![Multivariate analysis of LC-MSE estimates as shown by condition plots illustrating the differences between the WT and Tsc1+/− mice with and without rapamycin treatment. X-axis is condition and y-axis is log ratio of endogenous (L light) over reference (H heavy) peptides. Dots represent the mean of the log2 ratio for each condition, and error bars indicate the confidence intervals with 0.95 significance. The interval is not related to the model based analysis. Significant changes as measured by LC-MSE are indicated below each protein. Illustrated are examples of proteins which are affected by rapamycin treatment and which are normalized by rapamycin treatment. Corrected p values (p*) were determined by post hoc correction after Benjamini-Hochberg [91]. CR = Wt + rapamycin, CS = Wt + vehicle, TR = Tsc1+/− + rapamycin, TS = Tsc1+/− − rapamycin](https://www.researchgate.net/publication/318839638/figure/fig3/AS:963405015957517@1606705063521/Multivariate-analysis-of-LC-MSE-estimates-as-shown-by-condition-plots-illustrating-the_Q320.jpg)
![Multivariate analysis of SRM estimates as shown by condition plots illustrating the differences between Wt and Tsc1+/− mice with and without rapamycin treatment. X-axis represents the condition and the y-axis the log ratio of endogenous (L light) over reference (H heavy) peptides. Dots represent the mean of log2 ratio for each condition, and error bars indicate the confidence intervals with 0.95 significance. The interval is not related to the model based analysis. Significant changes as measured by LC-MSE are indicated below each protein. SRM was able to validate protein abundance changes identified by label-free LC-MSE. Corrected p values (p*) were determined by post hoc correction after Benjamini-Hochberg [91]](https://www.researchgate.net/publication/318839638/figure/fig4/AS:963405015941148@1606705063631/Multivariate-analysis-of-SRM-estimates-as-shown-by-condition-plots-illustrating-the_Q320.jpg)
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R E S E A R C H Open Access
A brain proteomic investigation of
rapamycin effects in the Tsc1
+/−
mouse
model
Hendrik Wesseling
1
, Ype Elgersma
2
and Sabine Bahn
1,2*
Abstract
Background: Tuberous sclerosis complex (TSC) is a rare monogenic disorder characterized by benign tumors in
multiple organs as well as a high prevalence of epilepsy, intellectual disability and autism. TSC is caused by
inactivating mutations in the TSC1 or TSC2 genes. Heterozygocity induces hyperactivation of mTOR which can be
inhibited by mTOR inhibitors, such as rapamycin, which have proven efficacy in the treatment of TSC-associated
symptoms. The aim of the present study was (1) to identify molecular changes associated with social and cognitive
deficits in the brain tissue of Tsc1
+/−
mice and (2) to investigate the molecular effects of rapamycin treatment,
which has been shown to ameliorate genotype-related behavioural deficits.
Methods: Molecular alterations in the frontal cortex and hippocampus of Tsc1
+/−
and control mice, with or without
rapamycin treatment, were investigated. A quantitative mass spectrometry-based shotgun proteomic approach (LC-
MS
E
) was employed as an unbiased method to detect changes in protein levels. Changes identified in the initial
profiling stage were validated using selected reaction monitoring (SRM). Protein Set Enrichment Analysis was
employed to identify dysregulated pathways.
Results: LC-MS
E
analysis of Tsc1
+/−
mice and controls (n= 30) identified 51 proteins changed in frontal cortex and 108
in the hippocampus. Bioinformatic analysis combined with targeted proteomic validation revealed several dysregulated
molecular pathways. Using targeted assays, proteomic alterations in the hippocampus validated the pathways
“myelination”,“dendrite,”and “oxidative stress”, an upregulation of ribosomal proteins and the mTOR kinase. LC-MS
E
analysis was also employed on Tsc1
+/−
and wildtype mice (n= 34) treated with rapamycin or vehicle. Rapamycin
treatment exerted a stronger proteomic effect in Tsc1
+/−
mice with significant changes (mainly decreased expression)
in 231 and 106 proteins, respectively. The cellular pathways “oxidative stress”and “apoptosis”were found to be affected
in Tsc1
+/−
mice and the cellular compartments “myelin sheet”and “neurofilaments”were affected by rapamycin
treatment. Thirty-three proteins which were altered in Tsc1
+/−
mice were normalized following rapamycin treatment,
amongst them oxidative stress related proteins, myelin-specific and ribosomal proteins.
Conclusions: Molecular changes in the Tsc1
+/−
mouse brain were more prominent in the hippocampus compared to
the frontal cortex. Pathways linked to myelination and oxidative stress response were prominently affected and, at least
in part, normalized following rapamycin treatment. The results could aid in the identification of novel drug targets for
the treatment of cognitive, social and psychiatric symptoms in autism spectrum disorders. Similar pathways have also
been implicated in other psychiatric and neurodegenerative disorders and could imply similar disease processes. Thus,
the potential efficacy of mTOR inhibitors warrants further investigation not only for autism spectrum disorders but also
for other neuropsychiatric and neurodegenerative diseases.
