Search Results (69)

Background/Objectives: Cannabis sativa has been utilized for medical purposes for thousands of years. It continues to be recognized as a plant with an extensive variety of medicinal and nutraceutical uses today. In this study, a chemical investigation of the flowers of C. sativa isolated by using a variety of chromatographic techniques led to the isolation of eleven compounds. These purified compounds were evaluated for antitumor activity against SK-N-SH neuroblastoma cells. Methods: The compounds were isolated by using chromatographic techniques. Their structures were identified by the examination of spectroscopic methods, including 1D (¹H, ¹³C, and DEPT) and 2D (COSY, HSQC, HMBC, and NOESY) nuclear magnetic resonance (NMR) spectra and mass spectrum, together with the comparison to those reported previously in the literature. The evaluation of toxicity on SK-N-SH cells was performed by the MTT method. Results: Eleven compounds were isolated from the flowers of C. sativa, including two new compounds, namely cannabielsoxa (1), 13²-hydroxypheophorbide c ethyl ester (2), and six known cannabinoids (6–11), together with the first isolation of chlorin-type compounds: pyropheophorbide A (3), 13²-hydroxypheophorbide b ethyl ester (4), and ligulariaphytin A (5) from this plant. The results also demonstrated that cannabinoid compounds had stronger inhibitory effects on neuroblastoma cells than chlorin-type compounds. Conclusions: The evaluation of the biological activities of compounds showed that compounds 4–10 could be considered as the potential compounds for antitumor effects against neuroblastomas. This is also highlighted by using docking analysis. Additionally, the results of this study also suggest that these compounds have the potential to be developed into antineuroblastoma products. Full article

Background/Objectives: α-Synuclein (α-syn) protein is a major pathological agent of familial Parkinson’s disease (PD), and its levels and aggregations determine neurotoxicity in PD pathogenesis. Although the pathophysiological functions of α-syn have been extensively studied, its biological functions remain elusive, and there are reports of wild-type (WT) α-syn and two missense mutations of α-syn (A30P and A53T) inducing protective neuritogenesis through neurite outgrowth. However, the function of another α-syn mutation, E46K, has not been fully elucidated. Thus, we compared the effect of E46K α-syn with other types to identify the mechanisms underlying neurite outgrowth. Methods: We transfected SK-N-SH cells with WT and mutant (A53T and E46K) α-syn to investigate the effects of their overexpression on neurite outgrowth. Then, we compared the differential effects of α-syn on neurite outgrowth using microscopic analysis, including confocal microscopy. We also analyzed the differential regulation of cell division control 42 effector protein 2 (Cdc42EP2) using real-time quantitative polymerase chain reaction and western blot analysis. Finally, to confirm the implication of neurite outgrowth, we knocked down Cdc42EP2 using small interfering RNA. Results: Unlike WT and A53T α-syn, E46K α-syn failed to promote neurite outgrowth by not inducing Cdc42EP2 and subsequent βIII-tubulin expression. Cdc42EP2 knockdown impaired neurite outgrowth in WT and A53T α-syn transfectants. Conclusions: Our findings suggest that WT and mutant α-syn are linked to Cdc42EP2 production in neuritogenesis, implying α-syn involvement in the physiological function of axon growth and synapse formation. Thus, α-syn may be a potential therapeutic target for PD. Full article

