Diabetes Alters KIF1A and KIF5B Motor Proteins in The Hippocampus

Diabetes Alters KIF1A and KIF5B Motor Proteins in the
Hippocampus
Filipa I. Baptista
1,2
, Maria J. Pinto
3,4
, Filipe Elvas
1,2
, Ramiro D. Almeida
3
, Anto nio F. Ambro sio
1,2,3,5
*
1Centre of Ophthalmology and Vision Sciences, IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 2Pharmacology and Experimental Therapeutics, IBILI,
Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 3Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal, 4PhD Programme in
Experimental Biology and Biomedicine (PDBEB), Centre for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal, 5AIBILI, Coimbra, Portugal
Abstract
Diabetes mellitus is the most common metabolic disorder in humans. Diabetic encephalopathy is characterized by cognitive
and memory impairments, which have been associated with changes in the hippocampus, but the mechanisms underlying
those impairments triggered by diabetes, are far from being elucidated. The disruption of axonal transport is associated
with several neurodegenerative diseases and might also play a role in diabetes-associated disorders affecting nervous
system. We investigated the effect of diabetes (2 and 8 weeks duration) on KIF1A, KIF5B and dynein motor proteins, which
are important for axonal transport, in the hippocampus. The mRNA expression of motor proteins was assessed by qRT-PCR,
and also their protein levels by immunohistochemistry in hippocampal slices and immunoblotting in total extracts of
hippocampus from streptozotocin-induced diabetic and age-matched control animals. Diabetes increased the expression
and immunoreactivity of KIF1A and KIF5B in the hippocampus, but no alterations in dynein were detected. Since
hyperglycemia is considered a major player in diabetic complications, the effect of a prolonged exposure to high glucose on
motor proteins, mitochondria and synaptic proteins in hippocampal neurons was also studied, giving particular attention to
changes in axons. Hippocampal cell cultures were exposed to high glucose (50 mM) or mannitol (osmotic control; 25 mM
plus 25 mM glucose) for 7 days. In hippocampal cultures incubated with high glucose no changes were detected in the
fluorescence intensity or number of accumulations related with mitochondria in the axons of hippocampal neurons.
Nevertheless, high glucose increased the number of fluorescent accumulations of KIF1A and synaptotagmin-1 and
decreased KIF5B, SNAP-25 and synaptophysin immunoreactivity specifically in axons of hippocampal neurons. These
changes suggest that anterograde axonal transport mediated by these kinesins may be impaired in hippocampal neurons,
which may lead to changes in synaptic proteins, thus contributing to changes in hippocampal neurotransmission and to
cognitive and memory impairments.
Citation: Baptista FI, Pinto MJ, Elvas F, Almeida RD, Ambro sio AF (2013) Diabetes Alters KIF1A and KIF5B Motor Proteins in the Hippocampus. PLoS ONE 8(6):
e65515. doi:10.1371/journal.pone.0065515
Editor: Anna Dunaevsky, University of Nebraska Medical Center, United States of America
Received September 18, 2012; Accepted May 1, 2013; Published June 12, 2013
Copyright: 2013 Baptista et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by PEst-C/SAU/UI3282/2011 and PEst2/SAU/LA0001/2011 (FCT, Portugal, and COMPETE). Filipa I. Baptista and Maria J. Pinto
acknowledge fellowships from Fundacao para a Cie ncia e a Tecnologia, Portugal (SFRH/BD/35961/2007 and SFRH/BD/51196/2010, respectively). Ramiro D.
Almeida is supported by FCT and COMPETE (PTDC/SAU-NEU/104100/2008) and by Marie Curie Actions, 7th Famework programme. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: afambrosio@fmed.uc.pt
Introduction
Diabetes has been associated with cognitive and memory
impairments, indicating that the hippocampus can be affected by
this disease. Several studies have demonstrated that diabetes
impairs synaptic structure and function in the hippocampus both
presynaptically [1,2] and postsynaptically [3,4]. Previously, we
found that diabetes changes the levels of several synaptic proteins
involved in exocytosis in hippocampal and retinal nerve terminals,
suggesting that axonal transport of those proteins to distal synaptic
sites may be impaired under diabetes [2,5]. Moreover, in
hippocampal cell cultures, we also found that prolonged exposure
to high glucose leads to an accumulation of syntaxin-1, VGluT-1
and synaptotagmin-1 at the cell body of hippocampal neurons,
further suggesting that axonal transport may be affected [6].
Potential alterations in axonal transport can somehow contribute
to the development of cognitive impairment and memory loss
under diabetes.
The impairment of axonal transport is an early and perhaps
causative event in many neurodegenerative diseases, and might be
due to alterations and/or loss of motor proteins (kinesin and
dynein), microtubules, cargoes (by inhibiting their attachment to
motor proteins) and ATP fuel supply (mitochondria) which enables
molecular motors to undertake the axonal transport [7]. The
inhibition of axonal transport leads to a rapid loss of function in
the distal axon and to a dying back axonal degeneration. The
axonal transport is known to be affected in experimental models of
diabetes. Most studies regarding nerve dysfunction in diabetes
focus on the peripheral nervous system, however increasing
evidence also shows that the central nervous system can be
affected by diabetes. At peripheral nervous system level, a
reduction in retrograde transport has been reported, namely the
transport of nerve growth factor in the sciatic nerve of diabetic
rats, and endogenous neurotrophins on the cervical and vagus
nerve of diabetic rats [810]. Moreover, alterations in the axonal
caliber in nerves of diabetic animals are likely to be secondary to
PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e65515
the impairment of slow anterograde axonal transport, which is
correlated with reduced local levels of neurofilament [11,12].
Studies using fluoro-gold labelling showed that diabetes affects the
retrograde axonal transport in retinal ganglion cells [13,14], and
recently, a deficit in anterograde transport from the retina to the
superior colliculus was detected at 6 weeks of diabetes [15].
Furthermore, it was also shown that hyperglycemia impairs axonal
transport in olfactory receptor neurons in mice [16]. Nevertheless,
to our knowledge, no studies have been performed to analyze the
effect of diabetes on axonal transport in the hippocampus, or to
investigate local changes in motor proteins in hippocampal
neurons. Therefore, the goal of this work, was to evaluate the
impact of early diabetes in the hippocampus, namely in the
content and distribution of KIF1A (kinesin that transports synaptic
vesicle precursors containing synaptophysin and synaptotagmin),
KIF5B (kinesin that transports mitochondria and membrane
organelles that contain presynaptic membrane proteins such as
syntaxin-1 and SNAP-25) and dynein (motor protein responsible
for the retrograde axonal transport of organelles, such as
mitochondria). Moreover, since hyperglycemia has been consid-
ered the main pathogenic factor underlying the development of
diabetic complications, we aimed to evaluate whether high glucose
per se, giving particular attention to changes occurring in the axons,
could affect the levels and distribution of motor and synaptic
proteins, and the distribution of mitochondria in the axons of
hippocampal neurons.
