Species: Mouse
Genes: Park2
Mutations: Parkin Q311X
Modification: Park2: Transgenic
Disease Relevance: Parkinson's Disease
Strain Name: FVB/NJ-Tg(Slc6a3-PARK2*Q311X)AXwy/J
Genetic Background: The BAC was microinjected into fertilized FVB/NJ zygotes, then bred to FVB/NJ inbred mice.
Availability: Available through The Jackson Laboratory, Stock# 009090, cryopreserved or frozen embryo.

Summary

The Q311X mutation is a nonsense mutation that produces a C-terminally truncated parkin, just 155 amino acids long. Parkin-Q311X mice (line A) have two copies of the transgene (Tg) integrated in tandem in a bacterial artificial chromosome (BAC), although its integration site is unknown. Notably, as hemizygous mice age, they lose dopaminergic neurons and develop progressive changes in motor behavior, including lower activity levels (Lu et al., 2009).

This transgenic model of Parkinson’s disease expresses near-physiological levels of mutant parkin mRNA in dopaminergic neurons (Lu et al., 2009). However, it has been difficult to assess the expression level of the mutant protein since no parkin antibody detects this variant (all reliable antibodies for western blotting recognize the C-terminal domain of parkin; personal communication, Jenny Sassone, March 2024). Transgenic mRNA was confirmed within dopaminergic neurons of the substantia nigra (SN) and ventral tegmental area (VTA) by in situ hybridization. Transgenic protein (immunostaining for N-terminal FLAG) was observed in neurons co-staining with SLC6a3, a marker of dopaminergic neurons.

As they age, Parkin-Q311X mice lose dopaminergic neurons in the SN (Lu et al., 2009; Siddiqui et al., 2015). The number of nigral neurons staining positive for tyrosine hydroxylase (TH) was comparable to wild-type mice at 3 months of age, but reduced by about 40 percent by 16 months of age. This decrease was primarily due to a drop in neuronal numbers, but also reflected reduced TH expression in the remaining neurons. Not surprisingly, the striatum received fewer dopaminergic projections and contained less dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), a dopamine metabolite. Neurons in the VTA were relatively spared. A more recent study found that the number of nigral dopaminergic neurons was decreased as early as 6 months of age (and also at 12 months), but not at 1 month of age, based on unbiased stereological counts of TH-positive neurons (Regoni et al., 2021).

Although Parkin-Q311X mice do not develop Lewy body-like inclusions, they do accumulate cytoplasmic α-synuclein in the midbrain (Lu et al., 2009). At 3 months of age the number of α-synuclein-positive nigral neurons was comparable to non-Tg, but it was elevated by 16 months. The cytoplasmic α-synuclein often co-localized with 3-nitrotyrosine, a marker of nitrative damage. Another study similarly demonstrated an increase in proteinase K–insoluble α-synuclein in the SN of Parkin-Q311X mice versus wild-type mice at 16 months of age, by western blotting and immunostaining (Siddiqui et al., 2015).

Other neural perturbations have been observed in Parkin-Q311X mice. For instance, protein levels of glutamate ionotropic receptors were examined in the SN of 3- to 4-week-old mice (Maraschi et al., 2014). The GluK2 subunit (but not GluK3) of the kainate receptor was significantly increased in Parkin-Q311X mice, while expression of AMPA receptor (GluA1 and GluA2/3) and NMDA receptor subunits (GluN1 and GluN2B) did not differ from wild-type controls. These mice also demonstrated increased SN levels of cleaved spectrin and cleaved calcineurin A, which are markers for excitotoxic damage.

Dysfunction in the burst-firing pattern activity of dopaminergic SN neurons has also been observed in younger, 1-month-old Parkin-Q311X mice as compared with wild-type control mice (Regoni et al., 2021).

Hemizygous Parkin-Q311X mice behave normally at young ages, but exhibit lower levels of activity as they age and have other modest differences in locomotor behavior compared with non-Tg littermates (Lu et al., 2009). In the open-field test, both males and females exhibited hypoactivity at 16 and 21 months of age, but not at 6 and 12 months of age. They also exhibited deficits in the adhesive-removal test (which measures motor response to sensory stimuli) at 13 to 16 months. Motor behavior was studied in both male and female hemizygous mice and no significant sex differences were reported. In another study, deficits in motor coordination and balance and spontaneous activity were also found at an older age (16 months) in Parkin-Q311X versus wild-type mice (Siddiqui et al., 2015).

Autophagy was found to be perturbed in Parkin-Q311X mice at 16 to 17 months of age (Siddiqui et al., 2015). The number of LC3 puncta in the SNpc was increased in mutant versus wild-type control mice, and there were also increases in p62 puncta formation, suggesting downstream lysosomal dysfunction. The ratio of autophagosomes to autolysosomes was also increased, indicating a blockage in the formation of autolysosomes. Moreover, autolysosomes of Parkin-Q311X mice contained undigested mitochondria. Further data pointing to perturbed lysosomal function in Parkin-Q311X mice included a decrease in striatal levels of mature activated cathepsin D; a decrease in mRNA levels of TFEB, TFAM, Nrf1, and PGC1α; and an increase in protein levels of PARIS.

