Neuroscience 294 (2015) 29–37
SPATIAL COGNITIVE DEFICITS IN AN ANIMAL MODELOF WERNICKE–KORSAKOFF SYNDROME ARE RELATEDTO CHANGES IN THALAMIC VDAC PROTEIN CONCENTRATIONS
K. O. BUENO, a L. DE SOUZA RESENDE, a A. F. RIBEIRO, a
D. M. DOS SANTOS, c E. C. GONCALVES, b F. A. B. VIGIL, a
I. F. DE OLIVEIRA SILVA, d L. F. FERREIRA, a
A. M. DE CASTRO PIMENTA c AND A. M. RIBEIRO a*
aDepartamento de Bioquımica e Imunologia, Laboratorio de
Neurociencia Comportamental e Molecular, LaNeC, Instituto de
Ciencias Biologicas, Universidade Federal de Minas Gerais, Belo
Horizonte, Minas Gerais 31270-010, Brazil
bDepartamento de Genetica, Laboratorio de Polimorfismo de
DNA, Instituto de Ciencias Biologicas, Universidade Federal do
Para, Belem, Para 66075-000, Brazil
cDepartamento de Bioquımica e Imunologia, Laboratorio de
Venenos e Toxinas Animais, LVTA, Instituto de Ciencias
Biologicas, Universidade Federal de Minas Gerais, Belo
Horizonte, Minas Gerais 31270-010, BrazildDepartamento de Analises Clınicas e Toxicologicas – Faculdade
de Farmacia, Universidade Federal de Minas Gerais, Belo
Horizonte, Minas Gerais 31270-010, Brazil
Abstract—Proteomic profiles of the thalamus and the cor-
relation between the rats’ performance on a spatial learning
task and differential protein expression were assessed in
the thiamine deficiency (TD) rat model of Wernicke–
Korsakoff syndrome. Two-dimensional gel-electrophoresis
detected 320 spots and a significant increase or decrease
in seven proteins. Four proteinswere correlated to rat behav-
ioral performance in the Morris Water Maze. One of the four
proteins was identified by mass spectrometry as Voltage-
Dependent Anion Channels (VDACs). The association of
VDAC is evident in trials in which the rats’ performance
was worst, in which the VDAC protein was reduced, as con-
firmed by Western blot. No difference was observed on the
mRNA of Vdac genes, indicating that the decreased VDAC
expression may be related to a post-transcriptional process.
http://dx.doi.org/10.1016/j.neuroscience.2015.03.0010306-4522/� 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Address: Avenida Presidente Antonio Carlos,6627, FaFiCH, sala F1002, Pampulha, Belo Horizonte, Minas GeraisCEP: 31270-901, Brazil. Tel: +55-31-34092642; fax: +55-31-99838401.
E-mail addresses: [email protected] (K. O. Bueno),[email protected] (L. de Souza Resende), [email protected] (A. F. Ribeiro), [email protected] (D. M. dos Santos),[email protected] (E. C. Goncalves), [email protected](F. A. B. Vigil), [email protected] (I. F. de Oliveira Silva),[email protected] (L. F. Ferreira), [email protected](A. M. de Castro Pimenta), [email protected] (A. M. Ribeiro).Abbreviations: 2D SDS–PAGE, two-dimensional sodium dodecylsulfate–polyacrylamide gel electrophoresis; VDACs, Voltage-Dependent Anion Channels; ATN, anterior thalamic nuclei; IML,internal medullary lamina; IPG, immobilized pH gradient; MWM,Morris Water Maze; PCR, polymerase chain reaction; PTD,pyrithiamine-induced TD; TD, thiamine deficiency; WE, Wernicke’sEncephalopathy; WKS, Wernicke–Korsakoff syndrome.
29
The results show that TD neurodegeneration involves
changes in thalamic proteins and suggest that VDAC protein
activity might play an important role in an initial stage of the
spatial learning process. � 2015 IBRO. Published by Elsevier
Ltd. All rights reserved.
Key words: thiamine deficiency, spatial learning, thalamus,
proteomic, VDAC, rats.
