Chronic activation in presymptomatic amyotrophic lateral sclerosis (ALS) mice of a feedback loop involving Fas, Daxx, and FasL.

The reasons for the cellular specificity and slow progression of motoneuron diseases such as ALS are still poorly understood. We previously described a motoneuron-specific cell death pathway downstream of the Fas death receptor, in which synthesis of nitric oxide (NO) is an obligate step. Motoneurons from ALS model mice expressing mutant SOD1 showed increased susceptibility to exogenous NO as compared with controls. Here, we report a signaling mechanism whereby NO leads to death of mutant, but not control, motoneurons. Unexpectedly, exogenous NO triggers expression of Fas ligand (FasL) in cultured motoneurons. In mutant SOD1(G93A) and SOD1(G85R), but not in control motoneurons, this up-regulation results in activation of Fas, leading through Daxx to phosphorylation of p38 and further NO synthesis. This Fas/NO feedback amplification loop is required for motoneuron death in vitro. In vivo, mutant SOD1(G93A) and SOD1(G85R) mice show increased numbers of positive motoneurons and Daxx nuclear bodies weeks before disease onset. Moreover, FasL up-regulation is reduced in the presence of transgenic dominant-negative Daxx. We propose that chronic low-level activation of the Fas/NO feedback loop may underlie the motoneuron loss that characterizes familial ALS and may help to explain its slowly progressive nature.

Neural adhesion molecules L1 and CHL1 are survival factors for motoneurons.

Many neurotrophic factors with survival activity for motoneurons in vivo were first identified using cultures of purified embryonic motoneurons. The L1 neural cell adhesion molecule has multiple roles in brain development. We showed by in situ hybridization and RT-PCR that L1 mRNA was expressed at significant levels in motoneurons of embryonic and postnatal spinal cord. We therefore cultured purified motoneurons from E14 rat embryos in the absence of trophic factors but with L1-Fc and CHL1-Fc fusion proteins. L1-Fc prevented the death of approximately half of the motoneurons that were saved by BDNF in a dose-dependent manner (EC50 = 10 pM). CHL1-Fc saved the same number of motoneurons as did L1-Fc, whereas P0-Fc had little neurotrophic activity at the same concentrations. Survival induced by L1 and CHL1 was completely inhibited by 20 microM LY294002 and PD98059, indicating that both MEK and PI3K pathways are required for signaling by these molecules. L1 can signal in other cell types through the FGF receptor FGFR1. In cultures of motoneurons, effects of suboptimal concentrations of L1 and suboptimal concentrations of FGF-2 were additive, but the effects of optimal concentrations of FGF-2 (50 ng/ml) were not further increased in the presence of L1-Fc. Thus, in this system, too, FGF and L1 may use similar signaling pathways.

Expression of vesicular glutamate transporters, VGLUT1 and VGLUT2, in cholinergic spinal motoneurons.

Mammalian spinal motoneurons are cholinergic neurons that have long been suspected to use also glutamate as a neurotransmitter. We report that VGLUT1 and VGLUT2, two subtypes of vesicular glutamate transporters, are expressed in rat spinal motoneurons. Both proteins are present in somato-dendritic compartments as well as in axon terminals in primary cultures of immunopurified motoneurons and sections of spinal cord from adult rat. However, VGLUT1 and VGLUT2 are not found at neuromuscular junctions of skeletal muscles. After intracellular injection of biocytin in motoneurons, VGLUT2 is observed in anterogradely labelled terminals contacting Renshaw inhibitory interneurons. These VGLUT2- and VGLUT1-positive terminals do not express VAChT, the vesicular acetylcholine transporter. Overall, our study establishes for the first time that (i) mammalian spinal motoneurons express vesicular glutamate transporters, (ii) these motoneurons have the potential to release glutamate (in addition to acetylcholine) at terminals contacting Renshaw cells, and finally (iii) the VGLUTs are not present at neuromuscular synapses of skeletal muscles.

Responsiveness to neurturin of subpopulations of embryonic rat spinal motoneuron does not correlate with expression of GFR alpha 1 or GFR alpha 2.

Glial cell-line derived neurotrophic factor (GDNF) and its relative neurturin (NTN) are both potent trophic factors for motoneurons. They exert their biological effects by activating the RET tyrosine kinase in the presence of a GPI-linked coreceptor, either GFR alpha 1 (considered to be the favored coreceptor for GDNF) or GFR alpha 2 (the preferred NTN coreceptor). By whole-mount in situ hybridization on embryonic rat spinal cord, we demonstrate that, whereas Ret is expressed by nearly all motoneurons, Gfra1 and Gfra2 exhibit complementary and sometimes overlapping patterns of expression. In the brachial and sacral regions, the majority of motoneurons express Gfra1 but only a minority express Gfra2. Accordingly, most motoneurons purified from each region are kept alive in culture by GDNF. However, brachial motoneurons respond poorly to NTN, whereas NTN maintains as many sacral motoneurons as does GDNF. Thus, spinal motoneurons are highly heterogeneous in their expression of receptors for neurotrophic factors of the GDNF family, but their differing responses to NTN are not correlated with expression levels of Gfra1 or Gfra2.

