Species: Mouse
Genes: SNCA, SNCA
Mutations: SNCA A53T
Modification: SNCA: Transgenic; SNCA: Knock-Out
Disease Relevance: Parkinson's Disease
Strain Name: FVB;129S6-Snca^tm1Nbm Tg(SNCA*A53T)1Nbm Tg(SNCA*A53T)2Nbm/J
Genetic Background: The PAC transgene was injected into FVB/N oocytes and founder mice bred to FVB/N. The knockout mice were made in a 129S6/SvEvTac background.
Availability: Available through The Jackson Laboratory, Stock #010799; Live and frozen embryo.

Summary

The dbl-PAC-Tg(SNCA^A53T)^+/+;Snca^-/- model, referred to here as double-transgenic mice, are mice lacking endogenous α-synuclein (Snca knockout mice) that were crossed with two homozygous transgenic lines overexpressing a full-length mutant (A53T) human α-synuclein. These mice, which are homozygous for both transgenes and the endogenous knockout, are viable into adulthood and exhibit early onset abnormalities of the enteric nervous system leading to gastrointestinal dysfunction. Neurons in the enteric nervous system develop α-synuclein pathology, but neurons in the brain are spared and there is no evidence of dopaminergic neuron loss (Kuo et al., 2010).

These homozygous double-transgenic mice have 18 copies of the human transgene (six on chromosome 3 and two on chromosome 14) and robust expression in the brain and colon (The Jackson Laboratory, July 2022 update). At 6 weeks of age, the level of transgene RNA was about 10-fold greater than the RNA level for endogenous mouse α-synuclein. However, transgenic protein levels were only slightly above endogenous levels (1.3- to 2-fold increase).  In the colon, transgene expression at both RNA and protein levels was very high, with an approximately 80-fold increase (Kuo et al., 2010).

Motor behavior
As they aged, the double-transgenic mice became less active than control mice (Kuo et al., 2010). They moved around less than controls in the open-field test at 6, 12, and 18 months of age. This was not attributed to differences in anxiety levels. Both males and females also performed worse on the accelerating Rotarod, compared with controls at 6, 12, and 18 months of age. These findings were also supported by a more recent characterization of the model, where Rotarod performance was impaired relative to control wild-type mice as early as 6 weeks of age (Radisavljevic et al., 2022). Moreover, double-transgenic mice demonstrated impairments on the pole test (longer time to descend than wild-type control mice) and the hindlimb clasping test (higher score) at 6, 14, and 22 weeks of age. However, in contrast to the original report, no differences were observed in total distance travelled on the open field test. Additionally, on the inverted grid, no differences were observed between genotypes in latency to fall.

Non-motor behavior
In addition to motor defects, double-transgenic mice also display non-motor perturbations (Radisavljevic et al., 2022). Anxiety-like behavior was evaluated by assessing time spent in the open field zone of an open field test, and double-transgenic mice spent significantly more time in this zone than wild-type mice at 6 and 22 weeks of age, suggesting decreased anxiety-like behavior. Moreover, on the tail suspension test, double-transgenic mice at 22 weeks of age displayed greater immobility time than wild-type mice, indicating greater depression-like behavior. Also, spontaneous activity was reduced in double-transgenic mice at 22 weeks of age, as assessed by a reduced number of rears in the cylinder test. 

Gastrointestinal dysfunction
By 3 months of age, double-transgenic mice begin to show signs of gastrointestinal dysfunction, including abnormally small and hard fecal pellets (Kuo et al., 2010). Further analysis showed reduced overall fecal mass with reduced water content, as well as a decrease in motility through the colon and prolonged whole-gut transit time. In a more recent report, whole-gut transit time (as measured with the carmine red test) was similarly impaired, and this was observed as early as 6 weeks of age (Radisavljevic et al., 2022). In this study, fecal water content did not differ between genotypes at 6 weeks of age, but reduced water content was apparent by 14 and 22 weeks, together pointing to progressively slower gastrointestinal transit. Despite chronic impairments to gastrointestinal function, body weight in the double-transgenic mice did not significantly differ from non-transgenic mice out to 18 months of age (Kuo et al., 2010).

In an analysis of the gut microbiome using 16S rRNA sequencing of fecal samples, no changes were observed in the α-diversity (indicator of total species of flora) between 2-month-old double-transgenic mice and wild-type mice, but β-diversity (indicator of the microbiome’s composition) did differ (Yang et al., 2024). Namely, double-transgenic mice had an increased amount of Bacilli, Lactobacillales, Proteobacteria, and Desulfovibrio. In another report using the same methodology, α-diversity was reduced in double-transgenic mice and β-diversity differed from wild-type mice at both 4 and 24 weeks of age (Radisavljevic et al., 2022).

