A Molecular Tweezer Ameliorates Motor Deficits in Mice Overexpressing α-Synuclein - PubMed (original) (raw)

. 2017 Oct;14(4):1107-1119.

doi: 10.1007/s13311-017-0544-9.

Sudhakar R Subramaniam 1, Iddo Magen 1, Patrick Lee 1, Jane Hayes 1, Aida Attar 1 2, Chunni Zhu 1, Nicholas R Franich 1, Nicholas Bove 1, Krystal De La Rosa 1, Jacky Kwong 1, Frank-Gerrit Klärner 3, Thomas Schrader 3, Marie-Françoise Chesselet 4 5 6 7, Gal Bitan 8 9 10

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A Molecular Tweezer Ameliorates Motor Deficits in Mice Overexpressing α-Synuclein

Franziska Richter et al. Neurotherapeutics. 2017 Oct.

Abstract

Aberrant accumulation and self-assembly of α-synuclein are tightly linked to several neurodegenerative diseases called synucleinopathies, including idiopathic Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Deposition of fibrillar α-synuclein as insoluble inclusions in affected brain cells is a pathological hallmark of synucleinopathies. However, water-soluble α-synuclein oligomers may be the actual culprits causing neuronal dysfunction and degeneration in synucleinopathies. Accordingly, therapeutic approaches targeting the toxic α-synuclein assemblies are attractive for these incurable disorders. The "molecular tweezer" CLR01 selectively remodels abnormal protein self-assembly through reversible binding to Lys residues. Here, we treated young male mice overexpressing human wild-type α-synuclein under control of the Thy-1 promoter (Thy1-aSyn mice) with CLR01 and examined motor behavior and α-synuclein in the brain. Intracerebroventricular administration of CLR01 for 28 days to the mice improved motor dysfunction in the challenging beam test and caused a significant decrease of buffer-soluble α-synuclein in the striatum. Proteinase-K-resistant, insoluble α-synuclein deposits remained unchanged in the substantia nigra, whereas levels of diffuse cytoplasmic α-synuclein in dopaminergic neurons increased in mice receiving CLR01 compared with vehicle. More moderate improvement of motor deficits was also achieved by subcutaneous administration of CLR01, in 2/5 trials of the challenging beam test and in the pole test, which requires balance and coordination. The data support further development of molecular tweezers as therapeutic agents for synucleinopathies.

Keywords: Parkinson’s disease; drug testing; motor behavior; mouse model; synucleinopathies; α-synuclein aggregation.

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Figures

Fig. 1

Fig. 1

Intracerebroventricular CLR01 administration improves performance in the challenging-beam test. (a) Mean + SEM errors per step on the challenging-beam before (pre-) and after (post-) treatment [**p < 0.01 vs the respective wild-type (WT) groups; ## p < 0.01 vs the vehicle-treated and 1-μM-treated groups; ^ p < 0.01 vs vehicle-treated group before and after treatment, 2-way analysis of variance (ANOVA)]. (b) Errors per step (mean ± SEM) in individual trials for mice treated with 1-μM CLR01. (c) Errors per step in individual trials for mice treated with 10-μM CLR01 [**p < 0.01 vs the respective WT mice; # p < 0.05 and ## p < 0.01 vs the vehicle-treated mice, 2-way ANOVA (genotype × treatment) for each trial]. n (WT/vehicle) = 16, n (WT/1 μM) = 15, n (WT/10 μM) = 17, n (Thy1-aSyn/vehicle) = 13, n (Thy1-aSyn/1 μM) = 12, n (Thy1-aSyn/10 μM) = 11

Fig. 2

Fig. 2

The effect of CLR01 on brain α-synuclein. Brains from wild-type (WT) and Thy1-aSyn mice were extracted into buffer-soluble, detergent-soluble, and guanidine-soluble fractions. Each fraction was analyzed by Western blotting with an anti-α-synuclein antibody and bands corresponding to α-synuclein were quantified densitometrically and normalized to β-actin, which was used as a loading control. (a, b) Representative blots of buffer-soluble and detergent-soluble fractions. (c–h) Quantification of normalized α-synuclein. The data in panels (c–h) are presented as mean + SEM. Grubb’s test was performed to remove outliers and 1 outlier was removed from the striatal detergent-soluble fraction of Thy1-aSyn/1 μM (n = 6) group. The difference between the WT and transgenic mice was statistically significant (p < 0.001) in all cases (#p < 0.05 vs vehicle-treated Thy1-aSyn mice, 2-way ANOVA). n (WT/vehicle) = 5, n (WT/1 μM) = 3, n (WT/10 μM) = 7, n (Thy1-aSyn/vehicle) = 6, n (Thy1-aSyn/1 μM) = 6, n (Thy1-aSyn/10 μM) = 5

Fig. 3

Fig. 3

CLR01 effects on α-synuclein in tyrosine hydroxylase (TH) neurons of the substantia nigra (SN). (a) 1-μM and (b) 10-μM CLR01 decreased the percentage of neurons in the left SN, ipsilateral to the infusion site, with low α-synuclein immunofluorescent (IF) intensity while increasing the percentage of neurons with high α-synuclein IF intensity (bootstrapping analysis displayed as means + 95% confidence intervals, *p < 0.05, n = 5 per group)

Fig. 4

Fig. 4

CLR01 improves motor deficits following subcutaneous administration. (a) Mean ± SEM of 5 trials and single trials of errors per step on the challenging beam test [**p < 0.01 vs the respective wild-type (WT) mice; # p < 0.05 vs the vehicle-treated mice, 2-way analysis of variance genotype × treatment for each trial, Bonferroni t test, n = 16/group]. (b) Time to turn in the pole test (mean and single trials) and (c) time to descend in the pole test (mean and single trials) (**p < 0.01 vs the respective WT mice; # p < 0.05 and ## p < 0.01 vs the vehicle-treated mice, Mann–Whitney U test, n = 16/group)

Fig. 5

Fig. 5

CLR01 persists in the brain 8 h postadministration. Wild-type or Thy1-aSyn mice were administered 2 μCi of 3H-CLR01 spiked into unlabeled CLR01 and the radioactivity in the brain was measured at 1, 3, and 8 h (n = 3 mice per time point) using scintillation counting (mean + SEM).

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