Synaptogenic Proteins Research Articles

Astrocyte-secreted neurocan controls inhibitory synapse formation and function

Astrocytes strongly promote the formation and maturation of synapses by secreted proteins. Several astrocyte-secreted synaptogenic proteins controlling excitatory synapse development were identified; however, those that induce inhibitory synaptogenesis remain elusive. Here, we identify neurocan as an astrocyte-secreted inhibitory synaptogenic protein. After secretion from astrocytes, neurocan is cleaved into N- and C-terminal fragments. We found that these fragments have distinct localizations in the extracellular matrix. The neurocan C-terminal fragment localizes to synapses and controls cortical inhibitory synapse formation and function. Neurocan knockout mice lacking the whole protein or only its C-terminal synaptogenic domain have reduced inhibitory synapse numbers and function. Through super-resolution microscopy, invivo proximity labeling by secreted TurboID, and astrocyte-specific rescue approaches, we discovered that the synaptogenic domain of neurocan localizes to somatostatin-positive inhibitory synapses and strongly regulates their formation. Together, our results unveil a mechanism through which astrocytes control circuit-specific inhibitory synapse development in the mammalian brain.

Synaptogenesis by Cholinergic Stimulation of Astrocytes.

Astrocytes release numerous factors known to contribute to the process of synaptogenesis, yet knowledge about the signals that control their release is limited. We hypothesized that neuron-derived signals stimulate astrocytes, which respond to neurons through the modulation of astrocyte-released synaptogenic factors. Here we investigate the effect of cholinergic stimulation of astrocytes on synaptogenesis in co-cultured neurons. Using a culture system where primary rat astrocytes and primary rat neurons are first grown separately allowed us to independently manipulate astrocyte cholinergic signaling. Subsequent co-culture of pre-stimulated astrocytes with naïve neurons enabled us to assess how prior stimulation of astrocyte acetylcholine receptors uniquely modulates neuronal synapse formation. Pre-treatment of astrocytes with the acetylcholine receptor agonist carbachol increased the expression of synaptic proteins, the number of pre- and postsynaptic puncta, and the number of functional synapses in hippocampal neurons after 24h in co-culture. Astrocyte secretion of the synaptogenic protein thrombospondin-1 increased after cholinergic stimulation and inhibition of the receptor for thrombospondins prevented the increase in neuronal synaptic structures. Thus, we identified a novel mechanism of neuron-astrocyte-neuron communication, where neuronal release of acetylcholine stimulates astrocytes to release synaptogenic proteins leading to increased synaptogenesis in neurons. This study provides new insights into the role of neurotransmitter receptors in developing astrocytes and into our understanding of the modulation of astrocyte-induced synaptogenesis.

Observing single dynamin-mediated fission events in TIRFM-amenable synapses.

The presynapse features a unique tight coupling of exo- and endocytosis resulting in the formation of synaptic vesicles with uniform morphology. The difficulty in accessing the presynapse has hampered attempts to directly visualize the functionality of presynaptic proteins which enable such exquisite pairing. To this end our lab has developed “Xenapses”, TIRFM-able synapses, formed by mouse hippocampal neurons cultured on micropatterned host substrates coated with synaptogenic proteins. Xenapses show all the characteristics of a synapse and allow us to directly observe the behavior of proteins involved in compensatory endocytosis. Xenapses lend themselves to unroofing, revealing via SEM the membrane surface to be decorated with preassembled clathrin structures, concomitant with a pre-sorted and pre-assembled readily retrievable pool (RRetP) of endocytic structures. We have further combined this preparation with CRISPR-Cas9 to endogenously label dynamin I. This has enabled the study of triggered compensatory endocytosis at an unprecedented spatial and temporal resolution. Dynamin-dependent fission occurred as discreet, quantal events, demonstrating the rate of endocytosis to be determined by the number of vesicles retrieved within a unit of time rather than by an intrinsic value. While dynamin was recruited within subsecond timescale following a single action potential, the subsequent assembly and fission, denoted by gradual increase in fluorescence followed by rapid collapse, occurred over many seconds with a peak at 5 seconds at physiological temperature. This casts a shadow over the dynamin-dependent ultrafast endocytic mode proposed to predominate under such conditions. These observations advance dynamin fission as the rate-limiting step in endocytosis, with the preassembled RRetP providing an element of speed advantage for the rapid, high fidelity retrieval of synaptic vesicles. The full potential of our system will be realized by investigating the choreography of the key endocytic proteins relative to dynamin fission.

Instructive roles of astrocytes in hippocampal synaptic plasticity: neuronal activity-dependent regulatory mechanisms.

