Báo cáo khoa học: LRRK2 in Parkinson’s disease: in vivo models and approaches for understanding pathogenic roles pdf

LRRK2 in Parkinson’s disease: in vivo models andapproaches for understanding pathogenic roles Zhenyu Yue Department of Neurology and Neuroscience, Mount Sinai School of Medicine, New Yor

LRRK2 in Parkinson’s disease: in vivo models and

approaches for understanding pathogenic roles

Zhenyu Yue

Department of Neurology and Neuroscience, Mount Sinai School of Medicine, New York, NY, USA

Introduction

Clinical symptoms of patients carrying Parkinson’s

dis-ease (PD)-associated mutations of leucine-rich repeat

kinase 2 (LRRK2) are indistinguishable from typical

sporadic PD The spectra of neuropathological features

of PARK8 (LRRK2) patients is broad and appears to

encompass those associated with other familial PD

cases such as PARK1 (a-synuclein) and PARK2

(Parkin) However, the neuropathology of PARK8 is

variable and is not always associated with the presence

of intracellular inclusions (e.g Lewy body, tau tangles and ubiquitin inclusions) [1,2] Recent studies also suggest that the penetrance of LRRK2 pathogenic mutations is incomplete [3,4]

LRRK2 encodes a large complex protein consisting

of 2527 amino acids (285 kDa) It belongs to the ROCO family, which is defined by the presence of a Ras of complex proteins (ROC) domain followed by a C-terminal of ROC (COR) domain of unknown

Keywords

animal models; BAC transgenics; dopamine;

GTPase; kinase; leucine-rich repeat

kinase 2 (LRRK2); Parkinson’s disease;

pathogenesis; ROCO

Correspondence

Z Yue, Department of Neurology and

Neuroscience, Mount Sinai School of

Medicine, New York, NY 10029, USA

Fax: +1 212 241 3869

Tel: +1 212 241 3155

E-mail: zhenyu.yue@mssm.edu

(Received 30 May 2009, revised 30 July

2009, accepted 18 August 2009)

doi:10.1111/j.1742-4658.2009.07343.x

The recent discovery of the genetic causes for Parkinson’s disease (PD) is fruitful; however, the continuing revelation of PD-related genes is rapidly outpacing the functional characterization of the gene products Although the discovery of multiple PD-related genes places PD as one of the most complex multigenetic diseases of the brain, it will undoubtedly facilitate the unfolding of a central pathogenic pathway and an understanding of the eti-ology of PD Recent findings of pathogenic mutations in leucine-rich repeat kinase 2 (LRRK2) (PARK8) that are linked to the most common familial forms and some sporadic forms of PD provide a unique opportunity to gain insight into the pathogenesis of PD Despite rapid growth in biochem-ical, structural and in vitro cell culture studies of LRRK2, the in vivo char-acterizations of LRRK2 function generally fall short and are largely limited to invertebrates The investigation of LRRK2 or homologs of LRRK2 in nonmammalian models provides important clues with respect

to the cellular functions of LRRK2, but an elucidation of the physiology and pathophysiology of LRRK2 relevant to PD would still depend on mammalian models established by multiple genetic approaches, followed by rigorous examination of the models for pathological process This minire-view summarizes previous studies of genes for ROCO and LRRK2 homo-logs in slime mold, nematode worms and fruit flies It also discusses the results obtained from available mouse models of LRRK2 that begin to provide information for understanding LRRK2-mediated pathogenesis in PD

Abbreviations

BAC, bacterial artificial chromosome; COR, C-terminal of ROC; KO, knockout; LRCK, LRR-ROC-COR-kinase; LRRK2, leucine-rich repeat kinase 2; PD, Parkinson’s disease; ROC, Ras of complex proteins; TH, tyrosine hydroxylase.

