1Department of Medical Biology,
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Autism spectrum disorder (ASD) is characterized by impairments in social interaction and the presence of stereotypy and restrictive behavior. The clinical heterogeneity of ASD makes it difficult to explain the mechanisms underlying the disease. In recent years, the association between autophagy and neuropsychiatric diseases has been investigated. In this review, we aimed elucidate the relationship between autism and autophagy mechanism in well-known autism relevant animal models. Autophagy is a cell-protective mechanism that allows cell survival in low nutrient conditions, often through the degradation of aging and damaged proteins and organelles. The target of rapamycin (TOR) complex is activated for the activation of autophagy. Apart from mTOR animal models, the valproic acid model is frequently used in autism studies. The coiled-coil and C2 domain containing 1A (CC2D1A) gene is one of the new candidate genes associated with ASD. In a recent study that used Cc2d1a knock-out mice, microtubule-associated protein 1A/1B-light chain 3 (LC3) and Beclin 1 expression levels were dysregulated in the hippocampus. It is thought that the impaired autophagy mechanism contributes to the etiology of ASD. These results showed that CC2D1A acts as a new biological pathway in autophagy. Choosing the right model is crucial for ASD studies, and further progress will be made as these results become available in the clinic. In particular, it is expected that further studies on CC2D1A will provide new information in this field.
Autism, animal models, autophagy, CC2D1A, mammalian target of rapamycin, valproic acid
Autism spectrum disorders (ASD) is a lifelong disorder with onset during childhood which is characterized by rigidity and ritualistic/repetitive patterns of interest disturbing various brain regions including prefrontal, hippocampal, cerebellar, and striatal and other midbrain regions. In approximately half of the cases, ASD is associated with intellectual disability (ID), comorbidity with other conditions including epilepsy, attention-deficit hyperactivity disorder, anxiety, depression, tics, sleep disorders, and gastrointestinal problems[2-7]. ASD prevalence and diagnostic rates have risen significantly over the past two decades reaching 1.0%-2.5%, with a male to female ratio of 4 to 1[8,9]. The reasons for sex discrepancy are not yet clearly understood[8,9]. Some studies argue that the differential expressions of ASD between sexes may result in an underdiagnosis of females because male patients tend to have more external behaviors such as increased repetitive behavior or aggression while female patients show more internal behaviors including depression, anxiety, and other emotional symptoms[2,10]. Males and females show similar levels of depression in childhood, but those levels diverge in adolescence, becoming dramatically greater in females. During adolescence, females of autism are associated with high comorbidity including tic and eating disorders, high incidence of suicide, and high rates of other medical problems. Additionally, ASD is diagnosed later in life in girls than in boys and some studies have reported that ASD symptoms may be milder in females or are masked by the occurrence of other comorbidities, such as depression, ID, or anxiety. Girls are expected to be more verbal and social by society than boys, and they can be more skilled at hiding social deficits.
ASD was considered a complex disorder that includes the complex interaction of genetic, epigenetic, and environmental factors which may lead to the alteration of brain structures and functions[14-16]. Clinical heterogeneity is the characteristic of ASD cases, and this heterogeneity is present at different levels of analysis such as genetics, neural systems, cognition, development, and behavior, as well as in clinical features from response to treatment. Despite the current technological advances, the etiology of autism is still unknown and great efforts are being made to understand the nature of autism and its associated molecular pathways. Rett’s syndrome, fragile X syndrome, and tuberous sclerosis (TS) are classified as syndromic ASD. Patients with syndromic autism have chromosome structure abnormalities or mutations. These syndromes have a unique single gene mutation which is linked to the synaptic protein called as mammalian target of rapamycin (mTOR). mTOR is a critical protein responsible for dendritic plasticity and cell survival. mTOR may be implicated in disrupted cell signaling in idiopathic ASD. A unique gene has not been identified to cause idiopathic autism. Several candidate genes have been identified for autism[20-22]. Idiopathic autism that is not associated with a syndrome is called classic autism. It is estimated that rare genetic variants are responsible for about 10%-30% ASD cases. Autism has a strong genetic basis with a complex transmission model that is thought to be the result of at least 1000 interacting genes[23,24]. These genes are associated with pathways such as cell growth and proliferation, synaptic activity and organization, transcription regulation, ubiquitination, chromatin rearrangement, protein synthesis, and transcription factors[Figure 1]. Several autism susceptibility genes including NRXN1, NLGN3, NLGN4, GABRB3, SHANK2, SHANK3, SCN1A, and CNTNAP2 have been identified by sequencing technologies[1,4,15]. Dysregulation of transforming growth factor β, bone morphogenetic protein (BMP), WNT/β-catenin, fibroblast growth factor (FGF), sonic hedgehog (SHH), and retinoic acid (RA) signaling pathways have been implicated in the pathogenesis of ASD.
