Expression Of Neuronal Markers Research Articles

Mitochondrial dynamics and metabolic remodeling in xenograft of IPSC-derived human neural precursors

It is well recognized that the regulation of mitochondrial functions affects the differentiation and maturation of neurons. The study of these processes is of both fundamental and practical importance for regenerative neurobiology. Aim of the study: to characterize the mitochondrial fission changes and their relation to the activation of oxidative phosphorylation (metabolic shift) during maturation of human IPSC-derived neural precursors grafted into rat striatum. Wistar rats (n = 15) were unilaterally injected into the caudate nucleus with neural precursors derived from human IPSCs. Changes in localization and expression of neuronal differentiation markers: nestin, NeuN, neuronal enolase, as well as mitochondrial outer membrane protein, ATP synthase and mitochondrial fission protein Drp1 were assessed by immunostaining. Measurements were performed on graft cells 2 weeks, 3 and 6 months after surgery. Maturation of grafted neurons was associated with fluctuations morphometric parameters of the mitochondrial fraction and Drp1 levels. Increased mitochondrial fission was detected 3 months after transplantation, before an increase in ATP synthase staining by 6th month and a switch of transplanted cells to oxidative phosphorylation. The conducted experiment demonstrated a link between mitochondrial dynamics and changes in the metabolic profile and maturation of transplanted neurons. The regulation of mitochondrial dynamics may have future implications for developing methods to improve the integration of transplanted neurons into recepient brain structures.

Electroacupuncture alleviates neuropathic pain in a rat model of CCD via suppressing P2X3 expression in dorsal root ganglia.

Sciatica and low back pain are prevalent clinical types of neuropathic pain that significantly impair patients' quality of life. Conventional therapies often lack effectiveness, making these conditions challenging to treat. Electroacupuncture (EA) is an effective physiotherapy for pain relief. Prior research has demonstrated a relationship between the frequency of neuropathic pain and the analgesic impact of EA stimulation. This work aimed to assess the analgesic effects of EA in a rat model of chronic compression of the dorsal root ganglion (CCD) and to understand the underlying processes. We established a rat CCD model to simulate sciatica and low back pain. EA was applied to rats with CCD at various frequencies (2Hz, 100Hz, and 2/100Hz). The paw withdrawal threshold (PWT) was measured to assess analgesic effects. Additionally, protein levels of the purinergic receptor P2X3 (P2X3) and the expression of nociceptive neuronal markers were analyzed using immunohistochemistry and western blot (WB) techniques. The study also measured levels of proinflammatory cytokines TNF-α and IL-1β in the dorsal root ganglion (DRG). The involvement of P2X3 receptors was further investigated using the P2X3 agonist, α,β-methylene ATP (α,β-meATP). CCD rats developed pronounced mechanical allodynia. EA stimulation at all tested frequencies produced analgesic effects, with 2/100Hz showing superior efficacy compared to 2Hz and 100Hz. The expression of P2X3 was increased in ipsilateral DRG of CCD model rats. P2X3 were co-labeled with isolectin B4 (IB4) and transient receptor potential vanilloid (TRPV1), indicating their role in nociception. 2/100Hz EA treatment significantly reduced mechanical allodynia and inhibited the overexpression of P2X3, TRPV1, substance P (SP), and calcitonin gene-related peptide (CGRP) in the ipsilateral DRG of CCD model rats. Additionally, EA reduced the levels of proinflammatory cytokines TNF-α and IL-1β in the ipsilateral DRG, indicating an anti-inflammatory effect. The P2X3 agonist α,β-me ATP attenuated the analgesic effect of 2/100Hz EA in CCD rats. The WB and immunofluorescence results consistently demonstrated P2X3 inhibition contributed to the analgesic effects of 2/100Hz EA on CCD-induced neuropathic pain. Our findings suggest that 2/100Hz EA alleviates neuropathic pain in rats by inhibiting the upregulation of P2X3 receptors in the ipsilateral DRG. This study backs up EA as a viable treatment option for sciatica and low back pain in clinical settings.

