The autism-related gene SNRPN regulates cortical and spine development via controlling nuclear receptor Nr4a1.
ABSTRACT: The small nuclear ribonucleoprotein polypeptide N (SNRPN) gene, encoding the RNA-associated SmN protein, duplications or deletions of which are strongly associated with neurodevelopmental disabilities. SNRPN-coding protein is highly expressed in the brain. However, the role of SNRPN protein in neural development remains largely unknown. Here we showed that the expression of SNRPN increased markedly during postnatal brain development. Overexpression or knockdown of SNRPN in cortical neurons impaired neurite outgrowth, neuron migration, and the distribution of dendritic spines. We found that SNRPN regulated the expression level of Nr4a1, a critical nuclear receptor during neural development, in cultured primary cortical neurons. The abnormal spine development caused by SNRPN overexpression could be fully rescued by Nr4a1 co-expression. Importantly, we found that either knockdown of Nr4a1 or 3, 3'- Diindolylmethane (DIM), an Nr4a1 antagonist, were able to rescue the effects of SNRPN knockdown on neurite outgrowth of embryonic cortical neurons, providing the potential therapeutic methods for SNRPN deletion disorders. We thus concluded that maintaining the proper level of SNRPN is critical in cortical neurodevelopment. Finally, Nr4a1 may serve as a potential drug target for SNRPN-related neurodevelopmental disabilities, including Prader-Willi syndrome (PWS) and autism spectrum disorders (ASDs).
Project description:Deficits in neurite outgrowth, possibly involving dysregulation of risk genes neuregulin-1 (NRG1) and disrupted in schizophrenia 1 (DISC1) have been implicated in psychiatric disorders including schizophrenia. Electrical stimulation using conductive polymers has been shown to stimulate neurite outgrowth of differentiating human neural stem cells. This study investigated the use of the electroactive conductive polymer polypyrrole (Ppy) to counter impaired neurite outgrowth of primary pre-frontal cortical (PFC) neurons from NRG1-knock out (NRG1-KO) and DISC1-locus impairment (DISC1-LI) mice. Whereas NRG1-KO and DISC1-LI exhibited reduced neurite length and number of neurite branches compared to wild-type controls, this was not apparent for cultures on electroactive Ppy. Additionally, the use of the Ppy substrate normalised the synaptophysin and PSD95 protein and mRNA expression whereas both are usually reduced by NRG1-KO or DISC1-LI. Our findings support the utility of Ppy mediated electrical stimulation to prevent the reduction of neurite outgrowth and related synaptic protein expression in the primary PFC neurons from NRG1-KO and DISC1-LI mice, providing proof-of-concept for treating neurodevelopmental diseases including schizophrenia.
Project description:Axonal regeneration in the central nervous system is prevented, in part, by inhibitory proteins expressed by myelin, including myelin-associated glycoprotein (MAG). Although injury to the corticospinal tract can result in permanent disability, little is known regarding the mechanisms by which MAG affects cortical neurons. Here, we demonstrate that cortical neurons plated on MAG expressing CHO cells, exhibit a striking reduction in process outgrowth. Interestingly, none of the receptors previously implicated in MAG signaling, including the p75 neurotrophin receptor or gangliosides, contributed significantly to MAG-mediated inhibition. However, blocking the small GTPase Rho or its downstream effector kinase, ROCK, partially reversed the effects of MAG on the neurons. In addition, we identified the lipid phosphatase PTEN as a mediator of MAG's inhibitory effects on neurite outgrowth. Knockdown or gene deletion of PTEN or overexpression of activated AKT in cortical neurons resulted in significant, although partial, rescue of neurite outgrowth on MAG-CHO cells. Moreover, MAG decreased the levels of phospho-Akt, suggesting that it activates PTEN in the neurons. Taken together, these results suggest a novel pathway activated by MAG in cortical neurons involving the PTEN/PI3K/AKT axis.
Project description:Fungicides often cause genotoxic stress and neurodevelopmental disorders such as autism (ASD). Fungicide-azoxystrobin (AZOX) showed acute and chronic toxicity to various organisms, and remained a concern for ill effects in developing neurons. We evaluated the neurotoxicity of AZOX in developing mouse brains, and observed prenatal exposure to AZOX reduced neuronal viability, neurite outgrowth, and cortical migration process in developing brains. The 50% inhibitory concentration (IC50) of AZOX for acute (24 h) and chronic (7 days) exposures were 30 and 10 μM, respectively. Loss in viability was due to the accumulation of reactive oxygen species (ROS), and inhibited neurite outgrowth was due to the deactivation of mTORC1 kinase activity. Pretreatment with ROS scavenger- N-acetylcysteine (NAC) reserved the viability loss and forced activation of mTORC1 kinase revived the neurite outgrowth in AZOX treated neurons. Intra-amniotic injection of AZOX coupled with <i>in utero</i> electroporation of GFP-labelled plasmid in E15.5 mouse was performed and 20 mg/kg AZOX inhibited radial neuronal migration. Moreover, the accumulation of mitochondria was significantly reduced in AZOX treated primary neurons, indicative of mitochondrial deactivation and induction of apoptosis, which was quantified by Bcl2/Bax ratio and caspase 3 cleavage assay. This study elucidated the neurotoxicity of AZOX and explained the possible cure from it.
