Faten El-Sabeawy

MHCI negatively regulates synapse density during the establishment of cortical connections

Major histocompatibility complex class I (MHCI) molecules modulate activity-dependent refinement and plasticity. We found that MHCI also negatively regulates the density and function of cortical synapses during their initial establishment both in vitro and in vivo. MHCI molecules are expressed on cortical neurons before and during synaptogenesis. In vitro, decreasing surface MHCI (sMHCI) on neurons increased glutamatergic and GABAergic synapse density, whereas overexpression decreased it. In vivo, synapse density was higher throughout development in β2m−/− mice. MHCI also negatively regulated the strength of excitatory, but not inhibitory, synapses and controlled the balance of excitation and inhibition onto cortical neurons. sMHCI levels were modulated by activity and were necessary for activity to negatively regulate glutamatergic synapse density. Finally, acute changes in sMHCI and activity altered synapse density exclusively during early postnatal development. These results identify a previously unknown function for immune proteins in the negative regulation of the initial establishment and function of cortical connections.

Neuroligin1: a cell adhesion molecule that recruits PSD-95 and NMDA receptors by distinct mechanisms during synaptogenesis

BackgroundThe cell adhesion molecule pair neuroligin1 (Nlg1) and β-neurexin (β-NRX) is a powerful inducer of postsynaptic differentiation of glutamatergic synapses in vitro. Because Nlg1 induces accumulation of two essential components of the postsynaptic density (PSD) – PSD-95 and NMDA receptors (NMDARs) – and can physically bind PSD-95 and NMDARs at mature synapses, it has been proposed that Nlg1 recruits NMDARs to synapses through its interaction with PSD-95. However, PSD-95 and NMDARs are recruited to nascent synapses independently and it is not known if Nlg1 accumulates at synapses before these PSD proteins. Here, we investigate how a single type of cell adhesion molecule can recruit multiple types of synaptic proteins to new synapses with distinct mechanisms and time courses.ResultsNlg1 was present in young cortical neurons in two distinct pools before synaptogenesis, diffuse and clustered. Time-lapse imaging revealed that the diffuse Nlg1 aggregated at, and the clustered Nlg1 moved to, sites of axodendritic contact with a rapid time course. Using a patching assay that artificially induced clusters of Nlg, the time course and mechanisms of recruitment of PSD-95 and NMDARs to those Nlg clusters were characterized. Patching Nlg induced clustering of PSD-95 via a slow palmitoylation-dependent step. In contrast, NMDARs directly associated with clusters of Nlg1 during trafficking. Nlg1 and NMDARs were highly colocalized in dendrites before synaptogenesis and they became enriched with a similar time course at synapses with age. Patching of Nlg1 dramatically decreased the mobility of NMDAR transport packets. Finally, Nlg1 was biochemically associated with NMDAR transport packets, presumably through binding of NMDARs to MAGUK proteins that, in turn, bind Nlg1. This interaction was essential for colocalization and co-transport of Nlg1 with NMDARs.ConclusionOur results suggest that axodendritic contact leads to rapid accumulation of Nlg1, recruitment of NMDARs co-transported with Nlg1 soon thereafter, followed by a slower, independent recruitment of PSD-95 to those nascent synapses.

The dynamic distribution of TrkB receptors before, during, and after synapse formation between cortical neurons

