Review

The balancing act of GABAergic synapse organizers Jaewon Ko1,2, Gayoung Choii1, and Ji Won Um1,3 1

Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea Department of Psychiatry, Yonsei University College of Medicine, Seoul 120-751, Korea 3 Department of Physiology, Yonsei University College of Medicine, Seoul 120-751, Korea 2

GABA (g-aminobutyric acid) is the main neurotransmitter at inhibitory synapses in the mammalian brain. It is essential for maintaining the excitation and inhibition (E/I) ratio, whose imbalance underlies various brain diseases. Emerging information about inhibitory synapse organizers provides a novel molecular framework for understanding E/I balance at the synapse, circuit, and systems levels. This review highlights recent advances in deciphering these components of the inhibitory synapse and their roles in the development, transmission, and circuit properties of inhibitory synapses. We also discuss how their dysfunction may lead to a variety of brain disorders, suggesting new therapeutic strategies based on balancing the E/I ratio. Inhibitory synapses: a nexus for brain function and dysfunction Neurons in the mammalian brain are linked to other nerve cells via synapses (see Glossary) and are assembled into the specific neural circuits central to all brain functions. Synapses are morphologically classified as either type I (asymmetric; mainly on dendritic spines) or type II (symmetric; mainly on soma and dendritic shafts) [1]. Type I synapses use glutamate to mediate excitatory synaptic transmission, while type II synapses use GABA and glycine to mediate inhibitory synaptic transmission. During brain development, diverse types of inhibitory neurons with distinct morphological, electrophysiological, and neurochemical characteristics recognize their target neurons, and form and specify synapses that incorporate specific GABA receptors. The selective connectivity and validation of inhibitory synapses are mediated by a group of proteins here called ‘inhibitory synapse organizers’. Normal brain functions require a balance between the excitatory and inhibitory synapse types. From a neurophysiological perspective, this concept of E/I balance refers to the relative contributions of excitatory and inhibitory synaptic inputs, whose integration in the organization of neural circuits is instrumental for normal neural information processing. The appropriate E/I ratio in this regard is dictated by various factors, particularly from various classes of Corresponding authors: Ko, J. ([email protected]); Um, J.W. ([email protected]). Keywords: inhibitory synapse; GABA; synaptic adhesion; excitation–inhibition balance; synapse organizer. 1471-4914/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2015.01.004

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inhibitory neurons across diverse brain regions. The importance of E/I ratio is reinforced by the fact that synaptic imbalances appear to underlie many brain disorders. Indeed, ‘synaptopathy’ is often used to describe key features of many brain disorders, including autism, schizophrenia, and epilepsy [2] (Box 1), reflecting that disruptions in crucial synaptic processes (e.g., synaptic adhesion and signaling)

Glossary Allosteric modulator: ligand that binds to the receptor at sites other than the active site, changing the shape or activity of the bound protein. Alzheimer’s disease: the most common form of dementia; damage to nerve cells of the brain results in impaired thinking, behavior, and memory. Autism spectrum disorders (ASDs): a term that encompasses autism and similar disorders. Endocannabinoid-mediated long-term depression (eCB-LTD): long-term depression induced by endocannabinoid (CB1) receptor activation. Electroencephalogram (EEG): records the massed electrical activity in the brain. Enriched environment: a specially designed multi-sensory space that contains equipment and materials designed to stimulate the senses. Epileptiform: an EEG pattern that shows abnormal electrical activity associated with an increased risk of seizures and particular types of epilepsy. Fragile X syndrome (FXS): an inherited condition in which a defect in the FMR1 gene on the X chromosome may cause a range of developmental problems, including learning disabilities and cognitive impairments. GABA: g-aminobutyric acid, the primary inhibitory neurotransmitter. Interneuron: a neuron that serves as a connection within the local circuit, without sending projections to other regions; it is typically inhibitory and uses the neurotransmitters GABA or glycine. Long-term potentiation (LTP): a long-lasting increase in synaptic strength in response to high-frequency stimulation, possibly the biological basis for learning and memory. Mammalian target of rapamycin (mTOR): a serine/threonine kinase that is activated by AKT and helps to control several cellular functions, including survival and cell division. Medial ganglionic eminence (MGE): a transient brain structure present during development; it gives rise to GABAergic stellate-shaped cells and directs the migration of cells to the neocortex. Metabotropic glutamate receptor (mGluR): a type of glutamate receptor that acts through second messengers. Orthosteric agonists: agonists that bind to the receptor at the primary and active sites. Perisomatic: the domain of the plasma membrane that includes proximal dendrites, the cell body, and the axon initial segment. Pyramidal neuron: a neuron with triangular-shaped cell body; it acts as the primary excitatory neuron in the cerebral cortex and hippocampus. Rett syndrome: a brain disorder, found almost exclusively in girls, characterized by autistic-like behaviors in its early stages. Schizophrenia: a psychotic disorder characterized by severely impaired thinking, emotions, and behaviors. Sensorimotor gating: state-dependent regulation of the transmission of sensory information. Synapse: a specialized anatomical site at which neurons communicate with each other. Synaptic transmission: the process by which presynaptic electrical activity is transduced into a chemical signal that transmits information to the postsynaptic neuron.

