Neuronal Cell Lineages Research Articles

NEUROD1: transcriptional and epigenetic regulator of human and mouse neuronal and endocrine cell lineage programs.

Transcription factors belonging to the basic helix-loop-helix (bHLH) family are key regulators of cell fate specification and differentiation during development. Their dysregulation is implicated not only in developmental abnormalities but also in various adult diseases and cancers. Recently, the abilities of bHLH factors have been exploited in reprogramming strategies for cell replacement therapy. One such factor is NEUROD1, which has been associated with the reprogramming of the epigenetic landscape and potentially possessing pioneer factor abilities, initiating neuronal developmental programs, and enforcing pancreatic endocrine differentiation. The review aims to consolidate current knowledge on NEUROD1's multifaceted roles and mechanistic pathways in human and mousecell differentiation and reprogramming, exploring NEUROD1 roles in guiding the development and reprogramming of neuroendocrine cell lineages. The review focuses on NEUROD1's molecular mechanisms, its interactions with other transcription factors, its role as a pioneer factor in chromatin remodeling, and its potential in cell reprogramming. We also show a differential potential of NEUROD1 in differentiation of neurons and pancreatic endocrine cells, highlighting its therapeutic potential and the necessity for further research to fully understand and utilize its capabilities.

Multi-omics technologies and molecular biomarkers in brain tumor-related epilepsy.

Brain tumors are one of the leading causes of epilepsy, and brain tumor-related epilepsy (BTRE) is recognized as the major cause of intractable epilepsy, resulting in huge treatment cost and burden to patients, their families, and society. Although optimal treatment regimens are available, the majority of patients with BTRE show poor resolution of symptoms. BTRE has a very complex and multifactorial etiology, which includes several influencing factors such as genetic and molecular biomarkers. Advances in multi-omics technologies have enabled to elucidate the pathophysiological mechanisms and related biomarkers of BTRE. Here, we reviewed multi-omics technology-based research studies on BTRE published in the last few decades and discussed the present status, development, opportunities, challenges, and prospects in treating BTRE. First, we provided a general review of epilepsy, BTRE, and multi-omics techniques. Next, we described the specific multi-omics (including genomics, transcriptomics, epigenomics, proteomics, and metabolomics) techniques and related molecular biomarkers for BTRE. We then presented the associated pathogenetic mechanisms of BTRE. Finally, we discussed the development and application of novel omics techniques for diagnosing and treating BTRE. Genomics studies have shown that the BRAF gene plays a role in BTRE development. Furthermore, the BRAF V600E variant was found to induce epileptogenesis in the neuronal cell lineage and tumorigenesis in the glial cell lineage. Several genomics studies have linked IDH variants with glioma-related epilepsy, and the overproduction of D2HG is considered to play a role in neuronal excitation that leads to seizure occurrence. The high expression level of Forkhead Box O4 (FOXO4) was associated with a reduced risk of epilepsy occurrence. In transcriptomics studies, VLGR1 was noted as a biomarker of epileptic onset in patients. Several miRNAs such as miR-128 and miRNA-196b participate in BTRE development. miR-128 might be negatively associated with the possibility of tumor-related epilepsy development. The lncRNA UBE2R2-AS1 inhibits the growth and invasion of glioma cells and promotes apoptosis. Quantitative proteomics has been used to determine dynamic changes of protein acetylation in epileptic and non-epileptic gliomas. In another proteomics study, a high expression of AQP-4 was detected in the brain of GBM patients with seizures. By using quantitative RT-PCR and immunohistochemistry assay, a study revealed that patients with astrocytomas and oligoastrocytomas showed high BCL2A1 expression and poor seizure control. By performing immunohistochemistry, several studies have reported the relationship between D2HG overproduction and seizure occurrence. Ki-67 overexpression in WHO grade II gliomas was found to be associated with poor postoperative seizure control. According to metabolomics research, the PI3K/AKT/mTOR pathway is associated with the development of glioma-related epileptogenesis. Another metabolomics study found that SV2A, P-gb, and CAD65/67 have the potential to function as biomarkers for BTRE. Based on the synthesized information, this review provided new research perspectives and insights into the early diagnosis, etiological factors, and personalized treatment of BTRE.

