Neuroprotective properties of Crocin on Glutamate

Excitotoxicity during ES Cell Neurogenesis

Atqiya Aishah, Sally Vuong and Annabelle Humphreys

Glutamate Crocin

★ Primary endogenous excitatory

neurotransmitter in CNS.

★ Excitotoxicity = neuronal injury

★ Elevated Glutamate in synaptic cleft leads to

influx of Ca2+ ions, inducing ROS production.

(stress)

★ Neuronal stress models the neuronal damage

in neurodegenerative disorders.

★ natural carotenoid found in flower

cronus (saffron)

★ potent neuronal antioxidant

★ activates notch signalling pathway

★ suppress inflammatory gene

expressions and ER stress →

axon demyelination and

degredation

★ induce anti-apoptotic proteins and

downregulate pro-apoptotic

(Syntichaki, 2003)

Aim and Hypothesis AIM: To determine the effects of glutamate and crocin on neuronal development

and test the neuroprotective properties of crocin against glutamate induced toxicity

as a model for neurodegenerative diseases and neuronal development.

HYPOTHESIS: Crocin is neuroprotective and reduces production of reactive

oxygen species induced by glutamate excitotoxicity during neuronal development.

Method

Neurogenesis

Three major types of neuroglia:

● astrocytes (structural support for CNS)

● Oligodendrocytes (form myelin in CNS)

● Microglia (act as macrophage in CNS)

Day 0

Day 1 Day 7 - Rosettes

Day 8- Yeast Contamination

1 µM Glutamate

1 µM Glutamate + 10 µM Crocin

Day 13

100 µM Glutamate 0.1 µM Crocin

100 µM Glutamate + 10 µm Crocin Untreated + FRR2 stain Untreated and no stain

Day 14

Crocin 10uM + Glutamate 100 uM

Glutamate 100 uM Crocin 0.1uM

Untreated

Effect of glutamate on proliferation, morphology and stress

(Day 13.3.9 imageJ ROS quantification,

Glutamate 100uM)

Effect of Glutamate on ROS levels

** p-value = 0.00025 (Significant p<0.05)

(Day 14.3.1, Glutamate 100uM Stained with

Phalloidin and Nuc Blue)

Apoptotic

necrosis

Unknown cells (Glutamate)

Day14, well 3 (Glutamate 100uM) stained with

Phalloidin and Nuc Blue (lKoskowski, 2012)

(Tai, 2003)

(Williams et al, 2013)

Glutamate induced glioma cell proliferation is prevented by

functional expression of the glutamate transporter GLT-1

In Vanhoutte’s 2008 paper: Astrocytes and C6 glioma cells were exposed to

Glutamate in order to assess the GLUT-1 transporter activity. It was noted when the

transporter was insufficiently expressed, the glial cells’ morphology was altered and

they began to resemble glioma cells. This suggests that an increased exposure to

glutamate promotes the formation of glioma cells.

GLUT-1 Transporter located in the membrane (of glial cells)

Glioma= primary brain tumor originating from supporting cells of the brain (neuroglia)

NOTE: 5-10 time more neuroglia than neurons

Effect of Crocin on ROS levels

** p-value = 0.04893 (Significant p<0.05)

Effect of Crocin on the proliferation and morphology of differentiating neurons

Day14, well 6 (Crocin 10uM) stained with Phalloidin Day13, well 6 (Crocin 10uM) stained with FRR2 ROS Probe

Day 14, well 5 (Crocin 1uM) stained with Phalloidin and NucBlue

(Dietrich, 2008)

(Stanford, 2015)

Effect of Crocin on Glutamate Excitotoxicity ROS

levels

** p-value = 0.00066 (significant P<0.05)

* p-value = 0.08360 (trending P≤0.1)

Effect of Crocin and glutamate-induced stress on proliferation and

morphology of neurons

Day 13, well 9 (100 uM Glutamate + 10 uM

Crocin) stained with Phalloidin and NucBlue

Day 13, well 9 (100 uM Glutamate + 10 uM

Crocin) stained with Phalloidin

Day 13, well 8 (10 uM Glutamate + 10 uM

Crocin) stained with Phalloidin and NucBlue

Day 13, well 8(10 uM Glutamate + 10 uM

Crocin) stained with Phalloidin

Effect on Neurogenesis (proliferation and differentiation)

Similar to Glutamate treated wells

GFAP= glial cell marker

GAP43= axonal outgrowth

NeuN= nucleus of neurons

Day 14, well 3

(Glutamate 100 uM)

(Gartner et al. 2013)

Conclusion

Day 14, Well 3 (100uM

Glutamate)

Day 14, Well 6 (10uM

Crocin)

Day 14, Well 8 (10uM

Glutamate + 10uM Crocin)

References (1) Byun, Nellie, and Eric Delpire. 'Axonal And Periaxonal Swelling Precede Peripheral Neurodegeneration In KCC3 Knockout Mice'.

Neurobiology of Disease 28.1 (2007): 39-51. Web. 28 Oct. 2015.

Deslauriers AM et al. (2011) Neuroinflammation and endoplasmic reticulum stress are coregulated by crocin to prevent demyelination

and neurodegeneration, The Journal of Immunology Available at:

http://www.jimmunol.org/content/early/2011/09/30/jimmunol.1004111.full.pdf (accessed 28th Oct 2015)

Fletcher, T.F. 'Lab 1 Neurohistology - Neuroglia'. Vanat.cvm.umn.edu. N.p., 2004. Web. 28 Oct. 2015.

