Efficient marking of neural stem cell-derived neurons with a modified murine embryonic stem cell virus, MESV2.

Treatments for nervous system disorders that involve transplanting genetically modified neural stem cells may ultimately be feasible. As a step towards this therapeutic approach, a novel murine embryonic stem cell gammaretroviral vector was developed with features designed to optimize transgene expression in neural stem cells and to increase vector safety. All potential start sites of translation in the 5' leader were removed. These sites may compete with an inserted transgene for translation initiation, and also produce potentially immunogenic peptides. Further, all of the gag gene sequences were replaced with a well-defined constitutive transport element from avian leukemia virus to promote nuclear export of viral RNA, and to eliminate any homology between the vector and a murine leukemia virus-derived gag-pol packaging plasmid. Two versions of the virus were made in which EGFP expression was driven either by the Rous sarcoma virus U3 enhancer or by a combination of sequences from the Syn1 and Pgk-1 promoters. Both of these viruses efficiently transduced neural stem cells isolated from embryonic rat hippocampus, and robust EGFP expression was observed in neurons derived from these cells following differentiation in vitro.

A 5' Leader of Rbm3, a Cold Stress-induced mRNA, Mediates Internal Initiation of Translation with Increased Efficiency under Conditions of Mild Hypothermia.

Although mild hypothermia generally reduces protein synthesis in mammalian cells, the expression of a small number of proteins, including Rbm3, is induced under these conditions. In this study, we identify an Rbm3 mRNA with a complex 5' leader sequence containing multiple upstream open reading frames. Although these are potentially inhibitory to translation, monocistronic reporter mRNAs containing this leader were translated relatively efficiently. In addition, when tested in the intercistronic region of dicistronic mRNAs, this leader dramatically enhanced second cistron translation, both in transfected cells and in cell-free lysates, suggesting that the Rbm3 leader mediates cap-independent translation via an internal ribosome entry site (IRES). Inasmuch as Rbm3 mRNA and protein levels are both increased in cells exposed to mild hypothermia, the activity of this IRES was evaluated at a cooler temperature. Compared to 37 degrees C, IRES activity at 33 degrees C was enhanced up to 5-fold depending on the cell line. Moderate enhancements also occurred with constructs containing other viral and cellular IRESes. These effects of mild hypothermia on translation were not caused by decreased cell growth, as similar effects were not observed when cells were serum starved. The results suggest that cap-independent mechanisms may facilitate the translation of particular mRNAs during mild hypothermia.

Identification of two short internal ribosome entry sites selected from libraries of random oligonucleotides.

Sequences that control translation of mRNA may play critical roles in regulating protein levels. One such element is the internal ribosome entry site (IRES). We previously showed that a 9-nt segment in the 5' leader sequence of the mRNA encoding Gtx homeodomain protein could function as an IRES. To identify other short sequences with similar properties, we designed a selection procedure that uses a retroviral vector to express dicistronic mRNAs encoding enhanced green and cyan fluorescent proteins as the first and second cistrons, respectively. Expression of the second cistron was dependent upon the intercistronic sequences and was indicative of IRES activity. B104 cells were infected with two retroviral libraries that contained random sequences of 9 or 18 nt in the intercistronic region. Cells expressing both cistrons were sorted, and sequences recovered from selected cells were reassayed for IRES activity in a dual luciferase dicistronic mRNA. Two novel IRESes were identified by this procedure, and both contained segments with complementarity to 18S rRNA. When multiple copies of either segment were linked together, IRES activities were dramatically enhanced. Moreover, these synthetic IRESes were differentially active in various cell types. These properties are similar to those of the previously identified 9-nt IRES module from Gtx mRNA. These results provide further evidence that short nucleotide sequences can function as IRESes and support the idea that some cellular IRESes may be composed of shorter functional modules. The ability to identify IRES modules with specific expression properties may be useful in the design of vectors for biotechnology and gene therapy.

Increased intracellular triglyceride in C(2)C(12) muscle cells transfected with human lipoprotein lipase.

