Species: Mouse
Genes: Pink1
Modification: Pink1: Knock-Out
Disease Relevance: Parkinson's Disease
Strain Name: B6.129S4-Pink1^tm1Shn/J
Genetic Background: Congenic C57BL/6J. The construct was introduced into 129S4/SvJae-derived J1 embryonic stem cells, which were injected into C57BL/6 blastocysts. The resulting chimeric animals were crossed to generate homozygotes and then backcrossed to C57BL/6J for >7 generations.
Availability: Available through The Jackson Laboratory, Stock# 017946; Live.

Summary

This mouse model was developed to investigate the effects of PINK1 deficiency (Kitada et al., 2007). The model involves a germline deletion of exons 4-7 of the endogenous Pink1 gene, creating truncated transcripts that are degraded. Mice homozygous for the null allele lack observable PINK1 protein. Homozygous mice have normal numbers of dopaminergic neurons and levels of striatal dopamine. However, they exhibit decreased evoked release of dopamine and other changes in striatal dopaminergic physiology.

Homozygous mice are viable and fertile. On average, they are heavier than wild-type mice at 5 months of age (Kelm-Nelson et al., 2018). Neuropathologically, these mice are grossly normal. However, behavioral deficits emerge at an early age.

Motor Behavior | Neuropathology | Mitochondrial Abnormalities | Other Cellular Changes | Other Organ System Changes | Modification Details

Motor Behavior

PINK1 KO mice showed reduced spontaneous locomotor activity at 3 to 6 months of age as assessed by the number of steps, rears, and landings in the cylinder test. They also took longer than wild-type mice to turn and climb down a pole, a test of locomotor skill. Moreover, modest deficits in ultrasonic vocalizations were observed at 4 to 6 months of age (Kelm-Nelson et al., 2018).

Neuropathology

Despite these behavioral alterations, the number of dopaminergic neurons in the substantia nigra and the levels of striatal dopamine at 2 to 3 months of age, as well as at 8 to 9 months of age, were comparable to those of wildtype mice (Kitada et al., 2007; also see Soman et al., 2021). The morphology of dopaminergic neurons also appeared to be grossly intact (Kitada et al., 2007), and there was no reduction in tyrosine hydroxylase immunolabeling (Kelm-Nelson et al., 2018). Likewise, homozygotes and controls had comparable levels of the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). Nonetheless, PINK1 KO mice were more sensitive than wild-type mice to MPTP-induced neural damage: following MPTP treatment, KO mice exhibited a greater loss of dopaminergic neurons in the substantia nigra pars compacta and a greater loss of terminal dopamine fiber density (as measured by dopamine transporter) in the striatum (Haque et al., 2012).

However, striatal slices from homozygous mice exhibited reduced dopamine release in response to electrical stimulation. Reduced evoked catecholamine release was also observed in disassociated adrenal chromaffin cells. The reduced transmission of dopamine was associated with plasticity abnormalities in the cortico-striatal pathway. High frequency stimulation in the absence of magnesium elicited weaker long-term potentiation in medium spiny neurons from KO mice compared to those of wildtype mice. Moreover, high-frequency stimulation in the presence of magnesium failed to evoke long-term depression. These impairments could be rescued by dopamine receptor agonists, suggesting dopamine receptors were functional. Quantitative analysis of dopamine binding in the striatum confirmed there was no difference in the density of D1 and D2 receptors (Kitada et al., 2007). In an analysis of freshly isolated striatal medium spiny neurons, the high voltage-activated Ca2⁺ current profile was similar between PINK1 KO and control mice, and no alterations were found in D2 receptor activation (Martella et al., 2011).

Moreover, dendritic health appears to be affected in PINK1 KO mice. The dendrites of midbrain dopaminergic neurons from 10-month-old KO mice were shorter than those of wild-type mice. Also, the dendrites of cultured cortical neurons isolated from embryonic mice grew more slowly, and harbored shorter mitochondria that occupied less dendritic volume and traveled less distance anterogradely. These alterations were accompanied by deficits in mitochondrial protein kinase A signaling, as suggested by reduced phosphorylation of the enzyme’s regulatory subunit β (DasBanerjee et al., 2017).

