Department of Neuroscience, King's College London, Institute of Psychiatry, De Crespigny Park, London, SE5 8AF, UK
* Author for correspondence (e-mail: b.anderton{at}iop.kcl.ac.uk)
Accepted 22 June 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Tau, -Synuclein, Axonal transport, Kinesin, Dynein, Glutathione S-transferase
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
-Synuclein is a pre-synaptic protein involved in maintenance of synaptic integrity and function and regulation of dopamine synthesis (Abeliovich et al., 2000
; Perez et al., 2002
; Cabin et al., 2002
; Chandra et al., 2004
). In vivo studies have shown that the majority of
-synuclein moves along axons in the slow component with the remainder travelling in the fast component of axonal transport (Jensen et al., 1998
; Jensen et al., 1999
). Aggregates of
-synuclein are present in brain in Parkinson's disease (Polymeropoulos et al., 1997
; Mezey et al., 1998
; Kruger et al., 1998
), dementia with Lewy bodies (Spillantini et al., 1997
), and 60-70% of AD cases (Mukaetova-Ladinska et al., 2000
; Hamilton, 2000
). Dysfunctional axonal transport has also been proposed as a mechanism involved in the pathogenesis of Parkinson's disease (Lach et al., 1992
). The co-occurrence of tau and
-synuclein aggregates in neurodegenerative diseases (Spillantini et al., 1998
; Iwai, 2000
; Mukaetova-Ladinska et al., 2000
; Hamilton, 2000
; Masliah et al., 2001
; Popescu et al., 2004
), and enhancement of tau pathology by
-synuclein (Giasson et al., 2003
), suggests that there may be a link between these two pathologies.
The paucity of protein synthesising machinery within axons means that neurons rely on an efficient axonal transport system and hence may be especially vulnerable to altered regulation of axonal transport. We have investigated the axonal transport of tau and -synuclein in neurons because defective transport has been implicated as a possible mechanism involved in diseases that exhibit tau and
-synuclein pathology. We found that both tau and
-synuclein can travel at speeds in the range of fast axonal transport. We show for the first time that the fast anterograde motor kinesin-1 may be involved in transport of both tau and
-synuclein, and this is supported by our finding of a direct interaction between tau and kinesin-1. Furthermore, we found that
-synuclein is associated with the retrograde motor dynein. These observations further our understanding of the mechanisms involved in tau and
-synuclein axonal transport, which may be important factors in development of neurodegenerative disease.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies
The rabbit polyclonal -synuclein antibody,
90, was raised against peptide C-AATGFVKKDQMGK, corresponding to amino acids 90-102 of rat
-synuclein with the addition of an N-terminal cysteine) conjugated to PPD with glutaraldehyde (Totterdell et al., 2004
). The rabbit polyclonal
-synuclein antibody,
C, was raised against a peptide C-EGYQDYEPEA, corresponding to amino acids 131-140 of human
-synuclein. For western blotting, mouse anti-kinesin-1 monoclonal antibody (MAB1614) and mouse anti-dynein, cytoplasmic (74 kDa intermediate chain) monoclonal antibody (MAB1618) were purchased from Chemicon International Inc., Temecula CA, USA. For immunofluorescence microscopy, mouse anti-kinesin-1 antibody (ab9097) was purchased from Abcam, Cambridge, UK. The monoclonal antibody to
-tubulin (DM1A) was purchased from Sigma Chemical Co., Poole, UK. The rabbit anti-human tau antibody (code no. A0024) was purchased from DAKO, Glostrup, Denmark. Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Amersham Biosciences UK Ltd, Little Chalfont, UK. Secondary antibodies linked to fluorescein isothiocyanate (FITC) or tetramethyl rhodamine isothiocyanate (TRITC) were purchased from Cambridge Biosciences, Cambridge, UK.
Purification of recombinant tau and -synuclein
Recombinant tau (2N4R isoform) was purified as previously described (Scott et al., 1991; Utton et al., 1997
). Samples were used from the pre-Mono-S purification stage.
Transfection of cortical cultures
DNA for transfection was prepared by an endotoxin-free maxiprep kit (Qiagen, Crawley, UK). Cortical neurons were obtained from embryonic day 18 (E18) rat embryos, cultured and transfected as previously described (Ackerley et al., 2000).
Nocodazole and cytochalasin treatment of transfected neurons
Neurons were transfected with either 0N4Rtau-EGFP or -synuclein and were treated 3 hours after transfection with either 5 µg/ml nocodazole (Tocris, Bristol, UK) or 25 µM cytochalasin B (Sigma Chemical Co., Poole, UK). Neurons were fixed 0, 30, 60 and 90 minutes following treatment. The
-synuclein transfected neurons were immunostained using
90. The analysis of overall slow axonal transport of tau-EGFP and
-synuclein was carried out as previously described (Ackerley et al., 2000
; Utton et al., 2002
). The distance travelled by the exogenous protein was measured from the perimeter of the cell body along the axon to the limit of the fluorescent front. Neurons were immunostained for tubulin using DM1A and FITC-conjugated secondary antibody and for actin using phalloidin-TRITC to ensure that breakdown of the microtubule and actin cytoskeletons, respectively, had occurred under the experimental conditions.
