Search Results (1,252)

Although calpain-5/CAPN5 is widely expressed in mammals, little is known regarding its functions. Pathogenic mutations of CAPN5 are causal for a devastating autoimmune eye disease, neovascular inflammatory vitreoretinopathy (NIV). To provide insight into both the physiological and pathological roles of CAPN5, it is essential to identify candidate interaction partners and possible substrates. Human SH-SY5Y neuroblastoma cells, transfected with full-length catalytically dead (Cys81Ala) CAPN5-3×FLAG, were used for anti-FLAG co-immunoprecipitation (co-IP) and quantitative proteomics using Sequential Window Acquisition of all THeoretical mass spectra (SWATH-MS). Fifty-one proteins were enriched at least four-fold, p < 0.01, relative to cells transfected with an empty FLAG vector. A high proportion (24/51) of candidate CAPN5 interaction partners are associated with protein quality control, including components of the chaperonin, chaperone, and ubiquitin–proteasome systems. Additional candidate interactors include tubulins, kinases, phosphatases, G proteins, and mitochondrial proteins. CAPN5 interactions for 14 of the candidate proteins were confirmed by co-IP and immunoblotting. Of these 14 proteins, 11 exhibited in vitro calcium-induced proteolysis following co-IP with WT CAPN5-3×FLAG. Impaired calcium-induced proteolysis of co-IP proteins was observed for the pathogenic CAPN5 variants R243L and R289W. Further studies are needed to validate the association of candidate CAPN5 interactors with proteins and complexes suggested by the SWATH-MS and co-IP results, and the possible role of CAPN5 within such complexes. The possible involvement of CAPN5 in protein quality control is relevant to NIV, as defects in protein quality control have been implicated in inherited retinal disorders. Proteomic data are available via ProteomeXchange with identifier PXD068008. Full article

Chalcone-based derivatives were designed as dual-acting ligands targeting the histamine H₃ receptor (H₃R) and monoamine oxidase B (MAO-B), based on the lead compound DL76. Three series of compounds (1–18) were synthesised and characterised, including simple chalcones (1–9) and piperidinyl chalcones (10–18). All piperidinyl derivatives exhibited nanomolar affinity for human H₃R (hH₃R), with compounds 10–12 achieving K_i values ≤ 30 nM. Simple chalcones showed potent human MAO-B (hMAO-B) inhibition (IC₅₀: 0.85–337 nM), especially 3,4-dichloro derivatives. Compound 15 was the most active hybrid, with a K_i of 46.8 nM for hH₃R and an IC₅₀ of 212.5 nM for hMAO-B. Molecular docking and 250 ns simulations revealed stabilising interactions at both binding sites and clarified structural features behind dual activity. Preliminary ADMET profiling showed low Caco-2 permeability and rapid microsomal metabolism, mainly via hydroxylation. Compound 15 exhibited micromolar cytotoxicity in SH-SY5Y and HepG2 cells, induced G2/M arrest, disrupted mitochondrial homeostasis, and was genotoxic in Peripheral Blood Mononuclear Cells (PBMCs). Additionally, for H₃R ligands (15, DL76, pitolisant), the study reports the first use of Surface Plasmon Resonance Microscopy (SPRM) to assess their interactions with this receptor. Therefore, piperidinyl chalcones show promise as ligands with dual action on H₃R and MAO-B, useful in the treatment of neurodegeneration and/or CNS cancers. Full article

Dysfunction of the membrane transporter P5B-ATPase 13A2 (ATP13A2) has been linked to neurodegenerative disorders, while its overexpression has been associated with colorectal cancer. ATP13A2 localizes to lysosomes and late endosomes, where it exports polyamines such as spermine into the cytosol. We previously showed that ATP13A2 expression alters lipid homeostasis and reduces the levels of bis(monoacylglycero)phosphate (BMP), an anionic phospholipid essential for lipid digestion in acidic compartments, suggesting that ATP13A2-mediated spermine export may affect lysosomal lipid catabolism. α/β-hydrolase domain-containing 6 (ABHD6), the enzyme responsible for BMP catabolism, was detected by immunofluorescence and immunoblot analysis in SH-SY5Y cells overexpressing human ATP13A2 and treated with spermine. The activities of the lipid-degrading hydrolases acid ceramidase (ACase) and glucocerebrosidase (GCase) were measured using specific fluorogenic substrates. ATP13A2-expressing cells showed higher ABHD6 expression, and spermine treatment promoted its translocation to the cytoplasm. Spermine induced a transient increase in ACase activity, followed by a stronger inhibition in ATP13A2-expressing cells. Moreover, GCase activity was elevated in these cells but also showed greater spermine-induced inhibition. Altogether, these results suggest that ATP13A2-mediated spermine export modulates the lipid digestion capacity of the endolysosomal system and support a functional interplay between polyamine and lipid metabolism in these organelles. Full article

