Supplementary Information (doc 1276K)

advertisement
Supplementary information
1. Supplementary material and methods
2. Supplementary references
3. Table S1: Primers used for quantitative PCR
4. Table S2: Body weight and novelty suppressed feeding test measurements
5. Figure S1: Correlations between novelty suppressed feeding test and expression of
myelination-related genes
6. Figure S2: Correlations between time spent in the corner of the open field and expression of
myelination-related genes
7. Figure S3: Correlations between time spent in the center of the open field and expression of
myelination-related genes
8. Figure S4: Potential pathway for fluoxetine affecting myelination-related genes
Supplementary materials and methods
RNA extraction and Double-Stranded cDNA Synthesis
Animals of group 2 were sacrificed at PND 128, brains were removed and immediately placed on dry
ice and stored at -80°C. Hippocampal tissue was dissected in 8 consecutive slices of 200 µm using a
2 mm punch needle. Tissue from 2 rats was pooled for total RNA isolation with QIAzol (RNeasy lipid
tissue kit; QIAGEN, Venlo, The Netherlands) according to the manufacturer's recommendations. Total
RNA was subjected to two rounds of poly(A) selection (Oligotex mRNA Mini Kit; QIAGEN), followed by
DNaseI treatment (QIAGEN) and fragmentation by hydrolysis (5× fragmentation buffer: 200mM Tris
acetate, pH8.2, 500mM potassium acetate and 150mM magnesium acetate) at 94°C for 90 s.
Fragmented mRNA was purified (RNeasy MinElute Kit; QIAGEN) and used for cDNA synthesis with 5
μg random hexamers by Superscript III Reverse Transcriptase (Invitrogen Life technologies, Bleiswijk,
The Netherlands). Double stranded cDNA synthesis was performed in second strand buffer
(Invitrogen) according to the manufacturer's recommendations and purified using the Minelute
Reaction Cleanup Kit (QIAGEN) according to the manufacturer's protocol.
Sequencing
DNA samples were prepared for RNA-seq by end repair of 20 ng DNA as measured by Qubit dsDNA
HS (Invitrogen). Adaptors were ligated to DNA fragments, followed by size selection (~300 bp) and 14
cycles of PCR amplification. Quality control of DNA libraries prepared for sequencing was made by
qPCR and by running the products on a Bioanalyzer (Bio-Rad, Veenendaal, The Netherlands). Cluster
generation and sequencing (36 bp, single read) was performed with the Illumina Genome Analyzer IIx
(GAIIx) platform according to standard Illumina protocols. Samples were sequenced to a depth of
approximately 15 million uniquely mapped tags per sample. Sequences were aligned to the rat rn4
reference genome with the Illumina Analysis Pipeline allowing one mismatch. Only the tags aligning to
one position on the genome were considered for further analysis. The output data were converted to
Browser Extensible Data (BED) files for downstream analysis and Wiggle (WIG) files for viewing.
Data analysis
RNA-seq data were analyzed using Genomatix software (www.genomatix.de). The number of
sequence reads for each transcript was quantified and additionally a standardized normalized
expression (NE) value per transcript was calculated (based on the number of reads located in the
exons of the transcript and normalized to the length of the transcript and the density of the data set).
NE values of vehicle and fluoxetine samples (group 2) were used to calculate fold change values for
each transcript. Genes were identified as differentially expressed if they showed a DESeq
1
P-value <
0.05 and fold change (FC) > 1.5 (among two biological replicates for both fluoxetine and vehicle
treatment). The Database for Annotation, Visualization and Integrated Discovery (DAVID,
http://david.abcc .ncifcrf.gov/) was used for gene ontology (GO) analysis.
Quantitative Reverse Transcription PCR
RNA-seq validation was performed by RT-qPCR analysis of selected genes using GoTaq® qPCR
Master Mix (Promega Benelux b.v. Leiden, The Netherlands). Primers were designed using Primer3
online software (http://frodo.wi.mit.edu). See table S1 for primer sequences. Complementary DNA
(cDNA) was synthesized using 1 μg of total RNA in a reverse transcription reaction using iScript cDNA
Synthesis Kit according to manufacturer’s protocol (Bio-Rad). qPCR reactions were performed in a
7500 Fast Real Time PCR System (Applied Biosystems, Foster City, CA, U.S.A) using the SYBR
Green fluorescence quantification system (GoTaq® qPCR Master Mix, Promega). Thermal cycling
was initiated with incubation at 95C for 10 min followed by 40 cycles of 95C for 30 sec and 60C for
1 min. To normalize the cDNA content of the samples, we used the comparative threshold cycle (CT)
method 2, which consists of the normalization of the number of target gene copies versus two
endogenous reference genes Ywhaz and Hprt1.
