Neuronal Differentiation of Human Mesenchymal Stem Cells Using Exosomes Derived from Differentiating Neuronal Cells

Exosomes deliver functional proteins and genetic materials to neighboring cells, and have potential applications for tissue regeneration. One possible mechanism of exosome-promoted tissue regeneration is through the delivery of microRNA (miRNA). In this study, we hypothesized that exosomes derived from neuronal progenitor cells contain miRNAs that promote neuronal differentiation. We treated mesenchymal stem cells (MSCs) daily with exosomes derived from PC12 cells, a neuronal cell line, for 1 week. After the treatment with PC12-derived exosomes, MSCs developed neuron-like morphology, and gene and protein expressions of neuronal markers were upregulated. Microarray analysis showed that the expression of miR-125b, which is known to play a role in neuronal differentiation of stem cells, was much higher in PC12-derived exosomes than in exosomes from B16-F10 melanoma cells. These results suggest that the delivery of miRNAs contained in PC12-derived exosomes is a possible mechanism explaining the neuronal differentiation of MSC.     

神经型一氧化氮合酶的活性调控

一氧化氮是重要的信使分子,在生物体内参与众多生理及病理过程。生物体内存在着复杂的一氧化氮合酶活性调控机制以精确调控一氧化氮的生成。在神经系统中,一氧化氮主要由神经型一氧化氮合酶催化生成。神经型一氧化氮合酶的活性主要受到翻译后水平上钙离子和钙调蛋白的调控,其调控方式包括二聚化、多位点的磷酸化和去磷酸化,以及主要由PDZ结构域介导的蛋白质-蛋白质相互作用。一氧化氮本身对其合酶的活性具有负反馈调控作用。近年来的研究提示,细胞质膜上的脂筏微区在神经性一氧化氮合酶的活性调控中也起到重要的调节作用。     

The Influence of Synaptic Weight Distribution on Neuronal Population Dynamics

The manner in which different distributions of synaptic weights onto cortical neurons shape their spiking activity remains open. To characterize a homogeneous neuronal population, we use the master equation for generalized leaky integrate-and-fire neurons with shot-noise synapses. We develop fast semi-analytic numerical methods to solve this equation for either current or conductance synapses, with and without synaptic depression. We show that its solutions match simulations of equivalent neuronal networks better than those of the Fokker-Planck equation and we compute bounds on the network response to non-instantaneous synapses. We apply these methods to study different synaptic weight distributions in feed-forward networks. We characterize the synaptic amplitude distributions using a set of measures, called tail weight numbers, designed to quantify the preponderance of very strong synapses. Even if synaptic amplitude distributions are equated for both the total current and average synaptic weight, distributions with sparse but strong synapses produce higher responses for small inputs, leading to a larger operating range. Furthermore, despite their small number, such synapses enable the network to respond faster and with more stability in the face of external fluctuations.     

The Effect of Correlated Neuronal Firing and Neuronal Heterogeneity on Population Coding Accuracy in Guinea Pig Inferior Colliculus

It has been suggested that the considerable noise in single-cell responses to a stimulus can be overcome by pooling information from a large population. Theoretical studies indicated that correlations in trial-to-trial fluctuations in the responses of different neurons may limit the improvement due to pooling. Subsequent theoretical studies have suggested that inherent neuronal diversity, i.e., the heterogeneity of tuning curves and other response properties of neurons preferentially tuned to the same stimulus, can provide a means to overcome this limit. Here we study the effect of spike-count correlations and the inherent neuronal heterogeneity on the ability to extract information from large neural populations. We use electrophysiological data from the guinea pig Inferior-Colliculus to capture inherent neuronal heterogeneity and single cell statistics, and introduce response correlations artificially. To this end, we generate pseudo-population responses, based on single-cell recording of neurons responding to auditory stimuli with varying binaural correlations. Typically, when pseudo-populations are generated from single cell data, the responses within the population are statistically independent. As a result, the information content of the population will increase indefinitely with its size. In contrast, here we apply a simple algorithm that enables us to generate pseudo-population responses with variable spike-count correlations. This enables us to study the effect of neuronal correlations on the accuracy of conventional rate codes. We show that in a homogenous population, in the presence of even low-level correlations, information content is bounded. In contrast, utilizing a simple linear readout, that takes into account the natural heterogeneity, even of neurons preferentially tuned to the same stimulus, within the neural population, one can overcome the correlated noise and obtain a readout whose accuracy grows linearly with the size of the population.     