Background: Neuroblastoma is the most common extracranial solid malignancy in infants and children. High-risk neuroblastoma patients are commonly treated with temozolomide (TMZ), which typically exhibits a poor therapeutic response. Serotonin, also known as 5-hydroxytryptamine (5-HT), plays various essential functions in the human body. In the central nervous system, it serves as a neurotransmitter. Beyond its physiological roles, 5-HT has recently been identified as a potential growth factor for several human tumors, including gliomas and carcinoid tumors. Recent literature has demonstrated that 5-HT receptor antagonists can inhibit the growth of cancer cells. Furthermore, both 5-HT receptors and their antagonists have been identified as potential anticancer agents, suggesting their significance in the development of new treatment strategies. Objectives: The primary aim of this study was to examine the effects of 5-HT and 5-HT antagonists on tumor (neuroblastoma (SH-SY5Y)) and healthy cells (microglia (HMC3)) and determine the impact of their interaction with the anticancer agent TMZ on cell proliferation/viability and migration. Methods: The study explored the interaction between 5-HT, the 5-HT antagonist granisetron (GRN), the anticancer agent TMZ, and their combinations, specifically assessing their influence on cell proliferation, viability, and migration. Results: As a result, the single and combined applications of 5-HT, TMZ, and GRN, a 5-HT antagonist, inhibited cell growth and proliferation in SH-SY5Y, causing decreased cell viability. Additionally, the combination of 5-HT and GRN increased the efficacy of TMZ. Conclusions: The study findings revealed that 5-HT and 5-HT antagonists may have therapeutic effects by exhibiting antiproliferative effects in SH-SY5Y cells at high concentrations. Full article

Altered protein synthesis plays a key role in ageing and multiple neurodegenerative diseases. In Alzheimer’s disease and other tauopathies, the intracellular accumulation of hyperphosphorylated Tau disrupts several cellular processes, including mRNA translation. Although Tau interacts with ribosomal proteins and modulates translational selectivity, its effects on global protein synthesis remain poorly understood. Studies report reduced translation in later disease stages but increased translation early in pathology. To clarify Tau’s impact in human neurons, we used SH-SY5Y cells overexpressing the P301L mutant form of Tau and quantified global protein synthesis using the SUnSET (Surface Sensing of Translation) puromycin-incorporation assay. We found that Tau-P301L expression greatly increased global translation by upregulating mTOR/S6 pathway. These effects were abolished by rapamycin treatment, indicating that Tau-driven translational upregulation is mTOR-dependent. Given that impaired translational control can disrupt synaptic plasticity and memory, Tau-induced alterations in protein synthesis may contribute to tauopathy progression and identify mTOR signalling as a potential therapeutic target. Full article

