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Identification of neurodegenerative factors using translatome–regulatory network analysis

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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Figure 1: Generation of Dat bacTRAP mice for the translational profiling of midbrain DA neurons.
Figure 2: TRAPseq analysis in a mouse model of Parkinson's disease and regulatory network analysis.
Figure 3: Analysis of MR expression patterns in midbrain DA neurons.
Figure 4: Virus-mediated knockdown of SATB1 in SNpc DA neurons.
Figure 5: Virus-mediated knockdown of ZDHHC2 in SNpc DA neurons.
Figure 6: Master regulator analysis on incipient Parkinson's disease and control expression signatures.

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Primary accessions

Gene Expression Omnibus

Referenced accessions

Gene Expression Omnibus

NCBI Reference Sequence

References

  1. Heiman, M. et al. A translational profiling approach for the molecular characterization of CNS cell types. Cell 135, 738–748 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Doyle, J.P. et al. Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell 135, 749–762 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Sanz, E. et al. Cell-type-specific isolation of ribosome-associated mRNA from complex tissues. Proc. Natl. Acad. Sci. USA 106, 13939–13944 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kanehisa, M. A database for post-genome analysis. Trends Genet. 13, 375–376 (1997).

    Article  CAS  PubMed  Google Scholar 

  5. Huang, D.W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    Article  CAS  Google Scholar 

  6. Huang, D.W., Sherman, B.T. & Lempicki, R.A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).

    Article  CAS  Google Scholar 

  7. Carter, R.J., Morton, J. & Dunnett, S.B. Motor coordination and balance in rodents. Curr. Protoc. Neurosci. Ch. 8, Unit 8.12 (2001).

  8. Dauer, W. & Przedborski, S. Parkinson′s disease: mechanisms and models. Neuron 39, 889–909 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Jackson-Lewis, V. & Przedborski, S. Protocol for the MPTP mouse model of Parkinson′s disease. Nat. Protoc. 2, 141–151 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Jackson-Lewis, V., Blesa, J. & Przedborski, S. Animal models of Parkinson′s disease. Parkinsonism Relat. Disord. 18 (suppl. 1): S183–S185 (2012).

    Article  PubMed  Google Scholar 

  11. Vila, M. et al. Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson′s disease. Proc. Natl. Acad. Sci. USA 98, 2837–2842 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Damier, P., Hirsch, E.C., Agid, Y. & Graybiel, A.M. The substantia nigra of the human brain. II. Patterns of loss of dopamine-containing neurons in Parkinson′s disease. Brain 122, 1437–1448 (1999).

    Article  PubMed  Google Scholar 

  13. Hirsch, E., Graybiel, A.M. & Agid, Y.A. Melanized dopaminergic neurons are differentially susceptible to degeneration in Parkinson′s disease. Nature 334, 345–348 (1988).

    Article  CAS  PubMed  Google Scholar 

  14. Jackson-Lewis, V., Jakowec, M., Burke, R.E. & Przedborski, S. Time course and morphology of dopaminergic neuronal death caused by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Neurodegeneration 4, 257–269 (1995).

    Article  CAS  PubMed  Google Scholar 

  15. Muthane, U. et al. Differences in nigral neuron number and sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in C57/bl and CD-1 mice. Exp. Neurol. 126, 195–204 (1994).

    Article  CAS  PubMed  Google Scholar 

  16. Bowenkamp, K.E. et al. 6-Hydroxydopamine induces the loss of the dopaminergic phenotype in substantia nigra neurons of the rat. A possible mechanism for restoration of the nigrostriatal circuit mediated by glial cell line-derived neurotrophic factor. Exp. Brain Res. 111, 1–7 (1996).

