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Forskolin suppresses Hypoxia-Induced Cognitive Dysfunction in Zebrafish
Quynh T N Nguyen , Jiwon Park , Dong Yeol Kim , Sang Min Kim , Hyun Jae Sung , Duong T T Tran , An Ngan Bui , Inn Oc Han ¹*
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Program in Biomedical Science and Engineering (BMSE)
Inha University, 100 Inha Ro, Michuholgu, Incheon 22212, South Korea
Abstract Forskolin activates PKA/CREB signaling and improved CREB-related synaptic plasticity
Repeated hypoxia (RH) exposure is reported to accelerate brain inflammation, affect the genes expression in RH-exposed zebrafish
initiation and progression of cognitive dysfunction. Forskolin, a diterpene natural plant A Con Fors B C
6h
24h
phytochemical obtained from (Coleus Forskohli), a potent direct activator of adenyl cyclase (AC) 4 Con Fors Con Fors 1.5 2.0
24h
6h
enzyme which further activates cAMP/PKA/CREB pathway. It has been shown that forskolin 3 C-fos Con Fors Con Fors 6h protein level (fold) 1.0 24h protein level (fold) 1.5
1.0
protects against various neuronal complications and induces long term memory; however, the substrate PKA substrate protein level (fold) 2 C-fos 0.5 0.5
PKA
underlying mechanism remains unclear. In this study, we showed that intraperitoneal 1 GAPDH c-Fos 0.0 Con Fors c-Fos 0.0 Con Fors
0
administration of 200 ng/g forskolin for 5 days on zebrafish notably recovered hypoxia-induced Con Fors 2.5 BDNF 1.5 0.6
social interaction impairment and learning memory deficit. Forskolin suppressed RH-induced 4 1.5 2.0 1.0 0.4
1.0
neuroinflammation indicated by the decrease of NFkB signaling and GFAP expression. RH p-CREB 3 6h mRNA level (fold) 0.5 24h mRNA level (fold) 1.5 β-actin BDNF 6h protein level (fold) 0.5 BDNF 24h protein level (fold) 0.2
1.0
triggered downregulated expression of CREB-dependent synaptic plasticity related genes, β-actin p-CREB protein level (fold) 2 c-Fos 0.0 c-Fos 0.5 0.0 0.0
1
0.0
including C-fos and BDNF, and this was restored by forskolin. As our previous study proposed 0 Con Fors Con Fors Con Fors Con Fors Con Fors
that RH-associated downregulation of O-GlcNAcylation flux could be an important causal factor Figure 3. Forskolin induces PKA/CREB signaling and upregulates transcription of c-Fos and BDNF in vitro
for cognitive defect, we further investigated whether O-GlcNacylation level is changed by
1.5
forskolin. Intriguingly, we observed that forskolin elevated O-GlcNAc level both in vitro and in Con Fors RH RH+Fors Con Fors RH RH+Fors
vivo. Under forskolin treatment, O-GlcNacylation flux on RH-exposed zebrafish was reserved in (fold) 1.0
line with the recovery of other inflammation and learning/memory markers. Thus, we are seeking DAPI DAPI C-fos 0.5
to study further about the potential regulation of O-GlcNAcylation on the neuroprotective effect of 0
Con Fors RH RH+Fors
forskolin.
c-Fos BDNF 2.5
2.0
BDNF (fold) 1.5
1.0
Materials and Methods Merge Merge 0.5
0
Con Fors RH RH+Fors
¶ Zebrafish ¶ Forskolin treatment ¶ Hypoxia
Figure 4. Forskolin restores RH-induced CREB-mediated genes downregulation in zebrafish brain
Forskolin suppressed RH-induced neuroinflammatory responses
Con Fors RH RH+Fors Con Fors RH RH+Fors 2.0
DL VD
1.5
DAPI DAPI DL (fold) 1.0
High genetic homology
≥ 85% with human Zebrafish GFAP - 0.5
• 200 ng/g ¶ T-maze 0.0 Con Fors RH RH+Fors
Large number of offspring • Intraperitonial injection GFAP GFAP 15
Short generation time
VD (fold) 10
Maintained at 28.0±1.0 C N2A cell Merge Merge GFAP - 5
o
with 12h light/12h dark cycle • Final concentration: 10 uM 0
Con Fors
• Diluted in DMSO Figure 5. Forskolin reduces RH-triggered microglial activation in zebrafish brain RH RH+Fors
¶ N2A cell
¶ Zebrafish Brain section Con Fors RH RH+Fors VD Con Fors RH RH+Fors 1.5
and Immunofluorescence 1.0
Social interaction and DAPI DAPI (fold)
Learning/Memory ability IkBα 0.5
¶ Passive avoidance test 0 Con Fors RH RH+Fors
IkBα p65 1.5
Mouse neuroblast 1.0
(fold)
Maintained in DMEM media 5% Merge Merge p65 0.5
FBS 0
4% PFA fixation Con Fors RH RH+Fors
Differentiated in serum-free DMEM 30% sucrose overnight Figure 6. Forskolin downregulates NFkB activity in zebrafish brain after hypoxia exposure
media Frozen in OCT, section 6 uM Learning/Memory ability
Forskolin upregulated HBP/O-GlcNacylation flux in vitro and in vivo
Results A C
Fors
Forskolin rescued RH-induced cognitive impaiment in zebrafish Con 3h 6h 24h Con Fors RH RH+Fors
OGT 2.0 DAPI
80 150 100 RL2 protein level (fold) 1.5 1.5 ✱ ✱
1.0
80
Running time (sec) 60 Interaction time (sec) 100 Latency (%) 60 RL2 0.5 Con 3h 6h 24h RL2 Merge RL2 (fold) 1.0
0.0
40
Fors
40
2.0
0.5
50
20
1.0
0
0 0 20 OGT protein level (fold) 1.5 0 Con Fors RH RH+Fors
1 2 3 Memory 1 2 3 Memory 1 2 3 Memory 0.5 Con Fors RH RH+Fors
Learning Learning Learning
β-actin
0.0
Figure 1. Effects of Forskolin on the RH-induced social interaction impairment in adult zebrafish using T- Con 3h 6h 24h DAPI
Fors
maze test
6h 24h
B Con Fors Con Fors
Con Fors RH RH+Fors 1.5
400 400 400 400 OGT OGT 1.0
Crossing time (s) 200 2h Crossing time (s) 300 2h Crossing time (s) 200 2h Crossing time (s) 300 2h GAPDH Merge OGT (fold) 0.5
300
300
200
200
100
100
100
100
0
1 st 2 nd 3 rd Memory 0 1 st 2 nd 3 rd 0 1 st 2 nd 3 rd 0 1 st 2 nd 3 rd 0 Con Fors RH RH+Fors
Learning Learning Memory Learning Memory Learning Memory Figure 7. Forskolin increases RL2 and OGT on translational level
Figure 2. Effects of Forskolin on the RH-induced learning/memory (L/M) impairment in adult zebrafish A, B. in vitro C. in vivo
using fear conditioning test
Conclussion
• Forskolin ameliorates RH-induced cognitive impairment by improving learning and memory ability and social interaction in zebrafish.
• Forskolin activates PKA/CREB signaling pathway and induce the transcription of c-Fos and BDNF, which is well-known to contribute to synaptic plasticity regulation.
• Forskolin also attenuates RH-triggered neuroinflammation through reducing activity of NFkB and brain astrocyte response.
• There is an upregulation of HBP/O-GlcNacylation flux under forskolin treatment both in vivo and in vitro. Further study is seeking to investigate whether this change of HBP pathway
play potential role in the neuroprotective effect of forskolin.
