We have previously shown that several ALS mutants of SOD1 can interact with the dynein-dynactin complex which is responsible for retrograde axonal transport [23
]. We have also demonstrated that the interaction between mutant SOD1 and dynein-dynactin plays a functional role in the formation of mutant SOD1 inclusions [24
]. In this study, we used the sciatic nerve double ligation technique, which has long been used to evaluate the rate of bi-directional transport under physiological and pathological conditions [36
], to determine whether the bi-directional axonal transport was impaired. In addition, we investigated whether mutant SOD1 could interact with and disrupt the anterograde axonal transport mediated by the kinesin-1 family members. Kinesin-1 proteins are abundant in neurons and have been shown to transport several cargos vital for neuronal function and survival. The goal of the study was to compare the interaction of mutant SOD1 with the dynein-dynactin and the kinesin-1 members, and to compare the effect of mutant SOD1 on the corresponding retrograde and anterograde axonal transport.
The interaction between mutant SOD1 and dynein-dynactin was again demonstrated in cultured cells and ALS mouse spinal cords in this study. Based on quantification of motor protein DHC accumulation at the distal side of the sciatic nerve ligation site 24 hour post-ligation, we showed that dynein-mediated axonal transport was significantly suppressed in 60 days old pre-symptomatic G93A mice compared to WT SOD1 mice ( and ). Interestingly, the data from the 48 hour post-ligation samples, showed no difference in dynein accumulation between G93A and WT SOD1 mice, suggesting that retrograde transport is not completely blocked but rather slowed down. These data together with our previous studies [23
] support the hypothesis that there is an early impairment of axonal retrograde transport in the G93A SOD1 transgenic mice, presumably due to the interaction between mutant SOD1 and the dynein-dynactin. This hypothesis is further supported by other studies that mutant SOD1 could alter the sub-cellular localization of dynein-dynactin [15
]. The same study also showed a decrease of retrograde transport in 50 days old G93A mice using a neurotracer approach.
The reduced retrograde transport could contribute to the motor neuron death by decreasing the removal of damaged mitochondria from the synapse, or by reducing the level of survival signals induced by neurotrophic factors secreted by the muscle [39
]. Since dynein-dynactin is the only major motor protein responsible for retrograde transport, the alterations in the retrogradely transported cargos may be an alternative mechanism contributing to motor neuron death. In fact a recent study reported a switch in retrograde signaling from pro-survival to stress in ALS mice [38
Besides its role in retrograde axonal transport, the dynein-dynactin complex also have many other functions, including participation in mitosis, endoplasmic reticulum to Golgi vesicular trafficking, neuronal migration, neurite outgrowth, synapse formation, formation of aggresomes and protein degradation by autophagy (for review see [40
]). The interaction between mutant SOD1 and dynein-dynactin could hence have various consequences. In two other recent studies from our lab we found that the amount of mutant SOD1 interacting with dynein-dynactin increases with disease progression and that this interaction between mutant SOD1 and dynein-dynactin plays a role in formation of large aggresome-like SOD1 inclusions [23
]. Increasing levels of dynein-dynactin interacting with mutant SOD1 and sequestration of dynein into aggresome-like inclusions could also lead to depletion of the pool of dynein available for axonal transport. Since dynein is also important for autophagic degradation of protein aggregates [41
], disruption of dynein function might also slow down the autophagic degradation of misfolded SOD1. This would further increase the cellular load of mutant SOD1 and the subsequent sequestration of dynein-dynactin.
