCS-SP2 AOBS microscope (Leica Microsystems,

Wetzlar, Germany) equipped with a glycerin immersion 63·objective with a 1.3 numerical aperture and Z-stack images were

taken every 30 s. During imaging, a stimulation medium contain-

ing 50 lM glutamate, 50 lM glycine and 2.7 mM CaCl2 was

added to the cells.

For immunostaining, neurons were grown on poly-L-lysine-

coated glass coverslips, infected as described above, and treated on

DIV 11–13 with control medium (50 lM glycine and 2.7 mM

CaCl2) or glutamate-containing medium (50 lM glutamate, 50 lMglycine and 2.7 mM CaCl2) for 20 min, then fixed with 4%

paraformaldehyde in PBS (pH 7.4) on ice for 15 min. After fixing,

cells were washed three times with ice-cold PBS and blocked with

10% NGS (Invitrogen) in PBS supplemented with 0.1% Triton X-

100 (Sigma). Primary monoclonal antibody to TGN38 (BD

Transduction) was diluted 1 : 100 in PBS with 5% NGS and

0.1% Triton X-100, and incubated with the cells overnight at 4�C.Cells were washed three times with PBS and incubated at RT with

secondary goat-anti-mouse Cy3-conjugated antibody (1 : 1000,

Invitrogen) in PBS with 1% NGS, followed by three washes with

PBS. Images were taken using the same inverted confocal laser-

scanning microscope TCS-SP2 AOBS with glycerin immersion 63·objective as above. All confocal images were deconvoluted using

HUYGENSPRO software (Scientific Volume Imaging, Hilversum,

the Netherlands) via the Huygens Remote Manager WEB-interface

developed at the EPFL.

ImmunoprecipitationAffinity-purified rabbit polyclonal anti-hippocalcin antiserum

HPCA2005 was conjugated to Protein A Sepharose 4 Fast

Flow� beads (GE Healthcare, Waukesha, WI, USA) and used for

immunoprecipitation. Using an immunoprecipitation protocol

described by (Steiner et al. 2002) hippocalcin-containing protein

complexes from transfected HEK293T cells were immunoprecip-

itated in the presence of CaCl2 or EDTA/EGTA. Briefly, 50 lg of

antibody were cross-linked to protein A sepharose beads using

20 mM dimethylpimelimidate in sodium borate buffer for 25 min

and the cross-linking reaction was quenched in ethanolamine for

1 h. Antibody-conjugated beads were washed with 0.2 M glycine-

HCl (pH 2.5) and in incubated with approximately 500 lg of

HEK293T cell protein extracted in presence of CaCl2 or EDTA/

EGTA for 4 h at 4�C. In the next step, beads were washed with

protein extraction buffer and hippocalcin-containing protein

complexes were eluted with 0.2 M glycine-HCl (pH 2.5). The

pH of the eluates was subsequently brought to 8.0 with Tris-HCl

(pH 10). Immunoprecipitated protein complexes were resolved by

12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis,

followed by analysis by Coomassie SimplyBlue SafeStain

(Invitrogen) and liquid-chromatography mass-spectrometric anal-

ysis (performed at the EPFL Proteomics Core Facility) or

immunoblotting.

Statistical analysisNumerical data represent the mean values ± SEM. The one-way

ANOVA was used to compare multiple conditions and the two-tailed

Student’s t-test was used for two-group comparisons. p < 0.05 was

set as the threshold for statistical significance.



Results

Hippocalcin RNA expression in human brain and itsdiminished expression in HD caudateOur previous results had demonstrated that decreased striatalhippocalcin expression was among the most reproduciblegene expression changes in brains of HD mice (Luthi-Carteret al. 2000). We therefore assessed whether decreasedhippocalcin RNA expression could also be detected inpostmortem brain tissues of human HD patients. An in situhybridization probe for hippocalcin was designed and testedin control human brain sections. Positive labeling in thehippocampus showed a discrete laminar pattern, with highexpression in Ammon’s horn and the dentate gyrus and lowerexpression in the subiculum; equivalently strong positivelabeling was also detected throughout the caudate andputamen; lower levels of specific labeling were also observedin the cerebral cortex (Fig. 1a). These regional distributionsare similar to those reported for rat brain (Paterlini et al.2000). Comparing caudate nucleus sections of 12 HD and 18control brains showed that hippocalcin mRNA was signifi-cantly down-regulated by 63% (Fig. 1b). These dataconfirmed that diminished hippocalcin expression was abona fide feature of human HD neuropathology.

