Lipid systems play a substantial role in the pathophysiological mechanisms underlying several brain disorders. However, the mechanisms underlying the lipid profile in stress-induced maladaptations and how these changes can impact distinct neuronal circuits and behavioral states remain to be fully elucidated. Here, we investigated the effects of p11 deficiency in combination with stress by using p11KO mice and chronic stress mouse models of depression to study spatial lipidomic and transcriptomic signatures in the mouse nucleus accumbens. Our results show that p11 deficiency and stress induce depression-related maladaptive phenotypes and provide novel evidence that these responses are associated with phospholipid dyshomeostasis in the nucleus accumbens. Phospholipid disturbances were predominantly related to phosphatidylethanolamine (PE) and ether PE metabolism, along with targets involved in the PE biosynthetic pathway. Moreover, chelerythrine administration, a compound reported to disrupt phospholipid balance, induces PE changes and depression-like behaviors. Altogether, the present study provides evidence that alterations in phospholipid-related pathways may alter reward/anti-reward circuits and how these changes might be implicated in stress-related disorders.

In this study, we demonstrate the complementary strengths of FMP-10 (4-(Anthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide) on-tissue derivatization with UV-MALDI MSI and IR-MALDESI MSI without derivatization for small molecule analysis. These MSI approaches were applied to the striatum of rats administered 6-OHDA to induce dopaminergic neurodegeneration, modeling Parkinson's disease. UV-MALDI MSI with FMP-10 derivatization enhanced the detection of dopaminergic and serotonergic metabolites, revealing clear lesion-related effects. IR-MALDESI MSI, in turn, expanded the molecular coverage by detecting several hundred additional small molecules, including neurotransmitters, amino acids, and lipids. The complementary application of these ionization strategies broadened metabolomic coverage, highlighting their potential for broader disease-related studies.

Parkinson's disease (PD) is characterized by Lewy body pathology, mainly consisting of accumulation of aggregated α-synuclein and lipid contents. Lipid dyshomeostasis has frequently been observed in PD, and compelling evidences indicate that lipids play critical roles in modulating α-synuclein toxicity. However, how α-synuclein regulates brain lipid composition remains poorly understood. Here, we used dual polarity matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to spatially profile brain lipids in a unilateral adeno-associated virus (AAV)-α-synuclein mouse model of Parkinsonism. We identified region-specific alterations in sphingolipids and glycerophospholipids in the substantia nigra and striatum of these mice. In sphingolipids, α-synuclein overexpression altered certain monosialotetrahexosylgangliosides (GM1s), sphingomyelins (SMs), and sulfated hexosyl ceramides (SHexCers; a.k.a. sulfatides). Glycerophospholipid changes followed a desaturation pattern: polyunsaturated fatty acid (PUFA)-containing species were decreased, while saturated and mono-unsaturated species were increased. Additionally, oxidized phosphatidylcholine (PC) lipids, such as SAzPC (1-stearoyl-2-azelaoyl-sn-PC) and PAzPC(1-palmitoyl-2-azelaoyl-sn-PC), were elevated in the substantia nigra, and ether phosphatidylethanolamines (PEs) were reduced in the substantia nigra but increased in the striatum. Our study provides a comprehensive, spatially resolved lipidomic landscape of α-synuclein-induced pathology in the nigrostriatal pathway, offering new insights into lipid dysregulation in experimental PD and a framework for future therapeutic exploration.

