Partial loss of Tip60 slows mid-stage
neurodegeneration in a spinocerebellar ataxia
type 1 (SCA1) mouse model
Kristin M. Gehrking1,2, J. Michael Andresen1,3, Lisa Duvick1, John Lough4,
Huda Y. Zoghbi5,6and Harry T. Orr1,2,3,∗
1Institute of Human Genetics and Institute of Translational Neuroscience,2Department of Biochemistry, Biophysics
and Molecular Biology and3Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis,
MN 55455, USA,4Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee,
WI 53226,5Department of Molecular and6Department of Human Genetics, Pediatrics, and Howard Hughes Medical
Institute, Baylor College of Medicine, Houston, TX 77030, USA
Received October 11, 2010; Revised and Accepted March 11, 2011
Spinocerebellar ataxia type 1 (SCA1) is one of nine dominantly inherited neurodegenerative diseases caused
by polyglutamine tract expansion. In SCA1, the expanded polyglutamine tract is in the ataxin-1 (ATXN1) protein.
ATXN1 is part of an in vivo complex with retinoid acid receptor-related orphan receptor alpha (Rora) and the
acetyltransferase tat-interactive protein 60 kDa (Tip60). ATXN1 and Tip60 interact directly via the ATXN1 and
HMG-box protein 1 (AXH) domain of ATXN1. Moreover, the phospho-mimicking Asp amino acid at position
776, previously shown to enhance pathogenesis, increases the ability of ATXN1 to interact with Tip60. Using
a genetic approach, the biological relevance of the ATXN1/Tip60 interaction was assessed by crossing
ATXN1[82Q] mice with Tip601/2animals. Partial Tip60 loss increased Rora and Rora-mediated gene expression
and delayed ATXN1-mediated cerebellar degeneration during mid-stage disease progression. These results
suggested a specific, temporal role for Tip60 during disease progression. We also showed that genetic back-
ground modulated ATXN1[82Q]-induced phenotypes. Of interest, these latter studies showed that some pheno-
types are enhanced on a mixed background while others are suppressed.
Spinocerebellar ataxia type 1 (SCA1) is one of nine inherited
polyglutamine diseases that cause neurodegeneration (1,2). In
these diseases, the mutant gene encodes an expanded gluta-
mine tract, which results in a polyglutamine expansion
within the protein. In SCA1, the mutant ATXN1 gene
encodes the protein ataxin-1 (ATXN1), which is widely
expressed (3,4); however, neurodegeneration is limited to cer-
ebellar Purkinje cells, brainstem and spinal cord (5). As with
most autosomal dominant ataxias, symptoms are characterized
by a progressive loss of motor coordination, neuropathies,
slurred speech, cognitive impairment and loss of other func-
tional abilities arising from deep cerebellar nuclei (6).
Evidence indicates that a normal function of ATXN1 is to
regulate gene expression. For example, ATXN1 interacts with
a variety of transcription factors, including the zinc-finger tran-
scription factors Drosophila Senseless and its mammalian
homologgrowth factor-independent 1(7),thetranscriptioncor-
epressor silencing mediator of retinoid and thyroid hormone
receptors (8), the human homolog of the Drosophila transcrip-
tion repressor Capicua (9), the transcription factor Sp1 (10),
the retinoid acid receptor-related orphan receptor alpha/
tat-interactive protein 60 kDa (Rora/Tip60) complex (11) and
tains a nuclear localization sequence (13). Wild-type (WT)
ATXN1 shuttles between the nucleus and cytoplasm (14).
Importantly, mutant ATXN1 must enter the nucleus to cause
∗To whom correspondence should be addressed. Email: email@example.com
# The Author 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
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Human Molecular Genetics, 2011, Vol. 20, No. 11
Advance Access published on March 22, 2011
disease (13) and its nuclear export is greatly reduced (14).
Within ATXN1 is the ATXN1 and HMG-box protein 1
(15). The ATXN1 AXH domain crystal structure reveals an
oligonucleotide-binding fold (16), which is likely the site of
the proposed RNA-binding activity of ATXN1 (17).
