Yoheved Berwald-Netter

Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.

A Novel CNS Gene Required for Neuronal Migration and Involved in X-Linked Subcortical Laminar Heterotopia and Lissencephaly Syndrome

X-SCLH/LIS syndrome is a neuronal migration disorder with disruption of the six-layered neocortex. It consists of subcortical laminar heterotopia (SCLH, band heterotopia, or double cortex) in females and lissencephaly (LIS) in males, leading to epilepsy and cognitive impairment. We report the characterization of a novel CNS gene encoding a 40 kDa predicted protein that we named Doublecortin and the identification of mutations in four unrelated X-SCLH/LIS cases. The predicted protein shares significant homology with the N-terminal segment of a protein containing a protein kinase domain at its C-terminal part. This novel gene is highly expressed during brain development, mainly in fetal neurons including precursors. The complete disorganization observed in lissencephaly and heterotopia thus seems to reflect a failure of early events associated with neuron dispersion.

Adenoviral Vector as a Gene Delivery System into Cultured Rat Neuronal and Glial Cells

Previous studies have demonstrated that a defective recombinant adenovirus can infect a wide range of postmitotic and slowly proliferating cell types such as hepatocytes, myotubes, pneumocytes and intestinal cells (Stratford‐Perricaudet et al., Hum. Gene Ther., 1, 241–256, 1990; Quantin et al., Proc. Natl. Acad. Sci. USA, 89, 2581–2584, 1992; Jaffe et al., Nature Genetics, 1, 372–378, 1992). We have used a defective recombinant adenovirus, Ad.RSVβgal, containing the Escherichia coliβ‐galactosidase gene targeted to the nucleus under the transcriptional control of the Rous sarcoma virus long terminal repeat promoter (Stratford‐Perricaudet et al., J. Clin. Invest., 90, 626–630, 1992) to infect non‐dividing neural cells in primary culture. We show that 80–100% of neuronal and astroglial cells infected with a viral titre lower than 109 p.f.u./ml express β‐galactosidase for at least 1 month without cell damage. These results demonstrate the potential usefulness of recombinant adenovirus infection for the analysis of brain‐specific gene regulation and for the transfer of genes into neural cells before their transplantation into the brain.

Neurotropism of rabies virus. An in vitro study.

The relative susceptibility of neurons and glia, grown as monolayers in vitro, to rabies virus infection was explored. Established cell lines of neuronal or glial phenotype and primary cultures of cells derived from mouse dorsal root ganglia (DRG) or brain were used as homologues of the targets of rabies virus in the nervous system. Fixed rabies virus (CVS) strain was used in most experiments; other fixed rabies strains (PV, HEP, ERA) and a street rabies virus isolate were used in some. Virus-cell tropism was determined by immunofluorescence assay for rabies nucleocapsid antigen and cell permissivity was assessed by titration of virus yields. Neuronal cells always exhibited a much greater susceptibility to infection and a greater propensity to sustain viral growth. By immunofluorescence, 90–100% of neurons commonly had viral inclusion bodies, while doses of the virus three to four orders of magnitude higher still left >99% of astrocytes, in brain cell cultures and 90 ± 5% of the non-neuronal cells in DRG cultures without any obvious signs of rabies virus. Neuroblastoma cells (95 ± 5% with viral antigens) produced viral yields about four orders of magnitude higher than glioma cells (10 ± 5% with viral antigens). Though the overall infectivity of street virus was lower than that of fixed virus strains, a significantly higher viral tropism for neurons than for glia was maintained. Thus, primary neuronal cultures offer a means of exploring molecular events in rabies virus infection and their role in pathogenesis.