The illicit use of anabolic androgenic steroids (AAS) to enhance physical strength, performance, and appearance is associated with severe physical and psychological adverse effects. Long-term AAS use has been associated with altered behavior and cognitive impairments. Nevertheless, the neurobiological effects of AAS use remain underexplored, especially in terms of brain neurotransmitter and lipid profiles. This study applied advanced Fourier-transform ion cyclotron resonance matrix-assisted laser desorption/ionization mass spectrometry imaging (FTICR-MALDI-MSI) to investigate the effects of three AAS decanoate prodrugs, nandrolone, testosterone, and trenbolone, on brain monoamine neurotransmitter systems and lipid composition in male rats (n = 32). The AAS administration was found to alter dopamine and serotonin levels, as well as the metabolism of these molecules across multiple brain regions; significant effects on neurotransmitter levels, associated metabolites, and turnover ratios were observed. Additionally, AAS treatment affected brain lipid composition, with nandrolone and testosterone increasing the levels of phosphatidylethanolamines, ether-linked phosphatidylethanolamines, and phosphatidylserines; in contrast, trenbolone demonstrated an opposite trend. The presented findings revealed distinct, steroid-specific alterations in brain neurotransmitters and lipid profiles; these changes were particularly strong in the tail of the striatum, where lipid dysregulation was linked to neurotransmitter dynamics. The potential neurotoxic effects further highlight the importance of understanding how steroid-specific mechanisms are linked to behavioral and neurobiological changes. This study advances our knowledge of the neurobiological effects, including the potential risks, of extended AAS use.

L-DOPA-induced dyskinesia (LID) is a significant and treatment-limiting complication in Parkinson’s disease (PD) therapy, yet its mechanisms remain poorly understood. We used high-resolution mass spectrometry imaging to map brain-region-specific alterations of glycerophospholipids and sphingolipids in a female macaque model of PD with and without LID following chronic L-DOPA treatment. LID was associated with depletion of antioxidant plasmalogen phosphatidylcholines in the globus pallidus interna, claustrum, and precentral gyrus—regions critical for motor function—and elevations of polyunsaturated fatty acid-containing glycerophospholipids, indicative of increased membrane fluidity. This lipid profile differed from similarly treated non-dyskinetic animals, suggesting lipid composition mediates differential susceptibility to LID. Lipid alterations correlated strongly with dyskinesia severity, dopamine, and L-DOPA concentrations, supporting a mechanistic link between lipid metabolism, neurotransmitter dysregulation, and LID. This comprehensive spatial lipidomic analysis identifies region-specific lipid dysregulation as a novel aspect of LID pathology, highlighting lipid pathways as potential therapeutic targets for mitigating dyskinesia.

The highly conserved CHCHD2 and CHCHD10 are small mitochondrial proteins residing in the intermembrane space. Recently, mutations in the genes encoding these proteins have been linked to severe disorders, including Parkinson's disease and amyotrophic lateral sclerosis. In cultured cells, a small fraction of CHCHD2 and CHCHD10 oligomerize to form a high molecular weight complex of unknown function. Here, we generated a whole-body Chchd2 knockout mouse to investigate the in vivo role of CHCHD2 and its protein complex. We show that CHCHD2 is crucial for sustaining full motor capacity, normal striatal dopamine levels, and lipid homeostasis in the brain of adult male mice. We also demonstrate that in mouse tissues, CHCHD2 and CHCHD10 exist exclusively as a high molecular weight complex, whose levels are finely tuned under physiological conditions. In response to mitochondrial dysfunction, the abundance and size of the CHCHD2-CHCHD10 complex increase, a mechanism conserved across different tissues. Although the loss of CHCHD2 does not abolish CHCHD10 oligomerization, it enhances cell vulnerability to mitochondrial stress, suggesting that CHCHD2 is protective against mitochondrial damage. Our findings uncover the role of CHCHD2 in preserving tissue homeostasis and provide important insights into the involvement of the CHCHD2-CHCHD10 complex in human diseases.

