The present study showed that in a neuronal cell line and in a transgenic mouse model of LBD, lentivirus mediated over-expression of beclin 1 reduced the abnormal accumulation of α-syn and related neuronal pathology by inducing autophagy. In neuronal cultures, α-syn aggregates co-localized with lysosomal markers and were associated with alterations in the autophagy pathway that lead to reduced neuronal plasticity. The effect of beclin 1 overexpression was related to the autophagy pathway because blocking with 3-MA or bafilomycin A1 resulted in greater accumulation of α-syn, while rapamycin had the opposite effect. Previous studies (Berger et al., 2006
; Sarkar et al., 2007
) have emphasized the upregulation of autophagy by pharmacological approaches (eg: rapamycin) but this is the first study to show a specific gene therapy directed method for upregulation of autophagy as a therapeutic tool for the treatment of accumulated α-syn. This approach can be specifically targeted to regions of the brain most affected by accumulation of α-syn aggregates.
In agreement with previous studies (Xilouri et al., 2009
) we found that neuronal cells with accumulated α-syn showed some increase of LC3 II levels but with impaired lysosomal function. More detailed analysis by confocal microscopy showed that the increased numbers of LC3-GFP autophagic structures were enlarged and irregular. Ultrastructural analysis revealed these electrodense structures to be extremely abnormal. In particular, there were numerous structures with single membranes and most structures were much larger than normal autophagic vesicles. This coupled with the alterations in LC3-GFP punctate staining suggest these may be immature autophagic vesicles. This suggests interference with the normal maturation pathway of the autophagic vesicles.
The mechanisms through which accumulation of wildtype α-syn might impair macroautophagy in our neuronal cells are not completely clear. However, a previous study using differentiated SH-SY5Y and primary neurons showed that the alterations in macroautophagy in wild type α-syn expressing cells were attributed to the blockage of CMA (Xilouri et al., 2009
). Moreover, studies have shown that mutant α-syn inhibits CMA and triggers increased macroautophagy and lysosomal dysfunction independent of CMA (Cuervo et al., 2004
; Xilouri et al., 2009
A therapeutic approach with rapamycin or beclin 1 might act to reestablish the physiological autophagy pathway. This view is supported by the studies with LC3-GFP, immunoblot with LC3 and ultrastructural studies in the α-syn tg mice treated with LV-Beclin 1 where we detected the presence of autophagic vesicles of normal appearance in contrast to α-syn tg mice treated with LV-control where abnormal and enlarged autophagic vesicles were observed.
This is consistent with recent studies showing that α-syn aggregates are cleared via autophagy and that alterations in the lysosomal pathway might participate in the mechanisms of α-syn-mediated neurodegeneration (Stefanis et al., 2001
; Meredith et al., 2002
; Webb et al., 2003
; Cuervo et al., 2004
; Rideout et al., 2004
; Nakajima et al., 2005
; Rockenstein et al., 2005b
). Mutant forms of α-syn found in familial PD patients have been shown to block autophagy and α-syn contains a consensus sequence for CMA targeting (Cuervo et al., 2004
). Further supporting a role for autophagy dysfunction in these disorders, recent studies have shown that in lysosomal storage diseases such as Gaucher’s disease (Tayebi et al., 2001
; Varkonyi et al., 2003
) and Niemann-Pick disease (Saito et al., 2004
), there is increased susceptibility to develop parkinsonism and α-syn accumulation.
Therefore, activation of the autophagy pathway by pharmacological means or gene transfer might reduce the pathology associated with the neuronal accumulation of α-syn aggregates. We have previously shown that beclin 1 is capable of inducing autophagy and reducing the neuropathological alterations in APP tg models of AD (Pickford et al., 2008
). In the present study we showed that beclin 1 gene transfer is also capable of reducing the accumulation of α-syn aggregates and associated neuronal deficits. Delivery of the LV-Beclin 1 virus in the limbic system of α-syn tg mice led to improvements in expression of the presynaptic marker, synaptophysin and the postsynaptic marker, MAP2. The decrease in accumulated α-syn was observed in areas of autophagy induction as determined by increases in expression of LC3. Ultrastructural analysis of neurons in the region of LV injection revealed decreased accumulated α-syn and increased markers of autophagy. Thus, the delivery of the beclin 1 by the lentivirus appears to reverse the accumulation of α-syn probably by increasing activation of the autophagy pathway.
These findings are in agreement with recent studies showing that rapamycin or rapamycin analogs are capable of reducing the accumulation of α-syn oligomers via the autophagy pathway (Sarkar et al., 2007
). Treatment of cells with rapamycin, which blocks the mTOR protein activity, results in upregulation of the autophagy pathway (Meijer and Codogno, 2004
). Rapamycin acts by binding to FK506 binding protein 12; which then inhibits phosphorylation of mTor. Moreover, rapamycin treatment has been shown to be protective in animal models of other neurodegenerative disorders such as Huntington Disease (Ravikumar et al., 2004
Similar to overexpression of beclin 1, rapamycin ultimately induces autophagy and reduces overall levels of misfolded proteins. However, in addition to inducing autophagy, rapamycin (via mTor) down-regulates overall protein synthesis and cell proliferation (Wang and Proud, 2006
; King et al., 2008
; Wyttenbach et al., 2008
). These undesirable side effects of rapamycin make its use in neurodegenerative diseases involving misfolded proteins problematic. This, coupled with the fact that rapamycin poorly crosses the blood-brain barrier to exert its effect on neurons (Pong and Zaleska, 2003
), makes the identification of alternative autophagy inducers desirable. Gene delivery of beclin 1 might alleviate some of these problems. The lentivirus, or other viral vector, containing the gene for beclin 1 can be delivered to the exact site of α-syn accumulation, for instance the caudate putamen or substantia nigra in PD or as in this study to the limbic system in case of LBD.
Although the role of beclin 1 in autophagy and cell maintenance is clear, ample evidence suggests that beclin 1 is required not only for cell viability but also for neuronal function in the face of aggregated proteins. Beclin 1 is a coiled-coil, 60kDa protein that is analogous to the yeast autophagy protein Apg6 (Liang et al., 1998
; Liang et al., 1999
). Beclin 1 binds Bcl2 to block apoptosis (Liang et al., 1998
; Takacs-Vellai et al., 2005
; Liang et al., 2006
) and functions through its interactions with Vps34 and PI3 kinase to induce autophagy in an mTor independent pathway (Furuya et al., 2005
; Takacs-Vellai et al., 2005
). Recently identified interacting proteins, UVRAG and Ambra1 also act as regulators of autophagy (Liang et al., 2006
; Fimia et al., 2007
). Beclin 1 has been localized in some instances to the Golgi apparatus of the ER and may play a role in sorting proteins for degradation via the autophagy pathway (Furuya et al., 2005
; Takacs-Vellai et al., 2005
). Thus, beclin 1 may be playing a role in neurons in PD and LBD in targeting excess α-syn to the autophagy pathway thereby reducing the potential for accumulation.
The specific molecular defects in the autophagy pathway leading to PD and LBD are not completely understood yet, however recent studies have shown that reduced Atg5 or 7 expression in knock out mice results in loss of neurons and accumulation of protein in the remaining neurons (Hara et al., 2006
; Komatsu et al., 2006
Taken together, these studies support the view that alterations in the autophagy pathway play an important role in the pathogenesis of disorders with parkinsonism, dementia and α-syn accumulation and that activation of the autophagy pathway might ameliorate the associated deficits. Thus, beclin 1 plays an important role in the intra-cellular degradation of α-syn either directly or indirectly through the autophagy pathway and may present a novel therapeutic target for LBD/PD.