Activity and MeCP2-dependent regulation of nNOS levels in enteric neurons.

BACKGROUND: Rett syndrome (RTT) is a neurological disorder characterized by severe cognitive impairment, motor dyspraxia, and seizures. Rett syndrome arises predominantly from mutations in MECP2, the gene coding for methyl-CpG-binding protein 2 (MeCP2). MeCP2 is an important mediator of synaptic development and is essential in regulating homeostatic synaptic plasticity (HSP) in the brain. In addition to demonstrating central nervous system impairment, RTT patients also suffer from gastrointestinal (GI) dysmotility. We hypothesize that this is due to a similar impairment of plasticity-dependent synaptic function in the enteric nervous system (ENS). We recently reported that MeCP2 is expressed in the ENS, providing evidence that neuronal dysfunction may mediate the GI pathology. METHODS: Baseline measures of MeCP2-KO vs wild-type (WT) GI neuronal nitric oxide synthase (nNOS) were assessed in tissue samples and in vitro. Experiments were carried out to measure nNOS in baseline vs activated plasticity states in vitro. Functional in vivo studies were carried out to determine whether MeCP2-KO mice reproduced the RTT GI hypomotility. KEY RESULTS: Methyl-CpG-binding protein 2-KO mice reproduced the GI hypomotility seen in RTT. MeCP2-KO GI tissue demonstrated elevated nNOS levels. Cultured WT enteric neurons showed upregulation of nNOS following moderate, prolonged stimulation by hyperkalemia; neurons from MeCP2-KO mice failed to show this nNOS upregulation. CONCLUSIONS & INFERENCES: MeCP2 is required for proper GI motility and normal nNOS levels. Neuronal nitric oxide synthase imbalances could mediate the GI dysmotility seen in RTT. Disruption of MeCP2-dependent HSP may be the basis for aberrant nNOS levels and hence GI dysmotility in MeCP2-KO and RTT.

MeCP2 in the enteric nervous system.

BACKGROUND: Rett syndrome (RTT) is an intellectual deficit and movement disorder that develops during early childhood in girls. Affected children are normal until 6-18 months of age, after which symptoms begin to appear. Most cases of RTT are due to mutations in the MeCP2 gene leading to disruption of neuronal communication in the central nervous system. In addition, RTT patients show peripheral ailments such as gastrointestinal (GI), respiratory, and cardiac dysfunction. The etiology of intestinal dysfunction in RTT is not well-understood. Reports on the presence of MeCP2 in the peripheral nervous system are scant. As such we examined the levels of MeCP2 in human and murine GI tissue and assessed MeCP2 expression at various developmental stages. METHODS: Immunohistochemistry for MeCP2, HuC/D, juvenile beta tubulin, and GFAP was performed on human and murine intestine. Western blots of these same tissues were probed with MeCP2, vAChT, nNOS, and beta-actin antibodies. KEY RESULTS: MeCP2 is expressed throughout the GI tract. MeCP2 is expressed specifically in the enteric nervous system of the GI tract. MeCP2 is expressed in the GI tract throughout development with appearance beginning at or before E11.5 in the murine intestine. CONCLUSIONS & INFERENCES: The proof of MeCP2 expression in enteric neurons suggests that the GI dysmotility in Rett may arise from enteric network dysfunction secondary to MeCP2 mutation.

The paralogous Hox genes Hoxa10 and Hoxd10 interact to pattern the mouse hindlimb peripheral nervous system and skeleton.

