Leucine-rich Repeat Kinase 2 Mice Research Articles

Modeling Parkinson's disease in LRRK2 mice: focus on synaptic dysfunction and the autophagy-lysosomal pathway.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with familial and sporadic forms of Parkinson's disease (PD), for which the LRRK2 locus itself represents a risk factor. Idiopathic and LRRK2-related PD share the main clinical and neuropathological features, thus animals harboring the most common LRRK2 mutations, i.e. G2019S and R1441C/G, have been generated to replicate the parkinsonian phenotype and investigate the underlying pathological mechanisms. Most LRRK2 rodent models, however, fail to show the main neuropathological hallmarks of the disease i.e. the degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of Lewy bodies or Lewy body-like aggregates of α-synuclein, lacking face validity. Rather, they manifest dysregulation in cellular pathways and functions that confer susceptibility to a variety of parkinsonian toxins/triggers and model the presymptomatic/premotor stages of the disease. Among such susceptibility factors, dysregulation of synaptic activity and proteostasis are evident in LRRK2 mutants. These abnormalities are also manifest in the PD brain and represent key events in the development and progression of the pathology. The present minireview covers recent articles (2018-2021) investigating the role of LRRK2 and LRRK2 mutants in the regulation of synaptic activity and autophagy-lysosomal pathway. These articles confirm a perturbation of synaptic vesicle endocytosis and glutamate release in LRRK2 mutants. Likewise, LRRK2 mutants show a marked impairment of selective forms of autophagy (i.e. mitophagy and chaperone-mediated autophagy) and lysosomal function, with minimal perturbations of nonselective autophagy. Thus, LRRK2 rodents might help understand the contribution of these pathways to PD.

Aberrant mitochondrial morphology and function associated with impaired mitophagy and DNM1L-MAPK/ERK signaling are found in aged mutant Parkinsonian LRRK2R1441G mice

ABSTRACT Mitochondrial dysfunction causes energy deficiency and nigrostriatal neurodegeneration which is integral to the pathogenesis of Parkinson disease (PD). Clearance of defective mitochondria involves fission and ubiquitin-dependent degradation via mitophagy to maintain energy homeostasis. We hypothesize that LRRK2 (leucine-rich repeat kinase 2) mutation disrupts mitochondrial turnover causing accumulation of defective mitochondria in aging brain. We found more ubiquitinated mitochondria with aberrant morphology associated with impaired function in aged (but not young) LRRK2R1441G knockin mutant mouse striatum compared to wild-type (WT) controls. LRRK2R1441G mutant mouse embryonic fibroblasts (MEFs) exhibited reduced MAP1LC3/LC3 activation indicating impaired macroautophagy/autophagy. Mutant MEFs under FCCP-induced (mitochondrial uncoupler) stress showed increased LC3-aggregates demonstrating impaired mitophagy. Using a novel flow cytometry assay to quantify mitophagic rates in MEFs expressing photoactivatable mito-PAmCherry, we found significantly slower mitochondria clearance in mutant cells. Specific LRRK2 kinase inhibition using GNE-7915 did not alleviate impaired mitochondrial clearance suggesting a lack of direct relationship to increased kinase activity alone. DNM1L/Drp1 knockdown in MEFs slowed mitochondrial clearance indicating that DNM1L is a prerequisite for mitophagy. DNM1L knockdown in slowing mitochondrial clearance was less pronounced in mutant MEFs, indicating preexisting impaired DNM1L activation. DNM1L knockdown disrupted mitochondrial network which was more evident in mutant MEFs. DNM1L-Ser616 and MAPK/ERK phosphorylation which mediate mitochondrial fission and downstream mitophagic processes was apparent in WT using FCCP-induced stress but not mutant MEFs, despite similar total MAPK/ERK and DNM1L levels. In conclusion, aberrant mitochondria morphology and dysfunction associated with impaired mitophagy and DNM1L-MAPK/ERK signaling are found in mutant LRRK2 MEFs and mouse brain. Abbreviations: ATP: adenosine triphosphate; BAX: BCL2-associated X protein; CDK1: cyclin-dependent kinase 1; CDK5: cyclin-dependent kinase 5; CQ: chloroquine; CSF: cerebrospinal fluid; DNM1L/DRP1: dynamin 1-like; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; LAMP2A: lysosomal-associated membrane protein 2A; LRRK2: leucine-rich repeat kinase 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MEF: mouse embryonic fibroblast; MFN1: mitofusin 1; MMP: mitochondrial membrane potential; PAmCherry: photoactivatable-mCherry; PD: Parkinson disease; PINK1: PTEN induced putative kinase 1; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RAB10: RAB10, member RAS oncogene family; RAF: v-raf-leukemia oncogene; SNCA: synuclein, alpha; TEM: transmission electron microscopy; VDAC: voltage-dependent anion channel; WT: wild type; SQSTM1/p62: sequestosome 1.

