Download Levels of kinesin light chain and dynein intermediate

Levels of kinesin light chain and dynein intermediate chain are reduced in the
frontal cortex in Alzheimer’s disease. Implications for axoplasmic transport.
Marina Morel, Céline Héraud, Charles Nicaise, Valérie Suain and Jean-Pierre Brion
Université Libre de Bruxelles, Faculty of Medicine, Laboratory of Histology, Neuroanatomy
and Neuropathology, 808 route de Lennik, B-1070 Brussels, Belgium.
Running title: Molecular Motors in Alzheimer’s disease
Send correspondence to:
Dr Jean-Pierre Brion
Laboratory of Histology, Neuroanatomy and Neuropathology
Université Libre de Bruxelles, Faculty of Medicine. 808, route de Lennik, Bldg GE
1070 – Brussels, BELGIUM
Tel: 32 - 2 – 5556505. Fax: 32 - 2 – 5556285. E-mail: [email protected]
Key-words: Kinesin light chain, Dynein intermediate chain, neurofibrillary tangles, GSK-3ß,
Alzheimer’s’ disease
Acta Neuropathologica (2012),123(1),71-84.
1
Abstract
Fast anterograde and retrograde axoplasmic transports in neurons rely on the activity of
molecular motors and are critical for maintenance of neuronal and synaptic functions.
Disturbances of axoplasmic transport have been identified in Alzheimer’s disease and in
animal models of this disease, but their mechanisms are not well understood. In this study we
have investigated the distribution and the level of expression of kinesin light chains (KLCs)
(responsible for binding of cargos during anterograde transport) and of dynein intermediate
chain (DIC) (a component of the dynein complex during retrograde transport) in frontal
cortex and cerebellar cortex of control subjects and Alzheimer’s disease patients. By
immunoblotting, we found a significant decrease in the levels of expression of KLC1 and 2
and DIC in the frontal cortex, but not in the cerebellar cortex, of Alzheimer’s disease patients.
A significant decrease in the levels of synaptophysin and of tubulin-!3 proteins, two neuronal
markers, was also observed. KLC1 and DIC immunoreactivities did not co-localize with
neurofibrillary tangles. The mean mRNA levels of KLC1,2 and DIC were not significantly
different between controls and AD patients. In SH-SY5Y neural cells, GSK-3!
phosphorylated KLC1, a change associated to decreased association of KLC1 with its
cargoes. Increased levels of active GSK-3! and of phosphorylated KLC1 were also observed
in AD frontal cortex. We suggest that reduction of KLCs and DIC proteins in AD cortex
results from both reduced expression and neuronal loss, and that these reductions and GSK3!-mediated phosphorylation of KLC1 contribute to disturbances of axoplasmic flows and
synaptic integrity in Alzheimer’s disease.
2
Introduction
Accumulations of organelles and of various proteins in axons and in cell body of neurons are
observed in a number of neurodegenerative diseases [11]. A disruption of axoplasmic
transport, an essential mechanism for maintenance of neuronal function is thought to underly
the formation of these lesions in many of these diseases [13, 35]. Axoplasmic transport
promotes the transport of proteins or organelles like mitochondria and vesicles along the
axon, and occurs along the cytoskeleton tracks, which provides the structural support. The
movement of cargos along microtubules requires the action of two main types of molecular
motors: kinesins and dyneins that are involved in anterograde and retrograde transport
respectively. Anterograde movement is responsible for traffic of organelles from their site of
biogenesis to synapses. On the other hand, retrograde movement allows the return of
organelles to the cell body for degradation. Conventional kinesin is composed of two heavy
chains (KHC) and two light chains (KLC) [7, 50]. The light chains bind the organelles,
whereas the heavy chains contain a binding domain with ATP providing energy for the
molecular motor. Cytoplasmic dynein is a complex of two heavy chains (DHC) associated
with intermediate chains (DIC) and light chains (DLC). The heavy chains contain the motor
domain using ATP hydrolysis. The other chains are involved in binding to vesicular cargo and
to dynactin [39]. Phosphorylation of molecular motors represents a regulatory mechanism of
axoplasmic transport [21].
Alzheimer’s disease is the most common neurodegenerative disease characterized by
progressive cognitive deterioration, and two brain lesions: senile plaques and neurofibrillary
tangles. Senile plaque is composed of an extracellular accumulation of A! peptide surrounded
by dystrophic neurites and reactive glial cells. The A! peptide is generated by cleavage of
APP (amyloid precursor protein). Neurofibrillary tangles correspond to intraneuronal
inclusions and are composed of abnormally and hyperphosphorylated forms of the
microtubule-associated protein tau [8]. Tau proteins stabilize microtubules and their
hyperphosphorylation leads to microtubule destabilisation [28], resulting in axoplasmic
transports disturbances. Ultrastructural studies in Alzheimer’s disease have documented the
presence in neurons of accumulations of degraded organelles [18] and of endoplasmic
reticulum profiles [43] strongly suggestive of disturbances of axoplasmic transport.
Disturbances of axonal transport have also been described in some animal models for AD and
tauopathies. Overexpression of mutant APP in transgenic mice caused abnormal transport
with axonal swellings [1, 48]. Mutant presenilin 1 reduced axonal transport in mice [40] and
3
oligomeric A! inhibited fast axonal transport in isolated axoplasm [41]. Transgenic mice
overexpressing human wild-type tau [45] or mutant tau [27] also developed axonal swellings
suggestive of defects in axonal transport. In vitro motility assays suggested that tau influences
the rate of attachment and detachment of motor proteins along microtubules [15, 47].
However, in a mice model overexpressing wild-type tau or in mice deleted for tau, axonal
transport rates in retinal ganglion cells were not affected [51]. Also tau binding to
microtubules in squid axoplasm did not affect fast axoplasmic transport [34]. In contrast,
aggregated filamentous forms of tau inhibited anterograde fast axonal transport in vivo [25]
by a mechanism involving activation of GSK-3! [23, 25].
