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Supplementary Information:
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Orexin signaling regulates both the hippocampal clock and the expression of
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Alzheimer's disease-risk genes
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Zhixiong Ma1, 2, Weiliang Jiang3, & Eric Erquan Zhang2*
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1
College of Life Sciences, Beijing Normal University, Beijing 100875, China.
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2
National Institute of Biological Sciences, Beijing 102206, China.
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3
Department of Gastroenterology, Shanghai First People’s Hospital, School of Medicine, Shanghai Jiao
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Tong University, Shanghai, 200080, China.
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*Corresponding Email:
[email protected]
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Supplementary Figure Legends:
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Supplementary Figure1: Orexin A shortens the period of hippocampal circadian
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clock.
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A and B：Orexin A shortens the period of hippocampal circadian clock in a dose
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dependent manner. (C) pre-orexin A treatment and post-orexin A shows the orexin A
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shortens the period of hippocampal circadian clock (n=4, * p< 0.05, paired T-test). D and
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E: The EMPA block the effect of orexin B induced shorted period in hippocampus
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(n=6-10, * p0.05,
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student’s T-test).
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Figure S1
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Figure S2
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Figure S3
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Supplementary Tables: Supplementary Table 1: qPCR primers of AD-risk genes A2m-1F TCAAAGTTGCAGTACCAGAG A2m-1R TCCCATAGGTGTATATGCCA Abca1-1F GTGTTTCTGGATGAACCAACC Abca1-1R CTTGACAATGCTTAGGGCAC Ache-1F CCTGAAGCCCTTAGAGGTG Ache-1R CTCGTCCAGAGTATCGGTG Adam10-1F CTGGACATATTTATGGTGAAGAAGG Adam10-1R CCACGAGTCTTGATGAAACC Adam9-1F AGATTGCCAGTTCCTTCCA Adam9-1R GAACCGTTGCAGTACTCAG Apbb1-1F ATTGTATCCGCCAGCTCTC Apbb1-1R GCAGATCCTTTCCCTCTCC Apbb2-1F GGGCAAAGACATGTACCTG Apbb2-1R GTAAGCAAAGTCCGTCTCAC APH1A-1F AGCTCCTTAAGAAGGCAGATG APH1A-1R TGATGATACCGAAGGACAGAC Aplp1-1F GCGAATGAATCAGAGCCTG Aplp1-1R AGAAGCTCCTGGATCTGTG Aplp2-1F GGAAATTGATGAGCTCCTTCAG Aplp2-1R GGGATCTCCTCACTCTCCT Apoa1-1F