The most 5' mouse Hoxa and Hoxd genes, which occupy positions 9-13 and which are related to the Drosophila AbdB gene, are all active in patterning developing limbs. Inactivation of individual genes produces alterations in skeletal elements of both forelimb and hindlimb; inactivation of some of these genes also alters hindlimb innervation. Simultaneous inactivation of paralogous or nonparalogous Hoxa and Hoxd genes produces more widespread alterations, suggesting that combinatorial interactions between these genes are required for proper limb patterning. We have examined the effects of simultaneous inactivation of Hoxa10 and Hoxd10 on mouse hindlimb skeletal and nervous system development. These paralogous genes are expressed at lumbar and sacral levels of the developing neural tube and surrounding axial mesoderm as well as in developing forelimb and hindlimb buds. Double-mutant animals demonstrated impaired locomotor behavior and altered development of posterior vertebrae and hindlimb skeletal elements. Alterations in hindlimb innervation were also observed, including truncations and deletions of the tibial and peroneal nerves. Animals carrying fewer mutant alleles show similar, but less extreme phenotypes. These observations suggest that Hoxa10 and Hoxd10 coordinately regulate skeletal development and innervation of the hindlimb.

Using smoothing spline anova to examine the relation of risk factors to the incidence and progression of diabetic retinopathy.

Smoothing spline ANOVA (ANalysis Of VAriance) methods provide a flexible alternative to the standard parametric GLIM (generalized linear models) methods for analysing the relationship of predictor variables to outcomes with data from large epidemiologic studies. These methods allow the visualization of relationships not readily fit by simple GLIM models, and provide for the ability to visualize interactions between the variables. At the same time, they reduce to GLIM models if the data suggest that the added flexibility is unwarranted. Using this method, we investigate risk factors for incidence and progression of diabetic retinopathy in a group of patients with older onset diabetes from the Wisconsin Epidemiological Study of Diabetic Retinopathy. We carry out four analyses to illustrate various properties of this class of methods. Some of the results confirm previous findings with use of standard methods, while others allow the visualization of more complex relationships not evident from the application of parametric methods.

Optimal use of sampled tissue sections for estimating the number of hepatocellular foci.

Statistical techniques were applied to computer-simulated data to evaluate the importance of various factors that may affect the estimation of the number of hepatocellular foci from foci transections. The simulations were modeled after mouse foci for which three-dimensional size distributions and densities were determined from serial section reconstructions. The foci had been induced in male C57BL/6 X C3H F1 mice at 20 and 28 weeks after a single injection, in infancy, of diethylnitrosamine. In an earlier report, we had shown that the number of foci per cu cm could be accurately estimated from profiles using a conditional estimator when the investigator is unable to identify profiles smaller than a certain size (epsilon). In the present study, emphasis was placed on assessing the value of step serial sections in order to make optimal use of the small tissue samples in mouse liver. Since all mathematical estimators of N3 are based on measurement of sampled profiles (n2') and ultimately derive from the fundamental relationship. N3' = N2'/2mu, (where N3' is the number of foci per cu cm, N2' is the number of profiles per sq cm, and mu, is the average adjusted focus radius), the relative importance of N2' and mu, on the conditional estimator was evaluated. This was accomplished by comparing the errors resulting from use of the conditional estimator with those resulting from use of two other estimators. The latter two estimators consisted of a "sampled focus estimator," which used sampled intact foci to estimate N3, and a "reference estimator," which used profiles from foci with a mu, that was known. Additionally, in order to provide a stable variance for the conditional estimator, we adopted a simple smoothing procedure. As expected, none of the estimators showed any significant bias. However, somewhat surprising was the finding that the standard deviations from use of all three estimators were almost identical. Consequently, it appears that the variance resulting from application of the smoothed conditional estimator to large sets of profile data is not due to difficulty in estimating mu epsilon. In these instances, the faulty estimates of N2' resulted almost entirely from the variability in sampling foci from tissue blocks which constituted only about 1/25 of the liver volume. In addition, the ability to reduce the error in estimating the number of foci by increasing the number of profiles was also evaluated. We simulated a sectioning protocol for the 28-week mouse livers in which the distances between the 5-micron-thick step sections in a 1-mm block of liver were progressively decreased from 1000 to 50 micron.(ABSTRACT TRUNCATED AT 400 WORDS)