Published as doi: 10.1096/fj.06-6679fje.
(The FASEB Journal. 2006;20:2573-2575.)
© 2006 FASEB
Unloading kinesin transported cargoes from the tubulin track via the inflammatory c-Jun N-terminal kinase pathway
Massimiliano Stagi*,
,
,
,1,
Philipp Gorlovoy*,,1,
Sergey Larionov*,
Kazuya Takahashi*, and
Harald Neumann*,,,2
* Neural Regeneration Unit, Institute of Reconstructive Neurobiology, University Bonn Life and Brain Center and Hertie Foundation, University Bonn, Bonn, Germany;
Institute of Multiple Sclerosis Research, University Göttingen and Hertie-Foundation, Göttingen, Germany;
Neuroimmunology Unit, European Neuroscience Institute Göttingen, Waldweg, Göttingen, Germany; and
Yale University, Department of Molecular Biophysics and Biochemistry, Sterling Hall of Medicine, New Haven, Connecticut, USA
2Correspondence: Neural Regeneration Unit, Institute of Reconstructive Neurobiology, University Bonn Life and Brain Center, University Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany. E-mail: hneuman1{at}uni-bonn.de
SPECIFIC AIMS
Axonal transport impairments are early signs of a variety of neurodegenerative and neuroinflammatory diseases. The molecular mechanisms of axonal transport dysfunction in neurodegenerative diseases and inflammatory brain diseases, such as multiple sclerosis, are not known. We asked whether the proinflammatory cytokine tumor necrosis factor-
(TNF), which is prevalent in all neuroinflammatory and neurodegenerative diseases, induces cellular stress response through the c-Jun N-terminal kinase (JNK) signaling cascade, and impairs axonal transport of synaptic precursor vesicles or mitochondria, and whether JNK signaling is involved in this process.
PRINCIPAL FINDINGS
1. Phosphorylated JNK in axons after treatment with TNF
Analysis of cultured hippocampal neurons identified by tau immunolabeling showed immunostaining for TNF receptors I (82.0%±7.2%; mean±SEM) and TNF receptors II (88.1%±1.3%). Furthermore, after acute treatment with TNF (20 ng/ml) for 20 min, strong immunolabeling of neurites with specific antibodies directed against phosphorylated JNK was detected by confocal microscopy. Quantification of tau-positive neurites showed that after TNF treatment the majority of them (84%±13%; mean±SEM) expressed phosphorylated JNK, while in untreated neurons a minority of tau-positive neurites (11%±7%) showed immunolabeling for phosphorylated JNK.
2. Dissociation of KIF5B from ßbeta;-tubulin-III after TNF treatment via JNK
Western blot analysis of protein lysates from cultured hippocampal neurons showed that acute treatment with TNF increased the amount of phosphorylated JNK in the cultured neuronal cells. Furthermore, immunoprecipitation by ßbeta;-tubulin-III-specific antibodies of cultured neurons demonstrated that kinesin family member 5B (KIF5B) and JNK were binding to ßbeta;-tubulin-III. Treatment with TNF (20 ng/ml) for 20 min reduced the amount of KIF5B and JNK bound to ßbeta;-tubulin-III in immunoprecipitated lysates of cultured neurons, suggesting that TNF affected the complex between ßbeta;-tubulin-III, KIF5B and JNK.
Lifetime-based Förster resonance energy transfer (FRET) analysis was performed to allow subcellular localization of the molecular interaction between KIF5B and ßbeta;-tubulin-III. Cultured hippocampal neurons were double-immunolabeled with monoclonal antibodies directed against KIF5B marked with the fluorophore FITC (KIF5B-FITC) and monoclonal antibodies directed against ßbeta;-tubulin-III marked with the fluorophore Cy3 (ßbeta;-tubulin-III-Cy3). A strong FRET interaction was observed in untreated neurons colabeled with ßbeta;-tubulin-III-Cy3 and KIF5B-FITC (Fig. 1
), showing a shifted distribution toward shorter lifetimes with a peak value of 1.5 ± 0.18 ns. Treatment with TNF (20 ng/ml) for 20 min abolished the FRET interaction specifically in neurites, but not in cell bodies (Fig. 1)
.
