Current Biology, Vol. 15, 1961–1967, November 8, 2005, ©2005 Elsevier Ltd All rights reserved. DOI 10.1016/j.cub.2005.09.043

The HMG-CoA Reductase Inhibitor Lovastatin
Reverses the Learning and Attention Deficits
in a Mouse Model of Neurofibromatosis Type 1
Weidong Li,1,2,3,4 Yijun Cui,1,2,3,4
Steven A. Kushner,1,2,3,4 Robert A.M. Brown,1,2,3,4
J. David Jentsch,3,4 Paul W. Frankland,1,2,3,4,5
Tyrone D. Cannon,2,3,4 and Alcino J. Silva1,2,3,4,*
1
Department of Neurobiology
2
Neuropsychiatric Institute3
Department of Psychology
4Brain Research Institute
University of California, Los Angeles
Los Angeles, California 90095

Summary
Neurofibromatosis Type 1 (NF1) is a common neurological disorder caused by mutations in the gene encoding Neurofibromin, a p21Ras GTPase Activating
Protein (GAP) [1]. Importantly, NF1 causes learning
disabilities and attention deficits [2, 3]. A previous
study showed that the learning and memory deficits
of a mouse model of NF1 (nf1+/−) appear to be causedby excessive p21Ras activity leading to impairments
in long-term potentiation (LTP) [4], a cellular mechanism of learning and memory [5–7]. Here, we identify
lovastatin as a potent inhibitor of p21Ras/Mitogen
Activated Protein Kinase (MAPK) activity [8, 9] in thebrain. Lovastatin is a specific inhibitor of three-hydroxy3-methylglutaryl coenzyme A (HMG-CoA) reductase,used commonly for the treatment of hypercholesterolemia [10]. We report that lovastatin decreased the enhanced brain p21Ras-MAPK activity of the nf1+/− mice,
rescued their LTP deficits, and reversed their spatial
learning and attention impairments. Therefore, theseresults demonstrate that lovastatin may prove usefulin the treatment of Neurofibromatosis Type 1.
Results and Discussion
The key pathophysiologic mechanism underlying NF1
mutations in both mice [4, 11–13] and humans [14, 15]
is increased p21Ras activity. Therapeutic interventions
designed to inhibit p21Ras function have been proposed as treatments of NF1 [16]. Posttranslational farnesylation is required for the membrane localization andfunction of p21Ras, and farnesylation provides a potential target for NF1 pharmacotherapy [17]. Indeed, pharmacologic inhibitors of farnesyltransferase downregulate p21Ras activity. It is unknown, however, whether
any of these inhibitors have the in vivo pharmacokinetics, biodistribution, and safety profile required for the
long-term treatment of cognitive dysfunction in NF1 [18].
Lovastatin, a specific inhibitor of the rate-limiting enzyme in cholesterol biosynthesis (HMG-CoA reductase), is widely used to treat hyperlipidemia in humans*Correspondence: silvaa@mednet.ucla.edu
5
Present address: Department of Integrative Biology, Hospital for
Sick Children Research Institute, 555 University Avenue, Toronto,
Ontario M5G 1X8, Canada.