TGTGGATGCGGTCAAAGACA Apoa1-1R TCCCAGAAGTCCCGAGTCAA Apoe-1F CACATTGCTGACAGGATGC Apoe-1R TAATCCCAGAAGCGGTTCAG Apba1-1F GAATGGAATTATCTGCAGCCT Apba1-1R ATTTCGATGATCCGATGTCC Apba3-1F CTCACAGAGACAAGAGAAATCC Apba3-1R TACTGGTTGTTCCTGTCCC App-1F ATGTCCCAGGTCATGAGAG App-1R GATAACGGCCTTCTTGTCAG Bace1-1F GTTTCCAAGCTCAACATCCTG Bace1-1R AGTAGCGATGCAGGAAAGG Bace2-1F CACGAACATCTCTGATTCCA Bace2-1R GCGTTTCAGAATTTGTCCAG Bche-1F AAATATGGACATCCCAATGGG Bche-1R GGGAGCACGAAGTTTAGAG BDNF-1F TCATACTTCGGTTGCATGAAGG 6
BDNF-1R Capn1-1F Capn1-1R Casp3-1F Casp3-1R Casp4-1F Casp4-1R CDC2-1F CDC2-1R Cdk5-1F Cdk5-1R Cdkl1-1F Cdkl1-1R Chat-1F Chat-1R Clu-1F Clu-1R Ctsb-1F Ctsb-1R Ctsc-1F Ctsc-1R Ctsd-1F Ctsd-1R Ctsg-1F Ctsg-1R CTSL1-1F CTSL1-1R Ep300-1F Ep300-1R Ern1-1F Ern1-1R Gap43-1F Gap43-1R Gnao1-1F Gnao1-1R GNAZ-1F GNAZ-1R Gnb1-1F Gnb1-1R
ACACCTGGGTAGGCCAAGTT CTATGAGTGGAACAAAGTGGAC AACGACATCCAGAACTCCC AATGGATTATCCTGAAATGGGC GAGCGAGATGACATTCCAG CAACAATTGCCACTGTCCA TTCTCCAGAGTTCCCACCT AAATTGGAGAAGGTACTTACGG CTCCTTCTTCCTCGCTTTC GATTGTGAAGTCATTCCTCTTCC TTCAGGTCCCTATGTAGCAC GCACCAGCAAGTATTTAGC CAACTCAAGTGTTTCCATGTC TGGGATCTGGCAACTTCGTC AGCACCTCCTCACAGCTAGA ATACCTGCATGAAGTTCTATGC GGTTTAGAAACTCCTCTAGCTG TATCCCTCTGGAGCATGGA TGGTAAGCAGCCTACATGAG CACCTACCCTGATCTGCTG CTTCTGTTGCTTCCATCACC GATTATCAGAATCCCTCTGCG ATCAGGTCTTCCACAGAGC CTCCAGGAGGTGCAGCTAAG TCCATAGGAGACGATGCCCT GACTGTATGGCACGAATGAG TCTTCTCCCATATCGCTCTC TGCGTCTGTAGAGCTGTGAG TCTCTTCTCCGAAACGGGGT AAGAAGATCCAGTCCTGCA AAAGGGAAGTTTCGTCAGG GGAGAAAGACGCTGTAGAC CATGTTCTTGGTCAGCCTC CACTTCAGGCTGTTTGACG TGGAGTCGAAGAGCATGAG GGCAGAGGTCAGAACGCAAA GCTCGTCTGGTTGTCCTCATA CCACAAACAAGGTTCATGC CCACATAATTCCCAGAAGGAG
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Gnb2-1F Gnb2-1R Gnb4-1F Gnb4-1R Gnb5-1F Gnb5-1R Gng11-1F Gng11-1R Gng3-1F Gng3-1R Gng4-1F Gng4-1R Gngt1-1F Gngt1-1R Gngt2-1F Gngt2-1R GSK3A-1F GSK3A-1R Gsk3b-1F Gsk3b-1R Hsd17b10-1F Hsd17b10-1R Ide-1F Ide-1R Il1a-1F Il1a-1R Insr-1F Insr-1R Lpl-1F Lpl-1R Lrp1-1F Lrp1-1R Lrp6-1F Lrp6-1R Ntrk1-1F Ntrk1-1R Ntrk2-1F Ntrk2-1R Pkp4-1F
ACACTGACCCAGATCACAG ACCTAGACATACTAAAGCAGGG GGGCTCCAGCTCTTCACTTG GAGGGATGGCGTGCATCTTA GTCTCCATCCTGTTTGGAC ATGCCCAAACTCTTAGGGT CTTCACATCGAGGATCTGC TTTAGATACCTGTTGTCTCTGC TATGAGTATTGGTCAAGCACG GCCTTGGACACCTTTATCC GACCCTGAGTCAGCTTCTC TTCATTCCTGCACTCCCTG AAAAACAGCTTCCCTGACAGAA AGCCTCCTTTGAGTTCCTTGA TGAGAAGGAGCTGTTGAGG CCTGTCTTGGAAATCAGATCAC AAGCTCTGCGATTTTGGCAGT GAGTTCTGGAGCACGGTAGTA CTTGGACAAAGGTCTTCCG AATGTGCACAAGCTTCCAG GTGGCCATTAAGACATACCAC AAGATTCACATTGATAACCCGC TGCATCAGGGATGAATGCA AAGAGATCGCATATAGGCCTC ATGATCTGGAAGAGACCATCC CGAGCTTCATCAGTTTGTATCTC TCAGAAAGTTTGCCCAACC GTCATCAGGTTCCGAACAG AGAATTACTGGTTTGGATCCAG AAATCAGCGTCATCAGGAG TGTATGAAGGTGGAGAGCC AGTTGGTAGGCTTGTCAGG GAGGAAGATCTTGATTTCAGAGG GTCCAATACATGTACCCAACC CATACACAGACGCTTCATGAC TGTGGGTACACCACAACTG AGAACGAGTATGGGAAGGA TTGGGTTTGTCTCGTAGTC TCCATGAAAGTGAGGGATCG
