Cellular and Molecular Life Sciences

Lysophosphatidic acid (LPA) is widely recognized as an extracellular lipid mediator; however, the functional significance of intracellularly produced LPA remains poorly understood. Here, we investigated the regulatory mechanism and functional role of a LPA-producing lysophospholipase D GDE4, also known as GDPD1, in prostate cancer cells. GDE4 expression is induced under ER stress conditions in a PERK-dependent manner and requires the transcription factor ATF3. Disruption of GDE4 expression resulted in altered intracellular levels of LPA and LPA precursor lysophosphatidylethanolamine, accompanied by reduced cell proliferation. RNA sequencing and subsequent validation identified a set of genes downregulated in GDE4-depleted cells. Pharmacological inhibition experiments indicated that peroxisome proliferator-activated receptor and {gamma} (PPAR and PPAR{gamma}) signaling pathways contribute to the regulation of these GDE4-dependent genes. Collectively, our findings suggest that GDE4-dependent lipid remodeling is associated with PPAR/{gamma}-mediated transcriptional regulation under ER stress conditions. These results provide a potential framework for understanding the link between intracellular lipid metabolism and stress-responsive gene regulation.

The deformability of the red blood cell (RBC) is essential for microcirculatory flow and is profoundly altered in hemoglobinopathies and during infection with Plasmodium falciparum. While many mechanical tests have been developed to probe RBC-mechanics, the dynamics of cell shape recovery following large deformations remains poorly characterized. Here, we integrate microfluidic constriction assays, ultrafast imaging, and computer simulations to quantify time-resolved shape recovery of individual erythrocytes. We show that parasite infection is the primary determinant of RBC viscoelastic behavior. In wild-type (HbAA) erythrocytes, the relaxation time increases progressively from ring to trophozoite to schizont stages, consistent with parasite-induced membrane stiffening and enhanced membrane-cytoskeleton coupling. In contrast, sickle trait (HbAS) erythrocytes exhibit a distinct response: although deformation becomes increasingly irreversible during parasite maturation, the relaxation time after constriction remains largely unchanged. Analysis of a mutant parasite line with enlarged and sparsely distributed knobs revealed a significant increase in relaxation time, demonstrating that knob architecture modulates recovery kinetics. Together, these findings suggest that the coupling between membrane and cytoskeleton, which is strongly changed by the establishment of the knobs during an infection with Plasmodium falciparum, should have a strong detrimental effect on microcirculatory flow, which is however weakened by the sickle cell trait.

The cluster of differentiation 36 (CD36) is a membrane protein with broad physiological roles in health and disease, and its function is regulated in part by phosphorylation. Experimental evidence shows that phosphorylation of Thr92 reduces CD36 affinity for thrombospondin-1 (TSP-1), binding of which initiates antiangiogenic signaling, whereas phosphorylation of Ser237 decreases CD36-mediated fatty acid uptake, with implications for energy metabolism. However, the only available crystal structure of CD36 lacks phosphorylation, and the molecular mechanisms by which phosphorylation regulates CD36 function remain largely unknown. This study provides an atomically detailed computational characterization of CD36 in unphosphorylated and dual phosphorylated states, using molecular dynamics simulations with a total sampling time of 30 microseconds in combination with Markov state models. We present, to our knowledge, the first evidence of a cryptic pocket on CD36 surface that is formed by phosphorylation. This cryptic surface pocket and a loop spanning residues 121-131 form a high affinity binding site for TSP-1 derived ligands, shifting their binding away from the canonical site. We propose that this altered binding provides a molecular basis for the disruption of antiangiogenic signaling upon CD36 phosphorylation. Additionally, our data indicate that, phosphorylation increases helicity and compaction within the helix-loop region spanning residues 296-331, narrowing one of the entrances to the internal cavity and reducing its overall volume. These conformational changes provide a potential mechanistic explanation for the decrease in fatty acid uptake upon CD36 phosphorylation. Our findings provide structural insights that may inform the future design of CD36 modulators and emphasize the importance of targeting phosphorylation induced CD36 conformations in angiogenic and metabolic diseases.

