Species: Rat
Genes: Pink1
Modification: Pink1: Knock-Out
Disease Relevance: Parkinson's Disease
Strain Name: HsdSage:LE-Pink1^em1Sage; formerly LEH-Pink1^tm1Sage-/-
Genetic Background: Long Evans Hooded
Availability: Available through Inotiv. Live. (Previously available through Envigo (formerly Horizon Discovery and Sage Labs), Cat# TGRL4690) 

Summary

This knockout (KO) rat model was created at Sage Labs (acquired first by Horizon Discovery, then by Envigo, and most recently by Inotiv) in collaboration with the Michael J. Fox Foundation. The model carries a deletion of the Pink1 (PTEN-induced putative kinase 1) gene, which encodes a serine/threonine protein kinase. Homozygous Pink1 KO rats show both motor impairments and dopaminergic cell loss (Dave et al., 2014), although the findings are mixed, as discussed below.

General Health | Brain Volume Loss | Neurotransmitter Alterations | α-Synuclein Aggregates | Mitochondrial Abnormalities | Motor Impairment | Non-motor Impairment | Transcriptomic Alterations | Modification Details

General Health

Homozygous rats appear normal at birth and there is no increase in mortality up to 8 months of age. Both male and female KO rats are heavier than wild-type counterparts at 4, 6, and 8 months of age. (Marquis et al., 2020). However, in another study. female KO rats weighed less than wild-type female rats at 2 months of age (Lechner et al., 2022).

Brain Volume Loss

In 6- to 8-month old KO rats, many brain regions were reduced in volume compared to wild-type rats based on MRI (Cai et al., 2019). In addition, resting-state functional connectivity, also measured with MRI, was altered in KO rats, despite the KO rats exhibiting normal cognitive and motor function at this age (Cai et al., 2019). Moreover, in a different study, male but not female KO rats exhibited an increased ventricular volume based on MRI (Ren et al., 2019).

Neurotransmitter Alterations

The effects of Pink1 KO on striatal dopamine remain uncertain. Dave et al. found Pink1 KO rats had more dopamine in the striatum than wild-type rats. Levels were elevated two- to threefold at 8 months of age, as measured by ultra-high performance liquid chromatography-MS/MS (Dave et al., 2014). However, Kelm-Nelson et al. reported a slight decrease at the same age, as measured by ELISA, which correlated with slower traversals of the tapered balance beam (Kelm-Nelson et al., 2018). The authors speculate the inconsistent results may be due to differences in animal husbandry and/or experimental procedures. Dave et al. also found striatal serotonin levels were up two- to threefold. There were no significant differences in the turnover of either transmitter (Dave et al., 2014). Quantitative autoradiography revealed subtle changes in the density of dopaminergic receptor subtypes in the striatum. At six months of age, KO rats had a 26 percent increase in the D2 receptor and a 19 percent increase in the D3 receptor. Densities of the D1 receptor and the dopamine transporter were unchanged (Sun et al., 2013).

Dave et al. also reported that Pink1 KOs exhibited age-related decreases in the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (Dave et al., 2014). Specifically, compared with wild-type, there was a 25 percent reduction at six months and a reduction of more than 50 percent at eight months. Despite the robust and progressive loss of dopaminergic neurons in the substantia nigra, TH-positive cells in the striatum were intact at all ages tested. DeAngelo et al. also observed a loss of TH-positive cells in the substantia nigra pars compacta (27 percent) at 8 months of age in KO rats (DeAngelo et al., 2022). In contrast, they also observed a 15 percent loss of TH in the striatum (DeAngelo et al., 2022). Consistent with these findings, Villeneuve et al. reported a loss of TH-positive neurons in the substantia nigra pars compacta at nine months (Villeneuve et al., 2014). Loss of dopaminergic cells in the substantia nigra was observed as early as at 2.5 months of age in KO rats based on deficits in TH staining (Grigoruţă et al., 2020). In contrast, Grant et al. found no alterations in TH levels in either the substantia nigra or striatum at eight months, but observed TH reductions in the locus coeruleus which correlated with vocal loudness, tongue force, and chewing rate (Grant et al., 2015, Cullen et al., 2018). de Haas et al. also failed to observe reductions in dopaminergic neurons of the substantia nigra and changes in striatal neurotransmitter (dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid [HVA]) levels at 8 months of age (de Haas et al. 2019).