A75 ROLE OF LRRK2 IN INFLAMMATORY BOWEL DISEASE

Abstract Background Variants of the leucine-rich repeat kinase 2 (LRRK2) are associated with an increased susceptibility to Parkinson disease but also Crohn’s disease (CD). Aims The present research is designed to develop a comprehensive understanding of the role of LRRK2 in immune system modulation, and how dysfunction of this pathway may lead to the development of CD. Methods WT and LRRK2-deficient neutrophil were infected with Gram-positive Bacteria (Listeria monocytogenes-LM) in a gentamicin protection assays and colony-forming unit assessment will determine the competence of LRRK2 deficient cells for bacterial phagocytosis as well as killing capacity). To examine how LRRK2 is involved in the generation of ROS during the respiratory burst, we will first examine if neutrophil from LRRK2-KO mice have altered ROS generation upon infection with LM and addition of PMA. We evaluate in vitro the ability of neutrophils from LRRK2-KO versus WT mice to transmigrate in vitro in a transwell assay using fMLP as a chemattractant. Also, we investigate the peritoneal cells (by FACS analysis) after injection of different microbial stimuli including FK105 (NOD1 ligand), MDP (NOD2 ligand) and LPS (TLR4 ligand) and anti-cd3 model of ielitis. Results We found that LRRK2 KO mice have a defect in migration of neutrophils to the peritoneal cavity after injection of different microbial stimuli including FK10565 (NOD1 ligand), MDP (NOD2 ligand) and LPS (TLR4 ligand). Neutrophils from LRRK2 mice were compromised in their ability to transmigrate in vitro in a transwell assay using fMLP as a chemoattractant. Chemotaxis was also compromised. In parallel, we designed experiments to examine reactive oxygen species (ROS) produced in response to infection of myeloid cells with bacteria. Neutrophils from LRRK2 KO mice infected with Listeria monocytogenes were less able to restrict bacteria growth compared to WT cells. Consistent with these findings, cells from LRRK2 KO mice produced lower levels of ROS following bacterial infection. In order to determine whether myeloid cell migration is compromised in vivo during inflammation, we performed experiments in WT and KO mice looking at different models of ileitis/colitis. Conclusions With this work we will further characterize the role of LRRK2 in intestinal homeostasis and mucosal barrier maintenance, including how its deficiency may predispose an individual to developing CD. Funding Agencies CAG, CIHR

LRRK2 mouse models: dissecting the behavior, striatal neurochemistry and neurophysiology of PD pathogenesis.

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.

Effects of LRRK2 Inhibitors on Nigrostriatal Dopaminergic Neurotransmission.

SummaryIntroductionMutations in leucine‐rich repeat kinase 2 (LRRK2) are the most prevalent cause of familial and sporadic Parkinson's disease (PD). Because most pathogenic LRRK2 mutations result in enhanced kinase activity, it suggests that LRRK2 inhibitors may serve as a potential treatment for PD. To evaluate whether LRRK2 inhibitors are effective therapies for PD, it is crucial to know whether LRRK2 inhibitors will affect dopaminergic (DAergic) neurotransmission. However, to date, there is no study to investigate the impact of LRRK2 inhibitors on DAergic neurotransmission.AimsTo address this gap in knowledge, we examined the effects of three types of LRRK2 inhibitors (LRRK2‐IN‐1, GSK2578215A, and GNE‐7915) on dopamine (DA) release in the dorsal striatum using fast‐scan cyclic voltammetry and DA neuron firing in the substantia nigra pars compacta (SNpc) using patch clamp in mouse brain slices.ResultsWe found that LRRK2‐IN‐1 at a concentration higher than 1 μM causes off‐target effects and decreases DA release, whereas GSK2578215A and GNE‐7915 do not. All three inhibitors at 1 μM have no effect on DA release and DA neuron firing rate. We have further assessed the effects of the inhibitors in two preclinical LRRK2 mouse models (i.e., BAC transgenic hG2019S and hR1441G) and demonstrated that GNE‐7915 enhances DA release and synaptic vesicle mobilization/recycling.Conclusion GNE‐7915 can be validated for further therapeutic development for PD.