Effects of Erythropoietin on Neuronal Activity

Recently, erythropoietin (EPO) receptors and synthesis of EPO have been identified in the brain. To clarify the effects of EPO on neuronal cells, we investigated the effects of EPO on Ca2+ uptake, intracellular Ca2+ concentration, membrane potential, cell survival, release and biosynthesis of dopamine, and nitric oxide (NO) production in differentiated PC12 cells, which possess EPO receptors. EPO (10(-12)-10(-10) M) increased 45Ca2+ uptake and intracellular Ca2+ concentration in PC12 cells in a dose-related manner; these increases were inhibited by nicardipine (1 microM) or anti-EPO antibody (1:100 dilution). EPO induced membrane depolarization in PC12 cells. After a 5-day culture without serum and nerve growth factor (NGF), viable cell number decreased to 50% of that of the control cells cultured with serum and NGF. EPO (10(-13)-10(-10) M) increased the number of viable cells cultured without serum and NGF; this increase was blunted by nicardipine or anti-EPO antibody. Incubation with EPO (10(-13)-10(-10) M) stimulated mitogen-activated protein kinase activity in PC12 cells. EPO (10(-13)-10(-10) M) increased dopamine release from PC12 cells and tyrosine hydroxylase activity; these increases were sensitive to nicardipine or anti-EPO antibody. Following a 4-h incubation with EPO (10(-14)-10(-10) M), NO production was increased, which was blunted by nicardipine and anti-EPO antibody. In contrast, maximal NO synthase activity was not changed by EPO. These results suggest that EPO stimulates neuronal function and viability via activation of Ca2+ channels.     

The Role of Astrocytic Glycogen in Supporting the Energetics of Neuronal Activity

Energy homeostasis in the brain is maintained by oxidative metabolism of glucose, primarily to fulfil the energy demand associated with ionic movements in neurons and astrocytes. In this contribution we review the experimental evidence that grounds a specific role of glycogen metabolism in supporting the functional energetic needs of astrocytes during the removal of extracellular potassium. Based on theoretical considerations, we further discuss the hypothesis that the mobilization of glycogen in astrocytes serves the purpose to enhance the availability of glucose for neuronal glycolytic and oxidative metabolism at the onset of stimulation. Finally, we provide an evolutionary perspective for explaining the selection of glycogen as carbohydrate reserve in the energy-sensing machinery of cell metabolism.     

Emergence of Bursting Activity in Connected Neuronal Sub-Populations

Uniform and modular primary hippocampal cultures from embryonic rats were grown on commercially available micro-electrode arrays to investigate network activity with respect to development and integration of different neuronal populations. Modular networks consisting of two confined active and inter-connected sub-populations of neurons were realized by means of bi-compartmental polydimethylsiloxane structures. Spontaneous activity in both uniform and modular cultures was periodically monitored, from three up to eight weeks after plating. Compared to uniform cultures and despite lower cellular density, modular networks interestingly showed higher firing rates at earlier developmental stages, and network-wide firing and bursting statistics were less variable over time. Although globally less correlated than uniform cultures, modular networks exhibited also higher intra-cluster than inter-cluster correlations, thus demonstrating that segregation and integration of activity coexisted in this simple yet powerful in vitro model. Finally, the peculiar synchronized bursting activity shown by confined modular networks preferentially propagated within one of the two compartments (‘dominant’), even in cases of perfect balance of firing rate between the two sub-populations. This dominance was generally maintained during the entire monitored developmental frame, thus suggesting that the implementation of this hierarchy arose from early network development.     

