The Journal of Neuroscience, June 1, 2003, 23(11):4499-4508
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Alzheimer's Presenilin 1 Mutations Impair Kinesin-Based Axonal Transport
Gustavo Pigino,1
Gerardo Morfini,2
Alejandra Pelsman,1
Mark P. Mattson,3
Scott T. Brady,2 and
Jorge Busciglio1
1 Department of Neuroscience, University of Connecticut Health Center,
Farmington, Connecticut 06030,
2 Department of Cell Biology, University of Texas Southwestern Medical Center,
Dallas, Texas 75390-9039, and
3 Laboratory of Neurosciences, National Institute on Aging Gerontology Research
Center, Baltimore, Maryland 21224
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Abstract
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Several lines of evidence indicate that alterations in axonal transport
play a critical role in Alzheimer's disease (AD) neuropathology, but the
molecular mechanisms that control this process are not understood fully.
Recent work indicates that presenilin 1 (PS1) interacts with glycogen synthase
kinase 3
(GSK3). In vivo, GSK3 phosphorylates
kinesin light chains (KLC) and causes the release of kinesin-I from
membrane-bound organelles (MBOs), leading to a reduction in kinesin-I driven
motility (Morfini et al.,
2002b
). To characterize a potential role for PS1 in the regulation
of kinesin-based axonal transport, we used
PS1-/- and PS1 knock-inM146V
(KIM146V) mice and cultured cells. We show that relative levels of
GSK3 activity were increased in cells either in the presence of mutant
PS1 or in the absence of PS1 (PS1-/-).
Concomitant with increased GSK3 activity, relative levels of KLC
phosphorylation were increased, and the amount of kinesin-I bound to MBOs was
reduced. Consistent with a deficit in kinesin-I-mediated fast axonal
transport, densities of synaptophysin- and syntaxin-I-containing vesicles and
mitochondria were reduced in neuritic processes of KIM146V
hippocampal neurons. Similarly, we found reduced levels of PS1, amyloid
precursor protein, and synaptophysin in sciatic nerves of KIM146V
mice. Thus PS1 appears to modulate GSK3 activity and the release of
kinesin-I from MBOs at sites of vesicle delivery and membrane insertion. These
findings suggest that mutations in PS1 may compromise neuronal function by
affecting GSK-3 activity and kinesin-I-based motility.
Key words: Alzheimer's disease; presenilin; GSK3; kinesin; axonal transport; growth cones
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Introduction
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Most early-onset familial Alzheimer's disease (FAD) cases are associated
with mutations in presenilin 1 (PS1) and presenilin 2 (PS2;
Levy-Lahad et al., 1995;
Sherrington et al., 1995).
Presenilins are integral membrane proteins with eight transmembrane domains
and a hydrophilic loop between transmembrane domains 6 and 7
(Doan et al., 1996;
Li and Greenwald, 1998). In
neurons the presenilins localize in the endoplasmic reticulum (ER),
intermediate compartment, nuclear membranes, and growth cones
(Busciglio et al., 1997;
Capell et al., 1997;
Lah et al., 1997;
Annaert et al., 1999;
Pigino et al., 2001).
Presenilins undergo endoproteolytic cleavage, generating stable N- and
C-terminal fragments (NTF and CTF) that interact with other proteins to form a
macromolecular complex containing the 
-secretase activity that is
responsible for the regulated intramembrane proteolysis of the amyloid
precursor protein (APP), Notch 1, ErbB-4, and E-cadherin
(Ebinu and Yankner, 2002;
Marambaud et al., 2002).
Glycogen synthase kinase 3 (GSK3) is a component of the WNT
signaling pathway with a role in cell proliferation, differentiation,
adhesion, microtubule dynamics, apoptosis, and fast axonal transport
(Frame and Cohen, 2001;
Morfini et al., 2002b).
GSK3 is inactivated by phosphorylation at serine 9 (Ser9) in its N
terminus (Woodgett, 1994;
Frame and Cohen, 2001;
Frame et al., 2001). The
phosphorylation and dephosphorylation of Ser9 operate as a primary switch to
regulate GSK3 kinase activity. GSK3 can inhibit itself by
autophosphorylation (Wang et al.,
1994) or be inhibited by other kinases that phosphorylate Ser9
(Woodgett, 1994). In
vivo substrates for GSK3 include PS1, -catenin, glycogen
synthase, tau, and kinesin-I light chains
(Frame and Cohen, 2001;
Morfini et al., 2002b).
GSK3 interacts with PS1 heterodimeric complexes and
coimmunoprecipitates with PS1 in cell lines
(Takashima et al., 1998;
Tesco and Tanzi, 2000).
GSK3 binds to the hydrophilic loop of PS1
(Takashima et al., 1998),
phosphorylates PS1 at Ser397 within the loop domain, and regulates
the degradation of PS1 CTF (Kirschenbaum
et al., 2001). Previous results indicate that FAD-linked PS1
mutations affect GSK3 kinase activity in transfected cell lines
(Takashima et al., 1998;
Weihl et al., 1999). These
observations suggest the possibility of a molecular and functional interaction
between PS1 and GSK3 that is deregulated by PS1 mutations.
Recently, Morfini et al.
(2002b) showed that
phosphorylation of kinesin-I light chain (KLC) by GSK3 promotes the
release of kinesin-I from membrane-bound organelles (MBOs), leading to a
reduction in fast anterograde axonal transport. Retrograde transport is not
affected, and anterograde transport of vesicles is reduced, but not abolished,
indicating that GSK3 regulates anterograde trafficking of MBOs
associated with specific kinesin isoforms
(Morfini et al., 2002b). These
results suggest that the temporal and spatial regulation of GSK3 in
neurons is an important element in the control of neuronal membrane
trafficking. Relevant pathways for this regulation may involve proteins that
interact with microtubules (MTs), that bind to membranes or membrane proteins,
or that are components of signaling pathways
(Ratner et al., 1998), all of
which are characteristics of PS1. Taken together, the above data raise the
possibility that interactions between PS1 and GSK3 may play a role in
the regulation of vesicle transport and/or delivery. To test this possibility,
we examined the effects on GSK3 activity of either eliminating PS1 or
introducing the human PS1 gene containing the M146V mutation, which causes a
very aggressive early-onset form of FAD.
