Background
Methods
Ethics statement
Neuronal culture, tissue isolations, and RNA extraction
Transcriptome profiling using microarrays
Microarray analysis and pathway-level analysis
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A search for the term “MP:0005386” in the JAX lab mouse database (http://www.informatics.jax.org/marker/) returned 3005 unique gene symbols, which are 2622 unique genes mapped to Entrez Gene IDs, out of which 2462 were measured on the Affymetrix Mouse Genome 430 2.0 microarray.
Human brain transcriptome data processing and integration with mouse data
Results
Gene expression profiles in embryonic brain tissue capture fewer genotype-based differences than primary neuronal cultures
Enriched biological pathways converge on mGluR5-downstream signaling pathways in cultured neurons, while the pathways in embryonic brain tissues center on genes associated with immunological signaling
KEGG pathway (ENTREZ ID as input) |
p value | No. of genes | Fold enrichment |
---|---|---|---|
Hippocampal culture | |||
mmu04910:Insulin signaling pathway
|
0.001
|
35
|
1.748
|
mmu03040:Spliceosome
|
0.001
|
32
|
1.801
|
mmu00900:Terpenoid backbone biosynthesis
|
0.002
|
8
|
3.796
|
mmu04720:Long-term potentiation
|
0.003
|
20
|
2.013
|
mmu04120:Ubiquitin mediated proteolysis
|
0.008
|
32
|
1.586
|
mmu04360:Axon guidance
|
0.009
|
31
|
1.584
|
mmu03010:Ribosome
|
0.011
|
22
|
1.740
|
mmu05211:Renal cell carcinoma
|
0.013
|
19
|
1.803
|
mmu04540:Gap junction
|
0.017
|
21
|
1.701
|
mmu04722:Neurotrophin signaling pathway
|
0.017
|
29
|
1.541
|
mmu00230:Purine metabolism
|
0.022
|
33
|
1.461
|
mmu00650:Butanoate metabolism
|
0.029
|
11
|
2.088
|
mmu04012:ErbB signaling pathway
|
0.031
|
21
|
1.603
|
mmu05215:Prostate cancer
|
0.038
|
21
|
1.567
|
mmu03018:RNA degradation
|
0.039
|
15
|
1.748
|
mmu03020:RNA polymerase
|
0.040
|
9
|
2.214
|
mmu00790:Folate biosynthesis | 0.070 | 5 | 3.019 |
mmu04914:Progesterone-mediated oocyte maturation | 0.078 | 19 | 1.485 |
mmu04150:mTOR signaling pathway | 0.080 | 13 | 1.661 |
mmu00240:Pyrimidine metabolism | 0.088 | 20 | 1.444 |
mmu05220:Chronic myeloid leukemia | 0.088 | 17 | 1.506 |
Cortical culture | |||
mmu04622:RIG-I-like receptor signaling pathway
|
0.001
|
23
|
2.027
|
mmu03040:Spliceosome
|
0.001
|
37
|
1.686
|
mmu04722:Neurotrophin signaling pathway
|
0.006
|
36
|
1.548
|
mmu04144:Endocytosis
|
0.012
|
48
|
1.395
|
mmu04660:T cell receptor signaling pathway
|
0.013
|
31
|
1.529
|
mmu04010:MAPK signaling pathway
|
0.016
|
63
|
1.308
|
mmu04120:Ubiquitin mediated proteolysis
|
0.018
|
36
|
1.444
|
mmu05220:Chronic myeloid leukemia
|
0.029
|
22
|
1.577
|
mmu04720:Long-term potentiation | 0.052 | 19 | 1.548 |
mmu00534:Heparan sulfate biosynthesis | 0.062 | 9 | 2.016 |
mmu03450:Non-homologous end-joining | 0.076 | 6 | 2.481 |
mmu04062:Chemokine signaling pathway | 0.080 | 41 | 1.267 |
mmu05210:Colorectal cancer | 0.096 | 22 | 1.391 |
Hippocampus in situ | |||
mmu04144:Endocytosis
|
0.013
|
14
|
2.133
|
mmu04060:Cytokine-cytokine receptor interaction
|
0.013
|
16
|
1.986
|
mmu05322:Systemic lupus erythematosus
|
0.014
|
8
|
3.088
|
mmu04514:Cell adhesion molecules (CAMs)
|
0.017
|
11
|
2.348
|
mmu05320:Autoimmune thyroid disease
|
0.028
| 6 |
3.451
|
mmu05330:Allograft rejection | 0.051 | 5 | 3.523 |
mmu04672:Intestinal immune network for IgA production | 0.082 | 5 | 2.998 |
mmu04610:Complement and coagulation cascades | 0.099 | 6 | 2.415 |
Cortex in situ | |||
mmu00590:Arachidonic acid metabolism
|
0.016
|
9
|
2.727
|
mmu04210:Apoptosis
|
0.029
|
9
|
2.439
|
mmu04640:Hematopoietic cell lineage | 0.059 | 8 | 2.275 |