View larger version (36K):
[in this window]
[in a new window]
|
Figure 1. Effects of TNF on the molecular interaction between KIF5B and ßbeta;-tubulin-III, as determined by lifetime-based FRET analysis. Neurons were coimmunolabeled with antibodies directed against KIF5B marked with FITC and antibodies directed against ßbeta;-tubulin-III marked with Cy3. Lifetime images were acquired of KIF5B-FITC. Lifetimes from 1.0 to 1.9 ns were coded in red, representing expected signals from FRET. Lifetimes from 1.9 to 4.0 ns were coded in green, representing the expected lifetime of KIF5B-FITC in the absence of acceptor. In untreated neurons, direct association between ßbeta;-tubulin-III and KIF5B was observed as demonstrated by lifetime values coded in red. After treatment with TNF for 20 min, the conformation of the KIF5B-ßbeta;-tubulin-III complex changed in neurites as demonstrated by lifetime values coded in green. Specifically, TNF induced a breakage of the normal KIF5B-ßbeta;-tubulin-III complex in neurites, but not in cell bodies. Scale bars: 20 µm.
|
|
To quantify the effect of TNF on the interaction between KIF5B-FITC and ßbeta;-tubulin-III-Cy3, we determined the average of the peak lifetimes collected from several regions of interest in neurites. In detail, the average peak lifetime value in neurites was 2.3 ± 0.5 ns (mean±SD) after 20 min of TNF treatment, while in untreated neurites the average peak lifetime was 1.5 ± 0.18 ns. In untreated neurons, 93 ± 27% (mean±SD) of neurites showed shorter lifetimes compatible with FRET, while in TNF-treated neurons, the percentage of neurites showing FRET between KIF5B-FITC and ßbeta;-tubulin-III-Cy3 was reduced to 4 ± 19%. No FRET was observed in the controls by colabeling KIF5B-FITC and neurofilament-Cy3 or by imaging KIF5B-FITC in the absence of acceptors. Thus, treatment of hippocampal neurons with TNF changed the conformation of the KIF5B-tubulin complex, indicating that it broke apart the molecular interaction between KIF5B and ßbeta;-tubulin-III in axons.
Next, hippocampal neurons were treated with TNF for 20 min, and additionally, JNK was blocked with the JNK inhibitor SP600125 (100 nM). Additional treatment with the JNK inhibitor reduced the effect of TNF on the molecular interaction between KIF5B and ßbeta;-tubulin-III. After inhibition of JNK, low lifetimes were again detected in 60 ± 19% (mean±SD) neurites of TNF plus JNK inhibitor-treated neurons, while reduced lifetimes were observed in 4 ± 19% neurites treated with TNF alone.
3. Inhibition of axonal mitochondrial and synaptophysin transport by TNF via JNK
To study whether TNF disturbs axonal transport of mitochondria, we transfected cultured hippocampal neurons with a mitochondrial targeting sequence tagged with enhanced yellow fluorescent protein (YFP). Using time lapse imaging of living cultured neurons, we determined the number of moving mitochondria (mobile fraction) and their velocity. In total 20 ± 12% (mean±SD) of mitochondria moved anterogradely and 21 ± 17% retrogradely. After treatment with TNF for 20 min, mitochondria stopped moving. In total, the mobile fraction of mitochondria decreased from 41 ± 14% to 7 ± 4% after treatment with TNF for 20 min. Both anterograde and retrograde movements were substantially reduced to 3 ± 4% and 4 ± 4%, respectively (Fig. 2
). The velocity of the remaining mobile mitochondria was unchanged after TNF treatment (Fig. 2)
.
View larger version (39K):
[in this window]
[in a new window]
|
Figure 2. Inhibition of mitochondrial movements by TNF via JNK. The percentage of mitochondria moving along axons (mobile fraction) anterogradely and retrogradely was quantified in cultured neurons transfected with a mitochondrial targeting sequence tagged to YFP. Treatment of neurons with TNF for 20 min reduced the mobile fraction. Effect of TNF on mitochondria movement was neutralized by inhibition of JNK with SP600125 (JNK inhibitor). Furthermore, the effect of TNF on axonal mitochondria movement was inhibited by blocking TNF with a TNFRI-IgG-fusion protein (TNF inhibition). No change in velocity was observed after treatment with TNF or combined treatment with TNF and JNK inhibitor. Data are presented as mean± SD. For each experimental condition n > 8; ***P < 0.001, **P < 0.01 (two-tailed Mann Whitney U test).
|
|
After cotreatment of neurons with TNF for 20 min and the generalized JNK inhibitor (SP600125; 100 nM) 45 ± 9% of mitochondria were mobile, very similar to the untreated situation (Fig. 2)
. Both anterograde and retrograde movements were regained in TNF-treated axons after JNK inhibition. The inhibitory effect of TNF on axonal movement of mitochondria was transient, and after 360 min of TNF treatment the percentage of moving mitochondria was normal again (Fig. 2)
.