function [5] LRRK2 also contains armadillo-like

repeats, LRR, kinase and WD40 domains [6] In vitro

biochemical analysis demonstrates that LRRK2

con-tains kinase and GTPase activities that are apparently

altered by pathogenic mutations of LRRK2 [7–12] In

addition, studies using cultured cells or neurons show

that enhancement of kinase activity in PD-related

mutants of LRRK2 is correlated with increased

neuro-toxicity, thus implicating a causal role of aberrant

enzymatic activity of LRRK2 in neuropathogenesis

[9,13–15] However, whether this possible

gain-of-func-tion in the kinase activity of LRRK2 contributes to

the the pathological process of PD has yet to be shown

in mammalian models

Understanding the physiological function of LRRK2

under normal conditions and in the context of PD

remains a daunting task, especially given the

complex-ity of LRRK2 protein structure, which consists of

mul-tiple functional domains that are likely to be involved

in numerous cellular pathways There is clearly a need

to investigate LRRK2 structure⁄ function-related

pro-teins (e.g ROCO family propro-teins) in various model

systems, including lower eukaryotes and invertebrates,

in order to obtain clues for building important

hypoth-eses The rapidity and efficiency of in vivo studies in

many nonmammal models have already provided

timely information about molecular mechanisms of

many disease processes and will continue to impact

our understanding of disease pathogenesis This

mini-review will examine the previous studies of genes for

ROCO or LRRK2 homologs in slime mold

Dictyoste-lium discoideum, nematode worms Caenorhabditis

ele-gans and fruit flies Drosophila melanogaster It will

also discuss the available information reported in the

literature (albeit limited), as well as ongoing studies in

several laboratories that have created LRRK2 rodent

models

ROCO proteins in slime mold

D discoideum

The finding of the conserved ROC and COR domains

in LRRK2 has stirred particular interest with respect

to studying the functions of the known ROCO

pro-teins The first ROCO protein was identified in slime

mold Dictyostelium [5] and, so far, at least two ROCO

proteins, GbpC and Pats1, have been characterized

in vivo in this species [16,17] Similar to LRRK2,

GbpC and Pats1 both have LRR and kinase domains

flanking the central ROCO sequence The sequence

arrangement of these functional motifs

‘LRR-ROC-COR-kinase’ (LRCK) is also found in LRRK2

homologs of nematode worms and fruit flies The

inves-tigation of ROCO structure of a prokaryotic protein in Chlorobium tepidum revealed mechanistic insight into protein dimerization and the regulation of ROC GTPase activity [18], which may be involved in intramolecular control of the kinase activity [9,19] It is possible that the subgroup of ROCO proteins (including mammalian LRRK2), which contain the conserved functional motifs

‘LRCK’, may adopt a similar structural mechanism to regulate enzymatic activities and their cellular functions (Fig 1)

A series of in vivo studies have revealed that the ROCO proteins, GbpC and Pats1, are involved in mul-tiple cellular processes: chemotaxis, cell division and development Deletion of GbpC in D discoideum was shown to cause a reduction of chemotactic reaction towards cAMP [20] In addition, loss of GbpC was associated with a decrease in phosphorylation of sin II and a change in subcellular localization of myo-sin heavy chain [21] The chemotactic ‘rescue’ experiment showed that the kinase domain alone is insufficient to complement the chemotactic defect in the GbpC-deletion mutant Furthermore, the study indicated that the LRR, ROC and kinase domains are all required for chemotaxis [16] These results thus sug-gest that the functional integrity of GbpC protein requires all subdomains in the core ‘LRCK’ sequence ROCO protein Pats1 was originally found in a genetic screen of cellular defect in cytokinesis of D dis-coideum [17] Mutant cells with Pats1 deletion exhibit abnormal cell morphology and division deficits It was shown that the WD40 domain of Pats1 interacted with myosin heavy chain, whereas the deletion of Pats1 caused an alteration in the localization of myosin heavy chain [17] Moreover, over-expression of the kinase domain alone resulted in a similar phenotype to that of the deletion mutants, suggesting that deregulation of kinase activity underlies the mechanism of cytokinesis impairment Taken together, the studies of mutant phe-notypes for the two ROCO proteins in D discoideum indicate their roles in regulating cytoskeleton structures Interestingly, it was previously noted that human LRRK2 binds to cytoskeleton-related proteins [22], microtubules [23] and phosphorylates moesin, a protein that anchors the actin cytoskeleton to the plasma mem-brane [24] Although cytoskeleton proteins are known

as frequent ‘contaminants’ in the process of searching binding proteins, the in vivo evidence for the relation-ship between ROCO proteins and cytoskeletons in

D discoideum suggests a need to further investigate the possibility that cytoskeleton proteins are the physiological targets of LRRK2 It is possible that LRRK2 regulates cytoskeletal mobility, which is linked

to various cellular vesicle trafficking events

The nematode worm models

Despite the fact that nematode worms are used

exten-sively as an in vivo model to study disease gene

func-tion, there are only a few reports on LRRK2 or LRK-1

(LRRK2 ortholog in C elegans) available to date

LRK-1 is the only ortholog of human LRRK2 found

in C elegans The product of LRK-1 shares a

con-served ‘LRCK’ core sequence with LRRK2 (Fig 1)

The first study characterized the phenotype of mutant

worms containing deletion of LRK-1 It provided

important evidence implicating a role for LRK-1 in

synaptic vesicle localization [25] It showed that

synap-tic vesicles and their associated proteins are exclusively

localized in the pre-synaptic regions but not in

dendrites By contrast, in mutant worms carrying

truncated LRK-1, synaptic vesicle proteins are located

in axons (pre-synaptic), as well as dendritic terminals

(post-synaptic) The localization of the synaptic vesicle

proteins in both pre- and post-synaptic regions as a

result of the lack of LRK-1 apparently is not a random

event because the mislocalization of the synaptic

pro-teins in dendrites depends on AP-1 clathrin adaptor,

which is known to be involved in dendritic transport,

but not on Unc104 kinesin, a motor protein required

for axonal transport Therefore, this study suggests

that LRK-1 protein, as a resident of Golgi apparatus,

controls the directionality of synaptic vesicle proteins

by restricting these proteins from going to the

dendrites This result further indicates a critical

func-tion of LRK-1 in establishing the polarity of synaptic

vesicle proteins and perhaps in regulating synaptic ves-icle life cycle in the axons However, this study did not reveal any results regarding the viability of neurons, especially dopaminergic neurons, or any functional consequence of the mislocalization of synaptic vesicle proteins

The above study also made reference to the partial defect of chemotaxis to volatile odorants in mutant worms carrying LRK-1 deletion [25] Although it is unknown whether the chemotaxis deficiency involves a dysfunctional cytoskeletal system (as found in mutant slime mold carrying an ROCO proteins deletion), it would be interesting to investigate the underlying mechanism associated with the loss of LRK-1 that could be related to the pre-motor symptom of hypo-smia in PD

The second study investigated how ectopic expres-sion of human LRRK2 wild-type or G2019S mutant

in worms modifies cellular responses to rotenone, a mitochondrial toxin in nematode worms [26] The results showed that over-expression of wild-type LRRK2 offers the transgenic worms a strong protection against rotenone toxicity, whereas over-expression of G2019S LRRK2 also protects, but to a lesser degree Furthermore, reduced endogenous LRK1-1 expression potentiates rotenone toxicity This report implicates a role for LRRK2 in cellular protec-tion against mitochondria-related stress This funcprotec-tion may be partially impaired by the PD mutation of G2019S Interestingly, over-expression of LRRK2 wild-type, but not the G2019S mutant, extended the