Keywords: Tuberous sclerosis, Rapamycin, Proteomics, SRM, Animal model
* Correspondence: sb209@cam.ac.uk
1
Department of Chemical Engineering and Biotechnology, University of
Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK
2
Department of Neuroscience, Erasmus Medical Center, Rotterdam 3000, CA,
The Netherlands
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Wesseling et al. Molecular Autism (2017) 8:41
DOI 10.1186/s13229-017-0151-y
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Background
Tuberous sclerosis complex (TSC) is a rare multi-system
monogenic hamartomatous disorder, which is caused by
mutations inactivating the TSC1 (hamartin) or TSC2
(tuberin) genes. TSC is characterized by benign tumors
in multiple organs, including the brain, kidneys, heart
and eyes [1]. Over 90% of TSC patients develop epilepsy,
and around 50% present with neuropsychiatric prob-
lems, such as intellectual disability (50%) [2, 3], autism
spectrum disorder (ASD) (17–68%), schizophrenia (10–
30%) and anxiety disorders (40%) [4], which account for
most of the mortality and morbidity [5].
At the molecular level, both Tsc1 and Tsc2 protein
products form hetero-dimers which inhibit the GTP-
binding protein RHEB (Ras homolog enriched in the
brain). Consequently, mutations within either Tsc1 or
Tsc2 lead to increased levels of activated RHEB [6],
which causes hyperactivation of mammalian target of
rapamycin (mTOR) signaling, a constitutive phosphoryl-
ation of eukaryotic translation initiation factor 4E-
binding protein 1 (4E-BP1) and activation of ribosomal
protein S6 through S6K1 phosphorylation [7, 8]. The net
effect is enhanced protein translation, cell proliferation
and growth [9]. Notably, increased mTOR signaling and
subsequent changes in global protein synthesis are
shared molecular mechanisms of several rare neurodeve-
lopmental disorders with an increased prevalence of
ASD, such as fragile X syndrome (FXS) [10].
The hyperactivation of mTOR induced by Tsc1 and
Tsc2 heterozygosity can be inhibited by mTOR inhibi-
tors, such as the macrolide rapamycin. Rapamycin is an
immunosuppressant, which is widely prescribed to pre-
vent rejection in organ transplantation and exerts anti-
tumor properties [11–13]. Rapamycin binds FK-binding
protein 12 (FKBP12), and as a complex, rapamycin-
FKBP12 directly binds to the mTOR complex 1
(mTORC1), thus reducing phosphorylation of down-
stream mTOR targets [14, 15]. Rapamycin and other
mTOR inhibitors have been shown to be efficacious in
the treatment of several TSC-associated tumors as well
as seizures [16–19] and may ameliorate the symptoms of
neurodevelopmental disorders in adults [20, 21]. In TSC
mouse models, rapamycin limits tumor growth [22, 23],
reduces neuropathology and ameliorates epileptic sei-
zures as well as learning deficits [24–26]. It was recently
reported that rapamycin normalizes social interaction
deficits relevant to core disabilities associated with ASD
in both Tsc1
+/−
and Tsc2
+/−
mice [27].
Here, we investigated the Tsc 1
+/−
mouse model, which
exhibits haploinsufficiency for the Tsc 1 gene, in an attempt
to identify molecular changes associated with the neuro-
psychiatric phenotype of TSC patients [5]. In this mouse
model, the typical human cerebral pathology of spontan-
eous seizures, cerebral lesions and giant dysmorphic cells
could not been detected using immuno-cytochemistry and
high resolution magnetic resonance imaging, respectively
[28]. Furthermore, spine number and dendritic branching
are normal [28]. However, the Tsc1
+/−
mouse shows prom-
inent behavioural deficits which mimic core symptoms of
ASD and other neuropsychiatric disorders [28]. Tsc1
+/−
mice show hippocampal learning deficits using the Morris
water maze test and contextual fear conditioning, as well
as social deficits indicated by reduced social interaction
and nest building [28]. Consequently, the Tsc1
+/−
mouse is
a suitable model to investigate aspects of the molecular
pathology associated with neuropsychiatric spectrum dis-
orders, especially in relation to ASD and intellectual
disability. In this study, we attempted to identify
changes in molecular pathways in the frontal cortex
and hippocampus of the Tsc1
+/−
mouse model using a
mass spectrometry-based proteomics approach. We
also investigated protein changes associated with
rapamycin treatment. Findings from this study could
aid in the identification of novel drug targets for the
treatment of cognitive, social and psychiatric symp-
toms in ASD.
Methods
A more detailed description of the materials and
methods used in this study can be found in the supple-
mentary methods section (Additional file 1).
