Depletion of CBP is directly linked with cellular toxicity caused by mutant huntingtin.

Abstract

Huntington's disease is a neurodegenerative disease caused by an expanded polyglutamine stretch within the huntingtin protein. Transfection of mutant huntingtin causes cell toxicity and depletion of CREB binding protein (CBP) or its recruitment into huntingtin aggregates. However, the role of CBP has been controversial and the relationship between polyglutamine-induced toxicity and CBP depletion has not been examined on an individual cell basis. Using a single-cell based assay, we found that, in HT22 cells or primary neurons transfected with mutant huntingtin, cell toxicity was accompanied by CBP depletion, rather than merely recruitment. Transfection with a htt exon1 construct containing uninterrupted polyglutamine or a polyglutamine region engineered to form a compact beta structure resulted in cell toxicity. CBP depletion was accompanied by histone hypo-acetylation. CBP overexpression rescued both acetylated histone levels and cell toxicity. These data suggest that CBP dysfunction and altered gene transcription contribute to mutant htt-induced neurotoxicity.

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Depletion of CBP is directly linked with cellular toxicity
caused by mutant huntingtin
Haibing Jiang,aMichelle A. Poirier,aYideng Liang,aZhong Pei,aCharlotte E. Weiskittel,a
Wanli W. Smith,aDonald B. DeFranco,c,dand Christopher A. Rossa,b,*
aDivision of Neurobiology, Department of Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
bDepartments of Neurology and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
cDepartment of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
dDepartment of Neuroscience, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
Received 27 January 2006; revised 26 March 2006; accepted 24 April 2006
Available online 12 June 2006
Huntington’s disease is a neurodegenerative disease caused by an
expanded polyglutamine stretch within the huntingtin protein. Trans-
fection of mutant huntingtin causes cell toxicity and depletion of CREB
binding protein (CBP) or its recruitment into huntingtin aggregates.
However, the role of CBP has been controversial and the relationship
between polyglutamine-induced toxicity and CBP depletion has not
been examined on an individual cell basis. Using a single-cell based
assay, we found that, in HT22 cells or primary neurons transfected
with mutant huntingtin, cell toxicity was accompanied by CBP
depletion, rather than merely recruitment. Transfection with a htt
exon1 construct containing uninterrupted polyglutamine or a polyglut-
amine region engineered to form a compact beta structure resulted in
cell toxicity. CBP depletion was accompanied by histone hypo-
acetylation. CBP overexpression rescued both acetylated histone levels
and cell toxicity. These data suggest that CBP dysfunction and altered
gene transcription contribute to mutant htt-induced neurotoxicity.
D 2006 Elsevier Inc. All rights reserved.
Keywords: Huntingtin; Polyglutamine; CBP; HAT; Beta structure; Cell
death; Neurodegeneration; Gene transcription
Introduction
Huntington’s disease (HD) is caused by an expansion of a
glutaminerepeatwithinthehuntingtin(htt)protein(DiProsperoand
Fischbeck,2005;GatchelandZoghbi,2005;PaulsonandFischbeck,
1996; Ross, 2002). HD pathology is characterized by neuronal
degeneration in selected regions of the brain, including the striatum,
cerebral cortex, and other regions. In addition, intracellular
inclusions (or aggregates) formed by mutant htt protein have been
observed in the cortex and striatum of postmortem HD brain
(DiFiglia et al., 1997; Ross, 1997). Various model systems of htt
aggregation and toxicity have been developed using cell culture and
transgenicanimals,anddemonstratethatmutanthttexpressionoften
leads to cell death (Hackam et al., 1998; Saudou et al., 1998; Steffan
et al., 2001).