INTRODUCTION
Thiamine deficiency (TD) can lead to a neurological
disorder termed Wernicke’s Encephalopathy (WE),
characterized by acute confusion, ataxia, and eye
movement abnormalities (Homewood and Bond, 1999).
In some conditions, WE can evolve into a chronic syn-
drome named Wernicke–Korsakoff syndrome (WKS)
(Hazell and Butterworth, 2009).
The pathophysiology underlying TD is multifactorial in
nature, involving a broad cascade of events that ultimately
result in focal neuronal cell death similar to the
pathological mechanisms inherent in neurodegenerative
diseases (Jhala and Hazell, 2011). Focal brain lesions
similar to those that occur in patients with WE/WKS can
be experimentally induced in rodents using pyrithiamine-
induced TD (PTD) combined with a thiamine-deficient diet
(Watanabe, 1978; Beauchesne et al., 2010).
The results of previous studies carried out by our
group (Pires et al., 2005) and other authors (Langlais
and Savage, 1995), using the PTD model, have shown
that this vitamin deficiency induces spatial learning and
memory deficits. Diencephalic amnesia produced by TD
is consistently associated with neuropathology in the
anterior and midline thalamic nuclei, mammillary bodies,
and the mammillothalamic tract (Langlais et al., 1992).
The morphological injuries caused by TD are
preceded by functional changes in neurotransmission
systems, among them, the serotonergic (Nakagawasai
et al., 2007a), dopaminergic (Nakagawasai et al.,
2007b), glutamatergic (Savage et al., 1999), GABAergic
(Butterworth, 1989) and cholinergic (Savage et al.,
2003; Pires et al., 2005; Vetreno et al., 2008) systems.
However, the molecular mechanisms underlying the neu-
rodegeneration process associated with TD are still
unknown.
Proteomics is a high-throughput protein analysis that
enables us to detect, simultaneously, large numbers of
30 K. O. Bueno et al. / Neuroscience 294 (2015) 29–37
differentially expressed proteins in samples obtained from
several sorts of diseased tissue (Verrills, 2006). By
identifying changes in protein expression in the thalamus,
hypotheses may be drawn from molecular mechanisms
underlying these alterations as to how TD may cause
structural and functional brain damage.
The purposes of the present study were to verify the
effects of severe TD on the proteomic profile of the
thalamus and to assess the correlation between rat’s
performance on a spatial learning task and thalamic
protein level change. A second goal was to identify the
proteins that showed differential expression between
groups and to correlate them with the latency of finding
the platform of Morris Water Maze (MWM).
EXPERIMENTAL PROCEDURES
Animals and treatments
Thirty-two Wistar male rats, approximately 2 months old
were individually housed in a 12/12-h light/dark cycle,
and divided into two groups: (i) Control (C, n= 16), in
which the rats received a standard control diet
associated with daily saline i.p. injections and (ii)
pyrithiamine-induced TD (PTD, n= 16), in which the
rats received a thiamine-deficient diet associated with
daily i.p. injections of pyrithiamine (0.25 mg/kg), which is
an inhibitor of the enzyme that is responsible for the
production of the active form of thiamine. The episode
of TD was interrupted after the onset of the last
neurological signs, i.e., seizures and impaired righting
reflexes, by the administration of two i.p. injections of
thiamine (100 mg/kg each) at an 8-h interval. After
treatment, all subjects were housed two per cage with
unlimited access to water and regular chow. Following
three weeks of recovery, PTD and C rats were trained
and tested in the MWM task (Morris, 1984; Carvalho
et al., 2006). The data obtained from this spatial cognitive
test were published elsewhere (Vigil et al., 2010).
On the first day following the behavioral tests, rats
were decapitated, their brains removed, and the
thalamus dissected from one hemisphere and stored at
�80 �C for use in proteomic analysis, as described
below. One additional experiment (total rats, n= 14)
was carried out using other groups of rats, which were
submitted to the same treatments (Control and PTD).
The thalamus samples from one of the brain
hemispheres obtained from the animals of both groups
were used for Western blot (n= 14) and real-time
polymerase chain reaction (PCR) analyses (n= 10).
The care and use of rats were in accordance with the
National Institute of Health Guide for Care and Use of
Laboratory Animals (Institute of Laboratory Animal
Resources Committee, 1985).