Here, we introduce Met-ID, a graphical user interface software designed to efficiently identify metabolites from MALDI-MSI data sets. Met-ID enables annotation of m/z features from any type of MALDI-MSI experiment, involving either derivatizing or conventional matrices. It utilizes structural information for derivatizing matrices to generate a subset of targets that contain only functional groups specific to the derivatization agent. The software is able to identify multiple derivatization sites on the same molecule, facilitating identification of the derivatized compound. This ability is exemplified by FMP-10, a reactive matrix that assists the covalent charge-tagging of molecules containing phenolic hydroxyl and/or primary or secondary amine groups. Met-ID also permits users to recalibrate data with known m/z ratios, boosting confidence in mass match results. Furthermore, Met-ID includes a database featuring MS2 spectra of numerous chemical standards, consisting of neurotransmitters and metabolites derivatized with FMP-10, alongside peaks for FMP-10 itself, all accessible directly through the software. The MS2 spectral database supports user-uploaded spectra and enables comparison of these spectra with user-provided tissue MS2 spectra for similarity assessment. Although initially installed with basic data, Met-ID is designed to be customizable, encouraging users to tailor the software to their specific needs. While several MSI-oriented software solutions exist, Met-ID combines both MS1 and MS2 functionalities. Developed in alignment with the FAIR Guiding Principles for scientific software, Met-ID is freely available as an open-source tool on GitHub, ensuring wide accessibility and collaboration.

Multiomics analysis of single tissue sections using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) provides comprehensive molecular insights. However, optimizing tissue sample preparation for MALDI-MSI to achieve high sensitivity and reproducibility for various biomolecules, such as lipids, N-glycans, and tryptic peptides, presents a significant challenge. This study introduces a robust and reproducible protocol for the comprehensive sequential analysis of the latter molecules using MALDI-MSI in fresh-frozen rodent brain tissue samples. The optimization process involved testing multiple organic solvents, which identified serial washing in ice-cold methanol, followed by chloroform as optimal for N-glycan analysis. Integrating this optimized protocol into MALDI-MSI workflows enabled comprehensive sequential analysis of lipids (in dual polarity mode), N-glycans, and tryptic peptides within the same tissue sections, enhancing both the efficiency and reliability. Validation across diverse rodent brain tissue samples confirmed the protocol's robustness and versatility. The optimized methodology was subsequently applied to a transgenic Alzheimer's disease (AD) mouse model (tgArcSwe) as a proof of concept. In the AD model, significant molecular alterations were observed in various sphingolipid and glycerophospholipid species, as well as in biantennary and GlcNAc-bisecting N-glycans, particularly in the cerebral cortex. These region-specific alterations are potentially associated with amyloid-beta (Aβ) plaque accumulation, which may contribute to cognitive and memory impairments. The proposed standardized methodology represents a significant advancement in neurobiological research, providing valuable insights into disease mechanisms and laying the foundation for potential preclinical applications. It could aid the development of diagnostic biomarkers and targeted therapies for AD and other neurodegenerative diseases, such as Parkinson's disease.

Inhibitors targeting amyloids formed by the human Islet Amyloid Polypeptide (hIAPP) are promising therapeutic candidates for type 2 diabetes. Peptide formulations derived from the nonamyloidogenic rat IAPP (rIAPP) sequence are currently used as hIAPP mimetics to support insulin therapy. rIAPP itself acts as a peptide inhibitor; yet, the structural-level consequences of such inhibition, particularly its impact on amyloid polymorphism, have not been studied in detail. Here, we conduct coaggregation experiments with varying rIAPP-to-hIAPP concentration ratios and employ high-resolution cryo-electron microscopy (Cryo-EM) to elucidate the polymorphism of the resulting fibril structures. Our results demonstrate that the polymorphism of hIAPP amyloids is highly sensitive to the electrostatic environment, which can be modulated by buffer composition, the concentration of the inhibitor, and cosolvents such as hexafluoroisopropanol (HFIP). Under native conditions, rIAPP associates with hIAPP but does not cross-aggregate, resulting in fibrils primarily composed of hIAPP. Significant inhibition is observed at relatively high concentrations of rIAPP. However, trace amounts of HFIP disrupt this inhibition, leading to increased fibril concentrations due to the formation of cross-seeded products composed of both hIAPP and rIAPP, as evidenced by mass spectrometry and two-dimensional infrared (2D IR) spectroscopy. These findings highlight the critical role of experimental conditions, particularly the electrostatic environment, in modulating amyloid polymorphism, cross-seeding, and inhibition. By providing structural insights into these processes, this study advances our understanding of peptide aggregation and offers valuable guidance for the rational design of more effective therapeutic inhibitors targeting hIAPP-related amyloidosis.

