Cerebellar Purkinje cell p75 neurotrophin receptor and autistic behavior.

The p75 neurotrophin receptor (p75NTR) is normally expressed in cerebellar Purkinje cells throughout the lifespan. Children with autism spectrum behavior exhibit apparent cerebellar Purkinje cell loss. Cerebellar transcriptome changes seen in the murine prenatal valproate exposure model of autism include all of the proteins known to constitute the p75NTR interactome. p75NTR is a modulator of cytoplasmic and mitochondrial redox potential, and others have suggested that aberrant response to oxidant stress has a major role in the pathogenesis of autism. We have created Purkinje cell-selective p75NTR knockout mice that are the progeny of hemizygous Cre-Purkinje cell protein 2 C57Bl mice and p75NTR floxed C57Bl mice. These Cre-loxP mice exhibit complete knockout of p75NTR in ~50% of the cerebellar Purkinje cells. Relative to Cre-only mice and wild-type C57Bl mice, this results in a behavioral phenotype characterized by less allogrooming of (P<0.05; one-way analysis of variance) and socialization or fighting with (each P<0.05) other mice; less (1.2-fold) non-ambulatory exploration of their environment than wild-type (P<0.01) or Cre only (P<0.01) mice; and almost twofold more stereotyped jumping behavior than wild-type (P<0.05) or Cre (P<0.02) mice of the same strain. Wild-type mice have more complex dendritic arborization than Cre-loxP mice, with more neurites per unit area (P<0.025, Student's t-test), more perpendicular branches per unit area (P<0.025) and more short branches/long neurite (P<0.0005). Aberrant developmental regulation of expression of p75NTR in cerebellar Purkinje cells may contribute to the pathogenesis of autism.

Exploiting oxidative stress and signaling in chemotherapy of resistant neoplasms.

Neural crest tumors of childhood are particularly resistant to apoptosis induction by chemotherapeutic agents. Mechanisms of resistance include altered glutathione handling that accompanies up-regulation of Bcl-2 and its relatives. We have designed and tested in preclinical model systems approaches to this problem. These approaches include adjunctive use of oxygen radical-generating neurotransmitter analogs taken up by these neural crest tumor cells with scavenging (i.e., "rescue") agents that are selective for normal neural crest and the use of reduction-dependent prodrugs of apoptosis-inducing agents. Promising prototypes for these conceptual approaches include, respectively, adjunctive use of the oxygen radical generator, 6-hydroxydopamine, with the normal cell-selective antioxidant, Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), and use of the reduction-dependent chemotherapeutic prodrug neocarzinostatin.

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 Alzheimer's 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 Alzheimer's disease.

Early events in Bcl-2-enhanced apoptosis.

Transfection of PC12 pheochromocytoma cells with bcl-2 potentiates apoptosis induced by the antimitotic agent, neocarzinostatin (NCS). The mechanism of potentiation involves caspase 3-dependent cleavage of Bcl-2 to its pro-apoptotic counterpart, but the cellular events proximal to caspase 3 activation in this system are not known. Two min after initiation of NCS treatment, Bax begins to translocate from cytosol to the mitochondria; the mitochondrial localization of Bax persists for 30 min after NCS treatment. At the same time, cytochrome C is released from the mitochondria to cytosol. The mitochondrial membrane potential exhibits differential change in mock- and bcl-2 -transfected PC12 cells. In mock-transfected PC12 cells, the mitochondrial membrane potential increases immediately, peaks at 15 min following initiation of NCS treatment, and drops thereafter. In contrast, in bcl-2 -transfected PC12 cells, the membrane potential drops immediately following NCS treatment. Caspase 9 is activated and peaks at 10 min in both mock- and bcl-2 transfected PC12 cells, however, the peak activity of caspase 9 is higher and caspase 9 activation lasts longer (30 min) after the treatment in bcl-2 transfectants. Not until 30 min after initiation of a 1 h treatment with NCS is Bcl-2 protein cleaved in bcl-2 -transfected cells. Thus, in bcl-2 -transfected cells, the mitochondrial membrane potential drops and cytochrome C is released from the mitochondria despite the presence of large amounts of intact mitochondrial Bcl-2. This makes it unlikely that cleavage of Bcl-2 is the only factor involved in potentiation of NCS-induced apoptosis by Bcl-2.