BCL2L13 is a mitochondrial BCL2 family protein linked to mitophagy and ceramide metabolism, but its role in NSCLC metastatic plasticity remains unclear. Human lung cancer Tissue Microarray and matched patient specimens showed subtype and site dependent BCL2L13 expression, with higher cytoplasmic granular staining in primary NSCLC and reduced, heterogeneous staining in lymph node metastases, most evident in adenocarcinoma and squamous cell carcinoma. Because Epithelial mesenchymal transition and anoikis resistance are central requirements for metastatic dissemination, this primary to node attenuation provided the rationale to test BCL2L13 knockdown and overexpression in metastasis relevant NSCLC models. In A549 and LLC cell lines. TGF beta 1 induced coordinated mitophagy and EMT with mitochondrial enrichment of BCL2L13. BCL2L13 knockdown impaired TGF beta 1 and carbonyl cyanide m chlorophenyl hydrazone associated mitophagy, reducing LC3 beta mitochondria colocalization, TOMM20, LAMP1 overlap and mitochondrial LC3 II, p62, TOMM20 turnover; BNIP3 and NIX redistribution did not compensate. BCL2L13 loss enhanced EMT marker switching and migration, whereas overexpression partially opposed these changes. During detachment, BCL2L13 knockdown reduced anoikis associated apoptosis despite preserved mitochondrial recruitment of BAX, BAK, BNIP3,NIX, altered BID processing, non parallel caspase activity and shifted FAK phosphorylation. Pharmacological autophagy modulation did not reverse this anoikis phenotype. Lipidomics identified adhesion state dependent ceramide synthases CerS2, CerS6 linked sphingolipid remodeling: BCL2L13 knockdown increased C24 linked sphingolipid species in attached cells but reduced C16, C24 ceramide related profiles during anoikis. These findings identify BCL2L13 downregulation as a metastasis associated mitochondrial-lipid state that limits mitophagic quality control while favoring EMT and detachment survival in NSCLC adenocarcinoma.

The immune checkpoint protein PD-L1 plays a pivotal role in tumor immune evasion by binding to PD-1 on immune cells, including T lymphocytes. While the expression and function of PD-L1 have been well studied, the importance of its spatial organization on the cell surface of tumor cells remains poorly understood. In this study, we used super-resolution microscopy combined with biochemical perturbations to investigate the factors regulating PD-L1 clustering and its effects on PD-1 binding and T cell inhibition. We found that PD-L1 is organized into nanoscale clusters at the plasma membrane, with distinct regulatory roles for the actin cytoskeleton, galectin-3, and cholesterol. Disruption of cortical actin increased PD-L1 cluster size, while galectin-3 promoted smaller, denser clusters and increased PD-L1 lateral mobility. Cholesterol depletion reduced PD-L1 cluster size and number and impaired PD-1 binding. These findings indicate that PD-L1 surface organization is collectively regulated by the actin cytoskeleton, galectin-3, and membrane cholesterol within the plasma membrane of tumour cells. Our results provide new insights into the dynamic regulation of PD-L1 and its potential as a therapeutic target in cancer immunotherapy.

Objective White smoke inhalation injury (WSI) causes severe acute lung damage with no specific therapy currently available. Sphingolipid metabolism is implicated in pulmonary inflammation, but its transcriptional regulatory landscape in WSI remains unexplored. This study aimed to identify key sphingolipid metabolism related genes and evaluate their regulatory roles and therapeutic potential in WSI. Methods We established a rat model of WSI and performed integrated bulk RNA sequencing, weighted gene coexpression network analysis (WGCNA), and single-cell RNA sequencing (scRNAseq) to screen for differentially expressed sphingolipid metabolism-related genes (DESRGs). Protein-protein interaction (PPI) network with four centrality algorithms was used to prioritize hub genes. In silico gene knockout and molecular docking were conducted to assess regulatory functions and identify potential drug candidates. Results We identified 22 DESRGs that were predominantly enriched in DNA replication and cell cycle pathways rather than canonical sphingolipid metabolic processes. PPI consensus prioritized three hub genes--Top2a, Ttk, and Ccna2--with Top2a exhibiting the highest expression in epithelial cells and significant downregulation after smoke exposure. ScRNAseq revealed immune cell infiltration and epithelial differentiation trajectories. Virtual knockout showed that Top2a depletion affected the largest transcriptomic fraction (~0.4%) and was enriched in lysosome biogenesis, innate immunity, phagocytosis, and lipid catabolism. Molecular docking identified thalidomide as a high affinity ligand for Top2a (Vina score: -8.5 kcal/mol). Conclusion Our multiomics integrative framework identifies Top2a as a central regulatory hub linking sphingolipid associated inflammation to epithelial responses in WSI, and nominates thalidomide as a potential drug repurposing candidate. These findings provide prioritized targets for future translational investigation.

