Edited by: Touqeer Ahmed, National University of Sciences and Technology, Pakistan
Reviewed by: Katsuhiko Tabuchi, Shinshu University, Japan; Shuzo Sugita, University Health Network (UHN), Canada
This article was submitted to Neuroplasticity and Development, a section of the journal Frontiers in Molecular Neuroscience
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Chemical synapses provide a vital foundation for neuron-neuron communication and overall brain function. By tethering closely apposed molecular machinery for presynaptic neurotransmitter release and postsynaptic signal transduction, circuit- and context- specific synaptic properties can drive neuronal computations for animal behavior. Trans-synaptic signaling via synaptic cell adhesion molecules (CAMs) serves as a promising mechanism to generate the molecular diversity of chemical synapses. Neuroligins (Nlgns) were discovered as postsynaptic CAMs that can bind to presynaptic CAMs like Neurexins (Nrxns) at the synaptic cleft. Among the four (Nlgn1-4) or five (Nlgn1-3, Nlgn4X, and Nlgn4Y) isoforms in rodents or humans, respectively, Nlgn3 has a heterogeneous expression and function at particular subsets of chemical synapses and strong association with non-syndromic autism spectrum disorder (ASD). Several lines of evidence have suggested that the unique expression and function of Nlgn3 protein underlie circuit-specific dysfunction characteristic of non-syndromic ASD caused by the disruption of Nlgn3 gene. Furthermore, recent studies have uncovered the molecular mechanism underlying input cell-dependent expression of Nlgn3 protein at hippocampal inhibitory synapses, in which trans-synaptic signaling of specific alternatively spliced isoforms of Nlgn3 and Nrxn plays a critical role. In this review article, we overview the molecular, anatomical, and physiological knowledge about Nlgn3, focusing on the circuit-specific function of mammalian Nlgn3 and its underlying molecular mechanism. This will provide not only new insight into specific Nlgn3-mediated trans-synaptic interactions as molecular codes for synapse specification but also a better understanding of the pathophysiological basis for non-syndromic ASD associated with functional impairment in Nlgn3 gene.
Proper brain function requires the orchestration of 1015 synaptic connections that provide platforms for intercellular communication between 1011 neurons (
Neuroligin (
What trans-synaptic signaling is critical for the unique expression and function of Nlgn3?
Genomic and protein structures of Neuroligin 3 (Nlgn3). Upper schema showing the organization of human Nlgn3 and splicing patterns at exons 3 and 4. Bars indicate exons with the coding and untranslational regions colored in dark and bright gray, respectively. Lower schema showing the domain structure of Nlgn3. Bars indicate positions of the mutations associated with ASD and schizophrenia. ChE, cholinesterase-like domain, Gph_BD, gephyrin binding domain; O-glyco, O-linked glycosylation sites; PDZ_BM, PDZ domain-binding motif.
Nlgn3 is a single membrane spanning protein with an N-terminal hydrophobic sequence with the characteristics of a cleaved signal peptide followed by a large extracellular domain, a highly conserved single transmembrane region, and a short cytoplasmic domain (
Nlgn3 molecule forms homodimers and heterodimers with other Nlgn isoforms in the secretory pathway and translocate to the plasma membrane (
Nlgn3 protein can be cleaved on its juxtamembrane domain in an activity-dependent manner which is conserved from rodents to humans (
Deletions or frameshift mutations in the coding region of
Dysregulation of mTOR (mammalian target of rapamycin) and MAPK (mitogen-activated protein kinase) pathways are strongly implicated in ASD (
Recently,
Consistent with the expression pattern of
The extracellular domain of Nlgn3 protein trans-synaptically binds to that of Nrxns in a Ca2+-dependent manner (
Pre- and postsynaptic Neuroligin 3 (Nlgn3) binding partners. Schematic diagram of the major Nlgn3 binding proteins. Shaded circles with dashed lines indicate the protein domains that interact with Nlgn3. HS, Heparin sulfate; LNS, laminin/neurexin/sex-hormone-binding globulin domain; EGFA, epidermal growth factor-like domains; PDZ_BM, PDZ domain-binding motif; ChE, Cholinesterase-like domain; Gph_BD, Gephyrin binding domain, ptp; protein tyrosine phosphatase domain; FN, fibronectin type III domain; Ig, Ig-like domain; A, acidic domain; FL, follistatin-like domain; EC, EF hand Ca2+ binding domain; G, G-domain; E, E-domain, PDZ, PDZ domain; SH3, Src-homology-domain-3; GK, guanylate kinase domain; MA, MAM domain; GPI, glycosylphosphatidylinositol anchor.
