Inhibition of RhoA Activity Does Not Rescue Synaptic Development Abnormalities and Long-Term Cognitive Impairment After Sevoflurane Exposure

General anesthetics interfere with dendritic development and synaptogenesis, resulting in cognitive impairment in the developing animals. RhoA signal pathway plays important roles in dendritic development by regulating cytoskeleton protein such as tubulin and actin. However, it’s not clear whether RhoA pathway is involved in inhaled general anesthetics sevoflurane-induced synaptic development abnormalities and long-term cognitive dysfunction. Rats at postnatal day 7 (PND7) were injected intraperitoneally with RhoA pathway inhibitor Y27632 or saline 20 min before exposed to 2.8% sevoflurane for 4 h. The apoptosis-related proteins and RhoA/CRMP2 pathway proteins in the hippocampus were measured 6 h after sevoflurane exposure. Cognitive functions were evaluated by the open field test on PND25 rats and contextual fear conditioning test on PND32-33 rats. The dendritic morphology and density of dendritic spines in the pyramidal neurons of hippocampus were determined by Golgi staining and the synaptic plasticity-related proteins were also measured on PND33 rats. Long term potentiation (LTP) from hippocampal slices was recorded on PND34-37 rats. Sevoflurane induced caspase-3 activation, decreased the ratio of Bcl-2/Bax and increased TUNEL-positive neurons in hippocampus of PND7 rats, which were attenuated by inhibition of RhoA. However, sevoflurane had no significant effects on activity of RhoA/CRMP2 pathway. Sevoflurane disturbed dendritic morphogenesis, reduced the number of dendritic spines, decreased proteins expression of PSD-95, drebrin and synaptophysin, inhibited LTP in hippocampal slices and impaired memory ability in the adolescent rats, while inhibition of RhoA activity did not rescue the changes above induced by sevoflurane. RhoA signal pathway did not participate in sevoflurane-induced dendritic and synaptic development abnormalities and cognitive dysfunction in developing rats.

     

EphB receptors regulate dendritic spine morphogenesis through the recruitment/phosphorylation of focal adhesion kinase and RhoA activation

Dendritic filopodia are small protrusions on the surface of neuronal dendrites that transform into dendritic spines upon synaptic contact with axon terminals. The formation of dendritic spines is a critical aspect of synaptic development. Dendritic spine morphogenesis is characterized by filopodia shortening followed by the formation of mature mushroom-shaped spines. Here we show that activation of the EphB receptor tyrosine kinases in cultured hippocampal neurons by their ephrinB ligands induces morphogenesis of dendritic filopodia into dendritic spines. This appears to occur through assembly of an EphB-associated protein complex that includes focal adhesion kinase (FAK), Src, Grb2, and paxillin and the subsequent activations of FAK, Src, paxillin, and RhoA. Furthermore, Cre-mediated knock-out of loxP-flanked fak or RhoA inhibition blocks EphB-mediated morphogenesis of dendritic filopodia. Finally, EphB-mediated RhoA activation is disrupted by FAK knock-down. These data suggest that EphB receptors are upstream regulators of FAK in dendritic filopodia and that FAK-mediated RhoA activation contributes to assembly of actin filaments in dendritic spines.     

Different Rho GTPase–dependent signaling pathways initiate sequential steps in the consolidation of long-term potentiation

The releasable factor adenosine blocks the formation of long-term potentiation (LTP). These experiments used this observation to uncover the synaptic processes that stabilize the potentiation effect. Brief adenosine infusion blocked stimulation-induced actin polymerization within dendritic spines along with LTP itself in control rat hippocampal slices but not in those pretreated with the actin filament stabilizer jasplakinolide. Adenosine also blocked activity-driven phosphorylation of synaptic cofilin but not of synaptic p21-activated kinase (PAK). A search for the upstream origins of these effects showed that adenosine suppressed RhoA activity but only modestly affected Rac and Cdc42. A RhoA kinase (ROCK) inhibitor reproduced adenosine''s effects on cofilin phosphorylation, spine actin polymerization, and LTP, whereas a Rac inhibitor did not. However, inhibitors of Rac or PAK did prolong LTP''s vulnerability to reversal by latrunculin, a toxin which blocks actin filament assembly. Thus, LTP induction initiates two synaptic signaling cascades: one (RhoA-ROCK-cofilin) leads to actin polymerization, whereas the other (Rac-PAK) stabilizes the newly formed filaments.     