Serotonin levels in the gut were reduced in double-transgenic versus wild-type mice (Radisavljevic et al., 2022).

α-Synuclein aggregation
Neurons of the enteric nervous system expressed mutant α-synuclein and accumulated α-synuclein aggregates in the cytoplasm and nucleus with age in double-transgenic mice (Kuo et al., 2010). Also, In red blood cells, an elevated level of α-synuclein aggregation was observed in double-transgenic mice at 1 and 12 (but not 6) months of age (Yang et al., 2024). However, the brain was remarkably intact. Double-transgenic mice did not exhibit Lewy body-like pathology or signs of α-synuclein aggregation (Kuo et al., 2010).

Neuropathology
There was no evidence of neurodegeneration in tyrosine hydroxylase (TH)-positive cells of the substantia nigra at 11 and 18 months of age (Kuo et al., 2010). A few dystrophic neurites were observed in the hippocampus at 12 to 22 months, but otherwise the brain structure of double-transgenic mice was comparable to that of control mice.

Dopamine Levels
The striatal dopaminergic concentration was comparable in double-transgenic and non-transgenic mice at 11 and 18 months of age, as were levels of dopaminergic metabolites homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC; Kuo et al., 2010).

Other phenotypes
The morphology of red blood cells (RBCs) was found to differ in double-transgenic versus wild-type mice (Yang et al., 2024). With a progressive worsening over time, double-transgenic mice first exhibited normal-shaped RBCs at 1 month of age, but had a significantly reduced proportion of normal-shaped RBCs compared to wild-type mice at 3, 6, and 12 months of age. This was accompanied by a gradual increase in the proportion of acanthocytes (cells with surface membrane protrusions) relative to wild-type mice.

In monocytes isolated from the peripheral blood of 7-month-old double-transgenic mice, mRNA and protein levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) were increased at baseline (except for IL-1β protein) compared to control cells (Liu et al 2022).

Phenotype Characterization

Q&A with Model Creator

Q&A with Robert Nussbaum

What would you say are the unique advantages of this model?
Early onset of gastrointestinal manifestations so the mice do not have to be aged beyond a few months. The signs of neuronal dysfunction can be easily assayed if one is doing preclinical testing of therapies designed to prevent α-synuclein aggregation and neuronal damage.

What do you think this model is best used for?
Studying genetic/environmental interactions involving gut flora and their metabolites to find out if there are particular bacterial species or bacterial products that are required for, exacerbate, or mitigate the onset of gastrointestinal dysfunction.
Preclinical studies of compounds that can slow or prevent development of neuronal dysfunction and damage.

What caveats are associated with this model?
There is very little evidence that the expression levels in the brain reach levels needed to cause brain disease. The absence of mouse α-synuclein may actually prevent rather than promote the spread of α-synuclein aggregation from the gut up the vagus, if indeed Braak's hypothesis is correct.

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References

Research Models Citations

1. α-synuclein KO Mouse

Paper Citations

1. Kuo YM, Li Z, Jiao Y, Gaborit N, Pani AK, Orrison BM, Bruneau BG, Giasson BI, Smeyne RJ, Gershon MD, Nussbaum RL. Extensive enteric nervous system abnormalities in mice transgenic for artificial chromosomes containing Parkinson disease-associated alpha-synuclein gene mutations precede central nervous system changes. Hum Mol Genet. 2010 May 1;19(9):1633-50. PubMed.
2. Radisavljevic N, Cirstea M, Bauer K, Lo C, Metcalfe-Roach A, Bozorgmehr T, Bar-Yoseph H, Brett Finlay B. Effects of Gut Microbiota Alterations on Motor, Gastrointestinal, and Behavioral Phenotype in a Mouse Model of Parkinson's Disease. J Parkinsons Dis. 2022;12(5):1479-1495. PubMed.
3. Yang Y, Stewart T, Zhang C, Wang P, Xu Z, Jin J, Huang Y, Liu Z, Lan G, Liang X, Sheng L, Shi M, Cai Z, Zhang J. Erythrocytic α-Synuclein and the Gut Microbiome: Kindling of the Gut-Brain Axis in Parkinson's Disease. Mov Disord. 2024 Jan;39(1):40-52. Epub 2023 Oct 5 PubMed.
4. Liu Z, Chan RB, Cai Z, Liu X, Wu Y, Yu Z, Feng T, Yang Y, Zhang J. α-Synuclein-containing erythrocytic extracellular vesicles: essential contributors to hyperactivation of monocytes in Parkinson's disease. J Neuroinflammation. 2022 Feb 22;19(1):53. PubMed.

External Citations

1. The Jackson Laboratory
2. The Jackson Laboratory, Stock #010799
3. The Jackson Laboratory

Further Reading