Parkinson's disease (PD) is the second most common age-related neurodegenerative disease after Alzheimer's disease (AD) 12. Like many other neurodegenerative diseases, PD and Lewy body dementia (LBD) are increasingly recognized as disorders of protein aggregation and inclusion body formation 3; in particular, PD is defined by the presence of Lewy bodies and Lewy neurites 45. Cortical Lewy bodies also constitute the defining neuropathological characteristics of LBD, a common form of dementia that exists in a "pure" form or overlaps with AD neuropathology 4. α-Synuclein is the major component of the Lewy body in PD and LBD 6 and is currently used as a diagnostic marker of Lewy bodies. Mutations in α-synuclein are also described in a subset of familial PD kindreds, whereas aggregated α-synuclein occurs in both familial and sporadic PD lesions 7. Besides α-synuclein, many molecules such as 14-3-3 protein and ubiquitin have been found in Lewy bodies 8910. It is also suggested that 14-3-3 protein which shares amino acid sequence homology with α-synuclein may be associated with Lewy body formation 8.

It is generally accepted that progressive, irreversible and regionally specific neurodegeneration, with Lewy bodies and Lewy neurites are the essential pathological hallmarks of idiopathic PD. The precise etiology, however, is unclear. One of the leading hypotheses suggests that oxidative stress and generation of reactive oxygen species damages macromolecules, resulting ultimately in cell death 11. In fact, Lewy bodies themselves have been shown to contain adducts induced by oxidative stress 12.

The forkhead box transcription factor, class O (FOXO) is the mammalian homologue of DAF-16, which is known to regulate life span of Caenorhabditis elegans 1314. In mammals, the FOXO class of transcription factors are key players in the regulation of cell-fate decisions, such as cell death, cell proliferation, and cell metabolism 15. It has been reported that phosphorylation/dephosphorylation of FOXO protein, which results in the translocation between cytoplasm and nucleus, is a major regulatory mechanism of FOXO-dependent-transcription 16. A recent study indicated that three FOXO family members including FOXO1, FOXO3, and FOXO4 play essential roles in the response to physiologic oxidative stress in hematopoietic stem cells of experimental mice 17. In the present study, we examined the expression of FOXO3a (also known as FKHRL1) in brain tissue from cases of PD and LBD.

The cellular localization of FOXO3a was examined in the hippocampus of LBD, AD/LBD, and age-matched control patients by immunohistochemical techniques using an antibody to FOXO3a. Whereas immunoreactivity of FOXO3a localized to the cytoplasm similarly in hippocampal neurons in control cases (Fig. 1A) as well as in cases of LBD (Fig. 1B), FOXO3a strongly and specifically localized to cortical Lewy bodies in all cases of pure LBD (Fig. 1C) and AD/LBD (Fig. 1D, case of age 64). Lewy neurites also contained FOXO3a in LBD and AD/LBD (Fig. 1E). Immunostaining these same cases for 14-3-3 protein also revealed specific localization in cortical Lewy bodies (Fig. 1F, case of age 59).

The significant overlap between the immunoreactivity to α-synuclein and FOXO3a further confirms the specific localization of FOXO3a in cortical Lewy bodies as well as in classical Lewy bodies in the brainstem of cases with PD (Fig. 2). Most Lewy bodies were found to be positive for α-synuclein and FOXO3a on adjacent serial sections, indicating the presence of FOXO3a in Lewy bodies in PD brain.

The localization of FOXO3a to Lewy bodies and Lewy neurites in PD and LBD indicates that FOXO3a is a potential component of Lewy bodies. While Lewy bodies are known also to be comprised of α-synuclein and ubiquitin 21 and occur in a variety of clinical disease states 7222324, the mechanism of α-synuclein accumulation in the Lewy body and its role in disease is poorly understood 7. The finding of FOXO3a in intimate association with PD/LBD lesions suggests that LB and Lewy neurite formation encompasses more basic cellular pathophysiology than simple "gain of function" toxicity. Given the role of FOXO in such processes as phosphorylation, acetylation/deacetylation, ubiquitination, and protein-protein interactions 25, the potential of multiple hits cannot be discounted 2627. In fact, FOXO3a has been shown to trigger the death of motoneurons in mice with the translocation from cytoplasm to nucleus 28, and a recent study also showed oxidative stress induces neuronal necrosis by activating FOXO3 29.

Interestingly, however, several lines of evidence now indicate that α-synuclein accumulation is fundamentally protective 3031. In our own studies, α-synuclein aggregation occurs in concert with microtubule polymerization and that an aggregate, once formed, is cytoprotective response against noxious stimuli 3233. This is compatible with the once controversial concept that neurodegenerative disease lesions are markers of pathogenic disease response rather than indicators of etiology 3435

Oxidative stress is believed to be an important factor in cell death induction and considerable evidence has accumulated that oxidative stress is involved in the pathogenesis of PD and α-synuclein aggregation 363738. As a transcription factor, it is necessary for FOXO3a to locate in nucleus to play its cell death-inducing role. In the present study, the high level expression of FOXO3a protein in an inclusion body in the neuronal cytoplasm suggests that FOXO3a may not reach the nucleus (e.g., to affect its known function in the apoptotic pathway). This finding adds further evidence to the notion that Lewy bodies may protect affected neurons from death and furthers the notion that profound alterations in cytoplasmic-nuclear trafficking are a key element of the neurodegenerative process 3940. The finding of the specific isoform FOXO3a in neurodegenerative disease as presented in this work, as well as its role in attenuating amyloid-like pathology in mouse models 41, clearly supports the investigation of the other members of the FOXO family transcription factor in aging and disease.

14-3-3 is a family of dimeric proteins that can modulate interaction between proteins and they are involved in cell signaling, regulation of cell cycle progression, intracellular trafficking/targeting, cytoskeletal structure and transcription 4243. In previous studies, phosphorylated FOXO3a has been shown to bind to 14-3-3 protein, causing cytoplasmic retention of phosphorylated FOXO3 and inhibition of FOXO3-induced transcriptional activation 16. Interestingly, 14-3-3 protein has also been localized to Lewy bodies 8. Therefore, while the mechanism of accumulation of FOXO3a in Lewy bodies awaits further study, a direct interaction between FOXO3a and 14-3-3 protein remains a distinct possibility. Since α-synuclein shares physical and functional homology with 14-3-3 proteins 44, it is reasonable to speculate that FOXO3a, α-synuclein, and 14-3-3 protein may form a complex, preventing the dephosphorylation and translocation of FOXO3a, and promote cell survival. This potential protein interaction is the subject of ongoing studies in our laboratory.

Our study demonstrates the localization of FOXO3a protein to Lewy bodies and Lewy neurites, suggesting a role of FOXO3a in the morphogenesis of inclusions in synucleinopathies. Given the pleiotrophic effects of FOXO3a in cellular pathophysiology, our results further the increasingly important concept that inclusion formation is a complex process, favoring an adaptive disease response over a primary deleterious process. Further studies investigating direct protein interaction in vitro and in vivo are in progress.

(AD): Alzheimer's disease; (FOXO): forkhead box transcription factor, class O; (LBD): Lewy body dementia; (PD): Parkinson's disease; (TBS): Tris-buffered saline.

The authors declare that they have no competing interests.

HL, SLS, RC, and BS carried out the experiments. XWZ, RJC, MAS and HGL designed the study, analyzed data, and wrote the manuscript. SGC, XW, EK, AT, and GP participated in the design of the study and helped analyze the data. All authors read and approved the final manuscript.