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Neuroligins, synapse balance and neuropsychiatric disorders
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PHAREP 120 1–6 Pharmacological Reports xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep 1 2
Review article
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Neuroligins, synapse balance and neuropsychiatric disorders
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Q1 Marzena
Mac´kowiak *, Patrycja Mordalska, Krzysztof We˛dzony
Laboratory of Pharmacology and Brain Biostructure, Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 January 2014 Received in revised form 17 April 2014 Accepted 22 April 2014 Available online xxx
Neuroligins are postsynaptic adhesion molecules that are involved in the regulation of synapse organisation and function. Four neuroligin proteins have been identified (neuroligin 1, 2, 3, 4), which are differentially enriched in the postsynaptic specialisation of synapses. Neuroligin 1 is localised on excitatory (glutamatergic) synapses, whereas neuroligin 2 is located on inhibitory (GABAergic/ glycinergic) synapses. Neuroligin 3 and 4 are present on both types of synapses. Recent data indicate that neuroligins are involved in synapse maturation and specification. Because of their synaptic localisation and function, neuroligins control the balance between excitatory and inhibitory synapses. Animal studies with neuroligin transgenic mice showed the involvement of neuroligin 1 in memory formation, and neuroligin 2, 3 or 4 in social behaviour. Interestingly, genetic analysis of humans showed a mutation in the neuroligin 2 gene in schizophrenic patients, while mutations in neuroligin 3 or 4 genes were found in autism. ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Keywords: Cell adhesion molecules Neuroligin Synapse Autism Schizophrenia
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Contents Neuroligin family . . . . . . . . . . . . . . . . . . . . . Neuroligins at the excitatory synapses . . . . Neuroligins at the inhibitory synapses . . . . Excitatory and inhibitory synapse balance . Neuropsychiatric disease . . . . . . . . . . . . . . . Autism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroligin 1 . . . . . . . . . . . . . . . . . . . . . . . . . Neuroligin 2 . . . . . . . . . . . . . . . . . . . . . . . . . Neuroligin 3 . . . . . . . . . . . . . . . . . . . . . . . . . Neuroligin 4 . . . . . . . . . . . . . . . . . . . . . . . . . Schizophrenia. . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .
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Neuroligin family
Abbreviations: ASDs, autism spectrum disorders; GABA, g-aminobutyric acid; LTP, long-term potentiation; mEPSC, miniature excitatory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; NMDA, N-methyl-D-aspartate; PSD-95, postsynaptic density protein-95; sPSC, spontaneous postsynaptic current. * Corresponding author. E-mail address: [email protected] (M. Mac´kowiak).
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Neuroligins are postsynaptic transmembrane adhesion pro- Q227 teins that are comprised of several domains including a cleaved 28 signal peptide, a cholinesterase-like domain, a carbohydrate 29 attachment region, a single transmembrane domain, and a short 30 C-terminal tail containing a type I PDZ-binding motif (for review 31 see Sudhof [1]). Neuroligin proteins have been found in several 32
http://dx.doi.org/10.1016/j.pharep.2014.04.011 1734-1140/ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Please cite this article in press as: Mac´kowiak M, et al. Neuroligins, synapse balance and neuropsychiatric disorders. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.011
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PHAREP 120 1–6 M. Mac´kowiak et al. / Pharmacological Reports xxx (2014) xxx–xxx
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Excitatory synapse
Inhibitory synapse
Neuroligins at the inhibitory synapses
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Presynapc membrane
Presynapc membrane
Neuroligin 2 is known to be constitutively and selectively present at inhibitory postsynaptic specialisations [11]. Neuroligin 2 is localised at both GABA-ergic and glycinergic inhibitory synapses [12,13]. Neuroligin 2 preferentially binds the inhibitory synapse scaffold protein gephyrin through a conserved cytoplasmic motif [13], and it is able to cluster GABAA receptors [14] (Fig. 1). In addition to neuroligin 2, neuroligin 3 and 4 were also found at inhibitory synapses [10,15]; however, they are not specific markers for inhibitory synapses (see above). Neuroligin 3 was observed at GABA-ergic synapses [10], while neuroligin 4 was localised at glycinergic synapses [15].
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Excitatory and inhibitory synapse balance
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Proper brain function is based on a balance between excitation and inhibition, which are mainly mediated by two major neurotransmitters, glutamate and GABA, respectively. The total number of synapses formed and ratio of excitatory to inhibitory synaptic inputs a neuron receives are factors critical for determining neuronal excitability [16]. Molecules that are involved in the control of a balance between excitatory and inhibitory synapse formation are important for proper neuronal excitability and function. Several findings indicate that neuroligins are involved in both excitatory and inhibitory synapse maturation and specificity, and they are able to control the balance between excitatory and inhibitory synapse formation [5]. An in vitro study showed that the suppression of single (neuroligin 1 or 2 or 3) or multiple neuroligin isoform (neuroligins 1–3) expression in cultured rat hippocampal neurons results in a loss of excitatory and inhibitory synapses. However, electrophysiological analysis demonstrated a predominant reduction of inhibitory synaptic function and alteration in normal excitatory/inhibitory balance in hippocampal neurons [6]. The largest changes in inhibitory synaptic transmission than excitatory transmission were also observed in an electrophysiological study in neuroligin knockout mice [7]. It was found that the deletion neuroligins 1–3 dramatically changed the balance between glutamatergic and GABAergic/glycinergic spontaneous postsynaptic currents (sPSC) in brainstem neurons, with a strong decrease in GABAergic/ glycinergic sPSC, without affecting the total synapse numbers [7]. In contrast, exogenous neuroligin 1 increased both excitatory and inhibitory presynaptic contacts and the frequency of miniature excitatory and inhibitory postsynaptic currents (mEPSC and mIPSC, respectively) in the cultured hippocampal neurons [17]. Thus, the above data indicate that the proper level of neuroligin proteins seems to be an important factor in the control of the excitatory and inhibitory balance in the brain, and the decrease in neuroligin levels mainly influence the inhibitory transmission. The fact that all single neuroligin knockout mice as well as all combinations of double neuroligin knockouts were viable, whereas neuroligin 1–3 triple knockout mice died shortly after birth, may indicate a significant degree of functional redundancy among neuroligins [7]. The above observation was also confirmed by the results from an in vitro study showing that the overexpression of all neuroligin isoforms is able to stimulate the formation of both excitatory and inhibitory terminals [6]. On the other hand, the study with transgenic mice showed that the overexpression of neuroligin 1 increased the maturation of excitatory synapses [18]. However, there were no differences in the number and size of glutamatergic and GABAergic hippocampal synapses in neuroligin 1 knockout mice when compared to wild-type animals [19]. In contrast, a decrease in GABAergic but not glutamatergic transmission was found in neuroligin 2 knockout mice [12,13]. The role of neuroligin 2 in the control of inhibitory synapse function was also
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Neurexin
Neurexin
NMDA-R
Neuroligin 1
GABAA-R
PSD-95 Postsynapc membrane
Neuroligin 2 Gephyrin
Postsynapc membrane
Fig. 1. Schematic illustration of neuroligin binding with neurexin (presynaptic membrane) and postsynaptic scaffolding proteins (PSD-95, gephyrin).
