Synapse alterations in autism : Review of animal model findings

Background. Recent research has produced an explosion of experimental data on the complex neurobiological mechanisms of developmental disorders including autism. Animal models are one approach to studying the phenotypic features and molecular basis of autism. In this review, we describe progress in understanding synaptogenesis and alterations to this process with special emphasis on the cell adhesion molecules and scaffolding proteins implicated in autism. Genetic mouse model experiments are discussed in relation to alterations to selected synaptic proteins and consequent behavioral deficits measured in animal experiments. Methods. Pubmed databases were used to search for original and review articles on animal and human clinical studies on autism. Results. The cell adhesion molecules, neurexin, neurolignin and the Shank family of proteins are important molecular targets associated with autism. Conclusion. The heterogeneity of the autism spectrum of disorders limits interpretation of information acquired from any single animal model or animal test. We showed synapse-specific/ model-specific defects associated with a given genotype in these models. Characterization of mouse models with genetic variations may help study the mechanisms of autism in humans. However, it will be necessary to apply new analytic paradigms in using genetically modified mice for understanding autism etiology in humans. Further studies are needed to create animal models with mutations that match the molecular and neural bases of autism.


INTRODUCTION
Autism spectrum disorder (ASD) is a neurodevelopmental disorder with heterogeneous phenotype predominantly affecting males 1 .According to the World Health Organization (WHO), there is a global median prevalence of autism spectrum disorder estimated at 62/10 000, that is one child in 160.The median rate of prevalence for Europe is 61.9/10 000 and for America is 65.5/10 000 (ref. 2 ).Although still a matter of debate, the most common symptoms of autism include social and communication impairments and repetitive behavior.Currently, no single cause of ASD is known and pharmacological treatments for the core symptoms are not available 1 .
Numerous animal models (lesion, transgenic, knockout, selective breeding, etc.) have been developed for a variety of psychiatric, neurodegenerative, and neurodevelopmental disorders.Animal models are often used to study maternal immune activation, genetic defects and environmental factors in autism too [3][4][5] .Mouse models for genetic defects include nonsyndromic autism associated mutations in single genes.The most common mutations in this context include cell adhesion molecules: neuroligins, neurexins and scaffolding proteins, i.e.Shank proteins.Neuroligins, neurexins and Shank proteins are synaptic proteins important for the formation of neural circuits and brain development.The other group of models represents syndromic autism associated mutations in a single gene, for example fragile X syndrome and Rett syndrome.Genetic mouse models of fragile X syndrome and Rett syndrome are experimentally used to examine the effects of environmental stimulation on behavioral deficits 6 .
In autistic subjects, abnormalities of brain growth and connectivity are usually apparent after the first year of life 7,8 .Early developmental stages are characterized by generation of new neurons, dendritic growth, synaptogenesis, neural circuit formation and experience-dependent remodeling 9 .Recent studies have suggested that early-life deprivation during a sensitive period can lead to commitments that are difficult to reverse at later ages 10 .The early years of development are crucial for the formation of neural circuits when there is a high predisposition to disruption.Thus, research on autism may benefit from transgenic mouse models and/or aversive stimuli at different stages of development.This review describes the most important mechanisms in synapse formation and synapse alterations in autism spectrum disorders, followed by a review of the results of experimental tests on animal models with mutations in neurexins, neuroligins and Shank genes.