[19]. Previous studies have shown that lovastatin caninhibit p21Ras isoprenylation and activity [8, 20]. Since
the cognitive deficits caused by mutations in the NF1gene may result from increased p21Ras activity, we hypothesized that lovastatin could rescue these deficits.
The NF1 null mutation mouse is lethal as a homozygote but is viable as a heterozygote [13]. Interestingly,
we have not detected neuroanatomic deficits in the
nf1+/− mice, although they do display a range of behavioral abnormalities that parallel the cognitive profile associated with NF1 patients [1].
We analyzed the effect of lovastatin treatment on
p21Ras/MAPK by using Western blotting. nf1+/− mice
and wild-type littermate controls (wt) were injected with
10 mg/kg lovastatin subcutaneously once per day for 4days, and they were sacrificed on the 4th day, 6 hr after
the final injection. Levels of p44/42 were examined inboth cortex and hippocampus by SDS-PAGE. Proteins
were transferred to membranes and hybridized with
anti-phospho p44/42 MAPK (Cell Signaling) antibody.
The results in Figure 1A show that the levels of phosphorylated p44/42 MAPK are higher in both the cortex(p < 0.05) and hippocampus (p < 0.05) of nf1+/− micecompared to wt. Several days of treatment with 10 mg/
kg of lovastatin decreased the levels of phosphorylated
p44/42 MAPK in nf1+/− mice, resulting in roughly equalamounts of phosphorylated p44/p42 MAPK as wt (cortex: p = 0.759, hippocampus: p = 0.850). Sample loading was controlled by reprobing the nitrocellulose membranes used for the analysis with an anti-p44/42
MAPK antibody.
Neurofibromin functions as a p21Ras GTPase-activating protein that catalyzes the conversion of active GTP
bound p21Ras to the inactive GDP bound form. We
therefore examined the impact of lovastatin treatment
on p21Ras activity directly. Cortical and hippocampal
extracts were reacted with GST-Raf1-RBD beads(Pierce Bio), which specifically bind p21Ras-GTP, theactive form of p21Ras. p21Ras-GTP was resolved by
SDS-PAGE and visualized with an anti-pan p21Ras
antibody (Sigma). Again, p21Ras-GTP levels were elevated in the nf1+/− compared to wt for both cortex (p <0.05) and hippocampus (p < 0.05). Lovastatin treatment
decreased the levels of p21Ras-GTP in both the cortex
and hippocampus of nf1+/− to the level of wt mice (cortex: p = 0.789, hippocampus: p = 0.195) (Figure 1B).
This result is consistent with decreased levels of MAPK
activity that we found in nf1+/− mice. Altogether, thesedata demonstrate that lovastatin can decrease p21Ras/
MAPK activity in the cortex and hippocampus and may
therefore be useful to treat cognitive deficits of the
nf1+/− mice.
NF1 in humans is associated with a broad spectrumof cognitive profiles, which may include learning disabilities, attentional disorders, and altered visuospatial
skills [2, 3, 21, 22]. We examined the effect of lovastatin
on nf1+/− mice in a variety of behavioral and cognitive
tests to assess how the loss of NF1 impacted on thesebehaviors and whether lovastatin could rescue any observed deficits.

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Figure 1. Downregulation of p21Ras-MAPK Activity in nf1+/− Mice by Lovastatin(A) Lovastatin was effective at decreasing active MAPK in the nf1+/− mice (4–9 mice per group).
(B) Lovastatin also decreased active p21Ras (p21Ras-GTP) in nf1+/− mice (6–7 mice for each group). Error bars represent ± one standard error.

To investigate altered attentional function in the nf1+/−
mice, we used the lateralized reaction-time task, a test
that measures divided visuospatial attention and is dependent upon prefrontal cortex. Animals nosepoke totrigger the delivery of a visual target stimulus on one
side of their visual field. The spatial location and timeof onset of the target is unpredictable and so attention
must be distributed across space and time. The taskdifficulty is altered by changing the duration that thetarget stimulus is visible. Wild-type and nf1+/− mice
were tested with lovastatin (nf1+/− = 7, wt = 7) or placebo (nf1+/− = 14, wt = 10). The rate of correct responses (an index of attention accuracy) revealed agenotype × treatment × target stimulus duration interaction (ANOVA, F2,70 = 3.200, p < 0.05). The correct

response rate of wt mice was significantly higher thanthat of nf1+/− mice (PLSD, p < 0.05; Figure 2A) at themost difficult stimulus duration (0.5 s), indicating thatthe nf1+/− mice have impaired attentional function.
In contrast, the performance of nf1+/− mice treatedwith lovastatin was indistinguishable from that of wt
mice at a target stimulus duration of 0.5 s (PLSD, p =0.148; Figure 2B) and was significantly higher thannf1+/− given placebo (PLSD, p < 0.05). These data demonstrate that nf1+/− mice exhibit substantial attention
deficits and that lovastatin treatment can reverse
these deficits.
We have previously shown that nf1+/− mice have abnormal spatial learning tested in the hidden version of
the water maze [4, 23], a task that is sensitive to lesions