    Article  CAS  PubMed  Google Scholar 

  17. Piovan, E. et al. Direct reversal of glucocorticoid resistance by AKT inhibition in acute lymphoblastic leukemia. Cancer Cell 24, 766–776 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Lefebvre, C. et al. A human B-cell interactome identifies MYB and FOXM1 as master regulators of proliferation in germinal centers. Mol. Syst. Biol. 6, 377 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Carro, M.S. et al. The transcriptional network for mesenchymal transformation of brain tumours. Nature 463, 318–325 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Aytes, A. et al. Cross-species regulatory network analysis identifies a synergistic interaction between FOXM1 and CENPF that drives prostate cancer malignancy. Cancer Cell 25, 638–651 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Margolin, A.A. et al. ARACNE: an algorithm for the reconstruction of gene regulatory networks in a mammalian cellular context. BMC Bioinformatics 7 (suppl. 1): S7 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Chen, J.C. et al. Identification of causal genetic drivers of human disease through systems-level analysis of regulatory networks. Cell 159, 402–414 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Repunte-Canonigo, V. et al. Identifying candidate drivers of alcohol dependence-induced excessive drinking by assembly and interrogation of brain-specific regulatory networks. Genome Biol. 16, 68 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Aubry, S. et al. Assembly and interrogation of Alzheimer′s disease genetic networks reveal novel regulators of progression. PLoS ONE 10, e0120352 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Chung, C.Y. et al. Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection. Hum. Mol. Genet. 14, 1709–1725 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Huang, Y. et al. Distribution of Satb1 in the central nervous system of adult mice. Neurosci. Res. 71, 12–21 (2011).

    Article  CAS  PubMed  Google Scholar 

  27. Close, J. et al. Satb1 is an activity-modulated transcription factor required for the terminal differentiation and connectivity of medial ganglionic eminence-derived cortical interneurons. J. Neurosci. 32, 17690–17705 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zheng, B. et al. PGC-1alpha, a potential therapeutic target for early intervention in Parkinson′s disease. Sci. Transl. Med. 2, 52ra73 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Grimm, J., Mueller, A., Hefti, F. & Rosenthal, A. Molecular basis for catecholaminergic neuron diversity. Proc. Natl. Acad. Sci. USA 101, 13891–13896 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Greene, J.G., Dingledine, R. & Greenamyre, J.T. Gene expression profiling of rat midbrain dopamine neurons: implications for selective vulnerability in parkinsonism. Neurobiol. Dis. 18, 19–31 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Phani, S., Gonye, G. & Iacovitti, L. VTA neurons show a potentially protective transcriptional response to MPTP. Brain Res. 1343, 1–13 (2010).

    Article  CAS  PubMed  Google Scholar 

  32. Greene, J.G., Dingledine, R. & Greenamyre, J.T. Neuron-selective changes in RNA transcripts related to energy metabolism in toxic models of parkinsonism in rodents. Neurobiol. Dis. 38, 476–481 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Miller, R.M. et al. Wild-type and mutant alpha-synuclein induce a multi-component gene expression profile consistent with shared pathophysiology in different transgenic mouse models of PD. Exp. Neurol. 204, 421–432 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Bifsha, P., Yang, J., Fisher, R.A. & Drouin, J. Rgs6 is required for adult maintenance of dopaminergic neurons in the ventral substantia nigra. PLoS Genet. 10, e1004863 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Ryan, S.D. et al. Isogenic human iPSC Parkinson′s model shows nitrosative stress-induced dysfunction in MEF2–PGC1alpha transcription. Cell 155, 1351–1364 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Okamoto, S., Krainc, D., Sherman, K. & Lipton, S.A. Antiapoptotic role of the p38 mitogen-activated protein kinase-myocyte enhancer factor 2 transcription factor pathway during neuronal differentiation. Proc. Natl. Acad. Sci. USA 97, 7561–7566 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Cho, E.G. et al. MEF2C enhances dopaminergic neuron differentiation of human embryonic stem cells in a parkinsonian rat model. PLoS ONE 6, e24027 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Gantz, S.C., Ford, C.P., Neve, K.A. & Williams, J.T. Loss of Mecp2 in substantia nigra dopamine neurons compromises the nigrostriatal pathway. J. Neurosci. 31, 12629–12637 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Panayotis, N. et al. Morphological and functional alterations in the substantia nigra pars compacta of the Mecp2-null mouse. Neurobiol. Dis. 41, 385–397 (2011).