Human LRRK2

Human LRRK1

C elegans LRK-1

Drosophila dLRRK

Dictyostelium Gbpc

Dictyostelium Pats1

2631

2527

2014

2393

3184

2351

RasGEFn RasGEF

cNB cNB GRAM DEP

LRCK

Fig 1 Schematic illustration of domain structures and alignment for LRRK2 and LRRK2 structure-related proteins ANK, N-terminal ankyrin repeat domain; DEP, Dishevelled, EGL-10, pleckstrin domain; GEF, guanine-exchange factor; GRAM, glucosyltransferases, Rab-like GTPase activators and myotublarins domain.

lifespan of worms, indicating a beneficial role of

LRRK2 in the ageing process This result suggests that

overproduction of LRRK2 (wild-type or G2019S

mutant) in worms, unlike in mammalian cell cultures

[9,19], does not cause toxicity [26]

The third report in nematode worms, however,

shows that over-expression of worm green flourescent

protein-tagged LRK-1 wild-type or LRK-1 G1876S

(corresponding to human G2019S) leads to an early

larval arrest [27] Although this result may suggest that

over-expresssion of worm LRK-1 is much more toxic

than human LRRK2, it also raises the question of

whether or not LRK-1 is a true ortholog of human

LRRK2 [6] Importantly, this study suggests a genetic

link between Lrk-1 and Pink-1, a C elegans homolog

of human PINK-1 that is associated with a recessive

form of PD [28] Mutant worms that lack Lrk-1 were

shown to have enhanced sensitivity to endoplasmic

reticulum stress induced by tunicamycin, a specific

inhibitor for N-linked glycosylation Interestingly, this

enhanced sensitivity is suppressed in mutant worms

with deletion of both Lrk-1 and Pink-1 genes On the

other hand, although Pink-1 mutant worms exhibit

increased vulnerability to paraquat, defects in

mito-chondrial cristae and impairment of axonal guidance,

a lack of Lrk-1 appeared to reverse the Pink-1

dele-tion-associated defects in double mutant Lrk-1 and

Pink-1 This study suggests an antagonistic role of

Lrk-1 and Pink-1 in stress response and neuronal

activities [27]

Fruit fly models of LRRK2

The fruit fly homolog of LRRK2 is dLRRK, which also

contains the conserved ‘LRCK’ core sequence (Fig 1)

To date, at least four studies have reported using fruit

fly D melanogaster to investigate the in vivo functions

of human LRRK2 or dLRRK The first study showed

that the mutant flies lacking dLRRK exhibited

impaired locomotive activity and a significant

reduc-tion of tyrosine hydroxylase (TH) immunostaining in

dopaminergic neurons Although the number of

dopa-minergic neurons appears unaltered, they display

abnormal morphology, suggesting that they are under

pathogenic stress or undergoing slow degeneration

[29] Two other studies, however, did not reproduce

the behavioral and TH deficits in mutant flies carrying

deletion of dLRRK Instead, they observed unchanged

numbers of TH+ neurons in these mutants, indicating

that dLRRK is dispensable for the survival of

dopami-nergic neurons [30,31] In addition, Wang et al [30]

showed that mutant flies containing C-terminal kinase

domain truncated dLRRK are selectively sensitive to

H2O2, but not to paraquat, rotenone or b-mercapto-ethanol By contrast, Imai et al [31] showed that dLRRK null flies are relatively resistant to general oxi-dative stress, such as paraquat and H2O2 treatment, compared to wild-type flies [31] Furthermore, dLRRK null flies have significant reduced levels of 4-hydroxy-2-nonenal of lipid peroxidation, an indication of oxida-tive damage Although the exact role of dLRRK in oxidative stress remains unclear, all studies in fly mod-els reported to date consistently demonstrate that dLRRK is not essential for the early development and viability of dopaminergic neurons

The results obtained from studies of transgenic flies over-expressing dLRRK or human LRRK2 have been somewhat inconsistent between the different groups Although Lee et al [29] indicated that over-expression

of a pathogenic mutant or wild-type dLRRK did not cause any significant defects in transgenic flies, two other independent reports demonstrated that express-ing mutants of dLRRK or LRRK2 in flies causes selec-tive degeneration of dopaminergic neurons as well as motor function deficits [31,32] Of these two reports, however, one showed that even over-expressing wild-type human LRRK2 led to the toxicity of dopaminer-gic neurons and impairment of motor function (although to a lesser degree than LRRK2 G2019S) [32], whereas the other indicated that over-expressing wild-type dLRRK did not affect the number of dopa-minergic neurons or motor function [31]

Interestingly, two studies have shown a relationship

of LRRK2 or dLRRK to the dopamine physiology Liu

et al [32] found that treatment of l-DOPA improved the motor impairment of transgenic flies caused by LRRK-G2019S but not the degeneration of TH+ neu-rons The results obtained by Imai et al [31] suggested that dLRRK is involved in negatively regulating homeostatic levels of dopamine They demonstrated that the over-expression of a PD-pathogenic mutant of dLRRK (but not wild-type dLRRK) resulted in a reduction in brain dopamine levels compared to that

of nontransgenic flies Conversely, dopamine content was elevated in mutant flies with a dLRRK deletion This increase in dopamine content is likely to be a result of dopamine release, uptake or metabolism, but not to an alteration of TH+ neuron numbers [31] Finally, Imai et al [31] provided evidence that both dLRRK and LRRK2 kinase can phosphorylate eukaryotic initiation factor 4E-binding protein, a nega-tive regulator of eukaryotic initiation factor 4E-medi-ated protein translation and a key mediator of various stress responses They proposed a model in which LRRK2 mediates the pathological effect in part through modulating translation initiation [31]