Hepatocellular carcinoma (HCC) is particularly aggressive and difficult to treat. Due to the lack of early clinical diagnosis and the unsatisfactory clinical treatment effect, it is particularly important to identify novel markers that can predict tumor behavior in HCC. biogenesis of ribosomes BRX1 (BRIX1) is abundant in various tissues of the human body. However, the regulatory mechanisms and its role in various tissues are not fully understood. Here, we analyzed the expression pattern of BRIX1 in HCC from public gene expression databases and tissue samples from clinical HCC. We confirmed that BRIX1 was upregulated in both HCC cell lines and HCC paraffin section samples. BRIX1 depletion significantly dicreased the capacity of cells to grow and migrate in vitro, and knockdown BRIX1 suppressed tumor growth in xenograft tumor model. Mechanistically, BRIX1 depletion suppressed the MAPK/ERK pathway, as reflected by reduced phosphorylated ERK (p-ERK) levels. In summary, we provide a rational clue for the further investigation of BRIX1 as an invaluable biological marker for diagnosing and predicting prognosis of patients with HCC.

O-linked {beta}-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole writer for intracellular O-GlcNAcylation. It catalyzes O-GlcNAcylation of thousands of protein substrates, but relatively less is known about the post-translational modifications that occur on OGT itself. Herein, we demonstrate that OGT is S-palmitoylated at Cys-472 and Cys-477, which is mediated by the S-acyltransferase Zinc Finger DHHC-Type Palmitoyl transferase 14 (zDHHC14) and removed by acyl protein thioesterase 2 (APT2). S-Palmitoylation stabilizes OGT by shunting it away from the lysosomal chaperone-mediated autophagy (CMA) pathway, as S-palmitoylation decreases the interaction between OGT and heat shock cognate 70 kDa protein (HSC70), the CMA chaperone. Via label-free quantitative mass spectrometry, we find that S- palmitoylation elevates the affinity between OGT and protein phosphatase 1 catalytic subunit gamma (PPP1CC), but not PPP1CB. We further demonstrate that S-palmitoylation of OGT augments binding with Yes-associated protein-1 (YAP), a protein that associates with PPP1CC, and subsequently enhances YAP O-GlcNAcylation. Our work unearths S-palmitoylation of OGT and CMA-mediated degradation of lysosomal OGT, the orchestration of which finetunes the activity of key OGT complexes, such as OGT-PPP1CC, and contributes to OGT substrate selectivity.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by high metastatic dissemination, therapy resistance, and poor clinical outcome. Inhibitor of differentiation 1 or ID1, is frequently overexpressed in PDAC and is associated with tumour progression and adverse clinical outcome. However, the mechanisms governing its post-transcriptional regulation remain insufficiently characterized. Here, we identify tumour-suppressive miR-615-5p as a regulator of ID1 expression in PDAC. Integrative in-silico target prediction prioritized miR-615-5p based on seed complementarity and thermodynamic stability with the ID1 3' -UTR. Expression analysis of available PDAC clinical datasets revealed reduced miR-615-5p expression associated with increased ID1 expression. Direct association was validated using luciferase reporter assays, where miR-615-5p suppressed 3' -UTR reporter activity of ID1 in a sequence dependent manner, while mutation of the predicted binding site attenuated this effect. Further biotinylated-RIP and AGO2-RIP assays demonstrated the co-enrichment of ID1 transcripts and miR-615-5p with AGO2 associated RISC complexes, while AntimiR mediated inhibition of miR-615-5p perturbs association between miR/ID1 to AGO2, supporting interaction specificity. Functionally, modulation of miR-615-5p altered ID1 expression and impacted PDAC cell migration in vitro. Mechanistic analyses further indicated that the miR-615-5p/ID1 axis influences autophagic flux where miR-615-5p mediated inhibition of autophagy suppresses ID1 dependent cellular migration. Collectively, these findings define a previously uncharacterized miRNA-dependent regulation of ID1 expression and link this axis to autophagy-associated migratory responses in PDAC cells. The study expands the post-transcriptional regulatory landscape of ID1 and provides a possible mechanism where suppression of miR-615-5p leads to ID1 overexpression and subsequent poor clinical outcome in PDAC cells.

