Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of familial Parkinson's disease (PD), resembling the sporadic disorder. Intensive effort has been directed toward LRRK2 mouse modeling and investigation, aimed at reproducing the human disease to inform mechanistic studies of pathogenesis and design of neuroprotective therapies. The physiological function of LRRK2 is still under exploration, but a clear role in striatal neurophysiology and animal behavior has emerged. Alterations in LRRK2 impair dopamine (DA) transmission, regulation and signaling, in addition to corticostriatal synaptic plasticity. Consistently, several subtle abnormalities in motor and nonmotor abilities have been demonstrated in LRRK2 genetic mouse models, generally paralleling preclinical symptoms of early DA dysfunction. However, the variability in model design and phenotypes observed requires a critical approach in interpreting the results, adapting the model used to the specific research question. Etiologically appropriate knockin mice might represent the ultimate animal model in which to study early disease mechanisms and therapies as well as to investigate drug effectiveness and off-target consequences.

Introduction

Parkinson's disease (PD) has long been considered a sporadic disorder in which genetics played hardly any role. This view was changed by the discovery of familial forms linked to precise gene alterations indicating a causal hereditary component [1]. Up to 14 genes have now been reported to carry Mendelian mutations in families with a history of parkinsonism [2], including the leucine-rich repeat kinase 2 (LRRK2) gene. Several mutations linked to autosomal dominant, late-onset PD have been described in this gene and coded protein. All major pathogenic mutations reside in the enzymatic domains, with R1441G/C/H and Y1699C affecting the GTPase domain and G2019S and I2020T substitutions located in the kinase domain. LRRK2-PD patients are clinically indistinguishable from sporadic PD (the vast majority of patients) [3]. Diagnosis of parkinsonism is based on motor impairment, characterized by bradykinesia, rigidity, tremor and postural instability [4], commonly accompanied by several nonmotor symptoms [5] including cognitive dysfunction and dementia [6]. Neuropathology features the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc) leading to DA depletion in the striatum, and the development of intracytoplasmic inclusions, termed Lewy bodies and neurites, mainly composed of α-synuclein [7]. Of note, at autopsy, examination of LRRK2-associated parkinsonism presents with pleomorphic neuropathology, ranging from pure nigral degeneration to accumulation of tau neurofibrillary tangles or classic Lewy pathology [3,8,9].

The p.G2019S substitution is the most common pathogenic mutation in LRRK2 and accounts for up to 40% of PD patients in specific populations [10,11], with ethnicity and age affecting penetrance [12]. This mutation resides in the kinase domain, and it has been shown to increase its activity, which prompted the development of selective LRRK2 kinase inhibitors as a disease-modifying treatment [13]. To gain insights into disease-causing mechanisms and to test molecules targeting LRRK2, several rodent models have been developed and aimed at reproducing the progressive behavioral impairments and neurodegeneration. In this review, we discuss the key features of mouse models with a focus on the role of LRRK2 on neurotransmission and neurotransmitter release in the striatum, a brain area that is critically involved in pathogenesis and symptom manifestation, and how the results from different genetic approaches could inform on the mechanisms underlying pathogenesis and aid therapeutic testing.

Striatal neurotransmission and neurotransmitter release in PD: role of LRRK2

As the target area of SNc DA neurons, the striatum has been the subject of intense studies. Pharmacological or toxic depletion of striatal DA in animal models leads to l-DOPA-responsive motor impairments (see ref. [14] for a review). The striatal neuronal population is represented for >90% by inhibitory spiny projection neurons (SPNs), with GABAergic and cholinergic interneurons constituting the remaining portion. The SPNs receive DA innervation from nigral neurons and glutamate inputs from the cerebral cortex and thalamus (Figure 1), and are divided into two subpopulations based on their target nuclei: in the rodent brain, striatonigral neurons project directly to the substantia nigra pars reticulata (SNr), whereas striatopallidal neurons synapse onto globus pallidus neurons, only indirectly communicating to the SNr [15]. The co-ordinated activity of these two pathways forms the basis of voluntary motor control [16] where DA is critically involved [17,18]. In addition, DA acts as a necessary neuromodulator of corticostriatal synaptic plasticity at SPNs [19]. Finally, synaptic and axon terminal dysfunction in the striatum have been proposed to be the first trigger to neurodegeneration, occurring as a ‘dying-back’ process and affecting the neuronal cell body at later stages [20,21].

