Alpha-Synuclein transgenic Mouse Models
PD is a neurodegenerative disease characterized by loss of dopaminergic neurons in the mesencephalon and by progressing reduction of dopaminergic innervation of the dorsal striatum. Symptoms of PD are impairments of motor and cognitive functions, and vegetative disturbances. Among other findings, PD is associated with increased ubiquitination and with the accumulation of alpha-synuclein in Lewy bodies and dystrophic neurites.
Some mutations of alpha-synuclein have been identified that increase the risk for familial PD. These findings have led to the generation of several transgenic mouse models that express different isoforms of human alpha-synuclein under control of different promoters, in order to investigate pathological changes and test the efficacy of pharmaceutical compounds in preclinical drug development studies in vivo. Consequently, interference with aggregation of alpha-synuclein is a common target in the development of pharmaceutical approaches to prevent or ameliorate PD.
QPS Austria has a license to Patent Rights owned by the Regents of the University of California for providing commercial testing services with several human alpha-synuclein transgenic mouse lines developed by Dr. Eliezer Masliah of UCSD. The 3 alpha-synuclein transgenic mouse lines available at QPS Austria feature different cellular expression patterns of human alpha-synuclein, age at onset and progression of pathology. Animals express different isoforms of human alpha-synuclein (wildtype or A53T-mutated). These animals constitute suitable models to study the influence of drugs on alpha-synuclein-related brain pathology and behavior.
D-Line transgenic Mouse Model
This transgenic (Tg) mouse line overexpresses wildtype human alpha-synuclein under the regulatory control of the human platelet-derived growth factor–β (PDGF-β) promoter (D-line; Masliah et al. 2000, Science 287: 1265-9). Mice are bred in-house on a C57BL/6 background. The model is well-established, and a large body of publications is available. These mice replicate features of human synucleinopathies such as abnormal accumulation of alpha-synuclein, increased phosphorylation of alpha-synuclein and high levels of ubiquitin in cortical and subcortical regions of the brain. Specifically, transgenic human alpha-synuclein mRNA is expressed at high levels in the hippocampus, neocortex, olfactory bulb and the substantia nigra, whereas expression is largely absent from other parts of the brain (Rockenstein et al. 2002, J Neurosci Res 68: 568-78).
Strong immunoreactivity of human alpha-synuclein protein is evident in different subsets of neuronal somata in the above-mentioned areas, and both expression and aggregation of alpha-synuclein increase over age (Amschl et al. 2013, BMC Neurosci 9;14:6). This distinct histopathological pattern allows testing the efficacy of pharmaceutical compounds to interfere with, or even reverse, the progression of alpha-synuclein accumulation. Additional immunoreactivity occurs in presynaptic cortical terminals, consistent with its role as a presynaptic protein.
Figure: Quantitative immunofluorescence (top) reveals increases in human α-synuclein expression over age in D-Line mice in both neocortex and hippocampus. Double immunofluorescence labeling (bottom) shows human α-synuclein expression in a subset of NeuN-positive pyramidal neurons in the hippocampus (left), co-immunoreactivity with phosphorylated α-synuclein (middle), and strong ubiquitin expression in D-Line mice (right).
Line 61 (TNWT61) transgenic Mouse Model
An excellent review discussed many features of this mouse line, concluding that it “provides a useful platform to elucidate the mechanism of early pathological mechanisms in sporadic PD and test new approaches for neuroprotection” (Chesselet et al. 2012, Neurotherapeutics 9: 297–314). This transgenic mouse model overexpresses wildtype human alpha-synuclein under the regulatory control of the murine Thy-1 promoter. The cellular expression is more widespread compared to the D-Line model, featuring high human alpha-synuclein mRNA levels in most brain areas. Line 61 (TNWT61) mice show abnormal accumulation of human alpha-synuclein protein in cortical and subcortical regions of the brain, including the substantia nigra. Immunoreactivity is present in both neuronal somata and presynaptic terminals (Rockenstein et al. 2002, J Neurosci Res 68: 568-78).
