We have previously demonstrated that dLRRK/LRRK2 phosphorylates and stimulates FoxO, which confers neurotoxic activity to FoxO, activating the expression of pro-apoptotic proteins such as Bim/Hid 
. Searching for LRRK2-FoxO signaling components, we found that Drosophila
cGK DG2 also exacerbates FoxO-mediated neurotoxicity. The current study suggests that cGKII/DG2 activates FoxO similar to, but independently of, LRRK2. However, in spite of the similar activation mechanism, the genetic results suggested that the Hid-DIAP-Dronc pathway is not a major cause of the optic degeneration by DG2-FoxO (Fig. S8A
–D). Supporting this result, a quantitative RT-PCR analysis showed that DG2 or DG2/dFoxO does not effectively stimulate FoxO-mediated transactivation of hid
as well as 4E-BP
(Fig. S8E and F
). We attempted to determine downstream effector(s) of DG2-dFoxO using a combination of microarrays, real-time PCR and Drosophila
genetic screening, but could not identify any candidate genes, suggesting that DG2 has more complex functions in gene regulation. For example, DG2 might modulate another transcription regulator through phosphorylation along with dFoxO.
Activation of the NOS-sGC pathway leads to increased cGMP levels 
, which in turn has physiological consequences by regulating cGMP effector proteins such as cGMP-regulated ion channels, cGMP-regulated phosphodiesterases, and cGKs 
. It is widely appreciated that cGKs have a variety of roles in tissues, and in the central nervous system. For instance, cGKs regulate neurotransmitter release/uptake and receptor trafficking, neuronal differentiation and axon guidance, synaptic plasticity and memory through the phosphorylation of substrates 
. There are two cGK isoforms, cGKI α/β and cGKII, in vertebrates. While cGKI α/β is cytosolic and mainly found in the cerebellum, cerebral cortex, hippocampus, hypothalamus, and olfactory bulb of the brain, cGKII is located in the cellular membranes and widely distributed in the brain 
. Here, we demonstrated that cGKII is abundantly expressed in DA neurons in the substantia nigra of the murine midbrain, suggesting that cGKII has a pathogenic role similar to DG2.
What signal mediates stimulation of cGMP synthesis and subsequent cGKII activation in PD remains unclear. The activation of microglia is believed to be one of the pathological processes 
, which might begin with the release of aggregated proteins such as oligomeric α-synuclein from neurons into the extracellular space 
. Inflammation will be amplified by microglial activation and the release of proinflammatory cytokines and inducible NOS 
. Similarly, dNOS, the only NOS orthologue in Drosophila
, is involved in an immune response 
. Thus, inducible NOS responding early to inflammation could be a trigger of the cGKII-FoxO-mediated neurotoxic pathway in humans. In this context, pathogenic LRRK2 with increased kinase activity might potentiate the above pathogenic mechanism. We found that cGKII physically interacts with LRRK2 (Fig. S9
), and that they are co-localized at the endosomes (Fig. S10
) although our current study suggests LRRK2 and cGKII act independently in the context of FoxO activation. However, we observed that co-expression of cGKII KD and LRRK2 3KD partially stimulates FoxO (). These kinases have been reported to form a dimmer when activated 
. Thus overexpression of kinase-dead forms of cGKII and LRRK2 may accidentally recruit and activate the endogenous kinases in 293T cells although we could not detect the endogenous expression of cGKII in this cell line.
The involvement of NO signaling in PD has been suggested by the findings of higher levels of nNOS and iNOS in the nigrostriatal region and basal ganglia in post mortem PD brains 
. The emerging evidence for pathogenic roles of microglia and astrocytes in PD now supports the idea that glia-induced inflammation and NO production promote the disease's development. However, most studies with post mortem samples or PD models showed only that NO could be a generator of oxidative stress since NO is a free radical involved in a wide range of physiologic events 
. A very recent study on rodent models of PD have shown that specific inhibition of sGC-cGMP signaling improves basal ganglia dysfunction and motor symptoms, suggesting that NO signaling could act specifically on PD etiology 
. Our study here provides the possibility that NO signaling downstream to cGK along with FoxO has a pathogenic role in PD.
The relationship between the NO signal and FoxO has been pointed out in a report on a tail suspension-induced model of muscle atrophy, where nNOS-NO is suggested to induce muscle atrophy by upregulating the muscle-specific E3 ubiquitin ligases MuRF-1 and atrogin-1/MAFbx through FoxO activation. Since, the AKT signal is not involved in this mechanism, the molecular mechanism by which FoxO is regulated by nNOS-NO remains unknown 
. Considering our finding regarding neurodegeneration, cGK may regulate FoxO as a mediator of the NO signal in the atrophic muscles as well. Studies have shown that cGK indirectly activates FoxO4 through activation of the JNK pathway 
, which provides anti-tumor effects in colon cancer cells. Although the proposed sites of phosphorylation by JNK do not appear to be conserved in dFoxO, there is substantial evidence that JNK-FoxO regulates different cellular processes including anti-aging and cell death in Drosophila
. Thus, DG2 could also stimulate the JNK pathway in conjunction with FoxO, widely affecting a variety of cellular mechanisms. This idea could explain why the FoxO SA mutant failed to suppress the DG2-mediated decrease in lifespan of Drosophila
Although more studies are needed in mammalian systems, our finding of a novel link between the NO signal and FoxO in neurodegeneration suggests that appropriate pharmacological control of the NO pathway would prevent or diminish pathological problems in PD.