**3.2 Fruit fly models**

*The Drosophila melanogster* paralogue of human LRRK2, dLRRK (CG5483) has similar GTPase and kinase domains among others, with 46% and 44% homology respectively. Expression of LRRK is ubiquitous in the adult fly brain, which suggests that *Drosophila* is a good model for *LRRK2* (Imai et al., 2008). As with the worm models, fly modelers have employed a number of similar experimental approaches including deletion and manipulation of dLRRK and overexpression of WT and mutant human LRRK2.

## **3.2.1 dLRRK loss of function**

Several groups have used the loss of function e03680 line which has a piggyback transposon that results in kinase-null dLRRK2 (Imai et al., 2008; Kanao et al., 2010; Lee et al., 2010b; Lee et al., 2007; Lin et al., 2010; Tain et al., 2009; Wang et al., 2008) or a chromosomally deficient line dLRRKdf (Imai et al., 2008; Lee et al., 2007). Additionally, loss of dLRRK activity has been achieved by RNAi knock-down (Gehrke et al., 2010; Imai et al., 2008) and by 3KD kinase-dead mutation (Imai et al., 2008; Kanao et al., 2010; Lee et al., 2010b). Results have been contrasting, even between groups using the same line. For example, the e03680 line was reported to have no loss of dopaminergic neurons (Wang et al., 2008, Imai et al , 2008), however locomotive impairment (Lee et al., 2007; Tain et al., 2009), degenerative changes in DA neurons (Lee et al., 2007), irregular nerve terminal morphology/overgrowth and changes in neurotransmission (Lee et al., 2010b) were also reported in this line. Interestingly, Imai and colleagues did report increased levels of dopamine in the e03680 line, supporting neurotransmission or storage changes (Imai et al., 2008). Additionally, heterozygous e03680 flies expressing toxic Tau-GFP had longer dendrites and branching and decreased Tau inclusions (Lin et al., 2010).

Data on second hit response has also been contradictory, with the e03680 flies exhibiting increased (Wang et al., 2008) and decreased sensitivity (Imai et al., 2008) although both groups reported resistance of this line to paraquat. Chromosomally deficient line dLRRKdf

fluorescent tags (Sakaguchi-Nakashima et al., 2007; Samann et al., 2009) driven by tissuespecific promoters whereas others have over-expressed human LRRK2 WT and pathogenic mutants via the dopamine neuron-specific DAT promoters (Saha et al., 2009; Wolozin et al.,

Over-expression of endogenous WT and mutant LRK-1 in neurons resulted in axon guidance defects and embryonic lethality (Samann et al., 2009). Surprisingly, worms expressing human LRRK2 had increased longevity and reduced vulnerability to mitochondrial toxicity, with human WT LRRK2 offering more protection than mutant G2019S, R1441C or R1441C kinase dead LRRK2 (Saha et al., 2009; Wolozin et al., 2008). Despite this protection, transgenic LRRK2 WT and G2019S worms exhibited DA neuron degeneration and decreased dopamine levels, with G2019S mutants exhibiting more severe phenotypes (Saha et al., 2009). These findings were confirmed by Yao et al, who also demonstrated that G2019S and R1441C mutant worms displayed progressive behavioral and locomotor dysfunction, which could be rescued by

Taken together, the data from worm models of LRRK2 suggests that WT LRK/LRRK2 may work in a gain-of-function manner which is antagonistic to DA neuronal survival. While it does seem likely that LRK-1 is linked to mitochondrial pathways in some manner, the

*The Drosophila melanogster* paralogue of human LRRK2, dLRRK (CG5483) has similar GTPase and kinase domains among others, with 46% and 44% homology respectively. Expression of LRRK is ubiquitous in the adult fly brain, which suggests that *Drosophila* is a good model for *LRRK2* (Imai et al., 2008). As with the worm models, fly modelers have employed a number of similar experimental approaches including deletion and manipulation of dLRRK and

