**4. Rodent LRRK2 models**

While invertebrate LRRK2 models have been and will continue to be highly informative, it is important to remember that the LRRK genes are not true orthologues of LRRK2 (Marin, 2008). In addition, physiological limitations include not only the lack of basal ganglia but also their short lifespan, leaving the effects of aging impossible to determine. Given that age is the biggest risk factor for development of LRRK2 parkinsonism, the development of mammalian LRRK2 models is key for understanding pathophysiology and for drug screening.

To date a number of rodent models have been published in the literature, with experimental approaches Including knock-out of LRRK2, over-expression of human wild-type and

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

*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

While invertebrate LRRK2 models have been and will continue to be highly informative, it is important to remember that the LRRK genes are not true orthologues of LRRK2 (Marin, 2008). In addition, physiological limitations include not only the lack of basal ganglia but also their short lifespan, leaving the effects of aging impossible to determine. Given that age is the biggest risk factor for development of LRRK2 parkinsonism, the development of mammalian LRRK2 models is key for understanding pathophysiology and for drug

To date a number of rodent models have been published in the literature, with experimental approaches Including knock-out of LRRK2, over-expression of human wild-type and

**3.2.3 Fly interactor studies** 

LRRK2 and these other PD-associated genes.

in LRRK2 can upset a delicate balance.

**4. Rodent LRRK2 models** 

screening.

mutant LRRK2, over-expression of mouse wild-type and mutant LRRK2 and targeted knock-in of human mutations into the murine genome.

#### **4.1 Murine LRRK2 knockouts**

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

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.

#### **4.2 Over-expression of LRRK2**

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 striatal abnormalities including phospho-tau increases.

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 presented, however no overt phenotypes were noted.

Modeling LRRK2 Parkinsonism 79

In a different transgenic approach Lin et al generated hemagglutinin tagged conditional human WT and G2019S using cDNA fragments inserted into the mouse prion protein expression vector controlled by the tetracycline-responsive promoter (tetP) (Lin et al., 2009). Expression levels were between 8-16 fold for the human WT and G2019S lines and mice were on a C57BL/6 background. Human WT mice were behaviorally normal, but G2019S mice were found to gain less weight than controls and had increased ambulatory and rearing activities. One month old human WT and G2019S and mice were also reported to have perturbed microtubule dynamics, evidenced by a dramatic shift of β-tubulin from soluble to insoluble fractions prepared from brain lysates. Finally, ubiquitin staining increased in cortical neurons of 6 month old human WT and G2019S mice and by 20 months this increase became more pronounced and was clustered with LRRK2 staining, which the authors suggest may hint to inhibition of ubiquitin-proteasome activities. Interestingly, both the human WT and G2019S mice enhanced α-synuclein pathology when crossed with inducible A53T mutant mice. Curiously, double A53T/ kinase dead G2019S mice also exhibited similar enhanced phenotype, suggesting that kinase activity was not involved in α-synuclein mediating toxicity. The authors instead attributed the synergistic effects to impairment of golgi function, microtubule transport and the ubiquitin-proteasomal pathway, which occurred when LRRK2 was co-expressed, regardless of the transgene type

Two recently described viral models are the only LRRK2 models to date that document dopamine neuronal loss. Lee et al (Lee et al., 2010a) developed herpes simplex virus (HSV) amplicon-based mouse models expressing either human WT, G2019S and G2019S-D1994A (kinase dead) LRRK2, in which up to 75% of nigral neurons were infected after injection. HSV amplicon–mediated delivery of LRRK2 G2019S induced significant loss (~50%) of tyrosine hydroxylase–positive neurons 3 weeks after stereotaxic injection into the ipsilateral striatum of mice compared to control viruses expressing WT LRRK2 and eGFP and G2019S-D1994A. This model is the first model that directly implicates kinase activity being responsible for toxicity *in vivo* in a mammalian model, supported by the lack of effect in the kinase dead model, as well as attenuation of the neuronal loss by kinase inhibitors GW5074 and indirubin-3′-monooxime. The second viral model, the only published rat model to date, used a second generation adeno-viral serotype 5 vector, with transgene expression driven by the neuronal-specific human synapsin-1 promoter to express human WT and G2019S LRRK2 in rat brain (Dusonchet et al., 2011). Injections were delivered to the striatum and retrograde expression in the nigra was around 2 fold expression level overall, although with only 30% of all neurons transduced, this suggested very high levels in individual neurons. G2019S, but not WT mice exhibited a progressive neuronal loss that reached ~20% by 42 days as well as a 10% decrease in tyrosine hydroxylase fiber density. No alpha- synuclein pathology was observed but the authors noted some transient abnormal phosphorylation of tau for both WT and G2019S which did not correlate with neuronal cell death in G2019S.