Solution structure and dynamics of the central CCP module pair of a poxvirus complement control protein.

The complement control protein (CCP) module (also known as SCR, CCP or sushi domain) is prevalent amongst proteins that regulate complement activation. Functional and mutagenesis studies have shown that in most cases two or more neighbouring CCP modules form specific binding sites for other molecules. Hence the orientation in space of a CCP module with respect to its neighbours and the flexibility of the intermodular junction are likely to be critical for function. Vaccinia virus complement control protein (VCP) is a complement regulatory protein composed of four tandemly arranged CCP modules. The solution structure of the carboxy-terminal half of this protein (CCP modules 3 and 4) has been solved previously. The structure of the central portion (modules 2 and 3, VCP approximately 2,3) has now also been solved using NMR spectroscopy at 37 degrees C. In addition, the backbone dynamics of VCP approximately 2,3 have been characterised by analysis of its (15)N relaxation parameters. Module 2 has a typical CCP module structure while module 3 in the context of VCP approximately 2,3 has some modest but significant differences in structure and dynamics to module 3 within the 3,4 pair. Modules 2 and 3 do not share an extensive interface, unlike modules 3 and 4. Only two possible NOEs were identified between the bodies of the modules, but a total of 40 NOEs between the short intermodular linker of VCP approximately 2,3 and the bodies of the two modules determines a preferred, elongated, orientation of the two modules in the calculated structures. The anisotropy of rotational diffusion has been characterised from (15)N relaxation data, and this indicates that the time-averaged structure is more compact than suggested by (1)H-(1)H NOEs. The data are consistent with the presence of many intermodular orientations, some of which are kinked, undergoing interconversion on a 10(-8)-10(-6) second time-scale. A reconstructed representation of modules 2-4 allows visualisation of the spatial arrangement of the 11 substitutions that occur in the more potent complement inhibitor from Variola (small pox) virus.

Specific expression of the urotensin II gene in sacral motoneurons of developing rat spinal cord.

The neuropeptide urotensin II (UII) is expressed in motoneurons of the brainstem and spinal cord in adults. Here, the expression pattern of the UII gene was studied in the developing rat spinal cord. UII mRNA was detected by reverse-transcription-polymerase chain reaction (RT-PCR) as early as E10. From E14 to E21, in situ hybridization revealed intense expression of the UII gene specifically in sacral motoneurons, while only faint expression was detected at cervical and thoracic levels. After birth (P0, P4), the expression of UII mRNA increased in motoneurons at all rostrocaudal levels. Thus, UII is the first gene reported to show expression limited to the sacral pool of motoneurons, which are known to have particular properties in terms of targets and programmed cell death.

Predicting asthma severity from allergic sensitivity to cockroaches in pregnant inner city women.

OBJECTIVE: To measure and compare cockroach (CR)-specific immunoglobin E (IgE) in sera from pregnant women with mild, moderate and severe asthma. STUDY DESIGN: CR IgE levels were measured in stored sera collected during the Collaborative Perinatal Project. Three matched groups of 93 women were formed: group I (mild), history of asthma but no acute exacerbation; group II (moderate), acute asthma exacerbation; group III (severe), required hospitalization for a diagnosis of status asthmaticus. ANOVA was used to compare the three means. RESULTS: Mean CR IgE paralleled prenatal asthma severity. Mean values were 6.50, 13.12 and 28.99 kU/L for groups I, II and III, respectively (P = .06). High allergen sensitivity, defined as CR IgE > 60 kU/L, was identified in 8 of the 93 study samples. The prevalence of high allergen sensitivity increased as clinical asthma became more severe. Sixty-two percent (5/8) of the high allergen sensitivity occurred in group III. CONCLUSION: There appears to be a positive correlation between sensitivity to CR allergens and asthma severity during pregnancy, and these findings support further evaluation of CR allergen sensitivity as a predictor of asthma severity in pregnancy.

Efficient gene transfer and expression of biologically active glial cell line-derived neurotrophic factor in rat motoneurons transduced wit lentiviral vectors.

Several studies have shown the ability of human immunodeficiency virus type 1 (HIV1)-based lentiviral vectors to infect nondividing brain and retinal neurons with high efficiency and long-term expression of the transduced gene. We show that purified embryonic motoneurons can be efficiently (>95%) transduced in culture using an HIV1-based lentiviral vector encoding LacZ. Expression of beta-galactosidase was observed for at least 9 days in these conditions. Furthermore, motoneurons transduced with a lentiviral vector expressing glial cell line-derived neurotrophic factor survived in the absence of additional trophic support, showing that the overexpressed protein was biologically active. Our results demonstrate the potential of lentiviral vectors in studying the biological effects of proteins expressed in motoneurons and in the development of future gene therapy for motoneuron diseases.

Active killing of neurons during development and following stress: a role for p75(NTR) and Fas?

Evidence for active triggering of neuronal death continues to accumulate. The transmembrane receptors p75(NTR) and Fas can trigger (and in some cases are required for) programmed cell death of the neurons that express them, through signalling pathways that are regulated by a variety of cytoplasmic effectors. Neuronal death induced by trophic deprivation often requires Fas signalling, further blurring the boundaries between naturally occurring and stress-induced neuronal death.