In the dorsal striatum, KO rats show significant differences compared to wild-type rats in basal neurotransmitter levels, as well as age-dependent effects (Creed et al., 2019). Basal levels of dopamine and acetylcholine were lower and glutamate levels were higher at 12 versus 4 months of age in KO rats. Moreover, 3,4-dihydroxyphenylacetic acid (DOPAC) and HVA were reduced at 12 versus 8 months of age in KO rats. Compared to wild-type rats, KO rats had greater basal levels of glutamate at 8 months of age, as well as trends to increased levels of basal HVA and glycine at 4 and 8 months of age, respectively. Evoked neurotransmitter release also differed in the dorsal striatum by age in KO rats. Evoked dopamine release was greater at 8 months and acetylcholine was greater at 12 months versus 4 months of age in KO rats. On the other hand, glutamate and GABA release were decreased in KO rats at 12 months compared to 4 months and 8 months, respectively. In a different study, striatal metabolites were also observed to differ between wild-type and KO rats in an age- and sex-dependent manner (Ren et al., 2019). Together, these data suggest that Park1 KO results in perturbations of neurotransmitter release, both under basal and evoked conditions, as well as defects in metabolites in the striatum.

With regard to norepinephrine, Kelm-Nelson et al. reported increased levels of this transmitter in the substantia nigra, and reduced levels in the locus coeruleus, which positively correlated with the number of complex calls (Kelm-Nelson et al., 2018).

α-Synuclein Aggregates

Brain α-synuclein staining was note overtly different in KO rats compared with wild-type rats in one study (Dave et al., 2014). However, others have found insoluble, proteinase K-resistant aggregates that stained positive for α-synuclein, thioflavin S, and ubiquitin in tissue sections from several brain regions. Aggregates were detected as early as 4 months of age and increased in number up to 12 months (Creed and Goldberg, 2019). In 8-month-old KO rats, aggregates were found in the periaqueductal gray, substantia nigra pars compacta, locus coeruleus, and nucleus ambiguous, which are regions that project to muscles involved in vocalization and swallowing (Grant et al., 2015). In the periaqueductal gray, where aggregates were particularly dense, the expression of α-synuclein remained unchanged, but mRNA levels of Atp13a2, a lysosomal ATPase, were reduced (Kelm-Nelson et al., 2016). At 12 months, inclusions were reported in the cortex, thalamus, striatum, and ventral midbrain (Creed and Goldberg, 2019). Also, an analysis of cortical homogenates at this age revealed an increase in the ratio of α-synuclein in synaptic vesicle-enriched fractions versus cytosolic fractions.

Mitochondrial Abnormalities

Metabolomic and proteomic analyses of mitochondria-associated factors in the brain revealed differences between Pink1 KO rats and wild-type controls as early as 10 days of age (Villeneuve et al., 2014, Villeneuve et al., 2015, Stauch et al., 2016). Several alterations suggested mitochondrial dysfunction, such as a reduction of mitochondrial complex I subunits, particularly in the striatum of 4-month-old rats. In addition, respiration driven by complex I was reduced in mitochondria isolated from striatal synapses of 3-month-olds, while that driven by complex II was increased, possibly as a compensatory mechanism. In 9-month-old rats, the oxygen consumption ratios of striatal mitochondria were actually elevated. In contrast, in non-synaptic striatal samples, mitochondrial respiration was not affected in 3-month-old Pink1 KO rats compared with wild-type rats (Stauch et al., 2016). Moreover, in Pink1 KO rats, the mitochondrial proteome in non-synaptic striatal samples exhibited 15 differentially expressed proteins compared with DJ-1 and Parkin KO rats at 3 months of age.

Decreased levels of mitochondrial content and antioxidant proteins (e.g., catalase, SOD2) have also been observed in the midbrain and prefrontal cortex of 9- to 11-week-old PINK1 KO rats (Grigoruţă et al., 2020).