Biomarkers in Alzheimer's Disease: Are Olfactory Neuronal Precursors Useful for Antemortem Biomarker Research?

Alzheimer's disease (AD), as the main cause of dementia, affects millions of people around the world, whose diagnosis is based mainly on clinical criteria. Unfortunately, the diagnosis is obtained very late, when the neurodegenerative damage is significant for most patients. Therefore, the exhaustive study of biomarkers is indispensable for diagnostic, prognostic, and even follow-up support. AD is a multifactorial disease, and knowing its underlying pathological mechanisms is crucial to propose new and valuable biomarkers. In this review, we summarize some of the main biomarkers described in AD, which have been evaluated mainly by imaging studies in cerebrospinal fluid and blood samples. Furthermore, we describe and propose neuronal precursors derived from the olfactory neuroepithelium as a potential resource to evaluate some of the widely known biomarkers of AD and to gear toward searching for new biomarkers. These neuronal lineage cells, which can be obtained directly from patients through a non-invasive and outpatient procedure, display several characteristics that validate them as a surrogate model to study the central nervous system, allowing the analysis of AD pathophysiological processes. Moreover, the ease of obtaining and harvesting endows them as an accessible and powerful resource to evaluate biomarkers in clinical practice.

Sex linked behavioral and hippocampal transcriptomic changes in mice with cell-type specific Egr1 loss

The transcription factor EGR1 is instrumental in numerous neurological processes, encompassing learning and memory as well as the reaction to stress. Egr1 complete knockout mice demonstrate decreased depressive or anxiety-like behavior and impaired performance in spatial learning and memory. Nevertheless, the specific functions of Egr1 in distinct cell types have been largely underexplored. In this study, we cataloged the behavioral and transcriptomic character of Nestin-Cre mediated Egr1 conditional knockout (Egr1cKO) mice together with their controls. Although the conditional knockout did not change nociceptive or anxiety responses, it triggered changes in female exploratory activity during anxiety testing. Hippocampus-dependent spatial learning in the object location task was unaffected, but female Egr1cKO mice did exhibit poorer retention during testing on a contextual fear conditioning task compared to males. RNA-seq data analyses revealed that the presence of the floxed Egr1 cassette or Nestin-Cre driver alone exerts a subtle influence on hippocampal gene expression. The sex-related differences were amplified in Nestin-Cre mediated Egr1 conditional knockout mice and female mice are more sensitive to the loss of Egr1 gene. Differentially expressed genes resulted from the loss of Egr1 in neuronal cell lineage were significantly associated with the regulation of Wnt signaling pathway, extracellular matrix, and axon guidance. Altogether, our results demonstrate that Nestin-Cre and the loss of Egr1 in neuronal cell lineage have distinct impacts on hippocampal gene expression in a sex-specific manner.

Nose-to-brain translocation and nervous system injury in response to indium tin oxide nanoparticles of long-term low-dose exposures

Indium tin oxide (ITO) is a semiconductor nanomaterial with broad application in liquid crystal displays, solar cells, and electrochemical immune sensors. It is worth noting that, with the gradual increase in worker exposure opportunities, the exposure risk in occupational production cannot be ignored. At present, the toxicity of ITO mainly focuses on respiratory toxicity. ITO inhaled through the upper respiratory tract can cause pathological changes such as interstitial pneumonia and pulmonary fibrosis. Still, extrapulmonary toxicity after nanoscale ITO nanoparticle (ITO NPs) exposure, such as long-term effects on the central nervous system, should also be of concern. Therefore, we set up exposure dose experiments (0 mg·kg−1, 3.6 mg·kg−1, and 36 mg·kg−1) based on occupational exposure limits to treat C57BL/6 mice via nasal drops for 15 weeks. Moreover, we conducted a preliminary assessment of the neurotoxicity of ITO NPs (20–30 nm) in vivo. The results indicated that ITO NPs can cause diffuse inflammatory infiltrates in brain tissue, increased glial cell responsiveness, abnormal neuronal cell lineage transition, neuronal migration disorders, and neuronal apoptosis related to the oxidative stress induced by ITO NPs exposure. Hence, our findings provide useful information for the fuller risk assessment of ITO NPs after occupational exposure.