Live microscopy of neuronally expressed tau-EGFP and -synuclein-EGFP
Rat cortical neurons were transfected with either 0N4R tau-EGFP or -synuclein-EGFP. Live image analysis of neurons was performed 48 hours after transfection (Ackerley et al., 2003
) and images were collected at 0.5-2.5 second intervals for 60-200 seconds. The kinetic parameters of moving fluorescent structures were analysed by manual tracking using MetaMorph software. Fluorescent structures were judged to be moving if they were displaced by more than 0.3 µm between consecutive images, this value was determined empirically by manually determining the average apparent `movement' of stationary fluorescent structures in the neurons. Displacement of less than 0.3 µm was considered most likely to be due to fluctuations in the conditions surrounding the cells rather than being due to axonal transport. Pause times were measured only for those structures that moved, paused and moved off again during the observation time.
Immunofluorescence labelling of neurons
Rat cortical neurons were fixed in 4% (w/v) paraformaldehyde/phosphate-buffered saline (PBS), permeabilised in 0.5% (v/v) Triton X-100 in PBS for 10 minutes and non-specific sites were blocked with 1% (w/v) bovine serum albumin (BSA) in PBS. Neurons were immunostained for tau using the rabbit polyclonal antibody (DAKO) and for -synuclein with
90. For co-localisation of tau and
-synuclein with kinesin-1, neurons were co-stained for kinesin-1 using a monoclonal antibody (Abcam, ab9097). Transfected neurons were immunostained 48 hours after transfection. Images were collected using a Zeiss Axiovert 200M microscope with Zeiss LSM 5 image software.
Immunoprecipitation
Adult rat brains or rat cortical cultures were homogenised in RIPA buffer (20 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol-bis (2-aminoethyl)-N,N,N',N'-tetraacetic acid, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 1% (v/v) Triton X-100, 0.1% (w/v) sodium dodecyl sulphate, 0.5% (w/v) sodium deoxycholate, Complete protease inhibitor (Roche, Welwyn Garden City, UK)), incubated on ice for 30 minutes and centrifuged at 100,000 g for 30 minutes at 4°C. The supernatant was removed and protein content was assayed (BioRad Protein Assay, Hemel Hempstead, UK). For immunoprecipitation, 1-5 µl rabbit antibody against human tau (DAKO, Glostrop, Denmark) or 16 µl rabbit antibody against -synuclein (
90) were added to the supernatant, and immunoprecipitates were isolated by the addition of protein A/G agarose (Amersham Biosciences UK Ltd, Little Chalfont, UK). Immunoprecipitates were washed three times with RIPA buffer and associated proteins were detected by immunoblotting the precipitates with either mouse anti-kinesin-1 monoclonal antibody or mouse anti-dynein, cytoplasmic (74 kDa intermediate chain) monoclonal antibody.
Purification of GST fusion proteins
The GST purification method was based on the manufacturer's instructions (Amersham Biosciences UK Ltd, Little Chalfont, UK). Expression of GST and GST-fusion proteins was induced with 0.5 mM isopropyl ß-D-1-thiogalactopyranoside for 2-3 hours. Bacterial pellets were resuspended in PBS containing Complete protease inhibitor and sonicated. Triton X-100 was added to a final concentration of 1% (v/v) and the suspension was incubated for 30 minutes at 4°C under constant agitation. Following centrifugation at 10,000 g for 10 minutes at 4°C the resulting supernatant, containing GST-fusion proteins, was incubated with glutathione Sepharose 4B for 1 hour at 4°C under constant agitation. GST-Sepharose beads were collected by centrifugation at 500 g for 1 minute at 4°C and washed three times by re-suspension and centrifugation with PBS containing Complete protease inhibitor. GST-protein was quantified by comparison with standard amounts of bovine serum albumin on Coomassie-Blue-R-stained polyacrylamide gels.
GST-binding assay
Purified GST-KLC proteins (10 nM) bound to glutathione Sepharose 4B beads were incubated with adult rat brain homogenate or purified recombinant tau (10 nM) in modified RIPA buffer (mRIPA) (20 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid, 0.2 mM PMSF, 1% (v/v) Nonidet 40 (NP40), Complete protease inhibitor). The GST-Sepharose beads were pelleted at 500 g for 1 minute at 4°C, the beads were washed three times with mRIPA buffer and resuspended in sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE) sample buffer containing 40 mM dithiothreitol. GST-bound proteins were analysed on western blots with antibodies to kinesin-1, dynein or tau.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The kinetic parameters of particles containing tau-EGFP or -synuclein-EGFP were determined using MetaMorph image analysis software (Table 1). These results show some similarities in the movement characteristics of tau-EGFP and
-synuclein-EGFP-containing structures, for example, the time spent pausing was approximately 70-80% for both proteins. The mean rate of transport between pauses for tau-EGFP structures was 31.4 mm/day, thus resembling that of neurofilaments at
45 mm/day (Wang et al., 2000
; Roy et al., 2000
; Wang and Brown, 2001
) and peripherin at 27 mm/day (Helfand et al., 2003
). The range of transport rates determined for tau-EGFP-containing structures, 14-124 mm/day, is also similar to that reported for peripherin (7-129 mm/day). When the tau-EGFP-containing structures did move they did so in an anterograde direction for 57% of their motile time and in a retrograde direction for 43% of their movement time.