Motor Impairment

Motor behavior in male PINK1 KO rats has been assessed systematically at 4, 6, and 8 months of age (Dave et al., 2014). Notably, Pink1 KO rats show abnormalities in gait, coordination, and strength. Deficits in coordination were reported as early as five weeks, with KO rats having increased foot slips on the tapered balance beam compared with wild-type animals (Inotiv Model Information Sheet, Jan 2023). Deficits in the tapered balance beam were similarly observed in a more recent study, where 2.5-month-old male KO rats exhibited more foot slips than wild-type rats (Grigoruţă et al., 2020). Female KO rats also displayed deficits in crossing the tapered balance beam at 2 months of age, but not at older ages (4, 6, 8 months) (Marquis et al., 2020). At 4 months of age, male KO rats exhibited abnormal paw positioning and a shorter stride than wild-type rats. By 8 months, male Pink1 KO rats had three- to fivefold more foot slips (both fore-and hind limb) on the balance beam (Dave et al., 2014), and took longer to traverse a tapered beam (Grant et al., 2015; de Haas et al. 2019). Male KO rats at this age also took more hind limb steps in the cylinder test for spontaneous movement, analogous to a shuffling gait (Grant et al. 2015). In another study, however, male KO rats exhibited deficits in gait at 5 months, but not at 8 months of age compared with wild-type rats (DeAngelo et al., 2022). Female KO rats, in contrast, did not display differences compared with wild-type rats in the cylinder test at 2, 4, 6, and 8 months of age (Marquis et al., 2020). On the accelerating Rotarod, Dave et al. reported that male KO rats performed normally at 4, 6, and 8 months of age (Dave et al., 2014). Motor impairment, as assessed by the beam balance assay in 14-month-old rats, can be temporarily reversed with intraperitoneal administration of Levodopa, although this treatment did not influence hindlimb strength (Vazquez-Mayorga 2022).

In terms of strength, both homozygous and heterozygous KO rats traveled less distance than wild-type rats in an open field after a hind limb fatigue challenge at 7 weeks of age (Inotiv Model Information Sheet, Jan 2023). At five months, 30 percent of KO rats were dragging their hind limbs (Inotiv Model Information Sheet, Jan 2023) and by eight months the percentage rose to 87 (Dave et al., 2014), although some have observed that this finding is transient (Cynthia A. Kelm-Nelson, personal communication). In addition, at 8 months of age, KO rats showed reduced overall muscle tone, less rearing behavior, and a 70 percent decrease in open-field mobility (Dave et al., 2014), although this latter measure may also be reflective of anxiety-like behavior. In more recent studies, deficits in total distance travelled in the open field and rearing behavior were similarly observed by 8 months of age, as was reduced muscle force (laryngeal) by 4 months of age (de Haas et al. 2019; Glass et al., 2019). Grip strength was impaired as early as 4 months of age. Another study reported that PINK1 KO rats exhibit significant muscle fatigue by 10 months of age (Vazquez-Mayorga 2022).

In contrast to the above, at 2 months of age, KO rats travelled a greater distance than wild-type rats on the open field and had more forelimb movements in the cylinder test (Lechner et al., 2022). Moreover, in this study, sex differences were observed in certain behaviors, and 2-month-old female KO rats spent more time in the center of the open field than males of either genotype or wild-type females, indicating that young KO females show less anxiety-like behavior.

KO rats do not exhibit convulsions or tremors. Nor do they exhibit grooming deficits or differences in startle response or body temperature. However, abnormalities in cranial sensorimotor and oromotor behaviors, including alterations in licking and biting (chewing and swallowing), have been detected at 2 to 8 months of age (Grant et al. 2015). Chewing and swallowing deficits may potentially be explained by extrinsic tongue muscle dysfunction. Extrinsic tongue muscles were found to have mild abnormalities in younger (4- to 6-month-old) KO rats compared to wild-type rats based on tongue press force, myosin heavy chain expression, and α-synuclein protein expression (Glass et al., 2020). Functionally, by 4 months of age, KO male rats show slower mastication rates and pharyngoesophageal bolus speeds compared to wild-type rats based on imaging following the ingestion of a peanut-butter-barium mixture, as well as abnormal swallowing behaviors (Krasko et al., 2023).