Orthodontic tooth movement relies on a tightly regulated pro-inflammatory and pro resorptive mechanoresponse of local periodontal ligament fibroblasts (PdLFs). Dysregulation is linked to complications such as root resorption and tooth loss. Hyperlipidemic conditions promote excessive PdL mechanoresponses, with growth differentiation factor 15 (GDF15) acting as potential regulator. This study examined the contribution of the inflammasome/pyroptosis pathway as underlying mechanism for dysregulated mechanoresponses. Human PdLFs were treated with palmitic acid (PA) or oleic acid (OA) for six days before 24 hours of compressive loading. PA increased CASP1, CASP4, and CASP3 activity, secretion of IL-1{beta}, IL-18, and HMGB1, and LDH release. Pharmacological blockade and siRNA-mediated knockdown of inflammasome- and pyroptosis-related targets revealed that NLRP3, CASP1, CASP4, and GSDMD partially contributed to monocyte and osteoclast overactivation. Silencing PA-increased GDF15, partially normalized the phenotype, at least in part by inflammasome/pyroptosis regulation. GDF15 acted through extracellular, and a nuclear signaling route, each accounting partially to this phenotype. Together, GDF15 partially regulates the PA-induced, pyroptosis-associated overactivated mechanoresponse alongside pyroptosis-independent mechanisms suggesting it as an interesting target for potential clinical interventions.

Proteins that distinguish quiescent cells from other non-proliferative states and actively regulate their return to proliferation remain poorly understood. Here, we combined quantitative proteomics with functional image-based screening to identify regulators of the quiescence-to-proliferation transition. Amongst the functional quiescence signature proteins we identified, we focussed on integrin 11 (ITGA11) which is induced across multiple models of reversible quiescence in distinct cell types and that has low expression in proliferating and senescent cells. Although ITGA11 is dispensable for proliferation of asynchronously cycling cells, it is required for efficient cell-cycle re-entry from quiescence. Mechanistically, ITGA11 promotes YAP accumulation and nuclear localization, thereby sustaining SKP2 expression and p27 degradation during cell cycle re-entry. Depletion of p27, or pharmacological activation of YAP signalling rescues the cell-cycle re-entry defect caused by ITGA11 depletion. Together, these findings identify ITGA11 as a functional quiescence signature protein that couples extracellular matrix sensing to YAP-dependent regulation of the Skp2-p27 axis, revealing a mechanism that controls the transition from quiescence to proliferation.

IgM is the most ancient antibody isotype, playing an important role in both circulatory and mucosal immune responses across vertebrates, yet structural characterization of its polymeric forms is limited outside of mammals. Here, we report the cryo-electron microscopy structure of mallard duck secretory (S) IgM at 3.37-[A] resolution. The structure revealed a pentameric core globally similar to human SIgM, supporting the view that pentameric IgM is subject to strong evolutionary constraints. However, compared to mammalian structures, we observed species-specific differences at molecular interfaces. Surface plasmon resonance binding assays characterizing secretory component (SC)-IgM interactions supported structural observations and, when compared to IgA binding, revealed isotype-specific contributions from the avian SC N-terminal extension. Together, these findings establish a comparative structural framework for polymeric IgM across vertebrates and provide insight into how avian SIgM-specific features may support mucosal immunity in birds.

Plasmodium falciparum heat shock protein 90 (PfHsp90) is a promising antimalarial target, but the molecular pathways influenced by its inhibition remain poorly understood. Herein, we leveraged chemoproteomic profiling employing geldanamycin and XL888 Hsp90 inhibitors to investigate proteins and pathways dependent on the chaperone during the Plasmodium blood stage. This study revealed 131 proteins reduced in abundance after inhibition, of which 40% co-immunoprecipitated with PfHsp90. Bioinformatic analyses identified DNA replication as the most enriched pathway. This link was investigated in phenotypic studies demonstrating reduced parasite DNA content after PfHsp90 inhibition. To assess nascent DNA synthesis, we utilized a 7-deaza-7-ethynyl-2'-deoxyadenosine (EdA) assay, yielding dual-stage attenuation of nucleoside incorporation following Hsp90 inhibition. We further show that parasite co-treatment with Hsp90 and DNA replication inhibitors produces synergistic interactions, highlighting the therapeutic potential of the discovered link. Overall, these findings expand our understanding of PfHsp90 function and uncover novel PfHsp90-dependent pathways.