Protein separation by two-dimensional sodiumdodecyl sulfate–polyacrylamide gel electrophoresis(2D-SDS–PAGE)
The proteomic profile of the thalamus was analyzed using
2D SDS–PAGE. Samples were prepared as described
previously with some modifications (Paulson et al.,
2004). Briefly, eight thalamus samples selected randomly
(PTD, n= 4; Control, n= 4) were individually homoge-
nized, delipidized in chloroform/methanol/water (4/8/3 v/
v/v), mixed continuously for 1 h, and centrifuged (2000g,10 min). The supernatant, containing the lipids, was dis-
carded and the extraction procedure was repeated twice.
The pellet was air-dried in ±4 �C overnight, and then dis-
solved in a100-ll sample buffer (7 M urea, 2 M thiourea,
1% C7bZO� (Sigma) and 40 mM Tris). The samples were
incubated for 4 h, centrifuged (5000g, 5 min), and the
supernatant was collected. The total protein concentration
of the supernatant of each sample was measured using
the Lowry assay (Lowry et al., 1951).
A 2D SDS–PAGE protocol was followed as described
previously (Wildgruber et al., 2000). Immobilized pH
gradient strips (IPG strips, 7 cm, pH 3–11 NL) (GE
Healthcare, Pittsburgh, Pennsylvania, USA) were
passively rehydrated with samples containing 250 lg of
protein in 130 -ll rehydration buffer (Urea 7 M, Thiourea
2 M, 2% 3-[(3-cholamidopropyl)-dimethylammonio]-1-
propanosulfonate hydrate (CHAPS), 0.5% IPGs buffer,
0.002% Bromophenol Blue) for 12 h at room temperature.
The proteins in the rehydrated strips were focused using
the IPGphor isoelectric focusing (IEF) system
(Pharmacia Biotech, San Francisco, CA, USA). Prior to
the second dimension, the IPG strips were equilibrated
for 15 min in a 5 -ml equilibration buffer (75 mM Tris–
HCl, pH 8.8, 6 M Urea, 30% Glycerol, 2% SDS, 0.002%
Bromophenol Blue) containing 1% Dithiothreitol
(Promega�) and then with another 5 mL of equilibration
buffer containing 2.5% Iodoacetamide (Sigma�) for
15 min.
Second-dimension separation was performed on
12.5% SDS–PAGE using the miniVE Vertical
Electrophoresis System (Amersham Biosciences, San
Francisco, CA, USA). The gel was stained using
colloidal Coomassie Blue Protein (G250-Sigma�)
according to the supplier’s protocol.
Image analysis
The gels were scanned on the Image Master Labscan v.
3.01 (with a resolution of 300 dpi) and Image Master 2D
Platinum (Amersham Biosciences) software was used
for matching and analyzing protein spots on 2D SDS
gels. The gel with the highest number of spots was
defined as the ‘‘reference gel’’ which was used to create
a master image for the matching of corresponding
protein spots. Background subtraction was performed
and the intensity volume of each individual spot was
normalized. In the normalization process the optical
intensity of each spot was measured by the sum of
‘‘pixels’’ within the area of the spot (spot volume) and
then converted to a percentage relative to the total
intensity of the spots detected in the corresponding gel.
Thus, the amount of protein was expressed as the
relative percentage of spot volume.
In-gel tryptic digestion
The protein spots were manually excised from stained 2D
SDS gels with a scalpel blade and the spots were
K. O. Bueno et al. / Neuroscience 294 (2015) 29–37 31
incubated in 200 ll of 200 mM Ammonium bicarbonate/
Acetonitrile solution (ACN; 40:60) twice at 37 �C for
30 min each. The gel pieces were dried for 30 min in a
vacuum-centrifuge, rehydrated in a 20-ll chilled
digestion buffer (20 lg/ml trypsin (Sigma) in 100 lMHCl; 40 mM Ammonium bicarbonate in 9% Acetonitrile
solution) for 60 min and then incubated at 37 �C in this
same solution overnight. Peptides were extracted with
50% Acetonitrile/0.1% Trifluoroacetic acid and desalted
and concentrated using C18 ZipTips (Millipore, Billerica,
MA, USA).
