Pickens, A. G. | bioRxiv Science

bioRxiv2024

N-Aldehyde-Modified Phosphatidylethanolamines generated by lipid peroxidation are robust substrates of N-Acyl Phosphatidylethanolamine Phospholipase D

N-acyl phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) hydrolyzes phosphatidylethanolamines (PE) where the headgroup nitrogen has been enzymatically modified with acyl chains of four carbons or longer (N-acyl-PEs or NAPEs). The nitrogen headgroup of PE can also be non-enzymatically modified by reactive lipid aldehydes, thus forming N-aldehyde modified-PEs (NALPEs). Some NALPEs such as N-carboxyacyl-PEs are linked to PE via amide bonds similar to NAPEs, but others are linked by imine, pyrrole, or lactam moieties. Whether NAPE-PLD can hydrolyze NALPEs was unknown. We therefore characterized the major NALPE species formed during lipid peroxidation of arachidonic acid and linoleic acid and generated various NALPEs for characterization of their sensitivity to NAPE-PLD hydrolysis by reacting synthesized aldehydes with PE. We found that NAPE-PLD could act on NALPEs of various lengths and linkage types including those derived from PE modified by malondialdehyde (N-MDA-PE), butane dialdehyde (N-BDA-PE), 4-hydroxynonenal (N-HNE-PE), 4-oxo-nonenal (N-ONE-PE), 9-keto-12-oxo-dodecenoic acid (N-KODA-PE), and 15-E2-isolevuglandin (N-IsoLG-PE). To assess the relative preference of NAPE-PLD for various NALPEs versus its canonical NAPE substrates, we generated a substrate mixture containing roughly equimolar concentrations of the seven NALPEs as well as two NAPEs (N-palmitoyl-PE and N-linoleoyl-PE) and measured their rate of hydrolysis. Several NALPE species, including the N-HNE-PE pyrrole species, were hydrolyzed at a similar rate as N-linoleoyl-PE and many of the other NALPEs showed intermediate rates of hydrolysis. These results significantly expand the substrate repertoire of NAPE-PLD and suggest that it may play an important role in clearing products of lipid peroxidation in addition to its established role in the biosynthesis of N-acyl-ethanolamines.

biochemistry↗

bioRxiv2021

Single-Cell RNA Sequencing Characterizes the Molecular Heterogeneity of the Larval Zebrafish Optic Tectum

The optic tectum (OT) is a multilaminated midbrain structure that acts as the primary retinorecipient in the zebrafish brain. Homologous to the mammalian superior colliculus, the OT is responsible for the reception and integration of stimuli, followed by elicitation of salient behavioral responses. While the OT has been the focus of functional experiments for decades, less is known concerning specific cell types, microcircuitry, and their individual functions within the OT. Recent efforts have contributed substantially to the knowledge of tectal cell types; however, a comprehensive cell catalog is incomplete. Here we contribute to this growing effort by applying single-cell RNA-sequencing (scRNA-seq) to characterize the transcriptomic profiles of tectal cells labeled by the transgenic enhancer trap line y304Et(cfos:Gal4;UAS:Kaede). We sequenced 13,320 cells, a 4X cellular coverage, and identified 25 putative OT cell populations. Within those cells, we identified several mature and developing neuronal populations, as well as non-neuronal cell types including oligodendrocytes, microglia, and radial glia. Although most mature neurons demonstrate GABAergic activity, several glutamatergic populations are present, as well as one glycinergic population. We also conducted Gene Ontology analysis to identify enriched biological processes, and computed RNA velocity to infer current and future transcriptional cell states. Finally, we conducted in situ hybridization to validate our bioinformatic analyses and spatially map select clusters. In conclusion, the larval zebrafish OT is a complex structure containing at least 25 transcriptionally distinct cell populations. To our knowledge, this is the first time scRNA-seq has been applied to explore the OT alone and in depth.

neuroscience↗