Accepted Manuscript

Title: Advances in Understanding the Pathophysiology of

Autism Spectrum Disorders

Author: Konstantin Yenkoyan Artem Grigoryan Katarine

Fereshetyan Diana Yepremyan

PII: S0166-4328(17)30431-X
DOI: http://dx.doi.org/doi:10.1016/j.bbr.2017.04.038
Reference: BBR 10831

To appear in: Behavioural Brain Research

Received date: 10-3-2017

Revised date: 16-4-2017
Accepted date: 18-4-2017

Please cite this article as: Yenkoyan K, Grigoryan A, Fereshetyan K, Yepremyan

D, Advances in Understanding the Pathophysiology of Autism Spectrum Disorders,
Behavioural Brain Research (2017), http://dx.doi.org/10.1016/j.bbr.2017.04.038

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Highlights

Overview and targeted discussion of current and most prominent modern theories of
origin and development of ASD.

Discussion on existing and proposed rodent models of ASD.

Sum up of the pathogenesis of autism and provision of the hints for future research in the

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field.

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 Advances in Understanding the Pathophysiology of Autism Spectrum Disorders
Konstantin Yenkoyana*, Artem Grigoryanb, Katarine Fereshetyana, Diana
Yepremyanac
Keywords: Autism, Pathophysiology, Neurons, Models
a
Yerevan State Medical University, Biochemistry Department, Yerevan, Armenia

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b
Yerevan State Medical University, Pathophysiology Department, Yerevan, Armenia

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c
Yerevan State Medical University, Biochemistry Department, Yerevan, Armenia

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Address for correspondence:

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Dr. Konstantin Yenkoyan
Biochemistry Department
Yerevan State Medical University
2 Koryun Street, Yerevan, 0025, Armenia,
Tel: +374 11 621 214
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E-mail: konstantin.yenkoyan@meduni.am; enkoyan@yahoo.com

Abstract
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Autism spectrum disorders (ASD) are common heterogeneous neurodevelopmental

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disorders with typical triad of symptoms: impaired social interaction, language and
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communication abnormalities, and stereotypical behavior. Despite extensive research, the

etiology and pathogenesis of ASD remain largely unclear. The lack of solid knowledge on the
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mechanisms of these disorders decreases the opportunities for pathogenetic treatment of autism.
Various theories where proposed in order to explain the pathophysiology underlying ASD.
Despite the fact that none of them is able to completely explain the impairments in the nervous
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system of ASD patients, these hypotheses were instrumental in highlighting the most important
mechanisms in the development of this complex disorder. Some new theories are based on
neurovisualization studies, others on the data from genomic studies, which become increasingly
available worldwide. As the research in this field is largely dependent on the animal models,
there is an ongoing discussion and search for the most appropriate one adequately reproducing

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the pathology. Here we provide an overview of current theories of the origin and development of
ASD discussed in the context of existing and proposed rodent models of ASD.

1. Introduction
Autism spectrum disorders (ASD) are one of the biggest challenges of modern medicine

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with yet unexplained increase in prevalence. About 1 in 68 children has been identified with

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ASD according to the estimates from CDC's Autism and Developmental Disabilities Monitoring
Network. ASD represents a heterogeneous set of neurodevelopmental disorders with typical triad

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of symptoms: impaired social interaction, language and communication abnormalities, and

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stereotypical behavior, the latter being characterized as ritualistic, repetitive, restrictive patterns
of activities, behaviors and interests. Since its first description by Austrian-American psychiatrist
and physician Leo Kanner in 1943 [1], a number of interesting theories attempting to explain
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etiology and pathogenesis have been suggested. The aim of the current review is to summarize
the most prominent modern theories, such as impairment in neural connectivity, neural
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migration, imbalance in excitatory-inhibitory neural activity, damaged synaptogenesis and
dendritic morphogenesis, disturbances in neuroimmunity, with a special focus on the
involvement of glia in the disorder and broken mirror neuron theory. We have also included
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insights from the single gene disorders with autism symptoms, as well as, discussion on the
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rodent models of ASD.

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Since ASD was defined in DSM-5 broader than in DSM-4, here in the review we will
mostly use the term ASD as an umbrella, which seems to be a better reflection of the state of
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knowledge about autism (DSM-5 Autism Spectrum Disorder Fact Sheet).

2. Modern theories of ASD development

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2.1. Neural connectivity

Impairment of neural connectivity and damaged synaptogenesis are perhaps the most
validated hypotheses that are able to provide adequate description to the pathogenesis of autism.
The significantly increased numbers of neurons in the autistic patients [2,3], may apparently
impair the process of shaping and fine-tuning of neural circuits. In order to create useful neural
circuits, the system should remove the non-functional, unnecessary neurons to increase the

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power of the working ones. Thus, in the normally developing brain the number of neurons is
being decreased while the connectivity of the remaining neurons is building up with each day.
There is evidence that this process in impaired in ASD children [2,3]. Courshesne et al. state that
early brain overgrowth produces defects in neural patterning and wiring, with the exuberant local
and short-distance cortical interactions impeding the function of large-scale, long-distance

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interactions between brain regions [4]. One of the outcomes of such impaired connectivity could

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also be the impaired lateralization, which is particularly important for the proper language
function [5].

