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The brain’s high demand for energy necessitates tightly regulated metabolic pathways to sustain physiological activity. Glucose, the primary energy substrate, undergoes complex metabolic transformations, with mitochondria playing a central role in ATP production via oxidative phosphorylation. Dysregulation of this metabolic interplay is implicated in Alzheimer’s disease (AD), where compromised glucose metabolism, oxidative stress, and mitochondrial dysfunction contribute to disease progression. This review explores the intricate bioenergetic crosstalk between astrocytes and neurons, highlighting the function of mitochondrial uncoupling proteins (UCPs), particularly UCP4, as important regulators of brain metabolism and neuronal function. Predominantly expressed in the brain, UCP4 reduces the membrane potential in the inner mitochondrial membrane, thereby potentially decreasing the generation of reactive oxygen species. Furthermore, UCP4 mitigates mitochondrial calcium overload and sustains cellular ATP levels through a metabolic shift from mitochondrial respiration to glycolysis. Interestingly, the levels of the neuronal UCPs, UCP2, 4 and 5 are significantly reduced in AD brain tissue and a specific UCP4 variant has been associated to an increased risk of developing AD. Few studies modulating the expression of UCP4 in astrocytes or neurons have highlighted protective effects against neurodegeneration and aging, suggesting that pharmacological strategies aimed at activating UCPs, such as protonophoric uncouplers, hold promise for therapeutic interventions in AD and other neurodegenerative diseases. Despite significant advances, our understanding of UCPs in brain metabolism remains in its early stages, emphasizing the need for further research to unravel their biological functions in the brain and their therapeutic potential.

Abstract Extracellular vesicles (EVs) are shed from the plasma membrane, but the regulation and function of these EVs remain unclear. We found that oxidative stress induced by H2O2 in Hela cells stimulated filopodia formation and the secretion of EVs. EVs were small (150 nm) and labeled for CD44, indicating that they were derived from filopodia. Filopodia‐derived small EVs (sEVs) were enriched with the sphingolipid ceramide, consistent with increased ceramide in the plasma membrane of filopodia. Ceramide was colocalized with neutral sphingomyelinase 2 (nSMase2) and acid sphingomyelinase (ASM), two sphingomyelinases generating ceramide at the plasma membrane. Inhibition of nSMase2 and ASM prevented oxidative stress‐induced sEV shedding but only nSMase2 inhibition prevented filopodia formation. nSMase2 was S‐palmitoylated and interacted with ASM in filopodia to generate ceramide for sEV shedding. sEVs contained nSMase2 and ASM and decreased the level of these two enzymes in oxidatively stressed Hela cells. A novel metabolic labeling technique for EVs showed that oxidative stress induced secretion of fluorescent sEVs labeled with NBD‐ceramide. NBD‐ceramide‐labeled sEVs transported ceramide to mitochondria, ultimately inducing cell death in a proportion of neuronal (N2a) cells. In conclusion, using Hela cells we provide evidence that oxidative stress induces interaction of nSMase2 and ASM at filopodia, which leads to shedding of ceramide‐rich sEVs that target mitochondria and propagate cell death.

Abstract In Alzheimer’s disease (AD), reactive astrocytes produce extracellular vesicles (EVs) that affect mitochondria in neurons. Here, we show that Aβ-induced generation of the sphingolipid ceramide by acid sphingomyelinase (A-SMase) triggered proinflammatory cytokine (C1q, TNF-α, IL-1α) release by microglia, which induced the reactive astrocytes phenotype and secretion of EVs enriched with ceramide. These EVs impeded the capacity of neurons to respond to energy demand. Inhibition of A-SMase with Arc39 and Imipramine reduced the secretion of cytokines from microglia, prompting us to test the effect of Imipramine on EV secretion and AD pathology in the 5xFAD mouse model. Brain derived-EVs from 5xFAD mice treated with Imipramine contained reduced levels of the astrocytic marker GFAP, ceramide, and Aβ and did not impair mitochondrial respiration when compared to EVs derived from untreated 5xFAD brain. Consistently, Imipramine-treated 5xFAD mice showed reduced AD pathology. Our study identifies A-SMase inhibitors as potential AD therapy by preventing cyotokine-elicited secretion of mitotoxic EVs from astrocytes.