Project description:Phospholipase C?3 (PLC?3) is a key enzyme regulating phosphoinositide metabolism; however, its physiological function remains unknown. Because PLC?3 is highly enriched in the cerebellum and cerebral cortex, we examined the role of PLC?3 in neuronal migration and outgrowth. PLC?3 knockdown (KD) inhibits neurite formation of cerebellar granule cells, and application of PLC?3KD using in utero electroporation in the developing brain results in the retardation of the radial migration of neurons in the cerebral cortex. In addition, PLC?3KD inhibits axon and dendrite outgrowth in primary cortical neurons. PLC?3KD also suppresses neurite formation of Neuro2a neuroblastoma cells induced by serum withdrawal or treatment with retinoic acid. This inhibition is released by the reintroduction of wild-type PLC?3. Interestingly, the H393A mutant lacking phosphatidylinositol 4,5-bisphosphate hydrolyzing activity generates supernumerary protrusions, and a constitutively active mutant promotes extensive neurite outgrowth, indicating that PLC activity is important for normal neurite outgrowth. The introduction of dominant negative RhoA (RhoA-DN) or treatment with Y-27632, a Rho kinase-specific inhibitor, rescues the neurite extension in PLC?3KD Neuro2a cells. Similar effects were also detected in primary cortical neurons. Furthermore, the RhoA expression level was significantly decreased by serum withdrawal or retinoic acid in control cells, although this decrease was not observed in PLC?3KD cells. We also found that exogenous expression of PLC?3 down-regulated RhoA protein, and constitutively active PLC?3 promotes the RhoA down-regulation more significantly than PLC?3 upon differentiation. These results indicate that PLC?3 negatively regulates RhoA expression, inhibits RhoA/Rho kinase signaling, and thereby promotes neurite extension.
Project description:<h4>Background</h4>Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.<h4>Methodology/principal findings</h4>Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR(-/-) mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR(-/-)) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR(-/-) neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.<h4>Conclusions</h4>proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.
Project description:Neurogenesis, especially neurite outgrowth is an essential element of neuroplasticity after cerebral ischemic injury. Mitochondria may supply ATP to power fundamental developmental processes including neuroplasticity. Although rosuvastatin (RSV) displays a potential protective effect against cerebral ischemia, it remains unknown whether it modulates mitochondrial biogenesis and function during neurite outgrowth. Here, the oxygen-glucose deprivation (OGD) model was used to induce ischemic injury. We demonstrate that RSV treatment significantly increases neurite outgrowth in cortical neurons after OGD-induced damage. Moreover, we show that RSV reduces the generation of reactive oxygen species (ROS), protects mitochondrial function, and elevates the ATP levels in cortical neurons injured by OGD. In addition, we found that, under these conditions, RSV treatment increases the mitochondrial DNA (mtDNA) content and the mRNA levels of mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF-1). Furthermore, blocking Notch1, which is expressed in primary cortical neurons, reverses the RSV-dependent induction of mitochondrial biogenesis and function under OGD conditions. Collectively, these results suggest that RSV could restore neurite outgrowth in cortical neurons damaged by OGD in vitro, by preserving mitochondrial function and improving mitochondrial biogenesis, possibly through the Notch1 pathway.
Project description:Neuritic degeneration and synaptic loss are features of both neuroinflammation and neurodegenerative disease. The tricyclic antidepressant amitriptyline has neurotrophic and anti-inflammatory properties and acts as a novel agonist of the neurotrophin Trk receptors. Primary cortical neurons were treated with amitriptyline, nortriptyline and NGF and tested for neuronal complexity by Sholl analysis, protein expression by Western immunoblotting, and synapse number by colocalization of pre and postsynaptic makers. Amitriptyline (500 nmol/L) and its active metabolite nortriptyline (50 nmol/L) are found to induce neurite outgrowth in rat primary cortical neurons. Amitriptyline-induced neurite outgrowth is blocked by inhibition of Trk signaling using Trk antagonist K252a (200 nmol/L) but not by the neurotrophin inhibitor Y1036 (40 μmol/L), indicating that amitriptyline binds directly to the Trk receptor to initiate neurite outgrowth. MEK inhibitor PD98059 (10 μmol/L) also blocks amitriptyline-induced neurite outgrowth, implicating activation of the MAPK signaling pathway downstream of Trk receptor activation. Furthermore, pretreatment of primary cortical neurons with amitriptyline and nortriptyline prevents the effects of the proinflammatory cytokine TNF-α (10 ng/mL) on neurite outgrowth and colocalization of synaptic proteins. These findings suggest that amitriptyline and nortriptyline can exert neurotrophic effects in primary cortical neurons via activation of a Trk/MAPK signaling pathway. These compounds therefore have significant potential to be used in the treatment of neurodegenerative conditions where atrophy and loss of synaptic connections contribute to progression of disease.