Nrxn has been considered the sole presynaptic binding partner of Nlgn for decades, however, a recent study identified Nlgn3 as a selective ligand for presynaptically-expressed type IIA receptor protein tyrosine phosphatase delta (PTPδ) (
MAM domain-containing glycosylphosphatidylinositol anchor 1 (MDGA1) and 2 (MDGA2) proteins also compete for the Nrxn binding site on the extracellular domain of Nlgn3 via a
Secreted protein acidic and rich in cysteine-like 1 (SPARCL1 also known as Hevin) is an extracellular matrix released from astrocytes and important for excitatory synaptogenesis (
The short intracellular domain of Nlgn3 contains the PDZ- (
Nlgn3 protein has been characterized as a synaptic organizer, akin to other Nlgn isoforms. Nlgn3 can induce presynaptic differentiation in co-culture assay with primary neurons and heterologous cells (
One of the most important outcomes obtained by Nlgn3 mutant studies is the heterogeneity of mutation impact in the brain circuits. Three mutant mouse models, including knockout and missense ASD mutations, have revealed that different Nlgn3 mutations cause distinct abnormalities at synapses. This section highlights the differential impact of Nlgn3 mutations in excitatory and inhibitory synapses (
Summary of electrophysiological and other phenotypes in Nlgn3 mutant mouse lines in the hippocampus and cerebellum.
Mouse (source) | Region | Electrophysiological phenotype | Other phenotypes | References |
KO (Sud2) | Hi Py | mEPSC freq ↓ /amp ↔, NMDA/APMA ↔, PPR ↔ | ||
mIPSC freq ↑ /amp ↔ | ||||
Global cKO (Sud3) | Hi Py* | mEPSC freq ↔ /amp ↔ | ||
mIPSC freq ↔ /amp ↔ | ||||
P0 cKO (Sud3) | Hi Py | NMDA/AMPA ↔ | ||
mIPSC freq ↑ /amp ↔ | ||||
P21 cKO (Sud3) | Hi Py | NMDA/AMPA ↔, PPR ↔ | ||
mIPSC freq ↔ /amp ↔ | ||||
R451C KI (Sud1) | Hi Py | mEPSC freq ↑ /amp ↔, NMDA/AMPA ↑, PPR ↔, | GluN2B ↑, PSD95 ↑, dendritic branching ↑ | |
LTP ↑, NMDA-EPSC amp ↑ /decay ↑ | spine size ↓, terminal size ↓ | |||
mIPSC freq ↔ /amp ↔ | ||||
R451C KI (Hei) | Hi Py | NMDA/AMPA ↑ | ||
R451C KI (Sud1) | Hi CA3 Py | mEPSC freq ↔ /amp ↔ | ||
mIPSC freq ↑ /amp ↔ | ||||
R704C KI (Sud4) | Hi Py | mEPSC freq ↓ /amp ↔, NMDA/AMPA ↑, PPR ↔, LTP ↔ | VGluT1 ↔, GluA1 ↑, GluA3 ↑ | |
mIPSC freq ↔ /amp ↔ | VIAAT ↔ | |||
KO (Sud2) | Hi Py | Pv-IPSC amp ↓ | Normal morphology in Pv+ In | |
Hi Py | Cck-IPSC amp ↑ | No morphology in Cck+ In | ||
Impaired tonic eCB signaling | ||||
Pv+ cKO (Sud3) | Hi Pv+ | mEPSC freq ↔ /amp ↔, NMDA/AMPA ↓, PPR ↓ | PSD95 ↔ | |
sEPSC freq ↑ /amp ↔, eEPSC amp ↑ | ||||
Impaired Group III mGluR activity | ||||
mIPSC freq ↔ /amp ↔ | ||||
KO (Tan) | Cb PC | mEPSC freq ↔ /amp ↓ | PF synapse morphology →, ectopic CF synapse ↑, ( |
|
PF-EPSC mGluR-LTD ↓, PPR ↔ | GluA2 phosphorylation ↓, mGluR1a ↓ | |||
PC cKO (Sud3) | Cb PC | CF-EPSC amp ↓ /PPR ↔ | ||
PF-EPSC PPR ↔ | ||||
R451C KI (Sud1) | Cb PC | mEPSC freq ↑ /amp ↔ | ( |
|
R451C KI (Sud1) | Cb PC | mEPSC freq ↔ /amp ↔ | PC number ↔ | |
Impaired synaptic elimination | Normal morphology of PC dendrites | |||
