The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
Cyclocreatine treatment improves cognition
in mice with creatine transporter deficiency
Yuko Kurosawa,1 Ton J. DeGrauw,2 Diana M. Lindquist,3 Victor M. Blanco,4 Gail J. Pyne-Geithman,5
Takiko Daikoku,6 James B. Chambers,7 Stephen C. Benoit,7 and Joseph F. Clark1,2
1Department of Neurology, University of Cincinnati, Cincinnati, Ohio, USA. 2Division of Neurology and 3Department of Radiology and Imaging Research Center,
Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA. 4Department of Emergency Medicine and 5Department of Neurosurgery,
University of Cincinnati, Cincinnati, Ohio, USA. 6Division of Reproductive Science, Cincinnati Children’s Hospital Medical Center,
Cincinnati, Ohio, USA. 7Department of Psychiatry and Behavior Neuroscience, University of Cincinnati, Cincinnati, Ohio, USA.
The second-largest cause of X-linked mental retardation is a deficiency in creatine transporter (CRT; encoded
by SLC6A8), which leads to speech and language disorders with severe cognitive impairment. This syndrome,
caused by the absence of creatine in the brain, is currently untreatable because CRT is required for creatine
entry into brain cells. Here, we developed a brain-specific Slc6a8 knockout mouse (Slc6a8–/y) as an animal
model of human CRT deficiency in order to explore potential therapies for this syndrome. The phenotype of
the Slc6a8–/y mouse was comparable to that of human patients. We successfully treated the Slc6a8–/y mice with
the creatine analog cyclocreatine. Brain cyclocreatine and cyclocreatine phosphate were detected after 9 weeks
of cyclocreatine treatment in Slc6a8–/y mice, in contrast to the same mice treated with creatine or placebo.
Cyclocreatine-treated Slc6a8–/y mice also exhibited a profound improvement in cognitive abilities, as seen with
novel object recognition as well as spatial learning and memory tests. Thus, cyclocreatine appears promising
as a potential therapy for CRT deficiency.
Creatine transporter (CRT; encoded by SLC6A8; geneID 6535)
transports creatine with high specificity into cells via second-
ary active transport, using the Na+ gradient to drive the trans-
port against creatine’s concentration gradient. Creatine and its
phosphorylated form, phosphocreatine, are used as an energy
reservoir by buffering ATP concentration via the creatine kinase
(CK) reaction (1). This high-energy phosphate-buffering system
is essential to maintain ATP levels. CK and CRT are coexpressed
in many tissues with a dynamic and/or fluctuating energy
demand. In the CNS, the creatine/phosphocreatine/CK system
plays an important role in neurotransmitter release, membrane
potential maintenance, Ca2+ homeostasis, and/or ion gradient
restoration (1–3). SLC6A8, a member of the solute carrier 6 fam-
ily, is expressed in many tissues, such as CNS, heart, and skeletal
muscle (4), and is highly specific for creatine (5–7). Interestingly,
the CNS is the main organ affected by creatine deficiency syn-
dromes, including CRT deficiency. There appears to be normal
cardiac function and normal creatine levels in the muscles of
patients with CRT deficiency (8).
Of the 3 creatine deficiency syndromes, l-arginine:glycine amid-
inotransferase (AGAT) deficiency and guanidinoacetate n-meth-
yltransferase (GAMT) deficiency are caused by defects in the
enzymes that synthesize creatine, whereas CRT deficiency results
from a defect in CRT caused by SLC6A8 deficiency, such that cre-
atine cannot enter the brain’s cells. In patients, creatine deficiency
syndromes have several common clinical manifestations, includ-
ing cognitive dysfunction with mental retardation, poor language
skills, and autism spectrum disorders (9–15). Proton magnetic res-
onance spectroscopy (MRS) of affected patients shows an absence
or dramatic diminution of the creatine peak, with relatively nor-
mal levels of n-acetyl aspartate (9, 16, 17).
Whereas AGAT and GAMT deficiencies have been identified in
about 100 patients worldwide, CRT deficiency is described as the
second-most common cause of X-linked mental retardation, with
an estimated 42,000 individuals affected in the US and approxi-
mately 1 million worldwide (17–20). Because SLC6A8 is located
on human chromosome Xq28, mutations in this gene result in
a more severe syndrome in males than in female carriers. Patients
with AGAT deficiency or GAMT deficiency have been successfully
treated with creatine supplementation, which reverses symptoms,
as well as other supplements, which manage buildup of intermedi-
ate metabolites (13–15, 21, 22); however, patients with CRT defi-
ciency are not successfully treated with creatine supplementation
(10, 16, 23–25). Creatine is found in blood and cerebrospinal fluid
(CSF), but is not able to enter brain cells — the cell membranes are
an effective barrier to creatine transport.
Some previous studies suggest that the rodent brain has the
enzymes to synthesize creatine (4, 5, 26, 27). However, it appears
that the synthesis system does not work in mice or humans in vivo,
because no detectable brain creatine was observed in the Slc6a8
whole-body knockout mouse (28) or in human patients (9, 16, 17),
as assessed by biochemical assays or MRS.
Normal brain function might primarily depend on its ability to
transport creatine into neurons, as suggested by the presence of
creatine in the CSF of patients with CRT deficiency. Despite its
presence in the CSF, however, patients’ brain creatine levels are
markedly reduced or not detectable when measured by 1H-MRS.
Yet a lack of MRS signal does not prove that creatine is absent;
it could be present at levels below the detection limit. SLC6A8
is highly expressed in neurons and oligodendrocytes, but not
in astrocytes, and is present in microcapillary endothelial cells
(MCECs), which form part of the blood-brain-barrier (BBB) (5).
An additional part of the BBB is a barrier between the periph-
ery and the CNS, formed by astrocytic end-feet around MCECs,
which regulate water and metabolite exchange. Passive creatine
diffusion into brain cells — against its concentration gradient —
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2012;122(8):2837–2846. doi:10.1172/JCI59373.
