Robert A. Lazzarini

CNS Myelin and Sertoli Cell Tight Junction Strands Are Absent in Osp/Claudin-11 Null Mice

Oligodendrocyte-specific protein (OSP)/claudin-11 is a recently identified transmembrane protein found in CNS myelin and testis with unknown function. Herein we demonstrate that Osp null mice exhibit both neurological and reproductive deficits: CNS nerve conduction is slowed, hindlimb weakness is conspicuous, and males are sterile. Freeze fracture reveals that tight junction intramembranous strands are absent in CNS myelin and between Sertoli cells of mutant mice. Our results demonstrate that OSP is the mediator of parallel-array tight junction strands and distinguishes this protein from other intrinsic membrane proteins in tight junctions. These novel results provide direct evidence of the pivotal role of the claudin family in generating the paracellular physical barrier of tight junctions necessary for spermatogenesis and normal CNS function.

The glial cells missing-1 protein is essential for branching morphogenesis in the chorioallantoic placenta

Trophoblast cells of the placenta are established at the blastocyst stage and differentiate into specialized subtypes after implantation. In mice, the outer layer of the placenta consists of trophoblast giant cells that invade the uterus and promote maternal blood flow to the implantation site by producing cytokines with angiogenic and vasodilatory actions. The innermost layer, called the labyrinth, consists of branched villi that provide a large surface area for nutrient transport and are composed of trophoblast cells and underlying mesodermal cells derived from the allantois. The chorioallantoic villi develop after embryonic day (E) 8.5 through extensive folding and branching of an initially flat sheet of trophoblast cells, the chorionic plate, in response to contact with the allantois. We show here that Gcm1, encoding the transcription factor glial cells missing-1 (Gcm1), is expressed in small clusters of chorionic trophoblast cells at the flat chorionic plate stage and at sites of chorioallantoic folding and extension when morphogenesis begins. Mutation of Gcm1 in mice causes a complete block to branching of the chorioallantoic interface, resulting in embryonic mortality by E10 due to the absence of the placental labyrinth. In addition, chorionic trophoblast cells in Gcm1-deficient placentas do not fuse to form syncytiotrophoblast. Abnormal development of placental villi is frequently associated with fetal death and intrauterine growth restriction in humans, and our studies provide the earliest molecular insight into this aspect of placental development.

The human mid-size neurofilament subunit: a repeated protein sequence and the relationship of its gene to the intermediate filament gene family.

We report the isolation and sequence of cDNA and genomic clones for one of the two large subunits of human neurofilament, NF‐M. Analysis of the sequence has allowed us to investigate the structure of the carboxy‐terminal tail of this protein, and to compare it to that of the small neurofilament as well as to other intermediate filaments. The carboxy‐terminal region of the protein contains a 13 amino acid proline‐ and serine‐rich sequence repeated six times in succession. Within each repeat unit are two smaller repeats of the sequence Lys‐Ser‐Pro‐Val. The four amino acid repeat may represent a kinase recognition site in a region of the protein that is known to be highly phosphorylated. We also note the presence of an additional heptad repeat at the extreme carboxy terminus of the protein. This region of 60 amino acids may be involved in coiled‐coil interactions similar to those that facilitate the filament formation in the rod region. The human gene contains only two introns. Their positions correspond to two of the three introns found in the small neurofilament of the mouse. Thus, two of the three neurofilament genes of mammals have similar structures which are quite different from those of the other intermediate filaments. This finding suggests a common origin of the neurofilament subunits, whose evolutionary relationship to other intermediate filament genes is uncertain.

A cellular mechanism governing the severity of Pelizaeus–Merzbacher disease

Pelizaeus–Merzbacher disease (PMD) is a leukodystrophy linked to the proteolipid protein gene (PLP). We report a cellular basis for the distinction between two disease subtypes, classical and connatal, based on protein trafficking of the two PLP gene products (PLP and DM20). Classical PMD mutations correlate with accumulation of PLP in the ER of transfected COS–7 cells while the cognate DM20 traverses the secretory pathway to the cell surface. On the other hand, connatal PMD mutations lead to the accumulation of both mutant PLP and DM20 proteins in the ER of COS–7 cells with little of either isoform transported to the cell surface. Moreover, we show that transport–competent mutant DM20s facilitate trafficking of cognate PLPs and hence may influence disease severity.

