Region-specific deficits in dopamine, but not norepinephrine, signaling in a novel A30P α-synuclein BAC transgenic mouse

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder classically characterized by the death of dopamine (DA) neurons in the substantia nigra pars compacta and by intracellular Lewy bodies composed largely of α-synuclein. Approximately 5-10% of PD patients have a familial form of Parkinsonism, including mutations in α-synuclein. To better understand the cell-type specific role of α-synuclein on DA neurotransmission, and the effects of the disease-associated A30P mutation, we generated and studied a novel transgenic model of PD. We expressed the A30P mutant form of human α-synuclein in a spatially-relevant manner from the 111kb SNCA genomic DNA locus on a bacterial artificial chromosome (BAC) insert on a mouse null (Snca-/-) background. The BAC transgenic mice expressed α-synuclein in tyrosine hydroxylase-positive neurons and expression of either A30P α-synuclein or wildtype α-synuclein restored the sensitivity of DA neurons to MPTP in resistant Snca-/- animals. A30P α-synuclein mice showed no Lewy body-like aggregation, and did not lose catecholamine neurons in substantia nigra or locus coeruleus. However, using cyclic voltammetry at carbon-fiber microelectrodes we identified a deficit in evoked DA release in the caudate putamen, but not in the nucleus accumbens, of SNCA-A30P Snca-/- mice but no changes to release of another catecholamine, norepinephrine (NE), in the NE-rich ventral bed nucleus of stria terminalis. SNCA-A30P Snca-/- mice had no overt behavioral impairments but exhibited a mild increase in wheel-running. In summary, this refined PD mouse model shows that A30P α-synuclein preferentially perturbs the dopaminergic system in the dorsal striatum, reflecting the region-specific change seen in PD.

Keywords: Behavior; Dopamine; Norepinephrine; Parkinson's disease; Voltammetry; α-Synuclein.

© 2013.

Figures

Fig. 1

Expression of human wild-type or A30P mutant α-synuclein in SNCA-BAC transgenic mice. A, Schematic representation of the human SNCA BAC transgene constructs carrying either wild-type or A30P mutant SNCA. B, Confirmation of the SNCA BAC transgene integrity by PCR in both SNCA lines. Human-specific PCR primers spanning each of the SNCA coding exons were used (exons 1–6). C, Location of transgene integration in SNCA-BAC lines. Fluorescent in situ hybridization (FISH) confirmed a single integration site for both transgenic lines using BAC probes in combination with chromosome painting. The SNCA-A30P transgene integration was observed near the telomere on one copy of chromosome 15; the SNCA-WT (hα-syn) transgene integration occurred near the telomere on one copy of chromosome 2. D, Expression of α-synuclein was quantified by western blot of whole brain homogenates from 3 month-old SNCA BAC mice. α-synuclein expression was quantified relative to actin levels and normalized to expression in mice expressing wildtype human α-synuclein (hα-syn). The difference in expression between A30P and hα-syn lines is not significant. n = 3, **p < 0.01. E, Immunohistochemical analysis in coronal brain sections of 3-month old animals revealed α-synuclein transgene expression in the cortex, SNpc, and VTA in hα-syn and SNCA-A30P mice recapitulating a spatial pattern of expression almost identical to that of endogenous α-synuclein protein in wild-type C57Bl6 mice. α-synuclein expression was not seen in Snca −/− KO animals.

Fig. 2

Immunohistochemical analysis of α-synuclein expression in the SNpc in SNCA-BAC transgenic mice. Immunofluorescence shows human α-synuclein protein expression in 3 month-old A30P and hα-syn mice that accurately recapitulates the pattern of endogenous α-synuclein expression in TH-positive neurons in the SNpc, seen as yellow cells in the merged frame and clearly visible in the high-magnification insets. Age-matched Snca −/− KO animals display no α-synuclein expression.

Fig. 3

Expression of wild-type or A30P α-synuclein restores susceptibility of DA neurons in Snca −/− mice to MPTP in 6-month old mice. A, TH immunoreactivity in striatum and SNpc in hα-syn, A30P, and Snca −/− KO mice 7 days following saline or acute dosing of MPTP free base. The A30P animals are similarly susceptible to MPTP lesion as hα-syn mice, as evidenced by a reduction of TH staining in the striatum and SNpc. Representative sections are shown. B, Quantitative unbiased stereological cell counts show a 30% reduction in TH + neurons in the SNpc of hα-syn and A30P animals. The Snca −/− KO animals were resistant to MPTP treatment. Results represent mean ± SEM for 3 animals per genotype and treatment *p < 0.05. C, D The level of striatal TH was diminished in hα-syn and A30P animals after exposure to MPTP as measured by western blot analysis of TH levels in striatal tissue, while TH expression remained unchanged in Snca −/− KO animals. TH band intensity was controlled for β-actin expression and normalized, within-genotype, to saline-treated controls (n = 3, *p < 0.05).