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Figure 2. Rescue by Lovastatin of Attention Deficits in nf1+/− Mice(A) nf1+/− mice have deficits compared to wt animals in the 0.5 ms target stimulus duration condition of the lateralized reaction time test. The
deficit is rescued by lovastatin treatment.
(B) The deficit is seen only at the most difficult interval. Longer intervals produce equivalent performance between the groups (wt = 10,
nf1+/− = 14, wt with lovastatin = 7, nf1+/− with lovastatin = 7). Error bars represent ± one standard error.

of the hippocampus and multiple cortical sites including retrosplenial, orbitofrontal, medial prefrontal cortices, and the cingulum bundle [24–27]. To test the hypothesis that lovastatin can rescue the deficits of
nf1+/− mice in this task, we injected 10 mg/kg lovastatin
subcutaneously for 3 days before the first training day,and then 6 hr before behavioral training daily. Mice weretrained with two trials per day. No differences were observed between genotypes and/or treatment groups inmeasures of acquisition, floating, thigmotaxic behavior,or swimming speed (data not shown).
Spatial learning was assessed in probe trials given at
the end of water maze training on days 5 and 7, sinceprevious studies have shown that probe trial performance is the most faithful measure of spatial learningin the Morris maze [28]. In the probe trials, the platform
was removed from the pool and the mice were allowed
to search for it for 60 s. Neither genotype had learned
the task by the day 5 probe (Figure 3A). Differences
emerged between the genotypes and treatments(ANOVA, F1,82 = 4.415, p < 0.05) after 2 more days of
training. Wild-type mice spent significantly more time
searching in the target quadrant than nf1+/− mice
(PLSD, p < 0.05; Figure 3B), confirming that the nf1+/−

mutants have impaired spatial learning. In contrast, themutants treated with lovastatin spent as much time as
wt in the target quadrant (p = 0.862; Figure 3B) andsignificantly more time than mutants given placebo (p <0.05). These results demonstrate that the spatial learning deficits of the nf1+/− mice are not caused byirreversible developmental abnormalities since they arereversed with acute lovastatin treatment in adult mutant mice.
Previous studies report a high incidence of attentiondeficit hyperactivity disorder (ADHD) in NF1 patientsand support an association between ADHD and learning problems in these children [2, 29]. Children with
ADHD are reported to have significantly reduced prepulse inhibition (PPI) [30]. PPI assays sensory “gating”
of environmental stimuli. A sudden acoustic stimulus
will normally elicit a whole-body startle response. If thestartle-producing stimulus is preceded by a weak
prestimulus, the startle response is inhibited in normal
persons and animals [31]. We tested whether nf1+/−
mice have deficits in this task and whether these deficits could also be reversed by lovastatin treatment (asdescribed above). A two-way repeated measures
ANOVA revealed significant main effects of genotype

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Figure 5. Rescue by Lovastatin of nf1+/− Deficits in Long-Term Potentiation

Figure 3. Lovastatin Rescue of Spatial Learning Deficits in nf1+/− Mice(A) Percent time spent in each quadrant during a water maze probe
trial on day 5.
(B) Percent time spent in each quadrant during a probe trial on
day 7. Quadrants are target quadrant (TQ), adjacent left, opposite
quadrant (OP), and adjacent right (wt = 24, nf1+/− = 21, wt withlovastatin = 21, nf1+/− with lovastatin = 20). Error bars represent ±one standard error.