A developmental increase in cerebrospinal fluid (CSF) production during development is essential for neuronal growth and ventricular expansion. A key regulator of CSF production is the specialized sensory multicilia of the choroid plexus (ChP), which mediate non-canonical Sonic hedgehog (Shh) signaling to suppress water channel and ion transporter expression, thereby limiting CSF production. ChP multicilia progressively shortens during development, attenuating Shh signaling and promoting CSF production. Here, we identify miR-34/449 miRNAs as essential regulators of ChP multiciliogenesis. Whereas mutations in canonical ciliogenesis genes elevate CSF production and contribute to hydrocephaly, deletion of miR-34/449 reduces CSF volume and causes microcephaly. Loss of miR-34/449 miRNAs causes excessive basal body amplification, defective basal body docking, and failure of developmental multiciliary shortening. Consequently, miR-34/449-deficient ChP cilia remain abnormally long and fail to attenuate Shh signaling, resulting in sustained repression of water channel and ion transporter expression and reduced CSF production. Mechanistically, miR-34/449 miRNAs directly target Gmnc, a master transcriptional regulator of multiciliogenesis, to restrain basal body amplification and promote basal body docking. Together, our findings identify miR-34/449 miRNAs as critical regulators of ChP multiciliogenesis and establish the developmental remodeling of ChP multicilia as a mechanism to couple Shh signaling dynamics to developmental control of CSF production.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

Inadequate autophagy promotes smoking-induced cellular senescence involved in chronic obstructive pulmonary disease (COPD) pathogenesis. Transcription factor EB (TFEB) is a master regulator of the autophagy-lysosome axis. For the first time, we investigated the therapeutic potential of pemafibrate, a putative TFEB inducer. COPD lung epithelial cells showed reduced TFEB expression. Pemafibrate enhanced autophagy/mitophagy flux and restored lysosomal acidification observed during cigarette smoke (CS) extract exposure in human bronchial epithelial cells, resulting in reduced cellular senescence. TFEB knockdown demonstrated involvement of pemafibrate-induced TFEB in these effects. Pemafibrate induced TFEB expression, mitigated alveolar enlargement and airflow obstruction, and attenuated the CS-induced increase in static lung compliance in a long-term CS-exposed mouse model. It reduced the CS exposure-induced cellular senescence, possibly through autophagy/mitophagy, as suggested by bulk RNA sequencing of mouse lungs. A retrospective cohort study showed that patients given pemafibrate displayed attenuated FEV1.0 decline compared with those given bezafibrate or fenofibrate. In conclusion, pemafibrate is a promising therapeutic agent for COPD, potentially exerting its effects through the regulation of the TFEB-autophagy/mitophagy-lysosome axis.

Skin, the bodys largest mechanosensitive organ, relies on a tension gradient across epidermal layers to maintain structure and function, but how mechanical force contributes to epidermal development and disease pathogenesis is poorly understood. By anchoring intermediate filaments (IF) to the plasma membrane, desmosomes, the most abundant intercellular junctions in the epidermis, help create a supracellular scaffolding that provides mechanical resilience to the tissue. However, the contribution of the desmosome-IF network to the epidermal response to mechanical strain remains unknown. Here we show that the desmosome-IF connection is not only required to induce a proper cellular mechano-response but is actively strengthened in response to stretch through the PP2A-mediated phospho-regulation of the cytoskeletal linker protein desmoplakin (DP). Additionally, we show in human skin dephosphorylated DP localizes to high tension layers, suggesting this mechano-response mechanism is coordinated with the epidermal tension gradient. Furthermore, in models of Carvajal syndrome, a cardio-cutaneous disorder caused by truncating DP mutations, cells lose mechano-responsive behavior and exhibit abnormal morphology in high-tension epidermal layers. Together, these findings identify the DP-IF network as a key component of the response to mechanical strain and show that its disruption compromises epidermal homeostasis and contributes to disease pathogenesis.

We present the solution structures of HIV-1 proteins NC(p7)1-55 corresponding to the full-length NC(p7) and mature p6. The studies were carried in water and, to mimic the membrane, in micellar DPC (Dodecylphosphocholine) conditions. Our results unravel for the first time the structure adopted by the N-terminal amino acids of the free NC(p7)1-55, with the formation of a small helix spanning residues F6 to R10. Our NMR and Fluorescence Anisotropy data disclose an interaction between NC(p7)1-55 and p6 both in water and DPC, with respective Kd of 2.5mM and 370 mM at 23{degrees}C. The interaction is thus strengthened in lipidic conditions. Protein p6 stabilizes the N-terminus of NC(p7)1-55 while increasing at the same time the dynamic of the first zinc finger. Although the entire p6 sequence is involved in the interaction, we show that its C-terminal region is particularly sensitive to the presence of NC(p7)1-55, with a propensity of forming a a helix ranging from amino acids S111 to F116. This study brings experimental evidence of a direct protein-protein interaction between p6 and the N-terminal region of NC(p7)1-55. We further show that such interaction is readily accommodated within the NC(p15) framework and hypothesize that it may facilitate the selective assembly of assembly of the viral genomic RNA (gRNA) in the cell.