Contemporary enterovirus-D68 isolates infect human spinal cord organoids.

Enterovirus D68 (EV-D68) is a nonpolio enterovirus associated with severe respiratory illness and acute flaccid myelitis (AFM), a polio-like illness causing paralysis in children. AFM outbreaks have been associated with increased circulation and genetic diversity of EV-D68 since 2014, although the virus was discovered in the 1960s. The mechanisms by which EV-D68 targets the central nervous system are unknown. Since enteroviruses are human pathogens that do not routinely infect other animal species, establishment of a human model of the central nervous system is essential for understanding pathogenesis. Here, we describe two human spinal cord organoid (hSCO)-based models for EV-D68 infection derived from induced, pluripotent stem cell (iPSC) lines. One hSCO model consists primarily of spinal motor neurons, while the another model comprises multiple neuronal cell lineages, including motor neurons, interneurons, and glial cells. These hSCOs can be productively infected with contemporary strains, but not a historic strain, of EV-D68 and produce extracellular virus for at least 2 weeks without appreciable cytopathic effect. By comparison, infection with hSCO with another enterovirus, echovirus 11, causes significant structural destruction and apoptosis. Together, these findings suggest that EV-D68 infection is not the sole mediator of neuronal cell death in the spinal cord in those with AFM and that secondary injury from the immune response likely contributes to pathogenesis. IMPORTANCE AFM is a rare condition that causes significant morbidity in affected children, often contributing to life-long sequelae. It is unknown how EV-D68 causes paralysis in children, and effective therapeutic and preventative strategies are not available. Mice are not native hosts for EV-D68, and thus, existing mouse models use immunosuppressed or neonatal mice, mouse-adapted viruses, or intracranial inoculations. To complement existing models, we report two hSCO models for EV-D68 infection. These three-dimensional, multicellular models comprised human cells and include multiple neural lineages, including motor neurons, interneurons, and glial cells. These new hSCO models for EV-D68 infection will contribute to understanding how EV-D68 damages the human spinal cord, which could lead to new therapeutic and prophylactic strategies for this virus.

Sex-Linked Growth Disorder and Aberrant Pituitary Gene Expression in Nestin-Cre-Mediated Egr1 Conditional Knockout Mice.

Genes that regulate hormone release are essential for maintaining metabolism and energy balance. Egr1 encodes a transcription factor that regulates hormone production and release, and a decreased in growth hormones has been reported in Egr1 knockout mice. A reduction in growth hormones has also been observed in Nestin-Cre mice, a model frequently used to study the nervous system. Currently, it is unknown how Egr1 loss or the Nestin-Cre driver disrupt pituitary gene expression. Here, we compared the growth curves and pituitary gene expression profiles of Nestin-Cre-mediated Egr1 conditional knockout (Egr1cKO) mice with those of their controls. Reduced body weight was observed in both the Nestin-Cre and Egr1cKO mice, and the loss of Egr1 had a slightly more severe impact on female mice than on male mice. RNA-seq data analyses revealed that the sex-related differences were amplified in the Nestin-Cre-mediated Egr1 conditional knockout mice. Additionally, in the male mice, the influence of Egr1cKO on pituitary gene expression may be overridden by the Nestin-Cre driver. Differentially expressed genes associated with the Nestin-Cre driver were significantly enriched for genes related to growth factor activity and binding. Altogether, our results demonstrate that Nestin-Cre and the loss of Egr1 in the neuronal cell lineage have distinct impacts on pituitary gene expression in a sex-specific manner.