mIPSC freq ↔ /amp ↑ | Molecular layer In number ↔ | |||
PC cKO (Sud3) | Cb PC | mIPSC freq ↔ /amp ↔ |
Summary of electrophysiological and other phenotypes in Nlgn3 mutant mouse lines in other brain regions.
Mouse (source) | Region | Electrophysiological phenotype | Other phenotypes | References |
R451C KI (Sud1) | SCx Py | mEPSC freq ↔ /amp ↔ | VGT1 ↔, synapse number ↔ | |
R451C KI (Sud1) | mEPSC freq ↓ /amp ↔, NMDA/APMA ↔ | |||
R451C KI (Sud1) | SCx Py | Pv-IPSC amp ↓ /PPR ↑ | ||
KO (Sud2) | SCx Py | mIPSC freq ↔ /amp ↔ | VIAAT ↑ | |
R451C KI (Sud1) | SCx Py | mIPSC freq ↔ /amp ↑ | VIAAT ↑, synapse number ↔ | |
mIPSC freq ↑ /amp ↔ | ||||
R451C KI (Sud1) | SCx Py | mIPSC freq ↑ /amp ↔ | ||
Impaired tonic eCB signaling | ||||
Sst-IPSC amp ↔ | Sst+ In number ↔ | |||
Pv-IPSC amp ↔ | Pv+ In number ↔ | |||
R451C KI (Sud1) | SCx Pv+ | eEPSC amp ↔ | ||
R451C KI (Sud1) | SCx Sst+ | Py-EPSC amp ↔ | ||
SCx Pv+ | Py-EPSC amp ↔ | |||
R451C KI (Sud1) | BA Py | mEPSC freq ↔ /amp ↑ | ||
mIPSC freq ↔ /amp ↓ | ||||
KO (Sud2) | St D1 | mEPSC freq ↔ /amp ↔ | ||
St D1 | mIPSC freq ↓ /amp ↔ | |||
St D2 | mEPSC freq ↔ /amp ↔ | |||
St D2 | mIPSC freq ↔ /amp ↔ | |||
KO (Sud2) | MNTB | Calyx-EPSC amp ↔, PPR ↔, RT ↔, DT ↔ | ||
R451C KI (Sud1) | Calyx-EPSC amp ↓, PPR ↔, RT ↔, DT ↔ | |||
R704C KI (Sud4) | Calyx-EPSC amp ↑, PPR ↔, RT ↔, DT ↔ | |||
Krox20 cKO (Sud3) | Calyx-EPSC amp ↓, RT ↔, DT ↔ | |||
Pv cKO (Sud3) | Calyx-EPSC amp ↓, RT ↑, DT ↑ | |||
KO (Tan) | VTA DA | GluA2-lacking AMPA-transmission ↑ | ||
DAN-KD | GluA2-lacking AMPA-transmission ↑ |
A major function of Nlgn3 protein at synapses is to control AMPAR-mediated basal excitatory transmission. Excitatory synapses on hippocampal CA1 pyramidal neurons have been the best characterized so far. Overexpression of Nlgn3 enhances AMPAR-mediated excitatory transmission and expression of presynaptic vesicular glutamate transporter 1 regardless of the specific Nlgn3 splice isoform (
Interestingly, the impact of Nlgn3 mutations in the calyx-MNTB synapses are distinct from hippocampal synapses. The calyx synapse is a large excitatory synapse in the MNTB that functions in the auditory system. Nlgn3 KO has no effect on excitatory synaptic transmission at the calyx synapse. In contrast, conditional deletion of
Nlgn3 protein contributes to NMDAR-mediated basal synaptic transmission at excitatory synapses on hippocampal CA1 inhibitory interneurons (
These results indicate that ASD-associated Nlgn3 mutations cause circuit-dependent abnormal excitatory synaptic efficacy. The molecular mechanism underlying the differential roles of Nlgn3 on AMPARs and NMDARs is not elucidated. Post-translational modifications of Nlgn3 may contribute to synaptic specification, leading to distinct effects of Nlgn3 on AMPAR- and NMDAR-mediated excitatory transmission. Further analysis is required to highlight the functional impact of Nlgn3 on receptor-mediated synaptic function.