Neuropeptide orphanin FQ inhibits dendritic morphogenesis through activation of RhoA

Brain‐derived neurotrophic factor (BDNF) plays a facilitatory role in neuronal development and promotion of differentiation. Mechanisms that oppose BDNF's stimulatory effects create balance and regulate dendritic growth. However, these mechanisms have not been studied. We have focused our studies on the BDNF‐induced neuropeptide OrphaninFQ/ Nociceptin (OFQ); while BDNF is known to enhance synaptic activity, OFQ has opposite effects on activity, learning, and memory. We have now examined whether OFQ provides a balance to the stimulatory effects of BDNF on neuronal differentiation in the hippocampus. Golgi staining in OFQ knockout (KO) mice revealed an increase in primary dendrite length as well as spine density, suggesting that endogenous OFQ inhibits dendritic morphology. We have also used cultured hippocampal neurons to demonstrate that exogenous OFQ has an inhibitory effect on dendritic growth and that the neuropeptide alters the response to BDNF when pre‐administered. To determine if BDNF and OFQ act in a feedback loop, we inhibited the actions of the BDNF and OFQ receptors, TrkB and NOP using ANA‐12 and NOP KO mice respectively but our data suggest that the two factors do not act in a negative feedback loop. We found that the inhibition of dendritic morphology induced by OFQ is via enhanced RhoA activity. Finally, we have evidence that RhoA activation is required for the inhibitory effects of OFQ on dendritic morphology. Our results reveal basic mechanisms by which neurons not only regulate the formation of proper dendritic growth during development but also control plasticity in the mature nervous system. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 769–784, 2013     

Signaling mechanisms mediating BDNF modulation of memory formation in vivo in the hippocampus

Given that brain-derived neutrophic factor (BDNF) modulates both short-term synaptic function and activity-dependent synaptic plasticity in the adult hippocampus, here we examined signaling mechanisms in vivo in the hippocampus mediating BDNF modulation of long-term memory (LTM) formation of a one-trial fear-motivated learning task in rats. Bilateral infusions of function-blocking anti-BDNF antibody into the CA1 region of the dorsal hippocampus decreased extracellular-signal regulated kinase 2 (ERK2) and CREB activation and impaired LTM retention scores. Inhibition of ERK1/2 activation by PD098059 produced similar effects and also reduced CREB phosphorylation. In contrast, intrahippocampal administration of recombinant human BDNF increased ERK1/2 and CREB activation and facilitated LTM. Activated-p38, activated-PKC isoforms, and activated-AKT were unaltered after BDNF or anti-BDNF infusion. In addition, no changes were found on PKA and PKA catalytic subunits in nuclear samples. Thus, our results suggest that BDNF exerts its role in LTM formation in vivo in CA1 region of the hippocampus, at least in part, via CREB activation. Moreover, BDNF-induced CREB activation appears to be mediated mainly through the activation of ERK1/2 signaling pathway.     

Brain-derived Neurotrophic Factor (BDNF)-induced Mitochondrial Motility Arrest and Presynaptic Docking Contribute to BDNF-enhanced Synaptic Transmission

Appropriate mitochondrial transport and distribution are essential for neurons because of the high energy and Ca²⁺ buffering requirements at synapses. Brain-derived neurotrophic factor (BDNF) plays an essential role in regulating synaptic transmission and plasticity. However, whether and how BDNF can regulate mitochondrial transport and distribution are still unclear. Here, we find that in cultured hippocampal neurons, application of BDNF for 15 min decreased the percentage of moving mitochondria in axons, a process dependent on the activation of the TrkB receptor and its downstream PI3K and phospholipase-Cγ signaling pathways. Moreover, the BDNF-induced mitochondrial stopping requires the activation of transient receptor potential canonical 3 and 6 (TRPC3 and TRPC6) channels and elevated intracellular Ca²⁺ levels. The Ca²⁺ sensor Miro1 plays an important role in this process. Finally, the BDNF-induced mitochondrial stopping leads to the accumulation of more mitochondria at presynaptic sites. Mutant Miro1 lacking the ability to bind Ca²⁺ prevents BDNF-induced mitochondrial presynaptic accumulation and synaptic transmission, suggesting that Miro1-mediated mitochondrial motility is involved in BDNF-induced mitochondrial presynaptic docking and neurotransmission. Together, these data suggest that mitochondrial transport and distribution play essential roles in BDNF-mediated synaptic transmission.     