-Synuclein-containing structures moved in an anterograde direction for 48% of the movement time and in a retrograde direction for 52% of the movement time. The mean rate of transport of
-synuclein-containing structures was much faster than tau at 161 mm/day and the range of transport rates also differed at 111-645 mm/day. These results suggest that different molecular mechanisms might be involved in the transport of
-synuclein compared to that of cytoskeletal proteins, such as tau and neurofilaments.
|
Tau-EGFP and -synuclein-EGFP containing structures colocalise with kinesin-1
Since both tau-EGFP and -synuclein-EGFP-containing structures were observed moving at rates comparable with fast transport, we determined whether known fast motors, such as kinesin-1, can be involved with this movement, because it has been previously shown for proteins such as peripherin (Helfand et al., 2003
). Co-localisation with kinesin-1 was investigated in rat cortical neurons transfected with tau-EGFP or
-synuclein-EGFP, fixed after 48 hours and immunostained for kinesin-1 (Fig. 3). A proportion of the structures containing tau-EGFP or
-synuclein-EGFP were identified by laser confocal immunofluorescence microscopy as being associated with kinesin-1 (Fig. 3, arrows). Not all of the tau- or
-synuclein-containing structures colocalised with kinesin-1 but, since the majority of the structures are paused at any given time, this observation was to be expected.
|
Tau and -synuclein both co-immunoprecipitate with kinesin-1, whereas only
-synuclein co-immunoprecipitates with dynein
Since both tau-EGFP and -synuclein-EGFP exhibited co-localisation with kinesin-1, we next determined if tau or
-synuclein are present in complexes with known molecular motors of fast axonal transport, such as kinesin and dynein. Immunoprecipitation experiments from adult rat brain were conducted using antibodies recognising tau (Fig. 4, left panels) or
-synuclein (Fig. 4, right panels). Immunoprecipitates were analysed on western blots using antibodies to kinesin-1 (Fig. 4, upper panels) or dynein intermediate chain (Fig. 4, lower panels). Tau and
-synuclein co-immunoprecipitated with kinesin-1, the tau antibody bringing down
15% of total tau and
1% of total kinesin-1, and the
-synuclein antibody bringing down
60% of total
-synuclein and
1% of total kinesin-1. The co-immunoprecipitation of tau and kinesin-1 was not significantly affected by the addition of 500 mM NaCl, indicating that the interaction is robust (data not shown). Only
-synuclein co-immunoprecipitated with dynein intermediate chain, with
0.5% of total dynein intermediate chain co-immunoprecipitating under these experimental conditions. Thus, both tau and
-synuclein can exist in a complex with kinesin-1 in the adult rat brain. These results are in agreement with previous studies suggesting direct or indirect interactions between tau and kinesin (Jancsik et al., 1996
) and between
-synuclein and dynein (Zhou et al., 2004
).
|
|
Further assessment of the components of the kinesin-1 complex that interact with tau
Kinesin-1 light chains have been shown to bind many of the cargoes transported by kinesin, such as amyloid precursor protein (Kamal et al., 2000). To assess the ability of GST-kinesin-1 light chain proteins to interact with tau and
-synuclein, purified GST, GST-kinesin-1 light-chain 1 (GST-KLC1) or GST-kinesin-1 light-chain 2 (GST-KLC2) bound to glutathione-Sepharose beads was incubated with adult rat brain homogenate. Bound proteins were separated by SDS-PAGE and analysed on western blots with antibodies against tau (Fig. 6). In rat brain homogenate, tau appears as multiple bands representing different splice isoforms in various phosphorylation states (Fig. 6A). Multiple tau species were also detected in pull-downs from rat brain homogenate using either GST-KLC1 or GST-KLC2 (Fig. 6A). No tau immunoreactivity was observed in controls with purified GST. These experiments indicate that tau exists in a complex with kinesin-1 but they do not address whether tau binds directly to kinesin-1 or whether this is an indirect interaction through other proteins. To investigate this, recombinant tau protein was incubated with purified GST, GST-KLC1 or GST-KLC2, and bound tau was detected on western blots. Significant binding of recombinant tau to both GST-KLC1 and GST-KLC2 was observed (Fig. 6B), with no detectable binding of tau to purified GST. These results indicate that tau can bind directly to both KLC1 and KLC2. By contrast, we could detect no binding of
-synuclein from rat brain homogenate or recombinant
-synuclein to GST-KLC1, or GST-KLC2 under these conditions (data not shown). These results serve as an additional control, indicating that the binding of tau to KLC1 and KLC2 is specific, and suggest that the association of
-synuclein with kinesin-1 in rat brain is mediated through additional proteins. Further analysis is necessary to determine the interaction of
-synuclein with potential accessory protein-components within a motor complex responsible for
-synuclein transport.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Other groups have demonstrated that the neurofilaments peripherin and tubulin, which like tau and -synuclein are transported principally in the slow component, exhibit rapid bidirectional movement of a sub-fraction with the bulk of the proteins remaining stationary during the period of observation (Prahlad et al., 1998
; Yabe et al., 1999
; Prahlad et al., 2000
; Wang et al., 2002; Helfand et al., 2003
; Brown, 2003
). The net transport rate is thus a summation of a small fraction moving rapidly in both directions for short periods while most of the protein is paused, i.e. the duty ratio determines the net transport rate (Brown, 2003
). We therefore undertook live-cell video-microscopy to investigate transport of tau and
-synuclein. Using video imaging, we were able to observe directly the movement of individual particles of tau and
-synuclein during the process of axonal transport in live neurons transfected with plasmids expressing fluorescently tagged proteins. In the case of both tau and
-synuclein, we detected structures that moved infrequently, but bi-directionally, along axons at rates consistent with fast transport; however, particle movement was interrupted by frequent pauses. By immunoprecipitation, we also found that tau and
-synuclein are each associated, either directly or indirectly with complexes containing the fast anterograde motor kinesin-1, and
-synuclein is also present in a complex with the fast retrograde motor dynein. We were unable to detect co-immunoprecipitation of dynein with tau under the experimental conditions used here. However, because we observed retrograde movement of tau in live neurons, it seems likely that tau does associate with factors controlling retrograde transport. We therefore cannot exclude the possibility that, compared to
-synuclein, there may be weak and/or transient interactions of tau with dynein or with dynein-linker proteins that are disrupted during immunoprecipitation. In the case of
-synuclein, it is of interest that recent reports have indicated an association of dynein with
-synuclein-containing inclusions (Zhou et al., 2004
; Hasegawa et al., 2004
). Notably, cellular aggregates of
-synuclein share some properties with aggresomes, these structures originate in the cytoplasm and subsequently form larger perinuclear inclusions after microtubule-based, dynein-mediated transport to the centrosome (Seo et al., 2002
; Tanaka et al., 2004
).