Experimental Procedure
Animals
All animals were handled according to the EU guidelines for the
use of experimental animals (86/609/EEC), and the experiments
were approved by our Institutional Ethics Committee (Comissao
de E
tica da Faculdade de Medicina da Universidade de Coimbra).

Approval ID: FMUC/08/11. Male Wistar rats (Charles River
Laboratories), eight weeks-old, were randomly assigned to control
or diabetic groups. Diabetes was induced with a single intraper-
itoneal injection of streptozotocin (STZ; 65 mg/kg, freshly
dissolved in 10 mM sodium citrate buffer, pH 4.5) (Sigma, St.
Louis, MO, USA). Hyperglycemic status (blood glucose levels
exceeding 250 mg/dl) was confirmed two days later with a
glucometer (Elite, Bayer, Portugal). Before sacrifice, rats were
weighted and blood samples were collected to measure glucose
levels. Diabetic rats and age-matched controls were anesthetized
with halothane and then sacrificed, two and eight weeks after the
onset of diabetes.
Immunohistochemistry in Brain Slices
Brain slices preparation. Rats from each experimental
group were deeply anesthetized with ketamine/xylazine and
transcardially perfused with 0.1 M phosphate-buffered saline
solution (PBS, in mM: 137 NaCl, 2.7 KCl, 4.3 Na
2
HPO
4
, 1.47
KH
2
PO
4
; pH 7.4) followed by 4% paraformaldehyde (PFA) in
0.1 M PBS. The brains were removed and post-fixed for 24 h in
4% PFA and then dehydrated in 20% sucrose in 0.1 M PBS for
24 h. Brain slices (30 mm thickness) were cut in a cryostat (Leica
CM3050S, Nussloch, Germany) and collected in 0.1 M PBS with
0.01% sodium azide. Brain slices were used for free-floating
immunohistochemistry.
Free-floating immunohistochemistry. Slices were washed
twice with 0.1 M PBS, blocked with 0.25% Triton X-100 and 5%
normal fetal bovine serum (FBS) in 0.1 M PBS for 1 h at room
temperature, and then incubated with the appropriate primary
antibodies (listed in Table 1) for 24 h at 4uC. Incubation with
primary antibodies was followed by incubation with conjugated
secondary antibody Alexa Fluor-488 (donkey anti-goat IgG,
1:250), for sections stained for KIF1A and KIF5B, or Alexa
Fluor-568 (goat anti-mouse IgG, 1:250), for sections stained for
Table 1. List of primary antibodies.
Primary Antibody Sample Antibody Dilution Protein (mg) Source
Mouse anti-KIF1A Total Extracts Hippocampus 1:1,000 20 BD Biosciences
Total Extracts Primary cultures 1:1,000 80
Goat anti-KIF1A Immunocytochemistry 1:50 _ Santa Cruz
Immunohistochemistry 1:50 _
Goat anti-KIF5B Total Extracts Hippocampus 1:2,000 10 Abcam
Immunocytochemistry 1:100 _
Mouse anti-Dynein Total Extracts Hippocampus 1:2,000 20 Abcam
Mouse anti-Tau Total Extracts Primary cultures 1:1,000 30 Cell Signaling
Rabbit anti-TUJ-1 Immunocytochemistry 1:1,000 _ Covance
Mouse anti-SNAP-25 Immunocytochemistry 1:100 _ SYSY
Mouse anti-Synaptophysin Immunocytochemistry 1:50 _ Chemicon
Mouse anti-Syntaxin-1 Immunocytochemistry 1:100 _ SYSY
Mouse anti-Synaptotagmin-1 Immunocytochemistry 1:200 _ SYSY
doi:10.1371/journal.pone.0065515.t001
Effect of Diabetes on Hippocampal Motor Proteins
dynein, plus DAPI (1:5,000), to stain cell nuclei, for 2 h 30 min at
room temperature. From this point forward, the slices were
protected from light. Sections were then washed three times with
0.1 M PBS in the dark and then mounted on slides with glycergel
(Dako mounting medium). Sections were examined with a LSM
710 Meta Confocal laser scanning microscope (Zeiss, Germany).
Immunofluorescence quantification in hippocampal
subregions. A semi-quantitative determination of immunore-
active product densities at the level of the dorsal hippocampus was
performed using ImageJ 1.42 software. In order to determine the
fluorescence intensity of motor proteins (KIF1A, KIF5B and
dynein), slides containg hippocampal slices from control and
diabetic groups were blind coded. Sections from each immuno-
histological experiment, consisting of samples from control and
diabetic group, were captured under identical conditions. Typi-
cally, four sections from each animal brain were used and the
CA1, CA3 and DG subregions were imaged for quantification.
Random window sampling within the subregions was carried out
for quantification so that the intrinsic variability in the expression
was appropriately quantified. To remove tissue background, for
each image, a negative control (primary antibody omitted) of
coverslipped tissue at the similar location was imaged, and
background values were then subtracted from the experimental
values, which were expressed in fluorescence arbitrary units (AU).
The product densities were averaged across the four sections from
each brain and expressed as mean percentage change; the
percentage change across the control and diabetic groups was
obtained and expressed as mean 6SEM. Although the intensity of
staining varied from one experiment to another, within a single
experiment the application of primary and secondary antibodies,
exposure times and acquisition image settings were uniform. This
approach provides a measurement of the relative percentage
change among control and diabetic groups based on the density of
staining in a given brain region.
Preparation of Total Hippocampal Extracts
After dissection, the hippocampi from each rat were homoge-
nized in lysis buffer (50 mM Tris-HCl, pH 7.4, 0.5% Triton X-
100, supplemented with complete miniprotease inhibitor cocktail
tablets and 1 mM DTT). The resulting homogenate was sonicated
(4 pulses, 2 seconds each) and then centrifuged at 16,1006g for
10 min. All procedure was performed at 4uC. The supernatant
was stored at 280uC until use.
