Synapse Formation In Hippocampal Neurons Research Articles

Activity-regulated E3 ubiquitin ligase TRIM47 modulates excitatory synapse development.

The Ubiquitin Proteasome System (UPS) has been shown to regulate neuronal development and synapse formation. Activity-dependent regulation of E3 ligase, a component of the UPS that targets specific proteins for proteasome-mediated degradation, is emerging as a pivotal player for the establishment of functional synapses. Here, we identified TRIM47 as a developmentally regulated E3 ligase that is expressed in rat hippocampus during the temporal window of synapse formation. We have demonstrated that the expression of TRIM47 is regulated by the glutamate-induced synaptic activity of hippocampal neurons in culture. In addition, the activity-dependent enhancement of TRIM47 expression is recapitulated following the object location test, a hippocampus-dependent spatial memory paradigm. We observed that this enhancement of TRIM47 expression requires NMDA receptor activation. The knockdown of TRIM47 leads to an enhancement of spine density without affecting dendritic complexity. Furthermore, we observed an increase in excitatory synapse development upon loss of TRIM47 function. Comprehensively, our study identified an activity-regulated E3 ligase that drives excitatory synapse formation in hippocampal neurons.

Glyphosate exposure induces synaptic impairment in hippocampal neurons and cognitive deficits in developing rats.

Glyphosate is the active ingredient of several widely used herbicide formulations. Studies based on Glyphosate exposure in different experimental models have suggested that the nervous system represented a key target for its toxicity. Previously, we demonstrated that exposure to glyphosate during gestation induces deficits on behavioral and cognitive function in rats. The aim of the present work was to examine whether cognitive dysfunction induced by Glyphosate was connected to changes on synapse formation and maturation. To understand how glyphosate affects synaptic assembly, we performed in vitro assays on cultured hippocampal neurons that were exposed to the herbicide (0.5 or 1mg/mL) for 5 or 10days. Biochemical and immunocytochemical approaches revealed that Glyphosate treated neurons showed a decrease on dendritic complexity and synaptic spine formation and maturation. Moreover, results indicated that Glyphosate decreased synapse formation in hippocampal neurons. To evaluate these effects in vivo, pup rats were treated with 35 or 70mg/kg of Glyphosate from PND 7 to PND 27, every 48h. Results indicated that Glyphosate postnatal exposure induced cognitive impairments, since recognition and spatial memory were altered. To go further, we evaluated synaptic protein expression and synaptic organization in hippocampus. Images revealed that Glyphosate treatment downregulates synapsin-1, PSD-95, and CaMKII expression, and also decreased PSD-95 clustering in hippocampus. Taken together, these findings demonstrate for the first time that Glyphosate exposure affects synaptic assembly and reduced synaptic protein expression in hippocampus and that likely triggers the impairment of cognitive function and neuronal connectivity.

Huntingtin-Interacting Protein 1-Related Protein Plays a Critical Role in Dendritic Development and Excitatory Synapse Formation in Hippocampal Neurons.

Huntingtin-interacting protein 1-related (HIP1R) protein is considered to be an endocytic adaptor protein like the other two members of the Sla2 family, Sla2p and HIP1. They all contain homology domains responsible for the binding of clathrin, inositol lipids and F-actin. Previous studies have revealed that HIP1R is highly expressed in different regions of the mouse brain and localizes at synaptic structures. However, the function of HIP1R in the nervous system remains unknown. In this study, we investigated HIP1R function in cultured rat hippocampal neurons using an shRNA knockdown approach. We found that, after HIP1R knockdown, the dynamics and density of dendritic filopodia, and dendritic branching and complexity were significantly reduced in developing neurons, as well as the densities of dendritic spines and PSD95 clusters in mature neurons. Moreover, HIP1R deficiency led to significantly reduced expression of the ionotropic glutamate receptor GluA1, GluN2A and GluN2B subunits, but not the GABAA receptor α1 subunit. Similarly, HIP1R knockdown reduced the amplitude and frequency of the miniature excitatory postsynaptic current, but not of the miniature inhibitory postsynaptic current. In addition, the C-terminal proline-rich region of HIP1R responsible for cortactin binding was found to confer a dominant-negative effect on dendritic branching in cultured developing neurons, implying a critical role of cortactin binding in HIP1R function. Taken together, the results of our study suggest that HIP1R plays important roles in dendritic development and excitatory synapse formation and function.