In this study, we observed marked differences in the relative associations of tau and -synuclein directly with molecular motors because tau interacts with a kinesin-1 complex binding to KLC1 and KLC2, whereas
-synuclein did not bind to either KLC1 or KLC2 under our experimental conditions. These results demonstrate that although both tau and
-synuclein are most probably transported through fast motors, there are clear differences between the mechanisms of their interactions with such motors, with tau being capable of directly binding to kinesin-1 whereas
-synuclein probably requires other accessory proteins to form a motile complex.
The rapid, yet infrequent, motility of tau-containing structures is consistent with reports of other non-membrane bound particles containing cytoskeletal proteins, such as neurofilaments (Prahlad et al., 2000), vimentin (Prahlad et al., 1998
), peripherin (Helfand et al., 2003
) and tubulin (Wang and Brown, 2002
), that also display this behaviour and move rapidly along microtubules. These recent findings show that cytoskeletal proteins, which were previously thought to be transported only in the slow component of axonal transport, are probably moved by fast motors including kinesin and dynein (Yabe et al., 1999
; Yabe et al., 2000
; Shah et al., 2000
; Wang et al., 2002; He et al., 2005
; Theiss et al., 2005
). Other studies of neurofilament transport show that, despite these rapid movements, neurofilaments spend
80% of the time in a paused state and hence their net overall rate of transport is slow (Wang et al., 2000
; Roy et al., 2000
; Wang et al., 2001; Brown, 2003
). Similarly, we found here that tau was paused for 73% of the time. The kinetic parameters of EGFP-tau movement in neurons bear some similarity to those of neurofilaments and peripherin. In particular, the reported velocity between pauses for EGFP-tau ranges from 14-124 mm/day, which is comparable to transport of neurofilaments at 2-160 mm/day (Wang and Brown, 2001
), indicating the existence of similar mechanisms for the transport of cytoskeletal components.
The rapid movement of -synuclein is also consistent with earlier reports showing that a proportion of synuclein can move in the fast component of axonal transport (Jensen et al., 1999
). Since
-synuclein can be associated with rat brain vesicles (Jensen et al., 1998
), it is not surprising that its transport parameters differ from those of cytoskeletal proteins. In particular, the average velocity of
-synuclein-containing particles (160 mm/day) is considerably faster than that of tau-containing structures (30 mm/day), further suggesting that different mechanisms, for example variable association with specific molecular motors, might underlie the regulation of
-synuclein and tau axonal transport. The variation in transport rates for proteins that have been shown to associate with kinesin is very wide, for example, the average transport rate for neurofilaments is approximately 30 mm/day (Wang et al., 2000
), whereas for amyloid precursor protein the average transport rate approaches 400 mm/day (Kaether et al., 2000
), hence the variation between the rates of tau and
-synuclein transport seen here is not unexpected.
Although this study has focused on the axonal transport of tau and -synuclein themselves, recent studies have brought to the forefront the importance of tau in the regulation of axonal transport of other organelles. For example, overexpression of tau in culture models results in inhibition of transported components such as mitochondria (Sato-Harada et al., 1996
; Ebneth et al., 1998
; Trinczek et al., 1999
; Stamer et al., 2002
; Seitz et al., 2002
). It has been proposed that the mechanism involved in this inhibition of transport by tau may be caused by competition of tau with kinesin for binding sites on microtubules (Stamer et al., 2002
; Seitz et al., 2002
). Since our data suggest that tau and kinesin can interact directly, an alternative explanation is that, when overexpressed, tau could sequester kinesin, preventing the motor from binding to other cargoes, and allowing the accumulation of neuronal components such as mitochondria in the cell body. Indeed when tau is overexpressed, although mitochondrial transport is ablated, tau does not inhibit its own transport along axons (Stamer et al., 2002
; Utton et al., 2002
), suggesting that movement of tau is not inhibited by excessive coating of the microtubules by the overexpressed tau.