Fig. 4

Electrically evoked dopamine transients and regulation of [DA]o by firing frequency in 3-month old hα-syn, SNCA A30P and Snca −/− KO measured by FCV. A, Mean profiles of [DA]oversus time (mean ± SEM) after a single pulse (0.2 μs; arrow) in the dorsal striatum (CPu). In CPu, there is no difference in peak [DA]o transiently evoked by single pulses in hα-syn mice compared to age-matched KO littermates (p = 0.2265, n = 60 (5 animals per genotype)). B, No significant differences seen in mean peak [DA]o evoked by a single pulse (1p) in hα-syn transgenic mice compared to KO littermates in the NAc. (p = .8680, n = 18 (5 animals per genotype)). C, Mean profiles of [DA]oversus time (mean ± SEM) after a single pulse (0.2 μs; arrow) in the dorsal striatum (CPu). In CPu, peak [DA]o transiently evoked by single pulses is slightly less in A30P than age-matched KO littermates (*p < 0.05, KO, n = 96; A30P, n = 92 (9 animals per genotype)). D, No significant differences seen in mean peak [DA]o evoked by a single pulse (1p) in A30P transgenic mice compared to KO littermates in the NAc. E, Cumulative histogram of peak [DA]o of evoked DA transients in the CPu in KO vs A30P mice for individual sampling sites. F, Comparison of the rates of decay of concentration-matched DA transients suggests that DA uptake rates are not significantly different between the two genotypes (n = 5, p > 0.05). G, No differences were observed in DA content (pmol per μg of protein) in the CPu or NAc dissected from striatal slices. Data shown represent mean ± SEM from Snca KO and SNCA A30P mouse samples (n = 8–10, p > 0.05). H, Regulation of [DA]o by firing frequency in the CPu. Mean peak [DA]o during 5 pulse trains (1–100 Hz) in the CPu varied with frequency in both genotypes in control conditions, with a slightly greater frequency sensitivity observed in A30P transgenic mice at 25 Hz. Data were normalized within genotype to the 1p value (n = 3, *p < 0.05).

Fig. 5

Electrically evoked noradrenergic transients and regulation of norepinephrine (NE) signals by firing frequency in 3-month old SNCA-A30P and Snca −/− KO mice measured by FCV. A, Cyclic voltammograms from a calibration using 2 μM NE (black), and 30 pulses at 50 Hz in Snca KO (red) and A30P (blue) slices. B, Maximal NE release as a function of current amplitude. Each data point was obtained with a 30-pulse, 50 Hz stimulation (n = 6 C57Bl/6 animals). C, Maximal release as a function of stimulation frequency. Each data point was obtained with a 30-pulse stimulation, and a current amplitude of 0.65 mA (n = 5 C57Bl/6 animals). D, Maximal release as a function of pulse number. Each data point was obtained with a stimulation frequency of 50 Hz, and current amplitude of 0.65 mA (n = 6 C57/Bl6 animals). E, Mean [NE]oversus time evoked by 30 pulses at 50 Hz in vBNST of C57/Bl6 mice in control conditions, in the presence of the α2 adrenergic antagonist, idazoxan (1 μM). Idazoxan significantly increased peak [NE]o in the vBNST (n = 3 animals, **p < 0.01). F, Mean [NE]oversus time evoked by 30 pulses at 50 Hz in vBNST of C57Bl/6 mice in control conditions and in the presence of 300 nM desipramine. Desipramine significantly increased peak [NE]o in the vBNST (n = 3 animals, *p < 0.05). G, Mean [DA]oversus time evoked by 30 pulses at 50 Hz in the CPu of C57/Bl6 mice in control conditions and in the presence of α2 adrenergic antagonist, idazoxan (1 μM). Unlike in the vBNST, idazoxan produces no change in peak [DA]o in the CPu (n = 3 animals). H, Mean profiles of [NE]oversus time (mean ± SEM) after a 30 pulse, 50 Hz stimulation (arrow) in the ventral bed nucleus of the stria terminalis (BNST). In vBNST, a very slight 8% difference was observed in peak [NE]o transiently evoked by 30 pulses at 50 Hz in A30P compared to age-matched KO littermates which was not significant (p > 0.05; KO, n = 79 sites; A30P, n = 82 sites (8 animals per genotype)). I, Mean peak [NE]o ± SEM vs. frequency during 30 pulse trains (10–100 Hz) in the vBNST varied with frequency in both genotypes in control conditions. No differences in frequency sensitivity were seen in A30P transgenic mice compared to KO animals (p > 0.05; 8 animals per genotype). J, Cumulative histogram of peak [NE]o of evoked NE transients in the vBNST in KO vs A30P mice for individual sampling sites. K, Immunohistochemical analysis in coronal mouse sections confirmed α-synuclein transgene expression in the LC in SNCA-A30P mice; α-synuclein expression was not seen in Snca −/− KO animals.

