Leslayann C. Schecterson

Novel roles for neurotrophins are suggested by BDNF and NT-3 mRNA expression in developing neurons

The results of our in situ hybridization experiments demonstrate that sensory neurons, sympathetic neurons, and motoneurons express brain-derived neurotrophic factor and/or neurotrophin-3 mRNAs during development in mouse. In accordance with previous data, we also find neurotrophins in the targets of sensory neurons (skin) and motoneurons (muscle) and the neurotrophin receptors p75, trkA, and trkB in sensory and sympathetic ganglia. These results suggest that neurotrophins have roles other than being target-derived factors that support neuron survival during developmental cell death (neurotrophic hypothesis), but may be transported in an orthograde fashion in neurons and released from axon terminals. We discuss several novel roles for neurotrophins, including autocrine/paracrine regulation of neuron survival, regulation of Schwann cell activity, and neuron to target signaling.

Neurotrophin and neurotrophin receptor mRNA expression in developing inner ear

Receptors which bind the neurotrophins NGF, BDNF, NT-3 and NT-4/5 were shown to be present in cochlear and vestibular ganglion cells during development, implying a neurotrophic role for these molecules in the inner ear. We have found by in situ hybridization that cochlear and vestibular sensory epithelial cells express BDNF and NT-3 mRNAs, but neither NGF or NT-4 mRNAs, in mouse embryos from embryonic day (E)11.5 through postnatal day (P)1. NT-3 mRNA was expressed throughout the sensory epithelium whereas BDNF mRNA appeared to be localized in hair cells (vestibular) and epithelial precursors of hair cells (cochlea). BDNF mRNA was also expressed in a subpopulation of cells in the cochleovestibular ganglion at E11.5 and E12.5. Additionally, cochlear and vestibular neurons contained mRNAs encoding the neurotrophin receptors p75 and trkB. TrkA mRNA was transiently expressed in cochleovestibular ganglion cells at E12.5. These data suggest that BDNF and NT-3 play a role in cochleovestibular neuron survival and neurite outgrowth during development in the inner ear.

Expression of BDNF and NT-3 mRNA in hair cells of the organ of Corti: quantitative analysis in developing rats.

Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are synthesized by inner and outer hair cells of the developing organ of Corti. This raises the possibility that the reorganization of cochlear innervation patterns that occurs postnatally may be influenced by changing levels of neurotrophin expression. To determine if differential expression of BDNF or NT-3 in the inner and outer hair cells correlates with the reorganization of afferent and efferent innervation, we used in situ hybridization techniques to quantify relative levels of transcript biosynthesis in hair cells of developing rats. BDNF transcripts decreased in inner and outer hair cells from E17 to insignificant levels at P4. NT-3 expression was high at E17 in inner and outer hair cells, decreased in outer hair cells by E21, in inner hair cells by P1, remained low during the first postnatal week and was increased in the adult. The decreases in expression of both neurotrophins at birth precede the retraction of afferent nerve terminals from outer hair cells. BDNF and NT-3 transcription decreases substantially in outer hair cells between E21 and P4 when efferent innervation begins, indicating target biosynthesis of these neurotrophins is not likely to be instrumental in efferent target selection.

Neurotrophin Receptors: Old Friends with New Partners

Neurotrophins are important regulators of embryonic development and adult function of most populations of neurons in vertebrate nervous systems. This signaling system regulates many diverse activities, including survival, axon outgrowth, and synaptic plasticity. In mammals, neurotrophin action is mediated by four receptors, p75NTR, TrkA, TrkB, and TrkC. Although early studies viewed these receptors as solitary agents in the cells outer membrane, recent discoveries reveal that the cell outer membrane is a crowded and highly interactive neighborhood. Neurotrophin receptors partner with a diverse array of membrane proteins, dramatically expanding their functional repertoire. This review will focus on some of the most recent discoveries concerning the promiscuous partnering of neurotrophin receptors.

Phenylbutyric acid rescues endoplasmic reticulum stress-induced suppression of APP proteolysis and prevents apoptosis in neuronal cells.