2838 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
is not expected to generate significant brain creatine levels, and
this is supported by data in patients.
Currently, there is no approved treatment for patients with CRT
deficiency; treatment strategies are palliative for managing seizure
and related sequelae. In order to identify a strategy for treatment
that focuses on the cognitive deficiencies, we needed a valid animal
model with the phenotype of the human disease. Here, we gen-
erated a brain-specific Slc6a8 knockout mouse (referred to herein
as Slc6a8–/y) with specific knockout of target areas of cortex and
hippocampus — the predominant areas of the brain for cognition
and memory — and treated those mice with the creatine analog
Cyclocreatine is a nearly planar creatine analog (Figure 1) with a
maximum kinetic velocity approaching that of creatine itself (29).
Because its planar characteristics aid in passive transport across
membranes, cyclocreatine might therefore be useful in the treat-
ment of CRT deficiency. Moreover, cyclocreatine is phosphorylated
and dephosphorylated by mitochondrial and cytosolic CK (30).
It has already been demonstrated that cyclocreatine phosphate
can indeed function as a phosphagen in mouse brain in vivo (31).
Cyclocreatine was given to humans when it was investigated as
a chemotherapeutic adjunct under an investigational new drug
phase I safety study; thus, toxicology and cGMP data are known
(32, 33). Here, we assessed the ability of cyclocreatine to treat
Slc6a8–/y mice. We hypothesized that cyclocreatine would cross the
BBB and pass through brain cell membranes, improving cognitive
function in treated mice.
Our results showed that Slc6a8 was efficiently deleted in Slc6a8–/y
mouse brains, and creatine content in these brains was somewhat
consistent with that seen in human patients. Slc6a8–/y mice had
impaired cognitive function, but normal balance and musculo-
skeletal control systems, also similar to human patients. Cognitive
abilities were improved after 9 weeks of cyclocreatine treatment
compared with Slc6a8fl/y control littermates, with cyclocreatine and
cyclocreatine phosphate seen in the mouse brain. Our results con-
firmed that in mice, cognitive deficiencies were caused by a lack of
CRT in the brain, and that this cognitive deficit could be reversed
by 9 weeks of treatment with cyclocreatine.
Creatine concentrations in brains and other organs. The knockout strate-
gy is outlined in Figure 2A. We found substantially decreased levels
of creatine in the brains of Slc6a8–/y mice compared with littermate
Slc6a8fl/y controls (2.8 ± 0.11 versus 11.2 ± 0.74 mmol/kg wet wt;
P ≤ 0.001; Table 1), a diminution close to that measured in patients
by 1H-MRS. The relative reduction of brain creatine was similar to
the reduced Slc6a8 mRNA expression in Slc6a8–/y mice (Figure 2B
and Supplemental Figure 1; supplemental material available
online with this article; doi:10.1172/JCI59373DS1). Furthermore,
there were no differences in any of the tissues studied, except-
ing a statistically significant increase in the amount of creatine
in the urine (Slc6a8–/y, 10.2 ± 2.57 mM; Slc6a8fl/y, 4.0 ± 0.66 mM;
P ≤ 0.05; Table 1).
Body morphometry. Although no significant difference in body
weight or percent lean body mass was observed in Slc6a8–/y com-
pared with Slc6a8fl/y mice before or after treatment, percent body
fat was significantly lower at baseline (Slc6a8–/y, 15.8% ± 1.3%;
Slc6a8fl/y, 19.8% ± 1.4%; P ≤ 0.05), a trend that was still seen after
treatment (Supplemental Figure 2, A–C). This may be due to
increased activity in Slc6a8–/y compared with Slc6a8fl/y mice (Sup-
plemental Figure 2D). Treatment with cyclocreatine, creatine, or
placebo did not significantly change morphometric parameters in
Slc6a8–/y or Slc6a8fl/y mice.
Cyclocreatine. We observed successful cyclocreatine entry into
the brains of all mice treated with cyclocreatine for 9 weeks, with
higher levels in Slc6a8fl/y than in Slc6a8–/y brains (3.1 ± 0.34 vs.
1.7 ± 0.2 mmol/kg wet wt; P ≤ 0.001; Figure 3). Brain cyclocreatine
was also observed by chemical analysis as well as by nuclear MRS
(Figure 4 and Supplemental Figure 3). We also found cyclocre-
atine in mouse hair (Slc6a8fl/y, 34.8 ± 3.7 mmol/kg dry wt; Slc6a8–/y,
39.9 ± 6.7 mmol/kg dry wt; Supplemental Figure 4) and claws
(data not shown). There were no deaths or overt health problems
observed in any of the groups studied.
31P-MRS. We observed a decrease in 31P phosphocreatine in Slc6a8–/y
versus control Slc6a8fl/y brains; however, there was a profound lack
of phosphocreatine peak, with no detectable inorganic phosphate,
in the former group (Supplemental Figure 3 and Figure 4, A and
B). Treatment with cyclocreatine did result in metabolite changes
(Figure 4B), and we observed a substantial peak corresponding to
cyclocreatine phosphate (and/or phosphocreatine) in Slc6a8–/y mice
(Supplemental Figure 3). The peak positions of phosphocreatine
and cyclocreatine phosphate are separated by less than 0.2 ppm
(34), which means that they cannot be resolved by in vivo nuclear
MRS. Because our chemical analysis showed no significant change
in creatine, we interpret this peak to represent cyclocreatine phos-
phate. The presumed cyclocreatine phosphate levels appeared com-
parable in Slc6a8–/y and Slc6a8fl/y mice, which suggests that cyclocre-
atine is phosphorylated in the brains of these mice.