Signal transduction for clinicians: why should we care?

Neurons must respond to a bewildering array of external and internal stimuli and must distinguish among them to generate an appropriate response or change in metabolic or electrical activity. Furthermore, the response of a cell to a given stimulus must depend on what else is happening inside and outside the cell at the time of arrival of that stimulus. The process of signal transduction is what gives the cell and organism the flexibility and "knowledge base" to carry out these functions. Conversely, aberrations of signal transduction underlie an increasing array of developmental, genetic, and acquired diseases and conditions of the nervous system. Pharmacological modulation of signal transduction pathways and their effectors holds great promise for the remediation of these neurologic disorders.

Apoptosis in the absence of caspase 3.

MCF-7 human breast cancer cells do not express caspase 3, thought by some to be a critical component of the apoptosis cascade. Nonetheless, both mock- and bcl-2-transfected MCF-7 cells undergo apoptosis after treatment with a variety of stimuli, including the DNA-cleaving antimitotic agent, neocarzinostatin (NCS). Transfection with bcl-2 shifts the concentration-response curve to NCS but does not change the phenomenology of apoptosis when it occurs. In both cases, NCS treatment results in condensation and fragmentation of MCF-7 cell nuclei and release of cytochrome c from the mitochondria to the cytosol. This apoptosis is accompanied by decreased levels of Bcl-2 and increased levels of Bax. Using a series of caspase inhibitors with overlapping specificities, enzyme-specific chromogenic substrates, and an antibody specific for activated caspase 7, we have determined that apoptosis in MCF-7 cells proceeds via sequential activation of caspases 9, 7 and 6. P21 is detected only after activation of caspase 7, and P53 is neither expressed at baseline nor up-regulated with apoptosis induction. This pathway bypasses the need for activated caspase 3 in these cells.

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.

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.

Neurology of systemic autoimmune disorders: a pediatric perspective.

Autoimmune disorders can involve patients of any age and organs of any organ system. The central and peripheral nervous systems are frequently among the targets of these diseases. Immune dysfunction often presents in childhood or adolescence. Among the autoimmune disorders that present during childhood and adolescence, systemic lupus erythematosus, dermatomyositis, and Behcet's disease affect the nervous system with some degree of frequency. Furthermore, although juvenile rheumatoid arthritis only rarely affects the nervous system during childhood, it and its adult-onset counterpart may have profound long-term neurological consequences. Both symptomatic and pathophysiologically aimed therapies are important in the treatment of the nervous system sequelae of systemic autoimmune disorders.

Interaction between 6-hydroxydopamine and transferrin: "Let my iron go".

The dopamine analogue 6-hydroxydopamine (6-OHDA) is selectively toxic to catecholaminergic neurons. Because of its selectivity for neuroblastic cells in the sympathetic nervous system lineage, 6-OHDA has been suggested as a chemotherapeutic agent for targeted treatment of patients with neuroblastoma. We tested the hypothesis that the toxicity of 6-OHDA is caused by its interaction with serum ferric transferrin (Fe-TF) resulting in release of iron. We further hypothesized that this iron, through its redox-cycling by 6-OHDA, triggers generation of reactive oxygen species. 6-OHDA-induced release of iron from Fe-TF was demonstrated by: (1) low-temperature EPR spectroscopic evidence for decay of the characteristic Fe-TF signal (g = 4.3) and appearance of the high-spin signal from iron chelated by 6-OHDA oxidation products; (2) spectrophotometric detection of complexing of iron with the Fe(2+) chelator ferrozine; (3) redox-cycling of ascorbate yielding EPR-detectable ascorbate radicals; and (4) generation of hydroxyl radicals as evidenced by EPR spectroscopy of their adduct with a spin trap, 5, 5'-dimethylpyrroline oxide (DMPO) (DMPO-OH). Our low-temperature EPR studies showed that in human plasma, 6-OHDA caused iron release only under nitrogen gas but not under air or oxygen. The absence of a 6-OHDA effect in plasma under aerobic conditions was most likely due to its ferroxidase activity [with consequent reuptake of Fe(III) by apoTF] and catalytic oxidation of 6-OHDA by ceruloplasmin. Modeling of these plasma activities by a stable nitroxide radical, 2,2,6, 6-tetramethyl-1-piperidinyloxy (TEMPOL), resulted in protection of plasma Fe-TF against iron release under nitrogen. Parenteral administration of 6-OHDA to mice resulted in iron release from Fe-TF as evidenced by transformation of the Fe-TF low-temperature EPR signal that was indistinguishable from that seen in in vitro models. In addition, administration of the iron chelator deferoxamine (DFO) to mice prior to administration of toxic doses of 6-OHDA resulted in a decrease in activity impairment of mice as compared to that seen with 6-OHDA alone. These findings underscore the physiological and pharmacological relevance of 6-OHDA-mediated iron release from Fe-TF and suggest that iron chelators (DFO) may be used for prevention of 6-OHDA toxicity.