Defects in ultrasonic vocalization (USV) have been observed from 2 to 8 months of age in male KO rats (Grant et al. 2015). USV abnormalities are also present in female KO rats; in particular, across 2, 4, 6, and 8 months of age, KO rats produced USV calls of lower intensity than wild-type rats (Marquis et al., 2020). Further examination of USV calls in a more recent study found that 2-month-old KO rats (male and female) exhibited a higher percent of complex USV calls than wild-type rats, but they had a shorter duration, bandwidth, and peak frequency of frequency-modulated calls (Lechner et al., 2022). USVs from 6-month-old male KO rats presented to female wild-type rats resulted in perturbed responses compared to USVs from male wild-type rats, indicating that USV defects due to Pink1 KO leads to compromised inter-sexual communication (Pultorak et al, 2016).

Muscles potentially responsible for vocal deficits in KO rats have been examined to understand the underlying pathophysiology. Using RNA sequencing, the vocal fold thyroarytenoid muscle was found to have 134 differentially expressed genes in 8-month-old male KO animals compared to wild-type rats (Lechner et al., 2021). In 8-month-old female KO rats, gene expression was also perturbed for 437 genes in the thyroarytenoid muscle, pointing to defects in neuromuscular synaptic transmission (Barnett et al., 2023).

It is interesting to note that variability appears to be a phenotype of this KO model—increased variability is seen in tongue force and USV acoustic parameters (Cynthia A. Kelm-Nelson, personal communication).

Non-motor Impairment

Observations from several behavioral paradigms indicate KO rats express anxiety-like behaviors in KO rats. Male KO rats demonstrated more anxiety-like behavior across 4, 8, and 12 months of age based on the elevated plus maze; they spent more time in and had a greater preference for the closed arms compared with wild-type rats (Hoffmeister et al., 2021). This increase in anxiety was also associated with greater vocal deficits in KO rats. In female KO rats, anxiety was assessed via the light/dark box and elevated plus maze (Marquis et al., 2020). While KO rats spent less time in the dark of the light/dark box than wild-type rats up to 7 months of age, their anxiety-like behavior increased starting at 8 months of age, based on the pattern of behavior on the elevated plus maze. Female KO rats also ingested less sucrose on the sucrose preference test, pointing to increased anhedonia-like behavior compared with wild-type rats.

Male KO rats exhibit additional non-motor impairments. KO rats have abnormalities in nociception at 6 to 10 months of age, showing faster thermal withdrawal latencies compared with wild-type rats, which suggests thermal hyperalgesia (Johnson et al., 2020). In contrast, female KO rats at 4, 6, and 8 months of age did not differ from wild-type rats in their withdrawal latencies from a hot stimulus, except at 2 months of age when they were quicker to withdraw (Marquis et al., 2020). Ventilation is also affected, with 6- to 10-month-old male KO rats exhibiting higher breathing frequencies at baseline (Johnson et al., 2020). In contrast, under maximal chemoreceptor stimulation, ventilation frequency decreased with age in KO rats but not in wild-type rats. Together, these findings indicate that KO rats have abnormal sensorimotor processing, but this is sex-dependent.

Renal function appears intact in 2-month-old KO rats as compared with wild-type rats based on levels of plasma creatinine, blood urea nitrogen, neutrophil gelatinase-associated lipocalin, and kidney injury molecule 1 (Li et al., 2020).

In contrast, gastrointestinal dysfunction occurs in KO male rats (Krasko et al., 2023). Namely, by 4 months of age, KO rats exhibit delayed cecum and colon motility, and by 6 months of age they have a lower fecal pellet count, and higher pellet weight in comparison with wild-type rats.

Transcriptomic Alterations

High-throughput RNA sequencing was conducted to assess the effects of Pink1 KO on gene expression in the periaqueductal gray in 8-month-old rats (Kelm-Nelson et al., 2020). Compared to wild-type rats, KO rats had alterations in genes encoding solute carriers and glutamate metabotropic receptors as well as protein localization genes, suggesting these genes may contribute to the vocal deficits observed in this model. RNA sequencing was also conducted on whole blood samples in KO rats, and inflammation- and interferon-related signaling genes were upregulated in 3-month-old rats, indicating early gene expression changes may be able to predict future behavioral outcomes in this model (Lechner et al., 2023).

