KRIT1 plays a key role in regulating the barrier function of endothelial cells, where it localizes to the adherens junctions, cytoplasm, and nucleus. However, how subcellular localization may regulate KRIT1 remains unclear. Here, we investigate what effect nuclear localization has on its ability to stabilize the endothelial barrier. We generated a KRIT1 mutant lacking the endogenous nuclear localization signal (NLS) and a nuclear-targeted KRIT1 isoform created by attaching the NLS of simian virus 40 and expressed these constructs in cells depleted of endogenous KRIT1. After confirming the relative non-nuclear (KRIT1ΔNLS) and nuclear (KRIT1NLS+) enrichment of these constructs using confocal microscopy and cellular fractionation, we assessed whether these constructs were able to functionally rescue phenotypes characteristic of KRIT1-depleted cells. Our results showed that nuclear localized KRIT1 remains fully functional. However, the KRIT1ΔNLS construct failed to rescue the KRIT1 depletion phenotype. KRIT1ΔNLS also disrupted binding to ICAP1α, as shown by co-immunoprecipitation. To determine whether the loss of function was due to loss of ICAP1α-mediated conformational change or altered localization, we incorporated two mutants known to disrupt the N- to C-terminal interaction in KRIT1, 192NPXY195 → APAA or W688A into our KRIT1ΔNLS and KRIT1NLS+ constructs. The presence of these additional mutations had unexpected effects on nuclear localization of KRIT1ΔNLS and KRIT1NLS+ but were able to restore barrier-stabilizing function to KRIT1ΔNLS. Overall, our findings provide key insights into the role of ICAP1α binding and nuclear localization in the regulation of KRIT1 and raise new questions regarding potentially novel functions of KRIT1 in the nucleus.
Protein misfolding of pancreatic secretory enzymes is increasingly recognized as a contributor to chronic pancreatitis. Recently, missense pancreatic lipase (PNLIP) mutations that impair lipase secretion and cause intracellular aggregation and endoplasmic reticulum (ER) stress have been linked to the disease. Among these, A174P, G233E, and C254R are located in the N-terminal domain, whereas V454F is situated in the C-terminal domain of PNLIP. To elucidate how these mutations disrupt PNLIP structure, we performed detailed molecular dynamics simulations and structural modeling of human PNLIP variants. Evolutionary conservation analyses suggested that the mutation sites contribute to PNLIP structural stability. All mutations, regardless of location, altered the residue interaction network within the N-terminal PNLIP domain, consistently affecting Glu270. Detailed structural analysis revealed that the A174P substitution disrupted several helix-stabilizing hydrogen bonds, whereas the G233E mutation introduced multiple local hydrogen bonds and caused structural distortions. The C254R mutation abolished the native Cys254-Cys278 disulfide bond and instead formed an alternative salt bridge destabilizing the lid region. All three mutations altered stabilizing interactions mainly within the N-terminal domain of PNLIP. Interestingly, the V454F substitution exerted long-range allosteric effects, modulating key interactions also within the N-terminal domain. Further structural analysis showed that Phe454 in the variant became solvent-exposed, potentially leading to aggregate formation through the mutation site. These observations provide a molecular basis for PNLIP misfolding and its downstream proteotoxic effects, supporting the development of targeted therapies for chronic pancreatitis.
IRE1α (hereafter referred to as IRE1) is one of the sensors implicated in the unfolded protein response that controls the ER protein homeostasis (also known as proteostasis). Alteration of proteostasis is observed in many diseases, making IRE1 a central element of cell adaptability upon disease onset and progression. Upon ER stress, IRE1 initially promotes cell adaptation. Conversely, when proteostasis cannot be restored, IRE1 activation can lead to cell death. IRE1 activity mainly regulates two pathways: the formation of the transcription factor XBP1s; and the regulated IRE1-dependent decay (RIDD) of RNA, which can contribute to both cell adaptation and death. Hence, on one hand, IRE1 favors gene expression, while on the other hand it induces transcript degradation. We have recently identified two genes, CD95 and UBE2D3, which are targeted by both signaling branches downstream of IRE1 RNase's activity, resulting in a dual and opposing regulation of their expression. We propose naming these targets 'DIT' for Dual IRE1 Targets. Interestingly, other IRE1 targets, such as BiP and DGAT2, have previously been reported to be regulated by XBP1s and RIDD in separate studies. We hypothesize that regulation of DIT could be crucial to tilt the balance between the pro-adaptative and pro-death outcomes of IRE1, especially in pathological contexts. Therefore, understanding this regulation could be key to unraveling the IRE1/XBP1s/RIDD signaling network. Here, we explore these hypotheses by highlighting various aspects of the regulation of IRE1 branches, and reviewing the DIT identified in the literature so far.