Mass spectrometric analysis and proteinidentification
Mass spectrometry (MS) analysis was performed on
a MALDI-TOF-TOF Autoflex III spectrometer (Bruker-
Daltonics, Bremen, Germany). Extracted peptides
(0.5 ll) were spotted onto an AnchorChip� 600/384
(BrukerDaltonics, Bremen, Germany) target microtiter
plate (MTP), mixed with a saturated solution of
a-cyano-4-hydroxycinnamic acid (0.5 ll) and allowed
to crystallize at room temperature. Samples were
analyzed by matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF/TOF). The MS and the MS/
MS spectra were acquired in the reflector mode with
external calibration, using the calibration mixture
Peptide Calibration Standard II (BrukerDaltonics,
Bremen, Germany). The MS and MS/MS data were
submitted to a MASCOT (http://www.matrixscience.com,
downloaded, May 20, 2013). The search parameters
were as follows: database, NCBI non redundant:
taxonomy, Rattus: type of search, peptide mass
fingerprint combined with MS/MS ion search; amino acid
sequence, enzyme, trypsin; fixed modification,
carbamidomethylation (Cys); variable modifications,
oxidation (Met); mass values, monoisotopic; peptide
charge state, (1) maximum missed cleavages, (2) and a
peptide mass tolerance of 0.05% Da (50 ppm).
Western blotting
After electrophoresis, proteins were transferred to a
nitrocellulose membrane (Millipore, Billeria, MA, USA).
Following, blocking non-specific binding with 10% non-fat
milk in TBS with 0.05% Tween-20 (TBS-T) overnight,
membranes were incubated 2 h at room temperature with
anti-VDAC (0.1:1000) antibodies (Catalog Number-
AB10527 Millipore). After three washes with TBS-T, blots
were incubated 1 h at room temperature with HRP-
conjugated anti-rabbit (0.2:1000) antibodies (Catalog
Number-AQ132P Millipore) (Ribeiro et al., 2010). After
three washes with TBS-T, blots were developed with ECL
and exposed to films. The density of the spots was ana-
lyzed using the Image J program (http://imagej.nih.gov/ij).
Molecular analysis
For the RNA extraction, the samples (PTD, n= 5; C,
n= 5) were thawed, and total RNA was extracted using
TRIzol� according to the manufacturer’s protocol
(Invitrogen, Sao Paulo, Brazil). Samples were quantified
using NanoDrop� ND-2000v3 1.0 (Thermo Fisher
Scientific, Waltham, MA, USA). The minimum
acceptable 260:280 nm ratio was >1.6.
The reverse transcription was performed in a total
volume of 20 ll using 2 lg of total RNA and oligo (dT20)
primers (ExxtendBiotecnologia Ltda., Paulinia, Brazil).
RevertAid� H Minus (Fermentas, Sao Paulo, Brazil) was
used according to the manufacturer’s protocol.
To the real-time PCR, the reactions from the extracted
samples of the thalamus of control and deficient rats were
made in 7900HT� Fast Real Timer PCR (Applied
Biosystems, Sao Paulo, Brazil) utilizing SYBR� Green
PCR Master Mix (Applied Biosystems, Sao Paulo,
Brazil). PCR amplification was performed without the
extension step (95 �C for 10 min, followed by 40 cycles
of 95 �C for 15 s and 60 �C for 60 s). Fluorescence
acquisition was measured in the last step of each cycle
(60 �C).Data were analyzed using 7900HT (Applied
Biosystems) software and a Microsoft Excel spreadsheet.
The minimum acceptable correlation coefficient was 0.98.
In all reactions, a negative control without sample was
tested. Melting curves were examined to guarantee the
absence of any spurious products. To normalize mRNA
levels, two reference genes (Ppia, peptidylprolyl
isomerase A; Hprt, hypoxanthine phosphoribosyltrans-
ferase) were used, and the relative quantity was
calculated following Vandesompele et al. (2002).