Neuronal connections in the striatum influenced by LRRK2.

Figure 1.
Neuronal connections in the striatum influenced by LRRK2.

Simplified cartoon depicting the major neuronal pathways and connections in the dorsolateral striatum. Glutamate neurons (red) from cortex (and thalamus) send their projections to the striatum through the corpus callosum, of which constitute the white matter. These axon terminals synapse onto the dendritic spines of SPNs (green), characterized by a large dendritic arborization. The DA neurons in the SNc also send their projections to the dorsal striatum, where they give rise to highly ramified axon terminals that capillary innervate the area. Along these ramifications, several varicosities release DA and constitute their presynaptic terminals. Here, DA acts on receptors located on both SPN populations, modulating their activity: stimulation of D1Rs expressed mainly on direct pathway SPNs leads to neuronal activation, whereas D2Rs on indirect pathway SPNs inhibit neuronal activity. In addition, DA plays an important role as a neuromodulator regulating the strength of corticostriatal synapses and is required for synaptic plasticity at these contacts. In addition, D2Rs expressed on cortical neuron presynaptic terminals modulate their activity. The result of all these co-ordinated connections is the control of voluntary movement. LRRK2 is proposed to act in the striatum at different levels: 1 — LRRK2 affects DA machinery and DA release from nigral neuron axon terminals, possibly through a direct mechanism, with PD-linked mutations generally causing a reduction in DA release; 2 — LRRK2 modulates plasticity of corticostriatal synapses, with high LRRK2 protein levels altering short-term plasticity in a D2R-dependent manner, and overexpression of the G2019S mutation preventing corticostriatal long-term synaptic plasticity; 3 — related to the function in synaptic plasticity, overexpression of LRRK2 influences the probability of release of glutamate from cortical terminals in the striatum after repeated stimulation and the G2019S mutation enhances glutamate activity in cortical neuron cultures; 4 — based on these findings, it can be argued that the activity of SPNs changes depending on LRRK2, thus affecting the striatal output pathways and the regulation of target nuclei with downstream consequences on modulation of motor behavior.

Figure 1.
Neuronal connections in the striatum influenced by LRRK2.

Simplified cartoon depicting the major neuronal pathways and connections in the dorsolateral striatum. Glutamate neurons (red) from cortex (and thalamus) send their projections to the striatum through the corpus callosum, of which constitute the white matter. These axon terminals synapse onto the dendritic spines of SPNs (green), characterized by a large dendritic arborization. The DA neurons in the SNc also send their projections to the dorsal striatum, where they give rise to highly ramified axon terminals that capillary innervate the area. Along these ramifications, several varicosities release DA and constitute their presynaptic terminals. Here, DA acts on receptors located on both SPN populations, modulating their activity: stimulation of D1Rs expressed mainly on direct pathway SPNs leads to neuronal activation, whereas D2Rs on indirect pathway SPNs inhibit neuronal activity. In addition, DA plays an important role as a neuromodulator regulating the strength of corticostriatal synapses and is required for synaptic plasticity at these contacts. In addition, D2Rs expressed on cortical neuron presynaptic terminals modulate their activity. The result of all these co-ordinated connections is the control of voluntary movement. LRRK2 is proposed to act in the striatum at different levels: 1 — LRRK2 affects DA machinery and DA release from nigral neuron axon terminals, possibly through a direct mechanism, with PD-linked mutations generally causing a reduction in DA release; 2 — LRRK2 modulates plasticity of corticostriatal synapses, with high LRRK2 protein levels altering short-term plasticity in a D2R-dependent manner, and overexpression of the G2019S mutation preventing corticostriatal long-term synaptic plasticity; 3 — related to the function in synaptic plasticity, overexpression of LRRK2 influences the probability of release of glutamate from cortical terminals in the striatum after repeated stimulation and the G2019S mutation enhances glutamate activity in cortical neuron cultures; 4 — based on these findings, it can be argued that the activity of SPNs changes depending on LRRK2, thus affecting the striatal output pathways and the regulation of target nuclei with downstream consequences on modulation of motor behavior.