Skeletal muscle exhibits a remarkable level of plasticity that enables it to adapt to exercise training, as well as the deleterious effects of aging. Fundamental to this malleability are epigenetic processes, which collectively enhance chromatin remodeling and subsequently alter DNA availability for gene expression. A growing body of evidence has demonstrated that acute exercise is a powerful inducer of epigenetic remodeling, capable of stimulating gene-specific alterations, which transcriptionally activate exercise-responsive genes. These epigenetic processes, including DNA methylation and various histone modifications, are highly responsive to exercise-induced signaling cascades and mitochondrially-related metabolites, together indicating that exercise can modulate the nuclear and mitochondrial epigenome as a mechanism to regulate gene expression. However, aging is characterized by a unique epigenetic signature, which likely supports the alterations in gene expression observed with age. Yet, the effects of exercise on epigenetic regulation with age remain underexplored. To investigate the intersectionality of these two phenotypes and highlight significant gaps within the literature, this review aimed to discuss the different types of epigenetic modifications that have been reported within skeletal muscle and how they are altered with acute and chronic exercise. Furthermore, we aimed to analyze mitochondrial epigenetics and their role in mediating alterations in mitochondrial-nuclear crosstalk observed with exercise and age. Elucidating age-dependent adaptations in the epigenome and the differential effects of exercise in these populations will help uncover the complexity of gene regulation with age, and importantly, reveal how exercise can regulate many of these processes to improve muscle health.
Pericytes (PCs) are perivascular cells that lie in close association with endothelial cells (ECs), with both cell types embedded within a shared basement membrane (BM), a specialised form of extracellular matrix (ECM). PCs regulate vascular integrity, angiogenesis and capillary blood flow and are capable of differentiating into other cell types including fibroblasts and smooth muscle cells. In recent years, a central role for PCs in regulating the development and maturation of the vasculature, maintaining tissue homeostasis and directing the pleiotropic remodelling of tissues during regeneration has emerged. Here, we review how PCs contribute to the synthesis and remodelling of the ECM in different pathophysiological conditions. Moreover, we provide an atlas of the PC matrisome, the complex of ECM molecules expressed by PCs, based on recent transcriptomics (in particular single-cell RNA sequencing) and proteomics datasets, with the caveat that such an entity does not exist in isolation due to the physical and paracrine interactions between PCs, ECs and other cell types. Understanding the role of PCs in modulating their microenvironment through active synthesis and degradation of specific matrisome components is essential to understand the role these plastic cells play in angiogenesis and in different pathologic conditions, including stroke, Alzheimer's disease and cancer.
The endoplasmic reticulum (ER) is a cellular organelle frequently subjected to stress under both physiological and pathological circumstances, associated with the accumulation of mis/unfolded proteins in its lumen. To cope with this stress, cells have evolved an adaptive program called the unfolded protein response (UPR), whose primary function is to restore ER proteostasis. When the stress is prolonged, the UPR can also trigger cell death. The UPR controls multiple machineries involved in pre-emptive quality control (QC) of proteins prior to ER entry, ribosome-associated QC, protein folding within the ER, protein degradation through various processes, and export from the ER for secretion. Because the UPR and the machineries it controls play fundamental roles in determining cell fate, they are finely regulated, including through post-translational modifications (PTMs). In this review, we focus on the role of the ubiquitin and ubiquitin-like PTMs in the regulation and mediation of ER proteostasis. We specifically focus on three core processes: the UPR, ER-associated ribosome QC and ER-associated degradation. Lastly, we briefly discuss how Ub and Ubl also control the integrated stress response and the formation of inter-organelle membrane contact sites and thus act as general regulators of responses to cellular stresses beyond ER proteotoxicity.