Assessment of hippocalcin expression in primary striatalneuronsThe reported prosurvival effects of hippocalcin in otherneurodegenerative conditions and the decreased hippocalcinexpression in HD suggested to us that deficient hippocalcinexpression might sensitize neurons to HD-related neuro-toxicites. Hippocalcin mRNA expression is known to bedevelopmentally regulated in rats and to be abundant in ratstriatum (Paterlini et al. 2000). Before undertaking studies ofHD models using primary rat striatal cultures, however, anassessment of hippocalcin expression in these cells waswarranted. Under our previously characterized culture con-ditions, dissociated E16 ganglionic eminence-derived cellsexpress many neurochemical features of striatal outputneurons including DARPP-32, dopamine receptors, andadenosine A2A receptors, within 1 week in vitro. To oursurprise, however, we measured little to no endogenoushippocalcin protein (by western blotting) within 3 weeksin vitro. Hippocalcin protein was only minimally detectablebetween 24 and 83 days after plating, even when varying thepotassium concentration in or adding brain-derived neuro-trophic factor (BDNF) to the medium (Fig. 2). Although thelow hippocalcin expression in our cultures was surprising tous, this hippocalcin-deficient condition also presented anexcellent opportunity to assess hippocalcin’s cellular effectsby controlled manipulation of its levels through hetero-logous expression. We therefore produced lentiviral vec-tors encoding hippocalcin and other hippocalcin-derived

sequences and confirmed the successful expression of thecorresponding proteins in cultured striatal neurons withintwo weeks after infection by western blotting (Fig. 2c). Thelentiviral-mediated expression of hippocalcin in culturedstriatal neurons was comparable to the expression ofendogenous hippocalcin in the adult rat striatum.

In order to assess calcium-dependent hippocalcin com-partmentalization in striatal cells, the trafficking of hippo-calcin-ECFP (versus ECFP alone) was recorded using livefluorescence confocal microscopy. Under basal conditions,hippocalcin-ECFP fluorescence was essentially uniformthroughout the nuclear and cytoplasmic compartments.Following a Ca2+ influx induced by glutamate application(Fig. 3a), nuclear localization of the fusion protein dimin-ished and extranuclear compartmentalization showed a

(a)

(b)

Fig. 1 Hippocalcin expression in human brain. (a) Hippocalcin

expression in human striatum and hippocampus, measured by radio-

active in situ hybridization histochemistry and film autoradiography. (b)

A significant decrease in hippocalcin expression was demonstrated in

the caudate nucleus of HD cases versus controls (*p < 0.005; n = 12

HD, 18 controls), normalized to b-actin.



corresponding increase in intensity. Following live cellmicroscopy, immunostaining was used to identify thecompartment to which hippocalcin translocates followingcalcium exposure. Immunostaining for the trans-Golgi net-work (TGN) marker TGN38 co-localized well with thecompartment of calcium-dependent hippocalcin-ECFP trans-location (Fig. 3b), whereas mitochondrial and endoplasmicreticulum markers did not co-localize with the areas of

increased ECFP fluorescence in the stimulated neurons (datanot shown). No change in the distribution of ECFP alone(control) fluorescence was observed following glutamatestimulation (data not shown). We therefore conclude that themajor calcium-dependent features of hippocalcin compart-mentalization in striatal neurons are its translocation out ofthe nucleus and into the TGN. These data are in agreementwith previous studies showing calcium-dependent traffickingof hippocalcin-EYFP to the TGN in non-neuronal cells(O’Callaghan et al. 2002, 2003).

Effects of hippocalcin on polyglutamine toxicityWe next tested the hypothesis that increased hippocalcinexpression would protect striatal neurons against the toxicity

(a)

(b)

Fig. 3 Hippocalcin-ECFP fusion protein localization in cultured striatal

neurons. (a) Confocal time-lapse microscopy of living striatal neurons

expressing hippocalcin-ECFP fusion protein shows that hippocalcin-

ECFP is abundant in the nucleus and translocates out of the nucleus

following glutamate-induced calcium influx. The calcium-induced

compartment of hippocalcin distribution is indicated by a white

arrowhead. Bar = 10 lm. (b) Immunostaining of glutamate- versus

mock-stimulated striatal neurons with a trans-Golgi network (TGN)

marker TGN38 demonstrates that hippocalcin-ECFP fusion protein

translocates to the TGN following glutamate stimulation. Bar = 10 lm.

