Species: Mouse
Genes: Lrrk2
Modification: Lrrk2: Knock-Out
Disease Relevance: Parkinson's Disease
Strain Name: B6.129X1(FVB)-Lrrk2^tm1.1Cai/J
Genetic Background: C57BL/6J (but may be mixed with C57BL/6N)
Availability: Available through The Jackson Laboratory, Stock# 012453, Cryopreserved or frozen embryo.

Modification Details:

Lrrk2 KO mice were generated by Cre-mediated deletion of coding exon 2 of the Lrrk2 gene, which results in a premature stop codon in exon 3 (Lin et al., 2009). Heterozygous KO mice were intercrossed to generate homozygous KO mice (Parisiadou et al., 2009).

Summary

This knock-out (KO) model was generated by deleting exon 2 of the Lrrk2 gene, which results in a premature stop codon in exon 3. Brain tissue from Lrrk2 KO mice does not express full-length LRRK2 protein. KO mice are viable, fertile, and do not exhibit any developmental or gross physical abnormalities (The Jackson Laboratory; Parisiadou et al., 2009).

The body weight of Lrrk2 KO mice does not differ from littermate controls from 3 and up to 18 months of age (Lin et al., 2009). Kidneys from KO mice are larger, heavier, and darker at 6 months of age compared with wild-type controls (Pellegrini et al., 2018). Markers of bone strength, including cortical mass, are increased in the tibiae of aged female KO mice compared to wild-type mice, as are measures of resistance to torsion and fracture (Berwick et al., 2017).

Motor Behavior

Motor behavior of Lrrk2 KO mice is intact compared with littermate controls up to 18 months of age, based on the latency to fall on a Rotarod test as well as ambulatory and rearing activity on an open field test (Lin et al., 2009).

However, when other parameters are examined on the open field test, differences emerge between genotypes, and these differences are age-dependent. For instance, 12-month-old mice traveled longer distances and had a higher walking speed compared to controls, but these genotype differences were not present at younger (3-month-old) and older (24-month-old) ages (Chen et al., 2020). In another study, at postnatal day 21, KO mice were more active than wild-type mice on the open field test, as demonstrated by the increased ambulatory activity, fine movement (such as grooming), and rearing (Parisiadou et al., 2014). However, not all studies have found age-dependent effects on motor function in Lrrk2 KO mice (Mazza et al., 2021).

With regard to motor skill learning (which can also be assessed with the Rotarod test), 24-month-old KO mice showed deficits that were not apparent at younger ages (Chen et al., 2020).

Non-Motor Behaviors

No differences were observed between Lrrk2 KO mice and wild-type controls across 6 to 24 months of age on several behavioral tests, including the elevated plus maze for anxiety-like behavior, the buried treat test to measure hyposmia, the grip strength test for forelimb strength, or working memory as measured by spontaneous alternation (Mazza et al., 2021).

Neuropathology

No differences were observed between Lrrk2 KO and wild-type mice (9- to 23-week-old) in the number of tyrosine hydroxylase–positive cells in the substantia nigra pars compacta (Singh et al., 2021). This was also true in 18- to 24-month-old mice, and no differences were observed in the striatum either (Mazza et al., 2021).

No abnormal accumulation of α-synuclein in the cell bodies of striatal neurons was observed in 20-month-old KO mice (Lin et al., 2009). Moreover, cortical stem cell–derived astrocytes from Lrrk2 KO mice that were treated with α-synuclein preformed fibrils did not differ from cells derived from wild-type mice in terms of the types of α-synuclein inclusions formed (Streubel-Gallasch et al., 2021).

No neurodegeneration was observed in the brains (striatum and cortex) of 20-month-old KO mice, based on staining for cleaved-caspase-3 and Jade C (Lin et al., 2009). Indeed, the numbers of spiny projection neurons (SPNs) in the dorsal striatum did not differ between KO and control mice at 12 months of age (Chen et al., 2020). However, the volumes of the cerebral cortex and dorsal (but not ventral) striatum were reduced in 12-month-old KO mice compared with controls.