Limited Clinical Utility of Non-invasive Prenatal Testing for Subchromosomal Abnormalities

The use of massively parallel sequencing of maternal cfDNA for non-invasive prenatal testing (NIPT) of aneuploidy is widely available. Recently, the scope of testing has increased to include selected subchromosomal abnormalities, but the number of samples reported has been small. We developed a calling pipeline based on a segmentation algorithm for the detection of these rearrangements in maternal plasma. The same read depth used in our standard pipeline for aneuploidy NIPT detected 15/18 (83%) samples with pathogenic rearrangements > 6 Mb but only 2/10 samples with rearrangements < 6 Mb, unless they were maternally inherited. There were two false-positive calls in 534 samples with no known subchromosomal abnormalities (specificity 99.6%). Using higher read depths, we detected 29/31 fetal subchromosomal abnormalities, including the three samples with maternally inherited microduplications. We conclude that test sensitivity is a function of the fetal fraction, read depth, and size of the fetal CNV and that at least one of the two false negatives is due to a low fetal fraction. The lack of an independent method for determining fetal fraction, especially for female fetuses, leads to uncertainty in test sensitivity, which currently has implications for this technique’s future as a clinical diagnostic test. Furthermore, to be effective, NIPT must be able to detect chromosomal rearrangements across the whole genome for a very low false-positive rate. Because standard NIPT can only detect the majority of larger (>6 Mb) chromosomal rearrangements and requires knowledge of fetal fraction, we consider that it is not yet ready for routine clinical implementation.     

A Refined Neuronal Population Measure of Visual Attention

Neurophysiological studies of cognitive mechanisms such as visual attention typically ignore trial-by-trial variability and instead report mean differences averaged across many trials. Advances in electrophysiology allow for the simultaneous recording of small populations of neurons, which may obviate the need for averaging activity over trials. We recently introduced a method called the attention axis that uses multi-electrode recordings to provide estimates of attentional state of behaving monkeys on individual trials. Here, we refine this method to eliminate problems that can cause bias in estimates of attentional state in certain scenarios. We demonstrate the sources of these problems using simulations and propose an amendment to the previous formulation that provides superior performance in trial-by-trial assessments of attentional state.     

Unique Responses of Differentiating Neuronal Growth Cones to Inhibitory Cues Presented by Oligodendrocytes

During central nervous system development, neurons differentiate distinct axonal and dendritic processes whose outgrowth is influenced by environmental cues. Given the known intrinsic differences between axons and dendrites and that little is known about the response of dendrites to inhibitory cues, we tested the hypothesis that outgrowth of differentiating axons and dendrites of hippocampal neurons is differentially influenced by inhibitory environmental cues. A sensitive growth cone behavior assay was used to assess responses of differentiating axonal and dendritic growth cones to oligodendrocytes and oligodendrocyte- derived, myelin-associated glycoprotein (MAG). We report that >90% of axonal growth cones collapsed after contact with oligodendrocytes. None of the encounters between differentiating, MAP-2 positive dendritic growth cones and oligodendrocytes resulted in growth cone collapse. The insensitivity of differentiating dendritic growth cones appears to be acquired since they develop from minor processes whose growth cones are inhibited (nearly 70% collapse) by contact with oligodendrocytes. Recombinant MAG(rMAG)-coated beads caused collapse of 72% of axonal growth cones but only 29% of differentiating dendritic growth cones. Unlike their response to contact with oligodendrocytes, few growth cones of minor processes were inhibited by rMAG-coated beads (20% collapsed). These results reveal the capability of differentiating growth cones of the same neuron to partition the complex molecular terrain they navigate by generating unique responses to particular inhibitory environmental cues.     