Spinocerebellar ataxia type 3 (SCA3), caused by the abnormal expansion of polyglutamine (polyQ) in the ataxin-3 protein, is one of the inherited polyQ neurodegenerative diseases that share similar genetic and molecular features. Mutant polyQ-expanded ataxin-3 protein is prone to aggregation in affected neurons and is predominantly degraded by autophagy, which is beneficial for neurodegenerative disease treatment. Not only does mutant polyQ-expanded ataxin-3 increase susceptibility to oxidative cytotoxicity, but it also hampers antioxidant potency in neuronal cells. Nuclear factor erythroid-derived 2-like 2 (Nrf2), a master transcription factor that controls antioxidant and detoxification gene expression, plays a crucial role in neuroprotection in SCA3 and other neurodegenerative diseases. The present data showed that treatment with erinacine A-enriched Hericium erinaceus mycelium ethanol extract (HEME) extended longevity and improved locomotor activity in ELAV-SCA3tr-Q78 transgenic Drosophila. Moreover, HEME treatment enhanced antioxidant potency and autophagy, which, in turn, corrected levels of mutant polyQ-expanded ataxin-3 and restrained protein aggregation in both cell and Drosophila models of SCA3. Markedly, HEME increased the activation of Nrf2. Silencing Nrf2 protein expression negated most of the promising effects of HEME on SK-N-SH-MJD78 cells, highlighting the critical role of increased Nrf2 activation in the efficacy of HEME treatment. These findings suggest that HEME has therapeutic potential in SCA3 by enhancing autophagic and Nrf2-mediated antioxidant pathways, which may also influence neurodegenerative progression in other polyQ diseases. Full article

Background: The level of the regulator of G-protein signaling 4-1 (RGS4-1) isoform, the longest RGS4 isoform, is significantly reduced in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia. However, the mechanism behind this has not been clarified. The 3′untranslated regions (3′UTRs) are known to regulate the levels of their mRNA splice variants. Methods: We constructed recombinant pmir-GLO vectors with a truncated 3′ regulatory region of the RGS4 gene (3R1, 3R2, 3R3, 3R4, 3R5, and 3R6). The dual-luciferase reporter assay was conducted to find functional regions in HEK-293, SK-N-SH, and U87cells and then predicted miRNA binding to these regions. We performed a dual-luciferase reporter assay and a Western blot analysis after transiently transfecting the predicted miRNAs. Results: The dual-luciferase reporter assay found that regions +401–+789, +789–+1152, and +1562–+1990 (with the last base of the termination codon being +1) might be functional regions. Hsa-miR-874-3p, associated with many psychiatric disorders, might target the +789–+1152 region in the 3′UTR of the RGS4 gene. In the dual-luciferase reporter assay, the hsa-miR-874-3p mimic, co-transfected with 3R1, down-regulated the relative fluorescence intensities. However, this was reversed when the hsa-miR-874-3p mimic was co-transfected with m3R1 (deletion of +853–+859). The hsa-miR-874-3p mimic significantly decreased the endogenous expression of the RGS4-1 isoform in HEK-293 cells. Conclusions: Hsa-miR-874-3p inhibits the expression of the RGS4-1 isoform by targeting +853–+859. Full article

Natural killer (NK) cells can be effective immunotherapeutic anti-cancer agents due to their ability to selectively target and kill tumor cells. This activity is modulated by the interaction of NK cell receptors with inhibitory ligands on the surface of target cells. NK cell inhibitory ligands can be upregulated on tumor cell surfaces in response to interferon-gamma (IFN-γ), a cytokine which is produced by activated NK cells. We hypothesized that the resistance of tumor cells to NK cell killing could be overcome by expression of the parainfluenza virus 5 (PIV5) V protein, which has known roles in blocking IFN-γ signaling. This was tested with human PM21-NK cells produced through a previously developed particle-based method which yields superior NK cells for immunotherapeutic applications. Infection of human SK-N-SH neuroblastoma cells with PIV5 blocked IFN-γ-mediated upregulation of three NK cell inhibitory ligands and enhanced in vitro killing of these tumor cells by PM21-NK cells. SK-N-SH cells transduced to constitutively express the V protein alone were resistant to IFN-γ-mediated increases in cell surface expression of NK cell inhibitory ligands. Real-time in vitro cell viability assays demonstrated that V protein expression in SK-N-SH cells was sufficient to increase PM21-NK cell-mediated killing. Toward a potential therapeutic application, transient lentiviral delivery of the V gene also enhanced PM21-NK cell killing in vitro. Our results provide the foundation for novel therapeutic applications of V protein expression in combination with ex vivo NK cell therapy to effectively increase the killing of tumor cells. Full article

