Inhibition of Endoplasmic Reticulum Stress is Involved in the Neuroprotective Effect of bFGF in the 6-OHDA-Induced Parkinson’s Disease Model
Cai Pingtao1, Ye Jingjing1, Zhu Jingjing1, Liu Dan1, Chen Daqing2, Wei Xiaojie3, Johnson Noah R.4, Wang Zhouguang1, Zhang Hongyu1, Cao Guodong4, Xiao Jian1,*, Ye Junming5,*, Lin Li1,*
1School of Pharmaceutical Sciences, Key Laboratory of Biotechnology and Pharmaceutical Engineering, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China 2Emergency Department, the Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, China 3Department of Neurosurgery, Cixi People’s Hospital, Wenzhou Medical University, Ningbo, Zhejiang, 315300, China 4Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA. 5Department of Anesthesia, the First Affiliated Hospital, Gannan Medical College, Ganzhou, 341000, China
Parkinson's disease (PD) is a progressive neurodegenerative disorder with complicated pathophysiologic mechanisms. Endoplasmic reticulum (ER) stress appears to play a critical role in the progression of PD. We demonstrated that basic fibroblast growth factor (bFGF), as a neurotropic factor, inhibited ER stress-induced neuronal cell apoptosis and that 6-hydroxydopamine (6-OHDA)-induced ER stress was involved in the progression of PD in rats. bFGF administration improved motor function recovery, increased tyrosine hydroxylase (TH)-positive neuron survival, and upregulated the levels of neurotransmitters in PD rats. The 6-OHDA-induced ER stress response proteins were inhibited by bFGF treatment. Meanwhile, bFGF also increased expression of TH. The administration of bFGF activated the downstream signals PI3K/Akt and Erk1/2 in vivo and in vitro. Inhibition of the PI3K/Akt and Erk1/2 pathways by specific inhibitors partially reduced the protective effect of bFGF. This study provides new insight towards bFGF translational drug development for PD involving the regulation of ER stress.
Cai Pingtao,Ye Jingjing,Zhu Jingjing, et al. Inhibition of Endoplasmic Reticulum Stress is Involved in the Neuroprotective Effect of bFGF in the 6-OHDA-Induced Parkinson’s Disease Model[J]. Aging and disease,
2016, 7(4): 336-449.
Figure 1. Effects of bFGF infusions on amphetamine-induced rotation and neurotransmitter levels in the striatum of PD model rats. (A) Effects of bFGF administration on the apomorphine (APO)-induced ipsilateral rotations measured at 1, 2 and 3 weeks after lesion. **P < 0.01 versus sham group, #P < 0.05 and ##P < 0.01 versus PD group. (B), (C), (D) The levels of monoamine neurotransmitters in the striatum detected by HPLC-ED at 3 weeks post-lesion. **P < 0.01 versus sham group, #P < 0.05 and ##P < 0.01 versus PD group. Values are presented as the mean ± SD (n = 10).
Figure 2. Effects of bFGF on TH levels and ER stress-related proteins at 3 weeks post-lesion in PD rats. (A) Immunohistochemistry of TH-positive cells in the right and left nigra. (B) TH levels analyzed by Western blot. **P < 0.01 versus sham group, ##P < 0.01 versus PD group. (C) Immunohistochemical analysis of GRP78, CHOP and caspase-12 in the left nigra. (D) GRP78, CHOP and caspase-12 levels analyzed by Western blot. (E) Erk1/2 and Akt analyzed by Western blot. **P < 0.01 versus sham group, #P < 0.05 and ##P < 0.01 versus PD group (n = 6).
