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DOI: 10.1126/science.1172871, 1327 (2009);
324
Science 
 
et al.
Srinivasa Subramaniam,
Mutant-Huntingtin CytotoxicityRhes, a Striatal Specific Protein, Mediates
 
www.sciencemag.org (this information is current as of June 7, 2009 ): The following resources related to this article are available online at 
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Supporting Online Material
 http://www.sciencemag.org/cgi/content/full/324/5932/1327#otherarticles, 11 of which can be accessed for free:
cites 29 articles
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2009 by the American Association for the Advancement of Science; all rights reserved. The titleCopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005.(print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the
Science 
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Boehringer Ingelheim. J.F., S.S., E.C., and T.C. were supportedby Wiener Wissenschafts, Forschungs und Technologiefonds;A.S. by the Christian-Doppler-Society; K.T. by the DeutscheForschungsgemeinschaft; and T.C. by the EuropeanMolecular Biology Organization Young InvestigatorProgram. This work was further supported by the AustrianProteomics Platform (GEN-AU). The Protein Data Bankaccession number for the CtsR
2
 /DNA complex is 3H0D.
Supporting Online Material
www.sciencemag.org/cgi/content/full/324/5932/1323/DC1Materials and MethodsFigs. S1 to S8Table S1References22 December 2008; accepted 13 April 200910.1126/science.1170088
Rhes, a Striatal Specific Protein,MediatesMutant-HuntingtinCytotoxicity
Srinivasa Subramaniam, Katherine M. Sixt, Roxanne Barrow, Solomon H. Snyder
*Huntington
’
s disease (HD) is caused by a polyglutamine repeat in the protein huntingtin (Htt) withmutant Htt (mHtt) expressed throughout the body and similarly in all brain regions. Yet, HDneuropathology is largely restricted to the corpus striatum. We report that the small guaninenucleotide
–
binding protein Rhes, which is localized very selectively to the striatum,binds physiologically to mHtt. Using cultured cells, we found Rhes induces sumoylation of mHtt,which leads to cytotoxicity. Thus, Rhes-mHtt interactions can account for the localizedneuropathology of HD.
H
untington
’
s disease (HD), a geneticallydominant neurodegenerative disorder, re-flects expansion of a polyglutamine re- peat in the protein huntingtin (Htt) (
1
). Mutant Htt(mHtt)occursuniformlythroughoutthebrainand peripheral tissues. Yet, HD is brain-specificwith profound abnormal movements related toselective, gross degeneration of the corpus stri-atum and lesser damage to the cerebral cortexeliciting dementia (
2
,
3
). Molecular mechanismscausingmHttcytotoxicityareunclear.mHttforms proteinaggregates,whichmaybeneuroprotectivewith soluble mHtt linked to cytotoxicity (
4
 – 
7 
).mHtt is sumoylated, which increases the solubleform of mHtt and elicits cytotoxicity and neu-rotoxicity in a 
Drosophila
model of HD (
8
).Rhes (Ras homolog enriched in striatum) isa small guanine nucleotide
 – 
 binding protein (G protein) very selectively localized to thestriatum (
9
). To determine whether Rhes bindsto Htt, we overexpressed Rhes in HEK293cells where it bound to both wild-type (wt) Htt and mHtt (Fig. 1A) (
10
). In conditionallyimmortalized Htt knock-in striatal neuronal cells(
11
), which lack endogenous Rhes (fig. S1C),overexpressed Rhes bound robustly to endoge-nousmHtt(Fig.1B).InHDtransgenicmice(
12
),endogenous striatal mHtt coprecipitated withRhes (Fig. 1C). In the presence of purified Rhesand Htt, Rhes bound much more to mHtt thanwtHtt protein (fig. S1A). Rhes did not bind toataxin (fig. S1B), a polyglutamine-repeat proteininvolved in another neurodegenerative disorder,spinocerebellar ataxia.To ascertain whether Rhes influences mHtt cytotoxicity, we used several cell lines. In
The Solomon H. Snyder Department of Neuroscience, JohnsHopkins University School of Medicine, 725 North WolfeStreet, Baltimore, MD 21205, USA.*To whom correspondence should be addressed. E-mail:ssnyder@jhmi.edu
Fig. 1.
Rhes binds Htt and affects cell survival. (
A
)Rhes interacts with N-terminal Htt. HEK293 cells weretransfected with glutathione
S 
-transferase (GST) orGST-Rhes together with Flag-tagged Htt or the N-terminal fragment containing 171 amino acids and 18glutamines (wtHtt) or 82 glutamines (mHtt). After 48hours, cell lysates were glutathione (GSH) precipitatedand immunoblotted (IB) for Flag. (
B
) Rhes interactswith full-length Htt. Striatal cells expressing wild-typeHtt (ST
Hdh
Q7/Q7 
) or mutant Htt (ST
Hdh
Q111/Q111
) weretransfected with GST or GST-Rhes. After 48 hours, celllysates were GSH-precipitated and immunoblotted forHtt. Htt and GST inputs are shown. (
C
) Rhes interactswith mHtt in striatum. Striatum of transgenic miceexpressing mHtt was lysed and immunoprecipitatedwith Rhes antibody or immunoglobulin IgG alone(bead control). Immunoprecipitates were probed withan N-terminal
–
specific Htt antibody (N-Htt).