Primary Cultures of Rat Hippocampal Neurons
Primary cultures of rat hippocampal neurons were prepared
from the hippocampi of E17E19 Wistar rat embryos. The
hippocampi were dissected under sterile conditions, using a light
microscope, in Ca
2+
- and Mg
2+
-free Hanks solution (in mM:
5.36 KCl, 0.44 KH
2
PO
4
, 137 NaCl, 4.16 NaHCO
3
, 0.34
Na
2
HPO
4
.2H
2
O, 5 glucose, 1 sodium piruvate, 10 HEPES and
0.001% phenol red, pH 7.4). The hippocampi were digested with
trypsin (0.06%, 15 min, at 37uC; Gibco Invitrogen, Life Tech-
nologies, Scotland, UK), in Ca
2+
- and Mg
2+
-free Hanks solution.
The hippocampi were then washed with Hanks solution
containing 10% fetal bovine serum (Biochrom, Cambridge, UK)
to stop digestion. The cells were dissociated in Neurobasal
medium (Gibco Invitrogen) supplemented with B27 (1:50 dilution;
Gibco Invitrogen), 0.5 mM glutamine, 25 mM glutamate and
50 mg/ml gentamycin. The cells were plated in six-well plates
(8.75610
4
cells/cm
2
) or in coverslips (2.25610
4
cells/cm
2
) coated
with poly-D-lysine (0.1 mg/ml). For experiments in which axon
segments were analyzed, neurons were plated in the center of the
coverslip (approximately 10,000 cells). Under these conditions,
axons grow outward the center (were soma and dendrites are
located) and away from the dense neuronal network, where they
can be imaged and analyzed independently. The cultures were
Table 2. Primer sequences.
Gene Forward primer (59-39) Reverse primer (59-39) Amplicon size (bp)
Reference genes
GAPDH GACTTCAACAGCAACTCC GCCATATTCATTGTCATACCA 105
HPRT ATGGGAGGCCATCACATTGT ATGTAATCCAGCAGGTCAGCAA 77
YWHAZ CAAGCATACCAAGAAGCATTTGA GGGCCAGACCCAGTCTGA 76
Target genes
KIF1A CATTAGTTAGTGGCGTTGA TACCTGGAGGCATTAGAAA 91
KIF5B GTGATGATTGCGTCCAAG CTTCTTTGCACAATCGTTG 90
DYNEIN TTCTGGCGTAGTCCTATT ACACCACATCTCAAGTCT 104
GAPDH - glyceraldehyde-3- phosphate dehydrogenase;
HPRT - human hypoxanthine phosphoribosyltransferase;
YWHAZ - tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide;
KIF1A - kinesin family member 1A;
KIF5B - kinesin family member 5B;
DYNEIN - dynein cytoplasmic 1 intermediate chain.
Table 3. Average weight and blood glucose levels of diabetic
and aged-matched control rats.
Diabetes duration Weight (g) Blood Glucose (mg/dL)
2 Weeks
Control 327.664.9 104.463.2
Diabetic 224.468.4*** 379.4618.7***
8 Weeks
Control 399.7610.3 90.262.0
Diabetic 247.7610.3*** 498.1628.6***
Measurements were made immediately before the sacrifice of the animals.
***p,0.001.
maintained in a humidified incubator with 5% CO
2
/95% air at
37uC for 14 days. The concentration of glucose in control
conditions was 25 mM. After seven days in culture, half of the
medium was replaced by fresh medium, and cells were incubated
with 25 mM of glucose (yielding a total of 50 mM glucose) or with
25 mM mannitol (plus 25 mM glucose in normal medium), which
was used as an osmotic control, and maintained for further seven
days.
RNA Extraction and cDNA Synthesis
Total RNA was isolated using the RNeasy Mini Kit (Qiagen,
Germany) according to the manufacturers instructions. Briefly,
one hippocampi, from control and diabetic rats, was mechanically
disrupted using a lysis buffer and subsequently homogenized in a
QIAshredder homogenizer. The sample was then transferred to an
RNeasy spin column, to yield a RNA-enriched solution. RNA
concentration was measured using a NanoDrop ND-1000
Spectrophotometer (NanoDrop Technologies, USA) in 2 mL
volume. First strand cDNA synthesis was performed using random
primers, 0.5 mg total RNA and SuperScript II Reverse Transcrip-
tase (Life Technologies, USA), according to the manufacturers
instructions. Additionally, the resulting cDNA (2 ml) was subjected
to a 35-cycle polymerase chain reaction (PCR) amplification using
26 MyTaq Red Mix (BIOLINE, UK), 200 nM of forward
(GCTCCTCCTGAGCGCAAG) and reverse (CATCTGCTG-
GAAGGTGGACA) b-actin primers. PCR products were visual-
ized after electrophoresis on 1.5% (w/v) agarose gels containing
0.005% (v/v) EtBr in 16TAE buffer to evaluate genomic DNA
contamination (data not shown).
Primer Design and Evaluation
Primers for quantitative real time polymerase chain reaction
(qRT-PCR) were designed using the Beacon Designer 6 software
(PREMIER Biosoft International, USA) for the amplification of
gene fragments between 70110 bp in length and an annealing
temperature of 56uC. An intron-spanning amplicon was chosen in
order to avoid amplification of genomic DNA in the cDNA
samples. Amplification efficiency of target and reference genes was
evaluated using a cDNA ten-fold dilution series and plotting
threshold cycle (Ct) values against cDNA dilution (data not
shown). Furthermore, a melting curve was performed at the end of
the cycling program to assess the primer specificity, represented by
a single peak at the melting temperature of the PCR-product.
Primers with amplification efficiency outside of 90110% range or
primer pairs generating multiple peaks were discarded. Final
primer sequences and amplicon lengths are shown in Table 2.
Quantitative Real Time Polymerase Chain Reaction
qRT-PCR was performed using 20 mL total reaction volume
containing 10 mL 26 iTaqTM SYBRH Green Supermix with
ROX (BioRad, USA), 200 nM of forward and reverse primers and
2 mL of 1:2 diluted cDNA in a StepOne Plus system (Life
Technologies, USA). PCR conditions were: 10 min 95uC for
initial denaturation; 40 x 15 sec 95uC for denaturation, 45 sec
56uC for primer annealing and 30 sec 72uC for elongation.