The soma and nuclear size of SPNs from Lrrk2 KO mice were enlarged, a characteristic of aging, compared to controls at 12 months of age (Chen et al., 2020). In addition, an increased frequency of nuclear invaginations, which also increases with age, was observed in SPNs from 12-month-old KO mice compared with controls. Dendritic morphology was also perturbed in the SPNs of 12-month-old (but not 2-month-old) KO mice versus controls, with KO mice exhibiting reduced numbers of dendritic branches and length of dendrites (Chen et al., 2020). In another study, striatal neurite complexity in younger (3-month-old) KO mice also did not differ from controls (Civiero et al., 2015). At very young ages (postnatal day 15), however, dendrites were perturbed in SPNs where there was a decrease in dendritic spines and an increase in dendritic filopodia in KO versus wild-type control mice (Parisiadou et al., 2014). In KO mice at this age, the dendritic spines were also less mature. Functionally, synaptic transmission was impaired in synaptic slices from KO mice, as observed by a reduction in the frequency and an increase in the amplitude of glutamatergic miniature excitatory postsynaptic currents (Parisiadou et al., 2014).

Neuroinflammation

Striatal staining of GFAP, a marker of reactive astrocytosis, did not differ between control and KO mice, but cells positive for Iba1 staining, a marker of activated microglia, were moderately enlarged in the striatum of 20-month-old KO mice (Lin et al., 2009).

Cx3cr1 encodes chemokine (C-X3-C) receptor 1, which is involved in promoting microglial migration and suppressing inflammation. CX3CR1 mRNA and protein levels were higher in primary microglia cultures of Lrrk2 KO mice compared with wild-type cells, and the mRNA finding was confirmed in vivo in mouse brains (Ma et al., 2016). Correspondingly, cultured primary microglia from Lrrk2 KO mice showed enhanced motility (faster and travelled more distance) when exposed to CX3CL1, an endogenous ligand of CX3CR1, as compared with microglia from wild-type mice.

Cytoskeletal Dynamics

LRRK2 has been proposed to be a kinase of the ERM proteins (ezrin, radixin, and moesin), which are responsible for linking cytoplasmic membrane proteins to the actin cytoskeleton. In primary cortical neurons from Lrrk2 KO mice, phosphorylation of ERM proteins was reduced compared with littermate controls (Parisiadou et al., 2009). This reduction was also observed in the filopodia of cultured hippocampal neurons, both in axonal growth cones and in dendritic filopodia. In addition, the length of neurites and total neurite outgrowth was increased in Lrrk2 KO mice compared with littermate controls, although the number of primary neurites did not differ. Moreover, F-actin levels in the growth cones were lower in cultured hippocampal neurons from Lrrk2 KO mice. And in striatal samples from developing (postnatal day 5, 15, and 21) mice, phosphorylation of the actin-disassembling enzyme cofilin was increased in KO compared with wild-type mice (Parisiadou et al., 2014).

LRRK2 has been suggested to interact with PAK6, a protein that can regulate actin cytoskeleton dynamics and is involved in dendrite development. Brain lysates from 12-month-old Lrrk2 KO mice have reduced PAK6 phosphorylation at site S560 and reduced phosphorylation of LIMK1 (at T508), a downstream effector of PAK6 (Civiero et al., 2015).

In primary bone marrow–derived macrophages, lipopolysaccharide stimulation upregulated Rapgef3 (encoding Epac-1) expression in cells from KO compared to wild-type mice (Levy et al., 2020). Epac-1 promotes cell adhesion and polarization, as well as enhanced leukocyte chemotaxis.

Endocytic Pathway

Lysosomal dysregulation was found in kidneys from 9-month-old Lrrk2 KO mice, with the size of late endosomes/lysosomes increased compared with wild-type controls, based on Lamp1-positive staining (Beilina et al., 2020). Moreover, in these same animals, precursor and mature cathepsin D accumulation was observed, as was increased Vps52 protein levels and increased levels of CI-M6pr, all of which further point to lysosomal dysregulation. Lysosomal dysregulation was also observed in kidneys and primary kidney cells from KO mice (Pellegrini et al., 2018). Similarly, cultured striatal astrocytes have fewer, but larger, lysosomal-like structures in KO versus wild-type mice, based on transmission electron microscopy (Streubel-Gallasch et al., 2021). Staining for Lamp2A, a marker of late endosomes/lysosomes, in cultured striatal astrocytes from KO mice further revealed fewer lysosomes than in wild-type cells.

In another study, phagosome proteolysis was enhanced in macrophages derived from KO mice, although phagocytic uptake was unaffected (Härtlova et al., 2018). Proteomic analysis of phagosomes from KO mice indicated an increase of lysosomal hydrolases, including cathepsin D.

In addition, kidney lysates from 1-year-old Lrrk2 KO mice exhibit dysregulation in components of clathrin-mediated endocytosis, namely lower levels of the clathrin adaptor protein complex 2 (AP2), as well as decreases in clathrin heavy chain (Heaton et al., 2020).

Rab10, a protein involved in membrane delivery and recycling, is phosphorylated in wild-type but not KO mouse embryonic fibroblasts (Ito et al., 2016).

Basal mitophagy was increased in cultured primary mouse embryonic fibroblasts (MEFs) from Lrrk2 KO mice compared to that observed in wild-type cells (Singh et al., 2021). In vivo, in adult (9- to 23-week-old) Lrrk2 KO mice, mitophagy was also increased compared to wild-type mice in dopaminergic neurons of the substantia nigra pars compacta, as detected by an increase in the number of mitolysosomes. In contrast, Purkinje cells of the cerebellum did not exhibit this alteration in mitophagy. Within glia, mitophagy increases were found in Iba1-positive microglia cells, but not in GFAP-positive astrocytes. No differences in macroautophagy were observed between Lrrk2 KO and wild-type mice in peripheral tissues, including lung and kidney.

Intracellular Signaling Pathways

RNA sequencing of dorsal striatal tissue from Lrrk2 KO mice and wild-type controls revealed that gene expression differences between genotypes were larger in younger (2-month-old) than in older (12-month-old) mice (Chen et al., 2020). Moreover, the genes affected by Lrrk2 KO were different at different ages. For instance, in younger mice, differentially regulated genes were related to potassium ion transport, response to calcium ion, nucleosome assembly, and DNA methylation while those regulated in older mice were related to ubiquitination, actin filament–based movement, leukocyte chemotaxis, and myeloid cell differentiation. Aligned with the alteration in nuclear assembly pathway genes during older ages in Lrrk2 KO mice, it was found that genomic stability and epigenetic modification functions were perturbed during aging. In striatal samples from aged (12- and 24-month-old) but not young (2-month-old) mice, levels of γH2AX—a proxy for DNA double-strand breaks and damage—were increased.

In a proteomic analysis, 1-year-old Lrrk2 KO mice kidneys were found to be enriched for protein degradation pathways compared to wild-type littermates (Pellegrini et al., 2018). In addition, cytoskeletal proteins—many related to exosome formation—were differentially expressed, as were proteins related to translational and lysosomal function.

Pharmacological studies in cultured cortical neurons from Lrrk2 KO mice revealed an aberrant elevation of basal PKA activity (Parisiadou et al., 2014). The underlying cause of this is proposed to be an abnormal localization of PKARIIβ within the dendritic spines (versus dendritic shafts) in KO cells.

Phosphorylation (at S845) of GluR1, a subunit of the AMPA glutamate receptor, was increased in the forebrain of very young (postnatal day 5 and 15) KO mice compared with controls, but this difference disappeared by postnatal day 30 (Parisiadou et al., 2014). A functional implication of this elevation may be increased GluR1 receptors at dendritic spines.

Canonical Wnt activity was increased in vivo in the brain as well as in fibroblasts from Lrrk2 KO mice compared with that observed in wild-type cells (Berwick et al., 2017).

Immune Function

When exposed to Mycobacterium tuberculosis infection, bone marrow-derived mouse macrophages from Lrrk2 KO mice were able to better control the infection than cells from wild-type mice, which was possibly due to controlling phagosome maturation and cytokine responses (Härtlova et al., 2018). Infection of mice in vivo supported this finding, with KO mice having lower growth of M. tuberculosis in the lung and spleen compared with wild-type controls. However, lesions in the lung (although fewer) were larger in KO mice versus wild-type mice, and levels of inflammatory cytokines were elevated in KO mice. Together, this suggests that adaptive immune responses may be dysregulated in KO mice.

In a subclinical infection model with Salmonella enterica, Lrrk2 KO mice had an enhanced immune response compared with wild-type mice, as observed by the increase in inflammatory markers IL-18 and IFN-γ (Levy et al., 2020). 

Following immunization with interphotoreceptor retinoid-binding protein, Lrrk2 KO mice were less likely than wild-type mice to develop severe experimental autoimmune uveitis, which is driven by the adaptive immune system (Wandu et al., 2015). Moreover, KO mice had lower “delayed type hypersensitivity” responses, and levels of splenocyte cytokine production were also lower. 

In a model of dextran sodium sulfate–induced colitis, Lrrk2 KO mice had less severe responses, including lower proinflammatory cytokine secretion, than wild-type mice (Takagawa et al., 2018).

When bone marrow–derived dendritic cells from Lrrk2 KO mice were exposed to irradiated Mycobacterium leprae, a marker of autophagy (LC3-I to LC3-II conversion) was increased compared to that in cells from control mice (Takagawa et al., 2018).

Phenotype Characterization

Q&A with Model Creator

Q&A  with Model Expert Mark R. Cookson

What would you say are the unique advantages of this model?
This model lacks endogenous Lrrk2 but is viable and fertile as a homozygous line and so can be propagated easily.

What do you think this model is best used for?
To examine the endogenous physiological function of LRRK2, which is easiest in tissues such as kidney that have high LRRK2 and low LRRK1.

What caveats are associated with this model?
The model is not a model of Parkinson’s disease, which is expected given that human mutations are gain of function. The model does have LRRK1, which might compensate for some effects of LRRK2 deficiency.

Anything else useful or particular about this model you think our readers would like to know?
This was one of several independent knockout models that were shown to have age-dependent alterations in the autophagy lysosome system.