Summary: Background: Previously, we showed that the sphingosine-1-phosphate (S1P) transporter spinster 2 (Spns2) mediates activation of microglia in response to amyloid β peptide (Aβ). Here, we investigated if Ponesimod, a functional S1P receptor 1 (S1PR1) antagonist, prevents Aβ-induced activation of glial cells and Alzheimer's disease (AD) pathology. Methods: We used primary cultures of glial cells and the 5XFAD mouse model to determine the effect of Aβ and Ponesimod on glial activation, Aβ phagocytosis, cytokine levels and pro-inflammatory signaling pathways, AD pathology, and cognitive performance. Findings: Aβ42 increased the levels of TLR4 and S1PR1, leading to their complex formation. Ponesimod prevented the increase in TLR4 and S1PR1 levels, as well as the formation of their complex. It also reduced the activation of the pro-inflammatory Stat1 and p38 MAPK signaling pathways, while activating the anti-inflammatory Stat6 pathway. This was consistent with increased phagocytosis of Aβ42 in primary cultured microglia. In 5XFAD mice, Ponesimod decreased the levels of TNF-α and CXCL10, which activate TLR4 and Stat1. It also increased the level of IL-33, an anti-inflammatory cytokine that promotes Aβ42 phagocytosis by microglia. As a result of these changes, Ponesimod decreased the number of Iba-1+ microglia and GFAP+ astrocytes, and the size and number of amyloid plaques, while improving spatial memory as measured in a Y-maze test. Interpretation: Ponesimod targeting S1PR1 is a promising therapeutic approach to reprogram microglia, reduce neuroinflammation, and increase Aβ clearance in AD. Funding: NIH R01AG064234, RF1AG078338, R21AG078601, VA I01BX003643.

Abstract Introduction There is an urgent need for biomarkers identifying individuals at risk of early‐stage cognitive impairment. Using cross‐sectional data from The Maastricht Study, this study included 197 individuals with mild cognitive impairment (MCI) and 200 cognitively unimpaired individuals aged 40 to 75, matched by age, sex, and educational level. Methods We assessed the association of plasma sphingolipid and ceramide transfer protein (CERT) levels with MCI and adjusted for potentially confounding risk factors. Furthermore, the relationship of plasma sphingolipids and CERTs with magnetic resonance imaging brain volumes was assessed and age‐ and sex‐stratified analyses were performed. Results Associations of plasma ceramide species C18:0 and C24:1 and combined plasma ceramide chain lengths (ceramide risk score) with MCI were moderated by sex, but not by age, and higher levels were associated with MCI in men. No associations were found among women. In addition, higher levels of ceramide C20:0, C22:0, and C24:1, but not the ceramide risk score, were associated with larger volume of the hippocampus after controlling for covariates, independent of MCI. Although higher plasma ceramide C18:0 was related to higher plasma CERT levels, no association of CERT levels was found with MCI or brain volumes. Discussion Our results warrant further analysis of plasma ceramides as potential markers for MCI in middle‐aged men. In contrast to previous studies, no associations of plasma sphingolipids with MCI or brain volumes were found in women, independent of age. These results highlight the importance of accounting for sex‐ and age‐related factors when examining sphingolipid and CERT metabolism related to cognitive function.

