The herpes simplex virus type 2 R1 protein kinase (ICP10 PK) blocks apoptosis in hippocampal neurons, involving activation of the MEK/MAPK survival pathway.

Herpes simplex virus type 1 (HSV-1) and HSV-2 trigger or counteract apoptosis by a cell-specific mechanism. Our studies are based on previous findings that the protein kinase (PK) domain of the large subunit of HSV-2 ribonucleotide reductase (ICP10) activates the Ras/MEK/MAPK pathway (Smith et al., J. Virol. 74:10417, 2000). Because survival pathways can modulate apoptosis, we used cells that are stably or transiently transfected with ICP10 PK, an HSV-2 mutant deleted in ICP10 PK (ICP10DeltaPK) and the MEK-specific inhibitor U0126 to examine the role of ICP10 PK in apoptosis. Apoptosis was induced by staurosporine or D-mannitol in human (HEK293) cells or HEK293 cells stably transfected with the ICP10 PK-negative mutant p139 (JHL15), as determined by morphology, DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage. HEK293 cells stably transfected with ICP10 (JHLa1) were protected from apoptosis. ICP10 but not p139 protected neuronally differentiated PC12 cells from death due to nerve growth factor withdrawal, and apoptosis (determined by TUNEL) and caspase-3 activation were seen in primary hippocampal cultures infected with ICP10DeltaPK but not with HSV-2 or a revertant virus [HSV-2(R)]. The data indicate that ICP10 has antiapoptotic activity under both paradigms and that it requires a functional PK activity. The apoptotic cells in primary hippocampal cultures were neurons, as determined by double immunofluorescence with fluorescein-labeled dUTP (TUNEL) and phycoerythrin-labeled antibodies specific for neuronal proteins (TuJ1 and NF-160). Protection from apoptosis was associated with MEK/MAPK activation, as evidenced by (i) increased levels of activated (phosphorylated) MAPK in HSV-2- but not ICP10DeltaPK-infected cultures and (ii) inhibition of MAPK activation by the MEK-specific inhibitor U0126. MEK and MAPK were activated by infection with UV-inactivated but not antibody-neutralized HSV-2, suggesting that activation requires cellular penetration but is independent of de novo viral protein synthesis.

Role of founder cell deficit and delayed neuronogenesis in microencephaly of the trisomy 16 mouse.

Development of the neocortex of the trisomy 16 (Ts16) mouse, an animal model of Down syndrome (DS), is characterized by a transient delay in the radial expansion of the cortical wall and a persistent reduction in cortical volume. Here we show that at each cell cycle during neuronogenesis, a smaller proportion of Ts16 progenitors exit the cell cycle than do control, euploid progenitors. In addition, the cell cycle duration was found to be longer in Ts16 than in euploid progenitors, the Ts16 growth fraction was reduced, and an increase in apoptosis was observed in both proliferative and postmitotic zones of the developing Ts16 neocortical wall. Incorporation of these changes into a model of neuronogenesis indicates that they are sufficient to account for the observed delay in radial expansion. In addition, the number of neocortical founder cells, i.e., precursors present just before neuronogenesis begins, is reduced by 26% in Ts16 mice, leading to a reduction in overall cortical size at the end of Ts16 neuronogenesis. Thus, altered proliferative characteristics during Ts16 neuronogenesis result in a delay in the generation of neocortical neurons, whereas the founder cell deficit leads to a proportional reduction in the overall number of neurons. Such prenatal perturbations in either the timing of neuron generation or the final number of neurons produced may lead to significant neocortical abnormalities such as those found in DS.

Interphase FISH for rapid identification of a down syndrome animal model.

Mouse models of Down syndrome (trisomy 21, DS) have been developed to study the consequences of triplication of regions syntenic to distal human chromosome 21. We report a technique for the identification of segmental trisomy 16 (Ts65Dn) and diploid progeny at any age using interphase fluorescence in situ hybridization (FISH) of uncultured tail fibroblasts. Our technique is faster and less expensive than blood karyotyping.

Impaired spatial working and reference memory in segmental trisomy (Ts65Dn) mice.