Furthermore, at the end of the PCR a melting curve analysis was
performed to evaluate unspecific products and primer-dimer
formation. Three technical replicates for each biological replicate
per group were performed. A non-template control was included
for each gene. Ct values were obtained during the exponential
amplification phase using automatic threshold option in StepOne
Software (Life Technologies, USA).
qRT-PCR Data Analysis
Reference gene expression stability between different groups
was evaluated using the NormFinder analysis algorithm for
Microsoft Excel [17], which identified Ywhaz as the most stable
gene (stability value: 0.002). Ywhaz gene was selected as our
reference gene for normalization of axonal transport protein gene
expression in all groups. Relative gene expression data was
analyzed using 2
2DDCt
method [18], where DDCt =(Ct gene of
interest-Ct reference gene) diabetes group - (Ct gene of interest-Ct
reference gene) control group. The data analysis was based on 5
independent biological replicates per group. The results were
expressed as the mean 6 SEM. Data were analyzed by the
unpaired Students t-test (IBM SPSS Statistics, USA) to determine
differences in gene expression between groups. Differences were
considered statistically significant when the p,0.05.
Preparation of Extracts of Cultured Hippocampal
Neurons
Cells were rinsed twice with ice-cold PBS and then lysed with
RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM
EDTA, 1% Triton X-100, 0.5% DOC, 0.1% SDS, 1 mM DTT)
supplemented with complete miniprotease inhibitor cocktail
tablets and phosphatase inhibitors (10 mM NaF and 1 mM
Na
3
VO
4
). The lysates were incubated on ice for 30 min and then
centrifuged at 16,1006g for 10 min at 4uC. The supernatant was
collected and stored at 280uC until use.
Western Blot Analysis
The protein concentration of each sample was determined by
the bicinchoninic acid (BCA) protein assay (Pierce Biotechnology,
Rockford, IL, USA). The samples were denaturated by adding 66
concentrated sample buffer (0.5 M Tris, 30% glycerol, 10% SDS,
0.6 M DTT, 0.012% bromophenol blue) and heating for 5 min at
95uC. Equal amounts of protein were loaded into the gel and
proteins were separated by sodium dodecyl sulphate-polyacryl-
amide gel electrophoresis (SDS-PAGE), using 68% gels. Then,
proteins were transferred electrophoretically to PVDF membranes
(Millipore, Billerica, Massachusetts, USA). The membranes were
blocked with 5% low-fat milk in Tris-buffered saline (137 mM
NaCl, 20 mM Tris-HCl, pH 7.6) containing 0.1% Tween-20
(TBS-T) for 1 h at room temperature. The membranes were then
Figure 1. Diabetes induces changes in kinesin KIF1A in the hippocampus. (A) Diabetes upregulates KIF1A mRNA expression, as assessed by
RT-qPCR. The results are expressed relatively to age-matched controls, and data are presented as mean 6 SEM of 5 animals. *p,0.5; ***p,0.001
compared to age-matched control analyzed by the unpaired Students t-test. (B) KIF1A protein levels were analyzed by immunoblotting in total
extracts of hippocampus isolated from control and STZ-induced diabetic animals (2 and 8 weeks of diabetes). Representative Western blots are
presented above the graphs, with the respective loading controls (b-III tubulin), to confirm that identical amounts of protein from control and
diabetic samples were loaded into the gel. The results are expressed as percentage of age-matched controls, and data are presented as mean 6 SEM
of 45 animals. **p,0.01 compared to age-matched control using Students t-test. (C) The distribution of KIF1A protein in hippocampal subregions of
control and STZ-induced diabetic animals was also analyzed by immunohistochemistry. KIF1A immunoreactivity increased at 8 weeks of diabetes. The
preparations were visualized under a laser scanning confocal microscope LSM 710 META (Zeiss, Germany). Scale bar: 50 mm. Insets shows the
expression pattern of KIF1A in hippocampal slices. The quantification of immunoreactivity (fluorescence intensity arbitrary units) was performed and
presented below the images. **p,0.01, ***p,0.01, significantly different from control as determined by the unpaired Students t-test.
doi:10.1371/journal.pone.0065515.g001
incubated with primary antibodies (listed in Table 1) overnight at
4uC. After washing for 1 h in TBS-T with 0.5% low-fat milk, the
membranes were incubated with an anti-mouse or anti-goat
alkaline phosphatase-linked IgG secondary antibody (1:10,000;
GE Healthcare, Buckinghamshire, UK) in TBS-T with 1% low-fat
milk for 1 h at room temperature. The membranes were processed
for protein detection using the enhanced chemifluorescence
substrate (ECF; GE Healthcare). Fluorescence was detected on
an imaging system (Thyphoon FLA 9000, GE Healthcare) and the
digital quantification of bands immunoreactivity was performed
using ImageQuant 5.0 software (Molecular Dynamics, Inc.,
Sunnyvale. CA, USA). The membranes were then reprobed and
tested for b-actin immunoreactivity (1:5,000) or b-III tubulin
(1:5,000) to prove that similar amounts of protein were applied to
the gels.
Immunocytochemistry
Hippocampal cell cultures were washed three times with PBS
and fixed with 4% paraformaldehyde and 4% sucrose for 10 min
at room temperature. Cells were then washed three times with
PBS and permeabilized with 0.25% Triton X-100 in PBS for
5 min at room temperature. Non-specific binding was prevented
incubating cells with 3% BSA/0.2% Tween-20 in PBS for 30 min.
Cells were then incubated with the primary antibodies (listed in
Table 1) for 2 h at room temperature. After incubation, cells were
rinsed three times with PBS and incubated with the secondary
antibodies for 1 h at room temperature in the dark. The nuclei
were stained with DAPI (1:5,000). Upon rinsing three times with
PBS, the coverslips were mounted on glass slides using Dako
Fluorescent mounting medium (Dako, Denmark). The prepara-
tions were visualized under a confocal laser scanning microscope
LSM 710 META (Zeiss, Germany) or under an inverted
microscope Zeiss Axiovert 200 (Zeiss, Germany), as indicated in
the corresponding figure legends. Neurons were plated at the
center of the coverslip to concentrate cell bodies in a limited
region, allowing the axons to grow out of this area. This made
possible to acquire two sets of images, an area comprising the cell
bodies, dendrites and axons and another area with isolated axons
(images of this were taken where only tau-positive neurites were
present). Quantitative analysis of immunocytochemistry data was
performed using ImageJ 1.42 software. In order to determine the
ratio between the fluorescence intensity of motor proteins (KIF1A,
KIF5B and dynein) and the area of tau fluorescence, 8-bit images
(10 per condition) were randomly selected and each channel
manipulated separately. Background signal of each channel image
was subtracted by adjusting the minimum grey value of the
greyscale (greyscale adjustment was equal for all images in each
single experiment). Channel images were then thresholded and the
signal of the motor proteins expressed in intensity per area (in mm
2
)
of tau fluorescence (threshold values were conserved in single
experiments). For each image, the ratio between the fluorescence
intensity of motor proteins (KIF1A, KIF5B or dynein) and the
area of tau fluorescence was calculated.
