A Complex Interaction Between Reduced Reelin Expression and Prenatal Organophosphate Exposure Alters Neuronal Cell Morphology.

Genetic and environmental factors are both likely to contribute to neurodevelopmental disorders including schizophrenia, autism spectrum disorders, and major depressive disorders. Prior studies from our laboratory and others have demonstrated that the combinatorial effect of two factors-reduced expression of reelin protein and prenatal exposure to the organophosphate pesticide chlorpyrifos oxon-gives rise to acute biochemical effects and to morphological and behavioral phenotypes in adolescent and young adult mice. In the current study, we examine the consequences of these factors on reelin protein expression and neuronal cell morphology in adult mice. While the cell populations that express reelin in the adult brain appear unchanged in location and distribution, the levels of full length and cleaved reelin protein show persistent reductions following prenatal exposure to chlorpyrifos oxon. Cell positioning and organization in the hippocampus and cerebellum are largely normal in animals with either reduced reelin expression or prenatal exposure to chlorpyrifos oxon, but cellular complexity and dendritic spine organization is altered, with a skewed distribution of immature dendritic spines in adult animals. Paradoxically, combinatorial exposure to both factors appears to generate a rescue of the dendritic spine phenotypes, similar to the mitigation of behavioral and morphological changes observed in our prior study. Together, our observations support an interaction between reelin expression and chlorpyrifos oxon exposure that is not simply additive, suggesting a complex interplay between genetic and environmental factors in regulating brain morphology

Human Astrocytes Exhibit Tumor Microenvironment-, Age-, and Sex-Related Transcriptomic Signatures

: Astrocytes are critical for the development and function of synapses. There are notable species differences between human astrocytes and commonly used animal models. Yet, it is unclear whether astrocytic genes involved in synaptic function are stable or exhibit dynamic changes associated with disease states and age in humans, which is a barrier in understanding human astrocyte biology and its potential involvement in neurological diseases. To better understand the properties of human astrocytes, we acutely purified astrocytes from the cerebral cortices of over 40 humans across various ages, sexes, and disease states. We performed RNA sequencing to generate transcriptomic profiles of these astrocytes and identified genes associated with these biological variables. We found that human astrocytes in tumor-surrounding regions downregulate genes involved in synaptic function and sensing of signals in the microenvironment, suggesting involvement of peri-tumor astrocytes in tumor-associated neural circuit dysfunction. In aging, we also found downregulation of synaptic regulators and upregulation of markers of cytokine signaling, while in maturation we identified changes in ionic transport with implications for calcium signaling. In addition, we identified subtle sexual dimorphism in human cortical astrocytes, which has implications for observed sex differences across many neurological disorders. Overall, genes involved in synaptic function exhibit dynamic changes in the peritumor microenvironment and aging. This data provides powerful new insights into human astrocyte biology in several biologically relevant states, that will aid in generating novel testable hypotheses about homeostatic and reactive astrocytes in humans.SIGNIFICANCE STATEMENTAstrocytes are an abundant class of cells playing integral roles at synapses. Astrocyte dysfunction is implicated in a variety of human neurological diseases. Yet our knowledge of astrocytes is largely based on mouse studies. Direct knowledge of human astrocyte biology remains limited. Here, we present transcriptomic profiles of human cortical astrocytes, and we identified molecular differences associated with age, sex, and disease state. We found that peritumor and aging astrocytes downregulate genes involved in astrocyte-synapse interactions. These data provide necessary insight into human astrocyte biology that will improve our understanding of human disease

Mechanisms of olfactory ensheathing cell-enhanced neurite outgrowth and axon regeneration after spinal cord injury