Figure 3. Effects of bFGF on 6-OHDA-induced apoptosis in primary hippocampal neurons. (A) Primary hippocampal neurons were treated with different concentrations of 6-OHDA for 24 h, and then cell viability was assessed by MTT assay. (B) Primary hippocampal neurons were treated with 6-OHDA (150 µM) and different concentrations of bFGF for 24 h, and then cell viability was assessed by MTT assay. (C) Primary hippocampal neurons were treated with 6-OHDA (150 µM) and bFGF (20 ng/ml) for 24 h, and then cells were stained with annexin V-FITC/propidium iodide and detected by flow cytometry; the lower right panel indicates the apoptotic cells. (D) Levels of cell apoptosis. *P < 0.05 versus control group, **P < 0.01, ***P< 0.001, #P < 0.05 versus 6-OHDA group (n = 3).
Figure 4. Effect of bFGF on 6-OHDA-induced ER stress and Erk/Akt phosphorylation in primary hippocampal neurons. (A) Primary hippocampal neurons were treated with 6-OHDA (150 µM) and bFGF (20 ng/ml) for 24 h. Cells were then collected, and ATF6,GRP78, XBP-1, caspase12, CHOP were analyzed by Western blot. (B) Optical density analysis of ER stress-related proteins. (C) p-Erk1/2, Erk1/2, p-Akt and Akt were analyzed by Western blot. (D) Optical density analysis of p-Erk/Erk and p-Akt/Akt. *P < 0.05 and **P < 0.01 versus control group, #P < 0.05 versus 6-OHDA group (n = 3).
Figure 5. Inhibition of PI3K/Akt and Erk1/2 pathways partially attenuates the reduction of the ER stress by bFGF in primary hippocampal neurons. (A) Primary hippocampal neurons were treated with or without the specific inhibitors LY294002 (20 μM) and U0126 (20 μM). Cell lysates were then analyzed by Western blotting for the expression of p-Akt, Akt, p-Erk, Erk, GRP78, XBP-1, ATF6, cleaved-caspase-12, and TH. Bar diagrams of (B) p-Akt/Akt and p-Erk/Erk; (C) GRP78, XBP-1, and ATF6; (D) cleaved caspase-12 and CHOP; and (E) TH from three Western blot analyses. *P < 0.05 versus control group, #P < 0.05 versus 6-OHDA group (n = 3).
Figure 6. Inhibition of PI3K/Akt and Erk1/2 pathways partially impairs the protective effects of bFGF in 6-OHDA-induced primary hippocampal neurons. (A) Primary hippocampal neurons were collected and stained with annexin V-FITC/PI and detected by flow cytometry; the lower right panel indicates apoptotic cells. (B) Cell apoptosis levels from three separate experiments. ***P < 0.001 versus control group, #P < 0.05 versus 6-OHDA group (n = 3).
Redgrave P, Rodriguez M, Smith Y, Rodriguez-Oroz MC, Lehericy S, Bergman H, et al. (2010). Goal-directed and habitual control in the basal ganglia: implications for Parkinson's disease. Nat Rev Neurosci, 11: 760-772
Schwarz J, Storch A (2010). Transplantation in Parkinson's disease: will mesenchymal stem cells help to reenter the clinical arena. Transl Res, 155: 55-56
Aminzadeh MA, Sato T, Vaziri ND (2012). Participation of endoplasmic reticulum stress in the pathogenesis of spontaneous glomerulosclerosis-Role of intra-renal angiotensin system. Transl Res, 160: 309-318
Li S, Yang L, Selzer ME, Hu Y (2013). Neuronal endoplasmic reticulum stress in axon injury and neurodegeneration. Ann Neurol, 74: 768-777
Cheng B, Gong H, Xiao H, Petersen RB, Zheng L, Huang K (2013). Inhibiting toxic aggregation of amyloidogenic proteins: a therapeutic strategy for protein misfolding diseases. Biochim Biophys Acta, 1830: 4860-4871
