Species: Mouse
Genes: LRRK2
Modification: LRRK2: Knock-In
Disease Relevance: Parkinson's Disease
Strain Name: B6.Cg-Lrrk2^tm1.1Shn/J
Genetic Background: The mutant colony was established in B6/129 F1 mice, but backcrossed to C57BL/6J wild-type mice for at least 12 generations.
Availability: Available through The Jackson Laboratory, Stock# 009346, Cryopreserved or frozen embryo.

Overview

This knock-in (KI) mouse model was generated by introducing a R1441C missense mutation into the GTPase domain at exon 31 of the mouse Lrrk2 (leucine-rich repeat kinase 2) gene (Tong et al., 2009). As such, this mutation is expressed through the control of endogenous regulatory elements. R1441C KI mice produce LRRK2 protein and mRNA in the brain at levels equal to that of wild-type controls. Homozygous mutant mice appear grossly normal, are viable and fertile, and have a similar body mass to wild-type mice (The Jackson Laboratory; Tong et al., 2009). Unless specified otherwise, the summary below refers to homozygous mice.

Neural Architecture | Neuroinflammation | Immune Functioning |Motor Behavior | Dopamine Levels and Proxy Measures for Dopamine | Affected Signaling Pathways | Synaptic Function | Intracellular Transport | Modification Details

Neural Architecture

In the substantia nigra pars compacta, dopaminergic neurons (as detected by tyrosine hydroxylase [TH] staining) did not differ in number between R1441C KI and wild-type mice at 12 and 22 months of age (Tong et al., 2009). The morphology of the neurons and their projections was also grossly normal in KI mice. In the locus coeruleus, the morphology and number of TH-immunoreactive neurons were also similar between wild-type and KI mice.

Aggregation of α-synuclein, ubiquitin, and tau was examined in the substantia nigra pars compacta and locus coeruleus, and no abnormal accumulation or phosphorylation was found in 3-, 12-, and 22-month-old R1441C KI mice.

Neuroinflammation

GFAP immunoreactivity, a marker of gliosis, was normal at 12 and 22 months of age in R1441C KI mice (Tong et al., 2009).

Compared to wild-type controls, R1441C KI mice exhibited increased infiltration of pro-inflammatory monocytes into the brain after α-synuclein fibril injection (Xu et al., 2022). Levels of microglia, on the other hand, did not differ in response to this injection.

Immune Functioning

In a preprint from Wallings et al., R1441C KI mice were reported to exhibit immune hyperresponsiveness (i.e., increased effector functions) at younger (2-3-month-old), but not older (18-21-month-old), ages (Wallings et al., 2023). Importantly, these findings were dependent on sex, as female mice showed this pattern, but males did not. The specific findings observed in primary peritoneal macrophages from young female KI mice were increased antigen presentation, cytokine release, phagocytosis, and lysosomal function. Moreover, in aged female mice, macrophages exhibited an exhausted phenotype that was characterized by suppressed antigen presentation and hypophagocytosis.

Motor Behavior

Spontaneous locomotor activity was assessed using the open field test in R1441C KI mice at 3, 12, and 24 months of age, and it was found to be equal to that of wild-type mice across all recorded measures, including total distance travelled, vertical beam breaks (rears), and stereotyped behaviors (e.g., scratching, grooming) (Tong et al., 2009). Involuntary motor movement, as assessed by the accelerating Rotarod at 3 and 12 months of age, was also similar between KI and control mice (Tong et al., 2009; also see Xenias et al., 2022).

Dopamine Levels and Proxy Measures for Dopamine

Levels of basal striatal dopamine and its major metabolites (dihydroxylphenylacetic acid, DOPAC; homovanillic acid, HVA) did not differ between KI and wild-type mice, as measured by HPLC, at 3, 12, and 23 months of age (Tong et al., 2009). However, levels of evoked dopamine release in the striatum, as measured by fast-scanning cyclic voltammetry, were reduced in heterozygous KI mice compared to wild-type controls (Xenias et al., 2022).

In addition, the acoustic startle reflex, which can serve as a proxy for detecting alterations in dopamine, was assessed in R1441C KI and wild-type mice at 12 months of age and the two performed similarly (Tong et al., 2009). 

Amphetamine is known to induce synaptic dopamine release through its activity on the dopamine transporter and the vesicular monoamine transporter 2. Amphetamine-stimulated locomotor activity differed between KI and wild-type mice, unlike spontaneous activity (Tong et al., 2009). Whereas wild-type mice responded to amphetamine with increased locomotor activity, activity was unchanged in KI mice after injection with amphetamine. Thus, the failure of amphetamine to induce locomotor activity may reflect impairments in psychostimulant-induced dopamine release. The release of other catecholamines—epinephrine and norepinephrine—was assessed in cultured chromaffin cells from KI mice. Upon stimulation with high levels of K⁺, deficits in catecholamine release, quantal size, and frequency of released events were observed in cells derived from KI mice as compared to those from wild-type mice.