Table S1: Primers used for quantitative PCR
Gene symbol
Ref-seq number
Forward primer
Reverse primer
Olfm1
NM_053573.1
AGACCTCAGGCTCAAGGTTC
CACCATGGACTTGTACTCACG
Grina
NM_153308.4
AGGCTCTTCTGCGTCTTCC
AACTCTTTTCATGGGACATGG
Syn2
NM_001034020.1
ATGCGGATGGAACCTACG
GGATGAGCACGAAGTCTGG
Adcy1
NM_001107239.1
CTGTGTGGAGATGGGACTTG
CACACGCATGTTCAGGTCTAC
Src
NM_031977.1
GGAATCAGAGCGGCTACTTC
TTTCACATTTAGGCCCTTGG
Tspan2
NM_022589.1
CAGCTCATTGGAATTGTTGG
AGTTCCGTATTGCACAGCAG
Prkcd
NM_133307.1
GCCTTTGTCCTGAATGTGG
CCTTCCTCACCCATCTCATC
Cldn11
NM_053457.2
CGCATACAGGAAACCAGATG
CTGGGGTGCTCCTTATTCTG
Cpne4
NM_001109003.1
TCATCCTCAAGATGCAATCC
CCACCGTAAACAGCTTTGAG
Nts
NM_001102381.1
CTGCTTGTCAGAAGGCTGAG
GATCTGCCTCCAGGACTCTC
Sult5a1
NM_001201369.1
CAGAGTCACCCATCTTGGAC
ACCAGAGTCAGGGCAAGTTC
Cntf
NM_013166.1
CTTGCCACTGGTACACCATC
TCGTTCAGACCTGACTGCTC
Egr2
NM_053633.1
TGCCCATGTAAGTGAAGGTC
TGATCAGATGAACGGAGTGG
Igf1
NM_001082479.1
AAAGTCAGCTCGTTCCATCC
TCTTGTTTCCTGCACTTCCTC
Plp1 (ex2-3)
NM_030990.2
TCTCCAAAAACTACCAGGACTATG
GGCCCCATAAAGGAAGAAG
Plp1 (ex3-5)
NM_030990.2
TTTGGGAAAATGGCTAGGAC
TGCAGATGGACAGAAGGTTG
Olig2
NM_001100557.1
TCACAGGAGGAACCGTGTC
TGCTGGAGGAAGATGACTTG
Sox10
NM_019193.2
TCTTTGGGGTGGTTGGAG
GCTGCTATCCAGGCTCACTAC
Mag
NM_017190.4
AGACAATGGCAATCAGGATG
TTTGTACCTCCAGGAACCTCTAC
Tf
NM_001013110.1
GGAAAGTGCAGGCTTCTAGG
CAGAGATGACACCAAGTGTTTG
Mog
NM_022668.2
GAGGTTCTCGGATGAAGGAG
CAGGGTTGATCCAGTAGAAGG
Cnp
NM_012809.2
GGCAGAAGAATATGCCCAAC
TCACAAAGAGGGCAGAGATG
Hprt1
NM_012583.2
GCAGACTTTGCTTTCCTTGG
CGAGAGGTCCTTTTCACCAG
Ywhaz
NM_013011.3
TTGAGCAGAAGACGGAAGGT
GAAGCATTGGGGATCAAGAA
Table S2: Body weight and novelty suppressed feeding test measurements
76
75
74
73
72
71
70
69
68
67
66
65
64
63
62
61
60
59
58
57
56
55
54
Fluo xetine
Fluoxetine
Fluoxetine
Fluoxetine
Fluoxetine
Fluoxetine
Fluoxetine
Fluoxetine
Fluoxetine
Fluoxetine
Fluoxetine
Fluoxetine
Vehicle
Vehicle
Vehicle
Vehicle
Vehicle
Vehicle
Vehicle
Vehicle
Vehicle
Vehicle
Vehicle
Vehicle
294
267
117
160
166
203
207
165
169
176
81
269
234
96
246
442
184
235
258
277
407
289
292
284
294
283
305
293
303
300
305
300
290
300
293
290
294
295
292
300
300
297
301
296
288
287
294
283
290
279
302
285
300
290
300
299
289
301
301
290
300
299
294
295
300
297
301
294
287
BW
(g) 2
288
296
280
289
282
299
293
302
298
302
298
294
305
304
296
302
301
297
299
304
300
303
299
288
BW
(g) 3
290
300
285
293
283
307
290
305
295
300
300
300
310
309
297
311
303
302
307
311
303
305
302
293
BW
(g) 4
291
300
288
291
287
313
294
307
299
299
299
298
316
312
306
316
304
304
313
315
306
309
307
300
BW
(g) 5
297
300
290
293
285
307
294
308
295
304
299
299
319
312
306
315
307
306
313
313
307
314
305
300
BW
(g) 6
296
306
292
295
290
316
300
312
301
302
305
305
321
319
313
322
317
314
321
323
315
319
317
308
BW
(g) 7
294
307
293
292
287
314
302
317