Mercado G, Valdés P, Hetz C (2013). An ERcentric view of Parkinson's disease. Trends Mol Med, 19: 165-175
Galehdar Z, Swan P, Fuerth B, Callaghan SM, Park DS, Cregan SP (2010). Neuronal apoptosis induced by endoplasmic reticulum stress is regulated by ATF4-CHOP-mediated induction of the Bcl-2 homology 3-only member PUMA. J Neurosci, 30: 16938-16948
Ryu EJ, Harding HP, Angelastro JM, Vitolo OV, Ron D, Greene LA (2002). Endoplasmic reticulum stress and the unfolded protein response in cellular models of Parkinson's disease. J Neurosci, 22: 10690-10698
Hoozemans J, Van Haastert E, Eikelenboom P, De Vos R, Rozemuller J, Scheper W (2007). Activation of the unfolded protein response in Parkinson’s disease. Biochem Biophys Res Commun, 354: 707-711
Chadi G, Silva C, Maximino JR, Fuxe K, da Silva GO (2008). Adrenalectomy counteracts the local modulation of astroglial fibroblast growth factor system without interfering with the pattern of 6-OHDA-induced dopamine degeneration in regions of the ventral midbrain. Brain Res, 1190: 23-38
Hsuan SL, Klintworth HM, Xia Z (2006). Basic fibroblast growth factor protects against rotenone-induced dopaminergic cell death through activation of extracellular signal-regulated kinases 1/2 and phosphatidylinositol-3 kinase pathways. J Neurosci, 26: 4481-4491
Zhang HY, Zhang X, Wang ZG, Shi HX, Wu FZ, Lin BB, et al. (2013). Exogenous basic fibroblast growth factor inhibits ER stress-induced apoptosis and improves recovery from spinal cord injury. CNS Neurosci Ther, 19: 20-29
Wang ZG, Zhang HY, Xu X, Shi HX, Yu X, Wang X, et al. (2012). bFGF inhibits ER stress induced by ischemic oxidative injury via activation of the PI3K/Akt and ERK1/2 pathways. Toxicol Lett, 212: 137-146
Zhang HY, Wu FZ, Kong XX, Yang J, Chen H, Deng L, et al. (2014). Nerve growth factor improves functional recovery by inhibiting endoplasmic reticulum stress-induced neuronal apoptosis in rats with spinal cord injury. J Transl Med, 12: 130
Xu R, Chen J, Cong X, Hu S, Chen X (2008). Lovastatin protects mesenchymal stem cells against hypoxia and serum deprivation induced apoptosis by activation of PI3K/Akt and ERK1/2. J Cell Biochem, 103: 256-269
Zhou JX, Zhang HB, Huang Y, He Y, Zheng Y, Anderson JP, et al. (2013). Tenuigenin Attenuates α-Synuclein Induced Cytotoxicity by Down-Regulating Polo-Like Kinase 3. CNS Neurosci Ther, 19: 688-694
Hoozemans JJ, Scheper W (2012). Endoplasmic reticulum: the unfolded protein response is tangled in neurodegeneration. Int J Biochem Cell Biol, 44: 1295-1298
Holtz WA, O'Malley KL (2003). Parkinsonian mimetics induce aspects of unfolded protein response in death of dopaminergic neurons. J Biol Chem, 278: 19367-19377
Silva RM, Ries V, Oo TF, Yarygina O, Jackson Lewis V, Ryu EJ, et al. (2005). CHOP/GADD153 is a mediator of apoptotic death in substantia nigra dopamine neurons in an in vivo neurotoxin model of parkinsonism. J Neurochem, 95: 974-986
Zhang HY, Wang ZG, Lu XH, Kong XX, Wu FZ, Lin L, et al. (2014). Endoplasmic reticulum stress: relevance and therapeutics in central nervous system diseases. Mol Neurobiol, 51: 1343-1352