To evaluate the cognitive phenotype of the segmental trisomy 16 (Ts65Dn) mouse, a model of Down Syndrome (DS, trisomy 21), we assessed spatial working and reference memory using a 12-arm radial maze (RAM). Ts65Dn mice made a greater number of reference memory errors across trials compared to control mice. Both genotypes showed improvement across trials, although improvement was slower in Ts65Dn mice. Ts65Dn mice also made a greater number of working memory errors on the RAM, and in contrast to control mice, did not improve across trials, always performing at near-chance levels. These results provide evidence for both spatial working and reference memory deficits in Ts65Dn mice, characteristics of cognitive dysfunction.

Basic fibroblast growth factor-2 and interleukin-1 beta regulate S100 beta expression in cultured astrocytes.

Basic fibroblast growth factor and interleukin-1 beta are known to regulate the expression of other trophic factors and to stimulate reactive gliosis in vivo. S100 beta is a glial-specific putative neurotrophic factor and has been considered a marker of the reactive status of astrocytes. Therefore, we tested the hypothesis that basic fibroblast growth factor-2 and interleukin-1 beta achieve their effects by altering S100 beta gene expression in cultured rat astrocytes using an RNase protection assay. Short-term treatment with basic fibroblast growth factor-2 produced a transient decrease in S100 beta messenger RNA which was followed by an increase after longer term treatment. In contrast, both short- and long-term treatment with interleukin-1 beta suppressed S100 beta messenger RNA. We measured levels of S100 beta nuclear primary transcript to assess whether alterations in transcriptional rate explain the changes in messenger RNA. Our results indicate that changes in transcription account for changes in steady state levels of messenger RNA since basic fibroblast growth factor-2-induced changes in S100 beta primary transcript temporally preceded changes in messenger RNA. We further measured intracellular S100 beta protein levels by enzyme-linked immunosorbent assay to determine whether changes in gene expression were translated into parallel changes in protein. Our results clearly demonstrate that basic fibroblast growth factor-2 and interleukin-1 beta influence the expression of the S100 beta gene, that this regulation appears to occur at the level of transcription, and that alterations in messenger RNA are sometimes, but not always, reflected in changes at the level of protein. These observations suggest that basic fibroblast growth factor-2 may amplify its trophic effects, in part, by influencing the expression of another trophic factor.

Abnormal calcium homeostasis in astrocytes from the trisomy 16 mouse.

The regulation of intracellular Ca2+ was investigated in cultured astrocytes from the trisomy 16 (Ts16) mouse, an animal model for Down syndrome and Alzheimer's disease (AD). The cytoplasmic ionized Ca2+ concentration ([Ca2+]cyt) was determined using digital imaging of fura-2-loaded cells. The relative Ca2+ content of internal endoplasmic reticulum (ER) stores was estimated from the magnitude of the transient increase in [Ca2+]cyt induced by cyclopiazonic acid (CPA), an inhibitor of Ca2+ sequestration into ER stores. At rest, the average [Ca2+]cyt was 140 nM in euploid (normal) astrocytes, but over twice as high, 320 nM, in Ts16 cells. In the absence of extracellular Ca2+, CPA induced a transient increase in [Ca2+]cyt to over 1200 nM in Ts16 astrocytes as compared to only 500 nM in euploid cells, indicating an increased amount of Ca2+ in the Ts16 astrocyte ER. In contrast to euploid astrocytes, both resting [Ca2+]cyt and the amount of Ca2+ in the ER stores varied widely among individual Ts16 astrocytes. These results show that Ts16 produces a dysregulation of Ca2+ homeostasis leading to increased cytoplasmic and stored Ca2+. Since increases in [Ca2+]cyt have been implicated in the etiology of neurodegenerative diseases, including AD, this finding of abnormal Ca2+ homeostasis in a genetic model of human neurological disorders suggests that Ca2+ dysregulation may be a common feature underlying neurodegenerative processes.

Spatial memory deficits in segmental trisomic Ts65Dn mice.