Neurogenic Stem Cell Niche in the Auditory Thalamus: In Vitro Evidence of Neural Stem Cells in the Rat Medial Geniculate Body.

The medial geniculate body (MGB) is a nucleus of the diencephalon representing a relevant segment of the auditory pathway and is part of the metathalamus. It receives afferent information via the inferior brachium of the inferior colliculus and transmits efferent fibers via acoustic radiations to the auditory cortex. Neural stem cells (NSCs) have been detected in certain areas along the auditory pathway. They are of great importance as the induction of an adult stem cell niche might open a regenerative approach to a causal treatment of hearing disorders. Up to now, the existence of NSCs in the MGB has not been determined. Therefore, this study investigated whether the MGB has a neural stem cell potential. For this purpose, cells were extracted from the MGB of PND 8 Sprague-Dawley rats and cultured in a free-floating cell culture assay, which showed mitotic activity and positive staining for stem cell and progenitor markers. In differentiation assays, the markers β-III-tubulin, GFAP, and MBP demonstrated the capacity of single cells to differentiate into neuronal and glial cells. In conclusion, cells from the MGB exhibited the cardinal features of NSCs: self-renewal, the formation of progenitor cells, and differentiation into all neuronal lineage cells. These findings may contribute to a better understanding of the development of the auditory pathway.

BMPRII+ neural precursor cells isolated and characterized from organotypic neurospheres: an in vitro model of human fetal spinal cord development.

Roof plate secretion of bone morphogenetic proteins (BMPs) directs the cellular fate of sensory neurons during spinal cord development, including the formation of the ascending sensory columns, though their biology is not well understood. Type-II BMP receptor (BMPRII), the cognate receptor, is expressed by neural precursor cells during embryogenesis; however, an in vitro method of enriching BMPRII+ human neural precursor cells (hNPCs) from the fetal spinal cord is absent. Immunofluorescence was undertaken on intact second-trimester human fetal spinal cord using antibodies to BMPRII and leukemia inhibitory factor (LIF). Regions of highest BMPRII+ immunofluorescence localized to sensory columns. Parenchymal and meningeal-associated BMPRII+ vascular cells were identified in both intact fetal spinal cord and cortex by co-positivity with vascular lineage markers, CD34/CD39. LIF immunostaining identified a population of somas concentrated in dorsal and ventral horn interneurons, mirroring the expression of LIF receptor/CD118. A combination of LIF supplementation and high-density culture maintained culture growth beyond 10 passages, while synergistically increasing the proportion of neurospheres with a stratified, cytoarchitecture. These neurospheres were characterized by BMPRII+/MAP2ab+/-/βIII-tubulin+/nestin-/vimentin-/GFAP-/NeuN- surface hNPCs surrounding a heterogeneous core of βIII-tubulin+/nestin+/vimentin+/GFAP+/MAP2ab-/NeuN- multipotent precursors. Dissociated cultures from tripotential neurospheres contained neuronal (βIII-tubulin+), astrocytic (GFAP+), and oligodendrocytic (O4+) lineage cells. Fluorescence-activated cell sorting-sorted BMPRII+ hNPCs were MAP2ab+/-/βIII-tubulin+/GFAP-/O4- in culture. This is the first isolation of BMPRII+ hNPCs identified and characterized in human fetal spinal cords. Our data show that LIF combines synergistically with high-density reaggregate cultures to support the organotypic reorganization of neurospheres, characterized by surface BMPRII+ hNPCs. Our study has provided a new methodology for an in vitro model capable of amplifying human fetal spinal cord cell numbers for > 10 passages. Investigations of the role BMPRII plays in spinal cord development have primarily relied upon mouse and rat models, with interpolations to human development being derived through inference. Because of significant species differences between murine biology and human, including anatomical dissimilarities in central nervous system (CNS) structure, the findings made in murine models cannot be presumed to apply to human spinal cord development. For these reasons, our human in vitro model offers a novel tool to better understand neurodevelopmental pathways, including BMP signaling, as well as spinal cord injury research and testing drug therapies.

