p75ECD-Fc reverses neonatal hypoxic-ischemic encephalopathy-induced neurological deficits and inhibits apoptosis associated with Nestin.

A recent study has introduced a recombinant fusion protein, consisting of the extracellular domain (ECD) of p75 and the Fc fragment of human immunoglobulin IgG1 (p75ECD-Fc), as a multifaceted agent within the nervous system. This research aimed to assess the effects of p75ECD-Fc on neuronal growth and the restoration of neurological functions in rats afflicted with neonatal hypoxic-ischemic encephalopathy (NHIE). In vitro analyses revealed that 1 µM p75ECD-Fc treatment markedly increased cell viability and facilitated neurite outgrowth in neurons exposed to oxygen-glucose deprivation (OGD). Subsequent in vivo studies determined that a dose of 78.6 µg/3 µl of p75ECD-Fc significantly mitigated brain damage and both acute and long-term neurological impairments, outperforming the therapeutic efficacy of hypothermia, as evidenced through behavioral assessments. Additionally, in vivo immunostaining showed that p75ECD-Fc administration enhanced neuronal survival and regeneration, and reduced astrocytosis and microglia activation in the cortex and hippocampus of NHIE rats. A noteworthy shift from A1 to A2 astrocyte phenotypes and from M1 to M2 microglia phenotypes was observed after p75ECD-Fc treatment. Furthermore, a co-expression of the p75 neurotrophin receptor (p75NTR) and Nestin was identified, with an overexpression of Nestin alleviating the neurological dysfunction induced by NHIE. Mechanistically, the neuroprotective effects of p75ECD-Fc, particularly its inhibition of neuronal apoptosis post-OGD, may be attributed to Nestin. Taken together, these results highlight the neuroprotective and anti-inflammatory effects of p75ECD-Fc treatment through the modulation of glial cell phenotypes and the Nestin-mediated inhibition of neuronal apoptosis, positioning it as a viable therapeutic approach for NHIE.

Corrigendum: MicroRNA339 Targeting PDXK Improves Motor Dysfunction and Promotes Neurite Growth in the Remote Cortex Subjected to Spinal Cord Transection.

[This corrects the article DOI: 10.3389/fcell.2020.00577.].

Treadmill training improves cognitive function by increasing IGF2 targeted downregulation of miRNA-483.

Optimal exercise can promote the development of cognitive functions. Nevertheless, mechanisms that elicit these positive effects of exercise still need to be elucidated. Insulin-like growth factor 2 (IGF2) is known to act as a potent enhancer of memory and cognitive functions, whereas the mechanism by which IGF2 regulates cognitive functions in terms of moderate treadmill exercise remains largely vague. In the study, rats were subjected to low-, moderate-, and high-intensity treadmill training for 6 weeks. Then, the Morris water maze test was used to investigate spatial learning and memory ability in rats subjected to treadmill exercises of different intensities. Subsequently, gene chip and bioinformatics analyses were used to explore IGF2 and predict target microRNAs (miRNAs). Quantitative real-time polymerase chain reaction, western blot, and immunofluorescence analysis were performed to detect the levels of IGF2. Furthermore, IGF2-small interfering RNA, the miRNA-483-mimic, and the miRNA-483-inhibitor were transfected to determine the role of IGF2 and miRNA-483 in the growth of hippocampal neurons. The results of the Morris water maze test showed that moderate-intensity treadmill training enhanced cognitive functions; meanwhile, the expression of IGF2 was significantly upregulated in the hippocampus after moderate-intensity treadmill exercise. From databases, miRNA-483 was screened and predicted as the target gene of IGF2. Moreover, silencing IGF2 inhibited neurite growth in the hippocampus of rats, the miRNA-483-inhibitor ameliorated silencing IGF2 induced impairment of hippocampal neurons. These findings suggested that treadmill training could enhance cognitive functions, wherein the underlying mechanism involved an increase in the expression of IGF2 and downregulation of miRNA-483.

A New Approach to Model Sporadic Alzheimer's Disease by Intracerebroventricular Streptozotocin Injection in APP/PS1 Mice.