The mechanisms by which axoplasmic transports are disturbed in AD might thus have several
origins, including microtubule dysfunction, altered binding of molecular motors by
competition, physical or biochemical disturbances induced by aggregated tau and
modification of signalling pathways controlling molecular motors activities. These
disturbances might also result from defects of the level of expression of molecular motors.
Many of these mechanisms have been analyzed mainly in experimental in vitro and in vivo
systems, and their relevance for axoplasmic transport deficits in AD needs to be further
assessed. To further document the abnormalities of axoplasmic flows disturbances in AD, we
have investigated in this study the phosphorylation and the level of expression of kinesin light
chain (KLCs) and of dynein intermediate chain (DIC) in brain tissue of AD patients.
4
Material and methods
Brain tissue
Human brain tissue samples taken at autopsy from 5 control subjects and 8 demented patients
clinically diagnosed as Alzheimer’s disease were fixed with formalin 10% and embedded in
paraffin. Some samples of the frontal cortex and cerebellum were kept at -80°C. The
neuropathological examination confirmed the presence of numerous senile plaques and
neurofibrillary tangles. Table 1 summarizes the clinical data and the neuropathological
stageing of control subjects and AD patients according to Braak and Braak stageing for NFT
[6] and CERAD plaque score for senile plaques [31]. All AD patients had a Braak’s NFT
stage V or VI, and a CERAD age-related plaque score C or B (for one patient). All studies on
post-mortem brain tissue were performed in compliance and following approval of the Ethical
committee of the medical School of the Free University of Brussels.
Preparation of tissue and cell homogenates and immunoblotting
Human brain samples were homogenized in a RIPA buffer containing Tris 50 mM, pH 7.4,
NaCl 150 mM, NP40 1%, deoxycholate-Na 0.25%, PMSF 1 mM, leupeptin 25 µg/ml,
pepstatin 25 µg/ml, Na4P2O7.10 H2O 10 mM, NaF 20 mM and Na3VO4 1 mM. After
centrifugation of homogenates (20000g, 20 min), the levels of proteins in supernatants were
estimated with the Bradford method. SH-SY5Y cells were scrapped and homogenized in a
buffer containing proteases and phosphatases inhibitors (Tris 50 mM, EDTA 10 mM, NaCl
100 mM, pH 7.4, PMSF 1 mM, leupeptin 25 µg/ml, pepstatin 25 µg/ml, Na3VO4 1 mM,
Na4P2O7.10 H2O 10 mM, NaF 20 mM). Samples were run on 10% (w/v) sodium dodecyl
sulphate polyacrylamide gels and proteins were electrophoretically transferred to
nitrocellulose membrane. Membranes were blocked in non-fat milk (10% (w/v) in TBS) and
then incubated with the primary antibody. For ECL detection, the secondary antibody was an
anti-mouse (Sigma) or an anti-rabbit (Cell Signaling) antibody conjugated to horseradish
peroxidase. After secondary antibody incubation, blots were developed using the ECL kit
Supersignal west Pico chemiluminescent Substrate (Pierce) and exposed to CL-Xposure film
(Pierce).
Antibodies
The B19 rabbit polyclonal antibody is a phosphorylation-independent tau antibody previously
5
described [10]. The mouse monoclonal antibody PHF-1 (kindly provided by Drs. P. Davies,
New York) is specific for tau phosphorylated at Ser396/404 [37]. The following antibodies
were also used: mouse monoclonal antibodies to Dynein intermediate chain (DIC)
(MAB1618, Chemicon), to tubulin-!3 (T8660, Sigma), to "-tubulin (T9026, Sigma, DM1A)
to GSK-3! (TPKI, Transduction Laboratories), to phosphoserine (abcam) and rabbit
polyclonal antibodies to KLC1 (H-75, Santa Cruz), to KLC2 (abcam), to A!42 (Biosource)
and to actin (A2066, Sigma). The rabbit polyclonal antibody to pY279/pY216-GSK-3"/!
detects the active form of GSK-3 phosphorylated on Tyrosine 279 (GSK-3") and on Tyrosine
216 (GSK-3!) (Biosource). The anti-synaptophysin antibody is a rabbit monoclonal antibody
against the C-terminus end of synaptophysin (clone YE269, Millipore).
Dephosphorylation of brain samples
To check for a possible reduction of affinity of KLC1 antibody to phosphorylated KLC1,
homogenates of frontal cortex were dephosphorylated with lambda phosphatase prior to SDSPAGE and immunoblotting. Samples containing 10 #g of proteins were incubated for 2h at
30°C with 2000 U of lambda phosphatase (New England BioLabs) in 50 mM Tris-HCL, 100
mM NaCl, 0.1 mM EGTA, 2 mM dithiotreitol, 2mM MnCl2, 0.1 % Brij 35, pH 7.5.
Immunohistochemistry
Tissues samples from the frontal cortex were fixed with 4% paraformaldehyde, cryo
preserved in 30% sucrose and embedded in tissue-Tek OCT compound. Cryosections (14µm
of thickness) were blocked in 10% serum with 0.1% Triton X-100 for 1h. Doubleimmunofluorescent labeling was performed with the anti-KLC1 and the phosphotau PHF1
antibodies, or the anti-DIC and the B19 tau antibodies. Primary antibodies were diluted in 1%
serum with 0.1% Triton X-100 and incubated overnight at room temperature. After washing
with 0.1% Triton X-100, cryosections were incubated with a secondary antibody conjugated
to biotin (Vector) and a secondary antibody conjugated to Alexa594 (Molecular Probes) for
30 minutes with the streptavidine-peroxidase complex (diluted in TNB buffer), followed by
10 min incubation with Tyramide-FITC diluted 1/50 in amplification buffer (Perkin Elmer).
Sections were washed and mounted using gelvatol medium. Sections were examined on an
Axiovert 200M Zeiss microscope using phase contrast optics and combination of fluorescence
filters for FITC and Texas Red. Digital images were acquired using a black and white camera
(Zeiss Axiocam MRm) and the Axiovision software (Zeiss) with an ApoTome system (Zeiss).
6
Paraffin sections were immunolabeled using the ABC method as previously described [26].