Much of the knowledge about the cell biology of lipoprotein lipase (LPL) in vitro has been gained from adipose tissue model systems. However, the importance of skeletal muscle lipoprotein lipase (SMLPL) to both lipoprotein and muscle metabolism remains unclear. Although the production of LPL in cultured myocytes has been documented, the amount of enzyme activity produced is small. To develop a more suitable tissue culture model for SMLPL, mouse C(2)C(12) myoblasts were stably transduced with a retroviral vector encoding the full-length human LPL (hLPL) cDNA. Control cells were transduced with a vector encoding beta-galactosidase. LPL expression was assayed as a function of cell growth by measuring LPL activity on days 3, 7, 9, 11, and 14 after subculture. The hLPL-transduced myoblasts increasingly overexpressed both heparin-releasable (HR) and intracellular (IN) LPL activity compared to nontransduced myoblasts (P < 0.001 at Day 11) and myoblasts transduced with the control vector (P < 0.001 at Day 11). This increase occurred while LPL mRNA levels remained stable between days 3 and 14. As expected, IN LPL activity was also increased in the transduced cells. High levels of LPL activity were also obtained after differentiating the C(2)C(12) cells into myotubes by serum deprivation. Additionally, throughout the time course, C(2)/LPL cells had greater amounts of intracellular triglyceride than both the C(2)C(12) and the C(2)/beta-GEO cells (P = 0.005 and P < 0.001, respectively) with the largest differences seen on day 14 of the time course (P = 0.001, C(2)/LPL vs C(2)C(12) (r) or C(2)/beta-GEO cells). Thus, C(2)C(12) myoblasts stably transduced with hLPL markedly overexpressed both HR and IN LPL activity compared to control cells which, in turn, was associated with increases in intracellular triglyceride content. Because LPL regulation in tissues is mostly posttranslational, this new in vitro model will permit the in-depth study of the posttranslational regulation of SMLPL and provide new insights into the fate of lipoprotein-derived fatty acids in muscle.

Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity.

Eukaryotic transcriptional regulation in different cells involves large numbers and arrangements of cis and trans elements. To survey the number of cis regulatory elements that are active in different contexts, we have devised a high-throughput selection procedure permitting synthesis of active cis motifs that enhance the activity of a minimal promoter. This synthetic promoter construction method (SPCM) was used to identify >100 DNA sequences that showed increased promoter activity in the neuroblastoma cell line Neuro2A. After determining DNA sequences of selected synthetic promoters, database searches for known elements revealed a predominance of eight motifs: AP2, CEBP, GRE, Ebox, ETS, CREB, AP1, and SP1/MAZ. The most active of the selected synthetic promoters contain composites of a number of these motifs. Assays of DNA binding and promoter activity of three exemplary motifs (ETS, CREB, and SP1/MAZ) were used to prove the effectiveness of SPCM in uncovering active sequences. Up to 10% of 133 selected active sequences had no match in currently available databases, raising the possibility that new motifs and transcriptional regulatory proteins to which they bind may be revealed by SPCM. The method may find uses in constructing databases of active cis motifs, in diagnostics, and in gene therapy.

Paucity of retinoic acid receptor alpha (RAR alpha) nuclear immunostaining in gliomas and inability of retinoic acid to influence neural cell adhesion molecule (NCAM) expression.

Neural cell adhesion molecule (NCAM) is down-regulated during periods of embryological cell migration and may be important in local tumor migration or metastases. Conflicting information exists in the literature about NCAM expression in human glial tumors and little is known about its expression in human brain metastases. We immunohistochemically stained a panel of 43 primary human brain tumors and their cultured counterparts for NCAM including glioblastoma multiformes, anaplastic astrocytomas, oligodendrogliomas, and contrasted their staining with a panel of 3 meningiomas, 11 brain metastases, and 5 normal brain samples utilizing the monoclonal antibody NKH-1. Most gliomas and metastatic melanomas and lung carcinomas showed a high percentage of cells positive for NCAM expression while NCAM staining was negative for other carcinomas. No difference was seen between intensity or percentage of cells that were NCAM positive, based on tumor grade or type. In glioma cell lines, NCAM expression was lost upon passage. In 15 glioma cell lines we also determined NCAM isoform expression by reverse transcription/polymerase chain reaction (RT/PCR) and found that 6 of 15 had message for NCAM 180, 8 of 15 for NCAM 140, and only 3 of 15 had message for NCAM 120. Normal brains always contained message for the 180 isoform and usually had mRNA for all 3 isoforms. Using monoclonal antibodies for retinoic acid receptor alpha (RAR alpha), we found nuclear staining in melanomas and lung carcinomas metastatic to brain and only rarely in gliomas. Neither the relative antigen density of NCAM nor the percent of NCAM-positive cells appreciably changed upon incubation with retinoic acid (RA), as measured by flow cytometry. RAR alpha was not found at a level measurable by immunohistochemistry in nuclei of most glial tumors, providing an explanation for why RA might not induce NCAM expression. Whether paucity of RAR alpha on primary gliomas might also correlate with results from clinical trials showing limited efficacy of RA in treatment of human gliomas awaits further study.