PS1-/- and PS1 knock-inM146V
(KIM146V) mice and cultured cells were used to characterize the
effect of PS1 and PS1 mutations on the regulation of kinesin-based transport
in neurons. We show that PS1 mutations increase GSK3 activity and
kinesin-I phosphorylation, leading to reduced levels of kinesin-I bound to
MBOs. These observations suggest that PS1 mutations may compromise neuronal
function by deregulating GSK3 activity and kinesin-I phosphorylation,
leading to impaired transport and/or targeting of MBOs essential for neuronal
function such as synaptic vesicles and mitochondria.
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Materials and Methods
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Cell culture. Neuronal cultures were prepared from PS1 wild-type
(WT) or PS1 KI M146V (Guo et
al., 1999) mouse embryos at day 16 of gestational age. After
dissection the cortical or hippocampal tissue was incubated in 0.25% trypsin
in Hank's for 20 min at 37°C, followed by dissociation and plating of the
cell suspension in culture dishes or glass coverslips covered with
poly-D-lysine (500 µg/ml), at a density of 16,000 cells/cm
2 for immunocytochemistry or 53,000 cell/cm 2 for
biochemical analysis. The cultures initially were plated in DMEM plus 10%
iron-supplemented calf serum (HyClone, Logan, UT) for 2 hr and then switched
to defined media consisting of Neurobasal media plus N2 and B27 supplements
(Life Technologies, Grand Island, NY). NT2 cells were cultured as described
(Pleasure et al., 1992).
Fibroblast cultures were generated from E16 PS1 WT, KO, and KI
M146V embryos as described
(Taccioli et al., 1998).
Antibodies. The following antibodies (Ab) were used: PSN2, a
monoclonal Ab that recognizes the NTF of PS1
(Pigino et al., 2001); 2025, a
polyclonal Ab raised against a synthetic peptide corresponding to PS1 amino
acid residues 1–20 (Pigino et al.,
2001); AD3L, a polyclonal Ab that recognizes the PS1 C terminus
(AD3L) (Pigino et al., 2001);
anti--tubulin isotype III (clone SDL 310; Sigma, St. Louis, MO);
anti--tubulin (clone DM1B; Sigma); anti-GSK3 monoclonal Ab (BD
Transduction Laboratories, San Diego, CA); anti-GSK3 polyclonal Ab
(334–348; Calbiochem, San Diego, CA); anti-GSK3 phosphorylated at
serine 9 (GSK3-Pser9; Cell Signaling, Boston, MA), which recognizes the
inactive form of GSK3; anti-cytochrome c (clone 6H2.B4;
PharMingen, San Diego, CA); H2, a monoclonal Ab that recognizes kinesin-I
heavy chain (Pfister et al.,
1989); 63–90, a monoclonal Ab that preferentially recognizes
dephosphorylated kinesin-I light chains
(Stenoien and Brady, 1997);
kinesin-I ALL, a monoclonal Ab that recognizes KLC irrespective of its
phosphorylation state (Stenoien and Brady,
1997); anti-synaptophysin (SY38) and anti-APP (Alz-90; both from
Chemicon, Temecula, CA); anti-SNAP25 (Alomone Labs, Jerusalem, Israel); and
anti-syntaxin-I (BD Transduction Laboratories).
Immunofluorescence. Immunocytochemical staining was performed as
described (Pigino et al.,
2001). Briefly, cells were fixed for 30 min at 37°C in 4%
paraformaldehyde/0.12 M sucrose in PBS and permeabilized with 0.2%
Triton X-100 in PBS for 10 min. Then the cultures were blocked for 1 hr in 5%
normal goat serum in PBS and incubated overnight at 4°C in a humid chamber
with the primary Ab, followed by incubation with the appropriate secondary Abs
conjugated with Alexa fluoro-red and fluoro-green (Molecular Probes, Eugene,
OR). The fluorescence was visualized with a Zeiss LSM 510 confocal microscope
or an Olympus IX-70 inverted microscope.
Preparation of brain and sciatic nerve homogenates. Adult brains
from PS1 WT and KI M146V mice were removed, homogenized in 1 ml of
radio immunoprecipitation assay (RIPA) buffer
(Pigino et al., 2001), and
processed for Western blot analysis. Sciatic nerves were dissected from adult
mice as described (Kasa et al.,
2001). Briefly, the sciatic nerves were exposed and removed with
surgical scissors. Segments 2 cm in length were excised, placed in ice-cold
Hank's for 1 min, and processed for SDS-PAGE and Western blot.
Western blot and immunoprecipitation. Changes in protein
expression level and association of PS1 and GSK3 with vesicular
fractions were analyzed by Western blot as described
(Busciglio et al., 2002;
Grace and Busciglio, 2003).
Immunoprecipitation experiments were performed and controlled as described
(Pigino et al., 2001).
Preparation of MBO-enriched fractions and subcellular
fractionation. MBOs were obtained as described
(Tsai et al., 2000;
Morfini et al., 2002b).
Briefly, the cells were washed twice with warm PHEM buffer [containing (in
mM): 60 PIPES, 25 HEPES, 10 EGTA, and 2 MgCl2, pH 7.4]
and extracted for 4 min at 37°C with 0.05% Triton X-100 in PHEM
supplemented with 1 mM GTP, 10 µM Taxol, and a
mixture of protease inhibitors (Complete; Roche Bioscience, Palo Alto, CA).
Then the cells were fixed for immunofluorescence or harvested for Western blot
analysis.