Briefly, tissue sections, after inhibition of endogenous peroxidase with H2O2, were
sequentially treated with the blocking solution (10% (v/v) normal horse serum in TBS (0.01
M Tris, 0.15 M NaCl, pH 7.4), incubated overnight with the diluted primary antibody, and
then with goat anti-rabbit antibodies conjugated to biotin (Vector) followed by the ABC
complex
(Vector
Laboratories).
The
peroxidase
activity
was
developed
using
diaminobenzidine as chromogen. Digital images were acquired using a Zeiss Axiocam) and
the Axiovision software on an AxioImagerM1 Zeiss microscope.
Quantification of Aß load
Paraffin sections of frontal cortex from control and AD patients were immunolabeled with the
anti-Aß42 antibody (ABC method) after pretreatment of sections with formic acid as
described [5]. Black and white digital images of four fields of views (each 669 x 844 #m)
randomly distributed along the cortical ribbon were acquired for each case. The Aß
immunlabelled area was determined in each image by detecting positive pixels using a
constant threshold (manually optimized) with the Image J program, and expressed as a
percentage of the total surface of the field of view. The mean value of the four fields of views
was determined for each case.
Quantitative PCR
Brain samples were homogenized with Magna Lyser Beads (Roche) and total RNA was
extracted using Tripure lysis solution and High Pure RNA Tissue Kit (Roche Diagnostics,
Belgium) according to the manufacturer’s protocol, which included DNase treatment. One
microgram of total RNA was reverse transcribed into cDNA with SuperScript®II enzyme,
oligo(dT), dNTP mix, First-Strand Buffer, DTT, RnaseOUT (Invitrogen, Belgium). The
mRNA quantification was performed using a real-time polymerase chain reaction on a
LightCycler 480 (Roche Diagnostics) with the MESA GREEN MasterMixes Plus for SYBR®
(Eurogentec) and cycling programme was 5 min at 95°C and then 40 cycles, each consisting
of 15 sec at 95°C, and 35 sec at 60°c. The primers (Supplementary Table I) for human actin,
kinesin light chain 1 (KLC1), kinesin light chain 2 (KLC2), dynein intermediate chain (DIC)
and hypoxanthine-guanine phosphoribosyltransferase (HPRT) were designed with Primer3
software (White head Institute for Biomedical Research, Cambridge, MA). Gene expression
level of each mRNA was calculated in duplicate, and then normalized to actin or HPRT
mRNAs levels. Specific amplifications were confirmed by melting curve analysis and
7
migration on agarose gel.
Plasmids vectors
The pcDNA5/FRT GSK-3!-EGFP encodes wild-type GSK-3! and the pcDNA5/FRT GSK3!-S9A-EGFP encodes an active form of GSK-3! with a Ser to Ala mutation that avoid
inactivating phosphorylation at Ser9 [16, 32]. The pcDNA5/FRT GSK-3!-K85A, kindly
provided by Dr Doble, encodes a kinase-dead GSK-3! with a Lys to Ala mutation. To
generate an EGFP-GSK-3!-K85A fusion protein we used a PCR-based strategy. The
pcDNA5/FRT GSK-3!-EGFP was first cleaved by KpnI and ApaI to release the wild-type
GSK-3!. Oligonucleotide primers were designed to amplify the sequence encoding for GSK3!-K85A from the pcDNA5/FRT GSK-3!-K85A and to introduce a KpnI site to its 5’ end
and an ApaI site to its 3’end. The KpnI/ApaI-digested PCR products were then ligated into
the pcDNA5/FRT downstream the EGFP sequence resulting in the pcDNA/FRT GSK-3!K85A-EGFP encoding for EGFP-GSK-3!-K85A fusion protein. The positive recombinant
was identified by PCR analysis and restriction endonuclease analysis. The pcDNA3-EGFP
plasmid (Clontech) is a cloning vector encoding a variant of wild-type enhanced green
fluorescent protein (EGFP). Preparations of stock plasmids solutions were performed using
the Nucleobond PC500 kit (Stratagene).
Transfection of SH-SY5Y cells
The SH-SY5Y cells were purchased from the EORTCC collection and were cultured in F12
medium added with 10% v/v foetal bovine serum, 100 IUs penicillin and 100 µg/ml
streptomycin. Cells were cultured at a density of 200,000 cells/ml for western blot analysis
Cells cultures were maintained at 37 °C in presence of 5% CO2. Transfection of SH-SY5Y
was performed 24 hours after the plating using Lipofectamine 2000 reagent (Gibco BRL)
with
a
ratio
of
3µg
Lipofectamine/1µg
DNA
and
they
were
analyzed
by
immunocytochemistry or western blotting. SH-SY5Y were transfected with either the EGFP,
the GSK-3!-EGFP, the GSK-3!-S9A-EGFP or the GSK-3!-K85A-EGFP vectors. Cell death
and suffering induced by expression of the transfected proteins was estimated by staining of
cells with DAPI (1µg/ml of culture medium) to visualize nuclear morphology [4]. The
proportion of cells with condensed and fragmented chromatin was estimated in the different
transfection conditions. Viability of SH-SY5Y cells was also estimated 24 hours after
transfection with the MTT assay. In some experiments, transfected cells were incubated with
8
the GSK-3ß inhibitor SB216763 (Tocris, UK) at a concentration of 10 #m for 6 hours before
homogenization and immunoprecipitation.
KLC1 immunoprecipitation
The anti-KLC1 antibody (2µg) was incubated with 50µl of protein A sepharose beads (Sigma)
and rotated at 4 °C for 1 h. The tissue samples were homogenized in a lysis buffer (HEPES
0.03M, NaCl 0.03M, Triton X-100 0.1 % (v/v)) and aliquots (100 #g of proteins) were
incubated with beads coupled with anti-KLC1 and rotated overnight at 4 °C. Controls were
performed by incubating samples with protein A sepharose beads alone. The beads were then
washed three times in lysis buffer and resuspended in an adjusted volume of protein sample
buffer. Fractions were then heated at 70°C for 10 min, separated by 10% gradient
SDS/PAGE, and transblotted to nitrocellulose membranes for immunodetection with the antiphosphoserine antibody or the anti-KLC1 antibody. For the anti-phosphoserine antibody, the
blocking reagent was BSA 10%. For ECL detection, the secondary antibody was an antimouse conjugated to biotin (Vector) and Avidin Biotin Complex (Vectastain ABC Kit,
Vector).
