High-Content Screening of Synaptic Density Modulators in Primary Neuronal Cultures.

The synapse, which represents the structural and functional basis of neuronal communication, is one of the first elements affected in several neurodegenerative diseases. To better understand the potential role of gene expression in synapse loss, we developed an original high-content screening (HCS) model capable of quantitatively assessing the impact of gene silencing on synaptic density. Our approach is based on a model of primary neuronal cultures (PNCs) from the neonatal rat hippocampus, whose mature synapses are visualized by the relative localization of the presynaptic protein Synaptophysin with the postsynaptic protein Homer1. The heterogeneity of PNCs and the small sizes of the synaptic structures pose technical challenges associated with the level of automation necessary for HCS studies. We overcame these technical challenges, automated the processes of image analysis and data analysis, and carried out tests under real-world conditions to demonstrate the robustness of the model developed. In this article, we describe the screening of a custom library of 198 shRNAs in PNCs in the 384-well plate format. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture of primary hippocampal rat neurons in 384-well plates Basic Protocol 2: Lentiviral shRNA transduction of primary neuronal culture in 384-well plates Basic Protocol 3: Immunostaining of the neuronal network and synaptic markers in 384-well plates Basic Protocol 4: Image acquisition using a high-throughput reader Basic Protocol 5: Image segmentation and analysis Basic Protocol 6: Synaptic density analysis.

Alzheimer's genetic risk factor FERMT2 (Kindlin-2) controls axonal growth and synaptic plasticity in an APP-dependent manner.

Although APP metabolism is being intensively investigated, a large fraction of its modulators is yet to be characterized. In this context, we combined two genome-wide high-content screenings to assess the functional impact of miRNAs and genes on APP metabolism and the signaling pathways involved. This approach highlighted the involvement of FERMT2 (or Kindlin-2), a genetic risk factor of Alzheimer's disease (AD), as a potential key modulator of axon guidance, a neuronal process that depends on the regulation of APP metabolism. We found that FERMT2 directly interacts with APP to modulate its metabolism, and that FERMT2 underexpression impacts axonal growth, synaptic connectivity, and long-term potentiation in an APP-dependent manner. Last, the rs7143400-T allele, which is associated with an increased AD risk and localized within the 3'UTR of FERMT2, induced a downregulation of FERMT2 expression through binding of miR-4504 among others. This miRNA is mainly expressed in neurons and significantly overexpressed in AD brains compared to controls. Altogether, our data provide strong evidence for a detrimental effect of FERMT2 underexpression in neurons and insight into how this may influence AD pathogenesis.

Testing for SGT-151 (CUMYL-PEGACLONE) and its Metabolites in Blood and Urine after Surreptitious Administration.

The authors aim to report a case of surreptitious administration of a synthetic cannabinoid (SC) and the subsequent toxicological investigations to be able to document accurately the case for submission at a hearing. A dealer gave surreptitiously a substance to two juvenile migrants who experienced shakings and faintness. The laboratory received a blood sample from each of the two victims, who, according to the investigators, were probably exposed to SGT-151, a SC, also known as CUMYL-PEGACLONE. Blood and urine specimens from the dealer, who claimed being a user of SGT-151 were received at the same time. To characterize the metabolites of SGT-151, the drug was incubated with a pool of human hepatic microsomes and the cofactors required to ensure the functioning of the main Phase I and Phase II enzymes. The incubation media were analyzed by liquid chromatography coupled to high-resolution mass spectrometry. The metabolites identified following transformation by hepatic microsomes were mostly N-dealkylated SGT-151, mono-hydroxylated SGT-151 and di-hydroxylated SGT-151. The presence of SGT-151 (5.4 ng/mL) and its metabolite, N-dealkyl SGT-151, was confirmed in the dealer's blood. Two metabolites of SGT-151 (OH-SGT-151, diOH-SGT-151) were detected in the dealer's urine. SGT-151 (~1 ng/mL) and its metabolite N-dealkyl SGT-151 were detected in the blood samples of the two victims.