Endothelial senescence is increasingly recognized as a driver of vascular pathology, while immunoglobulin G (IgG) has recently been reported to accumulate in aging tissues and induce senescence in macrophages and microglia. In cerebral cavernous malformations (CCMs), IgG accumulation has been obviously observed in CCM lesions, but the contribution of IgG to endothelial injury remains unclear. Using multi-omic profiling, endothelial models, and CCM mice, we identified IgG-secreting plasma cells enriched in lesions associated with endothelial senescence, hemorrhage, and disease severity. CCM loss-associated mTOR activation impaired lysosomal acidification and IgG processing, promoting intracellular IgG accumulation. IgG, in turn, induced NF-kB-dependent endothelial senescence. In vivo, BCMA-mediated plasma cell depletion attenuated lesion progression, whereas IgG supplementation partially restored disease severity. Anti-CD38 treatment likewise reduced IgG accumulation, endothelial senescence, hemorrhage, and lesion progression. These findings identify lysosomal dysfunction-mediated IgG as a pathogenic trigger of endothelial senescence and support targeting the plasma cell-IgG axis in CCM.

Background. GBA2-associated hereditary spastic paraplegia (SPG46) is a rare autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in GBA2, encoding the non-lysosomal glucocerebrosidase 2. GBA2 deficiency leads to glucosylceramide (GlcCer) accumulation and glucosylated cholesterol (GlcChol) depletion, causing cytoskeletal defects in immature neurons. However, the mechanisms linking lipid dysregulation to neuronal dysfunction remain poorly understood. Methods. We modelled GBA2 loss of function by chronic pharmacological inhibition in mouse cerebellar granule neurons (CGNs) and assessed neuronal morphology, synaptic organization, Ca2+ dynamics, mitochondrial function and actin cytoskeleton during maturation. Proteomic profiling was performed in GBA2-inhibited and GlcChol-supplemented neurons. Findings were validated in a zebrafish gba2 crispant model by evaluating motor behavior, cerebellar development, neuronal organization and mitochondrial function, and in patient-derived fibroblasts carrying a homozygous pathogenic GBA2 variant (NM_020944). The role of RAC1 was studied in both neurons and patients' cultured skin fibroblasts, and upon rac1 pharmacological inhibition in zebrafish crispants. Results. Chronic GBA2 inhibition impaired axonal outgrowth in immature CGNs but not neurite complexity in mature neurons, suggesting morphological compensation. Nevertheless, mature neurons displayed enlarged presynaptic terminals, impaired synaptic vesicle clustering and altered Ca2+ responses to potassium and glutamate, the latter associated with NMDA receptor redistribution without changes in total receptor levels. Mitochondrial alterations were observed in CGNs, patient fibroblasts and zebrafish, consistent with defective architecture of the mitochondrial network. Proteomics revealed convergent alterations in actin cytoskeleton, synaptic pathways and cellular metabolism following both GBA2 inhibition and GlcChol supplementation. GlcChol bidirectionally regulated RAC1 function, likely altering its spatial distribution rather than its global activation. Confocal imaging confirmed abnormal RAC1 and F-actin localization in patient fibroblasts. Zebrafish gba2 crispants recapitulated motor deficits, Purkinje cell loss, motor neuron disorganization and mitochondrial abnormalities. Pharmacological Rac1 inhibition rescued motor behavior and neuronal organization, linking cytoskeletal disorganization to the observed phenotype in the zebrafish model. Conclusions. Our findings identify a pathogenic GlcChol-RAC1-actin signalling axis linking lipid imbalance to synaptic disorganization, NMDA receptor redistribution and mitochondrial dysfunction in SPG46. The selective vulnerability of corticospinal neurons, cerebellar granule neurons and Purkinje cells may reflect their dependence on this pathway. Rac1 inhibition rescues disease phenotypes in vivo, highlighting this pathway as a promising therapeutic target.