Modification Details

The rat Pink1 gene was disrupted using zinc finger nuclease (ZFN) technology, in which targeted ZFN RNA was injected into fertilized rat oocytes. The ZFNs were engineered to bind to a recognition site in exon 4 of Pink1 and cleave the DNA. When the resulting double strand break was repaired by non-homologous end joining, a deletion of 26 base pairs was created. This deletion lead to a frameshift and the creation of a premature stop codon. Pink1 mRNA is virtually undetectable in homozygous rats.

Phenotype Characterization

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References

Paper Citations

1. Dave KD, De Silva S, Sheth NP, Ramboz S, Beck MJ, Quang C, Switzer RC 3rd, Ahmad SO, Sunkin SM, Walker D, Cui X, Fisher DA, McCoy AM, Gamber K, Ding X, Goldberg MS, Benkovic SA, Haupt M, Baptista MA, Fiske BK, Sherer TB, Frasier MA. Phenotypic characterization of recessive gene knockout rat models of Parkinson's disease. Neurobiol Dis. 2014 Oct;70:190-203. Epub 2014 Jun 24 PubMed.
2. Marquis JM, Lettenberger SE, Kelm-Nelson CA. Early-onset Parkinsonian behaviors in female Pink1-/- rats. Behav Brain Res. 2020 Jan 13;377:112175. Epub 2019 Sep 19 PubMed.
4. Cai X, Qiao J, Knox T, Iriah S, Kulkarni P, Madularu D, Morrison T, Waszczak B, Hartner JC, Ferris CF. In search of early neuroradiological biomarkers for Parkinson's Disease: Alterations in resting state functional connectivity and gray matter microarchitecture in PINK1 -/- rats. Brain Res. 2019 Mar 1;1706:58-67. Epub 2018 Oct 31 PubMed.
5. Ren X, Hinchie A, Swomley A, Powell DK, Butterfield DA. Profiles of brain oxidative damage, ventricular alterations, and neurochemical metabolites in the striatum of PINK1 knockout rats as functions of age and gender: Relevance to Parkinson disease. Free Radic Biol Med. 2019 Nov 1;143:146-152. Epub 2019 Aug 8 PubMed.
6. Kelm-Nelson CA, Trevino MA, Ciucci MR. Quantitative Analysis of Catecholamines in the Pink1 -/- Rat Model of Early-onset Parkinson's Disease. Neuroscience. 2018 May 21;379:126-141. Epub 2018 Feb 27 PubMed.
7. Sun J, Kouranova E, Cui X, Mach RH, Xu J. Regulation of dopamine presynaptic markers and receptors in the striatum of DJ-1 and Pink1 knockout rats. Neurosci Lett. 2013 Oct 21; PubMed.
8. DeAngelo VM, Hilliard JD, McConnell GC. Dopaminergic but not cholinergic neurodegeneration is correlated with gait disturbances in PINK1 knockout rats. Behav Brain Res. 2022 Jan 24;417:113575. Epub 2021 Sep 14 PubMed.
9. Villeneuve LM, Purnell PR, Boska MD, Fox HS. Early Expression of Parkinson's Disease-Related Mitochondrial Abnormalities in PINK1 Knockout Rats. Mol Neurobiol. 2014 Nov 25; PubMed.
10. Grigoruţă M, Martínez-Martínez A, Dagda RY, Dagda RK. Psychological Stress Phenocopies Brain Mitochondrial Dysfunction and Motor Deficits as Observed in a Parkinsonian Rat Model. Mol Neurobiol. 2020 Apr;57(4):1781-1798. Epub 2019 Dec 14 PubMed.
11. Grant LM, Kelm-Nelson CA, Hilby BL, Blue KV, Paul Rajamanickam ES, Pultorak JD, Fleming SM, Ciucci MR. Evidence for early and progressive ultrasonic vocalization and oromotor deficits in a PINK1 gene knockout rat model of Parkinson's disease. J Neurosci Res. 2015 Nov;93(11):1713-27. Epub 2015 Jul 31 PubMed.
