Taken together, our data support a central role for LRRK2 in canonical Wnt signaling (summarized as a schematic in Fig. ). In particular, our data are consistent with a model where under basal cell conditions without measurable canonical Wnt signaling activity, LRRK2 is associated with the BDC. Following Wnt stimulation, LRRK2 is recruited to the plasma membrane and directly associates with the intracellular domain of LRP6. LRRK2 also interacts with DVL proteins and proteins of the BDC, which are also recruited to the cell membrane after Wnt signal stimulation. Therefore, LRRK2 is able to assist in the formation of LRP6 signalosomes at the cell membrane. As such, despite the observed importance of GTP-binding and kinase activity, the role of LRRK2 is likely to be primarily as a scaffold for Wnt signaling complexes. This scaffold function takes place under basal conditions in the cytoplasmic BDC and after signal activation in association with LRP6 at membranes. LRP6 signalosomes are then internalized into the endosomal system, leading to the sequestration of GSK3β and other Wnt signaling components into multi-vesicular bodies. While it remains to be determined whether efficient intracellular trafficking of LRP6 signalosomes requires LRRK2, it is intriguing to note that both LRRK proteins are implicated in endocytosis (33
). The interaction of LRRK2 with DVL proteins, β-arrestin and GSK3β also suggests the possibility that LRRK2 participates in non-canonical Wnt signaling, although this possibility requires further investigation.
Figure 7. Schematic summary of the main study findings. (A1) In the basal state, LRRK2 is associated with the BDC. The distribution of BDCs between membrane and cytosol is in dynamic equilibrium dependent on interactions between Fz receptors (FzR) and DVL proteins, (more ...)
mutations to the LRRK2 Roc, COR and kinase domains all weaken the activation of canonical Wnt signaling elicited by the overexpression of DVL1 and LRRK2 (Fig. F), although whether these mutations disrupt Wnt signaling in vivo
remains to be determined. However, given that LRRK2 interactions with LRP6 (Fig. E), DVLs (21
) and GSK3β (22
) are all altered by pathogenic PARK8
mutations, it seems unlikely that β-catenin activation will remain unaffected. Such in vivo
studies might be difficult however, since PARK8
mutations are not fully penetrant—in fact, the most prevalent G2019S mutation is thought to have a penetrance of around 30% at the age of seventy (2
)—suggesting that observed effects on Wnt signaling might only be obvious in aged animals or following additional insults, e.g. challenge with agents causing oxidative stress or co-existing genetic variables.
Wnt ligands, Wnt antagonists and β-catenin modulators are expressed in overlapping patterns throughout the developing central nervous system (37
), demonstrating the redundancy of function for some as well as specific function for other Wnt signaling components. The pattern of Wnt β-catenin activity in the adult brain is also complex mirroring suggested Wnt signaling functions in neuronal maintenance, including synapse formation, synaptic plasticity and cell proliferation, and is further complicated by the observation that the expression of some Wnt components is age- and sex-dependent (39
). The most evident Wnt-responsive tissues in the adult brain include the dentate gyrus, hippocampus, sensory telencephalic cortex, several thalamic nuclei, collicula and cerebellar cortex (40
). Importantly, Wnt signaling pathways have also been demonstrated to be of fundamental importance for the biology of dopaminergic neurons in the ventral midbrain (42
). For example, impaired dopaminergic development and altered midbrain morphology is seen upon the disruption of Wnt1
) and Lrp6
) genes encoding two proteins that function specifically within the canonical Wnt pathway. A recent study also suggested an interaction between dopamine D2 receptors and β-catenin in the adult brain (48
LRRK2 is also a ubiquitously expressed protein, found in relative abundance in most brain regions, including the substantia nigra, thalamus, striatum, cortex, olfactory tubercle, nucleus accumbens, hippocampus and cerebellum (49
). Interestingly, LRRK2 was also shown to be present in the subventricular zone (SVZ), suggesting a role for LRRK2 in adult neurogenesis (53
). Deregulated Wnt signaling has also been shown to play an important role in impaired adult neurogenesis seen in PD. In particular, reactive astrocytes and microglia were shown to protect dopaminergic neurons in animal models of PD by activating canonical Wnt signaling and promoting neurogenesis from adult SVZ neuroprogenitor cells, by a mechanism based on the interplay between inflammation and canonical Wnt signaling (55
). Investigation of an interaction between LRRK2 and Wnt signaling contributing to adult neurogenesis in the SVZ would be of great interest to further research into the pathogenesis of PD and the identification of therapeutic targets.