In summary, these studies in fruit flies have provided

important in vivo information regarding the potential

function of LRRK2 (Table 1) Indeed, certain

observa-tions reported in fly models may appear to be

conflict-ing However, it is possible that the different genetic

backgrounds, the genomic locus of insertion for gene

disruption, transgenic expression levels and nutrient

conditions are responsible for the divergent results

These issues need to be resolved in the future in order

to understand better the physiological function of

LRRK2, as well as the pathogenic effect of PD

muta-tions of LRRK2 Once validated in mammalian

models, selected models could serve as a robust system

for revealing the genetic pathways of LRRK2 in PD

and for screening chemical compounds to intervene

with the LRRK2-mediated pathogenesis

Rodent models of LRRK2 and bacterial artificial

chromosome (BAC)-mediated LRRK2 transgenic

mice

Although there have been several studies reporting the

generation of genetically engineered LRRK2 mice

(including targeted deletion and transgenic expression),

no systematic investigation of these mice has been

described to date (one report was published recently

during the preparation of this manuscript [33])

There-fore, the physiological role of LRRK2 in the

mamma-lian central nervous system remains largely elusive

Analysis of LRRK2 expression in mouse brain shows

that it is broadly distributed in many regions, including

the cerebral cortex, hippocampus, striatum, amygdala,

cerebelluam and olfactory bulb, as well as in ventral

tegamental area and substantia nigra (albeit at low

lev-els) [11,34–40] Analysis of LRRK2 expression levels

during pre- and post-natal stages reveals that the

LRRK2 protein appears at embryonic day 17 (E17)

and is increasingly produced over the early post-natal

stage [11,34], reaching peak levels by 2 months [11]

LRRK2 knockout (KO) mice

Biskup et al [34] were the first to report the generation

of LRRK2 KO mice Taking advantage of the lack of

LRRK2 expression in these mice, they performed the

comprehensive evaluation of a panel of commercial

antibodies against LRRK2 for their staining specificity

However, no characterization of these mice was shown

[34] Although without showing any experimental data,

a study by Wang et al [41] indicated that LRRK2 KO

mice survive normally, and that they do not develop

any obvious neuropathological abnormalities or motor

dysfunctions up to 12 months of age Indeed, no loss

of dopaminergic neurons or motor behavioral deficits was observed even at 24 months of age in LRRK2 KO mice (Dr H Cai, personal communication) This result, along with the study showing the developmental expression levels of LRRK2, suggests that the role of LRRK2 in early embryonic development is negligible, but may be important for cellular function at the adult stage Furthermore, although it is possible that the lack of LRRK2 function can be compensated for by LRRK2 function-related molecules (e.g LRRK1), this observation in LRRK2 KO mice is consistent with the findings in nematode worms and fruit flies that the deletion of the single homolog of LRRK2 in either species has no effect on the viability of dopaminergic neurons Therefore, we propose that LRRK2 (as well

as LRRK2 homologs dLRRK and LRK-1) does not play a major role in a cellular pathway that is critical for neuronal survival Rather, it is involved perhaps in specific neuronal functions that can only distantly modulate neuronal survival or death in an age-depen-dent manner

It is also not surprising that the deletion of the LRRK2gene does not lead to degeneration of dopami-nergic neurons in mice, given that disruption of all known PD-related genes, such as a-synuclein, Parkin, DJ-1 and PINK-1, has not been associated with any obvious loss of dopaminergic neurons in mice It is intriguing to note that none of these PD-related genes are essential for neural development and differentia-tion, which is also in support of the hypothesis that dysfunction of these genes only leads to disruption of neuronal functions mostly at the adult stage in PD

BAC transgenic mice of LRRK2

To date, three laboratories have reported the availabil-ity of LRRK2 transgenic mice without providing details of the characterization of these mice Two labo-ratories, including ours, generated BAC-transgenic mice expressing murine FLAG-tagged LRRK2 [11] and human LRRK2 [42], whereas the third indicated the usage of a tetracyline-regulated system for the transgenic expression of human G2019S LRRK2 [41] The application of BAC-transgenic mice was initially described in 1997 [43] and has grown significantly over the past decade because of its usefulness in studying gene function in vivo, particularly in the central ner-vous system [44] Growing evidence demonstrates the power of this transgenic approach in conferring correct transgene expression under endogenous promoter con-trol with little concern about positional effect [45] The BAC transgenic approach has been successfully used

in establishing mouse models for neurodegenerative

diseases [46] and is expected to contribute to an

under-standing of the disease mechanisms in vivo

The application of BAC transgenics is especially

advantageous over conventional transgenics for

study-ing LRRK2 The main reasons are: (1) generation of

LRRK2 BAC transgenic mice does not involve the

synthesis of full-length LRRK2 cDNA, which is a

> 7 kb nucleotide and technically difficult to

manipu-late as a result of the large size; (2) the entire genomic

sequence of mouse or human LRRK2 is approximately

180 kb, which is the average length of BAC clones that are readily available in public domains; and (3) LRRK2 BAC transgenes with introduced PD muta-tions are suitable for modeling the LRRK2-mediated pathological process as a result of the dominant disease transmission for LRRK2 mutations Our laboratory has previously generated numerous BAC transgenic lines expressing FLAG-tagged LRRK2 wild-type Examination of the transgene expression in the brain shows a similar distribution pattern in all

Table 1 In vivo models for LRRK2 and LRRK2 homologs.