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species, including humans, rodents, and chickens. Five genes encoding neuroligin proteins have been identified in the human genome (NLGN1, NLGN2, NLGN3, NLGN4, NLGN4Y), and at least four genes coding neuroligin family members have been known in rodents (NLGN1-4). Sequence comparisons indicate that neuroligins 1, 3, and 4 are more similar to each other than to neuroligin 2 [1,2]. Neuroligins link the presynapse to the postsynaptic density by binding through the extracellular cholinesterase-like domain to their presynaptic partners, neurexins, in an alternative splicedependent manner. At the postsynaptic portion, neuroligins bind by their C-terminus with the third PDZ domain of a postsynaptic scaffold protein, such as the postsynaptic density protein-95 (PSD95), which anchors a variety of signalling molecules and surface receptors [1–3] (Fig. 1). It has been suggested that the extracellular domain of neuroligin is sufficient to induce the assembly of functional presynaptic terminals, while the intracellular domain is required for terminal maturation [4]. Several findings indicate that neuroligins are present at developing and mature synapses of the brain and play an important role in synapse organisation and function [1,3,5]. They were initially thought to be required for synapse formation [6], but recent findings indicate their crucial involvement in synapse maturation and specification [7]. It was found that neuroligins are differentially enriched in the postsynaptic specialisations of synapses, and they are found on either excitatory or inhibitory synapses.
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Neuroligins at the excitatory synapses
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Neuroligin 1 proteins are exclusively localised on excitatory synapses [8]. This observation is supported by the facts that the neuronal localisation, subcellular distribution and developmental expression of this protein are connected with the excitatory postsynaptic marker protein PSD-95 and N-methyl-D-aspartate (NMDA) receptor (Fig. 1). Moreover, electron microscopy study demonstrated that only asymmetric synapses contain neuroligin 1, and immunofluorescence labelling showed that neuroligin 1 colocalises with glutamatergic but not with g-aminobutyric acid (GABA)-ergic synapses [8]. An in vitro study also showed the coaggregation of neuroligin 1 with PSD-95 (scaffold protein of excitatory synapses), but not gephyrin (scaffold protein of inhibitory synapses) in cultured neurons [9]. A similar effect was observed for neuroligin 3 and neuroligin 4, which suggests that these two proteins are also expressed on excitatory synapses [9]. Additional studies confirmed the presence of neuroligin 3 on glutamatergic synapses in the brain and coimmunoprecipitation studies revealed the occurrence of neuroligin1–neuroligin3 complexes in the brain extracts [10].
Please cite this article in press as: Mac´kowiak M, et al. Neuroligins, synapse balance and neuropsychiatric disorders. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.011
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confirmed by the data from a study of transgenic mice that demonstrated that the enhanced expression of neuroligin 2 results in a significant increase in the density of symmetric (inhibitory) synapses in cortical areas, followed by a small significant change in excitatory synapse morphology [20]. The above results demonstrate that although the expression of neuroligin isoforms is specific for the type of synapse, they are also able to functionally replace each other. The differences in the synaptic localisation of distinct neuroligins suggest the presence of cellular processes involved in maintaining the excitatory/inhibitory balance by controlling the trafficking of different neuroligin isoforms to specific synaptic sites. Current evidence indicates an important role for both the extracellular and intracellular domains of neuroligins in their synaptic localisation. Several findings suggest that neuroligins mediate their effects on excitatory or inhibitory synapse development through trans-synaptic interaction with neurexins. Alterations in the extracellular domains of neuroligins (1, 2, 3) or neurexin-1b affected excitatory and inhibitory synapse formation [9,21,22]. However, recent data have also shown that the targeting of neuroligins to specific postsynaptic sites is determined by postsynaptic scaffolding molecules, which bind to the C-terminal PDZ-binding domain of neuroligins [23]. It was found that the postsynaptic complex of scaffolding protein PSD-95 and neuroligin can modulate the release of transmitter from vesicles at the synapse in a retrograde way [24] and link postsynaptic and presynaptic function. It was also suggested that not only the expression of neuroligins but also the interaction between neuroligins and PSD-95 regulate the balance of excitatory and inhibitory synapse formation [17]. PSD-95 restricts the localisation and effects of neuroligin 1 to excitatory synapses [17]. The overexpression of PSD-95 increased the level of neuroligin 1, but it could also shift the localisation of neuroligin 2 from inhibitory to excitatory contacts, reducing mIPSC frequency [9,17,22]. In addition, a decrease of the endogenous level of PSD-95 by siRNA administration shifts neuroligin 1 from excitatory to inhibitory synapses [25], while the knockdown of PSD-95 leads only to a partial shift of neuroligin 2 and neuroligin 3 from excitatory to inhibitory synapses [26], most likely because these neuroligins are already expressed on inhibitory synapses. The above data indicate that the PSD-95 level might be a main factor in determining the recruitment of neuroligins to excitatory versus inhibitory synapses, especially neuroligin 1. Recent evidence indicates that neuroligin 2 preferentially binds to inhibitory postsynaptic scaffolding molecules, such as gephyrin and collybistin, and the deletion of neuroligin 2 in mice disturbs GABAergic and glycinergic transmission [13]. Moreover, the siRNA-mediated deactivation of gephyrin leads to a shift of endogenous neuroligin 2 from inhibitory to excitatory synaptic contacts [26]. The above results might suggest that neuroligin 2 can bind to both inhibitory (gephyrin) and excitatory (PSD-95) postsynaptic scaffolding proteins, but it preferentially binds to gephyrin, most likely due to its higher affinity to inhibitory than excitatory scaffolding proteins. Interestingly, it was also shown that knockdown of neuroligin 2 evoked a reduction in glutamatergic events (i.e., the decrease in mEPSC) and dendritic spines, which was rescued by the overexpression of a chloride transporter, KCC2, together with neuroligin 2 shRNA [27]. KCC2 controls intracellular Cl concentration by pumping Cl outside neurons (for review, see Blaesse et al. [28]). Moreover, independent of its Cl transport function, KCC2 was also found to regulate dendritic spine maturation and excitatory synapse development by interacting with the spine cytoskeleton [29]. It was shown that the KCC2 C-terminal domain binds to the cytoskeleton-associated protein 4.1 N [29], which is known to play an important role in mediating structural interactions between the cytoskeleton,
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transmembrane proteins and adhesion molecules [30]. The above data suggest that neuroligin 2 not only regulates GABAergic synapses but also influences glutamatergic synapses by switching the bond with inhibitory to excitatory postsynaptic scaffolding proteins or by regulating KCC2 function. It seems that neuroligin 2 might be an important regulator in balancing GABAergic and glutamatergic functions in the brain. The last observation might be supported by data showing that the chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin 2, whereas the selective chronic inhibition of glutamatergic synapse components, such as NMDA receptors or their downstream signals, suppresses the synaptic-boosting activity of neuroligin 1 [31]. Thus, neuroligins specify and validate synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses.
