3. 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)
4. 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.
6. Neurogenesis
Three major types of neuroglia:
● astrocytes (structural support for CNS)
● Oligodendrocytes (form myelin in CNS)
● Microglia (act as macrophage in CNS)
9. Day 13
100 µM Glutamate 0.1 µM Crocin
100 µM Glutamate + 10 µm Crocin Untreated + FRR2 stain Untreated and no stain
10. Day 14
Crocin 10uM + Glutamate 100 uM
Glutamate 100 uM Crocin 0.1uM
Untreated
11. 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
12. Unknown cells (Glutamate)
Day14, well 3 (Glutamate 100uM) stained with
Phalloidin and Nuc Blue
(lKoskowski, 2012)
(Tai, 2003)
(Williams et al, 2013)
13. 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
14. Effect of Crocin on ROS levels
** p-value = 0.04893 (Significant p<0.05)
15. Effect of Crocin on the proliferation and morphology of differentiating neurons
Day14, well 6 (Crocin 10uM) stained with PhalloidinDay13, well 6 (Crocin 10uM) stained with FRR2 ROS Probe
Day 14, well 5 (Crocin 1uM) stained with Phalloidin and NucBlue
(Dietrich, 2008)
(Stanford, 2015)
17. 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
18. 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)
19. Conclusion
Day 14, Well 3 (100uM
Glutamate)
Day 14, Well 6 (10uM
Crocin)
Day 14, Well 8 (10uM
Glutamate + 10uM Crocin)
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