MET and the HGF pathway. Both genetic and protein expression studies have associated the receptor MET and its ligand HGF with ASD. A recent case–control study demonstrated a strong association of a single nucleotide polymorphism (G-to-C) in a common 5′ promoter of the MET gene with ASD. The relative risk of ASD diagnosis was 2.27 in subjects with the C/C as compared with the G/G genotype (32). This study is especially relevant because the MET gene is located at 7q31 in one of the regions most commonly associated by genetic linkage studies with ASD (104, 220). MET is a transmembrane receptor that possesses tyrosine kinase activity (14, 24) and is activated by binding to HGF, also termed scatter factor or hepatopoietin A. HGF and MET, are present in both developing and adult mammalian brains, suggesting important functions across a broad range of neurodevelopment (115). HGF acts as a neurotrophic factor for motor, sensory and parasympathetic neurons (203), and influences neuronal migration (169, 170) and dendritic development (91). The HGF/MET pathway also plays a role in regulating dendritic morphology in the developing cerebral cortex and promoting neurite outgrowth (170). Decreased levels of MET itself and altered levels of mRNA of proteins associated with the HGF/MET pathway have been documented in brain tissues from patients with ASD (33). In addition to these genetic observations and brain tissue findings, we have documented increased levels of HGF in cerebrospinal fluid (CSF) of patients with autism (211), suggesting a potential compensatory feedback mechanism.
Interestingly, the multifunctional roles of the HGF/MET pathway also involve the immune system, as studies have demonstrated expression of MET in dendritic cells (161) and during activation of monocytes (12). HGF-stimulated monocytes increased the expression of chemoattractant factors including MCP-1, MIP-2β, MIP-1α and IL-8 (13). HGF also exhibited immunosuppressive effects without up-regulation of IL-10 or TGF-β(161), findings that suggest HGF/MET signaling is involved in regulation of the inflammatory responses. Because some of the non-neurological manifestations of ASD include immune and gastrointestinal problems, the dysregulation of HGF/MET may provide a link between dysfunction of the CNS and other organs.
Reelin. RELN, which encodes the protein Reelin is another gene playing a critical role in cortical patterning that may be involved in autism. Reelin is a secreted extracellular matrix protein that controls neuronal migration, cortical layering and other aspects of brain development via interactions with lipoprotein receptors (reviewed by Forster (77)). It was initially implicated in ASD based on associations between a polymorphic GCG repeat immediately 5′ of the RELN gene and autism in both case-control and family-based studies in an Italian population (166). The fact that RELN is located on the distal long arm of chromosome 7 at a locus (7q22) associated with autism susceptibility added further support to the concept that Reelin function might be important, as did the reduced levels of Reelin found in post-mortem studies of autistic brains (73). Attempts to confirm these intriguing preliminary findings have yielded varied results. Some reports have supported an association between genetic changes in the RELN locus and autism (196, 199, 224), while others have not (22, 66, 118).Transgenic mouse studies are also suggestive, but not definitive, with some social changes and defects in cortical layering observed in mice mutant in RELN alleles (186).
Neurotrophins. Neurotrophic growth factors, or neurotrophins, are good candidates for involvement in ASD because of their fundamental roles in guiding CNS development and cortical organization, and their abnormal expression patterns in autistic individuals. The core functions of neurotrophins during neurodevelopment include regulation of cell proliferation, migration and survival, and extend to include the modulation of axonal and dendritic outgrowth, synapse formation and other neuroplastic processes (5). The neurotrophin family consists of at least four proteins, including nerve growth factor, BDNF, neurotrophin-3 and neurotrophin-4 (92). Their potential role in pathogenic ASD pathways has been examined in several studies involving a heterogeneous groups of neurodevelopmental disorders (146, 155, 179).
Neurotrophins and their receptors are expressed in the neocortex and hippocampus (102) and these patterns of neurotrophin expression are activity-dependent and regulated by sensory inputs, electrical activity and stimulation (102)(138). BDNF and its receptor, trkB, are densely expressed on cortical and hippocampal neurons, and influence both axonal and dendritic growth in a highly neuron-specific and age-dependent manner (139). In rodents, the expression of the trkB receptor peaks in the first 2 weeks postnatally, but BDNF action on cortical plasticity continues into adulthood (119, 139). With maturation, trkB becomes enriched at the site of glutamatergic synapses and therefore uniquely able to modulate experience-dependent plasticity (85).