(a)

(b)

(c)

Fig. 2 Assessment of hippocalcin expression in striatal neurons cul-

tured from embryonic rat brain. (a) Time course of hippocalcin

expression in striatal cells with and without varying KCl concentration

in the culture medium. Weak hippocalcin immunoreactivity was ob-

served only at the latest time points of 24 and 83 DIV; decreasing the

KCl concentration had no effect. (b) Treatment of neurons with BDNF

(25 ng/mL) did not induce hippocalcin expression; neither was it was

detectable in the embryonic rat brain. (c) Lentiviral-mediated expres-

sion of hippocalcin in cultured striatal neurons is comparable to the

level of endogenous hippocalcin in the adult rat striatum.



of mutant huntingtin fragments. NeuN-positive cell countswere used to assess neurotoxicity after 3 weeks’ exposure tolentiviral vectors encoding 171aa fragments of mutant versuswild-type huntingtin under control of the tetracyclineresponse element (TRE) promoter (TRE-htt171-82Q vs.TRE-htt171-18Q). This in vitro model of HD shows progres-sive neuropathology including the appearance of intracellularhuntingtin inclusions and decreased numbers of neuronalnuclear antigen (NeuN)-positive cells by 2–3 weeks post-infection (Fig. 4a). To determine whether hippocalcin wouldprotect striatal neurons from mutant huntingtin-inducedtoxicity, cultured neurons were co-infected with a hippocal-cin-expressing lentiviral vector and TRE-htt171-18Q or TRE-htt171-82Q lentiviral vectors on DIV 1. Negative controls forhippocalcin expression included ECFP and the non-calcium-

binding hippocalcin truncation mutant H2-72 (Palmer et al.2005). Counter to our original hypothesis, increasing hippo-calcin expression did not rescue striatal neurons from htt171-82Q toxicity (as assessed by the counting of NeuN-positivecells after 3 weeks in culture; Fig. 4a).

Effects of hippocalcin on glutamate-induced excitotoxicityStriatal neurons are not exposed to the same brain circuitryand cellular stressors in vitro as they are in vivo in the HDbrain. In the intact brain, striatal cells receive a constantexposure to glutamate, and excitotoxicity is believed to be amajor pathogenic mechanism in HD. To determine whetherhippocalcin would protect striatal neurons against excitotox-icity, cultured neurons were infected with hippocalcin, H2-72or EYFP expression vectors, and subsequently treated on

(a)

(b)

(c)

Fig. 4 Hippocalcin does not protect med-

ium spiny neurons against HD-related

neurotoxicities. (a) Hippocalcin does not

protect medium spiny neurons from mutant

huntingtin-induced neurotoxicity. Cultured

striatal neurons were infected with SIN-W-

TRE-htt171-18/82Q and SIN-W-PGK-tTA

vectors on DIV 1 and with hippocalcin, H2-

72 and ECFP in SIN-W-PGK vector on DIV

1. Bar = 50 lm. (b) Hippocalcin does not

protect striatal neurons from glutamate-

nduced neurotoxicity. Striatal neuron

cultures were infected with SIN-W-PGK

lentiviral vectors expressing hippocalcin or

controls H2-72 or ECFP on DIV 1 and were

subjected on DIV 12 to the glutamate- or

mock-stimulation for 15 min. Neurons were

fixed 48 h later, stained with anti-NeuN

antibody and counted. Bar = 50 lm. (c)

Hippocalcin does not protect cultured stria-

tal neurons from 3-NP-induced neurotoxic-

ity. Striatal neuron cultures were infected

with SIN-W-PGK lentiviral vectors

expressing hippocalcin, H2-72 or ECFP on

DIV 1 and 3-NP was added to the culture

medium on DIV 12. Neurons were fixed

48 h later, stained with the anti-NeuN anti-

body and counted. Bar = 50 lm.



DIV 12 with 10 lM glutamate, 50 lM glycine and 1.35 mMCaCl2 for 15 min; control cells were treated with glycine andcalcium without glutamate. Neuronal survival was assessed48 h later by counting NeuN-positive cells. Again, counter toour hypothesis, hippocalcin exhibited no significant neuro-protection (Fig. 4b).