Review Box 1. The synaptopathy hypothesis ‘Synaptopathy’ refers to a brain disorder arising from dysfunctions in synapse formation, function, and/or plasticity. A major hypothesis explaining how defects in synaptic proteins cause brain disorders proposes that the balanced E/I of the brain is disrupted at the synaptic level, leading to abnormal neural circuit activity, aberrant neural processing, and pathogenic networking. Over the past decade numerous synaptic genes have been linked to various brain disorders, thanks to next-generation sequencing technologies [113]. Some of these proteins are crucial for synapse formation, maintenance, or elimination at excitatory synapses. For example, the scaffold protein Shank3 regulates excitatory synapse structure and function; its deletion in mice alters the postsynaptic density and causes morphological and functional defects in the medium spiny neurons of the striatum, which are thought to be dysfunctional in ASDs [114]. Notably, Shank3-KO mice display repetitive grooming and social-interaction deficits, suggesting that this model may elucidate pathological mechanisms underlying a subset of ASDs. Similar analyses have targeted a variety of other synaptic proteins implicated in ASDs, schizophrenia, and mental retardation. These proteins are largely localized to excitatory synapses, and their dysfunction affects excitatory synapse structure, transmission and/ or plasticity, causing an imbalanced circuit-level shift toward inhibition that may form the basis for some brain disorders. Inhibitory synaptic proteins contribute equally to balancing the E/I ratio in healthy brains. The number and distribution of excitatory and inhibitory inputs onto a single neuron dictate the integration of synaptic inputs and their output. Dysfunctions of key inhibitory synaptic proteins can shift the balance toward excitation, leading to distinct types of brain disorders (e.g., epilepsy). Several inhibitory synaptic molecules have been associated with epilepsy in animal studies or genome-wide genetic screens, but the systematic examination of their potential links to brain disorders is only beginning. Identifying a full catalog of inhibitory synapse organizers should be a first step in our efforts to extract general organizing principles underlying synapse development and function. Moreover, we need to understand how these proteins shape neural circuit properties to accurately interpret the data from the transgenic animal models that mimic human disorders.

can lead to the degeneration of specific neural circuits and aberrant neural network activity. However, the therapeutic potential of first-generation receptor blockers/modulators has been limited by unpredictable side effects that may arise from excessive excitation or inhibition of neuronal activity [3]. The development of more-effective drugs is therefore crucial. Recent advances in understanding synaptic receptor structures and the pathophysiological mechanisms underlying some synaptopathies have led to the introduction of new disease models and candidate therapeutics [4,5]. Moreover, advanced technologies such as next-generation sequencing and induced pluripotent stem cells offer new opportunities for target gene identification, drug screening, and diagnostics [4,5]. The development of better therapeutic strategies will require a more complete understanding of synapse organization. Unlike the extensive information now available on excitatory synapse proteins, our understanding of inhibitory synapse organization remains limited [6,7]. An obstacle to this goal is the need to determine how synaptic- and circuitlevel inhibition is achieved by diverse interneurons that display distinct morphologies, physiological properties, connectivity patterns, and gene expression profiles. Although some organizing principles governing the spatiotemporal patterns of inhibitory activity and diversity in the cortex have been proposed [8], the molecular and biophysical

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properties of different types of interneurons should be explored further. The molecular mechanisms governing inhibitory synapse development and function need to be better understood because proper brain function requires a balance between excitatory and inhibitory operations [9]. This review discusses recent advances in elucidating the roles of inhibitory synapse organizers in the development, plasticity, and neural circuit activity of inhibitory synapses, and their molecular-level implications in various brain disorders. We deal with the development of inhibitory synapses with a focus on relatively unexplored molecules. We also summarize recent regenerative approaches aimed at recovering synapse-impairment-related disease conditions. Some of the recently identified inhibitory synaptic proteins may help researchers to identify general principles of inhibitory synapse development, pointing to new therapeutic strategies. Regulation of synapse development by inhibitory synapse organizers Inhibitory synapse development is initiated by the collaborative actions of various classes of proteins (Figure 1). In this section we describe the known roles of key inhibitory synapse organizers (Table 1). Synapse formation by synaptic adhesion molecules Synaptic adhesion molecules physically connect the pre- and postsynaptic compartments, and orchestrate transsynaptic recognition and signaling processes that are essential for synaptogenesis [10]. Neuroligin-2 (NL-2) was the first adhesion molecule reported to be selectively located at inhibitory synapses [11]. Numerous other inhibitory synaptic adhesion molecules have since been identified, often as serendipitous findings from studies analyzing the functional properties or synaptic localization of new neuronal transmembrane proteins. Some of these proteins appear to be crucial for inhibitory synapse structure and function (Figure 2). NL-2 interacts with other adhesion (e.g., neurexins and the MAM domain-containing GPI anchor proteins – MDGAs) and scaffold (gephyrin, collybistin, and S-SCAM) proteins, and its overexpression promotes inhibitory synapse development in an activity- and alternative splicing-dependent manner in hippocampal and cerebellar neurons [12–14]. GABAA receptors and gephyrin are known to be dispensable for the synaptic clustering of NL-2 [15]. NL-2 forms complexes with gephyrin and collybistin to cluster GABAA receptors and recruit inhibitory postsynaptic machineries [16], and interacts with other scaffolds to negatively modulate inhibitory synapse development. Phosphorylation of NL-2 by proline-directed kinases recruits the peptidyl-prolyl isomerase Pin1 to negatively regulate its interaction with gephyrin [17]. NL-2 deficiency in mice does not abrogate inhibitory synapse formation in the retina, but specifically impairs inhibitory synaptic transmission and decreases inhibitory synapse number at perisomatic synapses of the hippocampus, and increases dentate gyrus (DG) excitability [18–20]. Two studies recently documented that the MDGAs directly interact with NL-2 but not with other NLs [21,22]. MDGA1 and MDGA2 are glycosylphosphatidylinositol 257

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Schemacs of inhibitory synapse organizers Key:

Transmembrane proteins Dystroglycan N

C C

IgSF9 N

C

IgSF9b N C

MDGA N α-Neurexin N

C C

Neuroligin-2 N Neuroplasn-65 N

C

PlexinB1 N

C

PTPδ N

C

Sema4D N

C C

Slitrk3 N TrkB N

C

Non-membrane proteins C

BRAG3 N Collybisn N Gephyrin N S-SCAM N

C

Leucine-rich repeats (LRR) domain Cysteine-rich domain Ig-like domain Fibronecn type III (FNIII) repeat Phosphatase domain LNS domain EGF-like domain PDZ domain Guanylate kinase-like domain WW domain Cholinesterase-like domain MAM domain Coiled-coil mof IQ domain SEC7 domain PH domain G domain C domain E domain SH3 domain RhoGEF Protein tyrosine kinase domain Sema GAP domain Mucin-like domain GPI anchor Transmembrane segment

C C

100 amino acids TRENDS in Molecular Medicine

Figure 1. Schematic showing the domain structures of inhibitory synapse organizers. The domain structures of the key inhibitory synaptic molecules reviewed here. The relevant domains are illustrated (right, box). The scale bar indicates 100 amino acids (for all proteins). Abbreviations: BRAG3, brefeldin A-resistant guanine nucleotide exchange factor 3; C, carboxyl terminal; IgSF9, immunoglobulin superfamily member 9; MDGAs, MAM-domain-containing glycosylphosphatidylinositol-anchored proteins; N, amino terminal; PTPd, protein tyrosine phosphatase receptor type, D polypeptide; S-SCAM, synaptic scaffolding molecule; TrkB, tropomyosin-related kinase B.