and treatment (Figure 4). The nf1+/− animals have deficient PPI (F1,30 = 7.42, p < 0.05), and lovastatin treatment resulted in an increase in performance (F1,30 =6.61, p < 0.05). Importantly, the performance of nf1+/−
animals on lovastatin is indistinguishable from that ofwt animals on placebo (PLSD, p = 0.877), demonstrating that lovastatin can reverse the PPI deficits of thesemutants. The increased PPI of the wt animals on lovastatin may be attributed to Ras inhibition. Current mod-Figure 4. Lovastatin Rescues Sensory Gating Deficits as Measured
with PPI
PPI was examined via prepulses at three different stimulus intensities (70, 75, and 80 dB) (wt = 8, nf1+/− = 8, wt with lovastatin = 9,
nf1+/− with lovastatin = 9). Error bars represent ± one standard error.

Percentage of baseline fEPSP is plotted over time. A five thetaburst induction protocol was delivered at time 0 (wt = 8, nf1+/− = 7,
wt with lovastatin = 8, nf1+/− with lovastatin = 7). For clarity purposes, error bars (standard error of the mean) are shown in only
one direction. Representative traces are shown from left to right:
wt off drug, nf1+/− off drug, wt on lovastatin, nf1+/− on lovastatin.
Horizontal bar represents 2 ms. Vertical bar represents 0.5 mV.

els of PPI attribute the mechanism to a presynaptic inhibition of release [32]. Given that Ras activity is known
to increase presynaptic release of transmitter [33], theeffects of lovastatin and PPI would work in concert todecrease presynaptic release. In the case of the nf1+/−
mutants, which have upregulated Ras function, the decrease merely brings them back into the normal range.
In wt animals, release would be greatly curtailed, resulting in the enhancement of PPI.
We have previously shown that the learning deficitsof the nf1+/− mice are likely caused by impairments in
LTP [4], a stable long-lasting change in synaptic
strength widely believed to be a key cellular mechanism for learning and memory that is dependent upon
Ras/MAPK function [5–7, 34]. We therefore determined
whether the LTP deficits in nf1+/− mice could be reversed by lovastatin. Mice were injected with 10 mg/kg
of lovastatin as described above. We examined LTP in
hippocampal slices at the Schaffer collateral/CA1 synapse, since LTP at this synapse has served as a model
system for associative changes in synaptic strength
between neurons [5–7]. LTP was measured after a 5
theta-burst stimulation protocol (TBS, five bursts 200
ms apart, each burst of 4 pulses at 100 Hz), which mimics in vivo activity of hippocampal neurons during exploratory behavior [35]. Figure 5 shows that there wasa difference among the genotypes and treatments(ANOVA, F1,26 = 8.55, p < 0.05). The LTP measured in
nf1+/− mutants was significantly lower than in wt mice
(PLSD, p < 0.05; Figure 5), a result consistent with previously published findings [4]. The amount of LTP induced in nf1+/− mutants treated with lovastatin was sig-