Animals
Tsc1
+/−
mice were generated by replacing exons 6
through to 8 of the Tsc1 gene with a selection cassette,
as described previously [29]. This leads to the generation
of Tsc1 null embryos which express Tsc1 transcripts in
which exon 5 and 9 are fused, leading to a premature
TGA stop codon. Consequently, any protein translated
from this allele lacks all of the known functional do-
mains of hamartin including the putative Rho activation
domain. The Tsc1
+/−
mutant mouse was crossed six
times into the C57BL/6J OlaHsD background and then
at least three times into the C57BL/6N/HsD back-
ground. The offspring consisted of Tsc1
+/−
mice and
wildtype littermates. Mice were genotyped when they
were about 7 days old. They were housed in groups and
kept on a 12-h light/dark cycle, with food and water
available ad libitum. Mice were culled when they were
6–8 weeks old and genotype groups were sex- and age-
matched for the experiments for consistency with the
published behavioural data [28]. Mouse genotypes were
blinded using codes all the way through to the sample
preparation stage. The codes were un-blinded for the
mass spectrometry analysis since samples had to be dis-
tributed evenly to avoid run time biases. All animal exper-
iments were approved by the Dutch Ethical Committee or
Wesseling et al. Molecular Autism (2017) 8:41 Page 2 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
in accordance with Institutional Animal Care and Use
Committee guidelines.
Rapamycin treatment
Mice were injected intraperitoneally with 5 mg/kg rapamy-
cin or vehicle for 5 days and culled 24 h after the last injec-
tion [27]. Rapamycin was dissolved in 5% dimethyl sulfoxide
diluted with saline to 5 ml/kg. Mice were 5–7 weeks old at
thetimeofinjection.
Proteomic sample preparation
Sample preparation was carried out as described previ-
ously [30–32]. Based on the lysates, two randomized,
blinded, independent sample preparations were prepared
for liquid chromatography mass spectrometry (in expres-
sion mode; LC-MS
E
) and selected reaction monitoring
mass spectrometry to avoid bias in sample preparations.
Label-free LC-MS
E
proteomic profiling of brain tissue
Brain tissue analysis and data processing were performed
as described previously [31, 33, 34]. The Swiss-Prot hu-
man reference proteome (Uniprot release March 2013,
20,252 entries) was used for protein identification
searches. Protein abundance changes for the compari-
sons between Tsc1
+/−
and wildtype were determined by
the MSstats package [35] based on mixed-effect models
using the peptide intensities, following log
2
transform-
ation and exclusion of intensity values deviating by more
than 3 standard deviations from the mean of each
group.
Protein set enrichment analysis
Significantly changed proteins were partitioned into
three bins, according to their ratio: proteins decreased in
abundance (ratio < 1.0), proteins increased in abundance
(ratio > 1.0) and a bin to identify general disturbed path-
ways which included all proteins with increased and de-
creased abundance (ratio > 1 and <1). The Rpackage
database org.mouse.eg.db version 2.8.0 was used for
gene ontology (GO) term annotation based on entrez
gene identifiers and GO-term enrichment analysis was
performed using GOstats.
Label-based SRM mass spectrometry
Abundance alterations of a panel of 43 candidate pro-
teins previously implicated in the Tsc1
+/−
mouse path-
ology were measured using a targeted SRM mass
spectrometry approach as described previously [32, 36]
following the guidelines of Lange et al. [37]. SRMstats
was used at default settings [37]. The final transitions,
collision energy and retention time windows used for
each peptide can be requested.
Results
Label-free LC-MS
E
proteomic profiling of Tsc1
+/−
mouse
brains
We investigated protein abundance changes in the
frontal cortex and hippocampus of the Tsc1
+/−
mouse.
LC-MS
E
analysis resulted in the identification of 522
proteins (7071 peptides) in the frontal cortex and 463
proteins (5149 peptides) in the hippocampus. Of these,
the levels of 51 proteins were altered in the frontal cor-
tex (FDR-adjusted *p< 0.05) and 108 in the hippocam-
pus (FDR-adjusted *p< 0.05). In the frontal cortex, 17 of
the changed proteins were altered by more than 10%, as
were 49 of the 108 changed hippocampal proteins
(Additional file 2). In the case of the frontal cortex, this
included adenylyl cyclase-associated protein 2 (CAP2,
ratio = 0.89, FDR-adjusted *p= 0.013), elongation factor
1–α2 (EIF1A2, ratio = 0.97, FDR-adjusted *p = 0.03),
eukaryotic translation initiation factor 3 subunit L (eIF3l,
ratio = 1.17, FDR-adjusted *p = 0.03) and elongation fac-
tor 2 (Eef2, ratio = 0.95, FDR-adjusted *p = 0.05), which
are all regulators of translation. Copine 6 (ratio = 1.1, FDR-
adjusted p= 0.0097) and copine 8 (ratio = 0.8, FDR-
adjusted p=3.9×10
−6
), which are associated with synaptic
plasticity, were changed in the hippocampus. Nine proteins
(NCDN⬇⬇, MAP2⬆⬆,SUCB1⬇⬇,MYPR⬆⬇,NDUS7⬇⬇,
DPYL2⬇⬇,AT1A2⬇⬆,CRYM⬇⬆,ARP3⬆⬇)werefoundto
be changed in both frontal cortex (first arrow) and hippo-
campal tissue (second arrow) (Additional file 2).