For large proteins such as LRRK2, the over-expression approach is not without caveat and within the field there has been much concern about over-expession artifacts. To circumvent this, targeting the models' own genome, known as "knockin" is an alternative and potentially more physiological approach, since gene expression is recapitulated in the correct temporal, anatomical and quantitative manner and under the endogenous

or expression level.

**4.3 Targeted knockin of LRRK2** 

Two groups have since published data on BAC models expressing the kinase domain mutant G2019S. Li et al expressed a murine wild type and mutant G2019S BAC on a C57BL/6 background, with expression levels around 6 fold over endogenous LRRK2 (Li et al., 2010). Total dopamine content in the striatum was normal in WT BAC mice and G2019S mice at 6 months but declined by 25% in G2019S BAC mice by 12 months, suggesting an age related decline. Despite normal striatal dopamine levels, voltammetry in brain slices derived from 12 month old wild-type BAC transgenic mice showed a 25% increase in evoked dopamine release following a single pulse, whereas the decline in DA release following repeated stimulation was comparable to non-transgenic controls, as was their dopamine transporter (DAT)-mediated uptake. Conversely, the G2019S BAC mice displayed a 35% decrease in dopamine release by voltammetry, poorly sustained evoked dopamine release after repeated stimulation and a lower rate of dopamine uptake, presumably as a compensatory mechanism. No dopamine neuronal loss was seen in either model. Behaviorally the wild-type transgenic mice displayed enhanced ability on the beam test and increase spontaneous activity, whereas the G2019S BAC mice did not display any abnormalites. While no overt pathological lesions were reported, the authors did reveal that phospho- tau was decreased in the WT transgenic mice.

Our group (Melrose et al., 2010) also reported WT and mutant G2019S BAC mice, utilizing a human LRRK2 BAC rather than a murine BAC. Using *in vivo* microdialysis we demonstrated that basal extracellular dopamine levels were reduced by ~33% in G2019S BAC mice at ~8 months and an enhanced response to amphetamine challenge was noted. Total striatal dopamine, dopamine neuronal number, DAT levels and D1 and D2 receptor density were all unchanged up to 22 months. Behaviorally, the G2019S mice displayed some abnormal open field behavior but no motor deficits. Surprisingly, and in contrast to Li et al, we found that our human WT BAC mice also displayed reduced basal dopamine levels, even more pronounced (~66% versus 33%) than in our G2019S BAC mice. While this is likely attributed to differential expression levels in the two lines (WT BAC mice around 3.5 fold overexpression in half brain extracts and 2.5 fold in G2019S mice) it nevertheless supports a gain of function mechanism. Interestingly, D1 receptors were slightly upregulated in aged human WT BAC mice, although no changes were found in D2 receptors or DAT levels. Pathologically we did not detect any alpha-synuclein pathology in either line, but we did see changes in the phosphorylation of tau. Modest phosphorylation changes, detected by immunohistochemistry, were restricted to the hippocampus in the human WT BAC mice (the area of highest expression). In the G2019S mice however, tau alterations were much more pronounced and widespread. Biochemical analysis of tau revealed enhanced phosphorylation at several phospho-epitopes and upon dephosphorylation the tau species distribution was still different from the non-transgenic controls, suggesting that other post-translational modifications may be altered in G2019S mice and we hypothesize that these processing changes may increase the likelihood of abberant phosphorylation. One final observation worth noting is that we recently showed that neurogenesis is altered in our G2019S mice (Winner et al., 2011). Proliferation and migration of new neurons was impaired in the subventricular zone/rostral migratory stream and in the subgranular zone of the hippocampus. Furthermore, new neurons in the hippocampus had impaired outgrowth and a diminished number of spines. Curiously, exercise was found to partially recover the neuroblast deficit in the hippocampus, which may suggest that exercise induced signaling can at least partially compensate abberant LRRK2 activity caused by the G2019S mutation.