The structure and organization of the human heavy neurofilament subunit (NF-H) and the gene encoding it.

Genomic clones for the largest human neurofilament protein (NF‐H) were isolated, the intron/exon boundaries mapped and the entire protein‐coding regions (exons) sequenced. The predicted protein contains a central region that obeys the structural criteria identified for alpha‐helical ‘rod’ domains typically present in all IF protein components: it is approximately 310 amino acids long, shares amino acid sequence homology with other IF protein rod domains and displays the characteristic heptad repeats of apolar amino acids which facilitate coiled‐coil interaction. Nevertheless, anomalies are noted in the structure of the NF‐H rod which could explain observations of its poor homopolymeric assembly in vitro. The protein segment on the carboxy‐terminal side of the human NF‐H rod is uniquely long (greater than 600 amino acids) compared to other IF proteins and is highly charged (greater than 24% Glu, greater than 25% Lys), rich in proline (greater than 12%) and impoverished in cysteine, methionine and aromatic amino acids. Its most remarkable feature is a repetitive sequence that covers more than half its length and includes the sequence motif, Lys‐Ser‐Pro (KSP) greater than 40 times. Together with the recent identification of the serine in KSP as the main target for NF‐directed protein kinases in vivo, this repetitive character explains the massive phosphorylation of the NF‐H subunit that can occur in axons. The human NF‐H gene has three introns, two of which interrupt the protein‐coding sequence at identical points to introns in the genes for the two smaller NF proteins, NF‐M and NF‐L.(ABSTRACT TRUNCATED AT 250 WORDS)

The nucleotide sequence of the mRNA encoding the fusion protein of measles virus (Edmonston strain): A comparison of fusion proteins from several different paramyxoviruses

Membrane fusion is the primary cytopathic effect observed in cells infected with measles virus. The viral protein responsible for this process has previously been defined as the fusion (F) protein. Fusion is activated by the proteolytic cleavage of a precursor molecule (F0) to yield two disulfide-linked polypeptides (F1 and F2). In this paper the mRNA for the membrane fusion protein has been cloned and the resulting cDNAs were sequenced. A mRNA composed of 2377 nucleotides was found to contain one open reading frame which could potentially code for a protein of 550 amino acids. This corresponding gene product was identified as the fusion protein through use of antibodies directed against a synthetic peptide which was constructed from the deduced amino acid sequence. A long and rather G-C rich 5 terminus was found on the mRNA and this noncoding region may play some role in regulation of protein synthesis at the translational level. Protein sequence data derived from the cDNA clones revealed a highly conserved F1 amino terminus which is characteristic of most paramyxoviruses. Very little amino acid homology (except for the conservation of the F1 terminus and 9 cysteines) was evident when the sequence was compared to other paramyxovirus fusion proteins. However an overall hydrophobic nature was characteristic of all the F proteins and hydrophobicity plots for the fusion proteins of 4 different paramyxoviruses were very similar. Computer analysis was also employed to analyze the secondary structure of the measles virus F protein. Large stretches of alpha helix were characteristic of the regions which purportedly interact with membranes. The functional domains of the F protein and their similarity to those of the influenza hemagglutinin protein are discussed in this communication. We concluded that the distribution of hydrophobic regions capable of spanning biological membranes determines the fusogenic nature of the F protein.

Murine Gcm1 gene is expressed in a subset of placental trophoblast cells

The gcm gene of Drosophila melanogaster encodes a transcription factor that is an important component in cell fate specification within the nervous system. In the absence of a functional gcm gene, progenitor cells differentiate into neurons, whereas when the gene is ectopically expressed the cells produce excess glial cells at the expense of neuronal differentiation. Recent searches of databases have uncovered high sequence similarity between the Drosophila gcm gene and an anonymous human placental cDNA clone (Altschuller et al., 1996 ; this communication). Here we report the molecular organization of the murine Gcm1, its spatio‐temporal pattern of expression in developing placenta, and its map position at E1‐E3 on murine chromosome 9. The murine gene is composed of at least 6 exons. The promoter region contains an “initiation sequence” and is GC rich, characteristics of the promoters of several transcription factors. The mRNA has a modest 5UTR (ca. 200 bases) but an extensive 3 UTR (ca. 2kb). Northern blot and mRNA in situ hybridization studies showed that Gcm1 expression was readily detectable only in the placenta. It began at embryonic day 7.5 within trophoblast cells of the chorion and continued to about embryonic day 17.5 within a subset of labyrinthine trophoblast cells. Comparison with other transcription factors revealed that Gcm1 expression defines a unique subset of trophoblast cells. Dev Dyn 1999;214:303–311.

