Toward a conceptual framework for early brain and behavior development in autism.

This corrects the article DOI: 10.1038/mp.2017.131.

Toward a conceptual framework for early brain and behavior development in autism.

Studies of infant siblings of older autistic probands, who are at elevated risk for autism, have demonstrated that the defining features of autism are not present in the first year of life but emerge late in the first and into the second year. A recent longitudinal neuroimaging study of high-risk siblings revealed a specific pattern of brain development in infants later diagnosed with autism, characterized by cortical surface area hyper-expansion in the first year followed by brain volume overgrowth in the second year that is associated with the emergence of autistic social deficits. Together with new observations from genetically defined autism risk alleles and rodent model, these findings suggest a conceptual framework for the early, post-natal development of autism. This framework postulates that an increase in the proliferation of neural progenitor cells and hyper-expansion of cortical surface area in the first year, occurring during a pre-symptomatic period characterized by disrupted sensorimotor and attentional experience, leads to altered experience-dependent neuronal development and decreased elimination of neuronal processes. This process is linked to brain volume overgrowth and disruption of the refinement of neural circuit connections and is associated with the emergence of autistic social deficits in the second year of life. A better understanding of the timing of developmental brain and behavior mechanisms in autism during infancy, a period which precedes the emergence of the defining features of this disorder, will likely have important implications for designing rational approaches to early intervention.

Cutaneous sensory neurons expressing the Mrgprd receptor sense extracellular ATP and are putative nociceptors.

Sensory neurons expressing the Mrgprd receptor are known to innervate the outermost living layer of the epidermis, the stratum granulosum. The sensory modality that these neurons signal and the stimulus that they respond to are not established, although immunocytochemical data suggest they could be nonpeptidergic nociceptors. Using patch clamp of dissociated mouse dorsal root ganglion (DRG) neurons, the present study demonstrates that Mrgprd+ neurons have several properties typical of nociceptors: long-duration action potentials, TTX-resistant Na(+) current, and Ca(2+) currents that are inhibited by mu opioids. Remarkably, Mrgprd+ neurons respond almost exclusively to extracellular ATP with currents similar to homomeric P2X3 receptors. They show little or no sensitivity to other putative nociceptive agonists, including capsaicin, cinnamaldehyde, menthol, pH 6.0, or glutamate. These properties, together with selective innervation of the stratum granulosum, indicate that Mrgprd+ neurons are nociceptors in the outer epidermis and may respond indirectly to external stimuli by detecting ATP release in the skin.

A diverse family of GPCRs expressed in specific subsets of nociceptive sensory neurons.

In vertebrates, peripheral chemosensory neurons express large families of G protein-coupled receptors (GPCRs), reflecting the diversity and specificity of stimuli they detect. However, somatosensory neurons, which respond to chemical, thermal, or mechanical stimuli, are more broadly tuned. Here we describe a family of approximately 50 GPCRs related to Mas1, called mrgs, a subset of which is expressed in specific subpopulations of sensory neurons that detect painful stimuli. The expression patterns of mrgs thus reveal an unexpected degree of molecular diversity among nociceptive neurons. Some of these receptors can be specifically activated in heterologous cells by RFamide neuropeptides such as NPFF and NPAF, which are analgesic in vivo. Thus, mrgs may regulate nociceptor function and/or development, including the sensation or modulation of pain.

mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop.

We determined that two mouse cryptochrome genes, mCry1 and mCry2, act in the negative limb of the clock feedback loop. In cell lines, mPER proteins (alone or in combination) have modest effects on their cellular location and ability to inhibit CLOCK:BMAL1 -mediated transcription. This suggested cryptochrome involvement in the negative limb of the feedback loop. Indeed, mCry1 and mCry2 RNA levels are reduced in the central and peripheral clocks of Clock/Clock mutant mice. mCRY1 and mCRY2 are nuclear proteins that interact with each of the mPER proteins, translocate each mPER protein from cytoplasm to nucleus, and are rhythmically expressed in the suprachiasmatic circadian clock. Luciferase reporter gene assays show that mCRY1 or mCRY2 alone abrogates CLOCK:BMAL1-E box-mediated transcription. The mPER and mCRY proteins appear to inhibit the transcriptional complex differentially.

Expression of basic helix-loop-helix/PAS genes in the mouse suprachiasmatic nucleus.

