Niemann-Pick type C1 (NPC1) disease is a rare neurodegenerative disorder linked to defective cholesterol biosynthesis. Because olfactory dysfunction frequently precedes clinical manifestations in neurodegenerative conditions, we investigated whether the olfactory system might provide early insights into disease processes in NPC1. Using a knock-in mouse model carrying the human I1061T mutation, we investigated the olfactory epithelium in wild-type, heterozygous, and mutant mice to analyze neurodegeneration at 36 and 60 days after birth. Mutant mice showed reduced density of mature olfactory sensory neurons, increased apoptosis, and infiltration of macrophage/microglia. These structural alterations were accompanied by reduced odor-evoked responses and impaired odor-guided behavior. Finally, we assessed olfaction in a family with a child carrying biallelic NPC1 mutations and two heterozygous carrier parents, finding hyposmia of different severity in all three individuals. Our work extensively characterized the OE structurally and functionally, proposing it as a sentinel to monitor disease progression.

Resistance to drugs is one of the major issues affecting the response to pharmacological treatments for tumors. Different mechanisms have been proposed to explain the development of cancer drug resistance (CDR), and several approaches to overcome it have been suggested. However, the biological basis of CDR remains unclear. Here, we investigated whether mitochondrial damage and consequent mitochondrial dysfunction are major causes of drug resistance in different tumors. To this end, we used cell lines from three tumors: hepatocellular carcinoma, breast cancer, and colon cancer. We then applied a protocol that recapitulates chemotherapy regimens in patients, rendering each cell line resistant to the drug commonly used in their respective treatments. The combination of cellular respiration analysis, gene expression analysis of cytochrome c oxidase isoforms, and mass spectrometry assessment of cardiolipin reveals that mitochondrial dysfunction is the underlying cause of the resistant phenotype. Importantly, we disclosed for the first time the rapid inhibition of oxidative phosphorylation (OXPHOS) by L-lactate, the major product of fermentation. Finally, we demonstrated that inhibition of lactic acid fermentation and activation of OXPHOS can increase drug sensitivity in all tested drug-resistant cancer cells. Taken together, our results suggest that inhibiting fermentation and enhancing mitochondrial function in cancer cells may be a concrete option to control the worrisome phenomenon of CDR.

Exosomes are small, membrane-bound vesicles secreted by most cell types into the extracellular environment. They play a crucial role in intercellular communication by transporting bioactive molecules, including proteins, lipids, and RNAs, thereby influencing the phenotype and potentially the genotype in recipient cells. In recent years, exosomes have gained increasing attention in the study of pathophysiological conditions and numerous diseases, including multiple sclerosis (MS), an autoimmune disorder with myelin sheath and neuroaxonal damage in the central nervous system. In this study, we isolated and purified serum-derived exosomes from patients with relapsing remitting MS (RR-MS) and characterized their lipid profiles using matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS). Lipid analysis was performed in both negative and positive ion modes on intact exosomes, bypassing lipid extraction steps and significantly reducing sample-processing time. The lipid profiles of RR-MS exosomes were compared to those of exosomes isolated from the serum of healthy subjects (HS), and statistical analysis was applied to mass spectra to identify potential lipid biomarkers. The specific phospholipid marker of exosomal membranes, bis(monoacylglycero)phosphate (BMP), was clearly detected in both MALDI lipid profiles, with no significant differences in its content between the two sample groups. However, RR-MS exosomes exhibited significantly lower levels of phosphatidic acid (PA) compared to HS exosomes, despite PA being a key structural component of extracellular vesicles. Notably, comparative analysis revealed an enrichment of several lysophosphatidylcholine (LPC) species in RR-MS exosome membranes, aligning with their known proinflammatory role in MS pathology. Our most significant finding was a markedly lower phosphatidylcholine (PC) to LPC ratio in the pathological group indicating potential alterations in membrane lipid homeostasis. To the best of our knowledge, this study is the first to report a distinct lipid signature in serum-derived exosomes from RR-MS patients using direct MALDI-TOF/MS analysis.