LRRK2 is expressed in axons and dendrites of striatum and cortex [22,23], has low expression in nigral cell bodies [24] and regulates presynaptic vesicle dynamics [25]. Transgenic LRRK2 mouse models were initially developed using bacterial artificial chromosomes (BAC, see Table 1), which contains all the promoters and regulatory sequences of the gene of interest and thus allows a pattern of expression that resembles the physiologic localization [26]. Consistently with LRRK2 expression sites, showing a relative low abundance in SNc, initial reports did not show nigral neurodegeneration or Lewy-like pathology, but demonstrated reduction in striatal extracellular DA levels and alterations in presynaptic regulation of DA release together with axonal swelling and tau accumulation [2729]. Specifically, overexpression of mutant LRRK2 impaired DA transmission, while the effect of the wild-type (WT) version was inconsistent across studies. One caveat is that two studies overexpressed a human LRRK2 BAC [28,29], while the third utilized a murine BAC [27]. In addition, different background strains were used to develop these models. However, recent work from our laboratories confirmed that BAC mice expressing human WT-LRRK2 display lower striatal DA [30], but it cannot be excluded that overexpression of human or mouse transgene (on the top of endogenous LRRK2) might lead to different phenotypes.

Table 1
Transgenic LRRK2 rodent models

Synopsis of reports studying transgenic LRRK2, which have been grouped according to the genetic strategy utilized. We included a selection of studies in order to compare BAC- and cDNA-based models, as we discussed in the manuscript, which could help the understanding of early effects of LRRK2 on neurotransmission.

Study Mutation DA system Behavior phenotype 
BAC models 
 Li et al. 2009 [28]; Sanchez et al. 2014 [33hR1441G 35% reduction in extracellular DA release (after nomifensine) in 10-month-old mice ([28]) (no change at 2 months, [33]) Reduced rearing, hypokinesia 
hWT Not studied None 
 Li et al. 2010 [27mG2019S 35% reduction in extracellular DA (single pulse evoked) None 
mWT 25% increase in extracellular DA (single pulse evoked) Enhanced spontaneous activity, increased rearing 
 Melrose et al. 2010 [29]; Beccano-Kelly  et al. 2015 [30];  Volta et al. 2015 [38hG2019S 30% decrease in basal extracellular DA release Mild thigmotaxis in the open field [29]; early mild hyperactivity, late cognitive deficit [38
hWT 66% decrease in basal extracellular DA release None [29]; motor and cognitive impairments [30
 Walker et al. 2014 [64hG2019S (rat) No changes in extracellular DA release Impaired motor co-ordination on rotarod 
 Sloan et al. 2016 [65hWT (rat) Age-dependent reduction of single-pulse evoked DA release None 
 hG2019S (rat) Age-dependent reduction of single-pulse evoke DA release Age-dependent, L-DOPA responsive reduction in rotarod performance and spatial memory deficits 
 hR1441C (rat) Age-dependent reduction of single-pulse evoked DA release; age-dependent reduction in burst firing in SNc neurons Age-dependent, L-DOPA responsive reduction in rotarod performance and spatial memory deficits 
cDNA models 
 Zhou et al. 2011 [66Inducible and constitutive hG2019S rats under TRE promoter Elevated DA basal release in dox suppressed rats (LRRK2 expressed only in adulthood). Reduced DA release after nomifensine. No changes in constitutive hG2019S rats Increased locomotor activity 
 Maekawa et al. 2012 [31hI2020T cDNA Reduced total striatal DA levels Impaired locomotor activity 
 Chen et al. 2012 [51]; Chou et al. 2014 [46CMV enhancer/PDGF-b promoter hG2019S Decrease in the spontaneous firing rate and a reduction in evoked DA release Reduced locomotor activity 
CMV enhancer/PDGF-b promoter hWT None None 
 Liu et al. 2015 [36Pitx3-IRES2-tTA/tetO-LRRK2 hG2019S and hWT bigenic mice (expression is directed to nigra) Decreased total striatal DA and decreased extracellular DA release in G2019S. Increased total striatal DA and extracellular DA release in hWT None 
Study Mutation DA system Behavior phenotype 
BAC models 
 Li et al. 2009 [28]; Sanchez et al. 2014 [33hR1441G 35% reduction in extracellular DA release (after nomifensine) in 10-month-old mice ([28]) (no change at 2 months, [33]) Reduced rearing, hypokinesia 
hWT Not studied None 
 Li et al. 2010 [27mG2019S 35% reduction in extracellular DA (single pulse evoked) None 
mWT 25% increase in extracellular DA (single pulse evoked) Enhanced spontaneous activity, increased rearing 
 Melrose et al. 2010 [29]; Beccano-Kelly  et al. 2015 [30];  Volta et al. 2015 [38hG2019S 30% decrease in basal extracellular DA release Mild thigmotaxis in the open field [29]; early mild hyperactivity, late cognitive deficit [38
hWT 66% decrease in basal extracellular DA release None [29]; motor and cognitive impairments [30
 Walker et al. 2014 [64hG2019S (rat) No changes in extracellular DA release Impaired motor co-ordination on rotarod 
 Sloan et al. 2016 [65hWT (rat) Age-dependent reduction of single-pulse evoked DA release None 
 hG2019S (rat) Age-dependent reduction of single-pulse evoke DA release Age-dependent, L-DOPA responsive reduction in rotarod performance and spatial memory deficits 
 hR1441C (rat) Age-dependent reduction of single-pulse evoked DA release; age-dependent reduction in burst firing in SNc neurons Age-dependent, L-DOPA responsive reduction in rotarod performance and spatial memory deficits 
cDNA models 
 Zhou et al. 2011 [66Inducible and constitutive hG2019S rats under TRE promoter Elevated DA basal release in dox suppressed rats (LRRK2 expressed only in adulthood). Reduced DA release after nomifensine. No changes in constitutive hG2019S rats Increased locomotor activity 
 Maekawa et al. 2012 [31hI2020T cDNA Reduced total striatal DA levels Impaired locomotor activity 
 Chen et al. 2012 [51]; Chou et al. 2014 [46CMV enhancer/PDGF-b promoter hG2019S Decrease in the spontaneous firing rate and a reduction in evoked DA release Reduced locomotor activity 
CMV enhancer/PDGF-b promoter hWT None None 
 Liu et al. 2015 [36Pitx3-IRES2-tTA/tetO-LRRK2 hG2019S and hWT bigenic mice (expression is directed to nigra) Decreased total striatal DA and decreased extracellular DA release in G2019S. Increased total striatal DA and extracellular DA release in hWT None 