In contrast to the interaction with and reduction of dynein-dynactin retrograde transport, we could not find any evidence for an interaction between mutant SOD1 and the members of the kinesin-1 family (KIF5A, 5B, or 5C) in cell culture or in mice spinal cords ( and ). Immunostaining of the neuron specific KIF5A in both spinal cords and sciatic nerves of transgenic ALS mice failed to reveal any co-localization of KIF5A and mutant SOD1 aggregates in motor neurons (). Furthermore, we could see no decrease in KIF5A-mediated anterograde transport in 60 days old G93A mice using KIF5A accumulation as a surrogate measurement. This was unexpected since reduced anterograde transport has previously been shown to be an early event in ALS. In fact, a study using an antibody recognizing all three KIF5 subtypes showed reduced proximal accumulation of KIF5 after sciatic nerve ligations in pre-symptomatic low copy version of G93A [21
]. This difference could be due to usage of the different G93A lines or different antibodies recognizing all KIF5 subtypes versus the antibody specific for neuronal KIF5A used in our study. Perlson et al
recently showed that both retrograde and anterograde transport were suppressed in 85 days old G93A mice by measuring accumulation of DIC and kinesin heavy chain at the sciatic nerve ligation sites [38
]. The age of mice used in this study was 60 days, younger than those used in [38
]. It is possible that the anterograde transport is not altered in 60 days old mice while impairment of transport becomes detectable in 85 days old mice that are at the verge of developing clinical symptoms. In addition, the antibodies used in the two studies were different, particularly the kinesin antibodies. We used an antibody specific for neuronal isoform KIF5A. It is possible that the anterograde transport mediated by KIF5A motor protein is not changed in G93A mice while the transport mediated by other kinesin family members is changed.
A recent study showed that misfolded SOD1 interacted with the kinesin-2 motor complex via the KAP3 subunit. Furthermore, sequestering of KAP3 by mutant SOD1 was suggested to result in inhibition of ChAT transport [26
]. In addition, a genome-wide SNP analysis in a large set of sporadic ALS cases in U.S. and Europe revealed that a variant within the KAP3 gene was associated with decreased KAP3 expression and increased survival in sporadic ALS [27
]. The above two independent studies suggest that interference with kinesin-2 might contribute to both familial and sporadic ALS.
Interestingly, we observed a decrease in proximal dynein accumulation 24 hours post-sciatic nerve ligation in G93A mice compared to the WT mice ( and ). Accumulation of dynein at the proximal side has also previously been shown and is most likely due to kinesin-mediated anterograde transport of dynein as a cargo toward the synapse [21
]. Thus, our data suggest that dynein might be a cargo whose anterograde transport is affected by mutant SOD1 in ALS. Using live imaging of primary DRG neurons isolated from ALS mice, De Vos et al
examined axonal transport of other cargos and showed that transport of membrane bound organelles was affected in both anterograde and retrograde directions whereas transport of mitochondria was only affected in the anterograde direction [19
]. A separate study however showed that axonal transport of mitochondria in differentiated NSC34 cells was affected in both anterograde and retrograde directions in the presence of mutant SOD1 [22
]. The specific kinesin motor protein(s) transporting these cargos which showed reduced transport was not investigated in those studies. Furthermore the mechanism(s) underlying the decreased anterograde transport by mutant SOD1 is also unclear. It is possible that the reduced transport of these cargos could depend on reduced binding of these cargos to the motors rather than decreased motor transport/activity. In fact, reduced cargo-binding have been suggested in ALS disease (for review see [43
]). Studies have suggested that pathogenic proteins implicated in various neurodegenerative diseases can alter kinase signaling cascades and cause disruption in kinesin-mediated fast axonal transport [44
]. A hypothesis suggesting changes in the p38 signaling in the presence of mutant SOD1 has also been proposed [18
In our study looking directly at the KIF5A kinesin motor transport, we could not see decrease in anterograde movement of the motor itself. However, there could still be reduced transport of certain KIF5A cargos if cargo attachments where problematic. The observation of the impaired anterograde transport of DHC as a cargo provides an example for such disruptive events. Taken together, the results of this study and previous studies suggest that mutant SOD1 might only interact with and interfere with specific kinesin members which in turn could result in the impairment of a selective subset of cargos. Thus, it is critically important to examine the effect of mutant SOD1 on specific kinesin subtypes as well as different cargos using both in vitro live imaging and in vivo approaches to further our understanding of axonal transport defects in ALS disease.