Synapse formation
The formation of the nervous system requires multiple developmental events such as neuronal cell fate specification, cell migration, dendritic growth, axon guidance, synaptic target selection and synaptogenesis [11][12][13] .All of these developmental steps have to be completed for both interconnections of microcircuits in the brain and peripheral innervations of a specific tissue.By mediating the information flow between neurons, synaptogenesis is the final step in the development of the nervous system that provides regulation of intercellular communications within the nervous system.After the completion of the process of axon guidance and target recognition, synapse formation proceeds.One of the first steps in synaptogenesis is the induction and adhesion of opposed presynaptic and postsynaptic domains.This is mediated by cell adhesion molecules which trigger the events that lead to the assembly and differentiation of pre-and postsynaptic specializations 13 .These molecules can have several different functions.They promote stability of neuronal connections by linking synaptic partners.Furthermore, the cell adhesion molecules regulate differentiation of pre-and postsynaptic specialization and they participate in modulation of synaptic structure and function 14 .
The second step in synaptogenesis is organization of pre synaptic and postsynaptic specializations by scaffolding proteins.These proteins are critical mediators in assembling synaptic proteins into domains specialized for neurotransmitter release and reception.Scaffolds form a link between the adhesion proteins, ion channels, neurotransmitter receptors, intracellular signaling cascades, and the actin cytoskeleton 15 Synaptogenesis includes regulation of growth by intercellular signaling pathways.Synaptic growth is regulated by anterograde and retrograde signaling molecules, including the wingless family proteins (Wnt) and the bone morphogenetic protein pathway.The Wnt signaling molecules are important for a broad range of processes, from neurogenesis to synaptic plasticity 16 .8] ).This cytoplasmic pathway component interacts with actin and the microtubules.Postsynaptically, Wnt signaling promotes the growth of the postsynaptic membrane by translocation of the cleaved receptor Frizzled into the nucleus 19 .The retrograde bone morphogenetic protein pathway includes transcription factors which target multiple genes, including Rac GTPase activator 20 .This type of GTPase regulates the actin cytoskeleton and it is important for the growth of dendritic spines 21 .Impairment in the bone morphogenic protein has been found in fragile X syndrome 22 .When growth signals are released, such as Wnts and bone morphogenetic proteins, they bind to the synaptic transmembrane receptors and are internalized as receptor-ligand signaling complexes.These complexes are transported within the endosomal system, which in synaptic development is critical for activity-dependent circuit refinement 15 .

Role of the cell adhesion molecules in synapse formation
Many cell adhesion molecules are localized at synaptic sites in neuronal axons and dendrites.These include neurexins and neuroligins.These molecules form the trans-synaptic adhesion complex molecules that connect presynaptic and postsynaptic neurons at synapses and mediate trans-synaptic signaling 23 .Neurexins and neuroligins interact with each other in a Ca 2+ dependent manner (Fig. 1).
Neurexins act predominantly on the presynaptic terminal in neuronal cells and play essential roles in the neurotransmission and differentiation of synapses 24 .Each of three human neurexin genes (neurexin 1-3) generates transcripts under the control of two separate promoters for two neurexin isoforms, the longer neurexin-α form and the shorter neurexin-β form (ref. 25 ).Another important member of the neurexin family of proteins includes contactin-associated protein-like 2 (CNTNAP2), which is involved in neuron-glia interactions and clustering K + channels in myelinated axons 26 .
Neuroligins are synaptic cell adhesion molecules that are enriched in postsynaptic membranes where they may recruit receptors, channels, and signal-transduction molecules 27 .Neuroligins are encoded by four human genes (neuroligin 1-4), and like neurexins, undergo alternative splicing 25 .Interestingly, mammals express only three or four genes 28 .
In the rodent brain, all three neuroligin (neuroligin 1-3) proteins are detected at low levels before birth and increase rapidly during the first three postnatal days.The plateau of these proteins culminates during the period when most synapses are formed and they remain at high levels during adulthood 29 .The expression of neuroligin 1 and neuroligin 2 in the CNS is restricted to the excitatory and inhibitory synapses.Although neuroligin 3 is expressed mainly by neurons, a few studies suggest that neuroligin 3 can be expressed by the glial cells, astrocytes and Schwann cells in the developing embryo 30 .

Role of the scaffolding proteins in synapse formation
As mentioned above, the interactions between the cell adhesion molecules and the scaffolding proteins are essential for functional synapse formation.Scaffolding proteins include a large group of mutually binding syn- aptic proteins.In the developing brain, space and time regulation of expression of scaffolding proteins determine the morphology of neuronal processes and consequently synaptogenesis.A number of structurally related forms of scaffolding proteins have been identified.The most important group is the ankyrin related proteins.These proteins contain multiple domains for protein-protein interaction, including ankyrin repeats, and the SH3 domain.Particular members of this family are Shank proteins.Shank1-3 are synaptic scaffolding proteins that interact with a variety of membrane and cytoplasmic proteins.These proteins bind to neurexin-neuroligin complexes at postsynaptic excitatory glutamatergic synapses 26 .Specific positioning of Shank proteins at the postsynaptic sites of excitatory synapses suggests a role for this protein family in the organization of cytoskeletal/signaling complexes at specialized cell junctions 31 .Moreover, the neurexin-neuroligin-Shank pathway is required during the stabilization phase of the synapse in response to neuronal activity 32 .
Formation of trans-synaptic neurexin-neuroligin complex is essential for neural circuit function resulting in transforming an assembly of synapses into fully functional units.Moreover, neuroligins and neurexins are involved in maintaining the excitatory/inhibitory balance within the nervous system.Mutations in the genes of cell-adhesion and scaffolding molecules might be important in the development of autism spectrum disorders.