Some of these ATXN1-interacting factors, in a dosage-
dependent fashion, modify pathogenesis of SCA1 in mouse
and fly models (7–9,11). Notably, Rora haploinsufficiency
results in enhanced pathogenesis in SCA1 transgenic mice
(11). Previous work demonstrates that in the murine cerebel-
lum ATXN1, Tip60 and Rora exist in an endogenous
complex. The ability of GST–ATXN1 bind to in vitro tran-
scribed and translated Tip60 indicates that these proteins inter-
act directly (11). To examine the biological import of the
ATXN1/Tip60 interaction in vivo, we utilized a genetic
approach to investigate the impact of Tip60 haploinsufficiency
in an SCA1 mouse model.
Mapping the Tip60-binding region of ATXN1
Previously using co-immunoprecipitations from WT cerebel-
lar extracts, we showed that ATXN1, Rora and the coregulator
Tip60 exist in a complex in which ATXN1 interacts directly
with Tip60 (11). To examine the Tip60/ATXN1 interaction
in more detail, a series of ATXN1 deletion constructs were
generated and used in a GST-pull-down assay to assess their
ability to interact with Tip60 (Fig. 1A). The results showed
that an ATXN1 deletion construct that contained the AXH
domain alone was sufficient to promote interaction with
Tip60 (Fig. 1B, lane 9). In addition, all of the ATXN1 con-
structs containing the AXH domain [full length, full length
without the self-association region (SAR) (18), fragment IV
and fragment V] bound to Tip60 (Fig. 1B, lanes 3, 4, 6 and
7), while those lacking the AXH domain (fragment I and the
SAR domain alone) did not bind Tip60 (Fig. 1B, lanes 5
and 8). Thus, we conclude that Tip60 binds to ATXN1 via
the AXH domain.
Several lines of evidence indicate that phosphorylation of
Ser776 is critical for the induction of neuronal dysfunction
in SCA1 (12,19–21). Among these are data that a S776D sub-
stitution mimics phosphorylation and enhances disease sever-
ity (12,21). To examine whether D776 might also affect the
interaction of ATXN1 with Tip60, GST pull-downs were per-
formed using ATXN1–fragment V with either WT phosphor-
ylatable Ser, phospho-resistant Ala or phospho-mimicking
Asp at position 776. While substituting an Ala for the Ser at
position 776 had no effect on the ability of ATXN1–fragment
V to interact with Tip60, a phospho-mimicking Asp at residue
776 increased the ability of ATXN1–fragment V to interact
with Tip60 (Fig. 1C), suggesting that the ATXN1/Tip60 inter-
action is modulated by S776 phosphorylation.
ATXN1[82Q] mice with haploinsufficiency of Tip60
As a means to examine the biological relevance of the ATXN1/
gene dosage impacts the SCA1 phenotype. The approach
involved crossing Tip60+/2animals with ATXN1[82Q] mice
from the B05 line in which an expanded ATXN1 transgene is
driven by the Purkinje cell protein 2 (Pcp2/L7) promoter (22).
Heterozygous Tip60 mice were generated using homologous
recombination to replace exons 1–9 with a neomycin-targeting
vector (23). Heterozygous mice with a null Htatip allele
(Tip60+/2) are viable and phenotypically normal. However,
complete Tip60 loss of function (Tip602/2) causes embryonic
lethality near the blastocyst stage (23). The Tip60 heterozygous
mice were on the SV-129;C57BL/6 genetic background,
and the ATXN1[82Q] mice were on the FVB background.
Thus, offspring of this cross had genotypes of WT,
Tip60+/2all on a mixed FVB;SV-129;C57BL/6 background
(Supplementary Material, Fig. S1A).
that Tip60+/2mice had a partial loss of function, we measured
Tip60 mRNA levels in cerebellar lysates. Heterozygous Tip60
null mice expressed approximately half of WT Tip60 levels
(Supplementary Material, Fig. S1B). To assess whether partial
Tip60 loss affected ATXN1[82Q] transgene expression, we
compared transgene expression between ATXN1[82Q] and
ATXN1[82Q] protein cannot be quantitatively solubilized from
Purkinje cells of SCA1 transgenic mice (22), we used RNA
levels to quantify transgene expression. By quantitative-PCR,
ATXN1 transgene expression was found to be similar regardless
of Tip60 gene dosage (Supplementary Material, Fig. S1C). In
addition, ATXN1 immunostaining revealed no detectable differ-
ence in amount or deposition of ATXN1 in Purkinje cells
between ATXN1[82Q] and ATXN1[82Q]:Tip60+/2mice (Sup-
plementary Material, Figs S1D and S1E).
