The active biolipid sphingosine-1-phosphate (S1P) regulates cellular survival and stress responses, emerging also as a potential modulator of autophagy and mitophagy. Given the well-established role of S1P signalling in hypoxia, this study investigated how S1P modulates autophagic processes in a neuronal hypoxic model, mimicking such process in vitro through the chemical stabilization of the Hypoxia-inducible factor 1-alpha (HIF-1α) subunit via DMOG-mediated prolyl hydroxylase inhibition. Hypoxia-like conditions reshaped S1P metabolism by downregulating its major degradative enzymes, Sphingosine-1-phosphate phosphatase 1 (SGPP1) and Sphingosine-1-phosphate lyase 1 (SGPL1), potentially prolonging its signalling. Conversely, exogenous S1P administration determined a compensatory upregulation of these catabolic genes, likely promoting signal termination. After characterizing the distinct morphological rearrangements induced by DMOG and S1P, we examined their combined impact on the autophagic machinery. While DMOG alone activated autophagy, exogenous S1P acted as an inhibitor. Notably, selective activation of S1PR1 and S1PR5 showed divergent effects; however, their co-administration with DMOG led to a significant accumulation of Microtubule-associated protein 1 light chain 3 (LC3) and SQSTM1/p62. This molecular signature, further supported by mitochondrial-lysosomal colocalization analyses and thresholded Manders’ coefficients, is suggestive of altered mitophagy. Our findings demonstrate that S1P signalling is a key modulator of the neuronal hypoxic response, acting also mitochondrial quality control. The convergence between hypoxia-induced stress and S1P receptor activation during mitophagy suggests a primary role for S1P in protecting cells from metabolic exhaustion. Consequently, S1P biosynthesis and S1P receptors (S1PR) emerge as promising therapeutic targets for neurological conditions associated with hypoxic stress, dysregulated autophagy, and mitochondrial dysfunction. Divergent roles of S1PR1 and S1PR5 in the neuronal hypoxic response. HIF-1α upregulates GLUT1, GLUT3, BNIP3, and SPHK1. Under hypoxic conditions, exogenous S1P downregulates SPHK2, but also SGPP1 and SGPL1, which are involved in S1P catabolism, thus prolonging S1P signalling. Selective activation of S1PR1 and S1PR5 receptors induces divergent responses in cytoskeletal dynamics and degradative pathways compared to the pan-receptor effect of S1P. Concomitant HIF-1α stabilization further reprogrammes these responses
Cerebral ischemia-reperfusion injury (CIRI) significantly exacerbates neuronal damage during ischemic stroke. Longhu Xingnao Granules (LHXNG) has been used clinically for stroke-related disorders and has shown neuroprotective potential against CIRI. However, the pharmacological basis and underlying molecular mechanisms remain to be clarified. This study aimed to explore the neuroprotective effects and potential mechanisms of LHXNG against CIRI. Ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to characterize representative chemical constituents of LHXNG. Neuroprotective efficacy was assessed in a rat middle cerebral artery occlusion/reperfusion (MCAO/R) model. An in vitro erastin-induced ferroptosis model in PC12 cells, combined with small interfering RNA (siRNA)-mediated NUDT1 knockdown, was established. UPLC-Q-TOF/MS identified gastrodin, chlorogenic acid, paeoniflorin, ferulic acid, and astragaloside IV as representative constituents. In vivo, pretreatment with LHXNG improved neurological function, reduced cerebral infarct volume, and alleviated histopathological injury. Biochemically, LHXNG decreased ferrous iron (Fe²⁺), malondialdehyde (MDA), reactive oxygen species (ROS), and lipid peroxidation (LPO) levels while restoring glutathione (GSH) content, indicating the suppression of ferroptosis-related oxidative damage after CIRI. At the molecular level, LHXNG treatment was associated with increased nudix hydrolase 1 (NUDT1) and glutathione peroxidase 4 (GPX4) expression and decreased acyl-CoA synthetase long-chain family member 4 (ACSL4) expression, together with changes in ferroptosis-associated injury markers. Importantly, NUDT1 knockdown weakened the anti-ferroptotic effects of LHXNG-medicated serum in PC12 cells, suggesting a possible contribution of NUDT1 to this process. In conclusion, LHXNG pretreatment attenuates experimental CIRI in association with reduced ferroptosis-related oxidative injury and changes in NUDT1, GPX4, and ACSL4 expression. The potential contribution of NUDT1 warrants further investigation.