Other transgenic models have been developed using different genetic strategies, that is, using LRRK2 cDNA under the control of heterologous promoters that either direct pan-neuronal expression or restrict it specifically to DA neurons (Table 1). Overexpression of I2020T-LRRK2 caused reduction in striatal DA tissue content [31], whereas G2019S and R1441C mutation did not lead to any difference [32]. To increase complexity, voltammetric determination of action potential-induced DA overflow in striatal slices was not changed in R1441G-LRRK2 BAC mice [33], but was diminished in cDNA-based R1441C transgenic mice [34]. Of note, conditional overexpression of R1441C-LRRK2 in midbrain DA neurons did not significantly affect striatal DA content [35], while the same approach applied to G2019S mutation resulted in age-dependent decrease in DA content and release [36]. It should be noted that DA impairments in cDNA models are usually coupled to some degree of nigral neurodegeneration (with the exception of Thy1-LRRK2 mice which present normal neuron number but where assessment of DA release was not carried out [37]), whereas in BAC mice no DA loss has been reported, pointing to localized DA terminal dysfunction rather than a direct consequence of neuronal death. This could reflect the tendency of BAC transgenes to recapitulate gene expression levels, timing and patterns that are closer to the physiologic situation, whereas higher levels of differential anatomic gene expression are typically achieved with heterologous promoters. In support of this, we did not observe alterations in stimulated release of tritiated DA from striatal axon terminals in synaptosomes prepared from BAC human G2019S-LRRK2 mice; however, synaptosomes did display reduced sensitivity to pharmacological inhibition of DA release via DA D2 receptor (D2R) agonism [38], suggesting that DA terminal number is normal but function and regulation are indeed altered.