Population Status of Two Wedgefish Species in Western Indonesian Inner Waters,Inferred from Demographic Models with Limited Data

Journal of Ichthyology - Wedgefish (family Rhinidae) is a group of elasmobranchs that experience a global threat due to its highly valued fins. Similar condition happens to most species of...     

计算机模拟在野生动物种群遗传学研究中的应用

野生动物进化是遗传变异和环境变化相互作用的结果,其过程复杂、难以预测。进化过程包括基因突变、疾病、自然选择、空间扩散等自然因子,也包括驯化、栖息地破碎化、人工选择等人为干扰因子。计算机模拟能够揭示该复杂过程的历史特征,同时也能预测环境变化造成的遗传影响,正成为种群遗传学领域关注的研究方向。随着计算机模拟技术的发展,各种模拟软件层出不穷,功能全面且易操作,越来越多的计算机模拟软件被应用于野生动物的保护与管理。对计算机模拟应用于野生动物保护进行综述,总结了simu POP、ms、CDPOP、Sim Coal、Bayes SSC、DIYABC等近20种计算机模拟软件的功能,包括统计预测功能、统计推理功能和统计验证功能,并筛选了约20个典型的野生动物研究案例,展示了多达30个具体功能;同时,梳理了它们在推测生活史特征、推测进化历史特征、预测种群管理、预测遗传多样性受环境变化的影响、验证取样和验证统计方法等六大方面的适用性;从计算机模拟面临的挑战(准确率的监测)和机遇(多学科合作),对计算机模拟应用于未来野生动物保护提出了展望。     

Targeting Cdk5 Activity in Neuronal Degeneration and Regeneration

The major priming event in neurodegeneration is loss of neurons. Loss of neurons by apoptotic mechanisms is a theme for studies focused on determining therapeutic strategies. Neurons following an insult, activate a number of signal transduction pathways, of which, kinases are the leading members. Cyclin-dependent kinase 5 (Cdk5) is one of the kinases that have been linked to neurodegeneration. Cdk5 along with its principal activator p35 is involved in multiple cellular functions ranging from neuronal differentiation and migration to synaptic transmission. However, during neurotoxic stress, intracellular rise in Ca²⁺ activates calpain, which cleaves p35 to generate p25. The long half-life of Cdk5/p25 results in a hyperactive, aberrant Cdk5 that hyperphosphorylates Tau, neurofilament and other cytoskeletal proteins. These hyperphosphorylated cytoskeletal proteins set the groundwork to forming neurofibrillary tangles and aggregates of phosphorylated proteins, hallmarks of neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease and Amyotropic Lateral Sclerosis. Attempts to selectively target Cdk5/p25 activity without affecting Cdk5/p35 have been largely unsuccessful. A polypeptide inhibitor, CIP (Cdk5 inhibitory peptide), developed in our laboratory, successfully inhibits Cdk5/p25 activity in vitro, in cultured primary neurons, and is currently undergoing validation tests in mouse models of neurodegeneration. Here, we discuss the therapeutic potential of CIP in regenerating neurons that are exposed to neurodegenerative stimuli.     

Phospholipase D Activity of Rat Brain Neuronal Nuclei

Abstract: Phospholipase D activity of rat brain neuronal nuclei, measured with exogenous phosphatidylcholine as substrate, was characterized. The measured activity of neuronal nuclei was at least 36-fold greater than the activity in glia nuclei. The pH optimum was 6.5, and unsaturated but not saturated fatty acids stimulated the enzyme. The optimal concentration of sodium oleate for stimulation of the enzyme activity was 1.2 m M in the presence of 0.75 m M phosphatidylcholine. This phospholipase D activity was cation independent. In the absence of NaF, used as a phosphatidic acid phosphatase inhibitor, the principal product was diglyceride; whereas in the presence of NaF, the principal product was phosphatidic acid. The phospholipase D, in addition to having hydrolytic activity, was able to catalyze a transphosphatidylation reaction. Maximum phosphatidylethanol formation was seen with 0.2–0.3 M ethanol. GTPγS, ATPγS, BeF₂, AIF₃, phosphatidic acid, and phosphatidylethanol inhibited the neuronal nuclei phospholipase D activity. The addition of the cytosolic fraction of brain, liver, kidney, spleen, and heart to the incubation mixtures resulted in inhibition of the phospholipase D activity. Phospholipase D activity was detectable in nuclei prepared from rat kidney, spleen, heart, and liver.     