Lipoprotein lipase is expressed in cultured Schwann cells and functions in lipid synthesis and utilization.

We have previously demonstrated that lipoprotein lipase (LPL; triacylglycero-protein acylhydrolase, EC 3.1.1.34) is most likely expressed in the non-neuronal cells of the spinal cord, and glial cells may thus be the site of expression in the peripheral nervous system as well. We investigated the expression of LPL in cultured 1. 17 cells, an immortalized rat sciatic nerve Schwann cell line. The 1. 17 cells were shown to express LPL mRNA by reverse transcriptase-polymerase chain reaction analysis. The 1.17 Schwann cells demonstrated heparin-releasable lipolytic activity that was inhibited by the lipase inhibitor tetrahydrolipstatin in a dose-dependent manner. Preincubation of 1.17 cells with an anti-rat LPL antiserum reduced the heparin-releasable lipolytic activity to <10% of that measured in untreated cells. To investigate the role of LPL in Schwann cell lipid metabolism, 1.17 cells were incubated for up to 24 h with an emulsified [14C]triolein substrate and the incorporation of [14C]triolein radioactivity into various cellular lipids was examined in the presence of either anti-rat LPL antiserum or preimmune serum. Inhibiting LPL activity reduced the incorporation of 14C into cellular polar lipids, diacylglycerol, and cholesteryl esters by >80% at 2 and 6 h after addition of the radiolabeled substrate. At 24 h, radioactivity in diacylglycerol and cholesteryl esters was similar in cells treated with anti-LPL antiserum or preimmune serum, whereas 14C incorporation into polar lipids was still reduced by >60%. Separation of the polar lipids into individual lipid species revealed no specific changes in triolein-derived radioactivity incorporation across the phospholipid species examined. These results suggest that LPL-mediated hydrolysis of exogenous triacylglycerol is an important source of free fatty acids for the Schwann cell and thus may play a critical role in myelin biosynthesis in the peripheral nervous system.

Expression of mRNA for the elav-like neural-specific RNA binding protein, HuD, during nervous system development.

The expression of mRNA for the neuronal antigen HuD (Elavl4) associated with paraneoplastic encephalomyelitis and sensory neuronopathy was evaluated in the developing and adult rat nervous system. Using RNase protection assay and non-radioactive in situ hybridization histochemistry HuD expression was shown to be expressed at high levels at the earliest time point observed (E15), but declined significantly during the first postnatal week to levels which were maintained into adulthood. In the adult, HuD expression became restricted primarily to large pyramidal-like neurons. Exceptions of note were many smaller neurons within a variety of thalamic nuclei. Expression of HuD was observed to be coincident with terminal differentiation of all neuronal structures evaluated regardless of the timing of their development, providing correlative evidence for a role in neuronal differentiation or the maintenance of neuronal phenotype. The marked restriction of HuD mRNA expression with maturity suggests that its functional role in adult neurons varies significantly throughout the CNS.

Ontogeny of cation-Cl- cotransporter expression in rat neocortex.