(GPI)-anchor-containing members of the immunoglobulin (Ig) superfamily of adhesion molecules [23]. Their exact localizations have not been determined, but gain- and loss-of-function assays suggest that they may be localized to inhibitory synapses [21,22]. Inhibitory synapse development was suppressed by MDGA1 overexpression, whereas it was enhanced by MDGA knockdown in hippocampal neurons, suggesting that MDGAs function as negative regulators [21,22]. MDGAs are likely to interact with NL-2 in a cisconfiguration, inhibiting the NL2–neurexin synaptic adhesion to suppress NL-2-dependent inhibitory synapse development [21]. Thus, the interaction of MDGAs with NL-2 may control E/I balance in a subset of neural circuits. As inhibitory synapse brakes, the MDGAs are reminiscent of some excitatory synapse brakes, such as major histocompatibility complex class I (MHCI) molecules and members of the Nogo receptor family (NgR) [24,25]. Neurexins are cell surface molecules crucial for presynaptic functions [10]. Both endogenous and recombinant neurexins are targeted to the presynaptic terminals of inhibitory synapses in an activity-dependent manner in conjunction with particular postsynaptic ligands [26,27]. The a-neurexins show functional links to inhibitory synapses, as documented by the severe impairment of inhibitory synaptic transmission seen in knockout (KO) mice 258

deficient for all three a-neurexins, and their specific induction of inhibitory postsynaptic differentiation [28,29]. To support this notion, a-neurexin-unique sequences are involved in its inhibitory synaptogenic activity [29]. The aneurexins share a subset of ligands with the b-neurexins, but the different extracellular domains of the a-neurexins mediate distinct interactions with specific proteins (e.g., neuroligins, dystroglycans, neurexophilins, and cerebellin2) [10] and independently affect diverse functions under the control of alternative splicing at six canonical sites [30]. Intriguingly, the expression of alternative splicing variants of neurexins in inhibitory neurons dictate the actions of neuroligins in specific synapse types of target neurons [31]. Slit and Trk-like proteins (Slitrks) and leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) organize excitatory and inhibitory synapse development through a combination of isoformspecific synaptic adhesion pathways [32–34]. In particular, Slitrk3 is selectively localized to and promotes (together with presynaptic PTPd) the development of inhibitory synapses [35,36]. Slitrk3-deficient mice exhibit decreased inhibitory synapse density and transmission [36], while PTPd-deficient mice exhibit impaired learning and enhanced long-term potentiation (LTP) [37]. PTPd has not

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Table 1. Summary of synaptic functions, binding proteins, and animal models of inhibitory synapse organizers Organizer Membrane proteins Calsyntenin-3

Dystroglycan

IgSF9

Function(s)

Binding protein(s)

Neural phenotypes of animal model

Refs

Involved in inhibitory synapse development in a subset of interneurons Required for homeostatic regulation at inhibitory GABAergic synapses Regulates inhibitory synapse development

Neurexins (indirect)

Knockout (KO) mice: decreased inhibitory synapse number and transmission

[48]

a-Neurexins

KO mice: no reported inhibitory synapse phenotypes

IgSF9 S-SCAM

KO mice: decreased inhibitory synapse number and function

[42] [42]

IgSF9b

Neuroligin-2

Required for the development of inhibitory synapses onto interneurons Directs inhibitory synapse development and function in an activity-dependent fashion

IgSF9b S-SCAM Neurexins MDGAs Gephyrin Collybistins S-SCAM

Knock-in (KI) mice expressing a mutant lacking the cytoplasmic domain: no defect in inhibitory synapse development Not reported

KO mice: decreased inhibitory synaptic transmission in the hippocampus; increased DG excitability; increased anxiety-like behavior; reduced ultrasonic vocalizations

[20] [115]

Transgenic (overexpression) mice: impaired social interactions, stereotyped jumping behavior, anxiety; enhanced spike-wave discharges seen on EEG Neuroligin-3

Directs excitatory and inhibitory synapse development and function

Neurexins PSD-95

KO mice: impaired tonic endocannabinoid signaling; increased inhibitory synaptic transmission from CCK basket cells to pyramidal neuron synapses

[73] [73] [106,111]

R451C KI mice: increased inhibitory synaptic transmission in the somatosensory cortex; increased excitatory synaptic transmission in the hippocampus; increased inhibitory synaptic transmission from CCK basket cells to pyramidal neuron synapses; decreased inhibitory synaptic transmission from PV basket cells to pyramidal neuron synapses; increased LTP in the hippocampus; altered social behavior and spatial learning; smaller white matter structures

Neuroplastin-65

PTPd Sema4D Slitrk3

TrkB

Secreted proteins FGF7

Neurexophilin-1

Cytosolic proteins BRAG3

Regulates inhibitory synapse structure and GABAAR localization Required for inhibitory synapse development Promotes inhibitory synapse development Promotes inhibitory synapse development and function

GABAAR a subunits

Slitrk3 Liprin-a PlexinB1 PTPd

Regulates the synaptic localization of gephyrin in a subset of interneurons

BDNF

Promotes inhibitory synaptic differentiation

Not reported

Modulates short-term plasticity at inhibitory synapses

a-Neurexins

Contributes to inhibitory synapse development

Dystrophin S-SCAM ARF6

R704C KI mice: impaired excitatory synaptic transmission in the hippocampus Not reported

KO mice: impaired spatial learning with enhanced hippocampal LTP KO mice: no reported inhibitory synapse phenotypes KO mice: decreased inhibitory synapse number and function in hippocampus; increased seizure susceptibility; spontaneous epileptiform activity on EEG KO mice: impaired inhibitory synapse structures

