Neurobiology of Disease 82 (2015) 66–77
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Neurobiology of Disease
journal homepage: www.elsevier.com/locate/ynbdi
Ectopic expression of the striatal-enriched GTPase Rhes elicits cerebellar
degeneration and an ataxia phenotype in Huntington's disease
Supriya Swarnkar, Youjun Chen, William M. Pryor, Neelam Shahani, Damon T. Page, Srinivasa Subramaniam ⁎
Department of Neuroscience, The Scripps Research Institute, Jupiter, Florida 33458, USA
a r t i c l e
i n f o
Article history:
Received 4 November 2014
Revised 2 April 2015
Accepted 26 May 2015
Available online 3 June 2015
Keywords:
Cerebellar pathology
Ectopic expression
Soluble Huntingtin
Neuronal death
Stereotaxy
Knock-in HD mice
Transgenic HD mice
Rhes
Huntington's disease
Neurodegeneration
Ataxia
a b s t r a c t
Huntington's disease (HD) is caused by an expansion of glutamine repeats in the huntingtin protein (mHtt) that
invokes early and prominent damage of the striatum, a region that controls motor behaviors. Despite its ubiquitous
expression, why certain brain regions, such as the cerebellum, are relatively spared from neuronal loss by mHtt
remains unclear. Previously, we implicated the striatal-enriched GTPase, Rhes (Ras homolog enriched in
the striatum), which binds and SUMOylates mHtt and increases its solubility and cellular cytotoxicity, as the
cause for striatal toxicity in HD. Here, we report that Rhes deletion in HD mice (N171-82Q), which express the
N-terminal fragment of human Htt with 82 glutamines (Rhes−/−/N171-82Q), display markedly reduced HDrelated behavioral deficits, and absence of lateral ventricle dilatation (secondary to striatal atrophy), compared to
control HD mice (N171-82Q). To further validate the role of GTPase Rhes in HD, we tested whether ectopic Rhes expression would elicit a pathology in a brain region normally less affected in HD. Remarkably, ectopic expression of
Rhes in the cerebellum of N171-82Q mice, during the asymptomatic period led to an exacerbation of motor deficits,
including loss of balance and motor incoordination with ataxia-like features, not apparent in control-injected N17182Q mice or Rhes injected wild-type mice. Pathological and biochemical analysis of Rhes-injected N171-82Q mice
revealed a cerebellar lesion with marked loss of Purkinje neuron layer parvalbumin-immunoreactivity, induction of
caspase 3 activation, and enhanced soluble forms of mHtt. Similarly reintroducing Rhes into the striatum of Rhes
deleted Rhes−/−Hdh150Q/150Q knock-in mice, elicited a progressive HD-associated rotarod deficit. Overall, these studies establish that Rhes plays a pivotal role in vivo for the selective toxicity of mHtt in HD.
© 2015 Elsevier Inc. All rights reserved.
Introduction
Huntington's disease (HD) is a Mendelian-dominant condition that, in
the majority of HD patients, has symptoms-onset in middle age. In 1993,
cloning of the affected gene that codes for the protein huntingtin (Htt) offered the possibility of genetic diagnosis (The Huntington's Disease
Collaborative Research Group, 1993). Normal Htt protein contains
11–34 tandem glutamines near the N-terminus of the protein (commencing at the 18th amino acid position). Expansion of glutamines in
Htt to more than 37 glutamines (mHtt) causes HD, with a larger numbers
correlating with younger age of onset. Early symptoms of HD include psychiatric disturbances, but the predominant symptomatology is motoric
and leads to the earlier designation of the disease as Huntington's chorea.
The motor disabilities stem from massive damage to the corpus striatum,
which can shrink to as little as 10% of its normal volume in advanced disease. Cortical abnormalities are seen in early Huntington's disease
(Nopoulos et al., 2010). Surprisingly, the cerebellum is fairly impervious
to damage from HD (Gutekunst et al., 2002; Jackson et al., 1995; Tabrizi
⁎ Corresponding author. Fax: +1 561 228 2107.
E-mail address: ssubrama@scripps.edu (S. Subramaniam).
Available online on ScienceDirect (www.sciencedirect.com).
http://dx.doi.org/10.1016/j.nbd.2015.05.011
0969-9961/© 2015 Elsevier Inc. All rights reserved.
et al., 1999). Within the striatum, most of the medium spiny neurons
(MSN) are destroyed, but small interneurons, enriched in neuronal nitric
oxide synthase (nNOS), remain normal, even in advanced disease
(Ferrante et al., 1985). Despite the ubiquitous expression of mHtt in all organs of the body, why the pathology and the symptoms of HD appear to
be predominantly limited to the brain and its specific regions such as striatum remains unclear.
Over the last few years, several important findings about how mHtt
might kill MSN have emerged. Both cell-autonomous and nonautonomous mechanisms contribute to striatal damage in HD
(reviewed, (Ehrlich, 2012)). Numerous investigators have sought proteins that might interact uniquely with mHtt to cause damage. Many
Htt protein interactors and a number of mechanisms linking these
interactors to HD pathogenesis have been described (Kaltenbach et al.,
2007; Landles and Bates, 2004; Li and Li, 2004; Ross, 2004). mHtt forms
aggregates in tissues and were first thought to be the cause of the cellular
toxicity. However, several studies have now discovered that the soluble,
non-aggregated (or oligomeric) forms of mHtt are most toxic culprits
(Arrasate et al., 2004; Kitamura et al., 2006; Poirier et al., 2005;
Ratovitski et al., 2009; Saudou et al., 1998). Despite such increased knowledge about the mechanisms, it is less clear why selective regions such as
the corpus striatum, but not the cerebellum, are vulnerable to damage.