Primer design – Exon sequences were obtained from
the National Center for Biotechnology Information
database (http://www.ncbi.nlm.nih.gov, Vdac1: NM
031353.1, Vdac2: NM 031354.1, Vdac3: NM 031355.1,
accessed 16 September 2013). Primer sequences were
designed using Primer3 v.0.4.0 (Rozen and Skaletsky,
2000), and the quality and specificity of the primer
pairs were examined using NetPrimer (http://www.
premierbiosoft.com/netprimer/netprlaunch/netprlaunch.
html) and Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/
primer-blast/index.cgi?LINK_LOC=BlastHome)), respec-
tively. All primers were positioned in inter-exon regions.
Primers were synthesized by Exxtend (ExxtendBio
tecnologia, Paulinia, Brazil). The primers had the following
sequences: Vdac1F 50-TGGCTACAAGACGGACGAAT-
30; Vdac1R 50-CAGGCGAGATTGACAGCAG-30; Vdac2F50-GGCTACAGGACTGGGGACTT-30; Vdac2R 50-CCTG
ATGTCCAAGCAAGGTT-30; Vdac3F 50-TTGACACAGCC
AAATCCAAA-30 and Vdac3R 50-CCTCCAAATTCAGTG
CCATC-30. For the reference genes, primers had the
following sequences: PpiaF, 50-CAAACACAAATGGTTC
CCAGT-30; PpiaR 50-GCTCATGCCTTCTTTCACCT-30;
HprtF 50-CAGGCCAGACTTTGTTGGAT-30; HprtR 50-TCC
ACTTTCGCTGATGACAC-30.
Statistical analysis
Significant differences in protein expression between PTD
and C were assessed using the Student t-test. The
behavioral performance in the spatial navigation task
was analyzed using a two-way ANOVA with repeated
measures on the last element (2 � 5 factorial design:
two kinds of diet and five training sessions). Regression
analyses were used to determine the degree to which
Fig. 1. Two-dimensional gel-electrophoresis assays illustrating the
qualitative analysis of the rat thalamus proteins. The sixteen encircled
proteins indicate qualitative differences and the arrows indicate seven
proteins differentially expressed between C (n= 4) and PTD groups
(n= 4). The proteins were separated on pH 3–11 nonlinear immobi-
lized pH gradient strips, followed by a second-dimension separation
on 12.5% SDS–polyacrylamide gels.
32 K. O. Bueno et al. / Neuroscience 294 (2015) 29–37
biochemical and behavioral performance in each session
was linearly correlated. The normal distribution was
tested using the Kolmogorov–Smirnov test. For real-time
PCR data, besides the Kolmogorov–Smirnov test,
homoscedasticity of variance by the Lilliefors test was
also used. The relative amount of mRNA normalized for
each candidate gene was compared using a t-test for
independent samples. All values are expressed as
mean and the level of significance was p< 0.05. Real-
time PCR data analyses were performed with the
STATISTICA v. program. 6.1 (Stasoft, Sao Caetano do
Sul, Brazil).
RESULTS
MWM task
As published elsewhere (Vigil et al., 2010), significant
main effects of TD (F(1,30) = 6.00; p= 0.02) and sessions
(F(4,120) = 27.06; p= 0.00) were observed. Statistical
analysis also showed that the performance of rats from
the control group was significantly better compared to that
of the thiamine-deficient rats in the third (t= 2.7,
p= 0.01) and the fourth (t= 3.6, p= 0.001) sessions.
Although thiamine-deficient rats showed a slower acquisi-
tion performance than control rats, they were able to learn
the task, as in the fifth session there was no significant dif-
ference (t= 1.0, p= 0.33) between the performances of
rats from the two groups.
Image analysis
The thalamic proteomic data showed that 320 spots were
expressed in the average gel of each group. This means
that in the range of the analysis employed here there was
no difference between the numbers of proteins expressed
in the thalamus of C and PTD rats (Fig. 1).
Seven thalamic proteins were found to be differentially
regulated as a function of TD. Among them, the levels of
three increased and the levels of four decreased (Fig. 2).