KO mice: enhanced neurogenesis in the DG of the hippocampus; impaired inhibitory synapse development; increased seizure susceptibility KO mice: impaired GABAB receptor-dependent short-term depression of inhibitory synapses in the nucleus reticularis thalami

[37]

[36]

[51]

[53,54]

[76,84]

Not reported

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Table 1 (Continued ) Organizer Collybistin

Function(s) Required for inhibitory receptor clustering and function, via the recruitment of gephyrin

Binding protein(s) Gephyrin Neuroligin-2 GABAAR a2 subunit

Gephyrin

Essential for clustering of glycine and GABAA receptors (GlyRs and GABAARs, respectively) at inhibitory synapses Regulates inhibitory synapse development through cell typespecific gene transcription programs

GlyR b subunit GABAARa subunit Collybistin Pin1 Neuroligin-2 Various target genes

Npas4

yet been examined in the context of inhibitory synapse development. Given that only the initial crucial functions of PTPd or Slitrk3 are revealed by the currently available KO mice, conditional KO mice of these proteins would help to elucidate their precise roles at inhibitory synapses [36,37]. Intriguingly, both Slitrk3 and PTPd have been implicated in obsessive–compulsive disorder (OCD) [38]. A co-crystal structure of the Slitrk1/PTPd complex has allowed analysis of the discrete molecular steps that drive Slitrk-mediated presynaptic differentiation [39]. Slitrk3

Neural phenotypes of animal model KO mice: altered hippocampal synaptic plasticity; region-specific loss of postsynaptic gephyrin and GABAAR clusters in the hippocampus and the basolateral amygdala; reduced dendritic inhibition KO mice: abolished clustering of glycine receptors but not GABAA receptors; early postnatal death (immediately after birth with a stiff musculature due to impaired GlyR function)

Refs [59]

KO mice: impaired contextual memory; hyperactivity; higher social dominance; cognitive and sensorimotor gating deficits

[81,99]

[116]

transgenic mice in which the interaction with PTPd is specifically perturbed might trigger an imbalance the E/I ratio; if so, this model could provide new insights into the pathophysiological mechanisms underlying neuropsychiatric disorders such as OCD. The evolutionarily conserved immunoglobulin superfamily (IgSF) member 9 transmembrane proteins, IgSF9 and IgSF9b [40], are highly expressed in the brain and localized to dendrites. Though IgSF9 was initially reported to be important in excitatory synapse development based

Presynapc neuron

Glycine

GABA PTPδ IgSF9b Neurexin

Inhibitory synapse

Slitrk3

Neurexin Sema4D

MDGA1

Neurexin

Calsyntenin-3

IgSF9b NL-2

Dystroglycan Neuroplasn-65

BDNF

GABAR TrkB

GlyR

Key: Acn BRAG3 Cdc42 Collybisn Dystrophin Gephyrin Neurexophilin-1 S-SCAM

PlexinB1 Acn Microtubule

Postsynapc neuron TRENDS in Molecular Medicine

Figure 2. Schematic diagram of inhibitory synapse organization by key synaptic adhesion, scaffold, and signaling proteins. Diagrams depicting an overview (left) and zoomed-in molecular view (right) of inhibitory synapse organization. Inhibitory neurotransmitters GABA (blue) and glycine (green) are released from presynaptic neurons and bind to their respective receptors (GABAA receptors and glycine receptors) in the dendritic shaft or soma of postsynaptic neurons. The main organizers of an inhibitory synapse are shown. These include synaptic adhesion, scaffold, and signaling proteins that are exclusively localized to GABAergic synapses. Specific protein–protein interactions are indicated by overlap. Future work will be necessary to clarify whether the inhibitory synapse organizers function separately or together in a single synapse. Most of the intracellular signaling pathways in the pre- and postsynaptic neurons of inhibitory synapses are unknown.

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with only a1 or a2 subunit-containing GABAA receptors at the synapse, and with those containing the a5 subunit outside of synapses. Knockdown of neuroplastin-65 decreases GABAA receptors at synaptic sites, suggesting that it is essential for the maintenance of a subset of GABAA receptors [45]. In neuroplastin-65-deficient mice, both excitatory and inhibitory synapses are influenced in CA1 and the DG of the hippocampus [46], and GABAA receptor composition is drastically altered in neuroplastin65-KO mice [46]. The calsyntenins are a family of evolutionarily conserved synaptic adhesion molecules. Calsyntenin-3 induces presynaptic differentiation in heterologous synapse-formation assays: it requires presynaptic neurexins for its presynaptic induction activity, but whether it directly interacts with neurexins is controversial [47,48]. The calsyntenins are required for proper inhibitory synapse structure and function in hippocampal neurons and layer II/III of the somatosensory cortex [47]. It remains unknown whether calsyntenin-3 is mainly localized to inhibitory synapses and how it regulates inhibitory synapse development, particularly in interneurons (where it is strongly expressed) [47,48] (Figure 3). Such studies could add to our understanding of the pathophysiological mechanisms underlying calsyntenin-related brain disorders. The class IV semaphorin Sema4D promotes inhibitory synapse development, working in tandem with its receptor PlexinB1 [49,50]. Tropomyosin-related kinase B (TrkB) contributes to inhibitory synapse assembly by controlling the synaptic localization of gephyrin in a subset of interneurons [51]. Consistent with this, the TrkB ligand

on an IgSF9 RNAi strategy [40], IgSF9-deficient mice did not show any defect in hippocampal dendrite growth or branching [41]. In addition, application of IgSF9 RNAi in IgSF9-KO mice produced a decrease in excitatory synapse density, suggesting that the previous result may have been an off-target effect. Instead, IgSF9 appears to be expressed in a subset of interneurons, where it regulates inhibitory synapse development [42]. IgSF9-KO mice show decreased numbers of inhibitory synapses and impaired inhibitory synaptic transmission [42]. Intriguingly, knock-in (KI) mice expressing IgSF9 lacking the entire cytoplasmic domain had no defects, suggesting that IgSF9 regulates inhibitory synapse development independently of its intracellular domain [42]. IgSF9b is required at inhibitory synapses projecting to inhibitory interneurons, where it is mainly expressed (Figure 3), localizing to a subdomain of the inhibitory synapse distinct from the GABAA/gephyrincontaining subdomain [43]. IgSF9b, by contrast, couples with NL-2 via its intracellular domain [43]. Thus, it appears that IgSF9 and IgSF9b both contribute to organizing inhibitory synapse development, and that this occurs through different molecular mechanisms in distinct neuron types. The IgSF protein neuroplastin-65, specifically expressed in the forebrain and enriched in postsynaptic density (PSD) fractions, has been shown to regulate LTP at hippocampal synapses [44]. Although neuroplastin-65 was initially recognized to regulate excitatory synapse development, recent reports suggest that it is also required for inhibitory synapse development [44]. Neuroplastin-65 directly interacts with GABAA receptors [45]: it associates