In the adult hippocampus, synaptic plasticity is important for information processing, learning, and memory encoding. Astrocytes, the most common glial cells, play a pivotal role in the regulation of hippocampal synaptic plasticity. While astrocytes were initially described as a homogenous cell population, emerging evidence indicates that in the adult hippocampus, astrocytes are highly heterogeneous and can differentially respond to changes in neuronal activity in a subregion‐dependent manner to actively modulate synaptic plasticity. In this review, we summarize how local neuronal activity changes regulate the interactions between astrocytes and synapses, either by modulating the secretion of gliotransmitters and synaptogenic proteins or via contact‐mediated signaling pathways. In turn, these specific responses induced in astrocytes mediate the interactions between astrocytes and neurons, thus shaping synaptic communication in the adult hippocampus. Importantly, the activation of astrocytic signaling is required for memory performance including memory acquisition and recall. Meanwhile, the dysregulation of this signaling can cause hippocampal circuit dysfunction in pathological conditions, resulting in cognitive impairment and neurodegeneration. Indeed, reactive astrocytes, which have dysregulated signaling associated with memory, are induced in the brains of patients with Alzheimer's disease (AD) and transgenic mouse model of AD. Emerging technologies that can precisely manipulate and monitor astrocytic signaling in vivo enable the examination of the specific actions of astrocytes in response to neuronal activity changes as well as how they modulate synaptic connections and circuit activity. Such findings will clarify the roles of astrocytes in hippocampal synaptic plasticity and memory in health and disease.

A new co-culture method for identifying synaptic adhesion molecules involved in synapse formation

The proper formation of synapses is essential for brain function. Synaptic cell adhesion molecules (CAMs) are thought to play essential roles in the initiation of the synapse formation process. The artificial synapse formation assay, in which non-neuronal cells and neurons are co-cultured, has been shown to be a powerful system for screening CAMs. However, controlling a large number of cell pools in co-culture is complicated, creating a potential barrier for high-throughput screening. This protocol describes a new co-culture method in which cDNA plasmid is transfected into human embryonic kidney 293T cells using polyetherimide 24 h after cells were mixed with neurons, and immunostaining and confocal imaging are employed for analyzing synaptogenesis. This optimized method is simpler and easier to perform than the traditional method for the examination of the synaptogenic activities of individual cell-surface proteins in isolation, and provides an unbiased screening platform for synaptogenic proteins.

Subretinal Human Umbilical Tissue-Derived Cell Transplantation Preserves Retinal Synaptic Connectivity and Attenuates Müller Glial Reactivity.

Human umbilical tissue-derived cells (hUTC or palucorcel) are currently under clinical investigation for the treatment of geographic atrophy, a late stage of macular degeneration, but how hUTC transplantation mediates vision recovery is not fully elucidated. Subretinal administration of hUTC preserves visual function in the Royal College of Surgeons (RCS) rat, a genetic model of retinal degeneration caused by Mertk loss of function. hUTC secrete synaptogenic and neurotrophic factors that improve the health and connectivity of the neural retina. Therefore, we investigated the progression of synapse and photoreceptor loss and whether hUTC treatment preserves photoreceptors and synaptic connectivity in the RCS rats of both sexes. We found that RCS retinas display significant deficits in synaptic development already by postnatal day 21 (P21), before the onset of photoreceptor degeneration. Subretinal transplantation of hUTC at P21 is necessary to rescue visual function in RCS rats, and the therapeutic effect is enhanced with repeated injections. Synaptic development defects occurred concurrently with morphological changes in Müller glia, the major perisynaptic glia in the retina. hUTC transplantation strongly diminished Müller glia reactivity and specifically protected the α2δ-1-containing retinal synapses, which are responsive to thrombospondin family synaptogenic proteins secreted by Müller glia. Müller glial reactivity and reduced synaptogenesis observed in RCS retinas could be recapitulated by CRISPR/Cas9-mediated loss-of-Mertk in Müller glia in wild-type rats. Together, our results show that hUTC transplantation supports the health of retina at least in part by preserving the functions of Müller glial cells, revealing a previously unknown aspect of hUTC transplantation-based therapy.SIGNIFICANCE STATEMENT Despite the promising effects observed in clinical trials and preclinical studies, how subretinal human umbilical tissue-derived cell (hUTC) transplantation mediates vision improvements is not fully known. Using a rat model of retinal degeneration, the RCS rat (lacking Mertk), here we provide evidence that hUTC transplantation protects visual function and health by protecting photoreceptors and preserving retinal synaptic connectivity. Furthermore, we find that loss of Mertk function only in Müller glia is sufficient to impair synaptic development and cause activation of Müller glia. hUTC transplantation strongly attenuates the reactivity of Müller glia in RCS rats. These findings highlight novel cellular and molecular mechanisms within the neural retina, which underlie disease mechanisms and pinpoint Müller glia as a novel cellular target for hUTC transplantation.