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Another assumption based on this theory is that one of the primarily affected areas is the

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intrahemispheric connectivity [6]. Here the most significant contribution was the report of
minicolumn abnormalities in autism [7,8]. Prefrontal cortical microcircuits are assumed to play a
key role in the perception to action cycle that integrates relevant information about the
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environment, and then select and enact the behavioral responses. Minicolumns are composed of
radially oriented arrays of pyramidal neurons (layers II-VI), interneurons (layers I-VI), axons,
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and dendrites. Minicolumns assemble into macrocolumns, which form receptive fields.
Corresponding morphological studies imply that increased density and multiple wiring at the
minicolumnar scale might create the noise in the circuit [8], which disables efficient
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processing of the information. Thus the impaired connectivity and imperfect synaptic plasticity
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seems to be one of the central mechanisms in ASD development.

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2.2. Impaired Neural Migration

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The neural migration hypothesis closely resembles the previous one. However, it also
includes as an underlying event the impairment in neuronal migration during the antenatal
period. Initial misplacement of neurons may disable the further maturation of the brain. Several
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lines of evidence are supporting this hypothesis. Recently published meta-analysis shows that
reelin gene (RELN) mutation (rs362691) might contribute significantly to ASD risk [9]. Reelin is
one of the most important crucial proteins involved in migration and proper positioning of
neurons in the neocortex. This and other genetic factors might be responsible for the increased
thickness of the cortex and smudged boundaries with white matter tracts, a common observation
in ASD. Nevertheless, not all the parts of the brain are similarly affected. The most prominent

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areas are temporal and frontal lobes, whereas the occipital lobe seems to be less involved in these
derangements. Since the first years of life head circumference begins to correlate with the brain
size in both normal and autistic children, the index (i.e. head circumference) was used as an
indicator of relative brain size in autism [10]. Later studies showed that the peak overgrowth is
reached at 1-2 years of life [4], which corresponds to the age, at which the first clinical

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symptoms usually develop.

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2.3. Impaired Synaptogenesis and Dendritic Morphogenesis

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Early in central nervous system (CNS) development there is usually an overabundance of

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initial synapse formation followed by selective synapse elimination. Development and ongoing
regulation of synapses throughout the postnatal life are crucial for proper brain maturation. There
are several lines of evidence supporting the theory, according to which impaired synaptogenesis
is a key feature in ASD.
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One of those evidences is a morphological abnormality seen in ASD as an impaired
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dendritic morphology, including abnormalities in dendritic spines. This feature is best manifested
in Retts syndrome. This rare genetic disease is exceptional in sense that it falls into ASD in
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100% of cases. In this disease the mutated gene encodes for a methyl CpG binding protein 2
(MeCP2), which is an important silencer of genes. Moreover, mutant MeSP2 has a crucial role in
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the brain in terms of causing an impairment in synaptic maturation and pruning deficit during
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development [11]. It may be speculated, that, when suppression mechanisms are insufficient, lots
of unnecessary synaptic contacts are less effectively eliminated.
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Another research group recently reported increased dendritic spine density with reduced
developmental spine pruning in layer V pyramidal neurons in ASD temporal lobes in a
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postmortem study [12]. Layer V pyramidal neurons are the major excitatory neurons that form
cortical-cortical and cortical-subcortical projections, which correlate with the connectivity
problems in ASD. They also show that these spine deficits correlate with hyperactivated mTOR
and impaired autophagy. The latter may be considered as the most important fine-tuning
mechanism for dendritic spine formation. Recently, Kim et al. found that deletion of atg7, which
is vital for autophagy, from myeloid cell-specific lysozyme M-Cre mice resulted in social

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behavioral defects and repetitive behaviors, characteristic features of ASDs [13]. As neuronal
autophagy is responsible for majority of postnatal net spine elimination, it is likely that basal
autophagy regulates the synaptic strength and adjusts the connectivity of the neurons. The
reduction of mTOR-regulated neuronal autophagy is further consistent with other recent findings
of the same group, indicating increased mitochondrial mass and a lack of autophagic

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mitochondrial turnover in ASD brains [12].

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The number, size, shape and strength of synapses are regulated by close interaction of

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pre- and postsynaptic neurons and corresponding astrocytes and microglia [14]. Therefore, the
support for the role of impaired synaptogenesis in ASD comes not only from the studies,

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investigating the postsynaptic neuron, but also from the known structural and functional
interactions between pre- and postsynaptic membranes with involvement of glia [see later in
section 2.6]. an
Different types of impairments are detected also on the presynaptic membrane. Notably,
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as we will discuss later [section 2.8], the scaffolding proteins neuroligin 3 and 4 (NLGN3 and 4)
and SHANK [14] were found to be linked with autism [15], supporting this theory of ASD
pathogenesis.
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2.4. The Excitation-Inhibition Imbalance

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The excitatory/inhibitory (E/I) balance represents a critical condition for the proper
functioning of neuronal networks and it is essential for nearly all brain functions, including
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representation of sensory information and cognitive processes. The E/I balance is maintained via
highly regulated homeostatic mechanisms [16]. Neurons are able to compensate for experimental
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perturbations by modulating ion channels, receptors, signaling pathways, and neurotransmitters.

At the molecular level, these processes require chromatin remodeling, changes in gene
expression and repression, changes in protein synthesis, turnover and cytoskeleton rearrangement
[17]. Therefore, the E/I imbalance is not a standalone theory, but appears to be closely related to
other theories of ASD development.
This theory is also based on the genetic findings connecting disturbances in both GABA-
ergic and glutamatergic receptors with ASD. These are adhesion molecules which, by regulating
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Page 6 of 33
transsynaptic signaling, contribute to maintain a proper E/I balance at the network level.
Furthermore, GABA, the main inhibitory neurotransmitter in adult life, has been shown to
depolarize and excite targeted cells at late embryonic/early postnatal stages through an outwardly
directed flux of chloride. The depolarizing action of GABA and associated calcium influx
regulate a variety of developmental processes from cell migration and differentiation to synapse

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formation.

