**2. Background**

### **2.1 LRRK2 mutations**

There are five LRRK2 mutations (R1441C, R1441G; Y1699C; G2019S; I2020T) proven to segregate with disease and cause pathogenicity (Paisan-Ruiz et al., 2004; Zimprich et al., 2004) and two variants that are considered risk factors (LRRK2 G2385R and R1628P) (Farrer et al., 2007; Ross et al., 2008; Tan, 2006). While all leading to the same disease, the substitutions are not all found in the same functional domain. LRRK2 R1441C and G substitutions affect the Roc domain, a 'Ras-like' part of the protein (Mata et al., 2005). The LRRK2 Y1699C substitution and the R1628P risk factor are found in the COR domain and LRRK2 G2019S and I2020T are encoded within exon 41 at the 'activation hinge' of the MAPKKK domain (Mata et al., 2005). To date, the only substitution affecting risk of disease in the WD40 domain is the G2385R risk factor. The most common substitution, *LRRK2* G2019S, originates from a common founder and presents with variable, age-dependent penetrance (Healy et al., 2008; Hulihan et al., 2008; Kachergus et al., 2005).

Modeling LRRK2 Parkinsonism 73

act through a dominant "gain-of-function", data on kinase activity across the different mutations has been controversial, with only the G2019S mutation consistently reported to enhance kinase activity compared to wild-type activity (Adams et al., 2005; Jaleel et al., 2007;

LRRK2 has been characterized extensively in vitro. Although it is beyond the scope of this review to discuss these studies detail, several lines of evidence several lines of evidence point to a role in synaptic function (possibly by regulation of vesicle synthesis or transport and/or regulation of membranous structure) and as a regulator of neuronal outgrowth and guidance.

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

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

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

Luzon-Toro et al., 2007; MacLeod et al., 2006; Smith et al., 2006; West et al., 2005).

transgenic insertion of human wild type and mutant LRRK2.

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

**3. Invertebrate models 3.1 LRRK2 worm models** 

phenotype (Yao et al., 2010)

**3.1.2 Transgenic worm models** 