mmu04060:Cytokine-cytokine receptor interaction | 0.064 | 16 | 1.623 |
mmu00601:Glycosphingolipid biosynthesis | 0.083 | 4 | 3.838 |
Differentially expressed genes in Fmr1KO in cultured neurons are preferentially expressed in early human brain development, whereas genes in embryonic hippocampal and cortical tissues correspond more closely to later developmental stages
Case/cluster | H0 | H1 | H2 | Case/cluster | C0 | C1 | C2 |
---|---|---|---|---|---|---|---|
All genes | 5808 | 4242 | 3780 | All genes | 5572 | 4217 | 4041 |
% | 42 | 31 | 27 | % | 40 | 30 | 29 |
Differentially expressed genes in cultured cells | |||||||
Hippo culture KO/WT down | 239 | 200 | 260 | Cortex culture KO/WT down | 428 | 461 | 408 |
% | 34 | 29 | 37 | % | 33 | 36 | 31 |
Hippo culture KO/WT up | 821 | 191 | 298 | Cortex culture KO/WT up | 732 | 268 | 168 |
% | 63 | 15 | 23 | % | 63 | 23 | 14 |
Hippo culture KO/WT combined | 1060 | 391 | 558 | Cortex culture KO/WT combined | 1160 | 729 | 576 |
% | 53 | 19 | 28 | % | 47 | 30 | 23 |
Differentially expressed genes in primary brain tissue | |||||||
Hippo brain KO/WT down | 92 | 126 | 65 | Cortex brain KO/WT down | 102 | 52 | 57 |
% | 32.5 | 44.5 | 23 | % | 48.3 | 24.6 | 27.1 |
Hippo brain KO/WT up | 61 | 71 | 39 | Cortex brain KO/WT up | 96 | 81 | 162 |
% | 35.7 | 41.5 | 22.8 | % | 28.3 | 24 | 47.7 |
Hippo brain KO/WT combined | 153 | 197 | 104 | Cortex brain KO/WT combined | 198 | 133 | 219 |
% | 33.7 | 43.4 | 22.9 | % | 36 | 24.2 | 39.8 |
Discussion
Conclusions
Differentially expressed genes in Fmr1KO vs WT (disease genes) | ||||
---|---|---|---|---|
Culture | Brain | |||
Hippocampus | Cortex | Hippocampus | Cortex | |
Number of differentially expressed genes | 2648 | 3372 | 726 | 866 |
Distance between KO and WT clusters on PCA (PC1/PC2) | 3.73/0.7 | 3.31/3.20 | 0.99/0.6 | 0.39/0.32 |
Pathway enrichment | Neuronal processes | Immune processes | ||
Human development | Early (H0 cluster) | Early (C0 cluster) | Later (H1 cluster) | Later (C2 cluster) |
Up-regulated | Up-regulated | Down- and up- regulated | Up-regulated | |
SFARI human genes | Significantly enriched in down-regulated genes | Significantly enriched in down-regulated genes | Not significant | Not significant |
MGI mouse genes associated with behavioral/neurological phenotype | Significantly enriched in down-regulated genes | Significantly enriched in down-regulated genes | Not significant | Not significant |
FMRP targets | Significantly enriched in down-regulated genes | Significantly enriched in down- and up- regulated genes | Not significant | Not significant |
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Importance: In addition to neuronal pathology, research on disease treatments should also consider the impact of immune dysregulation, which is comparatively less studied.
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Importance: Cultured cells may recapitulate an early phase of the disease, which is also less obscured with “immunological” phenotype and in vivo compensatory mechanisms.
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Importance: These findings suggest that there are potential shared disease-related mechanisms in fragile X and other autism phenotypes in mice.
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Better captures the difference between genotypes in comparison to embryonic brain tissue.
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Bears greater resemblance to earlier stages of human development
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Bears the significant resemblance to the genes previously implicated with autism in human in comparison to brain tissue