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Review
. 2012 Apr;9(2):297-314.
doi: 10.1007/s13311-012-0104-2.

A progressive mouse model of Parkinson's disease: the Thy1-aSyn ("Line 61") mice

Marie-Francoise Chesselet  1 , Franziska Richter, Chunni Zhu, Iddo Magen, Melanie B Watson, Sudhakar R Subramaniam
Affiliations
Review

A progressive mouse model of Parkinson's disease: the Thy1-aSyn ("Line 61") mice

Marie-Francoise Chesselet et al. Neurotherapeutics. 2012 Apr.

Abstract

Identification of mutations that cause rare familial forms of Parkinson's disease (PD) and subsequent studies of genetic risk factors for sporadic PD have led to an improved understanding of the pathological mechanisms that may cause nonfamilial PD. In particular, genetic and pathological studies strongly suggest that alpha-synuclein, albeit very rarely mutated in PD patients, plays a critical role in the vast majority of individuals with the sporadic form of the disease. We have extensively characterized a mouse model over-expressing full-length, human, wild-type alpha-synuclein under the Thy-1 promoter. We have also shown that this model reproduces many features of sporadic PD, including progressive changes in dopamine release and striatal content, alpha-synuclein pathology, deficits in motor and nonmotor functions that are affected in pre-manifest and manifest phases of PD, inflammation, and biochemical and molecular changes similar to those observed in PD. Preclinical studies have already demonstrated improvement with promising new drugs in this model, which provides an opportunity to test novel neuroprotective strategies during different phases of the disorder using endpoint measures with high power to detect drug effects.