Differentiating Intracellular from Extracellular Alkaline Phosphatase Activity in Soil by Sonication

Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio  =  1/8 (w/v) and power density  =  15 watt ml^-1], the activity of alkaline phosphomonoesterase (phosphatase) in a Haplic Cambisol soil increased with sonication time in two distinct steps. A first plateau of enzyme activity was reached between 60 and 100 s, and a second higher plateau after 300 s. We also found that sonication for 100 s under optimal conditions activated most (about 80%) of the alkaline phosphatase that was added to an autoclaved soil, while total bacteria number was not affected. Sonication for 300 s reduced the total bacteria number by three orders of magnitude but had no further effects on enzyme activity. Our results indicate that the first plateau of alkaline phosphatase activity was derived from extracellular enzymes attached to soil particles, and the second plateau to the combination of extracellular and intracellular enzymes after cell lysis. We conclude that our adjusted sonication method may be an alternative to the currently used physiological and chloroform-fumigation methods for differentiating intracellular from extracellular phosphatase activity in soil. Further testing is needed to find out whether this holds for other soil types.     

The Concept of Data Utility in Health Risk Assessment: A Multi-Disciplinary Perspective

Human and ecological health risk assessments and the decisions that stem from them require the acquisition and analysis of data. In agencies that are responsible for health risk decision-making, data (and/or opinions/judgments) are obtained from sources such as scientific literature, analytical and process measurements, expert elicitation, inspection findings, and public and private research institutions. Although the particulars of conducting health risk assessments of given disciplines may be dramatically different, a common concern is the subjective nature of judging data utility. Often risk assessors are limited to available data that may not be completely appropriate to address the question being asked. Data utility refers to the ability of available data to support a risk-based decision for a particular risk assessment. This article familiarizes the audience with the concept of data utility and is intended to raise the awareness of data collectors (e.g., researchers), risk assessors, and risk managers to data utility issues in health risk assessments so data collection and use will be improved. In order to emphasize the cross-cutting nature of data utility, the discussion has not been organized into a classical partitioning of risk assessment concerns as being either human health- or ecological health-oriented, as per the U.S. Environmental Protection Agency's Superfund Program.     

Akt, a Target of Phosphatidylinositol 3-Kinase, Inhibits Apoptosis in a Differentiating Neuronal Cell Line

Phosphatidylinositol (PI) 3-kinase has been suggested to mediate cell survival. Consistent with this possibility, apoptosis of conditionally (simian virus 40 T^ts) immortalized rat hippocampal H19-7 neuronal cells was increased in response to wortmannin, an inhibitor of PI 3-kinase. Downstream effectors of PI 3-kinase include Rac1, protein kinase C, and the serine-threonine kinase Akt (protein kinase B). Here, we show that activation of Akt is one mechanism by which PI 3-kinase can mediate survival of H19-7 cells during serum deprivation or differentiation. While ectopic expression of wild-type Akt (c-Akt) does not significantly enhance survival in H19-7 cells, expression of activated forms of Akt (v-Akt or myristoylated Akt) results in enhanced survival which can be comparable to that conferred by Bcl-2. Conversely, expression of a dominant-negative mutant of Akt accelerates cell death upon serum deprivation or differentiation. Finally, the results indicate that Akt can transduce a survival signal for differentiating neuronal cells through a mechanism that is independent of induction of Bcl-2 or Bcl-x_L or inhibition of Jun kinase activity.