Neuronal differentiation and functional maturation of neurons from neural stem cells induced by bFGF-chitosan controlled release system.

Available methods for differentiating stem cells into neurons require a large number of cytokines and neurotrophic factors, with complex steps and slow processes, and are inefficient to produce functional neurons and form synaptic contacts, which is expensive and impractical in clinical application. Here, we demonstrated a bioactive material, basic fibroblast growth factor (bFGF)-chitosan controlled release system, for facilitating neuronal differentiation from NSCs and the functional maturation of the induced neurons with high efficiency. We illustrated by immunostaining that the neurons derived from NSCs expressed mature immunomarkers of interneurons and excitatory neurons. And we found by patch-clamp that the induced neurons exhibited diverse electrophysiological properties as well as formed functional synapses. In vivo, we implanted bFGF-chitosan into lesion area in traumatic brain injury (TBI) mice and similarly observed abundance of neuroblasts in SVZ and the presence of newborn functional neurons in injury area, which integrated into synaptic networks. Taken together, our efficient and rapid tissue engineering approach may be a potential method for the generation of functional neuronal lineage cells from stem cells and a therapy of brain injury and disease.

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor.

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor. In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

Repeated administration of acrylamide for 28 days suppresses adult neurogenesis of the olfactory bulb in young-adult rats

Acrylamide (AA) is a neurotoxicant that inhibits synaptic function in distal axons. We previously found that AA decreased neural cell lineages during late-stage differentiation of adult hippocampal neurogenesis and downregulated genes related to neurotrophic factor, neuronal migration, neurite outgrowth, and synapse formation in the hippocampal dentate gyrus in rats. To investigate whether olfactory bulb (OB)-subventricular zone (SVZ) neurogenesis is similarly affected by AA exposure, AA was administered to 7-week-old male rats via oral gavage at doses of 0, 5, 10, and 20 mg/kg for 28 days. Immunohistochemical analysis revealed that AA decreased the numbers of doublecortin-positive (+) cells and polysialic acid-neural cell adhesion molecule+ cells in the OB. On the other hand, the numbers of doublecortin+ cells and polysialic acid-neural cell adhesion molecule+ cells in the SVZ did not change with AA exposure, suggesting that AA impaired neuroblasts migrating in the rostral migratory stream and OB. Gene expression analysis in the OB revealed that AA downregulated Bdnf and Ncam2, which are related to neuronal differentiation and migration. These results suggest that AA decreased neuroblasts in the OB by suppressing neuronal migration. Thus, AA decreased neuronal cell lineages during late-stage differentiation of adult neurogenesis in the OB-SVZ, similar to the effect on adult hippocampal neurogenesis.

Instant formation of horizontally ordered nanofibrous hydrogel films and direct investigation of peculiar neuronal cell behaviors atop