Two groups have since published data on BAC models expressing the kinase domain mutant G2019S. Li et al expressed a murine wild type and mutant G2019S BAC on a C57BL/6 background, with expression levels around 6 fold over endogenous LRRK2 (Li et al., 2010). Total dopamine content in the striatum was normal in WT BAC mice and G2019S mice at 6 months but declined by 25% in G2019S BAC mice by 12 months, suggesting an age related decline. Despite normal striatal dopamine levels, voltammetry in brain slices derived from 12 month old wild-type BAC transgenic mice showed a 25% increase in evoked dopamine release following a single pulse, whereas the decline in DA release following repeated stimulation was comparable to non-transgenic controls, as was their dopamine transporter (DAT)-mediated uptake. Conversely, the G2019S BAC mice displayed a 35% decrease in dopamine release by voltammetry, poorly sustained evoked dopamine release after repeated stimulation and a lower rate of dopamine uptake, presumably as a compensatory mechanism. No dopamine neuronal loss was seen in either model. Behaviorally the wild-type transgenic mice displayed enhanced ability on the beam test and increase spontaneous activity, whereas the G2019S BAC mice did not display any abnormalites. While no overt pathological lesions were reported, the authors did reveal that

Our group (Melrose et al., 2010) also reported WT and mutant G2019S BAC mice, utilizing a human LRRK2 BAC rather than a murine BAC. Using *in vivo* microdialysis we demonstrated that basal extracellular dopamine levels were reduced by ~33% in G2019S BAC mice at ~8 months and an enhanced response to amphetamine challenge was noted. Total striatal dopamine, dopamine neuronal number, DAT levels and D1 and D2 receptor density were all unchanged up to 22 months. Behaviorally, the G2019S mice displayed some abnormal open field behavior but no motor deficits. Surprisingly, and in contrast to Li et al, we found that our human WT BAC mice also displayed reduced basal dopamine levels, even more pronounced (~66% versus 33%) than in our G2019S BAC mice. While this is likely attributed to differential expression levels in the two lines (WT BAC mice around 3.5 fold overexpression in half brain extracts and 2.5 fold in G2019S mice) it nevertheless supports a gain of function mechanism. Interestingly, D1 receptors were slightly upregulated in aged human WT BAC mice, although no changes were found in D2 receptors or DAT levels. Pathologically we did not detect any alpha-synuclein pathology in either line, but we did see changes in the phosphorylation of tau. Modest phosphorylation changes, detected by immunohistochemistry, were restricted to the hippocampus in the human WT BAC mice (the area of highest expression). In the G2019S mice however, tau alterations were much more pronounced and widespread. Biochemical analysis of tau revealed enhanced phosphorylation at several phospho-epitopes and upon dephosphorylation the tau species distribution was still different from the non-transgenic controls, suggesting that other post-translational modifications may be altered in G2019S mice and we hypothesize that these processing changes may increase the likelihood of abberant phosphorylation. One final observation worth noting is that we recently showed that neurogenesis is altered in our G2019S mice (Winner et al., 2011). Proliferation and migration of new neurons was impaired in the subventricular zone/rostral migratory stream and in the subgranular zone of the hippocampus. Furthermore, new neurons in the hippocampus had impaired outgrowth and a diminished number of spines. Curiously, exercise was found to partially recover the neuroblast deficit in the hippocampus, which may suggest that exercise induced signaling can at least partially compensate abberant LRRK2 activity caused by the

phospho- tau was decreased in the WT transgenic mice.

G2019S mutation.

In a different transgenic approach Lin et al generated hemagglutinin tagged conditional human WT and G2019S using cDNA fragments inserted into the mouse prion protein expression vector controlled by the tetracycline-responsive promoter (tetP) (Lin et al., 2009). Expression levels were between 8-16 fold for the human WT and G2019S lines and mice were on a C57BL/6 background. Human WT mice were behaviorally normal, but G2019S mice were found to gain less weight than controls and had increased ambulatory and rearing activities. One month old human WT and G2019S and mice were also reported to have perturbed microtubule dynamics, evidenced by a dramatic shift of β-tubulin from soluble to insoluble fractions prepared from brain lysates. Finally, ubiquitin staining increased in cortical neurons of 6 month old human WT and G2019S mice and by 20 months this increase became more pronounced and was clustered with LRRK2 staining, which the authors suggest may hint to inhibition of ubiquitin-proteasome activities. Interestingly, both the human WT and G2019S mice enhanced α-synuclein pathology when crossed with inducible A53T mutant mice. Curiously, double A53T/ kinase dead G2019S mice also exhibited similar enhanced phenotype, suggesting that kinase activity was not involved in α-synuclein mediating toxicity. The authors instead attributed the synergistic effects to impairment of golgi function, microtubule transport and the ubiquitin-proteasomal pathway, which occurred when LRRK2 was co-expressed, regardless of the transgene type or expression level.

