The work by De Vos and colleagues (27
) and our unpublished data indicate that mitochondrial axonal transport is impaired in mutant SOD1 neurons. Mutant SOD1 can potentially affect mitochondrial transport by multiple, nonexclusive mechanisms, resulting in dysfunctional mitochondria that can no longer reach the cellular sites where they are most needed. First, abnormal accumulation of mutant SOD1 around or inside mitochondria could trigger mitochondrial damage and metabolic dysfunction. Second, aggregates of mutant SOD1 with other abundant axonal proteins, such as neurofilaments, could physically block axonal transport or disrupt the cytoskeleton, or both, thereby impairing mitochondria ability to move normally. Mutant SOD1 could also interact with molecular motors or cargo adaptors or both involved in mitochondrial axonal transport. Finally, mutant SOD1 could interfere with cell-signaling pathways that regulate cytoskeleton stability and motor activity ().
FIG. 5. Mutant SOD1 can perturb mitochondrial dynamics at multiple levels: (a) SOD1 accumulates inside mitochondria and induces mitochondrial damage and metabolic dysfunction. Mutant SOD1 accumulation in the outer membrane and the intermembrane space can interfere (more ...)
As described earlier, ample evidence indicates that mutant SOD1 affects normal mitochondria function (69
). However, the molecular mechanisms underlying the mitochondrial damage remain to be identified. One possibility involves aberrant interactions of mutant SOD1 with mitochondrial proteins, resulting in disruption of their normal folding or import (29
). Thus, it was reported that mutant SOD1 interacts with proteins that may affect mitochondria directly or indirectly, including Hsps (78
), members of the Bcl-2 family (22
), and components of the protein translation machinery (55
). We and others demonstrated that in yeast (96
), rats (78
), and in transgenic mice expressing WT or mutant human SOD1 (44
), a substantial amount of SOD1, estimated at between 1 and 2% of total SOD1, is localized in various mitochondria compartments. It has been suggested that mutant SOD1 preferentially accumulates within mitochondria of neuronal tissues (67
). The accumulation of mutant SOD1 occurred before the appearance of mitochondria vacuolization, which suggests that the leakage or translocation of mutant SOD1 into mitochondria may be the primary event triggering their further degeneration (49
). SOD1 mutants associate with mitochondria isolated from spinal cord and motor neuronal cells to a much greater extent than WT SOD1 or endogenous SOD1 (33
), suggesting that this accumulation may represent a common toxic property of various SOD1 mutants.
In NSC-34 cells, SOD1 mutants localized into mitochondria and caused mitochondrial impairment, even when a relatively low level of mutant protein was expressed (33
). Mitochondrial localization of SOD1 was essential for mutant SOD1-mediated neurotoxicity in another motor neuron–like cell model of fALS, but no effects were observed when SOD1 mutants were targeted to nuclei or the endoplasmic reticulum (98
). Our unpublished data from NSC34 neuronal cells stably overexpressing either cytosol-targeted or mitochondria-targeted SOD1 (WT, G93A, and G85R) suggest that SOD1 targeted to mitochondria is sufficient to cause cell toxicity similar to that of untargeted SOD1, under a variety of stress conditions (I. Hervias and G. Manfredi, unpublished observations). Furthermore, overexpression of human copper chaperone for SOD1 (CCS) in G93A SOD1 mice highly enriched the amount of mutant SOD1 within mitochondria and caused a remarkably accelerated disease phenotype accompanied by very early mitochondrial abnormalities (92
). Because of the effects of mutant SOD1, mitochondria may become bad cargos for the transport machinery and deliver a reduced ATP supply to molecular motors, resulting in abnormal mitochondrial dynamics. However, a lack of ATP locally available to the motors would not be sufficient to explain the apparently selective impairment of anterograde transport in ALS motor neuron axons (27
). Therefore, it is likely that factors other than bioenergetic impairment contribute to mitochondrial motility defects.
Aberrant SOD1 aggregation affects cytoskeleton integrity and may also hinder mitochondrial transport. Indeed, neurofilament proteins inclusions appear in the axons of motor neurons (115
). Abnormal activation of p38 or cdk5/p35 kinases or both by mutant SOD1 can phosphorylate neurofilaments and impair their transport. Axonal transport was impaired in the ventral roots of G93A mice coincidental with the appearance of neurofilament inclusions and vacuoles in the proximal axons and soma of motor neurons (115
). Perturbations of normal transport can affect mitochondria distribution along neurites and around synapses, which are highly dependent on mitochondria to maintain their structure and function.
It is likely that SOD1 also interacts with components of the mitochondrial transport machinery and that these aberrant interactions lead to dysfunctional mitochondrial dynamics. Topologically, all of the proteins involved in mitochondrial transport and dynamics hold the potential to interact with mutant SOD1, because we have demonstrated that SOD1 resides both on the cytosolic side of the outer membrane and in the intermembrane space (104
Kinesin-1, the principal motor for mitochondrial anterograde transport, is an interesting candidate for interaction with mutant SOD1, because it has been shown to play an important role in retrograde movement of organelles (80
), although mitochondrial transport was not investigated specifically in this study. In particular, the heavy-chain kinesin family-5B (KIF5B) has been implicated in mitochondrial and membrane-bound organelle transport in axons (45
). Therefore, aberrant SOD1-kinesin-1 interactions may underlie both anterograde and retrograde transport defects in motor neurons in a nonmitochondria-specific manner. However, mutant SOD1 interactions with specific mitochondrial cargo adaptors would point toward a specific alteration of transport. For example, interactions of SOD1 with either Milton or Miro-1, two known mitochondrial cargo adaptors, would have an impact on mitochondrial docking to kinesin heavy chain.
The only mutant SOD1 interaction with the axonal transport machinery demonstrated so far is with the dynein complex, the motor responsible for retrograde transport (116
). These aberrant interactions between dynein and several SOD1 mutants, but not WT SOD1, which were also found in ALS transgenic mice, add one more piece to the puzzle and suggest that the axonal transport machinery is a target of SOD1 toxicity (95
Our finding of shorter, hyperfragmented mitochondria in the processes of mutant SOD1 neurons () could be the result of either defective transport, because mitochondria cannot establish proper contacts with transport-based machinery, or impairment of the fusion/fission machinery. It has been shown that SOD1 aggregates with Bcl-2 in spinal cord mitochondria (79
), and that members of the Bcl-2 family control mitochondrial fusion (90
). Moreover, mutant SOD1 interacts with the dynein complex, and dynein may also mediate the recruitment of the fission machinery to mitochondria (102
Another possibility is that SOD1 may target or activate signaling pathways that affect anterograde or retrograde motors, mitochondrial transport, and cytoskeleton stability. Inflammatory signals from neighboring glial cells (such as TNF-α) can activate p38 stress-activated protein kinase in mutant SOD1 transgenic mice (1
). Furthermore, glutamate levels are increased in mutant SOD1 mice (11
) and can activate JNK, p38, and cdk5/p35 kinases (1
). Activated p38 not only can phosphorylate and inhibit kinesin-1 activity (26
), but also can phosphorylate neurofilaments (1
). Activated JNK can phosphorylate and damage kinesin-1 (75
), and cdk5/p35 can phosphorylate neurofilaments (97
) and promote neurofilament accumulation, a hallmark of ALS pathology.