Spatial memory was assessed in the segmental trisomic 16 mouse (Ts65Dn), a potential model for Down syndrome (DS), using the 12-arm radial maze (RAM). Ts65Dn mice have a portion of mouse chromosome 16 syntenic to the distal end of human chromosome 21 triplicated. On each of 8 daily trials of the RAM, Ts65Dn mice made fewer correct choices than control mice and performed at or near chance levels, indicating a deficit in spatial working memory. On trials 9 and 10, Ts65Dn mice performed as well as control mice on the initial 12 choices, but required a greater number of choices to complete the RAM. The improved performance of Ts65Dn mice on trials 9 and 10 was lost when the animals were retested after a 50-day retention period, suggesting that long-term memory is also defective. These results are not likely explained by differences in either response bias or perceptual discrimination. Ts65Dn and control mice displayed comparable levels of performance in spontaneous alternation in a T-maze, demonstrating that simple spatial memory was not impaired. In the elevated plus maze, Ts65Dn mice did not display higher anxiety levels which could affect their performance in the RAM. In fact, Ts65Dn mice visited open arms on the elevated plus maze more frequently and spent more time on open arms than did control mice. Taken together, these results provide evidence for short- and long-term spatial memory deficits in Ts65Dn mice.

Expression of glial antigens in mouse astrocytes: species differences and regulation in vitro.

Expression of developmentally regulated antigens was used to characterize glial cells in cultures from embryonic mouse cerebral cortex. Over 90% of the cells had a flat morphology, and about 50% of these flat cells also expressed the ganglioside GD3. Up to 40% of all the GD3 expressing cells also expressed A2B5 antigen. Flat cells expressing either glial fibrillary acidic protein (GFAP), or GD3 or both were present at all times in vitro. These three populations of flat cells could not be further distinguished on the basis of NG2 or fibronectin expression, or with respect to their responses to the mitogens FGF-2, PDGF, or EGF. The glial cultures also contain a small number (approximately 5%) of process bearing cells with the morphological and immunocytochemical characteristics of oligodendrocyte precursors. The expression of GD3 by flat cells changed with time in culture as the fraction of flat cells expressing only GD3 declined and the fraction of cells expressing GFAP (with or without GD3) increased. The data are consistent with those flat cells expressing only GD3 being astrocyte precursors. Furthermore, between 1 and 3 weeks in vitro GD3/GFAP cells lose GD3 while retaining GFAP. Cells expressing only GFAP could be induced to express GD3 and A2B5 by treatment with FGF-2. The widespread and regulated expression of GD3 and A2B5 by murine glia is different from the restricted pattern of expression previously reported for these antigens in rat brain cell cultures. These results demonstrate that expression of GD3 and A2B5 by murine astrocytes depends on both culture age and extracellular signals and that these gangliosides are not markers for cell lineage in the mouse.

Consequences of trisomy 16 for mouse brain development: corticogenesis in a model of Down syndrome.

We have studied abnormalities in the tangential and radial expansion of the cerebral cortex during fetal development in the trisomy 16 (Ts16) mouse, a model for human trisomy 21 (Down syndrome). Slowed tangential expansion of the neuroepithelium in Ts16 resulted in a reduction of final telencephalic size and is predicted to decrease the number of radial cortical units in the mature brain. In addition, radial growth of the Ts16 cortex was delayed at the time of peak cortical neurogenesis in normal mice, but by embryonic day 18 the cortex reached normal thickness. Because mouse chromosome 16 shares many genes with human chromosome 21, abnormalities in Ts16 brain development may parallel abnormalities in trisomy 21.

Characterization of sensorimotor performance, reproductive and aggressive behaviors in segmental trisomic 16 (Ts65Dn) mice.

In the present study, segmental trisomy 16 (Ts65Dn) mice, an animal model of Down Syndrome (DS), were examined for sensorimotor, reproductive, and aggression abnormalities associated with DS. The Ts65Dn mice exhibited no sensorimotor deficits in olfactory sensitivity, visual abilities, orientation reactions, forelimb strength, postural skills, balance/ coordination, climbing, or locomotion compared to genetically matched control B6EiC3HF1 mice. In mating tests, the percentage of Ts65Dn mice displaying intromissions when paired with estrous females was significantly less than that in controls. Although the percentage of Ts65Dn mice that mounted and ejaculated with an estrous female was marginally less than in controls, there were no significant differences on the other measures of reproductive behavioral performance. In aggression tests, Ts65Dn males showed increased offensive aggression in a neutral arena both when paired and among grouped males. Conversely, Ts65Dn mice exhibited less offensive aggression against an intruder in their home cage than control males. In sum, these mice possess some of the adaptive behavior abnormalities observed in DS patients; however, because the Ts65Dn mice do not have any observed sensorimotor deficits that could interfere with behavioral assessments, they may serve as a useful model for the study of behavioral impairments associated with DS.