We developed a new method to isolate small extracellular vesicles (sEVs) from male and female wild-type and 5xFAD mouse brains to investigate the sex-specific functions of sEVs in Alzheimer’s disease (AD). A mass spectrometric analysis revealed that sEVs contained proteins critical for EV formation and Aβ. ExoView analysis showed that female mice contained more GFAP and Aβ-labeled sEVs, suggesting that a larger proportion of sEVs from the female brain is derived from astrocytes and/or more likely to bind to Aβ. Moreover, sEVs from female brains had more acid sphingomyelinase (ASM) and ceramide, an enzyme and its sphingolipid product important for EV formation and Aβ binding to EVs, respectively. We confirmed the function of ASM in EV formation and Aβ binding using co-labeling and proximity ligation assays, showing that ASM inhibitors prevented complex formation between Aβ and ceramide in primary cultured astrocytes. Finally, our study demonstrated that sEVs from female 5xFAD mice were more neurotoxic than those from males, as determined by impaired mitochondrial function (Seahorse assays) and LDH cytotoxicity assays. Our study suggests that sex-specific sEVs are functionally distinct markers for AD and that ASM is a potential target for AD therapy.

Abstract The formation of extracellular vesicles (EVs) is induced by the sphingolipid ceramide. How this pathway is regulated is not entirely understood. Here, we report that the ceramide transport protein (CERT) mediates a non‐vesicular transport of ceramide between the endoplasmic reticulum (ER) and the multivesicular endosome at contact sites. The process depends on the interaction of CERT's PH domain with PI4P generated by PI4KIIα at endosomes. Furthermore, a complex is formed between the START domain of CERT, which carries ceramide, and the Tsg101 protein, which is part of the endosomal sorting complex required for transport (ESCRT‐I). Inhibition of ceramide biosynthesis reduces CERT‐Tsg101 complex formation. Overexpression of CERT increases EV secretion while its inhibition reduces EV formation and the concentration of ceramides and sphingomyelins in EVs. In conclusion, we discovered a function of CERT in regulating the sphingolipid composition and biogenesis of EVs, which links ceramide to the ESCRT‐dependent pathway.

Abstract Background Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer’s disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. Methods A plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay. Results Here, we report that CERTL binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype. Conclusion Our results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.

Abstract Amyloid-β (Aβ) associates with extracellular vesicles termed exosomes. It is not clear whether and how exosomes modulate Aβ neurotoxicity in Alzheimer’s disease (AD). We show here that brain tissue and serum from the transgenic mouse model of familial AD (5xFAD) and serum from AD patients contains ceramide-enriched and astrocyte-derived exosomes (termed astrosomes) that are associated with Aβ. In Neuro-2a cells, primary cultured neurons, and human induced pluripotent stem cell-derived neurons, Aβ-associated astrosomes from 5xFAD mice and AD patient serum were specifically transported to mitochondria, induced mitochondrial clustering, and upregulated the fission protein Drp-1 at a concentration corresponding to 5 femtomoles Aβ/L of medium. Aβ-associated astrosomes, but not wild type or control human serum exosomes, mediated binding of Aβ to voltage-dependent anion channel 1 (VDAC1) and subsequently, activated caspases. Aβ-associated astrosomes induced neurite fragmentation and neuronal cell death, suggesting that association with astrosomes substantially enhances Aβ neurotoxicity in AD and may comprise a novel target for therapy.

Apolipoprotein ε4 (APOE)4 is a strong risk factor for the development of Alzheimer’s disease (AD) and aberrant sphingolipid levels have been implicated in AD. We tested the hypothesis that the APOE4 genotype affects brain sphingolipid levels in AD. Seven ceramides and sphingosine-1-phosphate (S1P) were quantified by LC-MSMS in hippocampus, cortex, cerebellum, and plasma of 5 months old human APOE3 and APOE4-targeted replacement mice with or without the familial AD (FAD) background of both sexes (145 animals). APOE4 mice had higher Cer(d18:1/24:0) levels in the cortex (1.7-fold, p = 0.002) than APOE3 mice. Mice with AD background showed higher levels of Cer(d18:1/24:1) in the cortex than mice without (1.4-fold, p = 0.003). S1P levels were higher in all three brain regions of older mice than of young mice (1.7-1.8-fold, all p ≤ 0.001). In female mice, S1P levels in hippocampus (r = −0.54 [−0.70, −0.35], p < 0.001) and in cortex correlated with those in plasma (r = −0.53 [−0.71, −0.32], p < 0.001). Ceramide levels were lower in the hippocampus (3.7–10.7-fold, all p < 0.001), but higher in the cortex (2.3–12.8-fold, p < 0.001) of female than male mice. In cerebellum and plasma, sex effects on individual ceramides depended on acyl chain length (9.5-fold lower to 11.5-fold higher, p ≤ 0.001). In conclusion, sex is a stronger determinant of brain ceramide levels in mice than APOE genotype, AD background, or age. Whether these differences impact AD neuropathology in men and women remains to be investigated.