The Therapeutic Potential of mGlu3 Antagonism in Correcting Hippocampal Long‐term Depression Impairments in a Mouse Model of MECP2 Duplication Syndrome

Precise levels of the transcriptional regulator Methyl CpG Binding Protein 2 (MeCP2) are known to be a key determinant for proper neurological function. Mutations in the MECP2 gene results in a disease known as Rett syndrome; in contrast, genetic duplication of MECP2 leads to a disorder known as MECP2 Duplication syndrome. The latter genetic neurodevelopmental disorder has a wide symptomology including stereotypy, seizures, cognitive deficits, and autism. In rodent models of MECP2 Duplication Syndrome in which MeCP2 is overexpressed, increases in glutamatergic synapse formation in hippocampal neurons occur, as well as impairments in hippocampal synaptic plasticity, a neurological phenomenon thought to be involved in memory and cognition. In accordance with this notion, MeCP2 overexpression causes memory and cognitive deficits in mice. Interestingly, the metabotropic glutamate receptor 3 (mGlu3) has been shown to be involved in regulating synaptic plasticity and cognitive behaviors in mice, and mGlu3 expression appears to correlate with MeCP2 expression levels in the brain. We hypothesized that that an upregulation of mGlu3 leads to an impairment in synaptic plasticity and cognitive deficits in MeCP2 overexpressing mice. To investigate the mechanisms by which MeCP2 overexpression induces neurophysiological impairments, a MeCP2‐Tg1 mouse model was used. Our results indicate that mGlu3 is upregulated the hippocampus of MeCP2‐Tg1 mice, correlating with an increase in MeCP2 protein expression. Electrophysiological experiments found that paired‐pulse facilitation was enhanced and input‐output intensity was reduced at the SC‐CA1 hippocampal synapse in the MeCP2‐Tg1 mice compared to wild‐type controls. Furthermore, (S)‐3,5‐Dihydroxyphenylglycine (DHPG) induced‐long term depression (LTD) was impaired in MeCP2‐Tg1 mice. Interestingly, an mGlu3 negative allosteric modulator, VU0650786, was capable of normalizing DHPG‐induced LTD to the level of that observed in wild‐type controls. These results suggest that overexpression of mGlu3 may contribute to synaptic plasticity impairments within the hippocampus of MeCP2‐Tg1 mice. Future experiments are aimed at elucidating behavioral correlates of these neurophysiological abnormalities and their underlying mechanisms.Support or Funding InformationT32NS007491 (BJS), rettsyndrome.org postdoctoral fellowship (RGG), Weatherstone predoctoral fellowship (BKS), T32 GM07628 (NF), R01 MH099269 (KAE), U54 MH084659 (CWL), R01 NS031373 (PJC), R21 MH102548 (CMN), Autism Speaks Treatment Award (CMN).

Urocortin 2 But Not Urocortin 3 Promotes the Synaptic Formation in Hipppocampal Neurons via Induction of NGF Production by Astrocytes.

CRH family peptides play differential role during various physiological and pathophysiological responses, such as stress. Urocortins (UCNs) have been implicated to play complementary or contrasting actions for the effects of CRH during stress. It has been shown that activation of CRH receptor type 1 (CRHR1) results in decreased synapse formation in hippocampus. We therefore explored the effect of UCN2 and UCN3, the exclusive CRHR2 agonists, on synaptic formation in hippocampus. In hippocampal slices cultures, UCN2 but not UCN3 treatment increased the levels of presynaptic protein synapsinI and postsynaptic protein postsynaptic density 95 (PSD95), which was reversed by CRHR2 antagonist astressin 2B. In isolated hippocampal neurons, however, UCN2 decreased the numbers of synapsinI- and PSD95-labeled terminals/clusters via CRHR2. Treatment of hippocampal neurons with the media of UCN2-treated astrocytes led to an increase in synapsinI- and PSD95-labeled terminals. In neuron-astrocyte cocultures, UCN2 also enhanced the numbers and level of synapsinI- and PSD95-labeled terminals. These effects did not occur if glial cells were transfected with CRHR2 small interfering RNA. UCN2 but not UCN3 treatment induced nerve growth factor (NGF) production in astrocytes via CRHR2. The effects of the media of UCN2-treated glial cells on synapse formation in hippocampal neurons were prevented by administration of NGF receptor antagonists. Our data indicate that UCN2 promotes synapse formation in hippocampus via induction of NGF secretion from astrocytes. CRHR2 in glial cells mediates the stimulatory effects of CRH. Glia-neuron communication is critical for neuronal circuits remodeling and synaptic plasticity in response to neurohormones or neuromodulators.