Effect of genetic background on SCA1 phenotypes
Morphological and neurological assessments were used to
examine the extent to which SCA1 phenotypes varied on the
FVB and FVB;SV-129;C57BL/6 mixed backgrounds. Progress-
we determined the molecular layer thickness in WT-FVB and
FVB;SV-129;C57BL/6 mice and whether it varied with age.
WT-FVB cerebella had a relatively stable molecular layer thick-
ness between 5 and 20 weeks of age. On the other hand,
WT-FVB;SV-129;C57BL/6 mice had a slightly thinner molecu-
lar layer that decreased somewhat with age (Fig. 2A). Consistent
with previous data (22,23,25), ATXN1[82Q] on an FVB back-
ground induced a slow, progressive Purkinje cell degeneration
from 5 to 20 weeks of age. In contrast, the ATXN1[82Q]-
FVB;SV-129;C57BL/6 mice had a slightly thinner molecular
layer that decreased rapidly by 12 weeks of age and remained
at 115 mM from 16 to 20 weeks of age (Fig. 2B).
Next, we assessed the motor phenotype using the accelerat-
ing rotarod paradigm. ATXN1[82Q] mice on the FVB genetic
background showed a rotarod deficit as early as 5 weeks of age
and had a more pronounced phenotype at 12 weeks (22,25). In
contrast, ATXN1[82Q] mice on the FVB;SV-129;C57BL/6
genetic background (ATXN1[82Q]-mix) did not show a
rotarod deficit until 30 weeks of age (Fig. 2C). Thus, while
Human Molecular Genetics, 2011, Vol. 20, No. 112205
SCA1 molecular layer pathology was more pronounced on the
mixed background, development of the neurological pheno-
typeas determinedby the
Partial loss of Tip60 slows mid-stage ATXN1[82Q]-induced
Purkinje cell pathology
To determine whether a partial loss of Tip60 affected the SCA1
phenotype, several parameters were compared in WT,
ATXN1[82Q] and ATXN1[82Q]:Tip60+/2mice all on the
mixed FVB;SV-129;C57BL/6 background. We first examined
the effect of Tip60+/2on SCA1 pathology in vivo by calbindin
immunostaining and measurement of the molecular layer thick-
ness (Fig. 3A). As quantified in Figure 3B, partial Tip60 loss
mice relative to ATXN1[82Q]-mix mice during the mid-stage
of disease. While the molecular layer was reduced in both
ATXN1[82Q]-mix and ATXN1[82Q]:Tip60+/2-mix mice at 5
weeks of age, at 12 and 16 weeks ATXN1[82Q]:Tip60+/2-mix
Figure 1. The interaction of ATXN1 with Tip60 requires the AXH domain of ATXN1 and is enhanced by a phospho-mimicking Asp at residue 776. (A) Dia-
grams of the GST–ATXN1 constructs used to pull-down His-tagged35S-labeled Tip60. (B) Representative polyacrylamide gel showing the ability of GST–
ATXN1 to pull-down His-tagged35S-labeled Tip60. Upper panel depicts an autoradiograph of35S-labeled Tip60 pulled down by each GST–ATXN1 construct.
Lower panel shows Coomassie stained GST–ATXN1 constructs. (C) Quantification of Tip60 pulled down by GST–ATXN1–fragment V in which the amino
acid at position 776 was either a Ser (S776), a phosphorylation resistant Ala (S776A) or phospho-mimicking Asp (S776D).
2206Human Molecular Genetics, 2011, Vol. 20, No. 11
cerebella had significantly thicker molecular layer than their
ATXN1[82Q]-mix mice had an average molecular layer
thickness of 115 mM. In contrast, at 12 and 16 weeks,
ATXN1[82Q]:Tip60+/2-mix molecular layer thickness was
134 and 143 mM, respectively. At 20 weeks, ATXN1
[82Q]:Tip60+/2-mix molecular layer thickness thinned to
114 mM, identical to ATXN1[82Q]-mix littermates (115 mM,
unchanged from 12 and 16 weeks) (Fig. 3B). Thus, partial
loss of Tip60 resulted in a protective window between 12 and
16 weeks during which molecular layer thinning was slowed.