Spatial learning and memory. Spatial learning and memory are
executive functions for mice, with consequences for environment
familiarity and food finding; therefore, we used the established
Morris water maze and radial arm maze tests to assess these cogni-
tive functions. In the Morris water maze, Slc6a8–/y mice took sig-
nificantly longer to find the hidden platform in the last 3 training
trials than did Slc6a8fl/y controls before treatment; 9 weeks of cyclo-
creatine treatment improved the performance of Slc6a8–/y mice so
that they were not significantly impaired compared with controls,
whereas creatine or placebo did not significantly change Slc6a8–/y
mouse performance (Figure 5A). The percentage of time spent
in platform area during the probe trial also improved in Slc6a8–/y
mice after 9 weeks of cyclocreatine treatment (Figure 5B). The
velocity of swimming in the platform area during the probe trial
was not significantly different between groups before and after
each treatment (Figure 5C), suggestive of normal motor function
in the Slc6a8–/y group. We concluded that the impaired spatial
learning and memory in Slc6a8–/y mice was normalized by 9 weeks
of cyclocreatine treatment.
Chemical structural formulas of cyclocreatine and creatine. Cyclocre-
atine is a kinetically similar analog of creatine that is phosphorylated
and dephosphorylated by mitochondrial and cytosolic CKs. As a small,
relatively planar molecule, cyclocreatine has the chemical characteris-
tics to cross membranes.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
In the radial arm maze task, Slc6a8–/y mice consistently performed
worse than their controls in all parameters measured at the base-
line assessment (i.e., working memory error and reference memory
error in both original baited arms and reversed baited arms; Sup-
plemental Figure 5). In Slc6a8fl/y mice, working and reference errors
decreased significantly in reversed baited arms compared with
original baited arms (P < 0.001 for both). However, no significant
difference was seen in Slc6a8–/y mice between original and reversed
baited arms with respect to either error, suggestive of impairment.
Novel object recognition. Slc6a8–/y mice showed significant impair-
ment in the novel object recognition test that was normalized
after 9 weeks of cyclocreatine treatment (Figure 6). Whereas the
discrimination index was significantly lower in Slc6a8–/y than in
Slc6a8fl/y mice before treatment (–0.07 ± 0.06 versus 0.41 ± 0.06;
P ≤ 0.001), cyclocreatine-treated Slc6a8–/y mice showed a discrimi-
nation index of 0.53 ± 0.16, not significantly different from that of
littermate controls receiving the same treatment. The other treat-
ments showed no significant change from pretreatment levels in
the Slc6a8–/y mice (creatine, –0.03 ± 0.06; placebo, 0.05 ± 0.13).
Activity. Home cage activity increased in Slc6a8–/y mice, which
were significantly more active than Slc6a8fl/y controls, especially in
the dark phase (P < 0.01; Supplemental Figure 2D). The trend was
still seen after cyclocreatine treatment (P = 0.06).
Motor function. All motor function parameters — duration to fall
from a rotarod with maximal speed, latency to fall in the hang-
ing wire test, and time taken to cross a 50-cm-long beam — were
essentially identical between Slc6a8–/y and Slc6a8fl/y mice and were
not significantly changed after cyclocreatine treatment (Supple-
mental Figure 6). Moreover, no missteps were observed with the
beam walk test.
Brain-specific Slc6a8 knockout. (A) Strategy for recombination and deletion of Slc6a8. The structure of the wild-type Slc6a8 locus, the targeted
locus after recombination in embryonic stem cells, and the floxed allele after Flp-mediated deletion of the neo cassette are shown. Cre expression
results in deletion of exons 2–4 (E2–E4). Exons are denoted by boxes; neo, loxP sequences, and FRT sequences are denoted by triangles. (B)
Quantitation of Slc6a8 mRNA levels. Band intensities in gel images were measured and corrected by the intensity of Actb.
2840 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
In the present study, we validated our brain-specific CRT-knock-
out Slc6a8–/y mouse model of CRT deficiency as being comparable
to the human disease. We also reported the effective treatment of
cognitive deficits in Slc6a8–/y mice with 9 weeks of therapy with
the repurposed drug cyclocreatine. We propose a 2-part model in
which creatine and phosphocreatine prove central to the energy
metabolism and function of brain cells (Figure 7, A and B): the
creatine transport mechanism is interrupted, leading to impaired
energy metabolism along with impaired function.
At baseline, the phenotype in Slc6a8–/y mice was pronounced
for cognitive and executive function deficits, but normal for bal-
ance and musculoskeletal control systems. Creatine and phos-
phocreatine in the brain was significantly decreased, whereas cre-
atine in skeletal muscle, heart, and serum was normal (Table 1).
Unlike whole-body Slc6a8-knockout mice, in which body weight
is normal and urine creatine is high (9, 11, 8, 35, 36), we observed
normal creatine in Slc6a8–/y muscle, also modeling what is seen
in patients. After 9 weeks of cyclocreatine treatment, cyclocre-
atine entered the brain and was phosphorylated, and the cogni-
tive deficits of Slc6a8–/y mice were normalized compared with
Slc6a8fl/y mice. Creatine or placebo treatment did not show any
cognitive or musculoskeletal benefits for Slc6a8–/y mice. These
data suggest that cyclocreatine is a potential therapeutic for
patients with CRT deficiency.
Justification of the Slc6a8–/y mouse model. A mouse model that
resembles the human condition is an important tool with which
to validate cyclocreatine as a therapy for patients with CRT defi-
ciency. We had previously reported whole-body Slc6a8-knockout
mice using different Cre recombinase (CMV promoter; ref. 28);
these had a very severe phenotype distinct from that of patients
with CRT deficiency, including motor deficits (8). We felt that
cyclocreatine treatment of the whole-body knockout might pro-
duce results in which it would be difficult to distinguish cogni-
tive and motor benefits. Thus, we here generated brain-specific
Slc6a8–/y mice in order to focus on the cognitive and executive
function deficiencies key to the patient pathology we sought to
address. Slc6a8–/y mice had a phenotype with markedly reduced
levels of creatine in the brain, similar to what is seen in human
patients (37), as well as impaired cognitive function, normal
motor function, and normal creatine levels in skeletal muscles,
heart, and serum. With the current model and the results report-
ed therein, we believe that cyclocreatine treatment improves
brain function independent of muscle effects and therefore
should be considered as a viable treatment of human CRT defi-
ciency. However, the data do not tell us whether cyclocreatine
enters the brain cells by diffusion or through an alternate trans-
Biochemical phenotype and human CRT deficiency. CRT deficiency
was originally characterized as lacking creatine in the brain (9, 16);
similarly, we reported here a substantial decrease in creatine in
mice lacking CRT, as assessed by biochemical analysis (Table 1).