Synaptic alterations in autism
Autism spectrum disorder is a pervasive neurodevelopmental disorder characterized by marked disruption in information processing and cognition.Many studies have found altered synaptic plasticity in the brains of affected individuals [33][34][35] .The early onset of ASD and relatively small pathological changes in the brain of patients with ASD led to the hypothesis that impaired synapse formation, neuronal connectivity and circuit stabilization may explain the pathogenesis of this disease 7,36,37 .
Altered neuronal activity in ASD might be a reflection of disturbed synaptic integrity due to altered expression of cell adhesion and scaffolding molecules involved in synaptic development.Many of these molecules are associated with ASD, including neuroligins, neurexins, and Shank3 26 .In particular, alterations in the genes encoding neuroligins 3 and 4, their binding partners neurexins 1 and 3, Shanks and CNTNAP2 are implicated in synaptic changes in autism 38 .Many other genes encoding synaptic proteins involved in cell adhesion and scaff olding are associated with autism as can be seen from Table 1.

Alterations in genes encoding cell adhesion molecules and scaffolding proteins implicated in autism
Patients with mutations in the genes encoding neuroligins suffer from disruption in excitatory/inhibitory balance in terms of increasing the excitatory/inhibitory ratio, which in turn may disrupt the sensory, social and emotional systems 40 .One type of neuroligin mutation that led to autism in a pair of Swedish twins was found to be R451 substitution in neuroligin 3.This mutation led to a 90% loss of total neuroligin 3 levels and also to a pre-dominant endoplasmic reticulum retention of neuroligin 3 (ref. 41,42).In a large French family, a two-base pair deletion (1253delAG) was found that resulted in a premature stop codon in the sequence of the normal neuroligin 4 gene.Two members of the family suff ered from autism, other members had non-specifi c X-linked mental retardation or pervasive developmental disorder 43 .Rare copy number variations and/or point mutations in the neurexin gene have been reported in the context of autism pathology.Deletion of the neurexin 1α promoter and exons 1-5 in a boy with autistic characteristics has been described in one study 44 .In another study, a deletion that eliminated several neurexin 1 exons, including 1α and 1β, in two female siblings was found in one ASD family 45 .Deletions of neurexin 3 were found in four ASD individuals.One was a de novo mutation, another one was inherited from a nonaff ected parents and the third was inherited from a father with subclinical autism 46 .Rare non-synonymous variants of mutation of CNTNAP2, a member of the neurexin family 47 , have been found in ASD patients.9][50] ).
Shank3 was the first gene from the Shank family reported to be associated with ASD.The Shank3 gene is located on chromosome 22q13.3.Three families with ASD were observed to carry alterations of 22q and/or the Shank3 gene 51 .In the first family, an individual carried a de novo deletion of 22q13.In the second family, two affected siblings carried also a de novo deletion, but they were heterozygous for an insertion of a guanine nucleotide in exon 21.In the last family, a terminal 22q deletion was identified in a girl affected autism who exhibited language delay.De novo deleterious mutations of Shank2 in ASD have also been identified [52][53][54] .Shank1 gene rare mutation has also been associated with ASD (ref. 55).