    Article  CAS  PubMed  Google Scholar 

  40. Chan, C.S. et al. 'Rejuvenation' protects neurons in mouse models of Parkinson′s disease. Nature 447, 1081–1086 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Guzman, J.N. et al. Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1. Nature 468, 696–700 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Mosharov, E.V. et al. Interplay between cytosolic dopamine, calcium, and alpha-synuclein causes selective death of substantia nigra neurons. Neuron 62, 218–229 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Yasui, D., Miyano, M., Cai, S., Varga-Weisz, P. & Kohwi-Shigematsu, T. SATB1 targets chromatin remodelling to regulate genes over long distances. Nature 419, 641–645 (2002).

    Article  CAS  PubMed  Google Scholar 

  44. Roth, A.F. et al. Global analysis of protein palmitoylation in yeast. Cell 125, 1003–1013 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Kostiuk, M.A., Keller, B.O. & Berthiaume, L.G. Palmitoylation of ketogenic enzyme HMGCS2 enhances its interaction with PPARalpha and transcription at the Hmgcs2 PPRE. FASEB J. 24, 1914–1924 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wang, K. et al. Genome-wide identification of post-translational modulators of transcription factor activity in human B cells. Nat. Biotechnol. 27, 829–839 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Gong, S. et al. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425, 917–925 (2003).

    Article  CAS  PubMed  Google Scholar 

  48. Heiman, M., Kulicke, R., Fenster, R.J., Greengard, P. & Heintz, N. Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP). Nat. Protoc. 9, 1282–1291 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Gentleman, R.C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Trapnell, C., Pachter, L. & Salzberg, S.L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Liberatore, G.T. et al. Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease. Nat. Med. 5, 1403–1409 (1999).

    Article  CAS  PubMed  Google Scholar 

  55. Przedborski, S. et al. Role of neuronal nitric oxide in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity. Proc. Natl. Acad. Sci. USA 93, 4565–4571 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ashburner, M. et al. Gene ontology: tool for the unification of biology. Nat. Genet. 25, 25–29 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Brown, R.E. & Wong, A.A. The influence of visual ability on learning and memory performance in 13 strains of mice. Learn. Mem. 14, 134–144 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  59. McFadyen, M.P., Kusek, G., Bolivar, V.J. & Flaherty, L. Differences among eight inbred strains of mice in motor ability and motor learning on a rotorod. Genes Brain Behav. 2, 214–219 (2003).

    Article  CAS  PubMed  Google Scholar 

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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

Authors and Affiliations

Authors

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.

Corresponding authors

Correspondence to Lars Brichta or Paul Greengard.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Brain morphology, body weight, food and water intake of Dat bacTRAP mice.

(a) Hematoxylin and eosin staining of coronal brain sections from TG Dat bacTRAP mice and WT littermates.

(b) Body weight of TG Dat bacTRAP mice and WT littermates at the ages of 3 months (TG, n=31 and WT, n=28), 6 months (TG, n=16 and WT, n=18), 9 months (TG, n=16 and WT, n=18) and 12 months (TG, n=41 and WT, n=45). Overall effect of genotype: P<0.0001. Two-way ANOVA.

(c) Analysis of total body water (P=0.405), extracellular fluid (P=0.851), intracellular fluid (P=0.327), fat-free mass (P=0.469) and fat mass (P=0.469) in 12 month-old TG Dat bacTRAP mice (n=8) and WT littermates (n=8). Unpaired t-test.

(d) Food intake in TG Dat bacTRAP mice (n=8) and WT littermates (n=7) at 3 months of age. Red lines and error bars indicate means ± SEM. P=0.722. Unpaired t-test.

(e) Water intake in TG Dat bacTRAP mice (n=8) and WT littermates (n=8) at 3 months of age. Red lines and error bars indicate means ± SEM. P=0.088. Unpaired t-test.

n-values indicate biological replicates. All data represent means ± SEM. ****P<0.0001.

Supplementary Figure 2 Copy number and chromosomal location of BAC transgenes in Dat bacTRAP mice.