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Neuropsychiatric disease
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Cell adhesion molecules are known to be involved in the pathomechanism of some neuropsychiatric disorders [1,32]. Several neurodevelopmental disorders, such as autism spectrum disorders (ASDs), some forms of mental retardation and schizophrenia, are known to be caused by shifts in excitatory and inhibitory balance [1]. The factors involved in the regulation of excitatory and inhibitory synapse balance might play an important role in disease development and also might be potential targets for new therapeutic strategies. As it was mentioned above, neuroligins participate in the regulation of excitatory and inhibitory balance, and because of that, several genetic and functional studies have been performed to determine the role of neuroligins in some neuropsychiatric diseases.
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Autism
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ASDs include idiopathic autism, Asperger’s syndrome or Rett syndrome [1]. The diagnosis of ASDs is based on the presence of two major symptoms: social-communication deficits and restricted and repetitive interests/behaviours. Moreover, ASDs are also associated with sensory and motor abnormalities, sleep disturbance, attention deficits, intellectual disability and mood disorders such as anxiety (for review, see Grzadzinski et al. [33]). The first symptoms of ASDs are typically observed in children before 2–3 years of age. ASDs are heritable in 80% of cases, which suggest that ASDs are largely determined by genes [1]. Genetic studies discovered mutations in the genes encoding neuroligin 3 and neuroligin 4 in patients with ASDs. A single mutation in the neuroligin 3 gene (the R451C substitution) and ten different mutations in the neuroligin 4 gene were detected. In addition, five different larger deletions of X-chromosomal DNA that included the neuroligin 4 locus were found in autism patients [1,34–39]. It is of interest that an in vitro study also showed that neuroligin 4X deletion resulted in neurodevelopmental defects and decreased gene expression of other neuroligins (neuroligin 1 and 3) [40]. Thus, the mutation in neuroligin genes may affect the neurodevelopmental process inducing disease symptoms, for example ASDs. Many genetic and non-genetic animals models of ASDs have been characterised and used to identify the aetiology of ASDs and to develop novel treatment (for review, see Won et al. [41]). Using these models, some animal behaviour similar to the symptoms observed in ASDs have been analysed, such as sociability and social novelty recognition, ultrasonic vocalisation showing the communication patterns, restricted interest or repetitive behaviour [41]. The role of neuroligins in ASD development was investigated in transgenic animals (Table 1).
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Please cite this article in press as: Mac´kowiak M, et al. Neuroligins, synapse balance and neuropsychiatric disorders. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.011
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Table 1 The phenotypes of transgenic mice with altered neuroligin expression. The behavioural parameters were measured in knockout (KO), knockin (KI) or mice with neuroligin overexpression (OE); N/A – not analysed. Neuroligins
Transgenic mice
Autism spectrum disorder-like phenotype Social interaction
Social communication
Repetitive behaviour
Other functional impairments
References
Neuroligin 1 (NLG1)
NLG1- KO NLG1-OE
Minimal impairment N/A
N/A N/A
Enhancement N/A
Learning and memory deficits Learning and memory deficits
[19,42] [18]
Neuroligin 2 (NLG2)
NLG2-KO NLG2-OE
Normal Impairment
Reduced calls N/A
Normal Enhancement
Anxiety Anxiety
[43,44] [20]
Neuroligin 3 (NLG3)
NLG3-KO
Impairment
Reduced calls
Normal
[47]
NLG3R451C-KI
Impairment
Increased calls
N/A
Hyperactivity Alterations in learning and memory processes Enhanced learning ability
Neuroligin 4 (NLG4)
NLG4-KO
Impairment
Reduced calls
Normal
N/A
[54]
[49]
such as reduction in ultrasound vocalisation and a lack of social novelty preference [47]. A decrease in the freezing response in contextual and cued fear conditioning, but increased learning ability during the reversal task of the Morris water maze, was also observed in neuroligin 3 knockout mice [47]. Recent experiments with mice having a R451C mutation in neuroligin 3 previously found in patients with autism disorders [37,38] showed that the R451C mutation of neuroligin 3 does not alter neuroligin 3 mRNA levels, but decreases the export of the neuroligin 3 protein from the endoplasmic reticulum [48]. Therefore, a decrease in neuroligin 3 concentration was observed in the synapses of mice with the R451C mutation [49]. The animals with the neuroligin 3 R451C mutation showed impaired social interaction [49,50], but enhanced spatial learning abilities [49]. They also showed increased inhibitory synaptic transmission in the hippocampus in early postnatal life [51] and in the adult somatosensory cortex [49]. However, in the adult hippocampus of neuroligin 3 R451C mutated mice, an increase in AMPA receptor-mediated excitatory synaptic transmission, an alteration in the kinetics of NMDA receptordependent synaptic response, and the enhancement of LTP were observed [50]. In contrast, the neuroligin 3 R704C mutation selectively decreased the AMPA receptor-mediated synaptic transmission in hippocampus, without changing NMDA or GABA receptor-mediated synaptic transmission [52]. Thus, neuroligin 3 is involved in the regulation of both inhibitory and excitatory transmission by changing AMPA receptor function. By controlling the recruitment of AMPA receptors to the postsynaptic membrane, neuroligin 3 is involved in a critical process in excitatory synapse maturation [53]. These results are also in line with immunohistochemical data showing the presence of the neuroligin 3 protein of either glutamatergic or GABAergic synapses [10], and they also confirm the role of neuroligin 3 in the control of excitatory/ inhibitory balance, which is impaired in ASDs.
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Neuroligin 4
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Available data indicate that neuroligin 4 knockout mice also exhibit highly selective deficits in reciprocal social interactions and communications reminiscent of autism in humans. They also showed a slight reduction in the total brain volume [47,54]. Such knockout mice are considered to be a mouse model of monogenic heritable autism [54]. The animal study might be supported by genetic findings showing the possibility of the involvement of neuroligin 4 gene mutations in autism and cognitive disability [38].