Mitochondrial Abnormalities

Mitochondrial transmembrane potential was examined in primary cardiomyocytes from PINK1 KO mice, and cells from KO mice showed perturbations at baseline and were further susceptible to reactive oxygen species–induced depolarization compared to cells from wild-type mice (Billia et al., 2011). Moreover, mitochondrial copy number (a marker of mitochondrial biogenesis) and mitochondrial gene cytochrome b (a marker of mitochondrial capacity) were reduced in PINK1 KO mice at 2 months of age versus controls, and this deficit was progressive since it worsened at 6 months of age. ATP production and complex I (NADH dehydrogenase) activity were also impaired in left ventricular extracts from 2-month-old PINK1 KO mice. In addition, the expression of many mitochondrial proteins was reduced in the cardiac ventricles of 2-month-old KO mice.

In murine embryonic fibroblasts isolated from embryonic PINK1 KO mice, the mitochondrial aspect ratio was higher than in cells derived from wild-type mice (Li et al., 2022).

In frozen liver sections of 3-month-old PINK1 KO mice, accumulation of ubiquitin on mitochondria did not differ compared to control mice (Yamada 2019 31339428). SQSTM1, a protein that functions like an autophagy receptor, also did not accumulate on mitochondria of KO mice, as was the case for control mice.

Moreover, mitochondrial dysfunction induced by a high-fat diet was greater in PINK1 mice than in wild-type mice. The altered diet was fed for a period of 6 months, starting at 2 months of age. Mitophagy markers, mitochondrial ultrastructure, oxygen consumption rate, and reactive oxygen species production were monitored to assess mitochondrial function (Mu et al., 2020).

In addition, quiescent muscle stem cells of PINK1 KO mice exhibited reduced mitophagy, increased release of mitochondrial reactive oxygen species, and reduced mitochondrial volume, as reported in a preprint (Cairns 2023).

Other Cellular Changes

Cytokine levels in PINK1 KO mice serum were similar to those in wild-type mice, a surprising finding given the role PINK1 plays in mitophagy which, by removing damaged mitochondria, mitigates inflammatory responses. However, following exhaustive exercise, which acutely stresses mitochondria, cytokine concentrations shot up in the serum of KO, but not wild-type, mice (Sliter et al., 2018).

Midbrain sections from 10-month-old PINK1 KO mice had reduced intracellular levels of BDNF versus those in wild-type mice, based on immunohistochemical staining (Soman et al., 2021). The cortex of 10-month-old PINK1 KO mice also showed reduced levels of mature and immature BDNF by western blot analysis.

Other Organ System Changes

Cardiac hypertrophy was observed at 2 and 6 months of age, but not at 1 week of age, in PINK1 KO mice versus control mice (Billia et al., 2011). Left ventricular dysfunction was also observed, based on fractional shortening, in 2- and 6-month-old KO mice. This dysfunction worsened with age. Moreover, the mRNA expression of hypertrophic markers (ANF, BNP, and β-MHC, but not α-MHC) was increased in 2- and 6-month-old PINK1 KO mouse. PINK1 KO mice also expressed higher levels of oxidative stress markers in left ventricular extracts from 2-month-old mice. This was associated with an increased susceptibility to apoptosis in the hearts of PINK1 KO mice.

In PINK1 KO mice who were fed a high-fat diet for 4 or 6 months starting at 2 months of age, cardiac outcomes related to diabetic cardiomyopathy were exacerbated and also appeared earlier than they did in wild-type mice (Mu et al., 2020).

Modification Details

A targeting vector containing a PGK-Neo cassette was used to disrupt exons 4 through 7 of the endogenous PINK1 gene. This creates a nonsense mutation at the beginning of exon 8; truncated RNA is degraded.

Phenotype Characterization

Q&A with Model Creator

Q&A with Ruben Karim Dagda.

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

Unlike the PINK1-KO rat model, which shows an overt loss of dopamine neurons by 2.5 months of age and is an excellent model for clinical translational research (CTR), the PINK1- KO mice are an excellent model to study the role of endogenous PINK1 on signaling pathways in the brain, for identifying interactors of PINK1, and for assessing changes in neurochemistry (e.g., levels of neurotrophic factors) in the absence of neuronal degeneration. In addition to using these mice to conduct, in vivo work (e.g., how stressors can induce Parkinsonian phenotypes in PINK1- KO mice compared to WT mice), PINK1-KO mice are ideal and amenable for culturing PINK1-deficient primary neurons, including midbrain, cortical, and hippocampal neurons to study molecular pathways, mitochondrial structure, and dynamics and function regulation by PINK1.