To study whether TNF also inhibits synaptophysin transport in axons, we transfected cultured hippocampal neurons with synaptophysin tagged with green fluorescent protein (GFP) and performed fluorescence recovery after photobleaching (FRAP) analysis. After acute TNF treatment, a significant reduction in the axonal transport of synaptophysin-GFP was observed. The relative FRAP intensity at 300 s was reduced from 0.32 ± 0.14 (mean±SD) to 0.11 ± 0.04 after 20 min treatment with TNF. The value returned back at 360 min of TNF treatment to normal levels comparable to untreated neurons. Furthermore, there was a significant decrease in the mobile fraction of synaptophysin-GFP from 39 ± 14% (mean±SD) to 13 ± 4% after 20 min treatment with TNF. The velocity of synaptophysin-GFP appeared to be unchanged since the half-time value t 
of the FRAP was not modulated by the TNF treatment. Inhibition of JNK by the inhibitor SP600125 completely neutralized the effect of TNF on axonal movement of synaptophysin-GFP.
CONCLUSIONS AND SIGNIFICANCE
The results elucidate the mechanism of inflammatory axonal transport impairment (Fig. 3
). First, data demonstrate that TNF breaks apart the molecular interaction between KIF5B and tubulin via JNK activity. Thus, JNK forms a complex with kinesin and directly modulate the function of kinesin. Second, results show that TNF inhibits anterograde and retrograde axonal movement of mitochondria via JNK. Third, TNF impairs axonal movement of synaptophysin via JNK. Thus, TNF acts on the kinesin-tubulin complex via JNK activity. Thereby, TNF has important functional consequences leading to impairment in axonal transport of mitochondria and synaptic vesicle precursors.
View larger version (60K):
[in this window]
[in a new window]
|
Figure 3. Hypothetical model of inflammation-mediated axonal transport impairment. TNF released by activated microglia during neuroinflammatory or neurodegenerative disease causes activation of JNK (JNK phosphorylated; pJNK), which subsequently acts on the conformation of the kinesin motor complexes and detaches kinesin from microtubule. Breakage of the kinesin-tubulin complex will lead to reversible cessation of the axonal transport and decrease of the mobile fraction of mitochondria and synaptic vesicle precursors moving along axons. Our data suggest that intact axonal transport is a sign of healthy neurons and that stress signaling over JNK acts on the motor transport possibly to reduce energy expenditure during inflammation.
|
|
The JNK is an attractive pathway as a regulator of the axonal transport. JNKs are linked in neurons to the kinesin motor proteins via JIPs (c-Jun N-terminal kinase interacting proteins). The indirect linkage of motors, JNK, and cargo via the JIP scaffolding proteins would allow JNK to act as signal transducer to modulate the motor activity and axonal transport of cargoes, such as synaptophysin and mitochondria (Fig. 3)
.
Principally, JNK kinases might phosphorylate either kinesin itself or an associated protein, thereby inactivating the motor and leading to dissociation of kinesin from tubulin. Alternatively, activity of JNK might be indirectly linked to the dissociation of the kinesin-tubulin complex. Western blot analysis of cultured neurons showed that JNK is binding to the kinesin-tubulin complex and that acute TNF treatment reduced the amount of JNK bound to tubulin. Furthermore, we now demonstrated by lifetime-based FRET analysis that TNF changed the conformation of the kinesin-tubulin complex in the axons, but not the cell bodies. This TNF-mediated effect on the conformation of the kinesin-tubulin complex was partially reverted by inhibition of JNK. Data of axonal transport analysis indicate that the kinesin dissociates from the microtubules, since the immobile fraction increased after TNF treatment without interference in the velocity of the remaining mobile particles. Furthermore, we now demonstrate that TNF-induced JNK activity inhibits synaptophysin transport. Thus, the JNK signaling pathway appears to be widely involved in axonal transport of organelles and vesicular structures.
Disturbances of axonal transport are observed in a variety of neurodegenerative and neuroinflammatory diseases. The molecular mechanisms of axonal transport dysfunction in neurodegenerative diseases such as Alzheimer’s disease and inflammatory brain diseases, such as multiple sclerosis are not known. Several studies observed a close association between activated microglial cells and neuronal injury in Alzheimer’s disease, multiple sclerosis, microbial infection, and brain injury. Activated microglial cells produce substantial amounts of TNF and are highly motile under normal and disease conditions. In neuroinflammatory diseases, they are activated and migrate to sites of neuronal lesions, where they bind and ensheathe naked or demyelinated axons.
In summary, TNF impairs the axonal transport system, which is required to maintain function, integrity, and viability of axonal and synaptic structures. The findings, which also show that the JNK signaling pathway interferes with the kinesin-mediated transport system, open new avenues for therapeutic intervention.
FOOTNOTES
1 These authors equally contributed to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6679fje
This article has been cited by other articles:
|
|
|
|
 
N. Hirokawa and Y. Noda
Intracellular Transport and Kinesin Superfamily Proteins, KIFs: Structure, Function, and Dynamics
Physiol Rev,
July 1, 2008;
88(3):
1089 - 1118.
[Abstract]
[Full Text]
[PDF]
|
|
|
Copyright © 2006 by The Federation of American Societies for Experimental Biology.