298
305
302
305
327
320
311
325
316
314
320
318
311
322
314
305
BW
(g) 8
291
303
292
294
284
310
299
310
300
299
304
304
324
319
313
328
320
314
322
324
315
320
317
310
BW
(g) 9
296
309
295
295
288
317
302
316
300
303
300
307
330
325
317
324
318
320
323
324
314
317
317
308
297
307
297
295
285
316
306
322
308
302
307
305
333
325
322
328
324
321
328
328
318
330
320
316
299
312
295
298
290
307
309
323
305
305
308
308
337
330
321
331
328
325
327
335
323
333
320
314
290
312
298
300
290
324
310
323
303
304
310
315
342
335
324
336
329
333
331
334
329
332
322
322
302
317
302
300
289
321
307
329
307
306
312
315
341
337
327
340
332
329
332
342
327
339
330
323
306
320
305
305
290
323
317
328
308
306
315
315
345
341
331
341
334
332
341
346
328
340
333
325
301
314
307
307
290
323
321
331
306
307
322
319
345
341
335
343
341
335
337
345
336
345
334
329
308
320
307
307
294
328
320
332
317
311
319
320
348
345
336
346
342
341
346
351
342
347
342
335
306
323
312
309
295
329
326
339
313
313
327
320
348
348
339
343
342
343
346
355
338
351
338
337
307
322
314
310
294
330
327
336
312
311
324
319
351
353
337
351
346
346
349
351
348
353
342
334
307
324
314
311
297
332
328
337
314
314
317
326
355
353
342
346
344
349
352
356
342
357
345
344
310
331
314
313
300
322
325
342
315
315
333
322
352
352
348
354
352
348
353
359
348
355
345
338
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
(g) 10 (g) 11 (g) 12 (g) 13 (g) 14 (g) 15 (g) 16 (g) 17 (g) 18 (g) 19 (g) 20 (g) 21
NSF
BW
Rat nr Treatm ent
Latency (s) (g) 1
77
Fig. S1: Correlations between latency to start eating in the novelty suppressed feeding test
(NSFT) and expression of myelination-related genes in adult fluoxetine-exposed rats. Spearman
correlations between latency to start eating (s) and expression of myelination-related genes (2ΔCT). A
significant negative correlation was found for latency to start eating and expression of Cldn11 (p<0.05)
and a trend for a negative correlation was found for latency to start eating and expression of Tspan2
(p<0.1).
Fig. S2: Correlations between time spent in the corner (OFC) and expression of myelinationrelated genes in neonatally fluoxetine-exposed rats. Spearman correlations between OFC (s) and
expression of myelination-related genes (2ΔCT). A significant negative correlation was found for OFC
and expression of Cldn11, Cnp, Plp1_3_5 and Plp1_2_3 (p<0.05) and a trend for a negative
correlation was found for Mag (p<0.1).
Fig. S3: Correlations between time spent in the center (OFCe) and expression of myelinationrelated genes in neonatally fluoxetine-exposed rats. Spearman correlations between OFCe (s) and
expression of myelination-related genes (2ΔCT). A trend for a positive correlation was found for OFCe
and expression of Cldn11, Plp1_3_5, Plp1_2_3 and Mog (p<0.1).