(a) Fluorescent in situ hybridization (FISH) analysis of metaphase chromosomes from primary fibroblasts derived from Dat bacTRAP mice using a probe detecting the Slc6a3 locus. The analysis revealed three independent signals, showing that TG Dat bacTRAP mice carry a tandem insert of BAC transgenes in a single location on chromosome 9q in addition to the two endogenous Slc6a3 loci on both chromosomes 13q.

(b-d) Number of genomic Slc6a3 copies in TG Dat bacTRAP mice (n=6) and WT littermates (n=4) in mouse generations F1 (b), F2 (c) and F3 (d). The detected copies include both the two endogenous Slc6a3 alleles and the Slc6a3 copies located on the BAC transgene.

n-values indicate biological replicates. Red lines and error bars indicate means ± SEM.

Supplementary Figure 3 Morphological, stereological and biochemical characterization of the midbrain DA neurons in Dat bacTRAP mice.

(a) Coronal brain sections from a WT littermate and a TG Dat bacTRAP mouse after immunohistochemistry for TH (brown) and staining for Nissl substance (blue) at the level of the midbrain DA system at a lower magnification (upper images) and a higher magnification (lower images).

(b) Number of TH+ neurons in the SNpc and the VTA in TG Dat bacTRAP mice (n=8) and WT littermates (n=6). SNpc: P=0.133; VTA: P=0.051. Unpaired t-test.

(c) Striatal levels of dopamine and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in TG Dat bacTRAP mice (n=4) and WT littermates (n=3). Dopamine: P=0.244; DOPAC: P=0.149; HVA: P=0.292. Unpaired t-test.

(d) Immunoblot analysis of TH, DAT and GAPDH protein levels in striatal tissue from TG Dat bacTRAP mice (n=6) and WT littermates (n=6). The molecular weight standard is shown on the right of the blot. Blot images are cropped. Full-length blots are presented in Supplementary Figure 9a-c.

(e) Normalized TH and DAT protein levels relative to GAPDH for panel (d). TH: P=0.262; DAT: P=0.414. Unpaired t-test.

Scale bars, 500 µm in (a) (upper images) and 50 µm in (a) (lower images). n-values indicate biological replicates. All data represent means ± SEM.

Supplementary Figure 4 Rotarod test, pole test and wire hang test in Dat bacTRAP mice.

(a) Rotarod test in TG Dat bacTRAP mice (n=16) and WT littermates (n=18) at three months of age (Day 1: P=0.553; Day 2: P=0.532; Day 3: P=0.489). One-Way ANCOVA.

(b) Rotarod test in TG Dat bacTRAP mice (n=16) and WT littermates (n=18) at the age of 6 months (Day 1: P=0.808; Day 2: P=0.384; Day 3: P=0.227). One-Way ANCOVA.

(c) Rotarod test in TG Dat bacTRAP mice (n=16) and WT littermates (n=18) at the age of 9 months (Day 1: P=0.888; Day 2: P=0.507; Day 3: P=1.000). One-Way ANCOVA.

(d) Rotarod test in TG Dat bacTRAP mice (n=15) and WT littermates (n=18) at the age of 12 months (Day 1: P=0.588; Day 2: P=0.367; Day 3: P=0.393). One-Way ANCOVA.

(e) Pole test in TG Dat bacTRAP mice (n=15) and WT littermates (n=17) at three months of age (Turning: P=0.338; Climbing down: P=0.849). Unpaired t-test.

(f) Pole test in TG Dat bacTRAP mice (n=15) and WT littermates (n=17) at the age of 6 months (Turning: P=0.210; Climbing down: P=0.570). Unpaired t-test.

(g) Pole test in TG Dat bacTRAP mice (n=15) and WT littermates (n=17) at the age of 9 months (Turning: P=0.055; Climbing down: P=0.113). Unpaired t-test.

(h) Pole test in TG Dat bacTRAP mice (n=15) and WT littermates (n=18) at the age of 12 months (Turning: P=0.203; Climbing down: P=0.167). Unpaired t-test.