After cyclocreatine treatment, Slc6a8–/y mouse brains showed sub-
stantial increases in cyclocreatine and cyclocreatine phosphate.
This suggests that cyclocreatine phosphate is effectively replacing
phosphocreatine in the brain and reversing the metabolic deficits,
which results in cognitive normalization.
Mitochondrial CK is thought to modulate mitochondrial respi-
ration by shifting the mitochondria’s sensitivity to ADP (38). Brain
mitochondria are essential for energy metabolism and normal
cellular function. In CNS, the creatine/phosphocreatine/CK sys-
tem is involved in neurotransmitter release, membrane potential
maintenance, Ca2+ homeostasis, and/or ion gradient restoration
Subcortical (mmol/kg wet wt)
Cortex (mmol/kg wet wt)
Gastrocnemius (mmol/kg wet wt)
Soleus (mmol/kg wet wt)
Heart (mmol/kg wet wt)
Bladder (mmol/kg wet wt)
Kidney (mmol/kg wet wt)
Liver (mmol/kg wet wt)
Lung (mmol/kg wet wt)
(n = 10)
(n = 9)
12.3 ± 1.01
11.2 ± 0.74
2.8 ± 0.20A
2.8 ± 0.11A
37.6 ± 3.13
30.2 ± 2.04
13.5 ± 0.70
6.1 ± 0.33
1.1 ± 0.04
0.24 ± 0.02
0.55 ± 0.04
0.14 ± 0.01
4.0 ± 0.66
38.8 ± 2.92
29.9 ± 2.24
13.5 ± 1.97
5.5 ± 0.29
1.1 ± 0.10
0.27 ± 0.02
0.49 ± 0.04
0.12 ± 0.004
10.2 ± 2.57B
Values are mean ± SEM. AP ≤ 0.001, BP < 0.05 vs. Slc6a8fl/y, 2-tailed
Student’s t test.
Increased cyclocreatine content in brain after cyclocreatine treatment. (A) Cyclocreatine (cCr) and (B) creatine (Cr) content in brains of Slc6a8–/y
mice (n = 7 [cyclocreatine]; 5 [creatine and placebo]) and Slc6a8fl/y littermate controls (n = 5 per group) after 9 weeks of treatment, measured by
biochemical assays. P, placebo. Data are mean ± SEM. ***P ≤ 0.001 vs. Slc6a8fl/y; ###P ≤ 0.001 as indicated by brackets.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
(1–3). One of the major energy-demanding activities of the CNS
is action potentials. After depolarization of an action potential,
which uses stored energy, the constitutive repolarization requires
resynthesis of energy in the form of ATP and phosphocreatine.
The ATP is used to reestablish the ionic gradients across the mem-
brane. Action potentials often travel together along an axon as a
train; the faster and longer the action potential train, the more
energy the neuron uses (39). In Slc6a8–/y animals, it is possible that
the ATP buffering is inadequate to meet the energy demands for
certain action potential trains. Lack of phosphocreatine means
the ATP concentration cannot be buffered, which could result
in truncated or altered action potential trains with concomitant
loss of message. Previous studies have suggested a relationship
between impaired/improved cognitive function or hippocampal
neurogenesis and changed brain energy metabolism that occurs
upon consumption of a high-fat, low-carbohydrate diet (40, 41).
Although we have not measured action potential trains or conduc-
tion velocities, our present studies demonstrate cognitive benefits
manifest in Slc6a8–/y mice after cyclocreatine treatment. This is
consistent with other reports that creatine supplementation can
improve cognitive performance parameters of healthy human sub-
jects when brain creatine is increased (42, 43). It may be that with
cyclocreatine phosphate, the ATP concentration was buffered; this
was demonstrated by Woznicki et al., who showed that cyclocre-
atine phosphate indeed functions as an ATP buffer, like phospho-
creatine, in the mouse brain in vivo (31).
The brain’s dependence on the CK system has been previous-
ly demonstrated, to a certain extent, with knockout of the 2 CK
isoenzymes expressed in brain, ubiquitous mitochondrial CK
(UMTCK) and brain CK (CKBB) (44). These mice show cognitive
deficits and abnormalities in the formation and maintenance of
hippocampal fiber connections. The cause of the morphological
changes in the absence of the CK is unclear. In our model, however,
it seems apparent that the creatine/phosphocreatine/CK system
plays a critical role in hippocampal function, which shows con-
comitant impairment with CRT knockout.
CRT therapies. Some groups — citing reports that the rodent
brain has the enzymes to synthesize creatine (5, 4, 26, 27) — have
attempted to treat patients with CRT deficiency by supplementing
the synthesis precursors for creatine, but this approach did not
succeed (45, 46). Even if creatine were synthesized in the brain, the
synthesis enzymes appear to be in different cell types and unable
to make enough creatine for normal function, because creatine
was not detected or extremely low in patients with CRT deficiency
and in Slc6a8 whole-body knockout mice (28). Thus, creatine is
not entering or being made in the brain, or at least in the brain
regions in which it is required. Importantly, our present study also
showed no significant changes in brain creatine levels or in func-
tional parameters in creatine-treated Slc6a8–/y mice, which demon-
strated that the CRT and creatine present in the animals’ brains
was inadequate to reverse the cognitive deficits.
Strikingly, cyclocreatine treatment normalized several of the
cognitive parameters in adult Slc6a8–/y mice, suggestive of not just
a palliative improvement, but the return of substantial neurocog-
nitive function. This is quite intriguing, given that the animals
were adults when they started cyclocreatine treatment. It is also
noteworthy that home cage activity did not significantly change,
an observation that requires further investigation.