PATH-17. MOLECULAR CLASSIFICATION OF H3K27-WILDTYPE SPINAL CORD GLIOMAS: IMPLICATIONS FOR PROGNOSIS AND THERAPEUTICS

Abstract BACKGROUND H3K27-wildtype spinal cord gliomas (H3WT-SCGs) are rare and heterogeneous tumors that present significant challenges in pathological classification. Traditional histologic diagnoses often do not align well with the underlying molecular characteristics of these tumors. METHODS We characterized 55 cases of H3WT-SCGs using a comprehensive approach that included DNA methylation array, whole-exome sequencing, transcriptome sequencing, proteomics, and single-cell RNA sequencing. Unsupervised clustering analysis was performed to identify distinct molecular subtypes. RESULTS The analysis identified seven distinct spinal cord methylation (SCM) clusters: pilocytic astrocytomas (PA-SPINE), PA-immune enriched (PA-IME), diffuse leptomeningeal glioneuronal tumor (DLGNT), subependymoma (SUBEPN-SPINE), glioblastoma-like (GBM-like), and pleomorphic xanthoastrocytoma-like (PXA-like). There was a 61.8% discordance between SCM classes and traditional histologic diagnoses. The new molecular classification provided a better correlation with molecular features and patient prognosis. BRAF fusions were enriched in PA-SPINE and DLGNT (p = 0.04), and DLGNT showed significant chromosomal alterations, including 1p loss (p = 6.5e-08) and 1q gain (p = 3.6e-05). Notably, we identified two distinct classes of PA-SPINE: adult (older than 18 years) and pediatric. Adult PAs exhibited lower frequencies of BRAF fusions (p = 1.4e-03), shorter tumor lengths (p = 3.7e-04), and longer progression-free survival (p = 0.062). Adult PA neoplastic cells were less differentiated, with higher expression of EGFR and neuronal markers, and lower cytokine and chemokine expression compared to pediatric cases. Single-cell analysis revealed reduced infiltration of monocyte-derived macrophages and proliferative myeloid cells in adult cases. Both PA-IME and PA-pediatric clusters were characterized as immune-hot tumors with high immune cell enrichment. CONCLUSION This study defined clinically relevant molecular classes of H3WT-SCGs that better reflect patient prognosis and molecular characteristics compared to traditional histologic diagnoses. The findings suggest potential therapeutic implications, especially in immune-hot tumors like IME and PA-pediatric, which could benefit from targeted immunotherapies.

Integrative analysis of long isoform sequencing and functional data identifies distinct cortical layer neuronal subtypes derived from human iPSCs.

Generation of human induced pluripotent stem cells (iPSCs) through reprogramming was a transformational change in the field of regenerative medicine that led to new possibilities for drug discovery and cell replacement therapy. Several protocols have been established to differentiate hiPSCs into neuronal lineages. However, low differentiation efficiency is one of the major drawbacks of these approaches. Here, we compared the efficiency of two methods of neuronal differentiation from iPSCs cultured in two different culture media, StemFlex Medium (SFM) and Essential 8 Medium (E8M). The results indicated that iPSCs cultured in E8M efficiently generated different types of neurons in a shorter time and without the growth of undifferentiated nonneuronal cells in the culture as compared with those generated from iPSCs in SFM. Furthermore, these neurons were validated as functional units immunocytochemically by confirming the expression of mature neuronal markers (i.e., NeuN, β tubulin, and Synapsin I) and whole cell patch-clamp recordings. Long-read single-cell RNA sequencing confirms the presence of upper and deep layer cortical layer excitatory and inhibitory neuronal subtypes in addition to small populations of GABAergic neurons in day 30 neuronal cultures. Pathway analysis indicated that our protocol triggers the signaling transcriptional networks important for the process of neuronal differentiation in vivo.NEW & NOTEWORTHY Low differentiation efficiency is one of the major drawbacks of the existing protocols to differentiate iPSCs into neuronal lineages. Here, we present time-efficient and robust approach of neuronal differentiation leading to the generation of functional brain units, cortical layer neurons. We found iPSCs cultured in Essential 8 media (E8M) resulted in neuronal differentiation without the signs of growth of spontaneously differentiated cells in culture at any point in 35 days compared with Stemflex media (SFM).