Alzheimer’s disease (AD) is the most common neurodegenerative disorder in the aging population. An accumulation of amyloid plaques and neurofibrillary tangles causes degeneration of neurons, leading to neuronal cell death. The anthocyanin-rich fraction of black rice (Oryza sativa L. variety “Luem Pua”) bran (AFBRB), extracted using a solution of ethanol and water and fractionated using Amberlite XAD7HP column chromatography, contains a high anthocyanin content (585 mg of cyanidin-3-O-glucoside and 24 mg of peonidin-3-O-glucoside per gram of the rich extract), which has been found to reduce neurodegeneration. This study focused on the neuroprotective effects of AFBRB in Aβ_25–35-induced toxicity in the human neuroblastoma cell line (SK-N-SH). SK-N-SH was exposed to Aβ_25–35 (10 µM) to induce an AD cell model in vitro. Pretreatment with AFBRB (0.1, 1, or 10 µg/mL) or C3G (20 µM) was conducted for 2 h prior to the treatment with Aβ_25–35 (10 µM) for an additional 24 h. The results indicate that AFBRB can protect against the cytotoxic effect of Aβ_25–35 through attenuation of intracellular ROS production, downregulation of the expression of the proteins Bax, cytochrome c, cleaved caspase-9, and cleaved caspase-3, upregulation of the expression of Bcl-2 in the mitochondrial death pathway, and reduction in the expression of the three major markers of ER stress pathways in similar ways. Interestingly, we found that pretreatment with AFBRB significantly alleviated Aβ-induced oxidative stress, ER stress, and apoptosis in SK-N-SH cells. This suggests that AFBRB might be a potential therapeutic agent in preventing neurodegenerative diseases. Full article

Background: The regulation of divalent metal transporter-1 (DMT1) by insulin has been previously described in Langerhans cells and significant neuroprotection was found by insulin and insulin-like growth factor 1 treatment during experimental cerebral ischemia in acute ischemic stroke patients and in a rat 6-OHDA model of Parkinson’s disease, where DMT1 involvement is described. According to the regulation of DMT1, previously described as a target gene of NF-kB in the early phase of post-ischemic neurodegeneration, both in vitro and in vivo, and because insulin controls the NFkB signaling with protection from ischemic cell death in rat cardiomyocytes, we evaluated the role of insulin in relation to DMT1 expression and function during ischemic neurodegeneration. Methods: Insulin neuroprotection is evaluated in differentiated human neuroblastoma cells, SK-N-SH, and in primary mouse cortical neurons exposed to oxygen glucose deprivation (OGD) for 8 h or 3 h, respectively, with or without 300 nM insulin. The insulin neuroprotection during OGD was evaluated in both cellular models in terms of cell death, and in SK-N-SH for DMT1 protein expression and acute ferrous iron treatment, performed in acidic conditions, known to promote the maximum DMT1 uptake as a proton co-transporter; and the transactivation of 1B/DMT1 mouse promoter, already known to be responsive to NF-kB, was analyzed in primary mouse cortical neurons. Results: Insulin neuroprotection during OGD was concomitant to the down-regulation of both DMT1 protein expression and 1B/DMT1 mouse promoter transactivation. We also showed the insulin-dependent protection from cell death after acute ferrous iron treatment. In conclusion, although preliminary, this evaluation highlights the peculiar role of DMT1 as a possible pharmacological target, involved in neuroprotection by insulin during in vitro neuronal ischemia and acute ferrous iron uptake. Full article