The physiological and functional significance of the directly observed fast transport of tau and -synuclein has yet to be fully determined. In disease conditions, however, where the amount of tau may be increased (Khatoon et al., 1992
; Savaskan et al., 2001), or the ratio of tau bound to microtubules may be altered, for example due to altered tau phosphorylation or tau mutations, the regulatory effects of tau as well as the transport of tau itself may be affected. In the case of
-synuclein, it has already been shown that mutant (A30P)
-synuclein, which causes a form of familial Parkinson's disease, abolishes
-synuclein-binding to vesicles (Jensen et al., 1998
). We have reported that A30P
-synuclein exhibits significantly reduced transport in cultured neurons compared to wild-type
-synuclein (Saha et al., 2004
), although another recent study did not detect any effects on slow axonal transport in peripheral nerves of transgenic mice expressing human A30P
-synuclein (Li et al., 2004
). Perturbations in the axonal transport rates of tau and
-synuclein, such as may occur in neurodegenerative disease, could therefore result in the formation of intracellular aggregates and eventual cell death in affected neurons.
In conclusion, this study has begun to elucidate the mechanisms involved in the axonal transport of two proteins, tau and -synuclein, both of which are known to be involved in neurodegenerative disease. It has also highlighted the fact that, similar to several other transported proteins, the overall slow rate of axonal transport of tau and
-synuclein may be mediated through fast transport motors, including kinesin and dynein.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abeliovich, A., Schmitz, Y., Farinas, I., Choi-Lundberg, D., Ho, W. H., Castillo, P. E., Shinsky, N., Verdugo, J. M., Armanini, M., Ryan, A. et al. (2000). Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron 25, 239-252.[CrossRef][Medline]
Ackerley, S., Grierson, A. J., Brownlees, J., Thornhill, P., Anderton, B. H., Leigh, P. N., Shaw, C. E. and Miller, C. C. (2000). Glutamate slows axonal transport of neurofilaments in transfected neurons. J. Cell Biol. 150, 165-176.
Ackerley, S., Thornhill, P., Grierson, A. J., Brownlees, J., Anderton, B. H., Leigh, P. N., Shaw, C. E. and Miller, C. C. (2003). Neurofilament heavy chain side arm phosphorylation regulates axonal transport of neurofilaments. J. Cell Biol. 161, 489-495.
Bendiske, J., Caba, E., Brown, Q. B. and Bahr, B. A. (2002). Intracellular deposition, microtubule destabilization, and transport failure: an "early" pathogenic cascade leading to synaptic decline. J. Neuropathol. Exp. Neurol. 61, 640-650.[Medline]
Brandt, R., Léger, J. and Lee, G. (1995). Interaction of tau with the neural plasma membrane mediated by tau's amino-terminal projection domain. J. Cell Biol. 131, 1327-1340.[Abstract]
Brown, A. (2003). Axonal transport of membranous and nonmembranous cargoes: a unified perspective. J. Cell Biol. 160, 817-821.
Cabin, D. E., Shimazu, K., Murphy, D., Cole, N. B., Gottschalk, W., McIlwain, K. L., Orrison, B., Chen, A., Ellis, C. E., Paylor, R. et al. (2002). Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking alpha-synuclein. J. Neurosci. 22, 8797-8807.
Chandra, S., Fornai, F., Kwon, H. B., Yazdani, U., Atasoy, D., Liu. X., Hammer, R. E., Battaglia, G., German, D. C. et al. (2004). Double-knockout mice for alpha- and beta-synucleins: effect on synaptic functions. Proc. Natl. Acad. Sci. USA 101, 14966-14971.
Cleveland, D. W., Hwo, S. Y. and Kirschner, M. W. (1977). Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly. J. Mol. Biol. 116, 227-247.[CrossRef][Medline]
Dai, J., Buijs, R. M., Kamphorst, W. and Swaab, D. F. (2002). Impaired axonal transport of cortical neurons in Alzheimer's disease is associated with neuropathological changes. Brain Res. 948, 138-144.[CrossRef][Medline]
Dayanandan, R., Van Slegtenhorst, M., Mack, T. G., Ko. L., Yen, S. H., Leroy, K., Brion, J. P., Anderton, B. H., Hutton, M. and Lovestone S. (1999). Mutations in tau reduce its microtubule binding properties in intact cells and affect its phosphorylation. FEBS Lett. 446, 228-232.[CrossRef][Medline]
Drubin, D. G. and Kirschner, M. W. (1986). Tau protein function in living cells. J. Cell Biol. 103, 2739-2746.[Abstract]
Ebneth, A., Godemann, R., Stamer, K., Illenberger, S., Trinczek, B. and Mandelkow, E. (1998). Overexpression of tau protein inhibits kinesin-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum: implications for Alzheimer's disease. J. Cell Biol. 143, 777-794.
Flament-Durand, J. and Couck, A. M. (1979). Spongiform alterations in brain biopsies of presenile dementia. Acta Neuropathol. (Berl.) 46, 159-162.[CrossRef][Medline]
Giasson, B. I., Forman, M. S., Higuchi, M., Golbe, L. I., Graves, C. L., Kotzbauer, P. T., Trojanowski, J. Q. and Lee, V. M. (2003). Initiation and synergistic fibrillization of tau and alpha-synuclein. Science 300, 636-640.