Distinct In Vitro Differentiation Protocols Differentially Affect Cytotoxicity Induced by Heavy Metals in Human Neuroblastoma SH-SY5Y Cells.

The SH-SY5Y cell line is widely used in neurotoxicity studies. However, the effects of inducing cell differentiation on the cytotoxic effects of heavy metals are unclear. Therefore, we investigated the effects of mercuric chloride (HgCl2), cadmium chloride (CdCl2), arsenic trioxide (As2O3), and methylmercury (MeHg) on SH-SY5Y cells differentiated in the presence of insulin-like growth factor-I (IGF-I) or all-trans retinoic acid (ATRA). Neurite outgrowth with distinct changes in neuronal marker expression, phenotype, and cell cycle was induced in SH-SY5Y cells by IGF-I treatment for 1day or ATRA treatment for up to 7days. The cytotoxic effects of HgCl2 decreased at lower concentrations and increased at higher concentrations in both IGF-I- and ATRA-differentiated cells compared with those in undifferentiated cells. Differentiation with IGF-I, but not with ATRA, increased the cytotoxic effects of CdCl2. Decreased cytotoxic effects of As2O3 and MeHg were observed at lower concentrations in IGF-I-differentiated cells, whereas increased cytotoxic effects of As2O3 and MeHg were observed at higher concentrations in ATRA-differentiated cells. Changes in the cytotoxic effects of heavy metals were observed even after 1day of ATRA exposure in SH-SY5Y cells. Our results demonstrate that the differentiation of SH-SY5Y cells by IGF-I and ATRA induces different cellular characteristics, resulting in diverse changes in sensitivity to heavy metals, which depend not only on the differentiation agents and treatment time but also on the heavy metal species and concentration.

Hippocampal neurogenesis modulated by Quinic acid: A therapeutic strategy for the neurodegenerative disorders.

Neural progenitor cells (NPCs) reside in the brain and participate in the mechanism of neurogenesis that permits the brain to generate the building blocks for enhancement of cognitive abilities and acquisition of new skills. The existence of NPCs in brain has opened a novel dimension of research to explore their potential for treatment of various neurodegenerative disorders. The present study provides novel insights into the intracellular mechanisms in neuronal cells proliferation, maturation and differentiation regulated by Quinic acid (QA). Furthermore, this study might help in discovery and development of lead molecule that can overcome the challenges in the treatment of neurodegenerative diseases. The growth supporting effect of QA was studied using MTT assay. For that purpose, hippocampal cell cultures of neonatal rats were treated with different concentrations of QA and incubated for 24, 48 and 72 h. Gene and protein expressions of the selected molecular markers nestin, neuron-specific class III beta-tubulin (Tuj-1), neuronal nuclear protein (NeuN), neuronal differentiation 1 (NeuroD1), glial fibrillary acidic protein (GFAP), neuroligin (NLGN) and vimentin were analyzed. QA-induced cell proliferation and differentiation of hippocampal progenitor cells was also accompanied by significantly increased expression of progenitor and immature neuronal marker, mature neuronal marker and differentiating factor, that is, nestin, Tuj-1, NeuN and NeuroD1, respectively. On the other hand, vimentin downregulation and constant GFAP expression were observed following QA treatment. Additionally, the effects of QA on the recovery of stressed cells was studied using in vitro model of oxygen glucose deprivation (OGD). It was observed that hippocampal cells were able to recover from OGD following the treatment with QA. These findings suggest that QA treatment promotes hippocampal neurogenesis by proliferating and differentiating of NPCs and recovers neurons from stress caused by OGD. Thus, the neurogenic potential of QA can be explored for the treatment of neurodegenerative disorders.