S. Swarnkar et al. / Neurobiology of Disease 82 (2015) 66–77
67
were crossed with heterozygous Hdh150Q (B6.129P2-Htttm2Detl/150J,
004595) mice from Jackson Laboratories and maintained in our state-ofthe-art animal breeding facility. We obtained in first-generation three
heterozygous male mice positive for Rhes+/−/Hdh150Q/−, which were
backcrossed Rhes+/−/Hdh150Q/− X Rhes−/− and resulted in 5 heterozygous (Rhes−/−/Hdh150Q/−) mice (3♂ and 2♀). Rhes-deleted Hdh150Q heterozygous mice were crossed (Rhes−/−/Hdh150Q/− X Rhes−/−/Hdh150Q/−)
to obtain Rhes deleted Hdh150Q homozygous mice (Rhes−/−/Hdh150Q/150Q)
along with littermate controls. Genotyping was confirmed for Hdh150Q
as per genotyping protocol from Jackson Laboratories and for Rhes as
per the standardized protocol as described before (Errico et al., 2008).
Previously, we have implicated Rhes as a mediator of mHtt toxicity.
Rhes, a 266-amino acid protein, contains a GTP-binding domain and a
SUMO E3 ligase domain, and is highly expressed in the striatum with
much lower levels in other regions, such as the cortex, which has little
or no expression in the cerebellum (Falk et al., 1999; Jiang et al., 2013;
Usui et al., 1994). Human expression data from Allen Brain atlas
confirms with striking enrichment in the striatum with little to no
expression in cerebellum. Although its role in the striatum is not yet
clear, several studies have demonstrated a role for Rhes in regulating
the G-protein-coupled receptor signaling of thyroid hormones and
dopamine (Errico et al., 2008; Vargiu et al., 2004). Behavioral analysis
of Rhes knockout mice, using dopamine-related drugs, suggests that
Rhes may have a role in motor coordination (reviewed, (Harrison,
2012)). We found that Rhes binds to mHtt and promotes cellular toxicity
in cell culture models by acting as SUMO-E3 ligase, which is confirmed by
a recent study (O'Rourke et al., 2013; Subramaniam et al., 2010;
Subramaniam et al., 2009). In addition, the role of Rhes in HD has also
been supported by other recent reports (Baiamonte et al., 2013; Lu and
Palacino, 2013; Mealer et al., 2013; Okamoto et al., 2009; Sbodio et al.,
2013; Seredenina et al., 2011), yet its definitive role in HD pathogenesis
in vivo remains unclear. Here, using knockout and overexpression strategies, we show that Rhes plays a major role in phenotypic and pathological
deficits of transgenic and knock-in HD mouse models being pivotal for
the selective regional toxicity of mHtt.
Stereotaxic injection of Rhes into the cerebellum of N171-82Q mice
The N171-82Q mice and wild-type littermates were injected in deep
cerebellar nuclei of cerebellum with either adenovirus-CMV-null
(abmgood) or adenovirus-CMV-Rhes (4 μl/injection, 1 × 1010 opu/μl)
(n = 7/group/mixed sex), using coordinates (anteriorposterior, −5.75
from bregma; mediolateral, − 1.8 from bregma; dorsoventral, − 2.6
from dura; incisor bar, 0.0), with a 10 μl Hamilton syringe at a rate of
0.5 μl/min (Dodge et al., 2005) for a total of 4 μl (1 × 1010 opu/μl) per injection site. The stereotaxic coordinates were validated using a fast
green dye, showing an effective distribution throughout the cerebellum
(Supplementary Fig. 1).
Stereotaxic injection of Rhes into the striatum of Rhes−/−/Hdh150Q/150Q mice
The Rhes deleted Hdh150Q mice and wild-type littermates
(~ 6–7 months old) were injected in striatum (n = 4/group/mixed
sex) with either adenovirus-CMV-null (abmgood) or adenovirusCMV-Rhes (0.5 μl/injection, 1 × 1010 opu/μl) and adenovirus-CMVGFP (0.8 μl/injection, 5.6 × 109 opu/μl) (n = 4/group/mixed sex). The
viral particle were stereotactically injected bilaterally in the dorsal striatum at the coordinates ML = ±1.50, AV = + 1.2, DV = − 3.25/3.75
and ML = ± 2.25, AV = 0, DV = − 3.25/3.75 from Bregma, with a
10 μl Hamilton syringe at a rate of 0.2 μl/min (Pryor et al., 2014). Proper
postoperative care was done till the animals recovered completely.
Materials and methods
Reagents and chemicals
Unless otherwise specified, chemicals and reagents were purchased
from Sigma (St. Louis. MO). EM-48 antibody was obtained from
Millipore (MAB5374). Antibodies against mTOR (2972S), pS6K T389
(9234S), S6K (9202S), Cleaved Caspase-3 (9661S) and huntingtin
(5656S) were from Cell Signaling Technology, Inc. Antibody for actin
was from Santa-Cruz. Pre-made adenoviral-control (null), adenoviralGFP, and adenoviral-Rhes particles were purchased from Applied Biologicals (Abm good, British Columbia, Canada).
Behavior analysis
Behavioral testing was performed as described in our previous work
(Pryor et al., 2014) during the light phase of the light–dark cycle between
8:00 am and 12:00 pm. For Rhes KO/transgenic N171-82Q mice, behavior
was recorded at 8, 12, and 16 weeks. Each set of behavioral testing
included body weight measurement and open-field test on the first
day, and rotarod on the second. For cerebellar injections, seven-week
old animals were pre-trained to stay on an accelerating rotarod starting
with 2–4 rpm and increased up to 40 rpm in 300 s (cut-off time) before
surgery. On the 4th, 9th, and 14th day post injection/surgery, all the behavioral tests were, sequentially carried out, including body weight and
open field tests in the morning, and rotarod and behavioral scoring in
the evening (Pryor et al., 2014).