Gene set enrichment analysis was employed to investi-
gate if the altered 108 and 51 proteins were enriched in
biological pathways and cellular compartments. Based on
GO enrichment analysis, proteins responsible for the bio-
logical pathways “reproductive behaviour”(p=0.008),
“neurological system process”(p=0.010)and“visual
learning”(p= 0.028) were altered in the frontal cortex of
the Tsc1
+/−
mouse. In the hippocampus, the proteins were
related to the biological pathways “ribonucleotide energy
metabolism”(p= 0.0097), “protein polymerisation”
(p=0.005)and“oxidative stress”(p=0.009).Onepath-
way, “visual learning”, was identified in both the frontal
cortex and hippocampus proteomic analyses. Cellular
compartment GO association enrichment revealed that
the altered proteins were associated with “myelination”
and “dendrite”in the frontal cortex and “myelin sheet”
and “endoplasmic reticulum-Golgi intermediate compart-
ment”in the hippocampus.
Selected reaction monitoring (SRM) validation of Tsc1
+/−
brain proteomic alterations
For orthogonal proteomic validation of the proteomics
results, we employed a targeted label-based LC-SRM ap-
proach to specifically quantify the levels of 43 candidate
proteins derived from LC-MS
E
profiling, subsequent path-
way analysis, literature findings and already established in-
Wesseling et al. Molecular Autism (2017) 8:41 Page 3 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
house assays. Label-based SRM assays are highly specific
and sensitive and outperform immunoblotting analyses for
validation [38–41]. This analysis showed that the levels of 5
and 20 of the targeted proteins were significantly changed
in the frontal cortex and hippocampus, respectively
(p< 0.05, Table 1). Specifically, two proteins (MYPR,
PURA) out of five LC-MS
E
candidates were validated in the
frontal cortex and six (NSF, MYPR, MBP, CPNE6, SODC,
MARCS) out of ten LC-MS
E
-derived protein candidates
were validated in the hippocampus (Table 1). Using tar-
geted SRM-assays, we confirmed the more prominent
proteomic alterations in the hippocampus and further vali-
dated GO-terms “myelination”,“dendrite”and “oxidative
stress”through confirmation of changes in MYPR, MBP,
TSN2 (all myelin specific proteins), MAP2 (dendritic
marker) and SODC (oxidative stress marker). We further
detected an upregulation in ribosomal proteins and mTOR
kinase in the Tsc 1
+/−
mice (Table 1).
Label-free LC-MS
E
proteomic profiling of the Tsc1
+/−
hippocampus following rapamycin treatment
To investigate the effects of rapamycin on the brain
proteome, label-free LC-MS
E
analysis was employed on
Tsc1
+/−
and wildtype mice treated with rapamycin or ve-
hicle (Fig. 1a). Only the hippocampus was studied in this
case as this brain region was more affected with regard
to significantly changed proteins (Table 1). The hippo-
campus plays not only an important role in cognition,
but hippocampal dysfunction has also been linked to a
wide range of neuropsychiatric symptoms [42, 43]. Defi-
cits in consolidating short- and long-term memory and
spatial navigation have been shown to be impaired in
Tsc1
+/−
and Tsc2
+/−
mice and were reversed by rapamy-
cin treatment in Tsc2
+/−
mice [24]. LC-MS
E
analysis led
to the identification of 8648 total peptides which trans-
lated to 597 proteins, which were detected across all
samples. Interestingly, rapamycin treatment exerted a
stronger proteomic effect in Tsc1
+/−
compared to wild-
type mice (Fig. 1c (2 and 4)) with significant changes in
231 and 106 proteins, respectively. An overall decrease
in protein levels was found in both Tsc1
+/−
and wildtype
mice.
Next, proteins were tested which were affected in all
four comparisons. This showed that 9 proteins were
changed in common (FRM4A, PEA15, PERQ1, MAP2,
BASP, CLD11, ALBU, TCAL3, CLH) and that the levels
of 54 proteins were affected by rapamycin treatment in
both wildtype and Tsc1
+/−
mice; of these proteins, 52
corresponded in their fold change direction (37 of the 52
proteins were decreased in abundance and 15 increased,
respectively). Pathway analysis linked the 52 overlapping
proteins to the biological process of “translation”
(p= 0.00082), “macromolecule biosynthetic process”
(p= 0.005) and “gene expression”(p= 0.014). Using
KEGG (Kyoto Encyclopedia of Genes and Genomes) an-
notation, “ribosome”was the most significant pathway
(p= 1.7 × 10
−7
) in the enrichment analysis.