12. Cullen KP, Grant LM, Kelm-Nelson CA, Brauer AF, Bickelhaupt LB, Russell JA, Ciucci MR. Pink1 -/- Rats Show Early-Onset Swallowing Deficits and Correlative Brainstem Pathology. Dysphagia. 2018 Apr 30; PubMed.
13. de Haas R, Heltzel LC, Tax D, van den Broek P, Steenbreker H, Verheij MM, Russel FG, Orr AL, Nakamura K, Smeitink JA. To be or not to be pink(1): contradictory findings in an animal model for Parkinson's disease. Brain Commun. 2019;1(1):fcz016. Epub 2019 Sep 13 PubMed.
14. Creed RB, Menalled L, Casey B, Dave KD, Janssens HB, Veinbergs I, van der Hart M, Rassoulpour A, Goldberg MS. Basal and Evoked Neurotransmitter Levels in Parkin, DJ-1, PINK1 and LRRK2 Knockout Rat Striatum. Neuroscience. 2019 Jun 15;409:169-179. Epub 2019 Apr 25 PubMed.
15. Creed RB, Goldberg MS. Analysis of α-Synuclein Pathology in PINK1 Knockout Rat Brains. Front Neurosci. 2018;12:1034. Epub 2019 Jan 9 PubMed.
16. Kelm-Nelson CA, Stevenson SA, Ciucci MR. Atp13a2 expression in the periaqueductal gray is decreased in the Pink1 -/- rat model of Parkinson disease. Neurosci Lett. 2016 May 16;621:75-82. Epub 2016 Apr 4 PubMed.
17. Villeneuve LM, Purnell PR, Stauch KL, Fox HS. Neonatal mitochondrial abnormalities due to PINK1 deficiency: Proteomics reveals early changes relevant to Parkinson׳s disease. Data Brief. 2016 Mar;6:428-32. Epub 2015 Dec 17 PubMed.
18. Stauch KL, Villeneuve LM, Purnell PR, Ottemann BM, Emanuel K, Fox HS. Loss of Pink1 modulates synaptic mitochondrial bioenergetics in the rat striatum prior to motor symptoms: concomitant complex I respiratory defects and increased complex II-mediated respiration. Proteomics Clin Appl. 2016 Dec;10(12):1205-1217. Epub 2016 Sep 21 PubMed.
19. Stauch KL, Villeneuve LM, Purnell PR, Pandey S, Guda C, Fox HS. SWATH-MS proteome profiling data comparison of DJ-1, Parkin, and PINK1 knockout rat striatal mitochondria. Data Brief. 2016 Dec;9:589-593. Epub 2016 Sep 23 PubMed.
21. Glass TJ, Kelm-Nelson CA, Russell JA, Szot JC, Lake JM, Connor NP, Ciucci MR. Laryngeal muscle biology in the Pink1-/- rat model of Parkinson disease. J Appl Physiol (1985). 2019 May 1;126(5):1326-1334. Epub 2019 Mar 7 PubMed.
26. Hoffmeister JD, Kelm-Nelson CA, Ciucci MR. Quantification of brainstem norepinephrine relative to vocal impairment and anxiety in the Pink1-/- rat model of Parkinson disease. Behav Brain Res. 2021 Sep 24;414:113514. Epub 2021 Aug 4 PubMed.
27. Johnson RA, Kelm-Nelson CA, Ciucci MR. Changes to Ventilation, Vocalization, and Thermal Nociception in the Pink1-/- Rat Model of Parkinson's Disease. J Parkinsons Dis. 2020;10(2):489-504. PubMed.
28. Li HB, Zhang XZ, Sun Y, Zhou Q, Song JN, Hu ZF, Li Y, Wu JN, Guo Y, Zhang Y, Shi J, Yu JB. HO-1/PINK1 Regulated Mitochondrial Fusion/Fission to Inhibit Pyroptosis and Attenuate Septic Acute Kidney Injury. Biomed Res Int. 2020;2020:2148706. Epub 2020 Oct 22 PubMed.

External Citations

1. Inotiv Model Information Sheet
2. I

Further Reading