Hamilton, R. L. (2000). Lewy bodies in Alzheimer's disease: a neuropathological review of 145 cases using alpha-synuclein immunohistochemistry. Brain Pathol. 10, 378-384.[Medline]
Hasegawa, T., Matsuzaki, M., Takeda, A., Kikuchi, A., Akita, H., Perry, G., Smith, M. A. and Itoyama, Y. (2004). Accelerated alpha-synuclein aggregation after differentiation of SH-SY5Y neuroblastoma cells. Brain Res. 1013, 51-59.[CrossRef][Medline]
He, Y., Francis, F., Myers, K. A., Yu, W., Black, M. M. and Baas, P. W. (2005). Role of cytoplasmic dynein in the axonal transport of microtubules and neurofilaments. J. Cell Biol. 168, 697-703.
Helfand, B. T., Loomis, P., Yoon, M. and Goldman, R. D. (2003). Rapid transport of neural intermediate filament protein. J. Cell Sci. 116, 2345-2359.
Iwai, A. (2000). Properties of NACP/alpha-synuclein and its role in Alzheimer's disease. Biochim. Biophys. Acta 1502, 95-109.[Medline]
Jancsik, V., Filliol, D. and Rendon, A. (1996). Tau proteins bind to kinesin and modulate its activation by microtubules. Neurobiology 4, 417-429.[Medline]
Jensen, P. H., Nielsen, M. S., Jakes, R., Dotti, C. G. and Goedert, M. (1998). Binding of alpha-synuclein to brain vesicles is abolished by familial Parkinson's disease mutation. J. Biol. Chem. 273, 26292-26294.
Jensen, P. H., Li, J. Y., Dahlstrom, A. and Dotti, C. G. (1999). Axonal transport of synucleins is mediated by all rate components. Eur. J. Neurosci. 11, 3369-3376.[CrossRef][Medline]
Kaether, C., Skehel, P. and Dotti, C. G. (2000). Axonal membrane proteins are transported in distinct carriers: a two-color video microscopy study in cultured hippocampal neurons. Mol. Biol. Cell 11, 1213-1224.
Kamal, A., Stokin, G. B., Yang, Z., Xia, C. H. and Goldstein, L. S. (2000). Axonal transport of amyloid precursor protein is mediated by direct binding to the kinesin light chain subunit of kinesin-I. Neuron 28, 449-459.[CrossRef][Medline]
Kawamata, T., Taniguchi, T., Mukai, H., Kitagawa, M., Hashimoto, T., Maeda, K., Ono, Y. and Tanaka, C. (1998). A protein kinase, PKN, accumulates in Alzheimer neurofibrillary tangles and associated endoplasmic reticulum-derived vesicles and phosphorylates tau protein. J. Neurosci. 18, 7402-7410.[Abstract]
Khatoon, S., Grundke-Iqbal, I. and Iqbal, K. (1992). Brain levels of microtubule-associated protein are elevated in Alzheimer's disease: A radioimmuno-slot-blot assay for nanograms of the protein. J. Neurochem. 59, 750-753.[Medline]
Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek, H., Epplen, J. T., Schols, L. and Riess, O. (1998). Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. Nat. Genet. 18, 106-108.[CrossRef][Medline]
Lach, B., Grimes, D., Benoit, B. and Minkiewicz-Janda, A. (1992). Caudate nucleus pathology in Parkinson's disease: ultrastructural and biochemical findings in biopsy material. Acta Neuropathol. 83, 352-360.[CrossRef][Medline]
Lee, G., Newman, S. T., Gard, D. L., Band, H. and Panchamoorthy, G. (1998). Tau interacts with src-family non-receptor tyrosine kinases. J. Cell Sci. 111, 3167-3177.
Lee, V. M., Goedert, M. and Trojanowski, J. Q. (2001). Neurodegenerative tauopathies. Annu. Rev. Neurosci. 24, 1121-1159.[CrossRef][Medline]
Li, W., Hoffman, P. N., Stirling, W., Price, D. L. and Lee, M. K. (2004). Axonal transport of human alpha-synuclein slows with aging but is not affected by familial Parkinson's disease-linked mutations. J. Neurochem. 88, 401-410.[CrossRef][Medline]
Liao, H., Li, Y., Brautigan, D. L. and Gundersen, G. G. (1998). Protein phosphatase 1 is targeted to microtubules by the microtubule-associated protein Tau. J. Biol. Chem. 273, 21901-21908.
Ligon, L. A. and Steward, O. (2000). Role of microtubules and actin filaments in the movement of mitochondria in the axons and dendrites of cultured hippocampal neurons. J. Comp. Neurol. 427, 351-361.[CrossRef][Medline]
Masliah, E., Rockenstein, E., Veinbergs, I., Sagara, Y., Mallory, M., Hashimoto, M. and Mucke, L. (2001). beta-amyloid peptides enhance alpha-synuclein accumulation and neuronal deficits in a transgenic mouse model linking Alzheimer's disease and Parkinson's disease. Proc. Natl. Acad. Sci. USA 98, 12245-12250.
