Geoffrey C. Owens

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 CLC-2 IN THE RAT NERVOUS SYSTEM

Abstract Regulation of expression of the voltage-gated chloride channel, ClC-2, was investigated during development and adult life in rat brain. RNase protection assays demonstrated a marked increase in levels of expression of ClC-2 in brain during early postnatal development which was also detected in adult brain. In situ hybridization of E15 and E18 rat brains demonstrated ClC-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, ClC-2 was highly expressed in most neurons. By P7 a pattern of differential expression emerged with evidence of decreased expression of ClC-2 mRNA in many neuronal populations. In adult rat brain, ClC-2 was expressed at highest levels in large neurons as found within layer V of cortex, Ammons 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 ClC-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.

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.

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

SummarySix 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.

Paucity of Retinoic Acid Receptor Alpha (RARα) 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 (RARα), 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. RARα 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 RARα on primary gliomas might also correlate with results from clinical trials showing limited efficacy of RA in treatment of human gliomas awaits further study.

In utero gene delivery by intraamniotic injection of a retroviral vector producer cell line in a nonhuman primate model.

In utero gene therapy (IUGT) offers the promise of treating a wide variety of genetic diseases before the development of disease manifestations. The most convenient and potentially easiest method of targeting the fetus is through injection into the amniotic cavity. For long-term correction of genetic defects, retroviral vectors have great potential as a tool for gene therapy strategies. However, retroviral vectors are limited by growth to low titers. In an attempt to increase the amount of vector particles delivered and assess the potential of intraamniotic administration, we injected a retroviral vector producer cell line encoding the lacZ gene into the amniotic fluid of a nonhuman primate model. After birth the infants were analyzed for vector-mediated transduction. Two of four fetuses were successfully transduced, with transgene expression detected in the esophagus, trachea, and stomach. In some sections of tissue, nearly 100% of the cells lining the lumen of these tissues were positive for transduction. Although successful, the limited number of tissues in which transduction was observed led to an in vitro analysis of the effects of amniotic fluid (AF). The presence of amniotic fluid inhibited transduction by 99%. AF affected both the transducing activity of the vector and the health of the packaging cells. The negative effects of AF were gestational age dependent; greater inhibition was observed from AF collected at later stages of pregnancy. The fact that transduction was successful despite these negative effects indicates that this approach is a promising strategy for gene therapy.