Synapse Elimination Triggered by BMP4 Exocytosis and Presynaptic BMP Receptor Activation

Invitro screening of signaling molecules involved in neural circuit formation has identified a large numberof synaptogenic proteins. However, factors that drive synapse elimination remain elusive. Here, we report that bone morphogenetic protein 4 (BMP4) released from axons has the ability to eliminate synapses. We found fast axonal transport of BMP4 in dense-core vesicles, its exocytosis, and subsequent cell surface clustering via type I BMP receptors near synapses. BMP4 overexpression or knockout in culture reduced or increased presynaptic structures, respectively. The destabilizing effect of surface BMP4 clusters was limited to nearby synapses. Invivo knockout of BMP4 and subsequent two-photon imaging of synapse dynamics confirmed its critical role in maintaining an appropriate density ofpresynaptic components along the axon. These results suggest an essential role for perisynaptic clustering of BMP4 during development in the construction of functional neuronal circuits.

Excitatory Synaptic Drive and Feedforward Inhibition in the Hippocampal CA3 Circuit Are Regulated by SynCAM 1.

Select adhesion proteins control the development of synapses and modulate their structural and functional properties. Despite these important roles, the extent to which different synapse-organizing mechanisms act across brain regions to establish connectivity and regulate network properties is incompletely understood. Further, their functional roles in different neuronal populations remain to be defined. Here, we applied diffusion tensor imaging (DTI), a modality of magnetic resonance imaging (MRI), to map connectivity changes in knock-out (KO) mice lacking the synaptogenic cell adhesion protein SynCAM 1. This identified reduced fractional anisotropy in the hippocampal CA3 area in absence of SynCAM 1. In agreement, mossy fiber refinement in CA3 was impaired in SynCAM 1 KO mice. Mossy fibers make excitatory inputs onto postsynaptic specializations of CA3 pyramidal neurons termed thorny excrescences and these structures were smaller in the absence of SynCAM 1. However, the most prevalent targets of mossy fibers are GABAergic interneurons and SynCAM 1 loss unexpectedly reduced the number of excitatory terminals onto parvalbumin (PV)-positive interneurons in CA3. SynCAM 1 KO mice additionally exhibited lower postsynaptic GluA1 expression in these PV-positive interneurons. These synaptic imbalances in SynCAM 1 KO mice resulted in CA3 disinhibition, in agreement with reduced feedforward inhibition in this network in the absence of SynCAM 1-dependent excitatory drive onto interneurons. In turn, mice lacking SynCAM 1 were impaired in memory tasks involving CA3. Our results support that SynCAM 1 modulates excitatory mossy fiber inputs onto both interneurons and principal neurons in the hippocampal CA3 area to balance network excitability. This study advances our understanding of synapse-organizing mechanisms on two levels. First, the data support that synaptogenic proteins guide connectivity and can function in distinct brain regions even if they are expressed broadly. Second, the results demonstrate that a synaptogenic process that controls excitatory inputs to both pyramidal neurons and interneurons can balance excitation and inhibition. Specifically, the study reveals that hippocampal CA3 connectivity is modulated by the synapse-organizing adhesion protein SynCAM 1 and identifies a novel, SynCAM 1-dependent mechanism that controls excitatory inputs onto parvalbumin-positive interneurons. This enables SynCAM 1 to regulate feedforward inhibition and set network excitability. Further, we show that diffusion tensor imaging is sensitive to these cellular refinements affecting neuronal connectivity.

γ-Aminobutyric Acid Type A (GABAA) Receptor Subunits Play a Direct Structural Role in Synaptic Contact Formation via Their N-terminal Extracellular Domains