When looking into the distribution and content of motor and
synaptic proteins, specifically in axons, quantitative analysis was
performed for images taken to isolated axons only. Quantification
of the ratio between motor proteins fluorescence intensity (KIF1A
or KIF5B) and the area of tau was performed as described above.
In order to determine the number of accumulations, both for
KIF1A and synaptic proteins, per area of tau or tuj-1, respectively,
8-bit images (12 per condition) were randomly selected. For each
protein of interest, background signal was subtracted (as described
before) and threshold values were applied so that only accumu-
lations are taken in consideration (threshold values were conserved
in single experiments). Particle analysis was performed and the
number of accumulations per axonal area calculated for each
image.
The values obtained for each image were normalized against
the control mean of that single experiment and for all analysis,
results are presented as normalized values 6 SEM of the number
of images analyzed (1012 per condition in each experiment). The
number of independent experiments is indicated above the graphs.
Statistical Analysis
For Western blotting analysis, statistical comparisons between
diabetic animals and respective age-matched controls were
performed using the unpaired Students t-test (variance analysis
was not undertaken since the effect of age on the content of motor
proteins was not the aim of this study). Thus, gels were always
loaded with samples from age-matched animals and not from
animals with different ages.
In order to quantitatively analyze immunofluorescence in brain
slices, all values were compiled for statistical analysis and
significant difference between control and diabetic animals was
performed using the unpaired Students t-test. Statistical analysis
for data obtained from hippocampal cell cultures was performed in
Graph Pad Prism 5 software. Statistical significance was assessed
by one-way ANOVA analysis followed by Dunnetts post hoc test
or unpaired Students t-test. Differences were considered signifi-
cant for p,0.05.
Results
Animals
Before diabetes induction, the body weight of animals assigned
for control and diabetic groups was similar (257.763.9 g for
control animals and 255.363.6 g for diabetic animals). The
glucose levels were also similar in both groups (87.161.2 mg/dl
for controls and 86.363.7 mg/dl for diabetic animals). Average
weight and blood glucose levels for both diabetic and aged-
matched control rats at the time of death are given in Table 3. A
marked impairment in weight gain occurred in diabetic rats
comparing to age-matched controls in all time points analyzed.
Figure 2. Diabetes induces changes in KIF5B in the hippocampus. (A) Diabetes upregulates KIF5B mRNA expression, as assessed by RT-qPCR.
The results are expressed relatively to age-matched controls, and data are presented as mean 6 SEM of 5 animals. *p,0.5; **p,0.01 compared to
age-matched control and analyzed by the unpaired Students t-test. (B) KIF5B protein levels were analyzed by immunoblotting in total extracts of
hippocampus isolated from control and STZ-induced diabetic animals (2 and 8 weeks of diabetes). Representative Western blots are presented above
the graphs, with the respective loading controls (b-III tubulin), to confirm that identical amounts of protein from control and diabetic samples were
loaded into the gel. The results are expressed as percentage of age-matched controls, and data are presented as mean 6 SEM of 5 animals. *p,0.05
compared to age-matched control using Students t-test. (C) The distribution of KIF5B protein in hippocampal subregions of control and STZ-induced
diabetic animals was also analyzed by immunohistochemistry. KIF5B immunoreactivity increased at 8 weeks of diabetes. The preparations were
visualized under a laser scanning confocal microscope LSM 710 META (Zeiss, Germany). Scale bar: 50 mm. Insets shows the expression pattern of
KIF5B in hippocampal slices. The quantification of immunoreactivity (fluorescence intensity arbitrary units) was performed and presented below the
images.*p,0.05, significantly different from control as determined by the unpaired Students t-test.
Diabetic animals also presented significantly higher blood glucose
levels when compared to age-matched controls.
Diabetes Increases mRNA Expression, Protein Levels and
Immunoreactivity of KIF1A and KIF5B in the
Hippocampus at 8 Weeks of Diabetes
KIF1A is an anterograde motor protein that transports
membranous organelles along axonal microtubules [19]. It is
thought that this protein may play a critical role in the
development of axonal neuropathies, which may result from
impaired axonal transport. Our previous results suggest that
hyperglycemic conditions might impair axonal transport in
hippocampal neurons [2,6]. To our knowledge, the effect of
diabetes on the expression and content of motor proteins in the
hippocampus has never been addressed, and so we analyzed
mRNA levels and the content of KIF1A and KIF5B in
hippocampal total extracts by western blotting, as well as the
immunoreactivity of both proteins in hippocampal Cornu Ammonis
(CA1 and CA3) and dentate gyrus (DG) subregions. Increased
mRNA expression of KIF1A (increase to 1.9260.6 of the control)
and KIF5B (increase to 1.3960.2 of the control) were detected
after 2 weeks of diabetes. Nevertheless, no significant changes were
detected neither in the levels of KIF1A in hippocampal total
extracts, nor in its immunoreactivity in hippocampal subregions at
2 weeks of diabetes (Figure 1A and B). At 8 weeks of diabetes,
KIF1A mRNA levels significantly increased to 2.2460.6 of the
control, as well as KIF1A protein levels in hippocampal total
extracts (increase to 140.364.0%) compared to age-matched
control animals (Figure 1A and B, respectively). A significant
increase in the immunoreactivity of this protein was also observed
by immunohistochemistry at 8 weeks of diabetes in CA1, CA3 and
DG hippocampal subregions (increase to 150.3612.3%,
200.9624.2% and 208.4612.8% of the control, respectively;
Figure 1C).
KIF5B is a microtubule-dependent motor protein required for
normal distribution of presynaptic cargoes and mitochondria. At 2
weeks of diabetes, KIF5B mRNA levels were also increased
(increase to 1.3960.3), but no significant changes were observed in
KIF5B immunoreactivity in the hippocampus by Western blotting
(Figure 2A and B). However, at 8 weeks of diabetes, the mRNA
expression significantly increased to 1.5660.3, and KIF5B protein
levels increased to 127.769.3% of the control (Figure 2A and B).
Moreover, by immunohistochemistry, it was also detected an
increase in the immunoreactivity of this protein in CA1, CA3 and
DG hippocampal subregions (increase to 127.168.5%,
166.9617.3% and 143.4622.4% of the control, respectively;
Figure 2C).
Diabetes does not Affect the Content of Dynein in the
Hippocampus
Dynein is the major molecular motor protein that moves
cargoes such as mitochondria, organelles and proteins towards the
minus end of microtubules, thus being responsible for retrograde
transport in neurons. In hippocampus, dynein mRNA and protein
levels remained similar to those found in control samples at 2 and
8 weeks of diabetes (Figure 3A and B). Similarly, no changes were
detected in CA1, CA3 and DG hippocampal subregions by
immunohistochemistry in both time-points of diabetes, 2 and 8
weeks (Figure 3C).