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Additional evidence of E/I theory comes from genetic studies. It was suggested that the
gene polymorphisms involved in E/I imbalance have consequences only in some brain regions,

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such as cerebellum, where the imbalance could result in excitotoxic cell death, while in many

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other synapses the situation remains under control. It was also suggested the involvement of
Bergmann glia in the process [18]. An indirect evidence of such mechanism was recently
provided by a number of human studies. The shift in glutamate/GABA ratio is related to the
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neuroinflammatory changes in the brain [19]. Excessive glutamatergic excitation may lead to
excitotoxic cell death which then triggers involvement of glial cells eventually propagating
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neuroinflammation.
Milestones of Glutamate/GABA based E/I theory is that the evidence for the involvement
of excitotoxicity in ASD pathogenesis comes mostly from in vivo studies where glutamate or
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GABA levels are measured in serum, which may not adequately explain the mechanisms of
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neuronal damage [20,21]. However, there is no further evidence on E/I imbalance in autistic
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brain, so that additional support of E/I imbalance theory has to come from further optogenetic
studies of ASD models.
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2.5. The Broken Mirror Theory

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Recently discovered mirror systems in brain could be involved in learning by imitation

and development of other neural circuits [22]. These neuronal networks that are localized in the
parietal lobe, premotor cortex, the insula and anterior cingulate cortex make possible the
recognition and understanding of the actions and emotions of other people without the higher
order cognitive mediation. Therefore, limited development of the mirror mechanism might
determine some of the core aspects of ASDs [23]. This concept is fundamental to the

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understanding of learning process by itself, since the most of human learning tools are mediated
by mirror neurons: whenever individuals observe an action being performed by someone else, a
set of neurons that code for that action is activated in the observers motor system. Since the
observers are aware of the outcome of their motor acts, they also understand what the other
individual is doing without the need for intermediate cognitive mediation [2325], so that mirror

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mechanism is involved in understanding the action and intention of other individuals [26]. Mirror

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neuron theory is closely related to the theory of mind (ToM) [27]. ToM is the capacity to
mentally understand subjective mental states, including thoughts and desires, regardless of

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whether or not the circumstances involved are real [28]. ToM develops early in children

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without disabilities, but it is significantly delayed in children with ASD [28,29]. Interesting
example of behavior, which is thought to be mediated by mirror neurons, is contagious yawning
[30]. It is well known that viewing others yawn commonly elicits spontaneous yawning in
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oneself. Notably, contagious yawning is decreased in children with ASD [31].
The comprehension of observed action was found to be affected in patients with ASD
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based on the functional magnetic resonance imaging data [32]. Another study showed the
reduction of gray matter in ASD patients in areas belonging to the mirror neuron system that are
suggested to be the basis of empathic behavior [33]. Cortical thinning of the mirror system
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correlates with ASD symptom severity. Cortical thinning was also observed in the areas involved
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in emotion recognition and social cognition [33]. However, some later studies did not confirm
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these findings while still reporting structural changes in corresponding regions of the brain based
on cortical thickness analysis and voxel-based morphometry [34]. Another elegant study
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supported the significant behavioral evidence for mirror neuron system dysfunction in ASD and
suggested that the lack of understanding of other individuals movements might have an
upstream effect leading to the poor development of communication skills [35].
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However, this theory could not be unequivocally supported in a number of other studies.
Some authors state that the evidence for the primary role of the mirror neuron system in ASD is
weak [36]. No significant differences in mirror neuron recordings in at least 24 to 48 month old
children were observed [37]. These findings, although challenging the "broken mirror"
hypothesis and suggesting that the impaired neural mirroring is not a major feature of ASD, still
acknowledge its role in the ASD puzzle.

Page 8 of 33
 2.6. Impaired Immunity and Neuroinflammation

The alterations of immune system including both acquired and innate immunity remain
one of most studied concepts in ASD [38]. Despite considerable research efforts, there is still
lack of data pointing towards etiological role of immune disturbances found in patients with

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ASD. One of the first valuable reports published in mid 1980s in this field was based on the
study of 31 patients with ASD, which revealed several abnormalities of the immune system,

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including reduced responsiveness in the lymphocyte blastogenesis assay to phytohemagglutinin,
concanavalin A, and pokeweed mitogen [39]. Recent studies revealed a systemic deficit of

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Foxp3+ T regulatory cells, increased RORt+ (Th17), T-bet+ (Th1), GATA-3+ (Th2) CD4+ T
helper cells in patients with ASD as compared to normally developed children [40]. Several
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reports using different methods and small ASD patient populations have shown an increase in
pro-inflammatory cytokines in peripheral blood [41]. Molloy et al. found an increased leukocyte
production of Th2-associated cytokines in autistic subjects [42]. However, there are no strong
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evidence on changes in total amount of T cells in peripheral blood in children with ASD. Instead,
it becomes clear that there is an imbalance in T cells subpopulations, as well as natural killer
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cells [43]. Altogether the current data confirm the presence of immune dysregulation in autism
[44].
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Meanwhile, studies have showed some benefits of intravenous IgG injections to ASD
patients: improvements in eye contact, speech and echolalia, calmer behavior [45] and transient
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improvements in attention span and hyperactivity were reported [46]. Since then, various
research groups have identified a range of immune functions that are atypical in ASD; yet, not
surprisingly, these findings are often as heterogeneous as the behavioral phenotypes, which make
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up ASD [47]. However, it seems that the most consistent finding is neuroinflammation with the
strong involvement of neuroglia [4749]. Considering the fact that immune system and glia are
involved in the proper development of nervous system and aid in shaping the neurons and
synapses of the developing brain [50,51], immune disturbances might not only accompany the
primary disorder but possibly contribute to the etiology of the disease.