Behavioral scoring was adapted from assessment previously reported
(Guyenet et al., 2010). The scoring consisted of a ledge test, clasping, gait,
kyphosis, and tremor. An unbiased person who was blind for animal's
genotype performed all the scoring. Individual measures were scored
on a scale of 0–3, with 0 representing an absence of the relevant phenotype and 3 representing the most severe manifestation. Each test was
Generation of Rhes-deleted N171-82Q mice
Homozygous Rhes KO mice, used in our earlier work (Mealer et al.,
2014; Subramaniam et al., 2010), were crossed with N171-82Q mice
from Jackson Laboratories and maintained by crossing C57BL6 × C3H/
B6-F1 females with hemizygous N171-82Q (HD) mice (strain 003627)
in our state-of-the-art animal breeding facility. We obtained eight
first-generation heterozygous mice positive for Rhes+/−/N171-82Q,
which were backcrossed Rhes+/−/N171-82Q X Rhes+/−/N171-82Q to
obtain homozygous Rhes-deleted Rhes−/−/N171-82Q mice. A total of
six groups of mice, shown in Table 1, were used for the longitudinal
longitudinal behavioral, striatal morphology, and biochemical analysis. Genotyping was confirmed by transnetyx (Cordova, TN).
Generation of Rhes-deleted Hdh150Q knock-in mice
(Rhes−/−/Hdh150Q/150Q)
Homozygous Rhes KO mice, used in our early work (Mealer et al.,
2014; Subramaniam et al., 2010), and wild-type (WT) littermates
Table 1
Mice used for the longitudinal behavioral, and striatal morphology, and biochemical analysis.
Rhes−/−
Rhes+/−
Genotype
WT
Sex
♂
♀
♂
♀
♂
Cohort 1
Cohort 2
Cohort 3
Cohort 4
Total
0
0
0
6
6
0
0
0
6
6
3
1
2
0
6
1
1
4
0
6
1
3
2
0
6
Rhes+/−/N171-82Q
Rhes−/−/N171-82Q
N171-82Q
♀
♂
♀
♂
♀
♂
♀
3
2
1
0
6
2
3
1
0
6
1
4
1
0
6
2
3
1
0
6
1
2
3
0
6
0
5
1
6
12
4
0
2
6
12
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S. Swarnkar et al. / Neurobiology of Disease 82 (2015) 66–77
performed in triplicate. To determine tremor, mice were placed in a clean
cage and observed for 30 seconds. Each mouse was scored as follows: 0,
no signs of tremor, 1, present but mild tremor, 2, severe intervals of tremor or constant moderate tremor, and 3, outrageous chronic tremor. All
protocols were approved by The Scripps Research Institute Florida Institutional Animal Care and Use Committee.
Immunohistochemistry
Animals were anesthetized with a 250 mg/kg dose of Avertin (2,2,2Tribromoethanol, T48402, Sigma-Aldrich) [Stock solution: 1 g/ml in
Tert-Amyl-Alcohol (2-Methyl-2-butanol, 152463, Sigma-Aldrich); working solution: 25 mg/ml in PBS] and perfused with 4% paraformaldehyde,
PFA (Sigma, St Louis, MO) in PBS (pH 7.4) through the left cardiac ventricle, as described. The brains were removed and post-fixed in 4% PFA overnight, before transfer into 20% sucrose in PBS for 48 h at 4 °C. The coronal
brain sections were collected using a cryostat (Leica) on + charged slides
(Fisher superfrost plus), 40 μm thick sections at 200 μm intervals for the
striatum, and 30 μm thick sections at 150 μm intervals for the cerebellum.
Histopathological changes were detected by immunostaining of frozen
sections with parvalbumin (P3088, Sigma) at 1:2000 dilutions,
NeuroTraceTM Fluorescent Nissl Stains (Green-N21480, Molecular
Probes) at 1:50 dilution, and DAPI at a 1:1000 dilution. Alexa Fluor secondary antibodies (Molecular Probes) were used to detect bound primary antibodies. Brain sections were mounted in Vectashield hard-set and
images were acquired using VS120 (Olympus) scanner. The average for
total area of lateral ventricle on both left and right hemisphere was calculated using VS-Desktop-V2.4 Olympus software for all the groups. The
confocal high-resolution images of the cerebellar regions were captured
using Leica TCS SP8 confocal microscope. The number of Parvalbuminpositive cells was manually counted on each confocal image.
Western blotting
Mouse brain cerebellum was homogenized in RIPA buffer (50 mM
Tris, pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, and
0.1% SDS) with a protease inhibitor cocktail (Roche) and phosphatase
inhibitor II (Sigma). The homogenate of HEK-293 cells infected with
Adeno Rhes virus (5–10 μg of protein) was loaded as a positive control.
Then, 50–100 μg proteins were separated by electrophoresis in 4–12%
Bis–Tris Gel (Invitrogen), transferred to PVDF membranes, and Western
blotting carried out as previously described (Pryor et al., 2014; Shahani
et al., 2014). An apparent molecular weight of Rhes is 28-32 kDa. In addition, a prominent Rhes band was seen at 38-42 kDa in WT but not in
KO Rhes striatum (data not shown), which is consistent with previous
report (Errico et al., 2004). Ectopically expressed Rhes, either in the striatum or cerebellum, also showed a prominent band at 38-42 kDa suggesting that Rhes in vivo may exist in a posttranslationally
(presumably SUMO) modified form (Subramaniam, 2009).
Statistics
The statistical significance was determined using the two-way
ANOVA Newman–Keuls multiple comparisons post-hoc test or in some
cases (example, western blots) Student's t-test to compare the two
groups (***p b 0.001, **p b 0.01 and *p b 0.05).