Neurofilament-M Interacts with the D1 Dopamine Receptor to Regulate Cell Surface Expression and Desensitization

We used the yeast two-hybrid assay to identify novel proteins that interact with the D1 dopamine receptor. The third cytoplasmic loop (residues 217–273) of the rat D1 receptor was used as bait to identify clones encoding interacting proteins from a rat brain cDNA library. This identified two clones encoding the C terminus of rat neurofilament-M (NF-M) (residues 782–846). The NF-M clone did not interact with the third cytoplasmic loops of the rat D2, D3, or D4receptors, but showed weak interaction with that of the D5receptor. Coexpression of full-length NF-M with the D1receptor in HEK-293 cells resulted in >50% reduction of receptor binding accompanied by a reduction in D1 receptor-mediated cAMP accumulation. NF-M had no effect on the expression of other dopamine receptor subtypes. Using a D1 receptor-green fluorescent protein chimera and confocal fluorescence microscopy, we found that NF-M reduced D1 receptor expression at the cell surface and promoted accumulation of the receptor in the cytosol. Interestingly, the D1 receptors that were expressed at the cell surface in the presence of NF-M were resistant to agonist-induced desensitization. Cellular colocalization of NF-M and the D1 receptor in the rat brain was examined by epifluorescence microscopy. These experiments showed that ∼50% of medium-sized striatal neurons expressed both proteins. Colocalization was also observed in pyramidal cells and interneurons within the frontal cortex. Similar immunohistochemical analyses using NF-M-deficient mice showed decrements in D1 receptor expression compared with control mice. These results suggest that NF-M interacts with the D1 receptor in vivo and may modify its expression and regulation.

mRNA levels of all three neurofilament proteins decline following nerve transection

The control of neurofilament (NF) protein gene expression was studied by determining and comparing the levels of mRNA to the heavy (NF-H), mid-sized (NF-M) and light (NF-L) NF protein subunits in rat dorsal root ganglia (DRG) following sciatic nerve transection. mRNA to NF-H (4.5 kb), to NF-M (3.4 kb) and to NF-L (2.5 and 4.0 kb) were identified in Northern blots and quantitated in dot blot analyses, using specific cDNA probes for each NF protein. Following transection and continuing for at least 28 days. The early and co-terminal fall in mRNAs suggests that the 3 NF genes are regulated by common factor(s) and that the function of these factor(s) is influenced by the state of axonal continuity with the target organ.

Cytoplasmic and nuclear localization of myelin basic proteins reveals heterogeneity among oligodendrocytes.

Myelin basic proteins (MBPs) are major proteins of central nervous system (CNS) myelin, where they facilitate the apposition of cytoplasmic faces of myelin lamellae. Myelin‐bearing oligodendrocytes transport MBP mRNA to myelin, where newly translated protein is directly inserted into the myelin sheath. An apparent absence of MBPs in oligodendrocyte perikarya has suggested that MBP localized to the soma is translationally inert. We now demonstrate by confocal immunofluorescence microscopy that not only are MBPs present in the majority of oligodendrocyte perikarya but oligodendrocytes are heterogeneous with respect to their localization of MBPs; MBPs are concentrated in some cells at the plasmalemma and distributed in others throughout the cytoplasm and, surprisingly, the nucleus. MBPs are present in the nuclei of over half of oligodendrocytes in the adult, but in almost all MBP+ oligodendrocytes during myelinogenesis. Transport of MBPs into nuclei appears to be a regulated process since some cells exhibit robust MBP accumulation in their cytoplasm but exclude MBPs from their nuclei. We show that oligodendrocyte nuclei contain all four major MBP isoforms, but that in transgenic mice, the epitope‐tagged 14 kD MBP isoform preferentially segregates to the plasmalemma. Our data demonstrate that oligodendrocytes are not required to exclude MBPs from their perikarya and suggest that MBPs have a specific function in the oligodendrocyte perikarya and nucleus.