Olfactory ensheathing cells (OECs) provide a pro-regenerative environment for the axons of olfactory receptor neurons and therefore are a promising candidate for cell transplantation therapy following spinal cord injury. We previously showed that OEC transplantation supports axon regeneration and functional re-connectivity following complete spinal cord injury, yet lack of an OEC-specific marker limited our ability to determine how they promoted these beneficial effects. Using both in vitro and in vivo models, we investigated the mechanisms by which OECs mediate axon regeneration. OECs enhance neurite outgrowth of postnatal cortical neurons in a scar-like culture model. We provide strong evidence that direct OEC-neurite alignment is critical to enhance neurite outgrowth in scar-like astrocyte and meningeal fibroblast inhibitory environments. We also tested eGFP-OECs from transgenic rats and showed that they facilitate neurite outgrowth in vitro. Then in a short-term study, we analyzed OEC survival, migration, and distribution within the lesion site of complete spinal cord transected rats. We found that rats transplanted with OECs preserve and associate with axons and neurons in the lesion core, reduce the presence of inhibitory CSPGs and myelin debris, and reduce secondary tissue damage due to microglial and macrophage activation and infiltration post-injury. Collectively, these data support a neuroprotective and proregenerative role of OECs through the modulation of glial scar formation following a complete spinal cord transection

Reelin expression and secretion by olfactory ensheathing cells

Olfactory ensheathing cells (OECs) contribute to axon guidance and fascicle organization during the cyclical degeneration and regeneration in the olfactory system throughout life. OECs also support neurite outgrowth in vitro and axon regeneration following spinal cord injury. Reelin, a large extracellular matrix protein, is responsible for proper neuronal positioning of migrating neurons in the neocortex and spinal cord of the developing nervous system. Because OECs share phenotypic characteristics with Schwann cells and the absence of Reelin may impair peripheral nerve regeneration (Lorenzetto et al., 2008; Vincent et al., 2005), we asked if OECs express Reelin to mediate neural repair after injury. Here we show that olfactory bulb OECs produce and secrete Reelin. We also determined that Reelin secretion is a potential mechanism by which OECs can mediate process outgrowth. OEC-secreted Reelin did not enhance neurite outgrowth of postnatal cerebral cortical neurons, but did mediate enhanced outgrowth of DRG axons

Olfactory Ensheathing Cells Express α7 Integrin to Mediate Their Migration on Laminin.

The unique glia located in the olfactory system, called olfactory ensheathing cells (OECs), are implicated as an attractive choice for transplantation therapy following spinal cord injury because of their pro-regenerative characteristics. Adult OECs are thought to improve functional recovery and regeneration after injury by secreting neurotrophic factors and making cell-to-cell contacts with regenerating processes, but the mechanisms are not well understood. We show first that α7 integrin, a laminin receptor, is highly expressed at the protein level by OECs throughout the olfactory system, i.e., in the olfactory mucosa, olfactory nerve, and olfactory nerve layer of the olfactory bulb. Then we asked if OECs use the α7 integrin receptor directly to promote neurite outgrowth on permissive and neutral substrates, in vitro. We co-cultured α7+/+ and α7lacZ/lacZ postnatal cerebral cortical neurons with α7+/+ or α7lacZ/lacZ OECs and found that genotype did not effect the ability of OECs to enhance neurite outgrowth by direct contact. Loss of α7 integrin did however significantly decrease the motility of adult OECs in transwell experiments. Twice as many α7+/+ OECs migrated through laminin-coated transwells compared to α7+/+ OECs on poly-L-lysine (PLL). This is in contrast to α7lacZ/lacZ OECs, which showed no migratory preference for laminin substrate over PLL. These results demonstrate that OECs express α7 integrin, and that laminin and its α7 integrin receptor contribute to adult OEC migration in vitro and perhaps also in vivo

Expression of α7 integrin and β-gal in the olfactory nerve.