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Neuroligin 3
Schizophrenia
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Neuroligin 3 knockout mice showed a slight reduction in the total brain volume [47]. Neuroligin 3 deficient mice display a behavioural phenotype reminiscent of the main symptoms of ASD,
Similar to ASDs, schizophrenia is a neurodevelopmental disorder with cognitive disabilities [55]. The onset of schizophrenia is usually observed in late adolescence or early adulthood; however,
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Neuroligin 1
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Neuroligin 1 knockout mice exhibited an increase in repetitive, stereotyped grooming behaviour, most likely associated with a reduced NMDA/AMPA receptor ratio in cortico-striatal synapses [19]. Moreover, a slight decrease in social interaction was also found [19]. Neuroligin 1 knockout mice also display deficits in spatial learning and memory that correlate with impaired hippocampal long-term potentiation (LTP) [19]. In addition, deficits in the storage of associative fear memory in fear conditioning tasks were discovered in neuroligin 1 knockout mice [42]. The transgenic mice with neuroligin 1 overexpression showed deficits in memory acquisition, but not retrieval. Moreover, a shift in the synaptic activity towards increased excitation as well as the impairment in LTP induction was observed in electrophysiological studies [18]. The obtained date indicated that the proper neuroligin 1 level, especially in the hippocampus, was critical for memory formation. Moreover, the results also suggest that the impairment of the neuroligin 1 level might induce the development of symptoms specific for autism (repetitive behaviour) but also might be involved in the cognitive disability observed in ASDs.
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Neuroligin 2
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Neuroligin 2 knockout mice showed an increase in anxiety-like behaviour, a decrease in pain sensitivity and a slight deficit in motor coordination [43]. The mice with neuroligin 2 deletions displayed normal social behaviour, but developmental delays and a reduction in pup ultrasonic vocalisation was observed [44]. The above changes in the mouse behaviour might be related to modifications in inhibitory transmission because the deletion of neuroligin 2 evoked alterations in GABAergic and glycinergic synaptic transmission and led to a loss of postsynaptic specialisations, specifically at perisomatic inhibitory synapses [13]. However, neuroligin 2 overexpression in transgenic mice resulted in stereotypical jumping behaviour, anxiety and impairment in social interaction [20]. Moreover, neuroligin 2 overexpression in the rat hippocampus led to reduced aggression and novel reactivity, but had no impact on sociability and memory [45]. Some evidence also indicates that the reduced sociability and increased aggression observed in rats following chronic restraint stress is induced by a decrease in the hippocampal neuroligin 2 but not neuroligin 1 expression [46]. Thus, the above results indicate that the modulation of the neuroligin 2 protein level might affect social and emotional behaviour.
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Please cite this article in press as: Mac´kowiak M, et al. Neuroligins, synapse balance and neuropsychiatric disorders. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.011
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the symptoms are considered to be the result of early life developmental disruptions. The sings of schizophrenia include both positive (delusion, hallucination) and negative symptoms (poor speech, social withdrawal, blunt affect). Genetic epidemiological studies showed that schizophrenia is a complex disease [56], and the meta-analytic results from twin studies showed heritability at approximately 70–80% [57]. Among several investigated genes, mutations in the neuroligin 2 [58] and neuroligin 4 [59] genes were found in schizophrenia patients. Screening for mutations in the exon and promoter of the neuroligin 2 gene in schizophrenic patients identified six point mutations: R215H, V510M, R621H, A637T, P800L and A819S [58]. Functional studies in the HEK 293T-neuron co-culture system using immunocytochemistry and electrophysiological recordings revealed that the R215H mutant was unable to develop GABAergic synapse formation due to a severe defect in intracellular trafficking [58]. The above finding indicates the importance of neuroligin 2 in the formation of functional inhibitory synapses. Moreover, the disruption of GABAergic transmission is observed in schizophrenia and defects in inhibitory circuit function contribute to working memory impairments observed in schizophrenia [56]. In addition, a recent genetic study also noted rare exonic deletions in the gephyrin gene as a risk factor for schizophrenia [60]. As it was mentioned above, gephyrin is an inhibitory postsynaptic scaffolding protein that is involved with the neuroligin 2 protein in GABAergic synapse formation. Thus, this might suggest that changes in the neuroligin 2 and gephyrin protein levels due to genetic mutations affect the proper development of inhibitory synapses in the schizophrenic brain. The preliminary study of neuroligin 2 in an animal neurodevelopmental model of schizophrenia based on the postnatal administration of an NMDA receptor antagonist showed the changes in neuroligin 2 protein levels in the medial prefrontal cortex in adolescent, but not adult rats [61]. An example of neuroligin 2 immunostaining in the rat medial prefrontal cortex is shown in Fig. 2. The above evidence indicates that alterations in the neuroligin 2 protein level induced by genetic or environmental (pharmacological) factors might have resulted in impairments in inhibitory synapse development, most likely during adolescence, and might have caused the deficit in GABAergic transmission observed in the brains of adult schizophrenic patients [56]. The screening for neuroligin 4 gene truncating and transmembrane domain mutations in schizophrenic patients was also investigated [59]. The results showed that the transmembrane domain is more likely involved in the aetiology of schizophrenia than the truncating mutations of the neuroligin 4 gene [59]. It is also of interest that the deletion in neuroligin 4 gene was also found in autistic patients [36]. The same relationship was found in relation to the gephyrin gene [60]. Moreover, the mice with altered neuroligin 2 protein levels showed some autistic-like symptoms [20]. Recent clinical data indicate that some patients with treatment-resistant schizophrenia have autistic symptoms, and these symptoms co-vary with negative symptoms, but not positive
Fig. 2. Neuroligin 2 immunostaining in the rat medial prefrontal cortex (mPC). (A) The distribution of neuroligin 2 in the mPFC. (B) The examples of neuroligin 2 immunoreactive cell (asterisk) and puncta in the neuropil (arrows). The scale bars are 500 mm (A) or 50 mm (B).
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symptoms of schizophrenia [36,62]. Thus, it is conceivable that alterations in the same genes (i.e., neuroligin 4, neuroligin 2 or gephyrin) might be involved in the symptoms of schizophrenia and autism, which are both neurodevelopmental disorders with cognitive deficits.
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Conclusions
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The collected data indicate that neuroligins are involved in maturation and synapse specification. The main factors determining their synaptic localisation are presynaptic neurexins and postsynaptic scaffolding proteins (PSD-95, gephyrin). The neuroligins’ ability to control the balance between excitatory and inhibitory synapses makes them an interesting target in the investigation of the pathophysiology of neuropsychiatric disorders. So far, a functional study using a transgenic animal model with impairments of neuroligin protein levels (knockout, knockin and overexpressed mice) showed the involvement of neuroligins in memory processes, and also in social and emotional behaviour. Moreover, genetic analysis of people showing symptoms of some neurodevelopmental disorders indicate that alterations of the neuroligin structure might be involved in the pathomechanism of psychotic disorders such as autism or schizophrenia.
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Funding
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This work was supported by Grant No. POIG.01.01.02.-12-004/ 09 ‘‘Depression-Mechanism-Therapy’’ (part 2.2. to MM).