Effects of hippocalcin on neurotoxicity associated withmitochondrial insufficiencyDysfunction of the mitochondrial electron transport systemactivity, most prominently, diminished complex II/IIIactivity, is also believed to be an important contributor toHD pathogenesis, leading to decreased ATP production andincreased oxidative stress (Benchoua et al. 2006). Thus, HDhas also been modeled using chemical agents such as 3-nitropropionic acid, an inhibitor of succinate dehydrogenase,which recapitulates the mitochondrial respiratory deficitscaused by mutant huntingtin. We therefore assessed whetherhippocalcin could diminish 3-NP-induced toxicity in culturedstriatal neurons. As for the other models of HD, hippocalcinwas negative for neuroprotection in this system (Fig. 4c).

Assessment of potential effectors of hippocalcin activity instriatal neuronsGiven that NCS proteins have no inherent enzymaticactivities, it is expected that their interactions with othercellular proteins will be necessary for their calcium-depen-dent activities. Therefore, one potential explanation for thelack of hippocalcin’s neuroprotective activity in the aboveexperiments could be the insufficiency of a necessary effectorin our model system. In order to identify and consider theimportance of specific candidate effectors, we assessedpreviously identified and novel hippocalcin binding proteins.We first used a yeast-2-hybrid screen to identify novelcandidate hippocalcin interactors from a brain cDNA library.This approach returned the ephrin EA4 receptor (or Tyro1),GABAA receptor-associated-like protein 2 (GABARAPL2),kinesin 2, BCS1L, NADH subunit 1, and glutathioneperoxidase 4 as putative hippocalcin effectors. We alsoperformed an immunoprecipitation-based screen in mamma-lian cells and identified calcium-dependent hippocalcin-binding proteins using a liquid chromatography-massspectrometry based strategy. These experiments demon-strated an association between hippocalcin and four mito-chondrial proteins, 3-hydroxyacyl-CoA dehydrogenasesubunits A and B, ATP synthase a and prohibitin (Fig. 5a).Based on the literature, we also considered two hippocalcininteractors previously reported by other groups: the NAIP(Mercer et al. 2000), and the b2-subunit of the adaptorprotein complex 2 (Palmer et al. 2005). In addition, weconsidered the G-protein-coupled receptor kinases (GRKs 2,3, 4, 5 and 6), which have been shown to interact with otherNCSs (Iacovelli et al. 1999; Sallese et al. 2000), and toregulate the function of important striatally-expressed neu-

rotransmitter receptors (Kabbani et al. 2002). We tested thesteady-state binding of a number of these candidate hippo-calcin interactors by co-immunoprecipitation (Table 1,Fig. 5b) which showed that the binding of ATP synthase a,prohibitin, 3-hydroxyacyl-CoA dehydrogenase subunit B andNAIP was readily detected, whereas no binding was detectedbetween hippocalcin and the G-protein-coupled receptorkinases, GABAA receptor-associated-like proteins 1 and 2,glutathione peroxidase 4, kinesin 2 or b2-adaptin. [Interest-ingly, however, we were able to show the latter to be a

(a)

(b)

Fig. 5 Identification of novel hippocalcin interactors. (a) SDS–PAGE

of hippocalcin-containing complexes immunoprecipitated from hippo-

calcin-expressing cells in presence or absence of calcium. Simple-

Blue� staining of the gel allowed to identify four bands that were

present only in the calcium-containing samples. These bands were

excised from the gel and analyzed by LC-MS. They were identified as:

(i) 3-hydroxyacyl-CoA dehydrogenase subunit A; (ii) ATP synthase a;

(iii) 3-hydroxyacyl-CoA dehydrogenase subunit B; (iv) prohibitin. (b) IP

with an anti-hippocalcin antibody (HPCA2005). ATP synthase a and

prohibitin co-immunoprecipitated with hippocalcin from rat striatal ly-

sate in a calcium-dependent manner. BIR1–3 domains of NAIP and

3-hydroxyacyl-CoA dehydrogenase subunit B but not subunit A co-

immunoprecipitated with hippocalcin from mammalian cell lysate in a

calcium-dependent manner.



substrate for calcium-dependent hydrolysis by calpains underthe conditions in which its interaction with hippocalcin wasoriginally described (Palmer et al. 2005), as we recentlyreported in (Rudinskiy et al. 2009)]. We nonethelessincluded all of the above proteins in our subsequentneurodegeneration assays, with the rationale that eventransient interactions that may have escaped detection byour immunoprecipitation protocol might be relevant formediating hippocalcin’s neuroprotective activity.