Statistical analysis
Statistical significance was determined by Student’s t test and by one-way Anova (with
Bonferroni post tests) and correlation analysis performed with Spearman’s coefficient using
Prism 4 software (Graphpad).
9
Results
Levels of abnormal PHF tau proteins and of Aß in frontal cortex of controls and AD
patients
We checked for the presence of phosphotau proteins with the PHF-1 antibody in control and
AD samples of frontal cortex used for assessing the levels of kinesin light chain and dynein
intermediate chain (Supplementary Fig. S1). As described previously [9], we observed in the
8 AD samples three main PHF-1 positive bands between 50 kD and 70 kD corresponding to
hyperphosphorylated tau proteins, a smear of higher molecular weight tau species
corresponding to aggregated species and lower molecular weight tau degradation products.
We observed also a weak PHF-1 immunoreactivity in one control homogenate (Braak Stage
II-III). No PHF-1 immunoreactivity was detected in the other samples from control subjects.
The mean level of A! load in the frontal cortex samples (as estimated by quantification of
immunolabelled Aß areas) was significantly higher in AD patients by comparison with
controls (p< 0.05, by Student t test) (data not shown).
Levels of kinesin light chains and of dynein intermediate chain are reduced in AD
frontal cortex but not in the cerebellar cortex
We then investigated whether expression levels of kinesin light chain and dynein intermediate
chain were modified in the same homogenates of the frontal cortex. The anti-KLC1 antibody
detected a 61kD protein (Fig. 1a), the expected molecular weight of KLC1, and the anti-DIC
antibody detected a single species of 74kD (Fig. 1b), corresponding to the dynein
intermediate chain. Although some variations were observed between AD cases,
quantification of KLC1 immunoreactivity normalized with actin revealed that the average
KLC1 levels in AD reached only 48.76% ± 8.55 (mean +/- SEM) of control levels (Fig. 1d), a
difference statistically significant (p=0.0159, by Student t test). The levels of KLC2
immunoreactivity normalized with actin were also significantly decreased in the frontal cortex
of AD patients (67.62 +/- 5.30, mean+/-SEM) (p=0.0058, by Student t test) (data not shown).
Quantification of DIC reactivity normalized with actin showed that the protein levels in AD
reached 44.95% ± 10.97 (mean +/- SEM) of control levels (Fig. 1e), a reduction also
significantly different (p=0.0176, by Student t test).
We then investigated whether expression levels of KLC1 and dynein were modified in
samples of the cerebellar cortex from the same control and AD cases. Quantification of KLC1
and DIC immunoreactivity normalized with actin revealed that the average KLC1 and DIC
10
levels in cerebellum were similar in AD patients and in control cases (Fig. 1i and 1j).
Furthermore, the reduction of KLC1 and DIC in the frontal cortex of AD patients was not
significantly correlated (Spearman’s test) with the postmortem delays.
We next analyzed the levels of two neuronal proteins, i.e. tubulin-!3 (a neuron specific
tubulin isoform) and synaptophysin (a glycoprotein present in the membrane of presynaptic
vesicles) in these samples. The anti-synaptophysin antibody detected the glycosylated form of
synaptophysin at 40kD (Fig. 2b). We observed a decrease of the level of expression of
tubulin-!3 (Fig. 2a), of synaptophysin (Fig. 2b) and of "-tubulin (Fig 2c), but not of actin
(Fig 2d), in AD frontal cortex. The percentage of tubulin-!3 expression levels in AD
normalized with actin was decreased to 42.53% ± 9.53 of levels in control subjects (p=0.0215,
by Student t test). For synaptophysin, the percentage of expression levels in AD patients
normalized with actin was decreased to 63.55% ± 14.76 of levels in control subjects
(p=0.0328, by Student t test). The levels of tubulin-!3 were not decreased in the cerebellar
cortex of AD patients (data not shown). We then estimated the levels of KLC1 and of DIC in
control and AD samples by normalizing them with the levels of tubulin-!3 and synaptophysin
(Fig. 3): no significant differences were observed in mean levels between control and AD
patients, both in the frontal cortex (Fig. 3) and the cerebellar cortex (data not shown). The
reduction of KLC1 and of DIC in the frontal cortex of AD patients was correlated with the
reduction of tubulin-!3 (Spearman’s test, p=0.028 and p=0.048, respectively), but not with the
reduction of synaptophysin. The reductions of KLC1 and DIC were highly correlated between
them (p<0.001).
Reductions of KLC1 and DIC levels are not correlated with loads of A! or PHF-1
positive tau
We next compared the levels of KLC1 and DIC with the load of Aß or PHF-1 positive tau in
the corresponding cases. The levels of KLC1 (p= 0.59) and DIC (p= 0.13) were not
significantly correlated with the levels of PHF-positive tau in the frontal cortex of AD cases
(Spearman’s test). The levels of KLC1 (p=0.55) and DIC (p= 0.33) were also not significantly
correlated with the levels of A! in the frontal cortex of AD cases or when including control
cases in this analysis.
11
Levels of KLC and DIC mRNAs in control and AD frontal cortex
We next analyzed by quantitative PCR the levels of KLC1, DIC, and KLC2 in samples of the
frontal cortex of control subjects and AD patients (Fig. 4). The mean levels of KLC1 and DIC
mRNAs normalized to actin mRNAs tended to be lower in AD patients, although not
significantly. These mean levels were similar between controls and AD patients when
normalized to HPRT mRNAs. The mean levels of KLC2 mRNAs were also similar between
controls and AD patients.