Neuronal precursors and immature cortical neurons actively accumulate Cl- and as a consequence depolarize in response to GABAA receptor activation. With maturity, intracellular Cl- decreases resulting in a shift towards GABAA inhibition. These observations suggest that changes in expression of cation-Cl- cotransporters may have a significant role in the ontogeny of neuronal Cl- homeostasis. Using ribonuclease protection analysis and in situ hybridization we examined the developmental expression of all presently known members of the cation-Cl- cotransporter gene family in rat brain. Of the inwardly directed cotransporters, NKCC-1, NKCC-2, and NCC-1, only NKCC-1 was detected at significant levels in brain. NKCC-1 was expressed in neurons, appearing first in cortical plate but not in ventricular or subventricular zone. Expression levels peaked by the third postnatal week and were maintained into adulthood. The outwardly directed cotransporters, KCC-1 and KCC-2, demonstrated significantly different levels and time courses of expression. KCC-1 was expressed prenatally at very low levels which increased little over the course of development. In contrast, KCC-2 expression appeared perinatally and increased dramatically after the first week of postnatal life. Differential changes in expression of this gene family occurred during periods of critical shifts in chloride homeostasis and GABA response suggestive of a role in these processes. Furthermore the absence of expression of known inwardly directed cotransporters in Cl- accumulating neuroepithelia and lack of evidence for glial expression suggests that as yet unidentified members of this gene family may be involved in chloride homeostasis in immature neuronal precursors and neuroglia.

Developmental expression of C1C-2 in the rat nervous system.

Regulation of expression of the voltage-gated chloride channel, C1C-2, was investigated during development and adult life in rat brain. RNase protection assays demonstrated a marked increase in levels of expression of C1C-2 in brain during early postnatal development which was also detected in adult brain. In situ hybridization of E15 and E18 rat brains demonstrated C1C-2 expression in deep brain nuclei and scattered cells within the neuroepithelial layers, but not in the regions of subventricular zone that primarily give rise to glial populations. By E18 all neurons within the emerging cortical plate and its equivalent in other areas of the CNS were heavily labeled. During the first postnatal week, C1C-2 was highly expressed in most neurons. By P7 a pattern of differential expression emerged with evidence of decreased expression of C1C-2 mRNA in many neuronal populations. In adult rat brain, C1C-2 was expressed at highest levels in large neurons as found within layer V of cortex, Ammon's Horn of hippocampus, or mitral cells of the olfactory bulb and Purkinje cells within the cerebellum. Many smaller neurons within the diencephalon maintained significant levels of expression. A functional conductance was readily detected in hippocampal neurons during the first postnatal week, which had the same characteristic properties as the conductance observed in adult neurons. The observed expression and functional presence of C1C-2 suggest a widespread role in neuronal chloride homeostasis in early postnatal life, and demonstrated that cell specific shut-down resulted in the adult pattern of expression.

Receptor expression, cytogenetic, and molecular analysis of six continuous human glioma cell lines.

Six human glioma cell lines were established from tissues obtained from five patients diagnosed with Kernohan grade IV glioblastoma multiforme and one from a patient with a grade II astrocytoma. One line was from a recurrent patient who had received prior therapy; the other lines were derived from patients at initial diagnosis and/or before cytoreductive therapies other than surgery were given. Considerable variability in phenotypic, karyotypic, and cell surface marker expression was displayed between the six human glioma cell lines. The karyotypes ranged from apparently normal (grade II astrocytoma) to those with complex rearrangements. Trisomy of chromosome 7 was the most common abnormality. The extensive cytogenetic and molecular characterization of these lines may facilitate their utilization in cellular and molecular biologic studies.

The effect of the mouse mutation claw paw on myelination and nodal frequency in sciatic nerves.

Despite the biophysical and clinical importance of differentiating nodal and internodal axolemma, very little is known about the process. We chose to study myelination and node of Ranvier formation in the hypomyelinating mouse mutant claw paw (clp). The phenotype of clp is delayed myelination in the peripheral nervous system. The specific defect is unknown but is thought to arise from a breakdown in the complex signaling mechanism between axon and Schwann cell. Myelination was assessed in sciatic nerve cross sections from adult and postnatal day 14 (P14) heterozygous and homozygous clp mice. Antibodies to P0, myelin-associated glycoprotein (MAG), and neural cell adhesion molecule were used to assess the stage of myelination. P14 homozygous clp mice showed an atypical staining pattern of immature myelin, which resolved into a relatively normal pattern by adulthood. Sodium channel clustering and node of Ranvier frequency were studied in whole-mount sciatic nerves with sodium channel and MAG antibodies. P14 homozygous clp nerves again showed an atypical, immature pattern with diffuse sodium channel clusters suggesting nodal formation was delayed. In the adult, homozygous clp sciatic nerves displayed dramatically shortened internodal distances. The data from this study support the hypotheses that node of Ranvier formation begins with the onset of myelination and that the number and location of nodes of Ranvier in the sciatic nerve are determined by myelinating Schwann cells.