Figure 1. Genetic architecture of ASD. Genetic contributions to ASD can also be caused by direct or indirect effects on genes and proteins by environmental influences. ASD: autism spectrum disorder
Several alterations have been detected in both the macro and microscopic structures of the brain in autism. In most ASD cases, the cerebral cortex is qualitatively similar to typically developing subjects in its general organization[26-29]. Abnormalities of brain development may contribute to the autism pathology. Neuritogenesis may also represent the structural basis of autism pathology. Abnormal functioning and changes of the cerebellum have been revealed in the postmortem brains of autism patients. There is a decrease in the number of Purkinje cells in the cerebellum. Neuropsychiatric developmental disorders including schizophrenia and autism are associated with synapse development and abnormal dendritic spine formation. Pathological events affecting the temporal lobe, especially the amygdala and the hippocampus, are thought to be related to the development of symptoms similar to autism. Bilateral disorders in the cerebellum, thalamus, hippocampus, and amygdala regions were detected in autism.
Autophagy is a type of cell death mechanism that functions as an intracellular quality control system to maintain homeostasis by removing damaged proteins. Autophagy is also a cell-protective mechanism that allows cell survival in low nutrient conditions and controls cell quality under stress conditions, often through degradation of aging and damaged proteins and organelles. Autophagy is carried out by at least three different mechanisms, microautophagy, macroautophagy, and chaperone dependent autophagy. Macroautophagy occurs at the basic level in most cells, playing an important role in the breakdown of damaged organelles and proteins. Microautophagy is the event of lysis of the cytoplasm directly by the lysosome and digestion of the cytoplasm content within the lysosome with the lysosome membrane collapsing inward. And, chaperone-mediated autophagy selectively transfers proteins with KFERQ motifs to the lysosome membrane.
Autophagy is induced by starvation, oxygen deficiency, and various stress conditions. In these cases, the target of rapamycin (TOR) complex is activated for the initiation of autophagy. The TOR complex is the key protein that controls the cell’s energy metabolism, protein synthesis, and cell growth. It was first identified as the target molecule of rapamycin, an immunosuppressive agent developed for use against yeast in fungi. The isoform in mammals is known as mTOR, and suppression of this protein or silencing of the mTOR gene through various gene modifications provides stimulation of autophagy. It is also known that the dysregulation of the autophagy mechanism is associated with human diseases such as cancer and neurodegeneration. Therefore, autophagy has attracted considerable interest in the biological sciences. Several autophagic proteins control this pathway. BECN1 is a principal player in autophagy, triggering autophagosome formation. LC3, serving as another important marker of autophagy, undergoes cleavage to become LC3-I and is then conjugated to phosphatidylethanolamine (PE), to produce LC3-II[Figure 2].