Flotillin-1 is an evolutionary-conserved memory-related protein up-regulated in implicit and explicit learning paradigms

Introduction. Studies of synaptic plasticity using the marine mollusk Aplysia californica as model system have been successfully used to identify proteins involved in learning and memory. The importance of molecular elements regulated by the learning- related neurotransmitter serotonin in Aplysia can then be explored in rodent models and finally tested for their relevance for human physiology and pathology.Materials and methods. Herein, 2-DE gel-based electrophoresis has been used to investigate protein level changes after treatment with serotonin in Aplysia abdominal ganglia.Results. Twenty-one proteins have been found to be regulated by serotonin, and protein level changes of actin depolymerizing factor (ADF), deleted in azoospermia associated protein (DAZAP-1), and Flotillin-1 have been verified by Western blotting.Discussion. Flotillin-1, a member of the flotillin/reggie family of scaffolding proteins, has been previously found to be involved in neuritic branching and synapse formation in hippocampal neurons in vitro. However, its importance for hippocampal- dependent learning and memory in the mouse has not been examined. Here, elevated levels of Flotillin-1 in hippocampal tissue of mice trained in the Morris water maze confirmed the relevance of Flotillin-1 for memory-related processes in a mammalian system. Thus, a translational approach—from invertebrates to rodents—led to the identification of Flotillin-1 as evolutionary-conserved memory-related protein.

Wnt-5a Is a Synaptogenic Factor with Neuroprotective Properties against Aβ Toxicity

Background: We have recently found that Wnt-5a regulates the synaptic structure and function in hippocampal neurons. This ligand is expressed in the hippocampus, stimulates dendritic spine morphogenesis and increases glutamatergic neurotransmission. Moreover, we have also shown that Wnt-5a induces the clustering of PSD-95. Objective: To explore the role of Wnt-5a in the formation of synaptic contacts. Methods: Primary rat hippocampal neurons were exposed to a formylated hexapeptide (Foxy-5) derived from the sequence of Wnt-5a to study synapse formation and function. Results: In short-term experiments, Wnt-5a only induced the clustering of PSD-95 but had no effect on the density of presynaptic puncta, while in long-term experiments, it induced both pre- and postsynaptic protein clustering and the number of synaptic contacts, in agreement with electrophysiological studies. In long-term experiments, Foxy-5 increased miniature excitatory postsynaptic current amplitude and frequency. Conclusion: Our findings indicate that Wnt-5a induces synapse formation in hippocampal neurons. In addition, we discuss recent findings indicating a neuroprotective action of Wnt-5a against Aβ neurotoxicity.

A versatile valve-enabled microfluidic cell co-culture platform and demonstration of its applications to neurobiology and cancer biology

A versatile microfluidic platform allowing co-culture of multiple cell populations in close proximity with separate control of their microenvironments would be extremely valuable for many biological applications. Here, we report a simple and compact microfluidic platform that has these desirable features and allows for real-time, live-cell imaging of cell-cell interactions. Using a pneumatically/hydraulically controlled poly(dimethylsiloxane) (PDMS) valve barrier, distinct cell types can be cultured in side-by-side microfluidic chambers with their optimum culture media and treated separately without affecting the other cell population. The platform is capable of both two-dimensional and three-dimensional cell co-culture and through variations of the valve barrier design, the platform allows for cell-cell interactions through either direct cell contact or soluble factors alone. The platform has been used to perform dynamic imaging of synapse formation in hippocampal neurons by separate transfection of two groups of neurons with fluorescent pre- and post-synaptic protein markers. In addition, cross-migration of 4T1 tumor cells and endothelial cells has been studied under normoxic and hypoxic conditions, which revealed different migration patterns, suggesting the importance of the microenvironments in cell-cell interactions and biological activities.