It has been suggested that mutant huntingtin can cause cell toxicity
by interfering with the function of CREB binding protein (CBP),
yielding alterations in gene transcription. CBP can interact with
mutant htt and with mutant atrophin-1, the protein implicated in
Dentatorubropallidoluysian Atrophy (DRPLA), in both cell culture
and transgenic mouse models (Nucifora et al., 2001). Colocalization of
CBP with polyQ aggregates has also been observed in cells in culture,
transgenic mice, and postmortem HD brain tissue (McCampbell et al.,
2000; Nucifora et al., 2001). Moreover, CBP function may also be
compromised by the loss of soluble levels of nuclear CBP (Jiang et al.,
2003; McCampbell et al., 2000; Nucifora et al., 2001). CBP can act as
a histone acetyltransferase (HAT) to acetylate nucleosome-bound
histones, a process that is required for remodeling of chromatin
structure, and regulation of gene transcription (Bannister and
Kouzarides, 1996). It has been suggested that mutant huntingtin
expression reduces the acetylated level of histones, which correlates
with toxicity, possibly through its effects on HATs such as CBP
(McCampbell et al., 2001; Steffan et al., 2001).
However, the role of CBP in htt toxicity has been controversial
(Obrietan and Hoyt, 2004; Tallaksen-Greene et al., 2005; Yu et al.,
2002). Recently, other transcription factors have been implicated
(Bae et al., 2005; Dunah et al., 2002; Li et al., 2002; Schaffar et al.,
2004). Furthermore, the relative contribution of CBP recruitment
(i.e. incorporation into aggregates whether or not altered from its
normal location) versus nuclear depletion to polyQ-induced cell
toxicity is uncertain. For example, Li et al. reported that CBP
0969-9961/$ - see front matter D 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.nbd.2006.04.011
* Corresponding author. Division of Neurobiology, Department of
Psychiatry, Departments of Neurology and Neuroscience, The Johns
Hopkins University School of Medicine, Baltimore, MD 21287, USA.
Fax: +1 410 614 0013.
E-mail address: caross@jhu.edu (C.A. Ross).
Available online on ScienceDirect (www.sciencedirect.com).
www.elsevier.com/locate/ynbdi
Neurobiology of Disease 23 (2006) 543 – 551

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recruitment was not observed in two different HD mouse models
(Yu et al., 2002).
We reported previously that endogenous CBP can be depleted
from nuclei of HT22 cells or Neuro2a cells following transfection
with mutant htt (Jiang et al., 2003). This observation may result
from either CBP recruitment into insoluble htt aggregates or from
enhanced degradation of CBP via the ubiquitin–proteosome
pathway. In addition, htt-induced toxicity was observed in HT22
cells containing nuclear or perinuclear aggregates (Jiang et al.,
2003). However, these studies did not definitively establish the role
of CBP depletion in polyQ-induced cell death at a single cell level.
In the present study, a single-cell based assay was used to examine
the relationship between CBP depletion and mutant htt-induced
cell death in HT22 cells and in mouse primary cortical neurons,
and the impact of CBP depletion on histone acetylation levels in
HT22 cells. The data indicate that depletion of CBP from the
nucleus of individual cells results in histone hypo-acetylation and
parallels an increase in cytotoxicity.
Materials and methods
Plasmids and antibodies
The following constructs have been described previously: Htt-
exon1-76Q, Htt-exon1-PGQ9, Htt-exon1-PGQP (Poirier et al.,
2005), and Htt-N63-148Q-myc (Cooper et al., 1998). A mouse
full-length-CBP-Flag (Fl-CBP-Flag) construct was a gift from Dr.
Richard Goodman of Oregon Health Sciences University. To
generate an in-frame deletion (amino acids 1446 to 1886) of the
HAT domain of CBP (i.e. CBPDHAT-Flag), Fl-CBP-Flag was
digested with BspE1/SgrAI and re-ligated. Primary antibodies used
include mouse anti-flag (M2) (Sigma, MO) at 1:1000, mouse anti-
myc (9E10) (Roche, IN) at 1:500, mouse anti-CBP (C-1) (Santa
Cruz, CA) at 1:50, rabbit anti-CBP (A-22) (Santa Cruz) at 1:200,
rabbit anti-Sp1 (PEP 2) (Santa Cruz) at 1:200, rabbit anti-TBP (SI-
1) (Santa Cruz) at 1:200, and rabbit anti-acetylated H4 (Lys5)
(Santa Cruz) at 1:200. Secondary antibodies include FITC anti-
mouse (Chemicon, CA) at 1:300, Rhodamine-anti-rabbit at 1:300,
Alexa Fluor 350 anti-mouse (Molecular probe Invitrogen, CA) at
1:500. Nuclei were stained with Hoescht (Sigma).