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References

Paper Citations

1. Parisiadou L, Xie C, Cho HJ, Lin X, Gu XL, Long CX, Lobbestael E, Baekelandt V, Taymans JM, Sun L, Cai H. Phosphorylation of ezrin/radixin/moesin proteins by LRRK2 promotes the rearrangement of actin cytoskeleton in neuronal morphogenesis. J Neurosci. 2009 Nov 4;29(44):13971-80. PubMed.
2. Lin X, Parisiadou L, Gu XL, Wang L, Shim H, Sun L, Xie C, Long CX, Yang WJ, Ding J, Chen ZZ, Gallant PE, Tao-Cheng JH, Rudow G, Troncoso JC, Liu Z, Li Z, Cai H. Leucine-rich repeat kinase 2 regulates the progression of neuropathology induced by Parkinson's-disease-related mutant alpha-synuclein. Neuron. 2009 Dec 24;64(6):807-27. PubMed.
3. Pellegrini L, Hauser DN, Li Y, Mamais A, Beilina A, Kumaran R, Wetzel A, Nixon-Abell J, Heaton G, Rudenko I, Alkaslasi M, Ivanina N, Melrose HL, Cookson MR, Harvey K. Proteomic analysis reveals co-ordinated alterations in protein synthesis and degradation pathways in LRRK2 knockout mice. Hum Mol Genet. 2018 Sep 15;27(18):3257-3271. PubMed.
4. Berwick DC, Javaheri B, Wetzel A, Hopkinson M, Nixon-Abell J, Grannò S, Pitsillides AA, Harvey K. Pathogenic LRRK2 variants are gain-of-function mutations that enhance LRRK2-mediated repression of β-catenin signaling. Mol Neurodegener. 2017 Jan 19;12(1):9. PubMed.
5. 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.
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. Mazza MC, Nguyen V, Beilina A, Karakoleva E, Coyle M, Ding J, Bishop C, Cookson MR. Combined Knockout of Lrrk2 and Rab29 Does Not Result in Behavioral Abnormalities in vivo. J Parkinsons Dis. 2021;11(2):569-584. PubMed.
8. Singh F, Prescott AR, Rosewell P, Ball G, Reith AD, Ganley IG. Pharmacological rescue of impaired mitophagy in Parkinson's disease-related LRRK2 G2019S knock-in mice. Elife. 2021 Aug 3;10 PubMed.
9. Streubel-Gallasch L, Giusti V, Sandre M, Tessari I, Plotegher N, Giusto E, Masato A, Iovino L, Battisti I, Arrigoni G, Shimshek D, Greggio E, Tremblay ME, Bubacco L, Erlandsson A, Civiero L. Parkinson's Disease-Associated LRRK2 Interferes with Astrocyte-Mediated Alpha-Synuclein Clearance. Mol Neurobiol. 2021 Jul;58(7):3119-3140. Epub 2021 Feb 24 PubMed.
10. Civiero L, Cirnaru MD, Beilina A, Rodella U, Russo I, Belluzzi E, Lobbestael E, Reyniers L, Hondhamuni G, Lewis PA, Van den Haute C, Baekelandt V, Bandopadhyay R, Bubacco L, Piccoli G, Cookson MR, Taymans JM, Greggio E. Leucine-rich repeat kinase 2 interacts with p21-activated kinase 6 to control neurite complexity in mammalian brain. J Neurochem. 2015 Dec;135(6):1242-56. Epub 2015 Oct 19 PubMed.
11. Ma B, Xu L, Pan X, Sun L, Ding J, Xie C, Koliatsos VE, Cai H. LRRK2 modulates microglial activity through regulation of chemokine (C-X3-C) receptor 1 -mediated signalling pathways. Hum Mol Genet. 2016 Aug 15;25(16):3515-3523. Epub 2016 Jul 4 PubMed.
12. Levy DR, Udgata A, Tourlomousis P, Symmons MF, Hopkins LJ, Bryant CE, Gay NJ. The Parkinson's disease-associated kinase LRRK2 regulates genes required for cell adhesion, polarization, and chemotaxis in activated murine macrophages. J Biol Chem. 2020 Jul 31;295(31):10857-10867. Epub 2020 Feb 28 PubMed.
13. 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.
15. Heaton GR, Landeck N, Mamais A, Nalls MA, Nixon-Abell J, Kumaran R, Beilina A, Pellegrini L, Li Y, International Parkinson Disease Genomics Consortium (IPDGC), Harvey K, Cookson MR. Sequential screening nominates the Parkinson's disease associated kinase LRRK2 as a regulator of Clathrin-mediated endocytosis. Neurobiol Dis. 2020 Jul;141:104948. Epub 2020 May 17 PubMed.
16. Ito G, Katsemonova K, Tonelli F, Lis P, Baptista MA, Shpiro N, Duddy G, Wilson S, Ho PW, Ho SL, Reith AD, Alessi DR. Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors. Biochem J. 2016 Sep 1;473(17):2671-85. Epub 2016 Jul 29 PubMed.
17. Wandu WS, Tan C, Ogbeifun O, Vistica BP, Shi G, Hinshaw SJ, Xie C, Chen X, Klinman DM, Cai H, Gery I. Leucine-Rich Repeat Kinase 2 (Lrrk2) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU) and the Adaptive Immune Response. PLoS One. 2015;10(6):e0128906. Epub 2015 Jun 11 PubMed.
18. Takagawa T, Kitani A, Fuss I, Levine B, Brant SR, Peter I, Tajima M, Nakamura S, Strober W. An increase in LRRK2 suppresses autophagy and enhances Dectin-1-induced immunity in a mouse model of colitis. Sci Transl Med. 2018 Jun 6;10(444) PubMed.

External Citations

1. The Jackson Laboratory
2. The Jackson Laboratory, Stock# 012453

Further Reading