(i) Wire hang test in TG Dat bacTRAP mice (n=16) and WT littermates (n=18) at three months of age (P=0.684). Red lines and error bars indicate means ± SEM. Unpaired t-test.

(j) Wire hang test in TG Dat bacTRAP mice (n=16) and WT littermates (n=18) at the age of 6 months (P=0.125). Red lines and error bars indicate means ± SEM. Unpaired t-test.

(k) Wire hang test in TG Dat bacTRAP mice (n=16) and WT littermates (n=18) at the age of 9 months (P=0.145). Red lines and error bars indicate means ± SEM. Unpaired t-test.

(l) Wire hang test in TG Dat bacTRAP mice (n=15) and WT littermates (n=18) at the age of 12 months (P=0.174). Red lines and error bars indicate means ± SEM. Unpaired t-test.

n-values indicate biological replicates. All data represent means ± SEM.

Supplementary Figure 5 Analysis of the footprint patterns of Dat bacTRAP mice.

(a) Gait analysis in TG Dat bacTRAP mice (n=15) and WT littermates (n=16) at the age of 3 months. Representative footprint patterns are shown together with the calculations of forelimb stride length (P=0.960), hind limb stride length (P=0.773), forelimb width (P=0.894), hindlimb width (P=0.153) and forelimb/hindlimb overlap (P=0.143). Unpaired t-test. Orange traces are from front paws and purple traces are from back paws.

(b) Gait analysis in TG Dat bacTRAP mice (n=14) and WT littermates (n=18) at the age of 6 months. Representative footprint patterns are shown together with the calculations of forelimb stride length (P=0.317), hind limb stride length (P=0.466), forelimb width (P=0.570), hindlimb width (P=0.026) and forelimb/hindlimb overlap (P=0.012). Unpaired t-test. Orange traces are from front paws and purple traces are from back paws.

(c) Gait analysis in TG Dat bacTRAP mice (n=16) and WT littermates (n=18) at the age of 9 months. Representative footprint patterns are shown together with the calculations of forelimb stride length (P=0.845), hind limb stride length (P=0.649), forelimb width (P=0.520), hindlimb width (P=0.003) and forelimb/hindlimb overlap (P=0.011). Unpaired t-test. Orange traces are from front paws and purple traces are from back paws.

(d) Gait analysis in TG Dat bacTRAP mice (n=15) and WT littermates (n=16) at the age of 12 months. Representative footprint patterns are shown together with the calculations of forelimb stride length (P=0.108), hind limb stride length (P=0.178), forelimb width (P=0.233), hindlimb width (P=0.038) and forelimb/hindlimb overlap (P=0.011). Unpaired t-test. Orange traces are from front paws and purple traces are from back paws.

n-values indicate biological replicates. All data represent means ± SEM. *P<0.05, **P<0.01.

Supplementary Figure 6 Evaluation of MPTP potency.

Number of TH+ neurons in the SNpc and the VTA in TG Dat bacTRAP mice and WT littermates that were injected with either saline (TG, n=8 and WT, n=6) or MPTP in saline (TG, n=12 and WT, n=12) using the subacute dosing regimen consisting of one daily intraperitoneal injection of MPTP-HCl (30 mg/kg free base per day) for five consecutive days. Animals were perfused 21 days after the last injection. P=0.0003 (SNpc, WT); P=0.000008 (SNpc, TG); P=0.014 (VTA, WT); P=0.0001 (VTA, TG). Unpaired t-test.

n-values indicate biological replicates. All data represent means ± SEM. * P <0.05, ***P<0.001, *****P<0.00001.

Supplementary Figure 7 Comparative MR expression analysis in SNpc and VTA DA neurons.

(a) Comparative TRAPseq analysis of SNpc DA neurons and VTA DA neurons in Dat bacTRAP mice (n=6). Mean gene expression values for VTA DA neurons were plotted against mean gene expression values for SNpc DA neurons. Lines to each side represent 1.5fold enrichment in either sample. The center line represents equal expression. Red dots represent transcripts enriched in SNpc DA neuron TRAP samples by ≥1.5fold (P<0.05). Green dots represent transcripts enriched in VTA DA neuron TRAP samples by ≥1.5fold (P<0.05). Black dots represent non-significant transcripts. Blue triangles depict select marker genes.