Aminoacylation of the lipid head group in many bacteria is carried out by bi-functional enzymes called MprF, which encode a soluble synthase domain that typically transfers lysine or alanine from a tRNA to lipid head groups. The modified lipid is subsequently translocated across the leaflets by a transmembrane domain. This modification of lipids probably evolved to adapt to the environment where the microbes reside. Here, we describe the cryo-EM structures of MprF enzyme from Pseudomonas aeruginosa, revealing a dimeric enzyme with a distinct architecture when compared with the homologous Rhizobium enzymes, and validate this arrangement with biochemical analyses. The cryo-EM maps and the models in detergent micelle and nanodisc reveal a conformational change of the terminal helix of the synthase domain, highlighting the dynamic elements in the enzyme that might facilitate catalysis. Several lipid-like densities are observed in the cryo-EM maps, which might indicate the path taken by the lipids, coupling the function of the two domains. The structures allow postulation of the binding modes of tRNA and lipid transport, and suggest that the mobile secondary structural elements in the synthase domain might play a mechanistic role in these functions.
β-1,2-Glucans are glucose polymers widely distributed in nature and play various physiological roles in the interactions between organisms such as pathogenicity and symbiosis. While various β-1,2-glucan-degrading enzymes have been identified recently, transporters incorporating β-1,2-glucans are still poorly characterized. In this study, we have found a β-1,2-glucan binding protein of ABC transporter from Chloroflexus aurantiacus Y-400-fl, a filamentous anoxygenic phototrophic bacterium. The protein showed a clear affinity for linear β-1,2-glucan in the gel shift assay. Isothermal titration calorimetric analysis revealed high binding affinities for both linear and cyclic β-1,2-glucans, unlike for the barley β-glucan. The recorded binding constants were high for the binding of the ABC transporter to β-1,2-glucans. The observed unfavorable negative entropy change may have resulted from conformational restraints upon complex formation. Complex structures with linear β-1,2-glucan and cyclic β-1,2-glucans with degrees of polymerization of 17-20 were obtained using X-ray crystallography. Ten glucose units, designated A-J from the nonreducing end, were shared among the substrates in the complexes. Unit G is recognized by W74, W308, and D336, which are highly conserved residues within the phylogenetic group Chy400_4166. The substrate-binding mode of Chy400_4166 is completely different from that of the β-1,2-glucooligosaccharide-binding protein from Listeria innocua. The discovery of a new type of β-1,2-glucan-related binding protein has expanded our understanding of the metabolism of β-1,2-glucans.
17β-oestradiol (E2) is essential for ovarian development. In teleosts, the current understanding of oestrogen synthesis primarily focuses on Cyp19a, which catalyses the synthesis of E2 from testosterone (T). In contrast, the conversion of oestrone (E1)-to-E2, mediated by Hsd17b12, and the role of this process in ovarian development remain understudied. This study investigated two Hsd17b12 isoforms in the commercially cultured fish, olive flounder (Paralichthys olivaceus). In vitro ovarian assays revealed isoform-specific functions. Hsd17b12a preferentially mediated E2 biosynthesis, whereas Hsd17b12b regulated T metabolism. Subsequent detection in HEK293T cells indicated that Hsd17b12a catalyses E1-to-E2 conversion, whereas Hsd17b12b mediates T-to-androstenedione (A) conversion. Site-directed mutagenesis targeting the conserved YxxxK catalytic motif showed that an alanine-to-serine substitution in Hsd17b12a and a serine-to-threonine substitution in Hsd17b12b significantly reduced enzymatic activity. In vivo overexpression of Hsd17b12a and -12b in the flounder ovaries revealed distinct phenotypes. Hsd17b12a overexpression elevated A and E1 levels without inducing histological changes. In contrast, Hsd17b12b overexpression induced proliferation of oogonium-like cells, significantly upregulated the expression of cyp26b1 and vasa, and increased A and E2 levels. Co-immunoprecipitation assays showed an interaction between Cyp19a and Hsd17b12a, and in vitro experiments showed co-expression of Hsd17b12b and cyp19a. These findings clarify the roles of the flounder Hsd17b12a and -12b in steroidogenesis, and, for the first time, their interaction with Cyp19a in fish.