1 Inducon excitaon onto inhibitory neurons

(B/C/D)

(A)

Inhibitory neuron #1

PTPδ Neurexin Neurexin Sema4D

IgSF9b Neurexin

Nptx2 Gpr3 Osgin2 Frmpd3 Ddhd1 Kcna1 Slc25a36

Inhibitory neuron #3 ?

(A) Inhibitory neuron #2

NPAS4

(D)

(B)

1 2

(C)

Excitatory neuron Inhibitory neuron #1: somatostan Inhibitory neuron #2: parvalbumin Inhibitory neuron #3: cholecystokinin

Slitrk3

Calsyntenin-3 IgSF9b

NL-2 Dystroglycan NL-3 PlexinB1

2 Inducon inhibion onto excitatory neurons

Inhibitory synapc transmission phenotype Mice/Synapse

B

C

NL-2 KO NL-3 KO NL-3 R451C KI

Decrease No change Decrease

No change

Nptx2 Gpr3 Osgin2 BDNF

D Increase Increase

NPAS4

TRENDS in Molecular Medicine

Figure 3. Inhibitory synapse organizers have distinct actions on neural circuit activities in distinct neuron types. The figure presents a pyramidal neuron (orange) interconnected to interneurons (green, blue or violet). The pyramidal neuron also receives an excitatory input from another excitatory neuron (not shown). Potential inhibitory synaptic pathways are based on their subcellular localizations (boxes). (A) Inhibitory synapse formed on an interneuron. Note that parvalbumin (PV)-positive interneurons also innervate the axon initial segments of pyramidal neurons (not shown here). (B–D) Inhibitory synapses formed on the pyramidal neuron. Npas4 activates distinct sets of inducible genes in pyramidal (i.e., excitatory; nucleus labeled 2) and inhibitory neurons (e.g., somatostatin-positive interneurons; nucleus labeled 1) of the medial ganglionic eminence, which is highly enriched for inhibitory neurons [86]. Notably, NL-2 specifies distinct inhibitory neural circuits involving PV-positive interneurons (synapse B), but not those that involve somatostatin (SOM)-positive interneurons (synapse C) [21]. The changes in inhibitory synaptic transmission observed in NL-3-KO and NL-3 R451C-KI mice are diagrammed. Abbreviations: BDNF, brain-derived neurotrophic factor; Ddhd1, DDHD domain-containing 1; Frmpd3, FERM and PDZ domain-containing 3; Gpr3, G protein-coupled receptor 3; Kcna1, potassium voltage-gated channel, shaker-related subfamily, member 1; Nptx2, neuronal pentraxin 2; Osgin2, oxidative stress-induced growth inhibitor family member 2; Slc25a36, solute carrier family 25, member 36.

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Review brain-derived neurotrophic factor (BDNF) regulates gephyrin clustering via mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways [52]. Lastly, the fibroblast growth factor 7 (FGF7) is a target-derived presynaptic organizer that selectively promotes inhibitory synaptic differentiation. FGF7-KO mice show selective impairments among the inhibitory nerve terminals that innervate CA3 pyramidal neurons [53], and are prone to epileptic seizures induced by chemical kindling; the latter effect is due to defects in inhibitory synapse formation, mossy fiber sprouting, and enhanced neurogenesis, likely reflecting E/I alterations in these mice [54]. Future studies should examine how Sema4D interacts with PlexinB1 to mediate inhibitory synapse development in vivo and investigate the molecular mechanisms underlying developmental changes related to FGF7-KO-induced seizure vulnerability. Synapse formation by scaffold proteins, signaling proteins, and transcription factors Scaffolding proteins, signaling proteins, cytoskeletal proteins, and transcription factors also coordinate inhibitory synapse development and function [55]. We describe recent progress in our understanding of gephyrin, collybistin, BRAG3, and Npas4 as examples of intracellular elements that mediate inhibitory synapse development (Figures 2 and 3). The scaffold molecule gephyrin is essential for glycine and GABAA receptor clustering, inhibitory synaptic transmission, and plasticity (reviewed in [56]). The topology, stoichiometry, and absolute densities of inhibitory receptors and gephyrin were recently determined using singlemolecule imaging [57]. Similar to the case of PSD-95, the clustering of gephyrin at postsynaptic membranes is regulated by BDNF-, glycogen synthase kinase 3b (GSK3b)-, cyclin-dependent kinase 5 (CDK5)-, and collybistindependent phosphorylation [56]. Both the S-nitrosylation and palmitoylation of gephyrin can also influence its clustering, suggesting that multiple mechanisms oversee gephyrin clustering and (possibly) inhibitory synapse development [56]. Collybistin is another protein required for inhibitory receptor clustering and function [58]. The major brain isoforms of collybistin adopt closed confirmations due to the presence of the src homology 3 domain (SH3), but binding to NL-2 or the Rho-like GTPase TC10 switches them to open forms and drives inhibitory synaptic differentiation [59]. Thus, the collybistins may link transsynaptic neuroligin-dependent synaptic adhesion with gephyrin recruitment during the development of inhibitory synapses. BRAG3 [also known as synArfGEF(P0) or IQSEC3] is a guanine nucleotide exchange factor for ADP-ribosylation factor 6 (ARF6). BRAG3 was initially identified as an excitatory synapse component, but was later demonstrated to be exclusively localized to inhibitory synapses [60,61]. Moreover, BRAG3 associates with dystrophin-associated glycoprotein complex and S-SCAM [60]; however, it is not yet known whether these protein complexes physiologically contribute to inhibitory synapse development. 262