Two recently described viral models are the only LRRK2 models to date that document dopamine neuronal loss. Lee et al (Lee et al., 2010a) developed herpes simplex virus (HSV) amplicon-based mouse models expressing either human WT, G2019S and G2019S-D1994A (kinase dead) LRRK2, in which up to 75% of nigral neurons were infected after injection. HSV amplicon–mediated delivery of LRRK2 G2019S induced significant loss (~50%) of tyrosine hydroxylase–positive neurons 3 weeks after stereotaxic injection into the ipsilateral striatum of mice compared to control viruses expressing WT LRRK2 and eGFP and G2019S-D1994A. This model is the first model that directly implicates kinase activity being responsible for toxicity *in vivo* in a mammalian model, supported by the lack of effect in the kinase dead model, as well as attenuation of the neuronal loss by kinase inhibitors GW5074 and indirubin-3′-monooxime. The second viral model, the only published rat model to date, used a second generation adeno-viral serotype 5 vector, with transgene expression driven by the neuronal-specific human synapsin-1 promoter to express human WT and G2019S LRRK2 in rat brain (Dusonchet et al., 2011). Injections were delivered to the striatum and retrograde expression in the nigra was around 2 fold expression level overall, although with only 30% of all neurons transduced, this suggested very high levels in individual neurons. G2019S, but not WT mice exhibited a progressive neuronal loss that reached ~20% by 42 days as well as a 10% decrease in tyrosine hydroxylase fiber density. No alpha- synuclein pathology was observed but the authors noted some transient abnormal phosphorylation of tau for both WT and G2019S which did not correlate with neuronal cell death in G2019S.

#### **4.3 Targeted knockin of LRRK2**

For large proteins such as LRRK2, the over-expression approach is not without caveat and within the field there has been much concern about over-expession artifacts. To circumvent this, targeting the models' own genome, known as "knockin" is an alternative and potentially more physiological approach, since gene expression is recapitulated in the correct temporal, anatomical and quantitative manner and under the endogenous

Modeling LRRK2 Parkinsonism 81

tau and LRRK2 protein, there appears to be an indirect link that warrants further investigation. If LRRK2 regulates tau physiology, this could have important implication for

Less consistent/investigated *in vivo*, but nevertheless still compelling are the changes in the inflammatory, autophagy/lysosomal, apoptotic, mitochondrial and proteosomal pathways observed in some models. These pathways may be particularly important in unraveling how LRRK2 leads to α-synuclein pathology in humans. While studies in invertebrates are suggestive of a potential role for LRRK2, more mammalian studies are needed into the role of LRRK2 in mitochrondrial/stress pathways. As it stands, there is no evidence for a direct interaction between LRRK2 and α-synuclein and the only instance of *in vivo* α-synuclein pathology in a LRRK2 model is in the kidneys, but not brain, of the LRRK2 knockout model (Tong et al., 2010). Notably, accumulation was accompanied by autophagic, proteasomal and inflammatory changes. Furthermore in double LRRK2 and α-synuclein A53T mice, enhancement of α-synuclein pathology was attributed to impairments in microtubule dynamic, golgi organization, mitochondrial toxicity and ubiquitin-proteasomal pathway (Lin et al., 2009). What is difficult to resolve though, is that double LRRK2 KO/ A53T mice have ameliorated α-synuclein brain pathology (Lin et al., 2009). These complex findings reiterate the notion that LRRK2 is likely a multi-faceted protein, which may have distinct