As an additional assessment of ATXN1[82Q]-induced path-
ology, we examined placement of excitatory synaptic terminals
onto Purkinje cells in SCA1 mice. Purkinje cells receive affer-
mate transporter VGluT2 in their synaptic vesicles (26).
At 12 and 16weeks,
Previously, we showed that the extension of climbing fiber
terminals along the Purkinje cell dendritic tree is compromised
in ATXN1[82Q]-mix mice (21). Using calbindin immunofluor-
escence to visualize Purkinje cell dendrites and VGluT2 immu-
nofluorescence to visualize climbing fiber terminals, the extent
drites was measured in ATXN1[82Q]:Tip60+/2-mix cerebella
(Fig. 3C). At all ages, WT mice had a significantly greater
with ATXN1[82Q]-mix expressing mice (Fig. 3D). However, at
12 and 16 weeks, partial loss of Tip60 resulted in a reduction in
the extent to which climbing fiber terminal extension was
compromised by ATXN1[82Q]. At 12 and 16 weeks,
ATXN1[82Q]:Tip60+/2-mix mice had a significantly greater
climbing fiber extension along Purkinje cell dendrites than
their ATXN1[82Q]-mix littermates. By 20 weeks of age, exten-
sion of climbing fiber terminals in ATXN1[82Q]:Tip60+/2-mix
mice was as compromised as in ATXN1[82Q]-mix animals
(Fig. 3D). Thus, by a second measure of ATXN1[82Q]-induced
pathology, partial loss of Tip60 afforded a protective period
during the mid-stage, 12–16 weeks of age, of disease.
Restoration of ATXN1[82Q]-induced changes in Rora, and
Rora-mediated gene expression by a partial loss of Tip60
In ATXN1[82Q]-mix mice, mutant ATXN1 depletes Rora from
cerebellar Purkinje cells and Rora haploinsufficiency results in
enhanced pathogenesis in SCA1 transgenic mice (11). For the
following set of experiments, we focused on a group of genes
downregulated in both SCA1 and staggerer mice. Within this
group, a subset of genes is known to bind Rora at the promoter
(11,27–30). We sought to determine whether the protective
effects on ATXN1[82Q]-induced pathology by Tip60+/2cor-
related with restoration of Rora and Rora-mediated gene
Western blot analysis revealed that partial Tip60 loss
increased Rora protein expression relative to ATXN1[82Q]-mix
littermates (Fig. 4A, lanes 3 versus 4). Next, we characterized
the effect of partial Tip60 loss on those Rora-mediated genes
whose expression was previously found decreased in SCA1
and staggerer mouse cerebella (11). These include Pcp2 (Pur-
kinje cell protein 2), Pcp4 (Purkinje cell protein 4), Slc1a6
(solute carrier family 1-high affinity aspartate/glutamate trans-
porter 6) and Itpr1 (Inositol 1,4,5-triphosphate receptor, type
1). Gene expression was assessed at three ages: 8 weeks, an
early stage with intermediate Purkinje cell atrophy; 12 weeks,
mid-stage during the period of the Tip60+/2-induced protec-
tion; and 20 weeks, late stage when both ATXN1[82Q]-mix
and ATXN1[82Q]:Tip60+/2-mix Purkinje cell atrophy reached
the same advanced level. At all ages, WT-mix and
Tip60+/2-mix gave the same results (data not shown).
In 8-week-old mice, there were no significant differences
Tip60+/2-mix expression of Slc1a6, Pcp4, Pcp2 or Itpr1
Figure 2. GeneticbackgroundaffectsSCA1phenotypesinATXN1[82Q]trans-
genic mice. (A) Cerebellar molecular layer thickness in aging WT-FVB and
FVB;SV-129;C57BL/6 (mix) mice (n ¼ 3–7 mice/genotype/age). (B) Cerebel-
lar molecular layer thickness in aging FVB and FVB;SV-129;C57BL/6 (mix)
mice expressing the ATXN1[82Q] transgene (n ¼ 3–7 mice/genotype/age).