BackgroundHydrogels have been widely used in many research fields owing to optical transparency, good biocompatibility, tunable mechanical properties, etc. Unlike typical hydrogels in the form of an unstructured bulk material, we developed aqueous dispersions of fiber-shaped hydrogel structures with high stability under ambient conditions and their application to various types of transparent soft cell culture interfaces with anisotropic nanoscale topography.MethodNanofibers based on the polyvinyl alcohol and polyacrylic acid mixture were prepared by electrospinning and hydrogelified to nano-fibrous hydrogels (nFHs) after thermal crosslinking and sulfuric acid treatment. By modifying various material surfaces with positively-charged polymers, negatively-charged superabsorbent nFHs could be selectively patterned by employing micro-contact printing or horizontally aligned by applying shear force with a wired bar coater.ResultsThe angular distribution of bar-coated nFHs was dramatically reduced to ± 20° along the applied shear direction unlike the drop-coated nFHs which exhibit random orientations. Next, various types of cells were cultured on top of transparent soft nFHs which showed good viability and attachment while their behaviors could be easily monitored by both upright and inverted optical microscopy. Particularly, neuronal lineage cells such as PC 12 cells and embryonic hippocampal neurons showed highly stretched morphology along the overall fiber orientation with aspect ratios ranging from 1 to 14. Furthermore, the resultant neurite outgrowth and migration behaviors could be effectively controlled by the horizontal orientation and the three-dimensional arrangement of underlying nFHs, respectively.ConclusionWe expect that surface modifications with transparent soft nFHs will be beneficial for various biological/biomedical studies such as fundamental cellular studies, neuronal/stem cell and/or organoid cultures, implantable probe/device coatings, etc.

Hypoxia Induces Early Neurogenesis in Human Fetal Neural Stem Cells by Activating the WNT Pathway.

Fetal neural stem cells (FNSCs) present in the human fetal brain differentiate into cells of neuronal and glial lineages. The developing fetus is exposed to lower oxygen concentrations than adults, and this physiological hypoxia may influence the growth and differentiation of the FNSCs. This study aimed to evaluate the effect of hypoxia on the differentiation potential of human FNSCs isolated from the subventricular zone of aborted fetal brains (n = 5). FNSCs were isolated, expanded, and characterized by Nestin and Sox2 expression using immunocytochemistry and flow cytometry, respectively. These FNSCs were exposed to 20% oxygen (normoxia) and 0.2% oxygen (hypoxia) concentrations for 48h, and hypoxia exposure (n = 5) was validated. Whole transcriptome analyses (Genespring GX13) of FNSCs exposed to hypoxia (Agilent 4 × 44K human array slides) highlighted that genes associated with neurogenesis were enriched upon exposure to hypoxia. The pathway analysis of these enriched genes (using Metacore) showed the involvement of the WNT signaling pathway. Microarray analyses were validated using neuronal and glial lineage commitment markers, namely, NEUROG1, NEUROG2, ASCL1, DCX, GFAP, OLIG2, and NKX2.2, using qPCR (n = 9). DCX, ASCL1, NGN1, and GFAP protein expression was analyzed by Western blotting (n = 3). This demonstrated upregulation of the neuronal commitment markers upon hypoxia exposure, while no change was observed in astrocytic and oligodendrocyte lineage commitment markers. Increased expression of downstream targets of the WNT signaling pathway, TCF4 and ID2, by qPCR (n = 9) and increased protein expression of CTNNB1 (β-catenin) and ID2 by Western blot (n = 3) indicated its involvement in mediating neuronal differentiation upon exposure to hypoxia.

RNA Helicase DDX6 Regulates A-to-I Editing and Neuronal Differentiation in Human Cells

The DEAD-box proteins, one family of RNA-binding proteins (RBPs), participate in post-transcriptional regulation of gene expression with multiple aspects. Among them, DDX6 is an essential component of the cytoplasmic RNA processing body (P-body) and is involved in translational repression, miRNA-meditated gene silencing, and RNA decay. In addition to the cytoplasmic function, DDX6 is also present in the nucleus, but the nuclear function remains unknown. To decipher the potential role of DDX6 in the nucleus, we performed mass spectrometry analysis of immunoprecipitated DDX6 from a HeLa nuclear extract. We found that adenosine deaminases that act on RNA 1 (ADAR1) interact with DDX6 in the nucleus. Utilizing our newly developed dual-fluorescence reporter assay, we elucidated the DDX6 function as negative regulators in cellular ADAR1p110 and ADAR2. In addition, depletion of DDX6 and ADARs results in the opposite effect on facilitation of RA-induced differentiation of neuronal lineage cells. Our data suggest the impact of DDX6 in regulation of the cellular RNA editing level, thus contributing to differentiation in the neuronal cell model.