High Glucose Induces a Mild Decrease in KIF5B, but does
not Affect the Overall Content and Distribution of Tau,
KIF1A and Dynein in Hippocampal Cultures
Hyperglycemia has been considered the main pathogenic factor
underlying the development of diabetic complications, triggering
several processes that may induce cell dysfunction. Here, we
evaluated whether high glucose per se, mimicking hyperglycemic
conditions, changes the content of proteins involved in axonal
transport in hippocampal neuronal cultures. Exposure of hippo-
campal neurons to high glucose, or mannitol (osmotic control), did
not affect the total protein content of tau, KIF1A, KIF5B and
dynein, as it can be observed by western blotting in Figure 4A. By
immunocytochemistry, we can observe that high glucose and
mannitol did not induce any significant change in the distribution
and content of KIF1A and dynein in the overall culture when
compared to control conditions (Figure 4B). However, a significant
decrease in the fluorescence intensity of KIF5B per tau area was
found in hippocampal neurons exposed to elevated glucose
(78.563.9% of control, Figure 4B). For both KIF1A and KIF5B,
no changes in the content of anterograde motor proteins were
observed in the osmotic control (mannitol).
High Glucose Levels alter the Content and Distribution of
Anterograde Motor Proteins in Axons
Although only mild (KIF5B) or no changes (KIF1A) were
detected in the content of motor proteins in hippocampal cultures
exposed to high glucose levels, changes can be occurring
specifically in axons and so being masked in the overall context.
In fact, previous data obtained in our lab show that diabetic
conditions affects the content of several presynaptic proteins in
nerve terminals [2,5,6], thus suggesting that their transport to
those distal sites might be impaired. Taken this into account, we
hypothesized that exposure to high glucose induces changes in
motor proteins specifically along axons. For that, we evaluated
their content and distribution in the axons of hippocampal
neurons. Exposure of hippocampal neurons to elevated glucose for
7 days increased the immunoreactivity of KIF1A in axons (KIF1A
intensity/axonal area; increase to 144.365.5% compared to
control conditions; Figure 5A). As it was already reported [20],
and as it can be observed in the axons segments shown in
Figure 5A, KIF1A has a characteristic punctate pattern along
axons of hippocampal neurons. To investigate whether high
glucose levels would affect this distribution pattern, we measured
the number of KIF1A accumulations along axons. An increase to
149.467.4% of control in the number of KIF1A accumulations
per axonal area compared to control conditions was observed
Figure 3. Diabetes does not induce changes in dynein in the hippocampus. (A) Diabetes does not change dynein mRNA expression, as
assessed by RT-qPCR. The results are expressed relatively to age-matched controls, and data are presented as mean 6 SEM of 5 animals as assessed
by the unpaired Students t-test. (B) Dynein protein levels were analyzed by immunoblotting in total extracts of hippocampus isolated from control
and STZ-induced diabetic animals (2 and 8 weeks of diabetes). Representative Western blots are presented above the graphs, with the respective
loading controls (b-III tubulin), to confirm that identical amounts of protein from control and diabetic samples were loaded into the gel. The results
are expressed as percentage of age-matched controls, and data are presented as mean 6 SEM of 7 animals. (C) The distribution of dynein in
hippocampal subregions of control and STZ-induced diabetic animals was also analyzed by immunohistochemistry. The preparations were visualized
in a laser scanning confocal microscope LSM 710 META (Zeiss, Germany). Scale bar: 50 mm. Insets shows the expression pattern of dynein in
hippocampal slices. The quantification of immunoreactivity (fluorescence intensity arbitrary units) was performed and presented below the images.
(Figure 5A). Also evident was a decrease (64.164.3% of control,
Figure 5B) of KIF5B intensity in the axons of hippocampal cells
incubated with high glucose for 7 days. These results are in
agreement with the decrease observed in Figure 4B, thus showing
that changes in the content of anterograde motor proteins are
more evident when looking specifically to distant isolated axons.
Due to the weak signal for dynein in axons, it was not possible to
quantify this protein in hippocampal axons. These results show
that hyperglycemia has a considerable effect on the axonal content
and distribution of motor proteins involved in the anterograde
transport.
High Glucose Decreases SNAP-25 and Synaptophysin
Immunoreactivity and Increases the Number of
Accumulations of Synaptotagmin-1 in Hippocampal
Axons
KIF1A is a neuron-specific kinesin motor protein that transports
synaptic vesicle precursors containing synaptophysin and synapto-
tagmin, whereas KIF5B is known to transport mitochondria and
synaptic vesicle precursors containing syntaxin-1 and SNAP-25
[19]. Since we detected changes in the number of KIF1A
accumulations and changes in KIF1A and KIF5B immunoreac-
tivity in hippocampal axons, we evaluated the effect of elevated
glucose on the immunoreactivity/intensity of fluorescence and on
the number of accumulations of synaptic proteins and mitochon-
dria (stained with mitotracker, a fluorescent dye that stains
mitochondria in live cells; Invitrogen, Life Technologies, Scotland,
UK). No significant changes were observed in the intensity of
fluorescence or in the number of fluorescent accumulations related
to mitochondria in the axons of cultured hippocampal neurons
exposed to high glucose compared to control (Figure 6A and B).
Nevertheless, regarding synaptic proteins, we found that SNAP-25
and synaptophysin immunoreactivity was significantly decreased
(70.565.8% and 61.166.8% of the control, respectively) in the
axons of hippocampal neurons exposed to elevated glucose (Fig. 6A
and B). Concerning the number of fluorescent accumulations of
synaptic proteins in the axons of hippocampal neurons exposed to
elevated glucose, we found that the number of accumulations of
SNAP-25, syntaxin-1 and synaptophysin were similar to the
control condition, with the exception of synaptotagmin-1, since the
number of accumulations of this protein was significantly
increased to 143.2618.1% of the control.
Discussion
In the present study, we addressed whether early diabetes can
affect axonal motor proteins that are important for adequate
transport of synaptic proteins and mitochondria in the hippocam-
pus. Specifically, we showed that diabetes alters the mRNA levels,
and immunoreactivity of KIF1A and KIF5B motor proteins in the
hippocampus of diabetic rats. Moreover, in hippocampal neuronal
cultures, we demonstrated that elevated glucose is able to change
the immunoreactivity and number of fluorescent accumulations of
motor and synaptic proteins in axons.