Cell culture and transfection
HT22 cells were maintained and transfected as described
previously (Jiang et al., 2003). Fl-CBP-Flag and Htt-N63-148Q-
myc plasmids were cotransfected at a ratio of 3:1 (Figs. 4C–E).
Mouse primary cortical neurons were isolated from CD-1 outbred
mice (Charles River, MA) at embryonic day 16. Primary neurons
were transfected with plasmid DNA by electroporation using
Nucleofector (Amaxa Inc., MD) as previously described (Poirier
et al., 2005).
Indirect immunofluorescence
HT22 cells and neurons were fixed 48 h after transfection/
electroporation for indirect immunofluorescence staining. Images
were taken using a Zeiss conventional fluorescence microscope.
CBP depletion was measured by direct comparison of soluble
nuclear CBP levels with background fluorescence staining. Cell
death in HT22 cells was determined via Hoescht staining of a
condensedorfragmentednucleus,oftenaccompaniedbyashrunken
cellbodyasindicatedbyphasecontrast.Viabilityofprimarycortical
neurons wasmonitoredbyaneuritemorphology assayincludingthe
following criteria: a viable neuron must have at least one healthy
neuritethatisatleasttwotimesthelengthofthesameneuronalbody,
with smooth (nonfragmented) neurites (Poirier et al., 2005).
Statistical analysis
Approximately 100 cells containing mutant htt aggregates were
counted from randomly selected fields in each experiment (Figs.
1B, 2B, 3B, D–E, and 4B, D–E). Data shown are the average of
six separate experiments and are presented as mean T SEM with
statistical analysis performed using Standard Student’s t test.
Results and discussion
CBP depletion is associated with htt-induced toxicity in HT22 cells
and primary neurons
Transient transfection experiments were carried out to express a
htt-exon1 protein containing 76 glutamine residues (i.e. Htt-exon1-
76Q) in HT22 cells. Endogenous CBP can be depleted from the
nucleus and recruited into a perinuclear htt aggregate, as shown in
Fig. 1A (top panels, arrowhead). In response to various insults,
HT22 cells undergo cell death that cannot be characterized as
strictly apoptotic or necrotic (Tan et al., 1998). As shown in Fig.
1A, a cell expressing polyQ-expanded htt (arrowhead) contains a
shrunken nucleus and retracted cell body with no detectable
cytoplasm. This cell is unlikely to be viable. Previously, we have
shown that HT22 cells with such morphological features were
often TUNEL positive (Jiang et al., 2003). These cells will be
referred to as nonviable throughout the text.
In contrast, images shown in the bottom panels depict HT22
cells containing htt aggregates and normal levels of endogenous
CBP within the nuclei. Despite recruitment of CBP into aggregates
(bottom panels, pointed by arrows), these cells appear viable.
Quantification of these results (Fig. 1B) demonstrates that nuclear
depletion of endogenous CBP is more often associated with the
loss of viability compared to HT22 cells with no detectable
changes in endogenous nuclear CBP levels. Moreover, diffuse
nuclear CBP expression in HT22 cells (despite partial recruitment
into htt aggregates) was observed in cells that exhibit no
morphological features of viability loss. Similar results were
observed in cells transfected with an N-terminal 63-residue
fragment of htt containing 148 glutamines and a C-terminal myc
tag, Htt-N63-148Q-myc (data not shown). Taken together, these
data indicate that CBP recruitment into htt aggregates is not
sufficient for toxicity, but that recruitment accompanied by nuclear
depletion of CBP correlates with cytotoxicity in HT22 cells.