Actin rings form plasma membrane diffusion barriers.

In a fluid system like the plasma membrane, molecules will not enrich spontaneously. Since a high local concentration of specific molecules is vital for many membrane protein functions, a domain enriched in these molecules must be formed. This can be achieved by the generation of a physical diffusion barrier to the lateral movement of membrane components. One such barrier exists in the axon initial segment (AIS) of neurons, but the mechanism of the barrier so far remained elusive. Two candidates have emerged to be the cause of the membrane compartmentalization in the AIS: periodic actin rings (fence model) or large immobile transmembrane voltage-gated sodium channels (picket model). Here, we show that the diffusion barrier in the AIS is caused by the actin cytoskeleton. To do so, we combined in silico experiments with high-speed, high-density single-particle tracking and superresolution microscopy. In silico single-particle tracking (SPT) experiments clearly showed that the compartmentalization is compatible with the fence model but incompatible with the picket model. Furthermore, we show via SPT of membrane proteins in progenitor-derived neuronal cells that the diffusion barrier is also present in cells that do not exhibit an ion-channel enriched AIS, but do show actin rings along their processes. Further experiments using dual-colour live-cell stimulated emission depletion microscopy (STED) showed that periodic actin rings in cells are near (20 nm) the plasma membrane. Lastly, we show via correlative SPT/STORM that membrane molecules accumulate between actin rings. Taken together, we present here evidence for the compartmentalization of the plasma membrane by the actin cytoskeleton. Membrane proximal actin structures are a common feature of many cell types. Therefore, we speculate that the observed membrane compartmentalization is not specific to the neuronal cell lineage but rather a general mechanism of large-scale membrane organization.

NPS-Crosslinked Fibrin Gels Load with EMSCs to Repair Peripheral Nerve Injury in Rats.

Neural tissue engineering has been introduced as a novel therapeutic strategy for trauma-induced sciatic nerve defects. Here, a neuropeptide S (NPS)-crosslinked fibrin scaffolds (NPS@Fg) loaded with an ectomesenchymal stem cell (EMSC) system to bridge an 8-mm sciatic nerve defect in rats are reported. The Schwann cell-like and neural differentiation of the EMSCs on the engineered fibrin scaffolds are also assessed in vitro. These results show that the NPS@Fg promotes the differentiation of EMSCs into neuronal lineage cells, which may also contribute to the therapeutic outcome of the NPS@Fg+EMSCs strategy. After transplantation NPS@Fg+EMSCs into sciatic nerve defects in rats, nerve recovery is assessed up to 12 weeks postinjury. In vivo experiments show that the combination of NPS crosslinked fibrin scaffolds with EMSCs can significantly accelerate nerve healing and improve morphological repair. In the study, NPS@Fg+EMSCs may represent a new potential strategy for peripheral nerve reconstruction.

Single-cell transcriptomic analysis reveals the adverse effects of cadmium on the trajectory of neuronal maturation.

Cadmium (Cd) is an extensively existing environmental pollutant that has neurotoxic effects. However, the molecular mechanism of Cd on neuronal maturation is unveiled. Single-cell RNA sequencing (scRNA-seq) has been widely used to uncover cellular heterogeneity and is a powerful tool to reconstruct the developmental trajectory of neurons. In this study, neural stem cells (NSCs) from subventricular zone (SVZ) of newborn mice were treated with CdCl2 for 24h and differentiated for 7days to obtain neuronal lineage cells. Then scRNA-seq analysis identified five cell stages with different maturity in neuronal lineage cells. Our findings revealed that Cd altered the trajectory of maturation of neuronal lineage cells by decreasing the number of cells in different stages and hindering their maturation. Cd induced differential transcriptome expression in different cell subpopulations in a stage-specific manner. Specifically, Cd induced oxidative damage and changed the proportion of cell cycle phases in the early stage of neuronal development. Furthermore, the autocrine and paracrine signals of Wnt5a were downregulated in the low mature neurons in response to Cd. Importantly, activation of Wnt5a effectively rescued the number of neurons and promoted their maturation. Taken together, the findings of this study provide new and comprehensive insights into the adverse effect of Cd on neuronal maturation.