Microarray analysis of cultured rat hippocampal neurons treated with brain derived neurotrophic factor

Brain derived neurotrophic factor (BDNF) has been shown to exert multiple actions on neurons. It plays a role in neuronal growth and maintenance and use-dependent plasticity, such as long-term potentiation and learning. This neurotrophin is believed to regulate neuronal plasticity by modifying neuronal excitability and morphology. There is experimental evidence for both an acute and a long-term effect of BDNF on synaptic transmission and structure but the molecular mechanisms underlying these events have not been completely clarified. In order to study the BDNF-induced molecular changes, the set of genes modulated in cultured hippocampal neurons by BDNF treatment was investigated after subchronic treatment with the neurotrophin. Microarray analysis performed with these cells, revealed increased expression of mRNA encoding the neuropeptides neuropeptide Y and somatostatin, and of the secreted peptide VGF (non acronymic), all of which participate in neurotransmission. In addition, the expression of genes apolipoprotein E (ApoE), delta-6 fatty acid desaturase (Fads2) and matrix metalloproteinase 14 (Mmp14), which play a role in neuronal remodelling, was also enhanced. More studies are needed to investigate and confirm the role of these genes in synaptic plasticity, but the results reported in this paper show that microarray analysis of hippocampal cultures can be used to expand our current knowledge of the molecular events triggered by BDNF in the hippocampus.     

Bidirectional regulation of hippocampal mossy fiber filopodial motility by kainate receptors: a two-step model of synaptogenesis

The rapid motility of axonal filopodia and dendritic spines is prevalent throughout the developing CNS, although the function of this motility remains controversial. Using two-photon microscopy, we imaged hippocampal mossy fiber axons in slice cultures and discovered that filopodial extensions are highly motile. Axonal filopodial motility is actin based and is downregulated with development, although it remains in mature cultures. This motility is correlated with free extracellular space yet is inversely correlated with contact with postsynaptic targets, indicating a potential role in synaptogenesis. Filopodial motility is differentially regulated by kainate receptors: synaptic stimulation of kainate receptors enhances motility in younger slices, but it inhibits it in mature slices. We propose that neuronal activity controls filopodial motility in a developmentally regulated manner, in order to establish synaptic contacts in a two-step process. A two-step model of synaptogenesis can also explain the opposite effects of neuronal activity on the motility of dendritic protrusions.     

Geranylgeranyltransferase I mediates BDNF‐induced synaptogenesis

Geranylgeranyltransferase I (GGT) is a prenyltransferase that mediates lipid modification of Rho small GTPases, such as Rho, Rac, and Cdc42, which are important for neuronal synaptogenesis. Although GGT is expressed in brain extensively, the function of GGT in central nerves system is largely unknown so far. We have previously demonstrated that GGT promotes the basal and neuronal activity and brain‐derived neurotrophic factor (BDNF)‐induced dendritic morphogenesis of cultured hippocampal neurons and cerebellar slices. This study is to explore the function and mechanism of GGT in neuronal synaptogenesis. We found that the protein level and activity of GGT gradually increased in rat hippocampus from P7 to P28 and subcellular located at synapse of neurons. The linear density of Synapsin 1 and post‐synaptic density protein 95 increased by over‐expression of GGT β, while reduced by inhibition or down‐regulation of GGT. In addition, GGT and its known substrate Rac was activated by BDNF, which promotes synaptogenesis in cultured hippocampal neurons. Furthermore, BDNF‐induced synaptogenesis was eliminated by GGT inhibition or down‐regulation, as well as by non‐prenylated Rac1 over‐expression. Together, our data suggested that GGT mediates BDNF‐induced neuronal synaptogenesis through Rac1 activation.     

Pragmin, a novel effector of Rnd2 GTPase, stimulates RhoA activity

The Rho family of small GTPases has been implicated in the reorganization of actin cytoskeleton and subsequent morphological changes in various cells. Rnd2 is a member of the Rnd subfamily, comprising Rnd1, Rnd2, and Rnd3. In contrast to Rnd1 and Rnd3, displaying an antagonistic action for RhoA signaling, signaling pathways of Rnd2 are not well known. Here we have performed a yeast two-hybrid screen using Rnd2 as bait and identified a novel Rnd2 effector protein, predominantly expressed in neurons, including cortical and hippocampal neurons. We named it Pragmin (pragma of Rnd2). In in vivo and in vitro binding assays, Pragmin specifically binds to Rnd2 among the Rho family GTPases in a GTP-dependent manner. Rnd2-bound Pragmin significantly stimulates RhoA activity and induces cell contraction through RhoA and the Rho-kinase pathway in HeLa cells. In PC12 cells, expressing Pragmin inhibits nerve growth factor-induced neurite outgrowth in response to Rnd2, and knock-down of Pragmin by Pragmin-specific small interfering RNA enhances neurite elongation. Therefore, Rnd2 regulates neurite outgrowth by functioning as the RhoA activator through Pragmin, in contrast to Rnd1 and Rnd3 inhibiting RhoA signaling.     