Wan XS, Lu XH, Xiao YC, Lin Y, Zhu H, Ding T, et al. (2014). ATF4-and CHOP-dependent induction of FGF21 through endoplasmic reticulum stress. Biomed Res Int, 2014
Tanaka KI, Fukuoka S, Kawahara S, Kimoto N, Ogawa N (2013). Effect of cabergoline on increase of several ER stress-related molecules in 6-OHDA-lesioned mice. Neurol Sci, 34: 259-261
Luo F, Wei L, Sun C, Chen X, Wang T, Li Y, et al. (2012). HtrA2/Omi is involved in 6-OHDA-induced endoplasmic reticulum stress in SH-SY5Y cells. J Mol Neurosci, 47: 120-127
Soto-Otero R, Méndez-Álvarez E, Hermida-Ameijeiras Á, Muñoz-Patiño AM, Labandeira-Garcia JL (2000). Autoxidation and Neurotoxicity of 6-Hydroxydopamine in the Presence of Some Antioxidants. J Neurochem, 74: 1605-1612
Glinka YY, Youdim MB (1995). Inhibition of mitochondrial complexes I and IV by 6-hydroxydopamine. Eur J Pharmacol, 292: 329-332
Egawa N, Yamamoto K, Inoue H, Hikawa R, Nishi K, Mori K, et al. (2011). The endoplasmic reticulum stress sensor, ATF6α, protects against neurotoxin-induced dopaminergic neuronal death. J Biol. Chem, 286: 7947-7957
Bhandary B, Marahatta A, Kim HR, Chae HJ (2012). An involvement of oxidative stress in endoplasmic reticulum stress and its associated diseases. Int J Mol Sci, 14: 434-456
Bouman L, Schlierf A, Lutz A, Shan J, Deinlein A, Kast J, et al. (2011). Parkin is transcriptionally regulated by ATF4: evidence for an interconnection between mitochondrial stress and ER stress. Cell Death Differ, 18: 769-782
Kosuge Y, Taniguchi Y, Imai T, Ishige K, Ito Y (2011). Neuroprotective effect of mithramycin against endoplasmic reticulum stress-induced neurotoxicity in organotypic hippocampal slice cultures. Neuropharmacology, 61: 252-261
Takano K, Tabata Y, Kitao Y, Murakami R, Suzuki H, Yamada M, et al. (2007). Methoxyflavones protect cells against endoplasmic reticulum stress and neurotoxin. Am J Physiol Cell Physiol, 292: C353-C361
Lin L, Yang J, Lin R, Yu L, Gao H, Yang S, et al. (2013). In vivo study on the monoamine neurotransmitters and their metabolites change in the striatum of Parkinsonian rats by liquid chromatography with an acetylene black nanoparticles modified electrode. J Pharm Biomed, 72: 74-79
Wang L, Yang HJ, Xia YY, Feng ZW (2010). Insulin-like growth factor 1 protects human neuroblastoma cells SH-EP1 against MPP+-induced apoptosis by AKT/GSK-3β/JNK signaling. Apoptosis, 15: 1470-1479
Zhang Q, Zhang J, Jiang C, Qin J, Ke K, Ding F (2014). Involvement of ERK1/2 pathway in neuroprotective effects of pyrroloquinoline quinine against rotenone-induced SH-SY5Y cell injury. Neuroscience, 270: 183-191
Fieblinger T, Sebastianutto I, Alcacer C, Bimpisidis Z, Maslava N, Sandberg S, et al. (2014). Mechanisms of dopamine D1 receptor-mediated ERK1/2 activation in the parkinsonian striatum and their modulation by metabotropic glutamate receptor type 5. J Neurosci, 34: 4728-4740
Sanchez A, Tripathy D, Yin X, Luo J, Martinez J, Grammas P (2012). Pigment epithelium-derived factor (PEDF) protects cortical neurons in vitro from oxidant injury by activation of extracellular signal-regulated kinase (ERK) 1/2 and induction of Bcl-2. Neurosci Res, 72: 1-8
Xu Y, Zhang Q, Yu S, Yang Y, Ding F (2011). The protective effects of chitooligosaccharides against glucose deprivation-induced cell apoptosis in cultured cortical neurons through activation of PI3K/Akt and MEK/ERK1/2 pathways. Brain Res, 1375: 49-58
