Nina Felice Schor

Oxidative signaling pathway for externalization of plasma membrane phosphatidylserine during apoptosis

Active maintenance of membrane phospholipid asymmetry is universal in normal cell membranes and its disruption with subsequent externalization of phosphatidylserine is a hallmark of apoptosis. Externalized phosphatidylserine appears to serve as an important signal for targeting recognition and elimination of apoptotic cells by macrophages, however, the molecular mechanisms responsible for phosphatidylserine translocation during apoptosis remain unresolved. Studies have focused on the function of aminophospholipid translocase and phospholipid scramblase as mediators of this process. Here we present evidence that unique oxidative events, represented by selective oxidation of phosphatidylserine, occur during apoptosis that could promote phosphatidylserine externalization. We speculate that selective phosphatidylserine oxidation could affect phosphatidylserine recognition by aminophospholipid translocase and/or directly result in enzyme inhibition. The potential interactions between the anionic phospholipid phosphatidylserine and the redox‐active cationic protein effector of apoptosis, cytochrome c, are presented as a potential mechanism to account for selective oxidation of phosphatidylserine during apoptosis. Thus, cytochrome c‐mediated phosphatidylserine oxidation may represent an important component of the apoptotic pathway.

Phospholipid signaling in apoptosis: peroxidation and externalization of phosphatidylserine

The role of phospholipids in apoptosis signaling and the relationship between oxidation of phosphatidylserine and its redistribution in the plasma membrane were studied. A novel method for detection of site-specific phospholipid peroxidation based on the use of cis-parinaric acid as a reporter molecule metabolically integrated into membrane phospholipids in living cells was employed. When several tissue culture cell lines and different exogenous oxidants were used, the relationship between the oxidation of phosphatidylserine and apoptosis has been revealed. The plasma membrane was the preferred site of phosphatidylserine oxidation in cells. It was shown that selective oxidation of phosphatidylserine precedes its translocation from the inside to the outside surface of the plasma membrane during apoptosis. A model is proposed in which cytochrome c released from mitochondria by oxidative stress binds to phosphatidylserine located at the cytoplasmic surface of the plasma membrane and induces its oxidation. Interaction of peroxidized phosphatidylserine with aminophospholipid translocase causes inhibition of the enzyme relevant to phosphatidylserine externalization.

Presenilin-1-Dependent Transcriptome Changes

Familial forms of Alzheimers disease (FADs) are caused by the expression of mutant presenilin 1 (PS1) or presenilin 2. Using DNA microarrays, we explored the brain transcription profiles of mice with conditional knock-out of PS1 (cKO PS1) in the forebrain. In parallel, we performed a transcription profiling of the hippocampus and frontal cortex of the FAD-linked ΔE9 mutant transgenic (TG) mice and matched controls [TG mice expressing wild-type human PS1 (hPS1)]. When the TG and cKO datasets were cross-compared, the majority of the 30 common expression alterations were in opposite direction, suggesting that the FAD-linked PS1 variant produces transcriptome changes primarily by gain of aberrant function. Our microarray studies also revealed an unanticipated inverse correlation of transcript levels between the brains of mice that coexpress ΔE9 hPS1+ amyloid precursor protein (APP)695 Swe and ΔE9 hPS1 single transgenic mice. The opposite directionality of these changes in transcript levels must be a function of APP and/or APP derivatives.

Neuroblastoma as a Neurobiological Disease

While neuroscientists are often involved in the assessment and care of patients with central nervous system tumors, they are only rarely involved in the case of peripheral nervous system neoplasia. Neuroblastoma is a childhood tumor of the primitive sympathetic nervous system. It is at once one of the most common and one of the most deadly tumors of childhood. The prognosis for children with this tumor has not changed in the past two decades. Clearly, a fresh approach to neuroblastoma is needed. The neuroscientist has much to add to our understanding and treatment of neuroblastoma and its sequelae. Conversely, neuroblastoma has much to teach us regarding the normal development of the neural crest and the aberrant loss of neurons in this lineage. A neuroscientists approach to neuroblastoma, its biology and clinical features, is presented herein.