Cyclocreatine treatment. CRT deficiency is caused by a mutation
of CRT, but it is also a disease of the 2 CKs. In the absence of its
substrate, mitochondrial CK can crystallize (47) and lead to mito-
chondrial paracrystalline inclusions and concomitant pathology.
Therefore, any treatment of CRT deficiency needs to address CRT,
UMTCK, and CKBB in the cytosol. Cyclocreatine is known to
interact with both of the CKs, and our present observations sug-
gest that it effectively bypasses the transporter, making it an ideal
drug to treat the disease.
Cyclocreatine was first synthesized by Kenyon and colleagues in
1971 (48). It was then evaluated for its biochemical properties in
vitro and in vivo by Walker and others (29, 31). These investigators
revealed that cyclocreatine is the most kinetically similar analog of
creatine in vitro, with Vmax approaching that of creatine itself (rela-
tive Vmax, 90 and 100 nmol/s; Km, 25 and 5 mM, for cyclocreatine
and creatine, respectively; ref. 29).
In mice fed cyclocreatine orally, the drug has been shown to be
taken up by brains, be phosphorylated to cyclocreatine phosphate,
and buffer ATP in vivo (31). These authors also demonstrated that
maximal value for phosphorylation in the mouse brain is mark-
edly higher for cyclocreatine than for the natural phosphagen,
phosphocreatine. When mice were fed a cyclocreatine-containing
diet, 98% of cyclocreatine observed in the brain was in the form of
cyclocreatine phosphate, whereas 70% of creatine was phosphocre-
atine, a reflection of a distinct Keq for the compounds.
Chemically, our present data demonstrated 2 things: (a) accu-
mulation of cyclocreatine in the brain was slower in Slc6a8–/y than
in Slc6a8fl/y mice, likely due to lack of CRT; and (b) cyclocreatine
Phosphorylated metabolites in the brain, measured by 31P-MRS. An indirect measure of the phosphate metabolites was determined by taking
the peak height of the metabolite of interest and dividing by the sum of all the other phosphate metabolites. (A) Baseline measurements (n = 3
per group) of inorganic phosphate (Pi), phosphocreatine (PCr), and β-adenosine triphosphate (β-ATP). (B) Ratio of phosphocreatine plus
phosphorylated cyclocreatine (PcCr) to total phosphorylated metabolites after 9 weeks of treatment (n = 3 per group). Data are mean ± SEM.
*P ≤ 0.05, **P ≤ 0.01 vs. Slc6a8fl/y.
2842 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
entered the Slc6a8–/y brain and was phosphorylated at levels similar
to those in Slc6a8fl/y mice. One possible reason why cyclocreatine
was taken up by the brains of Slc6a8–/y mice is that cyclocreatine
is a nearly planar 5-membered ring. Although our studies cannot
definitively prove how or where cyclocreatine entered the brain in
our Slc6a8–/y model, they demonstrated that it was in fact entering
and had a therapeutic effect.
Walker and colleagues also showed that in mice fed a diet con-
taining large amounts of cyclocreatine, intraperitoneally inocu-
lated Ehrlich ascites tumor cells grew more slowly than did those
in mice fed a control diet (49). Studies followed concerning the
effects of cyclocreatine on proliferation of various cancer cells,
in vitro and in vivo, but did not lead to an approved drug. Rein-
forcing the possibility that cyclocreatine may be used to treat
patients with CRT deficiency is the fact that it has already been
given to humans; thus, the manufacture and toxicology have
been established (32, 33). Using a repurposed drug may prove
a faster and more effective method of getting the treatment to
patients. Some side effects have been reported for cyclocreatine
in humans (1, 32, 33); we believe these can be mitigated with
various monitoring strategies, such as assaying for cyclocre-
atine in the hair to monitor compliance or excess accumulation
(Supplemental Figure 4). In human studies, hypoglycemia was
reported when cyclocreatine was administered intravenously (1);
we believe that this can be monitored and
mitigated, should such compilations arise in
patients with CRT deficiency.
Future studies on cyclocreatine are required to
determine the timing of the treatment and at what
age it should be administered. Because cyclocre-
atine phosphate has a half-life of 17–28 days in the
mouse brain in vivo (50), daily dosing may not be
needed. The doses to be used in humans and the
target cyclocreatine phosphate concentrations in
the brain are also yet to be determined.
A word of caution concerning future clinical studies.
The cyclocreatine we obtained was cGMP grade,
used in the previous clinical studies and highly
pure. A quick Internet search showed that cyclo-
creatine is available for sale, but at 98% and 99%
pure. As the manufacture of cyclocreatine involves
cyano compounds (30), it is possible that the
impurities in reagent-grade cyclocreatine may
be cyanohydrins or other toxins. Moreover, apo-
cyclocreatine was marketed on one Internet site as
cyclocreatine. Therefore, any human studies using
cyclocreatine must be performed with great care
using high-quality cyclocreatine. Patients with
CRT deficiency and their families are not advised
to try cyclocreatine treatment without consulting
their physicians and ensuring that FDA drug safe-
ty standards for such treatments are met.
Conclusion. Our present findings showed that
Slc6a8–/y mice demonstrated the clinical manifes-
tations of human CRT deficiency and were suc-
cessfully treated by cyclocreatine. Further studies
using these mice to assess pharmacodynamics,
pharmacokinetics, and dose ranging are needed
to better guide future clinical trials.
Materials. B. Tsao-Nivaggioli (Avicena Group, Palo Alto, California, USA)
provided the cGMP-grade cyclocreatine used for this study. All other chem-
icals were reagent grade.
Generation of Slc6a8–/y mice. A Cre-lox system (51) was used to generate a
conditional Slc6a8 knockout in the brain. We first generated Slc6a8 floxed
mice. Homologous recombination in C57BL/6N cells was carried out by
transfecting a targeting vector containing a loxP site within intron 1 and
a positive selection cassette containing the neomycin phosphotransferase
gene (neo) flanked by Frt sites and the second loxP site within intron 4
(Figure 2A). Neomycin-resistant ES cell clones were screened for homolo-
gous recombination by PCR, followed by Southern blot analysis of ES cell
genomic DNA using probes located outside the 5′ and 3′ homology arms.
