Species: Mouse
Genes: Park2
Mutations: Park2 S65A
Modification: Park2: Knock-In
Disease Relevance: Parkinson's Disease
Strain Name: C57BL/6-Prkn^tm1.1Muqit/J
Genetic Background: C57BL/6J
Availability: Available through The Jackson Laboratory, Stock# 029247, Cryopreserved.

Modification Details:

This constitutive KI mouse model was generated by Flp-mediated homologous recombination in mouse embryonic stem cells to introduce an S65A point mutation in exon 3 of the Park2 (Parkin) gene (McWilliams et al., 2018).

Summary
These knock-in (KI) mice carry a serine to alanine mutation (S65A) in the N-terminal ubiquitin-like domain of the Park2 gene that encodes Parkin, a ubiquitin E3 ligase (McWilliams et al., 2018). Serine 65 is the site at which PINK1 (PTEN-induced kinase 1) phosphorylates Parkin to induce mitophagy. This mutation is relevant to Parkinson’s disease as PARK2 mutations (including at serine 65) are associated with early-onset disease in humans.

Homozygous Parkin S65A KI mice are viable and fertile, and have a grossly normal appearance, growth, weight, and development relative to wild-type mice (McWilliams et al., 2018; The Jackson Laboratory). Mutant Parkin protein expression was stable and levels were similar to those in wild-type mice throughout the brain and also in other organs (heart, spleen, lung, pancreas), as measured by western blot. Functionally, primary cultured cortical neurons from Parkin S65A KI mice had lost the ability to ubiquitylate CISD1 (a canonical Parkin substrate) following mitochondrial depolarization (which was triggered by antimycin A and oligomycin [A/O] treatment), indicating a loss of function that was not observed in cells from wild-type mice. Phosphorylation of Parkin and ubiquitin at serine 65 was also lost in cortical KI cells following A/O treatment. Together, these results indicate that PINK1-dependent phosphorylation of Parkin at serine 65 is essential for activating Parkin’s E3 ligase activity as well as phosphorylating ubiquitin at serine 65. Of note, in a subsequent analysis using more sensitive approaches to detect phospho-ubiquitin, a significant reduction, but not complete loss in parkin knockout neurons, was observed (Antico et al., 2021).

Motor Function
At 12 and 18 months of age, homozygous Parkin S65A KI mice had impaired performance on the raised balance beam—which measures voluntary locomotor function, balance, and coordinated limb use—relative to wild-type controls (McWilliams et al., 2018). In particular, KI mice made more errors and took longer to cross the beam. The balance beam test also revealed a potential cognitive deficit, as KI mice took longer to turn. In contrast to the sensitive beam test, no differences were observed in Rotarod testing and gait analysis between KI and wild-type mice at 12 or 18 months of age.

Neuropathology
Striatal anatomy and volume did not differ between 18-month-old homozygous Parkin S65A KI mice and wild-type mice (McWilliams et al., 2018). In addition, tyrosine hydroxylase immunostaining did not differ in the midbrain or striatum of KI mice, pointing to a lack of effect of the mutation on striatal innervation. To assess the structure of the nigrostriatal pathway in more detail, aged adult mouse brains were optically cleared using iDISCO+ and analyzed with confocal microscopy, and innervation and overall connectivity also did not differ between wild-type and KI mice.

Immunolabeling of astrocytes (using GFAP) and microglia (using Iba1) to assess neuroinflammation did not reveal any differences between homozygous Parkin S65A KI mice and wild-type mice (McWilliams et al., 2018).

Levels of dopamine and 3,4-dihydroxyphenylacetic acid (3,4-DOPAC) from striatal samples, as well as their ratio, were comparable between 18-month-old homozygous Parkin S65A KI mice and wild-type mice, based on HPLC analysis (McWilliams et al., 2018).

Mitochondrial Abnormalities
Mitochondrial respiration in the striatum, as measured by high-resolution (Oroboros Oxygraph-2k) respirometry, was impaired in homozygous Parkin S65A KI mice relative to wild-type controls at 12 months of age (McWilliams et al., 2018). This deficit in respiratory control ratio was not apparent at younger ages (3 months) nor in other brain regions (cortex, midbrain). In contrast, basal mitophagy did not differ between homozygous Parkin S65A KI mice and wild-type controls (as assessed through crossing them to the mito-QC transgenic line) in nigrostriatal dopaminergic cell bodies and projections (McWilliams et al., 2018).

Phenotype Characterization

Q&A with Model Creator

Q&A with Miratul Muqit

What would you say are the unique advantages of this model?
This is a parkin model that is catalytically inactive. This may confer an advantage over the parkin knockout where other non-catalytic functions of parkin may also be affected. It is therefore a more precise model to use if researchers want to specifically prevent parkin activity.

What do you think this model is best used for?
Analysis of the signal transduction pathway of PINK1/parkin activation.

What caveats are associated with this model?
In this paper, we reported complete loss of phospho-ubiquitin in neurons in parkin knockout mice and parkin S65A knock-in mice. However, as noted in the summary, in a subsequent study, we observed a substantial reduction but not complete loss in parkin knockout neurons (Antico et al., 2021).

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References

Paper Citations

1. McWilliams TG, Barini E, Pohjolan-Pirhonen R, Brooks SP, Singh F, Burel S, Balk K, Kumar A, Montava-Garriga L, Prescott AR, Hassoun SM, Mouton-Liger F, Ball G, Hills R, Knebel A, Ulusoy A, Di Monte DA, Tamjar J, Antico O, Fears K, Smith L, Brambilla R, Palin E, Valori M, Eerola-Rautio J, Tienari P, Corti O, Dunnett SB, Ganley IG, Suomalainen A, Muqit MM. Phosphorylation of Parkin at serine 65 is essential for its activation in vivo. Open Biol. 2018 Nov 7;8(11) PubMed.
2. Antico O, Ordureau A, Stevens M, Singh F, Nirujogi RS, Gierlinski M, Barini E, Rickwood ML, Prescott A, Toth R, Ganley IG, Harper JW, Muqit MM. Global ubiquitylation analysis of mitochondria in primary neurons identifies endogenous Parkin targets following activation of PINK1. Sci Adv. 2021 Nov 12;7(46):eabj0722. PubMed.

External Citations

1. The Jackson Laboratory
2. The Jackson Laboratory, Stock# 029247

Further Reading

No Available Further Reading