We further employed enrichment analysis for the
genotype comparisons and the treatment comparisons
(Fig. 1c (1–4)). This associated the biological pathways
“oxidative stress”and “apoptosis”with the significantly
changed proteins identified in the Tsc1
+/−
vs Wt com-
parison (Fig. 1c (1)). Furthermore, cellular compart-
ments of myelin sheet and neurofilaments were affected
by rapamycin treatment in both Tsc1
+/−
and Wt mice
(Fig. 1c (2 and 4)). Proteins with decreased levels due to
rapamycin treatment mostly related to the biological
pathways “translation”,“macromolecular complex as-
sembly”and “chromosome organization”(Fig. 1c (2 and
4)). Downregulation of the pathway “chromosome
organization”was specifically observed in Tsc1
+/−
mice
following rapamycin treatment (Fig. 1c (4)).
Importantly, 41 proteins which were altered in vehicle-
treated Tsc1
+/−
mice were normalized following rapamy-
cin treatment. These proteins include a set of proteins
where rapamycin treatment normalizes the genotype-
induced protein alterations to wildtype levels (33 pro-
teins) and a set of proteins where rapamycin normalizes
the genotype-effect below or above baseline levels (8
proteins). The former include the Glycine receptor sub-
unit alpha-4 (GLRA4), the Calcium-dependent secretion
activator 1 (CAPS1), Rod cGMP-specific 3′,5′cyclic
phosphodiesterase beta (PDE6B) and Guanine deami-
nase (GUAD) (Fig. 2 (N)); the latter include Rho-
associated protein kinase 2 (ROCK2) and ribosomal pro-
teins (RS18, RL4, RS9). All ribosomal proteins affected
by rapamycin treatment were decreased in their abun-
dance levels. Furthermore, proteins were identified that
are affected by rapamycin treatment in both wildtype
and mutant mice, although there was no difference in
their abundance levels between vehicle-treated mutant
and wildtype mice (Fig. 2 (R)). This set was comprised of
41 proteins. Amongst them are the anaphase promoting
complex s7 (APC7), calcineurin subunit B type 1
(CANB1) and the GABA aminotransferase (GABT).
Finally, the levels of six proteins were found to be
altered between mutant and wildtype but did not change
following rapamycin treatment. Neuromodulin (NEUM),
the excitatory amino acid transporter 2 (EAAT2) and
SMP25 are examples.
SRM validation of rapamycin treatment effects in the Tsc1
+/−
hippocampus
The next phase of the study involved a targeted prote-
omic approach to validate the findings of the rapamycin
study (see Fig. 1c (1–4) [targeted]). This focused on
myelination deficits, alterations in the translational ma-
chinery and proteins found to be altered in the label-free
Wesseling et al. Molecular Autism (2017) 8:41 Page 4 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Table 1 Significantly changed proteins identified by label-based LC-SRM in the frontal cortex and hippocampus of Tsc1
+/−
mice
compared to wildtype mice
The first stage of the analysis consisted of a global profiling approach, followed by validation with a specific and sensitive label-based assay panel. pvalues were
determined using SRMstats (linear model with fixed subject effects) and corrected (p*) to control for multiple hypothesis testing (Benjamini-Hochberg) [90]. For
reasons of clarity, only ratios and significance levels of significantly changing proteins are shown. For full information, see Additional file 3.n.s. not significant, n.d.
not detected. Validated findings are in gray shading
Wesseling et al. Molecular Autism (2017) 8:41 Page 5 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
LC-MS
E
discovery study of the Tsc 1
+/−
mouse (SODC,
NSF, MAP2, PKCG). The rapamycin-target mTOR was
included as a positive control.
This analysis resulted in validation of increased
myelin-associated proteins in Tsc1
+/−
compared to wild-
type mice (Fig. 1c (1 and 3)). Furthermore, the decrease
a
b1
b2
c
Fig. 1 Label-free LC-MSE and Label-based SRM analysis of Tsc1+/−and wildtype mice under rapamycin and vehicle treatment identifies distinct proteomic
changes. a) Flow chart of the experimental design. b) Significant protein changes in the hippocampus of rapamycin-treated WT and Tsc1
+/−
mice identified
by label-free LC-MS
E
.Orange dots refer to proteins that are increased in abundance, and green dots represent downregulated proteins following rapamycin
treatment. c2and 4(see below) refer to significantly changed proteins following rapamycin treatment in wildtype (“treatment effect in Wt mice”)orTsc1
+/−
mice (“treatment effect in Tsc1
+/−
mice”). Protein enrichment analysis was performed on the identified proteins. Yellow and blue dots represent the TSC
genotype effect following rapamycin or vehicle treatments (b1and 2) and are linked to c1and 3. Proteins changing due to a combination of
TSC genotype and rapamycin treatment are labeled black or purple, respectively. b1Rapamycin induced changes in wildtype mice as
compared to Tsc1
+/−
mice following vehicle treatment. b2Rapamycin induced changes in Tsc 1
+/−
mice compared to Tsc1
+/−
alterations
following rapamycin treatment. c) Bar plots of genotype and treatment effects identified through global protein profiling and significantly
changed proteins identified in the targeted SRM analysis. Number of significant proteins, percentage of up- and downregulated proteins
and enriched pathways linked to the up- and downregulated proteins are displayed
Wesseling et al. Molecular Autism (2017) 8:41 Page 6 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
in oxidative stress-related proteins in the Tsc1
+/−
mouse
was validated by confirming decreased levels of SODC
(Fig. 1c (1)). Additionally, altered levels of NSF and
PKCG were also validated (Fig. 1c [targeted] and Fig. 3).