Background The familial and sporadic forms of Alzheimers disease (AD) have an identical pathology with a severe disparity in the time of onset [1]. The pathological similarity suggests that epigenetic processes may phenocopy the Familial Alzheimers disease (FAD) mutations within sporadic AD. Numerous groups have demonstrated that FAD mutations in presenilin result in ‘loss of function’ of γ-secretase mediated APP cleavage [2], [3], [4], [5]. Accordingly, ER stress is prominent within the pathologically impacted brain regions in AD patients [6] and is reported to inhibit APP trafficking through the secretory pathway [7], [8]. As the maturation of APP and the cleaving secretases requires trafficking through the secretory pathway [9], [10], [11], we hypothesized that ER stress may block trafficking requisite for normal levels of APP cleavage and that the small molecular chaperone 4-phenylbutyrate (PBA) may rescue the proteolytic deficit. Methodology/Principal Findings The APP-Gal4VP16/Gal4-reporter screen was stably incorporated into neuroblastoma cells in order to assay γ-secretase mediated APP proteolysis under normal and pharmacologically induced ER stress conditions. Three unrelated pharmacological agents (tunicamycin, thapsigargin and brefeldin A) all repressed APP proteolysis in parallel with activation of unfolded protein response (UPR) signaling—a biochemical marker of ER stress. Co-treatment of the γ-secretase reporter cells with PBA blocked the repressive effects of tunicamycin and thapsigargin upon APP proteolysis, UPR activation, and apoptosis. In unstressed cells, PBA stimulated γ-secretase mediated cleavage of APP by 8–10 fold, in the absence of any significant effects upon amyloid production, by promoting APP trafficking through the secretory pathway and the stimulation of the non-pathogenic α/γ-cleavage. Conclusions/Significance ER stress represses γ-secretase mediated APP proteolysis, which replicates some of the proteolytic deficits associated with the FAD mutations. The small molecular chaperone PBA can reverse ER stress induced effects upon APP proteolysis, trafficking and cellular viability. Pharmaceutical agents, such as PBA, that stimulate α/γ-cleavage of APP by modifying intracellular trafficking should be explored as AD therapeutics.

Transgenic quail as a model for research in the avian nervous system: A comparative study of the auditory brainstem

Research performed on transgenic animals has led to numerous advances in biological research. However, using traditional retroviral methods to generate transgenic avian research models has proved problematic. As a result, experiments aimed at genetic manipulations on birds have remained difficult for this popular research tool. Recently, lentiviral methods have allowed the production of transgenic birds, including a transgenic Japanese quail (Coturnix coturnix japonica) line showing neuronal specificity and stable expression of enhanced green fluorescent protein (eGFP) across generations (termed here GFP quail). To test whether the GFP quail may serve as a viable alternative to the popular chicken model system, with the additional benefit of genetic manipulation, we compared the development, organization, structure, and function of a specific neuronal circuit in chicken (Gallus gallus domesticus) with that of the GFP quail. This study focuses on a well‐defined avian brain region, the principal nuclei of the sound localization circuit in the auditory brainstem, nucleus magnocellularis (NM), and nucleus laminaris (NL). Our results demonstrate that structural and functional properties of NM and NL neurons in the GFP quail, as well as their dynamic properties in response to changes in the environment, are nearly identical to those in chickens. These similarities demonstrate that the GFP quail, as well as other transgenic quail lines, can serve as an attractive avian model system, with the advantage of being able to build on the wealth of information already available from the chicken. J. Comp. Neurol.5–23, 2013.

Trk activation in the secretory pathway promotes Golgi fragmentation.

Activation of nascent receptor tyrosine kinases within the secretory pathway has been reported, yet the consequences of intracellular activation are largely unexplored. We report that overexpression of the Trk neurotrophin receptors causes accumulation of autoactivated receptors in the ER-Golgi intermediate compartment. Autoactivated receptors exhibit inhibited Golgi-mediated processing and they inhibit Golgi-mediated processing of other co-expressed transmembrane proteins, apparently by inducing fragmentation of the Golgi apparatus. Signaling from G protein-coupled receptors is known to induce Trk transactivation. Transactivation of nascent TrkB in hippocampal neurons resulting from exposure to the neuropeptide PACAP caused Golgi fragmentation, whereas BDNF-dependent activation of TrkB did not. TrkB-mediated Golgi fragmentation employs a MEK-dependent signaling pathway resembling that implicated in regulation of Golgi fragmentation in mitotic cells. Neuronal Golgi fragments, in the form of dendritically localized Golgi outposts, are important determinants of dendritic growth and branching. The capacity of transactivated TrkB to enhance neuronal Golgi fragmentation may represent a novel mechanism regulating neural plasticity.