Reduced Expression of Prion Protein With Increased Interferon-β Fail to Limit Creutzfeldt-Jakob Disease Agent Replication in Differentiating Neuronal Cells.

Immortalized uninfected septal (SEP) neurons proliferate but after physiological mitotic arrest they express differentiated neuronal characteristics including enhanced cell-to-cell membrane contacts and ≥ 8 fold increases in host prion protein (PrP). We compared proliferating uninfected and Creutzfeldt-Jakob Disease (CJD) agent infected cells with their arrested counterparts over 33 days by quantitative mRNA and protein blot analyses. Surprisingly, uninfected arrested cells increased interferon-β (IFN-β) mRNA by 2.5–8 fold; IFN-β mRNA elevations were not previously associated with neuronal differentiation. SEP cells with high CJD infectivity titers produced a much larger 40–68-fold increase in IFN-β mRNA, a classic host anti-viral response that is virucidal for RNA but not DNA viruses. High titers of CJD agent also induced dramatic decreases in host PrP, a protein needed for productive agent replication. Uninfected arrested cells produced large sustained 20–30-fold increases in PrP mRNA and protein, whereas CJD arrested cells showed only transient small 5-fold increases in PrP. A > 10-fold increase in infectivity, but not PrP misfolding, induced host PrP reductions that can limit CJD agent replication. In contrast to neuronal lineage cells, functionally distinct migratory microglia with high titers of CJD agent do not induce an IFN-β mRNA response. Because they have 1/50th of PrP of an average brain cell, microglia would be unable to produce the many new infectious particles needed to induce a large IFN-β response by host cells. Instead, microglia and related cells can be persistent reservoirs of infection and spread. Phase separations of agent-associated molecules in neurons, microglia and other cell types can yield new insights into the molecular structure, persistent, and evasive behavior of CJD-type agents.

Transplanted neural lineage cells derived from dental pulp stem cells promote peripheral nerve regeneration.

Cell therapy for peripheral nerve injury is a promising strategy as regenerative medicine that restores neurological function. However, challenges remain in producing suitable and sufficient amounts of autologous cells for promoting nerve regeneration. This study aimed to identify the characteristics of neural lineage cells (NLCs) differentiated from dental pulp stem cells (DPSCs) and reveal their effect on functional recovery and nerve regeneration after cell transplantation into an immunodeficient rat using a nerve guide conduit. Here we report a protocol of neural induction in monolayer culture and characterize NLCs in vitro. Furthermore, NLCs were transplanted into an immunodeficient rat model with a 10-mm sciatic nerve defect, and cell survival and differentiation were investigated in vivo. Outcomes of nerve regeneration were also assessed using the remyelinated axon numbers, myelin sheath thickness, electrophysiological activities, and gastrocnemius muscle mass. NLCs comprised neuronal, astrocyte, oligodendrocyte, and neural crest lineage cells. NLCs enhanced the activities of endothelial cells, Schwann cells, and neurons in a paracrine-dependent manner in vitro. At 2weeks post-transplantation, numerous transplanted NLCs differentiated into platelet-derived growth factor receptor alpha (PDGFRα) + oligodendrocyte progenitor cells (OPCs) and a few PDGFRα + /p75 neurotrophin receptor + Schwann cell-like cells derived from OPCs were observed. At 12weeks post-transplantation, human Schwann cell-like cells survived, and axon growth, remyelination, electrophysiological activities, and muscle atrophy were improved. This study demonstrates the broad application of our protocol of neural induction of DPSCs and portrays the efficacy of transplantation of NLCs derived from human DPSCs as a promising strategy for peripheral nerve regeneration.