Overall expression levels are therefore higher compared to D-Line mice; however, the expression in individual neurons is moderate, avoiding common problems (e.g. cytotoxicity) associated with massive overproduction of transgenic protein when excessively strong promoters are used. The Line 61 mouse model is therefore well suited for studies that aim to reduce overall levels of human alpha-synuclein. Measurable behavioral differences to non-transgenic littermates start at about 2-4 four months of age, depending on the behavioral test (Fleming et al. 2004, J Neurosci 24: 9434-40).
Figure: Results from the challenging beam walk and the pole test indicate impaired motor function in Line 61 (TNWT61) mice compared to wildtype mice (t-test). Double immunofluorescence labeling shows strong human alpha-synuclein expression in a large number of NeuN-positive pyramidal neurons in the hippocampus (HC). Campbell-Switzer silver stains show aggregation of synuclein in somata and neuropil in the substantia nigra (SN).
A53T transgenic Mouse Model
The A53T transgenic mouse model mimics the most crucial phenotypic symptoms of alpha-synuclein aggregation and corresponding motor deficits and belongs therefore to the best PD models available. In contrast to the D-Line and TNWT61 mice, models in which all effects derive from increased levels of wildtype protein, additional effects related to the A53T mutation can be studied in this model. Point mutations in alpha-synuclein (A53T, A30P, E46K) have been implicated in human familial PD. A recent thorough investigation has shown that these mutations increase the neurotoxicity of alpha-synuclein but do not affect its physiological function as a presynaptic protein involved in vesicle release (Burré et al 2012, J Neurosci 32: 15227-42).
Consequently, this model is especially well-suited for studies on early onset PD or to test compounds that target pathophysiological effects related to the A53T mutation. The mice express human alpha-synuclein-A53T under control of the human PDGF-β promoter (Hashimoto et al. 2003, Ann NY Acad Sci 991: 171–88; Winner et al. 2008, Neurobiol Aging 29: 913-25), resulting in an age-dependent increase of alpha-synuclein positive inclusion bodies in the brain, a pathological relevant situation as described in PD. A53T mice start to develop Lewy body-like inclusions, consisting of alpha-synuclein, ubiquitin and other proteins at three months of age.
Starting at 6 months of age A53T alpha-synuclein transgenic mice show severe motor deficits as analyzed with the Beam Walk test and RotaRod test. Male and female mice are equally affected. Analysis of animals in the Two Choice Swim test up to an age of 12 months revealed no cognitive deficits. The A53T alpha-synuclein transgenic mice express the human transgene at high levels, resulting in an age-dependent increase of α-synuclein in the substantia nigra, cortex, striatum and hippocampus. In summary the A53T alpha-synuclein transgenic mouse is a suitable model for alpha-synuclein dependent Parkinson’s disease research since it illustrates major behavioral and histological hallmarks of PD.
Figure. (A) Beam Walk: Time to traverse the beam of the 3, 6, and 9 months old transgenic and non-transgenic animals (n = 12); one-way ANOVA with Dunn’s post hoc test. (B) Rotarod: Time on the rod of the 3, 6, and 9 months old transgenic and non-transgenic animals (n = 12); one-way ANOVA with Dunn’s post hoc test. *p<0.05; ***p<0.001. (C) Multichannel immuno-fluorescence labelling shows the brain region specific staining pattern of human α-synuclein (green channel), pan α-synuclein (red channel), and TH (blue channel) of a 3 months old A53T alpha-synuclein mouse. For a closer look the hippocampus , substantia nigra , thalamus , dorsal striatum  and olfactory bulb  are zoomed in. * marks artefacts.
QPS Austria offers custom tailored study designs for these models and we are flexible to customize to your special need. We are also happy to advice you and propose previously successful study designs. A typical turnaround time from agreement to the study plan and eventually to the final report is about 4 months. QPS Austria maintains its own colonies directly in its research facility, and animals of all age groups are typically available without any long delay. Non-transgenic control littermates are available as needed for proper study design. We would be happy to test your compounds in our alpha-synuclein transgenic mouse models! The most common readouts are:
- Motor function (Rotarod, Beam Walk)
- Murine, human, and phosphorylated alpha-synuclein in brain extracts or in immunohistology
- Integrity of the striatum and dopaminergic system (TH, DAT, DARPP-32, ChAT)
- Looking for something else? Please contact us!
As with all other in vivo models we are also ready to provide samples (brain tissue, CSF etc.) from these animals for analyses in your laboratory. We are happy to receive your inquiry.