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 The complement activation has been shown to regulate synaptic remodeling [50], and
microglia, TNF-, and other immune-related molecules have been implicated in normal synaptic
development and developmental apoptosis [52]. Although both astroglia and microglia are
fundamentally different in origin and function, they together orchestare the same developmental
processes such as neuro-/gliogenesis, angiogenesis, axonal outgrowth, synaptogenesis and

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synaptic pruning important mechanisms which are impaired in ASD. Therefore, independently

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of the ongoing discussion whether immune reactivity changes are primary etiological factors or
just epiphenomena, immunological mechanisms are directly involved in ASD.

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Neuroglial cells, such as astrocytes and microglia, along with perivascular macrophages

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and endothelial cells play important roles in neuronal function and homeostasis [5355]. Both
microglia and astroglia are fundamentally involved in cortical organization, neuroaxonal

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guidance and synaptic plasticity [56,57]. Neuroglial cells contribute in a number of ways to the
regulation of immune response in the CNS. Astrocytes, for example, play an important role in
the detoxification of the excessive amounts of excitatory amino acids [58], maintenance of the
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integrity of the blood-brain barrier [59], production of neurotrophic factors [54], metabolism of
glutamate [58], etc. Under the normal homeostatic conditions astrocytes facilitate neuronal
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survival by producing growth factors and mediating uptake/recycling of excitotoxic

neurotransmitters, such as glutamate, from the synaptic microenvironment [58]. However, during
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astroglial activation secondary to the injury or in response to neuronal dysfunction, astrocytes

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can produce several factors that may modulate inflammatory response. For instance, they secrete
pro-inflammatory cytokines, chemokines and metalloproteinases that can amplify immune
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reactions within the CNS [54]. Microglial and astroglial activation is an important factor in the
neuroglial response to injury or dysfunction of nervous system. Microglia is involved in synaptic
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stripping, cortical plasticity and immune surveillance [53,60]. The above-mentioned lines of
evidence suggest that immune dysfunction and abnormalities in synaptogenesis are only
seemingly distinct processes and within the developing brain they appear to be closely
interrelated mechanisms. Moreover, the connection between these mechanisms may be
bidirectional and mediated, at least in part, by glial cells.
Changes in astroglia and microglia can therefore produce marked neuronal dysfunction
that is likely to be associated with the mechanisms of neuronal dysfunction observed in autism.
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These neuroglial changes are mediated by the reactive oxidative species, cytokines, chemokines
and other neuroactive substances [54]. Understandably, all of these facts induce growing interest
to the role of immunity and immunological dysfunctions in the pathogenesis of ASD [41,61].
Several reports link the immunological dysfunction with autism, and some studies suggest that
up to 60% of patients with ASD have various types of systemic immune dysfunction, as part of

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either cellular or humoral immune responses [6264]. Pathomorphological evidence of

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immunological reactions within the CNS, such as lymphocyte infiltration and microglial nodules
were found in ASD patients [65]. Neuropathological studies of postmortem brain tissues from

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autistic patients demonstrate an active and ongoing neuroinflammatory process in the cerebral

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cortex and white matter characterized by astroglial and neuroglial activation. These findings
support a role for the neuroimmune response in the pathogenesis of ASD [66]. As both astroglia
and microglia are involved in pathogenic inflammatory mechanisms common to many different
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disorders of the CNS, it is possible that different factors (e.g. genetic susceptibility, maternal
factors, prenatal environmental exposures) may trigger the development of these neuroglial
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reactions. Furthermore, protein array technique used to establish the profiles of immune
mediators demonstrated that cytokines/chemokines, such as MCP-1 (monocyte chemoattractant
protein-1), IL-6 (interleukin-6) and TGFb1 (transforming growth factor b1), which are mainly
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derived from the activated glial cells, are the most prevalent cytokines in brain tissues, and
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similar shifts were found in the CSF (cerebrospinal fluid) from autistic patients [66]. Preliminary
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studies also show that the serum concentrations of subsets of cytokines and chemokines, such as
MCP-1 and IL-6, in parallel with their CSF levels may be useful as surrogate markers of the
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neuroinflammatory activity in autistic subjects [66].

There is an interesting cross-talk between the abovementioned theories of ASD that
focuses on the association of MET (mesenchymal-epithelial transition factor) receptor tyrosine
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kinase gene polymorphism and ASD [67]. Since MET signaling is involved in the neocortical
and cerebellar growth and maturation, immune function, and gastrointestinal repair, it is
reasonable to assume that immune dysfunction may contribute to both the etiology and
pathogenesis of ASD. Interestingly, autism has been reported to be associated with celiac
disease, suggesting that the shared genetic background may link the impairments in digestive,
immune and central nervous systems. This could be much more important in the light of the

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recent findings that gut permeability is not increased in patients with ASD as compared to
healthy controls [68,69].

The impaired immune activity observed in ASD spans both innate and adaptive levels of
the immune system, and suggests that disturbances in either of them may have profound effects
on neurodevelopment. Cytokines that have been observed at atypical levels in the brain tissue,

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CSF, circulating blood, and digestive system, can alter neuronal survival and proliferation. These
findings strongly suggest that neuroimmune reactions are part of the neuropathological processes

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in ASD, and that the immune response may contribute to CNS dysfunction [47]. However, the
contribution of the neuroinflammatory response to the specific neuropathologies and behavioral

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disturbances in ASD, and its relation in the etiology of ASD requires further exploration.

