We provide evidence of an abnormal distribution of mitochondria and mutant SOD1 in motor axons, forming a regular pattern of large clusters. Most likely, clustering would have escaped observation in conventional root cryosections that do not allow the analysis of long segments of axons in the same field. In addition, we show evidence that clustering is dependent on SOD1-G93A in motor axons, scarcely found in sensory axons, and appears early at asymptomatic stages of the disease. Colocalization of clusters with molecular markers such as ubiquitin, cytochrome c, nitrotyrosine, and nNOS allows us to suggest that mutant SOD1 and defective mitochondria create localized dysfunctional domains in motor axons, which may provide a clue to understanding progressive axonopathy in ALS.
Motor neurons are highly compartmentalized cells in which >95% of the cell volume is the axon (10
). It might be possible that a yet-unknown feature of motor-axon architecture facilitates the previously reported physical interactions between mutated SOD1 and mitochondria (23
). We attempted to demonstrate such discrete physical interactions by using whole-mount axoplasmic preparation, which was previously shown to provide detailed information of the subcellular localization of large molecules and organelles (20
). This technique allows the visualization of up to thousands of microns of linear axoplasm, preserving its structural features (21
) and its functionality, when performed in non-denaturing conditions (19
). However, the technique also may lead to potential artifacts. For this reason, we included different control experiments to confirm the occurrence of clustering. Thus, we show that clusters also can be found in conventional cryosections and are not associated with overt alteration of kinesin or NF-H distribution, as would be expected in an artifact condition. In addition, we found that SOD1/mitochondrial clustering in sensory axons was observed at very low frequency ( and ), and absent from axons containing WT SOD1, suggesting an increased vulnerability of motor axons.
The characteristic diffuse staining of cytochrome c
immunoreactivity surrounding the clusters suggests mitochondrial dysfunction. Cytochrome c
released in the axoplasm might elicit local activation of proteolytic cascades similar to those triggering apoptosis in the neuronal perikaryon. The implication of such biochemical pathways in the axon compartment is presently unknown. In agreement, nNOS and nitrotyrosine immunoreactivity were increased in axonal clusters, suggesting nitrative stress associated with dysfunctional mitochondria. Nitrotyrosine immunoreactivity in motor axons was previously reported in patients with sporadic ALS (1
). In particular, the mitochondrial/SOD-cluster environment could facilitate the formation of neurotoxic Zn-deficient SOD1 species (5
). Taken together, the characterization of clusters in motor axons indicates that mitochondria in clusters may become dysfunctional. Clusters may modify the function of restricted areas of the axoplasm, interfering, for example, with normal trafficking of organelles or trophic factors.
The SOD1 clusters in axons were preferentially localized in the axonal cortex in early stages and then invaded progressively the axonal core. This particular localization probably reflects the fact that clusters are displaced to the periphery of the axon, allowing the main axonal trafficking in the core. In addition, the increased binding of SOD1 to mitochondria in clusters seems to be specific for the SOD1-G93A mutation, because no evidence of preferential colocalization was observed for endogenous rat SOD1 and overexpressed wild-type human SOD1. Moreover, we provide evidence of a progressive ubiquitination of hSOD1/mitochondrial clusters, which becomes apparent in late stages of the disease. In agreement, previous reports showed increased levels of ubiquinated proteins in the sciatic nerve (14
) and spinal cord of ALS mice (4
Although SOD1 can accumulate in the mitochondrial matrix or interspace, it is likely that misfolded mutant SOD1 in clusters is preferentially associated with the outer membrane of mitochondria (35
). These authors propose that increasing recruitment of SOD1 to the mitochondrial outer membrane will increase as the disease progresses. Thus, at early stages of the disease, mitochondria may depart to the axon from the cell body with minimal content of mutant SOD1 on its outer membrane. It should be established whether SOD1 progressively increases in the outer membrane of mitochondria as they move anterogradely into the axoplasm. It is likely that SOD1 mutations provoke defective mitochondria traffic in axons. Depolarized mitochondria have been reported to be preferentially transported anterogradely (26
). In addition, in vitro
experiments tracking mitochondria in neurons from SOD1-G93A animals reported selective reduction of anterograde transport (11
), which may further reduce the number of axonal mitochondria.
The dynamics of the process of accumulation must be studied to establish whether mitochondria in clusters were being transported anterogradely or retrogradely. A recent study showed that in asymptomatic stages, at the ultrastructural level, mitochondria are accumulated in the axon hillock and initial segment (30
). Interestingly, aggreosome formation (18
) in culture cells expressing SOD1-G93A indicated that a tubulin minus end directed traffic of organelles ending in formation of abnormal accumulation in the centrosomal area. If this be the case in the motor neuron, a large proportion of damaged mitochondria could be targeted to the cell body in a slow process leading to the accumulation of vacuolated mitochondria in the cell perikaryon.
Taken together, our observations further emphasize the importance of the axonal compartment in ALS, as suggested in the dying-back model of progression of the disease (13
). In this regard, it was observed that SOD1 can be synthesized in axons (34
). This may result in local accumulation of misfolded mutant SOD1 in the axonal compartment. Further characterization of early axonal SOD1 homeostasis and mitochondrial dysfunction could establish new therapeutic targets and may contribute to understanding the nature and relevance of distal axonal degeneration in ALS.