The recent developments in mass spectrometry have revealed the importance of lipids as biomarkers in the context of different diseases and as indicators of the cell’s homeostasis. However, further advances are required to unveil the complex relationships between lipid classes and lipid species with proteins. Here, we present a new methodology that combines microarrays with mass spectrometry to obtain the lipid fingerprint of samples of a different nature in a standardized and fast way, with minimal sample consumption. As a proof of concept, we use the methodology to obtain the lipid fingerprint of 20 rat tissues and to create a lipid library for tissue classification. Then, we combine those results with immunohistochemistry and enzymatic assays to unveil the relationship between some lipid species and two enzymes. Finally, we demonstrate the performance of the methodology to explore changes in lipid composition of the nucleus accumbens from mice subjected to two lipid diets.

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Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells. K E Y WO R D S CD9, cytopermeable peptides, EV biogenesis, mitophagy, tetraspanin  INTRODUCTIONExtracellular Vesicles (EVs) have emerged as a potent mechanism of intercellular communication that can contain a complex message composed of several signalling factors, enzymatic activities and genetic material (Yáñez-Mó et al., 2015). They are present in all biological fluids, offering a great opportunity for non-invasive biomarker discovery in several clinical scenarios; and have therapeutic potential for a wide variety of diseases, including neurodegenerative disorders and cancer (Fais et al., 2016).Tetraspanins are commonly used as markers of EVs populations, being present on both microvesicles (EVs budding from the plasma membrane) and exosomes (EVs derived from late endosomal multivesicular bodies (MVB) compartments) (Andreu & Yáñez-Mó, 2014). However, no fundamental alterations in endosome biogenesis and maturation have been described in This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Highlights d Developmental deficiency in n-3 PUFA lipid species leads to motivational deficits d n-3 PUFA deficiency increases D2-MSN lateral inhibition in the nucleus accumbens d Rescue of n-3 PUFA levels in D2 neurons normalizes lateral inhibition of D2-MSNs d Rescue of PUFA levels in D2 neurons enhances motivation under n-3 PUFA deficiency

Acetylcholinesterase (AChE) is responsible for hydrolyzing the acetylcholine neurotransmitter, bringing an end point to cholinergic neurotransmission. Thus, AChE is the primary target of a wide spectrum of compounds used as pesticides, nerve agents or therapeutic drugs for neurodegenerative diseases such as Alzheimer’s disease (AD). This enzyme is heterogeneously distributed in the brain showing different activity depending on the nervous region. Therefore, the aim of this work is to report a novel technology that enables the simultaneous determination of tissue specific AChE activity, as well as the analysis and screening of specific inhibitors, by using cell membrane microarrays. These microarrays were composed of cell membranes, isolated from 41 tissues, organs and brain areas, that were immobilized over a slide, maintaining the functionality of membrane proteins. To validate this platform, demonstrating its usefulness in drug discovery as a high throughput screening tool, a colorimetric protocol to detect the membrane-bound AChE activity was optimized. Thus, rat cortical and striatal AChE activities were estimated in presence of increased concentrations of AChE inhibitors, and the donepezil effect was assessed simultaneously in 41 tissues and organs, demonstrating the major potential of this microarray’s technology.