Developmental axon regrowth and primary neuron sprouting utilize distinct actin elongation factors.

Intrinsic neurite growth potential is a key determinant of neuronal regeneration efficiency following injury. The stereotypical remodeling of Drosophila γ-neurons includes developmental regrowth of pruned axons to form adult specific connections, thereby offering a unique system to uncover growth potential regulators. Motivated by the dynamic expression in remodeling γ-neurons, we focus here on the role of actin elongation factors as potential regulators of developmental axon regrowth. We found that regrowth in vivo requires the actin elongation factors Ena and profilin, but not the formins that are expressed in γ-neurons. In contrast, primary γ-neuron sprouting in vitro requires profilin and the formin DAAM, but not Ena. Furthermore, we demonstrate that DAAM can compensate for the loss of Ena in vivo. Similarly, DAAM mutants express invariably high levels of Ena in vitro. Thus, we show that different linear actin elongation factors function in distinct contexts even within the same cell type and that they can partially compensate for each other.

A fly's view of neuronal remodeling.

Developmental neuronal remodeling is a crucial step in sculpting the final and mature brain connectivity in both vertebrates and invertebrates. Remodeling includes degenerative events, such as neurite pruning, that may be followed by regeneration to form novel connections during normal development. Drosophila provides an excellent model to study both steps of remodeling since its nervous system undergoes massive and stereotypic remodeling during metamorphosis. Although pruning has been widely studied, our knowledge of the molecular and cellular mechanisms is far from complete. Our understanding of the processes underlying regrowth is even more fragmentary. In this review, we discuss recent progress by focusing on three groups of neurons that undergo stereotypic pruning and regrowth during metamorphosis, the mushroom body γ neurons, the dendritic arborization neurons and the crustacean cardioactive peptide peptidergic neurons. By comparing and contrasting the mechanisms involved in remodeling of these three neuronal types, we highlight the common themes and differences as well as raise key questions for future investigation in the field. WIREs Dev Biol 2016, 5:618-635. doi: 10.1002/wdev.241 For further resources related to this article, please visit the WIREs website.

Nitric Oxide as a Switching Mechanism between Axon Degeneration and Regrowth during Developmental Remodeling.

During development, neurons switch among growth states, such as initial axon outgrowth, axon pruning, and regrowth. By studying the stereotypic remodeling of the Drosophila mushroom body (MB), we found that the heme-binding nuclear receptor E75 is dispensable for initial axon outgrowth of MB γ neurons but is required for their developmental regrowth. Genetic experiments and pharmacological manipulations on ex-vivo-cultured brains indicate that neuronally generated nitric oxide (NO) promotes pruning but inhibits regrowth. We found that high NO levels inhibit the physical interaction between the E75 and UNF nuclear receptors, likely accounting for its repression of regrowth. Additionally, NO synthase (NOS) activity is downregulated at the onset of regrowth, at least partially, by short inhibitory NOS isoforms encoded within the NOS locus, indicating how NO production could be developmentally regulated. Taken together, these results suggest that NO signaling provides a switching mechanism between the degenerative and regenerative states of neuronal remodeling.

Astrocytes play a key role in Drosophila mushroom body axon pruning.

Axon pruning is an evolutionarily conserved strategy used to remodel neuronal connections during development. The Drosophila mushroom body (MB) undergoes neuronal remodeling in a highly stereotypical and tightly regulated manner, however many open questions remain. Although it has been previously shown that glia instruct pruning by secreting a TGF-ß ligand, myoglianin, which primes MB neurons for fragmentation and also later engulf the axonal debris once fragmentation has been completed, which glia subtypes participate in these processes as well as the molecular details are unknown. Here we show that, unexpectedly, astrocytes are the major glial subtype that is responsible for the clearance of MB axon debris following fragmentation, even though they represent only a minority of glia in the MB area during remodeling. Furthermore, we show that astrocytes both promote fragmentation of MB axons as well as clear axonal debris and that this process is mediated by ecdysone signaling in the astrocytes themselves. In addition, we found that blocking the expression of the cell engulfment receptor Draper in astrocytes only affects axonal debris clearance. Thereby we uncoupled the function of astrocytes in promoting axon fragmentation to that of clearing axonal debris after fragmentation has been completed. Our study finds a novel role for astrocytes in the MB and suggests two separate pathways in which they affect developmental axon pruning.

Axon regrowth during development and regeneration following injury share molecular mechanisms.