Results
Rhes deletion ameliorates HD-related motor deficits in N171-82Q mice
To elucidate the genetic-basis for the role of Rhes in HD, we crossed
Rhes knockout mice (Rhes−/−) (Mealer et al., 2013; Spano et al., 2004;
Subramaniam et al., 2010, 2012) with N171-82Q mice, which expresses
an N-terminal fragment of human Htt containing 171 amino acids with
82 glutamines (Schilling et al., 1999). We derived six groups of mice,
and the distribution in each cohort is indicated in Table 1 (WT, Rhes+/−,
Rhes−/−, Rhes+/−/N171-82Q, Rhes−/−/N171-82Q and N171-82Q). Our
previous studies in a toxin-model of HD or L-DOPA Parkinson's disease
model indicated that Rhes−/− mice were protected from striatal-lesions
and motoric dysfunction compared to wild-type mice (Mealer et al.,
2014; Subramaniam et al., 2012). Also, previously we found no difference
in basal behavior/motor function of Rhes−/− mice compared to wild-type
controls (Mealer et al., 2014; Subramaniam et al., 2012), consistent with
previous reports (Errico et al., 2008; Lee et al., 2011; Quintero and Spano,
2011; Quintero et al., 2008), suggesting that that Rhes−/− or Rhes+/−
would also serve as additional controls for N171-82Q mice. This study included both male and female mice as N171-82Q mouse model is known
to exhibit gender-dependent phenotypic variations; males show reproducible and an early motor/behavioral deficits, whereas, females display
variable and late onset symptoms, consistent with previous reports
(Chen et al., 2013a; Duan et al., 2003; Masuda et al., 2008; Orr et al.,
2008; Schilling et al., 1999), beginning at 8-weeks of age (Fig. 1A).
N171-82Q mice showed a significant weight loss compared to WT,
Rhes−/− or Rhes+/− mice in an age-dependent manner, that weight loss
was not significantly protected in homozygous Rhes−/−/N171-82Q mice
or heterozygous Rhes+/−/N171-82Q mice (Fig. 1B). Compared to male
N171-82Q mice, female N171-82Q mice appear to resist loss of body
weight and are comparable to control groups in all ages tested (Fig. 1C).
Next, we subjected Rhes−/−/N171-82Q mice to a battery of
established behavioral tests, including rotarod, behavioral battery and
open-field analysis to assess motor and exploratory deficits. These deficits, as described in our recent work (Pryor et al., 2014), are reminiscent
of symptoms observed in HD and have been shown to be robust in N17182Q mice (Andreassen et al., 2001; Cheng et al., 2011; Chiu et al., 2011;
Figiel et al., 2012; Masuda et al., 2008; Rose et al., 2010; Schilling et al.,
1999; Stack et al., 2010). On the rotarod test, the N171-82Q male mice
performed poorly, as expected, with a progressive loss of ability to stay
on the rotarod at 12 and 16 weeks, compared to non-HD controls (WT,
Rhes−/−, or Rhes+/− males) (Fig. 1D). However, Rhes+/−/N171-82Q or
Rhes−/−/N171-82Q male mice performed significantly better when compared to N171-82Q (Fig. 1D). We did not observe any significant differences on the rotarod tests in female groups in any category at these
ages (Fig. 1E), consistent with previous findings (Duan et al., 2003; Jia
et al., 2012). Subsequently, we carried out a rapid and sensitive measure
of motor deficits in HD mouse models, as previously described (Guyenet
et al., 2010). Rhes+/−/N171-82Q or Rhes−/−/N171-82Q mice are also
protected from, ledge defect (which measures coordination), clasping,
gait abnormality, kyphosis and tremor defects observed in N171-82Q
mice, which are most robust in male mice (Fig. 1F) compared to female
mice (Fig. 1G).
Rhes deletion prevents psychiatric-like behavioral deficits in N171-82Q
mice
In the open-field test (OFT), which measures exploratory and
anxiety-like behavior (Denenberg, 1969), the N171-82Q mice at 8 and
12 weeks displayed a significantly higher distance travel compared to
WT, Rhes−/−, or Rhes+/− male (Fig. 2A) and female mice (Fig. 2B). Such
increased activity in N171-82Q mouse model compared to wild-type
control groups has been reported earlier (Chen et al., 2013a), and it
appears to be a common phenomenon even in other HD mouse model
as well (Waldron-Roby et al., 2012). Such enhanced distance travel in
OFT was markedly reduced in Rhes−/−/N171-82Q or Rhes+/−/N17182Q mice, both male (at 8 and 12 weeks, Fig. 2A) and female groups at
all ages (Fig. 2C). Estimation of velocity has revealed that all N171-82Q
male groups performed slightly albeit significantly worse than wildtype control groups (Fig. 2C). However, female N171-82Q groups had
higher velocity of movement, which is robustly attenuated in Rhes−/−/
N171-82Q and Rhes+/−/N171-82Q female groups (Fig. 2D). Other sex differences are also evident in open field tests; while N171-82Q male mice
showed a moderate but significantly (p b 0.05) higher center, sides and
corner frequency at 12 weeks (Figs. 2E, G), their female counterparts
exhibited highly significant (p b 0.001) higher center, sides and corner
frequency even at 8 weeks (Figs. 2F, H). Despite no observable motor
S. Swarnkar et al. / Neurobiology of Disease 82 (2015) 66–77
69
Fig. 1. Rhes deletion protects HD-related motor deficits in N171-82Q mouse model. (A) Longitudinal behavioral assays of the indicated genotypes/groups. (B and C) Depicts changes in
body weight in male and female. (D and E) Rotarod test in male and female mice. (F and G) Behavioral battery–ledge test, clasping, gait, kyphosis and tremor, scored from 0 to 3 (highest)
in male and female mice aged 16 weeks. Data are means ± SEM from 6 mice in each groups ***P b 0.001, **P b 0.01 and *P b 0.05, two-way ANOVA followed by Newman–Keuls multiple
comparisons post-hoc test.
deficits in female mice (such as those measured in the rotarod tests),
higher open-field activity may suggest that these mice have increased
anxiety-like behaviors. Interestingly, these altered behaviors are significantly lower in Rhes−/−/N171-82Q and Rhes+/−/N171-82Q mice, indicating that Rhes may have a gender-specific role in altering the anxiety-like
symptoms in the N171-82Q mouse model.