Altogether, studies in overexpressing models clearly involve LRRK2 in the regulation of DA neurotransmission and release; however, it is noteworthy that LRRK2 knockout (KO) animals do not show functional alterations in the nigrostriatal DA system [3941], suggesting that the absence of LRRK2 can be compensated by other players. Although being informative, overexpression of transgenic LRRK2 on an endogenous LRRK2 background may confound teasing out the precise consequences of LRRK2 mutations on DA transmission. In this regard, knockin (KI) mouse models represent the most appropriate context in which to study the effect of mutations, bearing physiologic levels of expression and presence of only the endogenous, mutated protein (Table 2). Early reports did not include measurement of extracellular striatal DA in LRRK2 KI mice [40,42], but the R1441C mutation was demonstrated to reduce the sensitivity of nigral neurons toward DAergic-mediated inhibition of firing (which was not different at basal conditions) [42]. Recently, we demonstrated that G2019S KI mice develop age-dependent reduction in striatal extracellular DA levels coupled to mitochondrial abnormalities and some degree of tau pathology [43]. It is important to mention that we recently reported increased (glutamatergic) synaptic transmission in primary cortical neurons prepared from the same mice [44]. Very recently, cortical inputs onto SPNs have been shown to be increased in a kinase-dependent manner in young G2019S KI mice [45], supporting an effect of this mutation on neurotransmission. Earlier, we also demonstrated that high levels of human WT-LRRK2 alter corticostriatal short-term synaptic plasticity and these alterations were dependent on D2Rs [30], and transgenic mice carrying G2019S-LRRK2 failed in expressing the long-term depression form of synaptic plasticity at these synapses [46]. Thus, a picture emerges whereby LRRK2 acts in the regulation of both DA release and DA-mediated plasticity at corticostriatal synapses, with pathogenic mutations probably affecting these mechanisms in an age-dependent manner. We hypothesized that these effects might be temporally separated and account for the progressive nature of PD. Consistently, overexpression of G2019S-LRRK2 also impairs hippocampal synaptic plasticity [47]. Further longitudinal studies are warranted to dissect the consequences of G2019S (KI) mutation on striatal physiology and the relation to the DA system. In addition, we posit that these changes might represent early alterations in humans that precede the onset of the disease. Indeed, the correct functioning of neurons is paramount to their health, and chronic dysfunction can cause sustained cellular stress ultimately leading to cell loss.

Table 2
LRRK2 KI mouse models carrying PD-causing mutations

Reports studying LRRK2 KI mice have been grouped according to the mouse line utilized. We have included mutant knock-in models that reported only in vitro data, in addition to those that extend to in vivo/ex vivo studies of the DA system and behavior.

NA, not applicable.