Expression and p75 neurotrophin receptor dependence of cholesterol synthetic enzymes in adult mouse brain

Normal brain function depends critically on cholesterol. Although cholesterol is synthesized locally in the adult brain, the precise anatomical localization of cholesterogenic enzymes is not known. Here we show that 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAred) and 7-dehydrocholesterol reductase (7dhcred), the first and last enzymes, respectively, in the cholesterol biosynthesis pathway, are co-expressed in neurons throughout adult murine brain. Co-localization is most prominent in cortical, hippocampal, and cholinergic neurons. Since adult hippocampal and cholinergic neurons express p75 neurotrophin receptors (p75NTR) we hypothesized that p75NTR regulates expression of cholesterogenic enzymes. Treatment of Neuro2a neuroblastoma cells or primary cerebellar cultures with siRNA downregulates p75NTR and decreases the expression level of HMG-CoAred and 7dhcred. Native neuroblastoma cell lines with differential expression of p75NTR differentially express 7dhcred; 7dhcred expression correlates with p75NTR expression. This suggests that, in p75NTR-expressing cells, p75NTR regulates cholesterol synthesis through regulation of HMG-CoAred and 7dhcred expression. The unexpected localization of cholesterogenic enzymes in adult neurons suggests that at least some adult neurons retain the ability to synthesize cholesterol.

DNA microarray profiling of developing PS1-deficient mouse brain reveals complex and coregulated expression changes.

Presenilin 1 (PS1) plays a critical role in the nervous system development and PS1 mutations have been associated with familial Alzheimers disease. PS1-deficient mice exhibit alterations in neural and vascular development and die in late embryogenesis. The present study was aimed at uncovering transcript networks that depend on intact PS1 function in the developing brain. To achieve this, we analyzed the brains of PS1-deficient and control animals at embryonic ages E12.5 and E14.5 using MG_U74Av2 oligonucleotide microarrays by Affymetrix. Based on the microarray data, overall molecular brain development appeared to be comparable between the E12.5 and E14.5 PS1-deficient and control embryos. However, in brains of PS1-deficient mice, we observed significant differences in the expression of genes encoding molecules that are associated with neural differentiation, extracellular matrix, vascular development, Notch-related signaling and lipid metabolism. Many of the expression differences between wild-type and PS1-deficient animals were present at both E12.5 and E14.5, whereas other transcript alterations were characteristic of only one developmental stage. The results suggest that the role of PS1 in development includes influences on a highly co-regulated transcript network; some of the genes participating in this expression network may contribute to the pathophysiology of Alzheimers disease.

Glutamate-induced cytotoxicity in PC12 pheochromocytoma cells: role of oxidation of phospholipids, glutathione and protein sulfhydryls revealed by bcl-2 transfection

Incubation of mock-transfected PC12 rat pheochromocytoma cells (PC12) for 2 h with increasing concentrations of glutamate caused progressive loss of viability (e.g., 67% with 15 mM glutamate). In contrast, the viability of bcl-2-transfected cells (PC12/bcl-2) was unaffected by glutamate. Neither PC12 nor PC12/bcl-2 cells showed a significant incidence of apoptosis in response to glutamate. Conventional phospholipid analysis by high-performance TLC and phosphorous determination showed no significant changes in the phospholipid composition of either cell line incubated with </=15 mM glutamate. Phospholipid peroxidation was quantified in the cells using our newly developed method based on fluorescence-HPLC analysis of metabolically incorporated oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid. Unlike previous studies that measured total phospholipid oxidation, this novel technology permitted quantitation of oxidative stress in different classes of labeled phospholipids (the amount of labeled phospholipids in the cells did not exceed 1% of total phospholipids). Significant peroxidation of phosphatidylcholine and phosphatidylethanolamine occurred in PC12 cells treated with >5 mM glutamate. The peroxyl radical initiator 2,2-azobis(2,4-dimethylvaleronitrile) caused a pronounced loss of all major phospholipid classes in PC12 cells, but no loss of cell viability. No phospholipid peroxidation was detected in PC12/bcl-2 cells incubated with </=15 mM glutamate or with 2, 2-azobis(2,4-dimethylvaleronitrile). These results directly demonstrate that peroxidation of membrane phospholipids is not responsible for the cytotoxicity of glutamate in PC12 cells. Total cellular thiol, protein thiol and GSH reserves were quantified by a previously described electron paramagnetic resonance spectrometric method. Total thiols were ca. 1.5-fold greater in PC12/bcl-2 than in PC12 cells. Glutamate (</=5 mM) caused a progressive and equally significant decrease in total thiols and GSH in both PC12 and PC12/bcl-2 cells. High glutamate concentrations caused oxidation of protein sulfhydryls in PC12 cells, but not in PC12/bcl-2 cells. The results suggest that the changes in cellular milieu caused by bcl-2 gene transfection protect PC12 cells from the toxic effects of glutamate in a manner consistent with prevention of protein sulfhydryl oxidation.

