At present, the diagnosis of ASD is still based on symptom evaluation, as the underlying pathological mechanism remains unclear. There are no blood-based diagnostic tools or approved drugs for ASD. Research to identify reliable biological markers of disease status and symptomology is therefore urgently needed. Neurobiological systems critical to social functioning are arguably the most promising biological sources for ASD biomarkers and therapeutic targets. However, existing methods for brain detection mostly relied on autopsy or animal models, which are limited because of poor timeliness and species differences. Most cells in the nervous system, including neurons, astrocytes, oligodendrocytes and microglia, secrete EVs under normal or pathological conditions. EVs can reflect the host cell proteins and nucleic acids at the time of secretion and can diffuse across the blood brain barrier into the periphery. Serum/plasma LCEVs can be captured by antibodies directed against the cell surface protein L1CAM embedded in the vesicle membrane(Goetzl et al., 2018; Goetzl et al., 2020; Nogueras-Ortiz et al., 2020). Although investigation of LCEVs is relatively novel, attractive evidence from other fields suggests that such investigation can afford insight into the pathological mechanisms and processes associated with Alzheimer's disease and depressive disorder(Kuwano et al., 2018; Song et al., 2020). A recent study developed a panel of single-molecule array assays to evaluate the use of L1CAM for neuron-derived EV (LCEVs) isolation, and demonstrated that L1CAM behaved as a soluble protein, not as an EV-associated protein, and therefore recommend against its use as a marker in LCEV isolation protocols(Norman et al., 2021). It is mentionable that they fractionated plasma and cerebrospinal fluid using size exclusion chromatography (SEC) and density gradient centrifugation (DGC), and found that L1CAM expression overlapped at the tails of earlier fractions with the later fractions. Actually, in this study, total EVs were extracted from serum preferentially and then were used to isolate LCEVs by immunoprecipitation to avoid soluble protein interference as much as possible. The average particle size of LCEVs was 61.50±20.71 nm in the ASD group and 62.07±20.75 nm in the TD group, which was consistent with a latest study that reported that LCEVs were smaller than other EVs isolated from plasma (p < 0.0001)(Saeedi et al., 2021). Overall, LCEVs can be enriched by L1CAM antibody in peripheral blood but the size was smaller than most EVs without L1CAM.
Thus far, a putative speech and language region at chromosome 7q31-q33 seems most strongly linked to autism. Cytogenetic abnormalities at the 15q11-q13 locus are fairly frequent in people with autism, and a "chromosome 15 phenotype" is described in individuals with chromosome 15 duplications(Nakatani et al., 2009). Some candidate genes are considered located at chromosomes 7q22-q33 and 15q11-q13(Muhle, Trentacoste, & Rapin, 2004), and 21 genes in chromosomal 8p region are identified as most likely to contribute to neuropsychiatric disorders and neurodegenerative disorders(Tabares-Seisdedos & Rubenstein, 2009). Variant alleles of the serotonin transporter gene (5-HTT) on chromosome 17q11-q12 are more frequent in individuals with autism than in healthy people(Nakatani et al., 2009). In addition, many mutations on NLGN4X, an X-linked cell adhesion molecule, result in ASD (Nguyen et al., 2020). In the present study, chromosome 17 was the commonly and mostly enriched chromosome for both DEmRs and DElnRs in ASD. A large portion of the DEmRs on chromosome 17 participates in cell communication and signal transduction, which are essential for synapse formation and neurotransmitter release. Abnormal expression of such mRNAs implies the abnormality of these functions in ASD.
Brain-derived EVs carry and release multiple molecules related to neuronal function and neurotransmission in the brain, which is beneficial for the reciprocal communication between neural cells (e.g., neuron−glia interactions), synaptic plasticity, neuronal development, and neuroimmune communication. In the present study, 104 DEmRs were annotated to be related to neuroactive ligand-receptor interaction, pathways of neurodegeneration, glutamatergic synapse, axon guidance, synaptic vesicle cycle, dendrite, neuron projection development, neuron migration and apoptotic process. Most (81.7%) of these neuron-related mRNAs were down-regulated in ASD. As demonstrated in the pathway of neuroactive ligand-receptor interaction (Fig. 3H), 5 receptors (e.g., EDNRA) were up-regulated and 19 (e.g., HTR3A) were down-regulated in ASD. A previous study reported that neuropeptide receptor gene expression was lower in children with autism and the lower neuropeptide receptor gene expression predicted greater social impairments and greater stereotyped behaviors(Oztan et al., 2018). We found that 5-hydroxytryptamine receptor 3A (HTR3A) significantly decreased in the ASD serum LCEVs in this study. HTR3A is one of the receptors for 5-hydroxytryptamine (serotonin), a biogenic hormone that also functions as a neurotransmitter and a mitogen. Ample evidence suggests that levels of serotonin and serotonin transporter (SERT) increase significantly in autistic children than in gender and age-matched non-autistic children (Abdulamir, Abdul-Rasheed, & Abdulghani, 2018; Meyyazhagan et al., 2020). It thus can be hypothesized that increase of serotonin and SERT may be a kind of cell self-help that compensates for the loss of receptors, but it needs to be experimentally confirmed in the future. Another specific signature is the decreased expression of vesicular glutamate transporter 2 (SLC17A6) in the ASD serum LCEVs. Receptors for glutamate (Glu), GRIK5, GRIK2 and GRIA4 were also down-regulated. Glu acts as an excitatory neurotransmitter at many synapses in the central nervous system. SLC17A6 mediates the uptake of Glu into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. The postsynaptic actions of Glu are mediated by a variety of receptors expressed on postsynaptic cell membrane. Emerging evidence suggests that imbalance between excitatory (Glu-mediated) and inhibitory (GABA-mediated) neurotransmission may be a common pathophysiological mechanism in ASD(Horder et al., 2018; Rojas, 2014). These studies, together with the findings in the present study, suggest that reduction in the expression of Glu transporter and receptors might be the main reason for the abnormalities of Glu-mediated neurotransmission and hence a therapeutic target in ASD.