Dopamine D2 receptor signaling, which is involved in the feedback regulation of dopamine release, was also impaired in KI mice (Tong et al., 2009). Following injection of quinpirole, a D2 receptor agonist, homozygous KI mice did not exhibit reductions in locomotor activity to the same extent as wild-type mice did. Moreover, antagonism of dopamine receptors (D1 and D2) during a striatal motor learning task (accelerated Rotarod) hindered motor performance in heterozygous KI mice to a greater extent than in wild-type mice (Xenias et al., 2022). Of note, dopamine receptor antagonism did not affect overall locomotor behavior (total distance travelled on an open field), and thus the effect was specific to impaired learning on the Rotarod training task.

Affected Signaling Pathways

The motor learning deficits in heterozygous KI versus wild-type mice were also associated with differential expression of 90 proteins in the striatum, including enrichment of those related to the cAMP-PKA signaling pathway (Xenias et al., 2022). PKA activity was also elevated in striatal synaptosomal samples (Chen et al., 2020) and in spiny projection neurons (SPNs; Parisiadou et al., 2014) of R1441C KI heterozygous mice. Moreover, levels of the PKA phospho-substrate GluA1 (AMPA receptor subunit) were also increased in the postsynaptic density fraction from striatal samples in heterozygous KI mice compared to wild-type controls.

Ciliation in striatal cholinergic neurons is important for responding to a Sonic hedgehog signal, which is involved in non–cell autonomous neuroprotection of dopaminergic circuits (Dhekne et al., 2018). While 7-month-old R1441C KI mice have similar overall levels of ciliation in the striatum compared to wild-type controls, ciliation specifically within cholinergic neurons of the striatum was decreased. This reduction of primary cilia in cholinergic neurons in the dorsal striatum was observed in KI mice as early as 10 weeks of age (Khan et al., 2021). Primary cilia formation is also perturbed in the somatosensory cortex of R1441C KI mice compared to wild-type controls, with KI animals showing fewer neurons having primary cilia, though the cilia that were present were equally long (Dhekne et al., 2018).

Levels of the autophagy marker LC3-II were reduced in cultured bone marrow–derived macrophages from KI mice (Hakimi et al., 2011).

Association with 14-3-3 proteins is reduced in the kidney, brain, and spleen of R1441C KI mice compared with wild-type controls, perhaps due to disrupted phosphorylation of the mutant LRRK2 at sites S910 and S935 (Nichols et al., 2010). Moreover, LRRK2 association with the PKA regulatory subunit IIb (PKARIIb) is also impaired, with higher levels of PKARIIb signal found in the dendritic spines of cultured hippocampal neurons from KI mice relative to wild-type controls (Parisiadou et al., 2014).

Synaptic Function

The electrical activity of dopaminergic neurons in the substantia nigra pars compacta was assessed through intracellular electrophysiology and the spontaneous, rhythmic firing activity was similar to that observed in wild-type control mice (Tong et al., 2009). However, dopamine, quinpirole, or amphetamine application did not result in the same levels of hyperpolarization in KI mice as it did in wild-type mice, further pointing to deficits in nigral dopamine signaling in KI mice.

Striatal dopamine signaling was also perturbed. Using whole-cell, current-clamp recordings of striatal SPNs, it was observed that indirect-pathway, but not direct-pathway, SPNs had decreased excitability in heterozygous KI mice compared to wild-type controls (Xenias et al., 2022). Moreover, the membrane capacitance and interspike interval of indirect- (but not direct-) pathway SPNs were increased in heterozygous KI versus control mice. On the other hand, both direct- and indirect-pathway SPNs had reduced miniature excitatory postsynaptic current (mEPSC) frequencies, and direct-pathway SPNs had larger amplitude mEPSCs in heterozygous KI mice (Chen et al., 2020).

Despite the observed differences in protein expression and function at striatal synapses, overall dendritic spine number and morphology of striatal SPNs in neonatal pups did not differ between heterozygous KI mice and wild-type controls (Chen et al., 2020). However, in homozygous KI mice, dendritic spine area in cultured hippocampal neurons was decreased compared with wild-type mice (Parisiadou et al., 2014). In addition, cultured striatal SPNs from R1441C KI mice exhibited an altered nuclear shape, though similar nuclear size, compared to wild-type control mice (Chen et al., 2020).

Intracellular Transport

Transport of proteins within R1441C mutant cells is also affected. For instance, in primary astrocytes cultured from R1441C KI mice, both retrograde and anterograde trafficking were increased between the plasma membrane and the trans-Golgi network (Beilina et al., 2020). Moreover, in primary fibroblast cells from R1441C KI mice, there was a more diffuse cytosolic staining of proteins involved in endoplasmic reticulum (ER)–Golgi transport, and transport from the ER to the cell surface was delayed compared with wild-type cells (Cho et al., 2014).

Modification Details

The KI mouse model was generated by homologous recombination in (C57BL/6×129)F1-derived embryonic stem cells. A 1.7-kb DNA fragment containing the R1441C missense mutation was electroporated using site-directed mutagenesis into the GTPase domain of exon 31 of the Lrrk2 locus (Tong et al., 2009; The Jackson Laboratory). The colony was established in B6/129 mice, but backcrossed to C57BL/6J wild-type mice for at least 12 generations.