Several groups have used the loss of function e03680 line which has a piggyback transposon that results in kinase-null dLRRK2 (Imai et al., 2008; Kanao et al., 2010; Lee et al., 2010b; Lee et al., 2007; Lin et al., 2010; Tain et al., 2009; Wang et al., 2008) or a chromosomally deficient line dLRRKdf (Imai et al., 2008; Lee et al., 2007). Additionally, loss of dLRRK activity has been achieved by RNAi knock-down (Gehrke et al., 2010; Imai et al., 2008) and by 3KD kinase-dead mutation (Imai et al., 2008; Kanao et al., 2010; Lee et al., 2010b). Results have been contrasting, even between groups using the same line. For example, the e03680 line was reported to have no loss of dopaminergic neurons (Wang et al., 2008, Imai et al , 2008), however locomotive impairment (Lee et al., 2007; Tain et al., 2009), degenerative changes in DA neurons (Lee et al., 2007), irregular nerve terminal morphology/overgrowth and changes in neurotransmission (Lee et al., 2010b) were also reported in this line. Interestingly, Imai and colleagues did report increased levels of dopamine in the e03680 line, supporting neurotransmission or storage changes (Imai et al., 2008). Additionally, heterozygous e03680 flies expressing toxic Tau-GFP had longer dendrites and branching and decreased Tau

Data on second hit response has also been contradictory, with the e03680 flies exhibiting increased (Wang et al., 2008) and decreased sensitivity (Imai et al., 2008) although both groups reported resistance of this line to paraquat. Chromosomally deficient line dLRRKdf

2008; Yao et al., 2010).

**3.2 Fruit fly models** 

**3.2.1 dLRRK loss of function** 

inclusions (Lin et al., 2010).

exogenous dopamine (Yao et al., 2010).

contrasting data makes any inferences difficult.

overexpression of WT and mutant human LRRK2.

was also resistant to hydrogen peroxide, and RNAi knockdown of LRRK2 decreased sensitivity to both hydrogen peroxide and paraquat (Imai et al., 2008).

#### **3.2.2 Transgenic fly models**

Many transgenic models of *Drosophila* employ the bipartite GAL4/UAS system (Brand and Perrimon, 1993) giving rise to tissue-specific gene expression. Some groups have used dLRRK WT and comparable mutant ectopic expressors utilizing GAL4 drivers directed to pan neuronal- and dopaminergic neuronal-specific expression among others (Gehrke et al., 2010; Imai et al., 2008; Kanao et al., 2010; Lee et al., 2010b; Lee et al., 2007; Lin et al., 2010). Some studies showed no deleterious effects, such as with locomotion or DA neuronal changes (Lee et al., 2007), whereas others showed that dLRRK mutants and/or WT overexpressors had decrease in dopamine and DA neurons (Imai et al., 2008) decreased dendrite arborization (Lee et al., 2010b; Lin et al., 2010) and neuromuscular junction (NMJ) bouton loss (Lee et al., 2010b). Other groups have utilized transgenic human LRRK2 GAL4 driven *Drosophila* models (Gehrke et al., 2010; Kanao et al., 2010; Lee et al., 2010b; Lin et al., 2010; Liu et al., 2008; Ng et al., 2009; Venderova et al., 2009). Significant retinal degeneration and other eye abnormalities were observed in eye-specific hLRRK2 WT- and mutantexpressing cells (Liu et al., 2008; Venderova et al., 2009) whereas others did not observe this effect (Kanao et al., 2010; Ng et al., 2009).