Subcellular fractions were collected as described (Morfini et al.,
2001b,
2002b). Brains from adult mice
were homogenized in 5 ml of homogenization buffer [HB; containing (in
mM): 300 sucrose, 10 HEPES, pH 7.4, 5 EDTA, supplemented with
protease inhibitors]. Homogenates were spun at 12,500 x g to
eliminate cell debris, nuclei, and mitochondria. Supernatants were centrifuged
at 39,800 x g for 40 min to obtain a vesicle (V0) pellet. Then
the supernatant (S0) was recentrifuged for 40 min at 120,000 x
g to obtain a V1 pellet, and this supernatant (S1) was centrifuged
for 2 hr at 260,000 x g to obtain the V2 pellet. The remaining
supernatant was called cytosol. All vesicle pellets were resuspended in
homogenization buffer. Equal amounts of protein from each fraction were
processed for Western blot. Growth cone-enriched fractions were prepared as
described (Pfenninger et al.,
1983; Morfini et al.,
2002b). For phosphorylation assays the resuspended V1 vesicle
fractions were incubated with 1 mM ATP for 20 min at 37°C
(Morfini et al., 2002b). For
dephosphorylation experiments identical blotted membranes were blocked first
with 5% albumin at room temperature (RT) and then were incubated in
dephosphorylation buffer (10 mM HEPES, pH 8, 10 mM
MgCl2) with or without calf intestine alkaline phosphatase (200
U/ml; Calbiochem) for 1 hr at RT.
Transfection. Hippocampal neurons and NT2 neuronal cells were
transfected as previously described with the expression vectors PS1 wild-type
(WT) and PS1 bearing mutations M146V, I143T, and D9, respectively
(Pigino et al., 2001). To
control transfection efficiency, we used an enhanced green fluorescent protein
(EGFP) vector (Clontech, Palo Alto, CA). All constructs were cloned into
pcDNA3 (Invitrogen, Carlsbad, CA), and the identity and integrity of each
clone were confirmed by sequencing.
Cell viability and caspase activity assays. Neuronal viability was
assessed by a propidium iodide exclusion assay as previously described
(Busciglio and Yankner, 1995;
Grace et al., 2002;
Grace and Busciglio, 2003).
The activity of caspases 2, 3, 6, 8, and 9 was quantified in triplicate
cultures by the Apotarget Caspase Colorimetric Assay kit, following the
vendor's procedure (Biosource, Camarillo, CA).
Image analysis. Image analysis was performed as described
(Pigino et al., 2001;
Grace et al., 2002;
Grace and Busciglio, 2003).
For synaptic vesicle and mitochondrial density quantification the images of
immunostained neurons were visualized with an Olympus IX-70 inverted
microscope. Images were captured with a CCD camera (Spot, Diagnostic
Instruments, Sterling Heights, MI) driven by Spot image acquisition software
and analyzed with NIH Image software. The identity of cultures was coded to
avoid experimental bias. To determine the ratio of immunofluorescence (IF)
intensity in different subcellular compartments, we obtained confocal images
with a Zeiss LSM 510 confocal microscope, and ratio image analysis was
performed with the Metamorph/Metafluor software. Ratio analysis was performed
in identical volume areas, in the same cell, in at least 30 individual cells
per condition.
Statistical analysis. All experiments were repeated at least three
times, using different brain specimens or cultures derived from at least three
different embryos. Each individual experiment was performed in quadruplicate.
In most cases the data were analyzed by ANOVA, followed by post hoc
Student–Newman–Keuls test to make all possible comparisons. Data
were expressed as the mean ± SEM, and significance was assessed at
p < 0.05.
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Results
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Endogenous PS1 coimmunoprecipitates with GSK3 and colocalizes
with active GSK3 in growth cones
To characterize molecular interactions between PS1 and GSK3 in
primary neurons, we first analyzed the expression and localization of
endogenous PS1 and GSK3 in cultured hippocampal neurons. Western blot
analysis revealed that PS1 and GSK3 expression increased steadily during
neuronal development (Fig.
1A). Both proteins showed a marked enrichment in a MBO
fraction with neuronal maturation (Fig.
1A). Presence in this cellular fraction and resistance to
mild permeabilization are consistent with their association with membrane
organelles and cytoskeletal elements
(Pigino et al., 2001;
Morfini et al., 2002b). We
further analyzed the association of PS1 and GSK3 with MBOs by
subcellular fractionation. Three vesicle fractions and a soluble fraction were
obtained by differential centrifugation from mouse brain homogenates (see
Materials and Methods). As previously reported, GSK3 was highly enriched
in fractions V1 and V2 (Morfini et al.,
2002b), whereas PS1 was enriched in fractions V0 and V1
(Fig. 1B). A
significant amount of PS1 and GSK3 colocalized in V1 (100,000 x
g microsomal pellet), a subpopulation of MBOs, and both were enriched
in growth cone preparations (Fig.
1B). Consistent with previous studies
(Morfini et al., 2002b),
kinesin-I was concentrated in these same fractions also. GSK3 and PS1
coimmunoprecipitated from cell lysates of doubly transfected
CosPS1/GSK3 cells
(Fig. 1C). Similarly,
endogenous GSK3 and PS1 coimmunoprecipitated from mouse brain
homogenates (Fig. 1C).
Collectively, these results indicate that the cellular distribution of both
proteins overlaps in specific vesicular compartments, and the results provide
evidence for a molecular interaction between PS1 and GSK3 in nerve
tissue.
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Figure 1. Endogenous PS1 and GSK3 exhibit similar expression profiles during
neuronal development, partially colocalize in the same vesicular compartment,
and coimmunoprecipitate from brain homogenates. A, Western blot
analysis of MBO and soluble fractions during development of hippocampal
neurons in culture at 1, 3, and 10 d in vitro (DIV). The figure shows
samples of three different homogenates obtained at each time point. Note that
the expression pattern is similar for PS1 and GSK3. Both proteins are
highly enriched in the MBO fraction, particularly at 10 DIV. PS1 FL,
Full-length PS1; PS1 CTF, C-terminus fragment of PS1. The antibodies include
anti-GSK3 (334–348), 1:1000, and anti-PS1 (AD3L), 1:2000.