<p>A-C: Anti-α7 integrin (ITGA7, red) and Hoechst nuclear stain (blue) show the presence of α7 protein in <i>α7</i>^<i>+/+</i> (A) but not <i>α7</i>^{<i>lacZ/lacZ</i>} (B) olfactory nerve (ON, arrowheads), olfactory nerve layer (ONL) and glomerular layer (GL). Adjacent <i>α7</i>^{<i>lacZ/lacZ</i>} section (C) has <i>α</i>7/β-gal reactivity (green) in the ON and ONL (arrowheads). D-F: OECs in the ON are marked with arrowheads and show antibody expression in <i>α7</i>^<i>+/+</i> (D) or <i>α</i>7/β-gal in <i>α7</i>^{<i>lacZ/lacZ</i>} nerve (F). The <i>α7</i>^{<i>lacZ/lacZ</i>} negative control shows only Hoechst-labeled nuclei (E). Scale bar A-C: 100 μm; D-F: 20 μm.</p

α7 integrin does not facilitate OEC-induced neurite outgrowth.

<p>A-H: Representative images from the experimental groups. A-D show cultures seeded with <i>α7</i>^<i>+/+</i> cortical neurons, whereas E-H contained <i>α7</i>^{<i>lacZ/lacZ</i>} neurons. Neurons were cultured on laminin (A, E), PLL (B, F), PLL + <i>α7</i>^<i>+/+</i> OECs (C, G), and PLL + <i>α7</i>^{<i>lacZ/lacZ</i>} OECs (D, H). Neurons were visualized with β3-tubulin, and OECs marked with Cell Tracker Green. I: Total neurite outgrowth per well was normalized to culture-matched outgrowth on PLL (normalized mean ± SEM is plotted; See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153394#pone.0153394.s002" target="_blank">S1 Table</a> for raw data). Neuronal genotype did not affect neurite outgrowth on laminin or PLL (n = 3, <i>p</i> = 0.838). The laminin substrate induced more neurite growth than PLL (1.99 ± 0.07 units normalized; ***<i>p</i><0.0001), and OECs enhanced neurite outgrowth lengths at a level that did not differ from laminin. The addition of OECs significantly increased the neuronal outgrowth compared to PLL only levels (<i>α7</i>^<i>+/+</i> OECS: 1.60 ± 0.11 **<i>p</i> = 0.0003; <i>α7</i>^{<i>lacZ/lacZ</i>} OECs: 1.71 ± 0.14, ***<i>p</i><0.0001). J: For neuron-OEC co-cultures we sorted individual neurite lengths by the type of association they made with OECs (i.e., aligned, cross, no contact) and averaged them. The lengths of neurites that aligned with OECs were compared to neurites grown on laminin, PLL, and different OEC associations. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153394#pone.0153394.s003" target="_blank">S2 Table</a> for averages, further description, and <i>p</i>-values). Scale bar A-H: 50 μm.</p

OEC-rich areas of the olfactory system express β-galactosidase (β-gal) in <i>α7</i>^{<i>lacZ/+</i>} and <i>α7</i>^{<i>lacZ/lacZ</i>} mice.

<p>A: In the central (olfactory bulb, OB) and peripheral (nasal cavity, NC) olfactory areas, sagittal sections from <i>α7</i>^{<b><i>+/+</i></b>} mice have no β-gal reaction. B, C: Mice with one (<i>α7</i>^{<i>lacZ/+</i>}, B) or two copies (<i>α7</i>^{<i>lacZ/lacZ</i>}, C) of the <i>α7</i>^<i>lacZ</i> allele have similar patterns of β-gal histochemistry. <i>α</i>7/β-gal is within the olfactory nerve layer (ONL) and the lamina propria (LP), two areas heavily populated with OECs. Blood vessels also express <i>α</i>7/β-gal. D, E: Arrows point to the β-gal-labeled olfactory nerve (cranial nerve I) as it courses through the cribriform plate (C) that separates the nasal cavity from the olfactory bulbs. F: A horizontal section of <i>α7</i>^{<i>lacZ/lacZ</i>} olfactory mucosa shows intense β-gal expression in the LP (arrows) and light reactivity in the olfactory epithelium (OE). Scale bars for A-C: 200 μm; D-E: 50 μm; F: 100 μm.</p