(b) Schematic illustration of the location of the SNpc and the VTA in coronal mouse brain sections. Adapted from the Allen Mouse Brain Atlas (Website: ©2014 Allen Institute for Brain Science. Allen Mouse Brain Atlas [Internet]. Available from: http://mouse.brain-map.org.).

(c) Representative in situ hybridization images from adult coronal mouse brain sections for the MRs Satb1 and Zdhhc2 taken from the Allen Mouse Brain Atlas.

(d) Expression view of the in situ hybridization images for Satb1 and Zdhhc2 presented in panel (c) taken from the Allen Mouse Brain Atlas.

(e) Validation of a SATB1 antibody using lysates from HEK 293T cells transfected with a SATB1-FLAG construct (lanes 1 and 3) or empty vector (control; lanes 2 and 4), and brain lysates from mice that received either saline (lane 5) or MPTP (lane 6) according to Fig. 2a. Bands correspond to either full-length SATB1-FLAG (lanes 1 and 3) or endogenous full-length SATB1 (lanes 5 and 6). HEK 293T cell lysates were diluted at 1:200 for analysis. The molecular weight standard is shown on the left of the blot. Blot images are cropped. Full-length blots are presented in Supplementary Figure 9d.

(f) Validation of a ZDHHC2 antibody using lysates from HEK 293T cells transfected with a ZDHHC2-FLAG construct (lanes 1 and 3) or empty vector (control; lanes 2 and 4), and brain lysates from mice that received either saline (lane 5) or MPTP (lane 6) according to Fig. 2a. Bands correspond to either full-length ZDHHC2-FLAG (lanes 1 and 3) or endogenous full-length ZDHHC2 (lanes 5 and 6). HEK 293T cell lysates were diluted at 1:100 for analysis. The molecular weight standard is shown on the left of the blot. Blot images are cropped. Full-length blots are presented in Supplementary Figure 9e.

n-values indicate biological replicates.

Supplementary Figure 8 AAV1-mediated knockdown of SATB1 and ZDHHC2 in SNpc DA neurons.

(a) Number of Nissl+, THˉ neurons in the SNpc in WT mice (n=6) eight weeks after injection according to Fig. 4a. Red lines and error bars indicate means ± SEM. The same symbol shape was used for paired values obtained from the same animal. P=0.4198. Paired t-test.

(b) Number of TH+ neurons in the SNpc in WT mice (n=6) eight weeks after injection according to the scheme shown in Fig. 4a, using Satb1 shRNA #2 (target sequence distinct from that of the first Satb1 shRNA). Red lines and error bars indicate means ± SEM. The same symbol shape was used for paired values obtained from the same animal. P=0.0137. Paired t-test.

(c) Number of Nissl+, THˉ neurons in the SNpc in WT mice (n=6) eight weeks after injection according to the scheme shown in Fig. 4a, using Satb1 shRNA #2 (target sequence distinct from that of the first Satb1 shRNA). Red lines and error bars indicate means ± SEM. The same symbol shape was used for paired values obtained from the same animal. P=0.4857. Paired t-test.

(d) Coronal brain section from a WT mouse four weeks after stereotaxic injection according to Fig. 4a. Panels show staining for TH (red) and EGFP autofluorescence (green).

(e) Coronal brain sections from WT mice four weeks post-injection according to Fig. 4a. SNpc DA neurons were retrogradely labeled with FG prior to the viral injections. Panels show staining for TH (red) and autofluorescence of FG (blue) and EGFP (green).

(f) Number of Nissl+, THˉ neurons in the SNpc in WT mice (n=5) eight weeks after injection according to Fig. 5a. Red lines and error bars indicate means ± SEM. The same symbol shape was used for paired values obtained from the same animal. P=0.0008. Paired t-test.