Correctly targeted ES cell clones were injected into MF-1 blastocysts. Male
chimeras were crossed with C57BL/6J females to produce N1F0 offspring,
which was confirmed by Southern blot and PCR analysis. The conditional
allele was generated by breeding the heterozygous mice to a germline Flp
deleter strain (Jackson Laboratory) to delete the neo cassette, leaving a
single loxP and Frt sites in intron 4. The resulting mice were further bred
with C57BL/6J females to be free of flpase. We then crossed Slc6a8fl/fl female
mice with male mice (C57BL/6J) expressing a Cre recombinase driven by
the CamkIIα promoter in the brain (52) to generate Slc6a8–/y mice and
Slc6a8fl/y littermate controls. We chose this Cre construct because it drives
expression throughout the mouse brain, but shows particularly dramatic
Improved spatial learning and memory in Slc6a8–/y mice after cyclocreatine treatment. (A)
Latency to hidden platform in trials, (B) percentage of time spent in platform area in probe
trial, (C) and velocity of swimming in platform area in Morris water maze probe trial for
Slc6a8–/y (n = 7 [cyclocreatine]; 5 [creatine and placebo]) and Slc6a8fl/y (n = 5 per group)
mice before and after 9 weeks of treatment. Data are mean ± SEM. *P ≤ 0.05 vs. Slc6a8fl/y.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
expression in the hippocampus and cortex, areas controlling cognitive
function, which is an important aspect of the human phenotype. It is also
known that Slc6a8 is highly expressed in that area in mice (53). We did not
observe problems with the floxed mice, so we used Slc6a8fl/y animals as con-
trol littermates. This minimizes the influence of genetic background on
the observed phenotype. A total of 29 Slc6a8–/y mice and 28 male Slc6a8fl/y
controls were used in this study. At 6 months of age, baseline assessment
was performed for all mice. To assess the efficacy of cyclocreatine treat-
ment, 17 of the 29 Slc6a8–/y mice and 15 of the 28 Slc6a8fl/y controls were
further used for 9 weeks of treatment.
Experimental design. Mice were maintained on an ad libitum standard pel-
leted diet (Teklad irradiated standard diets; Harlan animal research labo-
ratory) and on ad libitum water intake with 12-hour dark/light cycles in
70°F ± 2°F through the study. At the conclusion of the study, mice were
analyzed for Slc6a8 mRNA levels. Other tests — such as baseline values for
working and reference memory, novel object recognition, Morris water
maze, body composition, home cage locomotor activity, rotarod, hanging
wire grip, and beam walk tests — were done before and after treatment. At
12 months of age, Slc6a8–/y and Slc6a8fl/y mice were randomly assigned to
groups and started on 1 of 3 treatments for 9 weeks: (a) cyclocreatine (n = 7
[Slc6a8–/y]; 5 [Slc6a8fl/y]), (b) creatine (n = 5 per group), and (c) maltodextrin
as placebo (n = 5 per group). Each treatment compound was supplement-
ed in the drinking water, and the concentration was adjusted to deliver
0.28 mg/g body weight/d. This is a standard creatine dose for human sub-
jects (20 g/70 kg body weight/d), intended to induce maximum creatine or
phosphocreatine concentrations inside the body. Body weight and water
and food intake volumes were monitored every other day throughout the
treatment period. After 9 weeks of treatment, each parameter was evalu-
ated and compared with baseline values or between groups.
Genotyping. Genotyping of the conditional allele was performed by PCR
with primers that flank the position of the loxP-Frt insertion with genomic
DNA from mouse tail and brain templates (VS747 in intron 4, 5′-AGGTC-
CAGACAGTAACTACCCTTC-3′; VS584 in intron 4, 5′-TGGGTTT-
GCAGCTTGGTGTTATTGC-3′; Pnew in intron 1, 5′-TCCTACAC-
CAATACCCCCATAAGC-3′) under the following conditions: 40 cycles of a
reaction consisting of 30 seconds of denaturation at 94°C, 30 seconds of
annealing at 58°C, and 1 minute of elongation at 72°C, followed by a final
extension for 10 minutes at 72°C. The expected product sizes were 422 bp
for the WT allele, 548 bp for the floxed allele, and 346 bp for the knockout
allele. PCR for Cre recombinase expression was performed with genomic
DNA from mouse tail templates (Cam k1, 5′-GGTTCTCCGTTTGCACT-
CAGGA-3′; Cam k2, 5′-CCTGTTGTTCAGCTTGCACCAG-3′; Cam k5,
5′-CTGCATGCACGGGACAGCTCT-3′) under the following conditions:
30 cycles of a reaction consisting of 30 seconds of denaturation at 94°C,
30 seconds of annealing at 67°C, and 1 minute of elongation at 72°C, fol-
lowed by a final extension for 10 minutes at 72°C. The expected product
sizes were 350 bp for Cre recombinase and 300 bp for the internal control.
PCR products were separated by electrophoresis on a 1.5% agarose gel and
visualized by ethidium bromide staining; images were taken with a Univer-
sal Hood II (Bio-Rad Laboratories).
Semiquantified RT-PCR. Total RNA was extracted from brain tissues
of Slc6a8–/y and Slc6a8fl/y mice at 6 months of age using TRIzol reagent
(Invitrogen) according to the manufacturer’s instruction and was quanti-
fied by determination of absorbance at A260. Then, total RNA was treat-
ed with RNase-free DNase (Ambion). RT priming with oligo (dT) prim-
ers (Invitrogen) was performed to generate cDNAs from 1 μg total RNA
using Superscript II (Invitrogen) following the manufacturer’s instruc-
tions. Equal amounts of cDNA from all samples were subjected to PCR.