KLC1 immunoreactivities are decreased in AD and do not co-localize with NFT in AD
brain
We performed then an immunolabeling with the anti-KLC1 antibody on paraffin tissue
sections of the frontal cortex in control and AD cases. Neurons in the frontal cortex showed a
granular and vesicular-like KLC1 immunoreactivity in cell bodies and dendrites (Fig 5a). This
vesicular immunoreactivity was decreased in most AD cases (Fig. 5b). Since a binding
between kinesin and tau has been reported [12], we assessed the relationship between tau and
KLC1 or DIC immunoreactivities in tangles-bearing neurons by performing a double
immunolabeling with the anti-tau and the anti-KLC1 or the anti-DIC antibodies on cryostat
sections (Fig. 6). A few KLC1 or DIC vesicular-like structures were occasionnally observed
in tangle-bearing neurons but tangles themselves were not or weakly KLC1 or DIC positive.
GSK-3! phosphorylates KLC1 in a cellular model
Since GSK-3! has been reported to phosphorylate KLC in squid axoplasm and KLCs from rat
brain in vitro [33], we studied the ability of GSK-3! to phosphorylate KLC1 in human SHSY5Y neuroblastoma cells. SH-SY5Y cells were transfected with GSK-3!-EGFP, GSK-3!S9A-EGFP, GSK-3!-K85A-EGFP or EGFP alone as a control. The expression of GSK-3!EGFP, GSK-3!-S9A-EGFP, GSK-3!-K85A-EGFP was detected by immunoblotting with an
antibody to total GSK-3! (Fig. 7a). The endogenous GSK-3! was detected in transfected cells
at a lower molecular weight (47kD) than the GSK-3! conjugated to EGFP (75kD). The
percentage of cells with a nuclear fragmentation was no significantly different between
transfection conditions (data not shown). MTT assays also did not show difference in the
percentage of viable cells between the different transfection conditions (data not shown). The
enzymatic activity of transfected GSK-3! was assessed by studying changes in
phosphorylation of tau, a substrate of GSK-3!, with the PHF-1 phosphotau antibody. An
12
increase of tau phosphorylation was observed in cells transfected with GSK-3!-EGFP or an
active form of GSK-3! (GSK-3!-S9A-EGFP) (x1.9 and x1.4 respectively, after normalization
with total tau and by comparison with cells transfected with EGFP alone) (Fig. 7b).
We then immunoprecipitated KLC1 from cells transfected with EGFP, GSK-3!-EGFP, GSK3!-S9A-EGFP or GSK-3!-K85A-EGFP (Fig. 7c), and tested the phosphorylation state of
immunoprecipitated KLC1 with an anti-phosphoserine antibody. The phosphorylation state of
KLC1, normalized to total KLC1, was significantly higher in cells expressing GSK-3!-EGFP
or GSK-3!-S9A-EGFP but not in cells expressing GSK-3!-K85A-EGFP or EGFP (Fig. 5c3).
KLC1 was not detected when samples were incubated with protein A-sepharose beads alone.
We next analyzed phosphorylation levels of immunoprecipitated KLC1 in transfected SHSY5Y cells treated with the GSK-3! inhibitor SB216763. Inhibition of GSK-3ß led to a
decrease of KLC1 phosphorylation by comparison with untreated cells (fig 7d). KLC1
immunoprecipitation was not affected by GSK-3ß inhibition, indicating that the anti-KLC1
antibody was not phosphosensitive. Inhibition of GSK-3ß was also effective in decreasing tau
phosphorylation (assessed with the PHF-1 phosphotau antibody) in transfected cells (data not
shown).
Phosphorylation of KLC1 is increased in AD frontal cortex
The active form of GSK-3! is increased in AD brain [26] and we confirmed this observation
in the human brain homogenates of the frontal cortex used in the present study (Fig. 8a-c). To
assess the status of phosphorylation of KLC1 in human homogenates of frontal cortex in
control and AD cases, KLC1 was immunoprecipitated with the anti-KLC1 antibody and its
phosphorylation state estimated with the anti-phosphoserine antibody, after normalization to
total KLC1. An increased phosphorylation of KLC1 was observed in the frontal cortex of AD
patients (Fig. 8d-f). Since increased phosphorylation of KLC1 might reduce affinity of the
anti-KLC1 antibody, we checked if dephosphorylation of brain samples did reduce the level
of labeling by the KLC1 antibody by Western blotting. Prior dephosphorylation did not
modify the labeling of KLC1 by the KLC1 antibody in homogenates of the frontal cortex of
controls and AD subjects (Supplementary Fig. S2), another indication that the anti-KLC1
antibody was not phosphosensitive.
13
Discussion
In this study, we examined the level of expression of the kinesin light chains (KLCs) and of
the dynein intermediate chain (DIC) in the frontal cortex and cerebellum of control subjects
and AD patients. The light chain of kinesin is responsible for binding of cargo during
anterograde transport. The intermediate chain of dynein is a component of the dynein
complex for retrograde transport. By immunoblotting, a significant reduction in mean levels
of KLCs and DIC was observed in the frontal cortex of patients with Alzheimer's disease by
comparison with control subjects, i.e. in a brain area affected in the disease. By
immunohistochemistry, we also observed a reduction of KLC1 immunoreactivity in neurons
in most AD patients. We also studied the protein expression level of KLC1 and DIC in the
cerebellar cortex of the same control subjects and Alzheimer patients, a brain area spared in
the disease. We did not detect change in the levels of KLC1 and DIC in homogenates of
cerebellum of AD patients, indicating that the reduction of KLC1 and DIC was specific for an
affected area, the frontal cortex.
These reduced levels of KLCs and DIC in AD could play a role in functional abnormalities in
the kinesin- and dynein-dependent transports, leading to the deficits of axoplasmic transport
observed in AD. We did not observe a significant reduction of KLCs and DIC mRNAs in
frontal cortex in AD patients, suggesting that reduction of KLC and DIC proteins did not
primarily result from reduced transcriptional activity. This reduction of KLCs and DIC
proteins could however result from reduced mRNA stability or translational activity in
neurons in AD, or from increased protein degradation. Increase of Aß peptides or PHF-tau
proteins might be linked to this reduction, but we could not find a significant correlation
between KLC1 or DIC reduction and Aß amyloid or PHF-tau proteins levels. However, if low
levels of PHF-tau proteins or Aß are sufficient to provoke significant reduction of the cellular
levels of KLCs and DIC, the heavy accumulation of PHF-tau in our AD cases, and of Aß,
might mask any potental correlation between these measures at least in advanced AD cases.