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nificantly higher than that induced in mutants (PLSD,
p < 0.05; Figure 5) and equivalent to that of wt (PLSD,
p = 0.602; Figure 5). These data demonstrate that thelovastatin treatment completely reversed the LTP deficits of the nf1+/− mice.
The present results demonstrate that lovastatin treatment can reverse the biochemical, electrophysiological, and cognitive deficits observed in a mouse modelof NF1 and that these deficits are not due to irreversible
developmental changes. Previous studies have shownthat an increase in p21Ras activity is central to thepathophysiology associated with NF1 [16], and our biochemical data demonstrate that lovastatin reverses theabnormally elevated p21Ras/MAPK activity in an animal model of NF1. Importantly, our studies demonstrated that the dose of lovastatin that is effective in
nf1+/− mice did not affect cognitive function in control
mice, a result consistent with randomized studies performed with human subjects that did not identify a reliable effect of lovastatin on cognitive function [36, 37].
Nevertheless, it is worth noting that there are sporadic
reports that statins can be associated with mild cognitive impairment [38]. Altogether, the studies reported
here demonstrate that the cognitive deficits associatedwith NF1 can be reversed by treatments with lovastatin,
a widely prescribed drug that is known to be well tolerated even in long-term treatments. Thus, these datasuggest that lovastatin could be used to treat the cognitive impairments associated with Neurofibromatosis
Type 1 in humans.
Experimental Procedures
Animal Experiments
All animal protocols were approved by the Chancellor’s Animal Research Committee at the University of California at Los Angeles, inaccordance with the National Institutes of Health guidelines.
Studies were performed in 129T2/SvEmsJ-C57BL/6 F1 hybrids
generated by an F1 cross between nf1+/− mice (maintained in the
C57BL/6 background for more than 11 generations) and wild-typemice on the 129T2/SvEmsJ background with wild-type littermatesused as controls. All experiments were carried out blind with respect to genotype and treatment.
Lovastatin Solution and Pellet
Because of the extended nature of the lateralized reaction timetask (see below), lovastatin was administered orally as pellets,
while in other tasks lovastatin was injected in the lactone form.
Mevinolin (lovastatin, Sigma) was dissolved in ethanol, then incubated at room temperature in 1N NaOH and water to mevinolin to
the sodium salt. The final mevinolin solution (4 mg/ml) was adjusted
to pH 7.5 with HCl [10, 39]. Pellets for oral administration wereprepared by mixing crushed lovastatin tablets (Eon Labs) (prescription formulation) with melted peanut butter chips (H.B. Reese
Candy Co.). The mixture was molded into 200 mg pellets containing
0.15 mg Lovastatin and were administered to mice once daily.
Western Blot Analysis for p44/42 MAP Kinase
Phosphorylation and p21Ras Activity
Cortical and hippocampal regions from 4 to 7 control and lovastatin-treated mice were isolated and homogenized in protein extraction buffer. Supernatant was collected after centrifugation, andproducts were separated by electrophoresis on a 4%–15% gradient or 12% SDS-PAGE (Bio-Rad Laboratories). Gels were blottedto nitrocellulose membranes and then blocked for 1 hr at room
temperature with Tris-buffered saline (TBS) containing 0.1% (v/v)
Tween-20 and 5% (w/v) nonfat dry milk. After washing in TBST,
membranes were hybridized at room temperature with anti-phos-