Clough RL, Stefanis L (2007). A novel pathway for transcriptional regulation of α-synuclein. FASEB J, 21: 596-607
Morishima N, Nakanishi K, Tsuchiya K, Shibata T, Seiwa E (2004). Translocation of Bim to the endoplasmic reticulum (ER) mediates ER stress signaling for activation of caspase-12 during ER stress-induced apoptosis. J Biol Chem, 279: 50375-50381
Jin K, LaFevre-Bernt M, Sun Y, Chen S, Gafni J, Crippen D, et al. (2005). FGF-2 promotes neurogenesis and neuroprotection and prolongs survival in a transgenic mouse model of Huntington's disease. Proc Natl Acad Sci U S A, 102: 18189-18194
Kiyota T, Ingraham KL, Jacobsen MT, Xiong H, Ikezu T (2011). FGF2 gene transfer restores hippocampal functions in mouse models of Alzheimer's disease and has therapeutic implications for neurocognitive disorders. Proc Natl Acad Sci U S A, 108: E1339-E1348
Bogousslavsky J, Victor SJ, Salinas EO, Pallay A, Donnan GA, Fieschi C, et al. (2002). Fiblast (trafermin) in acute stroke: results of the European-Australian phase II/III safety and efficacy trial. Cerebrovasc Dis, 14: 239-251
Paciaroni M, Bogousslavsky J (2011). Trafermin for stroke recovery: is it time for another randomized clinical trial. Expert Opin Biol Ther, 11: 1533-1541
Zhao YZ, Li X, Lu CT, Lin M, Chen LJ, Xiang Q, et al. (2014). Gelatin nanostructured lipid carriers-mediated intranasal delivery of basic fibroblast growth factor enhances functional recovery in hemiparkinsonian rats. Nanomedicine, 10: 755-764
Cuevas P, Carceller F, Muñoz-Willery I, Giménez-Gallego G (1998). Intravenous fibroblast growth factor penetrates the blood-brain barrier and protects hippocampal neurons against ischemia-reperfusion injury. Surg Neurol, 49: 77-84
Lee H, Pienaar IS (2013). Disruption of the blood-brain barrier in Parkinson's disease: curse or route to a cure. Front Biosci, 19: 272-280
Zhu G, Chen G, Shi L, Feng J, Wang Y, Ye C, et al. (2015). PEGylated rhFGF-2 Conveys Long-term Neuroprotection and Improves Neuronal Function in a Rat Model of Parkinson’s Disease. Mol Neurobiol, 51: 32-42
Wei X, He S, Wang Z, Wu J, Zhang J, Cheng Y, et al. (2014). Fibroblast growth factor 1attenuates 6-hydroxydopamine-induced neurotoxicity: an in vitro and in vivo investigation in experimental models of parkinson’s disease. Am J Transl Res, 6: 664
Timmer M, Cesnulevicius K, Winkler C, Kolb J, Lipokatic-Takacs E, Jungnickel J, et al. (2007). Fibroblast growth factor (FGF)-2 and FGF receptor 3 are required for the development of the substantia nigra, and FGF-2 plays a crucial role for the rescue of dopaminergic neurons after 6-hydroxydopamine lesion. J Neurosci, 27: 459-471
Yang F, Liu Y, Tu J, Wan J, Zhang J, Wu B, et al. (2014). Activated astrocytes enhance the dopaminergic differentiation of stem cells and promote brain repair through bFGF. Nat Commun, 17:5627.
Soto I, Rosenthal JJ, Blagburn JM, Blanco RE (2006). Fibroblast growth factor 2 applied to the optic nerve after axotomy up-regulates BDNF and TrkB in ganglion cells by activating the ERK and PKA signaling pathways. J Neurochem, 96: 82-96
Yoshida K, Gage FH (1991). Fibroblast growth factors stimulate nerve growth factor synthesis and secretion by astrocytes. Brain Res, 538: 118-126