2.7. The role of epigenetics an

Extensive studies of gene expression changes in autism led to important findings. One of
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them is that among other genetic disorders, the Rett syndrome was found to be related to ASD.
While other single gene disorders associated with ASD have various contribution to the autistic
phenotype (Table 1), Rett syndrome always produces ASD symptoms [70]. Therefore, the
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genetic defect in this disorder deserves special attention. The affected gene encodes methyl CpG
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binding protein-2 (MeCP2) which is an essential epigenetic regulator in the human brain
development [71]. Interestingly, this gene, as well as fragile X mental retardation 1 (FMR1) gene
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(responsible for fragile X syndrome), is located on the X chromosome, which may, at least in
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part, explain the predominance of males among ASD patients. Thus, multiple gene expression
requires proper expression of MeCP2. However, MeCP2 mutation is found in relatively small
proportion of ASD patients. Future research may provide additional clues elucidating
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transcriptional problems in ASD. A recent study using co-expression network analysis

demonstrated altered expression of yet another gene, C11orf30 that has been implicated in
chromatin modification, DNA repair and transcriptional regulation [72]. Therefore, it can be
suggested that epigenetics may also mediate the influence of environmental factors on the
developing brain.

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 Another reason for focusing on epigenetics is the proposed role of the impairments in
microbiota-gut-brain axis in the pathogenesis of ASD [73]. Bacteria-derived short chain fatty
acids (butyric acid in particular) are potent inhibitors of histone deacetylase, causing reactivation
of gene expression. Propionic acid (PPA) is produced endogenously in course of the fatty acid
metabolism, but it is also a fermentation end product in the antibiotic resistant enteric gut

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bacteria (e.g. Clostridia) [74]. PPA has broad effects on the nervous system physiology,

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including activation of specific G protein coupled receptors (GPCR), neurotransmitter synthesis
and release, intracellular pH/calcium gating, mitochondrial function, lipid metabolism, immune

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function, gap junction gating and gene expression [75]. Additionally, bacteria might regulate the

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genome functioning through chromatin rearrangements, alterations in non-coding RNAs, and
RNA splicing factors. Consequently, bacteria can be considered as potential epimutagens that are
able to reshape the epigenome [76], and therefore may potentially contribute to ASD. However,
this notion remains to be further established.
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2.8. Insights from Single Gene Disorders with High Rate of Autism
It is now clear that ASD has a genetic basis with significant contribution of
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environmental factors. Nonetheless, none of the genes and specific environmental factors can be
considered as the sole cause of such heterogeneous spectrum of diseases. Therefore, instead of
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looking for a single autism gene, the investigatory approach in ASD studies turns towards
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finding similarities in the function of genes, which are mostly associated with single gene
disorders, but the altered expression of which produces also autistic symptoms (Table 1). Such
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approach applies knowledge learned from the studies on primary hypertension. Several rare
single gene mutations are known to cause arterial hypertension. Some of the affected genes
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encode sodium transporters in kidneys, others are involved in aldosterone and glucocorticoids
metabolism. Despite the seeming diversity of the mutated gene products, one might argue that
these defects converge on impaired sodium excretion resulting in increased sodium retention.
Therefore, these findings serve as a solid argument or evidence for considering the impairment in
pressure natriuresis as the leading cause of primary hypertension pathogenesis [77]. However,
this approach seems not to be very productive in case of ASD, as it is difficult to find a common

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denominator function for all of the culprit genes in the single gene disorders presenting with
ASD symptoms, the same way as it appeared in case of hypertension [70].

Nevertheless, the functions of the affected genes fit into several clusters which deserve
special attention. One of these clusters is the synaptic transmission supporting system:
mutations in NLGN3/4, SHANK3, or NRXN1 genes alter the synaptic function and lead to

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mental retardation, typical autism or Asperger syndrome [7880]. PTEN mutations may be
assigned, at least partially, to the same group, since PTEN has been shown to regulate neuronal

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arborization and social interaction [81]. The second cluster is related to the abnormal
cellular/synaptic growth rate. While tumor suppressor genes in general prevent cells from

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unwanted proliferation, in neurons these genes function as negative regulators of neuronal
hypertrophy and excessive proliferation at the earliest stages of CNS development [82]. From

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this standpoint, it is not surprising that the mutations in a range of key suppressor genes may be
associated with the ASD risk. Among them the strongest contributors to ASD pathogenesis are
the suppressors, which are associated with autophagy and mTOR pathway: TSC1/2, PTEN, NF1
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[83]. It will be very interesting to see whether the inhibitors of mTOR pathway, that show
promising effects in oncology, could also be beneficial in ASD treatment [84,85].
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The third cluster contains genes controlling gene transcription and translation, such as
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FMR1 and MECP2. Kelleher and Bear hypothesized that loss of the normal constraints on
synaptic activity-induced protein synthesis may be one of the several mechanisms leading to
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autism [70,86]. Turner et al. added another gene to this cluster, indicating the loss of -catenin
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function in severe autism [87]. Despite the initially promising genome-wide association studies
[38], much more remains to be done to show the unequivocal involvement of genetics in ASD
etiology.
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As we can notice, very few of the presented theories provide specific pathomechanisms,
and most of them are based on impairments not specific to ASD: nevertheless, the specificity
may lie not in a specific mechanism itself, but in unique spatiotemporal characteristics, which
may produce ASD.
3. Animal models of ASD

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 Modeling of ASD on rodents is of special importance since the etiology of these diseases
remains largely unknown. Given the complex neuroanatomical derangements observed in the
postmortem examination of autistic patients, it is almost impossible to delineate the pathogenetic
mechanisms responsible for main behavioral manifestations in the patients with autism.
Nonetheless, no commonly accepted autism model exists up to date. Moreover, it is important to

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emphasize that all of the existing models are not models of autism but models for autism [88].

