Axonal swellings, including globules and spheroids, are characteristic features of axonopathies observed in a number of diseases, including ischemia, trauma, neuroaxonal dystrophy, neurodegenerative disorders, as well as in aging. A recent study suggested that dysfunction of the autophagy-lysosome pathway could be one major contributor to axonal swellings[30, 31]. Failure to degrade subcellular materials or organelles at distal axons and/or nerve terminals or failure to export these materials by axonal transport has been shown to produce swollen nerve terminals. Such a mechanism might be involved in formation of αS- and P123H βS-globules. In the present study, αS-globules in brains of αS tg mice were characterized by autophagosome-like membranous elements and were immunopositive for various minor gangliosides, which is reminiscent of some types of lysosomal storage disease. Consistent with this, lysosomal activity, as assessed by the activities of cathepsins B and -D, was significantly decreased in brain extracts of αS tg mice compared with those from non-tg littermates. Similar lysosomal dysfunctions were previously observed for P123H βS-globules in brains of P123H βS tg mice. Taken together, these results suggest that downregulation of the lysosome degradation pathway may be a common mechanism leading to globule formation in αS and P123H βS tg mice.
In contrast to the lysosomal pathology, mitochondria accumulated specifically in αS-globules. To the best of our knowledge, only one study has previously described abnormal mitochondria in the axonal pathology in tg mice expressing prion promoter-driven αS. In agreement with this study, immunoelectron microscopy of αS revealed abnormal accumulation of mitochondria in αS-globules. Some αS-globules displayed clustering of mitochondria, while others had swollen mitochondria in the peripheral regions. Immunoreactivities of mitochondrial markers such as VDAC1 and cytochrome C were also found in αS-globules. These results suggest that mitochondria clustering might become hyperactive in response to lysosomal dysfunction. Consistent with these findings, αS-globules were associated with oxidative stress, as assessed by staining of 4-HNE and nitrated αS. Conversely, no evidence of mitochondria was obtained in P123H βS-globules, hence oxidative stress (assessed by 4-HNE staining) was less than that in αS-globules. The mechanism through which P123H βS causes mild level of oxidative stress without mitochondria is unclear, but it is noteworthy that cholesterol staining was positive in P123H βS-globules but not in αS-globules. Given that cholesterol and its metabolites are implicated in oxidative stress in the pathogenesis of neurodegenerative diseases, the increased oxidative stress in P123H βS-globules could be partly due to accumulation of cholesterol. A further study is warranted to test this intriguing possibility.
LRRK2 was found to be located in αS-globules and may be actively involved in the axonal pathology. Indeed, it was previously shown that LRRK2 was crucial for regulation of neurite formation and length. Knockdown of LRRK2 led to long, highly branched neuritic processes, whereas constructs with increased kinase activity exhibited short simple processes in neuronal cultures (or transduced nigrostriatal models). More recently, LRRK2R1441G BAC tg mice were shown to have various characteristic axonal pathologies, including large tyrosine hydroxylase-positive spheroid-like structures, dystrophic neurites and enlarged axonal endings. Although the mechanisms are still unclear, the specific accumulation of LRRK2 in αS-globules naturally leads to the speculation that LRRK2 may cooperate with αS in the axonal pathology. In support of this possibility, both αS and LRRK2 have been shown to be commonly involved in pathologies such as impairment of cytoskeleton dynamics and dysregulation of the protein degradation system. Moreover, it was recently shown that various neuropathological features of A53T αS tg mice, such as impaired microtubule dynamics, Golgi disorganization, and decreased proteasomal activity, were worsened by cross-breeding with LRRK2 tg mice, but ameliorated by genetic ablation of LRRK2. Further investigation is required to determine whether αS and LRRK2 cooperate with each other to produce diverse pathologies, including axonal degeneration.
Finally, given that P123H βS may represent a rare familial case of DLB, it is important to consider whether wild type βS has any role in the formation of axonal globules in sporadic cases of α-synucleinopathies. In this context, neurite accumulation of βS has been demonstrated in various synucleinopathies, including PD, DLB, and neurodegeneration with brain iron accumulation, type I. Although wild type βS is neuroprotective, this molecule might become pathogenic during aging. It is also possible that wild type βS might become pathogenic under certain extreme conditions or through the action of specific environmental factors, leading to stimulation of globule formation. Thus, it is an intriguing possibility that the synuclein family of peptides might contribute to the formation of diverse axonal pathologies.