Phenotype Characterization

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References

Paper Citations

1. Tong Y, Pisani A, Martella G, Karouani M, Yamaguchi H, Pothos EN, Shen J. R1441C mutation in LRRK2 impairs dopaminergic neurotransmission in mice. Proc Natl Acad Sci U S A. 2009 Aug 25;106(34):14622-7. Epub 2009 Aug 10 PubMed.
2. Xu E, Boddu R, Abdelmotilib HA, Sokratian A, Kelly K, Liu Z, Bryant N, Chandra S, Carlisle SM, Lefkowitz EJ, Harms AS, Benveniste EN, Yacoubian TA, Volpicelli-Daley LA, Standaert DG, West AB. Pathological α-synuclein recruits LRRK2 expressing pro-inflammatory monocytes to the brain. Mol Neurodegener. 2022 Jan 10;17(1):7. PubMed.
3. Wallings R, McFarland K, Staley H, Neighbarger N, Schaake S, Brueggemann N, Zittel S, Usnich T, Klein C, Sammler E, Tansey MG. The R1441C-LRRK2 mutation induces myeloid immune cell exhaustion in an age- and sex-dependent manner. 2023 Dec 15 10.1101/2023.10.12.562063 (version 2) bioRxiv.
4. Xenias HS, Chen C, Kang S, Cherian S, Situ X, Shanmugasundaram B, Liu G, Scesa G, Savio Chan C, Parisiadou L. R1441C and G2019S LRRK2 knockin mice have distinct striatal molecular, physiological, and behavioral alterations. Commun Biol. 2022 Nov 10;5(1):1211. PubMed.
5. Chen C, Soto G, Dumrongprechachan V, Bannon N, Kang S, Kozorovitskiy Y, Parisiadou L. Pathway-specific dysregulation of striatal excitatory synapses by LRRK2 mutations. Elife. 2020 Oct 2;9 PubMed.
6. Parisiadou L, Yu J, Sgobio C, Xie C, Liu G, Sun L, Gu XL, Lin X, Crowley NA, Lovinger DM, Cai H. LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity. Nat Neurosci. 2014 Mar;17(3):367-76. Epub 2014 Jan 26 PubMed.
7. Dhekne HS, Yanatori I, Gomez RC, Tonelli F, Diez F, Schüle B, Steger M, Alessi DR, Pfeffer SR. A pathway for Parkinson's Disease LRRK2 kinase to block primary cilia and Sonic hedgehog signaling in the brain. Elife. 2018 Nov 6;7 PubMed.
8. Khan SS, Sobu Y, Dhekne HS, Tonelli F, Berndsen K, Alessi DR, Pfeffer SR. Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain. Elife. 2021 Oct 18;10 PubMed.
9. Hakimi M, Selvanantham T, Swinton E, Padmore RF, Tong Y, Kabbach G, Venderova K, Girardin SE, Bulman DE, Scherzer CR, LaVoie MJ, Gris D, Park DS, Angel JB, Shen J, Philpott DJ, Schlossmacher MG. Parkinson's disease-linked LRRK2 is expressed in circulating and tissue immune cells and upregulated following recognition of microbial structures. J Neural Transm (Vienna). 2011 May;118(5):795-808. Epub 2011 May 7 PubMed.
10. Nichols RJ, Dzamko N, Morrice NA, Campbell DG, Deak M, Ordureau A, Macartney T, Tong Y, Shen J, Prescott AR, Alessi DR. 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization. Biochem J. 2010 Sep 15;430(3):393-404. PubMed.
11. Chen X, Xie C, Tian W, Sun L, Zheng W, Hawes S, Chang L, Kung J, Ding J, Chen S, Le W, Cai H. Parkinson's disease-related Leucine-rich repeat kinase 2 modulates nuclear morphology and genomic stability in striatal projection neurons during aging. Mol Neurodegener. 2020 Feb 19;15(1):12. PubMed. Correction.
12. Beilina A, Bonet-Ponce L, Kumaran R, Kordich JJ, Ishida M, Mamais A, Kaganovich A, Saez-Atienzar S, Gershlick DC, Roosen DA, Pellegrini L, Malkov V, Fell MJ, Harvey K, Bonifacino JS, Moore DJ, Cookson MR. The Parkinson's Disease Protein LRRK2 Interacts with the GARP Complex to Promote Retrograde Transport to the trans-Golgi Network. Cell Rep. 2020 May 5;31(5):107614. PubMed.
13. Cho HJ, Yu J, Xie C, Rudrabhatla P, Chen X, Wu J, Parisiadou L, Liu G, Sun L, Ma B, Ding J, Liu Z, Cai H. Leucine-rich repeat kinase 2 regulates Sec16A at ER exit sites to allow ER-Golgi export. EMBO J. 2014 Oct 16;33(20):2314-31. Epub 2014 Sep 8 PubMed.

External Citations

1. The Jackson Laboratory
2. The Jackson Laboratory, Stock# 009346

Further Reading