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Conflict of interest
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All authors declare that they have no conflicts of interest.
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References
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PHAREP 120 1–6 Pharmacological Reports xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep 1 2
Review article
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Neuroligins, synapse balance and neuropsychiatric disorders
4 5
Q1 Marzena
Mac´kowiak *, Patrycja Mordalska, Krzysztof We˛dzony
Laboratory of Pharmacology and Brain Biostructure, Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 January 2014 Received in revised form 17 April 2014 Accepted 22 April 2014 Available online xxx
Neuroligins are postsynaptic adhesion molecules that are involved in the regulation of synapse organisation and function. Four neuroligin proteins have been identified (neuroligin 1, 2, 3, 4), which are differentially enriched in the postsynaptic specialisation of synapses. Neuroligin 1 is localised on excitatory (glutamatergic) synapses, whereas neuroligin 2 is located on inhibitory (GABAergic/ glycinergic) synapses. Neuroligin 3 and 4 are present on both types of synapses. Recent data indicate that neuroligins are involved in synapse maturation and specification. Because of their synaptic localisation and function, neuroligins control the balance between excitatory and inhibitory synapses. Animal studies with neuroligin transgenic mice showed the involvement of neuroligin 1 in memory formation, and neuroligin 2, 3 or 4 in social behaviour. Interestingly, genetic analysis of humans showed a mutation in the neuroligin 2 gene in schizophrenic patients, while mutations in neuroligin 3 or 4 genes were found in autism. ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Keywords: Cell adhesion molecules Neuroligin Synapse Autism Schizophrenia
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Contents Neuroligin family . . . . . . . . . . . . . . . . . . . . . Neuroligins at the excitatory synapses . . . . Neuroligins at the inhibitory synapses . . . . Excitatory and inhibitory synapse balance . Neuropsychiatric disease . . . . . . . . . . . . . . . Autism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroligin 1 . . . . . . . . . . . . . . . . . . . . . . . . . Neuroligin 2 . . . . . . . . . . . . . . . . . . . . . . . . . Neuroligin 3 . . . . . . . . . . . . . . . . . . . . . . . . . Neuroligin 4 . . . . . . . . . . . . . . . . . . . . . . . . . Schizophrenia. . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .
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Neuroligin family
Abbreviations: ASDs, autism spectrum disorders; GABA, g-aminobutyric acid; LTP, long-term potentiation; mEPSC, miniature excitatory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; NMDA, N-methyl-D-aspartate; PSD-95, postsynaptic density protein-95; sPSC, spontaneous postsynaptic current. * Corresponding author. E-mail address: [email protected] (M. Mac´kowiak).
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Neuroligins are postsynaptic transmembrane adhesion pro- Q227 teins that are comprised of several domains including a cleaved 28 signal peptide, a cholinesterase-like domain, a carbohydrate 29 attachment region, a single transmembrane domain, and a short 30 C-terminal tail containing a type I PDZ-binding motif (for review 31 see Sudhof [1]). Neuroligin proteins have been found in several 32
http://dx.doi.org/10.1016/j.pharep.2014.04.011 1734-1140/ß 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology, Polish Academy of Sciences.
Please cite this article in press as: Mac´kowiak M, et al. Neuroligins, synapse balance and neuropsychiatric disorders. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.011
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PHAREP 120 1–6 M. Mac´kowiak et al. / Pharmacological Reports xxx (2014) xxx–xxx
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Excitatory synapse
Inhibitory synapse
Neuroligins at the inhibitory synapses
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Presynapc membrane
Presynapc membrane
Neuroligin 2 is known to be constitutively and selectively present at inhibitory postsynaptic specialisations [11]. Neuroligin 2 is localised at both GABA-ergic and glycinergic inhibitory synapses [12,13]. Neuroligin 2 preferentially binds the inhibitory synapse scaffold protein gephyrin through a conserved cytoplasmic motif [13], and it is able to cluster GABAA receptors [14] (Fig. 1). In addition to neuroligin 2, neuroligin 3 and 4 were also found at inhibitory synapses [10,15]; however, they are not specific markers for inhibitory synapses (see above). Neuroligin 3 was observed at GABA-ergic synapses [10], while neuroligin 4 was localised at glycinergic synapses [15].
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Excitatory and inhibitory synapse balance
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Proper brain function is based on a balance between excitation and inhibition, which are mainly mediated by two major neurotransmitters, glutamate and GABA, respectively. The total number of synapses formed and ratio of excitatory to inhibitory synaptic inputs a neuron receives are factors critical for determining neuronal excitability [16]. Molecules that are involved in the control of a balance between excitatory and inhibitory synapse formation are important for proper neuronal excitability and function. Several findings indicate that neuroligins are involved in both excitatory and inhibitory synapse maturation and specificity, and they are able to control the balance between excitatory and inhibitory synapse formation [5]. An in vitro study showed that the suppression of single (neuroligin 1 or 2 or 3) or multiple neuroligin isoform (neuroligins 1–3) expression in cultured rat hippocampal neurons results in a loss of excitatory and inhibitory synapses. However, electrophysiological analysis demonstrated a predominant reduction of inhibitory synaptic function and alteration in normal excitatory/inhibitory balance in hippocampal neurons [6]. The largest changes in inhibitory synaptic transmission than excitatory transmission were also observed in an electrophysiological study in neuroligin knockout mice [7]. It was found that the deletion neuroligins 1–3 dramatically changed the balance between glutamatergic and GABAergic/glycinergic spontaneous postsynaptic currents (sPSC) in brainstem neurons, with a strong decrease in GABAergic/ glycinergic sPSC, without affecting the total synapse numbers [7]. In contrast, exogenous neuroligin 1 increased both excitatory and inhibitory presynaptic contacts and the frequency of miniature excitatory and inhibitory postsynaptic currents (mEPSC and mIPSC, respectively) in the cultured hippocampal neurons [17]. Thus, the above data indicate that the proper level of neuroligin proteins seems to be an important factor in the control of the excitatory and inhibitory balance in the brain, and the decrease in neuroligin levels mainly influence the inhibitory transmission. The fact that all single neuroligin knockout mice as well as all combinations of double neuroligin knockouts were viable, whereas neuroligin 1–3 triple knockout mice died shortly after birth, may indicate a significant degree of functional redundancy among neuroligins [7]. The above observation was also confirmed by the results from an in vitro study showing that the overexpression of all neuroligin isoforms is able to stimulate the formation of both excitatory and inhibitory terminals [6]. On the other hand, the study with transgenic mice showed that the overexpression of neuroligin 1 increased the maturation of excitatory synapses [18]. However, there were no differences in the number and size of glutamatergic and GABAergic hippocampal synapses in neuroligin 1 knockout mice when compared to wild-type animals [19]. In contrast, a decrease in GABAergic but not glutamatergic transmission was found in neuroligin 2 knockout mice [12,13]. The role of neuroligin 2 in the control of inhibitory synapse function was also
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Neurexin
Neurexin
NMDA-R
Neuroligin 1
GABAA-R
PSD-95 Postsynapc membrane
Neuroligin 2 Gephyrin
Postsynapc membrane
Fig. 1. Schematic illustration of neuroligin binding with neurexin (presynaptic membrane) and postsynaptic scaffolding proteins (PSD-95, gephyrin).