To confirm that CBP depletion correlates specifically with
mutant htt-induced cell death, CBP expression was examined in
nontransfected cells that underwent spontaneous cell death within
the same culture. As shown in Fig. 1C, an untransfected and
nonviable cell (1st panel, center) maintains diffuse CBP nuclear
staining. This observation demonstrates that CBP depletion is not a
universal feature of cell death. Quantification of these data is
shown in Fig. 3F (left columns) and indicates that approximately
80% of untransfected cells that died spontaneously showed no
evidence of CBP depletion. Moreover, expression of htt exon1
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Fig. 1. Depletion of endogenous CBP parallels toxicity in HT22 cells expressing Htt-exon1-76Q. (A) Cells were fixed 48 h after transfection and mutant htt
expression was detected by anti-Flag immuno-staining (green). Endogenous CBP is shown in red. A transfected cell containing a mutant htt aggregate (top row,
1st panel, arrowhead), shows nuclear depletion of endogenous CBP accompanied by recruitment into a perinulear aggregate (top row, 2nd panel, arrow). This
nonviable cell has condensed nuclear staining (top row, 3rd panel) and a rounded cell body (top row, 5th panel). Bottom panels show healthy cells with htt
aggregates and normal diffuse nuclear CBP staining, despite of CBP being partially recruited in aggregates (pointed by arrows). Scale bar: 10 Am. (B)
Quantitative analysis demonstrates that mutant htt expressing cells with nuclear depletion of CBP are more likely to undergo cell death, than cells with normal
levels of nuclear CBP. (C) An untransfected cell that died spontaneously (center) shows diffuse and more condensed nuclear staining of endogenous CBP. Scale
bar: 10 Am. (D) A cell containing a Htt-exon1-PGQ9aggregate shows evidence for nuclear depletion of endogenous CBP. This cell was nonviable at the time of
analysis. Scale bar: 10 Am. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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with a normal length polyQ region (i.e. Htt-exon1-16Q) results in
minimum cell death (Poirier et al., 2005), and of this small
percentage, endogenous CBP is not depleted (data not shown).
Therefore, CBP depletion in transfected HT22 cells was specifi-
cally correlated with mutant htt-induced cell death.
To further investigate a role for CBP dysfunction in mutant
htt-mediated cell death, transient transfection experiments were
performed using htt exon1 constructs with a modified expanded
polyQ region. PGQ9 has a polyQ sequence length of 77 with
Pro-Gly pairs interspersed every nine residues to induce an
alternating beta-strand/beta-turn structure. This design was
previously shown to induce aggregation in vitro in a synthetic
polyQ peptide (Thakur and Wetzel, 2002), and induced
aggregation and toxicity in a htt exon1 cell culture model
(Poirier et al., 2005). PGQP, designed with Pro insertions to
disrupt the putative compact beta-structure, was unable to
aggregate or cause cytotoxicity in the same cell model of
mutant htt aggregation (Poirier et al., 2005). As shown in Fig.
1D, Htt-exon1-PGQ9expression resulted in both CBP depletion
and in cell death. In contrast, expression of Htt-exon1-PGQP did
not lead to depletion of nuclear CBP, nor did it induce toxicity
in HT22 cells (data not shown). These results suggest that
formation of a compact beta-sheet structure within htt mutant
polyQ may be required for CBP depletion as well as the
resulting cell toxicity.
While HT22 cells are a hippocampal-derived cell line, cultured
primary neurons can more closely approximate in vivo conditions.