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Each of the existing models may help to reveal parts of the mechanisms involved in pathogenesis
of autism and to develop effective treatment for autism. An approach aiming at the behavioral

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phenotyping of autism core symptoms and comparing them with potential underlying deficits

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could be of significant benefit for the field. However, such state-of-the-art behavioral
phenotyping tasks to assess the autism core symptoms in rodent models became available only
recently [89].
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A number of animal models of and for ASD exist and are currently being used as a tool in
understanding the mechanisms of the disease. Most of these models can be grouped into the
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following types: environmentally induced, by exposure of the pregnant animals or neonates to
certain chemicals or infection/inflammation, lesion models and genetic models [90]. The validity
of most of these models still needs to be verified.
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The animal models must qualify on several criteria like a construct, face and predictive
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validity. First of all, it is the construct validity which is based on the degree of similarity between
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the mechanisms underlying the animal models and human disease. To detect these similarities
its necessary to study behavior and several biological factors, which may be involved in
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pathogenesis of human disease [91]. Face validity implies that the phenomenological aspect
observed in animals is similar to the one observed in patients [92]. Next and maybe the most
debated criterion is the predictive validity, which requires an animal model to demonstrate the
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similar response that humans have after administration of certain class of medications [93].
3.1. Models induced by environmental factors
3.1.1. Biological agents
There is a growing amount of evidence that maternal infection leads to early immune
dysregulation, which may be involved in the development of ASD. The dysregulation in the
brains of autistic patients comprises microglial and astrocyte activation [61], up-regulation of

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pro-inflamatory cytokines, such as interleukin (IL-6), tumor necrosis factor (TNF-), IL-1 in
the brain, cerebrospinal fluid and blood serum [94].
Maternal immune activation by many viruses and bacteria during pregnancy, including
influenza virus, cytomegalovirus, Borna disease virus and bacterial lipopolysaccharide, exhibits
behavior of the offspring similar to the core symptoms of autism: deficits in social interaction

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and communication, as well as increased repetitive/stereotyped motor behaviors [95,96].

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Therefore, the abovementioned viruses administration to the pregnant rats is often used as an
animal model for ASD.

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3.1.2. Chemical agents

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More than 200 chemicals have neurotoxic effect on the adult brain and an increasing
number of potential teratogens are being identified [97]. Valproic acid (VPA) is one of the most
extensively studied among these compounds and is widely used for ASD modeling. Clinically,
an
VPA has been classically used for epilepsy, but is increasingly used for psychiatric conditions,
such as bipolar disease, and mood swings. Due to its teratogenic effect, the use of VPA during
M
early pregnancy is associated with approximately threefold increased risk of ASD. VPA
exposure causes oxidative stress and histone deacetylase inhibition in embryonic brain [91], both
of which may lead to autistic behavior.
d

There is some evidence indicating the decrease in glutathione peroxidase activity in

e

children with ASD, which leads to high production of reactive hydroxyl radicals [98]. At the
pt

same time, other studies supposed disruption of methionine cycle, which results in decreased
synthesis of cysteine and glutathione [99], as one of the important components of antioxidant
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system.
VPA decreases the concentration of reduced glutathione, suppresses the activity of
glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase the
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main enzymes of antioxidant system [100]. Thus, high levels of reactive oxygen species and
immature embryonic antioxidant system promote the manifestation of teratogenic effect of VPA.
VPA is an inhibitor of histone deacetylase (HDAC) [101], which plays an important role
in gene transcription regulation. VPA inhibits HDAC1 activity by competitive binding with
active center and induces proteosomal degradation of HDAC2 [102]. HDAC1 is responsible for
neuro- and gliogenesis and is expressed in both neural and glial stem cells. HDAC2 is expressed

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in postmitotic neuroblasts, but not in fully differentiated glial cells [103]. HDAC modulation in
different cell types and at different time points may lead to different outcomes and may help to
explain why VPA exposure is associated with ASD-like symptoms.
3.2. Models induced by brain lesion/damage
As brain lesions are mostly irreversible, animal models are used to induce brain

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dysfunctions similar to a particular neurological disease. Postnatal time point has been chosen

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because it is a sensitive period, during which neuronal proliferation and organization are
essentially complete, but neuronal differentiation, myelination, synaptogenesis and glia genesis

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are still in process. There are different methods to induce brain tissue damage and the following

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are commonly used: mechanical lesion, electrolytic and radio frequency lesions, excitotoxic or
neurotoxic lesions. Various studies of ASD brains have shown abnormalities in different regions,
such as the frontoparietal cortex, amygdala, hippocampus, basal ganglia, anterior cingulate
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cortex (ACC) and cerebellum, which proves the involvement of these structures in ASD
pathogenesis. Thus, from the variety of proposed lesion models some are of particular interest:
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early and late periods of excitotoxic agent (ibotenic acid) injections into amygdala [104] and
medial prefrontal cortex [105], as well as cerebellar mechanical lesion [106].
Intraventricular infusion of short chain fatty acids (e.g. propionic acid) produced by
d

enteric gut bacteria is also used for modeling of experimental ASD and may be classified to brain
e

lesion/damage models [107].