Neurons are highly dependent on mitochondria to meet their bioenergetic needs and understanding the metabolic changes during the differentiation process is crucial in the neurodegeneration context. Several in vitro approaches have been developed to study neuronal differentiation and bioenergetic changes. The human SH-SY5Y cell line is a widely used cellular model and several differentiation protocols have been developed to induce a neuron-like phenotype including retinoic acid (RA) treatment. In this work we obtained a homogeneous functional population of neuron-like cells by a two-step differentiation protocol in which SH-SY5Y cells were treated with RA plus the mitotic inhibitor 2-deoxy-5-fluorouridine (FUdr). RA-FUdr treatment induced a neuronal phenotype characterized by increased expression of neuronal markers and electrical properties specific to excitable cells. In addition, the RA-FUdr differentiated cells showed an enrichment of long chain and unsaturated fatty acids (FA) in the acyl chain composition of cardiolipin (CL) and the bioenergetic analysis evidences a high coupled and maximal respiration associated with high mitochondrial ATP levels. Our results suggest that the observed high oxidative phosphorylation (OXPHOS) capacity may be related to the activation of the cyclic adenosine monophosphate (cAMP) pathway and the assembly of respiratory supercomplexes (SCs), highlighting the change in mitochondrial phenotype during neuronal differentiation.

Abstract Barth syndrome (BTHS) is a lethal rare genetic disorder, which results in cardiac dysfunction, severe skeletal muscle weakness, immune issues and growth delay. Mutations in the TAFAZZIN gene, which is responsible for the remodeling of the phospholipid cardiolipin (CL), lead to abnormalities in mitochondrial membrane, including alteration of mature CL acyl composition and the presence of monolysocardiolipin (MLCL). The dramatic increase in the MLCL/CL ratio is the hallmark of patients with BTHS, which is associated with mitochondrial bioenergetics dysfunction and altered membrane ultrastructure. There are currently no specific therapies for BTHS. Here, we showed that cardiac mitochondria isolated from TAFAZZIN knockdown (TazKD) mice presented abnormal ultrastructural membrane morphology, accumulation of vacuoles, pro-fission conditions and defective mitophagy. Interestingly, we found that in vivo treatment of TazKD mice with a CL-targeted small peptide (named SS-31) was able to restore mitochondrial morphology in tafazzin-deficient heart by affecting specific proteins involved in dynamic process and mitophagy. This agrees with our previous data showing an improvement in mitochondrial respiratory efficiency associated with increased supercomplex organization in TazKD mice under the same pharmacological treatment. Taken together our findings confirm the beneficial effect of SS-31 in the amelioration of tafazzin-deficient dysfunctional mitochondria in a BTHS animal model.

IntroductionSubtle cognitive dysfunction and mental fatigue are frequent after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, characterizing the so-called long COVID-19 syndrome. This study aimed to correlate cognitive, neurophysiological, and olfactory function in a group of subjects who experienced acute SARS-CoV-2 infection with persistent hyposmia at least 12 weeks before the observation.MethodsFor each participant (32 post-COVID-19 patients and 16 controls), electroencephalography (EEG) and functional near-infrared spectroscopy (fNIRS) data were acquired using an integrated EEG–fNIRS system during the execution of a P300 odd-ball task and a Stroop test. The Sniffin' Sticks test was conducted to assess subjects' olfactory performance. The Montreal Cognitive Assessment (MoCA) and the Frontal Assessment Battery (FAB) were also administered.ResultsThe post-COVID-19 group consisted of 32 individuals (20 women and 12 men) with an average education level of 12.9 ± 3.12 years, while the control group consisted of 16 individuals (10 women and 6 men) with an average education level of 14.9 ± 3.2 years. There were no significant differences in gender (X2 = 0, p = 1) or age between the two groups (age 44.81 ± 13.9 vs. 36.62 ± 11.4, p = 0.058). We identified a lower concentration of oxyhemoglobin (p < 0.05) at the prefrontal cortical level in post-COVID-19 subjects during the execution of the Stroop task, as well as a reduction in the amplitude of the P3a response. Moreover, we found that post-COVID-19 subjects performed worst at the MoCA screening test (p = 0.001), Sniffin's Sticks test (p < 0.001), and Stroop task response latency test (p < 0.001).ConclusionsThis study showed that post-COVID-19 patients with persistent hyposmia present mild deficits in prefrontal function, even 4 months after the end of the infection. These deficits, although subtle, could have long-term implications for quality of life and cognitive wellbeing. It is essential to continue monitoring and evaluating these patients to better understand the extent and duration of cognitive impairments associated with long COVID-19.