At 1 month of age, cytoplasmic vacuolization—a feature of many cell death pathways—was observed more frequently in dopaminergic cells of the SN (but not ventral tegmental area) in Parkin-Q311X mice than in that of wild-type controls (Regoni et al., 2021).

Mitochondrial dysfunction was observed in Parkin-Q311X mice (Siddiqui et al., 2015). At 16 to 17 months of age, mitochondrial volume fractions (in the SN) and complex I activity (in primary striatal cells) were reduced in Parkin-Q311X mice versus wild-type controls. In much younger Parkin-Q311X mice (1 month of age), cellular markers for mitochondrial dysfunction have also been observed (Regoni et al., 2021). For instance, while SDHA and VDAC mitochondrial proteins were unchanged, expression of the short isoform of OPA1 was increased, suggesting mitochondrial damage. Indeed, electron microscopy revealed that mitochondrial morphology in the SN showed evidence of damage (e.g., lacking an outer membrane, swollen) in 1-month-old Parkin-Q311X mice that was more common than that observed in wild-type mice.

Of note, in a study by The Jackson Laboratory, this Parkin-Q311X mouse line was found to carry the retinal degeneration 1 mutation (Pde6brd1), and thus it is recommended that this strain not be used to study new retinal disorders (Chang et al., 2013).

Modification Details

Parkin-Q311X mice have BAC-mediated expression of mutant parkin in dopaminergic neurons. The promoter and regulatory regions of the Slc6a3 gene (encoding a dopamine transporter) drive expression of parkin with the truncation mutation Q311X. The protein has an N-terminal FLAG-tag. Two copies of the transgene are integrated in tandem.

Phenotype Characterization

Q&A with Model Creator

Expert/Creator Q&A with Jenny Sassone

What would you say are the unique advantages of this model?

The unique advantage of this model is the progressive loss of dopaminergic neurons.

What do you think this model is best used for?

It is best used to test neuroprotective strategies.

What caveats are associated with this model?

This mouse model expresses the parkin variant under a DAT (dopamine transporter) promoter, so the parkin variant is only present in dopaminergic neurons. However, parkin is normally expressed in many cell types, so this model does not recapitulate abnormalities in other cell types (other brain cell types such as glial cells or other neuron types could also be affected by the parkin mutation).

Anything else useful or particular about this model you think our readers would like to know?

The parkin variant expressed in this model has been found in only one family to date. The mutation is a certain cause of the disease, but other models expressing more common parkin mutations should be considered in order to confirm results obtained in this model.

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References

Paper Citations

1. Lu XH, Fleming SM, Meurers B, Ackerson LC, Mortazavi F, Lo V, Hernandez D, Sulzer D, Jackson GR, Maidment NT, Chesselet MF, Yang XW. Bacterial artificial chromosome transgenic mice expressing a truncated mutant parkin exhibit age-dependent hypokinetic motor deficits, dopaminergic neuron degeneration, and accumulation of proteinase K-resistant alpha-synuclein. J Neurosci. 2009 Feb 18;29(7):1962-76. PubMed.
2. Siddiqui A, Bhaumik D, Chinta SJ, Rane A, Rajagopalan S, Lieu CA, Lithgow GJ, Andersen JK. Mitochondrial Quality Control via the PGC1α-TFEB Signaling Pathway Is Compromised by Parkin Q311X Mutation But Independently Restored by Rapamycin. J Neurosci. 2015 Sep 16;35(37):12833-44. PubMed.
3. Regoni M, Zanetti L, Comai S, Mercatelli D, Novello S, Albanese F, Croci L, Consalez GG, Ciammola A, Valtorta F, Morari M, Sassone J. Early Dysfunction of Substantia Nigra Dopamine Neurons in the ParkinQ311X Mouse. Biomedicines. 2021 May 5;9(5) PubMed.
4. Maraschi A, Ciammola A, Folci A, Sassone F, Ronzitti G, Cappelletti G, Silani V, Sato S, Hattori N, Mazzanti M, Chieregatti E, Mulle C, Passafaro M, Sassone J. Parkin regulates kainate receptors by interacting with the GluK2 subunit. Nat Commun. 2014 Oct 15;5:5182. PubMed.
5. Chang B, Hurd R, Wang J, Nishina P. Survey of common eye diseases in laboratory mouse strains. Invest Ophthalmol Vis Sci. 2013 Jul 24;54(7):4974-81. PubMed.

External Citations

1. The Jackson Laboratory, Stock# 009090

Further Reading