Oxidative stress plays a central role in the pathogenesis of neurodegenerative disorders, including Parkinson’s disease. Therefore, compounds with antioxidant and neuroprotective properties represent promising candidates for therapeutic development. N-pyrrolyl hydrazide-hydrazones, a class of pyrrole-based derivatives, have shown promising potential due to their diverse biological activities, including monoamine oxidase-B (MAO-B) inhibition. This study investigated the neuroprotective properties of 10 N-pyrrolyl hydrazide-hydrazones using experimental in vitro and in vivo models of neurodegeneration. The compounds were tested on SH-SY5Y neuroblastoma cells subjected to oxidative stress induced by 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP⁺). A battery of in vitro and in vivo experimental methods was used, including cell viability assay, reactive oxygen species (ROS) production, and apoptosis evaluation by quantifying the sub-G0/G1 cell population. In vivo neuroprotective efficacy was further tested in a rotenone-induced Parkinsonism mouse model by analyzing oxidative biomarkers and brain histopathology. Compounds 2, 4, 5, 6, and 10 significantly preserved cell viability in the 6-OHDA-induced toxicity model, while no protection was observed in the MPP⁺ model. Particularly compound 2 reduced ROS levels and apoptosis in SH-SY5Y cells. In vivo, compound 2 demonstrated strong antioxidant activity by maintaining glutathione levels and reducing lipid peroxidation. Histological analysis confirmed its protective effect against rotenone-induced neuronal damage. These results suggest that N-pyrrolyl hydrazide-hydrazones, especially compound 2, possess significant antioxidant and MAO-B inhibitory properties, supporting their potential as neuroprotective agents. Full article

Olive leaf phenols are recognized for their antioxidant and anti-inflammatory properties. A hydroalcoholic extract of Olea europaea L. cv. Ogliarola leaves was recovered with an ultrasound-assisted extraction using green solvents. Phenol content was investigated by means of liquid chromatography coupled with photodiode array and mass spectrometer detectors. Extract cytotoxicity was determined in SH-SY5Y neuroblastoma cells by the MTT assay to establish non-cytotoxic concentrations. The effects of the extract under lipopolysaccharide-induced conditions were investigated by assessing oxidative stress and lipid peroxidation through malondialdehyde quantification using the thiobarbituric acid assay. Antiglycation capacity was examined with a BSA methylglyoxal model. In parallel, quantitative real-time PCR was employed to assess the modulation of inflammation- and oxidative stress-related genes (TLR4, NF-κB, IL-6, IL-8, Nrf2, and HO-1), providing molecular insights into the extract’s bioactivity. The extract did not exert cytotoxic effects at the selected concentrations and with modulated oxidative stress, lipid peroxidation, protein glycation, and gene expression profiles associated with inflammatory and redox pathways in neuronal cells. These data demonstrated that olive leaf extract, rich in phenols, influenced multiple biochemical and molecular endpoints relevant to neuronal physiology, supporting its potential application as a nutraceutical ingredient for the modulation of oxidative and glycation-related processes. Full article

Neuroblastoma (NB), a pediatric cancer of sympatho-adrenal (SA) lineage, is marked by disrupted differentiation and cellular heterogeneity. INSM1, a zinc-finger transcription factor, is highly expressed in NB and developing SA tissues, where it regulates neuroendocrine differentiation, especially in chromaffin cells. We investigated INSM1’s role in maintaining an undifferentiated, progenitor-like state in NB and its regulation via metabolic and epigenetic mechanisms. Transcriptomic profiling, promoter assays, and metabolic flux analysis revealed that INSM1 expression correlates with methionine cycle activity, particularly the S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio. Disruption of SAM/SAH balance altered INSM1 promoter activity and histone methylation, implicating epigenetic control in NB cell fate. Retinoic acid-induced differentiation downregulated INSM1 and N-Myc, linking INSM1 to tumor cell immaturity. INSM1 overexpression in SH-SY-5Y cells upregulated neuroendocrine and thyroid hormone-related genes (CHGA, CHGB, DDC, NCAM1, DIO3, TH), while suppressing genes involved in cell cycle (RRM, CDC25A), methionine metabolism (AHCY, MAT2A), transcriptional regulation (MYBL2, EZH2), and oncogenic signaling (ALK, LINC011667). These findings suggest that INSM1 promotes NB aggressiveness by sustaining a neuroendocrine progenitor-like phenotype through metabolic-epigenetic coupling. Full article