The establishment of cell-cell contacts between presynaptic GABAergic neurons and their postsynaptic targets initiates the process of GABAergic synapse formation. GABAA receptors (GABAARs), the main postsynaptic receptors for GABA, have been recently demonstrated to act as synaptogenic proteins that can single-handedly induce the formation and functional maturation of inhibitory synapses. To establish how the subunit composition of GABAARs influences their ability to induce synaptogenesis, a co-culture model system incorporating GABAergic medium spiny neurons and the HEK293 cells, stably expressing different combinations of receptor subunits, was developed. Analyses of HEK293 cell innervation by medium spiny neuron axons using immunocytochemistry, activity-dependent labeling, and electrophysiology have indicated that the γ2 subunit is required for the formation of active synapses and that its effects are influenced by the type of α/β subunits incorporated into the functional receptor. To further characterize this process, the large N-terminal extracellular domains (ECDs) of α1, α2, β2, and γ2 subunits were purified using the baculovirus/Sf9 cell system. When these proteins were applied to the co-cultures of MSNs and α1/β2/γ2-expressing HEK293 cells, the α1, β2, or γ2 ECD each caused a significant reduction in contact formation, in contrast to the α2 ECD, which had no effect. Together, our experiments indicate that the structural role of GABAARs in synaptic contact formation is determined by their subunit composition, with the N-terminal ECDs of each of the subunits directly participating in interactions between the presynaptic and postsynaptic elements, suggesting the these interactions are multivalent and specific.

Robust Type-specific Hemisynapses Induced by Artificial Dendrites.

Type-specificity of synapses, excitatory and inhibitory, regulates information process in neural networks via chemical neurotransmitters. To lay a foundation of synapse-based neural interfaces, artificial dendrites are generated by covering abiotic substrata with ectodomains of type-specific synaptogenic proteins that are C-terminally tagged with biotinylated fluorescent proteins. The excitatory artificial synapses displaying engineered ectodomains of postsynaptic neuroligin-1 (NL1) induce the formation of excitatory presynapses with mixed culture of neurons in various developmental stages, while the inhibitory artificial dendrites displaying engineered NL2 and Slitrk3 induce inhibitory presynapses only with mature neurons. By contrast, if the artificial dendrites are applied to the axonal components of micropatterned neurons, correctly-matched synaptic specificity emerges regardless of the neuronal developmental stages. The hemisynapses retain their initially established type-specificity during neuronal development and maintain their synaptic strength provided live neurons, implying the possibility of durable synapse-based biointerfaces.

New perspectives on the involvement of mTOR in depression as well as in the action of antidepressant drugs.

Despite the revolution in recent decades regarding monoamine involvement in the management of major depressive disorder (MDD), the biological mechanisms underlying this psychiatric disorder are still poorly understood. Currently available treatments require long time courses to establish antidepressant response and a significant percentage of people are refractory to single drug or combination drug treatment. These issues, and recent findings demonstrating the involvement of synaptic plasticity in the pathophysiological mechanisms of MDD, are encouraging researchers to explore the molecular mechanisms underlying psychiatric disease in more depth. The discovery of the rapid antidepressant effect exerted by glutamatergic and cholinergic agents highlights the mammalian target of rapamycin (mTOR) pathway as a critical pathway that contributes to the efficacy of these pharmacological agents in clinical and pre-clinical research. The mTOR pathway is a downstream intracellular signal that transmits information after the direct activation of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) and neurotrophic factor receptors. Activation of these receptors is hypothesized to be one of the major axes involved in the synthesis of synaptogenic proteins underlying synaptic plasticity and critical to both the rapid and delayed effects exerted by classic antidepressants. This review focuses on the involvement of mTOR in the pathophysiology of depression and on molecular mechanisms involved in the activity of emerging and classic antidepressant agents.

The LGI1–ADAM22 protein complex directs synapse maturation through regulation of PSD-95 function

Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95.

Effect of Tocotrienol Rich Fraction (TRF) on synaptogenic molecules in aging Caenorhabdis elegans

Objective: This study was performed to determine the effect of tocotrienol rich fraction (TRF) on the expression of synaptogenic genes and proteins in aging C.elegans . Method: Age synchronized DP132 strain worms were divided into two groups; control and treatment where worms were grown in nematode growth medium enriched with 50 ?g/ml TRF. The RNA and proteins expressions of syd-2, sad-1 and aex-3 were determined at day 2, 8 and 13 by q-RTPCR and western blot respectively. Results: The RNA and proteins expressions of syd-2, sad-1 and aex-3 decreased significantly with age in C.elegans . Interestingly, TRF treatment successfully increased the expression of these molecules from day 2 to day 8 which is known to be the reproductive phase of C.elegans but showed limited effect on the expression of these proteins at day 13. Conclusion: TRF enhances the expression of molecules involved in synaptic function of young C.elegans .