Alzheimer's disease (AD) is the most common cause of dementia among elderly people. Majority of AD cases are sporadic (SAD) with unknown cause. Transgenic animal models closely reflect the familial (genetic) aspect of the disease but not the sporadic type. However, most new drug candidates which are tested positive in transgenic animal models failed in clinical studies so far. Herein, we aim to develop an AD animal model that combines most of the neuropathological features seen in sporadic AD in humans with amyloid plaques observed in transgenic mice. Four-month-old wild-type and APP/PS1 AD mice were given a single intracerebroventricular (ICV) injection of 3 mg/kg streptozotocin (STZ), a diabetogenic agent. Three weeks later, their cognitive behavior was assessed, and their brain tissues were collected for biochemical and histological analysis. STZ produced cognitive deficits in both non-transgenic mice and AD mice. Biochemical analysis showed a severe decline in synaptic proteins, increase in tau phosphorylation, oxidative stress, disturbed brain insulin signaling with extensive neuroinflammation, and cell death. Significant increase was also observed in the level of the soluble beta amyloid precursor protein (APP) fragments and robust accumulation of amyloid plaques in AD mice compared to the control. These results suggest that STZ ICV treatment causes disturbance in multiple metabolic and cell signaling pathways in the brain that facilitated amyloid plaque accumulation and tau phosphorylation. Therefore, this animal model can be used to evaluate new AD therapeutic agents for clinical translation.

Effect of Sutellarin on Neurogenesis in Neonatal Hypoxia-Ischemia Rat Model: Potential Mechanisms of Action.

To investigate the therapeutic efficacy of Scutellarin (SCU) on neurite growth and neurological functional recovery in neonatal hypoxic-ischemic (HI) rats. Primary cortical neurons were cultured to detect the effect of SCU on cell viability of neurons under oxygen-glucose deprivation (OGD). Double immunofluorescence staining of Tuj1 and TUNEL then observed the neurite growth and cell apoptosis in vitro,and double immunofluorescence staining of NEUN and TUNEL was performed to examine the neuronal apoptosis and cell apoptosis in brain tissues after HI in vivo. Pharmacological efficacy of SCU was also evaluated in HI rats by neurobehavioral tests, triphenyl tetrazolium chloride staining, Hematoxylin and eosin staining and Nissl staining. Astrocytes and microglia expression in damaged brain tissues were detected by immunostaining of GFAP and Iba1. A quantitative real-time polymerase chain reaction and western blot were applied to investigate the genetic expression changes and the protein levels of autophagy-related proteins in the injured cortex and hippocampus after HI. We found that SCU administration preserved cell viability, promoted neurite outgrowth and suppressed apoptosis of neurons subjected to OGD both in vitroand in vivo. Meanwhile, 20 mg/kg SCU treatment improved neurological functions and decreased the expression of astrocytes and microglia in the cortex and hippocampus of HI rats. Additionally, SCU treatment depressed the elevated levels of autophagy-related proteins and the p75 neurotrophin receptor (p75NTR) in both cortex and hippocampus. This study demonstrated the potential therapeutic efficacy of SCU by enhancing neurogenesis and restoring long-term neurological dysfunctions, which might be associated with p75NTR depletion in HI rats.

Urine stem cells are equipped to provide B cell survival signals.

The interplay between mesenchymal stem cells (MSCs) and immune cells has been studied for MSCs isolated from different tissues. However, the immunomodulatory capacity of urine stem cells (USCs) has not been adequately researched. The present study reports on the effect of USCs on peripheral blood lymphocytes. USCs were isolated and characterized before coculture with resting and with anti-CD3/CD28 bead stimulated lymphocytes. Similarly to bone marrow mesenchymal stem cells (BM-MSCs), USCs inhibited the proliferation of activated T lymphocytes and induced their apoptosis. However, they also induced strong activation, proliferation, and cytokine and antibody production by B lymphocytes. Molecular phenotype and supernatant analysis revealed that USCs secrete a range of cytokines and effector molecules, known to play a central role in B cell biology. These included B cell-activating factor (BAFF), interleukin 6 (IL-6) and CD40L. These findings raise the possibility of an unrecognized active role for kidney stem cells in modulating local immune cells.

Characterization of Urine Stem Cell-Derived Extracellular Vesicles Reveals B Cell Stimulating Cargo.