Neuronal acquisition of tetanus toxin binding sites: Relationship with the last mitotic cycle

In an earlier study on the developing nervous system, the existence of a temporal correlation between the appearance of tetanus toxin-binding cells and neurogenesis was reported (A. Koulakoff, B. Bizzini, and Y. Berwald-Netter (1982). Dev. Brain Res. 5, 139-147). Using a combined approach of immunocytochemistry and [3H]thymidine autoradiography it is shown that, in the fetal mouse central nervous system, dividing cells do not express membrane binding sites for tetanus toxin. A time-course quantitative autoradiography revealed that the toxin-binding sites become apparent within 7 +/- 1 hr, following the last S phase, on cells undergoing the conversion from dividing to postmitotic state. The acquisition of surface binding sites for tetanus toxin may thus be an early property of nascent central neurons, marking the transition from cycling precursor neuroblasts to postmitotic neuronal cells. Parallel studies on in vivo-developing dorsal root ganglia disclosed that at least some peripheral nervous system cells are endowed with tetanus toxin-binding capacity while still capable of DNA synthesis and undergo one or more divisions.

Localization of aldolase C mRNA in brain cells

The expression of aldolase C and aldolase A mRNA was assessed by Northern blot hybridization using RNAs purified from cultured rat and mouse brain neurons and astroglial cells. Neurons were found to contain about 4‐fold more aldolase C mRNA and about twice as much aldolase A mRNA than astroglia. Analysis of the cellular localization of aldolase C mRNA by in situ hybridization to brain slices showed a predominantly neuronal labeling with an irregular distribution. A strong signal was observed in Purkinje cell somata and a weaker signal in subpopulations of neurons in cerebral cortex, striatum, hippocampus, hypothalamic nuclei and primary olfactory cortex.

Posttranslational modifications of tubulin in cultured mouse brain neurons and astroglia

Posttranslational modifications of tubulin were analyzed in mouse brain neurons and glia developing in culture. Purified tubulin was resolved by isoelectric focusing. After 3 weeks of culture neurons were shown to express a high degree of tubulin heterogeneity (8 α and 10 β isoforms), similar to that found in the brain at the same developmental stage. Astroglial tubulin exhibits a less complex pattern consisting of 4 α and 4 β isoforms.

A correlation between the appearance and the evolution of tetanus toxin binding cells and neurogenesis

The ontogenesis of cells expressing surface membrane binding sites for tetanus toxin (Tt) was studied in the mouse nervous system. Cells were labeled shortly after the tissue dissociation and the toxin bound was revealed by immunofluorescence. In the brain, spinal cord and dorsal root ganglia the toxin binding cells (TBC) are found as of very early stages of nervous system organogenesis, i.e. at 10 days of gestation. There is a close temporal correlation between the pattern of emergence and accumulation of TBC and the known pattern of appearance of post-mitotic neurons in mouse cerebral cortex, cerebellum and spinal cord. The curves of TBC abundance as a function of fetal age in various nervous system areas are different. They show regional fluctuations in the proportion of TBC that reflect the cumulative changes in the dynamics of neuronal subpopulations. The results indicate that Tt can be used as an ontogenetically early marker of neuronal differentiation and that the acquisition of Tt receptors may represent one of the earliest detectable characteristics of the developing neurons.

Effects of dimethylsulfoxide on membrane currents of neuroblastoma × glioma hybrid cell

Giga-ohm seal whole cell recording technique was used to examine ionic currents changes induced by dimethylsulfoxide (DMSO) in neuroblastoma X glioma hybrid NG 108-15 cells. DMSO (0.5-1%) reversible blocks sodium, potassium and calcium currents and shifts by about 6 mV the sodium inactivation curve towards more negative voltages.

Ultrastructural visualization of Na+-channel associated[125]α-scorpion toxin binding sites on fetal mouse nerve cells in culture

Purified neurotoxin II from the scorpion Androctonus australis Hector (alpha-ScTx) has previously been shown to bind specifically to the voltage-sensitive Na+ channels of excitable cells. Recent studies, using high specific activity 125I-labeled alpha-ScTx, demonstrated specific binding to neuronal cells derived from fetal mouse brains. In the present study, 125I-labeled alpha-ScTx was used to localize the voltage-sensitive Na+ channels in cultured fetal mouse brain cells. By quantitative electron microscope autoradiography we demonstrate that specific alpha-ScTx binding sites are selectively located at the plasma membrane. Estimates of their density revealed that neurites at 13 days in vitro carry at least 6 X more specific alpha-ScTx sites than cell body membrane.