Similar to studies examining excitatory synaptic function, there are a number of studies that have elucidated the global roles of Nlgn3 protein on GABA
Interneurons exhibit extraordinary morphological, physiological and molecular diversity (
Input cell-specific stimulation in Nlgn3 KO mice shows a significant increase in synaptic strength at Cck+ synapses, but no alteration at Pv+ synapses, suggesting an input cell-dependent function of Nlgn3 at hippocampal inhibitory synapses. Interestingly, this selective strengthening of Cck+ synapses is caused by disruption of tonic endocannabinoid signaling via cannabinoid CB1 receptors which are expressed at Cck+ synapses (
Nlgn3 loss-of-function analyses also affect spontaneous miniature inhibitory synaptic events in other brain regions. In the somatosensory cortex, inhibitory synaptic transmission is significantly increased in Nlgn3-R451C KI mice but not in Nlgn3 KO mice (
In the nucleus accumbens, Nlgn3 KO selectively reduces mIPSC frequency by 50% in D1-MSNs but not in D2-MSNs, consistent with cell type-specific expression of
In cerebellar Purkinje cells, single KO of
A few studies have examined the impact of Nlgn3 protein on long-term synaptic plasticity. Parallel fiber-Purkinje cell synapses in the cerebellum can induce mGluR-mediated long-term depression (LTD) which underlies motor coordination. The effect of Nlgn3 KO on this form of LTD remains controversial.
In contrast to Nlgn3 KO or KD, Nlgn3-R451C mutation significantly affects long-term synaptic plasticity with variable phenotypes at distinct synapses. LTP is enhanced at hippocampal excitatory synapses (
A functional impact of Nlgn3 in remodeling of neuronal circuits has been reported. Nlgn3 can affect the turnover of spines that form synaptic structures with excitatory neurons. In layer 2/3 pyramidal neurons of the somatosensory cortex, Nlgn3-R451C KI mice show enhanced dynamics of PSD95+ spines, which may be subsequently associated with stable synaptic connectivity. Importantly, abnormal PSD95+ spine dynamics is shared with another mouse model of non-syndromic ASD in which the chromosomal region corresponding to human 15q11–13 is paternally duplicated (patDp/ + mice) (
Nlgn3 also contributes to the developmental elimination of climbing fiber-Purkinje cell synapses. Nlgn3-R451C KI mice impair elimination of redundant climbing fiber-Purkinje cell synapses from postnatal day 10–15 (
One prevailing mechanism describing the pathophysiology of ASD is the imbalance between excitation and inhibition in neurons, which can be caused by circuit dysfunction described above. Supporting this notion, emerging evidence has revealed that functional impairment in Nlgn3 gene generates dysfunction in specific circuits. Based on the DSM-5, the diagnostic criteria to characterize ASD are (1) persistent deficits in social communication and interaction and (2) restricted, repetitive patterns of behavior, interests, or activities (
Summary of behavioral tests in Nlgn3 mutant mouse lines.