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Pkp4-1R Plat-1F Plat-1R Plau-1F Plau-1R Plg-1F Plg-1R Prkca-1F Prkca-1R Prkcb-1F Prkcb-1R Prkcd-1F Prkcd-1R Prkce-1F Prkce-1R PRKCG-1F PRKCG-1R Prkci-1F Prkci-1R Prkcq-1F Prkcq-1R Prkcz-1F Prkcz-1R Psen1-1F Psen1-1R Psen2-1F Psen2-1R Snca-1F Snca-1R Sncb-1F Sncb-1R Ubqln1-1F Ubqln1-1R Uqcrc1-1F Uqcrc1-1R Uqcrc2-1F Uqcrc2-1R mINS-1 F1 mINS-1 R1
CTTCCTGGTACCCACTGTC TTATTGTCGGAATCCAGATGGT TCACAGTATTCCCACGTCAG ACAATTACTGCAGGAACCC ATTCTTGGACAAACTGCCT CCACGTACCCAACTACTCTC TCATCATTGTCTGGGTTCCT AGTCGGGAAATTTAAGGAGC TTCAGATCCCTGTAAATGATCC AGAACCACAAATTCACCGC AAAGCAGCAGACTTGACAC TGAGTTCTGGCTGGATCTG GTTTAATGGCTCCACGACG TTTGGCAAGGTCATGTTGG CGTCTTGTAGGATAACGTCCT CCTGCAATGTCAAGTCTGTAG ACACTCGAAGGTCACAAATTC ACGATGAGGATATCGATTGGG CTGGAAGCAAGAATGCAGC AACCGTGGAACTCTACTCC TGAGGTTTCAGCTCTAACCA GTGCAGCGAAAGGATATGG GACAGAATCCATATGCCTCCT GCTCTTATCTATTCCTCAACAATGG CTTGGGTACCCTCCTTTGG CAGAGATGGAAGAAGACTCCT AAGATGAAGTCTCCCAGGC GAGTCCTCTATGTAGGTTCCA CTTGCTCTTTGGTCTTCTCAG CTATGTCGGAAGCAAGACCA CAGAGAACACAGCTCCTCC AATCCTCAGCTGCAGAGTC TTCACGGTTCAAGAATCCCA CTTTCAAGGGAACAAAGAATCG TGTAGGCATTAAGATGAGCC AGCAGGCAGTAGATATGAGG CCTTTGGTAGTCAAACTAGATGC CACCCCACCTGGAGACCTTA CCACACACCAGGTAGAGAGC
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Supplementary Table 2: qPCR primers of core clock genes mDBP_mrna-f CCTGAGGAACAGAAGGATGA mDBP_mrna-r
ATCTGGTTCTCCTTGAGTCTTCTTG
mCRY2-F1
GCCTCTGCCTCCCTTGATTT
mCRY2-R1
TGACATAAGCAGCATGGGCA
mCLOCK-F1
CACGCGCTCCCGTGAAAG
mCLOCK-R1 mCRY1-F2
GTCCTTGTCATCTTCTTCCACCA AGCTCGTGTCCGTTCGTG
mCRY1-R2
CCTGCGCTTCCAAGAAAACC
mBMAL1_mrna-f
CTCGACACGCAATAGATGGGA
mBMAL1_mrna-r
CTTCCTTGGTCCACGGGTT
mPER1_mrna-f
TGAAGCAAGACCGGGAGAG
mPER1_mrna-r
CACACACGCCATCACATCAA
mPER2_mrna-f
GAAAGCTGTCACCACCATAGAA
mPER2_mrna-r
AACTCGCACTTCCTTTTCAGG
mNR1D1 qPCR F mNR1D1 qPCR R
TGGCATGGTGCTACTGTGTAAGG
mNR1D2 qPCR F
GGAGTTCATGCTTGTGAAGGCTGT
mNR1D2 qPCR R
CAGACACTTCTTAAAGCGGCACTG
ATATTCTGTTGGATGCTCCGGCG
Supplementary Table 3: qPCR primers of housekeeping genes, orexin precursor gene and orexin receptors mActin_mrna-nf GGCTGTATTCCCCTCCATCG mActin_mrna-nr CAGTTGGTAACAATGCCATGT mGapdh_mrna-nf GACCTCAACTACATGGTCTACA mGapdh_mrna-nr ACTCCACGACATACTCAGCAC mOrexin-PreF1 CCTGCCGTCTCTACGAACTG mOrexinPreR1 GGTGCTAAAGCGGTGGTAGT Hcrtr1 F3 CTGTGGCGCGATTATCTCTAC Hcrtr1 R3 GCCAGGGACAGGTTGACAA Hcrtr2 F3 GAGGATTCCCTCTCTCGTCG Hcrtr2 R3 GGTGTAGGTATTCCCTCCACA
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Supplementary Table 4: Primers for amplified the coding sequence of E4BP4 and the promotor regions of BACE1 and BACE2 Human E4BP4 F ACCGGCGGCCGCATGCAGCTGAGAAAAATGCA Human E4BP4 R ACCGGGCGCGCCTTACCCAGAGTCTGAAGCAG BACE1 pro gen-REV R AGCTAAGCTTTTGGGCCAAGGTGGGCCCCG BACE1 pro 1k ATCCGGTACCTTGCAGCCTGGAAAAACTCT BACE2 pro gen-REV R AGCTAAGCTTGTGCGCCCAAGCCCACGGCG BACE2 pro 1.4k ATCCGGTACCTTGTTTTTTAAAACAGCCAACATAA
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