1. In vitro studies – neurodegeneration, neuroinflammation
Identification and Assessment of New Psychoactive Substances: A European Network
WS2- Risk assessment and neurotoxicity studies of the NPS
In vitro studies – neurodegeneration, neuroinflammation
yeast model impact
Alexandre Quintas
Carla Ferreira
From Egas Moniz Cooperativa de Ensino Superior

2. Methodology
Assay
Yeast Growth during 24H

3. Growth kinetics

4. IC50 Molécula Nome IC50 4-FMC 53,62 mM ± 0,02 N-ethylhexedrone
4-FMC
53,62 mM ± 0,02 
N-ethylhexedrone
53,70 mM ± 0,001
 4-MEC
85,81 mM ± 0,01
Buphedrone
 ~275,00 mM ± 0,08
Methedrone 
~622,00 mM ± 0,27
IC50

5. Future work - Proteomics
2-D gels
N-ethylhexedrone;
4-FMC;
4-MEC.

6. In vitro studies – neurodegeneration, neuroinflammation
Identification and Assessment of New Psychoactive Substances: A European Network
WS2- Risk assessment and neurotoxicity studies of the NPS
In vitro studies – neurodegeneration, neuroinflammation
Dora BRITES lab – Faculdade de Farmácia Lisbon
Human neurons (cell line – SH-SY%Y) - NEURODEGENERATION
Cell survival (MTS)
Cell death by apoptosis and necrosis (PI-Annexin kit)
Lisotracker (lysosomal activity)
Human microglia (cell line – CHME3) - INFLAMMATION
Cell survival (MTS)
Cell death by apoptosis and necrosis (PI-Annexin kit)
Morphological alterations (Iba-1)
Lisotracker (lysosomal activity)

7. Neuron-Glia Communication
Astrocytes
Microglia
Neurons
Injury
Degeneration
Oligodendrocytes
Pro-inflammatory cytokines
Alarmins and MicroRNAs
Mediators of oxidative stress
In the intact brain, neurons and glia are extensively linked

8. Cell survival (MTS)

9. Cell death by apoptosis and necrosis (PI-Annexin kit) - Neurons
Incubation time = 24 hours
*p<0.05 vs. w/o

10. Cell death by apoptosis and
necrosis (PI-Annexin kit) - Microglia

11. Morphological alterations
(Iba-1) Microglia

12. Lisotracker (lysosomal activity)
Neurons

13. In vivo studies: behavioral and cognitive studies
Identification and Assessment of New Psychoactive Substances: A European Network
WS2- Risk assessment and neurotoxicity studies of the NPS
In vivo studies: behavioral and cognitive studies
Ex vivo hippocampal histopathological findings of the tested rodents Dora Brites & Cristina Sampayo
Neurodegeneration (Fluorojade)
Microgliosis (Iba1)

14. Animals study – 4-MEC Experimental design Drug administration: Saline
128 mg 4-MEC (2 x 64 mg)

15. Histopatological findings of sacrificed animals - Cortex
Control
2 weeks
4-MEC
Neuropil vacuolotion
spongiosis
Neuropil (or "neuropile") is any area in the nervous system composed of mostly unmyelinated axons, dendrites and glial cell processes that forms a synaptically dense region containing a relatively low number of cell bodies. The most prevalent anatomical region of neuropil is the brain which, although not completely composed of neuropil, does have the largest and highest synaptically-concentrated areas of neuropil in the body. For example, the neocortex and olfactory bulb both contain neuropil.[1]
White matter, which is mostly composed of axons and glial cells, is generally not considered to be a part of the neuropil.[citation needed]
Neuropil (pl. neuropils) comes from the Greek: neuro, meaning "tendon, sinew; nerve" and pilos, meaning "felt".[2] The term's origin can be traced back to the late 19th century.[3]
Luxol (myelin, blue) & Cresyl Violet (neurons, purple) staining

16. Histopatological findings of sacrificed animals - Cortex
Control
3 weeks
4-MEC
Luxol (myelin, blue) & Cresyl Violet (neurons, purple) staining

17. Inflammatory marker/alarmin HMGB1
(preliminary data)

18. Histopatological findings of sacrificed animals - Cortex
Control
3 weeks
4-MEC
GFAP (astrocytes-green), Iba1 (microglia) & DAPI (nuclei) staining – cortex

19. Ongoing and Programmed Work
Identification and Assessment of New Psychoactive Substances: A European Network
Ongoing and Programmed Work
In vitro studies
Further evaluation of 4-FMC and 4-EMC, just initiated
Lysosome formation using Lysotracker® in both neurons and microglia human cell lines
Mitocondria dynamic changes (Mitotracker)
Detection of synaptic proteins mRNA expression in neurons
Detection of gene expression of inflammatory-related markers in microglial cells (e.g. cytokines mRNA and inflammatory-microRNAs expression)
Yeast cell proteomics
Ex vivo studies (hippocampal histopathology in brain samples already collected after behavioural studies) - slices
Neurodegeneration and neuritic arborization
Glia activation (astrocytes and microglia)
Myelination deficits in brain samples collected after behavioural and cognitive
Detection of synaptic proteins mRNA expression