Study Mutation In vitro only? DA system Behavior phenotype 
Mouse line ‘Jie Shen’ 
 Tong et al. 2009 [42R1441C No Reduced sensitivity to DA-mediated inhibition of firing Reduced sensitivity to D2R-dependent locomotion inhibition 
 Nichols et al. 2010 [67R1441C Yes NA NA 
 Parisiadou et al. 2014 [52R1441C No NA Reduced sensitivity to D1R-mediated locomotion stimulation 
Mouse line ‘Novartis’ 
 Herzig et al. 2011 [40G2019S No No alterations No alterations 
 Longo et al. 2014 [58G2019S No NA Hyperkinetic phenotype 
Mouse line ‘Melrose and Farrer’ 
 Dächsel et al. 2010 [68G2019S Yes NA NA 
 Beccano-Kelly et al. 2014 [44G2019S Yes NA NA 
 Yue et al. 2015 [43G2019S No Age-dependent reduction in extracellular DA levels in the striatum Early hyperactivity followed by anxiety-like behaviors 
 Lin et al. 2016 [69G2019S Yes NA NA 
Mouse line ‘Shu-Leong Ho’ 
 Liu et al. 2014 [70R1441G No Increased sensitivity to reserpine-induced reduction in tritiated DA uptake from striatal synaptosomes Increased sensitivity to reserpine-induced motor deficits 
 Ito et al. 2016 [71R1441G Yes NA NA 
Mouse line ‘Eli-Lilly’ 
 Steger et al. 2016 [59G2019S Yes NA NA 
 Matikainen-Ankney  et al. 2016 [45G2019S No Not assessed Not assessed 
Mouse line ‘GlaxoSmithKline’ 
 Steger et al. 2016 [59G2019S Yes NA NA 
 Ito et al. 2016 [71G2019S Yes NA NA 
Study Mutation In vitro only? DA system Behavior phenotype 
Mouse line ‘Jie Shen’ 
 Tong et al. 2009 [42R1441C No Reduced sensitivity to DA-mediated inhibition of firing Reduced sensitivity to D2R-dependent locomotion inhibition 
 Nichols et al. 2010 [67R1441C Yes NA NA 
 Parisiadou et al. 2014 [52R1441C No NA Reduced sensitivity to D1R-mediated locomotion stimulation 
Mouse line ‘Novartis’ 
 Herzig et al. 2011 [40G2019S No No alterations No alterations 
 Longo et al. 2014 [58G2019S No NA Hyperkinetic phenotype 
Mouse line ‘Melrose and Farrer’ 
 Dächsel et al. 2010 [68G2019S Yes NA NA 
 Beccano-Kelly et al. 2014 [44G2019S Yes NA NA 
 Yue et al. 2015 [43G2019S No Age-dependent reduction in extracellular DA levels in the striatum Early hyperactivity followed by anxiety-like behaviors 
 Lin et al. 2016 [69G2019S Yes NA NA 
Mouse line ‘Shu-Leong Ho’ 
 Liu et al. 2014 [70R1441G No Increased sensitivity to reserpine-induced reduction in tritiated DA uptake from striatal synaptosomes Increased sensitivity to reserpine-induced motor deficits 
 Ito et al. 2016 [71R1441G Yes NA NA 
Mouse line ‘Eli-Lilly’ 
 Steger et al. 2016 [59G2019S Yes NA NA 
 Matikainen-Ankney  et al. 2016 [45G2019S No Not assessed Not assessed 
Mouse line ‘GlaxoSmithKline’ 
 Steger et al. 2016 [59G2019S Yes NA NA 
 Ito et al. 2016 [71G2019S Yes NA NA 

The role of LRRK2 in animal behavior

Along with assessment of nigrostriatal function, LRRK2 mouse models have been intensely utilized for behavioral studies with the hope of reproducing the progressive motor impairment that defines PD. Similar to DA transmission, behavioral results vary from model to model, increasing the difficulty of interpretation. The very first report showed striking progressive, l-DOPA-responsive motor deficits in BAC human R1441G-LRRK2 mice that rendered them almost immobile at old age, despite decreased DA levels being detectable only after blockade of DA transporter [28]; a subsequent study using the same animals confirmed the late-stage locomotion reduction but in a significantly lower order of magnitude [48]. At variance, BAC human G2019S-LRRK2 animals displayed mild hyperactivity at a young adult age point [29,38], which decreased with age but not reaching full-blown parkinsonism [38]. Of note, BAC murine G2019S-LRRK2 mice did not differ from controls, but animals overexpressing murine WT-LRRK2 were hyperactive [27]. This was not confirmed in BAC human WT-LRRK2 mice, which resulted either normal [29] or displayed motor and cognitive impairment [30,38]. Elsewhere, cDNA-based G2019S-LRRK2 mice showed increased motor activity in the rotarod test [49], while locomotion results in the open field were contrasting [49,50]. A separate mouse model expressing G2019S-LRRK2 showed late-age motor deficits, but it is important to note that in this case ∼30% DA neuron loss was observed [51]. In this respect, it is key to distinguish models in which behavioral alterations occur in the absence of neurodegeneration (but possibly in conjunction with DA transmission dysfunctions) in the interpretation of results. Indeed, recapitulation of neuronal cell death will lead to behavioral deficits as exemplified by toxin models, while the absence of neurodegeneration, despite limiting the usefulness in reproducing the human disease, could facilitate molecular and pharmacological dissection of early and possibly reversible events, rather than focusing on late-stage, inexorable symptomatology.

Although cognitive impairment often accompanies motor disorder in PD, learning and memory have not been extensively studied in LRRK2 overexpressing animals. We have observed alterations in spatial and recognition memories in BAC human WT- and G2019S-LRRK2 mice [30,38], which are consistent with abnormalities in striatal [30,46] and hippocampal synaptic plasticity [47].