Therefore, this model system was used to investigate a possible
correlation between CBP depletion and mutant htt-induced
toxicity. A neuritic morphology assay previously used to monitor
mutant htt-induced neuronal cell death in primary cortical neurons
(Poirier et al., 2005) was used in the current study. As shown in
Fig. 2A (top panels), depletion of endogenous CBP from the
nucleus is accompanied by recruitment into a Htt-exon1-76Q
aggregate (green). In this neuron, mutant htt expression resulted in
death as assessed by an abnormal nuclear morphology and the
absence of a healthy neurite (see Materials and methods). In
contrast, a healthy neuron (bottom panels) maintained relatively
normal CBP nuclear diffuse nuclear staining, despite an area of
localized condensed CBP, which may represent an early recruit-
ment event. Similar results were observed in Htt-N63-148Q-myc-
transfected primary cortical neurons (data not shown). Quantifica-
Fig. 2. Depletion of endogenous CBP parallels toxicity in primary mouse cortical neurons expressing Htt-exon1-76Q. (A) Mouse primary cortical neurons
transfected with Htt-exon1-76Q were fixed 48 h after transfection and stained with an anti-Flag antibody. A neuron expressing mutant htt shows nuclear
depletion of endogenous CBP with partial recruitment into a htt aggregate (top panels). This cell was nonviable at the time of analysis. A different neuron
positively stained for htt aggregates has relatively diffuse nuclear CBP staining (bottom panels) and was found to be healthy. Scale bar: 10 Am. (B) Quantitative
analysis demonstrates that the majority of neurons showing nuclear CBP depletion were nonviable at the time of analysis, while neurons with normal levels of
nuclear CBP were healthy.
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tion of these data (Fig. 2B) demonstrates a strong correlation
between CBP depletion and neuronal cell death.
The present data suggest that a minimal threshold level of
soluble nuclear CBP must be maintained in order to promote
neuronal survival in mutant htt-expressing cells. It is difficult to
precisely define within individual cells the minimum CBP
expression level required for neuronal cell survival. The severe
neurological phenotypes associated with Rubinstein-Taybe syn-
drome (Petrij et al., 1995), a genetic disease caused by CBP
haploinsufficiency, highlight the importance of maintaining appro-
priate levels of CBP, particularly in neurons. CBP homozygous
knockout mice die at embryonic day 10.5 due to neural tube
closure defects (Kung et al., 2000; Yao et al., 1998), suggesting a
specific role for CBP in neuronal development. In addition, CREB/
CREM conditional double knockout mice manifest forebrain
abnormalities including striatal cell apoptosis, strongly consistent
with the idea that CBP dysfunction may lead to neuronal death of
selected brain areas (Mantamadiotis et al., 2002).
In the current studies, both CBP depletion and cell death
were not typically observed in transfected HT22 cells or in
primary neurons lacking visible aggregates. However, this
observation does not suggest that visible htt aggregates must be
formed in cells undergoing toxicity. Previous studies have shown
that mutant htt-induced cell death is correlated with the level of
Fig. 3. Nuclear depletion of Sp1 and TBP is not specifically correlated with mutant htt toxicity in HT22 cells. (A) A cell containing mutant htt aggregates (1st
panel, green) has normal levels of endogenous Sp1 (2nd panel, red). This cell was nonviable at the time of analysis. Scale bar: 10 Am. (B) Quantitative analysis
indicates that nuclear Sp1 staining pattern is not affected by mutant htt expression. (C) A cell containing a mutant htt aggregate (1st panel, green) has normal
levels of endogenous TBP (2nd panel, red). This cell was nonviable at the time of analysis. Scale bar: 10 Am. (D) Quantitative analysis shows that nuclear TBP
can either be depleted (20%) or present (25%) in the nuclei of nonviable cells containing mutant htt. (E) Quantitative analysis of CBP/TBP depletion. Data
indicate that 45% of cells nonviable at the time of analysis showed depletion of TBP, while 90% of such cells had CBP depletion. This analysis demonstrates a
lower correlation between TBP depletion and mutant htt-induced toxicity than for depletion of CBP. (F) Quantitative analysis shows that normal levels of CBP
are present in the majority of untransfected cultured cells that died spontaneously. In contrast, no significant difference was observed between the percentage of
cells with normal levels of TBP and those showing TBP depletion. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
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