Protection from oxygen–glucose deprivation by neurosteroid treatment in primary neurons and oligodendrocytes

Preterm birth results in an increased risk of neonatal brain injury and neurobehavioural disorders. Despite the seriousness of these adverse outcomes, there are currently no effective therapies to protect the vulnerable developing brain. We propose that neurosteroid replacement therapy may be a novel approach in reducing detrimental neurological outcomes following preterm birth. The use of guinea pig primary neuronal and oligodendrocyte cultures with relevance to late gestation allows insight into the mechanisms behind the effectiveness of these treatments. Primary neuronal and oligodendrocyte cultures were derived from fetal guinea pig frontal cortex brain tissue at gestational age 62 (GA62). Cell cultures were pre-treated with either etifoxine (5 µM) or zuranolone (1 µm) for 24 h prior to insult. Cells were then exposed to either oxygen–glucose deprivation (OGD; 0% O2 and no glucose DMEM; preterm birth insult) or sham (standard cell culture conditions; 25 mM DMEM) for 2 h. Lactate dehydrogenase assay (LDH) was performed following OGD as a measure of cytotoxicity. Relative mRNA expression of key neuronal and oligodendrocyte markers, as well as neuronal receptors and transporters, were quantified using high throughput (Fluidigm) RT-PCR. OGD significantly increased cellular cytotoxicity in both neurons and oligodendrocytes. Additionally, key neuronal marker mRNA expression was reduced following OGD, and oligodendrocytes displayed arrested mRNA expression of key markers of lineage progression. Treatment with etifoxine restored a number of parameters back to control levels, whereas treatment with zuranolone provided a robust improvement in all parameters examined. This study has demonstrated the neuroprotective potential of neurosteroid replacement therapy in a model of hypoxia related to preterm birth. Neuroprotection appears to be mediated through glutamate reduction and increased brain derived neurotrophic factor (BDNF). Future work is warranted in examining these treatments in vivo, with the overall aim to suppress preterm associated brain damage and reduce long term outcomes for affected offspring.

Investigating the effect of pregabalin on neuronal development using ultrashort self-assembling peptides: Assessing 3D neuronal cultures with high throughput robotic 3D bioprinting

Pregabalin is a widely prescribed drug for various neurological disorders, yet its effects on embryonic cortical neuron development when given to pregnant women remain inadequately explored. In this study, we employed advanced three-dimensional (3D) culturing and in-house developed high-throughput robotic 3D bioprinting technologies to evaluate their potential in neuropharmacology applications, using pregabalin as a model compound. Using a robotic 3D bioprinter and tetrameric IIZK peptide hydrogel as bioink, we created constructs with pregabalin-treated and untreated primary mouse embryonic cortical neurons. This setup allowed us to study the drug’s effects on cell viability, expression of neuronal markers, and neuron development. Our comparative analysis between 2D and 3D peptide-based cell culture models revealed that at a therapeutic concentration of 10 μM, pregabalin does not affect neuronal viability or the morphogenesis of cortical neurons. However, it significantly alters adenosine triphosphate (ATP) release, suggesting potential disruptions in mitochondrial function. Moreover, gene expression analysis of key genes involved in the development of the forebrain and the differentiation and maturation of neurons revealed significant alterations, including the downregulation of Dlx2, Nhlh2, Otp, and Gad67. These findings, together with observed alterations in neuronal activity and oscillations, emphasize the complex impact of pregabalin on neuronal development and function. They highlight the necessity for comprehensive clinical evaluations of its use during pregnancy. Furthermore, our research demonstrates the feasibility and value of integrating 3D cultures with high-throughput 3D bioprinting in neuropharmacology, opening new avenues for investigating drug effects on neuronal development and function, and contributing to safer clinical practices.