pt

An important limitation of lesion models is that damage of specific region of the brain
affects the whole central nervous system, which makes the identification of causational
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relationship between the lesioned structure and the behavioral outcome difficult. Another
noteworthy problem in lesion models is the important ability of the brain plasticity, when other
areas compensate dysfunction of the damaged area. It can lead to the absence of abnormal
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behavioral phenotype.
3.3. Models induced by genetic modifications
Several human syndromes derived from a single gene mutation increase the risk for ASD.
The more common aberrations are Fragile X syndrome, a mutation in FMR1 [108], Rett
syndrome, a mutation in MECP2 [109], tuberous sclerosis, mutations in TSC1 or TSC2 [110] and
Timothy syndrome, a mutation in CACNA1C [111]. The identification of such genetic variations

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with the development of new strategies for genetic engineering facilitated the development of
genetic animal models of ASD [90].
Despite the fact that mice with single gene knockout dominate among genetic models of
ASD, they are not able to represent the whole complexity of the behavioral alteration. Genetic
mutations lead to the biochemical disturbances known at ASD, but do not produce the complex

t
structural, neuropathological abnormalities. Thus, genetic models provide face validity, but not

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construct validity [112].
Based on existing models of ASD and looking towards an optimal one, many scientists

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focus attention on combining some models with each other. For example, the combination of

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genetic and environmental factors may result in a better ASD model. Genetic models provide
construct validity, as far as they mimic the mechanism that leads to ASD development, whereas
environmental models provide face validity, as they primarily mimic the behavioral symptoms
common in ASD patients [112].
an
Given the overcomplexity and intricacy of the pathogenesis of ASD, it becomes obvious
M
that modeling the human disorders on animals has its certain limitations. However, by using the
same behavioral battery, it becomes possible to evaluate each of the existing models, and
moreover, it also becomes possible to delineate the neurochemical and neuroanatomical
d

alterations, which are typical for ASD. Thus, comparative analysis of the models will enable not
e

only evidence-based selection of the best known models, but will also assist to explore and
pt

develop current hypotheses on the pathophysiology of ASD (see Figure 1).

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4. Conclusion
Current evidence from animal models and clinical data indicate that ASD may be referred
to a group of disorders with complex etiology. An excellent review on the pathogenesis of ASD
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[38] raises a question: Will we have to understand ASD gene by gene in hundreds of models, or
might there be some convergent biology that will limit the search space in the quest for
mechanistic insight? Further studies will answer the question asked by Geschwind on the
etiology of ASD: Many genes, common pathways? [113]. Most likely, hundreds of genes are
involved in pathogenesis of the spectrum. For the most of the ASD patients none of those gene
polymorphisms is enough to explain the disease. Thus, several gene defects along with yet

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Page 18 of 33
poorly understood environmental stimuli may cause those mechanisms that lead to similar
behavioral manifestations of ASD. Therefore, in terms of etiology, ASD may be considered
similar to such common pathologies of humankind such as obesity, essential hypertension, type 2
diabetes, etc., in the sense that the variety of gene polymorphisms contributes to disease
manifestation. Pooling together recent advances in the genetics of ASD and the use of animal

t
models shed enough light on this issue suggesting the most common clusters of underlying

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pathomechanisms (Figure 2). One might argue that not only autistic patients have impaired social
communication and language disorders, but also their neurons fail to communicate with each

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other. Proper inter-neuronal communication requires normal functioning of several systems:

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structural backbone for synapse functioning, activity-dependent modifications in protein
synthesis and degradation, silencing of several genes, and up-regulation of expression of others.
In this sense, if we put together the known genetic disorders with high prevalence of ASD, we
an
identify several interconnected clusters, which represent the pathways to ASD:
(1) impaired synapse construction including derangements in neurexin/neuroligin
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interaction, SHANK and PSD 95;
(2) derangements in protein turnover: activity-dependent protein sythesis, PI3K/mTOR
signaling pathway, coupling synaptic activity to the translational machinery, and
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consequent protein synthesis-dependent long-term potentiation (LTP) and long-term

e

depression (LTD);
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(3) impaired epigenetic regulation of protein expression-dependent (e.g. FMRP and

MECP2) regulation of gene expression and abnormality of synaptic/cell growth;
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(4) translation of synaptic activity to corresponding structural modifications. These

include calcium channels, particularly, L-type voltage-gated calcium channel and
ionotropic receptors (AMPA and NMDA). These ionic flows are necessary to adapt
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through series of phosphorylations in order that the structural and functional activities
match the demands of ongoing circuitries.
Taken together all these cellular-molecular derangements that emerge from the
kaleidoscopic data in the field of autism research lead to the concept that autistic neuron is not
able to regulate the synaptic flow coming from different neurons and to adjust itself to the
ongoing demands, i.e. impairnment in synaptic plasticity develops (Figure 2, D). While adequate

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Page 19 of 33
functioning of these systems requires adjusting the synaptic ultrastructure to the demands of the
ongoing circuitries, the derangements in synaptic molecular machinery most likely result in
subthrheshold neuronal injury. As a result, cells respond not as much by appropriate synaptic
reorganisation, but more by release of injury/stress signals which eventually activate glial cells
towards the site of danger signal release. Involvement of local defense systems is manifested

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by activation of glial cells, and therefore, proinflammatory signaling becomes an important

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secondary pathogenetic mechanism in the series of events (Figure 2, C). The abovementioned
changes may also affect brain maturation, and more specifically, neuronal migration. The latter,

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along with certain known differences in vulnerability of various neuronal populations, may

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explain why specific brain regions (e.g. prefrontal cortex or cerebellum) are more frequently
affected in ASD (Figure 2, B).
However, while the whole puzzle of this disorder has not been solved yet, there are
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enough puzzle-pieces correctly placed to predict the future success in the field.
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Acknowledgements

We thank Dr. Souren Mkrtchian for his critical review and helpful comments to this
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manuscript.
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This work was supported by The State Committee of Science MES RA, in the frame of
the research project SCS 13YR-3A0058.
pt
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Table 1. Single gene disorders with the high rate of autism.