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species, including humans, rodents, and chickens. Five genes encoding neuroligin proteins have been identified in the human genome (NLGN1, NLGN2, NLGN3, NLGN4, NLGN4Y), and at least four genes coding neuroligin family members have been known in rodents (NLGN1-4). Sequence comparisons indicate that neuroligins 1, 3, and 4 are more similar to each other than to neuroligin 2 [1,2]. Neuroligins link the presynapse to the postsynaptic density by binding through the extracellular cholinesterase-like domain to their presynaptic partners, neurexins, in an alternative splicedependent manner. At the postsynaptic portion, neuroligins bind by their C-terminus with the third PDZ domain of a postsynaptic scaffold protein, such as the postsynaptic density protein-95 (PSD95), which anchors a variety of signalling molecules and surface receptors [1–3] (Fig. 1). It has been suggested that the extracellular domain of neuroligin is sufficient to induce the assembly of functional presynaptic terminals, while the intracellular domain is required for terminal maturation [4]. Several findings indicate that neuroligins are present at developing and mature synapses of the brain and play an important role in synapse organisation and function [1,3,5]. They were initially thought to be required for synapse formation [6], but recent findings indicate their crucial involvement in synapse maturation and specification [7]. It was found that neuroligins are differentially enriched in the postsynaptic specialisations of synapses, and they are found on either excitatory or inhibitory synapses.
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Neuroligins at the excitatory synapses
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Neuroligin 1 proteins are exclusively localised on excitatory synapses [8]. This observation is supported by the facts that the neuronal localisation, subcellular distribution and developmental expression of this protein are connected with the excitatory postsynaptic marker protein PSD-95 and N-methyl-D-aspartate (NMDA) receptor (Fig. 1). Moreover, electron microscopy study demonstrated that only asymmetric synapses contain neuroligin 1, and immunofluorescence labelling showed that neuroligin 1 colocalises with glutamatergic but not with g-aminobutyric acid (GABA)-ergic synapses [8]. An in vitro study also showed the coaggregation of neuroligin 1 with PSD-95 (scaffold protein of excitatory synapses), but not gephyrin (scaffold protein of inhibitory synapses) in cultured neurons [9]. A similar effect was observed for neuroligin 3 and neuroligin 4, which suggests that these two proteins are also expressed on excitatory synapses [9]. Additional studies confirmed the presence of neuroligin 3 on glutamatergic synapses in the brain and coimmunoprecipitation studies revealed the occurrence of neuroligin1–neuroligin3 complexes in the brain extracts [10].
Please cite this article in press as: Mac´kowiak M, et al. Neuroligins, synapse balance and neuropsychiatric disorders. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.011
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confirmed by the data from a study of transgenic mice that demonstrated that the enhanced expression of neuroligin 2 results in a significant increase in the density of symmetric (inhibitory) synapses in cortical areas, followed by a small significant change in excitatory synapse morphology [20]. The above results demonstrate that although the expression of neuroligin isoforms is specific for the type of synapse, they are also able to functionally replace each other. The differences in the synaptic localisation of distinct neuroligins suggest the presence of cellular processes involved in maintaining the excitatory/inhibitory balance by controlling the trafficking of different neuroligin isoforms to specific synaptic sites. Current evidence indicates an important role for both the extracellular and intracellular domains of neuroligins in their synaptic localisation. Several findings suggest that neuroligins mediate their effects on excitatory or inhibitory synapse development through trans-synaptic interaction with neurexins. Alterations in the extracellular domains of neuroligins (1, 2, 3) or neurexin-1b affected excitatory and inhibitory synapse formation [9,21,22]. However, recent data have also shown that the targeting of neuroligins to specific postsynaptic sites is determined by postsynaptic scaffolding molecules, which bind to the C-terminal PDZ-binding domain of neuroligins [23]. It was found that the postsynaptic complex of scaffolding protein PSD-95 and neuroligin can modulate the release of transmitter from vesicles at the synapse in a retrograde way [24] and link postsynaptic and presynaptic function. It was also suggested that not only the expression of neuroligins but also the interaction between neuroligins and PSD-95 regulate the balance of excitatory and inhibitory synapse formation [17]. PSD-95 restricts the localisation and effects of neuroligin 1 to excitatory synapses [17]. The overexpression of PSD-95 increased the level of neuroligin 1, but it could also shift the localisation of neuroligin 2 from inhibitory to excitatory contacts, reducing mIPSC frequency [9,17,22]. In addition, a decrease of the endogenous level of PSD-95 by siRNA administration shifts neuroligin 1 from excitatory to inhibitory synapses [25], while the knockdown of PSD-95 leads only to a partial shift of neuroligin 2 and neuroligin 3 from excitatory to inhibitory synapses [26], most likely because these neuroligins are already expressed on inhibitory synapses. The above data indicate that the PSD-95 level might be a main factor in determining the recruitment of neuroligins to excitatory versus inhibitory synapses, especially neuroligin 1. Recent evidence indicates that neuroligin 2 preferentially binds to inhibitory postsynaptic scaffolding molecules, such as gephyrin and collybistin, and the deletion of neuroligin 2 in mice disturbs GABAergic and glycinergic transmission [13]. Moreover, the siRNA-mediated deactivation of gephyrin leads to a shift of endogenous neuroligin 2 from inhibitory to excitatory synaptic contacts [26]. The above results might suggest that neuroligin 2 can bind to both inhibitory (gephyrin) and excitatory (PSD-95) postsynaptic scaffolding proteins, but it preferentially binds to gephyrin, most likely due to its higher affinity to inhibitory than excitatory scaffolding proteins. Interestingly, it was also shown that knockdown of neuroligin 2 evoked a reduction in glutamatergic events (i.e., the decrease in mEPSC) and dendritic spines, which was rescued by the overexpression of a chloride transporter, KCC2, together with neuroligin 2 shRNA [27]. KCC2 controls intracellular Cl concentration by pumping Cl outside neurons (for review, see Blaesse et al. [28]). Moreover, independent of its Cl transport function, KCC2 was also found to regulate dendritic spine maturation and excitatory synapse development by interacting with the spine cytoskeleton [29]. It was shown that the KCC2 C-terminal domain binds to the cytoskeleton-associated protein 4.1 N [29], which is known to play an important role in mediating structural interactions between the cytoskeleton,
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transmembrane proteins and adhesion molecules [30]. The above data suggest that neuroligin 2 not only regulates GABAergic synapses but also influences glutamatergic synapses by switching the bond with inhibitory to excitatory postsynaptic scaffolding proteins or by regulating KCC2 function. It seems that neuroligin 2 might be an important regulator in balancing GABAergic and glutamatergic functions in the brain. The last observation might be supported by data showing that the chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin 2, whereas the selective chronic inhibition of glutamatergic synapse components, such as NMDA receptors or their downstream signals, suppresses the synaptic-boosting activity of neuroligin 1 [31]. Thus, neuroligins specify and validate synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses.