Leucine-rich repeat kinase 2 interacts with p21-activated kinase 6 to control neurite complexity in mammalian brain.

Leucine‐rich repeat kinase 2 (LRRK2) is a causative gene for Parkinson's disease, but the physiological function and the mechanism(s) by which the cellular activity of LRRK2 is regulated are poorly understood. Here, we identified p21‐activated kinase 6 (PAK6) as a novel interactor of the GTPase/ROC domain of LRRK2. p21‐activated kinases are serine‐threonine kinases that serve as targets for the small GTP binding proteins Cdc42 and Rac1 and have been implicated in different morphogenetic processes through remodeling of the actin cytoskeleton such as synapse formation and neuritogenesis. Using an in vivo neuromorphology assay, we show that PAK6 is a positive regulator of neurite outgrowth and that LRRK2 is required for this function. Analyses of post‐mortem brain tissue from idiopathic and LRRK2 G2019S carriers reveal an increase in PAK6 activation state, whereas knock‐out LRRK2 mice display reduced PAK6 activation and phosphorylation of PAK6 substrates. Taken together, these results support a critical role of LRRK2 GTPase domain in cytoskeletal dynamics in vivo through the novel interactor PAK6, and provide a valuable platform to unravel the mechanism underlying LRRK2‐mediated pathophysiology. We propose p21‐activated kinase 6 (PAK6) as a novel interactor of leucine‐rich repeat kinase 2 (LRRK2), a kinase involved in Parkinson's disease (PD). In health, PAK6 regulates neurite complexity in the brain and LRRK2 is required for its function, (a) whereas PAK6 is aberrantly activated in LRRK2‐linked PD brain (b) suggesting that LRRK2 toxicity is mediated by PAK6.

(R1441C) LRRK2 induces the degeneration of SN dopaminergic neurons and alters the expression of genes regulating neuronal survival in a transgenic mouse model

Mutation of leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of both familial and sporadic Parkinson's disease (PD) cases. Several mutations in LRRK2 gene were reported in PD patients. R1441 is the second most frequent site of LRRK2 mutation. We generated (R1441C) LRRK2 transgenic mice that displayed motor deficits at the age of 16months. Compared with wild-type mice, 16-month-old (R1441C) LRRK2 mice exhibited a significant reduction in the number of substantia nigra (SN) dopaminergic neurons. To elucidate molecular pathogenic pathways involved in (R1441C) LRRK2-induced death of SN dopaminergic neurons, we performed microarray analysis to visualize altered mRNA expressions in the SN of (R1441C) LRRK2 mouse. In the SN of (R1441C) LRRK2 transgenic mouse, the mRNA expression of three genes that promote cell death was upregulated, while the mRNA expression of seven genes that contribute to neurogenesis/neuroprotection was significantly downregulated. Our results suggest that altered expression of these genes involved in regulating neuronal survival may contribute to the pathogenesis of (R1441C) LRRK2-induced PD.

Conditional expression of Parkinson's disease-related R1441C LRRK2 in midbrain dopaminergic neurons of mice causes nuclear abnormalities without neurodegeneration