Overexpression of a transmembrane isoform of neural cell adhesion molecule alters the invasiveness of rat CNS-1 glioma.

CNS-1 is a highly invasive neural cell adhesion molecule (NCAM)-positive rat glioma that exhibits similarities in its pattern of infiltration to human gliomas. To investigate whether increasing NCAM expression alters invasive behavior, retroviruses encoding human NCAM 140 and a cytoplasmic truncation of NCAM 140 were used to transduce a population of CNS-1 glioma cells that had a relatively low endogenous level of NCAM. Compared to cells transduced with a control virus, cells overexpressing either intact or truncated human NCAM 140 showed decreased invasion of a reconstituted basal lamina. Changes in growth rate or in key matrix metalloproteinase activities could not account for this result. In a migration assay on type IV collagen, cells exhibited a substrate concentration-dependent increase in the rate of migration; however, overexpression of NCAM 140 or truncated NCAM 140 inhibited motility at higher substrate concentrations. Consistent with these findings was the decreased spread of NCAM 140 overexpressers in vivo following instillation of cells into the right frontal cortex of rat brain. NCAM 140 overexpressers showed considerably more restricted perivascular and periventricular spread than cells transduced with a control virus. However, NCAM-140-overexpressing tumor exhibited a less cohesive pattern of growth near the site of tumor instillation and more individual cell infiltration of brain parenchyma with more pronounced perineuronal satellitosis. The stability of recombinant NCAM expression was confirmed by recovering tumor cells from tumor-bearing animals and measuring NCAM levels by flow cytometry. These observations show that overexpression of NCAM 140 decreases the long-range spread of CNS-1 glioma along basal lamina pathways but enhances local infiltration of neuropil.

Gene organization and chromosome location of the neural-specific RNA binding protein Elavl4.

We have isolated the gene that encodes the neural-specific RNA binding protein HuD in the mouse (Elavl4), and have mapped its location to the mid-distal region of chromosome 4, close to the neurological mutant clasper. The coding region of the Elavl4 gene covers approximately 44 kb; the first two RNA binding domains (RBDs) that are homologous to the two RBDs found in the Drosophila sex-lethal gene are each encoded in two exons, whereas the third RBD is encoded in a single exon. Elavl4 mRNAs are alternatively spliced in the region between RBDs 2 and 3 due to the variable use of two micro-exons, and RNase protection analysis indicates that two of four possible splice variants are the predominant isoforms expressed in the central nervous system. The high degree of sequence conservation between the Hu proteins suggests that the exon organization of all the Hu protein genes will be similar, if not identical, to the Elavl4 gene.

Schwann cells infected with a recombinant retrovirus expressing myelin-associated glycoprotein antisense RNA do not form myelin.

To elucidate the role of myelin-associated glycoprotein (MAG) in the axon-Schwann cell interaction leading to myelination, neonatal rodent Schwann cells were infected in vitro with a recombinant retrovirus expressing MAG antisense RNA or MAG sense RNA. Stably infected Schwann cells and uninfected cells were then cocultured with purified sensory neurons under conditions permitting extensive myelination in vitro. A proportion of the Schwann cells infected with the MAG antisense virus did not myelinate axons and expressed lower levels of MAG than control myelinating Schwann cells, as measured by immunofluorescence. Electron microscopy revealed that the affected cells failed to segregate large axons and initiate a myelin spiral despite having formed a basal lamina, which normally triggers Schwann cell differentiation. Cells infected with the MAG sense virus formed normal compact myelin. These observations strongly suggest that MAG is the critical Schwann cell component induced by neuronal interaction that initiates peripheral myelination.

Expressing antisense P0 RNA in Schwann cells perturbs myelination.

Primary Schwann cells were infected in vitro with a recombinant retrovirus expressing a dominant selectable marker, neomycin phosphotransferase (conferring resistance to the drug G418), and antisense P0 RNA under the control of the human beta-actin promoter. A proportion of the G418-resistant cells failed to form myelin when cocultured with dorsal root ganglion neurons under conditions that promote Schwann cell differentiation. These cells expressed high levels of P0 antisense RNA. Among the impaired cells, the majority had segregated and ensheathed individual axon but had not differentiated further. They did not express P0 but did express myelin- associated glycoprotein and galactocerebroside. A minority of partially inhibited Schwann cells were also observed that elaborated thin myelin sheaths containing variable numbers of compacted and noncompacted lamellae. These data indicate that restricting the level of P0 expression inhibits spiralling of the Schwann cell membrane and subsequent compaction.

