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Chronic Treatment with 17-DMAG Improves Balance and Coordination in A New Mouse Model of Machado-Joseph Disease

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Neurotherapeutics

Abstract

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disease currently with no treatment. We describe a novel mouse model of MJD which expresses mutant human ataxin-3 at near endogenous levels and manifests MJD-like motor symptoms that appear gradually and progress over time. CMVMJD135 mice show ataxin-3 intranuclear inclusions in the CNS and neurodegenerative changes in key disease regions, such as the pontine and dentate nuclei. Hsp90 inhibition has shown promising outcomes in some neurodegenerative diseases, but nothing is known about its effects in MJD. Chronic treatment of CMVMJD mice with Hsp90 inhibitor 17-DMAG resulted in a delay in the progression of their motor coordination deficits and, at 22 and 24 weeks of age, was able to rescue the uncoordination phenotype to wild-type levels; in parallel, a reduction in neuropathology was observed in treated animals. We observed limited induction of heat-shock proteins with treatment, but found evidence that 17-DMAG may be acting through autophagy, as LC3-II (both at mRNA and protein levels) and beclin-1 were induced in the brain of treated animals. This resulted in decreased levels of the mutant ataxin-3 and reduced intranuclear aggregation of this protein. Our data validate this novel mouse model as a relevant tool for the study of MJD pathogenesis and for pre-clinical studies, and show that Hsp90 inhibition is a promising therapeutic strategy for MJD.

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Acknowledgements

We would like to thank to Dr. Henry Paulson for providing the anti-ataxin-3 serum, Dr. Mónica Sousa for the pCMV vector and to Eng. Lucília Goreti Pinto, Luís Martins, Miguel Carneiro and Celina Barros for technical assistance. This work was supported by Fundação para a Ciência e Tecnologia through the projects FEDER/FCT, POCI/SAU-MMO/60412/2004 and PTDC/SAU-GMG/64076/2006. This work was supported by Fundação para a Ciência e Tecnologia through fellowships SFRH/BPD/91562/2012 to A.S-F., SFRH/BD/78388/2011 to S.D-S., SFRH/BD/51059/2010 to A.N-C., and SFRH/BPD/79469/2011 to A.T-C..

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Authors and Affiliations

  1. Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal

    Anabela Silva-Fernandes, Sara Duarte-Silva, Andreia Neves-Carvalho, Marina Amorim, Carina Soares-Cunha, Andreia Teixeira-Castro & Patrícia Maciel

  2. ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal

    Anabela Silva-Fernandes, Sara Duarte-Silva, Andreia Neves-Carvalho, Marina Amorim, Carina Soares-Cunha, Andreia Teixeira-Castro & Patrícia Maciel

  3. ICBAS - Abel Salazar Biomedical Sciences Institute, University of Porto, Porto, Portugal

    Pedro Oliveira

  4. Dept. of Neurodegeneration, H. Lundbeck A/S, Valby, Denmark

    Kenneth Thirstrup

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  1. Anabela Silva-Fernandes
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  2. Sara Duarte-Silva
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  3. Andreia Neves-Carvalho
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Correspondence to Patrícia Maciel.

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Anabela Silva-Fernandes and Sara Duarte-Silva contributed equally to this work

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Supplementary Fig. 1

Intergenerational instability of the CAG repeat in MJD mice. (A) Representative Genescan tract diagrams showing the variation of the CAG repeat tract in maternal and paternal meioses of CMVMJD135 mice. (B) Differential pattern of CAG repeat variation in CMVMJD135 male (n = 42) and female meioses (n = 24). Values are presented as frequency (%). (JPEG 50 kb)

High resolution image (TIFF 9127 kb)

Supplementary Fig. 2

Evaluation of the progression of the disease at 40 weeks of age in the SHIRPA protocol. Spatial cognitive performance in CMVMJD135 mice at 10 weeks of age. (A) Exploratory behavior given by the number of rears was dramatically decreased in CMVMJD135 mice as well as the spontaneous locomotor activity (B). In addition, at 40 weeks of age animals demonstrate severe tremors (C) and hindlimb tonus resistance decrease (n = 7–10) (D). (E) Morris water maze test (MWM) was performed in wt (n = 7) and CMVMJD135 mice (n = 20) in five consecutive days and the time and distance travelled was recorded. (F) To exclude a motor influence in this spatial memory task the open-field test was performed one week after the MWM test and no differences were found between controls and transgenic animals indicating that the cognitive function is preserved in this model, as in MJD patients. MWM testing at later ages was not possible due to motor impairments. *p < 0.05, **p < 0.01, ***p < 0.001. (JPEG 45 kb)

High resolution image (TIFF 16724 kb)

Supplementary Fig. 3

Neuropathology of substantia nigra in CMVMJD135 mice. Anti-GFAP immunohistochemistry in the substantia nigra of wt (A) and CMVMJD135 (B) mice (n = 3–4). Scale bar: 50 μm. Cresyl violet staining of the susbtantia nigra in wt (C) and transgenic mice (D). Scale bar: 20 μm. (JPEG 94 kb)

High resolution image (TIFF 29877 kb)

Supplementary Fig. 4

One-week of 17-DMAG treatment increases Hsp70 protein levels in the muscle (A) and in the brain (brainstem) (B) in a dose-dependent manner. Male wt mice were injected three times during a week with different concentrations of 17-DMAG: 5, 10 and 25 mg/kg (n = 4 for each dosage). 12 hours after the last injection mice were sacrificed and Hsp70 proteins levels were measured. In brain tissue, as in muscle, there was a dose-dependent response in Hsp70 induction by 17-DMAG, although the increase in expression was only statistically significant at 25 mg/kg (p < 0.05) which we selected for the therapeutic study. With systemic administration of this dosage, the concentration of 17-DMAG in the brain 1 hour after treatment was 42 ng/g (drug quantity/brain weight) with a plasma/brain ratio of 0,2. *p < 0.05, **p < 0.01, ***p < 0.001. (JPEG 27 kb)

High resolution image (TIFF 5885 kb)

Supplementary Fig. 5

Decreased neuropathology in CMVMJD135 mice treated with 17-DMAG. Comparative sections of pontine nuclei of male CMVMJD135 mice treated with vehicle (A and C) or 17-DMAG (B and D) at 16 weeks of age (A and B) and 30 weeks of age (C and D) stained with cresyl violet (n = 3 for each group); 17-DMAG-treated mice present a decrease in the number of pyknotic cells in the pontine nuclei at both ages. Scale bar: 20 μm. (JPEG 69 kb)

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Supplementary Table

Sample size calculations for each behavioral test assuming a power of 0.8 and a significance level of 0.05. (PDF 15 kb)

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Silva-Fernandes, A., Duarte-Silva, S., Neves-Carvalho, A. et al. Chronic Treatment with 17-DMAG Improves Balance and Coordination in A New Mouse Model of Machado-Joseph Disease. Neurotherapeutics 11, 433–449 (2014). https://doi.org/10.1007/s13311-013-0255-9

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