B, PS1 and GSK3 colocalize on a subpopulation of MBOs and are
enriched in a growth cone fraction. The Western blots show the localization of
PS1, GSK3, and kinesin-I heavy (KHC) and light (KLC) chains in three
vesicle fractions (V0, V1, and V2) and supernatant (Cyt). Significant amounts
of PS1 and GSK3 colocalize in V1 where kinesin-I is enriched. PS1 and
GSK3 are highly enriched in growth cone fractions (GCs). PS1 NTF,
N-terminus fragment of PS1. The antibodies include anti-PS1 (PSN2), 1:1000;
anti-KHC (H2), 1:2000; anti-KLC (63–90), 1:2000; and anti-KLC ALL,
1:2000. C, Coimmunoprecipitation of PS1 and GSK3 from Cos cell
lysates cotransfected with PS1 and GSK3 expression vectors (Cos Lys) and
mouse brain homogenates (Brain Lys). A sample of transfected Cos cell lysates
was included as a positive control for GSK3 staining (Lys). The Cos cell
lysate that was immunoprecipitated with anti-PS1 antibody (Anti-PS1) shows
immunoreactivity with anti-GSK3 in the Western blot. Similarly, anti-PS1
immunoprecipitate from mouse brain homogenate shows immunoreactivity for
GSK3. No GSK3 immunoreactivity is observed in immunoprecipitates
for nonimmune mouse IgG (Ctrl IgG) or protein A-Sepharose beads alone (Beads).
Immunoprecipitations were performed with anti-PS1 antibody PSN2. Western blot
was revealed with anti-GSK3 (Calbiochem).
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Analysis of PS1 and GSK3 localization in hippocampal neurons by IF
and confocal microscopy indicated that PS1 and GSK3 were present in the
cell body, neuritic shaft, and growth cones of cultured neurons
(Fig. 2A,B). Both
proteins were present after mild detergent extraction, which selectively
retains membrane-associated proteins, consistent with subcellular
fractionation studies (Fig.
2A,B, arrows). At higher magnification GSK3 and PS1
showed extensive colocalization in the central area of the growth cone as well
as in individual filopodia of extracted cells
(Fig. 2C–E,
arrows). Ratio image analysis showed maximum overlap between PS1 and
GSK3 in growth cones, strongly suggesting localization of both antigens
in the same subcellular structures (Fig.
2F, white pseudocolor). Thus membrane-associated PS1 and
GSK3 colocalize and are enriched in the growth cone, a neuronal
compartment in which active membrane delivery and insertion take place during
neurite extension.
FAD-linked PS1 mutations increase GSK3 activity, KLC
phosphorylation, and release of kinesin-I from MBOs
To evaluate the possibility of a role for PS1 in the regulation of
GSK3 activity, we analyzed fibroblast cultures derived from PS1 WT,
PS1-/-
(Wong et al., 1997), and PS1
KIM146V (Guo et al.,
1999) mice. In PS1 KIM146V mice the endogenous
PS1 gene was replaced by a human PS1 gene bearing the M146V
mutation, which causes a very aggressive early-onset form of FAD. Western blot
analysis of PS1 WT, KIM146V, and
PS1-/- fibroblasts showed similar levels of
PS1 expression in PS1 WT and KIM146V cells
(Fig. 3A), because
expression of PS1 KIM146V is driven by the mouse PS1 endogenous
promoter. As expected, no PS1 immunoreactivity was detected in
PS1-/- cells
(Fig. 3A). A marked
increase in GSK3 activity was observed in KIM146V and
PS1-/- cells, as reflected by the significant
reduction in GSK3-Pser9 immunoreactivity
(Fig. 3A). In
contrast, no significant changes were detected in the level of total
GSK3, kinesin-I heavy chain (KHC), and tubulin
(Fig. 3A). Similarly,
expression of FAD-linked PS1 mutations M146V, I143T, and deletion of exon 9
(D9) in NT2 human neuronal cells significantly increased the activation of
GSK3, indicated by a reduction in GSK3-Pser9 immunoreactivity,
with no changes in total GSK3 level
(Fig. 3B). None of the
mutations that were analyzed caused neuronal death under the conditions of the
assay, as assessed by a propidium iodide exclusion assay
(Fig. 3C). This rules
out the possibility that GSK3 activation was secondary to a
neurodegenerative process triggered by the expression of mutant forms of
PS1.
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Figure 3. Increased activation of GSK3 in PS1
-/- and PS1 KI M146V fibroblasts
and neuronal cells expressing PS1 mutations M146V, I143T, and D9. A,
Protein expression analysis of PS1 wild-type (WT), PS1
-/- (KO), and PS1 KI M146V (KI)
fibroblasts. The figure shows samples of two different WT, KO, and KI
cultures. Reduced levels of GSK3-Pser9 (inactive form) were detected in
PS1 KO and KI as compared with WT fibroblasts. No changes in the expression
levels of total GSK3, kinesin-I heavy chain (KHC), and tubulin were
observed. Note the absence of PS1 in KO fibroblast and similar levels of PS1
expression in WT and KI fibroblasts. B, NT2 neuronal cells were
transfected with PS1 WT and PS1 mutant M143T, M146V, and D9 constructs. No
significant changes were detected in total GSK3 (GSK3 total) and
tubulin levels (data not shown). The histogram represents the amount of
GSK3-Pser9 expressed as a percentage of the level in control cells
(transfected with GFP). Values are the mean ± SE; n = 3
independent experiments. *p < 0.02 relative to GFP by Student's
t test. Transfection and quantitative Western blot analysis were
performed as described in Materials and Methods. C, Expression of PS1
mutations does not affect cell viability. Sister cultures to the ones used for
the experiment shown in B were used to assess cell viability. The
results showed no changes in cell survival associated with the expression of
PS1 mutations. Viability was assessed by using a propidium iodide exclusion
assay as described in Materials and Methods. At least 200 cells were scored
per culture in triplicate cultures. The histogram represents the mean ±
SE.
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To examine the effect of PS1 mutations on kinesin-I phosphorylation, we
used a phosphorylation-sensitive Ab, 63–90, against KLC N-terminal
(Stenoien and Brady, 1997).