Mitochondrial proteotoxic stress activates the mammalian UPRmt through a multilayered mechanistic architecture rather than a linear pathway. At its core lies an import-gated sensing logic: reduced preprotein import and mito-nuclear stoichiometric imbalance activates the integrated stress response (ISR) toward the translation of ATF4, CHOP, and the mitochondria-targeted transcription factor ATF5. These factors cooperatively reprogram transcription to expand the chaperone-protease capacity while transiently reducing the nuclear-encoded OXPHOS load. Parallel translational mechanisms that include eIF2α-dependent repression, stress-granule triage, and miRNA-driven selective silencing reduce the mitochondrial precursor import and maintain proteostatic symmetry between the cytosol and mitochondria. Within the organelle, LONP1- and CLPP-dependent proteolysis, mitoribosome pausing, and tRNA-processing checkpoints further dampen nascent chain pressure. Epigenetic licensing by demethylases and acetyltransferases links metabolic and bioenergetic status to promoter accessibility at UPRmt loci. Together, these import-gated, translational, and epigenetic control layers form a coherent mechanistic circuit ensuring that mitochondrial recovery is matched to folding, assembly, and metabolic capacity. We propose a unified framework explaining how these layers cooperate to determine adaptive versus maladaptive outcomes.
Despite its clinical importance, the metabolic landscape of MYCN nonamplified neuroblastoma (MYCN-NA NB) remains poorly defined. In this study, we performed integrated metabolic characterization of 22 NB samples by combining single-cell RNA sequencing (scRNA-seq) with proteomics and metabolomics. This was followed by multi-omics integration, two-way orthogonal partial least squares analysis, and biomarker selection using a random forest classifier. The scRNA-seq analysis of NB tumors revealed that, compared to the MYCN-amplified (MYCN-A) group, the MYCN-NA group exhibited higher proportions of T cells, monocytes, and neurons, while lipid metabolism pathways were specifically enriched. Proteomics identified 821 differentially expressed proteins [27 upregulated and 794 downregulated in the high-risk (HR) NB group], and functional analysis indicated that these proteins were primarily involved in biological processes such as the tricarboxylic acid cycle (TCA) and lipid metabolism. Metabolomics further detected 15 significantly altered metabolites associated with pathways such as linoleic acid metabolism. Integrated multiomics analysis identified key molecules, including RPL9 and POLR2G, with potentially critical roles in HR NB. Multi-omics correlation and network analyses revealed significant interactions between key proteins (RPL9 and POLR2G) and metabolites (D-ribose and 9-oxoODE). The biomarker combination determined by random forest and bidirectional orthogonal partial least squares modeling underscored the central role of oxidative lipid metabolism remodeling in high-risk disease progression. This study systematically elucidated the crucial role of lipid metabolic reprogramming in the pathogenesis of HR MYCN-NA NB. These findings provide critical insight for uncovering the mechanisms underlying NB progression and for identifying potential therapeutic targets.
Coproheme decarboxylase (ChdC) is the terminal enzyme in Gram-positive heme b biosynthesis, an enzyme holding a special importance given its unique structure-function relationship and its necessity for bacterial survival. In the past, the enzyme has been shown to perform a double decarboxylation of two propionate groups on coproheme (Fe(III)-coproporphyrin III), subsequently converting it to heme b (iron-protoporphyrin IX). Notably, the active site of ChdCs is universally covered by a flexible loop. Its importance has not been fully studied but given its position it is assumed to provide steric hinderance for substrates or potential inhibitors to pass. This study aims to investigate its physiological role by introducing a histidine-to-alanine mutation located on the loop of ChdC from Listeria monocytogenes. Molecular dynamics simulation shows an increased flexibility of the structural element in the mutant. As a consequence, various kinetic studies at steady- and presteady-state conditions suggest coproheme binding and active site accessibility are improved. Using simulations and X-ray crystallography, we show evidence that the loop is originally stabilized by a hydrogen bond between S116 and the mutated H117. The higher accessibility also results in a higher susceptibility to damage from oxidative cosubstrates like H2O2, suggesting the loop in its wild-type conformation plays a key biological role in regulating the transfer of cosubstrates towards the main substrate coproheme.
Bacterial biofilms-structured communities of bacteria encased in self-produced polymeric matrices-present formidable challenges in clinical medicine. The resistance of biofilms to conventional antibiotics stems from multiple factors. These include limited drug penetration and the presence of metabolically dormant bacteria that survive treatments, despite their retaining sensitivity under standard laboratory conditions. Bacteriophages (phages), the viruses that infect and kill bacteria, have emerged as promising alternatives or adjuncts to antibiotic therapy, including against bacterial biofilms. Phages, nonetheless, likely evolved to optimize especially their dissemination between spatially separated bacteria, including spatially separated biofilms, rather than to become specialists at eradicating all targeted bacteria from biofilms. By contrast, complete bacterial elimination from the body is the standard goal of antibacterial therapies. Considered here is how an understanding of these competing goals-virion dissemination vs. complete bacterial eradication-can inform our development of phage-based anti-biofilm therapies.