Gartner, A., Pereira, T., Gomes, R., Luis, A. L., Lacueva Franca, M., Guena, S., Armada-Da-Silva, P. And Mauricio, A. C.

‘Mesenchymal Stem Cells From Extra-Embryonic Tissues For Tissue Engineering – Regeneration Of The Peripheral Nerve'. INTECh

51.1051-4 (2013): n. pag. Web.

Kloskowski, T., Gurtowska, N., Olkowska, J., Nowak, J. M., Adamowicz, J., Tworkiewicz, J., Dębski, R., Grzanka, A. And Drewa, T.

'Ciprofloxacin Is A Potential Topoisomerase II Inhibitor For The Treatment Of NSCLC'. International Journal of Oncology 41.6 (2012):

1943-1949. Web. 28 Oct. 2015.

M.O. Simoe, C., Silva, I., R. Teixeir, M., L. Lang, K., R. Guimara, T. D., E. Dudek, S., J. Duran, F., Ludwig, S., S.B. Caro, M. And P.

Schenke, E. 'Proliferative Inhibition And Apoptotic Mechanism On Human Non-Small-Cell Lung Cancer (A549 Cells) Of A Novel

Cucurbitacin From Wilbrandia Ebracteata Cogn'. International J. of Cancer Research 9.2 (2013): 54-68. Web.

References (2)

Maherally, Z., Smith, J. R., An, Q. And Pilkington, G. J. 'Receptors For Hyaluronic Acid And Poliovirus: A Combinatorial Role In Glioma

Invasion?'. PLoS ONE 7.2 (2012): e30691. Web.

New World Encyclopedia (2008) Glial Cells, Available at: http://www.newworldencyclopedia.org/entry/Glial_cell (accessed 28th Oct 2015)

Purves, D., Augustine, G., Fitzpatrick, D., Katz, L., Lamantia, A., Mcnamara, J. And Williams, S. 'Neuroglial Cells'. Sinauer Associates

(2001): n. pag. Web. 28 Oct. 2015.

Radak Z (2013) The complex role of physical exercise and reactive oxygen species on brain, Journal of Sport and Health Science, 2(2): 87-93

Rajeai Z et al. (2013) Antihyperglycemic and antioxidant of crocin in Streptozotocin-induced diabetic rats, J Med Food 16(3): 206 -210

Ransohoff RM, Cardona AE (2010) The myeloid cells of the central nervous system parenhyma. Nature 468(7321): 253-262

Ochiai T et al. (2004) Crocin prevents the death of rat pheochromyctoma (PC-12) cells by its antioxidant effects stronger than those of

alpha-tocopherol, Neuroscience Letters 362: 61 - 64

Seppa, Nathan. 'Glowing Amino Acid Lights Up Growing Brain Cancer'. ScienceNews February 2015 (2015): n. pag. Web.

Shen CN, David Tosh. 'Transdifferentiation, Metaplasia And Tissue Regeneration'. Organogenesis 1.2 (2004): 36. Web. 29 Oct. 2015.

Soeda S et al. (2001) Crocin suppresses tumor necrosis factor-alpha-induced cell death of neuronally differentiated PC-12 cells, Life

Sciences 69: 2887 - 2898

Stanford Medicine (2015) Giles Plant Lab Research, Available at: http://neurosurgery.stanford.edu/research/plant/research.html (accessed 28th

Oct 2015)

References (3)

Stephan, Dietrich A. 'Unraveling Autism'. The American Journal of Human Genetics 82.1 (2008): 7-9. Web. 28 Oct. 2015.

Syntichaki, Popi, and Nektarios Tavernarakis. 'The Biochemistry Of Neuronal Necrosis: Rogue Biology?'. Nature Reviews

Neuroscience 4.8 (2003): 672-684. Web.

Tai, Yu-Tzu. 'Cell Of The Month: Neural Progenitor Cells Expressing Tyrosine Hydrolase'. Nature Cell Biology 5.9 (2003): 779-779.

Web.

University of Pennsylvania,. 'Neural Tissue Engineering : Cullen Lab | School Of Medicine | University Of Pennsylvania'.

Med.upenn.edu. N.p., 2015. Web. 28 Oct. 2015.

Vanhoutte, Nicolas, and Emmanuel Hermans. 'Glutamate-Induced Glioma Cell Proliferation Is Prevented By Functional Expression Of

The Glutamate Transporter GLT-1'. FEBS Letters 582.13 (2008): 1847-1852. Web. 28 Oct. 2015.

Wang K et al. (2015) Neuroprotective effects of crocin against traumatic brain injury in mice: Involvement in Notch signalling pathway,

Neuroscience Letters 591: 53 - 58

Williams, Claire J., and David T. Dexter. 'Neuroprotective And Symptomatic Effects Of Targeting Group III Mglu Receptors In

Neurodegenerative Disease'. J. Neurochem. 129.1 (2013): 4-20. Web.

Yang, Y., Hou, L., Li, Y., Ni, J. And Liu, L. 'Neuronal Necrosis And Spreading Death In A Drosophila Genetic Model'. Cell Death Dis 4.7

(2013): e723. Web. 28 Oct. 2015.