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

Methodology Assay Yeast Growth during 24H

Growth kinetics

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

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

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)

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

Cell survival (MTS)

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

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

Morphological alterations (Iba-1) Microglia

Lisotracker (lysosomal activity) Neurons

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)

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

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

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

Inflammatory marker/alarmin HMGB1 (preliminary data)

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

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