This Ab preferentially recognizes dephosphorylated epitopes in KLCs (Morfini
et al., 2001a,
2002b). Western blot analysis
with Abs 63–90 (KLC) and H2 (KHC) of V1 vesicle fractions incubated with
or without ATP showed an increase in the apparent molecular weight of KLCs
after incubation with ATP (Fig.
4A). This shift is attributable to the phosphorylation of
KLCs by vesicle-associated kinase(s) and correlates with the detachment of
kinesin-I from membranes (Morfini et al.,
2002b). Immunoreactivity with 63–90, but not H2, antibody is
diminished significantly after incubation of the vesicles with ATP.
Dephosphorylation with alkaline phosphatase restores 63–90
immnunoreactivity, indicating that KLCs levels are not affected and consistent
with observations that 63–90 Ab immunoreactivity is sensitive to
phosphorylation (Fig.
4A). Western blot of the same fractions with anti-KLC ALL
Ab, which recognizes KLC irrespective of its phosphorylation state
(Stenoien and Brady, 1997),
confirmed that similar levels of KLC were present in all samples
(Fig. 4A). Western
blot analysis of brain homogenates revealed a significant reduction in the
immunoreactivity of KLCs detected by Ab 63–90 in KIM146V as
compared with WT brains (Fig.
4B), but no changes in protein levels of KHC, tubulin,
and GSK3 were observed (Fig.
4B). Analysis of PS1 WT and KIM146V cortical
culture homogenates showed a 49 ± 2% reduction (SEM; p <
0.01 by Student's t test) in 63–90 immunoreactivity of
63–90 for KIM146V cortical neurons
(Fig. 4C), with no
changes in levels of KLC, KHC, and tubulin
(Fig. 4C). A similar
result was observed in fibroblast from PS1-/-
and KIM146V mice (Fig.
4D) and with NT2 cell-expressing mutations M146V, I143T,
and D9 (data not shown). These experiments demonstrate increased
phosphorylation of KLCs in brain tissue and neurons expressing PS1 mutations
concurrent with the increased level of GSK3 kinase activity. To
establish the role of kinesin-I phosphorylation in the release of kinesin-I
from cargo vesicles, we performed a kinesin release assay
(Morfini et al., 2002b). KLC
and KHC were released from V1 vesicles (pellet) and were found in the
supernatant after incubation with ATP while the vesicle markers synaptophysin,
syntaxin-I, and APP remained in the pellet fraction
(Fig. 4E). Thus
kinesin-I is released from synaptophysin-, syntaxin-I-, and APP-containing
vesicles after phosphorylation by V1-associated kinase(s). Importantly,
63–90 immunoreactivity decreases after kinesin is phosphorylated and
detached from vesicles, similarly as observed with PS1 mutant cells and
tissues (Fig. 4).
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Figure 4. Enhanced KLC phosphorylation in PS1 KI M146V hippocampal neurons
and mouse brain. A, The 63–90 epitope on kinesin-I light chains
(KLC) is sensitive to phosphorylation. V1 vesicle fractions were incubated
without (Control) and with ATP. The samples were separated in an 8% gel and
transferred; immunoblots were prepared with the H2 (kinesin-I heavy chain) and
63–90 antibodies. Notice the increase in relative molecular weight of
KLCs after incubation with ATP. This shift is caused by the phosphorylation of
KLCs by vesicle-associated kinase(s) and correlates with the detachment of
kinesin-I from membranes (Morfini et al.,
2002b). Immunoreactivity with 63–90, but not H2, antibody is
diminished after the incubation of vesicles with ATP. When transfer membranes
are dephosphorylated with alkaline phosphatase (+AP), 63–90
immnunoreactivity is recovered, indicating that the epitope for the Ab
63–90 is sensitive to phosphorylation. The same fractions were separated
in a 4–20% gel, and Western blot was performed with the anti-KLC ALL
antibody, which recognizes KLC irrespective of its phosphorylation state.
Equal amounts of KLC were observed in all samples. B, C, Western blot
analysis of WT and PS1 KI M146V total brain homogenates and
cortical neuronal cultures. Samples from two WT and two KI brains and cortical
cultures are shown. Note the reduction in KLCs staining with Ab 63–90 in
PS1 KI M146V brain and cortical culture samples, indicating
increased phosphorylation of KLCs in PS1 KI brain and cortical culture
homogenates. Similar protein levels of KHC that have been detected with H2
antibody, which recognizes KHC, rule out changes in kinesin-I expression in
PS1 KI mouse brain tissue. Similar levels of tubulin and GSK3 rule out
general changes in protein expression. Similar levels of KLC immunoreactivity
with KLC (ALL) antibody also are observed in cortical cultures. D,
Western blot analysis of WT, PS1 KI M146V, and PS1 KO fibroblasts.
Note the reduction in Ab 63–90 immunoreactivity. E, Kinesin-I
release assay. V1 vesicles were incubated with 1 mM ATP for 30 min
at 37°C and centrifuged at 120,000 x g; pellet (P) and
supernatant (S) fractions were analyzed by Western blot. After incubation with
ATP (+) the KLC and KHC are released from the vesicle fraction (P) and found
in the supernatant while the vesicle markers synaptophysin (Syn) syntaxin-I
(Synt) and APP remain in the vesicle fraction (P). Omission of ATP (-) results
in the complete recovery of kinesin-I with the pellet fraction. Note the
reduction in Ab 63–90 immunoreactivity after kinesin-I light chain
phosphorylation.
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Perfusion of active GSK3 into squid axoplasm drastically reduces
anterograde transport of certain MBOs without affecting retrograde transport.
This inhibition in anterograde transport is associated with a release of
kinesin-I from MBOs, caused by increased phosphorylation of KLC by GSK3
(Morfini et al., 2002b). To
determine the effect of increased GSK3 activity and kinesin-I
phosphorylation on kinesin-I binding to MBOs in cells expressing PS1 mutants
or lacking PS1, we analyzed the association of kinesin-I with MBOs fractions
obtained from PS1 WT, KIM146V, and KO fibroblasts. Immunoblots
showed a marked reduction in the levels of KLC and KHC associated with
MBO-enriched fractions from KIM146V and KO as compared with WT
fibroblasts (Fig. 5A).