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Neuropsychiatric disease
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Cell adhesion molecules are known to be involved in the pathomechanism of some neuropsychiatric disorders [1,32]. Several neurodevelopmental disorders, such as autism spectrum disorders (ASDs), some forms of mental retardation and schizophrenia, are known to be caused by shifts in excitatory and inhibitory balance [1]. The factors involved in the regulation of excitatory and inhibitory synapse balance might play an important role in disease development and also might be potential targets for new therapeutic strategies. As it was mentioned above, neuroligins participate in the regulation of excitatory and inhibitory balance, and because of that, several genetic and functional studies have been performed to determine the role of neuroligins in some neuropsychiatric diseases.
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Autism
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ASDs include idiopathic autism, Asperger’s syndrome or Rett syndrome [1]. The diagnosis of ASDs is based on the presence of two major symptoms: social-communication deficits and restricted and repetitive interests/behaviours. Moreover, ASDs are also associated with sensory and motor abnormalities, sleep disturbance, attention deficits, intellectual disability and mood disorders such as anxiety (for review, see Grzadzinski et al. [33]). The first symptoms of ASDs are typically observed in children before 2–3 years of age. ASDs are heritable in 80% of cases, which suggest that ASDs are largely determined by genes [1]. Genetic studies discovered mutations in the genes encoding neuroligin 3 and neuroligin 4 in patients with ASDs. A single mutation in the neuroligin 3 gene (the R451C substitution) and ten different mutations in the neuroligin 4 gene were detected. In addition, five different larger deletions of X-chromosomal DNA that included the neuroligin 4 locus were found in autism patients [1,34–39]. It is of interest that an in vitro study also showed that neuroligin 4X deletion resulted in neurodevelopmental defects and decreased gene expression of other neuroligins (neuroligin 1 and 3) [40]. Thus, the mutation in neuroligin genes may affect the neurodevelopmental process inducing disease symptoms, for example ASDs. Many genetic and non-genetic animals models of ASDs have been characterised and used to identify the aetiology of ASDs and to develop novel treatment (for review, see Won et al. [41]). Using these models, some animal behaviour similar to the symptoms observed in ASDs have been analysed, such as sociability and social novelty recognition, ultrasonic vocalisation showing the communication patterns, restricted interest or repetitive behaviour [41]. The role of neuroligins in ASD development was investigated in transgenic animals (Table 1).
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Table 1 The phenotypes of transgenic mice with altered neuroligin expression. The behavioural parameters were measured in knockout (KO), knockin (KI) or mice with neuroligin overexpression (OE); N/A – not analysed. Neuroligins
Transgenic mice
Autism spectrum disorder-like phenotype Social interaction
Social communication
Repetitive behaviour
Other functional impairments
References
Neuroligin 1 (NLG1)
NLG1- KO NLG1-OE
Minimal impairment N/A
N/A N/A
Enhancement N/A
Learning and memory deficits Learning and memory deficits
[19,42] [18]
Neuroligin 2 (NLG2)
NLG2-KO NLG2-OE
Normal Impairment
Reduced calls N/A
Normal Enhancement
Anxiety Anxiety
[43,44] [20]
Neuroligin 3 (NLG3)
NLG3-KO
Impairment
Reduced calls
Normal
[47]
NLG3R451C-KI
Impairment
Increased calls
N/A
Hyperactivity Alterations in learning and memory processes Enhanced learning ability
Neuroligin 4 (NLG4)
NLG4-KO
Impairment
Reduced calls
Normal
N/A
[54]
[49]
such as reduction in ultrasound vocalisation and a lack of social novelty preference [47]. A decrease in the freezing response in contextual and cued fear conditioning, but increased learning ability during the reversal task of the Morris water maze, was also observed in neuroligin 3 knockout mice [47]. Recent experiments with mice having a R451C mutation in neuroligin 3 previously found in patients with autism disorders [37,38] showed that the R451C mutation of neuroligin 3 does not alter neuroligin 3 mRNA levels, but decreases the export of the neuroligin 3 protein from the endoplasmic reticulum [48]. Therefore, a decrease in neuroligin 3 concentration was observed in the synapses of mice with the R451C mutation [49]. The animals with the neuroligin 3 R451C mutation showed impaired social interaction [49,50], but enhanced spatial learning abilities [49]. They also showed increased inhibitory synaptic transmission in the hippocampus in early postnatal life [51] and in the adult somatosensory cortex [49]. However, in the adult hippocampus of neuroligin 3 R451C mutated mice, an increase in AMPA receptor-mediated excitatory synaptic transmission, an alteration in the kinetics of NMDA receptordependent synaptic response, and the enhancement of LTP were observed [50]. In contrast, the neuroligin 3 R704C mutation selectively decreased the AMPA receptor-mediated synaptic transmission in hippocampus, without changing NMDA or GABA receptor-mediated synaptic transmission [52]. Thus, neuroligin 3 is involved in the regulation of both inhibitory and excitatory transmission by changing AMPA receptor function. By controlling the recruitment of AMPA receptors to the postsynaptic membrane, neuroligin 3 is involved in a critical process in excitatory synapse maturation [53]. These results are also in line with immunohistochemical data showing the presence of the neuroligin 3 protein of either glutamatergic or GABAergic synapses [10], and they also confirm the role of neuroligin 3 in the control of excitatory/ inhibitory balance, which is impaired in ASDs.
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Neuroligin 4
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Available data indicate that neuroligin 4 knockout mice also exhibit highly selective deficits in reciprocal social interactions and communications reminiscent of autism in humans. They also showed a slight reduction in the total brain volume [47,54]. Such knockout mice are considered to be a mouse model of monogenic heritable autism [54]. The animal study might be supported by genetic findings showing the possibility of the involvement of neuroligin 4 gene mutations in autism and cognitive disability [38].