PCR primer pairs were as follows: Slc6a8 forward, 5′-CCATGAAGACT-
GTGCCAATG-3′; Slc6a8 reverse, 5′-CCCCTTCCACACACAGAAGT-3′;
Actb forward, 5′-GTGGGCCGCCCTAGGCACCAG-3′; Actb reverse,
5′-CTCTTTGATGTCACGCACGATTTC-3′. Reactions were performed in
25 μl total volume with 25 pM of each primer. Amplification conditions
were as follows: 95°C for 10 minutes, followed by 28 or 25 cycles (for
Slc6a8 or Actb, respectively) of 95°C for 30 seconds, 60°C for 30 seconds,
and 72°C for 30 seconds. The expected product size was 202 bp. Ampli-
fied fragments were separated by electrophoresis on 2% agarose gels and
visualized by ethidium bromide staining. The intensity of each band was
measured by Scion Image (Scion Corp.), and the intensity of Slc6a8 was
expressed relative to that of Actb.
Cyclocreatine and creatine assay. Animals were deeply anesthetized with
1%–2% isoflurane delivered with oxygen. Whole blood was harvested with
a 1-ml syringe and 27-gauge, 0.625-inch needle from the heart and centri-
fuged at 3,000 g for 20 minutes; serum was subsequently placed in Eppen-
dorf tubes. Brain, liver, heart, kidney, lung, bladder, and soleus and gas-
trocnemius muscles were rapidly removed and frozen in liquid nitrogen.
Urine and hair samples were also collected. The samples were each dropped
into 250 μl boiling water and boiled for 20 minutes to remove protein and
lyse cells. The total creatine content of the protein-free extract was assayed
using the fluorometric method of Conn (54). Total cyclocreatine content
of brain and hair was assayed using the method of Griffith (55). All chemi-
cals were obtained from Sigma-Aldrich unless otherwise stated.
In vivo MRS. All data were collected on a Bruker BioSpec 7T system
(Bruker BioSpec 70/30) equipped with 400 mT/m actively shielded gradi-
ents. In total, 6 mice were used at baseline assessment, and 18 mice after
9 weeks of treatment. Mice were anesthetized by 1%–2% isoflurane deliv-
ered in air, and the respiration rate was maintained at 60–100 breaths per
minute. Core temperature was maintained at 37°C by warm air circulated
through the magnet bore. All animals’ brains were scanned with a custom-
built radio frequency coil. T2-weighted axial and sagittal localizer images
were acquired with a fast-spin echo sequence. A proton double-spin echo
sequence was used to shim on a voxel approximately 6 mm × 4 mm × 6 mm,
covering most of the brain. The water proton line width averaged about
17 Hz. After shimming, 31P data were acquired with an ISIS sequence
from the same voxel using a 4-second repetition time, 2,048 complex
points, 8,000 Hz spectral width, and 4 repetitions of 480 averages each.
Total phosphorus acquisition time was 2 hours. Individual metabolites
Improved object recognition memory in Slc6a8–/y mice after cyclo-
creatine treatment. Novel object recognition tests were conducted
3 hours after familiarization in Slc6a8–/y (n = 7 [cyclocreatine]; 5 [cre-
atine and placebo]) and Slc6a8fl/y (n = 5 per group) mice before and after
9 weeks of treatment. The discrimination index was calculated as the
difference between new and familiar object exploration times divided
by total time spent in object zones. Data are mean ± SEM. **P ≤ 0.01,
***P ≤ 0.001 vs. Slc6a8fl/y.
2844 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
were identified by chemical shift. Ratios of metabolites to total phos-
phorus were calculated by comparing the total peak amplitude of all
phosphorylated metabolites with the individual peak heights. Metabolite
peak heights were averaged and reported.
Morris water maze. Before and after 9 weeks of treatment, a total of
32 mice were subjected to Morris water maze testing, a hippocampal-
dependent task of spatial learning and memory. The maze consisted of
a circular fiberglass pool (122 cm diameter, 75 cm high; Rowland Fiber-
glass Inc.) filled with water (18 ± 1°C, 43 cm deep). A clear glass platform
(10.5 cm × 10.5 cm square) was submerged 1 cm below the water surface.
The pool was situated in a room containing extramaze cues (42-cm × 76-cm
posters printed with contrasting patterns and shapes) that provide specific
visual reference points for locating the submerged platform. A video cam-
era mounted to the ceiling, directly above the center of the pool, was used
for recording the probe trial. The recording was digitized by a computer
and analyzed using CleverSystem Topscan software (Cleversys) for path
analysis (distance traveled and percentage of time in the target platform
area). Each mouse received 3 trials in the water maze on each of 5 days. The
submerged platform remained in one quadrant of the pool throughout all
trials, and latency to find the platform was recorded. If the mouse failed
to reach the platform within 60 seconds, the trial was terminated, and the
mouse was guided onto the platform for 5 seconds. On the sixth day, each
mouse received a final 60-second probe trial in which the platform was
removed from the pool.
Radial maze. An 8-arm radial maze was used to test spatial working and
reference memory at baseline with a total of 32 mice (n = 17 [Slc6a8–/y]; 15
[Slc6a8fl/y]). The maze consisted of an octagonal central platform (51.5 cm in
diameter) with 8 radial arms (61 cm long, 12 cm wide, 10 cm high) extending
outward (Lafayette Instrument Co.). The maze was elevated 70.5 cm above
the floor in a room containing many extramaze visual cues. Mice must use
these visual stimuli to navigate the maze, which lacked intramaze, nonspa-
tial cues for navigation. First, a food deprivation schedule was carried out to
reduce animals’ weight to 85% of baseline. During the entire training and
test periods, water was available ad libitum. Next, an acclimation trial was
conducted. On days 1 and 2, food pellets were placed near the end of all
arms. From day 3, food pellets were located only in arms 3, 5, 7, and 8; the
Creatine and cyclocreatine in brain function. (A)
The creatine/CK system is essential for shut-
tling energy from sites of energy production to
sites of energy use. Creatine and phosphocre-
atine can modulate energy metabolism at the
mitochondria and glycolytic pathways. When
creatine is absent, energy supply can be insuf-
ficient or slow during energy demands. Having
cyclocreatine and phospholylated cyclocreatine
keeps ATP levels more constant and decreases
pathophysiological consequences. (B) In con-
trast to the transport and use of creatine by
the normal brain, with CRT deficiency, creatine
cannot enter the brain, resulting in poor speech
and cognition. In the cyclocreatine-treated CRT
deficiency brain, cyclocreatine enters brain
cells and works with the cell’s metabolism to
improve speech and cognition.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
remaining 4 arms were empty. Mice received 1 or 2 training trials per day for
22 trials, and then were tested using reversed baited arms (food pellets in
arms 1, 2, 4, and 6 only) for 28 trials, for a total of 50 trials. At the begin-
ning of each trial, mice were placed in the central platform, then allowed to
explore the maze. Trials were completed when the 4 food pellets were eaten
or 15 minutes had elapsed. Arm entries for each mouse were recorded by an
investigator. The total number of entries into unbaited arms was tabulated
as an index of reference memory error, and the number of reentries into pre-
viously baited arms was used as an index of working memory error.