Fig. 6

Immunohistochemical and western blot analysis of striata in aged (18-month old) hα-syn, A30P, and Snca −/− KO mice. A, TH immunoreactivity was unchanged at 18 months of age. Western blot analysis of striatal lysates from hα-syn, A30P, and Snca −/− KO mice show also no differences in TH expression with age. B, DAT immunoreactivity was unchanged at 18 months of age. Similarly, western blot analysis of striatal lysates from hα-syn, A30P, and Snca −/− KO mice does not demonstrate a reduction in DAT expression with age. Analysis was performed on 3 animals per genotype at each age. Representative sections are shown. Columns represent percentage change from the 6-month old hα-syn control. Results represent the mean ± SEM for three animals per genotype at each age. For western blot analysis, 10 μg protein was loaded per lane.

Fig. 7

Stereological counts of SNpc and LC in aged hα-syn, A30P, and Snca −/− KO mice. A, B No differences were seen in TH-positive cells (A) or hematoxylin-positive (B) in the SNpc at 18 months of age in A30P animals compared with hα-syn and KO animals. C, D, Similarly, no differences were observed TH cell number (C) or hematoxylin-positive cell numbers (D) in the LC at 18 months of age between the three genotypes. Results represent the mean ± SEM for 5 animals per genotype at each age, p > 0.05.

Fig. 8

Absence of synuclein pathology in 23-month old A30P transgenic animals. A diffuse cytosolic α-synuclein immunostaining was seen in neuronal cells of the anterior olfactory nucleus and granular cell layer of the olfactory bulb with antibody LB509 (A), but not by Syn-1 (C), or pSyn #64 against the phosphorylated α-syn (E). Magnification in A-F × 40, inserts show higher-power magnified image × 200.

Fig. 9

A30P mice display a very mild behavioral phenotype. A, A30P mice have similar total locomotor activity at all ages when introduced into a novel environment as compared to age-matched hα-syn and Snca −/− KO controls. Results represent the mean number of ambulations ± SEM for 10–13 mice per genotype. B, No deficits in forepaw stride length were apparent at 3 months or 10–13 months of age in A30P mice, compared to age-matched hα-syn and Snca −/− KO controls. Results represent average stride length (cm) ± SEM for 8–10 animals per genotype. C, No differences were seen in motor learning or performance on the rotarod at 18 months of age. Results represent latency to fall (sec) ± SEM for 4–10 animals per genotype. D, At 6 months of age, the hα-syn mice spent a greater percentage of their time in the open arms of the elevated plus maze as compared to age-matched A30P and Snca KO animals over the 5 min test period. All genotypes display an increased anxiety-like phenotype with age. Results represent the mean percentage of time spent in the open arms ± SEM for 10–13 mice per genotype. **p < 0.01 E, All genotypes had similar immobility times in the tail suspension test, which remains unchanged with age. Results represent mean time (s) ± SEM for 10–13 mice per genotype. F, A30P mice display increased wheel running during hours 16–17 of their light–dark cycle compared to hα-syn and Snca −/− KO mice at 12 months of age. Results represent total activity counts ± SEM for 5 animals per genotype *p < 0.05, **p < 0.01. G, A30P mice display normal circadian activity levels compared to hα-syn and KO animals at 12 months of age. Results represent total ambulations ± SEM for 8 animals per genotype. H, A30P mice do not display differences in stool frequency compared to age-matched hα-syn and Snca −/− KO controls. Results represent average stool frequency ± SEM for 5–11 animals per genotype. I, A30P mice do not display deficits in spontaneous alternation as measured by the T-maze at 3, 10–13, or 15–18 months of age when compared to age-matched hα-syn or Snca KO mice. Results represent the mean alternation ± SEM for 10–13 animals per genotype at each age.