Elucidation of the biological functions of extracellular vesicles (EVs) and their potential roles in physiological and pathological processes is an expanding field of research. In this study, we characterized USC-derived EVs and studied their capacity to modulate the human immune response in vitro. We found that the USC-derived EVs are a heterogeneous population, ranging in size from that of micro-vesicles (150 nm-1 µm) down to that of exosomes (60-150 nm). Regarding their immunomodulatory functions, we found that upon isolation, the EVs (60-150 nm) induced B cell proliferation and IgM antibody secretion. Analysis of the EV contents unexpectedly revealed the presence of BAFF, APRIL, IL-6, and CD40L, all known to play a central role in B cell stimulation, differentiation, and humoral immunity. In regard to their effect on T cell functions, they resembled the function of mesenchymal stem cell (MSC)-derived EVs previously described, suppressing T cell response to activation. The finding that USC-derived EVs transport a potent bioactive cargo opens the door to a novel therapeutic avenue for boosting B cell responses in immunodeficiency or cancer.

MicroRNA339 Targeting PDXK Improves Motor Dysfunction and Promotes Neurite Growth in the Remote Cortex Subjected to Spinal Cord Transection.

Spinal cord injury (SCI) is a fatal disease that can cause severe disability. Cortical reorganization subserved the recovery of spontaneous function after SCI, although the potential molecular mechanism in this remote control is largely unknown. Therefore, using proteomics analysis, RNA interference/overexpression, and CRISPR/Cas9 in vivo and in vitro, we analyzed how the molecular network functions in neurological improvement, especially in the recovery of motor function after spinal cord transection (SCT) via the remote regulation of cerebral cortex. We discovered that the overexpression of pyridoxal kinase (PDXK) in the motor cortex enhanced neuronal growth and survival and improved locomotor function in the hindlimb. In addition, PDXK was confirmed as a target of miR-339 but not miR-124. MiR-339 knockout (KO) significantly increased the neurite outgrowth and decreased cell apoptosis in cortical neurons. Moreover, miR-339 KO rats exhibited functional recovery indicated by improved Basso, Beattie, and Bresnehan (BBB) score. Furthermore, bioinformatics prediction showed that PDXK was associated with GAP43, a crucial molecule related to neurite growth and functional improvement. The current research therefore confirmed that miR-339 targeting PDXK facilitated neurological recovery in the motor cortex of SCT rats, and the underlying mechanism was associated with regulating GAP43 in the remote cortex of rats subjected to SCT. These findings may uncover a new understanding of remoting cortex control following SCI and provide a new therapeutic strategy for the recovery of SCI in future clinical trials.

COX5A over-expression protects cortical neurons from hypoxic ischemic injury in neonatal rats associated with TPI up-regulation.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy (HIE) represents as a major cause of neonatal morbidity and mortality. However, the underlying molecular mechanisms in brain damage are still not fully elucidated. This study was conducted to determine the specific potential molecular mechanism in the hypoxic-ischemic induced cerebral injury. METHODS: Here, hypoxic-ischemic (HI) animal models were established and primary cortical neurons were subjected to oxygen-glucose deprivation (OGD) to mimic HIE model in vivo and in vitro. The HI-induced neurological injury was evaluated by Zea-longa scores, Triphenyte-trazoliumchloride (TTC) staining the Terminal Deoxynucleotidyl Transferased Utp Nick End Labeling (TUNEL) and immunofluorescent staining. Then the expression of Cytochrome c oxidase subunit 5a (COX5A) was determined by immunohistochemistry, western blotting (WB) and quantitative real time Polymerase Chain Reaction (qRT-PCR) techniques. Moreover, HSV-mediated COX5A over-expression virus was transducted into OGD neurons to explore the role of COX5A in vitro, and the underlying mechanism was predicted by GeneMANIA, then verified by WB and qRT-PCR. RESULTS: HI induced a severe neurological dysfunction, brain infarction, and cell apoptosis as well as obvious neuron loss in neonatal rats, in corresponding to the decrease on the expression of COX5A in both sides of the brain. What's more, COX5A over-expression significantly promoted the neuronal survival, reduced the apoptosis rate, and markedly increased the neurites length after OGD. Moreover, Triosephosephate isomerase (TPI) was predicted as physical interactions with COX5A, and COX5A over-expression largely increased the expressional level of TPI. CONCLUSIONS: The present findings suggest that COX5A plays an important role in promoting neurological recovery after HI, and this process is related to TPI up-regulation.