Although brain-derived neurotrophic factor (BDNF) potently regulates neuronal connectivity in the developing CNS, the mechanism by which BDNF influences the formation and/or maintenance of glutamatergic synapses remains unknown. Details about the subcellular localization of the BDNF receptor, TrkB, relative to synaptic and nonsynaptic proteins on excitatory neurons should provide insight into how BDNF might exert its effects during synapse formation. Here, we investigated the subcellular localization of tyrosine kinase receptor B (TrkB) relative to synaptic vesicle-associated proteins and NMDA receptors using immunocytochemistry, confocal microscopy, and time-lapse imaging in dissociated cultures of cortical neurons before, during, and after the peak of synapse formation. We find that TrkB is present in puncta on the surface and intracellularly in both dendrites and axons throughout development. Before synapse formation, some TrkB puncta in dendrites colocalize with NMDA receptors, and almost all TrkB puncta in axons colocalize with synaptic vesicle proteins. Clusters of TrkB fused to the enhanced green fluorescent protein (TrkB-EGFP) are highly mobile in both axons and dendrites. In axons, TrkB-EGFP dynamics are almost identical to vesicle-associated protein (VAMP2-EGFP), and these proteins are often transported together. Finally, surface TrkB is found in structures that actively participate in synapse formation: axonal growth cones and dendritic filopodia. Over time, surface TrkB becomes enriched at glutamatergic synapses, which contain both catalytic and truncated TrkB. These results suggest that TrkB is in the right place at the right time to play a direct role in the formation of glutamatergic synapses between cortical neurons.

MHC class I molecules are present both pre- and postsynaptically in the visual cortex during postnatal development and in adulthood

Immune molecules have been discovered recently to play critical roles in the development, function, and plasticity of the cerebral cortex. MHC class I (MHCI) molecules are expressed in the central nervous system and regulate activity-dependent refinement of visual projections during late postnatal development. They have also been implicated in neurodevelopmental diseases such as schizophrenia and autism. Despite the excitement generated by these unique roles for immune proteins in the brain, little is known about how these molecules regulate cortical connections. The first step toward elucidating the mechanism is to identify the spatial and temporal distribution of MHCI proteins throughout development. Using a pan-specific antibody that recognizes many MHCI variants for biochemistry and immunohistochemistry, we found that MHCI proteins are expressed in the rat visual cortex at all ages examined—during the peak of synaptogenesis, the critical period of synaptic refinement, and adulthood. Their abundance in the cortex peaked during early postnatal development, declining during periods of plasticity and adulthood. In contrast to current assumptions, pre- and postembedding immunogold electron microscopy (EM) revealed that MHCI proteins were present both pre- and postsynaptically at all ages examined. They were often found in the postsynaptic density and were closely associated with synaptic vesicles in the presynaptic terminal. These results suggest a previously undescribed model in which MHCI molecules function on both sides of the synapse to regulate connectivity in the mammalian visual cortex before, during, and after the establishment of connections.

Mechanisms of Gender-specific TCDD-induced Toxicity in Guinea Pig Adipose Tissue

After treatment with TCDD, the activities of cytosolic AhR-associated c-Src kinase, microsomal protein kinase C (nPKC epsilon), microsomal c-Src kinase, nuclear p44/42 MAPK, c-Jun N terminus kinase, and the amount of microsomal pan-Ras protein were different in males and females. TCDD did not decrease body or adipose tissue weights in transgenic src-deficient male mice as compared to their wild-type littermates, and the activity of AhR-associated c-Src kinase was not increased by TCDD in src-deficient male mice. Similar results were obtained when TCDD was given to male guinea pigs treated with the Src-kinase inhibitor, geldanamycin. Treatment with estradiol protected male guinea pigs from TCDD-induced wasting. TCDD induced similar changes in protein tyrosine kinase activity in adipose tissues of castrated male and intact female guinea pigs. The gender-specific mechanisms of TCDD-induced toxicity appear to involve c-Src kinase, nPKC epsilon, and pan-Ras, as well as overlap in the cytosolic signal transduction pathways of TCDD and sex steroids.

Biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in immature male and female chickens.