Rhes deletion prevents striatal atrophy and brain weight loss of N171-82Q
mice
Next, we investigated whether the protection against motor deficits in
Rhes−/−/N171-82Q or Rhes+/−/N171-82Q male mice is accompanied by
alterations in the anatomical deficits seen in N171-82Q mice. Consistent
with previous reports (Duan et al., 2008; Gardian et al., 2005), N17182Q mice showed an enlargement of the lateral ventricles compared
to controls, which is presumably due to striatal atrophy. Ventricular
enlargement is largely attenuated in Rhes deleted, Rhes+/−/N171-82Q
and Rhes−/−/N171-82Q, mice groups (Figs. 3A, B). Brain weight
loss observed in N171-82Q is significantly reduced in Rhes deleted
mice (Fig. 3C). Together these results suggest that the embryonic deletion (complete KO) or even a partial lowering (heterozygous knockout)
of Rhes is sufficient to protect against the behavioral and anatomical
deficits seen in HD. Thus, Rhes GTPase physiologically modulates
the striatal-related deficits in the N171-82Q transgenic mouse model.
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S. Swarnkar et al. / Neurobiology of Disease 82 (2015) 66–77
Fig. 2. Rhes deletion reduces anxiety-like behaviors in N171-82Q HD mouse model. Open-field test (OFT): showing (A–B) total distance traveled by male and female mice, (C–D) velocity
for movements in male and female mice, (E–F) thigmotaxis frequency from center point to center of male and female mice, and (G–H) thigmotaxis frequency from center point to sides
and corner of male and female mice. Data are means ± SEM from 6 mice in each groups ***P b 0.001, **P b 0.01 and *P b 0.05, two-way ANOVA followed by Newman–Keuls multiple
comparisons post-hoc test.
Cerebellar expression of Rhes causes early motor and locomotion deficits in
N171-82Q mice
Having established that striatal expression of Rhes promotes HDrelated deficits in an HD mouse model, we asked whether the ectopic
expression of Rhes in a brain region not associated with HD pathology
could elicit a regional-specific deficit. To answer this question, we
overexpressed Rhes into the cerebellum, a region usually not affected
in HD, and has little or no expression of Rhes (Falk et al., 1999; Figiel
et al., 2012; Jiang et al., 2013; Luthi-Carter et al., 2002; Schilling et al.,
1999). We chose ~8-week-old WT or N171-82Q mice for such injections
because at this age, there are no apparent motor phenotype (rotarod or
general motor activity) in any of the N171-82Q mice tested compared to
wild-type littermate controls, which is consistent with previous reports
S. Swarnkar et al. / Neurobiology of Disease 82 (2015) 66–77
71
Fig. 3. Rhes deletion prevents enlargement of lateral ventricles in N171-82Q HD mouse model. (A) Representative image of striatal section of indicated mouse group, stained for cresyl
violet. (B) Quantification of ventricle size [area in (μm2)]. (C) Quantification of brain weight (mg) of indicated mouse groups. Data are means ± SEM from 6 mice in each groups
***P b 0.001, **P b 0.01 and *P b 0.05, two-way ANOVA followed by Newman–Keuls multiple comparisons post-hoc test.
(Chen et al., 2013a; Cheng et al., 2011; Schilling et al., 1999). Stereotaxic
coordinates for cerebellar injections were validated using a fast green
dye that showed effective distribution throughout the cerebellum
(Supplementary Fig. 1). We then injected null control adenovirus
(Ad-null) or Rhes expressing adenovirus (Ad-Rhes) into littermate WT
control or N171-82Q transgenic mice (4 μl/injection, 1 × 1010 opu/μl). In
a separate cohort of mice, we confirmed Rhes overexpression levels that
were comparable between the WT and N171-82Q mice as well as its
marked activity measured by mTORC1 (mechanistic Target of
Rapamycin)-dependent phosphorylation of S6K (Subramaniam et al.,
2012) (Supplementary figure 2A, B). The four groups [wild-type mice
injected with adenovirus-Null (WT-Ad Null) or adenovirus-Rhes (WTAd Rhes); N171-82Q mice injected with adenovirus-Null (N171-82Q-Ad
Null) or adenovirus-Rhes (N171-82Q-Ad Rhes) (n = seven/per group,
mixed sex distribution)] were subjected to longitudinal behavioral tests,
on the 4th, 9th, and 14th day after injection, and sacrificed for biochemical
and histochemical analysis, on day 17 as indicated in Fig. 4A. Within four
days of injection, a sudden drop in body weight was observed in Ad Rhesinjected N171-82Q mice compared to Ad Null-injected N171-82Q or Ad
Rhes-injected WT mice (Fig. 4B). The body weight did not drop further,
but remained significantly low in Ad-Rhes-injected N171-82Q, compared to control-injected mice (Fig. 4B).
Rotarod test revealed that Ad-Rhes-injected HD mice exhibited a
progressive loss of ability to stay on the rotating rod, which worsened
with time, compared to control groups (Fig. 4C). Also we carried out behavioral tests for ataxia-like defects, as previously described (Guyenet
et al., 2010), with multitude of tests including: the ledge test, hind
limb clasping, gait analysis, kyphosis and tremor. All of these analyses
of cerebellar functions worsened with time in Ad-Rhes injected N17182Q mice compared to control groups (Fig. 4). The open-field test
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S. Swarnkar et al. / Neurobiology of Disease 82 (2015) 66–77
Fig. 4. Ectopic Rhes expression in the cerebellum of N171-82Q mice elicits ataxia-like deficits. (A) Depicts the plan for the longitudinal behavioral and pathological analysis for Adenovirus-null,
or Adenovirus Rhes, injected WT and N171-82Q mice. (B) Body weight changes, (C) rotarod, (D) behavioral battery–ledge test, clasping, gait, kyphosis and tremor, scored from 0 to 3 (highest).
Data are means ± SEM from 7 mice in each group. ***P b 0.001, **P b 0.01 and *P b 0.05, two-way ANOVA followed by Newman–Keuls multiple comparisons post-hoc test.
(OFT) also revealed a clear deficit in Rhes injected cerebellum of N17182Q mice, at 4th and 9th day post injection, with significantly less thigmotaxis as well as center and sides/corner frequency on 4th day (Fig. 5).