L-3,4-dihydroxyphenylalanine synthesis by genetically modified Schwann cells.

We have investigated whether Schwann cells can be modified by gene transfer to synthesize L-3,4-dihydroxyphenylalanine (L-DOPA), the immediate precursor in the formation of dopamine. By using a retrovirus containing a rat tyrosine hydroxylase (TH) cDNA, we established an immortalized rodent Schwann cell line that stably expressed high levels of TH and secreted L-DOPA in vitro when supplied with tyrosine and the essential cofactor biopterin. We also infected primary Schwann cells and demonstrated that cells expressing TH secreted L-DOPA while maintaining their capacity to myelinate neurons in vitro. This study indicate that it may be feasible to utilize autotransplantation of genetically modified Schwann cells to alleviate the movement disorders in Parkinson's disease.

The myelin-associated glycoproteins: membrane disposition, evidence of a novel disulfide linkage between immunoglobulin-like domains, and posttranslational palmitylation.

The myelin-associated glycoproteins (MAG) are members of the immunoglobulin gene superfamily that function in the cell interactions of myelinating glial cells with axons. In this paper, we have characterized the structural features of these proteins. The disposition of MAG in the bilayer as a type 1 integral membrane protein (with an extracellularly disposed amino terminus, single transmembrane segment, and cytoplasmic carboxy terminus) was demonstrated in protease protection studies of MAG cotranslationally inserted into microsomes in vitro and in immunofluorescent studies with site specific antibodies. A genetically engineered MAG cDNA, which lacks the putative membrane spanning segment, was constructed and shown to encode a secreted protein. These results confirm the identify of this hydrophobic sequence as the transmembrane segment. Sequencing of the secreted protein demonstrated the presence of a cleaved signal sequence and the site of signal peptidase cleavage. To characterize the disulfide linkage pattern of the ectodomain, we cleaved MAG with cyanogen bromide and used a panel of antibodies to coprecipitate specific fragments under nonreducing conditions. These studies provide support for a novel disulfide linkage between two of the immunoglobulin domains of the extracellular segment. Finally, we report that MAG is posttranslationally palmitylated via an intramembranous thioester linkage. Based on these studies, we propose a model for the conformation of MAG, including its RGD sequence, which is considered with regard to its function as a cell adhesion molecule.

Expression of recombinant myelin-associated glycoprotein in primary Schwann cells promotes the initial investment of axons by myelinating Schwann cells.

Myelin-associated glycoprotein (MAG) is an integral membrane protein expressed by myelinating glial cells that occurs in two developmentally regulated forms with different carboxyterminal cytoplasmic domains (L-MAG and S-MAG). To investigate the role of MAG in myelination a recombinant retrovirus was used to introduce a MAG cDNA (L-MAG form) into primary Schwann cells in vitro. Stably infected populations of cells were obtained that constitutively expressed MAG at the cell surface without the normal requirement for neuronal contact to induce expression. Constitutive expression of L-MAG did not affect myelination. In long term co-culture with purified sensory neurons, the higher level of MAG expression on infected Schwann cells was reduced to control levels on cells that formed myelin. On the other hand, unlike normal Schwann cells, infected Schwann cells associated with nonmyelinated axons or undergoing Wallerian degeneration expressed high levels of MAG. This suggests that a posttranscriptional mechanism modulates MAG expression during myelination. Immunostaining myelinating cultures with an antibody specific to L-MAG showed that L-MAG was normally transiently expressed at the earliest stages of myelination. In short term co-culture with sensory neurons, infected Schwann cells expressing only L-MAG segregated and ensheathed larger axons after 4 d in culture provided that an exogenous basal lamina was supplied. Similar activity was rarely displayed by control Schwann cells correlating with the low level of MAG induction after 4 d. These data strongly suggest that L-MAG promotes the initial investment by Schwann cells of axons destined to be myelinated.