Alternatively, the reduced levels of KLC1 and DIC observed in the frontal cortex of AD
patients could also be linked to neuronal loss. Neuronal loss in several brain regions
(especially in areas of association cortex, such as the frontal cortex) has been observed in AD
[36]. The levels of tubulin-!3 (a neuron specific tubulin isoform) and of synaptophysin were
reduced in the frontal cortex but not in the cerebellum in AD in our samples. The reduction of
these two markers in the frontal cortex is consistent with a neuronal and synaptic loss
14
affecting the frontal cortex. KLC1 and DIC levels normalized with the levels of tubulin-!3
and synaptophysin were not significantly different between AD patients and control subjects
in the frontal cortex (and cerebellum), an observation compatible with the hypothesis that the
decrease of KLC1 and DIC is a reflect of neuronal and synaptic loss. Synaptic loss is however
considered as occurring independently from neuronal loss and could even be greater than
neuronal loss in AD patients, leading to an underestimation of the reduction and DIC and
KLC1 after normalization with synaptophysin. Since the mean mRNAs level of KLC1, a
neuronal enriched form of KLC, was not significantly decreased, it also suggests that decrease
of KLC1 protein was not simply due to neuronal loss. Finally, it is possible that the reduction
of KLC1 and DIC in patients with AD results from a combination of reduced expression or
increased degradation of these proteins and of neuronal loss.
Expression of KLC1 variants might also play a role in susceptibility to development of
axoplasmic transport defects in AD. There is an alternative splicing of KLC1 which is the
basis of binding specificity with the cargo [30] and genetic variability of KLC1 has been
suggested to be a risk factor for early onset Alzheimer's disease [2, 14]. A change in the
expression of components of the kinesin or dynein complexes might also influence the
metabolism of tau and APP. For instance, a higher accumulation of A!40 and A!42 was
observed in mutant APP transgenic mice (TgAPPsw) crossed with KLC1wt/KLC1null mice
compared to single TgAPPsw mice [48]. In KLC1-/- mice, cytoskeletal disorganisation and an
accumulation of abnormally phosphorylated tau was also observed [19]. In neuroblastoma
cells, treatment with a siRNA against DIC induced abnormal localization and accumulation of
APP and tau [24].
We also observed an increased serine phosphorylation of KLC1 in AD frontal cortex. GSK3! plays a role in the regulation of kinesin-1 dependent fast anterograde transport by
phosphorylating KLCs, decreasing thereafter their association with cargoes and transport of
the latter [33]. We previously observed that GSK-3! negatively controls the movements of
mitochondria by increasing their pausing periods, depending of molecular motors activity
[32]. GSK-3! activity is increased in AD [26, 38] and thus this increased activity of GSK-3!
could directly impair kinesin-based transport by increasing the phosphorylation level of
KLC1 as we observed. Increased GSK-3ß activity could result from reduced negative
regulation by wnt and insulin signalling [22], by exposure to Aß [49] and from the action of
aggregated tau [25]. Oligomeric A! was also found to inhibit axonal transport in isolated
axoplasm, through casein kinase2 phosphorylation of KLC [41].
15
Interestingly, tau can interact with KLC1 and tau phosphorylation by GSK-3! increases its
association with kinesin and facilitates its transport, and might potentially induce an aberrant
axonal transport of tau [12]. We did not observe significant co-localization of KLC1 and DIC
immunoreactivity with tau immunoreactivity in NFT, suggesting that KLC1 and DIC do not
interact with the hyperphosphorylated tau protein in NFT, and were not trapped in NFT. NFT
are however composed of abnormally phosphorylated and aggregated PHF-tau, possibly less
prone to bind to kinesin.
In isolated axoplasm, recombinant tau filaments were reported to inhibit kinesin-dependent
anterograde transport through activation of GSK-3! [25], an effect mediated by exposure of a
phosphatase-activating domain in the aminoterminus of abnormally conformed tau,
independently of microtubule binding [23]. Our observation of increased KLC1 serine
phosphorylation in human samples containing high levels of PHF-tau proteins and increased
levels of active GSK-3ß is compatible with a mechanism in which abnormally conformed
PHF-tau proteins could drive inhibition of kinesin-dependent transport through KLC1
phosphorylation.
GSK-3! was previously found to phosphorylate KLC2 [17]. KLC2 is an ubiquitous form of
KLC whereas KLC1 is a neuronal enriched form of KLC [42]. Serine phosphorylation of
KLC1 was increased in the neuronal SH-SY5Y cells transfected with GSK-3ß and its active
form (and not with an inactive mutant). Pharmacological inhibition of GSK-3ß with
SB216763 also led to a decrease of KLC1 phosphorylation. Although the involvement of
other kinases is not completely excluded [3], these results nevertheless consistently suggest
that GSK-3ß is involved in phosphorylating KLC1 in SH-SY5Y cells.
These deficits in axoplasmic transport in AD might especially affect the transport of synaptic
vesicles and the integrity of synapses, dependent of an adequate transport of synaptic vesicles.
KLC1 has been detected in the synaptic fraction [42], suggesting that it contributes effectively
to the transport of synaptic vesicles. Several studies have indicated that changes in levels of
kinesin and dynein or in their transport abilities lead to a reduction of the transport of synaptic
vesicles. For instance, dissociation of KIF5B from tubulin-!3 led to a reduction in transport of
mitochondria and of vesicles containing synaptophysin [46]. A model of KIF1B knockout
mice showed reduced transport of synaptic vesicles [52]. A reduction of synaptophysin in AD
brain has been reported and interpreted as evidence for synaptic loss [20, 29]. We also
observed this reduction of synaptophysin in the frontal cortex of AD patients. The reduction
of KLCs and DIC in AD and increased serine phosphorylation of KLC1, leading to reduced
16
transport of synaptic vesicles, might thus contribute to the well-documented synaptic loss in
AD [29, 44].