Linear regression analysis
The results of linear regression between the performance
of the rats in the MWM task and the levels of each of the
seven altered thalamic proteins showed that four proteins
correlate with the latency to find the platform in the third
session of MWM (Fig. 3), the session in which the rats
showed the worst performance during the learning
process. Negative correlation was found between the
relative volume of spots 7868 (r= �0.77, p= 0.02),
7869 (r= �0.86, p= 0.005) and 8105 (r= �0.83,p= 0.009) and latency in the third session. On the
other hand, the protein spot 8096 was positively
correlated with the latency in the third section (r= 0.90,
p= 0.002). No significant correlation was found for the
other three proteins (spots 7858, 8123 and 8228).
Mass spectrometric analysis and proteinidentification
Through mass spectrometry the protein of the 8105 spot
was characterized. The MASCOT identified it as VDACs
(Voltage-Dependent Anion Channels) with score 50
(p< 0.005)/NCBI: 20130519. Mass: 30851. Type of
search: MS/MS Ion Search. Instrument type: MALDI-
TOF-TOF. Fig. 4A shows a descriptive table of the
identified protein. The remaining proteins could not be
identified.
Western blotting
To confirm 2D SDS–PAGE results, seven thalamus
samples from PTD rats and seven from control rats
were further analyzed by Western blot. The specificity of
the antibodies against VDAC was verified by Western
blot. Western blot results showed that the protein spot
corresponding to the protein spot features identified by
2D SDS–PAGE were decreased in the PTD rats
compared with the control rats (p= 0.029) (Fig. 4B, C).
These results confirmed the data obtained by 2D SDS–
PAGE analysis and protein identification by MS.
Molecular analysis
No differences in mRNA levels of Vdac genes were
observed between the groups (tVdac1 = �0.32,p> 0.05; tVdac2 = �0.43, p> 0.05; tVdac3 = �0.90,p> 0.05).
DISCUSSION
As described by Vigil et al. (2010), thiamine-deficient sub-
jects showed an impaired performance in the water maze
Fig. 2. Proteins with significant differences between C (n= 4) and PTD (n= 4) groups (p< 0.05).
K. O. Bueno et al. / Neuroscience 294 (2015) 29–37 33
spatial test, although these rats were able to learn the
task by the fifth session. This result is consistent with pre-
vious findings obtained by our group (Pires et al., 2005)
and other authors (Langlais and Savage, 1995; Mumby
et al., 1999) in which thiamine-deficient rats showed spa-
tial-learning deficits. The spatial cognitive performance is
mainly associated with the hippocampus (Morris et al.,
1990; Moser et al., 1995; Yu et al., 2013) and, as far as
we know, this study is the first to show that rat’s perfor-
mance in the MWM task is related to changes in the levels
of proteins in the thalamus.
According to Aggleton and Brown (1999) the link from
the hippocampus to the mammillary bodies and anterior
thalamic nuclei (ATN), via the fornix, is critical for normal
episodic memory. Moreover, damage to this axis is
responsible for the core deficits in anterograde amnesia
(Aggleton and Sahgal, 1993; Savage et al., 2011).
A number of studies have been conducted to
determine the role of the thalamus in diencephalic
amnesia produced by TD. Damage to internal medullary
lamina (IML) and ATN have been reported to underlie
learning and memory impairments observed in
Fig. 3. Scatter plot of the volume of each thalamic protein and the behavioral performance expressed as latency to find the platform in the third
session of MWM task. Negative correlations were found between the relative volume of spots (n= 8).
34 K. O. Bueno et al. / Neuroscience 294 (2015) 29–37
PTD-treated rats (Langlais et al., 1992; Langlais and
Savage, 1995). Langlais et al. (1992) showed that PTD
rats without IML gliotic scarring display a learning curve
in the MWM similar to the control rats, while PTD rats with
IML lesions have longer latencies. The ATN are also
important for learning and memory as damage to this
region produces a persistent amnestic syndrome. There
is evidence for considerable neuronal loss (50–90%) in
the ATN following acute PTD treatment (Langlais and
Savage, 1995). Complete lesions of the ATN impair perfor-
mance on two behavioral tests to assess hippocampal-
dependent learning and memory (spontaneous alternation
and delayed alternation tests). In contrast, incomplete
ATN lesions do not impair spontaneous alternation perfor-
mance (Savage et al., 2011).