Effects of heterologous expression of hippocalcin togetherwith its candidate effectorsTo ensure that concentrations of its putative effectors wouldnot be limiting in our assay, we reassessed hippocalcin’sneuroprotective activity after heterologous expression incombination with its reported interactor proteins. However,none of these proteins, either alone or in combination withhippocalcin, protected primary striatal neurons from htt171-82Q exposure or glutamate- or 3-NP-induced neurotoxicity(Figs 6 and 7; Tables S1–S3). Therefore, we conclude itunlikely that NAIP or any of these other candidate hippo-calcin effectors is a critical mediator of hippocalcin-mediatedneuroprotection in striatal cells. Moreover, these data moregenerally argue against hippocalcin’s being an importantsurvival factor in striatal neurons undergoing HD-relatedneurodegeneration.

Discussion

Previous data from our group (Luthi-Carter et al. 2000,2002) and others (Desplats et al. 2006) has demonstrated

large down-regulations of hippocalcin RNA in two differentmouse models of HD. In the present report, we demonstratethat hippocalcin RNA is also dramatically reduced in humanHD caudate. These data show that hippocalcin, like adeno-sine A2A, cannabinoid CB1, and dopamine receptor RNAs, isa consistent pathologic hallmark of mutant Huntington’seffects on the medium spiny neurons of the striatum.

The current study of hippocalcin as a potential HDmodifier was motivated by a strong rationale comprising thepreviously reported neuroprotective properties of hippocalcinand the observed down-regulation of hippocalcin in the HDstriatum. These findings compelled us to hypothesize andassess a role for hippocalcin in striatal neuron survival underHD-like conditions. However, lentiviral-mediated hippocal-cin expression in primary cultured striatal neurons failed toprotect them against any of the three HD-related neurotox-icities tested here, comprising mutant huntingtin-inducedneurodegeneration, mitochondrial dysfunction caused by 3-NP and glutamate-induced excitotoxicity. Overall, theseresults are contrary to our hypothesis and suggest thathippocalcin does not play a major role in governing thesurvival of striatal neurons in HD. In fact, heterologousexpression of hippocalcin alone tended to decrease, ratherthan increase, the survival of striatal neurons in culture.

We also addressed the possibility that hippocalcin-bindingeffector proteins might be necessary for hippocalcin-medi-ated neuroprotection. To this end, we re-assessed the effectsof hippocalcin over-expression in combination with variouscandidate interactors, analogous to previous experiments ofMercer and colleagues, who reported a synergistic protectionbetween hippocalcin and the neuronal apoptosis inhibitor

Table 1 Candidate hippocalcin interactors

Candidate interactor Function Origin

Interaction confirmed

by CO-IP

ATP synthase a ATP synthesis Mass-spectromerty, CO-IP Yes, Ca2+-dependent

Prohibitin Chaperon for proteins imported to

mitochondria

Mass-spectromerty, CO-IP Yes, Ca2+-dependent

3-Hydroxyacyl-CoA dehydrogenase

subunit A

b-Oxidation of long-chain fatty acids Mass-spectromerty, CO-IP No

3-Hydroxyacyl-CoA dehydrogenase

subunit B

b-Oxidation of long-chain fatty acids Mass-spectromerty, CO-IP Yes, Ca2+-dependent

GABAA receptor-associated-like

protein 2

GABAA-receptor trafficking Yeast-2-hybrid screen No

GABAA receptor-associated-like

protein 1

Intra-Golgi membrane transport Sequence homology to

GABARAPL1, TGN

association

No

Kinesin 2 Microtubule motor Yeast-2-hybrid screen No

Glutathione peroxidase 4 Negative regulator of oxidative stress Yeast-2-hybrid screen No

b2-Adaptin Clathrin adaptor protein Literature No

G-protein-coupled receptor

kinases 2/3/4/5/6

Regulate the activities of metabotropic

receptors

Interactors of other NCSs,

literature

No

Neuronal apoptosis inhibitory protein Negative regulator of neuronal apoptosis Literature Yes, Ca2+-dependent



protein (NAIP) (Mercer et al. 2000). These tests have alsoyielded negative results. One remaining possibility, however,is that we have not yet discovered the important hippocalcininteractor needed for its protection of striatal neurons, andthat this interactor, like hippocalcin, is not expressed (or isinsufficiently expressed) under our culture conditions.