In conclusion, our study indicates that the levels of KLCs and DIC are reduced and serine
phosphorylation of KLC1 is increased in AD frontal cortex and we suggest that these
phenomenoms contribute to the development of deficits of axoplasmic transport in this
disease.
17
Acknowledgements: This study was supported by the Interuniversity Attraction Poles
program (P6/43) of the Belgian Federal Science Policy Office (BELSPO), by the Diane
program (Walloon region), by grants from the Fonds de la Recherche Scientifique Médicale,
and the Fondation pour la Recherche sur la maladie d’Alzheimer/Stichting voor Alzheimer
Onderzoek. M. Morel was supported by a grant from the Van Buuren Foundation. The
authors thank Dr Doble (Mc Master University, Canada) for providing the GSK-3!-K85A
construct.
18
Legends of figures
Figure 1: KLC1 and DIC are decreased in AD frontal cortex but not in AD cerebellar cortex.
(a-c): Immunoblots of homogenates of the frontal cortex in 2 representative control cases and
2 AD patients with the anti-KLC1 (a), anti-DIC (b) and anti-actin (c) antibodies.
(d and e): Densitometry quantification (percentage of control) of KLC1 (d) and DIC (e)
immunoreactivities normalized with actin immunoreactivity in 5 control and 8 AD cases. A
significant decrease of KLC1 and DIC immunoreactivity was detected in AD frontal cortex.
(f-h): Immunoblots of homogenates of the cerebellar cortex in 2 representative control cases
and 2 AD patients with anti-KLC1 (f), anti-DIC (g), and anti-actin (h) antibodies.
(i-j): Densitometry quantification (percentage of control) of KLC1 (i) and DIC (j)
immunoreactivities normalized with actin, in control and AD subjects. KLC1 and DIC levels
are similar in control and AD subjects in the cerebellar cortex.
# p < 0.05 ; by Student t test.
Figure 2: Tubulin-!3, "-tubulin and synaptophysin, but not actin, are decreased in the frontal
cortex in AD.
(a-d): Immunoblots of homogenates of the frontal cortex in 2 representative control cases and
2 AD patients with antibodies to tubulin-!3 (a), synaptophysin (b), "-tubulin (c) and actin (d).
(e-h): Densitometry quantification (percentage of control) of tubulin-!3 (e), glycosylated
synaptophysin (f), and "-tubulin (g) immunoreactivity normalized with actin and of actin (h)
immunoreactivity in control and AD subjects. A decrease of tubulin-!3, of "-tubulin and of
synaptophysin, but not of actin, was detected in AD cases. # p < 0.05 ; by Student t test.
Figure 3: Densitometry quantification (percentage of control) of KLC1 (a and b) and DIC (c
and d) immunoreactivities in frontal cortex normalized with tubulin-!3 (a and c) or with
glycosylated synaptophysin (b and d) immunoreactivity. Levels of expression of KLC1 and
DIC are similar in control and AD cases when normalized with tubulin-!3 or synaptophysin.
Figure 4: qPCR quantification (percentage of controls) of mRNAs for KLC1, DIC and KLC2
in frontal cortex of control and AD subjects.
(a and b): KLC1 mRNAs levels normalized with actin (a) or HPRT (b) mRNAs.
(c and d): DIC mRNAs levels normalized with actin (c) or HPRT (d) mRNAs.
19
(e and f): KLC2 mRNAs levels normalized with actin (e) or HPRT (f) mRNAs.
The mean levels of these mRNAs are not significantly different between control and AD
cases.
Figure 5: KLC1 immunoreactivity is decreased in frontal cortex in AD.
Immunocytochemical labelling of paraffin tissue sections of the frontal cortex of a control
subject (a) and an AD subject (b) with the anti-KLC1 antibody. Neurons show a vesicular
KLC1 immunoreactivity that is decreased in AD patients.
Scale bar: 10 #m.
Figure 6: KLC1 and DIC immunoreactivities do not co-localize with tangles.
(a-c) Double immunofluorescence with anti-KLC1 (a) and anti-phosphotau (PHF-1) (b)
antibodies on cryostat tissue section of frontal cortex from an AD subject. The merged image
is shown in (c). The arrow in (a) and (c) points to a KLC1 positive vesicular structure in a
neuron containing a phosphotau positive tangle.
(d-f) Double immunofluorescence with anti-DIC (d) and anti-tau (B19) (e) antibodies on
cryostat tissue section of frontal cortex from an AD subject. The merged image is shown in
(f). The arrow in (d) and (f) points to a KLC1 positive vesicular structure in a neuron
containing a phosphotau positive tangle. DAPI counterstaining. Tangles do not show an
increased KLC1 or DIC immunoreactivity.
(g and h): Control sections incubated with secondary antibodies but without the anti-KLC1
(g) or the anti-DIC (h) antibodies.
Scale bar : 10 µm
Figure 7: GSK-3! phosphorylates KLC1 in SH-SY5Y cells.
(a): Immunoblotting analysis of SH-SY5Y cells transfected with EGFP (lane 1), with GSK3!-EGFP (lane 2), GSK-3!-S9A-EGFP (lane 3) or GSK-3!-K85A-EGFP (lane 4). Blots were
labeled with a total antibody to GSK-3!. The arrow points to the position of the transfected
proteins and the asterisk to the position of endogenous GSK-3!.
(b): Tau phosphorylation in transfected SH-SY5Y cells. (b1-2) Immunoblotting analysis of
SH-SY5Y cells transfected with EGFP, GSK-3!-EGFP, GSK-3!-S9A-EGFP or GSK-3!K85A-EGFP. Blots were labeled with the phosphorylation-independent B19 tau antibody (b1)
20
or the phosphotau antibody PHF-1 (b2). Tau phosphorylation is increased only in cells
transfected with GSK-3!-EGFP or GSK-3!-S9A-EGFP.