Truncation of endogenous

LRK-1

Worm Subtle defects in movement; partially defective in chemotaxis to volatile

odorants; impairment of polarized synaptic vesicle localization

[25]

Transgenic expression of

human LRRK2

Worm Over-expression of LRRK2 wild-type protects against rotenone toxicity and

extend life span; over-expression of LRRK2 G2019S also protects but to a lesser extent

[26]

Transgenic expression of

green flourescent

protein-tagged LRK-1 wild-type or

G1876S mutant

Worm Over-expression of either LRK-1 wild-type or G1876S (corresponding to

human G2019S) leads to an early larval arrest

[27]

Disruption of endogenous

LRK-1

Worm Antagonistic action of worm Lrk-1 versus Pink-1 in stress response and

neuronal activities

[27] Transgenic expression and

disruption of endogenous

dLRRK

Fly No obvious behavioral abnormality associated with transgenic over-expression

of dLRRK wild-type or mutant; deletion mutant shows impaired locomotive activity and a significant reduction of TH immunostaining in dopaminergic neurons

[29]

Disruption of endogenous

dLRRK

Fly No obvious behavioral deficits; unchanged TH+ neurons; enhanced sensitivity

to H2O2

[30] Disruption of endogenous

dLRRK

Fly Relatively resistant to general oxidative stress; reduced oxidative damage;

unchanged TH+ neurons; increased dopamine content

[31]

Transgenic expression of

dLRRK

Fly Over-expression of ‘pathogenic’ dLRRK mutant caused loss of TH+ neurons

in aged mice and reduced dopamine content; over-expression of wild-type dLRRK2 or kinase-dead mutant had no effect on viability of TH+ neurons

[31]

Transgenic expression of

human LRRK2

Fly Over-expression of LRRK2 wild-type or G2019S mutant causes loss of TH+

neurons and impairment of motor function (with worse phenotype in G2019S mutant flies); treatment of L -DOPA improves motor function but not neurodegeneration

[32]

LRRK2 KO Mouse Survived normally; display no overt behavioral abnormality; unaltered number

of dopaminergic neurons for up to 24 months

[41, and unpublished results] BAC transgenics ⁄ murine

LRRK2

Mouse One line expressing FLAG tagged LRRK2 wild-type (> 20-fold) shows regulated

expression pattern and unaltered TH+ neuron morphology or number

[11]

BAC transgenics ⁄ human

LRRK2

Mouse Human BAC mice show very similar expression pattern to mouse BAC

transgenic lines [11]; over-expression of LRRK2 wild-type (> 20-fold), G2019S or Y1699C (seven- to 11-fold and up to 12 months) did not cause overt behavioral abnormalities

[42,47]

Tetracycline-regulated

transgenics ⁄ human LRRK2

G2019S

Mouse No obvious neuropathologies or motor abnormalities at 12 months and older [41]

BAC transgenics ⁄ human

LRRK2 R1441C

Mouse BAC mice expressing LRRK2 R1441C develop typical motor function

abnormality related to PD; no obvious loss of midbrain TH+ cells; age-dependent and levodopa-responsive slowness of movement associated with reduced dopamine release and axonal pathology of nigrostriatal

dopaminergic projection

[33]

transgenic lines We identified one BAC line that

pro-duces FLAG-LRRK2 wild-type protein at a level

twenty-fold greater than the endogenous LRRK2

pro-tein [11] Unexpectedly, the transgenic mice overloaded

with the exogenous LRRK2 did not show obvious

neurotoxicity or motor function abnormalities over

20 months (X Li and Z Yue, unpublished results),

despite FLAG-LRRK2 purified from transgenic brain

displaying robust kinase and GTPase activity [11]

Melrose et al [42] previously reported the generation

of BAC transgenic mice producing human LRRK2

wild-type or mutants Although no information was

given about the viability of TH+ neurons, it was

indi-cated that BAC transgenic mice over-expressing

LRRK2 wild-type [20-fold for up to 24 months),

mutant G2019S or Y1699C (seven- to 11-fold for up

to 12 months) did not show an overt behavioral

phe-notype [47] Consistent with these observations,

tetra-cycline-regulated transgenic mice producing LRRK2

G2019S were also reported to be spared of obvious

neuropathologies or motor abnormalities at 12 months

and older [41]

Interestingly, a more recent study by Li et al [33]