The suprachiasmatic nuclei contain a circadian clock that drives rhythmicity in physiology and behavior. In mice, mutation of the Clock gene produces abnormal circadian behavior [Vitaterna M. H. et al. (1994) Science 264, 715-725]. The Clock gene encodes a protein containing basic helix-loop-helix and PAS (PER-ARNT-SIM) domains [King D. P. et al. (1997) Cell 89, 641-653]. The PAS domain may be an important structural feature of a subset of genes involved in photoreception and circadian rhythmicity. The expression and regulation of messenger RNAs encoding eight members of the basic helix-loop-helix/PAS protein superfamily were examined by in situ hybridization. Six of the genes studied (aryl hydrocarbon receptor nuclear transporter, aryl hydrocarbon receptor nuclear transporter-2, Clock, endothelial PAS-containing protein, hypoxia-inducible factor-1alpha and steroid receptor coactivator-1) were expressed in the suprachiasmatic nucleus of adult and neonatal mice. No evidence for rhythmicity of expression was observed when comparing brains collected early in the subjective day (circadian time 3) with those collected early in subjective night (circadian time 15). Neuronal PAS-containing protein-1 messenger RNA was expressed in the suprachiasmatic nucleus of adult (but not neonatal) mice, and a low-amplitude rhythm of neuronal PAS-containing protein-1 gene expression was detected in the suprachiasmatic nucleus. Neuronal PAS-containing protein-2 messenger RNA was not detected in adult or neonatal suprachiasmatic nucleus. Exposure to light at night (30 or 180 min of light, beginning at circadian time 15) did not alter the expression of any of the genes studied. The expression of multiple members of the basic helix-loop-helix/PAS family in the suprachiasmatic nucleus suggests a rich array of potential interactions relevant to the regulation of the suprachiasmatic circadian clock.

A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock.

We examined the transcriptional regulation of the clock-controlled arginine vasopressin gene in the suprachiasmatic nuclei (SCN). A core clock mechanism in mouse SCN appears to involve a transcriptional feedback loop in which CLOCK and BMAL1 are positive regulators and three mPeriod (mPer) genes are involved in negative feedback. We show that the RNA rhythm of each mPer gene is severely blunted in Clock/Clock mice. The vasopressin RNA rhythm is abolished in the SCN of Clock/Clock animals, leading to markedly decreased peptide levels. Luciferase reporter gene assays show that CLOCK-BMAL1 heterodimers act through an E box enhancer in the vasopressin gene to activate transcription; this activation can be inhibited by the mPER and mTIM proteins. These data indicate that the transcriptional machinery of the core clockwork directly regulates a clock-controlled output rhythm.

Discovery of a putative heme-binding protein family (SOUL/HBP) by two-tissue suppression subtractive hybridization and database searches.

In the domestic chicken, Gallus gallus, the retina and pineal gland contain circadian clocks that are directly entrained by environmental light-dark cycles. To identify novel genes that are expressed in the retina and pineal gland, we performed two-tissue suppression subtractive hybridization (SSH). Two-tissue SSH is designed to identify genes expressed in common between two RNA samples while at the same time subtracting out abundant transcripts. Using this method, we identified a novel chicken gene, named ckSoul, that is strongly expressed in the retina and pineal gland. The protein product of ckSoul is similar to a novel heme-binding protein (p22 HBP) and to an uncharacterized mammalian gene in the expressed sequence tag (EST) database. The mouse transcript of this new gene is expressed in the retina and may represent the mammalian ortholog of ckSoul. Molecular analysis of the mammalian and chicken proteins suggests SOUL and HBP are members of a new family of heme-binding proteins.

Molecular analysis of mammalian timeless.

We cloned the mouse cDNA of a mammalian homolog of the Drosophila timeless (tim) gene and designated it mTim. The mTim protein shows five homologous regions with Drosophila TIM. mTim is weakly expressed in the suprachiasmatic nuclei (SCN) but exhibits robust expression in the hypophyseal pars tuberalis (PT). mTim RNA levels do not oscillate in the SCN nor are they acutely altered by light exposure during subjective night. mTim RNA is expressed at low levels in several peripheral tissues, including eyes, and is heavily expressed in spleen and testis. Yeast two-hybrid assays revealed an array of interactions between the various mPER proteins but no mPER-mTIM interactions. The data suggest that PER-PER interactions have replaced the function of PER-TIM dimers in the molecular workings of the mammalian circadian clock.