Modulation of two functionally distinct Ca2+ stores in astrocytes: role of the plasmalemmal Na/Ca exchanger.

Mechanisms that regulate the amount of releasable Ca2+ in intracellular stores of cultured mouse astrocytes were investigated using digital imaging of fura-2 loaded cells. At rest, the cytoplasmic Ca2+ concentration, [Ca2+]cyt, was about 110 nM. In the absence of extracellular Ca2+, cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum (ER) Ca(2+)-ATPase, induced a transient, four-fold increase in [Ca2+]cyt due to the release of Ca2+ from inositol triphosphate (IP3) sensitive stores. Caffeine (CAF), which releases Ca2+ from Ca(2+)-sensitive stores, induced a two-fold increase in [Ca2+]cyt. The CPA- and CAF-sensitive stores could be released independently. Changes in the amplitudes of the Ca2+ transients were taken as a measure of changes in store content. Removal of extracellular Na+ or addition of ouabain, which inhibit Ca2+ extrusion and promote Ca2+ entry across the plasmalemma via the Na/Ca exchanger, caused minimal increases in resting [Ca2+]cyt but greatly potentiated both CPA- and CAF-induced Ca2+ transients. The amount of Ca2+ releasable from the IP3(CPA) sensitive store was directly proportional to cytosolic Na+ concentration (i.e., inversely proportional to the transmembrane Na+ electrochemical gradient). Under these reduced Na+ gradient conditions, little, if any, Ca2+ destined for the ER stores enters the cells through voltage-dependent Ca2+ channels. These results demonstrate that mouse astrocytes contain two distinct ER Ca2+ stores, the larger, IP3- (CPA-) sensitive, and the smaller, Ca(2+)- (CAF-) sensitive. The Ca2+ content of both ER stores can be regulated by the Na/Ca exchanger. Thus, the magnitude of cellular responses to signals that are mediated by Ca2+ release induced by the two second messengers, IP3 and Ca2+, can be modulated by factors that affect the net transport of Ca2+ across the plasmalemma.

Glutamate as a hippocampal neuron survival factor: an inherited defect in the trisomy 16 mouse.

The survival of cultured mouse hippocampal neurons was found to be greatly enhanced by micromolar concentrations of the excitatory neurotransmitter glutamate. Blockade of kainate/AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptors increased the rate of neuron death, suggesting that endogenous glutamate in the cultures promotes survival. Addition of glutamate (0.5-1 microM) further increased neuron survival, whereas glutamate in excess of 20 microM resulted in increased death. Thus, the survival vs. glutamate dose-response relation is bell-shaped with an optimal glutamate concentration near 1 microM. We found that hippocampal neurons from mice with the genetic defect trisomy 16 (Ts16) died 2-3 times faster than normal (euploid) neurons. Moreover, glutamate, at all concentrations tested, failed to increase survival of Ts16 neurons. In contrast, the neurotrophic polypeptide basic fibroblast growth factor did increase the survival of Ts16 and euploid neurons. Ts16 is a naturally occurring mouse genetic abnormality, the human analog of which (Down syndrome) leads to altered brain development and Alzheimer disease. These results demonstrate that the Ts16 genotype confers a defect in the glutamate-mediated survival response of hippocampal neurons and that this defect can contribute to their accelerated death.

A novel, abundant sodium channel expressed in neurons and glia.