Flies with ectopic neuronal expression of mutant and/or WT transgenes exhibited locomotive impairment and DA neuron loss (Lin et al., 2010; Liu et al., 2008; Ng et al., 2009; Venderova et al., 2009) and decreased boutons, NMJ length and arborization (Lee et al., 2010b). Endogenous dLRRK (Imai et al., 2008) and transgenic WT hLRRK2 (Ng et al., 2009) protein appeared punctate and localized to the cytoplasm and exogenous dLRRK colocalized to endosomal structures that partially overlapped with synaptic markers (Imai et al., 2008). Interestingly, transgenic mutant LRRK2 tended to form intracellular aggregates (Ng et al., 2009). Lin et al expressed hLRRK2 WT and mutants in *Drosophila* with GAL4 drivers specific for DA neuron dendrites and showed their degeneration (Lin et al., 2010). The hLRRK2 G2019S fly showed the most severe defect, including aberrant localization of axonal proteins, including microtubule-associated protein tau, to dendrites. Dendrite degeneration was exacerbated by overexpression of tau and conversely rescued by knockdown of tau in the transgenic mutant flies, this toxic effect being attributed to increased tau phosphorylation phosphorylation through Sgg (GSK3b homologue) (Lin et al., 2010). Both dLRRK mutants and/or WT overexpressors were significantly sensitive to oxidative stressors paraquat and hydrogen peroxide (Imai et al., 2008). Additionally, transgenic hLRRK WT (Venderova et al., 2009) and mutant (Ng et al., 2009) flies had increased sensitivity to mitochondria Complex I toxin rotenone and WT hLRRK2 flies had a decrease in the number of mitochondria at presynaptic nerve terminals (Lee et al., 2010b).

Taken together, dLRRK2 appears to have some role in the maintenance of dopaminergic neurons and the over-expression of LRRK2 results in locomotive impairment, DA neuron loss, decreased dendrite arborization, protein sorting or trafficking defects and increased sensitivity to oxidative stress. Although mortality rates differed among the studies, most groups saw that the hLRRK2 pathogenic mutants tended to have a more severe phenotype, providing evidence for the gain-of-function hypothesis. Like the worm models, the second hit data, while somewhat contrasting, do go some way to support LRRK2 involvement in stress response and mitochondrial pathways.

Modeling LRRK2 Parkinsonism 77

mutant LRRK2, over-expression of mouse wild-type and mutant LRRK2 and targeted

Several groups have reported LRRK2 knockout mice in varying detail. In 2009, Andreas-Mateo et al reported that mice with partial knockout of exon 39 and complete knockout of exon 40 are viable, grossly normal and have completely intact dopaminergic system in terms of dopamine levels and neuronal number up to 2 years of age and show no altered sensitivity to 1-methyl-4 phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Andres-Mateos et al., 2009). Around the same time Lin et al also reported that no pathological or behavioral abnormalities were observed in their knockout mice that lack LRRK2 exon 2 (Lin et al., 2010). Interestingly, when they crossed the LRRK2 knockout to conditional A53T mice, a complete amelioration of α-synuclein mediated neuropathological abnormalities was observed. The third report which described two lines of knockout mice (one with ablation of exon 1, the other ablation of exons 29/30) also reported a lack of any dopaminergic phenotypes or brain neuropathology (Tong et al., 2010). Surprisingly, a major renal phenotype was found in these mice which included dramatic morphological changes and shrinkage, accumulation of alpha-synuclein and pSer 129 alphasynuclein, impaired ubiquitin degradation, impaired autophagy, apoptosis and inflammation. Interestingly, in a different exon 1 knockout model, yet to be fully described, decreased phospho-tau levels and concurrent increased soluble tubulin levels were reported, implying a

Our group has also created LRRK2 KO mice, which are in the final stages of characterization. Microdialysis in these mice has revealed that while the dopamine release following KCl stimulated is not different in magnitude to the wild-type controls, it appears to be slightly delayed. Starting from about 4 months we have also seen progressive darkening of the kidneys and histopathological analysis reveals severe inflammation.