TrkB Downregulation Is Required for Dendrite Retraction in Developing Neurons of Chicken Nucleus Magnocellularis

The chick embryo (Gallus domesticus) is one of the most important model systems in vertebrate developmental biology. The development and function of its auditory brainstem circuitry is exceptionally well studied. These circuits represent an excellent system for genetic manipulation to investigate mechanisms controlling neural circuit formation, synaptogenesis, neuronal polarity, and dendritic arborization. The present study investigates the auditory nucleus, nucleus magnocellularis (NM). The neurotrophin receptor TrkB regulates dendritic structure in CNS neurons. TrkB is expressed in NM neurons at E7–E8 when these neurons have dendritic arbors. Downregulation of TrkB occurs after E8 followed by retraction of dendrites and by E18 most NM cells are adendritic. Is cessation of TrkB expression in NM necessary for dendritic retraction? To answer this question we combined focal in ovo electroporation with transposon mediated gene transfer to obtain stable expression of Doxycycline (Dox) regulated transgenes, specifically TrkB coexpressed with EGFP in a temporally controlled manner. Electroporation was performed at E2 and Dox added onto the chorioallointoic membrane from E7.5 to E16. Expression of EGFP had no effect on development of the embryo, or cell morphology and organization of auditory brainstem nuclei. NM cells expressing EGFP and TrkB at E17–E18 had dendrites and biophysical properties uncharacteristic for normal NM cells, indicating that cessation of TrkB expression is essential for dendrite retraction and functional maturation of these neurons. These studies indicate that expression of transposon based plasmids is an effective method to genetically manipulate events in mid to late embryonic brain development in chick.

An All-Purpose Tool for Axon Guidance

The p75 neurotrophin receptor functions as a co-receptor in three distinct systems that mediate repellant signals. The functions of the 75-kilodalton neurotrophin receptor p75NTR have remained enigmatic despite nearly three decades of study. Recent studies reveal that p75NTR is a versatile co-receptor that controls signaling by receptors for multiple ligands that provide repellant guidance cues to developing axons.

Postsynaptic FMRP Regulates Synaptogenesis In Vivo in the Developing Cochlear Nucleus

A global loss of the fragile X mental retardation protein (FMRP; encoded by the Fmr1 gene) leads to sensory dysfunction and intellectual disabilities. One underlying mechanism of these phenotypes is structural and functional deficits in synapses. Here, we determined the autonomous function of postsynaptic FMRP in circuit formation, synaptogenesis, and synaptic maturation. In normal cochlea nucleus, presynaptic auditory axons form large axosomatic endbulb synapses on cell bodies of postsynaptic bushy neurons. In ovo electroporation of drug-inducible Fmr1-shRNA constructs produced a mosaicism of FMRP expression in chicken (either sex) bushy neurons, leading to reduced FMRP levels in transfected, but not neighboring nontransfected, neurons. Structural analyses revealed that postsynaptic FMRP reduction led to smaller size and abnormal morphology of individual presynaptic endbulbs at both early and later developmental stages. We further examined whether FMRP reduction affects dendritic development, as a potential mechanism underlying defective endbulb formation. Normally, chicken bushy neurons grow extensive dendrites at early stages and retract these dendrites when endbulbs begin to form. Neurons transfected with Fmr1 shRNA exhibited a remarkable delay in branch retraction, failing to provide necessary somatic surface for timely formation and growth of large endbulbs. Patch-clamp recording verified functional consequences of dendritic and synaptic deficits on neurotransmission, showing smaller amplitudes and slower kinetics of spontaneous and evoked EPSCs. Together, these data demonstrate that proper levels of postsynaptic FMRP are required for timely maturation of somatodendritic morphology, a delay of which may affect synaptogenesis and thus contribute to long-lasting deficits of excitatory synapses. SIGNIFICANCE STATEMENT Fragile X mental retardation protein (FMRP) regulates a large variety of neuronal activities. A global loss of FMRP affects neural circuit development and synaptic function, leading to fragile X syndrome (FXS). Using temporally and spatially controlled genetic manipulations, this study provides the first in vivo report that autonomous FMRP regulates multiple stages of dendritic development, and that selective reduction of postsynaptic FMRP leads to abnormal development of excitatory presynaptic terminals and compromised neurotransmission. These observations demonstrate secondary influence of developmentally transient deficits in neuronal morphology and connectivity to the development of long-lasting synaptic pathology in FXS.