Mutations in the LRRK2 gene are among the most common genetic causes of PD, yet the clinical features of LRRK2 PD are largely indistinguishable from those of sporadic PD(6, 42). LRRK2 also reportedly plays a role in idiopathic PD with postmortem brain tissue from patients with idiopathic PD showing enhanced LRRK2 kinase activity(8). Understanding LRRK2 PD should therefore provide more insights into the underlying mechanisms and help create new therapeutic opportunities for idiopathic PD(42). However, there is no murine LRRK2 PD model that fully recapitulates human PD(43).
The G2019S mutation is one of the most common mutations of LRRK2 in PD and has activating and gain-of-function effects on LRRK2 kinase activity(6). Studies using LRRK2 G2019S Tg mice have shown some PD-related phenotypes. These include loss of dopaminergic neurons, disruption of dopamine homeostasis, which is accompanied by dopamine-dependent behavioral deficits, and α-synucleinopathies(44–46). A degenerative phenotype in the substantia nigra of these mice is typically observed in aged mice at approximately 15–20 months of age(44, 46). In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced models of PD, LRRK2 G2019S Tg mice were more susceptible to MPTP-mediated neurotoxicity(47, 48). Although LRRK2 G2019S Tg mice can display many of the cardinal features of PD, these models bear several key caveats due to overexpression artifacts or interspecies differences(43). LRRK2 G2019S KI models have been developed to overcome potentially confounding insertional effects of the Tg models(22, 49). However, LRRK2 G2019S KI mouse models have failed to exhibit dopaminergic neuron degeneration or α-synuclein pathology(22, 49, 50). This is probably due to the incomplete penetrance of the mutation(5). These findings suggest the involvement of other environmental or genetic factors in establishing PD models in LRRK2 G2019S KI mice.
Neuroinflammation is increasingly recognized as an essential process involved in PD pathogenesis(51). Injection of a sublethal dose of LPS reportedly resulted in the loss of dopaminergic neurons in LRRK2 G2019S Tg mice as early as seven days after treatment(21). However, we did not observe a similar phenotype in our LRRK2 G2019S KI mice receiving the same dose of LPS two weeks after LPS exposure. Although it is challenging to compare these two studies, we speculate that the reason for the difference might be the context of the G2019S mutation in transgenic versus endogenous LRRK2. The toxicity of LPS to dopaminergic neurons might have been intensified in the LRRK2 G2019S Tg mice utilized by Kozina and colleagues, which displayed a high expression level of LRRK2 G2019S(21). In the scope of our current study, we have not yet established a mouse model of inflammation-associated LRRK2 PD. Previous studies reported that the LPS paradigm in WT mice maintained for 10 months after inflammatory exposure was sufficient to cause dopaminergic neuronal death(20, 30). With astrocyte activation being exacerbated in LRRK2 G2019S KI mice, we anticipate that when we extend the duration of LPS exposure, LRRK2 G2019S KI mutant mice will potentially exhibit an accelerated neurodegenerative phenotype. This hypothesis merits further investigation.
The roles of LRRK2 in microglia have been extensively investigated(14, 52–54), but little is known about the functions of LRRK2 in astrocytes(55–57). Under physiological conditions, astrocytes are essential for brain homeostasis because they provide neurotrophic factors to support neurons and promote synapse formation and plasticity(58). However, astrocytes undergo sequential phenotypic changes called “reactive astrocytosis” in response to brain injuries and diseases. Reactive astrocytes are present and drive neuronal death in many neurodegenerative disorders, including PD(19, 39). Recent studies have reported alterations in astrocytes in LRRK2 PD patients. These include morphological and metabolic alterations, which are recognized as pathological features of PD(59, 60). It has been reported that induced pluripotent stem cell (iPSC)-derived astrocytes from PD patients carrying the LRRK2 G2019S mutation exhibit many PD features, such as increased expression of α-synuclein, thereby increasing the responsiveness of these cells to inflammatory stimuli(59). In another study, astrocytes generated from the iPSCs of PD patients with LRRK2 G2019S mutations exhibited irregular mitochondrial morphology, decreased mitochondrial activity and ATP production, and increased reactive oxygen species (ROS) production(60). These astrocytes thus fail to support neurons homeostatically and may have contributed to dopaminergic neurodegeneration. ]
In the model of neuroinflammation triggered by LPS, we observed an astrocyte activation-potentiating phenotype in LRRK2 G2019S KI mice. Further studies are needed to understand the toxicity of activated astrocytes to neurons and the underlying mechanisms involved. In addition, with a greater degree of astrocyte activation, LRRK2 G2019S KI mice could serve as a mouse model to study the anti-inflammatory effects of small molecules and their specificity for LRRK2.
Caffeine is an adenosine A2A receptor antagonist and the most widely consumed psychoactive substance(61). Epidemiological and metabolomic studies have shown that higher coffee and caffeine intake is associated with a reduced risk of PD(62, 63), and the serum levels of caffeine and its metabolites are lower in idiopathic PD patients(61, 63). Preclinical models also support the neuroprotective effects of caffeine in PD(40, 64, 65). In a metabolomic profiling study carried out to identify markers of resistance to developing PD among LRRK2 mutation carriers, our group identified caffeine and its related analytes as potential modulators(23). We observed evidence of protection by caffeine against LPS-potentiated astrocyte activation in mice bearing the pathogenic mutation G2019S of LRRK2 at a dosage producing concentrations achieved with typical human consumption(40, 41). The effects of caffeine in the CNS, including its psychostimulant and neuroprotective effects, are mediated by its antagonistic effects on adenosine receptors(66), most prominently on A2A receptors(41). LRRK2 expression is enriched in the striatum(67, 68), the brain area that is laden with A2A receptors(69), and is also known to play a role in developing striatal circuits(70). Thus, the involvement of LRRK2 in striatal neuroplasticity may underlie the potentiation of the neuroprotective effects of caffeine in the context of pathogenic LRRK2 mutations. Interestingly, caffeine’s effects appear to be specific to LRRK2 G2019S KI mice in our current study. The enhanced kinase activity of LRRK2 appears to underlie the toxicity of the protein in the model. However, we did not find any evidence that caffeine ameliorates the phosphorylation of the protein at its phosphorylation site or its substrate, which is a readout of its kinase activity(7, 32, 33). Further studies will be needed to unravel the molecular networks that are potentially altered by the G2019S mutation of LRRK2 and how they are modulated by caffeine treatment. As caffeine specifically attenuates a pathogenic phenotype in LRRK2 G2019S KI mice, the development of this relatively low-risk dietary and pharmacological agent as a candidate therapeutic for PD patients with LRRK2 mutations or as a preventive strategy for at-risk LRRK2 mutation carriers to reduce the penetrance of PD is possible.
In summary, our findings revealed that in a model of inflammation triggered by LPS, LRRK2 G2019S KI mice displayed increased LRRK2 kinase activity. The enhanced kinase activity of LRRK2 in LPS-challenged LRRK2 G2019S KI mice was likely accompanied by delayed weight recovery and increased astrogliosis, which was ameliorated by caffeine administration. Our findings thus add to the increasing body of evidence that the kinase activity of LRRK2 underlies its toxicity in PD pathogenesis and support the therapeutic potential of caffeine in LRRK2 PD.