Three period homologs in mammals: differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain.

We have cloned and characterized the mouse cDNA of a third mammalian homolog of the Drosophila period gene and designated it mPer3. The mPER3 protein shows approximately 37% amino acid identity with mPER1 and mPER2 proteins. The three mammalian PER proteins share several regions of sequence homology, and each contains a protein dimerization PAS domain. mPer3 RNA levels oscillate in the suprachiasmatic nuclei (SCN) and eyes. In the SCN, mPer3 RNA levels are not acutely altered by light exposure at different times during subjective night. This contrasts with the acute induction by light of mPer1 and mPer2 RNA levels during early and late subjective night. mPer3 is widely expressed in tissues outside of brain. In liver, skeletal muscle, and testis, mPer RNAs exhibit prominent, synchronous circadian oscillations. The results highlight the differential light responses among the three mammalian Per genes in the SCN and raise the possibility of circadian oscillators in mammals outside of brain and retina.

Archael phosphoproteins. Identification of a hexosephosphate mutase and the alpha-subunit of succinyl-CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus.

When soluble extracts from the extreme acidophilic archaeon Sulfolobus solfataricus were incubated with [gamma-32P]ATP, several radiolabeled polypeptides were observed following SDS-PAGE. The most prominent of these migrated with apparent molecular masses of 14, 18, 35, 42, 46, 50, and 79 kDa. Phosphoamino acid analysis revealed that all of the proteins contained phosphoserine, with the exception of the 35-kDa one, whose protein-phosphate linkage proved labile to strong acid. The observed pattern of phosphorylation was influenced by the identity of the divalent metal ion cofactor used, Mg2+ versus Mn2+, and the choice of incubation temperature. The 35- and 50-kDa phosphoproteins were purified and their amino-terminal sequences determined. The former polypeptide's amino-terminal sequence closely matched a conserved portion of the alpha-subunit of succinyl-CoA synthetase, which forms an acid-labile phosphohistidyl enzyme intermediate during its catalytic cycle. This identification was confirmed by the ability of succinate or ADP to specifically remove the radiolabel. The 50-kDa polypeptide's sequence contained a heptapeptide motif, Phe/Pro-Gly-Thr-Asp/Ser-Gly-Val/Leu-Arg, found in a similar position in several hexosephosphate mutases. The catalytic mechanism of these mutases involves formation of a phosphoseryl enzyme intermediate. The identity of p50 as a hexosephosphate mutase was confirmed by (1) the ability of sugars and sugar phosphates to induce removal of the labeled phosphoryl group from the protein, and (2) the ability of [32P]glucose 6-phosphate to donate its phosphoryl group to the protein.

Two period homologs: circadian expression and photic regulation in the suprachiasmatic nuclei.

We have characterized a mammalian homolog of the Drosophila period gene and designated it Per2. The PER2 protein shows >40% amino acid identity to the protein of another mammalian per homolog (designated Per1) that was recently cloned and characterized. Both PER1 and PER2 proteins share several regions of homology with the Drosophila PER protein, including the protein dimerization PAS domain. Phylogenetic analysis supports the existence of a family of mammalian per genes. In the mouse, Per1 and Per2 RNA levels exhibit circadian rhythms in the SCN and eyes, sites of circadian clocks. Both Per1 and Per2 RNAs in the SCN are increased by light exposure during subjective night but not during subjective day. The results advance our knowledge of candidate clock elements in mammals.

2D-PAGE analysis: adrenergically regulated pineal protein AIP 37/6 is a phosphorylated isoform of cytosolic malate dehydrogenase.

The adrenergic transmitter norepinephrine (NE) dramatically increases the prominence of only two out of the hundreds of [35S]methionine-labeled pineal proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). One of these regulated proteins is AIP 37/6 (37 kDa, pI approximately 6). The labeling of this protein is increased approximately 100-fold by NE. In the study presented here the identity of AIP 37/6 was investigated. The results of microsequencing, immunochemical analysis of 2D-PAGE blots and size exclusion chromatography indicate that AIP 37/6 is an isoform of cytosolic malate dehydrogenase (cMDH; approximately 36.3 kDa; pI approximately 6.5). Associated studies indicate that this isoform is phosphorylated whereas the bulk of cMDH is not. Cotranslational phosphorylation of cMDH is discussed.