Figure 2. Molecular mechanism of autophagosome formation in mammalian autophagy process. Three major steps of the autophagy are figured out as initiation (A), nucleation (B) and elongation (C). PM: plasma membrane; PE: phosphatidylethanolamine
Neuronal autophagy is crucial for the interaction, signaling, and development of neurons, and the alteration of autophagy negatively affects the growth and function of neurons. Proper growth of axons and dendrites is important for neuronal balance. Organelles or proteins with impaired functions are normally degraded for structural plasticity during development. Recent studies have shown that genes associated with autophagy are very important in the development and maturation of dendrites, axons, and synapses. ASD, as a neurodevelopmental disorder, may result in synaptic abnormalities. Autophagy and its related pathways are thought to be associated with the development of ASD, but the precise roles of neuronal autophagy in ASD were not fully understood[43,44]. Some studies have shown that activation of excess autophagy causes autism, while other studies have shown that deficiency of autophagy causes autism[24,29,45-48]. Recently, in the postmortem brains of patients with ASD, impaired autophagy, and hyperactive mTOR signaling were observed. Autophagy is inhibited in mice with phosphatase and tensin homolog (PTEN) mutation and these mice exhibit autistic behaviors and abnormal neuronal development. This result suggests that the dysregulation of autophagy in individuals with ASD. Mutation of the activity-dependent neuroprotector protein (ADNP) gene has also been identified as one of the causes of ASD. ADNP is a gene encoding a transcription factor located on the long arm of chromosome 20 (20q13.13). ADNP syndrome is characterized by global developmental delays, ID, speech barriers, and motor dysfunctions. Deletions in this chromosomal region cause ID. ADNP controls the expression of genes during embryonic and postnatal development, including neurogenesis-crucial genes and BECN1. Given the direct relationship between ADNP and MAP1-LC3B, as well as the connection of autism with eukaryotic initiation factor 4E (Eif4e) and its tight relationship with autophagy, ADNP is also inevitable to play a role in autism and autophagy[51,54]. ADNP knock-out mice resulted in embryonic death during the closure of the neural tube. ADNP +/- male mice showed cognitive deficiencies in behavior tests.
An ideal animal model of a human disorder should present some characteristics features of the disease. The model should resemble the symptoms of human disorder and can be genetically modified by specific stimulation. Both the models and human patients should respond similarly to certain treatments. Animal models can provide an advantage over human studies by allowing controlled testing of the effects of specific disease-causing factors on synaptic function and behavioral outcomes.
Many of the known genetic variations which contribute to the risk of ASD affect the expression of proteins that have roles in the chromatin remodeling or function, formation, and maintenance of synapses. Deletions of such genes in animals can cause a behavioral phenotype reminiscent of ASD with impairments in communication, social interaction, and repetitive behaviors. Knock-out models that include monogenic ASD genes are NRXN1, MECP2, NLGN3, SHANK3, NLGN4, FMR1, and TSC1/2[45,58-64]. Although there are few neuropathological data available in some models, there is growing interest of carrying specific ASD genes. The emerging risk genes are as follows: CHD8, SCN2A, SYNGAP1, DSCAM, and TBR1[65-69]. In the literature, there are syndromic animal models (i.e., Rett Syndrome, Prader-Willi, and Angelman Syndromes, fragile X, and Tuberous Sclerosis Complex) and environmental animal models [i.e., valproic acid (VPA), maternal autoantibodies, and maternal immune activation] that can be generated. In this review, the most studied mTOR related models and VPA models related to autophagy are discussed. Also, we explain the developments in the coiled-coil and C2 domain containing 1A (CC2D1A) animal models, which are not well-known in the literature, through the autophagy mechanism.
The correct protein level is provided by the delicate balance between protein synthesis and breakdown. High synaptic protein levels can occur as a result of high translation and/or accumulation of damaged protein. In neurons, mTORC1 takes the role of braking autophagy. It is strategically positioned in the presynaptic and postsynaptic regions. Under rich nutrient conditions, the target of mTORC1 is Unc-51-like autophagy-activating kinase 1 (ULK-1) which phosphorylates in Ser757, the anti-autophagy region. In this case, ULK-1 moves away from the AMP kinase (AMPK) and autophagy initiation is stopped. On the contrary, in the starvation conditions, AMPK phosphorylates and activates ULK-1 in Ser317 which mediates the phosphorylation and activation of Beclin-1 in Ser14, an important step in the “nucleation phase” of autophagy. Beclin-1 promotes the lipidization of LC3-I to achieve the lipid form LC3-II which enables membrane elongation and formation of autophagosomes. With lipidation, LC3-II is localized to the phagophore membrane and mediates the formation of mature LC3-II autophagosomes, limiting membrane elongation and cargo. Neuronal autophagy has a key role in protein balance and is an important regulator of memory formation, synaptic plasticity, and structural remodeling[73,74].
