**7. Translational regulation and the process of ubiquitination**

It has been seen that many synaptic proteins are critical to the formation and maintenance of proper synaptic function. The expression level of many of these proteins may be tightly controlled by the balance between translation and turnover. The growing number of devel‐ opmental cognitive diseases, whose underlying cause is a defect in the regulation of either translation or turnover, suggests that the equilibrium between these opposing processes is a sensitive point in establishing normal cognition and behavior [36].

Ubiquitination, the covalent attachment of ubiquitin to a target protein, regulates most cellular processes and is involved in several neurological disorders. Many genes in the ubiquitin pathway and neuronal proteins that are targeted by the ubiquitin-proteasome system have been linked to cognitive deficits [57, 82].

Studies have highlighted an important role for protein degradation by the ubiquitin protea‐ some system (UPS) in synaptic plasticity [126, 91]. These observations suggest that changes in synaptic transmission involve extensive regulation of the synaptic proteome. The synaptic proteome is also affected by nonsense-mediated mRNA decay (NMD) that provides a quality control linked to translation. NMD has a role in degradation of aberrant mRNAs with a premature termination codon and the regulation of the transcriptome [109]. CNVs and mutations in several genes associated to NMD such as *UPF3B, UPF3A, SMG6, EIF4A3, RNPS1* and *RBM8A* have been identified as probable causes or predisposing factors for neurodevelopmental disorders such as autism [5, 76, 108].

UPS consists of a group of enzymes, an ubiquitin activating enzyme (E1), an ubiquitin conjugating enzyme (E2) and an ubiquitin ligase (E3), which are associated with ubiquitin ligases and proteasomes to mediate protein degradation. The ubiquitinated target protein is subsequently shuttled to a protease complex known as the 26S proteasome and subjected to degradative proteolysis. They also play a role in the regulation of cell signaling and cell cycle progression, and are associated with cytoskeletal elements. Thus, posttranslational ubiquiti‐ nation modifies protein function and triggers the subsequent degradation of ubiquitinated proteins by the 26S proteasome. Several components of the UPS are required for proper brain development, axon guidance, and the development and plasticity of synapses. It has been shown that protein degradation via the UPS controls the appropriate synaptic balance, maintaining optimum levels of the protein, thereby promoting functional balance [23].

Several studies have shown a crucial role of UPS in neuronal transmission. For example, mutations in *UBE3A* have been associated with ASD. *UBE3A* encodes an ubiquitin E3 ligase that contains a domain that catalyzes the ubiquitination of target proteins. A reduction in density results in defects in synaptic plasticity [57]. UBE3A regulates the development of excitatory synapses by controlling the degradation of activity-regulated cytoskeletal protein (Arc or Arg3.1). Arc is critical for long-term memory formation and essentially every form of plasticity, including LTP, long-term depression (LTD), and homeostatic scaling (Greer et al., 2010). It has been shown that UBE3A-deficient mice, express high levels of Arc in response to synaptic activity, which coincides with severely impaired hippocampal LTP [78, 133]. Also, Arc regulates the ionotropic glutamate receptor (iGluRs) expression and trafficking. Findings from various experimental systems implicate iGluR dysfunction in ASD [148].

### **8. miRNAs**

phases of neuronal polarization; microtubules and their stability determine axon formation, they maintain the identity of axons and they regulate the dynamics of dendritic spines. Once the synapses have been formed, the neuronal cytoskeleton supports maturation and mainte‐ nance, and so the synaptic cytoskeleton is essential for the stabilization and remodeling of synaptic connections [40]. Actin filaments are the predominant component of the cytoskeleton in dendritic spines [20]. Changes in these key molecules mediating that bind to actin and members of the Rho family of small GTPases, such as RhoA, Rac and Cdc42 can disrupt this process [69]. These proteins play important roles in synaptic functions, dendritic branching, the formation and maintenance of dendritic spines and the growth and differentiation of neurites [133]. Their genes include *OPHN1, MEGAP, OCRL1, ARHGEF6, ARHGEF9, FGD1,*

It is known that genetic alterations in the pathways controlling local protein synthesis in neurons contribute to diverse intellectual disabilities and ASD. A set of cytoskeletal proteins has been reported as mutated in these individuals. The resulting disorders are called synap‐ topathies with dysgenesis of dendritic spines being a recurrent anatomical feature. These include factors that regulate the dynamics of the actin cytoskeleton, such as GAPs and guanosine factors [12]. Mutations in the tumor suppressor genes, *TSC1* and *TSC2*, are also connected to the ASD mutant proteins that seem to disturb the dynamics of the cytoskeleton and the structure of dendritic spines [61]. Moreover, the microtubule-associated protein, *KATNAL2*, has emerged as a risk factor for ASD [105]. But the best example of dendritic spine defects is Fragile X syndrome. This disease results from a loss of function of the RNA-binding protein, the fragile X mental retardation protein (FMRP), which regulates dendritic targeting of mRNAs and controls protein synthesis and mRNA decay in neuronal soma and at synapses. High-throughput screenings have revealed that a wide array of neuronal mRNAs is targeted by FMRP, suggesting that simultaneous dysregulation of many proteins contributes to the

syndrome, including cytoplasmic FMRP-interacting protein 1 (CYFIP1) [38].