Pro-BDNF Knockout Causes Abnormal Motor Behaviours and Early Death in Mice.

Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family, best characterized for its survival and differentiative effects in the central nervous system. Pro-BDNF, known as the precursor of BDNF, is believed to have opposite functions to mature BDNF (mBDNF). The opposing effects of Pro-BDNF and mBDNF have led researchers to propose a 'yin' (Pro-BDNF) and 'yang' (mBDNF) model of which, the specific mechanism of its opposing functions is unclear and requires further investigation. In order to elucidate pro-BDNF's explicit role, we established a pro-BDNF knockout (KO) mouse model. This BDNF pro-domain KO mouse model showed significant weight loss, impaired righting reflex, abnormal motor behaviours and short lifespan (less than 22 days), mimicking a Huntington's disease (HD)-like phenotype. ELISA results showed BDNF pro-domain KO not only blocked pro-BDNF, but also significantly affected the level of mBDNF. Abnormal morphologic changes were found in the dentate gyrus (DG) of the hippocampus in pro-BDNF KO mice, and western blot confirmed significant cell apoptosis in pro-BDNF KO mice brains. Furthermore, the expression of glutamic acid decarboxylase 65/67 (GAD65/67) was significantly reduced in pro-BDNF KO mice, indicating impaired inhibitory neurotransmission. Heterozygous (Het) mice showed impaired learning and memory capability and depressive-like behaviours, compared with wild type (WT) mice. Overall, these results support that pro-domain of BDNF is an indispensable part of the BDNF gene; without the proper formation of pro-BDNF, mBDNF cannot be produced successfully and function correctly on its own. Our study also supports the BDNF hypothesis in the pathogenesis of HD.

DPYSL2 is a novel regulator for neural stem cell differentiation in rats: revealed by Panax notoginseng saponin administration.

BACKGROUND: The limited neuronal differentiation of the endogenous or grafted neural stem cells (NSCs) after brain injury hampers the clinic usage of NSCs. Panax notoginseng saponins (PNS) were extensively used for their clinical value, such as in controlling blood pressure, blood glucose, and inhibiting neuronal apoptosis and enhancing neuronal protection, but whether or not it exerts an effect in promoting neuronal differentiation of the endogenous NSCs is completely unclear and the potential underlying mechanism requires further exploration. METHODS: Firstly, we determined whether PNS could successfully induce NSCs to differentiate to neurons under the serum condition. Mass spectrometry and quantitative polymerase chain reaction (Q-PCR) were then performed to screen the differentially expressed proteins (genes) between the PNS + serum and serum control group, upon which dihydropyrimidinase-like 2 (DPYSL2), a possible candidate, was then selected for the subsequent research. To further investigate the actual role of DPYSL2 in the NSC differentiation, DPYSL2-expressing lentivirus was employed to obtain DPYSL2 overexpression in NSCs. DPYSL2-knockout rats were constructed to study its effects on hippocampal neural stem cells. Immunofluorescent staining was performed to identify the differentiation direction of NSCs after 7 days from DPYSL2 transfection, as well as those from DPYSL2-knockout rats. RESULTS: Seven differentially expressed protein spots were detected by PD Quest, and DPYSL2 was found as one of the key factors of NSC differentiation in a PNS-treated condition. The results of immunostaining further showed that mainly Tuj1 and GFAP-positive cells increased in the DPYSL2-overexpressed group, while both were depressed in the hippocampal NSCs in the DPYSL2-knockout rat. CONCLUSIONS: The present study revealed that the differentiation direction of NSCs could be enhanced through PNS administration, and the DPYSL2 is a key regulator in promoting NSC differentiation. These results not only emphasized the effect of PNS but also indicated DPYSL2 could be a novel target to enhance the NSC differentiation in future clinical trials.

Conversion of human urine-derived cells into neuron-like cells by small molecules.