Intense pharmaceutical interest has surrounded LRRK2 since its discovery and many companies are developing inhibitors of LRRK2 and screening is already underway in many of the models described above. It is still not clear exactly what function of LRRK2 mediates its toxicity and data attributing it to enzymatic kinase activity is conflicting. Although the general consensus appears to be that LRRK2 pathogenicity is a toxic gain of function, the kidney phenotype in LRRK2 KO mice suggests that peripheral effects may be an important loss-of-function consideration. A highly selective LRRK2 inhibitor (LRRK2-IN1) was recently developed by Nathaniel Gray and colleagues (Deng et al., 2011) which abolished Ser910 and Ser935 phosphorylation of LRRK2 in the kidney of mice after 1 hour. No changes were observed in brain because the compound is unable to cross the blood brain barrier. Once CNS drug delivery issues are overcome, it will be interesting to see the effects

Aasly, J. O., et al., 2005. Clinical features of LRRK2-associated Parkinson's disease in central

Abeliovich, A., et al., 2000. Mice lacking alpha-synuclein display functional deficits in the

Adams, J. R., et al., 2005. PET in LRRK2 mutations: comparison to sporadic Parkinson's disease and evidence for presymptomatic compensation. Brain. 128, 2777-85. Andres-Mateos, E., et al., 2009. Unexpected lack of hypersensitivity in LRRK2 knock-out

mice to MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). J Neurosci. 29,

LRRK2 therapies in Alzheimer's disease.

**4.5 The future for LRRK2 therapeutics** 

of LRRK-IN1in in vivo LRRK2 models.

Norway. Ann Neurol. 57, 762-5.

nigrostriatal dopamine system. Neuron. 25, 239-52.

cellular specific roles.

**5. References** 

15846-50.

transcriptional and translational controls. Nevertheless, many would still argue, justifiably, that over-expression models and their phenotypes, even if exaggerated, often give vital clues to normal and pathological functions. Equally, the risk with the knockin approach is that the relatively low mutant expression levels, within a species that doesn't normally develop the disease, may yield no or very subtle phenotypes.

To date only one knockin model has been published, by Tong et al, who created a ROC domain R1441C mutant (Tong et al., 2009). Whilst the homozygous knockin mice were grossly normal and did not exhibit dopamine neuronal loss, amperometric recordings in chromaffin cells derived from the R1441C, stimulated with high KCl, revealed significant reductions in the total catecholamine release per cell, quantal size and the frequency of release events. Slice recordings from dopaminergic neurons in the nigra of R1441C mice also exhibited smaller amplitude and duration of hyperpolarization after dopamine application, as well as slower recover times, and reduced responses to amphetamine and the D2 agonist quinpirole, compared with WT controls. Locomotor testing in the open field also revealed the knockin mice had a reduced behavioral response to amphetamine and quinpirole, providing *in vivo* evidence that dopamine release and D2 autoceptor feedback mechanisms may be impaired. No neuropathology was noted in these mice.

#### **4.4 What do the models tell us about LRRK2?**

At first glance, the success of the LRRK2 models appears to be nominal, with not a single model recapturing all the key features desired for a PD model – dopamine cell loss, motor impairment and alpha-synculein pathology. However, independent of the approach, a unifying theme is emerging from both invertebrate and vertebrate LRRK2 models, suggesting an important role for LRRK2 in dopaminergic neurotransmission, even in the absence of dopamine neuronal loss. Several other genetic PD mouse models also have abnormalities in dopamine transmission without neuronal loss including PINK1, parkin, DJ-1 and SNCA knockouts and SNCA WT over-expression mice (Abeliovich et al., 2000; Goldberg et al., 2003; Goldberg et al., 2005; Kitada et al., 2009; Nemani et al., 2010). Imaging studies have long established that in asymptomatic PD, the earliest detectable changes occur in the dopamine transporter and the same holds true for asymptomatic LRRK2 (Nandhagopal et al., 2008; Sossi et al., 2010) and SNCA patients (Bostantjopoulou et al., 2008; Perani et al., 2006; Samii et al., 1999). Thus, the data evolving from LRRK2 models (and other PD genetic models) may be recapturing early preclinical events. In reality this may make the models more valuable because the onset of motor symptoms in PD is only after 50- 70% of the dopamine neurons are lost, by which point neuro-protective therapy would be futile. Understanding these early events in disease is key to allow us identify biomarkers to detect at risk patients and design neuro-protective therapies.

