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Identification of neurodegenerative factors using translatome–regulatory network analysis

Nature Neuroscience volume 18, pages 13251333 (2015) | Download Citation

Abstract

For degenerative disorders of the CNS, the main obstacle to therapeutic advancement has been the challenge of identifying the key molecular mechanisms underlying neuronal loss. We developed a combinatorial approach including translational profiling and brain regulatory network analysis to search for key determinants of neuronal survival or death. Following the generation of transgenic mice for cell type–specific profiling of midbrain dopaminergic neurons, we established and compared translatome libraries reflecting the molecular signature of these cells at baseline or under degenerative stress. Analysis of these libraries by interrogating a context-specific brain regulatory network led to the identification of a repertoire of intrinsic upstream regulators that drive the dopaminergic stress response. The altered activity of these regulators was not associated with changes in their expression levels. This strategy can be generalized for the identification of molecular determinants involved in the degeneration of other classes of neurons.

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Acknowledgements

We are grateful to N. Heintz, E. Schmidt and M. Heiman for consultations on TRAP analysis, to M. Kerner and J. Ni for assistance with stereotaxic injections, to B. Sezer, G. Bustamante, T. Kassel and K. Dam for assistance with genotyping, quantitative real-time PCR and behavioral experiments, to J. Gresack for consultations on behavioral experiments, to E. Griggs for assistance with the graphic design and to J. Terlizzi for commenting on the manuscript. We thank Transgenic Services and the Genomics Core at Rockefeller University as well as the Tri-Institutional Laboratory of Comparative Pathology and the Molecular Cytogenetics Core at Memorial Sloan-Kettering Cancer Center for technical support. This study was supported by US Army Medical Research contracts W81XWH-10-1-0640 and W81XWH-12-1-0039 (to L.B.), by US National Institutes of Health National Institute of Neurological Disorders and Stroke grants NS072428, NS088009 and NS078614, US Army Medical Research contracts W81XWH-08-1-0465, W81XWH-12-1-0431 and W81XWH-13-1-0416, the Parkinson's Disease Foundation and the Target-ALS program (to S.P.), and by US Army Medical Research contract W81XWH-09-1-0402 and the JPB Foundation (to P.G.).

Author information

Author notes

    • Andrea Califano
    • , Serge Przedborski
    •  & Paul Greengard

    These authors contributed equally to directing this work.

Affiliations

  1. Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York, USA.

    • Lars Brichta
    • , Ee-Lynn Yap
    • , Zachary Walker
    • , Jack Zhang
    • , Jean-Pierre Roussarie
    •  & Paul Greengard
  2. Department of Systems Biology, Columbia University, New York, New York, USA.

    • William Shin
    • , Mariano J Alvarez
    •  & Andrea Califano
  3. Department of Biological Sciences, Columbia University, New York, New York, USA.

    • William Shin
  4. Department of Neurology, Columbia University, New York, New York, USA.

    • Vernice Jackson-Lewis
    • , Javier Blesa
    •  & Serge Przedborski
  5. Department of Pathology and Cell Biology, Columbia University, New York, New York, USA.

    • Vernice Jackson-Lewis
    • , Javier Blesa
    •  & Serge Przedborski
  6. Center for Motor Neuron Biology and Disease, Columbia University, New York, New York, USA.

    • Vernice Jackson-Lewis
    • , Javier Blesa
    •  & Serge Przedborski
  7. Columbia Translational Neuroscience Initiative, Columbia University, New York, New York, USA.

    • Vernice Jackson-Lewis
    • , Javier Blesa
    •  & Serge Przedborski

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Contributions

L.B., M.J.A., A.C., S.P. and P.G. designed experiments; L.B., E.-L.Y. and Z.W. generated and characterized Dat bacTRAP mice and performed immunostaining; W.S. created the regulatory model under A.C.'s supervision and carried out the interrogation, leading to the identification of the 19 reported MR genes; L.B. carried out TRAPseq analyses and stereotaxic injections; V.J.-L. and J.B. were responsible for MPTP experiments, tissue dissection and stereology; L.B., J.Z. and J.-P.R. analyzed expression data; L.B., W.S., M.J.A., A.C., S.P. and P.G. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Lars Brichta or Paul Greengard.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–9 and Supplementary Tables 8 and 9

  2. 2.

    Supplementary Methods Checklist

Excel files

  1. 1.

    Supplementary Table 1

    Genes enriched in midbrain DA neuron TRAP samples (n = 4) at least 1.5-fold (P < 0.05) as compared to whole midbrain total RNA samples (n = 5).

  2. 2.

    Supplementary Table 2

    Genes depleted in midbrain DA neuron TRAP samples (n = 4) at least −1.5-fold (P < 0.05) as compared to whole midbrain total RNA samples (n = 5).

  3. 3.

    Supplementary Table 3

    Genes differentially expressed in midbrain DA neuron TRAP samples from MPTP-treated mice (n = 4) as compared to midbrain DA neuron TRAP samples from saline-treated mice (n = 4) (up- or downregulated at least 1.5-fold, P < 0.05).

  4. 4.

    Supplementary Table 4

    ARACNe-predicted target genes of statistically significant MRs determined by MARINa analysis of the saline- and MPTP-specific translatomes.

  5. 5.

    Supplementary Table 5

    Genes enriched in SNpc DA neuron TRAP samples (n = 6) at least 1.5-fold (P < 0.05) as compared to VTA DA neuron TRAP samples (n = 6).

  6. 6.

    Supplementary Table 6

    Genes enriched in VTA DA neuron TRAP samples (n = 6) at least 1.5-fold (P < 0.05) as compared to SNpc DA neuron TRAP samples (n = 6).

  7. 7.

    Supplementary Table 7

    Expression of ARACNe-predicted SATB1 target genes in midbrain DA neurons after SATB1 knockdown as compared to controls.

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DOI

https://doi.org/10.1038/nn.4070

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