Mercken, M., Fischer, I., Kosik, K. S. and Nixon, R. A. (1995). Three distinct axonal transport rates for tau, tubulin, and other microtubule-associated proteins: Evidence for dynamic interactions of tau with microtubules in vivo. J. Neurosci. 15, 8259-8267.[Abstract]
Mezey, E., Dehejia, A. M., Harta, G., Suchy, S. F., Nussbaum, R. L., Brownstein, M. J. and Polymeropoulos, M. H. (1998). Alpha synuclein is present in Lewy bodies in sporadic Parkinson's disease. Mol. Psychiatry 3, 493-499.[CrossRef][Medline]
Mori, H., Hamada, Y., Kawaguchi, M., Honda, T., Kondo, J. and Ihara, Y. (1989). A distinct form of tau is selectively incorporated into Alzheimer's paired helical filaments. Biochem. Biophys. Res. Commun. 159, 1221-1226.[CrossRef][Medline]
Mukaetova-Ladinska, E. B., Hurt, J., Jakes, R., Xuereb, J., Honer, W. G. and Wischik, C. M. (2000). Alpha-synuclein inclusions in Alzheimer and Lewy body diseases. J. Neuropathol. Exp. Neurol. 59, 408-417.[Medline]
Okabe, S. and Hirokawa, N. (1993a). Do photobleached fluorescent microtubules move?: re-evaluation of fluorescence laser photobleaching both in vitro and in growing Xenopus axon. J. Cell Biol. 120, 1177-1186.[Abstract]
Okabe, S., Miyasaka, H. and Hirokawa, N. (1993b). Dynamics of the neuronal intermediate filaments. J. Cell Biol. 121, 375-386.[Abstract]
Perez, R. G., Waymire, J. C., Lin, E., Liu, J. J., Guo, F. and Zigmond, M. J. (2002). A role for alpha-synuclein in the regulation of dopamine biosynthesis. J. Neurosci. 22, 3090-3099.
Polymeropoulos, M. H., Lavedan. C., Leroy, E., Ide, S. E., Dehejia, A., Dutra, A., Pike. B., Root, H., Rubenstein, J., Boyer, R. et al. (1997). Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science 276, 2045-2047.
Popescu, A., Lippa, C. F., Lee, V. M. and Trojanowski, J. Q. (2004). Lewy bodies in the amygdala: increase of alpha-synuclein aggregates in neurodegenerative diseases with tau-based inclusions. Arch. Neurol. 61, 1915-1919.
Prahlad, V., Yoon, M., Moir, R. D., Vale, R. D. and Goldman, R. D. (1998). Rapid movements of vimentin on microtubule tracks: kinesin-dependent assembly of intermediate filament networks. J. Cell Biol. 143, 159-170.
Prahlad, V., Helfand, B. T., Langford, G. M., Vale, R. D. and Goldman, R. D. (2000). Fast transport of neurofilament protein along microtubules in squid axoplasm. J. Cell Sci. 113, 3939-3946.
Praprotnik, D., Smith, M. A., Richey, P. L., Vinters, H. V. and Perry, G. (1996). Filament heterogeneity within the dystrophic neurites of senile plaques suggests blockage of fast axonal transport in Alzheimer's disease. Acta Neuropathol. 91, 226-235.[CrossRef][Medline]
Rahman, A., Friedman, D. S. and Goldstein, L. S. (1998). Two kinesin light chain genes in mice. Identification and characterization of the encoded proteins. J. Biol. Chem. 273, 15395-15403.
Richard, S., Brion, J.-P., Couck, A. M. and Flament-Durand, J. (1989). Accumulation of smooth endoplasmic reticulum in Alzheimer's disease: new morphological evidence of axoplasmic flow disturbances. J. Submicrosc. Cytol. Pathol. 21, 461-467.[Medline]
Roy, S., Coffee, P., Smith, G., Liem, R. K., Brady, S. T. and Black, M. M. (2000). Neurofilaments are transported rapidly but intermittently in axons: implications for slow axonal transport. J. Neurosci. 20, 6849-6861.
Saha, A. R., Ninkina, N. N., Hanger, D. P., Anderton, B. H., Davies, A. M. and Buchman, V. L. (2000). Induction of neuronal death by alpha-synuclein. Eur. J. Neurosci. 12, 3073-3077.[CrossRef][Medline]
Saha, A. R., Hill, J., Utton, M. A., Asuni, A. A., Ackerley, S., Grierson, A. J., Miller, C. C., Davies, A. M., Buchman, V. L., Anderton, B. H., Hanger, D. P. et al. (2004). Parkinson's disease alpha-synuclein mutations exhibit defective axonal transport in cultured neurons. J. Cell Sci. 117, 1017-1024.
Sato-Harada, R., Okabe, S., Umeyama, T., Kanai, Y. and Hirokawa, N. (1996). Microtubule-associated proteins regulate microtubule function as the track for intracellular membrane organelle transports. Cell Struct. Funct. 21, 283-295.[Medline]
Savaskan, N. E. and Nitsch, R. (2001). Molecules involved in reactive sprouting in the hippocampus. Rev. Neurosci. 12, 195-215.[Medline]
Scott, C. W., Blowers, D. P., Barth, P. T., Lo, M. M., Salama, A. I. and Caputo, C. B. (1991). Differences in the abilities of human tau isoforms to promote microtubule assembly. J. Neurosci. Res. 30, 154-162.[CrossRef][Medline]
Seitz, A., Kojima, H., Oiwa, K., Mandelkow, E. M., Song, Y. H. and Mandelkow, E. (2002). Single-molecule investigation of the interference between kinesin, tau and MAP2c. EMBO J. 21, 4896-4905.
