Michael W. Weible

In sympathetic but not sensory neurones, phosphoinositide-3 kinase is important for NGF-dependent survival and the retrograde transport of 125I-βNGF

The way in which the same ligands and receptors have different functional effects in different cell types must depend on subtle differences in the second messenger cascades. Sensory and sympathetic neurones both retrogradely transport nerve growth factor (NGF) and depend on NGF for their developmental survival. NGF binding to the high affinity tyrosine kinase (TrkA) receptors initiates second messenger signalling cascades, one of which includes the activation of phosphoinositide-3 kinase (PI3-kinase). We demonstrate that 100-fold higher concentrations of the PI3-kinase inhibitor, Wortmannin, are required to inhibit the survival effects and retrograde axonal transport of NGF in sensory neurones than in sympathetic neurones. Similarly, although less potently than Wortmannin, the PI3-kinase inhibitor LY294002 required a 10-fold higher concentration to inhibit the survival effects of NGF in sensory than in sympathetic neurones. Inhibitors of other second messengers, including staurosporine, pertussis and cholera toxins, failed to have an effect on the transport of the NGF receptor complex in both cell types. Also, Wortmannin did not affect the structural integrity of the sympathetic nerve terminals. As PI3-kinase is present in both neuronal populations, this suggests that the Wortmannin sensitive isoform of PI3-kinase (p110) is essential in sympathetic neurones both for survival and for NGF-TrkA receptor complex trafficking. As sensory neurones also depend on NGF for their developmental survival and endocytose and retrogradely transport the NGF-TrkA receptor complex, this population of neurones may either recruit a different isoform of PI3-kinase or utilize PI3-kinase independent signalling pathways for these cellular functions.

In vivo characterization of astrocyte precursor cells (APCs) and astrocytes in developing rat retinae: Differentiation, proliferation, and apoptosis

To characterize the timing of differentiation, antigenic expression, morphology, proliferative potential, and apoptosis during astrocyte differentiation in the rat retina in vivo.

Phenotypic characterization of neural stem cells from human fetal spinal cord: Synergistic effect of LIF and BMP4 to generate astrocytes

If cell based therapy for spinal cord injury is to become a reality, greater insights into the biology of human derived spinal cord stem cells are a prerequisite. Significant species differences and regional specification of stem cells necessitates determining the effects of growth factors on human spinal cord stem cells. Fetal spinal cords were dissociated and expanded as neurospheres in medium with bone morphogenetic protein 4 (BMP4), leukemia inhibitory factor (LIF) or BMP4 and LIF. First‐generation neurospheres comprised a heterogeneous population of neural cell types and after plating emergent cells included neurons, oligodendrocytes and GFAP+ cells which coexpressed stem cells markers and those of the neuronal lineage and were thus identified as GFAP+ neural precursor cells (NPC). When plated, neurospheres maintained in BMP4 demonstrated a reduced proportion of emergent oligodendrocytes from 13 to 4%, whereas LIF had no statistically significant effect on cell type distribution. Combining BMP4 and LIF reduced the proportion of oligodendrocytes to 3% and that of neurons from 37 to 16% while increasing the proportion of GFAP+ NPC from 45 to 79%. After 10 passages in control media aggregates gave rise to multiple neural phenotypes and only continued passage of neurospheres in the presence of BMP4 and LIF resulted in unipotent aggregates giving rise to only astrocytes. These results provide a means of obtaining pure populations of human spinal‐cord derived astrocytes, which could be utilized for further studies of cell replacement strategies or in vitro evaluation of therapeutics.

Signalling organelle for retrograde axonal transport of internalized neurotrophins from the nerve terminal.

The retrograde axonal transport of neurotrophins occurs after receptor‐mediated endocytosis into vesicles at the nerve terminal. We have been investigating the process of targeting these vesicles for retrograde transport, by examining the transport of [125I]‐labelled neurotrophins from the eye to sympathetic and sensory ganglia. With the aid of confocal microscopy, we examined the phenomena further in cultures of dissociated sympathetic ganglia to which rhodamine‐labelled nerve growth factor (NGF) was added. We found the label in large vesicles in the growth cone and axons. Light microscopic examination of the sympathetic nerve trunk in vivo also showed the retrogradely transported material to be sporadically located in large structures in the axons. Ultrastructural examination of the sympathetic nerve trunk after the transport of NGF bound to gold particles showed the label to be concentrated in relatively few large organelles that consisted of accumulations of multivesicular bodies. These results suggest that in vivo NGF is transported in specialized organelles that require assembly in the nerve terminal.

