A53Tα-syn overexpression causes subtle deficits in DA release in response to high versus low frequency stimulation, when nAChRs are inhibited.

<p>(<b>a</b>) Profiles of mean [DA]_o± SEM versus time, evoked by 4-pulse stimulation (1–100 Hz), with intact nAChR activity (upper), and with nAChR inhibition (DHβE, 1 µM) (lower)(6–14 months only shown). (<b>b</b>) Mean peak [DA]_o±SEM evoked by 4-pulse stimulation (1–100 Hz), normalised to single pulse release, with nAChR activity (upper) and with nAChR inhibition (in DHβE, 1 µM)(lower). With intact nAChR activity, there was no significant difference in frequency response between genotypes at 6–14 months (<i>n = 9–15</i>). At 18–24 months, normalised peak [DA]_o evoked by 10 Hz stimulation was slightly greater in line B versus WT (2-way ANOVA, <i>post hoc</i> Bonferroni t tests, <i>p<0.05</i>; <i>n = 9–15</i>). With nAChR inhibition, in DHβE, at 6–14 months, normalised peak [DA]_o evoked by 100 Hz stimulation was significantly lower in line B than WT (2-way ANOVA, <i>post hoc</i> Bonferroni t test, <i>p<0.001</i>; <i>n = 9–15</i>) and, at 18–24 months, normalised peak [DA]_o evoked by 100 Hz stimulation was significantly lower in both A53Tα-syn overexpressing lines compared to WT (2-way ANOVA, <i>post hoc</i> Bonferroni t test, <i>p<0.05</i>; <i>n = 9–15</i>).</p

A53Tα-syn overexpressors show enhanced recovery of DA release following prolonged stimulation.

<p>(<b>a</b>) Mean peak [DA]_o± SEM evoked during a prolonged 10 Hz train (60 s), normalised to mean peak [DA]_o evoked by a single pulse. There was no significant difference in normalised peak [DA]_o between genotypes (<i>n = 8–10</i>). (<b>b,c</b>) Mean peak [DA]_o± SEM evoked by a single stimulus pulse, against time after the end of initial prolonged stimulation. Values were normalised to (<b>b</b>) peak [DA]_o evoked by initial 10 Hz train or to (<b>c</b>) maximum [DA]_o evoked by subsequent single pulses. Curve fits are two phase exponential associations, <i>R²>0.80</i>. Both sets of curves are significantly different between genotypes. Inset is the expanded initial 5 minutes following the end of prolonged stimulation, showing the same curve fits as main graphs.</p

DA re-release following a single pulse stimulation is unchanged in A53Tα-syn overexpressors.

<p>(<b>a</b>) Profiles of mean [DA]_o± SEM versus time, evoked by 2 single pulse stimulations, separated by 4 s. There was no significant difference in normalised [DA]_o evoked by a second pulse between genotypes, at either age, in either region (CPu only shown)(<i>n = 15–40</i>). (<b>b</b>) Mean peak [DA]_o±SEM evoked by second stimulus pulse, normalised to peak [DA]_o evoked by first stimulus pulse.</p

The paternal and maternal transmission of the 42 CAG repeat is stable.

<p>(A) A CAG-repeat flanking PCR with one FAM-labelled primer was performed. As expected, wild-type (WT) animals showed one product at 94 bp, whereas heterozygotes (CAG1/CAG42) showed an additional larger product. In homozygous mutant mice (CAG42) only the larger product was detected. c: negative control. (B) The exact PCR product size was confirmed by fragment length analysis. The peak in the WT sample represents the 94 bp product, while the 217 bp peak appeared in CAG42 animals. Both peaks were detectable in CAG1/CAG42 mice.</p

Normal and expanded insoluble ATXN2 drives PABPC1 into insolubility.

<p>(A) Overexpression of CAG22-ATXN2 and CAG74-ATXN2 led to significantly decreased <i>PABPC1</i> transcript levels. (B) Overexpressed Q22-ATXN2 or Q74-ATXN2 reduced endogenous soluble PABPC1 levels, whereas endogenous insoluble PABPC1 levels were significantly increased in the presence of insoluble ATXN2 (n = 3).</p

Soluble ATXN2 protein levels are reduced and PABPC1 levels change.

<p>In cerebellar tissue, a trend in ATXN2 reduction was observed at 6 weeks and 6 months age, with significance in ATXN2 reduction being reached at 18 months of age. Also, a significant PABPC1 reduction was apparent in 6 weeks and 18 months old CAG42 mice in the cerebellum. In the cortex, a reduction of ATXN2 levels was observed in 6 weeks old animals as a trend and with significance from 6 months onwards. An elevation of PABPC1 levels became significant at 18 months of age (n = 10–12 mice/genotype/tissue).</p

Elevated FBXW8 diminishes specifically insoluble expanded ATXN2.

<p>(A) The protein levels of FBXW8 were significantly upregulated in the cerebellum of 18 months old CAG42 mice compared to wild-type (n = 9–11 mice/genotype). (B) Soluble levels of Q22-ATXN2 were unchanged after FBXW8 overexpression, while for Q74-ATXN2 a slight reduction was apparent. With respect to insoluble levels, Q22-ATXN2 was again not influenced by FBXW8 overexpression, while the Q74-ATXN2 reduction became significant. FBXW8 levels were significantly increased by its overexpression both in the soluble and insoluble fraction (n = 7).</p

Differentially regulated mRNAs in CAG42 mice at the age of 18 months.

<p>Genes with significant dysregulation and consistency between more than one oligonucleotide spot are shown (n = 4 mice/genotype).</p

Increased sequestration of PABPC1 by insoluble Q42-ATXN2 with age.

<p>(A) In the cerebellum, a progressive insolubility of Q42-ATXN2 from 6 to 12 and 24 months was detectable. The solubility of PABPC1 decreased from 6 to 24 months of age. Insoluble Q42-ATXN2 levels did not change in the cortex, however, the insolubility of PABPC1 increased over time in CAG42 mice. Insoluble PABPC1 levels in WT mice remained stably low in both tissues. (n = 3 mice/genotype/tissue). (B) In the soluble fraction, both wild-type and expanded ATXN2 were able to co-immunoprecipitate PABPC1, with slightly more PABPC1 being pulled down in CAG42 mice.</p

<i>Atxn2</i> and <i>Pabpc1</i> mRNA levels are elevated in specific ages and areas.

<p>In the cerebellum an <i>Atxn2</i> mRNA elevation was not detectable until 6 months, whereas the <i>Pabpc1</i> transcript levels remained unchanged. In the cortex an <i>Atxn2</i> mRNA elevation was already detected by 6 weeks and <i>Pabpc1</i> mRNA was upregulated by 6 months (n = 9–16 animals/genotype/tissue).</p