Immunomodulatory drugs (IMiDs), including lenalidomide and pomalidomide in combination with proteasome inhibitors, dexamethasone and anti-CD38 monoclonal antibodies, play a central role in the treatment of multiple myeloma (MM) across newly diagnosed and relapsed stages. These treatment regimens have significantly improved patient outcomes worldwide, establishing IMiDs as one of the backbones of MM therapy. A new generation of more potent compounds called cereblon E3 ligase modulators (CELMoDs) is now being developed to potentially replace the older IMiDs. In addition, novel immunotherapeutic approaches led by chimeric antigen receptor (CAR T), T-cell engagers and antibody-drug conjugates are also increasingly used in relapsed and refractory myeloma patient care. However, despite these advances, resistance to IMiD-based therapies inevitably develops and represents a major clinical challenge. Understanding the biological basis of resistance to IMiD-based therapy is crucial to plan and maximise treatment options for patients when they relapse on IMiD containing regimens. Emerging evidence underscores the role of genetic and epigenetic alterations, changes in downstream signalling, and dysregulation of the bone marrow immune microenvironment in driving therapeutic resistance. In this review, we explore current literature on the molecular and immune mechanisms related to the onset of therapeutic resistance. We then suggest ways to overcome resistance and exemplify options for the future, focusing on immunotherapy combinations with IMiDs or CELMoDs and novel agents.
Photodynamic therapy (PDT) is an innovative treatment option for cancer, but current approaches are limited by poor tumor selectivity and low uptake. Here, we introduce a novel concept for a targeted phototoxic peptide, in which a lysosomally activatable payload is delivered selectively into the cell by receptor-mediated endocytosis. For the phototoxic payload, 6-carboxytetramethylrhodamine (TMR) was attached to a cell-penetrating peptide (CPP), which is specifically activated after internalization in the endosome. The activity of the CPP was blocked by electrostatic interactions with a poly-glutamate sequence but could be restored through cleavage by the lysosomal protease cathepsin B, both in vitro and in cells. The unmasked CPP binds to the negatively charged lysosomal membrane and, upon irradiation, TMR generates reactive oxygen species (ROS) that disrupt the integrity of the membrane. This leads to a release of lysosomal contents into the cytosol, which subsequently induces cell death. To achieve targeted delivery, the activatable payload was additionally conjugated to chemerin-9, a high-affinity ligand for the chemokine-like receptor 1 (CMKLR1), a G protein-coupled receptor overexpressed in various cancers. Through this receptor-targeted approach, the peptide accumulates only in CMKLR1-expressing cells while the lysosomal activation completely prevented off-target toxicity. Notably, this strategy enables even a weak photosensitizer like TMR to achieve potent cytotoxicity through lysosomal targeting. Thus, this approach represents an advancement in the selectivity and efficacy of PDT and holds promise for the development of novel cancer therapies.
The nuclear receptor NR5A2 (Liver Receptor Homolog-1, LRH-1) has been well characterized in tissues of endodermal origin for the transcriptional control of development, metabolism, and steroidogenesis. In this minireview, we discuss the so far underappreciated expression and role of LRH-1 in hematopoietic cells. We further highlight how deregulation of LRH-1 may contribute to the pathogenesis of leukemia and immune cell-mediated diseases, and how targeting LRH-1 can be employed in immune cell-targeted therapies. Given that LRH-1 expression and function are highly tissue-specific, we further discuss how these contextual differences may be exploited to achieve therapeutic selectivity, especially focusing on the myeloid and T cell lineage. Although current evidence for LRH-1 functions in these immune cells is yet limited, its established role in the transcriptional regulation of development, differentiation, metabolism, proliferation, and cytokine expression of hematopoietic cells suggests a substantial and largely unexploited potential for therapeutic applications in leukemia and immunopathological diseases.
