Dona L. Wong

A Genomic Screen of Autism: Evidence for a Multilocus Etiology

We have conducted a genome screen of autism, by linkage analysis in an initial set of 90 multiplex sibships, with parents, containing 97 independent affected sib pairs (ASPs), with follow-up in 49 additional multiplex sibships, containing 50 ASPs. In total, 519 markers were genotyped, including 362 for the initial screen, and an additional 157 were genotyped in the follow-up. As a control, we also included in the analysis unaffected sibs, which provided 51 discordant sib pairs (DSPs) for the initial screen and 29 for the follow-up. In the initial phase of the work, we observed increased identity by descent (IBD) in the ASPs (sharing of 51.6%) compared with the DSPs (sharing of 50.8%). The excess sharing in the ASPs could not be attributed to the effect of a small number of loci but, rather, was due to the modest increase in the entire distribution of IBD. These results are most compatible with a model specifying a large number of loci (perhaps >/=15) and are less compatible with models specifying </=10 loci. The largest LOD score obtained in the initial scan was for a marker on chromosome 1p; this region also showed positive sharing in the replication family set, giving a maximum multipoint LOD score of 2.15 for both sets combined. Thus, there may exist a gene of moderate effect in this region. We had only modestly positive or negative linkage evidence in candidate regions identified in other studies. Our results suggest that positional cloning of susceptibility loci by linkage analysis may be a formidable task and that other approaches may be necessary.

Co-Localization of substance P- and phenylethanolamine-N-methyltransferase-like immunoreactivity in neurons of ventrolateral medulla that project to the spinal cord: Potential role in control of vasomotor tone

Both substance P (SP)- and epinephrine-containing neurons in the rostral ventrolateral medulla have been thought to play a role in regulating vasomotor tone. The combination of retrograde transport of a fluorescent dye (Fast Blue) and immunofluorescent staining for SP- and phenylethanolamine-N-methyltransferase (PNMT)-immunoreactivity was used to determine the relationships of these two groups of ventrolateral medullary neurons which project to the spinal cord. The majority of spinally projecting neurons in the rostral ventrolateral medulla contain both PNMT-like and SP-like immunoreactivity. The presence of PNMT-immunoreactive material in a neuron implies that epinephrine is a probable neurotransmitter for such a cell. Earlier work demonstrated that epinephrine and SP have opposite effects on the firing of sympathetic preganglionic neurons. Our results raise the possibility of a novel mechanism of synaptic regulation of the sympathetic preganglionic vasomotor neurons.

ADRENERGIC RESPONSES TO STRESS: TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL CHANGES

Stress effects on adrenergic responses in rats were examined in adrenal medulla, the primary source of circulating epinephrine (Epi). Irrespective of duration, immobilization (IMMO) increased adrenal corticosterone to the same extent. In contrast, Epi changed little, suggesting that Epi synthesis replenishes adrenal pools and sustains circulating levels for the heightened alertness and physiological changes required of the “flight or fight” response. IMMO also induced the Epi‐synthesizing enzyme, phenylethanolamine N‐methyltransferase (PNMT). The rise in its mRNA and protein was preceded by increases in Egr‐1 and Sp1 mRNA, protein, and protein‐DNA binding complex formation. With repeated and prolonged stress, PNMT protein did not reflect the magnitude of change in mRNA. The latter suggests that post‐transcriptional, in addition to transcriptional mechanisms, regulate PNMT responses to stress. To further reveal molecular mechanisms underlying stress‐induced changes in adrenergic function, the effects of hypoxia on PNMT promoter‐driven gene expression are being examined in adrenal medulla‐derived PC12 cells. Hypoxia activates the PNMT promoter to increase PNMT promoter‐driven luciferase reporter gene expression and endogenous PNMT in PC12 cells. Induction of both appear mediated via activation of multiple signaling pathways and downstream activation of hypoxia inducible factor and PNMT transcriptional activators, Egr‐1 and Sp1. Hypoxia generates both partially and fully processed forms of PNMT mRNA. The former reportedly is translated into a truncated, nonfunctional protein, and the latter into enzymatically active PNMT. Together, findings suggest that stress increases PNMT gene transcriptional activity but post‐transcriptional regulatory mechanisms limit the biological end‐point of functional PNMT enzyme and, thereby, Epi.