Fig S4: Potential pathway for fluoxetine affecting myelination-related genes. It has been shown
that fluoxetine can stimulate the 5-HT2B receptor on astrocytes, which results in activation of its
downstream signaling cascades3. The 5-HT2B receptor is Gq/11 protein coupled and stimulates the
diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) pathway (IP3-DAG). DAG is the
physiological activator of protein kinase C (PKC), which in turn can activate the mitogen-activated
protein kinase (originally called ERK, extracellular signal-regulated kinase) pathway (MAPK-ERK1/2)3.
A third signal transduction pathway activated by fluoxetine-induced stimulation of the 5-HT2B receptor
is the PI3K-AKT pathway3../../Users/Yvet/Downloads/fnbeh-09-00025.pdf. Activation of these signal
transduction pathways results in transcription factor activation and transcription of neurotrophic
factors. MAPK-ERK1/2 activation by fluoxetine results in transcription of glial-derived nerve factor
(GDNF)3, 4../../Users/Yvet/Downloads/fnbeh-09-00025.pdf. In addition, transcription of brain derived
neurotrophic factor transcription is induced, but not through the MAPK-ERK1/2 pathway4. Possibly
also transcription and release of ciliary neurotrophic factor (CNTF) is triggered by fluoxetine. The
released CNTF can bind to the CNTF receptor triggering intracellular signaling through three major
signal transduction pathways: JAK- STAT, MAPK-ERK1/2 and PI3K-AKT5. These signal transduction
pathways all mediate different responses. Studies have shown that CNTF-activated astrocytes release
an astrocyte specific factor (>30 kD), which promotes proliferation and survival of oligodendrocyte
precursor cells6. Furthermore, studies have shown that CNTF can induce maturation of
oligodendrocytes7,
8.
An increase in the number of oligodendrocyte precursors and mature
oligodendrocytes in our hippocampus tissue might explain the enhanced expression of myelinationrelated genes. It has been shown that the expression of Nkx6.2 in oligodendrocytes is strongly
induced after CNTF treatment (grey dotted line)9 and that Plp1 is required for transport and expression
of SIRT2 in myelin (grey dotted line)10.
Supplementary references
1.
Anders S, Huber W. Differential expression analysis for sequence count data.
Genome biology 2010; 11(10): R106.
2.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25(4): 402408.
3.
Hertz L, Rothman DL, Li B, Peng L. Chronic SSRI stimulation of astrocytic 5-HT2B
receptors change multiple gene expressions/editings and metabolism of glutamate,
glucose and glycogen: a potential paradigm shift. Frontiers in behavioral
neuroscience 2015; 9: 25.
4.
Mercier G, Lennon AM, Renouf B, Dessouroux A, Ramauge M, Courtin F, et al. MAP
kinase activation by fluoxetine and its relation to gene expression in cultured rat
astrocytes. J Mol Neurosci 2004; 24(2): 207-216.
5.
Askvig JM, Watt JA. The MAPK and PI3K pathways mediate CNTF-induced neuronal
survival and process outgrowth in hypothalamic organotypic cultures. Journal of cell
communication and signaling 2015.
6.
Albrecht PJ, Enterline JC, Cromer J, Levison SW. CNTF-activated astrocytes release
a soluble trophic activity for oligodendrocyte progenitors. Neurochemical research
2007; 32(2): 263-271.
7.
Mayer M, Bhakoo K, Noble M. Ciliary neurotrophic factor and leukemia inhibitory
factor promote the generation, maturation and survival of oligodendrocytes in vitro.
Development 1994; 120(1): 143-153.
8.
Stankoff B, Aigrot MS, Noel F, Wattilliaux A, Zalc B, Lubetzki C. Ciliary neurotrophic
factor (CNTF) enhances myelin formation: a novel role for CNTF and CNTF-related
molecules. J Neurosci 2002; 22(21): 9221-9227.
9.
Rivera FJ, Kandasamy M, Couillard-Despres S, Caioni M, Sanchez R, Huber C, et al.
Oligodendrogenesis of adult neural progenitors: differential effects of ciliary
neurotrophic factor and mesenchymal stem cell derived factors. J Neurochem 2008;
107(3): 832-843.
10.
Werner HB, Kuhlmann K, Shen S, Uecker M, Schardt A, Dimova K, et al. Proteolipid
protein is required for transport of sirtuin 2 into CNS myelin. J Neurosci 2007; 27(29):
7717-7730.
Download