Neuronal Distribution in Colorectal Cancer: Associations With Clinicopathological Parameters and Survival

Over the past years, insights in the cancer neuroscience field increased rapidly, and a potential role for neurons in colorectal carcinogenesis has been recognized. However, knowledge on the neuronal distribution, subtypes, origin, and associations with clinicopathological characteristics in human studies is sparse. In this study, colorectal tumor tissues from the Netherlands Cohort Study on diet and cancer (n = 490) and an in-cohort validation population (n = 529) were immunohistochemically stained for the pan-neuronal markers neurofilament (NF) and protein gene product 9.5 (PGP9.5) to study the association between neuronal marker expression and clinicopathological characteristics. In addition, tumor and healthy colon tissues were stained for neuronal subtype markers, and their immunoreactivity in colorectal cancer (CRC) stroma was analyzed. NF-positive and PGP9.5-positive nerve fibers were found within the tumor stroma and mostly characterized by the neuronal subtype markers vasoactive intestinal peptide and neuronal nitric oxide synthase, suggesting that inhibitory neurons are the most prominent neuronal subtype in CRC. NF and PGP9.5 protein expression were not consistently associated with tumor stage, sublocation, differentiation grade, and median survival. NF immunoreactivity was associated with a worse CRC-specific survival in the study cohort (P = .025) independent of other prognostic factors (hazard ratio, 2.31; 95% CI, 1.33-4.03; P = .003), but these results were not observed in the in-cohort validation group. PGP9.5, in contrast, was associated with a worse CRC-specific survival in the in-cohort validation (P = .046) but not in the study population. This effect disappeared in multivariate analyses (hazard ratio, 0.81; 95% CI, 0.50-1.32; P = .393), indicating that this effect was dependent on other prognostic factors. This study demonstrates that the tumor stroma of CRC patients mainly harbors inhibitory neurons and that NF as a single marker is significantly associated with a poorer CRC-specific survival in the study cohort but necessitates future validation.

Schizophrenia-Like Deficits and Impaired Glutamate/Gamma-aminobutyric acid Homeostasis in Zfp804a Conditional Knockout Mice.

Zinc finger protein 804A (ZNF804A) was the first genome-wide associated susceptibility gene for schizophrenia (SCZ) and played an essential role in the pathophysiology of SCZ by influencing neurodevelopment regulation, neurite outgrowth, synaptic plasticity, and RNA translational control; however, the exact molecular mechanism remains unclear. A nervous-system-specific Zfp804a (ZNF804A murine gene) conditional knockout (cKO) mouse model was generated using clustered regularly interspaced short palindromic repeat/Cas9 technology and the Cre/loxP method. Multiple and complex SCZ-like behaviors, such as anxiety, depression, and impaired cognition, were observed in Zfp804a cKO mice. Molecular biological methods and targeted metabolomics assay validated that Zfp804a cKO mice displayed altered SATB2 (a cortical superficial neuron marker) expression in the cortex; aberrant NeuN, cleaved caspase 3, and DLG4 (markers of mature neurons, apoptosis, and postsynapse, respectively) expressions in the hippocampus and a loss of glutamate (Glu)/γ-aminobutyric acid (GABA) homeostasis with abnormal GAD67 (Gad1) expression in the hippocampus. Clozapine partly ameliorated some SCZ-like behaviors, reversed the disequilibrium of the Glu/GABA ratio, and recovered the expression of GAD67 in cKO mice. Zfp804a cKO mice reproducing SCZ-like pathological and behavioral phenotypes were successfully developed. A novel mechanism was determined in which Zfp804a caused Glu/GABA imbalance and reduced GAD67 expression, which was partly recovered by clozapine treatment. These findings underscore the role of altered gene expression in understanding the pathogenesis of SCZ and provide a reliable SCZ model for future therapeutic interventions and biomarker discovery.