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Neuronal PAS domain protein 4 (Npas4) is a transcription factor encoded by a neuronal activity-dependent immediate-early gene and is required for activity-dependent inhibitory synapse development, activity-dependent changes in inhibitory synaptic connectivity, and neurite outgrowth [62–64]. Npas4 knockdown selectively reduces the number of inhibitory synapses formed on both the soma and dendrites, suggesting that Npas4 may regulate the development of inhibitory synapses originating from multiple interneurons [62]. Npas4 regulates numerous genes, including other immediately-early genes and those encoding transcription factors and signaling proteins that modulate inhibitory synaptic functions. Among the Npas4 target genes, BDNF is noteworthy in that its expression in cultured neurons is reduced by Npas4 knockdown, and Npas4 directly binds to the BDNF gene promoter in an activity-dependent manner [62]. Expression levels of Npas4 and BDNF are reduced in serotonin transporter (SERT) KO mice, but chronic treatment with the antidepressant, duloxetine, restores the levels of both proteins, consistent with a recent report that the level of Npas4 is low in the hippocampus of depression-model rats [65,66]. Regulation of inhibitory synaptic plasticity Inhibitory synapses exhibit diverse forms of short- and long-term synaptic plasticity, depending on the involved interneuron type and brain region [67,68]. Inhibitory synaptic plasticity, manifested as changes in the probability of GABA release, post-transcriptional modification, and lateral diffusion of GABA receptors, modifications in the number of functional inhibitory synapses, and differences in the reversal potential of postsynaptic responses, is crucial for E/I balance, neural circuit refinement, and most forms of experience-dependent learning and memory. Furthermore, pathological levels of neural activity can induce long-term changes in inhibitory synaptic plasticity and may manifest as schizophrenia [67]. A few of the molecular mechanisms that regulate inhibitory synaptic plasticity have been elucidated. Presynaptic activation of the type II mGluRs causes inhibitory longterm depression (iLTD) [69], while decreased postsynaptic protein kinase A (PKA) activity in conjunction with calcineurin expression via A-kinase anchoring protein (AKAP) regulates iLTD to trigger the internalization of GABAA receptors [70]. Retrograde endocannabinoid (eCB) signaling mediates presynaptic iLTD (eCB-LTD) via the presynaptic active zone proteins RIM1a/Rab3B and P/Q-type voltage-gated calcium channels (VGCCs; reviewed in [71]). Intriguingly, the P/Q-type VGCCs are functionally compromised in neurexin double KO mice, implying that the a-neurexin-mediated influx of Ca2+ may be important for eCB-mediated plasticity at inhibitory synapses [72]. However, NL-3 is not required for phasic-eCB signalingdependent eCB-LTD [73]. Homeostatic synaptic plasticity is essential to maintain stability in neuronal networks in the face of global changes in neuronal activity. Gephyrin is a central element of various signaling pathways that contribute to homeostatic synaptic plasticity at inhibitory synapses [56]. Changes in network activity and signaling trigger downstream molecules to regulate gephyrin cluster size/density and the

Review interactions of gephyrin with GABAA receptor. This affects particular forms of inhibitory synaptic plasticity and determines the strength of inhibition, which in turn adjusts the excitability of the neuron and network. Accordingly, gephyrin-binding proteins such as collybistin may induce changes in downstream signaling events, altering the structural and functional organizations of inhibitory synapses. The dystroglycans, which act as ligands for a-neurexins, are localized to inhibitory synapses where they are upregulated in an activity-dependent manner [74]. Though these proteins are not essential for inhibitory synapse formation, the glycosylated forms of these proteins are necessary for homeostatic synaptic plasticity at inhibitory synapses in the hippocampus. Thus, dystroglycans may be required to recruit or stabilize GABAA receptors during the homeostatic scaling-up of inhibitory synaptic strength [74,75]. Another a-neurexin-specific ligand, neurexophilin-1, which shows limited expression in subpopulations of inhibitory neurons, instructs short-term synaptic plasticity at inhibitory synapses, and maintains presynaptic GABAB and postsynaptic GABAA receptors [76]. Moreover, neurexophilin-1 competes with dystroglycans for binding to a-neurexins [77]. Thus, various classes of inhibitory synaptic molecules are involved in different forms of synaptic plasticity, cooperatively driving the activity-dependent rewiring of local microcircuits and maintaining the E/ I balance in the brain. Regulation of inhibitory neural circuits The development of functional inhibitory circuitry is a complex process that relies on numerous intrinsic and extrinsic factors to mediate synaptic inhibition. These factors are operated by distinct groups of inhibitory neurons acting on distinct compartments of other neurons (i.e., dendrite versus perisoma; reviewed in [78]). Particular inhibitory synapse proteins are important in shaping circuit development, as exemplified by studies showing that some neurodevelopmental disorders are associated with abnormal development of inhibitory neural circuits [79]. We discuss below NL-2 and Npas4 as examples. NL-2 deletion in mice impairs inhibitory synapse function [12] and uniformly decreases inhibitory synaptic transmission at unitary connections in response to synaptic activity [18]. Intriguingly, the latter effect was observed at inhibitory connections originating from fast-spiking interneurons (i.e., parvalbumin-positive cells) but not from burst-spiking interneurons (i.e., somatostatin-positive cells) [18] (Figure 3). The mechanisms underlying the different roles of NL-2 in distinct inhibitory interneurons are not yet clear; one possibility is that NL-2 and its specific ligands may show differential expression. Overall, NL-2 appears to function in the activity-dependent circuit refinement that regulates the strength of inhibitory connections. Npas4 regulates inhibitory synapse structure and function in cultured neurons [62]. Its conditional deletion in postnatal neurons decreases inhibitory synaptic transmission in vivo [62,80], whereas its induction by enriched environment regulates domain-specific synaptic inhibition. In Npas4-KO mice exposed to an enriched environment (but not those exposed to standard housing

