Anle138b modulates α-synuclein oligomerization and prevents motor decline and neurodegeneration in a mouse model of multiple system atrophy

Antonio Heras-Garvin, Daniel Weckbecker, Sergey Ryazanov, Andrei Leonov, Christian Griesinger, Armin Giese, Gregor K Wenning, Nadia Stefanova, Antonio Heras-Garvin, Daniel Weckbecker, Sergey Ryazanov, Andrei Leonov, Christian Griesinger, Armin Giese, Gregor K Wenning, Nadia Stefanova

Abstract

Background: MSA is a fatal neurodegenerative disease characterized by autonomic failure and severe motor impairment. Its main pathological hallmark is the accumulation of α-synuclein in oligodendrocytes, leading to glial and neuronal dysfunction and neurodegeneration. These features are recapitulated in the PLP-hαSyn mouse model expressing human α-synuclein in oligodendrocytes. At present, there is no effective disease-modifying therapy. Previous experiments have shown that the aggregation inhibitor, anle138b, reduces neurodegeneration and behavioral deficits in mouse models of other proteinopathies.

Objectives: To test the therapeutic potential of anle138b in a mouse model of MSA.

Methods: Two-month-old PLP-hαSyn mice were fed over a period of 4 months with pellets containing anle138b at two different doses (0.6 and 2 g/kg) and compared to healthy controls and PLP-hαSyn mice fed with placebo pellets. At the end of the treatment, behavioral and histological analyses were performed.

Results: We observed a reversal of motor function to healthy control levels when PLP-hαSyn mice were treated with both doses of anle138b. Histological and molecular analyses showed a significant reduction in α-synuclein oligomers and glial cytoplasmic inclusions in animals fed with anle138b compared to nontreated mice. These animals also present preservation of dopaminergic neurons and reduction in microglial activation in SN correlating with the α-synuclein reduction observed.

Conclusions: Anle138b reduces α-synuclein accumulation in PLP-hαSyn mice, leading to neuroprotection, reduction of microglial activation, and preservation of motor function supporting the use of anle138b in a future clinical trial for MSA. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Keywords: anle138b; movement disorders; multiple system atrophy; neurodegeneration; α-synuclein.

© 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Figures

Figure 1
Figure 1
Anle138b treatment prevents motor impairment in PLP‐hαSyn mice. Schematic representation of the beam challenge test used for behavioral analysis. The number of slips when the animals traverse the beam were counted and normalized per the number of steps. n = 8 to 10 per experimental group. Error bars indicate SEM. ANOVA, slips per step/genotype: **P < 0.01; Slips per step/treatment: #P < 0.05; ###P < 0.001 (Bonferroni's test). WT, wild‐type healthy control animals; TG, PLP‐hαSyn mice feed with placebo food pellets; TG + 0.6, PLP‐hαSyn mice feed with pellets containing anle138b at 0.6 g/kg of food; TG + 2, PLP‐hαSyn mice feed with pellets containing anle138b at 2 g/kg of food. [Color figure can be viewed at wileyonlinelibrary.com]
Figure 2
Figure 2
Anle138b prevents dopaminergic neuronal loss in the SNc of PLP‐hαSyn mice. (A) Representative images of SN sections stained against TH. Scale bar, 400 μm. (B) Stereological counting of the total number of dopaminergic (TH+) neurons in the entire SNc in the different groups. n = 4 to 5 per experimental group. Error bars indicate SEM. ANOVA, TH+ neurons/genotype: ****P < 0.0001; TH+ neurons/treatment: ###P < 0.001 (Bonferroni's test). (C), Correlation analysis of number of TH+ neurons in the SNc and number of slips per step. P = 0.0021; R2 = 0.4349. [Color figure can be viewed at wileyonlinelibrary.com]
Figure 3
Figure 3
Anle138b reduces α‐syn accumulation in PLP‐hαSyn mice (A) Representative images of striatal sections stained with the antibody 15G7 against human α‐syn. Red arrows indicate individual GCI. Scale bar, 25 μm. (B,C) GCI density in SNc and STR (striatum) of PLP‐hαSyn mice determined by stereological counting of brain sections stained with 15G7 and expressed in GCI/mm2. n = 8 to 10 per experimental group. Error bars indicate SEM. ANOVA, GCI density/treatment: ##P < 0.01; ###P < 0.001 (Bonferroni's test). (D) Correlation analysis between the density of GCIs in the SNc and the number of TH+ neurons in the same region. P = 0.0041; R2 = 0.5415. (E) Correlation analysis of density of GCIs in the SNc and number of slips per step. P = 0.0001; R2 = 0.4546. (F) Representative blots of midbrain samples obtained after sucrose‐gradient centrifugation. Total α‐syn (upper panel) and phosphorylated α‐syn (lower panel) levels were analyzed in the different fractions. (G) Quantification of total α‐syn (upper panel) and phosphorylated α‐syn (lower panel) levels in the oligomeric fractions. n = 4 per experiental group. Error bars indicate SEM. Groups were compared by unpaired two‐tailed t test. *P < 0.05. [Color figure can be viewed at wileyonlinelibrary.com]
Figure 4
Figure 4
Anle138b reduces microglial activation in PLP‐hαSyn mice. (A) Representative immunofluorescence images of SN sections stained against CD68. Scale bar, 150 μm. (B) CD68‐positive (CD68+) area in the different groups, determined by ImageJ analysis (National Institutes of Health, Bethesda, MD) and expressed as the % of the total area of the SN. n = 6 to 9 per experimental group. Error bars indicate SEM. ANOVA, CD68+ area/genotype: ****P < 0.0001; CD68+ area/treatment: ##P < 0.01; ####P < 0.0001 (Bonferroni's test). (C) Correlation analysis of CD68+ area in the SN and density of GCIs (15G7) in the same brain region. P = 0.0017; R2 = 0.4288. (D) Correlation analysis between the CD68+ area in the SN and the number of TH+ neurons in the SNc. P = 0.0027; R2 = 0.4862. (E) Correlation analysis of CD68+ area in the SN and number of slips per step. P = 0.0033; R2 = 0.2689. [Color figure can be viewed at wileyonlinelibrary.com]
Figure 5
Figure 5
Pathophysiological features of MSA and potential therapeutic effect of anle138b. (A) Schematic overview of the CNS in healthy conditions. (B) During MSA pathogenesis α‐syn accumulates in the cytoplasm of oligodendrocytes inducing oligodendroglial dysfunction. Toxic oligomeric species of α‐syn are also formed and spread through the brain parenchyma triggering microglial activation and neuroinflammation. All these events lead finally to demyelination and neurodegeneration. (C) Treatment with anle138b would reduce the formation of GCIs and other toxic α‐syn species and would attenuate neuroinflammation, demyelination, and neurodegeneration in MSA patients. [Color figure can be viewed at wileyonlinelibrary.com]

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