In the case of rapamycin treatment effects, decreased
levels of proteins involved in translation could be vali-
dated. Specifically, decreased levels of all three tested
ribosomal subunits as well as the mTOR-kinase were
identified. Moreover, the analysis showed that rapamycin
normalized the protein levels of SODC, NSF and PKCG
in Tsc1
+/−
mice (Fig. 1c [targeted]).
Discussion
The pathogenesis of psychiatric disorders such as ASD re-
mains elusive, and there is accumulating evidence that
several neuronal circuits and pathways are affected. This
is especially true for the social, cognitive and neuropsychi-
atric symptoms associated with these disorders. In an at-
tempt to gain further insight into these pathways, this
study combines unbiased and targeted proteomic ap-
proaches to investigate the hippocampus and frontal cor-
tex of a mouse model of TSC, which is one of the most
frequent causes of syndromic ASD [44]. The investigated
Tsc 1
+/−
mouse model exhibits social and cognitive deficits,
which are core behavioural symptoms of ASD in humans
[45] and other relevant rodent models [46], without any
obvious brain pathology (such as tumors or epilepsy). This
makes the Tsc 1
+/−
mouse an excellent model of pharma-
cologically treatable ASD. A previous study has demon-
strated the effectiveness of rapamycin to normalize
reciprocal social interactions in this model [27]. The aim
of the present study was to investigate proteins and path-
ways affected by rapamycin treatment, which could sup-
port drug discovery efforts and in turn the development of
improved treatments for TSC, ASD and possibly other
neuropsychiatric disorders.
Proteomic profiling of the frontal cortex and hippo-
campus brain tissue in this study identified and
Fig. 2 Multivariate analysis of LC-MS
E
estimates as shown by condition plots illustrating the differences between the WT and Tsc1
+/−
mice with and
without rapamycin treatment. X-axis is condition and y-axis is log ratio of endogenous (Llight) over reference (Hheavy) peptides. Dots represent the
mean of the log
2
ratio for each condition, and error bars indicate the confidence intervals with 0.95 significance. The interval is not related to the model
based analysis. Significant changes as measured by LC-MS
E
are indicated below each protein. Illustrated are examples of proteins which are affected
by rapamycin treatment and which are normalized by rapamycin treatment. Corrected pvalues (p*) were determined by post hoc correction after
Benjamini-Hochberg [91]. CR = Wt +rapamycin,CS=Wt + vehicle, TR = Tsc1
+/−
+rapamycin,TS=Tsc1
+/−
−rapamycin
Wesseling et al. Molecular Autism (2017) 8:41 Page 7 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
quantified a large number of significantly changed pro-
teins in the Tsc1
+/−
mouse model. Differentially
expressed proteins and altered molecular pathways were
identified and selected candidate proteins were validated
using SRM as a highly quantitative method. In a second
stage, proteomic analysis of rapamycin treatment effects
were investigated to identify down-stream effects of
mTOR-pathway inhibition in the hope to gain new in-
sights into the molecular underpinnings of social impair-
ments in ASD and other psychiatric disorders.
We were able to show that myelin proteins and the
translational machinery, specifically several ribosomal
subunits, were significantly altered in Tsc1
+/−
mice
treated with rapamycin. Our findings of lower ribosomal
subunit abundances are consistent with a rapamycin-
induced downregulation of ribosomal biogenesis [47].
Regarding the effects on myelination, previous research
has linked the mTOR pathway to oligodendrocyte differ-
entiation and axonogenesis [48]. Oligodendrocytes pro-
duce myelin, and this is specifically regulated at the late
progenitor to immature oligodendrocyte transition stage
(as shown by changes in expression of the myelin
marker proteins MYPR and MBP). We identified an
increase in myelin proteins in the Tsc1
+/−
mouse, but
not in Wt mice. A recent study has shown that ablation
of TSC1 is associated with oligodendrocyte-specific
over-activation and subsequent hypomyelination [49].
An increase in myelin proteins in the Tsc1
+/−
mouse
brain may be due to the globally enhanced (Table 1) pro-
tein translation and cell proliferation in the context of
mTOR hyperactivation. An increase in cell growth and
proliferation could interfere with oligodendrocyte matur-
ation and thus result in incomplete myelination as seen
in some demyelinating diseases, such as multiple scler-
osis [50].