Novel object recognition. Before and after 9 weeks of treatment, the novel
object recognition task was conducted in a Plexiglas open-field apparatus
(60 cm × 50 cm × 21 cm high) to test short-term recognition. Mice were
individually habituated to the open-field apparatus with no objects in the
cage for 5 minutes at a time on 3 consecutive days. On the fourth day,
2 identical objects (50-ml falcon tubes, 11 cm high, 3.5 cm diameter, cov-
ered with yellow tape) were placed symmetrically 12 cm away from the wall.
The mouse was placed near the wall at equal distance from both objects
and observed for 5 minutes. A second 5-minute trial was done 3 hours
later, in which one of the familiar objects was replaced by a novel one (tube
4 cm in diameter, covered with red tape). We defined exploration zones
around the objects (10 cm diameter), and time spent inside the zones was
used as an index of object exploration. The discrimination index was cal-
culated by dividing the difference between exploration times of the new
object and the familiar object by the total time spent in object zones.
Body composition analysis. Total lean tissue, fat tissue, and water of the
32 total Slc6a8–/y and Slc6a8fl/y mice was determined by MRI (56) before
and after 9 weeks of treatment. After system calibration, an unanesthe-
tized mouse was weighed, placed in the restraint tube, and inserted into the
Echo MRI whole-body composition analyzer (EchoMedical Systems) for 45
seconds. Fat mass and lean body mass were determined.
Home cage locomotor activity. We measured the locomotor activity
expressed by each animal in its home cage before and after 9 weeks of treat-
ment. This allowed us to capture general locomotor activity, unrelated to
the animals’ activity in a novel, anxiogenic environment. The cage rack
frame (Lafayette Instrument Co.) equipped with infrared photobeams was
placed around each animal’s standard shoebox home cage. Infrared pho-
tobeam interruption sensors (7X and 15Y) mounted in the frame detected
movement, which was recorded and analyzed using HMM100 Motor Mon-
itor software (Lafayette Instrument Co.). Vertical and horizontal activity
within the home cage was recorded for 48 hours, and events were collapsed
into 60-minute bins. In order to discern short- and long-term activity–
related circadian rhythms, results were expressed as the average number of
beam interruptions per group per hour.
Rotarod. The rotarod test of motor coordination and motor learning
was performed before and after 9 weeks of treatment. On day 1, mice
were placed on a stationary rod (3.2 cm diameter) of the apparatus (Type
ENV-576M; Med associates) for 30 seconds. Daily training trials were then
administered for the next 7 consecutive days: mice were placed on the rod
at increasing speeds, from 16 to 32 rpm, for up to 2 minutes. On day 9,
mice were placed on the stationary rod at maximum speed (32 rpm). The
duration in seconds until the animal fell from the rod was recorded as a
measure of motor coordination; cutoff time was 120 seconds.
Hanging wire grip. Before and after 9 weeks of treatment, the hanging wire
grip test was performed by placing a mouse on a wire net (2.5-cm × 2.5-cm
grid, 30 cm2 wire), then turning the net upside-down at approximately 50 cm
above the cage floor to prevent the animal from easily climbing down. The
elapsed time until the animal fell was recorded. Tests consisted of 3 trials
with 30-second intervals; cutoff time was 540 seconds.
Beam walk. The beam walk is a test of complex motor coordination.
Before and after 9 weeks of treatment, animals were trained to ambulate
across the beam (1 m long, 9 mm diameter) to a 14-cm2 platform for 7
consecutive days. On day 8, mice were placed on the beam 50 cm from the
platform, and latency to reach the platform was measured. Missed steps off
of the beam were also recorded.
Statistics. Data are expressed as mean ± SEM. Statistical significance
of mean differences for each parameter was determined by 2-tailed Stu-
dent’s t test; by ANOVA followed by post-hoc Tukey test (treatment group
as between-subject factor); or by repeated-measures ANOVA followed by
post-hoc Bonferroni test for multiple comparisons (treatment group and
treatment timing as between- and within-subject factors). A P value less
than 0.05 was considered significant.
Study approval. The experimental procedures performed herein were in
compliance with the NIH Guide for the Care and Use of Laboratory Animals
and were approved by the University of Cincinnati Institutional Animal
Care and Use Committee.
The authors thank Akiko Kuma (Tokyo Medical and Dental Uni-
versity, Tokyo, Japan) for her scientific advice. This work was sup-
ported by funding from the NIH (grant no. NS049172). T. Dai-
koku was supported by Cincinnati Children’s Hospital Medical
Center (Perinatal Institute Pilot/Feasibility Grant).
Received for publication February 15, 2012, and accepted in
revised form May 23, 2012.
Address correspondence to: Joseph F. Clark, University of Cin-
cinnati, 231 Albert Sabin Way, MSB Building Room 1055B,
Cincinnati, Ohio 45267-0536, USA. Phone: 513.558.7085; Fax:
513.558.7009; E-mail: firstname.lastname@example.org.
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