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). The clinical and neurochemical features of LRRK2-linked PD are similar to idiopathic disease although neuropathology is somewhat heterogeneous. Dominant mutations in LRRK2 precipitate neurodegeneration through a toxic gain-of-function mechanism which can be modeled in transgenic mice overexpressing human LRRK2 variants. A number of LRRK2 transgenic mouse models have been developed that display abnormalities in dopaminergic neurotransmission and alterations in tau metabolism yet without consistently inducing dopaminergic neurodegeneration. To directly explore the impact of mutant LRRK2 on the nigrostriatal dopaminergic pathway, we developed conditional transgenic mice that selectively express human R1441C LRRK2 in dopaminergic neurons from the endogenous murine ROSA26 promoter. The expression of R1441C LRRK2 does not induce the degeneration of substantia nigra dopaminergic neurons or striatal dopamine deficits in mice up to 2years of age, and fails to precipitate abnormal protein inclusions containing alpha-synuclein, tau, ubiquitin or autophagy markers (LC3 and p62). Furthermore, mice expressing R1441C LRRK2 exhibit normal motor activity and olfactory function with increasing age. Intriguingly, the expression of R1441C LRRK2 induces age-dependent abnormalities of the nuclear envelope in nigral dopaminergic neurons including reduced nuclear circularity and increased invaginations of the nuclear envelope. In addition, R1441C LRRK2 mice display increased neurite complexity of cultured midbrain dopaminergic neurons. Collectively, these novel R1441C LRRK2 conditional transgenic mice reveal altered dopaminergic neuronal morphology with advancing age, and provide a useful tool for exploring the pathogenic mechanisms underlying the R1441C LRRK2 mutation in PD.

LRRK2 R1441G mice are more liable to dopamine depletion and locomotor inactivity.

ObjectiveMutations in leucine-rich repeat kinase 2 (LRRK2) pose a significant genetic risk in familial and sporadic Parkinson's disease (PD). R1441 mutation (R1441G/C) in its GTPase domain is found in familial PD. How LRRK2 interacts with synaptic proteins, and its role in dopamine (DA) homeostasis and synaptic vesicle recycling remain unclear.MethodsTo explore the pathogenic effects of LRRK2R1441G mutation on nigrostriatal synaptic nerve terminals and locomotor activity, we generated C57BL/6N mice with homozygous LRRK2R1441G knockin (KI) mutation, and examined for early changes in nigrostriatal region, striatal synaptosomal [3H]-DA uptake and locomotor activity after reserpine-induced DA depletion.ResultsUnder normal conditions, mutant mice showed no differences, (1) in amount and morphology of nigrostriatal DA neurons and neurites, (2) tyrosine hydroxylase (TH), DA uptake transporter (DAT), vesicular monoamine transporter-2 (VMAT2) expression in striatum, (3) COX IV, LC3B, Beclin-1 expression in midbrain, (4) LRRK2 expression in total cell lysate from whole brain, (5) α-synuclein, ubiquitin, and tau protein immunostaining in midbrain, (6) locomotor activity, compared to wild-type controls. However, after a single intraperitoneal reserpine dose, striatal synaptosomes from young 3-month-old mutant mice demonstrated significantly lower DA uptake with impaired locomotor activity and significantly slower recovery from the effects of reserpine.InterpretationAlthough no abnormal phenotype was observed in mutant LRRK2R1441G mice, the KI mutation increases vulnerability to reserpine-induced striatal DA depletion and perturbed DA homeostasis resulting in presynaptic dysfunction and locomotor deficits with impaired recovery from reserpine. This subtle nigrostriatal synaptic vulnerability may reflect one of the earliest pathogenic processes in LRRK2-associated PD.

Gene and MicroRNA Transcriptome Analysis of Parkinson's Related LRRK2 Mouse Models

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of genetic Parkinson’s disease (PD). The biological function of LRRK2 and how mutations lead to disease remain poorly defined. It has been proposed that LRRK2 could function in gene transcription regulation; however, this issue remains controversial. Here, we investigated in parallel gene and microRNA (miRNA) transcriptome profiles of three different LRRK2 mouse models. Striatal tissue was isolated from adult LRRK2 knockout (KO) mice, as well as mice expressing human LRRK2 wildtype (hLRRK2-WT) or the PD-associated R1441G mutation (hLRRK2-R1441G). We identified a total of 761 genes and 24 miRNAs that were misregulated in the absence of LRRK2 when a false discovery rate of 0.2 was applied. Notably, most changes in gene expression were modest (i.e., <2 fold). By real-time quantitative RT-PCR, we confirmed the variations of selected genes (e.g., adra2, syt2, opalin) and miRNAs (e.g., miR-16, miR-25). Surprisingly, little or no changes in gene expression were observed in mice expressing hLRRK2-WT or hLRRK2-R1441G when compared to non-transgenic controls. Nevertheless, a number of miRNAs were misexpressed in these models. Bioinformatics analysis identified several miRNA-dependent and independent networks dysregulated in LRRK2-deficient mice, including PD-related pathways. These results suggest that brain LRRK2 plays an overall modest role in gene transcription regulation in mammals; however, these effects seem context and RNA type-dependent. Our data thus set the stage for future investigations regarding LRRK2 function in PD development.