suggests that BAC transgenic mice expressing the

human LRRK2 R1441C mutant develop typical motor

function deficit related to PD These mice are

associ-ated with the degeneration of TH+ axons and

tauopa-thy, as well as TH+ cell atrophy, despite lacking

obvious loss of midbrain TH+ cells Furthermore,

these BAC models develop an age-dependent and

levo-dopa-responsive slowness of movement associated with

diminished dopamine release and axonal pathology of

nigrostriatal dopaminergic projection [33] Although

this transgenic line provides a promising model for

fur-ther dissection of LRRK2-associated PD pathogenesis,

future experiments will be needed to resolve the

differ-ence in behavioral as well as possible pathological

phenotypes observed among different BAC transgenic

models Although it remains unclear at present, the

dif-ferent PD-related mutations examined and the distinct

genetic background of the host mice, as well as the

varied transgene expression levels, may be responsible

for the differential phenotype of these BAC models

It is mysterious that none of the reported LRRK2

transgenic mice show the loss of dopaminergic neurons

or the accumulation of a-synuclein at substantia nigra,

the hallmarks of PD pathology Alhough the

physio-logical function of LRRK2 has yet to be formally

dem-onstrated in these transgenic models, current evidence

suggests that the pathological consequence of

over-expressing only LRRK2 wild-type or PD-related

muta-tions in rodent model is mostly neuron dysfunction,

rather than degeneration One of the intriguing findings

reported by Li et al [47] is that the LRRK2-R1441C BAC mice show reduced dopamine release, which is consistent with previous studies conducted in fruit flies showing the connection of dLRRK to dopamine and movement control [31,32] In addition, we found that BAC transgenic mice expressing LRRK2-G2019S also displayed a decrease in dopamine release and striatal dopamine levels in the absence of obvious neuropathol-ogy (X Li, J C Patel and coworkers, unpublished results) These results suggest a pathogenic role of LRRK2 mutants in the deregulation of the striatal dopamine system Whether other PD-related mutants

of LRRK2 also have the same effect, and whether the normal function of LRRK2 is involved in striatal dopa-mine transmission, remains to be shown

The above observation, therefore, is in line with the previous evidence indicating that single genetic alter-ation of PD-related genes, such as the over-expression

of a dominant gene a-synuclein or the deletion of a recessive gene (DJ-1, Parkin or PINK1), is unlikely to recapitulate the full spectrum of PD The lack of mani-festation of the most important PD hallmarks in LRRK2 transgenic mice (e.g dopaminergic neuron loss and deposits of a-synuclein in Lewy body) is also not surprising considering that LRRK2 PD-mutations are not fully penetrant and that LRRK2 patients dis-play a broad range of clinical phenotypes [1–4] There-fore, the current challenges facing us are not only to

‘tease out’ LRRK2-associated neuronal functions that are perturbed as a result of PD-related mutations, but also to identify the cellular pathways or factors that cross-talk with and thus can significantly modify LRRK2-mediated phenotypic expressions

Concluding remarks

The invertebrate models including nematode worms and fruit flies have begun to unveil the functions of the orthologs of LRRK2 in vivo (Table 1) Although rapid analysis of these models will undoubtedly facili-tate an understanding of the function of LRRK2 func-tion in PD, the lack of a sophisticated structural and functional equivalent of the human central nervous system in these organisms limits their application when understanding the in vivo function of LRRK2 in humans The ultimate comprehension of LRRK2 phys-iology and pathophysphys-iology in PD will still depend on the establishment and detailed characterization of mammalian models of LRRK2 The collective data obtained from both KO and transgenic mouse models (albeit in preliminary form) suggest that LRRK2 is not essential for neural development and differentia-tion, and that it does not play a primary role in cell

death pathways However, these LRRK2 rodent

mod-els should provide valuable tools for dissecting the

specific neuronal functions of LRRK2 (e.g dopamine

transmission) and likely pre-symptomatic (or early)

events of the disease process They should also be

use-ful in testing the ‘two-hit’ or ‘multiple-hit’ hypothesis

proposing that LRRK2 and other genetic or

environ-mental factors are required to work together and

facilitate the pathological process of PD

Acknowledgements

I wish to thank Drs Chenjian Li, Huaibin Cai,

Xianting Li and Sarah Funderburk for their critical

comments, and Dr Huaibin Cai for sharing

unpub-lished results I am also grateful to Dr Nina Pan for

assisting in the preparation of Fig 1 and Table 1 This

work was supported by grants to Z.Y from the US

NIH⁄ NINDS NS061152, NS060809, RNS055683A,

the Michael J Fox Foundation, and the

Bachmann-Strauss Dystonia & Parkinson Foundation

References

1 Zimprich A, Biskup S, Leitner P, Lichtner P, Farrer M,

Lincoln S, Kachergus J, Hulihan M, Uitti RJ, Calne

DB et al (2004) Mutations in LRRK2 cause

autoso-mal-dominant parkinsonism with pleomorphic

pathology Neuron 44, 601–607

2 Paisan-Ruiz C, Jain S, Evans EW, Gilks WP, Simon J,

van der Brug M, Lopez de Munain A, Aparicio S, Gil

AM, Khan N et al (2004) Cloning of the gene

contain-ing mutations that cause PARK8-linked Parkinson’s

disease Neuron 44, 595–600

3 Healy DG, Falchi M, O’Sullivan SS, Bonifati V, Durr

A, Bressman S, Brice A, Aasly J, Zabetian CP,

Goldwurm S et al (2008) Phenotype, genotype, and

worldwide genetic penetrance of LRRK2-associated

Parkinson’s disease: a case-control study Lancet Neurol

7, 583–590

4 Ozelius LJ, Senthil G, Saunders-Pullman R, Ohmann E,

Deligtisch A, Tagliati M, Hunt AL, Klein C, Henick B,

Hailpern SM et al (2006) LRRK2 G2019S as a cause

of Parkinson’s disease in Ashkenazi Jews N Engl J

Med 354, 424–425

5 Bosgraaf L & Van Haastert PJ (2003) Roc, a Ras⁄

GT-Pase domain in complex proteins Biochim Biophys Acta

1643, 5–10

6 Marin I (2006) The Parkinson disease gene LRRK2:

evolutionary and structural insights Mol Biol Evol 23,

2423–2433

7 West AB, Moore DJ, Biskup S, Bugayenko A, Smith

WW, Ross CA, Dawson VL & Dawson TM (2005)