An APPL1/Akt signaling complex regulates dendritic spine and synapse formation in hippocampal neurons

The formation and plasticity of dendritic spines and synapses, which are poorly understood on a molecular level, are critical for cognitive functions, such as learning and memory. The adaptor protein containing a PH domain, PTB domain, and leucine zipper motif (APPL1) is emerging as a critical regulator of various cellular processes in non-neuronal cells, but its function in the nervous system is not well understood. Here, we show that APPL1 localizes to dendritic spines and synapses and regulates the development of these structures in hippocampal neurons. Knockdown of endogenous APPL1 using siRNA led to a significant decrease in the number of spines as well as synapses and this defect could be rescued by expression of siRNA-resistant APPL1. Expression of exogenous APPL1 increased the spine and synaptic density and the amount of surface GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). Deletion of the C-terminal phosphotyrosine binding domain of APPL1, which binds the serine/threonine kinase Akt, resulted in a significant decrease in the spine and synaptic density, suggesting a role for Akt in regulating the development of these structures. Consistent with this, knockdown of Akt with siRNA or expression of dominant negative Akt led to a dramatic decrease in spine and synapse formation. In addition, APPL1 increased the amount of active Akt in spines and synapses and the effects of APPL1 on these structures were dependent on Akt, indicating that Akt is an effector of APPL1 in the regulation of these processes. Moreover, APPL1 signaling modulates spine and synapse formation through p21-activated kinase (PAK). Thus, our results indicate that APPL1 signaling through Akt and PAK is critical for spine and synaptic development and point to a role for APPL1 and its effectors in regulating cognitive function.

Synapse formation regulated by protein tyrosine phosphatase receptor T through interaction with cell adhesion molecules and Fyn

The receptor-type protein tyrosine phosphatases (RPTPs) have been linked to signal transduction, cell adhesion, and neurite extension. PTPRT/RPTPrho is exclusively expressed in the central nervous system and regulates synapse formation by interacting with cell adhesion molecules and Fyn protein tyrosine kinase. Overexpression of PTPRT in cultured neurons increased the number of excitatory and inhibitory synapses by recruiting neuroligins that interact with PTPRT through their ecto-domains. In contrast, knockdown of PTPRT inhibited synapse formation and withered dendrites. Incubation of cultured neurons with recombinant proteins containing the extracellular region of PTPRT reduced the number of synapses by inhibiting the interaction between ecto-domains. Synapse formation by PTPRT was inhibited by phosphorylation of tyrosine 912 within the membrane-proximal catalytic domain of PTPRT by Fyn. This tyrosine phosphorylation reduced phosphatase activity of PTPRT and reinforced homophilic interactions of PTPRT, thereby preventing the heterophilic interaction between PTPRT and neuroligins. These results suggest that brain-specific PTPRT regulates synapse formation through interaction with cell adhesion molecules, and this function and the phosphatase activity are attenuated through tyrosine phosphorylation by the synaptic tyrosine kinase Fyn.

IgLON cell adhesion molecules regulate synaptogenesis in hippocampal neurons

IgLON cell adhesion molecules (CAMs) belonging to the immunoglobulin superfamily comprise of LAMP, neurotrimin (Ntm), OBCAM, and Kilon. In the present study, we performed the single and double transfection of IgLON gene constructs into hippocampal neurons in vitro and evaluated synaptic number. The quantitative analysis showed that the single over-expression of LAMP or OBCAM increased synaptic number, while the over-expression of Kilon reduced synaptic number and Ntm had no effects. The double over-expression of Kilon-Ntm, Kilon-OBCAM, LAMP-Ntm, and Ntm-OBCAM decreased synaptic number and that of Kilon-LAMP and LAMP-OBCAM had no effect. These results suggest that IgLON CAMs participate in regulating synapse formation in hippocampal neurons.

α5 Integrin Signaling Regulates the Formation of Spines and Synapses in Hippocampal Neurons

The actin-based dynamics of dendritic spines play a key role in synaptic plasticity, which underlies learning and memory. Although it is becoming increasingly clear that modulation of actin is critical for spine dynamics, the upstream molecular signals that regulate the formation and plasticity of spines are poorly understood. In non-neuronal cells, integrins are critical modulators of the actin cytoskeleton, but their function in the nervous system is not well characterized. Here we show that alpha5 integrin regulates spine morphogenesis and synapse formation in hippocampal neurons. Knockdown of alpha5 integrin expression using small interfering RNA decreased the number of dendritic protrusions, spines, and synapses. Expression of constitutively active or dominant negative alpha5 integrin also resulted in alterations in the number of dendritic protrusions, spines, and synapses. alpha5 integrin signaling regulates spine morphogenesis and synapse formation by a mechanism that is dependent on Src kinase, Rac, and the signaling adaptor GIT1. Alterations in the activity or localization of these molecules result in a significant decrease in the number of spines and synapses. Thus, our results point to a critical role for integrin signaling in regulating the formation of dendritic spines and synapses in hippocampal neurons.