MYPR and MBP as well as TSN-2 were amongst the
altered myelin proteins. These proteins play an import-
ant role in oligodendrocyte differentiation during devel-
opment. Furthermore, in vitro studies have shown that
MBP mRNA and protein expression are significantly de-
creased by mTOR inhibition [48, 51]. Inhibiting mTOR
in oligodendrocyte precursor cell/dorsal root ganglion
co-cultures potently abrogated oligodendrocyte differen-
tiation and reduced numbers of myelin segments. Disor-
ganized and structurally compromised axons with poor
myelination have already been found in TSC patients,
Fig. 3 Multivariate analysis of SRM estimates as shown by condition plots illustrating the differences between Wt and Tsc1
+/−
mice with and
without rapamycin treatment. X-axis represents the condition and the y-axis the log ratio of endogenous (Llight) over reference (Hheavy)
peptides. Dots represent the mean of log
2
ratio for each condition, and error bars indicate the confidence intervals with 0.95 significance. The
interval is not related to the model based analysis. Significant changes as measured by LC-MS
E
are indicated below each protein. SRM was able
to validate protein abundance changes identified by label-free LC-MS
E
. Corrected pvalues (p*) were determined by post hoc correction after
Benjamini-Hochberg [91]
Wesseling et al. Molecular Autism (2017) 8:41 Page 8 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
and this may at least to some extent explain the behav-
ioural and cognitive deficits associated with the disorder
[52]. Impaired adult myelination has been shown in the
prefrontal cortex of socially isolated mice [53]. Import-
antly, changes in oligodendrocyte function and myelin-
ation abnormalities are amongst the most consistent
hallmarks of psychiatric pathology in post-mortem brain
studies. Changes were reported for schizophrenia, bipo-
lar disorder, depression and ASD [36, 54–58]. In wild-
type mice, rapamycin treatment led to a reduction of
myelin and myelin protein expression [59, 60]. This is
consistent with our findings, where rapamycin affects
both wildtype (reduced) and mutant myelin (increased)
protein expression.
Interestingly, several proteins that we found altered in
the Tsc1
+/−
mouse model were reversed by rapamycin
treatment. One of these proteins, a glycine receptor sub-
unit, which abundance was decreased in the mutant and
normalized by rapamycin treatment, could be a potential
drug target for novel treatments of ASD and
schizophrenia-spectrum disorders. The glycine receptor
co-localizes with GABA
A
receptors on hippocampal
neurons [61]. A microdeletion at Xq22.2 implicates
GLRA4 to be involved in intellectual disability and be-
havioural problems [62]. In a case report, glycine recep-
tor antibodies could be detected in a patient with
treatment-resistant focal epilepsy, tantrums, clumsiness
and impaired speech [63] and in patients with progres-
sive encephalomyelitis with rigidity and myoclonus stiff
person syndrome [64]. Treatments targeting the glycine
transporter are under investigation as novel treatment
approaches for schizophrenia [65].
Other altered proteins, which are normalized by rapa-
mycin treatment, included the calcium-dependent secre-
tion activator 1 (CAPS1) (decreased in Tsc1
+/−
and
normalized by rapamycin), two guanine metabolism as-
sociated proteins (guanine deaminase and PDE6B; both
increased in Tsc1
+/−
and normalized by rapamycin) and
the vesicle-fusing ATPase NSF (increased in Tsc1
+/−
,
normalized by rapamycin), a molecular component of
the exocytosis machinery [66], which is required for
membrane fusion [67] and regulates the disassembly of
SNARE complexes on early endosomes [68]. The NSF
gene has also been linked with cocaine dependence [69]
and schizophrenia [36, 70]. Direct interactions with cell
surface receptors such as AMPA receptors [71, 72], β2-
adrenergic receptors [73], dopaminergic receptors [74]
and the adrenomedulin receptor [75] have been re-
ported. Interestingly, a coordinated action of NSF and
PKC regulates GABA
B
receptor signaling efficiency [76].
PKCG was found to be strongly downregulated by rapa-
mycin treatment in this study and is known to be in-
volved in the regulation of the neuronal receptors
GLUR4 and NMDAR1 [77]. It binds and phosphorylates
the GLUR4 glutamate receptor and regulates its function
by increasing membrane-associated GRIA4 expression
[78]. Several preclinical and clinical trials have investi-
gated mGLUR antagonists for the treatment of social
deficits in ASD [78, 79] and ASD associated with FXS
[80, 81] and PKCG inhibitors could represent a novel
treatment strategy to ameliorate cognitive and social def-
icits. Notably, Ketamine, which is thought to exert anti-
depressant action through modulation of mTOR
pathway activity [82], potentiates persistent learning and
memory impairment through the PKCG-ERK signaling
pathway [83].
Another protein strongly downregulated following rapa-
mycin treatment is the anaphase promoting complex S7
(APC7), which is a cell cycle-regulated E3 ubiquitin ligase
controlling progression through mitosis and the G1 phase
of the cell cycle. The control of APC7 through rapamycin
might be a major breakpoint in cell proliferation. Rapamy-
cin has already been shown to also downregulate the ex-
pression of the APC/C inhibitor Emi1 [84].
