DJ-1 knock-down impairs astrocyte mitochondrial function.

Mitochondrial dysfunction has long been implicated in the pathogenesis of Parkinson's disease (PD). PD brain tissues show evidence for mitochondrial respiratory chain Complex I deficiency. Pharmacological inhibitors of Complex I, such as rotenone, cause experimental parkinsonism. The cytoprotective protein DJ-1, whose deletion is sufficient to cause genetic PD, is also known to have mitochondria-stabilizing properties. We have previously shown that DJ-1 is over-expressed in PD astrocytes, and that DJ-1 deficiency impairs the capacity of astrocytes to protect co-cultured neurons against rotenone. Since DJ-1 modulated, astrocyte-mediated neuroprotection against rotenone may depend upon proper astrocytic mitochondrial functioning, we hypothesized that DJ-1 deficiency would impair astrocyte mitochondrial motility, fission/fusion dynamics, membrane potential maintenance, and respiration, both at baseline and as an enhancement of rotenone-induced mitochondrial dysfunction. In astrocyte-enriched cultures, we observed that DJ-1 knock-down reduced mitochondrial motility primarily in the cellular processes of both untreated and rotenone treated cells. In these same cultures, DJ-1 knock-down did not appreciably affect mitochondrial fission, fusion, or respiration, but did enhance rotenone-induced reductions in the mitochondrial membrane potential. In neuron-astrocyte co-cultures, astrocytic DJ-1 knock-down reduced astrocyte process mitochondrial motility in untreated cells, but this effect was not maintained in the presence of rotenone. In the same co-cultures, astrocytic DJ-1 knock-down significantly reduced mitochondrial fusion in the astrocyte cell bodies, but not the processes, under the same conditions of rotenone treatment in which DJ-1 deficiency is known to impair astrocyte-mediated neuroprotection. Our studies therefore demonstrated the following new findings: (i) DJ-1 deficiency can impair astrocyte mitochondrial physiology at multiple levels, (ii) astrocyte mitochondrial dynamics vary with sub-cellular region, and (iii) the physical presence of neurons can affect astrocyte mitochondrial behavior.

Cerebral air embolism complicating cardiac ablation procedures.

Two patients with similar courses of neurologic impairment and subsequent recovery after cerebral air embolism complicating cardiac ablation procedures are described. Hyperbaric oxygen therapy, combined with aggressive resuscitative efforts, appears to have contributed to each patient's recovery.

Disparity tuning in macaque area V4.

Neural processing of stereoscopic depth is conventionally associated with the dorsal (spatial) pathway in primate visual cortex. The role of depth information in the ventral (object) pathway has been less certain. We found prominent tuning for stereoscopic disparity in area V4, an intermediate stage in the ventral pathway. Eighty percent of the cells in our sample exhibited significant disparity tuning over the -1.0 degree to 1.0 degree range, and the majority showed > 2:1 response differences. Tuning function shapes were similar to those reported previously in other visual areas. We observed a significant tuning bias towards crossed (near) disparities. This could reflect an emphasis in the ventral pathway on foreground objects or parts of objects projecting towards the viewer.

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.

GFAP and S100beta expression in the cortex and hippocampus in response to mild cortical contusion.

We studied the acute response of glial fibrillary acidic protein (GFAP) and S100beta gene expression in the cerebral cortex and hippocampus to mild unilateral cortical contusion. Our goal was to evaluate and compare the expression patterns of each gene in the early stages of the astrocytic response to brain injury. RNA was extracted from the cerebral cortex and hippocampus of male rats at 0, 3, 12, 24, or 96 h after lesion or sham-operation, then quantified using an RNase protection assay. Contusion produced a robust elevation in GFAP mRNA by 12 h in both brain regions on the ipsilateral side to the contusion. In the cortex, but not the hippocampus, this elevation was sustained at 96 h. S100beta mRNA levels were elevated bilaterally in lesioned animals at 24 h in both brain regions. However, these data are difficult to interpret because sham mRNA levels decreased with time, making it unclear whether contusion stimulates S100beta gene expression or whether it mitigates the inhibitory effect of sham. We further analyzed the effect of contusion on GFAP and S100beta immunoreactive astrocyte density at 96 h postlesion or postsham by double-label immunocytochemistry. All detectable astrocytes under all conditions were S100beta immunoreactive in both brain regions. Furthermore, all S100beta immunoreactive astrocytes in the lesioned ipsilateral cortex were also GFAP immunoreactive, whereas only about 11% of S100beta positive cells were also GFAP labeled in the contralateral lesioned or the ipsilateral sham cortex. In the hippocampus, all S100beta immunoreactive cells were also GFAP immunoreactive under all conditions. These data correlate with the gene expression data at 96 h, and suggest that, at least in the cortex, resident S100beta-expressing astrocytes produce GFAP at levels that are undetectable by immunocytochemistry until they are activated in response to injury.