**7. Translational regulation and the process of ubiquitination**

sensitive point in establishing normal cognition and behavior [36].

New research has revealed many interactions associated with brain disorders, opening up new perspectives to define regulatory pathways shared by neurological disabilities characterized

It has been seen that many synaptic proteins are critical to the formation and maintenance of proper synaptic function. The expression level of many of these proteins may be tightly controlled by the balance between translation and turnover. The growing number of devel‐ opmental cognitive diseases, whose underlying cause is a defect in the regulation of either translation or turnover, suggests that the equilibrium between these opposing processes is a

Ubiquitination, the covalent attachment of ubiquitin to a target protein, regulates most cellular processes and is involved in several neurological disorders. Many genes in the ubiquitin

*LIMK1, PAK3*, and *IQSEC2* [133].

322 Autism Spectrum Disorder - Recent Advances

by dendritic spine dysmorphogenesis.

As previously reported, CNVs are recognized as important genetic factors in ASD, with a high prevalence of *de novo* CNVs in sporadic and familial cases compared with control subjects. However, studies conducted so far have highlighted a pathogenic role of CNVs in terms of changes in dosage of encoding protein genes without bearing in mind the potential involve‐ ment of non-encoded RNAs, particularly miRNAs, even with the inherent difficulties of this type of study [120, 150]. Generally these two themes (CNV and miRNAs) are investigated separately.

A few studies have investigated the transcriptome in ASD samples of postmortem brains and some of them used mRNA from peripheral blood of patients [1, 54, 71, 110]. More recently, disruption of miRNA expression has been repeatedly reported in microarray studies and it is believed to be linked to the pathogenesis of autism (Sarachana et al., 2010; [28, 55]. However, lymphoblastoid cells are not representative of neural tissue and very few miRNAs exhibit consistent deregulation between studies.

According to [93], miRNA loci are underrepresented in highly polymorphic and wellvalidated CNV regions. One study investigated the pathogenic role of miRNAs in autism by checking associations with *de novo* CNVs. Twenty-four miRNA genes likely to play a patho‐ genic role in autism were identified on chromosomes 1, 2 and 22. Two, *mir-HSA-4436b-1 and 4436b-HSA-mir-2*, appear to be strong candidates. Unfortunately, the targets of these miRNAs were not identified [94]. The difference in penetrance of the deleted/duplicated genes may be explained by a variety of factors including: (i) pre-natal exposure to environmental risk factors; (ii) the presence/absence of functional SNPs in genes that encode proteins related to suscept‐ ibility for autism; (iii) epistasis; (iv) epigenetic factors and (v) the number and type of genes encoding proteins co-existing in different CNVs and overlapping in the same miRNA.

The *HEY1, SOX9*, *miR-486* and *miR-181b* are some candidate genes. All of these are involved in the development and function of the nervous system, and some, such as *HEY1*, are involved in Notch signaling networks [55]. However, a systematic analysis of CNV-miRNAs based on their interactions with target genes identified other miRNAs such as hsa-miR-590-3p, hsamiR-944, miR-HSA-570, hsa-miR-34a, hsa-miR-124, hsa-miR-548f, hsa-miR-429, miR-HSA-200b, hsa-miR-195 and miR-497-HSA. Moreover, the miRNAs related with CNVs can explain the difference in levels of the important genes that are controlled by them. These CNVmiRNAs can also harm the overall biogenesis and processing of all miRNAs by targeting key molecules in the miRNA pathway [150].

On the other hand, dysfunction of neuronal miRNAs can result in a number of neuropatho‐ logical conditions. It has been reported that neural miRNAs and their target mRNAs are coexpressed, suggesting their participation in feedback mechanisms to connect the transcriptional activation with the control of local dendritic protein synthesis [145]. Interest‐ ingly, the functions attributed to miRNAs overlap with growth abnormalities, delays and the disruption of neuronal maturation observed in the brains of autistic individuals. Aberrations in the translational control of multiple mRNAs mediated by targets of each miRNA may lead to the difference in phenotypes observed in ASD. Moreover, multiple miRNAs may target the same mRNA leading to phenotypes resulting from the converging of several loci in CNVs. Thus, a change in expression or level of miRNA will affect the expression of target genes and might have a pleiotropic effect that would produce a more severe autistic phenotype. However, one can not underestimate the clinical relevance of the deregulation of a single or a subset of CNV-miRNAs. Based on this, it is clear that the characterization of this relationship may illustrate the complexity of the underlying neuronal development, function and dysfunction that will eventually help in the understanding and treatment of autism [150].