Parkinson’s disease-associated mutations in leucine-rich

repeat kinase 2 augment kinase activity Proc Natl Acad Sci USA 102, 16842–16847

8 Gloeckner CJ, Kinkl N, Schumacher A, Braun RJ, O’Neill E, Meitinger T, Kolch W, Prokisch H & Ueffing M (2006) The Parkinson disease causing LRRK2 mutation I2020T is associated with increased kinase activity Hum Mol Genet 15, 223–232

9 Smith WW, Pei Z, Jiang H, Dawson VL, Dawson TM

& Ross CA (2006) Kinase activity of mutant LRRK2 mediates neuronal toxicity Nat Neurosci 9, 1231–1233

10 Ito G, Okai T, Fujino G, Takeda K, Ichijo H, Katada

T & Iwatsubo T (2007) GTP binding is essential to the protein kinase activity of LRRK2, a causative gene product for familial Parkinson’s disease Biochemistry

46, 1380–1388

11 Li X, Tan YC, Poulose S, Olanow CW, Huang XY & Yue Z (2007) Leucine-rich repeat kinase 2

(LRRK2)⁄ PARK8 possesses GTPase activity that is altered in familial Parkinson’s disease R1441C⁄ G mutants J Neurochem 103, 238–247

12 Guo L, Gandhi PN, Wang W, Petersen RB, Wilson-Delfosse AL & Chen SG (2007) The Parkinson’s dis-ease-associated protein, leucine-rich repeat kinase 2 (LRRK2), is an authentic GTPase that stimulates kinase activity Exp Cell Res 313, 3658–3670

13 Smith WW, Pei Z, Jiang H, Moore DJ, Liang Y, West

AB, Dawson VL, Dawson TM & Ross CA (2005) Leucine-rich repeat kinase 2 (LRRK2) interacts with parkin, and mutant LRRK2 induces neuronal degeneration Proc Natl Acad Sci USA 102, 18676– 18681

14 Greggio E, Jain S, Kingsbury A, Bandopadhyay R, Lewis P, Kaganovich A, van der Brug MP, Beilina A, Blackinton J, Thomas KJ et al (2006) Kinase activity is required for the toxic effects of mutant LRRK2⁄ darda-rin Neurobiol Dis 23, 329–341

15 MacLeod D, Dowman J, Hammond R, Leete T, Inoue

K & Abeliovich A (2006) The familial Parkinsonism gene LRRK2 regulates neurite process morphology Neuron 52, 587–593

16 van Egmond WN, Kortholt A, Plak K, Bosgraaf L, Bosgraaf S, Keizer-Gunnink I & van Haastert PJ (2008) Intramolecular activation mechanism of the

Dictyosteli-um LRRK2 homolog Roco protein GbpC J Biol Chem

283, 30412–30420

17 Abysalh JC, Kuchnicki LL & Larochelle DA (2003) The identification of pats1, a novel gene locus required for cytokinesis in Dictyostelium discoideum Mol Biol Cell 14, 14–25

18 Gotthardt K, Weyand M, Kortholt A, Van Haastert PJ

& Wittinghofer A (2008) Structure of the Roc-COR domain tandem of C tepidum, a prokaryotic homo-logue of the human LRRK2 Parkinson kinase EMBO

J 27, 2352

19 West AB, Moore DJ, Choi C, Andrabi SA, Li X,

Dikeman D, Biskup S, Zhang Z, Lim KL, Dawson VL

et al.(2007) Parkinson’s disease-associated mutations in

LRRK2 link enhanced GTP-binding and kinase

activities to neuronal toxicity Hum Mol Genet 16,

223–232

20 Bosgraaf L, Russcher H, Smith JL, Wessels D, Soll DR

& Van Haastert PJ (2002) A novel cGMP signalling

pathway mediating myosin phosphorylation and

chemo-taxis in Dictyostelium EMBO J 21, 4560–4570

21 Bosgraaf L, Waijer A, Engel R, Visser AJ, Wessels D,

Soll D & van Haastert PJ (2005) RasGEF-containing

proteins GbpC and GbpD have differential effects on

cell polarity and chemotaxis in Dictyostelium J Cell Sci

118, 1899–1910

22 Dachsel JC, Taylor JP, Mok SS, Ross OA, Hinkle

KM, Bailey RM, Hines JH, Szutu J, Madden B,

Petrucelli L et al (2007) Identification of potential

protein interactors of Lrrk2 Parkinsonism Relat

Disord 13, 382–385

23 Gandhi PN, Wang X, Zhu X, Chen SG &

Wilson-Delf-osse AL (2008) The Roc domain of leucine-rich repeat

kinase 2 is sufficient for interaction with microtubules

J Neurosci Res 86, 1711–1720

24 Jaleel M, Nichols RJ, Deak M, Campbell DG,

Gillardon F, Knebel A & Alessi DR (2007) LRRK2

phosphorylates moesin at threonine-558:

characteriza-tion of how Parkinson’s disease mutants affect kinase

activity Biochem J 405, 307–317

25 Sakaguchi-Nakashima A, Meir JY, Jin Y, Matsumoto

K & Hisamoto N (2007) LRK-1, a C elegans

PARK8-related kinase, regulates axonal-dendritic polarity of SV

proteins Curr Biol 17, 592–598

26 Wolozin B, Saha S, Guillily M, Ferree A & Riley M

(2008) Investigating convergent actions of genes linked

to familial Parkinson’s disease Neurodegener Dis 5,

182–185

27 Samann J, Hegermann J, von Gromoff E, Eimer S,

Baumeister R & Schmidt E (2009) Caenorhabditits

ele-gans LRK-1 and PINK-1 act antagonistically in stress

response and neurite outgrowth J Biol Chem 284,

16482–16491

28 Valente EM, Abou-Sleiman PM, Caputo V, Muqit

MM, Harvey K, Gispert S, Ali Z, Del Turco D,

Bentivoglio AR, Healy DG et al (2004) Hereditary

early-onset Parkinson’s disease caused by mutations in

PINK1 Science 304, 1158–1160

29 Lee SB, Kim W, Lee S & Chung J (2007) Loss of

LRRK2⁄ PARK8 induces degeneration of dopaminergic

neurons in Drosophila Biochem Biophys Res Commun

358, 534–539

30 Wang D, Tang B, Zhao G, Pan Q, Xia K, Bodmer R &

Zhang Z (2008) Dispensable role of Drosophila

ortho-log of LRRK2 kinase activity in survival of

dopaminer-gic neurons Mol Neurodegener 3, 3

31 Imai Y, Gehrke S, Wang HQ, Takahashi R, Hasegawa

K, Oota E & Lu B (2008) Phosphorylation of 4E-BP

by LRRK2 affects the maintenance of dopaminergic neurons in Drosophila EMBO J 27, 2432–2443

32 Liu Z, Wang X, Yu Y, Li X, Wang T, Jiang H, Ren Q, Jiao Y, Sawa A, Moran T et al (2008) A Drosophila model for LRRK2-linked parkinsonism Proc Natl Acad Sci USA 105, 2693–2698

33 Li Y, Liu W, Oo TF, Wang L, Tang Y, Jackson-Lewis

V, Zhou C, Geghman K, Bogdanov M, Przedborski S

et al.(2009) Mutant LRRK2(R1441G) BAC transgenic mice recapitulate cardinal features of Parkinson’s disease Nat Neurosci 12, 826–828

34 Biskup S, Moore DJ, Rea A, Lorenz-Deperieux B, Coombes CE, Dawson VL, Dawson TM & West AB (2007) Dynamic and redundant regulation of LRRK2 and LRRK1 expression BMC Neurosci 8, 102

35 Westerlund M, Ran C, Borgkvist A, Sterky FH, Lindq-vist E, Lundstromer K, Pernold K, Brene S, Kallunki

P, Fisone G et al (2008) Lrrk2 and alpha-synuclein are co-regulated in rodent striatum Mol Cell Neurosci 39, 586–591

36 Galter D, Westerlund M, Carmine A, Lindqvist E, Sydow O & Olson L (2006) LRRK2 expression linked to dopamine-innervated areas Ann Neurol 59, 714–719

37 Melrose H, Lincoln S, Tyndall G, Dickson D & Farrer

M (2006) Anatomical localization of leucine-rich repeat kinase 2 in mouse brain Neuroscience 139, 791–794

38 Higashi S, Biskup S, West AB, Trinkaus D, Dawson

VL, Faull RL, Waldvogel HJ, Arai H, Dawson TM, Moore DJ et al (2007) Localization of Parkinson’s dis-ease-associated LRRK2 in normal and pathological human brain Brain Res 1155, 208–219

39 Simon-Sanchez J, Herranz-Perez V, Olucha-Bordonau

F & Perez-Tur J (2006) LRRK2 is expressed in areas affected by Parkinson’s disease in the adult mouse brain Eur J Neurosci 23, 659–666

40 Taymans JM, Van den Haute C & Baekelandt V (2006) Distribution of PINK1 and LRRK2 in rat and mouse brain J Neurochem 98, 951–961

41 Wang L, Xie C, Greggio E, Parisiadou L, Shim H, Sun

L, Chandran J, Lin X, Lai C, Yang WJ et al (2008) The chaperone activity of heat shock protein 90 is criti-cal for maintaining the stability of leucine-rich repeat kinase 2 J Neurosci 28, 3384–3391

42 Melrose HL, Kent CB, Taylor JP, Dachsel JC, Hinkle

KM, Lincoln SJ, Mok SS, Culvenor JG, Masters CL, Tyndall GM et al (2007) A comparative analysis of leucine-rich repeat kinase 2 (Lrrk2) expression in mouse brain and Lewy body disease Neuroscience 147, 1047– 1058

43 Antoch MP, Song EJ, Chang AM, Vitaterna MH, Zhao

Y, Wilsbacher LD, Sangoram AM, King DP, Pinto LH

& Takahashi JS (1997) Functional identification of the

mouse circadian Clock gene by transgenic BAC rescue.

Cell 89, 655–667

44 Heintz N (2001) BAC to the future: the use of bac

transgenic mice for neuroscience research Nat Rev

Neurosci 2, 861–870

45 Gong S, Zheng C, Doughty ML, Losos K, Didkovsky

N, Schambra UB, Nowak NJ, Joyner A, Leblanc G,

Hatten ME et al (2003) A gene expression atlas of the

central nervous system based on bacterial artificial

chromosomes Nature 425, 917–925

46 Gray M, Shirasaki DI, Cepeda C, Andre VM, Wilburn

B, Lu XH, Tao J, Yamazaki I, Li SH, Sun YE et al (2008) Full-length human mutant huntingtin with a stable polyglutamine repeat can elicit progressive and selective neuropathogenesis in BACHD mice J Neurosci

28, 6182–6195

47 Melrose H (2008) Update on the functional biology of Lrrk2 Future Neurol 3, 669–681