Neuronal Synapse Formation Induced by Microglia and Interleukin 10

Recently, it was found that microglia regulated synaptic remodeling of the developing brain, but their mechanisms have not been well understood. In this study, the action of microglia on neuronal synapse formation was investigated, and the primary target of microglial processes was discovered. When the developing microglia were applied to cultured hippocampal neurons without direct contact, the numbers of dendritic spines and excitatory and inhibitory synapses significantly increased. In order to find out the main factor for synaptic formation, the effects of cytokines released from microglia were examined. When recombinant proteins of cytokines were applied to neuronal culture media, interleukin 10 increased the numbers of dendritic spines in addition to excitatory and inhibitory synapses. Interestingly, without external stimuli, the amount of interleukin 10 released from the intact microglia appeared to be sufficient for the induction of synaptic formation. The neutralizing antibodies of interleukin 10 receptors attenuated the induction of the synaptic formation by microglia. The expression of interleukin 10 receptor was newly found in the hippocampal neurons of early developmental stage. When interleukin 10 receptors on the hippocampal neurons were knocked down with specific shRNA, the induction of synaptic formation by microglia and interleukin 10 disappeared. Pretreatment with lipopolysaccharide inhibited microglia from inducing synaptic formation, and interleukin 1β antagonized the induction of synaptic formation by interleukin 10. In conclusion, the developing microglia regulated synaptic functions and neuronal development through the interactions of the interleukin 10 released from the microglia with interleukin 10 receptors expressed on the hippocampal neurons.     

Protein kinase D promotes plasticity-induced F-actin stabilization in dendritic spines and regulates memory formation

Actin turnover in dendritic spines influences spine development, morphology, and plasticity, with functional consequences on learning and memory formation. In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways. Using in vitro models of neuronal plasticity, such as glycine-induced chemical long-term potentiation (LTP), known to evoke synaptic plasticity, or long-term depolarization block by KCl, leading to homeostatic morphological changes, we show that actin stabilization needed for the enlargement of dendritic spines is dependent on PKD activity. Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation. We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines.     

Important role of matrix metalloproteinase 9 in epileptogenesis

Temporal lobe epilepsy (TLE) is a devastating disease in which aberrant synaptic plasticity plays a major role. We identify matrix metalloproteinase (MMP) 9 as a novel synaptic enzyme and a key pathogenic factor in two animal models of TLE: kainate-evoked epilepsy and pentylenetetrazole (PTZ) kindling-induced epilepsy. Notably, we show that the sensitivity to PTZ epileptogenesis is decreased in MMP-9 knockout mice but is increased in a novel line of transgenic rats overexpressing MMP-9. Immunoelectron microscopy reveals that MMP-9 associates with hippocampal dendritic spines bearing asymmetrical (excitatory) synapses, where both the MMP-9 protein levels and enzymatic activity become strongly increased upon seizures. Further, we find that MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber sprouting. The latter observation provides a possible mechanistic basis for the effect of MMP-9 on epileptogenesis. Our work suggests that a synaptic pool of MMP-9 is critical for the sequence of events that underlie the development of seizures in animal models of TLE.     

Chondroitin sulfate proteoglycans down-regulate spine formation in cortical neurons by targeting tropomyosin-related kinase B (TrkB) protein

Chondroitin sulfate proteoglycans (CSPGs) are components of the extracellular matrix that inhibit axonal sprouting and experience-dependent plasticity. Although protein-tyrosine phosphatase σ (PTPσ) has been proven to be a receptor for CSPGs, its downstream signaling has remained a mystery. Here, we show that CSPGs target and dephosphorylate tropomyosin-related kinase B, the receptor of brain-derived neurotrophic factor (BDNF), via PTPσ in embryonic cortical neurons in vitro. Whereas BDNF promoted dendritic spine formation in embryonic cortical neurons, CSPGs abolished the effects of BDNF and eliminated existing dendritic spines when BDNF was present. The latter effect was dependent on the p75 receptor, presumably because BDNF binding to the p75 receptor elicits elimination of dendritic spines. These results suggest that the inhibitory activity of CSPGs on dendritic spine formation operates through the targeting of neurotrophins at the receptor level.     

Ectodomain shedding of nectin-1 regulates the maintenance of dendritic spine density

Synaptic remodeling has been postulated as a mechanism underlying synaptic plasticity and cell adhesion molecules are thought to contribute to this process. We examined the role of nectin-1 ectodomain shedding on synaptogenesis in cultured rat hippocampal neurons. Nectins are Ca(2+) -independent immunoglobulin-like adhesion molecules, involved in cell-cell adherens junctions. Herein, we show that the processing of nectin-1 occurs by multiple endoproteolytic steps both in vivo and in vitro. We identified regions containing two distinct cleavage sites within the ectodomain of nectin-1. By alanine scanning mutagenesis, two point mutations that disrupt nectin-1 ectodomain cleavage events were identified. Expression of these mutants significantly alters the density of dendritic spines. These findings suggest that ectodomain shedding of nectin-1 regulates dendritic spine density and related synaptic functions.