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Neuroligin 3
Schizophrenia
357
Neuroligin 3 knockout mice showed a slight reduction in the total brain volume [47]. Neuroligin 3 deficient mice display a behavioural phenotype reminiscent of the main symptoms of ASD,
Similar to ASDs, schizophrenia is a neurodevelopmental disorder with cognitive disabilities [55]. The onset of schizophrenia is usually observed in late adolescence or early adulthood; however,
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Neuroligin 1
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Neuroligin 1 knockout mice exhibited an increase in repetitive, stereotyped grooming behaviour, most likely associated with a reduced NMDA/AMPA receptor ratio in cortico-striatal synapses [19]. Moreover, a slight decrease in social interaction was also found [19]. Neuroligin 1 knockout mice also display deficits in spatial learning and memory that correlate with impaired hippocampal long-term potentiation (LTP) [19]. In addition, deficits in the storage of associative fear memory in fear conditioning tasks were discovered in neuroligin 1 knockout mice [42]. The transgenic mice with neuroligin 1 overexpression showed deficits in memory acquisition, but not retrieval. Moreover, a shift in the synaptic activity towards increased excitation as well as the impairment in LTP induction was observed in electrophysiological studies [18]. The obtained date indicated that the proper neuroligin 1 level, especially in the hippocampus, was critical for memory formation. Moreover, the results also suggest that the impairment of the neuroligin 1 level might induce the development of symptoms specific for autism (repetitive behaviour) but also might be involved in the cognitive disability observed in ASDs.
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Neuroligin 2
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Neuroligin 2 knockout mice showed an increase in anxiety-like behaviour, a decrease in pain sensitivity and a slight deficit in motor coordination [43]. The mice with neuroligin 2 deletions displayed normal social behaviour, but developmental delays and a reduction in pup ultrasonic vocalisation was observed [44]. The above changes in the mouse behaviour might be related to modifications in inhibitory transmission because the deletion of neuroligin 2 evoked alterations in GABAergic and glycinergic synaptic transmission and led to a loss of postsynaptic specialisations, specifically at perisomatic inhibitory synapses [13]. However, neuroligin 2 overexpression in transgenic mice resulted in stereotypical jumping behaviour, anxiety and impairment in social interaction [20]. Moreover, neuroligin 2 overexpression in the rat hippocampus led to reduced aggression and novel reactivity, but had no impact on sociability and memory [45]. Some evidence also indicates that the reduced sociability and increased aggression observed in rats following chronic restraint stress is induced by a decrease in the hippocampal neuroligin 2 but not neuroligin 1 expression [46]. Thus, the above results indicate that the modulation of the neuroligin 2 protein level might affect social and emotional behaviour.
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the symptoms are considered to be the result of early life developmental disruptions. The sings of schizophrenia include both positive (delusion, hallucination) and negative symptoms (poor speech, social withdrawal, blunt affect). Genetic epidemiological studies showed that schizophrenia is a complex disease [56], and the meta-analytic results from twin studies showed heritability at approximately 70–80% [57]. Among several investigated genes, mutations in the neuroligin 2 [58] and neuroligin 4 [59] genes were found in schizophrenia patients. Screening for mutations in the exon and promoter of the neuroligin 2 gene in schizophrenic patients identified six point mutations: R215H, V510M, R621H, A637T, P800L and A819S [58]. Functional studies in the HEK 293T-neuron co-culture system using immunocytochemistry and electrophysiological recordings revealed that the R215H mutant was unable to develop GABAergic synapse formation due to a severe defect in intracellular trafficking [58]. The above finding indicates the importance of neuroligin 2 in the formation of functional inhibitory synapses. Moreover, the disruption of GABAergic transmission is observed in schizophrenia and defects in inhibitory circuit function contribute to working memory impairments observed in schizophrenia [56]. In addition, a recent genetic study also noted rare exonic deletions in the gephyrin gene as a risk factor for schizophrenia [60]. As it was mentioned above, gephyrin is an inhibitory postsynaptic scaffolding protein that is involved with the neuroligin 2 protein in GABAergic synapse formation. Thus, this might suggest that changes in the neuroligin 2 and gephyrin protein levels due to genetic mutations affect the proper development of inhibitory synapses in the schizophrenic brain. The preliminary study of neuroligin 2 in an animal neurodevelopmental model of schizophrenia based on the postnatal administration of an NMDA receptor antagonist showed the changes in neuroligin 2 protein levels in the medial prefrontal cortex in adolescent, but not adult rats [61]. An example of neuroligin 2 immunostaining in the rat medial prefrontal cortex is shown in Fig. 2. The above evidence indicates that alterations in the neuroligin 2 protein level induced by genetic or environmental (pharmacological) factors might have resulted in impairments in inhibitory synapse development, most likely during adolescence, and might have caused the deficit in GABAergic transmission observed in the brains of adult schizophrenic patients [56]. The screening for neuroligin 4 gene truncating and transmembrane domain mutations in schizophrenic patients was also investigated [59]. The results showed that the transmembrane domain is more likely involved in the aetiology of schizophrenia than the truncating mutations of the neuroligin 4 gene [59]. It is also of interest that the deletion in neuroligin 4 gene was also found in autistic patients [36]. The same relationship was found in relation to the gephyrin gene [60]. Moreover, the mice with altered neuroligin 2 protein levels showed some autistic-like symptoms [20]. Recent clinical data indicate that some patients with treatment-resistant schizophrenia have autistic symptoms, and these symptoms co-vary with negative symptoms, but not positive
Fig. 2. Neuroligin 2 immunostaining in the rat medial prefrontal cortex (mPC). (A) The distribution of neuroligin 2 in the mPFC. (B) The examples of neuroligin 2 immunoreactive cell (asterisk) and puncta in the neuropil (arrows). The scale bars are 500 mm (A) or 50 mm (B).
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symptoms of schizophrenia [36,62]. Thus, it is conceivable that alterations in the same genes (i.e., neuroligin 4, neuroligin 2 or gephyrin) might be involved in the symptoms of schizophrenia and autism, which are both neurodevelopmental disorders with cognitive deficits.
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Conclusions
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The collected data indicate that neuroligins are involved in maturation and synapse specification. The main factors determining their synaptic localisation are presynaptic neurexins and postsynaptic scaffolding proteins (PSD-95, gephyrin). The neuroligins’ ability to control the balance between excitatory and inhibitory synapses makes them an interesting target in the investigation of the pathophysiology of neuropsychiatric disorders. So far, a functional study using a transgenic animal model with impairments of neuroligin protein levels (knockout, knockin and overexpressed mice) showed the involvement of neuroligins in memory processes, and also in social and emotional behaviour. Moreover, genetic analysis of people showing symptoms of some neurodevelopmental disorders indicate that alterations of the neuroligin structure might be involved in the pathomechanism of psychotic disorders such as autism or schizophrenia.
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Funding
435
This work was supported by Grant No. POIG.01.01.02.-12-004/ 09 ‘‘Depression-Mechanism-Therapy’’ (part 2.2. to MM).
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Conflict of interest
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All authors declare that they have no conflicts of interest.
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References
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Please cite this article in press as: Mac´kowiak M, et al. Neuroligins, synapse balance and neuropsychiatric disorders. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.04.011
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