BACKGROUND: The molecular mechanisms that determine axonal growth potential are poorly understood. Intrinsic growth potential decreases with age, and thus one strategy to identify molecular pathways controlling intrinsic growth potential is by studying developing young neurons. The programmed and stereotypic remodeling of Drosophila mushroom body (MB) neurons during metamorphosis offers a unique opportunity to uncover such mechanisms. Despite emerging insights into MB γ-neuron axon pruning, nothing is known about the ensuing axon re-extension. RESULTS: Using mosaic loss of function, we found that the nuclear receptor UNF (Nr2e3) is cell autonomously required for the re-extension of MB γ-axons following pruning, but not for the initial growth or guidance of any MB neuron type. We found that UNF promotes this process of developmental axon regrowth via the TOR pathway as well as a late axon guidance program via an unknown mechanism. We have thus uncovered a novel developmental program of axon regrowth that is cell autonomously regulated by the UNF nuclear receptor and the TOR pathway. CONCLUSIONS: Our results suggest that UNF activates neuronal re-extension during development. Taken together, we show that axon growth during developmental remodeling is mechanistically distinct from initial axon outgrowth. Due to the involvement of the TOR pathway in axon regeneration following injury, our results also suggests that developmental regrowth shares common molecular mechanisms with regeneration following injury.

Dexamethasone enhances the norepinephrine-induced ERK/MAPK intracellular pathway possibly via dysregulation of the alpha2-adrenergic receptor: implications for antidepressant drug mechanism of action.

Norepinephrine (NE) and glucocorticoids (GCs) have been shown to oppositely affect various aspects of neuronal plasticity. These findings provided the basis for the plasticity hypothesis of major depression, which suggests that the disease-related impairment in neuronal plasticity is associated with long-term increase in GCs and may be reconstituted by antidepressants and monoamines. To investigate the interaction between GCs and NE, the plasticity-relevant ERK/MAPK pathway was studied in SH-SY5Y neuroblastoma cells treated with dexamethasone (DEX), a synthetic GC, NE, or both. NE treatment activated ERK and c-Jun and increased AP-1 transcriptional activity. Although DEX had no effect, co-treatment caused a prolonged and robust activation of the ERK/AP-1 pathway beyond NE-induced activation. Co-treatment also induced hyperactivation of CREB as compared to NE activation while DEX decreased pCREB. Independent alterations of ERK and CREB suggest an upstream point of interaction. Yohimbine, an alpha(2)-adrenergic receptor (AR) antagonist, prevented the hyperactivation of the ERK/AP-1 pathway and CREB induced by co-treatment. Immunofluorescence showed that after 2h of NE treatment, beta-arrestin was co-localized with the alpha(2)-AR at the plasma membrane while following co-treatment beta-arrestin was diffused within the cell, suggesting that DEX delays AR downregulation by altering beta-arrestin translocation. These results show a novel complex interaction by which GCs augment NE-induced intracellular signaling that may be relevant to antidepressant mode of action.

Norepinephrine-glucocorticoids interaction does not annul the opposite effects of the individual treatments on cellular plasticity in neuroblastoma cells.

The plasticity hypothesis of major depression states that glucocorticoids may be detrimental to neuronal plasticity while monoamines and antidepressants may reconstitute cellular plasticity. The aim of the present study was to investigate how dexamethasone, a synthetic glucocorticoid, and norepinephrine, both of which are involved in depression, interact to affect aspects of neuronal plasticity. Dexamethasone and norepinephrine administered separately oppositely affected differentiation of human neuroblastoma SH-SY5Y cells, observed by both morphological alterations and gene expression, at the level of mRNA and protein of the differentiation markers Gap-43, L1 and laminin. Norepinephrine increased differentiation, manifested as an increase in neurite length, neurite number, and gene expression, while dexamethasone reduced these parameters. Opposite effects were also observed in the expression of the transcription factor CREB with norepinephrine upregulating phosphorylated CREB (pCREB) levels, while dexamethasone downregulated CREB mRNA and protein levels, as well as pCREB levels. Interestingly, co-administration of dexamethasone and norepinephrine resulted in morphology more differentiated than control and similar to that induced by norepinephrine, albeit to a lesser degree. The alterations in the expression of the differentiation markers induced by norepinephrine or dexamethasone treatments were mostly annulled by the co-treatment. However, pCREB levels were robustly enhanced by co-treatment, as compared to both control and norepinephrine treated cells, providing a possible explanation for the morphological increase in differentiation. These results suggest that in order for cells to combat the deleterious effects of glucocorticoids, a hyperactivation of pCREB may be necessary to restore differentiation and plasticity.

Convergent effects on cell signaling mechanisms mediate the actions of different neurobehavioral teratogens: alterations in cholinergic regulation of protein kinase C in chick and avian models.