Long-term neurochemical alterations within the
thalamus and its involvement in the cognitive
impairments in the PTD model have also been reported
after the restoration of thiamine levels. There are
significant reductions in overall GABA and glutamate
levels in the midbrain thalamus (Langlais et al.,
1988).Significant increases in serotonin and its metabolite
were reported by Langlais et al. (1988) after PTD treat-
ment. Vigil et al. (2010) demonstrated a correlation
between a serotonergic thalamic parameter and the
rodent performance in a spatial task, indicating that a
higher thalamic 5-hydroxyindolacetic acid (5-HIAA) con-
centration was associated with poorer performance in
the beginning of the acquisition process.
Although the role of the thalamus in the behavioral
deficits related to TD has been recognized for many
years, the molecular mechanisms underlying these
changes are still unknown. The data of the present
study, besides demonstrating the occurrence of
increased and decreased protein levels in the thalamus
of PTD rats, also indicate that some of these proteins
might have a role in the molecular mechanism involved
in the neurobiology of the spatial learning task.
It is interesting to mention that a significant correlation
between the volumes of the altered proteins was verified
only with the latency to find the platform in the third
session of the MWM task. Following recovery from an
acute bout of pyrithiamine-induced TD, rats are impaired
in their ability to learn a spatial navigation task (third
and fourth sessions) but with additional training, PTD
rats are able to attain latencies similar to those of the
control group. This learning curve showing a cognitive
deficit during the intermediate sessions of training,
followed by a performance recovery – similar to control
rats – in the last session, was previously found by our
group and other authors for PTD (Langlais et al., 1992;
Carvalho et al., 2006) and also for old rats (Oliveira-
Silva et al., 2007; Oliveira et al., 2010). One possible
explanation for these findings is that the learning, which
involves a neurobiological dynamic process, has a kinetic
mechanism in which the intermediate steps are essential
for encoding the information responsible for behavioral
changes. Hence, as a consequence of neurodegenerative
processes, the speed of learning is affected, but during
the training sessions all the rats are able to learn the task.
To our knowledge, this is the first time that the
identified protein, VDAC, has been shown to be related
to thiamin-deficiency dysfunction. However, it is known
that it is implicated in cognitive functions (Weeber et al.,
2002; Levy et al., 2003). VDAC, also known as Porins,
are the most abundant proteins in the mitochondrial outer
membrane (Linden et al., 1984). This protein has a major
function as part of the apoptosis mechanism (Zaid et al.,
2005; McCommis and Baines, 2012) and in the metabolite
transport between the mitochondria and the cytoplasm,
Fig. 4. Protein data. (A) The descriptive data of the identified proteins. (B) Representative image of the Western blot analysis confirming the identity
of spot 8105 as VDAC (arrow). The other spots presumably refer to isoforms of VDAC. (C) Quantitative results of analysis of relative density with a
significant difference between C (n= 7) and PTD (n= 7) groups (p= 0.029).
K. O. Bueno et al. / Neuroscience 294 (2015) 29–37 35
regulating energy metabolism in neurons by maintaining
cellular ATP levels (Colombini, 2004; Shoshan-Barmatz
et al., 2006). Kielar et al. (2009) showed level increases
of VDAC in the thalamus of mouse associated with neu-
rodegeneration in the Batten disease model. On the other
hand, Yoo et al. (2001), in postmortem studies, showed
that patients with Alzheimer’s disease had significantly
decreased levels of VDAC1 (Yoo et al., 2001). Weeber
et al. (2002) showed that the regulation of VDAC activity
may influence important synapse functions in the hip-
pocampus. They found that long-term potentiation
(LTP), a long-lasting enhancement in the amplitude of
synaptic replay that has been correlated with learning
and memory (Malenka and Nicoll, 1999) is significantly
impaired in Vdac1 knockout mice. It is possible that the
altered expression of VDAC modifies synapse function
with consequent interference in learning and memory pro-
cesses. VDAC-deficient mice exhibited significantly worse
performance in some steps of the training than those of
wild-type controls in the MWM task. These results are
similar to those obtained by our group using thiamine-
deficient rats. It is important to mention that in addition
to the hippocampal and thalamic areas, there are other
brain areas that play roles in memory and spatial learning
processes (Aggleton et al., 2010).Thus, additional pro-
teomic studies would be important in order to determine
if the observed changes shown here are brain region-
specific. Nevertheless, our finding that in severe TD there
is a negative correlation between the VDAC levels in the
thalamus and latency to find the platform in the MWM in
the third session of training indicates that this protein might
play an important role in the neurobiological mechanism
of the initial stage of the spatial acquisition task.