Glycans and their conjugates (glycoproteins, proteoglycans and glycolipids) are major constituents of the neural cell membrane and extracellular matrix (ECM). Glycans and glycoconjugates participate in nearly every biological process in the developing brain. A potential link between ASD and changes in glycosylation was first observed in patients with congenital glycosylation disorders (CDGs)(Freeze, Eklund, Ng, & Patterson, 2015). Recent advances in genome sequencing have identified many genetic variants that occur in genes encoding glycosylated proteins (proteoglycans or glycoproteins) or enzymes involved in glycosylation (glycosyltransferases and sulfotransferases)(Dwyer & Esko, 2016; Yu et al., 2013). However, it remains unknown whether “glycogene” variants cause changes in glycosylation and whether they contribute to the etiology and pathogenesis of ASDs. In the present study, we analyzed the whole transcriptome of serum LCEVs in ASD to screen potential biomarkers and explore the important molecular events in brain neurons of ASD children. Our results showed that a total of 54 DEmRs (3.8%) were glycogenes, and most of them (90.7%) were down-regulated in ASD. The 54 DEmRs mainly participated in carbohydrate metabolic process, protein N-linked glycosylation, carbohydrate binding, glycolysis, glycosaminoglycan metabolic process and glycolipid metabolic process. Thereinto, OSTC, MAN1B1 and MGAT5, translating to key enzymes for N-linked glycosylation, were significantly down-regulated in ASD. In our previous study, we found a significant decrease of STL binding glycans or glycoproteins that contain trimers and tetramers of GlcNAc (core structure of N-glycans) in ASD versus in TD (fold change = 0.54, p=0.0057) (Qin et al., 2017). In all, no matter at the gene level, the transcription level, or the level of translation and post-translation modification, abnormalities of glycosylation and carbohydrate metabolism might be an important molecular mechanism of ASD. Moreover, the decrease of receptors and transporters of neurotransmitters may be related with the decrease of glycogenes as most of the receptors and transporters are highly glycosylated. OSTC is a subunit of the oligosaccharyl transferase (OST) complex that catalyzes the initial transfer of a defined glycan (Glc3Man9GlcNAc2 in eukaryotes) from the lipid carrier dolichol-pyrophosphate to an asparagine residue within an Asn-X-Ser/Thr consensus motif in nascent polypeptide chains. In the present study, expression of OSTC significantly decreased in ASD serum LCEVs, suggesting it as a candidate biomarker for ASD diagnosis.
Recent studies have shown that abnormal expression of miRNAs could be involved in the underlying pathogenesis of ASD. miRNAs are small noncoding mRNAs that regulate gene expression and are often linked to biological processes and implicated in neurodevelopment. A dozen of miRNAs, such as miRNA-125b and miRNA-132, have been observed to regulate the expression of ASD risk genes, act differently on the morphology of the spine and synaptic plasticity in brain neurons, and participate in ASD etiopathogenesis(Schepici, Cavalli, Bramanti, & Mazzon, 2019). However, compared with mRNA and lncRNA, fewer miRNAs were found differentially expressed in ASD serum LCEVs in the present study. Among 11 DEmiRs, PC-5p-139289_26 was significantly up-regulated and hsa-miR-193a-5p was significantly down-regulated in ASD, and both of them had the largest number of predicted targets that were differentially expressed in ASD, indicating that these two miRNAs might play important roles in ASD. These targets were mostly involved in glutathione synthesis and recycling and mannosyltransferase activity, which are closely correlated with synthesis of Glu and glycans involved in the neuron- and glycan-related networks in ASD. However, the relationships between miRNAs and their target genes have not yet been verified.
This study might have some limitations that merit consideration. Firstly, we did not examine the correlation between the expression of candidate biomarkers and disease severity. This would be addressed in our future research. Secondly, we collected only one blood sample per participant (due to the invasive nature of venipuncture, particularly in children), which limited our ability to assess within-individual consistency of our biological measures. Thirdly, some of our participants were not medication-free. Even though their medications were stable (for at least four weeks) before blood collection, it is possible that our results might be influenced by the medication status. Fourthly, our samples were mainly from a single hospital. Although it is one of the few famous hospitals in the northwest of China that treat ASD children from five neighboring provinces, most of its patients are still from the local regions. Further research involving participants from multiple areas would be a great addition to the present study.