Aside from the effects on dopamine neurotransmission, the other consistent theme arising from LRRK2 *in vivo* data appears to be the impairment of cytoskeletal function, evidenced by the alterations observed in tau phosphorylation and perturbed microtubule dynamics. Although tau pathology is rare in LRRK2 brains, there is mounting evidence of the involvement of tau in PD in general, with over 15 genetic studies in small populations implicating variability in the tau gene with Parkinson's disease and a recent study of a large number of European PD samples unequivocally showing that tau gene (MAPT) variability is a major risk factor for PD, along with the alpha-synculein gene (SNCA) (Simon-Sanchez et al., 2009). Even though it has been proven already that there is no direct interaction between

transcriptional and translational controls. Nevertheless, many would still argue, justifiably, that over-expression models and their phenotypes, even if exaggerated, often give vital clues to normal and pathological functions. Equally, the risk with the knockin approach is that the relatively low mutant expression levels, within a species that doesn't normally

To date only one knockin model has been published, by Tong et al, who created a ROC domain R1441C mutant (Tong et al., 2009). Whilst the homozygous knockin mice were grossly normal and did not exhibit dopamine neuronal loss, amperometric recordings in chromaffin cells derived from the R1441C, stimulated with high KCl, revealed significant reductions in the total catecholamine release per cell, quantal size and the frequency of release events. Slice recordings from dopaminergic neurons in the nigra of R1441C mice also exhibited smaller amplitude and duration of hyperpolarization after dopamine application, as well as slower recover times, and reduced responses to amphetamine and the D2 agonist quinpirole, compared with WT controls. Locomotor testing in the open field also revealed the knockin mice had a reduced behavioral response to amphetamine and quinpirole, providing *in vivo* evidence that dopamine release and D2 autoceptor feedback mechanisms

At first glance, the success of the LRRK2 models appears to be nominal, with not a single model recapturing all the key features desired for a PD model – dopamine cell loss, motor impairment and alpha-synculein pathology. However, independent of the approach, a unifying theme is emerging from both invertebrate and vertebrate LRRK2 models, suggesting an important role for LRRK2 in dopaminergic neurotransmission, even in the absence of dopamine neuronal loss. Several other genetic PD mouse models also have abnormalities in dopamine transmission without neuronal loss including PINK1, parkin, DJ-1 and SNCA knockouts and SNCA WT over-expression mice (Abeliovich et al., 2000; Goldberg et al., 2003; Goldberg et al., 2005; Kitada et al., 2009; Nemani et al., 2010). Imaging studies have long established that in asymptomatic PD, the earliest detectable changes occur in the dopamine transporter and the same holds true for asymptomatic LRRK2 (Nandhagopal et al., 2008; Sossi et al., 2010) and SNCA patients (Bostantjopoulou et al., 2008; Perani et al., 2006; Samii et al., 1999). Thus, the data evolving from LRRK2 models (and other PD genetic models) may be recapturing early preclinical events. In reality this may make the models more valuable because the onset of motor symptoms in PD is only after 50- 70% of the dopamine neurons are lost, by which point neuro-protective therapy would be futile. Understanding these early events in disease is key to allow us identify biomarkers to

Aside from the effects on dopamine neurotransmission, the other consistent theme arising from LRRK2 *in vivo* data appears to be the impairment of cytoskeletal function, evidenced by the alterations observed in tau phosphorylation and perturbed microtubule dynamics. Although tau pathology is rare in LRRK2 brains, there is mounting evidence of the involvement of tau in PD in general, with over 15 genetic studies in small populations implicating variability in the tau gene with Parkinson's disease and a recent study of a large number of European PD samples unequivocally showing that tau gene (MAPT) variability is a major risk factor for PD, along with the alpha-synculein gene (SNCA) (Simon-Sanchez et al., 2009). Even though it has been proven already that there is no direct interaction between

develop the disease, may yield no or very subtle phenotypes.

may be impaired. No neuropathology was noted in these mice.

detect at risk patients and design neuro-protective therapies.

**4.4 What do the models tell us about LRRK2?** 

tau and LRRK2 protein, there appears to be an indirect link that warrants further investigation. If LRRK2 regulates tau physiology, this could have important implication for LRRK2 therapies in Alzheimer's disease.