(D
) Rhesreduces cell survival. HEK293 cells were transfectedwith Myc/Myc-Rhes and wtHtt
–
mHtt constructs. ***
P
<0.005versusmHtt alone.(
E
) Wild-type(ST
Hdh
Q7/Q7 
) ormutant (ST
Hdh
Q111/Q111
) striatal cells were transfectedwith Myc/Myc-Rhes. ***
P
< 0.005 versus Myc. (
F
)Depletion of Rhes prevents PC12 cell death. Controlshort hairpin
–
mediated (shRNA) or Rhes shRNA 1 to 4were cotransfected with mHtt. Only shRNA4 wassignificantly cytoprotective (**
P
< 0.01 versus controlshRNA). After 48 hours, cell survival was measured byMTT.
A CD
   M  y  c   M  y  c  -   R   h  e  s   M  y  c  +   m   H   t   t   M  y  c  +   w   t   H   t   t    M  y  c  -   R   h  e  s  +   w   t   H   t   t   M  y  c  -   R   h  e  s  +   m   H   t   t
   S  u  r  v   i  v  a   l   (   %  c  o  n   t  r  o   l   )   S  u  r  v   i  v  a   l   (   %  c  o  n   t  r  o   l   )
  S   T   H  d   h
  Q   7  /  Q   7
 +    M  y  c  S   T   H  d   h
  Q  1  1  1  /  Q  1  1  1
 +    M  y  c  S   T   H  d   h
  Q   7  /  Q
   7 +    M  y  c  -   R   h  e  s  S   T   H  d   h
  Q  1  1  1  /  Q  1  1  1
 +    M  y  c  -   R   h  e  s
E FB
IB: RhesmHttRhesmHttIB: Htt
   B  e  a   d  c  o  n   t  r  o   l   R   h  e  s   I  g   G
IPInputIB: HttHttHttGSTGST-Rhes-+GST-RhesGST-++-+-
  S   T   H  d   h
  Q   7  /  Q   7 
  S   T   H  d   h
  Q  1  1  1  /  Q  1  1  1
 
GSH beadsIB: HttIB: GSTIB: HttInputmHttFlag-mHtt+--+Flag-wtHttGST-RhesGSTmHttwtHttwtHtt+-+--+-+-++-GSH beadsIB: FlagInputIB: FlagIB: GSTGSTGST-Rhes
   S  u  r  v   i  v  a   l   (   %  c  o  n   t  r  o   l   )
  m   H   t   t  +   c  o  n   t  r  o   l   s   h   R   N  A  m   H   t   t  +    R   h  e  s   s   h   R   N  A   1  m   H   t   t  +    R   h  e  s   s   h   R   N  A   2  m   H   t   t  +    R   h  e  s   s   h   R   N  A   3  m   H   t   t  m   H   t   t  +    R   h  e  s   s   h   R   N  A   4   M  o  c   k
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HEK293 cells, overexpression of mHtt or Rhesalone did not decrease cell survival. However,overexpression of Rhes together with mHtt reduced cell survival by 50%, whereas survivalwas normal in cells containing wtHtt and Rhes(Fig.1D).WeconfirmedthatsurvivalofastriatalcelllinewithmHttisthesameasthatincellswithwtHtt (
13
) (Fig. 1E). Overexpression of Rhes inmHtt knock-in striatal cells (ST
 Hdh
Q111/Q111
)(
14
) reduced cell survivalby 60%, whereas over-expression of Rhes in wtHtt knock-in striatalcells (ST
 Hdh
Q7/Q7 
) had no effect (Fig. 1E).Rhes
’
s influences on striatal cell survival wereconcentration-dependent (fig. S2A). Cleavedcaspase-3, an index of apoptosis, was selectively
A
18
   H   t   t  a  g  g  r  e  g  a   t  e  s   (   F  o   l   d  c   h  a  n  g  e   )
3048 hmycMyc-RhesFlag-mHtt-+++-+-+++-++-+-++
01234567
05101520
   H   t   t   S  o   l  u   b   l  e   (   F  o   l   d  c   h  a  n  g  e   )
**************
TALONIB: FlagIP: FlagIB: Sumo1
+-++-+++6298188
kDa 
981886298188InputMycMyc-RhesHis-Sumo1Flag-mHtt+-++-+++**TALON/ IP
C
IB: Sumo1IB: MycIB: Flag
IP: FlagIB: Flag
62
    P  e   l   l  e   t
D
mHtt(aggregates)mHtt(SDS soluble)GSTGST-RhesHis-Sumo1Myc-mHttMyc-mHtt K6,9,15,91R+-++--+++-+-+-+-++-+TALONIB: Myc6298188
kDa 
IB: Myc
   S  u  r  v   i  v  a   l   (   %   c  o  n   t  r  o   l   )
***020406080100
IB: FlagIB: FlagIB: Rhes183048 h-+++-+-+++-++-+-++MycMyc-RhesFlag-mHtt
    P  e   l   l  e   t
mHtt(aggregates)mHtt(SDS soluble)mHtt( soluble)RhesmHtt( soluble)
B
Fig. 2.