BackgroundBarth syndrome is a rare genetic disease characterized by cardiomyopathy, skeletal muscle weakness, neutropenia, growth retardation and organic aciduria. This variable phenotype is caused by pathogenic hemizygous variants of the TAFAZZIN gene on the X chromosome, which impair metabolism of the mitochondrial phospholipid cardiolipin. Although most patients are usually diagnosed in the first years of life, the extremely variable clinical picture and the wide range of clinical presentations may both delay diagnosis. This is the case reported here of a man affected with severe neutropenia, who was not diagnosed with Barth syndrome until adulthood.Case presentationWe describe herein a family case, specifically two Caucasian male cousins sharing the same mutation in the TAFAZZIN gene with a wide phenotypic variability: an infant who was early diagnosed with Barth syndrome due to heart failure, and his maternal cousin with milder and extremely different clinical features who has received the same diagnosis only at 33 years of age.ConclusionsOur report supports the underestimation of the prevalence of Barth syndrome, which should be always considered in the differential diagnosis of male patients with recurrent neutropenia with or without signs and symptoms of cardiomyopathy.

Cardiolipin (CL), a dimeric phospholipid carrying four fatty acid chains in its structure, is the lipid marker of mitochondria, wherein it plays a crucial role in the functioning of the inner membrane. Its metabolite monolysocardiolipin (MLCL) is physiologically nearly absent in the lipid extract of animal cells and its appearance is the hallmark of the Barth syndrome (BTHS), a rare and often misdiagnosed genetic disease that causes severe cardiomyopathy in infancy. The method described here generates a "cardiolipin fingerprint" and allows a simple assay of the relative levels of CL and MLCL species in cellular lipid profiles. In the case of leukocytes, only 1 mL of blood is required to measure the MLCL/CL ratio via matrix-assisted laser desorption ionization - time-of-flight/mass spectrometry (MALDI-TOF/MS) just within 2 h from blood withdrawal. The assay is straightforward and can be easily integrated into the routine work of a clinical biochemistry laboratory to screen for BTHS. The test shows 100% sensitivity and specificity for BTHS, making it a suitable diagnostic test.

Lipids have been first described during 18th century and since then they have been described in all living tissues. The chemistry of lipids started when the French chemist Poultier de la Salle (1719–1788) for the first time isolated cholesterol crystals from bile. Although Poultier’s work was never published, his disciples had quoted it later. Recently, the advance in technology further improved the ability to characterize their highly variable structure and function. Almost 250 years later, this special volume of Frontiers in Physiology entitled “The multifaceted roles of lipids in physiological and pathophysiological states” aims to cover the last advances in lipid physiology. Because the human lipidome is made of thousands of lipid molecules, it is no wonder that their different chemical structures may exert an enormous variety of biological functions. These include energy production and membrane structural scaffold, sorting and regulation of membrane proteins, cellular signaling and vesicle trafficking. Lipidomics has been developed to study qualitatively and quantitatively these multifaceted molecules. This OMICS technology is a relatively young branch of analytical chemistry as well as an interdisciplinary field of study involving biochemistry and biophysics, applied mass spectrometry, complex statistical analyses, and miniaturization of the assays, which needs increased analytical standardization, especially in the quantification of individual lipid species. In addition, fine alterations of lipid composition can lead to a wide spectrum of human pathologies, ranging from cancer to metabolic, cardiovascular and neurodegenerative diseases. Enlightening this complexity, the various articles pinpoint some of these facts. The readers will thus find a Research Topic of articles on lipid functions ranging from oxysterols to cardiolipin, from fatty acids derivatives to protein lipidation and beyond. OPEN ACCESS