Neural cell transplantation is an effective way for treatment of neurological diseases. However, the absence of transplantable human neurons remains a barrier for clinical therapies. Human urine-derived cells, namely renal cells and urine stem cells, have become a good source of cells for reprogramming or trans-differentiation research. Here, we show that human urine-derived cells can be partially converted into neuron-like cells by applying a cocktail of small molecules. Gene expression analysis has shown that these induced cells expressed some neuron-specific genes, and a proportion of the cells are GABAergic neurons. Moreover, whole-cell patch clamping recording has shown that some induced cells have neuron-specific voltage gated Na+ and K+ currents but have failed to generate Ca2+ currents and action potentials. Taken together, these results suggest that induced neuronal cells from human urine-derived cells may be useful for neurological disease modelling, drug screening and cell therapies.

Coating Materials for Neural Stem/Progenitor Cell Culture and Differentiation.

Neural stem/progenitor cells (NSPCs) have a potential to treat various neurological diseases, such as Parkinson's Disease, Alzheimer's Disease, and Spinal Cord Injury. However, the limitation of NSPC sources and the difficulty to maintain their stemness or to differentiate them into specific therapeutic cells are the main hurdles for clinical research and application. Thus, for obtaining a therapeutically relevant number of NSPCs in vitro, it is important to understand factors regulating their behaviors and to establish a protocol for stable NSPC proliferation and differentiation. Coating materials for cell culture, such as Matrigel, laminin, collagen, and other coating materials, can significantly affect NSPC characteristics. This article provides a review of coating materials for NSPC culturing in both two dimensions and three dimensions, and their functions in NSPC proliferation and differentiation, and presents a useful guide to select coating materials for researchers.

Overexpression of miR-124 Protects Against Neurological Dysfunction Induced by Neonatal Hypoxic-Ischemic Brain Injury.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a major cause of lifelong disabilities worldwide, without effective therapies and clear regulatory mechanisms. MicroRNAs (miRNAs) act as a significant regulator in neuroregeneration and neuronal apoptosis, thus holding great potential as therapeutic targets in HIE. In this study, we established the hypoxia-ischemia (HI) model in vivo and oxygen-glucose deprivation (OGD) model in vitro. Zea-longa score and magnetic resonance imaging were applied to verify HI-induced neuronal dysfunction and brain infarction. Subsequently, a miRNA microarray analysis was employed to profile miRNA transcriptomes. Down-regulated miR-124 was found 24 h after HIE, which corresponded to the change in PC12, SHSY5Y, and neurons after OGD. To determine the function of miR-124, mimics and lentivirus-mediated overexpression were used to regulate miR-124 in vivo and in vitro, respectively. Our results showed that miR-124 overexpression obviously promoted cell survival and suppressed neuronal apoptosis. Further, the memory and neurological function of rats was also obviously improved at 1 and 2 months after HI, indicated by the neurological severity score, Y-maze test, open field test, and rotating rod test. Our findings showed that overexpression of miR-124 can be a promising new strategy for HIE therapy in future clinical practice.

Single-nucleotide polymorphism screening and RNA sequencing of key messenger RNAs associated with neonatal hypoxic-ischemia brain damage.

A single-nucleotide polymorphism (SNP) is an alteration in one nucleotide in a certain position within a genome. SNPs are associated with disease susceptibility. However, the influences of SNPs on the pathogenesis of neonatal hypoxic-ischemic brain damage remain elusive. Seven-day-old rats were used to establish a hypoxic ischemic encephalopathy model. SNPs and expression profiles of mRNAs were analyzed in hypoxic ischemic encephalopathy model rats using RNA sequencing. Genes exhibiting SNPs associated with hypoxic ischemic encephalopathy were identified and studied by gene ontology and pathway analysis to identify their possible involvement in the disease mechanism. We identified 89 up-regulated genes containing SNPs that were mainly located on chromosome 1 and 2. Gene ontology analysis indicated that the up-regulated genes containing SNPs are mainly involved in angiogenesis, wound healing and glutamatergic synapse and biological processing of calcium-activated chloride channels. Signaling pathway analysis indicated that the differentially expressed genes play a role in glutamatergic synapses, long-term depression and oxytocin signaling. Moreover, intersection analysis of high throughput screening following PubMed retrieval and RNA sequencing for SNPs showed that CSRNP1, DUSP5 and LRRC25 were most relevant to hypoxic ischemic encephalopathy. Significant up-regulation of genes was confirmed by quantitative real-time polymerase chain reaction analysis of oxygen-glucose-deprived human fetal cortical neurons. Our results indicate that CSRNP1, DUSP5 and LRRC25, containing SNPs, may be involved in the pathogenesis of hypoxic ischemic encephalopathy. These findings indicate a novel direction for further hypoxic ischemic encephalopathy research. This animal study was approved on February 5, 2017 by the Animal Care and Use Committee of Kunming Medical University, Yunnan Province, China (approval No. kmmu2019038). Cerebral tissue collection from a human fetus was approved on September 30, 2015 by the Ethics Committee of Kunming Medical University, China (approval No. 2015-9).