Human amniotic epithelial stem cell-derived dopaminergic neuron-like cells ameliorate motor dysfunction in a rat model of Parkinson's disease

AimsParkinson's disease (PD) remains a substantial clinical challenge due to the progressive loss of midbrain dopaminergic (DA) neurons in nigrostriatal pathway. In this study, human amniotic epithelial stem cells (hAESCs)-derived dopaminergic neuron-like cells (hAESCs-DNLCs) were generated, with the aim of providing new therapeutic approach to PD. Materials and methodshAESCs, which were isolated from discarded placentas, were induced to differentiate into hAESCs-DNLCs by following a “two stages” induction protocol. The differentiation efficiency was assessed by quantitative real-time PCR (qRT-PCR), immunocytochemistry (ICC), and ELISA. Immunogenicity, cell viability and tumorigenicity of hAESCs-DNLC were analyzed before in vivo experiments. Subsequently, hAESCs-DNLCs were transplanted into PD rats, behavioral tests were monitored after graft, and the regeneration of DA neurons was detected by immunohistochemistry (IHC). Furthermore, to trace hAESCs-DNLCs in vivo, cells were pre-labeled with PKH67 green fluorescence. Key findingshAESCs were positive for pluripotent markers and highly expressed neural stem cells (NSCs) markers. Based on this, we established an induction method reliably generates hAESCs-DNLCs, which was evidenced by epithelium-to-neuron morphological changes, elevated expressions of neuronal and DA neuronal markers, and increased secretion of dopamine. Moreover, hAESCs-DNLCs maintained high cell viability, no tumorigenicity and low immunogenicity, suggesting hAESCs-DNLCs an attractive implant for PD therapy. Transplantation of hAESCs-DNLCs into PD rats significantly ameliorated motor disorders, as well as enhanced the reinnervation of TH+ DA neurons in nigrostriatal pathway. SignificanceOur study has demonstrated evident therapeutic effects of hAESCs-DNLCs, and provides a safe and promising solution for PD.

A novel APP splice variant-dependent marker system to precisely demarcate maturity in SH-SY5Y cell-derived neurons.

SH-SY5Y, a neuroblastoma cell line, can be converted into mature neuronal phenotypes, characterized by the expression of mature neuronal and neurotransmitter markers. However, the mature phenotypes described across multiple studies appear inconsistent. As this cell line expresses common neuronal markers after a simple induction, there is a high chance of misinterpreting its maturity. Therefore, sole reliance on common neuronal markers is presumably inadequate. The Alzheimer's disease (AD) central gene, amyloid precursor protein (APP), has shown contrasting transcript variant dynamics in various cell types. We differentiated SH-SY5Y cells into mature neuron-like cells using a concise protocol and observed the upregulation of total APP throughout differentiation. However, APP transcript variant-1 was upregulated only during the early to middle stages of differentiation and declined in later stages. We identified the maturity state where this post-transcriptional shift occurs, terming it "true maturity." At this stage, we observed a predominant expression of mature neuronal and cholinergic markers, along with a distinct APP variant pattern. Our findings emphasize the necessity of using a differentiation state-sensitive marker system to precisely characterize SH-SY5Y differentiation. Moreover, this study offers an APP-guided, alternative neuronal marker system to enhance the accuracy of the conventional markers.

Anlotinib induces neuronal-like differentiation of neuroblastoma by downregulating CRMP5.

The therapeutic effect of anlotinib on neuroblastoma is still not fully understood. This study aims to explore the differentiation therapeutic effects of anlotinib on neuroblastoma and its potential association with the neural development regulatory protein collapsin response mediator protein 5 (CRMP5), both in vivo and in vitro. A patient-derived xenograft (PDX) model was established to observe the therapeutic effect of anlotinib. Neuroblastoma cell lines SK-N-SH and SK-N-AS were cultured to observe the morphological impact of anlotinib. Transwell assay was used to evaluate the cell invasion, and Western blot analysis and immunohistochemistry were employed to detect the expressions of neuronal differentiation-related proteins. Results indicate that anlotinib effectively inhibited tumor growth in the PDX model, modulated the expressions of neuronal differentiation markers. In vitro, anlotinib treatment induced neurite outgrowth in neuroblastoma cells and inhibited their invasive ability, reflecting a change in neuronal marker expression patterns consistent with the PDX model. Similarly, in the SK-N-AS mouse xenograft model, anlotinib demonstrated comparable tumor-suppressing effects and promoted neuronal-like differentiation. Additionally, anlotinib significantly downregulated CRMP5 expression in neuroblastoma both in vivo and in vitro. Overexpression of CRMP5 significantly reversed the differentiation therapy effect of anlotinib, exacerbating the aggressiveness and reducing the differentiation level of neuroblastoma. These findings highlight the potential of anlotinib as an anti-neuroblastoma agent. It may suppress tumor proliferation and invasion by promoting the differentiation of tumor cells towards a neuronal-like state, and this differentiation therapy effect involves the inhibition of CRMP5 signaling.