Interestingly, abnormalities in neurotrophins, especially BDNF, have been implicated in the etiology of several brain disorders that show altered cortical maturation and plasticity, such as schizophrenia and depression (158, 197). Genetic studies and expression of BDNF in serum of patients with ASD have pointed out potential links to the pathogenesis of autism. Nelson et al found elevated levels of BDNF and NT4/5 by assessment of archived neonatal blood samples of ASD patients (155). Elevation of BDNF was also reported in a study of 18 Japanese children with ASD as compared with controls (148), and the authors suggested hyperactivity of this growth factor may be involved in neurobiological abnormalities in autism. Similar findings were reported in a study of American children with ASD, where elevation of BDNF was demonstrated along with the presence of auto-antibodies against BDNF (47, 153, 206).
It is still unknown how these observations fit into the neurodevelopmental pathogenesis of ASD, and it is unclear whether the increase in BDNF is a primary pathogenic mechanism or a secondary reaction to cortical abnormalities in ASD. However, one report suggesting that genetic changes in autistic individuals account for altered neurotrophin levels supports the notion that BDNF dysregulation could be a primary factor in the development of autism. CADPS2 is a gene found in the AUTS1 susceptibility locus for autism on 7q31 (42). Sadakata et al have recently shown that CADPS2 is aberrantly spliced in some autistic patients, and that Cadps2 knockout mice have autistic-like phenotypes. CADPS2 regulates the exocytosis of dense-core vesicles, including BDNF-containing vesicles. In addition, the cellular distribution of BDNF in the brain largely overlaps with that of CADPS2 (183, 184).
Neurotransmitters. Several lines of research suggest that abnormalities in serotoninergic, GABAergic and glutamatergic pathways occur in autism (reviewed by Zimmerman (225)). Neurotransmitter function in the CNS is linked not only to synaptic neuronal interactions, but also to other roles including brain maturation and cortical organization. Neurotransmitters and their receptors may act as paracrine signaling molecules in the immature brain and help control mechanisms that govern neuronal migration and positioning (134). It is well known that activation of specific GABA and glutamate receptors (GluRs) occurs during cell migration, and is involved in regulating radial and tangential migration (134). Because of these diverse functions, neurotransmitters and their receptors are clearly capable of playing central roles in the wide variety of neurobiological alterations associated with ASD.
The role of serotonin in autism has been explored using biomarker, neuroimaging and genetic approaches (193). The most relevant brain imaging studies used positron emission tomography to show that young children with autism lacked the developmental peak in brain 5-HT synthesis capacity seen in typically developing infants (36)(41). Reduced synthesis of 5-HT was observed in dentatothalamocortical pathways, with simultaneous increases in the contralateral dentate cerebellar nucleus (41). More recently, SPECT studies demonstrated significant reductions in 5-HT2A binding in the cerebral cortex (152). Elevated levels of serotonin in the platelets of patients with autism has also been observed by a number of groups (29, 48, 123). In contrast, studies that assess changes in 5-HT receptors in platelets or whole blood of individuals with autism show decreased 5-HT2 receptor binding (51, 140).
Genetic studies have also identified abnormalities in serotonin-related genes. Tryptophan hydroxylase-2 (TPH2) is the rate-limiting enzyme in 5-HT synthesis in the CNS, and one group found a particular variant of TPH2 to be associated with autism (53). A second study, however, was not able to confirm this (181). Polymorphisms in the promoter region of the serotonin transporter gene SLC6A4 have also been reported to be associated with autism and cortical gray matter volume (39, 52, 67, 204, 213, 215). Finally, the gene ITGB3 has been proposed as a regulator of serotonin levels in autism based on genetic association studies (214, 215). Synergistic interaction between the SLC6A4 and ITGB3 loci has also been suggested (58).
Another line of research supporting serotonin as a neurobiological factor in ASD comes from pharmacological interventions. Drugs acting on the 5-HT2 receptor (28, 143) alter the serotonin system and have caused behavioral improvements in autistic patients (94, 101, 114, 150, 168). Specifically, the selective serotonin reuptake inhibitor fluoxetine causes improvements in social behavior while decreasing aggressive and stereotyped behaviors in children with autism (6, 27, 50, 64, 72, 82). Interestingly, approaches that decrease CNS serotonin such as tryptophan depletion exacerbated symptoms in patients with ASD (49, 142).