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conditions), inhibitory synaptic transmission is decreased upon stimulation of axons located in the stratum pyramidale layer of hippocampal CA1, but is increased upon stimulation of axons located in the stratum radiatum of CA1. Moreover, Npas4 and its target, BDNF, regulate somatic but not apical dendritic inhibition in neurons, suggesting that the Npas4-mediated signaling cascade reconfigures the mode of inhibition of a neural circuit that may underlie cognitive functions [80,81]. Npas4 also activates specific repertoires of late-response genes in multiple types of neurons following membrane depolarization and sensory stimulation [82] (Figure 3). Npas4 increases the excitation of somatostatin-containing interneurons and lowers their overall circuit activity by activating a host of late-response genes selectively expressed in interneurons, but increases the inhibition of excitatory neurons and lowers their overall circuit activity by activating a distinct set of late-response genes selectively expressed in excitatory pyramidal neurons [82]. Thus, neural activity appears to induce a common set of transcriptional regulators but distinct sets of late-response genes in excitatory and inhibitory neurons, and Npas4 may coordinate the activations of distinct sets of effector genes in both neuron types, thus providing unique circuit-wide homeostatic mechanisms (Figure 3). Dysfunction of inhibitory synaptic proteins in brain diseases Because perturbations in the GABA system can lead to severe neurological impairment [79], numerous orthosteric agonists, antagonists, and allosteric modulators of GABAA and GABAB receptors have been devised to correct the E/I imbalances manifested in various brain disorders. Drugs targeting the GABA-metabolizing enzymes (e.g., GABA transaminase) and neurotransmitter transporters (e.g., GAT1 and glycine transporters) have attracted therapeutic interest in the context of GABA-related brain disorders [83]. These drugs typically decrease the uptake of GABA into presynaptic terminals, whereas benzodiazepines, barbiturates, topiramate, and felbamate enhance inhibitory synaptic transmission by allosterically modulating GABAA receptor-mediated chloride currents [84]. Drugs in this latter group are widely prescribed to treat a subset of neurological disorders, but undesirable side effects limit their continuous use [85]. Given the important contributions of inhibitory synapse dysfunctions to neurodevelopmental disorders, neurological conditions that would benefit from drug development in this area include autism spectrum disorders (ASDs), schizophrenia, epilepsy, Down’s syndrome, fragile X syndrome, and Rett syndrome [86]. For ASDs and tuberous sclerosis, animal models that reproduce the genetic bases of the human disorders have strong construct and face validity. The autistic-like social behaviors of Shank2-KO mice are improved by the pharmacological restoration of NMDA-type glutamate receptor function with a positive allosteric modulator of mGluR5 [87]. Similarly, tuberous sclerosis complex (TSC) KO mice show hippocampal network hyperexcitability (including elevated spontaneous activity and increased seizure 263

Review

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susceptibility) at least in part due to reduced inhibition of KO pyramidal neurons [88]. NL-2-KO mice show abnormal anxiety-related behaviors, consistent with previous reports that pharmacological manipulation of GABAA receptor function in rodents alters anxiety levels, and that benzodiazepines effectively treat anxiety in humans [89]. Despite the normal social behaviors seen in NL-2-KO mice, NL-2 may still be associated with ASDs because increased anxiety is frequently associated with ASDs [90]. NL-3 localizes to GABAergic synapses; however, its roles at inhibitory synapses have not been fully explored [91] although they have clinical implications in ASDs, and several NL-3- and NL-4-KO and KI mice have been used to probe the candidate neural circuits and synapses responsible for idiopathic ASDs [92,93]. MDGA1 and MDGA2 have been associated with schizophrenia and ASDs, respectively [94,95]; however, relevant behavioral analyses of MDGA-KO mice are lacking. Neurexin-1a-deficient mice show normal anxiety-related behaviors, locomotor activity, spatial learning, and memory, but reveal repetitive grooming behaviors, impaired sensorimotor gating, and impaired nest-building behaviors. This suggests that neurexin-1a-KO mice may provide a model for schizophrenia [96]. Npas4-KO mice also replicate schizophrenia-like behaviors, including social anxiety, hyperactivity, and sensorimotor gating deficits [97]. Notably, repeated treatment with aripiprazole (a partial agonist of the dopamine D2 receptor) upregulates Npas4, indicating that augmentation of Npas4 using novel antipsychotic drugs might improve the cognitive defects of schizophrenia [98,99]. However, caution should be used when interpreting data from animal models

of neuropsychiatric disorders given the functional and anatomical differences between human and rodent brains. Numerous human genetic studies have linked single genes to multiple brain diseases. For example, some epilepsy-linked genes are also associated with ASDs. Loss of Slitrk3 in mice decreases glutamic acid decarboxylase (GAD) levels in a subset of hippocampal cell layers, increases seizure susceptibility, and enhances abnormal epileptiform activities on electroencephalogram (EEG) recordings [36]. Interestingly, altered expression of a human microRNA against Slitrk3 was observed in patients suffering from ASDs [100], suggesting that Slitrk3 may also be associated with ASDs. Interestingly, a recent report linked gephyrin to idiopathic generalized epilepsy, ASDs, and schizophrenia [101,102]. Notably, the severe symptoms of humans carrying a homozygous deletion of GPHN are consistent with the phenotypes observed by Feng et al. [103], further validating GPHN-KO mice as models for brain diseases. Historically, inhibitory synapses have been targeted in the development of antiepileptic drugs; thus, it is not surprising that key inhibitory synaptic molecules are involved in the etiology of epilepsy [83]. The introduction of two reported ASD-related NL-3 mutations into mice is associated with characteristic electrophysiological and behavioral phenotypes in various brain areas [73,92,93,104,105]. NL-3 R451C KI mice exhibit social-interaction deficits, increased synaptic inhibition in the somatosensory cortex, and decreased AMPA receptor-mediated synaptic transmission in the hippocampus [106]. NL-3 R451C KI mutations also impair inhibitory synaptic transmission in parvalbumin basket cell synapses, by lowering the probability of GABA release,