(g) Number of TH+ neurons in the SNpc in WT mice (n=3) eight weeks after injection according to the scheme shown in Fig. 5a, using Zdhhc2 shRNA #2 (target sequence distinct from that of the first Zdhhc2 shRNA). Red lines and error bars indicate means ± SEM. The same symbol shape was used for paired values obtained from the same animal. P=0.0675. Paired t-test.

(h) Number of Nissl+, THˉ neurons in the SNpc in WT mice (n=3) eight weeks after injection according to the scheme shown in Fig. 5a, using Zdhhc2 shRNA #2 (target sequence distinct from that of the first Zdhhc2 shRNA). Red lines and error bars indicate means ± SEM. The same symbol shape was used for paired values obtained from the same animal. P=0.1182. Paired t-test.

(i) Coronal brain section from a WT mouse three weeks after stereotaxic injection according to Fig. 5a. Panels show staining for TH (orange) as well as autofluorescence of RFP (red) and EGFP (green).

(j) Coronal brain sections from WT mice two weeks post-injection according to Fig. 5a. SNpc DA neurons were retrogradely labeled with FG prior to the viral injections. Panels show staining for TH (red) as well as autofluorescence of FG (blue), EGFP (green) and RFP (red).

Scale bars, 200 µm in (d) and (i) and 50 µm in (e) and (j). n-values indicate biological replicates. ***P<0.001, *P<0.05.

Supplementary Figure 9 Full-length western blots.

(a-c) Immunoblot analysis of DAT, TH and GAPDH protein levels in striatal tissue from TG Dat bacTRAP mice (n=6) and WT littermates (n=6). These blots correspond to the cropped images presented in Supplementary Figure 3d.

(d) Validation of a SATB1 antibody using lysates from HEK 293T cells transfected with a SATB1-FLAG construct (lanes 1 and 3) or empty vector (control; lanes 2 and 4), and brain lysates from mice that received either saline (lane 5) or MPTP (lane 6) according to Fig. 2a. Bands correspond to either full-length SATB1-FLAG (lanes 1 and 3) or endogenous full-length SATB1 (lanes 5 and 6). HEK 293T cell lysates were diluted at 1:200 for analysis. This blot corresponds to the cropped image presented in Supplementary Figure 7e.

(e) Validation of a ZDHHC2 antibody using lysates from HEK 293T cells transfected with a ZDHHC2-FLAG construct (lanes 1 and 3) or empty vector (control; lanes 2 and 4), and brain lysates from mice that received either saline (lane 5) or MPTP (lane 6) according to Fig. 2a. Bands correspond to either full-length ZDHHC2-FLAG (lanes 1 and 3) or endogenous full-length ZDHHC2 (lanes 5 and 6). HEK 293T cell lysates were diluted at 1:100 for analysis. This blot corresponds to the cropped image presented in Supplementary Figure 7f.

(f-i), Immunoblot analysis of SATB1 and ZDHHC2 protein levels in midbrain from WT mice injected with either saline (n=4) or MPTP (n=4) according to Fig. 2a. GAPDH and β-tubulin, respectively, were used as loading controls. These blots correspond to the cropped images presented in Figure 3f.

n-values indicate biological replicates. The molecular weight standard is shown on the right of each blot (in kDa).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Tables 8 and 9 (PDF 1265 kb)

Supplementary Methods Checklist (PDF 532 kb)

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). (XLS 607 kb)

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). (XLS 847 kb)

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). (XLS 58 kb)

Supplementary Table 4

ARACNe-predicted target genes of statistically significant MRs determined by MARINa analysis of the saline- and MPTP-specific translatomes. (XLS 609 kb)

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). (XLS 145 kb)

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). (XLS 394 kb)

Supplementary Table 7

Expression of ARACNe-predicted SATB1 target genes in midbrain DA neurons after SATB1 knockdown as compared to controls. (XLS 40 kb)

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Brichta, L., Shin, W., Jackson-Lewis, V. et al. Identification of neurodegenerative factors using translatome–regulatory network analysis. Nat Neurosci 18, 1325–1333 (2015). https://doi.org/10.1038/nn.4070

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