Leucine-rich repeat kinase 2 functionally interacts with microtubules and kinase-dependently modulates cell migration

Recent studies indicate that the Parkinson's disease-linked leucine-rich repeat kinase 2 (LRRK2) modulates cytoskeletal functions by regulating actin and tubulin dynamics, thereby affecting neurite outgrowth. By interactome analysis we demonstrate that the binding of LRRK2 to tubulins is significantly enhanced by pharmacological LRRK2 inhibition in cells. Co-incubation of LRRK2 with microtubules increased the LRRK2 GTPase activity in a cell-free assay. Destabilization of microtubules causes a rapid decrease in cellular LRRK2(S935) phosphorylation indicating a decreased LRRK2 kinase activity. Moreover, both human LRRK2(G2019S) fibroblasts and mouse LRRK2(R1441G) fibroblasts exhibit alterations in cell migration in culture. Treatment of mouse fibroblasts with the selective LRRK2 inhibitor LRRK2-IN1 reduces cell motility. These findings suggest that LRRK2 and microtubules mutually interact both in non-neuronal cells and in neurons, which might contribute to our understanding of its pathogenic effects in Parkinson's disease.

(G2019S) LRRK2 activates MKK4-JNK pathway and causes degeneration of SN dopaminergic neurons in a transgenic mouse model of PD.

(G2019S) mutation of leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of both familial and sporadic Parkinson's disease (PD) cases. Twelve- to sixteen-month-old (G2019S) LRRK2 transgenic mice prepared by us displayed progressive degeneration of substantia nigra pars compacta (SNpc) dopaminergic neurons and parkinsonism phenotypes of motor dysfunction. LRRK2 is a member of mixed lineage kinase subfamily of mitogen-activated protein kinase kinase kinases (MAPKKKs). We hypothesized that (G2019S) mutation augmented LRRK2 kinase activity, leading to overphosphorylation of downstream MAPK kinase (MKK) and resulting in activation of neuronal death signal pathway. Consistent with our hypothesis, (G2019S) LRRK2 expressed in HEK 293 cells exhibited an augmented kinase activity of phosphorylating MAPK kinase 4 (MKK4) at Ser(257), and protein expression of active phospho-MKK4(Ser257) was upregulated in the SN of (G2019S) LRRK2 transgenic mice. Protein level of active phospho-JNK(Thr183/Tyr185) and phospho-c-Jun(Ser63), downstream targets of phospho-MKK4(Ser257), was increased in the SN of (G2019S) LRRK2 mice. Upregulated mRNA expression of pro-apoptotic Bim and FasL, target genes of phospho-c-Jun(Ser63), and formation of active caspase-9, caspase-8 and caspase-3 were also observed in the SN of (G2019S) LRRK2 transgenic mice. Our results suggest that mutant (G2019S) LRRK2 activates MKK4-JNK-c-Jun pathway in the SN and causes the resulting degeneration of SNpc dopaminergic neurons in PD transgenic mice.

Mouse models for LRRK2 Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disease. Mutations in Leucine-rich-repeat-kinase 2 (LRRK2), the causative gene for PARK8 type PD with autosomal dominant inheritance, are the most prevalent genetic causes of both familial and sporadic PD. Animal models are critical tools in the attempt to understand the mechanisms of LRRK2-mediated pathogenesis. We have generated human Bacterial Artificial Chromosome (BAC) mediated transgenic mouse models expressing mutant LRRK2 that robustly recapitulate the behavioral, neurochemical and pathological features of PD. These mice develop an age-dependent decrease in motor activity that is progressive and responds to treatment with levodopa. Pathologically, the most salient phenotype is early axonopathy of nigrostriatal dopaminergic neurons, accompanied by hyperphosphorylated tau. The mice also exhibit a consistent dopamine transmission deficit in both acute brain slices and live freely moving animals. Here we will discuss LRRK2 mouse models from several laboratories, their commonalities and differences, and offer scientific insights drawn from these studies.

Dopaminergic Neuronal Loss, Reduced Neurite Complexity and Autophagic Abnormalities in Transgenic Mice Expressing G2019S Mutant LRRK2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD.