Species: Mouse
Genes: LRRK2
Modification: LRRK2: Knock-In
Disease Relevance: Parkinson's Disease
Strain Name: C57BL/6-Lrrk2^tm4.1Arte
Genetic Background: C57BL/6NTac
Availability: Available through Taconic, Cat#13940, Live. Research services with this model are available from Scantox Neuro.

This constitutive knock-in (KI) mouse model was generated by introducing the LRRK2 G2019S point mutation into exon 41 of the mouse LRRK2 gene (Matikainen-Ankney et al., 2016). Homozygous mutant mice appear grossly normal. They generate litters comparable in size to wild-type animals, and grow at a normal rate.

The cytoarchitecture of the neocortex and striatum appears normal in Nissl-stained brain sections. Striatal levels of tyrosine hydroxylase (TH) are similar at 3 weeks of age in KI mice compared to non-transgenic, wild-type controls (Matikainen-Ankney et al., 2016), but may be slightly lower at 2 months of age compared to controls (Pajarillo et al., 2023). Neither striatal dopamine levels, measured by microdialysis, nor TH levels in the substantia nigra, measured by immunostaining, differ between KI and wild-type mice at 2 months of age (Pajarillo et al., 2023). Immunostaining for the microglial marker Iba1 in the striatum or midbrain does not differ between 2-month-old wild-type and KI mice.

LRRK2 levels in both striatal and whole-brain lysates are similar in KI mice to those of wild-type controls. For example, in whole brain samples (at 10 weeks of age), in primary cultured astrocytes, and in striatal lysates taken from postnatal day (P) 14, P21, or P60 mice, total levels of LRRK2 do not differ between KI and wild-type mice (Wang et al., 2021; Scantox Neuro [formerly QPS Neuropharmacology] 2023; Matikainen-Ankney et al, 2016). However, analysis of the multimer-to-monomer ratio of LRRK2 in cortical samples show elevations in KI mice versus controls (Sarkar et al., 2021). Levels of LRRK2 auto-phosphorylation measured in whole brain samples are elevated about twofold at S1292, but are not different at S935, in KI versus non-transgenic mice (De Miranda et al., 2021; Scantox Neuro [formerly QPS Neuropharmacology] 2023). However, in primary astrocytes from KI mice, pS935 is reduced compared to wild-type mice (Wang et al., 2021). In that same study, levels of phosphorylated Rab10 (pT73), one of the substrates of LRRK2 phosphorylation, are twofold higher in KI mice compared with astrocytes from wild-type controls; this observation is also seen in whole brain tissue (De Miranda et al., 2021).

Another putative LRRK2 phospho-substrate, adaptor protein subunit AP2M1—a protein involved in clathrin-mediated endocytosis—shows increased levels of pThr156 in brain lysates from KI mice compared to wild-type mice (Liu et al., 2021). KI mice in this study also exhibit endocytosis defects in immature cultured hippocampal neurons, as measured by transferrin internalization at cell somata, but in studies of cultured striatal projection neurons, transferrin receptor internalization along dendrites is unaffected by G2019S (Gupta et al., 2023).

With regard to cytoskeletal effects of G2019S in the brain, levels of F-actin are greater in the cortex of 10- to 12-week-old KI mice compared to controls, though no actin rods are present (Sarkar et al., 2021).

Axonal transport of autophagic vesicles is perturbed based on live imaging of cortical neurons from KI mice in comparison with wild-type controls (Boecker et al., 2021). Defects in retrograde transport of autophagic vesicles include increased number of pauses, the fraction of total time paused, and the number of reversals. No differences are observed in microtubule dynamics (run length, run time, velocity, or density) between KI and wild-type mice, indicating that this likely does not account for the effects on transport. Furthermore, and in contrast to the effects observed for autophagic vesicles, transport of the cargo LAMP-1 (lysosomal-associated membrane protein 1) is not affected by G2019S KI.

Under baseline conditions, the motor phenotype of KI mice appears largely normal. Assessments of grip strength and locomotor activity, as well as performance on the pole test, balance beam, and static rods, revealed no motor impairments in KI mice at 3 to 4, 12 to 13, or 18 to 19 months of age (Pioli et al., 2011). KI mice show no differences with wild-type mice in latency-to-fall in an accelerating Rotarod test in young (3-4-month-old) or older (14-16-month-old) mice (Pioli et al., 2011; Matikainen-Ankney et al., 2018). Similarly, Rotarod performance in 2-, 8-, and 10-month-old KI mice is similar to that in wild-type mice (Crown et al., 2020; Pajarillo et al., 2023). In other studies, open-field exploration and locomotion in KI mice are similar to wild-type mice at 2 to 4 months of age (Matikainen-Ankney et al., 2018; Pajarillo et al., 2023). One study reports slightly but significantly slower walking speed in KI mice (Pajarillo et al., 2023).

KI mice performed similarly to wild-type mice in a variety of other behavioral tests measured under baseline conditions. They show no baseline anxiety-like behaviors as assessed in an elevated plus maze (Matikainen-Ankney et al., 2018), nor deficits in exploration of new environments, as assessed by the spontaneous alternation in the Y-maze, and their arousal and vigilance are normal, as assessed in a psychomotor-vigilance task. Moreover, KI mice score similarly to wild-type mice in SHIRPA, a standardized set of fairly gross measurements of muscular, cerebellar, sensory and neuropsychiatric function. Hippocampal-dependent recognition memory assessed by a novel object recognition test at 2 months of age also does not differ between KI and wild-type mice under basal conditions (Pajarillo et al., 2023).

Some measures of fronto-striatal based executive function, assessed by touchscreen-based operant tasks, are impaired in 2- to 6-month-old male KI mice (Hussein et al., 2022). For instance, visuospatial attention deficits and slower information processing speeds are observed in the 5-CSRT (choice serial reaction time) task, which are not due to reduced motivation, impaired visual sensory perception, or altered gross locomotor activity. Such deficits are rescued by systemic administration of the acetylcholinesterase inhibitor donepezil, implicating deficient cholinergic signaling. Consistent with this, the density of cholinergic innervation in prelimbic and infralimbic cortical areas and in the dorsomedial striatum of young adult male KI mice, assessed by immunolabeling for the vesicular acetylcholine transporter (VAChT), is significantly lower than that in wild-type mice, though no differences in VAChT density are found in the dorsal lateral geniculate nucleus (Hussein et al., 2022).

Goal-directed learning (measured by an instrumental conditioning and outcome devaluation paradigm) is also impaired in 2- to 6-month-old male KI mice, which is not due to differences in motivation. However, cognitive flexibility, measured by a visual discrimination and reversal learning task, is largely similar in KI and wild-type mice (Hussein et al., 2022).

Sleep behavior is perturbed in KI mice. In 8- to 10-month-old male KI mice, although total sleep time is similar to wild-type mice, sleep is more fragmented, with more numerous, but shorter, bouts of sleep (Crown et al., 2020). Additional data based on electrocorticography reveal that KI mice have more frequent and longer thalamocortical sleep spindles—oscillatory events involved in memory consolidation, which occur during the slow-wave phase of sleep—compared with wild-type controls.

Under conditions where KI mice are subjected to behavioral stress or pharmacological challenges, additional differences with wild-type mice emerge. Following manganese exposure, 2-month-old KI mice show lower levels of TH in the substantia nigra and perform worse than wild-type mice in a novel object recognition test (Pajarillo et al., 2023). At 18 months of age, an increased locomotor response after amphetamine challenge is observed (Pioli et al., 2011).

Social-defeat stress, used to measure depression-like behaviors, has significantly different effects on KI mice compared to wild-type mice (Matikainen-Ankney et al., 2018; Guevara et al., 2020). While young adult (3-4-month-old) male KI mice exhibit normal social behavior at baseline (unstressed conditions), they are unusually resilient to a depression-like syndrome promoted by chronic (10-day) social-defeat stress, which in wild-type mice can produce significant social avoidance. This lack of behavioral susceptibility to social stress correlates with a loss of bidirectional synaptic plasticity in the striatum. When subjected to a corticostriatal stimulation protocol that normally induces long-term potentiation in spiny projection neurons of the dorsomedial striatum in wild-type mice, both D1R (dopamine D1 receptor)- and D2R-expressing subtypes of striatal projection neurons in KI mice fail to produce potentiated responses, with D2R-expresssing neurons additionally developing an abnormal long-term depression. As reported in a preprint, KI mice have altered surface AMPA receptor subunit stoichiometry in D1R-expressing striatal projection neurons at baseline, where GluA1 incorporation is favored over GluA2, due in part to reduced endocytosis and cell-surface mobility (Gupta et al., 2023). A chemical-LTP (long-term potentiation) stimulation protocol applied to cultured spiny projection neurons also fails to promote the addition of synaptic GluA1 in KI neurons.

In contrast to the resilience observed in the chronic (10-day) social-defeat stress paradigm noted above, young (10- to 12-week-old) male KI mice that undergo acute (1-day) social-defeat stress exhibit greater social avoidance than wild-type mice (Guevara et al., 2020). KI mice also show an increase in sucrose consumption compared with wild-type mice following acute social stress, suggesting that depression-like and anhedonia-like behaviors are uncoupled in this mouse model. The basis for such behavioral differences compared with wild-type mice may lie in different capacities for synaptic and non-synaptic adaptations. For example, striatal projection neurons in the nucleus accumbens (NAc) of acutely-defeated wild-type mice, which are largely unaffected behaviorally by acute social-defeat stress, exhibit significant increases in intrinsic membrane excitability but no changes in synaptic properties. In contrast, striatal projection neurons in the NAc of acutely-defeated KI mice, which are socially avoidant, lack changes in intrinsic membrane excitability and exhibit changes in synaptic properties (increased amplitude and frequency of spontaneous excitatory post-synaptic currents [sEPSCs]). In the absence of social-defeat stress, measures of intrinsic excitability in the NAc (rheobase, resting membrane potential, membrane resistance, and number of action potentials following application of depolarizing currents) do not differ between genotypes.

Significant differences in structural and functional synaptic properties between KI and wild-type mice are evident in both dorsal and ventral striatum early in postnatal development (Matikainen-Ankney et al., 2016; Guevara et al., 2020). Whole-cell patch-clamp recordings from dorsomedial striatal projection neurons (in direct or indirect pathways) in acute brain slices at P21 show a fourfold increase in the frequency of sESPCs, an increase in the probability of larger sEPSC amplitudes, and no changes in synapse density or dendritic spine numbers, but the spine heads in KI spiny projection neurons are larger. Such findings apply to both heterozygous and homozygous mice, consistent with G2019S being an autosomal dominant mutation.

In male and female KI mice, amplitude and frequency of sESPCs in the NAc are greater than in wild-type controls, and evoked AMPAR-mediated responses are also greater, indicating that glutamatergic synapses are stronger in KI mice. Analysis of dendritic spine morphology in the NAc of KI mice at this early postnatal age also reveals that the spine heads are larger than in wild-type mice. These synaptic differences may be transient, however, since the sESPC amplitudes in the NAc (as measured with whole cell patch-clamp recordings of spiny projection neurons) do not differ between 10- to 12-week-old wild-type and KI male mice (Guevara et al., 2020).

Modification Details

Homologous recombination was used to introduce a mutation corresponding to human G2019S in exon 41 of the mouse LRRK2 gene (Matikainen-Ankney et al., 2016). This KI line also has 2 loxP sites surrounding exon 41, allowing it to be crossed with a Cre line to remove the mutated exon in a spatially selective manner, although this feature has not been verified (Taconic, 2023).

Phenotype Characterization

Q&A with Model Creator

Q&A with George W. Huntley.

What would you say are the unique advantages of this model?
The mutant mouse LRRK2 protein is expressed at normal, physiological levels in brain and body, obviating potential confounds of LRRK2 overexpression or the expression of both mouse and human forms of LRRK2 within single cells. The mutant protein is expressed endogenously throughout life, mimicking the expression profile that occurs naturally in humans.

What do you think this model is best used for?
Any analysis of cellular, synaptic, or behavioral outcomes that obligatorily require intact, physiologically-relevant brain structures, neurons and glia, and synaptic circuits. Also allows evaluation of brain-body interactions. Particularly well-suited for non-motor features of Parkinson’s, including psychiatric-like, cognitive-like, or other non-motor behavioral alterations and the underlying neurobiology. Allows longitudinal behavioral evaluation across a lifespan.

What caveats are associated with this model?
The KI mice do not exhibit typical, aging-related motor abnormalities and dopaminergic neuron degeneration that are the clinically-defining features of Parkinson’s.

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References

Paper Citations

1. Matikainen-Ankney BA, Kezunovic N, Mesias RE, Tian Y, Williams FM, Huntley GW, Benson DL. Altered Development of Synapse Structure and Function in Striatum Caused by Parkinson's Disease-Linked LRRK2-G2019S Mutation. J Neurosci. 2016 Jul 6;36(27):7128-41. PubMed.
4. Sarkar S, Bardai F, Olsen AL, Lohr KM, Zhang YY, Feany MB. Oligomerization of Lrrk controls actin severing and α-synuclein neurotoxicity in vivo. Mol Neurodegener. 2021 May 24;16(1):33. PubMed.
5. De Miranda BR, Castro SL, Rocha EM, Bodle CR, Johnson KE, Greenamyre JT. The industrial solvent trichloroethylene induces LRRK2 kinase activity and dopaminergic neurodegeneration in a rat model of Parkinson's disease. Neurobiol Dis. 2021 Jun;153:105312. Epub 2021 Feb 23 PubMed.
6. Liu Q, Bautista-Gomez J, Higgins DA, Yu J, Xiong Y. Dysregulation of the AP2M1 phosphorylation cycle by LRRK2 impairs endocytosis and leads to dopaminergic neurodegeneration. Sci Signal. 2021 Jul 27;14(693) PubMed.
7. Gupta S, Guevara CA, Tielemans A, Huntley GW, Benson DL. Parkinson's-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity. 2023 Oct 17 10.1101/2023.10.13.562231 (version 1) bioRxiv.
8. Boecker CA, Goldsmith J, Dou D, Cajka GG, Holzbaur EL. Increased LRRK2 kinase activity alters neuronal autophagy by disrupting the axonal transport of autophagosomes. Curr Biol. 2021 May 24;31(10):2140-2154.e6. Epub 2021 Mar 24 PubMed.
9. Pioli E, Murray TK, Buckner N, Cooper J, Mitchell SN, O'Neill MJ. Behavioral Phenotyping of G2019S Knock-In Leucine-Rich Repeat Kinase 2 (LRRK2) Transgenic Mice. Poster Presentation in 10th International Conference AD/PD, Barcelona, March 2011. Neurodegenerative Dis. Vol. 8, Suppl. 1, 2011.
10. Matikainen-Ankney BA, Kezunovic N, Menard C, Flanigan ME, Zhong Y, Russo SJ, Benson DL, Huntley GW. Parkinson's Disease-Linked LRRK2-G2019S Mutation Alters Synaptic Plasticity and Promotes Resilience to Chronic Social Stress in Young Adulthood. J Neurosci. 2018 Nov 7;38(45):9700-9711. Epub 2018 Sep 24 PubMed.
11. Crown LM, Bartlett MJ, Wiegand JL, Eby AJ, Monroe EJ, Gies K, Wohlford L, Fell MJ, Falk T, Cowen SL. Sleep Spindles and Fragmented Sleep as Prodromal Markers in a Preclinical Model of LRRK2-G2019S Parkinson's Disease. Front Neurol. 2020;11:324. Epub 2020 May 8 PubMed.
12. Hussein A, Tielemans A, Baxter MG, Benson DL, Huntley GW. Cognitive deficits and altered cholinergic innervation in young adult male mice carrying a Parkinson's disease Lrrk2G2019S knockin mutation. Exp Neurol. 2022 Sep;355:114145. Epub 2022 Jun 19 PubMed.
13. Guevara CA, Matikainen-Ankney BA, Kezunovic N, LeClair K, Conway AP, Menard C, Flanigan ME, Pfau M, Russo SJ, Benson DL, Huntley GW. LRRK2 mutation alters behavioral, synaptic, and nonsynaptic adaptations to acute social stress. J Neurophysiol. 2020 Jun 1;123(6):2382-2389. Epub 2020 May 6 PubMed.

External Citations

1. Scantox Neuro [formerly QPS Neuropharmacology] 2023
2. Taconic
3. Taconic, Cat#13940
4. Scantox Neuro

Further Reading

No Available Further Reading