A possible scenario is that over-activated mTOR from hippocampal neurons from fragile X mice results in decreased autophagy and accumulation of a particular group of synaptic proteins. Neurons from Fmr-1 knock-out mice showed the accumulation of ubiquitin-protein aggregates localized by p62, and autophagy was impaired in Fmr-1 knock-out neurons. This findings in this study showed that autophagy and protein degradation decreased in hippocampal neurons of this model. Excess mTOR activity is causally associated with decreased autophagy causing spinal defects, impaired cognition, and exaggerated synaptic plasticity in Fmr1- knock-out mice.
Interestingly, a recent study found that increased activity of mTOR and MAPK pathways in the peripheral blood samples of idiopathic ASD patients. ERK1-2, rpS6, p-eIF4E, and p-MNK1 (components of the mTOR and MAPK signaling pathways) showed a significant increase in patients with ASD compared to controls. TSC1, rpS6, p-eIF4E, and p-MNK1 protein expression discriminated patients according to their clinical severity. Overactive mTOR signals suppress autophagy in the brains of tuberous sclerosis complex (TSC) 1+/- and TSC2+/- mice during postnatal development and decreased autophagy causing an imbalance in the pruning of the spines in the cortical layer V pyramidal neurons. Dysregulation of autophagy and its association with impaired spinal pruning may be more common in at least one subset of ASD in Tsc mutant mice. TS is a genetic disease caused by mutations in the TSC1 or TSC2 genes and mTOR activity is regulated negatively with these genes. TSC1+/- mice showed impairments in social interactions and hippocampus-dependent contextual fear conditioning. TSC2 mutations created learning and memory deficits and contextual fear conditioning. Mice carrying a dominant-negative TSC2 mutation had reduced social interactions and preference for social innovations.
Human with mutations in the PTEN gene tend to develop ASD, macrocephaly, seizures, and ID. It is thought that neurological symptoms associated with the loss of PTEN and other “mTORopathies” are caused by hyperactivation of mTORC1-mediated protein synthesis. One study with Pten knock-out mice revealed that rapamycin-mediated inhibition of MTORC1 activity increased behavioral and neurophysiological abnormalities and showed a reduction in brain size. The group also found that genetic deletion of mTORC2 activity suppressed seizures, recovered ASD-like behavior and long-term memory, and normalized metabolic changes of Pten knock-out mice. They found that reducing mTORC2 rescued behavioral and neurophysiological abnormalities. The contactin associated protein 2 (CNTNAP2) is the first widely studied autism susceptibility gene. CNTNAP2 knock-out mice show core ASD-like phenotypes. By RNA sequencing of CNTNAP2 knock-out mouse, hyperactive Akt-mTOR signals were detected in the hippocampus. After treatment with mTOR inhibitor rapamycin or Akt inhibitor LY294002, it was reported that the social deficit was recovered in mice but had no effect on hyperactivity and recurrent/restricted behavior. Additionally, the effect of rapamycin and LY294002 on social behavior is reversible. Thus, the hyperactive Akt-mTOR signaling pathway has been identified as a therapeutic target for abnormal social behavior in patients with CNTNAP2 dysfunction.
According to latest study by Lieberman et al., autophagy is downregulated during postnatal development following the upregulation of mTOR activity in the mice striatum. In the same study, a VPA model has been also conducted; autophagy is specifically reduced. They concluded that the impairment of autophagy is accompanied by impairments in synaptic transmission and social behavior in the late postnatal development in this mouse model. In the TRIM32-/- model, the authors generated impaired GABAergic interneurons and leading autism relevant behaviors in mice, concomitant with an increased autophagy mechanism. Therefore, they suggested that theTRIM32-/- mouse is a novel autism mouse model.