Immunological isolation and characterization of neuronal progenitors from human dental pulp: A laboratory-based investigation.

Dental pulp stem cells (DPSCs) contain a population of stem cells with a broad range of differentiation potentials, as well as more lineage-committed progenitors. Such heterogeneity is a significant obstacle to experimental and clinical applications. The aim of this study is to isolate and characterize a homogenous neuronal progenitor cell population from human DPSCs. Polysialylated-neural cell adhesion molecule (PSA-NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic-activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non-neural markers was used to characterize the magnetically isolated PSA-NCAM+ fraction. PSA-NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic-AMP, neurotrophin-3, B27 and N2 supplements to induce neuronal differentiation. Both PSA-NCAM+ and differentiated PSA-NCAM+ cells were used in Ca2+ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization. PSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic-activated cell sorting and anti-PSA-NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA-NCAM+ cells. Immunofluorescent staining revealed expression of pan-neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP-induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast-voltage-responding fluorescent molecule, FluoVolt™ in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations. In summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural-like properties that can be effectively isolated with magnetic-activated cell sorting (MACS).

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

Effects of Anionic Liposome Delivery of All-Trans-Retinoic Acid on Neuroblastoma Cell Differentiation.

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-α-phosphatidylcholine (PC) and L-α-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.

Neuroendocrine modulation by metamizole and indomethacin: investigating the impact on neuronal markers and GnRH release.

Some evidence that non-steroidal anti-inflammatory drugs have neuroprotective effects indicates their potential for use in a new field. However, their effects on hormone secretion have yet to be adequately discovered. Therefore, we aimed to evaluate the effects of metamizole and indomethacin on neuronal markers as well as the GnRH expression in the GT1-7 cell line. The effects of these drugs on proliferation were evaluated by MTT analysis. The effect of 10-50-250 µM concentrations of the drugs also on the expression of neuronal factors and markers, including NGF, nestin and βIII Tubulin, and additionally GnRH, was determined by the RT-qPCR method. NGF and nestin mRNA expressions were increased in all concentrations of both metamizole and indomethacin. No changes were detected in βIII Tubulin. While metamizole showed an increase in GnRH mRNA expression, there was no change at 10 and 50 µM concentrations of indomethacin, but a remarkable decrease was observed at 250 µM concentrations. The results of our study showing an increase in the expression of neuronal factors reveal that metamizole and indomethacin may have possible neuroprotective effects. Moreover, the effects on the GnRH expression appear to be different. Animal models are required to confirm these effects of NSAIDs on neurons.

Morphological and electrophysiological characterization of a novel displaced astrocyte in the mouse retina.

Astrocytes throughout the central nervous system are heterogeneous in both structure and function. This diversity leads to tissue-specific specialization where morphology is adapted to the surrounding neuronal circuitry, as seen in Bergman glia of the cerebellum and Müller glia of the retina. Because morphology can be a differentiating factor for cellular classification, we recently developed a mouse where glial-fibrillary acidic protein (GFAP)-expressing cells stochastically label for full membranous morphology. Here we utilize this tool to investigate whether morphological and electrophysiological features separate types of mouse retinal astrocytes. In this work, we report on a novel glial population found in the inner plexiform layer and ganglion cell layer which expresses the canonical astrocyte markers GFAP, S100β, connexin-43, Sox2 and Sox9. Apart from their retinal layer localization, these cells are unique in their radial distribution. They are notably absent from the mid-retina but are heavily concentrated near the optic nerve head, and to a lesser degree the peripheral retina. Additionally, their morphology is distinct from both nerve fiber layer astrocytes and Müller glia, appearing more similar to amacrine cells. Despite this structural similarity, these cells lack protein expression of common neuronal markers. Additionally, they do not exhibit action potentials, but rather resemble astrocytes and Müller glia in their small amplitude, graded depolarization to both light onset and offset. Their structure, protein expression, physiology, and intercellular connections suggest that these cells are astrocytic, displaced from their counterparts in the nerve fiber layer. As such, we refer to these cells as displaced retinal astrocytes.