However, such difference was not apparent on 9th and 14th day, suggesting that the moderate OFT deficits in Rhes-injected N171-82Q
might be due to severe loss of motor co-ordination (see accompanied
Video), and not, presumably related to an anxiety-like behavior. These
results suggest that ectopic Rhes expression in cerebellum causes
mHtt-related deficits with rapid onset of ataxia-like motor imbalance
in N171-82Q mice, which would otherwise have undectable cerebellar
dysfunction at this age.
Cerebellar expression of Rhes elicits cerebellar degeneration, an increased level of soluble Htt and caspase-3 activation in the cerebellum of N171-82Q mice
The severe motor deficits with ataxia-like phenotype in Ad-Rhesexpressing N171-82Q mice (in cerebellum) prompted us to investigate
the cerebellar integrity. Parvalbumin, which is expressed in Purkinje
neurons and molecular layer interneurons (Caillard et al., 2000) as a
marker has shown to be reduced both in mouse models and post
mortem tissue of human neurodegeneration (spinocerebellar ataxia1) (Vig et al., 1996, 1998, 2012). In our immunohistochemical analysis,
the Nissl and DAPI co-stains clearly differentiated the granule layer and
S. Swarnkar et al. / Neurobiology of Disease 82 (2015) 66–77
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Fig. 5. Ectopic Rhes expression in the cerebellum of N171-82Q mice elicits moderate effects on anxiety-like behaviors. (A–D) Open field test showing total distance, velocity, thigmotaxis
frequency from center point to center and thigmotaxis frequency from center point to sides and corner. Data are means ± SEM from 7 mice in each group ***P b 0.001, and *P b 0.05, twoway ANOVA followed by Newman–Keuls multiple comparisons post-hoc test.
the molecular layer in cerebellar sections (Fig. 6A), with a clear
parvalbumin positive Purkinje-cell layer (P). The immunoreactivity in
this layer is markedly reduced in the Ad-Rhes-injected cerebellum of
N171-82Q mice compared to control groups (Figs. 6A, B). We found,
consistent with previous reports, parvalbumin staining in the molecular
layer (M) (Caillard et al., 2000), that also appears to be affected in AdRhes-injected cerebellum of N171-82Q mice, compared to control
groups (Figs. 6A, B, compare P and M layers). Of note, the Ad-Nullinjected wild type as well as N171-82Q mice displayed comparable and
intact Purkinje cell layers, at this time point, implying that the cerebellum
is not affected at this age. In addition, Rhes expression alone did not elicit
a cerebellar phenotype, indicating that Rhes itself is not toxic to the cerebellum, and that the Rhes/mHtt interaction is required for toxicity.
Western blot analysis revealed that the Rhes-injected cerebellum of
N171-82Q elicited robust increase in the level of soluble Htt, compared
to Ad-null-injected N171-82Q mice (Figs. 7A, B), and upregulation of
cleaved caspase 3 (Fig. 7), an apoptotic marker. All together these data
indicate that Rhes expression in the cerebellum of N171-82Q mice
promotes an ataxia-like phenotype, which is accompanied by a loss of
parvalbumin-immunoreactivity and biochemical upregulation of the cell
death pathways, as well as enhanced mHtt solubility. Thus, ectopic Rhes
expression can elicit both selective neuronal degeneration and specific
behavioral deficits in an N171-82Q mouse model.
Reintroducing Rhes into the striatum of Rhes-deleted Hdh150Q/150Q knock-in
mice accelerates motor deficits
Having established that the Rhes plays an important role in HD
transgenic N171-82Q mice, we wondered whether Rhes can also
play a similar role in HD knock-in mice, expressing full length Htt
containing (Hdh 150Q/150Q), which develop late onset HD-related
motor symptoms. Rhes-deleted Hdh150Q/150Q mice (Rhes −/− /
Hdh 150Q/150Q) appeared normal, and their rotarod performance was
comparable to that of Rhes-deleted WT mice (Rhes−/−/WT) at 6months of age. This is expected as Hdh150Q/150Q mice develop agedependent, behavioral phenotype with significant motor abnormalities
appearing between 12 and 25 months of age (Heng et al., 2007). Therefore, we hypothesized that if Rhes were necessary for striatal damage in
Hdh150Q/150Q mice, an overexpression of Rhes into the striatum of
Rhes−/−/Hdh150Q/150Q mice would promote motor deficits. In support
of this hypothesis, we found that Ad-Rhes injection, but not AdNull or Ad-GFP, into the striatum elicited a progressive motor deficits
in Rhes−/−/Hdh 150Q/150Q, but not in non-HD Rhes−/−/WT control
mice (Fig. 8). Together these data suggest that Rhes overexpression selectively in the striatum of Rhes-deleted Rhes −/−/Hdh150Q/150Q mice accelerates HD-related deficits. Thus, Rhes plays major role in striatal
damage in HD.
Discussion
Three major findings emerged from this report regarding the
physiological role of Rhes in HD: 1) genetic deletion of Rhes ameliorates
HD-associated behavioral and pathological deficits; 2) ectopic expression of Rhes in the cerebellum of transgenic N171-82Q mice can elicit
a robust cerebellar phenotype reminiscent of cerebellar ataxia, and
3) reintroducing Rhes in striatum of Rhes−/−/Hdh150Q/150Q knock-in
mice can elicit HD-related motor deficits (see graphical abstract). Together, these studies are consistent with the notion that Rhes confers
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S. Swarnkar et al. / Neurobiology of Disease 82 (2015) 66–77
Fig. 6. Rhes expression in the cerebellum of N171-82Q HD mice reduces parvalbumin-positive immunoreactivity in Purkinje-cell layer (P) and molecular layer (M). (A) Representative confocal
images of parvalbumin, Nissl and nuclear stain (DAPI) of adenovirus-Null (Ad-Null) or adeno virus-Rhes (Ad-Rhes) injected wild-type or HD mice. Granule layer is shown as G. (B) Highresolution representative pictures showing parvalbumin positive cells Purkinje-cell layer. (C) Relative percent of Parvalbumin-positive cells in the defined area of Purkinje-neuron layer.