Abstract: Microtubules are polymers composed of αβ-tubulin subunits that provide structure to cells and play a crucial role in in the development and function of neuronal processes and cilia, microtubule-driven extensions of the plasma membrane that have sensory (primary cilia) or motor (motile cilia) functions. To stabilize microtubules in neuronal processes and cilia, α tubulin is modified by the posttranslational addition of an acetyl group, or acetylation. We discovered that acetylated tubulin in microtubules interacts with the membrane sphingolipid, ceramide. However, the molecular mechanism and function of this interaction are not understood. Here, we show that in human induced pluripotent stem cell–derived neurons, ceramide stabilizes microtubules, which indicates a similar function in cilia. Using proximity ligation assays, we detected complex formation of ceramide with acetylated tubulin in Chlamydomonas reinhardtii flagella and cilia of human embryonic kidney (HEK293T) cells, primary cultured mouse astrocytes, and ependymal cells. Using incorporation of palmitic azide and click chemistry–mediated addition of fluorophores, we show that a portion of acetylated tubulin is S-palmitoylated. S-palmitoylated acetylated tubulin is colocalized with ceramide-rich platforms in the ciliary membrane, and it is coimmunoprecipitated with Arl13b, a GTPase that mediates transport of proteins into cilia. Inhibition of S-palmitoylation with 2-bromo palmitic acid or inhibition of ceramide biosynthesis with fumonisin B1 reduces formation of the Arl13b-acetylated tubulin complex and its transport into cilia, concurrent with impairment of ciliogenesis. Together, these data show, for the first time, that ceramide-rich platforms mediate membrane anchoring and interaction of S-palmitoylated proteins that are critical for cilium formation, stabilization, and function.

Ceramide-rich platforms (CRPs) mediate association of proteins with the sphingolipid ceramide and may regulate protein interaction in membrane contact sites to the cytoskeleton, organelles, and infectious pathogens. However, visualization of ceramide association to proteins is one of the greatest challenges in understanding the cell biology of ceramide. Here we introduce a novel labeling technique for ceramide-associated proteins (CAPs) by combining photoactivated cross-linking of a bioorthogonal and bifunctional ceramide analog, pacFACer with proximity ligation assays (PLAs). pacFACer cross-linked to CAPs is covalently attached to a fluorophore using click chemistry. PLAs use antibodies to: (1) the candidate CAP and the fluorophore (PLA1); and (2) the CAP and ceramide (PLA2). PLA1 shows the subcellular localization of a particular CAP that is cross-linked to pacFACer, while PLA2 tests if the cross-linked CAP forms a complex with endogenous ceramide. Two proteins, tubulin and voltage-dependent anion channel 1 (VDAC1), were cross-linked to pacFACer and showed PLA signals for a complex with ceramide and pacFACer, which were predominantly colocalized with microtubules and mitochondria, respectively. Binding of tubulin and VDAC1 to ceramide was confirmed by coimmunoprecipitation assays using anti ceramide antibody. Cross-linking to pacFACer was confirmed using click chemistry-mediated attachment of biotin and streptavidin pull-down assays. Inhibition of ceramide synthases with fumonisin B1 (FB1) reduced the degree of pacFACer cross-linking and complex formation with ceramide, while it was enhanced by amyloid beta peptide (Aβ). Our results show that endogenous ceramide is critical for mediating cross-linking of CAPs to pacFACer and that a combination of cross-linking with PLAs (cross-link/PLA) is a novel tool to visualize CAPs and to understand the regulation of protein interaction with ceramide in CRPs.