Mouse (source) | Social | Cognitive | Rep. | Motor | Other phenotypes | References |
KO (Bro) | Sociability: ↔ | FL :↓ | Activity: ↑ | USV: ↓ | ||
Social rew/mem: ↓ MWM: ↔ | Olfactory-dependent behavior: ↓ | |||||
KO (Bro) | NT | NT | NT | NT | Pheromone preference: ↓ | |
KO (Bro) | Abnormal visual transitive inference. | |||||
KO (Sud2) | NT | NT | ↑ | Activity: ↑ | ||
KO (Tan) | Sociability: ↓ | NT | NT | NT | Gamma osc: ↓ | |
Social rew/mem: ↓ | ||||||
KO (Tan) | NT | NT | NT | Motor coordination: ↓ | ||
(rescued by Nlgn3 OE in PC) | ||||||
KO (Tan) | Housing environment causes different behavioral phenotypes. | |||||
KO (Tan) | Sociability: ↓ | NT | NT | NT | ||
Social rew/mem: ↓ | ||||||
KD in DA | Sociability: ↓ | NT | NT | NT | ||
Social rew/mem: ↓ | ||||||
KO (Tan) | Sociability: ↔ | NT | NT | NT | ||
Social rew/mem: ↓ | ||||||
KD in DA | Sociability: ↔ | NT | NT | NT | ||
Social rew/mem: ↓ | ||||||
Rescued by MNK inhibition | ||||||
cKO (Sud3, D1) | NT | NT | ↑ | Activity: ↑ | ||
cKO (Sud3, D2) | NT | NT | ↔ | Activity: ↔ | ||
cKO (Sud3, PC) | NT | NT | ↔ | Activity: ↑ | ||
cKO (Sud3, Pv+) | NT | Abnormal fear extinction | Gamma osc: ↓ | |||
Rescued by Nlgn3 expression in CA1 Pv+ neurons | ||||||
R451C KI (Sud1) | Sociability: ↓ | MWM: ↑ | NT | NT | ||
R451C KI (Sud1) | Sociability: ↓ | ↔ | ↔ | ↔ | ||
R451C KI (Sud1) | Sociability: ↓ | MWM: ↑ | NT | NT | Anxiety: ↑ | |
Backcross w 129S2/SvPasCrl line | Locomotor activity: ↓ | |||||
R451C KI (Sud1) | NT | NT | ↑ | Activity: ↑ | ||
R451C KI (Sud1) | Sociability: ↔ | NT | ↑ | NT | Aggression: ↑ | |
R451C KI (Sud1) | Increased interest in mating and atypical aggressive behavior by social isolation ( |
|||||
R451C KI (Sud1) | Sociability: ↔ | NT | NT | NT | Gamma osc: ↓ | |
Social novelty: ↓ (Rescued by the optogenetic restoration of gamma osc) | ||||||
R451C KI (Sud1) | Abnormal visual transitive inference in both lines. | |||||
R451C KI (Sud1) | Environmental enrichment reduced anxiety and increased aggression | |||||
R451C KI (Hei) | Sociability: ↔ | ↔ | ↔ | |||
R451C KI (Hei) | Sociability: ↔ | ↔ | ↔ | Anxiety: ↔ | ||
Locomotor activity: ↔ |
Recent cell type-specific KD, KO, and rescue approaches, and the development of simultaneous pre- and post-synaptic gene introduction method allows us to deepen our knowledge of synapse-, cell type-, and brain region-specific roles of Nlgn3 on synaptic function and animal behaviors. In this section, we summarize the functional importance of Nlgn3 expressed in different cells and circuits in the brain (
Schematic diagram of the circuit-specific Nlgn3 functions in the mesolimbic pathway.