It is also possible that some of the important functionssubserved by hippocalcin in vivo are not required for thesurvival striatal neurons in vitro. The data of Tzingounis and

colleagues lab have implicated hippocalcin in the regulationof hippocampal neuron slow afterhyperpolarization (Tzingo-unis et al. 2007). This effect may mediated by interactionswith A-type potassium channels (such as KCNQ2 orKCNQ3) which are activated by rapid calcium entry(Tzingounis and Nicoll 2008). Interestingly, these suggestthat hippocalcin plays a role analogous to the structurallydivergent NCSs of class E, comprising the potassiumchannel-interacting proteins (KChIPs) (Buxbaum et al.1998; An et al. 2000). In the context of the present work,however, there would be no obvious role for plasticity-related influences on striatal neuron survival in vitro, as these

(a)

(b)

(c)

Fig. 6 Co-expression of hippocalcin with BIR domains 1–3 of NAIP

does not protect medium spiny neurons against three types of HD-

related neurotoxicity. (a) Co-expression of hippocalcin and BIR1–3

does not protect medium spiny neurons from mutant huntingtin-in-

duced neurotoxicity. (b) Co-expression of hippocalcin and BIR1–3

does not protect striatal neurons from glutamate-induced neurotoxic-

ity. (c) Co-expression of hippocalcin and BIR1–3 does not protect

cultured striatal neurons from 3-NP-induced neurotoxicity. Neuronal

survival was assessed by NeuN-positive cell counting as described in

the legend of Fig. 4.

(a)

(b)

(c)

Fig. 7 Hippocalcin’s potential effector 3-CoA-hydroxyacyl dehydro-

genase subunit B (ECHB) not protect medium spiny neurons from

mutant huntingtin- (a) glutamate- (b) or 3-NP- (c) induced neuro-

toxicity, either alone or in combination with hippocalcin. Neuronal

survival was assessed by NeuN-positive cell counting as described in

the legend of Fig. 4.



neurons are devoid of excitatory inputs and do not fire actionpotentials (Runne et al. 2008).

In the process of identifying potential hippocalcin effectorsthat might be critical mediators of its activities in striatalneurons, we provided evidence of novel hippocalcin interac-tors (Table 1), and confirmed hippocalcin’s previously re-ported interaction with NAIP (Mercer et al. 2000). Our newlyidentified hippocalcin interactors include three mitochondrialproteins whose steady state binding activities were strongenough to be confirmed by co-immunoprecipitation: 3-hydroxyacyl-CoA dehydrogenase subunit B, prohibitin anda subunit of ATP synthase (whose b subunit was also reportedin a proteomic screen for hippocalcin interactors by Hayneset al. 2006). 3-Hydroxyacyl-CoA dehydrogenase is a mem-brane-bound two-enzyme protein complex also known asmitochondrial trifunctional protein, which plays an importantrole in energy generation in the cell by catalyzing the b-oxidation of long-chain fatty acids. ATP synthase a is acatalytic core subunit of a well studied F1FO-ATP synthasedriven by the proton gradient across the mitochondrialmembrane. Prohibitins have been suggested to act as chaper-ones for the proteins imported in the mitochondria and protectthem from proteolytic degradation. Despite the strong inter-actions of these three proteins with hippocalcin in vitro,however, we have not yet been able to detect significant co-localization of hippocalcin-ECFP with mitochondrial markersusing confocal microscopy in cultured primary striatalneurons. Thus, the physiological relevance of these potentiallyinteresting interactions remains to be determined.

In some cases, we were not able to confirm interactions ofhippocalcin with proteins that were identified in our yeast-2-hybrid screen or based on previously published reports byassessing co-immunoprecipitation from mammalian celllysates. Beyond the possibility of false positives in theprimary assays, it may be that our immunoprecipitationconditions were unfavorable for these interactions. However,these experiments to not rule out the possibility that theseinteractions, even if weaker or more transient, mightnonetheless be physiologically relevant.