Calcium calmodulin-dependent protein kinase (CaMK) mediates calcium-induced neural gene activation. CaMK also inhibits the non-syndromic intellectual disability gene, Freud-1/CC2D1A, a transcriptional repressor of human serotonin-1A (5-HT1A) and dopamine-D2 receptor genes. The altered expression of these Freud-1-regulated genes is implicated in mental illnesses such as major depression and schizophrenia. We hypothesized that Freud-1 is blocked by CaMK-induced phosphorylation. The incubation of purified Freud-1 with either CaMKIIα or CaMKIV increased Freud-1 phosphorylation that was partly prevented in Freud-1-Ser644Ala and Freud-1-Thr780Ala CaMK site mutants. In human SK-N-SH neuroblastoma cells, active CaMKIV induced the serine and threonine phosphorylation of Freud-1, and specifically increased Freud-1-Thr780 phosphorylation in transfected HEK-293 cells. The activation of purified CaMKIIα or CaMKIV reduced Freud-1 binding to its DNA element on the 5-HT1A and dopamine-D2 receptor genes. In SK-N-SH cells, active CaMKIV but not CaMKIIα blocked the Freud-1 repressor activity, while Freud-1 Ser644Ala, Thr780Ala or dual mutants were resistant to inhibition by activated CaMKIV or calcium mobilization. These results indicate that the Freud-1 repressor activity is blocked by CaMKIV-induced phosphorylation at Thr780, resulting in the up-regulation of the target genes, such as the 5-HT1A receptor gene. The CaMKIV-mediated inhibition of Freud-1 provides a novel de-repression mechanism to induce 5-HT1A receptor expression for the regulation of cognitive development, behavior and antidepressant response. Full article

All–trans–retinoic acid (ATRA) has long been known to affect cell growth and differentiation. To improve ATRA’s therapeutic efficacy and pharmacodynamics, several delivery systems have been used. In this study, free ATRA and anionic–liposome–encapsulated ATRA were compared for their effects on SK–N–SH human neuroblastoma cell growth and differentiation. Anionic liposomes made of L–$\alpha$–phosphatidylcholine (PC) and L–$\alpha$–phosphatidic acid (PA), empty (PC–PA) and loaded with ATRA (PC–PA–ATRA), were characterized by dynamic light scattering (DLS) and electrophoretic mobility measurements, and drug entrapment efficiency (EE%) was measured to evaluate the applicability of the new colloidal formulation. The results of brightfield microscopy and cell growth curves indicated that ATRA, whether free or encapsulated, reduced growth and induced differentiation, resulting in SK–N–SH cells changing from epithelioid to neuronal–like morphologies, and producing a significant increase in neurite growth. To further characterize the neuro-differentiation of SK–N–SH cells, the expression of βIII–Tubulin and synaptophysin and mitochondria localization were analyzed via immunofluorescence. Increased expression of neuronal markers and a peculiar localization of mitochondria in the neuritic extensions were apparent both in ATRA– and PC–PA–ATRA–differentiated cells. As a whole, our results strongly indicate that ATRA treatment, by any means, can induce the differentiation of parent SK–N–SH, and they highlight that its encapsulation in anionic liposomes increases its differentiation ability in terms of the percentage of neurite–bearing cells. Interestingly, our data also suggest an unexpected differentiation capability of anionic liposomes per se. This work highlights the importance of developing and carefully testing novel delivery nanocarriers, which are a necessary first “step” in the development of new therapeutic settings. Full article

The spinocerebellar ataxias (SCA) comprise a group of inherited neurodegenerative diseases. Machado–Joseph Disease (MJD) or spinocerebellar ataxia 3 (SCA3) is the most common autosomal dominant form, caused by the expansion of CAG repeats within the ataxin-3 (ATXN3) gene. This mutation results in the expression of an abnormal protein containing long polyglutamine (polyQ) stretches that confers a toxic gain of function and leads to misfolding and aggregation of ATXN3 in neurons. As a result of the neurodegenerative process, SCA3 patients are severely disabled and die prematurely. Several screening approaches, e.g., druggable genome-wide and drug library screenings have been performed, focussing on the reduction in stably overexpressed ATXN3(polyQ) protein and improvement in the resultant toxicity. Transgenic overexpression models of toxic ATXN3, however, missed potential modulators of endogenous ATXN3 regulation. In another approach to identify modifiers of endogenous ATXN3 expression using a CRISPR/Cas9-modified SK-N-SH wild-type cell line with a GFP-T2A-luciferase (LUC) cassette under the control of the endogenous ATXN3 promotor, four statins were identified as potential activators of expression. We here provide an overview of the high throughput screening approaches yet performed to find compounds or genomic modifiers of ATXN3(polyQ) toxicity in different SCA3 model organisms and cell lines to ameliorate and halt SCA3 progression in patients. Furthermore, the putative role of cholesterol in neurodegenerative diseases (NDDs) in general and SCA3 in particular is discussed. Full article