There were also significant reductions in levels of both KHC and KLC in
MBO-enriched fractions prepared from NT2 neuronal cell cultures expressing
M146V, I143T, and D9 mutations as compared with PS1 WT
(Fig. 5B). Thus there
is a clear reduction in the amount of kinesin-I associated with MBOs in cells
lacking PS1 or expressing PS1 mutations. These results indicate that a lack of
PS1 and PS1 mutations results in increased GSK3 activity, KLC
phosphorylation, and release of kinesin-I from MBOs.
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Figure 5. PS1 mutations enhance the release of kinesin-I from MBO-enriched fractions.
A, Western blot analysis of KHC and KLC in MBO-enriched fractions
from PS1 WT, PS1 KO, and PS1 KI M146V fibroblasts. KHC and KLC were
detected with antibodies H2 and 63–90, respectively. Samples of two
different cultures were assayed. Note the reduction in KHC and KLC in PS1 KI
and KO as compared with WT fibroblasts. Similar tubulin levels were observed
in all samples. These results suggest a reduced amount of kinesin-I associated
with MBOs in KI and KO fibroblasts. B, Western blot shows reduced
levels of both kinesin-I heavy and light chains (KHC and KLC) in MBO fractions
prepared from NT2 neuronal cell cultures expressing the PS1 KI
M146V mutation. Similar tubulin levels were detected in PS1 WT and
M146V. KHC and KLC were detected with antibodies H2 and 63–90,
respectively. The histogram shows the result of the quantification of KHC
levels in MBO-enriched fractions prepared from transfected cultures expressing
PS1 WT and PS1 mutants M143T, M146V, and D9. The amount of KHC in MBO-enriched
fractions was expressed as a percentage of the level in control cells
(transfected with GFP). Values are the mean ± SE; n = 3
independent experiments. *p < 0.01 relative to GFP by Student's
t test. KHCs were detected with antibody H2; similar results were
obtained for KLCs (data not shown). Tubulin levels did not change
significantly among samples. MBO fractions, transfection, and quantitative
Western blot analysis were performed as described in Materials and
Methods.
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Reduced APP and synaptophysin immunoreactivity in nerves and cultured
neurons from PS1 KIM146V mice
Synaptic vesicle precursors are transported anterogradely by kinesin motors
along the axons, and disruption of kinesin-based vesicle motility alters their
transport, leading to reduced synaptic density
(Ferreira et al., 1992;
Yonekawa et al., 1998;
Zhao et al., 2001). To analyze
the effect of PS1 mutations on synaptic vesicle trafficking, we examined the
density of synaptophysin- and syntaxin-I-containing vesicles in neuritic
processes of PS1 WT and KIM146V neurons. Both synaptophysin and
syntaxin-I are associated with synaptic vesicle membranes and are transported
in synaptic vesicle precursors (Okada et
al., 1995; Yonekawa et al.,
1998). Immunofluorescence analysis showed a diminished number of
synaptophysin- and syntaxin-I-containing vesicles in processes of PS1
KIM146V hippocampal neurons
(Fig. 6A). Quanti
tative analysis confirmed a significant reduction in the density of
synaptophysin-immunoreactive dots in KIM146V as compared with WT
hippocampal neurons (Fig.
6B); a similar result was obtained when the density of
syntaxin-I-containing vesicles was assessed (data not shown). WT and
KIM146V neurons exhibited similar viability and caspase activity
levels, ruling out a potential effect of neuronal cell death on vesicle
transport (Fig. 6C,D).
A comparable reduction in synaptophysin immunoreactivity was detected in
transfected hippocampal neurons expressing M146V, I143T, and D9 mutations, but
not PS1 WT or GFP (Fig.
6E). Thus there is a reduction in the number of synaptic
vesicle markers in neurites of primary neurons and neuronal cell lines
expressing PS1 mutations.
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Figure 6. Reduced density of synaptic vesicles in hippocampal neurons expressing PS1
mutations. A, Immunofluorescence with anti-synaptophysin (Syn) and
anti syntaxin-I (Synt) antibodies of PS1 WT and KI hippocampal neurons at 4 d
in culture. Note the reduced number of vesicle clusters (arrows) in KI
processes. The gray scale in the images was inverted for clarity. Scale bar,
20 µm. B, Reduced density of synaptophysin-containing vesicles in
PS1 KI M146V hippocampal neurons. WT and PS1 KI M146V
hippocampal cultures were fixed and double-immunostained with anti-tubulin
class III and anti-synaptophysin antibodies. The density of
synaptophysin-immunoreactive vesicles was assessed as described
(Grace et al., 2002). The
numbers represent the mean ± SE; n = 4 independent
experiments. *p < 0.05 relative to WT by Student's t
test. C, WT and PS1 KI M146V hippocampal neurons exhibit
similar viability. Neuronal viability was assessed at 4 DIV by a propidium
iodide exclusion assay. No significant differences in the number of viable
neurons were detected between WT and PS1 KI M146V cultures. At
least 200 neurons were scored per culture in triplicate cultures. The numbers
represent the mean ± SE. D, WT and PS1 KI M146V
hippocampal neurons exhibit similar caspase activities. No significant
differences in caspase activity were observed between WT and PS1 KI
M146V cultures. The activity of caspases 2, 3, 6, 8, and 9 was
measured in triplicate cultures. Caspase activity in PS1 KI M146V
cultures was expressed as a percentage after the activity of each caspase in
WT cultures had been normalized as 100. E, A significant reduction in
synaptophysin-immunoreactive vesicles was observed in hippocampal neurons
expressing PS1 mutations. Transfected neurons were identified by
cotransfection with a GFP expression vector
(Pigino et al., 2001). Images
of transfected neurons were captured at a final magnification of 400x,
and the number of synaptophysin-immunoreactive dots was scored. At least 30
cells were analyzed per experimental condition. The numbers represent the mean
± SE; n = 3 independent experiments. *p < 0.02
relative to control (GFP) by Student's t test.