(C) Motor performance using the accelerating rotarod paradigm in aging
FVB;SV-129;C57BL/6 (mix) mice expressing the ATXN1[82Q] transgene
(n ¼ 6–12 mice/genotype).
Figure 3. Tip60 haploinsufficiency rescues SCA1 cerebellar pathology during the mid-stage of disease. (A) Calbindin immunofluorescence of Purkinje cells in
aging WT, ATXN1[82Q] and ATXN1[82Q]:Tip60+/2FVB;SV-129;C57BL/6 (mix) mice. (B) Quantitative analysis of the molecular thickness in aging WT,
ATXN1[82Q] and ATXN1[82Q]:Tip60+/2FVB;SV-129;C57BL/6 (mix) mice (n ¼ 3–7 mice/genotype/age). (C) Immunofluorescence of calbindin (red) and
VGluT2 (green) in WT, ATXN1[82Q] and ATXN1[82Q]:Tip60+/2FVB;SV-129;C57BL/6 (mix) mice. (D) Quantitative analysis of the climbing fiber extension
along Purkinje cell dendrites in aging WT, ATXN1[82Q] and ATXN1[82Q]:Tip60+/2FVB;SV-129;C57BL/6 (mix) mice (n ¼ 3–5 mice/genotype/age).
Human Molecular Genetics, 2011, Vol. 20, No. 112207
2208 Human Molecular Genetics, 2011, Vol. 20, No. 11
(Fig. 4B). At 12 weeks, Slc1a6 and Pcp4 expression were sig-
nificantly higher in ATXN1[82Q]:Tip60+/2-mix mice com-
pared with ATXN1[82Q]-mix mice (Fig. 4C). At 20 weeks in
addition to Slc1a6 and Pcp4 expression, Itpr1 was also signifi-
cantly higher in ATXN1[82Q]:Tip60+/2than ATXN1[82Q]
mice (Fig. 4D). Interestingly, Slca6 expression at 20 weeks
was significantly higher than in WT-mix cerebella. Thus, in
the presence of ATXN1[82Q] and with increasing age, the
mally elevated in Tip60 haploinsufficient mice. We also
examined the expression of three SCA1/staggerer downregu-
lated Rora-mediated genes (Calb1, Idb2 and Cals) at 20
weeks of age, which had previously been shown to not have
Tip60 at the promoter complex (29). This is in contrast to
Slca6 and Pcp4, which have Rora-dependent recruitment of
Tip60 to the promoter complex (29). In each case, expression
Previously, we showed that ATXN1, Rora and the coregulator
Tip60 exist in a complex in which ATXN1 interacts directly
with Tip60 (11). In the present study, we further examined
the interaction of ATXN1 and Tip60, showing that this inter-
action is both dependent on ATXN1’s AXH domain and
enhanced by a phospho-mimicking Asp at position 776.
Importantly, the AXH domain is required for mutant,
polyglutamine-containing ATXN1 to induce neurodegenera-
tion (7). Recently, we showed that a phospho-mimicking
Asp at residue 776 enhances ATXN1-induced pathogenesis
(21). Thus, Tip60 interacts with a region of ATXN1 crucial
for disease, and this interaction is promoted by an amino
acid substitution that enhances pathogenesis. Moreover, the
finding that D776 promotes the ATXN1/Tip60 interaction
strengthens the concept that the conformational change
induced by S776 phosphorylation includes the AXH domain.
Based on these features, we hypothesized that the ATXN1/
Tip60 interaction has biological relevance.
As a basis for examining the ATXN1/Tip60 interaction in
vivo, we reasoned that reducing Tip60 levels might modify
ATXN1[82Q]-induced disease. We found that a partial loss
of Tip60 slowed the progression of ATXN1[82Q]-induced
Purkinje cell atrophy. The protective effect of reduced Tip60
levels was associated with increased Rora protein and Rora-
mediated gene expression. These results indicate that Tip60-
mediated pathways contribute to SCA1.