L-lactate administration enhances functional recovery in several preclinical animal models of acute brain injury, and its protective effects have been mainly related to its metabolic functions. The neurovascular unit, consisting of endothelial cells, pericytes, glial cells and neurons, is central to brain metabolism and post-ischemic inflammation. Here, we analyzed the effect of L-lactate supplementation on the response to injury of cellular components of the neurovascular unit in the acute post-ischemic phase. When administered at reperfusion, L-lactate enhanced proliferation in the vascular compartment of the lesion. Furthermore, reactive glial cells from lactate-treated mice exhibited increased morphological complexity and differential expression of the gap-junction protein connexin 30 and the lysosomal protein CD68. Our results suggest that, in addition to its presumed ability to help overcome the hypoxic energy crisis and prevent brain cell death, L-lactate supplementation might contribute to stroke recovery by facilitating the execution and/or progression of signaling programs that promote proliferation, as well as by fine-tuning the efficiency with which glial cells confine and clear ischemic damage. L-lactate supplementation could fuel recovery after stroke and help mitigate damage extension. L-lactate could protect neurovascular unit cells from early death, promote proliferation within the lesion and refine the efficiency with which glial cells confine and clear ischemic damage, thereby preventing its spread
Neuromelanin (NM), a neuronal pigment linked to catecholamine metabolism, has been implicated in both protective and detrimental roles in neurodegenerative diseases, including Parkinson’s and Alzheimer’s diseases. Given the continuity between plasma and cerebrospinal fluid, we aimed to identify and characterize plasma proteins retained by synthetic catecholamine polymers used as neuromelanin analogs. Proteins retained or enriched on these matrices were operationally classified as candidate neuromelanin-binding plasma proteins (cNBPPs). cNBPPs in pooled plasma were identified using an exploratory affinity-proteomics approach with two NM analogs (poly-L-DOPA and poly-noradrenaline). Following stringent washing and LC-MS/MS analysis, the identified proteins were evaluated by structural bioinformatics, Gene Ontology, and protein–protein interaction network analyses. Raw data are available in PRIDE repository (PXD084910), and additional processed data at DOI: https://doi.org/10.4321/repisalud.27499 . We identified 98 cNBPPs, 51 (52%) of which were retained by both NM-analog matrices, suggesting shared physicochemical determinants. cNBPPs were more frequently annotated as glycosylated than non-NBPPs. Abundant cNBPPs were typically larger and displayed increased 3₁₀-helix content, whereas low-abundance cNBPPs exhibited reduced α-helix and increased β-sheet content. Functional enrichment associated cNBPPs with inflammation, immune regulation, complement activation, vascular biology, and amyloid-related processes. cNBPPs included hub proteins such as ApoE and adiponectin, while several served as highly connected network nodes, suggesting links between the cNBPP network and neuroinflammation or protein aggregation. These findings offer a hypothesis-generating perspective on potential relationships between NM and neurodegeneration involving plasma proteins and, potentially, their CSF or brain counterparts. This exploratory affinity-proteomics screening may help prioritize candidates for future mechanistic and diagnostic studies. Graphical Abstract Candidate neuromelanin-binding plasma proteins support a protein-corona model. Plasma affinity proteomics using synthetic neuromelanin (NM) analogs identified structurally selective candidate neuromelanin-binding plasma proteins (cNBPPs). Their enrichment in complement, vascular, extracellular proteostasis, and amyloid-related pathways supports a protein-corona hypothesis in which extracellular NM may recruit compatible proteins with potential relevance to neurodegeneration
γ-secretase is an intramembrane protease complex whose four human assemblies are defined by combinatorial association of PSEN1 or PSEN2 with APH1A or APH1B together with nicastrin and PEN-2. Rather than being functionally equivalent, the four complexes are now increasingly appreciated to differ in proteolytic output, substrate access, and disease susceptibility in ways that have direct therapeutic implications. Biochemical, structural, and genetic evidence collectively demonstrate that γ-secretase heterogeneity is not incidental, but both functional and consequential. Each complex produces a distinct amyloid-β profile reflecting differences in processive trimming efficiency, with PSEN2-containing complexes generating more aggregation-prone species than their PSEN1 counterparts. These intrinsic biochemical differences are compounded by differential subcellular sorting, with PSEN1 being trafficked to the plasma membrane and PSEN2 complexes directed to the endolysosomal compartment, creating spatially segregated proteolytic environments with distinct substrate repertoires and pH conditions. Complex composition varies across CNS cell types and is dynamically regulated by inflammatory and lipid signals, adding a contextual layer that cannot be reduced to subunit identity alone. In disease, the divergent clinical profiles of PSEN1 and PSEN2 familial Alzheimer’s Disease mutations, the non-redundant roles of APH1B complexes, and the selective therapeutic window achieved by PSEN1-specific inhibition in T-cell acute lymphoblastic leukaemia demonstrate that these distinctions have real pathological and pharmacological consequences. The failure of indiscriminate γ-secretase inhibitors in clinical trials reflects the cost of treating a heterogeneous system as uniform. The mechanistic framework now available, spanning structural biology, proteomics, transcriptomics and isoform-selective pharmacology, provides the tools to do better. Realising the therapeutic potential of γ-secretase will require heterogeneity to become a design principle rather than an afterthought.