Data are means ± SEM from 7 mice in each groups ***P b 0.001, **P b 0.01 and *P b 0.05, two-way ANOVA followed by Newman–Keuls multiple comparisons post-hoc test.
striatal vulnerability in HD, and that the cerebellum and other regions,
which are normally impervious to damage in HD, can become vulnerable
upon Rhes overexpression. Striatal atrophy is an early event in human
HD, which is correlated to the length of poly-Q repeats (Graveland
et al., 1985; Gutekunst et al., 2002; Jackson et al., 1995; Rosas et al.,
2003; Ruocco et al., 2006b; Tabrizi et al., 1999, 2011; Vonsattel et al.,
1985), and which is consistent with clinical symptoms, such as involuntary movements and exaggerated reflexes, abnormal gait, and dystonia
(Kalkhoven et al., 2014). Yet, damage outside the striatum, including in
the cerebellum, can occur in HD. Whether cerebellar damage is an
early event or late event in the disease progression remains controversial.
While some researchers claim, based on brain MRI studies, that cerebellar
damage is an early event others have suggested it is a late event in the
disease (Gomez-Anson et al., 2009; Hobbs et al., 2010; Rees et al., 2014;
Rosas et al., 2003). Cerebellar atrophy may occur because of retrograde
secondary changes due to massive neuronal loss in the striatum, or due
to the progressive loss of white matter during the course of the disease
(Ruocco et al., 2006b). Early striatal damage occurs in the majority of
adult-onset HD, which is correlated with the clinical motor symptoms.
However, the clinical features of ataxia, reflecting cerebellar damage,
can also occur in juvenile HD, which is characterized by dystonia, rigidity,
and early death (Ruocco et al., 2006a). Though, Rhes mRNA expression
is abundant in the striatum of human HD (source: Allen brain atlas),
(Rhes protein levels in different brain region remains unknown)
whether or how Rhes is regulated in different regions of the brain during
development or aging remains unclear. In rats, Rhes mRNA expression is
transiently upregulated in the cerebellum, during the period of post-natal
development (P15-P25) (Harrison et al., 2008). Thus, the possibility that
Rhes might also have a role in cerebellar damage in HD cannot be ruled
out. Nevertheless, this report—from knockout and ectopic expression of
S. Swarnkar et al. / Neurobiology of Disease 82 (2015) 66–77
75
Fig. 7. Rhes expression increases EM-48-positive soluble huntingtin and caspase-3 activation in N171-82Q mice. (A) Western blot for full-length (FL)-Htt, EM-48 positive Htt, Caspase-3
(p19 and p17 fragement), Rhes along with ponceau staining of adenovirus-null (Ad-null) or adeno virus-Rhes (Ad-Rhes) injected wild-type or HD (N171-82Q) mice.
(B) Quantification of soluble Htt in adenovirus-null (Ad-null) or adeno virus-Rhes (Ad-Rhes) injected HD mice. Data are means ± SEM from n = 3 (HD) mice. P = 0.0339,
Student's t test.
Rhes in transgenic and knock-in HD mouse models—strongly supports
the finding that Rhes GTPase plays an important role in regionalspecific damage of HD.
Fig. 8. Reintroducing Rhes in the striatum of Rhes-deleted Hdh150Q/150Q knock-in mice
(Rhes−/−/Hdh150Q/150Q, 6 months old) elicits early motor deficits. Rotarod test in Ad-null,
Ad-GFP or Ad-Rhes, injected in Rhes deleted WT or Hdh150Q/150Q mice. Data are means ±
SEM from 4 mice in each group. *P b 0.05, two-way ANOVA followed by Newman–Keuls
multiple comparisons post-hoc test.
How might Rhes elicit neuronal death in HD? Mechanistically, we
predict that Rhes enhances soluble forms of poly-Q expanded Htt,
through post-translation SUMO modification, which is toxic to cells.
This prediction is based on previous biochemical data, in which depletion of SUMO1 prevents Rhes-mediated poly-Q Htt toxicity in a cell
culture model (Subramaniam et al., 2009). This notion is further supported in the Rhes-injected cerebellum of HD mice, which displayed
higher soluble forms of Htt (Fig. 7). The downstream signals that are
recruited by SUMOylated mHtt remain unclear. These might involve
transcriptional dysregulation, mitochondrial stress, and caspase-3 activation [(Steffan et al., 2004; Subramaniam et al., 2009) and (Fig. 7)].
Our recent study implicates mHtt in the promotion of mTORC1 signaling
in HD pathogenesis (Pryor et al., 2014). We found that mHtt alters the
movement of mTOR kinase and binds to its regulator Rheb GTPase to
promote mTORC1 activity. When mTORC1 is genetically upregulated
in the striatum of N171-82Q mice, this caused HD pathogenesis and
the premature death of HD mice (Pryor et al., 2014). As Rhes is a prominent activator of mTORC1 (Subramaniam et al., 2012), we speculate
that Rhes-mediated SUMOylation of mHtt would further deregulate
mTORC1 signaling in the striatum, thus leading to early striatal motor
deficits, which remains to be investigated.
A bigger question emerges from this study—would Rhes be now beyond doubt an excellent drug target for HD? Although a complete
knockout of Rhes is relatively indistinguishable from the wild-type
mice at baseline, the behavioral deficits emerged when the mice were
challenged with dopamine-related drugs. For example, complete Rhes
knockout mice exhibit an increased D1 agonist-initiated locomotor activation and an increased D2 antagonist-initiated catalepsy (Errico et al.,
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S. Swarnkar et al. / Neurobiology of Disease 82 (2015) 66–77
2008). Similarly, D1/D2 receptor agonist stimulation induces a stereotypic behavior that is enhanced in Rhes KO mice (Quintero and Spano,
2011; Quintero et al., 2008). Recently, we found that L-DOPA-induced
dyskinetic movements were markedly reduced in Rhes KO mice, in a
Parkinson's disease model (Subramaniam et al., 2012). These studies
imply that Rhes plays an important role as a negative regulator of
dopamine-related movements, which may have implications in HD
(Chen et al., 2013b). Thus targeting Rhes might provide beneficial effects
on striatal-motor defects associated with both HD and PD. However, due
to its diverse yet incompletely understood roles blocking Rhes function in
adult animals and the biological consequences on striatal functions
remains unclear. Rhes acts as a SUMO-E3 ligase; activates mTORC1
signaling (Subramaniam et al., 2010, 2012); activates Akt (Bang et al.,
2012); modulates autophagy through Beclin-1, independent of mTOR
(Mealer et al., 2013); and interacts with golgi protein ACBD3 (Sbodio
et al., 2013).