By two measures of pathology, we found Purkinje cell
degeneration was slowed in ATXN1[82Q]:Tip60+/2-mix com-
pared with ATXN1[82Q]-mix animals. The protective effect of
a partial loss of Tip60 was restricted to the period between 12
and 16 weeks of age, i.e. a mid-stage of disease progression in
this model of Purkinje cell disease in SCA1. The observation
that Tip60+/2seems not to have affected disease onset and
that its protective effect diminished with age raises some inter-
esting points. First, the lack of a Tip60+/2effect on early
disease suggests that the ATXN1/Tip60 interaction does not
impact ATXN1’s postulated role in Purkinje cell development
and disease onset (11). Rather, the data suggest that the inter-
action of mutant ATXN1 with Tip60 plays a role in SCA1 pro-
gression after disease initiation. The pattern of Tip60+/2
protective effect adds to the concept that disease initiation
and progression result from mutant ATXN1’s effects on
various and complex cellular pathways. The Tip60+/2protec-
tive window corresponds to the time period previously shown
Figure 4. Tip60 haploinsufficiency restoration of Rora levels (n ¼ 3 mice) and Rora-mediated gene expression. (A) Western blot analysis of cerebellar Rora
levels in WT, ATXN1[82Q] and ATXN1[82Q]:Tip60+/2FVB;SV-129;C57BL/6 (mix) mice, and in homozygous staggerer (sg/sg) mice at 12 weeks of age.
COS cells transfected with Rora cDNA are shown as a positive control. (B) Quantitative PCR of four Rora-mediated genes in WT, ATXN1[82Q] and
ATXN1[82Q]:Tip60+/2FVB;SV-129;C57BL/6 (mix) mice at 8 weeks of age. (C) Quantitative PCR of four Rora-mediated genes in WT, ATXN1[82Q]
and ATXN1[82Q]Tip60+/2FVB;SV-129;C57BL/6 (mix) mice at 12 weeks of age. (D) Quantitative PCR of four Rora-mediated genes in WT, ATXN1[82Q]
and ATXN1[82Q]:Tip60+/2FVB;SV-129;C57BL/6 (mix) mice at 20 weeks of age.
Human Molecular Genetics, 2011, Vol. 20, No. 11 2209
to separate disease initiation and neuronal dysfunction from
subsequent Purkinje cell death (21), suggesting that perhaps
the pathways critical for progression to neuronal death
overlap with those that limit the extent of the Tip60+/2protec-
The pattern of the Tip60+/2effect on Rora-mediated gene
expression illustrates the complex role of this system in
SCA1. Both Rora and Rora-mediated gene expression are
decreased in ATXN1[82Q] mutant mice (11). Consistent with
Rora’s role in SCA1 pathogenesis, the ATXN1[82Q]-induced
loss of Rora was reversed at 12 weeks of age in
ATXN1[82Q]:Tip60+/2-mix animals. Moreover, Tip60+/2
was also associated with a restoration of certain Rora-mediated
genes at 12 weeks of age. Interestingly, not all Rora-mediated
gene expression levels were restored by partial Tip60 loss at
12 weeks of age. However, by 20 weeks of age, expression of
all four Rora-mediated genes examined was at or above the
WT level. Thus, the restoration of some Rora-mediated genes
(e.g. Slc1a6 and Pcp4) occurred early in the Tip60+/2protec-
tive window, while the restoration of others was delayed (e.g.
Pcp2 and Itpr1). This suggests that only a subset of Rora-
mediated genes may contribute to the Tip60+/2protective
effect. It is important to note that Pcp4 and Slc1a6 are not
only genes with Rora-mediated expression, but are also genes
with Rora-dependent recruitment of Tip60 to the promoter
complex (29). Conversely, the expression of three genes
examinedthat didnot show
ATXN1[82Q];Tip60+/2mice (Calb1, Grm1 and Cals) are
genes where Rora does not recruit Tip60 to the transcription
complex (29). Interestingly, expression of all of the four Rora-
mediated genes that were elevated in ATXN1[82Q]:Tip60+/2
mice remained elevated at 20 weeks of age when Tip60+/2
of certain Rora-mediated genes, e.g. Slc1a6, contributes to
Whether partial Tip60 loss enhances Rora-mediated gene
expression by relieving ATXN1-induced Rora depletion or
perhaps alters specific posttranslational modifications that
impact Rora-mediated transcription remains an open question.