Less consistent/investigated *in vivo*, but nevertheless still compelling are the changes in the inflammatory, autophagy/lysosomal, apoptotic, mitochondrial and proteosomal pathways observed in some models. These pathways may be particularly important in unraveling how LRRK2 leads to α-synuclein pathology in humans. While studies in invertebrates are suggestive of a potential role for LRRK2, more mammalian studies are needed into the role of LRRK2 in mitochrondrial/stress pathways. As it stands, there is no evidence for a direct interaction between LRRK2 and α-synuclein and the only instance of *in vivo* α-synuclein pathology in a LRRK2 model is in the kidneys, but not brain, of the LRRK2 knockout model (Tong et al., 2010). Notably, accumulation was accompanied by autophagic, proteasomal and inflammatory changes. Furthermore in double LRRK2 and α-synuclein A53T mice, enhancement of α-synuclein pathology was attributed to impairments in microtubule dynamic, golgi organization, mitochondrial toxicity and ubiquitin-proteasomal pathway (Lin et al., 2009). What is difficult to resolve though, is that double LRRK2 KO/ A53T mice have ameliorated α-synuclein brain pathology (Lin et al., 2009). These complex findings reiterate the notion that LRRK2 is likely a multi-faceted protein, which may have distinct cellular specific roles.

#### **4.5 The future for LRRK2 therapeutics**

Intense pharmaceutical interest has surrounded LRRK2 since its discovery and many companies are developing inhibitors of LRRK2 and screening is already underway in many of the models described above. It is still not clear exactly what function of LRRK2 mediates its toxicity and data attributing it to enzymatic kinase activity is conflicting. Although the general consensus appears to be that LRRK2 pathogenicity is a toxic gain of function, the kidney phenotype in LRRK2 KO mice suggests that peripheral effects may be an important loss-of-function consideration. A highly selective LRRK2 inhibitor (LRRK2-IN1) was recently developed by Nathaniel Gray and colleagues (Deng et al., 2011) which abolished Ser910 and Ser935 phosphorylation of LRRK2 in the kidney of mice after 1 hour. No changes were observed in brain because the compound is unable to cross the blood brain barrier. Once CNS drug delivery issues are overcome, it will be interesting to see the effects of LRRK-IN1in in vivo LRRK2 models.

#### **5. References**


Modeling LRRK2 Parkinsonism 83

Higashi, S., et al., 2007a. Localization of Parkinson's disease-associated LRRK2 in normal

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**5** 

*Slovenia* 

**Alpha-Synuclein Interactions with Membranes** 

Synucleinopathies are a group of neurodegenerative disorders that share common pathological intracellular deposits that contain aggregates of the protein α-synuclein. Substantial evidence suggests that fibril formation by α-synuclein is a critical step in the development of Parkinson's disease (PD). Indeed, *in vitro*, α-synuclein forms fibrils with morphologies and staining characteristics similar to those extracted from disease-affected brains. Also, three single-point mutations and duplication or triplication of the α-synuclein

However, the function of α-synuclein remains unknown. A significant portion of αsynuclein is localized within membrane fractions, and especially synaptic vesicles associated with vesicular transport processes. These observations suggest that α-synuclein has a role in vesicular trafficking. Although α-synuclein belongs to a group of natively unfolded proteins, there is strong evidence that the membrane affinity of the protein induces an αhelical conformation. A large number of studies have investigated α-synuclein–lipid interactions in the search for a physiological function, as well as to understand this potential membrane involvement in the pathogenesis of α-synuclein. In this review, we will predominantly focus on current opinion about the native wild-type α-synuclein–lipid interactions and the structure of α-synuclein that is established at the membrane surface. However, it should be noted that membranes have been reported to both accelerate and inhibit the fibril formation of α-synuclein, although this will not be the focus of the present

A significant number of proteins involved in protein deposition diseases have been seen to be intrinsically disordered proteins. Well-known examples include amyloid β-protein and tau protein in Alzheimer's disease, prion protein (PrP) in prion diseases, exon 1 region of

It has been estimated that more than 30% of eukaryotic proteins have disordered regions that are greater than 50 consecutive residues (Dunker et al., 2001). This term "disordered protein" refers to proteins that in their purified state at neutral pH, have been either shown experimentally or predicted to lack an ordered structure; such proteins are also known as natively unfolded, or intrinsically unstructured. Disordered proteins, or disordered regions

huntingtin in Huntington's disease, and α-synuclein in PD (Fink, 2005).

**1. Introduction** 

review.

locus correlate with early onset of PD.

**2. Intrinsically disordered proteins** 

Katja Pirc1 and Nataša Poklar Ulrih1,2 *1University of Ljubljana, Biotechnical faculty* 

*2Centre of excellence CIPKeBiP* 