Aging‐related changes in astrocytes in the rat retina: imbalance between cell proliferation and cell death reduces astrocyte availability

The aim of this study was to investigate changes in astrocyte density, morphology, proliferation and apoptosis occurring in the central nervous system during physiological aging. Astrocytes in retinal whole‐mount preparations from Wistar rats aged 3 (young adult) to 25 months (aged) were investigated qualitatively and quantitatively following immunofluorohistochemistry. Glial fibrillary acidic protein (GFAP), S100 and Pax2 were used to identify astrocytes, and blood vessels were localized using Griffonia simplicifolia isolectin B4. Cell proliferation was assessed by bromodeoxyuridine incorporation and cell death by TUNEL‐labelling and immunolocalization of the apoptosis markers active caspase 3 and endonuclease G. The density and total number of parenchymal astrocytes in the retina increased between 3 and 9 months of age but decreased markedly between 9 and 12 months. Proliferation of astrocytes was detected at 3 months but virtually ceased beyond that age, whereas the proportion of astrocytes that were TUNEL positive and relative expression of active caspase 3 and endonuclease G increased progressively with aging. In addition, in aged retinas astrocytes exhibited gliosis‐like morphology and loss of Pax2 reactivity. A small population of Pax2+/GFAP− cells was detected in both young adult and aged retinas. The reduction in the availability of astrocytes in aged retinas and other aging‐related changes reported here may have a significant impact on the ability of astrocytes to maintain homeostasis and support neuronal function in old age.

Prolonged recycling of internalized neurotrophins in the nerve terminal

BACKGROUND Neurons require contact with their target tissue in order to survive and make correct connections. The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. However, the mechanism by which the neurotrophin signal is propagated from axon terminal to cell body remains unclear. METHODS Retrograde axonal transport was examined using the transport of I(125)-labeled neurotrophins from the eye to sympathetic and sensory ganglia. The phenomena was further studied by adding rhodamine-labeled nerve growth factor (NGF) to cultures of dissociated sympathetic ganglia and the movement of organelles followed with the aid of video microscopy. RESULTS I(125)-labeled neurotrophins were transported from the eye to the sympathetic and sensory ganglia. A 100-fold excess of unlabeled neurotrophin, administered up to 4 h after the labeled material, completely prevented accumulation of labeled neurotrophin in the ganglia. The effect was specific for the labeled neurotrophin as administration of a high concentration of a different neurotrophin failed to inhibit the transport. In dissociated cultures, we found rapid binding of label, to surface membrane receptors, followed by an accumulation of labeled vesicles in the growth cone. Incubation of these cultures with unlabeled NGF led to a rapid loss of label in the growth cones. CONCLUSIONS These results suggest that there is a pool of internalized neurotrophin, in vesicles in the nerve terminal, which is in rapid equilibrium with the external environment. It is from this pool that a small fraction of the neurotrophin-containing vesicles is targeted for retrograde transport. Potential models for this system are presented.

P2X7 Receptors Mediate Innate Phagocytosis by Human Neural Precursor Cells and Neuroblasts

During early human neurogenesis there is overproduction of neuroblasts and neurons accompanied by widespread programmed cell death (PCD). While it is understood that CD68+ microglia and astrocytes mediate phagocytosis during target‐dependent PCD, little is known of the cell identity or the scavenger molecules used to remove apoptotic corpses during the earliest stages of human neurogenesis. Using a combination of multiple‐marker immunohistochemical staining, functional blocking antibodies and antagonists, we showed that human neural precursor cells (hNPCs) and neuroblasts express functional P2X7 receptors. Furthermore, using live‐cell imaging, flow cytometry, phagocytic assays, and siRNA knockdown, we showed that in a serum‐free environment, doublecortin+ (DCX) neuroblasts and hNPCs can clear apoptotic cells by innate phagocytosis mediated via P2X7. We found that both P2X7highDCXlow hNPCs and P2X7highDCXhigh neuroblasts, derived from primary cultures of human fetal telencephalon, phagocytosed targets including latex beads, apoptotic ReNcells, and apoptotic hNPC/neuroblasts. Pretreatment of neuroblasts and hNPCs with 1 mM adenosine triphosphate (ATP), 100 µM OxATP (P2X7 antagonist), or siRNA knockdown of P2X7 inhibited phagocytosis of these targets. Our results show that P2X7 functions as a scavenger receptor under serum‐free conditions resembling those in early neurogenesis. This is the first demonstration that hNPCs and neuroblasts may participate in clearance of apoptotic corpses during pre target‐dependent neurogenesis and mediate phagocytosis using P2X7 as a scavenger receptor. Stem Cells 2015;33:526–541