Phenylethanolamine N-methyltransferase gene expression: synergistic activation by Egr-1, AP-2 and the glucocorticoid receptor

The gene encoding the epinephrine synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT), is transcriptionally activated by Egr-1, AP-2, and the glucocorticoid receptor (GR). Stimulation by AP-2 requires its synergistic interaction with an activated GR. The present studies show that the GR also cooperates with Egr-1 or the combination of Egr-1 and AP-2 to activate the PNMT promoter. Together Egr-1, AP-2, and the GR can induce PNMT promoter-mediated luciferase reporter gene expression beyond the sum of their independent contributions as well as synergistically activate the endogenous PNMT gene leading to marked increases in PNMT mRNA. Examination of the effects of mutation of the AP-2 or Egr-1 binding sites on PNMT promoter activation by DEX and the factor binding to the remaining intact site or by all three transcriptional activators showed changes in luciferase reporter gene expression which suggest that DNA structure may be altered thereby reducing or enhancing synergistic activation. It also appears that the -165 bp Egr-1 site may not be critical for the synergism observed between Egr-1, AP-2 and the GR. When the glucocorticoid response element (GRE) within the PNMT promoter was mutated, PNMT promoter activation by Egr-1 and DEX, AP-2 and DEX or all three showed both inhibition and enhancement, even when the GRE was completely eliminated. These observations indicate that induction of PNMT gene transcription may occur either through GR interaction with other transcriptional proteins after binding to its cognate GRE or through direct protein-protein interaction in the absence of GRE binding. While the mechanisms by which Egr-1 and the GR and Egr-1, AP-2 and the GR function cooperatively to stimulate PNMT promoter activity remain to be elucidated, this synergistic stimulation of the PNMT promoter by these factors may provide important in vivo and in vitro regulatory control of the PNMT gene.

Glucocorticoid regulation of phenylethanolamine N-methyltransferase in vivo.

In vivo, supraphysiological doses of glucocorticoids are required to restore adrenal medullary phenylethanolamine N‐methyltransferase (PNMT, E.C. 2.1.1.28) activity after hypophysectomy. However, in vitro, phenylethanolamine N‐methyltransferase gene expression appears normally glucocorticoid‐responsive. To explore this paradox, rats were given dexamethasone or the type II‐specific glucocorticoid RU28362 (1–1000 μg/day), and adrenal phenylethanolamine N‐methyltransferase activity and mRNA levels were determined. At low doses (1–30 μg/day), neither steroid altered mRNA whereas at higher doses (100–1000 μg/day), mRNA rose 10‐ to 20‐fold, with dexamethasone ~ 3 times as potent as RU28362. In contrast, enzyme activity fell with low doses of either steroid, consistent with suppression of ACTH and endogenous steroidogenesis. At higher doses of RU28362, enzyme activity remained low and unchanged despite increased mRNA expression, whereas higher doses of dexamethasone progressively restored the enzyme to normal. These findings suggest 1) that glucocorticoid regulation of phenylethanolamine N‐methyltransferase activity occurs largely independent of gene expression; 2) that glucocorticoid effects on enzyme activity are primarily indirect, probably through cosubstrate regulation and/or enzyme stabilization; and 3) that these effects are not mediated via a classical (type II) glucocorticoid receptor mechanism, given the high doses of dexamethasone and corticosterone required and the inability of RU28362 to mimic the effects of these less selective steroids.— Wong, D. L., Lesage, A., Siddall, B., Funder, J. W. Glucocorticoid regulation of phenylethanolamine N‐methyltransferase in vivo. FASEB J. 6: 3310‐3315; 1992.

Epinephrine: A Short- and Long-Term Regulator of Stress and Development of Illness

Epinephrine (Epi), which initiates short-term responses to cope with stress, is, in part, stress-regulated via genetic control of its biosynthetic enzyme, phenylethanolamine N-methyltransferase (PNMT). In rats, immobilization (IMMO) stress activates the PNMT gene in the adrenal medulla via Egr-1 and Sp1 induction. Yet, elevated Epi induced by acute and chronic stress is associated with stress induced, chronic illnesses of cardiovascular, immune, cancerous, and behavioral etiologies. Major sources of Epi include the adrenal medulla and brainstem. Although catecholamines do not cross the blood–brain barrier, circulating Epi from the adrenal medulla may communicate with the central nervous system and stress circuitry by activating vagal nerve β-adrenergic receptors to release norepinephrine, which could then stimulate release of the same from the nucleus tractus solitarius and locus coeruleus. In turn, the basal lateral amygdala (BLA) may activate to stimulate afferents to the hypothalamus, neocortex, hippocampus, caudate nucleus, and other brain regions sequentially. Recently, we have shown that repeated IMMO or force swim stress may evoke stress resiliency, as suggested by changes in expression and extinction of fear memory in the fear-potentiated startle paradigm. However, concomitant adrenergic changes seem stressor dependent. Present studies aim to identify stressful conditions that elicit stress resiliency versus stress sensitivity, with the goal of developing a model to investigate the potential role of Epi in stress-associated illness. If chronic Epi over expression does elicit illness, possibilities for alternative therapeutics exist through regulating stress-induced Epi expression, adrenergic receptor function and/or corticosteroid effects on Epi, adrenergic receptors and the stress axis.

Phenylethanolamine N-methyltransferase gene expression. Sp1 and MAZ potential for tissue-specific expression.

