While recent breakthroughs have shed light on the cellular mechanisms involved in familial PD, little is understood about the causes underlying idiopathic PD pathogenesis. In the current study, we attempt to uncover how the loss of an essential catabolic pathway contributes to the pathological features associated with PD by examining the effects of impaired autophagy in DA neurons and its role in maintaining the levels of endogenous PD-related proteins in the brain. We show that the targeted deletion of Atg7 in TH+ neurons results in early axonal dystrophy and degeneration, striatal DA depletion, enlarged dendritic swellings, and delayed cell loss and locomotor dysfunction in mutant mice. Furthermore, we demonstrate a potential link between autophagy deficiency and the accumulation of α-syn and LRRK2 proteins.
Our results indicate that disrupted autophagy has distinct consequences in DA neurons compared to other neuron populations. While inactivation of Atg7
in Purkinje cells causes rapid cell death beginning at 8 weeks (Komatsu et al., 2007a
deletion in DA neurons leads to a delayed and moderate loss of cell bodies (~30%) as late as 9 months. Our stereological analysis suggests that the majority of TH-labeled cell bodies are surprisingly resistant to dysfunctional autophagy. On the other hand, Atg7
-deficient DAergic processes undergo substantial degeneration, which is likely associated with the reduced striatal DA levels observed at 4 months. Despite the severity of these early pathogenic events, locomotor function does not become impaired until 9 months of age, when there is significant DA neuron cell death in cKOTH
mice. Whether the onset of behavioral abnormalities is directly correlated to significant cell loss or reflects the point at which DA depletion or TH+ axon degeneration exceeds a certain threshold remains unclear. However, the gradual decline in locomotor activity observed in our mouse model suggests the latter.
α-Syn is the primary constituent of Lewy bodies and has a significant pathological role in both familial and idiopathic PD. A recent study showed that overexpression of wild-type α-syn impairs autophagic activity (Winslow et al., 2010
), and implies a functional relationship between α-syn and autophagic degradation. Conversely, growing evidence also indicates a role for autophagy in controlling α-syn protein levels (Spencer et al., 2009
; Yu et al., 2009
) and suggests that dysfunctional autophagic clearance may contribute to the development of α-syn inclusions in idiopathic PD. The extent to which autophagy regulates α-syn levels is still unclear, since other catabolic pathways have been implicated (Webb et al., 2003
; Cuervo et al., 2004
; Rideout et al., 2004
). A recent report suggests that although α-syn is mostly degraded by the ubiquitin-proteasome system (UPS) under normal conditions in vivo
, autophagy is recruited as the primary clearance system in transgenic mice expressing elevated levels of oligomeric α-syn (Ebrahimi-Fakhari et al., 2011
). In our study, DAergic axons from cKOTH
mice develop α-syn aggregates at 20 months, well after other neuropathology is observed and in contrast to the early and pervasive appearance of p62 and ubiquitinated inclusions. However, even at this late stage, α-syn deposits are not present in all affected DAergic axons. We detect similar α-syn localization in cerebellar Purkinje axons in cKONes
mice, but at a much younger age (P35) and higher frequency. Purkinje cells are particularly vulnerable to the loss of autophagy at earlier time points (Komatsu et al., 2007a
; Nishiyama et al., 2007
), which may be one of the factors contributing to the early appearance of α-syn aggregates. Presumably, additional neuron populations would display α-syn pathology if cKONes
mice lived beyond several months, therefore future studies should examine autophagy impairment in other cell-specific conditional models. In both cases, endogenous α-syn accumulates in Atg7
-deficient axons, but not within cell bodies. These findings suggest that autophagy is involved in axonal α-syn protein homeostasis in vivo
. While other degradation pathways, such as UPS or chaperone mediated-autophagy serve as the primary mechanisms for α-syn turnover in the soma, autophagy may play a more prominent role in clearing presynaptic α-syn, particularly when the cell is stressed.
We also asked whether autophagy deficiency would increase LRRK2 protein levels, as previous examination of postmortem PD brains revealed that LRRK2 is occasionally found in α-syn-containing Lewy bodies (Zhu et al., 2006
) and a recent study shows that LRRK2 exacerbates α-syn-mediated neuropathology (Lin et al., 2009
). We show for the first time that impaired autophagy leads to LRRK2 accumulation in certain brain regions (e.g. cerebellum), as well as in autophagy-deficient cell lines. Interestingly, our qPCR data in cell culture indicates that LRRK2 accumulation is not the consequence of impaired LRRK2 protein turnover per se, but rather may be related to the marked increase in LRRK2 mRNA. These intriguing results suggest that LRRK2 levels are up-regulated in certain cell types in response to the loss of autophagy. Several reports have shown that autophagic flux is impeded by familial LRRK2 mutations and indicates a role for LRRK2 in autophagy regulation (Plowey et al., 2008
; Alegre-Abarrategui et al., 2009
; Ramonet et al., 2011
). In light of these findings, LRRK2 up-regulation in our cell model may reflect a compensatory response to decreased autophagic activity. Alternatively, enhanced LRRK2 transcription may serve as a response to cellular stress, which suggests that LRRK2 accumulation is an indirect consequence of dysfunctional autophagy. Further studies should examine the mechanisms underlying increased LRRK2 levels induced by impaired autophagy and how it may, in turn, affect α-syn accumulation.
The current study suggests that autophagy-deficient DA neurons in aged animals are susceptible to presynaptic accumulation of α-syn, but not LRRK2. In addition, cerebellar Purkinje cells undergo early accumulation of both α-syn and LRRK2 in mice with brain-specific deletion of Atg7
, which reflects the unique vulnerability of Purkinje cells with impaired autophagy, as shown previously (Hara et al., 2006
; Komatsu et al., 2006
; Komatsu et al., 2007a
; Nishiyama et al., 2007
). Although the cerebellum is unaffected in PD, these findings highlight the potential consequences on PD-related protein levels when neuronal autophagy is severely compromised. While our mouse models do not recapitulate all of the pathogenic features in human PD, our study supports the notion that autophagy is one of several cellular systems that may deteriorate with age and contributes to PD pathogenesis. We propose that insufficient autophagy in CNS neurons, particularly midbrain DA neurons, represents a risk for the development of the disease, even in the absence of PD-related gene mutations. Consequently, the manipulation of autophagic activity should be explored as a potential therapeutic strategy for the treatment of PD.