Fetal grafts containing suprachiasmatic nuclei restore the diurnal rhythm of CRH and POMC mRNA in aging rats.

We assessed whether fetal tissue containing the suprachiasmatic nuclei (SCN) can restore age-related changes in the diurnal rhythm of hypothalamic corticotropin-releasing hormone (CRH) and anterior pituitary proopiomelanocortin (POMC) mRNA. Young, middle-aged, and middle-aged SCN-transplanted rats were killed at seven times of day. In young rats, CRH mRNA exhibited a diurnal rhythm in the dorsomedial paraventricular nuclei but not in other subdivisions of the nuclei. No rhythm was detected in aging rats. SCN transplants restored a rhythm in CRH mRNA, but the timing was not precisely the same as in young animals. POMC mRNA exhibited a daily rhythm in young rats. Aging abolished the rhythm and decreased the average mRNA level; fetal transplants restored the rhythm, but the amplitude remained attenuated. These data are the first demonstration that fetal tissue can restore the diurnal rhythm of a neuroendocrine axis that is driven by the SCN. We conclude that the neuroendocrine substrate from the aging host remains capable of responding to diurnal cues to express diurnal rhythmicity in CRH/POMC mRNA when fetal SCN transplants confer the appropriate signals.

Dexamethasone regulates basic fibroblast growth factor, nerve growth factor and S100beta expression in cultured hippocampal astrocytes.

Glucocorticoids regulate hippocampal neuron survival during fetal development, in the adult, and during aging; however, the mechanisms underlying the effects are unclear. Since astrocytes contain adrenocortical receptors and synthesize and release a wide variety of growth factors, we hypothesized that glucocorticoids may alter neuron-astrocyte interactions by regulating the expression of growth factors in hippocampal astrocytes. In this study, three growth factors, which are important for hippocampal neuron development and survival, were investigated: basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and S100beta. Enriched type I astrocyte cultures were treated with 1 microM dexamethasone (DEX), a synthetic glucocorticoid, for up to 120 h. Cells and culture medium were collected and total RNA and protein were measured at 6, 12, 24, 48, 72, 96 and 120 h after the initiation of hormone treatment. Growth factor mRNA levels were measured and quantified using solution hybridization-RNase protection assays and protein levels were quantified using ELISA methods. We report that DEX stimulates the bFGF mRNA levels over the 120-h treatment. In contrast, DEX suppresses NGF mRNA continuously over the same period of treatment. DEX induces a biphasic response in S100beta mRNA levels. In addition, some of the changes in gene expression are translated into parallel changes in protein levels of these growth factors. Our results demonstrate that dexamethasone can differentially regulate the expression of growth factors in hippocampal astrocytes in vitro. This suggests that one of the mechanisms through which glucocorticoids affect hippocampal functions may be by regulating the expression of astrocyte-derived growth factors.

Assessment of gene expression and peptide secretion from individual cells.

We have developed an assay that allows one to monitor gene expression in and peptide secretion from individual cells. By combining the reverse hemolytic plaque with in situ hybridization, investigators can quantitate simultaneously the level of gene expression and the level of secretion of a peptide. The method can be used in any system in which an appropriate antibody for the reverse hemolytic plaque assay and probes complementary to the mRNA of interest are available. It can be used to monitor the level of mRNA and secretion of the peptide product, or expression of one gene and the secretion of another peptide. In this paper we will describe the major steps of the method. We have used the pituitary lactotroph as a model to demonstrate the power of this technique. However, we believe that this method may be an important approach to answer many questions regarding the cellular and molecular mechanisms that regulate the coupling of peptide secretion and gene expression at the single cell level.

Simultaneous monitoring of pituitary hormone secretion and gene expression within individual cells.

We have recently developed a method to simultaneously quantitate the level of gene expression and the level of secretion of a peptide from individual cells. Our approach has been to combine the reverse hemolytic plaque assay sequentially with in situ hybridization. We present data to show how we have used the pituitary lactotroph as a model to demonstrate the power of this technique. However, we are particularly excited about the potential application of this strategy to approach a broad spectrum of questions regarding the cellular and molecular mechanisms that regulate the coupling of peptide secretion and gene expression at the single cell level. The method can be used in any system in which an appropriate antibody for the reverse hemolytic plaque assay and probes complementary to the mRNA of interest are available.