The estimated rate of autism spectrum disorders (ASDs) in each disease ("rate of autism"), and the estimated rate of each disease in children with
ASDs are indicated ("rate in autism"). All of them are associated with various severity of mental retardation. ND indicates that the prevalence of
ASDs among individuals carrying mutations in the specified gene has not been determined [70].
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Figure 1. Graphical illustration of hypothetic interrelation of autism spectrum disorders with its' different models (there is no direct
quantitative criteria showing prevalence of any single model).

Figure 2. Summary of proposed converging mechanisms of autism spectrum disorders.

Proposed mechanisms involved in autism spectrum disorders (ASD) pathophysiology are shown in this
figure as multilayer disturbances. At the tissue level it may involve altered minicolumns, impairment of
synaptogenesis with possible imbalance of excitation and inhibition, in temporal dimension resulting in a
decrease in neuronal numbers (box A) and decreased efficacy of interneuronal communication. These
shifts and neuronal injuries are accompanied by glial involvement (box C) resulting in increase in
inflammatory signaling. These might potentially interfere with impairments in neuronal migration (box B)
thus disabling proper neuronal connectivity. At the cellular-molecular level (box D), these changes are
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Page 20 of 33
considered to be the consequence of impairments in several clusters: impaired synapse construction
(shown in green), derangements in protein synthesis, quality control, ubiquitination and epigenetic
regulation of protein expression (violet dotted square) and abnormality of cellular growth and synaptic
plasticity (blue dotted square). The violet small circle denotes a protein, expression of which is regulated
by the above-mentioned pathways and which is necessary for the effective synaptic plasticity (e.g. AMPA
receptor subunit). Despite being critisized, its still possible that the abovementioned changes affect some
crucial areas in human brain (box A) more than others (e.g. prefrontal cortex with mirror neurons or
cerebellar Purkinje cells).

t
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Abbreviations: Cav - L-type voltage-gated calcium channel; NMDA - N-methyl-D-aspartate receptor;
AMPA - -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; mGluR metabotropic
glutamate receptor; CaMKII - Ca2+ /calmodulin-dependent protein kinase II; FMRP - fragile X mental

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retardation protein; MECP2 - methyl CpG binding protein 2; UBE3A - Ubiquitin-protein ligase E3A;
NF1- neurofibromin, Shank - SH3 and multiple ankyrin repeat domains 3, mTOR mammalian target of
rapamycin; Akt - Protein kinase B; PI3K - Phosphatidylinositol-4,5-bisphosphate 3-kinase; PTEN -

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Phosphatase and tensin homolog; TSC1/2 - tuberous sclerosis proteins 1 and 2; PSD95 - postsynaptic
density protein 95.

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M
e d
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Table 1. Single gene disorders with the high rate of autism.

Gene Protein Disorder Rate of Rate in Gene function

autism autism

FMR1 Fragile X mental Fragile X syndrome 15-30% 2-5% Translational

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retardation protein repressor

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(FMRP)

TSC1/2 Tuberous sclerosis Tuberous sclerosis 25%60% 1%4% Gene suppressor -

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proteins 1 and 2 complex inhibitor of mTOR

PTEN Phosphatase and PTEN hamartoma ND 1% Gene suppressor -

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tensin homolog syndrome (ASD with inhibitor of
macrocephaly) PI3K/mTOR signaling

NF1 Neurofibromin Neurofibromatosis 4% 0-4% Gene suppressor -

MECP2 Methyl-CpG-binding
type I

Retts syndrome
an
100% 2%
inhibitor of
PI3K/mTOR signaling

Global transcriptional
protein-2 repressor
M
UBE3A E6AP ubiquitin- Angelmans 40% 1% Ubiquitination
protein ligase syndrome

CACNA1C alpha-1 subunit of a Timothys syndrome 60% <1% L-type voltage-gated

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voltage-dependent calcium channel

calcium channel
e

NLGN3/4 Neuroligin Familial ASD ND <1% Synaptic adhesion

pt

NRXN1 Neurexin-1-alpha Familial ASD ND <1% Synaptic adhesion

CNTNAP2 Contactin-associated ND <1% Synaptic adhesion

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protein-like 2

SHANK3 SH3 and multiple Familial ASD (22q13 ND <1% PSD scaffolding,
ankyrin repeat domains microdeletion formation and
3 (proline-rich syndrome) maturation of
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synapse-associated dendritic spines

protein 2 )

CTNND2 Adhesive junction- ND <1% Participation in WNT

associated -catenin signaling, histone
protein modification,
dendritic
morphogenesis

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The estimated rate of ASDs in each disease ("rate of autism") and the estimated rate of each disease in children with ASDs are indicated ("rate in
autism"). All of them are associated with various degrees of mental retardation. ND indicates that the prevalence of ASDs among individuals
carrying mutations in the specified gene has not been determined [67].

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