Tip60 was shown to be a co-activator for the androgen nuclear
receptor (32). Moreover, androgen receptor activation by
Tip60 acetylation requires direct interaction via Tip60’s
LXXL motif (33). It is intriguing to speculate that perhaps
the acetyltransferase activity of Tip60 is in some way con-
nected to the decrease in cerebellar Rora induced by
ATXN1[82Q]. If this were to be the case, this would
support a model where the function of Tip60, coactivator
versus corepressor, varies depending on the nuclear receptor
and other proteins with which it associates.
Our results also showed that the time course and severity of
SCA1 symptoms in the mouse varied when the same mutant
ATXN1[82Q] transgene was expressed on two different
genetic backgrounds. Purkinje cell degeneration was more
rapid when ATXN1[82Q] was expressed on the mixed
genetic background versus the FVB background. Moreover,
development of motor performance deficit (ataxia) was
delayed on the mixed background compared with the FVB
background. This strongly suggests the presence of genetic
modifiers regulating the severity of SCA1 phenotypes in
mice. Disease presentation in SCA1 patients is also greatly
influenced by non-polyglutamine factors, which may include
genetic modifiers (34,35). Identifying the genetic modifiers
that influence SCA1 phenotypes in mice should illuminate
additional pathways that influence neurodegeneration.
In summary, we conclude the ATXN1/Tip60 interaction
contributes to SCA1 pathogenesis with a partial loss of
Tip60 delaying cerebellar degeneration in an SCA1 mouse
model, specifically during mid-stage disease. These findings,
taken with the observation that ATXN1[82Q]-mediated path-
ology and ATXN1[82Q]-mediated motor deficits are differen-
tially influenced by genetic background, indicate that not only
is the relationship between the molecular pathways that
underlie disease initiation and progression complex, so is the
relationship between pathology and neuronal dysfunction.
MATERIALS AND METHODS
In vitro transcription/translation GST pull-down assay
ATXN1 cDNAs were cloned into pGEX vectors (Amersham
Biosciences) and expressed in E. coli BL21(DE3) cells.
Human Tip60 was cloned into pCDNA3.1/His expression
vector (Invitrogen). In vitro transcription/translation and
GST pull-down were done as described (11). Samples were
run on NuPage 4–12% Bis–Tris polyacrylamide gels (Invitro-
gen), stained with SimplyBlue SafeStain colloidal Coomassie
(Invitrogen) and dried on Whatman 3MM paper. Amount of
ATXN1 was measured by densitometry of the Coomassie
staining, and bound radioactive Tip60 was determined by
autoradiography followed by band excision and scintillation
ATXN1[82Q]:Tip60+/2mice were the F1progeny (1:1:1:1)
resulting from breeding ATXN1[82Q] and Tip60+/2mice.
ATXN1[82Q] mice were maintained on the FVB background
and the Tip60+/2mice on a SV-129;C57BL/6 background.
Genotyping of mice. PCR was used to identify Tip60+/2and
ATXN1[82Q] transgenic animals as described by Hu et al.
(23) and Burright et al. (22), respectively.
Immunostaining and quantitative measurements
Animals were perfused with 10% formalin and 50 micron
vibratome sections cut as described (21). Floating cerebellar
slices were incubated with the following primary antibodies
in blocking buffer [2% donkey serum, 0.3% Triton X-100 in
1× phosphate buffered saline (PBS)] at dilutions indicated:
goat calbindin (SC-7691, Santa Cruz Biotechnology) at
1:500, rabbit 11750/ataxin-1 at 1:2500 and mouse VGLUT2
(MAB5504, Millipore) at 1:1000. Secondary antibodies used
were: donkey anti-goat Cy3 (#705-165-147, Jackson Immu-
noresearch) at 1:500, donkey anti-rabbit Cy2 (#711-225-152,
Jackson Immunoresearch) at 1:500 and donkey anti-mouse
Cy5 (#115-175-146, Jackson Immunoresearch) at 1:500. Sec-
tions were washed, mounted onto microscope slides with gly-
cerol–gelatin containing 4 mg/ml n-proplyl gallate (Sigma)
2210Human Molecular Genetics, 2011, Vol. 20, No. 11
laser-scanning microscope (#FV1000 IX2, Olympus). Molecu-
lar layer thickness was measured with FluoView software.