High-content imaging of presynaptic assembly

Presynaptic assembly involves the specialization of a patch of axonal membrane into a complex structure that supports synaptic vesicle exocytosis and neurotransmitter release. In mammalian neurons, presynaptic assembly is widely studied in a co-culture assay, where a synaptogenic cue expressed at the surface of a heterologous cell induces presynaptic differentiation in a contacting axon. This assay has led to the discovery of numerous synaptogenic proteins, but has not been used to probe neuronal mechanisms regulating presynaptic induction. The identification of regulatory pathways that fine-tune presynaptic assembly is hindered by the lack of adequate tools to quantitatively image this process. Here, we introduce an image-processing algorithm that identifies presynaptic clusters in mammalian co-cultures and extracts a range of synapse-specific parameters. Using this software, we assessed the intrinsic variability of this synaptic induction assay and probed the effect of eight neuronal microRNAs on presynaptic assembly. Our analysis revealed a novel role for miR-27b in augmenting the density of presynaptic clusters. Our software is applicable to a wide range of synaptic induction protocols (including spontaneous synaptogenesis observed in neuron cultures) and is a valuable tool to determine the subtle impact of disease-associated genes on presynaptic assembly.

N-cadherin induces partial differentiation of cholinergic presynaptic terminals in heterologous cultures of brainstem neurons and CHO cells

N-cadherin is a calcium-sensitive cell adhesion molecule commonly expressed at synaptic junctions and contributes to formation and maturation of synaptic contacts. This study used heterologous cell cultures of brainstem cholinergic neurons and transfected Chinese Hamster Ovary (CHO) cells to examine whether N-cadherin is sufficient to induce differentiation of cholinergic presynaptic terminals. Brainstem nuclei isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of choline acetyltransferase (ChAT) transcriptional regulatory elements (ChATBACEGFP) were cultured as tissue explants for 5 days and cocultured with transfected CHO cells for an additional 2 days. Immunostaining for synaptic vesicle proteins SV2 and synapsin I revealed a ~3-fold increase in the area of SV2 immunolabeling over N-cadherin expressing CHO cells, and this effect was enhanced by coexpression of p120-catenin. Synapsin I immunolabeling per axon length was also increased on N-cadherin expressing CHO cells but required coexpression of p120-catenin. To determine whether N-cadherin induces formation of neurotransmitter release sites, whole-cell voltage-clamp recordings of CHO cells expressing α3 and β4 nicotinic acetylcholine receptor (nAChR) subunits in contact with cholinergic axons were used to monitor excitatory postsynaptic potentials (EPSPs) and miniature EPSPs (mEPSPs). EPSPs and mEPSPs were not detected in both, control and in N-cadherin expressing CHO cells in the absence or presence of tetrodotoxin (TTX). These results indicate that expression of N-cadherin in non-neuronal cells is sufficient to initiate differentiation of presynaptic cholinergic terminals by inducing accumulation of synaptic vesicles; however, development of readily detectable mature cholinergic release sites and/or clustering of postsynaptic nAChR may require expression of additional synaptogenic proteins.

Synaptogenic Proteins and Synaptic Organizers: “Many Hands Make Light Work”

Synaptogenesis is thought to be mediated by cell adhesion proteins, which induce the initial contact between an axon and its target cell and subsequently recruit and organize the presynaptic and postsynaptic protein machinery required for synaptic transmission. A new study by Linhoff and colleagues in this issue of Neuron identifies adhesion proteins of the LRRTM family as novel synaptic organizers.

An Unbiased Expression Screen for Synaptogenic Proteins Identifies the LRRTM Protein Family as Synaptic Organizers

Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.

Unbiased expression screen for synaptogenic proteins based on fibroblast-neuron coculture

Unbiased expression screen for synaptogenic proteins based on fibroblast-neuron coculture

Interaction of the N-Terminal Domain of the AMPA Receptor GluR4 Subunit with the Neuronal Pentraxin NP1 Mediates GluR4 Synaptic Recruitment

Synaptogenesis requires recruitment of neurotransmitter receptors to developing postsynaptic specializations. We developed a coculture system reconstituting artificial synapses between neurons and nonneuronal cells to investigate the molecular components required for AMPA-receptor recruitment to synapses. With this system, we find that excitatory axons specifically express factors that recruit the AMPA receptor GluR4 subunit to sites of contact between axons and GluR4-transfected nonneuronal cells. Furthermore, the N-terminal domain (NTD) of GluR4 is necessary and sufficient for its recruitment to these artificial synapses and also for GluR4 recruitment to native synapses. Moreover, we show that axonally derived neuronal pentraxins NP1 and NPR are required for GluR4 recruitment to artificial and native synapses. RNAi knockdown and knockout of the neuronal pentraxins significantly decreases GluR4 targeting to synapses. Our results indicate that NP1 and NPR secreted from presynaptic neurons bind to the GluR4 NTD and are critical trans-synaptic factors for GluR4 recruitment to synapses.