In addition to modeling PD features (using mutant LRRK2 overexpression), research was aimed at elucidating the role of endogenous LRRK2 in these behaviors in KO mice. Again, variability between laboratories hampers a clear conclusion. Normal horizontal activity has been observed in KO mice up to 12 months of age [30,38,39] even in the presence of alterations in anxiety-like behaviors and enhanced rotarod performance [39]. In contrast, very young (P21) LRRK2 KO mice showed increased locomotion, in parallel to alterations in SPN's spine maturation and synaptic transmission [52]. However, such changes were not observed in much older animals [30,39], while mild decreases in neurotransmission were reported in cortical neurons prepared from LRRK2 KO mice [44]. Altogether, these data suggest that deletion of LRRK2 has an impact on neuronal development and leads to very early alterations that are compensated for in adulthood. One hypothesis posits that the paralog LRRK1, which shares some functions in endosomal trafficking [53] and dimerizes with LRRK2 [54], could compensate for LRRK2 dysfunctions. However, no changes in LRRK1 gene expression have been observed in BAC, KO or KI models [29,39,43], and the study of LRRK1 protein levels was precluded by lack of specific LRRK1 antibodies. Finally, acute silencing of striatal LRRK2 in adult mice was also tolerated in terms of DA terminal maintenance and behavior [38], further supporting the hypothesis that a degree of redundancy for the role of LRRK2 is developed with maturation.

The available evidence thus suggests that LRRK2 can be removed without overt negative consequences at the neurological level. However, overexpression and mutations generally cause motor and cognitive deficits that appear consistent with the alterations observed in DA and non-DA neurotransmission (see the previous section).

LRRK2 KI mouse models: modeling early PD?

Without doubt, transgenic and KO mouse models have greatly contributed to the understanding of the role of LRRK2 in neurobiology and behavior. That being said, LRRK2-PD is linked to single amino acid variations and, to date, there is no genetic evidence in humans that PD is caused by alterations in LRRK2 expression levels. Ideally, a disease model requires the exact genetic reproduction of the human disease in the mouse. With this in mind, several laboratories, including ours, are focusing their efforts on the study of gene-targeted LRRK2 KI models, which we have summarized in Table 2. A distinct physiological advantage of the KI models is they permit the investigation of the effects of gene dosage, logically relevant since LRRK2-PD patients can be found in both the heterozygous and homozygous state [55,56]. Similar to BAC-based models, the KI mutant mice did not display age-dependent neurodegeneration or α-synuclein pathology, somewhat limiting enthusiasm toward these models. Nevertheless, neurophysiology yielded interesting results that appear consistent with the general mechanisms proposed to be affected by LRRK2 (as discussed above). Studies accurately investigating behavioral phenotypes are still scarce, but have provided critical insights. Mice carrying R1441C or G2019S mutation were initially shown to feature normal motor abilities across a wide age range [40,42], but pharmacological stimulation of D2Rs in R1441C KI animals was less efficient in inducing locomotion inhibition [42]. Alteration of D2R-dependent signaling has become a recurring theme of LRRK2 mouse models, regardless of the genetic strategy employed. Indeed, we found increased D2R protein expression in the striatum of BAC human WT-LRRK2 mice, but decreased phosphorylation of the SPN-specific DA- and cAMP-regulated phosphoprotein of 32 kDa (DARPP32) protein in a site regulated by D2Rs [30]. Nevertheless, autoradiographic binding of tritiated D2R ligand [29] and D2R protein expression [27] were also reported as unaltered, perhaps suggesting that intracellular signaling rather than protein levels is the mechanism affected by LRRK2. Consistently, R1441C KI mice also showed alteration in signaling cascades downstream from DA D1 receptors (D1Rs) and differential behavioral response to stimulation of this receptor subclass [52]. These data further support the appropriateness of physiologic mouse models since changes in receptor protein expression are probably due to LRRK2 overexpression and dependent on the particular mouse line, while downstream signaling is consistently altered also in KI mice. Indeed, in the above section, we discussed the alteration in DA transmission and release in KI animals [43] pointing to early and subtle changes. Moreover, these deficits were observed in heterozygous KI mice, demonstrating that just one copy of the mutant, endogenous allele is sufficient to impair DA transmission.