For molecular layer measurements, calbindin-stained Pur-
kinje cells were analyzed from two sagittal sections that
were analyzed per mouse from at least five mice per genotype.
Using confocal laser-scanning microscopy, six measurements
were taken at the primary fissure and averaged to determine
molecular layer thickness (21,24). For climbing fiber-Purkinje
cell measurements, climbing fibers were similarly measured
with VGluT2 antibody staining from six measurements at
the primary fissures of two sagittal sections per mouse in at
least three mice per genotype. Purkinje cell dendrite lengths
were obtained as stated above, and the climbing fiber:Purkinje
cell ratio was calculated with averaged measurements. Data
are expressed as the mean+SEM. The P-value was calcu-
lated using Student’s t-test (two-tailed equal variance).
visualized ona FluoViewinverted confocal,
Accelerating Rotarod (Model 7650 Ugo Basile) analysis was
performed at 9, 12, 16, 20 and 30 weeks of age as described
(25). Student’s t-test was used to assess statistical significance.
Western blot analysis
Mouse cerebella were homogenized in brain extraction buffer:
0.25 M Tris–HCl pH 7.5 with phosphatase inhibitors cocktails
I and II (P2850, P5726 Sigma) and protease inhibitors
(1183617001, Roche). Forty milligrams of protein were
denatured, run on 4–12% Bis–Tris Gel (NP0321BOX, Invi-
trogen) and blotted on nitrocellulose membrane (Protran BA
85, Whatman/GE Healthcare). Membranes were blocked over-
night at 48C with 10% blotto (10% w/v milk in 1× tris buf-
fered saline) with 0.1% v/v Tween-20. Blocked membranes
were incubated with RORa antibody (H-65 #sc-28612, Santa
Cruz Biotechnology) for 1 h at 228C, washed three times
with 0.1% PBS + 0.1% tween and incubated with anti-goat
horse radisn peroxidase secondary antibody for 45 min.
Samples were probed with mouse anti-GAPDH (#MAB374,
Chemicon) as a loading control. Densitometry was used to
quantify protein levels.
Total RNA was isolated from the cerebella of ATXN1[82Q],
ATXN1[82Q]:Tip60+/2and WT littermates at 8, 12 and 20
weeks using the TRIZOL method (Invitrogen). A minimum of
three mice was used per genotype. Fifty nanograms of total
RNA were used per reaction in triplicate with the TaqMan
one-step reverse transcriptase (RT)–PCR kit (#4309169,
Applied Biosytems). The following probes were assayed from
(Mm00500973_m1), Slc1a6 (Mm00436591_m1) and Itpr1
(Mm01183049_m1). Samples were normalized to GAPDH.
Reactions were run on a Real-time Quantitative PCR System
ABI PRISM 7500.
Cell growth and transfection. For Rora experiments, CHO
cells were grown in GibcoTMMinimum Essential Medium
(MEM) and Alpha Medium (#32561037, Invitrogen) sup-
plemented with 10% FBS and Pen/strep. Cells were plated
in media without antibiotics 24 h before transfection at
105cells/ml unless otherwise specified. Cells were transfected
with Lipofectamine Plus (#11514-015, Invitrogen).
Cell culture lysates. At 48 h post-transfection, cells were lysed
in Tris-Triton lysis buffer (50 mM Tris–HCl pH 7.5, 2.5 mM
MgCl2, 100 mM NaCl, 0.5% Triton), with phosphatase inhibi-
tors, protease inhibitors and sodium butyrate. Protein was run
on 4–12% Bis–Tris gels.
Data were expressed as the mean+standard error of the
mean. Statistical comparisons were made with student’s
t-test unless otherwise noted.
Supplementary Material is available at HMG online.
We are grateful to Robert Ehlenfeldt and Orion Rainwater for
managing the SCA1 mouse colony.
Conflict of Interest statement. None declared.
This work was supported by National Institutes of Health
Funding to pay the Open Access publication charges for this
article was provided by a Ruth L. Kirschstein NRSA to K.M.G.
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