PhenylethanolamineN-methyltransferase (PNMT) promoter-luciferase reporter gene constructs (pGL3RP863, pGL3RP444, and pGL3RP392) transfected into COS1, RS1, PC12, NIH/3T3, or Neuro2A cells showed the highest basal luciferase activity in the Neuro2A cells. DNase I footprinting with Neuro2A cell nuclear extract identified protected PNMT promoter regions spanning the –168/–165 and –48/–45 base pair Sp1/Egr-1 binding sites. Gel mobility shift assays and transient transfection assays using site-directed mutant PNMT promoter-luciferase reporter gene constructs indicated that the elevated basal luciferase activity in the Neuro2A cells was mediated by Sp-1. Furthermore, activation of the PNMT promoter by Sp1 depends on both its binding affinity for its cognate target sequences and its intracellular concentrations. When Sp1 levels were increased through an expression plasmid, luciferase reporter gene expression rose well beyond basal wild-type levels, even with either Sp1 binding element mutated. Finally, another transcription factor expressed in the Neuro2A cells competes with Sp1 by interacting with DNA sequences 3′ to the –48 base pair Sp1 site to prevent Sp1 binding and induction of the PNMT promoter. The DNA consensus sequence, Southwestern analysis, and gel mobility shift assays with antibodies identify MAZ as the competitive factor. These findings suggest that Sp1 may potentially contribute to the tissue-specific expression of the PNMT gene, with the competition between Sp1 and MAZ conferring additional tissue-specific control.

Glucocorticoid Effects on Mesotelencephalic Dopamine Neurotransmission

Multiple neurochemical estimates were used to examine peripheral corticosterone (CORT) effects in dopaminergic terminal regions. Acute CORT administration, which elevated plasma CORT (5 h), slightly decreased dihydroxyphenylacetic acid (DOPAC) to dopamine (DA) ratios in the striatum but not in other regions examined. Two weeks of adrenalectomy (ADX) increased both medial prefrontal cortex DOPAC/DA and homovanillic acid (HVA)/DA and striatal HVA/DA. A reciprocal pattern of changes was observed with CORT replacement in ADX animals. In contrast, CORT replacement in ADX animals did not significantly influence tyrosine hydroxylase content, basal dihydroxyphenylalanine (DOPA) accumulation after NSD 1015 treatment or the decline in DA after alpha-methyl-para-tyrosine, suggesting that neither DA neuronal activity nor release are altered by CORT. Moreover, neither gamma-hydroxybutyric acid lactone-induced increases in DOPA accumulation or stress-induced increases in DA utilization were influenced by CORT replacement, indicating that neither autoreceptor regulation of DA synthesis nor acute stress regulation of DA utilization are changed by CORT. The findings are most consistent with direct inhibition of basal DA metabolism in the medial prefrontal cortex and striatum. The possible physiological and behavioral significance of this inhibition is being further explored.

Glucocorticoid‐Dependent Action of Neural Crest Factor AP‐2: Stimulation of Phenylethanolamine N‐Methyltransferase Gene Expression

Abstract: AP‐2 is a vertebrate transcription factor expressed in neural crest cells and their derivative tissues, including the adrenal medulla, where epinephrine is produced. AP‐2 is shown to stimulate expression of the gene encoding the epinephrine biosynthetic enzyme phenylethanolamine N‐methyltransferase (PNMT). However, stimulation of the PNMT gene by AP‐2 requires glucocorticoids and appears to be mediated through the interaction of AP‐2 with activated type II glucocorticoid receptors. Mutation of AP‐2 and/or glucocorticoid receptor binding elements within the PNMT promoter disrupts the ability of AP‐2 and glucocorticoids to induce PNMT promoter activity. These findings suggest, in the case of PNMT, that AP‐2 stimulates gene expression through a novel glucocorticoid‐dependent mechanism.

Stress-induced changes in epinephrine expression in the adrenal medulla in vivo

Immobilization (IMMO) stress was used to examine how stress alters the stress hormone epinephrine (EPI) in the adrenal medulla in vivo. In rats subjected to IMMO for 30 or 120 min, adrenal corticosterone increased to the same extent. In contrast, EPI changed very little, suggesting that EPI synthesis replenishes adrenal pools and sustains circulating levels for the heightened alertness and physiological responses of the ‘flight or fight’ response. In part, stress activates EPI via the phenylethanolamine N‐methyltransferase (PNMT) gene as single or repeated IMMO elevated PNMT mRNA. The rise in PNMT mRNA was preceded by induction of the PNMT gene activator, Egr‐1, with increases in Egr‐1 mRNA, protein, and protein–DNA binding complex apparent. IMMO also evoked changes in Sp1 mRNA, protein, and Sp1–DNA complex formation, although for chronic IMMO changes were not entirely coincident. In contrast, glucocorticoid receptor and AP‐2 mRNA, protein, and protein–DNA complex were unaltered. Finally, IMMO stress elevated PNMT protein. However, with seven daily IMMOs for 120 min and delayed killing, protein stimulation did not attain the highly elevated levels expected based on mRNA changes. The latter may perhaps suggest initiation of adrenergic desensitization to prolonged and repeated IMMO stress and/or dissociation of transcriptional and post‐transcriptional regulatory mechanisms.