Project description:The processing of amyloid precursor protein (APP) to Abeta is an important event in the pathogenesis of Alzheimer's disease, but the physiological function of APP is not well understood. Our previous work has shown that APP processing and Abeta production are regulated by the extracellular matrix protein Reelin. In the present study, we examined whether Reelin interacts with APP, and the functional consequences of that interaction in vitro. Using coimmunoprecipitation, we found that Reelin interacted with APP through the central domain of Reelin (repeats 3-6) and the E1 extracellular domain of APP. Reelin increased cell surface levels of APP and decreased endocytosis of APP in hippocampal neurons in vitro. In vivo, Reelin levels were increased in brains of APP knock-out mice and decreased in APP-overexpressing mice. RNA interference knockdown of APP decreased neurite outgrowth in vitro and prevented Reelin from increasing neurite outgrowth. Knock-out of APP or Reelin decreased dendritic arborization in cortical neurons in vivo, and APP overexpression increased dendritic arborization. APP and Reelin have previously been shown to promote neurite outgrowth through interactions with integrins. We confirmed that APP interacted with alpha3beta1 integrin, and alpha3beta1 integrin altered APP trafficking and processing. Addition of an alpha3beta1 integrin antibody prevented APP and Reelin-induced neurite outgrowth. These findings demonstrate that Reelin interacts with APP, potentially having important effects on neurite development.
Project description:Although recovery following a stroke is limited, undamaged neurons under the right conditions can establish new connections and take on-board lost functions. Sonic hedgehog (Shh) signaling is integral for developmental axon growth, but its role after injury has not been fully examined. To investigate the effects of Shh on neuronal sprouting after injury, we used an in vitro model of glial scar, whereby cortical astrocytes were mechanically traumatized to mimic reactive astrogliosis observed after stroke. This mechanical trauma impaired neurite outgrowth from post-natal cortical neurons plated on top of reactive astrocytes. Addition of Shh to the media, however, resulted in a concentration-dependent increase in neurite outgrowth. This response was inhibited by cyclopamine and activated by oxysterol 20(S)-hydroxycholesterol, both of which modulate the activity of the Shh co-receptor Smoothened (Smo), demonstrating that Shh-mediated neurite outgrowth is Smo-dependent. In addition, neurite outgrowth was not associated with an increase in Gli-1 transcription, but could be inhibited by PP2, a selective inhibitor of Src family kinases. These results demonstrate that neurons exposed to the neurite growth inhibitory environment associated with a glial scar can be stimulated by Shh, with signaling occurring through a non-canonical pathway, to overcome this suppression and stimulate neurite outgrowth.
Project description:FE65 is an adaptor protein that binds to the amyloid precursor protein (APP). As such, FE65 has been implicated in the pathogenesis of Alzheimer's disease. In addition, evidence suggests that FE65 is involved in brain development. It is generally believed that FE65 participates in these processes by recruiting various interacting partners to form functional complexes. Here, we show that via its first phosphotyrosine binding (PTB) domain, FE65 binds to the small GTPase ADP-ribosylation factor 6 (ARF6). FE65 preferentially binds to ARF6-GDP, and they colocalize in neuronal growth cones. Interestingly, FE65 stimulates the activation of both ARF6 and its downstream GTPase Rac1, a regulator of actin dynamics, and functions in growth cones to stimulate neurite outgrowth. We show that transfection of FE65 and/or ARF6 promotes whereas small interfering RNA knockdown of FE65 or ARF6 inhibits neurite outgrowth in cultured neurons as compared to the mock-transfected control cells. Moreover, knockdown of ARF6 attenuates FE65 stimulation of neurite outgrowth and defective neurite outgrowth seen in FE65-deficient neurons is partially corrected by ARF6 overexpression. Notably, the stimulatory effect of FE65 and ARF6 on neurite outgrowth is abrogated either by dominant-negative Rac1 or knockdown of Rac1. Thus, we identify FE65 as a novel regulator of neurite outgrowth via controlling ARF6-Rac1 signaling.