Modification: PINK1: Knock-Out
Disease Relevance: Parkinson's Disease
Strain Name: LEH-Pink1tm1Sage-/-
Genetic Background: Long Evans Hooded
Availability: Available through Horizon Discovery (formerly Sage Labs), Cat# TGRL4690; Live.
This knockout rat model was created at Sage Labs (now Horizon) 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).
The 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 leads to a frameshift and the creation of a premature stop codon. Pink1 mRNA is virtually undetectable in homozygous rats.
Homozygous rats appear normal at birth. Their behavior has been assessed systematically at four, six, and eight months of age. Notably, Pink1 KO rats show abnormalities in gait, coordination, and strength. Gait impairments appear as early as four months of age, with abnormal paw positioning and a shorter stride than wild-type rats. Deficits in motor coordination are seen by eight months of age. Compared with wild-type rats, Pink1 KOs had three- to fivefold more foot slips (both fore-and hind-limb) on the balance beam. They performed normally on the accelerating Rotarod at all ages tested. In terms of strength, 13 out of 15 Pink1 KOs showed reduced overall muscle tone by eight months of age and particular weakness in the hind limb extensor muscle, leading to dragging hind limbs. Hind limb grip strength was impaired as early as four months of age.
Other motor deficits include less rearing behavior and reduced total distance travelled, with a 70 percent decrease in mobility at eight months of age compared with wild-type rats.
The rats did not exhibit convulsions or tremors. Nor did they exhibit grooming deficits or differences in startle response or body temperature. There was no increase in mortality up to eight months of age.
Pink1 KOs were heavier than wild-type counterparts at four, six, and eight months of age. For example, at eight months of age Pink1 KO rats weighed 651 ± 27.8 grams; whereas wild-type rats weighed 587 ± 18.4 grams.
Interestingly, Pink1 KO rats had more dopamine in the striatum than wild-type rats. Levels were elevated two- to threefold at eight months of age. Similarly, 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).
Belaying the increased striatal dopamine concentrations, Pink1 KOs exhibited age-related decreases in the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. Specifically, compared to 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.
Staining for α-synuclein revealed no overt differences in the striatum or in any other brain region, compared with wild-type rats.
The rat Pink1 gene was disrupted using zinc finger nuclease (ZFN) technology. 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, a deletion of 26 base pairs was created. This deletion lead to a frameshift and the creation of a premature stop codon.
When visualized, these models will distributed over a 18 month timeline demarcated at the following intervals: 1mo, 3mo, 6mo, 9mo, 12mo, 15mo, 18mo+.
- Dopamine Deficiency
- α-synuclein Inclusions
- Mitochondrial Abnormalities
- Cognitive Dysfunction
Staining for α-synuclein revealed no increase in the striatum or in any other brain region tested.
Striatal dopamine level was increased 2-3 fold in Pink1 KO rats compared with wild-type levels at eight months of age.
Abnormalities in gait, coordination, and strength. As early as four months of age, KO’s have abnormal paw positioning and a shorter stride. By 8 months, they exhibit 3-5-fold more foot slips on the balance beam. Overall decrease in muscle tone, particularly in the hind limb extensor, leading to dragging hind limbs.
Age-related decrease in tyrosine hydroxylase (TH)-positive dopaminergic neurons in the substantia nigra. Twenty-five and 50 percent reduction at 6 months and 8 months, respectively. No change in TH-positive cells in the striatum.
- 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.
- 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.
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