Barth Syndrome (BTHS), a genetic disease associated with early-onset cardioskeletal myopathy, is caused by loss-of-function mutations of the TAFAZZIN gene, which is responsible for remodeling the mitochondrial phospholipid cardiolipin (CL). Deregulation of CL biosynthesis and maturation in BTHS mitochondria result in a dramatically increased monolysocardiolipin (MLCL)/CL ratio associated with bioenergetic dysfunction. One of the most promising therapeutic approaches for BTHS includes the mitochondria-targeted tetrapeptide SS-31, which interacts with CL. Here, we used TAFAZZIN knockdown (TazKD) mice to investigate for the first time whether in vivo administration of SS-31 could affect phospholipid profiles and mitochondrial dysfunction. The CL fingerprinting of TazKD cardiac mitochondria obtained by MALDI-TOF/MS revealed the typical lipid changes associated with BTHS. TazKD mitochondria showed lower respiratory rates in state 3 and 4 together with a decreased in maximal respiratory rates. Treatment of TazKD mice with SS-31 improved mitochondrial respiratory capacity and promoted supercomplex organization, without affecting the MLCL/CL ratio. We hypothesize that SS-31 exerts its effect by influencing the function of the respiratory chain rather than affecting CL directly. In conclusion, our results indicate that SS-31 have beneficial effects on improving cardiac mitochondrial dysfunction in a BTHS animal model, suggesting the peptide as future pharmacologic agent for therapy.

Melanoma, one of the most lethal cutaneous cancers, is characterized by its ability to metastasize to other distant sites, such as the bone. Melanoma cells revealed a variable in vitro propensity to be attracted toward bone fragments, and melanoma-derived exosomes play a role in regulating the osteotropism of these cells. We have here investigated the lipid profiles of melanoma cell lines (LCP and SK-Mel28) characterized by different metastatic propensities to colonize the bone. We have purified exosomes from cell supernatants by ultracentrifugation, and their lipid composition has been compared to identify potential lipid biomarkers for different migration and invasiveness of melanoma cells. Matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) lipid analysis has been performed on very small amounts of intact parental cells and exosomes by skipping lipid extraction and separation steps. Statistical analysis has been applied to MALDI mass spectra in order to discover significant differences in lipid profiles. Our results clearly show more saturated and shorter fatty acid tails in poorly metastatic (LCP) cells compared with highly metastatic (SK-Mel28) cells, particularly for some species of phosphatidylinositol. Sphingomyelin, lysophosphatidylcholine, and phosphatidic acid were enriched in exosome membranes compared to parental cells. In addition, we have clearly detected a peculiar phospholipid bis(monoacylglycero)phosphate as a specific lipid marker of exosomes. MALDI-TOF/MS lipid profiles of exosomes derived from the poorly and highly metastatic cells were not significantly different.

The reduction of sperm motility and count, or oligoasthenozoospermia, is one of the major causes of reduced fertility or infertility in men. Lipid composition of spermatozoa is important in determining their functional characteristics, in particular on motility, acrosomal exocytosis or fusogenic properties of the sperm. Here we investigated the levels of semen lipids in 11 infertile patients with severe oligoasthenozoospermia and 9 normozoospermic subjects with normal motility values. Sperm polar and neutral lipids were analyzed by thin-layer chromatography (TLC) and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Semen of patients with oligoasthenozoospermia showed a reduction of the degree of fatty acid unsaturation in the phospholipids chains that might affect the membrane fluidity. Furthermore, a significant higher cholesterol sulfate/seminolipid ratio was found in semen of oligoasthenozoospermic patients than in subjects with normal motility values, suggesting a critical role of sulfolipids in semen quality. The results may facilitate the understanding of the role of lipids on male fertility and offer interesting perspectives to find innovative treatments for oligoasthenozoospermia.