Apoptosis in development and disease of the nervous system: II. Apoptosis in childhood neurologic disease

The loss of cells in the human nervous system has long been known as the hallmark of incurable degenerative disease. Recent studies that began with attempts to understand cell loss during normal development have now begun to contribute to our understanding of the process of pathological cell loss. In many neurodegenerative conditions, it has become clear that apoptosis, or programmed cell death, plays a role in the diminution of cell number. In the cases of human immunodeficiency virus-associated encephalopathy and several of the hereditary neurodegenerative disorders, triggers and mediators of this process have been identified. This identification is not only the first step toward treatment of such disorders, but it also raises the possibility of exploiting this information to design targeted apoptosis-based therapies for tumors of the nervous system.

P75 neurotrophin receptor regulates expression of neural cell adhesion molecule 1

Our recent transcriptome profiling studies suggest that presenilin 1 (PS1) regulates expression of neural cell adhesion molecule (Ncam1) through p75 neurotrophin receptor. To better understand regulation of Ncam1 transcript and protein levels by p75, we performed a series of in vitro and in vivo experiments. The combined results suggest that p75 receptor is required for both resting and NGF-induced Ncam1 expression. Activation of TrkA receptors alone does not upregulate Ncam1. The normal Ncam1 expression depends on the relative ratio of TrkA and p75 receptors, and p75 extracellular domain is necessary for baseline Ncam1 expression. NGF-induced Ncam1 expression is dependent on the presence of an intact palmitoylation site within p75 receptor. Finally, we show that the expression of Ncam1 is altered in brains of two transgenic mouse lines that express familial Alzheimers disease (FAD)-linked PS1 variants, suggesting that expression of dominantly inherited mutant PS1 genes interferes with the normal Ncam1 expression via the p75 signaling pathway.

Cerebellar glutamine synthetase in children after hypoxia or ischemia.

Background Glutamate has been implicated in the pathophysiology of acute hypoxic-ischemic encephalopathy. Glutamine synthetase is an enzyme found in astrocytes that converts glutamate to its nontoxic analogue, glutamine. The present study tests the hypothesis that brain glutamine synthetase activity increases in response to acute hypoxic-ischemic insults and not in response to chronic hypoxia-ischemia or non-hypoxic-ischemic neurological disease. Summary of Report Frozen sections of cerebellum from children who died with acute or chronic hypoxic-ischemic insults or chronic non-hypoxic-ischemic neurological disease were spectrophotometrically assayed for glutamine synthetase activity by an observer who was blinded to the clinical group assignment of each specimen. Enzyme activity was elevated in specimens from children with acute hypoxic-ischemic insults (mean 6.5; range 5.4-7.2 units/g wet tissue wt) as compared with those from patients with chronic hypoxia-ischemia (mean 2.8; range 0.7-10.2 units/g wet tissue wt) or with non-hypoxic-ischemic neurological disease (mean 2.6; range 1.3-3.9 units/g wet tissue wt). This difference was not due to differences in the degree of histological astrocytosis or edema among the specimens. Statistical analysis by the Kruskal-Wallis one-way analysis of variance by ranks test indicates that the three data groups do not come from one population (p<0.05). Conclusions These results support the notion that glutamine synthetase activity increases in response to acute hypoxic-ischemic nervous system injury in children and that other compensatory mechanisms prevail in the case of chronic hypoxic-ischemic insults.