Seo, J. H., Rah, J. C., Choi, S. H., Shin, J. K., Min, K., Kim, H. S., Park, C. H., Kim, S., Kim, E. M., Lee, S. H. et al. (2002). a-Synuclein regulates neuronal survival via Bcl-2 family expression and PI3/Akt kinase pathway. FASEB J. 16, 1826-1828.
Shah, J. V., Flanagan, L. A., Janmey, P. A. and Leterrier, J. F. (2000). Bidirectional translocation of neurofilaments along microtubules mediated in part by dynein/dynactin. Mol. Biol. Cell 11, 3495-3508.
Sontag, E., Nunbhakdi-Craig, V., Lee, G., Brandt, R., Kamibayashi, C., Kuret, J., White, C. L., III, Mumby, M. C. and Bloom, G. S. (1999). Molecular interactions among protein phosphatase 2A, tau, and microtubules. Implications for the regulation of tau phosphorylation and the development of tauopathies. J. Biol. Chem. 274, 25490-25498.
Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R. and Goedert, M. (1997). Alpha-synuclein in Lewy bodies. Nature 388, 839-840.[CrossRef][Medline]
Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M. and Goedert, M. (1998). alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies. Proc. Natl. Acad. Sci. USA 95, 6469-6473.
Stamer, K., Vogel, R., Thies, E., Mandelkow, E. and Mandelkow, E. M. (2002). Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress. J. Cell Biol. 156, 1051-1063.
Tanaka, M., Kim, Y. M., Lee, G., Junn, E., Iwatsubo, T. and Mouradian, M. M. (2004). Aggresomes formed by alpha-synuclein and synphilin-1 are cytoprotective. J. Biol. Chem. 279, 4625-4631.
Theiss, C., Napirei, M. and Meller, K. (2005). Impairment of anterograde and retrograde neurofilament transport after anti-kinesin and anti-dynein antibody microinjection in chicken dorsal root ganglia. Eur. J. Cell Biol. 84, 29-43.[Medline]
Totterdell, S., Hanger, D. and Meredith, G. E. (2004). The ultrastructural distribution of alpha-synuclein-like protein in normal mouse brain. Brain Res. 1004, 61-72.[CrossRef][Medline]
Trinczek, B., Ebneth, A., Mandelkow, E. M. and Mandelkow, E. (1999). Tau regulates the attachment/detachment but not the speed of motors in microtubule-dependent transport of single vesicles and organelles. J. Cell Sci. 112, 2355-2367.
Utton, M. A., Vandecandelaere, A., Wagner, U., Reynolds, C. H., Gibb, G. M., Miller, C. C., Bayley, P. M. and Anderton, B. H. (1997). Phosphorylation of tau by glycogen synthase kinase 3beta affects the ability of tau to promote microtubule self-assembly. Biochem. J. 323, 741-747.[Medline]
Utton, M. A., Connell, J., Asuni, A. A., Van Slegtenhorst, M., Hutton, M., De Silva, R., Lees, A. J., Miller, C. C. and Anderton, B. H. (2002). The slow axonal transport of the microtubule-associated protein tau and the transport rates of different isoforms and mutants in cultured neurons. J. Neurosci. 22, 6394-6400.
Wang, L. and Brown, A. (2001). Rapid intermittent movement of axonal neurofilaments observed by fluorescence photobleaching. Mol. Biol. Cell 12, 3257-3267.
Wang, L. and Brown, A. (2002). Rapid movement of microtubules in axons. Curr. Biol. 12, 1496-1501.[CrossRef][Medline]
Wang, L., Ho, C. L., Sun, D., Liem, R. K. and Brown, A. (2000). Rapid movement of axonal neurofilaments interrupted by prolonged pauses. Nat. Cell Biol. 2, 137-141.[CrossRef][Medline]
Yabe, J. T., Pimenta, A. and Shea, T. B. (1999). Kinesin-mediated transport of neurofilament protein oligomers in growing axons. J. Cell Sci. 112, 3799-3814.
Yabe, J. T., Jung, C., Chan, W. K. and Shea, T. B. (2000). Phospho-dependent association of neurofilament proteins with kinesin in situ. Cell Motil. Cytoskeleton 45, 249-262.[CrossRef][Medline]
Yabe, J. T., Chan, W. K. H., Chylinski, T. M., Lee, S., Pimenta, A. F. and Shea, T. B. (2001). The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation. Cell Motil. Cytoskeleton 48, 61-83.[CrossRef][Medline]
Zhang, B., Higuchi, M., Yoshiyama, Y., Ishihara, T., Forman, M. S., Martinez, D., Joyce, S., Trojanowski, J. Q. and Lee, V. M. (2004). Retarded axonal transport of R406W mutant tau in transgenic mice with a neurodegenerative tauopathy. J. Neurosci. 24, 4657-4667.
Zhou, Y., Gu, G., Goodlett, D. R., Zhang, T., Pan, C., Montine, T. J., Montine, K. S., Aebersold, R. H. and Zhang, J. (2004). Analysis of alpha-synuclein-associated proteins by quantitative proteomics. J. Biol. Chem. 279, 39155-39164.
|