The brine shrimp Artemia is an interesting experimental system for studies of developmental processes. Hatching of dormant cysts gives rise to shrimp larvae called nauplii, characterized by numerous naupliar stages representing the first forms of brine shrimp life cycle. Here combined Thin Layer Chromatography (TLC) and Matrix-Assisted Laser Desorption Ionization-Time-of-Flight/Mass Spectrometry (MALDI-TOF/MS) analyses have been performed to gain information on the lipid profiles of cysts and two naupliar stages. Lipid bands isolated after preparative TLC of the lipid extracts have been analyzed to detect various species of each lipid class; in addition Post-Source Decay (PSD) analyses allowed the identification of phospholipid chains. We compared the relative abundance of various polar and neutral lipid species in the lipid extracts, proving for the first time that during the development of nauplii there is an increase of cardiolipin (CL) and lysophospholipid levels; in parallel, the amount of phosphatidylcholine (PC) decreases. In addition, as regards neutral lipids, we found an increase of diacylglycerols (DAGs) in correspondence of the decrease of triacylglycerols (TAGs). Data reflect the fact that naupliar stages, being an active form of life, are more metabolically active and offer a platform to develop further studies on the importance of lipid metabolic pathways and bioactive lipids during the development.

Study design, size, duration Sperm samples from 33 men undergoing IVF/ICSI treatment were collected of which 9 with normozoospermia (group A; control) and 11 with asthenospermia (group B) were included in this study. The total progressively motile sperm cells/ejaculate were 166.4 + 44.3 millions and 6.4 + 5.9 millions in group A and B respectively. Semen samples were collected, frozen, and stored at 80 °C until processed.

Barth syndrome is a rare X-linked disease affecting less than 200 individuals worldwide. Several comorbidities have been associated with the pathology and, among those, cardiac myopathy and neutropenia are the most life threatening. The appropriate nutritive support is important to sustain the everyday life of Barth syndrome patients given the chronic fatigue they experience. Since they often prefer salty and fried food, and avoid vegetables and fruits, their eating habit and food preferences do not always provide the proper amount of vitamins and amino acids. It has been indeed reported that Barth syndrome patients have altered taste sensitivity. As olfaction also contributes to food consumption and flavor perception, we decided to investigate their olfactory abilities using the "Sniffin' sticks' extended test". We found no significant difference in any of the tested olfactory abilities between the group of Barth syndrome patients and the healthy controls. In summary, altered food preference of Barth boys could not be easily explained with an altered olfactory perception.

Parkin mutations are a major cause of early‐onset Parkinson's disease (PD). The impairment of protein quality control system together with defects in mitochondria and autophagy process are consequences of the lack of parkin, which leads to neurodegeneration. Little is known about the role of lipids in these alterations of cell functions. In the present study, parkin ‐mutant human skin primary fibroblasts have been considered as cellular model of PD to investigate on possible lipid alterations associated with the lack of parkin protein. Dermal fibroblasts were obtained from two unrelated PD patients with different parkin mutations and their lipid compositions were compared with that of two control fibroblasts. The lipid extracts of fibroblasts have been analyzed by combined matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spectrometry (MALDI‐TOF/MS) and thin‐layer chromatography (TLC). In parallel, we have performed direct MALDI‐TOF/MS lipid analyses of intact fibroblasts by skipping lipid extraction steps. Results show that the proportions of some phospholipids and glycosphingolipids were altered in the lipid profiles of parkin ‐mutant fibroblasts. The detected higher level of gangliosides, phosphatidylinositol, and phosphatidylserine could be linked to dysfunction of autophagy and mitochondrial turnover; in addition, the lysophosphatidylcholine increase could represent the marker of neuroinflammatory state, a well‐known component of PD.

Detection, identification, characterization and quantitation of oxidized cardiolipin forms in vivo is considered a challenging task, because these lipid species exist in low abundance in the biological systems. Recently it has been shown that chain-shortened products of oxidized standard phosphatidylcholines could be successfully identified by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) (1). Here we have characterized oxidation products of standard tetra-linoleoyl cardiolipin, such as hydroxide- and hydroperoxide-derivatives and truncated forms, by MALDI-TOF/MS analysis. In addition, by following a recently developed protocol (2), we have analysed MALDI/TOF cardiolipin fingerprints of intact leukocytes isolated from controls and aged chronic heart failure patients, in order to check for the presence of oxidized cardiolipin species. The comparison of the two sets of analyses has showed MALDI signals potentially attributable to oxidized cardiolipin species in the mass spectra of patients. In conclusion, the use of MALDI-TOF/MS analyses on intact cells or organelles might offer novel opportunities to detect oxidized lipid species in native biomembranes.