Periodontal ligament (PDL) stem-like cells (PDLSCs) are promising for regeneration of the periodontium because they demonstrate multipotency, high proliferative capacity, and the potential to regenerate bone, cementum, and PDL tissue. However, the transplantation of autologous PDLSCs is restricted by limited availability. Since PDLSCs are derived from neural crest cells (NCs) and NCs persist in adult PDL tissue, we devised to promote the regeneration of the periodontium by activating NCs to differentiate into PDLSCs. SK-N-SH cells, a neuroblastoma cell line that reportedly has NC-like features, seeded on the extracellular matrix of PDL cells for 2 weeks, resulted in the significant upregulation of PDL marker expression. SK-N-SH cell-derived PDLSCs (SK-PDLSCs) presented phenotypic characteristics comparable to induced pluripotent stem cell (iPSC)-derived PDLSCs (iPDLSCs). The expression levels of various hyaluronic acid (HA)-related genes were upregulated in iPDLSCs and SK-PDLSCs compared with iPSC-derived NCs and SK-N-SH cells, respectively. The knockdown of CD44 in SK-N-SH cells significantly inhibited their ability to differentiate into SK-PDLSCs, while low-molecular HA (LMWHA) induction enhanced SK-PDLSC differentiation. Our findings suggest that SK-N-SH cells could be applied as a new model to induce the differentiation of NCs into PDLSCs and that the LMWHA–CD44 relationship is important for the differentiation of NCs into PDLSCs. Full article

Background: Neuroblastoma is the most common extracranial solid tumour in children, accounting for 15% of paediatric cancer deaths. Multiple genetic abnormalities have been identified as prognostically significant in neuroblastoma patients. Optical genome mapping (OGM) is a novel cytogenetic technique used to detect structural variants, which has not previously been tested in neuroblastoma. We used OGM to identify copy number and structural variants (SVs) in neuroblastoma which may have been missed by standard cytogenetic techniques. Methods: Five neuroblastoma cell lines (SH-SY5Y, NBLW, GI-ME-N, NB1691 and SK-N-BE2(C)) and two neuroblastoma tumours were analysed using OGM with the Bionano Saphyr^® instrument. The results were analysed using Bionano Access software and compared to previous genetic analyses including G-band karyotyping, FISH (fluorescent in situ hybridisation), single-nucleotide polymorphism (SNP) array and RNA fusion panels for cell lines, and SNP arrays and whole genome sequencing (WGS) for tumours. Results: OGM detected copy number abnormalities found using previous methods and provided estimates for absolute copy numbers of amplified genes. OGM identified novel SVs, including fusion genes in two cell lines of potential clinical significance. Conclusions: OGM can reliably detect clinically significant structural and copy number variations in a single test. OGM may prove to be more time- and cost-effective than current standard cytogenetic techniques for neuroblastoma. Full article

