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

  • Franziska Richter
  • Sudhakar R. Subramaniam
  • Iddo Magen
  • Patrick Lee
  • Jane Hayes
  • Aida Attar
  • Chunni Zhu
  • Nicholas R. Franich
  • Nicholas Bove
  • Krystal De La Rosa
  • Jacky Kwong
  • Frank-Gerrit Klärner
  • Thomas Schrader
  • Marie-Françoise Chesselet
  • Gal Bitan
Original Article

DOI: 10.1007/s13311-017-0544-9

Cite this article as:
Richter, F., Subramaniam, S.R., Magen, I. et al. Neurotherapeutics (2017). doi:10.1007/s13311-017-0544-9

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.

Key Words

Parkinson’s disease synucleinopathies mouse model motor behavior α-synuclein aggregation drug testing 

Supplementary material

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Fig. S1

Continuous weight gain under CLR01 intracerebroventricular (ICV) treatment. Body weight (g) after 28 days of ICV infusion with vehicle or CLR01 at 1 μM or 10 μM (n = 12–18/group). Mean ± SEM (GIF 26 kb)

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Fig. S2

CLR01 intracerebroventricular (ICV) treatment does not affect performance in the pole test. Pole test (time to turn, mean + SEM) in Thy1-aSyn and wild-type (WT) mice administered vehicle (n = 18 WT, n = 14 Thy1-aSyn) or CLR01 at 1 μM (n = 17 WT, n = 13 Thy1-aSyn) or 10 μM (n = 17 WT, n = 12 Thy1-aSyn) (a) preadministration and (b) postadministration; **p < 0.01 vs the corresponding WT mice, no significant differences between treatment groups in each genotype, or between pre -and postadministration (Mann–Whitney U test) (GIF 46 kb)

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Fig. S3

CLR01 intracerebroventricular (ICV) treatment does not affect proteinase-K-resistant α-synuclein aggregates. Number of (a, b) aggregates (n/area, area in μm2 × 100) and (c, d) percent surface area (% of area in μm2) occupied by these aggregates in the (a, c) left and (b, d) right substantia nigra in Thy1-aSyn mice treated with vehicle (saline, n = 7) or CLR01 at 1 μM or 10 μM intracerebroventricularly (n = 6 each); mean + SEM, 1-way ANOVA for each measure revealed no differences (GIF 92 kb)

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Fig. S4

CLR01 does not affect the α-synuclein oligomer-size distribution in the striatum. (a) Native polyacrylamide gel electrophoresis (PAGE)/Western blot analysis using an anti-α-synuclein antibody shows bands corresponding to a monomer, a small oligomer with an apparent mobility between dimer and trimer, and a high-molecular-weight (HMW) smear. The individual lanes shown for the vehicle- and CLR01-treated mice represent individual animals. Recombinant α-synuclein cross-linked using photo-induced cross-linking of unmodified proteins [47] is shown as a reference. (b, c) Densitometric analysis (mean + SEM) of the 3 bands observed in the native PAGE/Western blots of the buffer-soluble fraction extracted from the (b) substania nigra and (c) striatum of Thy1-aSyn mice (n = 6 each). GAPDH = glyceraldehyde 3-phosphate dehydrogenase (GIF 71 kb)

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Fig. S5

CLR01 subcutaneous treatment does not affect performance in the cylinder test. Mean + SEM of forelimb steps and hindlimb steps over 3 min in the cylinder test in Thy1-aSyn mice vs the respective wild-type mice, **p < 0.01, 2-way analysis of variance, Bonferroni t test (n = 16 for each group) (GIF 60 kb)

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Funding information

Funder NameGrant NumberFunding Note
National Institute of Neurological Disorders and Stroke
  • NS38367
  • ES016732
RJG Foundation
  • 20095024
Michael J. Fox Foundation for Parkinson's Research
  • RRIA Award
American Parkinson Disease Association
    Team Parkinson/Parkinson Alliance

      Copyright information

      © The American Society for Experimental NeuroTherapeutics, Inc. 2017

      Authors and Affiliations

      • Franziska Richter
        • 1
      • 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
        • 1
        • 2
        • 4
        • 5
      • Gal Bitan
        • 1
        • 2
        • 5
      1. 1.Department of Neurology, David Geffen School of MedicineUniversity of California, Los AngelesLos AngelesUSA
      2. 2.Brain Research InstituteUniversity of California, Los AngelesLos AngelesUSA
      3. 3.Institute of Organic ChemistryUniversity of Duisburg-EssenEssenGermany
      4. 4.Department of Neurobiology, David Geffen School of MedicineUniversity of California, Los AngelesLos AngelesUSA
      5. 5.Molecular Biology InstituteUniversity of California, Los AngelesLos AngelesUSA

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