Due to their high polarity, neurons are particularly dependent
on active intracellular transport. Deficits in this transport have
been considered to contribute to the pathogenesis of multiple
neurodegenerative diseases [21]. Direct evidence from genetic
studies demonstrates that mutations in major components of the
cytoskeleton and axonal transport result in axonal defects in
Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis and
Alzheimer disease [7]. Post-translational modifications of cyto-
skeleton proteins also result in axonal defects in diabetic
neuropathy [22]. In previous studies, we found that diabetes
changes the levels of several synaptic proteins involved in
exocytosis in hippocampal nerve terminals, with no changes in
total extracts, suggesting that axonal transport of those proteins to
distal synaptic sites may be impaired in diabetes [2,5]. In this
study, we found that there is an increase in KIF1A and KIF5B
levels in the hippocampus at 8 weeks after the onset of diabetes,
with no changes in dynein levels, suggesting that the anterograde
transport may be impaired in the hippocampus. An impairment of
axonal transport of certain cargoes may lead to their accumulation
in the cell body. In a rat model of a-synucleinopathy, elevated
levels of KIF1A were observed in substantia nigra [23], and the
authors suggested the possibility that accumulation of these motor
proteins may be due to the imbalance in protein degradation and
synthesis or to axonal transport deficit. Moreover, we found that
long-term exposure to elevated glucose induces an accumulation of
syntaxin-1, synaptotagmin-1 and VGluT-1 in the cell bodies of
cultured hippocampal neurons [6], further suggesting that axonal
transport may be impaired. These observations directed us to
further analyze the effect of hyperglycemia, which is considered
the main factor underlying the development of diabetic compli-
cations, on motor proteins, namely KIF1A, KIF5B and dynein.
KIF1A transports synaptic vesicle precursors of synaptophysin and
synaptotagmin-1, but does not transport organelles that contain
plasma membrane proteins, such as syntaxin-1 or SNAP-25.
These are transported by KIF5 motors. The number of fluorescent
accumulations of KIF1A increased in the axons of hippocampal
neurons exposed to elevated glucose for 7 days. Likewise,
increased number of accumulations of synaptotagmin-1 was also
detected. The accumulation of these particles may be due to
impairments at the microtubule network and/or impairment in
KIF1A motor function, leading to the accumulation of KIF1A.
KIF5B protein immunoreactivity in the axons of hippocampal
cells incubated with high glucose for 7 days decreased and
similarly SNAP-25 immunoreactivity was also decreased. Likewise,
in our previous studies, a significant decrease in the content of
SNAP-25 was detected in hippocampal cultures [6], as we had also
demonstrated in hippocampal nerve terminals from diabetic rats
[2]. These observations suggest that SNAP-25 appears to be
particularly affected by hyperglycemic conditions, at least in
hippocampal neurons, but the mechanisms underlying these
effects are unknown. The reduction in SNAP-25 levels might
Figure 4. High glucose induces a mild decrease in KIF5B but does not affect the overall content and distribution of tau, KIF1A and
dynein in hippocampal cultures. Cultured hippocampal neurons were exposed to 25 mM glucose (Control), 50 mM glucose (Glucose) and
25 mM mannitol (Mannitol) for 7 days. (A) The protein levels of tau, KIF1A, KIF5B and dynein were analyzed by western blotting. Representative
images of protein immunoreactive bands are presented above the graphs, with the respective loading control (b-actin or b-III tubulin). The
densitometry of each band was analyzed and the results are expressed as percentage of control 6 SEM of 5 independent experiments. Statistical
significance for the analysis of hippocampal cell cultures protein content was determined by using ANOVA, followed by Dunnetts post hoc test.
Differences were considered significant for p,0.05. (B) The immunoreactivity of KIF1A, KIF5B and dynein in the overall culture was analyzed by
immunocytochemistry. Magnification 6306; Scale bar 50 mm. Quantification of the ratio between the fluorescence intensity for motor proteins
(KIF1A, KIF5B or dynein) and the area of tau was performed for 3 independent experiments. *** p,0.001, significantly different from control as
determined by one-way-ANOVA followed by Dunnets post hoc test.
Figure 5. High glucose changes the number of KIF1A accumulated particles and KIF1A and KIF5B intensity in the axons of
hippocampal neurons. Cultured hippocampal neurons were exposed to 25 mM glucose (Control) and 50 mM glucose (Glucose) for 7 days. The
immunoreactivity and accumulated particles of KIF1A (A) and KIF5B (B), was analyzed by immunocytochemistry. Magnification 6306; Scale bar
10 mm. Quantification of accumulated particles and immunoreactivity was made for 3 independent cultures. *** p,0.001, significantly different from
control as determined by the unpaired Students t-test.
significantly impair neurotransmission. In SNAP-25 KO neuronal
cultures neurotransmitter release is almost abolished [24].
Moreover, synaptophysin immunoreactivity decreased in axons
of hippocampal neurons exposed to high glucose. When analyzing
the whole distribution of synaptophysin in hippocampal cultures
[6], we did not detect any change in the immunoreactivity of this
protein, but when we analyzed potential changes at hippocampal
axons, a significant decrease in the immunoreactivity of this
protein was detected. Since synaptophysin is an integral protein of
the synaptic vesicle membranes that has been correlated with
synaptic density and neurotransmitter release, this decrease may
contribute to impair neurotransmitter release. The changes
reported here in motor proteins, specifically those occurring in
axons, namely the increase in the number of fluorescent
accumulations of KIF1A and decreased immunoreactivity of
KIF5B strongly suggest that axonal transport may be compro-
mised. As a consequence, a decrease in the number of synaptic
vesicles and synaptic density may ultimately account for changes in
synaptic transmission in the hippocampus. Nevertheless, we must
also keep in mind that other KIFs might partially compensate for
the function of the kinesins here studied, and synaptic vesicle
precursors might be transported by KIFs other than KIF1A and
KIF5B [25]. Moreover, previous studies have shown that under
diabetes, axonal transport defects may be due to altered levels of
cargoes, as a result of decreased synaptic protein synthesis,
abnormal translational processing of the protein or impaired
incorporation of synaptic proteins into vesicles showing that
impaired axonal transport of proteins is observed, while the actual
rates of the axonal transport process are not affected [2629].
Retrograde transport is powered mainly by cytoplasmic dynein,
but some kinesins can also be involved in this transport [19].
