**3. Invertebrate models**

#### **3.1 LRRK2 worm models**

LRK-1, the paralogue of LRRK2 in *Caenorhabditis elegans,* shares ~35% identity and 57% similarity with the enzymatic GTPase and kinase domains (Samann et al., 2009) and is expressed ubiquitously, including in muscle, intestine and importantly in neurons (Sakaguchi-Nakashima et al., 2007; Samann et al., 2009). Experimental approaches to study LRRK2 in worms have included loss of LRK-1 function, over-expression of LRK-1 and transgenic insertion of human wild type and mutant LRRK2.

#### **3.1.1 LRK-1 loss of function worm models**

Sakaguchi-Nakashima et al first described loss-of-function LRK-1 mutants, the km17 line, which has a deletion in the kinase and WD40 domains, and km41 line that has the GTPase and kinase domain deleted (Sakaguchi-Nakashima et al., 2007). A third deletion strain, tm1898 ( also lacking the GTPAse and kinase domain) was later described (Saha et al., 2009). All three deletion mutants result in mislocalization of synaptobrevin to dendrites, suggesting that normally LRK-1 plays a role in sorting and/or localization of synaptic vesicle proteins (Sakaguchi-Nakashima et al., 2007; Samann et al., 2009) possibly by excluding them from dendrite-specific transport machinery in the Golgi (Sakaguchi-Nakashima et al., 2007). Loss-of-function LRK-1 (km17 and km41) worms also exhibited defects in movement and chemotaxis (Sakaguchi-Nakashima et al., 2007). Both the km41 and tm1898 mutants were reported to have increased sensitivity to endoplasmic reticiulum stress, following tunicamycin exposure but were resistant to mitochondrial stress via paraquat (Samann et al., 2009). Conversely, Saha reported that RNAi knockdown of LRK-1, while not affecting baseline viability, did sensitize nemotodes to mictochrondrial dysfunction via rotenone treatment, a response that was confirmed in the km17 deletion line (Saha et al., 2009). Interestingly, loss of LRK-1 has been found to suppress the neurite outgrowth defects, mitochondrial abnormalities and increased paraquat sensitivity in PINK-1 loss-of-function mutants. Additionally, the increased sensitivity of LRK-1 loss-of-function worms to tunicamycin was reduced in PINK-1 loss-of-function mutants, suggesting an antagonistic role of PINK-1 and LRK-1 (Samann et al., 2009). Curiously, loss of LRK-1 also attenuates transgenic LRRK2-induced DA neurodegeneration and basal slowing behavioral phenotype (Yao et al., 2010)

#### **3.1.2 Transgenic worm models**

Transgenic worms have been created by either overexpressing wild-type (WT) LRK-1 and/or comparable mutant LRK-1 or by overexpressing human WT or mutant LRRK2 with

Modeling LRRK2 Parkinsonism 75

was also resistant to hydrogen peroxide, and RNAi knockdown of LRRK2 decreased

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

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

sensitivity to both hydrogen peroxide and paraquat (Imai et al., 2008).

**3.2.2 Transgenic fly models** 

effect (Kanao et al., 2010; Ng et al., 2009).

stress response and mitochondrial pathways.

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., 2008; Yao et al., 2010).

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 exogenous dopamine (Yao et al., 2010).

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 contrasting data makes any inferences difficult.