What caveats are associated with this model?

Given the compensatory signaling pathways that are elicited in PINK1 KO mice, they are not ideal for CTR, including the identification of new therapeutics or biomarkers of Parkinson’s disease. The PINK1 KO rats would be the more ideal model for CTR.

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

Mini clinical trials can be modeled in PINK1 KO rats. Namely, “wash-out trials and cross-over trials can be performed to test the performance of candidate therapeutics on motor symptoms and muscle fatigue,” at least based on our experience testing two potential promising therapeutics (Vazquez-Mayorga et al., 2022; Dagda et al., 2022).

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References

Research Models Citations

1. Pink1 KO Rat

Paper Citations

1. Kitada T, Pisani A, Porter DR, Yamaguchi H, Tscherter A, Martella G, Bonsi P, Zhang C, Pothos EN, Shen J. Impaired dopamine release and synaptic plasticity in the striatum of PINK1-deficient mice. Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11441-6. PubMed.
2. Kelm-Nelson CA, Brauer AF, Barth KJ, Lake JM, Sinnen ML, Stehula FJ, Muslu C, Marongiu R, Kaplitt MG, Ciucci MR. Characterization of early-onset motor deficits in the Pink1-/- mouse model of Parkinson disease. Brain Res. 2018 Feb 1;1680:1-12. Epub 2017 Dec 8 PubMed.
3. Soman SK, Tingle D, Dagda RY, Torres M, Dagda M, Dagda RK. Cleaved PINK1 induces neuronal plasticity through PKA-mediated BDNF functional regulation. J Neurosci Res. 2021 Sep;99(9):2134-2155. Epub 2021 May 27 PubMed.
4. Haque ME, Mount MP, Safarpour F, Abdel-Messih E, Callaghan S, Mazerolle C, Kitada T, Slack RS, Wallace V, Shen J, Anisman H, Park DS. Inactivation of Pink1 gene in vivo sensitizes dopamine-producing neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and can be rescued by autosomal recessive Parkinson disease genes, Parkin or DJ-1. J Biol Chem. 2012 Jun 29;287(27):23162-70. Epub 2012 Apr 17 PubMed.
5. Das Banerjee T, Dagda RY, Dagda M, Chu CT, Rice M, Vazquez-Mayorga E, Dagda RK. PINK1 regulates mitochondrial trafficking in dendrites of cortical neurons through mitochondrial PKA. J Neurochem. 2017 Aug;142(4):545-559. Epub 2017 Jun 23 PubMed.
6. Billia F, Hauck L, Konecny F, Rao V, Shen J, Mak TW. PTEN-inducible kinase 1 (PINK1)/Park6 is indispensable for normal heart function. Proc Natl Acad Sci U S A. 2011 Jun 7;108(23):9572-7. Epub 2011 May 23 PubMed.
7. Li J, Dang X, Franco A, Dorn GW 2nd. Reciprocal Regulation of Mitofusin 2-Mediated Mitophagy and Mitochondrial Fusion by Different PINK1 Phosphorylation Events. Front Cell Dev Biol. 2022;10:868465. Epub 2022 May 12 PubMed.
8. Mu J, Zhang D, Tian Y, Xie Z, Zou MH. BRD4 inhibition by JQ1 prevents high-fat diet-induced diabetic cardiomyopathy by activating PINK1/Parkin-mediated mitophagy in vivo. J Mol Cell Cardiol. 2020 Dec;149:1-14. Epub 2020 Sep 15 PubMed.
9. Cairns G, Thumiah-Mootoo M, Abbasi MR, Racine J, Lariovov N, Prola A, Khacho M, Burelle Y. PINK1 Deficiency Alters Muscle Stem Cell Fate Decision And Muscle Regenerative Capacity. 2023 Jun 25 10.1101/2023.06.23.546123 (version 1) bioRxiv.
10. Sliter DA, Martinez J, Hao L, Chen X, Sun N, Fischer TD, Burman JL, Li Y, Zhang Z, Narendra DP, Cai H, Borsche M, Klein C, Youle RJ. Parkin and PINK1 mitigate STING-induced inflammation. Nature. 2018 Sep;561(7722):258-262. Epub 2018 Aug 22 PubMed.

Other Citations

1. Martella et al., 2011

External Citations

1. The Jackson Laboratory, Stock# 017946

Further Reading