Cell line dependence of Bcl-2-induced alteration of glutathione handling.

Bcl-2 has been associated with both oxidative and antioxidative effects in vivo. Moreover, despite evidence that Bcl-2 is antiapoptotic by virtue of its effect on reactive oxygen species and their scavengers, Bcl-2 exerts its antiapoptotic effects even under anaerobic conditions. The reasons for the variable relationship between Bcl-2 and reactive oxygen species are not clear. The present studies demonstrate that the impact of Bcl-2 on glutathione (GSH) metabolism is cell line-dependent. Bcl-2 overproduction in PC12 cells is associated with increased functional thiol reserves, increased reductive activation of chemotherapeutic prodrugs, and GSH accumulation after treatment with N-acetylcysteine. In contrast, Bcl-2-overproducing MCF-7 breast cancer cells demonstrate neither altered GSH handling nor potentiation of chemotherapeutic prodrug reduction. These findings indicate that the effects of Bcl-2 on GSH handling are millieu-dependent. This could account for the variable effects of Bcl-2 in in vivo systems. Furthermore, since our previous studies have demonstrated that reduction-dependent prodrugs may be useful chemotherapeutic agents against tumors that demonstrate altered GSH handling, screening in vitro for alteration of GSH handling may predict responsiveness of such tumors to these reduction-dependent agents.

Selective oxidation and externalization of membrane phosphatidylserine: Bcl-2-induced potentiation of the final common pathway for apoptosis.

The induction of apoptosis in PC12 cells by the enediyne neocarzinostatin (NCS) is paradoxically potentiated by overexpression of bcl-2. The enhanced activation of NCS seen in bcl-2-overexpressing cells cannot by itself be responsible for the potentiation of apoptosis, since Bcl-2 would be expected to block apoptosis at a point distal to NCS activation (e.g., in the apoptosis final common pathway). We now report that overexpression of bcl-2 in PC12 cells does not protect the cells from NCS-induced oxidation of membrane phosphatidylserine (PS), and results in potentiation of NCS-induced externalization of membrane PS, two events associated with the apoptosis final common pathway. The mechanism of potentiation of apoptosis by Bcl-2 is related to the enhanced reducing potential of bcl-2-overexpressing PC12 cells.

Differential membrane antioxidant effects of immediate and long-term estradiol treatment of MCF-7 breast cancer cells.

Previous studies have documented the direct antioxidant effects of estradiol, and it is tempting to ascribe the antiapoptosis effects of estradiol to its scavenging of reactive oxygen species. However, recent reports have also demonstrated that long-term exposure of MCF-7 human breast cancer cells to estradiol results in estrogen receptor- and estradiol dose-dependent overexpression of the antiapoptosis gene, bcl-2. We have used the pattern of protection of membrane phospholipids from oxidation as a probe to separate these direct and indirect effects of estradiol from one another. Immediate exposure to estradiol non-specifically protects all membrane phospholipids from oxidation by the diazo radical initiator, AMVN. This implies the direct antioxidant activity of estradiol in this system. In contrast, long-term exposure, with associated increased expression of bcl-2, protects only phosphatidylserine, the oxidation of which is a critical component of the final common pathway for apoptosis. This bcl-2-mediated indirect effect of estradiol is accompanied by prevention of apoptosis in MCF-7 cells.