Knockout of p75 neurotrophin receptor attenuates the hyperphosphorylation of Tau in pR5 mouse model.

p75 neurotrophin receptor (p75NTR) has been implicated in Alzheimer's disease (AD). However, whether p75NTR is involved in Tau hyperphosphorylation, one of the pathologies observed in AD, remains unclear. In our previous study, the extracellular domain of p75NTR blocked amyloid beta (Aß) toxicity and attenuated Aß-induced Tau hyperphosphorylation. Here we show that, in the absence of Aß, p75NTR regulates Tau phosphorylation in the transgenic mice with the P301L human Tau mutation (pR5). The knockout of p75NTR in pR5 mice attenuated the phosphorylation of human Tau. In addition, the elevated activity of kinases responsible for Tau phosphorylation including glycogen synthase kinase 3 beta; cyclin-dependent-kinase 5; and Rho-associated protein kinase was also inhibited when p75NTR is knocked out in pR5 mice at 9 months of age. The increased caspase-3 activity observed in pR5 mice was also abolished in the absence of p75NTR. Our study also showed that p75NTR is required for Aß- and pro-brain derived neurotrophin factor (proBDNF)-induced Tau phosphorylation, in vitro. Overall, our data indicate that p75NTR is required for Tau phosphorylation, a key event in the formation of neurofibrillary tangles, another hallmark of AD. Thus, targeting p75NTR could reduce or prevent the pathologic hyperphosphorylation of Tau.

A Novel Role of VEGFC in Cerebral Ischemia With Lung Injury.

Cerebral ischemia (CI) is a severe brain injury resulting in a variety of motor impairments combined with secondary injury in remote organs, especially the lung. This condition occurs due to insufficient blood supply to the brain during infancy. However, it has a molecular linkage that needs to be thoroughly covered. Here, we report on the role of vascular endothelial growth factor C (VEGFC) in lung injury induced by CI. The middle cerebral artery occlusion (MCAO) was depended to establish the animal model of CI. Rats were used and brain ischemia was confirmed through TTC staining. Serum was used for protein chip analysis to study the proteomic interaction. Immunohistochemistry analyses were used to quantify and locate the VEGFC in the lung and brain. The role of VEGFC was detected by siVEGFC technology in SY5Y, HUCEV, and A549 cell lines, under normal and oxygen glucose deprivation (OGD) conditions in vitro. As a result, the TTC staining demonstrated that the model of brain ischemia was successfully established, and MPO experiments reported that lung damage was induced in MCAO rats. VEGFC levels were up-regulated in serum. On the other hand, immunohistochemistry showed that VEGFC increased significantly in the cytoplasm of neurons, the endothelium of small trachea and the lung cells of CI animals. On a functional level, siVEGFC effectively inhibited the proliferation of SY5Y cells and decreased the viability of HUVEC cells in normal cell lines. But under OGD conditions, siVEGFC decreased the growth of HUVEC and increased the viability of A549 cells, while no effect was noticed on SYSY cells. Therefore, we confirmed the different role of VEGFC played in neurons and lung cells in cerebral ischemia-reperfusion injury. These findings may contribute to the understanding the molecular linkage of brain ischemia and lung injury, which therefore provides a new idea for the therapeutic approach to cerebral ischemia-reperfusion.