A novel, voltage-gated sodium channel cDNA, designated NaCh6, has been isolated from the rat central and peripheral nervous systems. RNase protection assays showed that NaCh6 is highly expressed in the brain, and NaCh6 mRNA is as abundant or more abundant than the mRNAs for previously identified rat brain sodium channels. In situ hybridization demonstrated that a wide variety of neurons express NaCh6, including motor neurons in the brainstem and spinal cord, cerebellar granule cells, and pyramidal and granule cells of the hippocampus. RT-PCR and/or in situ hybridization showed that astrocytes and Schwann cells express NaCh6. Thus, this sodium channel is broadly distributed throughout the nervous system and is shown to be expressed in both neurons and glial cells.

Sodium/calcium exchange in rat cortical astrocytes.

Regulation of the cytosolic free Ca2+ concentration ([Ca2+]cyt) by an Na/Ca exchanger was studied in primary cultured rat cortical astrocytes. [Ca2+]cyt was measured by digital imaging in cells loaded with fura-2. The resting [Ca2+]cyt, approximately 150 nM, was only slightly increased by reducing the extracellular Na+ concentration ([Na+]o) to 6.2 mM, or by treating the cells with ouabain for 15 min (to raise cytosolic Na+). Following treatment with ouabain, however, lowering [Na+]o caused [Ca2+]cyt to rise rapidly to approximately 1300 nM. When Ca2+ sequestration in intracellular stores was blocked by thapsigargin, lowering [Na+]o increased [Ca2+]cyt to approximately 1500 nM in the absence of ouabain. The low-[Na+]o-stimulated rise in [Ca2+]cyt was abolished by removal of external Ca2+, but was not blocked by the Ca2+ channel blocker verapamil, or by caffeine or ryanodine, which deplete an intracellular Ca2+ store responsible for Ca(2+)-induced Ca2+ release. These data suggest that Na+ gradient reduction promotes net Ca2+ gain via Na/Ca exchange. Normally, however, a large rise in [Ca2+]cyt is prevented by sequestration of the entering Ca2+; this buffering of cytosolic Ca2+ can be circumvented by blocking sequestration with thapsigargin, or overwhelmed by enhancing net Ca2+ gain by pretreating the cells with ouabain. The presence of Na/Ca exchanger protein and mRNA in the astrocytes was confirmed by Western and Northern blot analyses, respectively. Immunohistochemistry revealed that exchanger molecules are distributed in a reticular pattern over the astrocyte surface. We suggest that the Na/Ca exchanger plays a role in regulating both [Ca2+]cyt and the intracellular stores of Ca2+ in astrocytes, and may thus contribute to the control of astrocyte responsiveness to neurotransmitters and neurotoxins.

Mutually exclusive exon splicing of type III brain sodium channel alpha subunit RNA generates developmentally regulated isoforms in rat brain.

We have identified two exons of the type III rat brain sodium channel alpha subunit gene that undergo mutually exclusive alternative RNA splicing to produce mRNAs coding either for an isoform predominant in neonatal brain (IIIN) or a different isoform (IIIA) predominant in the adult. These exons are 92 base pairs in length and encode amino acids 203-232, which correspond to part of the S3 and most of the S4 transmembrane segments within domain I and the extracellular loop between them. Despite 21 nucleotide differences between the exons, only a single amino acid at position 209 is altered, specifying either aspartic acid (IIIA) or serine (IIIN). As evidence that these isoforms are generated via alternative splicing, we demonstrate that both exons are encoded within the type III gene. The nucleotide sequences of the neonatal and adult type III exons and the intervening intron as well as the developmental regulation of this splicing are nearly identical in the type II sodium channel gene. The conservation of the exon/intron structure and of the developmentally regulated patterns of expression of the type II and III sodium channel genes suggests that alternative mRNA splicing of this exon may play a substantial role in modulating sodium channel function during brain development by alteration of a single amino acid.

Brain and heart sodium channel subtype mRNA expression in rat cerebral cortex.