The first rodent over-expression models were described by McLeod et al in 2006 (MacLeod et al., 2006), whose group investigated both embryonic cDNA electroporation and adult adeno-associated viral expression of WT and mutant LRRK2 kinase domain constructs in rats. A reduction in neurite outgrowth and branching was observed in the embryos expressing mutant G2019S and Y1699C in periventricular layer neural progenitors. In adult rats, viral expression of WT and G2019S in the nigra resulted apotosis in nigral neurons and

Three years later, Chenjian Li's group published the first bacterial artificial chromosome (BAC) mouse models, using human WT and ROC domain mutant R1441G BACs (Li et al., 2009). The mice showed an expression pattern of transgenic LRRK2 that mirrored endogenous expression patterns at levels around 5–fold on an FVB/N background. Striking phenotypes were reported including progressive levodopa-responsive slowness of movement associated with ~33% decreased dopamine release following nomifensine treatment and axonal pathology of nigrostriatal dopaminergic projection. While nigral neuronal loss was absent, the R1441G mice displayed a marked reduction in the number of tyrosine hydroxylase–positive nigral dendrites and accumulation of microtubule associated protein tau pathology in the striatum. Extensive characterization of their expression matched human WT BAC model model was not

knock-in of human mutations into the murine genome.

role for LRRK2 in regulation microtubule dynamics (Gillardon, 2009).

striatal abnormalities including phospho-tau increases.

presented, however no overt phenotypes were noted.

**4.1 Murine LRRK2 knockouts** 

**4.2 Over-expression of LRRK2** 

### **3.2.3 Fly interactor studies**

Studies have been performed utilizing double transgenic hLRRK2 flies concomitantly expressing human PINK1, DJ-1 or Parkin. These fly models exhibited various eye abnormalities and some had decreased DA neuron survival (Venderova et al., 2009) whereas coexpression of Parkin with mutant LRRK2 provided significant protection against DA neuron degeneration that occurs with age or in response to rotenone (Ng et al., 2009) Also, loss-of-function dLRRK suppressed Parkin and PINK1 pathology in flies (Tain et al., 2009). Thus, this data further supports the observations that wild-type *d*LRRK negatively regulates DA neuron survival and provides evidence that there is some sort of relationship between LRRK2 and these other PD-associated genes.

*Drosophila* models have been utilized for the search and characterization of LRRK2 substrates and/or interacting proteins (Imai et al., 2008; Lee et al., 2010b; Tain et al., 2009; Venderova et al., 2009). Imai and colleagues showed that both *Drosophila* and human LRRK2 phosphorylate 4E-BP (a negative regulator of translation and mediator of stress response), and that pathogenic mutations caused hyper-phosphorylation of 4E-BP, which led to reduced resistance to oxidative stress and increased dopaminergic neurodegeneration in flies (Imai et al., 2008). Likewise, loss-of-function e03680 flies had a decrease in the amount of phosphorylated 4E-BP (Tain et al., 2009). This association was confirmed in a study that not only showed that LRRK2 interacted with 4E-BP at the postsynaptic compartment, but also found that hLRRK2 phosphorylated Futsch (MAP1b homolog) and possibly interacted with Tubulin (Lee et al., 2010b). Another study found that dLRRK/hLRRK2 phosphorylated FoxO (a transcription factor involved in cell metabolism and oxidative stress which regulates 4E-BP), which subsequently activated downstream cell death regulators and resulted in neurodegeneration (such as eye defects and DA neuron loss) in dFoxO overexpressing flies (Kanao et al., 2010). However, it has been found that LRRK2 phosphorylates itself and other substrates more strongly than 4E-BP in mammals (Kumar et al., 2010), so the negative effects may be *Drosophila* specific. Another study recently reported that dLRRK/hLRRK2 targeted microRNA pathways and that pathogenic LRRK2 antagonized these miRNAs, which negatively affected cell cycle and DA neuron survival (Gehrke et al., 2010). Additionally, the group found that LRRK2 associated with and negatively regulated Argonaute (dAgo1/hAgo2) RISC proteins and LRRK2 mutants promoted the association of phospho-4E-BP1 with hAgo2. Thus, it appears that WT LRRK2 can regulate protein expression by targeting microRNA silencing pathways, and mutations in LRRK2 can upset a delicate balance.