Taken together, we propose that the true potential of LRRK2 KI mice ultimately lies within the subtlety of the phenotypes displayed, in the perspective that the KI models allow the individuation of early disease mechanisms as well as exploration of the benefits of neuroprotection/neurointervention long before the onset of motor symptoms and pathology. This is paramount, since in PD patients, motor symptoms usually do not present until 40–50% of DA neurons are already lost [57], at which point intervention would probably be ineffective. Understanding the early mechanisms would allow the best chance to mitigate synaptic failure and prevent or slow the progression of axonopathy to neuronal death. In addition, investigating these early changes could also offer the possibility of nominating biomarkers that would allow the monitoring of both disease progression and drug effectiveness.

One caveat is that different KI lines for a single mutation have been developed and discordances in phenotyping have emerged. For example, G2019S-LRRK2 KI mice produced at Novartis have recently been shown to resist age-related motor decline and display a hyperactive phenotype [58], while mice carrying the same mutation developed at Mayo Clinic feature increased motor abilities when younger which decay by 1 year of age [43]. Of note, the same Novartis mice were initially reported to be comparable with WT littermates [40]. Assessment of animal behavior is influenced by many variables, most of which are not controllable by the operators. Subtle differences in the gene-targeting strategies, background strain, rederivation procedures, housing and experimental environment might all account for the slight variations between models.

In addition to disease modeling, LRRK2 KI mice represent a valuable resource for pharmacological studies. Animals carrying mutations that abolish LRRK2 kinase activity (kinase dead) or render it resistant to kinase inhibitors have been developed and are utilized to assess the involvement of the kinase activity in the phenotypes observed, measure the effectiveness of kinase inhibitors and reveal possible off-target effects. Indeed, the inclusion of KI G2019S and A2016T kinase inhibitor resistant mouse lines in a recent phosphoproteomic study facilitated the discovery of several members of the Rab GTPase family as the first ‘bona fide’ in vivo substrates of LRRK2 kinase activity, via comparative studies of KI mouse-derived cells with and without kinase inhibitor treatment [59]. Rab GTPases shuffle between the cytosol and membrane compartments, where they are activated and interact with their cellular effectors. Rab proteins play crucial roles in membrane trafficking between several membrane-bound organelles, regulating vesicle trafficking (for a review, see ref. [60]). Of interest, Rabs interact with SNARE complexes to mediate vesicle fusions to membranes, a process that is necessary for neurotransmitter release [61]. In their study, Steger et al. [59] identified Rab8a as a substrate of LRRK2 and, interestingly, Rab8 homeostasis has been previously reported to be disrupted by α-synuclein [62]. Thus, the identification of Rab proteins as LRRK2 substrates strongly supports the involvement of LRRK2 in fundamental processes ultimately affecting neurotransmission and neurotransmitter release (Figure 1).

Genetic studies have recently focused their attention on modifiers of LRRK2-PD penetrance and age of onset [63]. These lines of research will undoubtedly provide future opportunities to apply those (etiologically relevant) modifiers to existing KI models with the hypothesis that they will be ‘pushed’ from synaptic failure/dysfunction to progressive DA loss and Lewy body pathology. Such an accelerated model would then allow the ability to monitor and track key events participating in the switch from axonopathy to cell death and identify the ‘point of no return’, which would be critical for successful neuroprotective therapy.

Abbreviations

     
  • BAC

    bacterial artificial chromosome

  •  
  • CMV

    cytomegalovirus

  •  
  • D1R

    dopamine D1 receptor

  •  
  • D2R

    dopamine D2 receptor

  •  
  • DA

    dopamine

  •  
  • KI

    knockin

  •  
  • KO

    knockout

  •  
  • LRRK

    leucine-rich repeat kinase

  •  
  • PD

    Parkinson's disease

  •  
  • PDGF

    platelet-derived growth factor

  •  
  • SNc

    substantia nigra pars compacta

  •  
  • SNr

    substantia nigra pars reticulata

  •  
  • SPN

    spiny projection neuron

  •  
  • TRE

    tetracycline responsive element

  •  
  • WT

    wild type.

Funding

This work was supported by Parkinson Society Canada (M.V., 2014–2016 grant cycle), National Institute of Neurological Disorders and Stroke [5R01NS65860-2 to H.M.] and the Michael J. Fox Foundation LRRK2 consortium (H.M.).

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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