Transcriptional regulation of the rat Müllerian inhibiting substance type II receptor in rodent Leydig cells

Müllerian inhibiting substance (MIS) causes regression of the fetal Müllerian duct on binding a heteromeric complex of types I and II cell-surface receptors in the fetal urogenital ridge. The MIS type II receptor (MISRII), which provides specificity for MIS, is also expressed in the adult testis, ovary, and uterus. The rat MISRII promoter was cloned to study the molecular mechanisms underlying its temporal and cell-specific expression. The 1.6-kilobase (kb) promoter contained no recognizable TATA or CAAT box, but there was a consensus Sp1 site upstream of the transcription initiation site. Two binding sites for the orphan nuclear receptor steroidogenic factor-1 (SF-1) are occupied in vitro by using nuclear extracts from R2C cells, an MIS-responsive rat Leydig cell line that expresses endogenous MISRII, with differing affinities, indicating that the distal SF-1 site is bound more avidly than is the proximal SF-1 site. R2C cells transfected with MISRII promoter/luciferase reporter constructs show a 12-fold induction with the 1.6-kb fragment and deletion of sequences upstream of −282-bp lowered luciferase expression to one-third. Mutation of both SF-1 sites greatly inhibited luciferase expression, whereas mutation of either site alone resulted in continuing activation by endogenous SF-1, indicating redundancy. In vitro binding and transcriptional analyses suggest that a proximal potential Smad-responsive element and an uncharacterized element also contribute to activation of the MISRII gene. R2C cells and MISRII promoter regulation can now be used to uncover endogenous transcription factors responsible for receptor expression or repression.

An alternative pathway for rod signals in the rodent retina: Rod photoreceptors, cone bipolar cells, and the localization of glutamate receptors

In the mammalian retina, extensive processing of spatiotemporal and chromatic information occurs. One key principle in signal transfer through the retina is parallel processing. Two of these parallel pathways are the ON- and OFF-channels transmitting light and dark signals. This dual system is created in the outer plexiform layer, the first relay station in retinal signal transfer. Photoreceptors release glutamate onto ON- and OFF-type bipolar cells, which are functionally distinguished by their postsynaptic expression of different types of glutamate receptors, namely ionotropic and metabotropic glutamate receptors. In the current concept, rod photoreceptors connect only to rod bipolar cells (ON-type) and cone photoreceptors connect only to cone bipolar cells (ON- and OFF-type). We have studied the distribution of (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunits at the synapses in the outer plexiform layer of the rodent retina by immunoelectron microscopy and serial section reconstruction. We report a non-classical synaptic contact and an alternative pathway for rod signals in the retina. Rod photoreceptors made synaptic contact with putative OFF-cone bipolar cells that expressed the AMPA glutamate receptor subunits GluR1 and GluR2 on their dendrites. Thus, in the retina of mouse and rat, an alternative pathway for rod signals exists, where rod photoreceptors bypass the rod bipolar cell and directly excite OFF-cone bipolar cells through an ionotropic sign-conserving AMPA glutamate receptor.

Homeobox gene Prx3 expression in rodent brain and extraneural tissues

Different cDNA clones encoding a rat homeobox gene and the mouse homologue OG-12 were cloned from adult rat brain and mouse embryo mRNA, respectively. The predicted amino acid sequences of the proteins belong to the paired-related subfamily of homeodomain proteins (Prx homeodomains). Hence, the gene was named Prx3 and the mouse and rat genes are indicated as mPrx3 and rPrx3, respectively. In the mouse as well as in the rat, the predicted Prx3 proteins share the homeodomain but have three different N termini, a 12-aa residue variation in the C terminus, and contain a 14-aa residue motif common to a subset of homeodomain proteins, termed the “aristaless domain.” Genetic mapping of Prx3 in the mouse placed this gene on chromosome 3. In situ hybridization on whole mount 12.5-day-old mouse embryos and sections of rat embryos at 14.5 and 16.5 days postcoitum revealed marked neural expression in discrete regions in the lateral and medial geniculate complex, superior and inferior colliculus, the superficial gray layer of the superior colliculus, pontine reticular formation, and inferior olive. In rat and mouse embryos, nonneuronal structures around the oral cavity and in hip and shoulder regions also expressed the Prx3 gene. In the adult rat brain, Prx3 gene expression was restricted to thalamic, tectal, and brainstem structures that include relay nuclei of the visual and auditory systems as well as other ascending systems conveying somatosensory information. Prx3 may have a role in specifying neural systems involved in processing somatosensory information, as well as in face and body structure formation.

Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.

Neonatal lead exposure impairs development of rodent barrel field cortex

Childhood exposure to low-level lead can permanently reduce intelligence, but the neurobiologic mechanism for this effect is unknown. We examined the impact of lead exposure on the development of cortical columns, using the rodent barrel field as a model. In all areas of mammalian neocortex, cortical columns constitute a fundamental structural unit subserving information processing. Barrel field cortex contains columnar processing units with distinct clusters of layer IV neurons that receive sensory input from individual whiskers. In this study, rat pups were exposed to 0, 0.2, 1, 1.5, or 2 g/liter lead acetate in their dam's drinking water from birth through postnatal day 10. This treatment, which coincides with the development of segregated columns in the barrel field, produced blood lead concentrations from 1 to 31 μg/dl. On postnatal day 10, the area of the barrel field and of individual barrels was measured. A dose-related reduction in barrel field area was observed (Pearson correlation = −0.740; P < 0.001); mean barrel field area in the highest exposure group was decreased 12% versus controls. Individual barrels in the physiologically more active caudoventral group were affected preferentially. Total cortical area measured in the same sections was not altered significantly by lead exposure. These data support the hypothesis that lead exposure may impair the development of columnar processing units in immature neocortex. We demonstrate that low levels of blood lead, in the range seen in many impoverished inner-city children, cause structural alterations in a neocortical somatosensory map.

Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: Relevance to diseases of the nervous system

Many proteins contain reiterated glutamine residues, but polyglutamine of excessive length may result in human disease by conferring new properties on the protein containing it. One established property of a glutamine residue, depending on the nature of the flanking residues, is its ability to act as an amine acceptor in a transglutaminase-catalyzed reaction and to make a glutamyl–lysine cross-link with a neighboring polypeptide. To learn whether glutamine repeats can act as amine acceptors, we have made peptides with variable lengths of polyglutamine flanked by the adjacent amino acid residues in the proteins associated with spinocerebellar ataxia type 1 (SCA1), Machado–Joseph disease (SCA3), or dentato-rubral pallido-luysian atrophy (DRPLA) or those residues adjacent to the preferred cross-linking site of involucrin, or solely by arginine residues. The polyglutamine was found to confer excellent substrate properties on any soluble peptide; under optimal conditions, virtually all the glutamine residues acted as amine acceptors in the reaction with glycine ethyl-ester, and lengthening the sequence of polyglutamine increased the reactivity of each glutamine residue. In the presence of transglutaminase, peptides containing polyglutamine formed insoluble aggregates with the proteins of brain extracts and these aggregates contained glutamyl–lysine cross-links. Repeated glutamine residues exposed on the surface of a neuronal protein should form cross-linked aggregates in the presence of any transglutaminase activated by the presence of Ca2+.

Estrogen receptors α and β in the rodent mammary gland

An obligatory role for estrogen in growth, development, and functions of the mammary gland is well established, but the roles of the two estrogen receptors remain unclear. With the use of specific antibodies, it was found that both estrogen receptors, ERα and ERβ, are expressed in the rat mammary gland but the presence and cellular distribution of the two receptors are distinct. In prepubertal rats, ERα was detected in 40% of the epithelial cell nuclei. This decreased to 30% at puberty and continued to decrease throughout pregnancy to a low of 5% at day 14. During lactation there was a large induction of ERα with up to 70% of the nuclei positive at day 21. Approximately 60–70% of epithelial cells expressed ERβ at all stages of breast development. Cells coexpressing ERα and ERβ were rare during pregnancy, a proliferative phase, but they represented up to 60% of the epithelial cells during lactation, a postproliferative phase. Western blot analysis and sucrose gradient centrifugation confirmed this pattern of expression. During pregnancy, the proliferating cell nuclear antigen was not expressed in ERα-positive cells but was observed in 3–7% of ERβ-containing cells. Because more than 90% of ERβ-bearing cells do not proliferate, and 55–70% of the dividing cells have neither ERα nor ERβ, it is clear that the presence of these receptors in epithelial cells is not a prerequisite for estrogen-mediated proliferation.