A one-step, on-tissue chemical derivatisation method for MALDI mass spectrometry imaging was found to improve the detectability of aldehydes and ketones by charge-tagging. The developed reactive matrices, containing a UV-chromophore, ionisable moiety and hydrazide group, showed an equal or higher detection efficiency than Girard's reagent P, enabling improved imaging of brain metabolites without the need for additional co-matrices.

In an era when population aging is increasing the burden of neurodegenerative conditions, deciphering the mechanisms underlying brain senescence is more important than ever. Here, we present a spatial metabolomics analysis of age-induced neurochemical alterations in the mouse brain using negative ionization mode mass spectrometry imaging. The age-dependent effects of the acetylcholinesterase inhibitor tacrine were simultaneously examined. For ultrahigh mass resolution analysis, we utilized a Fourier-transform ion cyclotron resonance spectrometer. To complement this, a trapped ion mobility spectrometry time-of-flight analyzer provided high speed and lateral resolution. The chosen approach facilitated the detection and identification of a wide range of metabolites, from amino acids to sphingolipids. We reported significant, age-dependent alterations in brain lipids which were most evident for sulfatides and lysophosphatidic acids. Sulfatide species, which are mainly localized to white matter, either increased or decreased with age, depending on the carbon chain length and hydroxylation stage. Lysophosphatidic acids were found to decrease with age in the detailed cortical and hippocampal subregions. An age-dependent increase in the glutamine/glutamate ratio, an indicator of glia-neuron interconnection and neurotoxicity, was detected after tacrine administration. The presented metabolic mapping approach was able to provide visualizations of the lipid signaling and neurotransmission alterations induced by early aging and can thus be beneficial to further elucidating age-related neurochemical pathways.

Molecular catalysts based on abundant elements that function in neutral water represent an essential component of sustainable hydrogen production. Artificial hydrogenases based on protein-inorganic hybrids have emerged as an intriguing class of catalysts for this purpose. We have prepared a novel artificial hydrogenase based on cobaloxime bound to a de novo three alpha-helical protein, α3C, via a pyridyl-based unnatural amino acid. The functionalized de novo protein was characterized by UV-visible, CD, and EPR spectroscopy, as well as MALDI spectrometry, which confirmed the presence and ligation of cobaloxime to the protein. The new de novo protein produced hydrogen under electrochemical, photochemical and reductive chemical conditions in neutral water solution. A change in hydrogen evolution capability of the de novo enzyme compared with native cobaloxime was observed, with tunover numbers around 80% for that of cobaloxime, and hydrogen evolution rates of 40% of that of cobaloxime. We discuss these findings in the context of existing literature, and our study contributes important information about the functionality of cobaloxime HER catalysts in protein environments, and the feasibility of artificial enzymes to the field of artificial metalloenzymes. Small de novo proteins as enzyme scaffolds have the potential to function as upscaleable bioinspired catalysts thanks to their efficient atom economy, and the findings presented here show that this type of novel enzymes are a possible product.

Metabolism of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to the neurotoxin MPP+ in the brain causes permanent Parkinson's disease-like symptoms by destroying dopaminergic neurons in the pars compacta of the substantia nigra in humans and non-human primates. However, the complete molecular pathology underlying MPTP-induced parkinsonism remains poorly understood. We used dual polarity matrix-assisted laser desorption/ionization mass spectrometry imaging to thoroughly image numerous glycerophospholipids and sphingolipids in coronal brain tissue sections of MPTP-lesioned and control non-human primate brains (Macaca mulatta). The results revealed specific distributions of several sulfatide lipid molecules based on chain-length, number of double bonds, and importantly, hydroxylation stage. More specifically, certain long-chain hydroxylated sulfatides with polyunsaturated chains in the molecular structure were depleted within motor-related brain regions in the MPTP-lesioned animals, e.g., external and internal segments of globus pallidus and substantia nigra pars reticulata. In contrast, certain long-chain non-hydroxylated sulfatides were found to be elevated within the same brain regions. These findings demonstrate region-specific dysregulation of sulfatide metabolism within the MPTP-lesioned macaque brain. The depletion of long-chain hydroxylated sulfatides in the MPTP-induced pathology indicates oxidative stress and oligodendrocyte/myelin damage within the pathologically relevant brain regions. Hence, the presented findings improve our current understanding of the molecular pathology of MPTP-induced parkinsonism within primate brains, and provide a basis for further research regarding the role of dysregulated sulfatide metabolism in PD.