Real-time PCR for Vdac1, Vdac2 and Vdac3 genes
was carried out with the objective to verify whether the
levels of VDAC protein changes were related to the
levels of their corresponding mRNA. No significant
difference among the groups was observed. This result
suggests that the variation in the VDAC levels found in
the present study was not due to changes in the
regulation of RNA expression and, as a result it might
be a consequence of post-translational change. The
absence of a relationship between mRNA transcripts
and the protein expressions can be explained by a
36 K. O. Bueno et al. / Neuroscience 294 (2015) 29–37
post-transcriptional mechanism, which controls the
protein translation rate (Harford and Morris, 1997) and
by the differences in half-lives of specific proteins or
mRNAs (Varshavsky, 1996). One possible explanation
for the observed decrease in the VDAC level induced by
TD could be the occurrence of an increase in Parkin-
mediated Lys 27 poly-ubiquitylation of VDAC, which was
found to be related to neurodegeneration (Geisler et al.,
2010; Burte et al., 2014).
In short, the main findings of the present work are: (i)
TD increased and decreased thalamic protein levels; (ii)
four thalamic protein changes are related to spatial
cognitive performance of rats; (iii) changes in VDAC,
one of the four proteins altered by TD, relate to rats’
spatial learning; (iv) the association of VDAC is evident
in the session with the rats’ worst performance, and (v)
no difference was observed on the mRNA of Vdacgenes, indicating that the decreased VDAC expression
may be related to a post-transcriptional process.
CONCLUSIONS
The present results show for the first time that TD
neurodegeneration involves changes in thalamic
proteins that are related to rat’s performance on spatial
cognitive tasks. We hypothesize that, of the seven
proteins altered by TD described here, four – including
VDAC – might have a role as a molecular component of
the neurobiological substrate of the initial step of the
spatial learning process. In addition, if the training
persists, other biological mechanisms might come into
play to compensate for the early dysfunction and
consequently the behavioral deficits are minimized.
The present data provide a new pathway for the study
of molecular mechanisms related to thiamine-induced
learning and memory impairments. Further experiments
need to be designed to identify and characterize the
other three altered thalamic proteins. Regarding these
three proteins, the present findings allow prioritization of
targets for future mechanistic studies of thalamic protein
involvement in the TD neurodegenerative process.
AUTHOR CONTRIBUTIONS
Ribeiro, A.M., was the general coordinator of the
research, conceived the project, designed the
experiments and analyzed the data.
Bueno, K.O., and Resende, L.S., carried out all the
biochemical experiments.
Vigil, F.A.B., and Oliveira-Silva, I.F., and Ferreira,
L.F., carried out the behavioral tests.
Goncalves, E.C., collaborated with the 2D SDS–
PAGE experiments.
Ribeiro, A.F., was responsible for the real-time PCR
experiments and data analysis.
Pimenta, A.M.C., and Santos, D.M., collaborated on
the Mass Spectrometry analysis.
Ribeiro, A.M., Resende, S.R., and Bueno, K.O.,
conducted the data analysis and wrote the manuscript.
Acknowledgments—This research was supported by CAPESThe
role of VDAC in cell death: frien (Coordenacao de
Aperfeicoamento de Pessoal de Nıvel Superior) and CNPQ
(Conselho Nacional de Desenvolvimento Cientıfico e
Tecnologico). Leticia Resende received scholarship from
FAPEMIG (Fundacao de Amparo a Pesquisa do estado de
Minas Gerais) and Kenia de Oliveira Bueno and Andrea
Frozino Ribeiro from CAPES. The authors declare that there
are no conflicts of interest regarding the publication of this paper.
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(Accepted 2 March 2015)(Available online 09 March 2015)
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