Rhes inhibits mHtt aggregate formation. (
A
) HEK293 cells weretransfected with Myc or Myc-Rhes and Flag-mHtt. At the indicated time points,cells were lysed, the pellet fraction (containing mHtt aggregates plus SDS-soluble mHtt), and the soluble fractions (containing only soluble Htt) wereimmunoblotted (IB) for Flag or Rhes. (
B
) Quantification of aggregated andsoluble Htt. Fold change compared with results at 18 hours. (
C
) Rhes increasesmHttsumoylationincells.HEK293cellsweretransfectedwithMycorMyc-Rhes,His-SUMO1 and Flag-mHtt. After 36 hours, cell lysates were immunoprecipi-tated (IP) with antibody against Flag-IgG beads and immunoblotted for Flag.SUMO-tagged proteins were enriched with TALON metal-affinity resin andimmunoblotted for Flag or SUMO1. Input lysates were immunoblotted forSUMO1,Myc,orFlag.(
D
)EffectofmHtt-K6,9,15,91Rmutationonsumoylationand cell survival. HEK293 cells were transfected with GST or GST-Rhes, His-SUMO1, and Myc-tagged mHtt or Myc
–
mHtt-K6,9,15,91R. After 48 hours, theSUMO-tagged protein was enriched with TALON metal-affinity resin andimmunoblotted for Myc. The pellet fraction of the cell lysate was subjected toaggregate detection assay. Cell survival was measured by MTT.
Fig. 3.
Rhes enhances sumoylation. (
A
) Rhes interacts withUbc9. HEK293 cells were transfected with GST or GST-Rhes andMyc-Ubc9. After 48 hours, cells were lysed, precipitated withGSH beads and probed for Myc. Purified GST or GST-Rhes wasincubated with purified Ubc9 and precipitated with GSH beads.The precipitates and inputs were immunoblotted for Ubc9. (
B
)Rhes sumoylates mHtt. Four
m
l of in vitro translated mHtt(control) and its K6,9,15,91R mutant were subjected tosumoylation [1× RB buffer, 250 ng E1, 125 ng E2, 1.5
m
gSUMO1, 5 mM adenosine triphosphate (ATP), and 2 mMdithiothreitol (DTT)] in the presence of 500 ng Rhes (+) orbovine serum albumin (BSA) (
–
). Htt was detected by N-Httantibody. Rhes sumoylates Htt in a time- (Rhes, 500 ng) andconcentration-dependent manner. (
C
and
D
) Sumoylation ofmultiple substrates. Indicated substrates (500 ng) weresubjected to sumoylation assay as in (B). Rhes sumoylatessubstrates in a (C) time- (Rhes, 200 ng) and (D) concentration-dependent manner.
GSH beadsIB: MycInputIB: MycIB: GST
-+++-+
B
-+++-+
   I  n  p  u   t
Ubc9
GSH beadsIB: Ubc9GSH beadsIB: GST
A
GSTGST-RhesMyc-Ubc9
GSTGST-RhesUbc9
IB: GST
C
+ Sumo
- S  um o
RhesngSP100S*SP100IkBS*IkB1-+-+-+-30900minIkBS*IkBSP100S*SP100RanGAPS*RanGAPIB: GSTRhesE2-25KS*E2-25KE2-25KS*E2-25K
+ Sumo- SumoRhesmHtt
   1   0   0   3   0   0   6   0   0   1   2   0   0   0   1   2   0   0
Rhesng
- S  um o
+ Sumo901-+-+-+0minRhes-mHttS*mHtt30mHttS*mHtt
   K  6 ,   9 ,   1   5 ,   9  1   R
- +- +
  c  o  n   t  r  o   l
- +
  c  o  n   t  r  o   l
mHttS*mHtt90 min
90 min
In cellsIn vitro
D
   1   0   1   0   0   2   0   0   1   0   0   0   0   1   0   0   0
5 JUNE 2009 VOL 324
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