Species: Mouse
Genes: LRRK2
Mutations: LRRK2 G2019S
Modification: LRRK2: Transgenic
Disease Relevance: Parkinson's Disease
Strain Name: B6.Cg-Tg(Lrrk2*G2019S)2Yue/J
Genetic Background: A BAC construct was injected into B6C3 F1 oocytes. Founder line 2 was established and maintained by breeding to C57BL/6J inbred mice.
Availability: Available through The Jackson Lab, Stock# 012467, cryopreserved.

Summary

This transgenic (Tg) mouse overexpresses a mutant form of Lrrk2 (G2019S) using a bacterial artificial chromosome (BAC) (Li et al., 2010). Transgene expression is driven by the mouse Lrrk2 promoter sequence. Hemizygous mice develop an age-associated decrease in striatal dopamine, but no loss of dopaminergic neurons or behavioral motor deficits.

Hemizygous mice are viable and fertile and do not have any overt brain abnormalities, at least out to 18 months of age. Immunoblots of whole-brain lysate indicate that the mutant protein is present at least about sixfold above endogenous levels (Li et al., 2010) or as much as 20-30-fold (West et al., 2014). Lrrk2-G2019S expression is observed in the cerebral cortex, striatum, substantia nigra, internal capsule, and hippocampus (Li et al., 2010). The highest expression levels were observed in the cortex and striatum with clear expression in the substantia nigra pars compacta, but not in the substantia nigra pars reticulata. Overall, the pattern of Lrrk2 expression in this Tg mouse is consistent with the distribution of Lrrk2 protein observed in non-Tg mice (West et al., 2014).

Tg mice exhibit higher levels of plasma corticosterone at baseline compared with non-Tg animals at 6 to 8 months of age, indicating enhanced basal stress tone (Rudyk et al., 2019). However, at 12 to 15 weeks of age, this difference in plasma corticosterone is not yet apparent (Litteljohn et al., 2018).

Overexpression of mutant Lrrk2 in this model did not alter motor performance in hemizygous mice. They performed like wild-type controls in the open-field test and in a test of motor coordination involving walking across a beam at 6 or 12 months of age (Li et al., 2010). Home-cage activity also did not differ from non-Tg mice at 12 to 15 weeks of age (Litteljohn et al., 2018). However, at 18 months of age, Tg mice develop motor deficits based on Rotarod performance compared with wild-type controls (Liu et al., 2022).

Tg mice exhibit deficits in sleep, spending less time in the REM sleep phase than wild-type controls at 12 and 18 months of age (Liu et al., 2022). With regard to CLOCK protein expression, Tg mice express lower levels than wild-type controls in the midbrain at 18 months of age, but not at 12 months. Moreover, chronic sleep deprivation in Tg mice leads to an exacerbated Parkinson’s disease-like phenotype compared to that observed in sleep deprived wild-type mice.

Hemizygous BAC-Lrrk2-G2019S mice displayed no signs of neuronal or other cell death in any brain region, including the cortex, striatum, and hippocampus. There was no difference in the number of dopaminergic neurons in the substantia nigra compared to littermate controls at 6 or 12 months. In addition, nigrostriatal terminals appeared normal at six and 12 months. Lrrk2-G2019S protein purified from Tg brains had higher kinase activity than wild-type Lrrk2 (Li et al., 2010). By 18 months of age, dopaminergic neuron number in the substantia nigra remained the same between Tg mice and wild-type controls (Liu et al., 2022). Moreover, cultured primary hippocampal and midbrain neurons from homozygous Tg mice did not exhibit defects in neuron health or viability (Henderson et al., 2018).

The brains of BAC-Lrrk2-G2019S mice showed no evidence of α-synuclein aggregation or deposits (Li et al., 2010). Homozygous Tg mice also did not show α-synuclein deposits up to 12 months of age (Henderson et al., 2019). Moreover, homozygous mice do not accumulate tau pathology up to 12 months of age (Cornblath et al., 2021).

Hemizygous BAC-Lrrk2-G2019S mice develop an age-related decline in striatal dopamine content. Levels were not different from littermate controls at 5 or 6 months of age, but by 12 months of age, mutant mice had up to 25 percent less dopamine and less homovanillic acid (HVA), a dopamine metabolite (Xiao et al., 2015). However, a different study found that by 12 to 15 weeks of age, striatal dopamine was reduced compared to non-Tg controls (Litteljohn et al., 2018). Levels of tyrosine hydroxylase in the striatum were comparable to controls at 10 months of age; likewise, enzymatic activity levels were unchanged. Tyrosine hydroxylase in the substantia nigra also did not differ between Tg and non-Tg mice at 5 and 10 months of age (Xiao et al., 2015). Another study confirmed these changes, with levels of dopamine, 3,4-dihydroxy-phenylacetic acid (DOPAC), and HVA being lower in Tg versus wild-type controls at 12 months of age (Liu et al., 2022).

Levels of dopamine and DOPAC, but not HVA, were also lower than wild-type controls at 18 months. In this same study, tyrosine hydroxylase levels in the striatum did not differ between Tg and wild-type animals at 12 or 18 months of age, similar to that reported for the original description of the model. However, one study that examined dopaminergic neurons in the substantia nigra of 20-month-old Tg mice found that the amount of tyrosine hydroxylase and neuronal nuclei staining was reduced compared to non-Tg at this time point, but not at 12 months of age; this cell loss was selective to dopaminergic neurons and not observed in other neuron types (Chen et al., 2017). Moreover, this dopaminergic cell loss at 20 months of age was correlated with increased phosphorylation of amyloid precursor protein in the midbrain of Tg mice, which the authors hypothesize may be driving neurotoxicity in this model.

At 6 and 8 months of age, Tg mice have higher levels of hippocampal 5-HT1A and lower levels of striatal brain-derived neurotrophic factor compared with non-Tg mice (Rudyk et al., 2019). Another study found that Tg mice had higher levels of hippocampal norepinephrine, 5-HT, and 5-hydroxyindole acetic acid than non-Tg controls at 12 to 15 weeks of age (Litteljohn et al., 2018).

In striatal slices taken from mice at 1 year of age, the peak amount of evoked dopamine was lower in BAC-Lrrk2-G2019S mice than in littermate controls. This effect was not seen at six months of age. In addition, the decrease in evoked dopamine was accompanied by a decrease in dopamine reuptake, which may be a compensatory response (Li et al., 2010).

Levels of the chemokine receptor CX3CR1, which is expressed highly by microglia, did not differ in Tg versus non-Tg mice in the substantia nigra pars compacta at 6 and 8 months of age (Rudyk et al., 2019). Moreover, at 5 and 12 months of age, the number of activated microglia (as determined based on morphology of Iba-1-positive cells) in the substantia nigra did not differ between Tg and non-Tg mice (Xiao et al., 2015).

Electrophysiological studies in acute hippocampal slices have shown impaired synaptic plasticity in BAC-Lrrk2-G2019S mice. Specifically, LRRK2-G2019S mice showed a profound deficit in synaptically induced long-term depression whereas mice expressing wild-type Lrkk2 did not. Long-term potentiation was not affected. In terms of basal synaptic transmission, mutant mice had higher basal efficiency than mice expressing wild-type Lrrk2. These changes were observed in aged mice (8-12 months) but not in young mice (3-6 months). Presynaptic function appeared intact as assessed by measuring paired-pulse facilitation, post-tetanic potentiation, and the response to a train of stimuli (Sweet et al., 2015).

Lrrk2 protein levels were reported to be 10- to 12-fold higher in cultured mutant hippocampal neurons than in non-Tg neurons (Volpicelli-Daley et al., 2016), although a less than twofold elevation was detected in cultured midbrain neurons (Pan et al., 2017). In cultured primary hippocampal neurons from homozygous Tg mice, Lrrk2 expression was increased 25-fold, and a 50-fold increase was observed in phosphorylated (pS935) Lrrk2 (Henderson et al., 2018). It took years to breed these mice to compound homozygosity, and, importantly, there appear to be multiple insertions of the transgene, as validated by quantitative PCR and outbreeding (Michael X. Henderson, personal communication).

While some researchers observed that mutant hippocampal neurons had increased levels of α-synuclein protein (about 1.5-fold higher than non-Tg neurons) (Volpicelli-Daley et al., 2016), others have not. For instance, one study found that by 18 months of age, while α-synuclein protein levels were higher in Tg mice, this difference was not statistically significant (Liu et al., 2022). Another study, conducted in homozygous mice, demonstrated that soluble and insoluble α-synuclein proteins levels did not differ in primary hippocampal neurons in Tg versus non-Tg mice (Henderson et al., 2018). Furthermore, 18 days after exposure to exogenous α-synuclein fibrils, the mutant neurons developed more α-synuclein inclusions than non-transgenic neurons. The inclusions generally co-localized with tau along axons, but were also seen in the cell body. Inclusions were absent from neurons not exposed to fibrils. A different study found that following brain injection of α-synuclein fibrils, 8- to 16-week-old Tg mice had a greater number of pro-inflammatory infiltrating monocytes than non-Tg controls (Xu et al., 2022). These results suggest that mutant Lrrk2 may promote neuroinflammatory responses. To assess patterns of α-synuclein spread through the brain following injection of pre-formed fibrils in 3-month-old homozygous Tg mice, a network analysis was conducted (Henderson et al., 2019). The analysis revealed that compared with non-Tg mice, some brain areas exhibited higher pathology while others exhibited lower pathology in response to injection of α-synuclein pre-formed fibrils.

Rab10 phosphorylation by Lrrk2 may drive pro-inflammatory immune responses, and the pT73-Rab10/total Rab10 ratio was significantly higher in kidney lysates from 4- to 6-month-old Tg versus non-Tg mice (Wang et al., 2022). Of note, however, Rab10 phosphorylation and Lrrk2 autophosphorylation patterns in Tg mice differ by age and tissue type or brain area (Iannotta et al., 2020). Moreover, Lrrk2 autophosphorylation patterns also depend on the type of Lrrk2 mutation, which can also lead to changes in regulator protein binding (Iannotta et al., 2020; Li et al., 2011; Sheng et al., 2012). Innate immunity was also examined in thioglycollate-elicited primary macrophages from Tg mice, and expression of cytokines and chemokines did not differ from non-Tg mice, but chemotaxis was increased (Moehle et al., 2015).

There were no overt morphological differences in cultured neurons from Lrkk2-G2019S mice and non-Tg neurons and quantification of confocal images revealed no significant differences in the abundance of axons, dendrites, or total cells numbers in culture (Volpicelli-Daley et al., 2016). Although the mutant neurons develop elaborate arbors, their growth rate was found to be somewhat reduced compared to non-Tg neurons and time lapse imaging revealed reduced neurite motility (Sepulveda et al., 2013). Moreover, neurite length was reduced in primary cortical neurons from Tg mice compared with that in non-Tg neurons (Rassu et al., 2019 31560168).

Despite the overall lack of gross morphological defects in Tg cells, some organelles are affected by the mutation. Nuclear membrane circularity was decreased and the levels of 53BP1 (a nuclear protein) were higher in the cytosol in dopaminergic neurons from the substantia nigra in 12-month-old Tg mice, indicating that mutated Lrrk2 perturbs nuclear envelope integrity (Shani et al., 2019). In addition, cultured cortical astrocytes from Tg mice have fewer, but enlarged, lysosomes compared with those from non-Tg mice, and lysosomes from Tg mice have decreased lysosomal activity (Henry et al., 2015). Finally, synaptic vesicle endocytosis in some neurons appears to be impaired. Using pH-sensitive optical reporters coupled to vesicular transporters, the deficit was detected in cultured ventral midbrain neurons, including dopaminergic neurons, but not in neocortical neurons. Inhibiting LRRK2 activity rescued the phenotype (Pan et al., 2017). 

Mitochondrial function has been assessed in cells derived from Tg mice, and embryonic fibroblasts and bone marrow-derived macrophages exhibit mitochondrial fragmentation and membrane depolarization (Weindel et al., 2022). Moreover, the cortex and midbrain of Tg mice have FBXO7 aggregation, which is a protein that aggregates in mitochondria (Zhou et al., 2015).

The description above refers to hemizygous mice, unless otherwise noted. Homozygous mice are viable and have higher levels of mutant protein expression (personal communication, Zhenyu Yue, January 2017). Their behavior has not yet been phenotypically characterized. However, one study of homozygous Tg mice found a lack of α-synuclein pathology in cultured primary neurons (Henderson et al., 2018), and another found mixed pathology across brain regions following the injection of α-synuclein pre-formed fibrils (Henderson et al., 2019).

Modification Details

This model was generated using a BAC containing the entire mouse Lrrk2 gene modified to include the G2019S mutation. The BAC (~240 kb) contained the murine Lrrk2 promoter region (~35 kb) and a FLAG-tag downstream of the start codon. The transgene inserted at Chr18:44968085 (Build GRCm38/mm10), where it does not affect any known genes (Goodwin et al., 2017). 

Phenotype Characterization

Q&A with Model Creator

Q&A with Michael Henderson

What would you say are the unique advantages of this model?
This model shows a similar regional distribution of LRRK2 compared to wild-type mice. It has a large amount of overexpressed LRRK2, which can be advantageous when looking for protein localization or effect, since endogenous LRRK2 can be difficult to detect by immunolabeling.

What caveats are associated with this model?
Due to potential artifacts associated with overexpression and multiple transgene insertions, many labs have now shifted to using LRRK2*G2019S knock-in mice, such as LRRK2 G2019S KI and B6.Cg-Lrrk2tm1.1Hlme/J mice. The mice recapitulate many of the phenotypes of the LRRK2 G2019S (BAC Tg) BAC transgenic mice, without all the caveats associated with transgenic overesxpression.

COMMENTS / QUESTIONS

1. • June Kinoshita
     
   • Posted: 11 Feb 2010
   • Paper: Enhanced striatal dopamine transmission and motor performance with LRRK2 overexpression in mice is eliminated by familial Parkinson's disease mutation G2019S.

   At PDOnline Research, Zhenyu Yue of Mount Sinai School of Medicine discusses two new BAC Lrrk2 mouse models, recently published in Journal of Neuroscience.

   View all comments by June Kinoshita

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References

Research Models Citations

1. LRRK2 G2019S KI Mouse

Paper Citations

1. Li X, Patel JC, Wang J, Avshalumov MV, Nicholson C, Buxbaum JD, Elder GA, Rice ME, Yue Z. Enhanced striatal dopamine transmission and motor performance with LRRK2 overexpression in mice is eliminated by familial Parkinson's disease mutation G2019S. J Neurosci. 2010 Feb 3;30(5):1788-97. PubMed.
2. West AB, Cowell RM, Daher JP, Moehle MS, Hinkle KM, Melrose HL, Standaert DG, Volpicelli-Daley LA. Differential LRRK2 expression in the cortex, striatum, and substantia nigra in transgenic and nontransgenic rodents. J Comp Neurol. 2014 Aug 1;522(11):2465-80. Epub 2014 Apr 12 PubMed.
3. Rudyk C, Dwyer Z, Hayley S, CLINT membership. Leucine-rich repeat kinase-2 (LRRK2) modulates paraquat-induced inflammatory sickness and stress phenotype. J Neuroinflammation. 2019 Jun 7;16(1):120. PubMed.
4. Litteljohn D, Rudyk C, Dwyer Z, Farmer K, Fortin T, Hayley S, Canadian Lrrk2 in Inflammation Team (CLINT). The impact of murine LRRK2 G2019S transgene overexpression on acute responses to inflammatory challenge. Brain Behav Immun. 2018 Jan;67:246-256. Epub 2017 Sep 8 PubMed.
5. Liu X, Yu H, Wang Y, Li S, Cheng C, Al-Nusaif M, Le W. Altered Motor Performance, Sleep EEG, and Parkinson's Disease Pathology Induced by Chronic Sleep Deprivation in Lrrk2G2019S Mice. Neurosci Bull. 2022 Oct;38(10):1170-1182. Epub 2022 May 25 PubMed.
6. Henderson MX, Peng C, Trojanowski JQ, Lee VM. LRRK2 activity does not dramatically alter α-synuclein pathology in primary neurons. Acta Neuropathol Commun. 2018 May 31;6(1):45. PubMed.
7. Cornblath EJ, Li HL, Changolkar L, Zhang B, Brown HJ, Gathagan RJ, Olufemi MF, Trojanowski JQ, Bassett DS, Lee VM, Henderson MX. Computational modeling of tau pathology spread reveals patterns of regional vulnerability and the impact of a genetic risk factor. Sci Adv. 2021 Jun;7(24) Print 2021 Jun PubMed.
8. Xiao Q, Yang S, Le W. G2019S LRRK2 and aging confer susceptibility to proteasome inhibitor-induced neurotoxicity in nigrostriatal dopaminergic system. J Neural Transm (Vienna). 2015 Dec;122(12):1645-57. Epub 2015 Aug 8 PubMed.
9. Chen ZC, Zhang W, Chua LL, Chai C, Li R, Lin L, Cao Z, Angeles DC, Stanton LW, Peng JH, Zhou ZD, Lim KL, Zeng L, Tan EK. Phosphorylation of amyloid precursor protein by mutant LRRK2 promotes AICD activity and neurotoxicity in Parkinson's disease. Sci Signal. 2017 Jul 18;10(488) PubMed.
10. Sweet ES, Saunier-Rebori B, Yue Z, Blitzer RD. The Parkinson's Disease-Associated Mutation LRRK2-G2019S Impairs Synaptic Plasticity in Mouse Hippocampus. J Neurosci. 2015 Aug 12;35(32):11190-5. PubMed.
11. Volpicelli-Daley LA, Abdelmotilib H, Liu Z, Stoyka L, Daher JP, Milnerwood AJ, Unni VK, Hirst WD, Yue Z, Zhao HT, Fraser K, Kennedy RE, West AB. G2019S-LRRK2 Expression Augments α-Synuclein Sequestration into Inclusions in Neurons. J Neurosci. 2016 Jul 13;36(28):7415-27. PubMed. Correction.
12. Pan PY, Li X, Wang J, Powell J, Wang Q, Zhang Y, Chen Z, Wicinski B, Hof P, Ryan TA, Yue Z. Parkinson's Disease-Associated LRRK2 Hyperactive Kinase Mutant Disrupts Synaptic Vesicle Trafficking in Ventral Midbrain Neurons. J Neurosci. 2017 Nov 22;37(47):11366-11376. Epub 2017 Oct 20 PubMed.
13. 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.
14. Henderson MX, Cornblath EJ, Darwich A, Zhang B, Brown H, Gathagan RJ, Sandler RM, Bassett DS, Trojanowski JQ, Lee VM. Spread of α-synuclein pathology through the brain connectome is modulated by selective vulnerability and predicted by network analysis. Nat Neurosci. 2019 Aug;22(8):1248-1257. Epub 2019 Jul 25 PubMed.
15. Wang S, Unnithan S, Bryant N, Chang A, Rosenthal LS, Pantelyat A, Dawson TM, Al-Khalidi HR, West AB. Elevated Urinary Rab10 Phosphorylation in Idiopathic Parkinson Disease. Mov Disord. 2022 Jul;37(7):1454-1464. Epub 2022 May 6 PubMed.
16. Iannotta L, Biosa A, Kluss JH, Tombesi G, Kaganovich A, Cogo S, Plotegher N, Civiero L, Lobbestael E, Baekelandt V, Cookson MR, Greggio E. Divergent Effects of G2019S and R1441C LRRK2 Mutations on LRRK2 and Rab10 Phosphorylations in Mouse Tissues. Cells. 2020 Oct 22;9(11) PubMed.
17. Li X, Wang QJ, Pan N, Lee S, Zhao Y, Chait BT, Yue Z. Phosphorylation-dependent 14-3-3 binding to LRRK2 is impaired by common mutations of familial Parkinson's disease. PLoS One. 2011 Mar 1;6(3):e17153. PubMed.
18. Sheng Z, Zhang S, Bustos D, Kleinheinz T, Le Pichon CE, Dominguez SL, Solanoy HO, Drummond J, Zhang X, Ding X, Cai F, Song Q, Li X, Yue Z, van der Brug MP, Burdick DJ, Gunzner-Toste J, Chen H, Liu X, Estrada AA, Sweeney ZK, Scearce-Levie K, Moffat JG, Kirkpatrick DS, Zhu H. Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations. Sci Transl Med. 2012 Dec 12;4(164):164ra161. PubMed.
19. Moehle MS, Daher JP, Hull TD, Boddu R, Abdelmotilib HA, Mobley J, Kannarkat GT, Tansey MG, West AB. The G2019S LRRK2 mutation increases myeloid cell chemotactic responses and enhances LRRK2 binding to actin-regulatory proteins. Hum Mol Genet. 2015 Aug 1;24(15):4250-67. Epub 2015 Apr 29 PubMed.
20. Sepulveda B, Mesias R, Li X, Yue Z, Benson DL. Short- and long-term effects of LRRK2 on axon and dendrite growth. PLoS One. 2013;8(4):e61986. Print 2013 PubMed.
21. Shani V, Safory H, Szargel R, Wang N, Cohen T, Elghani FA, Hamza H, Savyon M, Radzishevsky I, Shaulov L, Rott R, Lim KL, Ross CA, Bandopadhyay R, Zhang H, Engelender S. Physiological and pathological roles of LRRK2 in the nuclear envelope integrity. Hum Mol Genet. 2019 Dec 1;28(23):3982-3996. PubMed.
22. Henry AG, Aghamohammadzadeh S, Samaroo H, Chen Y, Mou K, Needle E, Hirst WD. Pathogenic LRRK2 mutations, through increased kinase activity, produce enlarged lysosomes with reduced degradative capacity and increase ATP13A2 expression. Hum Mol Genet. 2015 Nov 1;24(21):6013-28. Epub 2015 Aug 6 PubMed.
24. Zhou ZD, Xie SP, Sathiyamoorthy S, Saw WT, Sing TY, Ng SH, Chua HP, Tang AM, Shaffra F, Li Z, Wang H, Ho PG, Lai MK, Angeles DC, Lim TM, Tan EK. F-box protein 7 mutations promote protein aggregation in mitochondria and inhibit mitophagy. Hum Mol Genet. 2015 Nov 15;24(22):6314-30. Epub 2015 Aug 26 PubMed.
25. Goodwin LO, Splinter E, Davis TL, Urban R, He H, Braun RE, Chesler EJ, Kumar V, van Min M, Ndukum J, Philip VM, Reinholdt LG, Svenson K, White JK, Sasner M, Lutz C, Murray SA. Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis. Genome Res. 2019 Mar;29(3):494-505. Epub 2019 Jan 18 PubMed.

External Citations

1. Liu et al., 2022
2. The Jackson Lab, Stock# 012467
3. B6.Cg-Lrrk2tm1.1Hlme/J

Further Reading

No Available Further Reading