Genetic mouse models with respect to alterations in cell adhesion molecules and scaffolding proteins
Neurobiological studies using animal models are important for defining the core characteristics of ASD.Mutations in neurexins, neuroligins and Shank genes might have impact on the development of complex behaviors.Neuroligin 1 mutant mice targeting the first two coding exons displayed a mild ASD-related phenotype 56 .They showed increased repetitive, stereotyped grooming behavior.The mice displayed impaired spatial learning and memory in a Morris water maze test.Interestingly, Neuroligin 1 mutant mice displayed few deficits in social behavior.In only one of several tasks, Neuroligin 1 knockout (KO) mice interacted less with a caged adult target.In other social tasks, these mice showed normal interaction and approach to social targets and normal social recognition.They also showed normal anxiety-like behavior, locomotor activity, motor coordination/learning, auditory startle responses, and sensitivity to sensory stimuli.Neuroligin 1 KO mice showed altered sensitivity to pain and heat stimuli but no change in the perception of auditory stimuli 57 .Neuroligin 2 mutant mice targeting the first coding exon displayed abnormal communication with decreased number of pup ultrasonic vocalizations.These mice displayed marked increase in anxiety-like behavior, a decrease in pain sensitivity, exploration and a slight decrease in motor coordination.The mutant mice displayed delays in several developmental milestones (growth parameters, eye opening, teeth eruption and the acquisition of several reflexes).In contrast, social interactions, and social behavior appeared normal.Acoustic, tactile, olfactory sensory information processing and sensorimotor gating (the state-dependent regulation of transmission of sensory information to the motor system) were not affected 58,59 .
Neuroligin 3 mutant mice, targeting exons 2-3 displayed behavioral phenotype reminiscent of the lead symptoms of ASD (ref. 56).These mice displayed reduced ultrasonic vocalization and a lack of social novelty preference.These observations might be related to the olfactory deficiency observed in the Neuroligin 3 mutants.The mice displayed enhanced motor activity in the open field, however anxiety-like behavior evaluated in the elevated plus maze test appeared normal.They also showed normal motor performance on the rotarod, and no gross performance deficits in a Morris water maze test.These mice displayed normal duration of time spent in social interaction, prepulse inhibition of the startle response (to access sensorimotor gating) and sucrose preference 60 .
Neuroligin 3 R451C knockin (KI) mice were also generated for illustrating the association of neuroligin 3 and ASD (ref. 61).This mutation substitutes cysteine for arginine at residue 451 in Neuroligin 3, a gene located on the X-chromosome.This results in neuroligin 3 retention in the endoplasmic reticulum and a decreased delivery of the protein to the cell surface 62,63 .Neuroligin 3 KI mice show impaired sociability and enhanced spatial learning and memory in a Morris water maze test 42,64 .In the study of Chadman et al. 62 , juvenile reciprocal social interaction, adult social approach and cognitive abilities were normal in these mice butit was also found that these mice showed from minor developmental differences including slightly different rates of somatic growth, slower righting reflexes, faster homing reflexes in females and more vocalizations.Longer latencies to fall from the rotarod and less vertical activity in the open field in Neuroligin 3 KI mice were observed 62 .
Neuroligin 4 mutant mice also display ASD-related behaviors.Deficits in social interactions and ultrasonic communications were observed in these mice 65 .Further, aggression, nest-building parameters, as well as selfgrooming and circling as indicators of repetitive stereotypes were explored in these mutant mice 66 .However, in another study, the authors failed to confirm the social or communication deficits.In the same study, anxiety-like behaviors, self-grooming, motor coordination and open field exploration did not differ across the genotypes.Measures of developmental milestones also remained unchanged 67 .
Neurexin 1α mutant mice represent mice lacking neurexin 1α -encoded isoforms targeting the first coding exon yields.These mice display a mild ASD-related phenotype.The authors of the study Etherton et al. 68 observed a decrease in prepulse inhibition, an increase in grooming behaviors, impaired nest-building activity, and enhanced motor learning in these mice 68 .In the same study, neurexin 1α deficient mice did not exhibit any obvious changes in social behaviors, anxiety-like behaviors, locomotor activity or spatial learning.However, the authors Grayton et al. 69 observed an altered social approach, reduced social investigation and reduced locomotor activity in a novel environment 69 .Males displayed increased aggressive behavior as well.
Neurexin 2 KO mice are generated by targeting exon 23, which is shared by Nrxn2α and Nrxn2β (ref. 70).These mice display behavioral abnormalities, characterized by deficits in social interaction and anxiety-like behavior 71 .Dachtler et al. 72 reported also deficits in social memory, but prepulse inhibition and passive avoidance learning were normal.
Genetic mouse models of different Shank protein mutations were produced to investigate their contribution to autistic pathology.Shank1 mutant mice with a deletion of exons 14 and 15 resulted in the knockout of all detectable Shank1 proteins in these animals 22 .Shank1 mutant mice showed decreased movement in the open field test, anxiety-like behavior measured by a light/dark test and open field test.They showed a deficit in motor learning measured by rotarod, also a deficit in contextual fear conditioning and communicative behaviors by ultrasonic vocalizations.Interestingly, spatial learning and memory were enhanced [73][74][75] .
Two different lines of Shank2 mutant mice were generated.Won et al. 76 described exons 6-7 deletion mice and Schmeisser et al. 77 reported on exon 7 deletion mutant mice.Mutant mice with exons 6-7 deletion exhibited ASD-like behaviors including reduced social interaction, reduced social communication by ultrasonic vocalizations, and repetitive jumping.These mice displayed impaired spatial learning and memory in a Morris water maze test, but novel object recognition memory was normal.Impaired nesting behavior, hyperactivity in the open field test and anxiety-like behavior in an elevated plus maze were also reported 76 .Animals with mutation targeting exon 7 also showed profound autistic-like behavioral alterations including repetitive grooming and abnormalities in vocal and social behaviors.Moreover, these mice showed anxiety-like behavior in the light/dark test, hyper- in the elevated plus maze or light/dark test.Impaired motor performance on the rotarod, impaired learning and memory in novel object recognition were also observed, however, acquisition of the Morris water maze test and fear conditioning were found to be normal 78,79 .Mutant mice with deletion of exons 4-9 (JAX 017442) were described by Wang and colleagues (2011) (ref. 80).These animals had impaired sociability, social novelty, social interaction in freely interacting pairs, abnormal ultrasonic vocalization, and increased self-grooming and stereotypic investigation of novel objects.Impaired motor performance on the rotarod and impaired learning and memory in the Morris water maze test as well as in novel object recognition test were also observed.However, their anxiety and locomotor activity were at normal levels.
Peça et al. 81 generated mouse line targeting exons 4-7 and another targeting exon 13-16.Because of the more profound phenotypes in the latter, the authors of this study focused attention and further study on exon 13-16 of the mutant mice.These mice displayed alterations in social behavior including impaired sociability and preference for social novelty, as well as decreased pair interaction, and profound self-grooming.They also showed increased anxiety in the elevated zero maze and light/dark test, normal activity in the open field test, and normal motor performance on the rotarod.In the Morris water maze test, mice displayed normal acquisition and flexibility in reversal learning.
Kouser et al. 82 targeted exon 21 of Shank3, reporting some ASD-related behaviors.These mice exhibited impaired social novelty in the three chamber test but normal social interaction or social learning during reciprocal social interaction with a juvenile mouse.Mice also showed impaired nest building, normal adult ultrasonic vocalizations, and increased self-grooming.In the Morris water maze test, impaired learning and memory was demonstrated.Anxiety-like behavior was manifested in the light/dark test but not in the elevated plus maze test and the open field test.They also reported a decreased activity in mice when initially presented to the open field and impaired motor performance on the rotarod.Table 2 summarizes the key behavioral changes in mouse autism model with respect to alterations in cell adhesion molecules and scaffolding proteins.