Copine 6, which we found upregulated in the hippo-
campus of Tsc1
+/−
compared to wildtype mice, is a
calcium-dependent regulator of the actin cytoskeleton in
neuronal spines and negatively regulates spine matur-
ation during neuronal development [85]. Changes in
copine 6 expression may be involved in neurodevelop-
mental disorders, as deformed dendritic spines and
changes in spine density are hallmarks of many neurode-
velopmental conditions, such as Down’s syndrome [86,
87] and FXS [88]. Interestingly, hippocampi from pa-
tients suffering from uncontrolled epileptic seizures, typ-
ically a problem in tuberous sclerosis patients, exhibit a
decrease in spine density [89]. We also found that
MAP2, a dendritic spine marker, was increased by Tsc1
heterozygocity and decreased by rapamycin treatment.
Interestingly, over twice as many significantly changed
proteins were identified in the Tsc1
+/−
hippocampus as
compared to control animals following rapamycin treat-
ment (Fig. 1c; 231 vs. 106 changed proteins, respect-
ively). TSC1 mutations are linked to numerous changes
in biochemical processes, including cell cycle regulation,
translational control and metabolism which are linked to
mTOR pathway hyperactivation. It can be speculated
that rapamycin-related inhibition of the mTORC1 com-
plex results in TSC genotype-dependent adaptations in a
wide range of molecular pathways. These adaptations
could be indirectly involved in the therapeutic effect of
rapamycin. A further explanation for the enhanced rapa-
mycin treatment effect in the Tsc1
+−/
mice is selective
vulnerability. Mutant mice might be more susceptible to
the treatment as mTOR hyperactivation modulated simi-
lar downstream molecular pathways during neurodeve-
lopment as are affected by the rapamycin-induced
mTOR hypoactivation.
Wesseling et al. Molecular Autism (2017) 8:41 Page 9 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Conclusions
Taken together, the results from this comprehensive
study represent the first proteomic characterization of
the Tsc1
+/−
mouse model to date. The findings yield
novel insights into the molecular Tsc1
+/−
mouse path-
ology as well as the molecular effects of rapamycin treat-
ment, which is an effective treatment for several clinical
symptoms of the tuberous sclerosis complex. Further-
more, the mTOR pathway, which is modulated by rapa-
mycin treatment, is a novel drug target for the treatment
of ASD, schizophrenia and affective disorders. We hope
that the findings from this study will provide evidence
and support for future clinical trials in the field of
neuropsychiatric disorders.
Additional files
Additional file 1: Supplementary methods. Detailed information of the
experimental methods. (DOCX 31 kb)
Additional file 2: Significantly altered proteins identified by label-free
LC-MS
E
analysis in the frontal cortex and hippocampus of the Tsc1
+/−
mice compared to Wt mice. Overlapping proteins between hippocampus
and frontal cortex are bold. (XLSX 23 kb)
Additional file 3: Full information for significantly changed proteins
identified by label-based LC-SRM in the frontal cortex and hippocampus
of Tsc1
+/−
mice compared to wildtype mice. (DOCX 31 kb)
Abbreviations
ASD: Autism spectrum disorder; LC: Liquid chromatography; MBP: Myelin
basic protein; mTOR: Mammalian target of rapamycin; MYPR: Myelin
proteolipid protein; NSF: Vesicle-fusing ATPase; PKCG: Protein kinase C
gamma; SODC: Superoxide dismutase C; SRM: Selected reaction monitoring;
TSC: Tuberous sclerosis complex; TSC1: Harmartin; TSC2: Tuberin; TSN-
2: Tetraspanin 2; Wt: Wildtype
Acknowledgements
We would like to thank Susan Gooden for the generation and treatment of
the Tsc1
+/-
mouse model and Jadviga Schreiber for proofreading the
manuscript.
Funding
This research was kindly supported by the Stanley Medical Research Institute
(SMRI) and the Dutch Fund for Economic Structure Reinforcement (#0908)
the NeuroBasic PharmaPhenomics project.
Availability of data and materials
The datasets presented in this study can be made available on reasonable
request.
Authors’contributions
HW carried out the label-free LC-MS
E
experiments, designed and carried out
the SRM experiments and performed all statistical and bioinformatic data
analyses. HW prepared the figures and tables and drafted the manuscript. SB
and YE conceived the study and participated in its design and coordination.
SB helped to interpret the results and to write the manuscript. All authors
read, edited and approved the final manuscript.
Ethics approval and consent to participate
All animal experiments were approved by the Dutch Ethical Committee or in
accordance with Institutional Animal Care and Use Committee guidelines.
All animal experiments were approved by the Dutch Animal Experiment
Committee (Dierexperimenten commissie [DEC]) and in accordance with
Dutch animal care and use laws.
Consent for publication
Not applicable.
Competing interests
S.B. is a director of Psynova Neurotech Ltd. and PsyOmics Ltd. The other
authors declare no conflict of interest.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Received: 21 September 2016 Accepted: 14 June 2017
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