Wolbachia influences the production of octopamine and affects Drosophila male aggression

ABSTRACT Wolbachia bacteria are endosymbionts that infect approximately 40% of all insect species and are best known for their ability to manipulate host reproductive systems. Though the effect Wolbachia infection has on somatic tissues is less well understood, when present in cells of the adult Drosophila melanogaster brain, Wolbachia exerts an influence over behaviors related to olfaction. Here, we show that a strain of Wolbachia influences male aggression in flies, which is critically important in mate competition. A specific strain of Wolbachia was observed to reduce the initiation of aggressive encounters in Drosophila males compared to the behavior of their uninfected controls. To determine how Wolbachia was able to alter aggressive behavior, we investigated the role of octopamine, a neurotransmitter known to influence male aggressive behavior in many insect species. Transcriptional analysis of the octopamine biosynthesis pathway revealed that two essential genes, the tyrosine decarboxylase and tyramine β-hydroxylase genes, were significantly downregulated in Wolbachia-infected flies. Quantitative chemical analysis also showed that total octopamine levels were significantly reduced in the adult heads.

Comparison of nerve terminal events in vivo effecting retrograde transport of vesicles containing neurotrophins or synaptic vesicle components

Although vesicular retrograde transport of neurotrophins in vivo is well established, relatively little is known about the mechanisms that underlie vesicle endocytosis and formation before transport. We demonstrate that in vivo not all retrograde transport vesicles are alike, nor are they all formed using identical mechanisms. As characterized by density, there are at least two populations of vesicles present in the synaptic terminal that are retrogradely transported along the axon: those containing neurotrophins (NTs) and those resulting from synaptic vesicle recycling. Vesicles containing nerve growth factor (NGF), NT‐3, or NT‐4 had similar densities with peak values at about 1.05 g/ml. Synaptic‐derived vesicles, labeled with anti‐dopamine β‐hydroxylase (DBH), had densities with peak values at about 1.16 g/ml. We assayed the effects of pharmacologic agents in vivo on retrograde transport from the anterior eye chamber to the superior cervical ganglion. Inhibitors of phosphatidylinositol‐3‐OH (PI‐3) kinase and actin function blocked transport of both anti‐DBH and NGF, demonstrating an essential role for these molecules in retrograde transport of both vesicle types. Dynamin, a key element in synaptic vesicle recycling, was axonally transported in retrograde and anterograde directions, and compounds able to interfere with dynamin function had a differential effect on retrograde transport of NTs and anti‐DBH. Okadaic acid significantly decreased retrograde axonal transport of anti‐DBH and increased NGF retrograde transport. We conclude that there are both different and common proteins involved in endocytosis and targeting of retrograde transport of these two populations of vesicles.

PtdIns 4-kinasebeta and neuronal calcium sensor-1 co-localize but may not directly associate in mammalian neurons.

It was recently demonstrated that the yeast homologue of phosphatidylinositol 4‐kinaseβ PIK1 is directly associated with frq1, the yeast homologue of mammalian neuronal calcium sensor‐1 (NCS‐1) (Hendricks et al., [1999] Nat. Cell Biol. 1:234– 241). This was a novel finding and suggests that a calcium binding protein activates and regulates PtdIns 4‐kinaseβ. This finding had not been shown in mammalian cells and both PtdIns 4‐kinaseβ and NCS‐1 have been shown to have important roles in the regulation of exocytotic release associated with neurotransmission. The aims of this study were to determine if PtdIns 4‐kinaseβ and NCS‐1 directly associate in mammalian neural tissues. We show that the immunostaining pattern for PtdIns 4‐kinaseβ and NCS‐1 is co‐localized throughout the neurites of newborn cultured dorsal root ganglia (DRG) neurons but not in E13 DRG neurons. We then provide biochemical evidence that PtdIns 4‐kinaseβ may not be in physical association with NCS‐1 in mammalian nervous tissue unlike that previously reported in yeast. J. Neurosci. Res. 62:216–224, 2000.