VPA is commonly used as an antiepileptic drug. Clinical studies have shown that exposure to VPA in utero is associated with cognitive deficits, birth defects, and an increased risk of ASD. Clinical evidence shows that there is a link between VPA exposure and both cognitive abnormalities and autism. Animal studies in recent years have investigated anatomical, behavioral, molecular, and physiological changes due to in utero VPA exposure. The behavioral tests revealed that VPA exposure causes autistic-like behavior in offspring; these include social behavioral deficits, increased repetitive behavior, and communication deficits in rodents. In the embryonic period, a single intraperitoneal VPA injection to 12.5-day-old female rats causes autism relevant symptoms in their offspring, and the brain structures and biomarker levels of the offspring are similar to those in autistic patients[87,88]. The VPA model is often used in ASD studies[89-91]. mTOR, the main marker of cellular metabolism, cell growth, and autophagy, has been reported to contribute to ASD development via the IGF1/PI3K/AKT/mTOR pathway. Accordingly, it was assumed that autophagy will increase or decrease through PI3K/AKT/mTOR, thereby contributing to the development of ASD. Based on this assumption, VPA was given to rats and it was observed that the rats had autistic-like behaviors. Zhang et al. rats were given VPA and autistic-like behaviors were observed. Various autophagic markers were examined in the hippocampus of these rats by the immunohistochemical method and mTOR inhibition was found to increase PI3K/AKT/mTOR-mediated autophagic activity.
Sphingosine 1-phosphate (S1P) is abundant in the brain tissue and plays an important role in brain development, regulation of neuronal differentiation, proliferation, survival, and apoptosis. There is growing evidence that abnormal S1P levels are associated with the pathogenesis of schizophrenia, Alzheimer’s disease, and anxiety disorder. In a study that investigated the pathogenesis of S1P in autism, a VPA rat model was used to evaluate S1P expression levels in the serum and brain tissue. Expression of autophagic proteins Beclin-1, LC3-II, and P62 were investigated in rats exposed to VPA. The investigators in the study found that increased S1P may be associated with decreased autophagy in this model. A study was carried out to illuminate the link between the Notch signaling pathway and the pathogenesis of autism. They also investigated whether 3,5-difluorophenacetyl-L-alanyl-S-phenylglycine-2-butyl ester (Dapt) caused autism relevant behavior in the VPA rat model by regulating autophagy and affecting the morphology of dendritic spines. Autophagy-dependent proteins LC3B, Becn1, and phospho-p62 were inhibited by Dapt in rat VPA model in the hippocampus, cerebellum, and prefrontal cortex. The Notch signaling pathway participates in the ASD pathogenesis by affecting dendritic spine growth and regulating autophagy.
The CC2D1A (coiled-coil and C2 domain-containing 1A) gene is one of the new candidate genes associated with autism. In humans, the CC2D1A gene mutation was first associated with autosomal recessive nonsyndromic intellectual disability. This means that the CC2D1A gene has a function in the central nervous system. Cc2d1a knock-out mouse model has been shown that regulates multiple intracellular signaling pathways, involved in neuronal differentiation and brain development, activators of Protein Kinase B (PKB), and NF-κB[98,99]. CC2D1A protein consists of a COOH-terminal C2 domain and NH2-terminal domains. The CC2D1A gene family consists of two homologous units, CC2D1A and CC2D1Bfor both humans and mice. The CC2D1A protein has two main isoforms containing 950 and 388 amino acids. The shorter isoform of the protein does not include exons 14 to 16, which are deleted in patients with ASD. Therefore, only the long protein isoform plays a central role in disease pathogenesis in the patients. The most conserved motif is a C2 domain and the other motif, DM14, is unique to the CC2D1A protein family, but its role is unknown. The C2 motif is located at the C-terminus of CC2D1A at positions 661-762, and is found in proteins that function in calcium-dependent phospholipid binding, where the C2 domain itself participates in the binding pocket of the Ca2+ cation. The DM14 motif repeats four times in the human CC2D1A sequence but only three times in the Caenorhabditis elegans orthologue sequence. The DM14 motif also appears only in the long isoform of CC2D1A, while the C2 domain is present in both isoforms, suggesting that it probably plays a central role in its protein activity[100-103]. The CC2D1A gene functions to bind to DNA and suppress transcription of serotonin receptor 1 A (HTR1A). The physiological role of the CC2D1A gene is not fully known. In a study, CC2D1A knock-out mice were produced to examine this role. Mice with the homozygous CC2D1A gene mutation died immediately after birth due to their inability to breathe, while mice with the heterozygous mutation remained alive and