The visualization of small metabolites by MALDI mass spectrometry imaging in brain tissue sections is challenging due to low detection sensitivity and high background interference. We present an on-tissue chemical derivatization MALDI mass spectrometry imaging approach for the comprehensive mapping of carboxyls and aldehydes in brain tissue sections. In this approach, the AMPP (1-(4-(aminomethyl)phenyl)pyridin-1-ium chloride) derivatization reagent is used for the covalent charge-tagging of molecules containing carboxylic acid (in the presence of peptide coupling reagents) and aldehydes. This includes free fatty acids and the associated metabolites, fatty aldehydes, dipeptides, neurotoxic reactive aldehydes, amino acids, neurotransmitters and associated metabolites, as well as tricarboxylic acid cycle metabolites. We performed sensitive ultrahigh mass resolution MALDI-MS detection and imaging of various carboxyl- and aldehyde-containing endogenous metabolites simultaneously in rodent brain tissue sections. We verified the AMPP-derivatized metabolites by tandem MS for structural elucidation. This approach allowed us to image numerous aldehydes and carboxyls, including certain metabolites which had been undetectable in brain tissue sections. We also demonstrated the application of on-tissue derivatization to carboxyls and aldehydes in coronal brain tissue sections of a nonhuman primate Parkinson's disease model. Our methodology provides a powerful tool for the sensitive, simultaneous spatial molecular imaging of numerous aldehydes and carboxylic acids during pathological states, including neurodegeneration, in brain tissue.

Prosaposin (PSAP) modulates glycosphingolipid metabolism and variants have been linked to Parkinson's disease (PD). Here, we find altered PSAP levels in the plasma, CSF and post-mortem brain of PD patients. Altered plasma and CSF PSAP levels correlate with PD-related motor impairments. Dopaminergic PSAP-deficient (cPSAPDAT) mice display hypolocomotion and depression/anxiety-like symptoms with mildly impaired dopaminergic neurotransmission, while serotonergic PSAP-deficient (cPSAPSERT) mice behave normally. Spatial lipidomics revealed an accumulation of highly unsaturated and shortened lipids and reduction of sphingolipids throughout the brains of cPSAPDAT mice. The overexpression of α-synuclein via AAV lead to more severe dopaminergic degeneration and higher p-Ser129 α-synuclein levels in cPSAPDAT mice compared to WT mice. Overexpression of PSAP via AAV and encapsulated cell biodelivery protected against 6-OHDA and α-synuclein toxicity in wild-type rodents. Thus, these findings suggest PSAP may maintain dopaminergic lipid homeostasis, which is dysregulated in PD, and counteract experimental parkinsonism.

Mass spectrometry imaging (MSI) is a powerful technique that combines the abil-ity of microscopy to provide spatial information about multiple molecular species with the specificity of mass spectrometry (MS) for unlabeled mapping of analytes in diverse biological tissues. Initial pharmacological applications focused on drug distributions in different organs, including the compartmentalized brain. However, recent technological advances in instrumentation, software, and chemical tools have allowed its use in quantitative spatial omics. It now enables visualization of distributions of diverse molecules at high lateral resolution in studies of the pharmacokinetic and neuropharmacodynamic effects of drugs on functional biomolecules. Therefore, it has become a versatile technique with a multitude of applications that have transformed neuropharmacological re-search and enabled research into brain physiology at unprecedented resolution, as described in this review.