(c): Immunoprecipitation with an anti-KLC1 antibody followed by western blot with an antiphosphoserine antibody (c1) or with the anti-KLC1 antibody (c2). SH-SY5Y cells were
transfected with EGFP (lane 1), with GSK-3!-EGFP (lane 2), GSK-3!-S9A-EGFP (lane 3) or
GSK-3!-K85A-EGFP (lane 4). A control lane (lane 5) shows the absence of non-specific
binding of KLC1 when using protein A-sepharose beads alone without the anti-KLC1
antibody. Lanes 5: control lanes using using protein A-sepharose beads alone.
(c3)
Densitometry
quantification
(percentage
of
control)
of
anti-phosphoserine
immunoreactivity normalized with KLC1 immunoreactivity. An increase of KLC1
phosphorylation was observed in cells transfected with GSK-3!-EGFP or with GSK-3!-S9AEGFP. The level of phosphorylation of KLC1 in cells transfected with GSK-3!-K85A-EGFP
was similar to the level of phosphorylation in cells transfected with EGFP. ## p < 0.01 ; by
one-way ANOVA.
(d) Immunoprecipitation with an anti-KLC1 antibody followed by western blot with an antiphosphoserine antibody (d1) or with the anti-KLC1 antibody (d2). SH-SY5Y cells were
transfected with EGFP (lane 1) or with GSK-3!-EGFP (lanes 2 and 3), and treated with
DMSO (lanes 1 and 2) or with the GSK-3ß inhibitor SB216763. The latter treatment
decreases the phosphorylation of KLC1. Lanes 4: control lanes using using protein Asepharose beads alone.
Figure 8: Active GSK-3! is increased in AD frontal cortex and phosphorylation of KLC1 is
increased in AD frontal cortex.
(a and b): Immunoblots of homogenates of the frontal cortex with the pY279/Y216-GSK3"/! antibody (a) and the total GSK-3! antibody (b) in 2 representative control cases and 2
AD cases. The anti-pY279/Y216-GSK-3"/! antibody recognizes the active form of GSK-3"
when phosphorylated on Tyrosine 279 (50kD, indicated by an arrow) and active form of
GSK-3! when phosphorylated on Tyrosine 216 (47kD, indicated by an asterisk).
(c):
Densitometry
quantification
(percentage
of
control)
of
anti-pY216-GSK-3!
immunoreactivity normalized with total GSK-3! immunoreactivity in 5 control cases and 8
AD patients. An increase of pY216-GSK-3! reactivity was detected in AD brain.
(d): Immunoprecipitation of KLC1 from homogenates of frontal cortex in 2 representative
control cases (lanes 1 and 2) and 2 AD cases (lanes 3 and 4) followed by western blot with the
21
anti-phosphoserine antibody. A control lane (lane 5) shows the absence of non-specific
binding of KLC1 when using protein A-sepharose beads alone without the anti-KLC1
antibody.
(e) Immunoprecipitation of KLC1 followed by western blot with the anti-KLC1 antibody of
control and AD cases shown in (d).
(f)
Densitometry
quantification
(percentage
of
control)
of
anti-phosphoserine
immunoreactivity normalized with KLC1 immunoreactivity. An increase of KLC1
phosphorylation was observed in frontal cortex of AD patients # p < 0.05 ; by Student t test.
22
Table 1
Control
Cases
Age at
Sex
death
(years)
Post Mortem
interval (hours)
Stageing
Braak &
Braak
CERAD
plaque score
1
M
72
24
I - II
0
2
F
71
7
I - II
B
3
M
81
16,5
I - II
0
4
F
77
48
II-III
B
5
M
67
24
I - II
0
1
F
81
8
V - VI
C
2
F
73
22
V - VI
C
3
F
70
21
V - VI
C
4
F
76
20
V - VI
C
5
F
91
5,5
V - VI
B
6
M
84
7
V - VI
C
7
F
89
7
V - VI
C
8
M
75
10
V - VI
C
AD Cases
Age at
Summary
death
Post Mortem
(years)
interval (hours)
Sex
mean +/-
F/M
SD
mean +/- SD
Control
2/3
73,6 +/- 7,3
23,9 +/- 15,9
AD
6/2
79,9 +/- 7,6
12,6 +/- 7,1
Table 1: Summary of the characteristics of control subjects and AD patients. The
neuropathological stageing of AD patients is determined according to the Braak and Braak
stageing and the CERAD plaque score.
23
Supplementary Figures and tables
Supplementary Figure S1: Detection of PHF-tau proteins in subjects used in this study.
(a and b): Immunoblots of homogenates of the frontal cortex in control subjects (a) and in AD
patients (b) with an anti-phosphotau antibody (PHF-1). Three main bands between 50kD and
70kD (PHF-tau proteins) were observed in AD frontal cortex. Control cases did not show
these PHF-1 positive bands with the exception of one case (Fig. 1a, line 4) showing faintly
reactive PHF-1 bands. Numbers on the left of blots refer to the positions of molecular weight
markers: 36kD (Carbonic anhydrase), 50kD (alcohol dehydrogenase), 64kD (glutamic
dehydrogenase), 98kD (BSA).
Supplementary Figure S2: Absence of phosphosensitivity of the KLC1 antibody
Homogenates of frontal cortex of the same subjects were treated (b) or not (a) with lambda
phosphatase before immunoblotting with the anti-KLC1 antibody.
Lanes 1 and 2: control subject. Lanes 3 and 4: AD patient.
Prior dephosphorylation did not affect the level of labelling of KLC1 with the anti-KLC1
antibody.
Supplementary Table I
Genes
KLC1
KLC2
DIC
HPRT
Actin
Sense
5'-aggtctcgtaaacagggtcttg-3'
5'-agaaggtcctgggcaagttt-3'
5'-tggacctctggaacctcaac-3'
5'-agtctggcttatatccaacacttcg-3'
5'-ggatgcagaaggagatcactg-3'
Antisense
5'-gccactctcgtacttgaccac-3'
5'-gttcttggtcttggccacat-3'
5'-ggaactgcaagctctccaac-3'
5'-gactttgctttccttggtcagg-3'
5'-cgatccacacggagtacttg-3'
Sequences of primers used for qPCR amplification of mRNAs.
24
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