Anthonoic acids A–C (1–3), the first representatives of sulfated and N-(2-hydroxyethyl)-substituted lipidic α-amino acids, were isolated along with their plausible precursor, anthamino acid A (4), from the marine sponge Antho ridgwayi. The structures of these compounds were determined using the analysis of 1D and 2D NMR and HR ESI mass spectra. A structural feature of 1–4, compared to all previously known lipidic amino acids, is the presence of a sulfate group near the end opposite the amino acid terminus. At a concentration of 1 µM, anthonoic acids A–C (1–3) effectively protected H9c2 and SH-SY5Y cells in biotests, which modeled hypoxia induced by the addition of CoCl₂ to the medium and damage caused by ischemia/reperfusion. These natural products act via the Nrf2-mediated pathway by reducing intracellular ROS levels, accompanied by the upregulation of SOD activity, which is controlled by the Nrf2 transcriptional factor. Anthonoic acids A–C (1–3) do not activate the transcriptional activity of NF-κB but inhibit ATP-induced cell damage and calcium influx, indicating the involvement of P2X7 receptors in the cytoprotective effect of anthonoic acids A–C. Full article

Background: Alzheimer’s disease and Parkinson’s disease are multifactorial disorders causing severe disability, rising with the increase in life expectancy. Currently, the identification of natural compounds useful against these disorders is becoming an urgent necessity. In this study, we used polyphenol-enriched extracts obtained from leaves of Mediterranean plants, which are important in animal feeding (Lotus ornithopodioides, Hedysarum coronarium, Medicago sativa) and in the human Mediterranean diet (Cichorium intybus). Objectives: The aims of this study were as follows: (i) tentative identification of the organic compounds present in the extracts; (ii) determination of their effect on the activity of monoamine oxidase (MAO)-A and MAO-B, key enzymes involved in the metabolism of aminergic neurotransmitters, as well as on protein expression level of these enzymes in cell lines expressing basal MAO-A and MAO-B. Methods: The ability of plant polyphenol extracts to inhibit MAO-A and MAO-B activity was assessed by in vitro enzyme assays. The protein expression level was analyzed by Western blotting. Results: Our data demonstrate that all the extracts behaved as MAO-A and MAO-B inhibitors, although to a different extent and enzyme inhibition mechanism; among them, the extract from L. ornithopodioides induced a decrease in MAO-A protein level in human AGS gastric adenocarcinoma and SH-SY5Y neuroblastoma cell lines. Conclusions: These data reinforce the hypothesis that a plant-based diet and/or integrative supplementation of pharmacological treatments can be considered for preventing and relieving symptoms of neurodegenerative diseases. Full article

Background/Objectives: Capsanthin is a xanthophyll carotenoid from Capsicum species with an extended conjugated polyene chain that underlies both its orange–red color and strong antioxidant potential. In this study, we investigated whether capsanthin protects RA-differentiated SH-SY5Y neuron-like cells against glutamate-induced stress. Methods: Neuronal dysfunction was induced by glutamate exposure, and capsanthin treatment was evaluated using cell viability, reactive oxygen species (ROS) production, antioxidant defense markers, inflammatory cytokines, mitochondrial energy status, and apoptosis-related endpoints. Antioxidant responses were assessed using superoxide dismutase, catalase, glutathione peroxidase activities, and total antioxidant capacity. Cytokine release (TNFα, IL-6, IL-8, IL-4, IL-10) was quantified by ELISA. Mitochondrial function was monitored using ATP content. Apoptosis-associated genes (BAX, BCL-2, CASP3, and CASP9) were analyzed using SYBR Green-based RT-qPCR, complemented by caspase-9 ELISA and caspase-3 Western blotting. Results: Glutamate increased oxidative stress and shifted the cytokine profile toward a pro-inflammatory state, accompanied by reduced ATP levels and a pro-apoptotic transcriptional pattern. Capsanthin significantly attenuated glutamate-induced ROS production, stabilized antioxidant enzyme activities and total antioxidant capacity, reduced pro-inflammatory cytokines while supporting anti-inflammatory signaling, and preserved ATP levels. Conclusions: Overall, capsanthin mitigated excitotoxic stress by maintaining redox balance, limiting inflammatory responses, and protecting mitochondrial energy metabolism in neuron-like cells, supporting its potential as a neuroprotective candidate for glutamate-induced neuronal stress. Full article

