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Identification of NUB1 as a suppressor of mutant Huntingtin toxicity via enhanced protein clearance

Nature Neuroscience volume 16, pages 562–570 (2013)Cite this article

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Abstract

Huntington's disease is caused by expanded CAG repeats in HTT, conferring toxic gain of function on mutant HTT (mHTT) protein. Reducing mHTT amounts is postulated as a strategy for therapeutic intervention. We conducted genome-wide RNA interference screens for genes modifying mHTT abundance and identified 13 hits. We tested 10 in vivo in a Drosophila melanogaster Huntington's disease model, and 6 exhibited activity consistent with the in vitro screening results. Among these, negative regulator of ubiquitin-like protein 1 (NUB1) overexpression lowered mHTT in neuronal models and rescued mHTT-induced death. NUB1 reduces mHTT amounts by enhancing polyubiquitination and proteasomal degradation of mHTT protein. The process requires CUL3 and the ubiquitin-like protein NEDD8 necessary for CUL3 activation. As a potential approach to modulating NUB1 for treatment, interferon-β lowered mHTT and rescued neuronal toxicity through induction of NUB1. Thus, we have identified genes modifying endogenous mHTT using high-throughput screening and demonstrate NUB1 as an exemplar entry point for therapeutic intervention of Huntington's disease.

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Figure 1: Primary genome-wide screens for soluble human mHTT clearance in Drosophila S2R cells.
Figure 2: Secondary validation screens in human Huntington's disease fibroblasts for genetic modifiers of endogenous mHTT protein abundance.
Figure 3: In vivo validation of the hits from the cell culture screen using Drosophila.
Figure 4: NUB1 suppresses mHTT-induced toxicity in Drosophila.
Figure 5: Overexpression of NUB1 reduces mHTT protein amounts and rescues from its toxicity.
Figure 6: NUB1 overexpression reduces mHTT and aggregation in hESC- and iPSC-derived neurons.
Figure 7: mHTT reduction by NUB1 occurs through enhanced ubiquitination and requires CUL3 ubiquitin E3 ligase.
Figure 8: Interferon-β reduces mHTT and rescues toxicity via NUB1.

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Acknowledgements

The authors wish to thank D. Curtis for discussions and suggestions during the project, X. Shi for performing the control screening on SMN level changes, C. Mazur for assistance with cloning and sample processing, P. Manos and C. Littlefield for assistance with the hESC and human iPSC experiments, and E. Kim for assistance with the fly experiments. I.A. was funded by a John J. Wasmuth postdoctoral fellowship from the Hereditary Disease Foundation. A.V. was funded by the Hereditary Disease Foundation. M.D. was funded by US National Institutes of Health grant NS074381.

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Authors and Affiliations

  1. School of Life Sciences, Fudan University, Shanghai, China

    Boxun Lu

  2. Developmental & Molecular Pathways, Novartis Institutes for BioMedical Research, Inc., Cambridge, Massachusetts, USA

    Boxun Lu, Qiong Wang, Frada Berenshteyn, Haidi Yang, Salah Ichcho, Arnaud Lacoste, Marc Hild & James Palacino

  3. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston Texas, USA

    Ismael Al-Ramahi, Tatiana Gallego-Flores & Juan Botas

  4. Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Houston, Texas, USA

    Ismael Al-Ramahi, Tatiana Gallego-Flores & Juan Botas

  5. Department of Neurology, Harvard Medical School, Boston, Massachusetts, USA

    Antonio Valencia & Marian DiFiglia

  6. MassGeneral Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Boston, Massachusetts, USA

    Antonio Valencia & Marian DiFiglia

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Contributions

B.L. and J.P. initiated the project and designed the experiments; B.L. and H.Y. carried out the primary S2 screen (Fig. 1); B.L. and Q.W. carried out the validation screen in human patient fibroblasts (Fig. 2); B.L., M.H. and J.P. analyzed the screen data; I.A.-R., T.G.-F. and J.B. performed in vivo experiments in the fly (Figs. 3 and 4); A.V. and M.D. did the rescue experiments in neurons (Figs. 5d and 8e); S.I. did the quantitative PCR for Nedd8 and FAT10 in HN10 cells (Supplementary Fig. 7); A.L. generated the human iPSC lines; F.B. and A.L. differentiated hESCs and iPSCs into neurons; B.L. performed all other experiments; B.L. and J.P. wrote the manuscript.

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Correspondence to Boxun Lu or James Palacino.

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Lu, B., Al-Ramahi, I., Valencia, A. et al. Identification of NUB1 as a suppressor of mutant Huntingtin toxicity via enhanced protein clearance. Nat Neurosci 16, 562–570 (2013). https://doi.org/10.1038/nn.3367

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