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We also examined synaptic protein levels in PS1 KIM146V axons
in vivo. The protein levels of PS1, synaptophysin, and APP were
reduced markedly in sciatic nerves of KIM146V mice
(Fig. 7). In contrast, the
levels of tubulin and SNAP25, another synaptic protein, were similar in WT and
KIM146V sciatic nerves (Fig.
7). GSK3-Pser9 and 63–90 immunoreactivity levels were
reduced in both sciatic nerves and spinal cord samples. No significant changes
were observed in PS1, APP, synaptophysin, SNAP25, or tubulin expression in the
KIM146V spinal cord segments, where the cell bodies of neurons
projecting their axons into the sciatic nerve are located
(Fig. 7), suggesting that there
was no decrease in PS1, APP, or synaptophysin protein expression in
KIM146V neurons. These results are consistent with a deficit in
transport of a population of vesicle precursors and/or specific synaptic
proteins associated with PS1 mutations in vitro and in
vivo.
Reduced density of mitochondria in neurites of PS1 KIM146V
hippocampal neurons
Mitochondria are transported anterogradely by kinesin motors, and defects
in kinesin-I transport have been shown to reduce mitochondrial movement along
axonal processes (Tanaka et al.,
1998). We examined mitochondrial density in cell bodies and
neurites of PS1 WT and KIM146V hippocampal neurons by using an
antibody against the mitochondrial marker cytochrome c. The result
was a significant decrease in mitochondrial density in the neurites, but not
in the cell bodies, of KI neurons (Fig.
8), indicating that PS1 mutations also significantly reduce
mitochondrial transport into neuritic processes.
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Figure 8. Reduced mitochondrial density in neuritic processes of PS1 KI
M146V hippocampal neurons. A–D, Immunofluorescence
of PS1 WT and KI M146V (KI) hippocampal neurons stained with an
antibody against cytochrome c. Merged images of phase contrast and
fluorescence are shown. The gray scale in the images was inverted for clarity.
A, B, Representative WT and KI hippocampal neurons fixed at 2 DIV.
Note the presence of mitochondria in cell bodies and neuritic processes.
C, D, Higher magnification images of neuritic processes. Note the
presence of mitochondria along the processes. Scale bars: (in B)
A, B, 10 µm; (in D) C, D, 5 µm. E,
Assessment of mitochondrial density in neuritic processes and cell bodies of
PS1 WT and KI hippocampal neurons. A significant decrease in mitochondrial
density was observed in neuronal processes, but not in cell bodies, of KI
neurons. PS1 WT and KI hippocampal neurons were fixed at day 2 in culture. To
visualize mitochondria, we immunostained the cultures with a monoclonal
antibody specific for cytochrome c (1:500), an electron-transporting
mitochondrial resident protein. Mitochondrial density in neurites and cell
bodies was assessed by image analysis. The images were captured at a final
magnification of 400 x, and the numbers of mitochondria were scored in
the cell body and neuritic processes of randomly selected neurons as described
in Materials and Methods. At least 20 cells were analyzed per experimental
condition. The numbers represent the mean ± SE; n = 3
independent experiments. *p < 0.01 relative to WT by Student's
t test.
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Discussion
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These experiments provide strong evidence for molecular and functional
interactions between PS1 and GSK3. Based on results with PS1 null and
mutant models, PS1 appears to modulate the activity of GSK3 in neurons.
The absence of both PS1 and PS1 mutations associated with FAD increases
GSK3 kinase activity, leading to increased KLC phosphorylation and
release of kinesin-I from MBOs. Consistent with increased GSK3 kinase
activity, hyperphosphorylation of tau also is associated with PS1 mutations
(Takashima et al., 1998;
Pigino et al., 2001) and is a
hallmark of Alzheimer's disease (AD). There is a precedent for proteins that
bind GSK3 and limit its activity. Previous studies indicate that
regulation of GSK3 activity can be mediated by proteins that bind
GSK3. For example, GPB/FRAT bind GSK3 in vivo and inhibit
its kinase activity (Yost et al.,
1998; Freemantle et al.,
2002). Moreover, mutations in FRAT that lead to the deregulation
of GSK3 have been associated with pathogenesis in leukemia
(Yuan et al., 1999;
Saitoh et al., 2001).
Survival and proper function of neurons depend on the efficient delivery of
proteins from the cell body to axonal and dendritic processes. Kinesin-I
(Morfini et al., 2002b), PS1,
and the active form of GSK3 are enriched and colocalize in growth cones
(Figs. 1,
2) where vesicle delivery and
membrane insertion are essential for axonal elongation. This distribution is
consistent with functional interactions among these proteins that are
important in the transport of membrane proteins to neurites. Disruption of
these functional interactions would be expected to compromise the efficient
delivery of these cargoes. Axons in particular are highly susceptible to
transport deficiencies because they lack the elements necessary for protein
synthesis. In this context, defects in protein transport have been suggested
to play a critical role in AD and other neurodegenerative conditions
(de Waegh et al., 1992;
Sheetz et al., 1998;
Williamson and Cleveland,
1999; Goldstein,
2001; Morfini et al.,
2002a), and several studies describe a progressive loss of motor
function in AD patients (Sica et al.,
1998; Goldman et al.,
1999; Waite et al.,
2000; Pettersson et al.,
2002). A role for PS1 in protein trafficking was suggested
initially by the analysis of protein metabolism in PS1-defficient cortical
cultures (Naruse et al.,
1998). Changes in membrane trafficking in
PS1-/- neurons include accumulation of APP
and APLP1 (a neuron-specific homolog of APP) C-terminal fragments and a
dramatic slowdown in the transit and maturation of TrkB receptors. PS1
mutations also alter intracellular trafficking of -catenin
(Nishimura et al., 1999),
leading to suggestions that PS1 is a component of the machinery involved in
the selection and sorting of cargo exiting the ER. However, alterations in the
delivery of membrane proteins are also consistent with changes in
kinesin-based motility in PS1-/- neurons (see
below), and these two possibilities are not mutually exclusive.