Usutu virus (USUV) is an emerging arbovirus recently associated with outbreaks in Western Europe. Although USUV is typically associated with asymptomatic or nonspecific febrile disease, the occurrence of severe neuroinvasive forms of disease has raised concern. There is currently no antiviral treatment available for USUV infection; therefore, we sought to investigate the protective effects of the nucleoside analogue 7DMA against USUV. Adding to 7DMA’s activity against USUV in vitro reported by us and others, we found that 7DMA inhibits USUV replication at multiple stages in mammalian cell lines Vero CCL81 and SH-SY5Y. In vivo testing of 7DMA using the susceptible IFNAR^-/- mouse model indicated that 7DMA treatment significantly reduced USUV viremia and viral load in tissues and prolonged mice survival. The characterization of the protective effects of 7DMA indicated that treatment also altered immunological aspects of disease development, further increasing the expression of mediators such as CXCL10, IL-15, and IFN-γ, and increasing neutrophil recruitment to target organs. We did not observe significant tissue damage or pathology in USUV-infected mouse brains, suggesting that systemic infection and disease are the major components leading to mortality in this model. We conclude that 7DMA exerts protective effects against USUV infection in the IFNAR^-/- model. Full article

Neuroblastoma (NB) is the most prevalent paediatric extracranial solid tumour, which remains a major therapeutic challenge, especially in cases of recurrent and disseminated disease. c-Jun N-terminal kinases (JNKs) are increasingly evidenced to play a key role in NB tumourigenesis and progression through apoptosis regulation, making selective JNK inhibitors promising candidates for use in targeted anticancer drugs in NB. Our study comprehensively investigated the acute antineoplastic potential of the selective JNK inhibitor AS601245 (JNK inhibitor V) on the human MYCN-non-amplified neuroblastoma cell line, SH-SY5Y, with particular focus on its effects on NB cell viability, proliferation, migration, apoptosis, gene and protein expression, and mitochondrial metabolism. JNK V selectively impaired NB cell survival and function, without exerting cytotoxicity toward normal human Schwann cells (HSC) and fibroblasts (BJ). Our findings highlighted a dose-dependent inhibition of proliferation (XTT assay), colony formation (clonogenic assay), and migration (wound healing assay), accompanied by increased caspase-3 activity (caspase-3 assay), pro-apoptotic genes (qRT-PCR) and protein (Western blotting) expression, and significant disruption of both oxidative phosphorylation and glycolysis (Agilent Seahorse XF Assay). These results provide new insights into the therapeutic potential of JNK inhibition as a targeted strategy for NB. Full article

Background/Objectives: Though ototoxic, cisplatin is a mainstay of chemotherapy for a variety of cancers. One suggested mechanism of cisplatin ototoxicity involves damage to the spiral ganglion afferent neurons in the inner ear. There is a need for a high-throughput model to screen medications for efficacy against cisplatin and to develop a local therapeutic to mitigate cisplatin’s debilitating side effects. Microparticles encapsulating a therapeutic medication are an injectable and tunable method of sustained drug delivery, and thus a promising treatment. Methods: SH-SY5y human neuroblastoma cells were used as a cell line model for the spiral ganglion neurons. The cells were dosed with cisplatin and four potential therapeutics (melatonin, metformin, cyclosporine, and N-acetylcysteine), with cell viability measured by CCK-8 assay. The most promising therapeutic, N-acetylcysteine (NAC), was then encapsulated into multiple poly(lactic-co-glycolic acid) (PLGA) microparticle subtypes of varied lactide–glycolide (L:G) ratios and NAC amounts. The elution profile of each microparticle subtype was determined over two months. Results: Of the therapeutics screened, only cells dosed with 1 or 10 mM NAC prior to cisplatin injury demonstrated an improvement in cell viability (73.8%, p < 1 × 10⁻⁸) when compared to cells dosed with cisplatin alone. The 75:25 L:G microparticles demonstrated an increase in the amount of NAC released compared to the 50:50 L:G microparticles. Conclusions: NAC is a potential therapeutic agent for cisplatin toxicity when tested in a neuronal cell line model. NAC was encapsulated into PLGA microparticles and eluted detectable concentrations of NAC for 6 days, which is a first step towards otoprotection for the weeks long duration of chemotherapy treatment. This work describes a method of screening potential therapeutics and a strategy to develop local drug eluting treatments to protect against cisplatin ototoxicity. Full article