Table 2. Summary of neurological disorders associated with inhibitory synapse organizersa Organizer Neuroligin-2 Neuroligin-3 a-Neurexins

MDGA1 MDGA2 Gephyrin

Collybistin

Slitrk3 PTPd

IgSF9b Neuroplastin-65 Calsyntenin-3 Dystroglycan a

Mutation(s) Missense mutations Missense mutation (R451C) CNV (truncations in a1- and a2-neurexin) SNPs (a1-neurexin) Missense mutations (a1-neurexin) CNV (deletions in a3-neurexin) SNPs CNV (duplication) CNV (hemizygous microdeletions) CNV (microdeletions in the G domain)

Disease(s) Schizophrenia Autism Schizophrenia Schizophrenia ASD ASD Schizophrenia and bipolar disorder Autism Autism, schizophrenia, and epilepsy Idiopathic generalized epilepsy

SNPs CNV (centromeric breakpoint and microdeletion) CNV (chromosomal translocation and microdeletion) Nonsense mutation

X-linked infantile spinal muscular atrophy X-linked mental retardation Epilepsy Syndromic X-lined mental retardation and epilepsy

Abnormal expression of miRNAs SNPs in 50 -UTR CNV (hemizygous deletion) CNV (deletion) CNV (duplication) SNPs

Autism Restless leg syndrome Attention deficit hyperactivity disorder Autism Bipolar disorder Obsessive–compulsive disorder

SNPs SNPs Not reported Missense mutation (T192M)

Major depressive disorder Intellectual disability Alzheimer disease Muscular dystrophies

Abbreviations: CNV, copy-number variation; miRNA, microRNA; SNP, single-nucleotide polymorphism; UTR, untranslated region.

264

Refs [117] [118] [119] [120] [121] [122] [95] [94] [101] [102] [123] [124] [125] [126] [127] [128] [129] [130] [131] [38] [132] [133] [134] [135]

Review but enhance inhibitory synaptic transmission in cholecystokinin (CCK) basket cell synapses [73]. Conversely, NL-3KO selectively abolishes CCK basket cell-mediated inhibitory synaptic transmission due to defects in tonic endocannabinoid signaling [73] (Figure 3). Both NL-3-KO and NL-3 R451C-KI mice develop stereotyped repetitive behaviors due to impaired synaptic inhibition of D1-dopamine receptor-expressing neurons in the striatum [93]. By contrast, NL-3 R704C-KI mice exhibit selective decreases in AMPA receptor-mediated synaptic transmission in the hippocampus [106]. Overall, these data suggest that ASDrelated NL-3 mutations differentially modulate synaptic properties via multiple mechanisms, each a potential therapeutic target for ASDs. Npas4 may also play an important role in cognitive functions. Its mRNA and protein levels are upregulated by fear conditioning [81], and Npas4 knockdown in the lateral nucleus of the amygdala abrogated learned (but not innate) fear-memory formation [81]. This knockdown also selectively impaired fear-memory retention following retrieval, suggesting that Npas4-mediated signaling is crucial for fear-memory reconsolidation [81]. Several behavioral abnormalities have been reported in Npas4-KO mice, which exhibit novelty-induced locomotor hyperactivity, high levels of aggressive/dominant behavior, and impaired long-term working memory and social interactions [97]. A dose-dependent sensorimotor gating deficit was also observed in Npas4-KO mice, suggestive of typical symptoms of schizophrenia and autism. Given that Npas4 is crucial for E/I balance, Npas4 could be a therapeutic target for these neuropsychiatric disorders. Neurological disorders associated with inhibitory synapse organizers are summarized in Table 2. Concluding remarks and future perspectives Various regenerative approaches, including GABAergic neuronal precursor grafting, are currently being investigated as therapeutic alternatives for the treatment of inhibitory synapse-related brain disorders. For example, delivery of the GAD-encoding gene into the subthalamic nucleus to promote GABA synthesis has been reported to relieve clinical symptoms of Parkinson’s disease [107]. Transplantation of medial ganglionic eminence (MGE)-derived GABAergic precursors appears to restore synaptic plasticity defects in the visual cortex [108]. Moreover, postnatal transplantation of MGE-derived GABAergic precursor cells reduces seizure activity in the hippocampus [109], suggesting that inhibitory interneuron grafting-based cell therapy could be a powerful therapeutic approach for some brain disorders. In addition to the available animal models, patient-derived induced neuronal (iN) cells can be used to model specific neural circuit dysfunctions that offer new insight into the pathological mechanisms of human brain disorders [110], especially because iN cells derived from embryonic fibroblasts of NL-3 R704C KI mice successfully recapitulated cellular and electrophysiological phenotypes of these mice [111]. However, caution should be used when seeking to match in vivo brain developmental processes using human neural progenitor cells, especially for potential clinical applications [112].

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Box 2. Outstanding Questions  How do excitatory and inhibitory synaptic proteins cooperate to organize synapse development and function?  What mechanisms underlie inhibitory synapse development?  How do inhibitory synaptic adhesion molecules cooperate with various intracellular proteins to mediate synaptic inhibition?  How does inhibitory synapse elimination occur?  Is inhibitory synapse formation, transmission, and plasticity relevant to cognitive behaviors?  Which brain circuits are responsible for inhibitory synapse-related diseases (e.g., epilepsy)?  How do inhibitory synapse organizers affect the functioning of these circuits?  Can we pharmacologically or optogenetically rescue diseaserelated phenotypes in the adult?

Better understanding of the inhibitory synapse organizers reviewed above may make important contributions to our understanding of how brains operate at the synaptic and circuit levels (Box 2). Future advances in our molecular understanding of inhibitory synapse organization may enable us to accurately control the E/I balance, thus opening the door to new therapeutic interventions against devastating brain disorders. Acknowledgments This work was supported by the Korean Healthcare Technology R&D Project, Ministry for Health and Welfare Affairs, Republic of Korea (A120590 to J.K.), Yonsei University Future-leading Research Initiative of 2014 (to J.K.), Basic Science Research Program through the National Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A6A3A04061338 to J.W.U.), and in part by the Brain Korea 21 (BK21) PLUS program. G.C. is a fellowship awardee of the BK21 PLUS program.

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