The neuronal trafficking of hippocalcin is another valuableissue addressed in this work. Although O’Callaghan andcolleagues have previously reported calcium-dependenttranslocation of hippocalcin to the trans-Golgi network ofHeLa cells (O’Callaghan et al. 2002, 2003), the presentstudy provides the first specific evidence of neuronalhippocalcin trafficking to the TGN in neurons. Taking intoaccount hippocalcin’s association with TGN and the fact thatstriatal neurons express GABA receptors, we tested whetherhippocalcin could interact with GABAA receptor-associated-like protein 1 (GABARAPL1), which is known to be anintra-Golgi membrane transport modulator and which ishighly homologous to GABARAPL2, one of proteins thatgave positive results in yeast-2-hybrid screen. However, wedid not find evidence of hippocalcin-GABARAPL1 interac-

tion by immunoprecipitation of hippocalcin-containing pro-tein complexes (with the caveats stated above). We were alsointerested by the calcium-regulated compartmentalization ofhippocalcin-ECFP in the nucleus, particularly given thepreviously reported role of the NCS calsenilin (also known asDREAM) as a transcriptional repressor (Carrion et al. 1999).However, further experiments failed to uncover evidence of arole for hippocalcin in the modulation of acute geneexpression responses to glutamate, forskolin and brain-derived neurotrophic factor (Rudinskiy and Luthi-Carter,unpublished data). Therefore, a specific nuclear or generegulatory role for hippocalcin remains to be established.

Another possible explanation for the fact that hippocalcinover-expression shows no neuroprotective activity in oursystem is that another member of neuronal calcium sensorfamily possesses a role redundant with that of hippocalcinin striatal neurons (or, at least, under our experimentalconditions). This perspective would also be consistent withthe mild phenotype of hippocalcin-deficient mice (Kobay-ashi et al. 2005). Given that little is known about functionalredundancy between different NCSs, however, the proba-bility of this effect remains uncertain. We find this to be anunlikely explanation of our results for the followingreasons. First, at the RNA level, other NCSs demonstratelittle or no expression in striatal neurons. Second, theantibody used for hippocalcin detection would also cross-react with NVP-3, the NCS protein most closely related tohippocalcin, ruling out the presence of NVP-3 as afunctionally redundant NCS in our cultures. Third, noother NCS has been shown to bind NAIP, which is arguablyhippocalcin’s most likely survival-related effector. There-fore, our interpretation based on the evidence available todate is that hippocalcin itself is unable to rescue striatalcells against neurotoxicity, rather than being replaced by analternate NCS in our system.

The data of the present study provide considerableevidence against a putative role of hippocalcin in regulat-ing striatal neuron vulnerability in Huntington’s disease.Nonetheless, we cannot definitively rule out this possibil-ity, particularly as it relates to a hippocalcin effector nottested in the present study [such as CAPS1 (Haynes et al.2006) or MLK2 (Nagata et al. 1998)], or conditions inthe HD brain that are not recapitulated faithfully in ourmodel systems. Thus, revisiting the issue may be war-ranted as hippocalcin’s striatal functions become betterunderstood.

Acknowledgements

We are grateful for the financial support from the Swiss National

Science Foundation and the EPFL. We also thank Anne B. Young

for sharing laboratory resources, Maria de Fatima Rey for the

production of lentiviral vectors, Meredith Dixon, Maria de Fatima

Rey and Marc Forrest for production and maintenance of cell



cultures, Patrick Schorderet for assistance in cloning cDNAs

encoding hippocalcin interactors, Marc Moniatte and Diego Chiappe

for mass-spectrometric analysis and Floyd J.-C. Sarria for assistance

with image processing.

Supporting Information

Additional Supporting Information may be found in the online

version of this article:

Figure S1. Western blotting using new rabbit-polyclonal antise-

rum (HPCA2005) against hippocalcin.

Table S1. Co-expression of hippocalcin with potential interactors

does not protect medium spiny neurons against mutant huntingtin

expression.


does not protect medium spiny neurons against toxicity associated

with succinate dehydrogenase expression.


does not protect medium spiny neurons against glutamate excito-

toxicity.

As a service to our authors and readers, this journal provides

supporting information supplied by the authors. Such materials are

peer-reviewed and may be re-organized for online delivery, but are

not copy-edited or typeset. Technical support issues arising from

supporting information (other than missing files) should be

addressed to the authors.

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Diminished hippocalcin expression in Huntington’s disease brain does not account for increased...

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