It has been reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced body wasting in mammals is associated with decreased adipose tissue lipoprotein lipase (LPL) and glucose transporting (GT) activity with differential sensitivity between genders. This study extends those findings to chickens as an avian model. A significant decrease in body weight gain was demonstrated in immature male and female chickens 10 days after treatment with a single intraperitoneal (i.p.) dose of 10 and 100 microg TCDD/kg. Body weight gain decrease was associated with hepatomegaly and induction of hepatic CYP1A enzymes in both genders. The increase in liver/body weight ratio (48%) and the decreased LPL activity (28%) were significant only in females at 10 microg TCDD/kg. However, the increase in liver/body weight ratio (31%) and the decrease in LPL activity (26%) were significantly demonstrated in males at 100 microg TCDD/kg. Levels of GT were significantly decreased in females (46%) and in males (48%) following treatment with 10 microg TCDD/kg and 100 microg TCDD/kg, respectively. Therefore, in chickens, as in mammals, the TCDD-induced body wasting is accompanied with decreased LPL activity and decreased GT activity and the magnitude of these changes is gender dependent. In contrast to mammals, this study suggests that female chickens are equally, if not more responsive to TCDD toxicity than males.

Interruption of Estradiol Signal Transduction by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Through Disruption of the Protein Phosphorylation Pathway in Adipose Tissues from Immature and Mature Female Rats

At doses of 10-115 microg/kg, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased body and adipose tissue weights of mature female rats. Doses below 10 microg TCDD/kg decreased body and adipose tissue weights of immature, but not mature females. Doses of 2 and 10 microg TCDD/kg decreased adipose tissue epidermal growth factor receptor (EGFR) binding activity 5 and 7 days later in immature and mature females, respectively. At these times, there was a decrease in the activities of tyrosine kinase (TK), mitogen-activated protein kinase (MAP2K), and protein kinase A (PKA). In mature females, estradiol (E2, 15 microg/kg) increased TK and PKA activities and decreased MAP2K activity. In immature females, E2 decreased TK and PKA activities but not MAP2K activity. TCDD abolished the stimulatory effect of E2 on TK and PKA in mature females, and in immature females TCDD potentiated the negative effect of E2 on all three kinases. TCDD decreased binding of [3H]E2 to cytosolic and nuclear estrogen receptors (ERs) of mature and immature females, and antagonized the stimulatory effect of E2 on ER binding activity. E2 increased DNA binding activity of the estrogen response element (ERE) and activator protein-1, and TCDD antagonized this effect. Geldanamycin, an inhibitor of Src tyrosine kinase, reduced the effects of TCDD on body and adipose tissue weights. Geldanamycin antagonized the effects of TCDD on EGFR binding activity and TK activity. In cell-free preparations, TCDD antagonized E2 action on TK activity in mature females, as well as E2 action on PKA activity in immature females. We hypothesize that TCDD antagonizes E2 action in female adipose tissues through disruption of common cytosolic signal transduction pathways.

Interaction of estrogen and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in immature male chickens (Gallus domesticus)

The interaction of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and estrogen was studied in chickens to more clearly define this relationship in an avian species and its role in the enhanced sensitivity of female chickens to TCDD-induced wasting syndrome. Twenty male chickens (7-9 weeks old) were divided evenly into four groups: control (CTL, received the same volume of vehicle); estrogen-treated (E2, 1 mg/kg estradiol cypionate injections on days 1, 2 and 3); TCDD-treated (TCDD, single 50 microg/kg injection on day 4); and estrogen plus TCDD (E2+TCDD, as above), with measurements taken on day 14. The E2 group compared with the CTL group had decreased comb height (24%), comb length (26%) and adipose tissue (AT) lipoprotein lipase (LPL) activity relative to AT mass (51%), while liver mass and body weight gain were each increased by 28%. The TCDD group had increased liver mass (62%), reduced comb length (17%), and reduced AT LPL activity indexed to AT mass (70%) compared with the CTL group. Finally, the E2+TCDD group had 37% lower body weight gain and 30% larger livers relative to body mass compared with the E2 group, but were not significantly different from the TCDD group. These data show that TCDD antagonized several effects of exogenous estrogen in male chickens, while estrogen enhanced TCDD toxicity in a tissue-specific manner.