Alzheimer's disease (AD), a progressive neurodegenerative disorder with a rising global prevalence, is pathologically characterised by the presence of amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs). These lesions lead to synaptic damage, neuronal loss, and cognitive impairment. Despite the recent approval of immunotherapies for AD treatment, their limited efficacy highlights the urgent need for exploring novel disease mechanisms and developing targeted therapeutic strategies. Annexin A2 (ANXA2), a calcium-dependent phospholipid-binding protein, participates in diverse physiological processes (e.g. membrane organisation, cytoskeleton linkage) and contributes to the pathogenesis of diseases such as cancer and Parkinson's disease. Emerging evidence indicates that ANXA2 interacts with AD-related pathological components (Aβ, tau) and regulates AD-associated inflammatory pathways, suggesting its potential role in AD. However, current evidence regarding ANXA2 in AD remains limited, and the molecular mechanisms underlying its contribution to AD pathogenesis remain unclear. This review comprehensively summarises the current knowledge on ANXA2's cellular and physiological functions in the central nervous system (CNS), as well as its involvement in AD pathology, aiming to provide guidance for research into ANXA2's therapeutic potential for AD prevention and treatment.
Xylan is the second most abundant polysaccharide in plant cell walls, consisting of a β-1,4-linked xylopyranosyl backbone that can be substituted with various side chains. Depolymerization of xylan is predominantly catalyzed by the coordinated activity of β-xylanases and β-xylosidases. In this study, we determined the cryo-electron microscopy (cryo-EM) structure of Enterobacter cloacae β-xylosidase (EcXyl43), a glycoside hydrolase family 43 (GH43) enzyme. Additionally, we resolved the X-ray crystal structure of a catalytically inactive F507A mutant of EcXyl43. Together, these structures represent the first structural characterization of a β-xylosidase from the Enterobacter genus using both X-ray diffraction and cryoEM. Furthermore, to elucidate the molecular basis of substrate recognition and specificity, we conducted molecular dynamics simulations of the enzyme. Structural and computational analysis of EcXyl43 identified key determinants of the enzyme's preference for longer xylooligosaccharides and revealed how noncatalytic residues within the auxiliary domain modulate its activity.
Painful chemotherapy-induced peripheral neuropathy (CIPN) is an increasingly common condition for which no adequate treatments are known. Prominent toxic effects of chemotherapeutic drugs such as paclitaxel include debilitating ongoing pain. Because of the complexity of the tissues involved in vivo, there is a need for simple models that capture essential features of CIPN for mechanistic analysis. Lamberti et al. employ long-term culture of dissociated nociceptors to begin defining physiological and molecular mechanisms underlying persistent spontaneous activity (SA) induced in vitro by repeated application of paclitaxel. They demonstrate major contributions to progressively increasing SA from potentiated depolarizing spontaneous fluctuations of membrane potential (DSFs, which were only recently recognized to control the timing and frequency of irregular spontaneous discharge in nociceptors), and they provide strong evidence that the SA and DSFs involve enhanced function and/or expression of Nav1.8, TRPV1, TRPA1, and TRPM8 channels. This model system offers considerable promise for further defining the mechanisms of SA important for driving ongoing pain after CIPN. Comment on: https://doi.org/10.1111/febs.70517.
Neuroregeneration is the ability of nervous tissue to renew itself after injury. This process is highly limited in mammals. In contrast, marine chordates such as ascidians display a remarkable regenerative capacity, making them valuable models to understand neural regeneration. This study investigated dermatan sulfate (DS) and chondroitin sulfate (CS) profiles during brain regeneration in the ascidian Styela plicata. Neurodegeneration was induced by 3-acetylpyridine (3-AP), after which neural complex (NC) analyses were conducted using histology, RT-qPCR, liquid chromatography, behavioral testing, and phylogenetic methods at 1,5 and 10-day postinjection. One day after treatment, the cerebral ganglion exhibited significant degeneration, followed by morphological and molecular recovery at 10 days, when neuronal and synaptic markers returned to control levels. Gene expression analyses revealed early upregulation of C-6, C-4, and C-2 sulfotransferases in the final stage. For the first time, the presence of dermatan 2,6 sulfate (D2,6S) and chondroitin 4 sulfate (C4S) in the cortex of the cerebral ganglion was described and 2,6 sulfate disaccharides were found to make up the majority of the NC, emphasizing their relationship with regeneration. A behavioral test revealed that co-injecting 3-AP and D2,6S restored the compromised siphon movements. Finally, ascidian DS epimerase (DSE) was most closely related to vertebrate DSE type 2, which is found in the brains of mammals. Together, these results indicate that specific glycosaminoglycan sulfation patterns are dynamically regulated during neural regeneration in ascidians. This study provides new insights into the molecules and strategies promoting neuroregeneration in vertebrates and advances the field of regenerative medicine.