Schematic diagram of the circuit-specific Nlgn3 functions in the hippocampal CA1 region.
Aberrant striatal development and connectivity are linked to repetitive behaviors and abnormal reward circuitry in ASD (
Both Nlgn3 KO and Nlgn3-R451C KI mice display enhanced rotarod performance and stereotypic behaviors (
In VTA DA neurons, cocaine, social reward, and novelty stimuli induce AMPAR-mediated synaptic plasticity by regulating GluR2-lacking AMPAR trafficking (
Recent studies have highlighted Nlgn3 function in VTA DA neurons on social behavior. Social reward, sociability, and social novelty were reduced by specific Nlgn3 KD in VTA DA neurons (
Gamma band oscillations implicated in various phases of hippocampal- and cortical-dependent cognitive behaviors (
Numerous
Importantly, a recent report supports that endogenous Nlgn3 regulates excitatory presynaptic release probability in the hippocampus.
The most fundamental and intriguing role of Nlgns is the impact of their trans-synaptic interactions on synaptic function. However, elucidating how trans-synaptic interactions shape functional synaptic properties has remained a challenge. To understand the physiological roles of trans-synaptic molecules one must be able to manipulate the expression of these molecules in pre- and post-synaptic neurons simultaneously and then elucidate the functional consequences of this manipulation. We have recently advanced methodology for performing non-overlapping gene transfections which allows us to express Nlgn and Nrxn isoforms in pre- and post-synaptic neurons simultaneously and decipher roles of specific Nlgn3 and Nrxn interactions on hippocampal inhibitory synaptic transmission (
What mechanism underlies the input cell- and splice isoform-dependent function of Nlgn3-A protein at inhibitory synapses on CA1 pyramidal cells? One promising mechanism is a trans-synaptic interaction of Nlgn3-A protein with a specific Nrxn isoform. Each Nrxn isoform has been hypothesized to regulate synaptic function via trans-synaptic interactions with postsynaptic binding partners in an isoform-dependent manner (
Although the function of Nlgns has been extensively examined in the context of neurons, recent evidence indicates that Nlgn proteins can regulate glial morphogenesis and differentiation. Astrocytic Nlgn1-3 proteins are required for astrocyte morphogenesis both
Nlgn3 protein can function as a cortical neuronal activity-regulated glioma mitogen (
A prevalent concern among people with ASD revolves around gastrointestinal (GI) symptoms such as constipation, diarrhea, and abdominal pain (
Given the heterogenous nature and presentation of ASD, identifying therapeutic targets and improving therapeutic effectiveness remains a challenge. Nlgn3 gene is strongly associated with a non-syndromic monogenic form of ASD and for the past decade behavioral analyses with animal models have revealed that Nlgn3 protein at distinct synapses underlie some abnormal phenotypes caused by the disruption of Nlgn3 gene. Knowledge about the molecular mechanisms underlying the heterogeneous expression and function of Nlgn3 protein will help us understand the complex pathophysiological basis of ASD with the potential to create novel therapeutical strategies.
Trans-synaptic signaling between presynaptic and postsynaptic CAMs is a promising mechanism to decipher molecular heterogeneity at synapses. One feasible approach to dissect specific trans-synaptic signaling with synaptic CAMs is based on cellular biological methods to manipulate the expression levels of both presynaptic and postsynaptic CAM variants at targeted synapses (
First, can trans-synaptic signaling be generalized to other synapses? Although single-cell transcriptomics has demonstrated cell type-specific expression patterns of synaptic CAMs (
MU, AC, and KF wrote the manuscript. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
This work was supported by grants from the National Institutes of Health Grants (R01NS085215 to KF, T32 GM107000, and F30MH122146 to AC), Grants-in-Aid for Scientific Research (20H03349, 20K21461, and 20H05918 to MU), and the Naito Foundation (to MU).