Cluster ion beam ToF-SIMS and/or MALDI-ToF mass spectrometry imaging (using 1,5-DAN matrix via sublimation) of a single coronal rat brain tissue section followed by classical- or immuno- histochemical staining faclilated a new multimodal chemical imaging workflow allowing complementary correlation of the lipid molecular ion images with the immuno/histological features within cerebellum region of the same brain tisue section.

There is emerging evidence that amyloid beta (A beta) aggregates forming neuritic plaques lead to impairment of the lipid-rich myelin sheath and glia. In this study, we examined focal myelin lipid alterations and the disruption of the myelin sheath associated with amyloid plaques in a widely used familial Alzheimer's disease (AD) mouse model; 5xFAD. This AD mouse model has A beta(42) peptide-rich plaque deposition in the brain parenchyma. Matrix-assisted laser desorption/ionization imaging mass spectrometry of coronal brain tissue sections revealed focal A beta plaque-associated depletion of multiple myelin-associated lipid species including sulfatides, galactosylceramides, and specific plasmalogen phopshatidylethanolamines in the hippocampus, cortex, and on the edges of corpus callosum. Certain phosphatidylcholines abundant in myelin were also depleted in amyloid plaques on the edges of corpus callosum. Further, lysophosphatidylethanolamines and lysophosphatidylcholines, implicated in neuroinflammation, were found to accumulate in amyloid plaques. Double staining of the consecutive sections with fluoromyelin and amyloid-specific antibody revealed amyloid plaque-associated myelin sheath disruption on the edges of the corpus callosum which is specifically correlated with plaque-associated myelin lipid loss only in this region. Further, apolipoprotein E, which is implicated in depletion of sulfatides in AD brain, is deposited in all the A beta plaques which suggest apolipoprotein E might mediate sulfatide depletion as a consequence of an immune response to A beta deposition. This high-spatial resolution matrix-assisted laser desorption/ionization imaging mass spectrometry study in combination with (immuno) fluorescence staining of 5xFAD mouse brain provides new understanding of morphological, molecular and immune signatures of A beta plaque pathology-associated myelin lipid loss and myelin degeneration in a brain region-specific manner.

Acute myocardial infarction (MI) is a cardiovascular disease that remains a major cause of morbidity and mortality worldwide despite advances in its prevention and treatment. During acute myocardial ischemia, the lack of oxygen switches the cell metabolism to anaerobic respiration, with lactate accumulation, ATP depletion, Na+ and Ca2+ overload, and inhibition of myocardial contractile function, which drastically modifies the lipid, protein, and small metabolite profile in the myocardium. Imaging mass spectrometry (IMS) is a powerful technique to comprehensively elucidate the spatial distribution patterns of lipids, peptides, and proteins in biological tissue sections. In this work, we demonstrate an application of multimodal chemical imaging using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), which provided comprehensive molecular information in situ within the same mouse heart tissue sections with myocardial infarction. MALDI-IMS (at 30 μm per pixel) revealed infarct-associated spatial alterations of several lipid species of sphingolipids, glycerophospholipids, lysophospholipids, and cardiolipins along with the acyl carnitines. Further, we performed multimodal MALDI-IMS (IMS3) where dual polarity lipid imaging was combined with subsequent protein MALDI-IMS analysis (at 30 μm per pixel) within the same tissue sections, which revealed accumulations of core histone proteins H4, H2A, and H2B along with post-translational modification products, acetylated H4 and H2A, on the borders of the infarcted region. This methodology allowed us to interpret the lipid and protein molecular pathology of the very same infarcted region in a mouse model of myocardial infarction. Therefore, the presented data highlight the potential of multimodal MALDI imaging mass spectrometry of the same tissue sections as a powerful approach for simultaneous investigation of spatial infarct-associated lipid and protein changes of myocardial infarction.