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 neuroscientist's approach to neuroblastoma, its biology and clinical features, is presented herein.

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 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

Whimpers and bangs: of death and dying in the nervous system.

Mechanisms of cell death in general and in the nervous system in particular have received increasing attention over the past several years. Recent studies have begun to address the pathogenetic and therapeutic implications of these mechanisms vis-a-vis human disease. This review is aimed at putting the rapidly evolving vocabulary and body of knowledge regarding cell death in the perspective of neurologic disease of childhood.

The roles of mitotic arrest and protein synthesis in induction of apoptosis and differentiation in neuroblastoma cells in culture.

Studies of the response of neural crest tumor cells to the DNA cleaving antimitotic agent, neocarzinostatin, have left unanswered the question of whether the DNA cleavage per se or the antimitotic effect is responsible for this response. Furthermore, they do not define the timeframe within which a cell commits to its fate. Using the reversible microtubule-active agent, vinblastine, we now demonstrate that mitotic arrest, even without DNA cleavage, results in the same cellular changes as those seen with neocarzinostatin treatment. The commitment of the cell to its fate occurs within a 15 min treatment with vinblastine, and requires new protein synthesis. The immediate early gene products, c-Fos and c-Jun, appear not to be determinants of this process.

Direct evidence for antioxidant effect of Bcl-2 in PC12 rat pheochromocytoma cells.

Mock-transfected PC12 rat pheochomocytoma cells and PC12 cells transfected with the bcl-2 gene, a gene associated with inhibition of apoptosis, were subjected to oxidative stress by incubation in the presence of the azo-initiator of lipid peroxyl radicals, 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). Extraction and chromatographic analysis by two-dimensional TLC of the major phospholipid classes showed no differences in the phospholipid composition between the mock- and bcl-2-transfected cell lines after incubation in the presence of 0.5 mM AMVN for 2 h at 37 degrees C. A method consisting of incorporation of cis-parinaric acid into the constituent membrane phospholipids before exposure to AMVN was developed to improve the sensitivity of detecting lipid peroxidation in PC12 cells. Analysis of the pattern of changes in parinaric acid-labeled phospholipids after exposure to 0.25 and 0.5 mM AMVN by HPLC showed significant oxidation of phosphatidylcholine (PC), phosphatidylethanolamine (PEA), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SPH) during a 2-h incubation. The extent of oxidation of each phospholipid class was dependent on the concentration of AMVN present up to 1 mM. Based on phospholipid fractional composition, the specific rates of PnA peroxidation in phospholipid classes were estimated. In mock-transfected PC12 cells, the order of AMVN-induced oxidation effectiveness was the same for both specific rates and relative rates: PC >> PEA > PS > SPH > PI. While a dramatic decrease in both relative and specific oxidation rates was observed for all phospholipid classes in bcl-2-transfected PC12 cells, the specific oxidation rates were higher for aminophospholipids (PEA and PS) than for other phospholipids. This suggests that antioxidant protection by bcl-2-related product(s) may be phospholipid-specific and that aminophospholipids are relatively less protected than the other phospholipids. The vitamin E analogue, 2,2,5,7,8-pentamethyl-6-hydrochromane, acted as an effective antioxidant in preventing oxidation of parinaric acid-labeled membrane phospholipids during incubation in the presence of AMVN and the extent of protection was approximately the same in both cell lines. Since, unlike the agents used to generate oxidative stress in other studies, temperature-driven generation of peroxyl radicals by AMVN is not dependent on intracellular metabolism, the results presented provide proof for antioxidant protection, rather than abrogation of radical generation afforded by bcl-2 transfection of PC12 cells.