Growth factor-mediated Fyn signaling regulates α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression in rodent neocortical neurons

Src-family protein tyrosine kinases (PTKs) transduce signals to regulate neuronal development and synaptic plasticity. However, the nature of their activators and molecular mechanisms underlying these neural processes are unknown. Here, we show that brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor enhance expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor 1 and 2/3 proteins in rodent neocortical neurons via the Src-family PTK(s). The increase in AMPA receptor levels was blocked in cultured neocortical neurons by addition of a Src-family-selective PTK inhibitor. Accordingly, neocortical cultures from Fyn-knockout mice failed to respond to BDNF whereas those from wild-type mice responded. Moreover, the neocortex of young Fyn mutants exhibited a significant in vivo reduction in these AMPA receptor proteins but not in their mRNA levels. In vitro kinase assay revealed that BDNF can indeed activate the Fyn kinase: It enhanced tyrosine phosphorylation of Fyn as well as that of enolase supplemented exogenously. All of these results suggest that the Src-family kinase Fyn, activated by the growth factors, plays a crucial role in modulating AMPA receptor expression during brain development.

Two different neurodegenerative diseases caused by proteins with similar structures

The downstream prion-like protein (doppel, or Dpl) is a paralog of the cellular prion protein, PrPC. The two proteins have ≈25% sequence identity, but seem to have distinct physiologic roles. Unlike PrPC, Dpl does not support prion replication; instead, overexpression of Dpl in the brain seems to cause a completely different neurodegenerative disease. We report the solution structure of a fragment of recombinant mouse Dpl (residues 26–157) containing a globular domain with three helices and a small amount of β-structure. Overall, the topology of Dpl is very similar to that of PrPC. Significant differences include a marked kink in one of the helices in Dpl, and a different orientation of the two short β-strands. Although the two proteins most likely arose through duplication of a single ancestral gene, the relationship is now so distant that only the structures retain similarity; the functions have diversified along with the sequence.

An N-methyl-d-aspartate receptor channel blocker with neuroprotective activity

Excitotoxicity, resulting from sustained activation of glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype, is considered to play a causative role in the etiology of ischemic stroke and several neurodegenerative diseases. The NMDA receptor is therefore a target for the development of neuroprotective agents. Here, we identify an N-benzylated triamine (denoted as NBTA) as a highly selective and potent NMDA-receptor channel blocker selected by screening a reduced dipeptidomimetic synthetic combinatorial library. NBTA blocks recombinant NMDA receptors expressed in Xenopus laevis oocytes with a mean IC50 of 80 nM; in contrast, it does not block GluR1, a glutamate receptor of the non-NMDA subtype. The blocking activity of NBTA on NMDA receptors exhibits the characteristics of an open-channel blocker: (i) no competition with agonists, (ii) voltage dependence, and (iii) use dependence. Significantly, NBTA protects rodent hippocampal neurons from NMDA receptor, but not kainate receptor-mediated excitotoxic cell death, in agreement with its selective action on the corresponding recombinant receptors. Mutagenesis data indicate that the N site, a key asparagine on the M2 transmembrane segment of the NR1 subunit, is the main determinant of the blocker action. The results highlight the potential of this compound as a neuroprotectant.

A role for intermolecular disulfide bonds in prion diseases?

The key event in prion diseases seems to be the conversion of the prion protein PrP from its normal cellular isoform (PrPC) to an aberrant “scrapie” isoform (PrPSc). Earlier studies have detected no covalent modification in the scrapie isoform and have concluded that the PrPC → PrPSc conversion is a purely conformational transition involving no chemical reactions. However, a reexamination of the available biochemical data suggests that the PrPC → PrPSc conversion also involves a covalent reaction of the (sole) intramolecular disulfide bond of PrPC. Specifically, the data are consistent with the hypothesis that infectious prions are composed of PrPSc polymers linked by intermolecular disulfide bonds. Thus, the PrPC → PrPSc conversion may involve not only a conformational transition but also a thiol/disulfide exchange reaction between the terminal thiolate of such a PrPSc polymer and the disulfide bond of a PrPC monomer. This hypothesis seems to account for several unusual features of prion diseases.