A Gelatin/Alginate Double Network Hydrogel Nerve Guidance Conduit Fabricated by a Chemical-Free Gamma Radiation for Peripheral Nerve Regeneration.

Nerve guidance conduits (NGCs) are widely developed using various materials for the functional repair of injured or diseased peripheral nerves. Especially, hydrogels are considered highly suitable for the fabrication of NGCs due to their beneficial tissue-mimicking characteristics (e.g., high water content, softness, and porosity). However, the practical applications of hydrogel-based NGCs are hindered due to their poor mechanical properties and complicated fabrication processes. To bridge this gap, a novel double-network (DN) hydrogel using alginate and gelatin by a two-step crosslinking process involving chemical-free gamma irradiation and ionic crosslinking, is developed. DN hydrogels (1% alginate and 15% gelatin), crosslinked with 30 kGy gamma irradiation and barium ions, exhibit substantially improved mechanical properties, including tensile strength, elastic modulus, and fracture stain, compared to single network (SN) gelatin hydrogels. Additionally, the DN hydrogel NGC exhibits excellent kink resistance, mechanical stability to successive compression, suture retention, and enzymatic degradability. In vivo studies with a sciatic defect rat model indicate substantially improved nerve function recovery with the DN hydrogel NGC compared to SN gelatin and commercial silicone NGCs, as confirm footprint analysis, electromyography, and muscle weight measurement. Histological examination reveals that, in the DN NGC group, the expression of Schwann cell and neuronal markers, myelin sheath, and exon diameter are superior to the other controls. Furthermore, the DN NGC group demonstrates increased muscle fiber formation and reduced fibrotic scarring. These findings suggest that the mechanically robust, degradable, and biocompatible DN hydrogel NGC can serve as a novel platform for peripheral nerve regeneration and other biomedical applications, such as implantable tissue constructs.

High OXPHOS efficiency in RA-FUdr-differentiated SH-SY5Y cells: involvement of cAMP signalling and respiratory supercomplexes

Neurons are highly dependent on mitochondria to meet their bioenergetic needs and understanding the metabolic changes during the differentiation process is crucial in the neurodegeneration context. Several in vitro approaches have been developed to study neuronal differentiation and bioenergetic changes. The human SH-SY5Y cell line is a widely used cellular model and several differentiation protocols have been developed to induce a neuron-like phenotype including retinoic acid (RA) treatment. In this work we obtained a homogeneous functional population of neuron-like cells by a two-step differentiation protocol in which SH-SY5Y cells were treated with RA plus the mitotic inhibitor 2-deoxy-5-fluorouridine (FUdr). RA-FUdr treatment induced a neuronal phenotype characterized by increased expression of neuronal markers and electrical properties specific to excitable cells. In addition, the RA-FUdr differentiated cells showed an enrichment of long chain and unsaturated fatty acids (FA) in the acyl chain composition of cardiolipin (CL) and the bioenergetic analysis evidences a high coupled and maximal respiration associated with high mitochondrial ATP levels. Our results suggest that the observed high oxidative phosphorylation (OXPHOS) capacity may be related to the activation of the cyclic adenosine monophosphate (cAMP) pathway and the assembly of respiratory supercomplexes (SCs), highlighting the change in mitochondrial phenotype during neuronal differentiation.