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Figures

Fig. 1
Fig. 1
Schematic diagram of Thy1-aSyn construct: over-expression of full length human wild-type alpha-synuclein under the murine Thy-1 promoter. (See Rockenstein, et al., 2002 [21] for details)
Fig. 2
Fig. 2
Brain sections (1 hemisphere) show increased immunostaining for mouse/human aSyn (mouse anti-alpha-synuclein); clone 42, 1:500 (BD Biosciences, San Jose, CA) (A) in Thy1-aSyn mice (5 months old) compared to wild-type (WT) littermates. (B) 3.4-fold increase in aSyn in the SN pars compacta of Thy1-aSyn mice (SNc; arrows in [A]) (Image J, mean + SEM; n = 5; **p < 0.01 Student’s t test)
Fig. 3
Fig. 3
Human alpha-synuclein (SNCA) is expressed in nigral dopaminergic neurons of Thy1-aSyn mice. (A) Gel electrophoresis of polymerase chain reaction (PCR) products (cDNA synthesized from amplified mRNA of 500 neurons per sample) from laser-captured microdissected tyrosine hydroxylase (TH)-positive neurons of the substantia nigra from wild-type (WT) and Thy1-aSyn transgenic (TG) mice. Human WT alpha-synuclein (SNCA 68 bp) is expressed in samples of TG mice only (positive control [+Ctrl] is cDNA from total human brain RNA). Laser captured nigral neurons used for this analysis express TH (Th, ~200 bp) and the housekeeping gene hypoxanthine guanine phosphoribosyl transferase (Hprt, ~200 bp), but not glutamic acid decarboxylase 1 (Gad1, gamma-aminobutyric acid (GABA)ergic neuron marker, ~200 bp) and glial fibrillary acidic protein (Gfap, glial cell marker, ~200 bp). Positive control for Th, Hprt, Gad1, and Gfap is cDNA from total mouse brain RNA. Negative control is a nontemplate control (water, H2O). (B) Relative expression of SNCA transcript in TH-positive neurons of the substantia nigra pars compacta (quantitative real-time PCR, n = 3 per group)
Fig. 4
Fig. 4
Alpha-synuclein protein over-expression in brain regions of Thy1-aSyn mice assessed by Western blotting using an antibody against mouse and human alpha-synuclein (mouse anti-alpha-synuclein); clone 42, 1:3000 (BD Biosciences, San Jose, CA). Left: Western blot of alpha-synuclein in the substantia nigra. Right: Alpha-synuclein expression (17 kDa) in fold change compared to wild-type (wt). Thy1-aSyn (tg) mice brain in comparison to wt. Normalized to alpha-tubulin for loading control. Substantia nigra (SN), striatum (str), thalamus (thal), cortex (cor), frontal cortex (f.cor), hippocampus (hipp), cerebellum (CB), and olfactory bulb (OB) (mean ± SEM of 3-6 mice; *p < 0.05; **p < 0.01; ***p < 0.001; 2-way repeated measures (RM) analysis of variance, main genotype effect p < 0.001, student’s t-test planned comparison for each subregion
Fig. 5
Fig. 5
Alpha-synuclein pathology in human (A, B) and mouse (C) substantia nigra neurons. (A) Lewy body (black arrow) in melanin containing nigral neuron of a Parkinson’s disease (PD) patient, hematoxylin & eosin stain. (B) Alpha-synuclein accumulated in melanin containing nigral neuron of a PD patient. (C) Proteinase K resistant alpha-synuclein aggregate in substantia nigra of a 5-months-old Thy1-aSyn mouse detected with mouse anti-alpha-synuclein; clone 42, 1:250 (BD Biosciences, San Jose, CA). Scale bar = 20 μm. (Figures [A] and [B] courtesy of Spencer Tung and Harry V. Vinters)
Fig. 6
Fig. 6
Serine 129 phosphorylated alpha-synuclein protein accumulation in brain regions of Thy1-aSyn mice (antibody is rabbit anti-phosphoser129-alpha-synuclein (Abcam, USA; at 1:500 dilution, ab59264). (A) Immunostaining in the substantia nigra of wild-type (WT) (black arrow points to dark brown stained fibers) and Thy1-aSyn mice (black arrow points to dark brown cytoplasmic and nuclear staining) at 5 months. Scale bar = 20 μm. (B) Western blot of phosphorylated alpha-synuclein in the substantia nigra. (C) Serine 129 phosphorylated alpha-synuclein protein as fold change compared to wild-type (wt). Thy1-aSyn (tg) mice brain in comparison to wt. Normalized to alpha-tubulin for loading control. Substantia nigra (SN), striatum (str), thalamus (thal), cortex (cor), frontal cortex (f.cor), hippocampus (hipp), cerebellum (CB), and olfactory bulb (OB) (mean ± SEM of 3-6 mice; *p < 0.05; **p < 0.01; ***p < 0.001; 2-way RM analysis of variance, main genotype effect p < 0.001, student's t-test planned comparison for each subregion
Fig. 7
Fig. 7
Parkinsonism-like phenotype in Thy1-aSyn mice aged 14 months. (A) Thy1-aSyn mice show a 40% loss of dopamine in the striatum compared to age matched wild-type (WT) mice (mean ± SEM; n = 8; *p < 0.05; Student’s t test). (B) Thy1-aSyn mice need longer to remove a sticker from their nose compared to wild-type. This behavioral deficit can be reversed by L-dopa (mean ± SEM; n = 6-7; **p < 0.01 compared to wild-type vehicle, Mann-Whitney U test). (See Lam, et al., 2011 [24] for details)
Fig. 8
Fig. 8
Hyperactivity at 4 to 5 months of age. Thy1-aSyn mice (A) travel more distance and (B) show higher velocity in the open field and show increased numbers of climbing in a wire cage cylinder compared to wild-type (WT) mice (mean ± SEM; *p < 0.05 Student’s t test; n = 10-11 per group)
Fig. 9
Fig. 9
Early nonmotor deficits in Thy1-aSyn (black) versus wild-type (grey). Colonic dysfunction at 4 to 5 months (n = 8-12) (mean + SEM; **p < 0.01 Student’s t test)
Fig. 10
Fig. 10
Early nonmotor deficits in Thy1-aSyn (black) versus wild-type (grey). Cognitive deficits in (A) One-trial object-place recognition test at 4 to 5 months (mean + SEM; n = 10-12; *p < 0.05 compared to wild-type; Student’s t test), (B) Y-maze at 7 to 9 months (mean + SEM; **p < 0.01; n = 10-12; Student’s t test), and (C) novel object recognition test at 4 to 5 months (mean + SEM; n = 17-18; *p < 0.01 Student’s t test).
Fig. 11
Fig. 11
Errors per step on the challenging beam. Thy1-aSyn (also known as ASO) mice make more errors compared to wild-type (WT) as early as 2 months of age. (A) Originally published (see Fleming, et al., 2004 [26] for details) (n = 7 Thy1-aSyn and n = 17 wild-type), and (B) observed in 2010 to 2011 (n = 19 at 2 and 4 months, n = 4-8 at 6 months) (mean ± SEM, **p < 0.01 compared to wild-type; ^^p < 0.01 compared to 2 months of age; 2-way RM analysis of variance). Note the high robustness of the progressive deficit before and after 6 years of breeding (more than 12 generations of mice). (C) This deficit persists and progresses to 14 months of age, when dopamine loss occurs. Female mice show a strong trend to increased errors per step at this age (mean ± SEM; n = 4-5 males; n = 7-11 females; **p < 0.01 Student’s t test)
Fig. 12
Fig. 12
Corticostriatal synapse alterations in 6 months old Thy1-aSyn mice, prior to dopamine loss. Terminals from nigral dopaminergic neurons and from cortical glutamatergic neurons project to the striatum and form synapses onto GABAergic medium spiny neurons. Increased extrasynaptic dopamine results in alteration of dopaminergic modulation of the corticostriatal synapse. Reduced spontaneous and evoked excitatory postsynaptic currents (EPSCs) indicate decrease in neurotransmitter release at the corticostriatal synapse. Increased microglia activation and tumor necrosis factor-α (TNF-alpha) release might contribute to neuronal dysfunction in the striatum. AMPAR = xxxx; D1/2R = xxxx; D2R = xxxx; DAT = xxxx; NMDAR = xxxx; VMAT = xxxx.
Fig. 13
Fig. 13
Iron accumulation and oxidative stress. (A, B) Progressive iron accumulation in the substantia negra (SN) of Thy1-aSyn mice (A) quantification of iron stain at 5, 12, and 22 months of age shown in (B) perl stain, total iron. Scale bar = 30 μm. Optical density (OD) in the SN (Image J) shows 2.6-fold increase of iron accumulation between 5 m and 12 m, and further 3.1-fold increase between 12 m and 22 m (**p < 0.01 compared to 5 m, ##p < 0.01 compared to 12 m; one-way analysis of variance followed by Fisher’s Least Significant Difference (LSD), n = 5 each). (C) Increased staining for DNA damage marker (anti-phospho-Histone H2A.X [Ser139] Millipore, green) in nigral TH (rabbit anti-TH antibody; 1:500, Millipore, red) positive neurons of Thy1-aSyn mice, but not wild-type (WT) at 5 months of age (40 μm brain sections, 100 x /1.6-fold zoom confocal image of 1 focal plane). Scale bar = 25 μm. TH = tyrosine hydroxylase
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