Increased estrogen receptor β expression correlates with decreased spine formation in the rat hippocampus

Estrogens play an important role in the brain function acting through two receptor types, ERalpha and ERbeta, both well-recognized as transcription factors. In this study, we investigated the ERbeta mRNA and protein levels in the rat hippocampus by using two in vivo models that are known to affect synapse formation. Natural estrous-proestrous cycle was used as a model in which a marked decrease in the density of hippocampal synapses was previously observed between proestrus and estrus. We have found that ERbeta mRNA and protein were displayed in high levels in the estrus and in low levels in the proestrous phase. By applying kainic acid (KA) to adult rats, we demonstrated that up-regulation of ERbeta mRNA and protein in hippocampal CA regions was vulnerable to KA-induced excitotoxicity. Furthermore, we note a concomitant decrease of ERbeta in the excitotoxicity-resistant denate gyrus that undergoes intense plastic changes, including synaptogenesis. These data suggested that decreases in ERbeta expression correlated with increase in synapse formation. This notion has been tested in vitro in hippocampal cultures, in which overexpression of ERbeta by means of gene transfection resulted in the lowering of the dendritic spine density that was elevated by estrogen. In summary, our results suggest that ERbeta inhibits synapse formation in hippocampal neurons.

Progesterone-induced agrin expression in astrocytes modulates glia–neuron interactions leading to synapse formation

Experimental evidence recently obtained suggests that synaptogenesis is a tripartite event in which not only pre- and post-synaptic neurons but also glial cells play a key role. However, the molecular mechanisms by which glia modulate the formation of synapses in the CNS remain poorly understood. In the present study, we analyzed the role of astrocytes in synapse formation in cultured hippocampal rat neurons. For these experiments, hippocampal neurons were cultured in the presence or absence of a monolayer of astrocytes. Our results indicated that hippocampal neurons cultured in the presence of astrocytes formed more synapses than the ones cultured in their absence only when kept in N2 serum-free medium. To get insights into the potential molecular mechanisms underlying this effect, we analyzed the expression of proteins known to induce synapse formation in hippocampal neurons. A significant increase in agrin expression was detected in astrocytes cultured in N2 serum-free medium when compared with the ones cultured in serum containing medium. Experiments performed using different components of the N2 mixture indicated that progesterone induced the expression of agrin in astrocytes. Taken collectively, these results provide evidence supporting a role for astrocytes in synapse formation in central neurons. Furthermore, they identified agrin as a potential mediator of this effect, and astrocytes as a bridge between the endocrine and nervous systems during synaptogenesis.

SynGAP regulates spine formation.

SynGAP is a brain-specific ras GTPase-activating protein that is an abundant component of the signaling complex associated with the NMDA-type glutamate receptor. We generated mutant mice lacking synGAP to study its physiological role. Homozygous mutant mice die in the first few days after birth; however, neurons from mutant embryos can be maintained in culture. Here, we report that spine and synapse formation are accelerated in cultured mutant neurons, and the spines of mature mutant neurons are significantly larger than those of wild type. Clusters of PSD-95 and subunits of AMPA-type and NMDA-type glutamate receptors accumulate in spines of mutant neurons by day 10 in vitro, whereas in wild-type neurons they are still mostly located in dendritic shafts. The frequency and amplitude of miniature EPSCs are larger in mutant neurons at day 10 in vitro, confirming that they have more functional synapses. At day 21 in vitro, the spines of mutant neurons remain significantly larger than those of wild type. The mutant phenotype at day 10 in vitro can be rescued by introduction of recombinant wild-type synGAP on day 9. In contrast, introduction of mutant synGAP with a mutated GAP domain or lacking the terminal domain that binds to PSD-95 does not rescue the mutant phenotype, indicating that both domains play a role in control of spine formation. Thus, the GAP activity of synGAP and its association with PSD-95 are important for normal regulation of spine and synapse formation in hippocampal neurons.