KIF1A is responsible for the transport of dense-core vesicles in the
axons of hippocampal neurons, remaining associated with dense-
core vesicles during retrograde axonal transport, demonstrating
that these vesicles retain the molecular machinery necessary for
transport in both directions [30]. Defects in axonal transport of
synaptophysin-containing vesicle precursors have been observed in
KIF1A mutant mice [25]. Recent findings also demonstrate that
KIF1A is necessary for BDNF-mediated hippocampal synapto-
genesis and learning enhancement induced by environmental
enrichment [31], which reinforces the importance of this kinesin in
the hippocampus.
Tau is a microtubule associated protein, whose main function is
to modulate the stability of axonal microtubules. Excessive tau
phosphorylation is known to disrupt its binding to microtubules
altering molecular trafficking, which ultimately may lead to
synaptic dysfunction [32,33]. Diabetes induces abnormal hyper-
phosphorylation of tau in the brain, including the hippocampus
[34], and proteolytic tau cleavage [35], both of which are
associated with Alzheimers disease [36]. In fact, tau modification
can be induced by insulin dysfunction and hyperglycemia, which
may contribute to the increased incidence of Alzheimers disease
in diabetic patients [37]. We did not detect any change in tau
immmunoreactivity in hippocampal cultures exposed to high
glucose. However, we cannot exclude the possibility of changes in
tau phosphorylation state. Evidence obtained in kinesin-1 deficient
mice suggested that defects in axonal transport can initiate
biochemical changes that induce the activation of axonal stress
kinase pathways leading to abnormal tau hyperphosphorylation.
This further impairs axonal transport by disrupting the microtu-
bule network and blocking axonal highways that ultimately will
give rise to compromised synapse function and neurodegeneration
[38,39].
KIF5 motors are also responsible for axonal transport of
mitochondria. In KIF5A
2/2
neurons, the velocity of mitochon-
drial transport is reduced both in anterograde and retrograde
direction [40]. Decreased number of mitochondria in axons will
likely decrease ATP supply to molecular motors leading to
decreased anterograde and retrograde movement of both mito-
chondria and vesicles [7]. Growing evidence suggests that
mitochondrial dysfunction play a significant role in neurodegen-
erative diseases like Huntingtons disease, Alzheimers disease and
amyotrophic lateral sclerosis [4143]. Mitochondrial dysfunction
has also been proposed as a mediator of neurodegeneration in
diabetes [44] but as far as we are concerned there are no studies
addressing the effect of diabetes in mitochondria transport in the
central nervous system. In our work, we did not detect changes in
the intensity of fluorescence, neither in distribution or number of
accumulations related with mitochondria in the axons of
hippocampal neurons exposed to elevated glucose when compared
to control. Nevertheless, we cannot exclude the possibility that
hippocampal axons are being affected by diabetes since probably
other factors, besides hyperglycemia, may also have an effect in
mitochondria transport.
Hyperglycemia appears to be an important determinant for the
changes observed in this study. However, under diabetic
conditions, the lack or reduced levels of insulin, a potent trophic
factor, might also play an important role in axonal transport
impairment and synaptic changes observed in diabetic animals
[45,46], thus contributing to changes in hippocampal physiology.
For instance, short-term replacement of insulin in type I diabetic
rats has shown to prevent cognitive deficits [47].
Inflammation may also be a factor contributing to changes in
axonal transport in diabetes. Previously, it was reported that pro-
inflammatory cytokines, such as tumor necrosis factor-a (TNF-a)
and interleukin-1b, are upregulated in the hippocampus of
diabetic BB/Wor rats [48] and STZ-induced diabetic animals
[49]. TNF induces perinuclear clustering of mitochondria caused
by impaired kinesin-mediated transport [50] and the activation of
TNF receptor-1 induces the activation of kinase pathways,
resulting in hyperphosphorylation of kinesin light chain (KLC)
and inhibition of kinesin activity, evidencing direct regulation of
kinesin-mediated organelle transport by extracellular stimuli via
cytokine receptor signaling pathways in L929 cells [51]. Moreover,
it was previously demonstrated that nitric oxide released from
activated microglia inhibits axonal movement of synaptic vesicle
precursors containing synaptophysin and synaptotagmin in
hippocampal neurons, suggesting that disturbance of axonal
transport by microglial nitric oxide may therefore be responsible
for axonal injury and synaptic dysfunction in brain diseases
characterized by neuroinflammation [52]. TNF produced by
activated glial cells in inflammatory or degenerative neurological
Figure 6. High glucose decreases SNAP-25 and synaptophysin immunoreactivity and increases the number of synaptotagmin-1
fluorescent accumulations. Cultured hippocampal neurons were exposed to 25 mM glucose (Control) and 50 mM glucose (Glucose) for 7 days.
(A) The fluorescence intensity and the number of fluorescent accumulations related to mitochondria and synaptic proteins were analyzed in the
axons of hippocampal neurons. The preparations were visualized under a laser scanning confocal microscope LSM 710 META (Zeiss, Germany). Scale
bar: 20 mm. (B) The quantification of the number of fluorescent accumulations and intensity of fluorescence of mitochondria or immunoreactivity of
synaptic proteins was performed and was expressed as percentage of the control..*p,0.05, **p,0.01, significantly different from control as
determined by the unpaired Students t-test.
diseases affects neurites by acting on the kinesin-tubulin complex
and inhibiting axonal mitochondria and synaptophysin transport
via JNK in hippocampal cultures [53]. Very recently, it was
demonstrated that hydrogen peroxide, a common reactive oxygen
species elevated during inflammation, also inhibits axonal trans-
port in hippocampal cultures [54]. Further studies will be needed
to determine if similar pathways may be active under diabetic
conditions, therefore contributing for the detected changes in the
levels of synaptic and motor proteins in the hippocampus.
In summary, our data demonstrate that the mRNA levels and
content of KIF1A and KIF5B motor proteins are altered in the
hippocampus of diabetic rats. Furthermore, we showed that high
glucose leads to an increase in the number of fluorescent
accumulations of KIF1A and synaptotagmin-1 and decreased
immunoreactivity of KIF5B, SNAP-25 and synaptophysin specif-
ically in the axons of hippocampal neurons. Altogether, these
changes suggest that the anterograde axonal transport may be
impaired in the hippocampus, which may lead to changes in the
content of synaptic proteins in nerve terminals, since their
transport is mediated by these kinesins, and ultimately contribute
to neural changes underlying diabetic encephalopathy.
Author Contributions
Conceived and designed the experiments: FIB MJP RDA AFA. Performed
the experiments: FIB MJP FE. Analyzed the data: FIB MJP FE RDA AFA.
Contributed reagents/materials/analysis tools: RDA AFA. Wrote the
paper: FIB FE MJP.
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