Impaired synaptic vesicle transport, reduced synaptic density, and altered
synaptic function occur in various experimental systems with altered kinesin-I
function. Synaptic function and density are affected specifically by kinesin-I
mutants in Drosophila (Hurd and
Saxton, 1996). Further, mutation in a kinesin-I gene has been
associated with spastic paraplegia in humans
(Reid et al., 2002).
A number of membrane proteins or MBOs exhibit altered distribution in the
PS1 KIM146V and KO cells. Mutations in both PS1 and PS2 reduce
levels of secreted APP in the conditioned media of cultured cells
(Ancolio et al., 1997;
Marambaud et al., 1998),
further suggesting that alterations in protein transport are associated with
the expression of presenilin mutations. PS1 has been found to undergo fast
axonal transport and to accumulate in ligated sciatic nerves
(Kasa et al., 2001), and
axonal transport of APP is mediated by kinesin-I
(Ferreira et al., 1993).
Axonal trans port of APP is decreased greatly in a gene-targeted mouse mutant
of the neuron-enriched kinesin light chain-1 gene
(Kamal et al., 2000).
Similarly, Kamal et al. (2001)
reported the presence of PS1 in cargo vesicles containing APP, BACE, and
kinesin-I. Thus APP and the secretases that generate amyloid (A)
appear to be transported in the same vesicular structures. The decreased
density of synaptic vesicles or their precursors observed in hippocampal
neurons expressing PS1 mutations (Fig.
6) is consistent with a reduction in their transport. This
possibility is favored by a marked decrease in the level of two synaptic
proteins, synaptophysin and APP, in sciatic nerves of KIM146V mice.
There was also a significant reduction in the levels of APP and synaptophysin
in PS1 KIM146V mouse sciatic nerves, but not in the spinal cord,
where neuronal cell bodies reside (Fig.
7).
Transport of other membrane proteins or organelles also may be altered by
the inhibition of kinesin-I-based transport. Previous reports indicate that
mitochondria are transported anterogradely by kinesin-I motors and that
disruption of mouse KHC by gene targeting leads to decreased mitochondrial
density in neuronal processes (Tanaka et
al., 1998). Reduced transport of mitochondria may lead to
depletion of ATP in axonal processes and synapses. In this regard, a number of
studies have shown impaired energy metabolism in the AD brain
(Fiskum et al., 1999;
Hirai et al., 2001;
Valla et al., 2001).
Similarly, mitochondrial density was reduced in neuronal processes, but not
cell bodies, of PS1 KIM146V neurons. Finally, a significant
reduction was seen in the amounts of kinesin-I bound to membrane structures
from neurons and fibroblasts from PS1 KIM146V mice. A model in
which misregulation of GSK3 activity by PS1 mutations causes premature
release of kinesin-I cargo or impaired transport of MBOs such as mitochondria
could account for all of these changes
(Morfini et al., 2002b). In
the Alzheimer's brain an initial misregulation of transport might lead to a
progressive increase in A production and accumulation in the axon and,
potentially, to a more generalized disruption and blockage of protein
transport. Thus in AD various pathological processes involving PS1, APP,
A, and tau may converge to produce a deleterious effect on axonal
transport and neuronal homeostasis.
Changes in kinase activities and phosphorylation patterns specifically
implicate GSK3 in different pathological conditions
(Mandelkow et al., 1992;
Eldar-Finkelman et al., 1999;
Summers et al., 1999). In
particular, a role for GSK3 in AD pathology is suggested by the fact
that GSK3 phosphorylates tau to produce AD-like immunoreactivity
(Mandelkow et al., 1992;
Mulot et al., 1994;
Wagner et al., 1996) and that
GSK3 expression levels are altered in the Alzheimer's brain
(Baum et al., 1996). The
discovery that GSK3 plays a role in regulating kinesin-based motility
suggests that axonal transport may be a vulnerable step in AD pathogenesis
(Morfini et al., 2002a).
Evidence that PS1 mutations produce a misregulation of GSK3 similar
to the one observed in PS1-/- cells suggests
that PS1 mutations are responsible for a loss of ability to modulate
GSK3. In this regard, all three PS1 mutations that were examined induced
similar increases on GSK3 activity. In contrast, the effect of different
FAD-linked PS1 mutations on -secretase activity appears to differ,
promoting either increased A42 production
(Borchelt et al., 1996) or
reduced A generation (Amtul et al.,
2002). Various experimental and clinical phenotypes associated
with different PS1 mutations may be the cumulative result of altered PS1
function on multiple cellular pathways, including -secretase activity
(Ebinu and Yankner, 2002),
regulation of intracellular calcium levels
(Mattson et al., 1998),
regulation of cell cycle protein expression
(Janicki et al., 2000), and
kinesin-based transport.
In summary, these observations suggest that PS1 affects kinesin-based
axonal transport, a phenomenon that correlates with local deregulation of
GSK3 activity. Thus PS1 mutations may compromise neuronal function by
altering kinesin-based transport mechanisms, including membrane protein
transport in the secretory/endocytic pathways and fast anterograde axonal
transport. The consequent reductions in efficiency of axonal transport in
affected neurons would make them more vulnerable and ultimately lead to
neurodegeneration.
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Footnotes
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|---|
Received Jan. 27, 2003;
revised Mar. 4, 2003;
accepted Mar. 12, 2003.
This work was supported by grants from the Alzheimer's Association, The
Patterson Trust, and the National Institutes of Health (HD38466) to J.B. We
are grateful to Dr. Sam Sisodia (University of Chicago) for providing PS1
+/- breeders.
Correspondence should be addressed to Jorge Busciglio, Department of
Neuroscience, University of Connecticut Health Center, 263 Farmington Avenue,
Farmington, CT 06030. E-mail:
busciglio{at}nso1.uchc.edu.
Copyright © 2003 Society for Neuroscience
0270-6474/03/234499-10$15.00/0
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Top
Abstract
Introduction