pho-p44/42 (Cell Signaling) antibody diluted 1:1000 in the blocking
solution. The membranes were visualized with the ECL Plus protocol (Amersham BioSciences) and quantified by a Storm System
phosphor imager (Amersham Biosciences). Membranes were strippedin stripping buffer and reprobed with anti-p44/42 (Cell Signaling) as
a control for protein loading. The amount of phosphorylated p44/
42 was expressed as a ratio of total p44/42. Other groups werenormalized to the wt ratio.
For p21Ras activity assay, p21Ras pull-down experiments were
performed with the EZ-detect p21Ras activation kit (Pierce Biotechnology), according to the manufacturer’s protocol. 25 ␮l ofsample per lane was loaded on SDS-PAGE. p21Ras-GTP was detected and quantified by Western blotting as described via an antipan-p21Ras antibody (Sigma). The ratio of Ras-GTP in each group
was normalized to wt.
Lateralized Reaction Time Task
Animals (placebo-treated: nf1+/− = 14, wt = 10; lovastatin treated:
nf1+/− = 7, wt = 7) were food deprived to 90% of their free-feedingweights. Mice were trained in a miniaturized versions of a “5choice” box (Med Associates, St Albans, VT). The chamber was
equipped with a horizontal array of five apertures that could beinternally illuminated and the opposite wall was fitted with a pellet
magazine for reward delivery. Animals were shaped to produce a“poke and hold” response in the central aperture during which aside aperture would be illuminated. A correct response was scoredwhen the animals correctly poked the aperture that had been illuminated. Animals that did not perform at 75% accuracy at 1 s target
stimulus duration during the training phase were dropped. Mice
were tested on a variable duration condition in which the target
aperture was illuminated for 0.5, 1.0, or 2.0 s (varied within session).
We measured correct responses/total trials, which vary as a function of the target stimulus duration and was therefore analyzed byuse of stimulus duration as a repeated measure [40].
Water Maze
The basic protocol for the water maze experiments has been previously described [11]. Mice from the 129T2/SvEmsJ-C57B/6 F1
genetic background were given two trials per day (30 s intertrial
intervals) with a probe trial (60 s) at the end of training days 5 and7. Mice were given subcutaneous injections of 10 mg/kg lovastatin
or vehicle for 3 days before the first training day and then 6 hr
before training every day.
Prepulse Inhibition
Mice were food deprived as described above. 5 min after placement in the conditioning chamber, mice were presented with 20
noise bursts (40 ms duration, 120 dB, <1 ms rise/fall time). In the
prepulse inhibition phase, mice were presented with a total of 90
trials (30 trials each at 70, 75, and 80 dB). For each prepulse intensity (20 ms duration, <1 ms rise/fall time), there were three types oftrial: prepulse alone, prepulse/startle stimulus, and startle stimulus
alone. In the prepulse/startle stimulus trial, the onset of the prepulse preceded the onset of the startle stimulus by 100 ms. Background noise levels were maintained at 68 dB throughout testing,and the trials were spaced 15 s [41].
Hippocampal LTP
Transverse hippocampal slices (400 mm thick) were placed in asubmerged recording chamber perfused (2 ml/min) with ACSF containing 120 mM NaCl, 3.5 mM KCL, 2.5 mM CaCl2, 1.3 mM MgSO4,
1.25 mM NaH2PO4, 26 mM NaHCO3, and 10 mM D-glucose at 34°C.
Field excitatory postsynaptic potentials (fEPSPs) were recordedwith a Pt/Ir electrode (FHC, Bowdoinham, ME) from the stratum
radiatum layer of the area CA1 elicited by stimulation of the Schaffer collateral/commissural afferents by two bipolar electrodes
placed 300 ␮m on either side of the recording electrode. Test
pulses of 60 ␮A alternated between the two electrodes every minute throughout the duration of the experiment. After 20 min of stable baseline, LTP was induced via a TBS protocol as described in
the text delivered to the test pathway. Multiple slices from a single
animal were averaged and entered into analysis as a single subject.

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The responses from the last 10 min block of recordings (40–50 min)were compared for statistical tests.
Statistical Analysis
Western blots were analyzed with the one-sample t test (mean =100, one tail upper). We analyzed percent time in training quadrant
for the different genotypes from the water maze by using 2-way
ANOVA. Attention data was analyzed with three-way repeatedmeasures ANOVA on the average of correct response rate. PPI data
was analyzed with two-way repeated-measures ANOVA. For theelectrophysiological experiments, the significance of differences
between the groups was determined by two-way ANOVA. Post-hoc
comparisons (Fisher’s PLSD) between groups were carried out
where appropriate.
Acknowledgements
We thank R. Chen, A. Matynia, and M.J. Sanders for technical assistance. We are grateful to R.M. Costa for invaluable discussionsof the experiment described here. This work was supported by
grants from the NIH (R01 NS38480), Neurofibromatosis, Inc. (National, Illinois, Mass Bay Area, Minnesota, Arizona, Kansas andCentral Plains, Mid-Atlantic, and Texas chapters), the Merck, andthe National Neurofibromatosis Foundation (NNF) to A.J.S. Y.C. was
supported by a Young Investigator Award from NNF.
Received: May 24, 2005
Revised: August 24, 2005
Accepted: September 19, 2005
Published: November 7, 2005
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