productive. Therefore, since the CC2D1A gene is vital, the absence of this gene as in the homozygous case is fatal. The absence of the CC2D1A gene primarily affects brain function. Studies with animal models have revealed that this gene has a role in the regulation of endosomal traffic and signaling pathways. Disruption of this gene affects numerous biochemical pathways including cAMP response element-binding protein (CREB), NF-κB, protein kinase A (PKA), protein kinase B (PKB/AKT), Notch, and BMP. CC2D1A plays a role in the transcriptional regulation o both dopamine and serotonin receptors in the brain. All studies have shown that CC2D1A regulated critical pathways for cognitive functions with neuronal differentiation[102,103]. Although there are not many studies in this animal model, abnormalities were found in the brain in the studies performed. Developmental changes in neurons of Cc2d1a knockout mouse brains were demonstrated during synapse maturation and induced neurotransmitter release. Based on these findings, it is thought that the CC2D1A gene serves as a developmental regulator of synapse function. CC2D1A knock-out or knock-down in vitro experiments showed a decrease in hippocampal neurons. Also, delays in synaptic maturation have been observed in cortical neurons. In these animal models, it has been determined that there is a lack of neuronal plasticity, spatial learning, and memory accompanying decreased socialization, hyperactivity, anxiety, and excessive self-care and Cc2d1a has been shown to control synaptic maturation of excitatory neurons. Further studies are needed to determine whether CC2D1A controls sex-specific circuit function. Conditional removal of CC2D1A from the dorsal raphe demonstrated increased anxiety and depression-like behavioral phenotypes which correlated with reduced serotonin levels and increased 5HT-1A autoreceptor in the raphe, in both males and females, suggesting that there may be regional specificity in the function of CC2D1A.
In vitro studies in hippocampal neurons and embryonic fibroblasts from CC2D1A knock-out mice showed that CC2D1A binds to phosphodiesterase 4D (PDE4D), an enzyme involved in cAMP degradation. In a recent study, PDE4D activation and downstream signaling molecules were tested in the hippocampus of Cc2d1a knock-out mice. Cc2d1a knock-out male mice were hyperactive and show a deficit in spatial memory, which led to a reduction in cAMP response element-binding protein signaling but this finding has not been correlated with female mice. These findings showed that CC2D1A regulates cAMP intracellular signaling in the male-specific regions of the hippocampus. In our recent study, we showed the dysregulation of autophagy with CC2D1A deficient mice in the hippocampus. We wanted to evaluate the severity of autism by creating two different groups and followed them over the next three generations. LC3 and Beclin gene and protein expression levels in the hippocampus tissues of male and female mice in both groups were examined. All of the animal groups were observed to be extremely aggressive and hyperactive. Overall decreases were observed in autophagy levels. In the literature, this was the first major study in the CC2D1A mouse model in which autism was associated with autophagy.
Genetic studies of autism have made surprising progress over the past 20 years. Our understanding of the genetic and epigenetic factors in ASD etiology and the interaction on the disease will be continued to improve with future studies and ongoing research results.
Animal models are used to study potential disorder mechanisms. The well-known causes of autism are commonly based on specific human genetic mutations; however, ASD pathogenesis is most likely shaped by a complex interaction between several genetic variants as well as environmental factors in humans. In the animal models, monogenic mutations can lead to milder phenotypes that might explain some of the differences observed in behavioral manifestations between ASD patients and animal models. Given the clinical heterogeneity of the ASD patients, it is controversial whether it is necessary or even possible to see all the human symptoms in rodent models. However, monogenic rodent models are a valuable resource for solving the cause-and-effect relationships of ASD since the majority of susceptibility genes appear to converge in shared biological pathways. Therefore, rodent models are important preclinical tools necessary to investigate the validation of pathophysiology, gene function, and therapeutic approaches in ASD.
Choosing the right model is of great importance for ASD studies and progress will be made in the reflection of the results to be obtained in the clinic. Especially, the studies to be done with CC2D1A models are expected to gain new information in this field.
Idea conception: Sener EF
Read, Literature research, manuscript writing and editing, adjusted and approved the final manuscript: Dana H, Tahtasakal R, Sener EFAvailability of data and materials
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