The impairment in canonical Wnt signaling activation caused by PARK8
mutations in our study (Fig. F) is in good accord with decreased Wnt signaling observed in neurodegeneration causing Alzheimer's disease and frontotemporal dementia (58
). Moreover, a general requirement for Wnt signaling for the development of dopaminergic neurons and negative effects of decreased Wnt signaling on neuronal survival is well established (42
). However, other studies show that increases in Wnt signaling might also lead to neurodegeneration, as observed in PARK2
early onset PD and JNPL3 frontotemporal dementia mouse models (20
). Thus, it is likely that neuronal Wnt signaling is subject to regulation within defined boundaries to permit normal neuronal and synaptic function without eliciting cell cycle reentry leading to cell death in postmitotic neurons. Thus, investigations into the effects of PARK8
mutations on Wnt signaling in patients with familial and idiopathic PD are a future priority.
Dysregulated canonical Wnt signaling represents an intriguing candidate mechanism for PD pathogenesis. The importance of Wnt signaling for basic neuronal functions is well established, not least synaptic plasticity (10
) and the control of microtubule stability (64
). It is important to note that not all of these events require β-catenin-dependent transcriptional regulation, indicating both genomic and non-genomic actions. Moreover, the development of ventral mid-brain dopaminergic neurons appears particularly dependent on canonical Wnt signaling (42
). Thus, dopaminergic neurons could be particularly sensitive to mild perturbations in canonical Wnt signaling that would elicit a slow but progressive loss of neuronal function ultimately ending in cell death. Furthermore, dysregulated Wnt signaling has the potential to explain the link between PD and variations in MAPT
(encoding the microtubule-binding protein, tau) linked to PD in numerous genomic linkage studies (68
). Tau is well described as a GSK3β substrate, and this phosphorylation event can be modulated by Wnt ligands (19
). Moreover, tau phosphorylation is a pathological hallmark of Alzheimer's disease (19
) and frontotemporal dementia and has been strongly linked to PARK8
mutations and increased LRRK2 expression in both PD patients and animal models (22
). Thus, in addition to suggesting an intriguing mechanism for PD pathogenesis, our data also suggest tantalizing links to Alzheimer's disease and frontotemporal dementia.
Our observations are also likely to have implications beyond neurodegeneration. For example, constitutive activation of β-catenin is well described in numerous types of cancer and we note that an increased risk of non-skin cancer has been observed for individuals with PARK8
). Moreover, the capacity of the LRRK2-IN-1 compound to inhibit canonical Wnt signaling (Fig. B) suggests the possibility of targeting LRRK2 kinase activity in the treatment of tumors linked to elevated β-catenin activation. Unfortunately, LRRK2 kinase inhibitors showed an inhibitory effect on Wnt signaling that was similar to that seen with pathogenic LRRK2 mutations. This suggests that the usefulness of LRRK2 inhibitors for the treatment of PD might be limited. In fact, LRRK2 inhibitors could have detrimental effects on disease progression and cognitive ability, since decreased Wnt signaling was observed in Alzheimer's disease (19
In conclusion, our data show LRRK2 to be a central component of canonical Wnt signaling. We propose that dysregulated Wnt signaling is a new potential pathomechanism leading to PARK8 PD and suggest that alterations in Wnt signaling pathways might also be a common cause of idiopathic PD. Our data also suggest that the targeting of LRRK2 kinase activity might not be the most suitable approach for the treatment of neurodegeneration. In contrast, controlled Wnt signaling activation—e.g. with small molecules targeting LRP6—appears a more promising therapeutic approach for PD.