A wide range of studies suggest that changes in serotonin and other neurotransmitters can result in aberrant cortical development. 5-HT afferents from the brainstem raphe nuclei innervate cerebral cortex during a critical time in cortical morphogenesis. Similar to the peak in serotonin synthesis at 2 years of age in humans, rodents show a transient peak in serotonin levels in the first few days after birth (46, 100). At this time, layer IV of the sensory areas of cortex exhibits dense patches of staining for serotonin and 5-HTTs, particularly in the “barrel field” in primary somatosensory cortex (18, 60, 78, 178). In vivo, it appears that too little or too much serotonin is detrimental to cortical development. Experimental approaches in rodents with neonatal systemic 5-HT depletion reveal delayed development of several cortical layers (162), the aberrant appearance of thalamocortical afferent patterning in the barrel field (18) and an ultimate decrease in the size of the barrel field (156, 165). Altered dendritic and synaptic development appears to be at the root of serotonin's effects (137, 219), as barrel formation is restored in MAOA and 5-HTT single and double knockouts by the blockade of serotonin synthesis, or the additional knockout of 5-HT1B receptors, which normally inhibit glutamate release (185).
The interaction of serotonin pathways with neurotrophins such as BDNF suggests a potential interplay between these factors in ASD pathogenesis. BDNF and serotonin show co-regulation in response to environmental factors (25, 136). During brain development, factors such as perinatal stress or environmental enrichment lead to long-term alterations in BDNF expression in brain and blood plasma (25, 79). In rodent models, maternal infection can cause long-term increases in BDNF within the cerebral cortex and other brain areas that eventually affect the development of serotoninergic pathways (83). Another example of this interaction comes from mice heterozygous for BDNF (BDNF+/–) that display premature, age-associated loss in forebrain serotonergic innervation (130). Similarly, 5-HTT function is impaired in the brains of BDNF+/– mice (61). Localized increases in BDNF expression promote 5-HT fiber sprouting after injury (88, 133). In turn, 5-HT depletion via inhibition of synthesis is accompanied by decreases in BDNF levels in the mature hippocampus (223). Such decreases in BDNF expression may be mediated by serotonergic mechanisms in that 5-HT2A receptor antagonists have been shown to block stress induced decreases in BDNF expression in the hippocampus and cortex (210).
Excitatory neurotransmitter signaling via glutamate receptors (GluRs) also likely plays a role in cortical development (134), and has the potential for involvement in the pathogenesis of ASD. Candidate genes-screening and association analyses showed that the kainate receptor GluR6 (105, 198, 202), metabotropic GluR8 (GRM8) (195) and one of four N-methyl-D-aspartate (NMDA) receptor 2 subunits, GRIN2A (8), appear to be associated with ASD. Interestingly, cDNA micro-array techniques along with other mRNA and protein studies of brain tissues from patients with autism identified significant increases in expression of several genes associated with glutamatergic pathways, including excitatory amino acid transporter 1 and glutamate receptor AMPA 1 (173). Such disturbances of the glutamatergic system may well affect cortical development and plasticity, as experimental evidence suggests that GluRs play roles in the activity-dependent refinement of synaptic connectivity (65). GluRs are classified broadly into two groups, ionotropic sites, linked to ion channels and metabotropic sites, linked to second messengers (144). The ionotropic sites include those activated by the exogenous agonists, NMDA, amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate (KA). NMDA receptors influence both the retraction of incorrectly placed axon arbors and synapses and the elaboration of correctly positioned terminals. NMDA receptors also have well-documented roles in cortical development and activity-dependent plasticity (89, 134).
GABAergic pathways also play important roles during brain development, and the interplay of glutamatergic and GABAergic systems facilitates modeling of the cerebral cortex by positioning of principal, pyramidal and interneurons (134). The establishment of the GABAergic system and the migration of GABAergic interneurons are crucial for the development of an inhibitory cortical system that regulates the excitatory processes mediated by glutamatergic pathways (127). A balance between excitation and inhibition is crucial for normal development, and its disruption may produce profound consequences for CNS function and homeostasis (126). GABAergic interneurons are also important for processing of information across cortical domains and are part of the structure of mini-columns, an essential module involved in the physiopathology of cortical dysfunction in autism (35). The potential involvement of the GABAergic system in the pathogenesis of ASD has been suggested by clinical, neuropathological and genetic studies. Elevated levels of GABA in platelets (180) and reduction in the GABAergic receptor system has been documented by studies of brain tissues from patients with autism (16, 17). The location of three genes for subunits of the GABAA receptor, GABRB3, GABRA5 and GABRG3 on the proximal 15q arm (189) prompted genetic studies in ASD that yielded inconsistent results (reviewed by Schmitz (188)). One study that evaluated fourteen GABA receptor subunit genes found an association between GABRA4 and a potential increase in the risk of autism through interaction with GABRB1(131).