The expression of mRNAs coding for the alpha subunit of rat brain and rat heart sodium channels has been studied in adult and neonatal rat cerebral cortex using the reverse transcription-polymerase chain reaction (RT-PCR). Rat brain sodium channel subtype I, II, IIA, and III sequences were simultaneously amplified in the same PCR using a single oligonucleotide primer pair matched to all four subtype sequences. Identification of each subtype-specific product was inferred from the appearance of unique fragments when the product was digested with specific restriction enzymes. By using this RT-PCR method, products arising from mRNAs for all four brain sodium channel subtypes were identified in RNA extracted from adult rat cerebral cortex. The predominant component was type IIA with lesser levels of types I, II, and III. In contrast, the type II and IIA sequences were the predominant RT-PCR products in neonatal rat cortex, with slightly lower levels of type III and undetectable levels of type I. Thus, from neonate to adult, type II mRNA levels decrease relative to type IIA levels. Using a similar approach, we detected mRNA coding for the rat heart sodium channel in neonatal and adult rat cerebral cortex and in adult rat heart. These results reveal that mRNAs coding for the heart sodium channel and all four previously sequenced rat brain sodium channel subtypes are expressed in cerebral cortex and that type II and IIA channels may be differentially regulated during development.

Development of saxitoxin-sensitive and insensitive sodium channels in cultured neonatal rat astrocytes.

Voltage-sensitive Na channels were studied in cultures of neonatal rat cortical astrocytes. These channels were present at all times in culture as determined by tracer 22Na+ influx in the presence of batrachotoxin (BTX) and sea anemone polypeptide toxin (AxTx). The affinity of saxitoxin (STX) binding and sensitivity to STX inhibition of sodium influx were utilized to characterize these channels. Up to 7 d in culture, high-affinity 3H-STX binding (Kd of 0.2 nM at 4 degrees C) was very low, and 22Na+ influx was inhibited only by high concentrations (Ki = 170 nM) of STX. From 7 to 14 d, total specific binding of STX increased to a maximum of over 2 pmol/mg protein and remained constant for 28 d. By 14 d, inhibition of 22Na+ influx by STX was clearly biphasic, indicating the presence of 2 populations of channels with Ki's of 0.2 nM and 150 nM. At 14 d in culture, binding of 3H-STX to astrocyte membranes revealed the presence of 2 specific sites. During this second week, increasing numbers of high-affinity STX binding sites and increasing sensitivity to the inhibition of BTX + AxTx-stimulated 22Na+ influx by STX coincided with the change in morphology of primitive flat polygonal cells to highly branched stellate forms characteristic of mature astrocytes in vivo. Changes in culture conditions modified the time course of the onset of high STX affinity binding. Twenty-four hours after changing to serum-free G5 medium, there was both an 8-fold increase in STX binding sites and a change to a stellate shape in all cells. The results suggest that although low-affinity STX Na channels are always present in astrocytes, after 7 d in culture a different population of channels appears with the high affinity for STX characteristic of adult neuronal sodium channels. This spontaneous process is greatly accelerated by changing to a chemically defined medium.

Ultrastructural metabolic activity following quick-freezing and freeze-substitution in tetrahydrofuran in the superior cervical ganglion.

A method of quick-freezing and freeze-substitution has been developed for localizing diffusible substances such as 2-deoxyglucose-6-phosphate (2-DG-6-P) ultrastructurally in neural tissue. Quick-freezing under pressure provides well preserved tissue down to 30-35 microns from the surface. This allows blocks of neural tissue to be quick-frozen and analysed for diffusible substances in areas removed from the freezing face. Freeze-substitution in tetrahydrofuran following quick-freezing was found to dissolve and remove 2-deoxyglucose (2-DG) but not 2-DG-6-P. Consequently, this technique extends the ability to analyse localization of glucose utilization to postsynaptic as well as presynaptic sites. We have applied the technique to isolated superior cervical ganglion while provoking selective increases in energy metabolism. Exposure to an elevated extracellular potassium (12 mM) concentration produced a pattern of metabolic activity with enhanced neuropil labelling (neuronal and glial processes). With antidromic stimulation of the external carotid nerves, deoxyglucose uptake in neuronal and glial soma in the caudal portion of the ganglion was enhanced more than neuropil labelling. This caudal region corresponds to the region of origin of the cell bodies of the external carotid nerve. Results from this technique suggest that the contribution of glia to overall rate of energy metabolism may be significant and that this is a promising method for correlating the relationship between functional activity and cellular electrical activity.