A recent study investigated the role of Rhes shRNA in an adult HD
mouse model (Lee et al., 2014). In that study, Rhes shRNA, which was
injected into seven-week-old N171-82Q or four-month-old BACHD
mice, did not alter the results of the rotarod, a widely used test to assess
motor defects in HD mouse models (Lee et al., 2014). This suggests that
i) depleting Rhes mRNA in the striatum of adult animals has no lethal
consequences, and ii) depleting Rhes at seven weeks or later does not alleviate the motor symptoms or the Rhes-mediated HD defects. The latter
possibility is particularly important as Rhes expression begins in the striatum as early as postnatal day 4, and gradually reaches a peak at day 15
and 24 (Harrison et al., 2008); thus suggesting that Rhes-mediated
striatal dysfunction might have begun before the Rhes shRNA injection.
This notion is further strengthened by our rapid cerebellar HD model, in
which we found, a robust deficit in motor functions, within four days of
Ad-Rhes injection. Moreover in Lee et al. (2014) model it is unclear
how much Rhes protein is depleted. As mRNA levels does not usually predict its protein levels (Liu and Tian, 2004; Lundberg et al., 2010;
Schwanhausser et al., 2011; Vogel et al., 2010), it is challenging to interpret the data just with reduced Rhes mRNA levels. Nevertheless, in the
Lee et al., 2014 model, Rhes mRNA depletion negatively affected mobility
in the open-field tests in Rhes shRNA-depleted HD mice (BACHD model).
This is similar to our model, in which Rhes-deleted N171-82Q mice but
not WT mice were less mobile than control N171-82Q mice (Figs. 2 A–
H), which may be attributable to the protective effects of Rhes on
anxiety-like hyperactive behaviors in HD. Thus, our data supports the hypothesis that Rhes offers protection against both motor and psychiatriclike deficits in HD, but the potential mechanisms by which Rhes differentially regulate motor vs. psychiatric-like deficits in HD pathogenesis remain to be investigated.
In another study Lee and colleagues (Lee et al., 2015) found that
AAV-Rhes overexpression into the striatum of N171-82Q mice improved the rotarod performance compared to GFP injected N171-82Q
mice. This appears to be contradictory with our study in HD-knock-in
mice (Fig. 8). But due to lack of control (WT) mice data in Lee et al.
(2015) study, it is difficult to interpret whether AAV-Rhes improved
rotarod deficits observed at 14 and 18 week is restricted to N171-82Q
mice or it has any effect on WT mice. Moreover, we have used untagged
Rhes in our study, whereas Lee et al. (2015) used Rhes, which is Cterminally tagged with flag epitope. As C-terminal region of Rhes processes SUMO-E3 ligase and the farnesylation site (C263), any mutation
or tag in C-terminal might affect Rhes' function by altering its intracellular localization (Subramaniam et al., 2009, 2010). For example, Rhes
C263S mutant is abnormally localized to the nucleus instead of its predominant perinuclear localization (Subramaniam et al., 2009). Thus the
apparent contradicting results of our study and that of Lee et al. (2015)
could be due to different HD models used for striatal Rhes overexpression and/or the biochemistry of Rhes.
Overall, our study demonstrates that Rhes is important for promoting
the tissue-specific damage of mHtt in HD. We found that Rhes deletion
protects against HD-related deficits in a well-characterized mouse
model of HD and ectopic expression of Rhes in the cerebellum, which is
normally not affected in HD, or in the striatum of HD-knock-in mice,
can elicit robust pathological changes accompanied by biochemical, morphological, and motor-behavioral deficits. Thus this study is consistent
with growing number of studies linking toxic role of Rhes in HD cell
and mouse models (Baiamonte et al., 2013; Lu and Palacino, 2013;
Mealer et al., 2013; Mealer et al., 2014; Okamoto et al., 2009; Sbodio et
al., 2013; Seredenina et al., 2011) Rather than depleting Rhes in the
adult, which might have untoward side effects due to its association
with multiple protein partners, we hypothesize that selective inhibition
of Rhes' activity or its interaction with mHtt might have better beneficial
effects in ameliorating or preventing symptoms in HD (Subramaniam
and Snyder, 2011).
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.nbd.2015.05.011.
Author contributions
S. Swarnkar and S. Subramaniam designed the study and S. Swarnkar
carried out all mice behavioral and brain-analysis work. Y.C. assisted in
mouse work and brain analysis. W. P performed a blind analysis of behavioral battery. N.S prepared the graphs and analyzed the data together with
S. Swarnkar and S. Subramaniam.. D.T.P. provided technical support for
brain imaging. S. Subramaniam provided further conceptual input, and
wrote the paper with input from all co-authors.
Funding
This work was supported by Scripps startup funds (to S.
Subramaniam).
Abbreviations
Rhes
(Ras homolog enriched in striatum)
HD
(Huntington's disease)
Htt
(huntingtin)
mHtt
(poly glutamine expanded Htt)
mTOR
(mammalian target of rapamycin)
S6K
(ribosomal S6 kinase)
Acknowledgments
We thank Melissa Benilous, Nancy Norton, and Trina Miles for
administrative support. We thank Robbert Mealer for his thoughtful insights/suggestions while writing this manuscript. We are grateful to the
people in The Scripps Research Institute, Jupiter, Florida especially in the
Department of Neuroscience, for their continuous support in setting up
the laboratory and providing technical support whenever needed.
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