CONCLUSIONS
For effective use of animals in brain research it is essential to determine the most appropriate animal model.However, for autism, its heterogeneity makes it difficult to generalize the information acquired from any single animal model or test.In this review, we aimed to show the importance of changes in synapses or synaptogenesis which may be involved in the pathogenesis of ASD.We describes animal genetic models of ASD with respect to alterations in cell adhesion molecules and scaffolding proteins.We also showed synapse-specific or model-specific defects associated with a given genotype in these models.Characterization of mouse models carrying genetic variations could help us study the mechanisms of autism development in humans more efficiently.However, it will be necessary to apply new analytic paradigms in using genetically modified mice for understanding autism etiology in humans.Further studies are needed to create animal models with mutations that match the molecular and neural bases of autism.

Fig. 1 .
Fig. 1.Interaction of neurexins and neuroligins in transsynaptic complex.Neurexins are located on the presynaptic terminal and neuroligins on the postsynaptic nerve terminal.

Table 1 .
39st of major genes involved in cell adhesion and scaffolding associated with autism39.

Table 2 .
77havioral changes resulting from alterations in cell adhesion molecules and scaffolding proteins in mouse autism model. in the open field test, but normal learning and memory in novel object recognition test77.Different lines of Shank3 mutant mice carrying different mutations in Shank3 gene have been reported.Mutant mice with exons 4-9 deletion (JAX 017890) displayed a mild ASD-related phenotype.These mice showed normal sociability and social novelty, impairment in juvenile or male-female pair interactions, normal pup ultrasonic vocalizations with decreased adult ultrasonic vocalizations, an increase in self-grooming and inflexibility in the Morris water maze test.These mice showed slight hypoactivity in the open field test, but no enhanced anxiety-like behavior activity ↑ (increased), ↓ (decreased), -/-(homozygote knockout), +/-(heterozygote knockout), NL (neuroligin), NRXN (neurexin)