Serotonin 1A (5-HT1A) autoreceptors located on serotonin neurons inhibit their activity, and their upregulation has been implicated in depression, suicide and resistance to antidepressant treatment. Conversely, post-synaptic 5-HT1A heteroreceptors are important for antidepressant response. The transcription factor deformed epidermal autoregulatory factor 1 (Deaf1) acts as a presynaptic repressor and postsynaptic enhancer of 5-HT1A transcription, but the mechanism is unclear. Because Deaf1 interacts with and is phosphorylated by glycogen synthase kinase 3β (GSK3β)—a constitutively active protein kinase that is inhibited by the mood stabilizer lithium at therapeutic concentrations—we investigated the role of GSK3β in Deaf1 regulation of human 5-HT1A transcription. In 5-HT1A promoter-reporter assays, human HEK293 kidney and 5-HT1A-expressing SKN-SH neuroblastoma cells, transfection of Deaf1 reduced 5-HT1A promoter activity by ~45%. To identify potential GSK3β site(s) on Deaf1, point mutations of known and predicted phosphorylation sites on Deaf1 were tested. Deaf1 repressor function was not affected by any of the mutants tested except the Y300F mutant, which augmented Deaf1 repression. Both lithium and the selective GSK3 inhibitors CHIR-99021 and AR-014418 attenuated and reversed Deaf1 repression compared to vector. This inhibition was at concentrations that maximally inhibit GSK3β activity as detected by the GSK3β-sensitive TCF/LEF reporter construct. Our results support the hypothesis that GSK3β regulates the activity of Deaf1 to repress 5-HT1A transcription and provide a potential mechanism for actions of GSK3 inhibitors on behavior. Full article

C9orf72 mutations are the most common form of familial amyotrophic lateral sclerosis (C9-ALS). It causes the production of proline–arginine dipeptide repeat proteins (PR-DPRs) in motor neurons (MNs), leading to the molecular pathology characteristic of ALS. UNC13A is critical for maintaining the synaptic function of MNs. Most ALS patients have nuclear deletion of the splicing repressor TDP-43 in MNs, which causes inclusion of the cryptic exon (CE) of UNC13A mRNA, resulting in nonsense-mediated mRNA decay and reduced protein expression. Therefore, in this study, we explored the role of PR-DPR in CE inclusion of UNC13A mRNA. Our results showed that PR-DPR (PR₅₀) induced CE inclusion and decreased the protein expression of UNC13A in human neuronal cell lines. We also identified an interaction between the RNA-binding protein NOVA1 and PR₅₀ by yeast two-hybrid screening. NOVA1 expression is known to be reduced in patients with ALS. We found that knockdown of NOVA1 enhanced CE inclusion of UNC13A mRNA. Furthermore, the naturally occurring triterpene betulin can inhibit the interaction between NOVA1 and PR₅₀, thus preventing CE inclusion of UNC13A mRNA and protein reduction in human neuronal cell lines. This study linked PR-DPR with CE inclusion of UNC13A mRNA and developed candidate therapeutic strategies for C9-ALS using betulin. Full article

The aim of this study was to elucidate the chemistry of cellular degeneration in human neuroblastoma cells upon exposure to outer-membrane vesicles (OMVs) produced by Porphyromonas gingivalis (Pg) oral bacteria by monitoring their metabolomic evolution using in situ Raman spectroscopy. Pg-OMVs are a key factor in Alzheimer’s disease (AD) pathogenesis, as they act as efficient vectors for the delivery of toxins promoting neuronal damage. However, the chemical mechanisms underlying the direct impact of Pg-OMVs on cell metabolites at the molecular scale still remain conspicuously unclear. A widely used in vitro model employing neuroblastoma SH-SY5Y cells (a sub-line of the SK-N-SH cell line) was spectroscopically analyzed in situ before and 6 h after Pg-OMV contamination. Concurrently, Raman characterizations were also performed on isolated Pg-OMVs, which included phosphorylated dihydroceramide (PDHC) lipids and lipopolysaccharide (LPS), the latter in turn being contaminated with a highly pathogenic class of cysteine proteases, a key factor in neuronal cell degradation. Raman characterizations located lipopolysaccharide fingerprints in the vesicle structure and unveiled so far unproved aspects of the chemistry behind protein degradation induced by Pg-OMV contamination of SH-SY5Y cells. The observed alterations of cells’ Raman profiles were then discussed in view of key factors including the formation of amyloid β (Aβ) plaques and hyperphosphorylated Tau neurofibrillary tangles, and the formation of cholesterol agglomerates that exacerbate AD pathologies. Full article