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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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Gene Expression Omnibus

Referenced accessions

Gene Expression Omnibus

NCBI Reference Sequence

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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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