Although the actions of heroin on central nervous system (CNS) development are mediated through opioid receptors, the net effects converge on dysfunction of cholinergic systems. We explored the mechanisms underlying neurobehavioral deficits in mouse and avian (chick, Cayuga duck) models. In mice, prenatal heroin exposure (10 mg/kg on gestation days 9-18) elicited deficits in behaviors related to hippocampal cholinergic innervation, characterized by concomitant pre- and postsynaptic hyperactivity, but ending in a reduction of basal levels of protein kinase C (PKC) isoforms betaII and gamma and their desensitization to cholinergic receptor-induced activation. PKCalpha, which is not involved in the behaviors studied, was unaffected. Because mammalian models possess inherent confounding factors from maternal effects, we conducted parallel studies using avian embryos, evaluating hyperstriatal nucleus (intermedial part of the hyperstriatum ventrale, IMHV)-related, filial imprinting behavior. Heroin injection to the eggs (20 mg/kg) on incubation days 0 and 5 diminished the post-hatch imprinting ability and reduced PKCg and bII content in the IMHV membrane fraction. Two otherwise unrelated agents that converge on cholinergic systems, chlorpyrifos and nicotine, elicited the same spectrum of effects on PKC isoforms and imprinting but had more robust actions. Pharmacological characterization also excluded direct effects of opioid receptors on the expression of imprinting; instead, it indicated participation of serotonergic innervation. The avian models can provide rapid screening of neuroteratogens, exploration of common mechanisms of behavioral disruption, and the potential design of therapies to reverse neurobehavioral deficits.

Prenatal heroin exposure alters cholinergic receptor stimulated activation of the PKCbetaII and PKCgamma isoforms.

Prenatal exposure of mice to heroin (SC injection of 10mg/kg to the dams on gestational days 9-18) resulted at adulthood in behavioral deficits related to septohippocampal cholinergic innervation accompanied with both presynaptic and postsynaptic cholinergic hyperactivity; including an increase membrane PKC activity, and a desensitization of PKC to cholinergic input which were highly correlated with the behavioral performance and were reversed by cholinergic grafting. Therefore, we studied the receptor induced activation of the behaviorally relevant PKCgamma and PKCbetaII isoforms and the less behaviorally relevant PKCalpha isoform. Time course studies revealed peak translocation after 40 min incubation with carbachol for PKCgamma (110% increase from basal, i.e. no carbachol level, P < 0.01), 30 min for phosphorylated PKCbetaII (130%, P < 0.05) and 5 min for non-phosphorylated PKCbetaII (64%, P < 0.05) with no peak for alpha. Prenatal heroin abolished the translocation of PKCgamma and PKCbetaII while PKCalpha remained unaffected. A decrease occurred in basal phosphorylated membrane (-45%, P < 0.01) and cytosol-associated (-29%, P < 0.01) PKCbetaII, in membrane-associated non-phosphorylated PKCbetaII (-32%, P < 0.01) and PKCgamma (-25%, P < 0.01) and in cytosolic PKCalpha (-27%, P < 0.01), while membrane-associated PKCalpha was slightly increased (11%, P < 0.05). The results suggest that prenatal heroin disrupts cholinergic receptor induced PKC translocation and activation with the underlying mechanism of neuroteratogenicity potentially lying in the PKCgamma and PKCbetaII, while PKCalpha remains unaffected.

Functional changes after prenatal opiate exposure related to opiate receptors' regulated alterations in cholinergic innervation.

Opioid drugs act primarily on the opiate receptors; they also exert their effect on other innervations resulting in non-opioidergic behavioural deficits. Similarly, opioid neurobehavioural teratogenicity is attested in numerous behaviours and neural processes which hinder the research on the mechanisms involved. Therefore, in order to be able to ascertain the mechanism we have established an animal (mouse) model for the teratogenicity induced by opioid abuse, which focused on behaviours related to specific brain area and innervation. Diacetylmorphine (heroin) and not morphine was applied because heroin exerts a unique action, distinguished from that of morphine. Pregnant mice were exposed to heroin (10 mg/kg per day) and the offspring were tested for behavioural deficits and biochemical alterations related to the septohippocampal cholinergic innervation. Some studies employing the chick embryo were concomitantly added as a control for the confounding indirect variables. Prenatal exposure to heroin in mice induced global hyperactivation both pre- and post-synaptic along the septohippocampal cholinergic innervation, including basal protein kinase C (PKC) activity accompanied by a desensitization of PKC activity in response to cholinergic agonist. Functionally, the heroin-exposed offspring displayed deficits in hippocampus-related behaviours, suggesting deficits in the net output of the septohippocampal cholinergic innervation. Grafting of cholinergic cells to the impaired hippocampus reversed both pre- and post-synaptic hyperactivity, resensitized PKC activity, and restored the associated behaviours to normality. Consistently, correlation studies point to the relative importance of PKC to the behavioural deficits. The chick model, which dealt with imprinting related to a different brain region, confirmed that the effect of heroin is direct. Taken together with studies by others on the effect of prenatal exposure to opioids on the opioidergic innervation and with what is known on the opioid regulation of the cholinergic innervation, it appears that heroin exerts its neuroteratogenicity by inducing alterations in the opioidergic innervation, which by means of its regulatory action, attenuates the functional output of the cholinergic innervation. In our model, there was hyperactivity mostly of the post-synaptic components of the cholinergic innervation. However, the net cholinergic output is decreased because PKC is desensitized to the effect of the cholinergic agonist, and this is further evidenced by the extensive deficits in the related behaviours.