Influence of factors secreted by wobbler astrocytes on neuronal and motoneuronal survival.

During late postnatal development, mice with the autosomal recessive wobbler mutation (wr/wr) develop motoneuron degeneration associated with astrogliosis in the spinal cord. In vitro, primary wobbler astrocytes are also affected, exhibiting abnormal cell-cell contacts. To characterize further the wobbler disease, we investigated the in vitro effects of wobbler astrocytes on primary neuronal cultures from the spinal cords of 15-day-old wild-type mouse and rat embryos. Cocultures with the wobbler astrocytes, or direct addition of wobbler astrocyte-conditioned medium, led to a decrease in neuron number in primary mixed neuronal cultures, containing motoneurons and interneuron-like cells. In contrast, wobbler astrocyte-conditioned medium enhanced survival of highly purified motoneurons. These in vitro results suggest the possibility that wobbler astrocytes act not on motoneurons directly but, rather, through other spinal neurons to induce motoneuron degeneration in the wobbler disease.

Reg-2 is a motoneuron neurotrophic factor and a signalling intermediate in the CNTF survival pathway.

Cytokines that are related to ciliary neurotrophic factor (CNTF) are physiologically important survival factors for motoneurons, but the mechanisms by which they prevent neuronal cell death remain unknown. Reg-2/PAP I (pancreatitis-associated protein I), referred to here as Reg-2, is a secreted protein whose expression in motoneurons during development is dependent on cytokines. Here we show that CNTF-related cytokines induce Reg-2 expression in cultured motoneurons. Purified Reg-2 can itself act as an autocrine/paracrine neurotrophic factor for a subpopulation of motoneurons, by stimulating a survival pathway involving phosphatidylinositol-3-kinase, Akt kinase and NF-kappaB. Blocking Reg-2 expression in motoneurons using Reg-2 antisense adenovirus specifically abrogates the survival effect of CNTF on cultured motoneurons, indicating that Reg-2 expression is a necessary step in the CNTF survival pathway. Reg-2 shows a unique pattern of expression in late embryonic spinal cord: it is progressively upregulated in individual motoneurons on a cell-by-cell basis, indicating that only a fraction of motoneurons in a given motor pool may be exposed to cytokines. Thus, Reg-2 is a neurotrophic factor for motoneurons, and is itself an obligatory intermediate in the survival signalling pathway of CNTF-related cytokines.

Programmed cell death of embryonic motoneurons triggered through the Fas death receptor.

About 50% of spinal motoneurons undergo programmed cell death (PCD) after target contact, but little is known about how this process is initiated. Embryonic motoneurons coexpress the death receptor Fas and its ligand FasL at the stage at which PCD is about to begin. In the absence of trophic factors, many motoneurons die in culture within 2 d. Most (75%) of these were saved by Fas-Fc receptor body, which blocks interactions between Fas and FasL, or by the caspase-8 inhibitor tetrapeptide IETD. Therefore, activation of Fas by endogenous FasL underlies cell death induced by trophic deprivation. In the presence of neurotrophic factors, exogenous Fas activators such as soluble FasL or anti-Fas antibodies triggered PCD of 40-50% of purified motoneurons over the following 3-5 d; this treatment led to activation of caspase-3, and was blocked by IETD. Sensitivity to Fas activation is regulated: motoneurons cultured for 3 d with neurotrophic factors became completely resistant. Levels of Fas expressed by motoneurons varied little, but FasL was upregulated in the absence of neurotrophic factors. Motoneurons resistant to Fas activation expressed high levels of FLICE-inhibitory protein (FLIP), an endogenous inhibitor of caspase-8 activation. Our results suggest that Fas can act as a driving force for motoneuron PCD, and raise the possibility that active triggering of PCD may contribute to motoneuron loss during normal development and/or in pathological situations.

Patterns of programmed cell death in populations of developing spinal motoneurons in chicken, mouse, and rat.

During embryonic development, approximately one-half of the spinal motoneurons initially generated are lost during a wave of programmed cell death (PCD). Classical studies in this system laid the basis of much work on the role and control of neuronal cell death during development. However, we have little information concerning the timing of cell death in motoneuron pools at different rostrocaudal levels, especially in rodents. We developed a novel protocol for whole-mount TUNEL labeling that allows apoptotic nuclei to be visualized in whole-mount preparations of embryonic spinal cord; double labeling with antibodies to Islet 1/2 showed that nearly all TUNEL-positive cells were motoneurons. In chicken and mouse embryos, the density of TUNEL-positive nuclei was specifically increased following target ablation. The pattern of naturally occurring motoneuron PCD was studied in spinal cords from different species and ages: chick (E4.5-E9.0), mouse (E11.5-E15.5), and rat (E13.5-E16. 5). In all species, motoneuron PCD is first apparent at cervical levels and last at sacral levels. However, motoneuron PCD does not follow a strict rostrocaudal sequence. Following cervical motoneuron PCD, TUNEL profiles are first observed at lumbar levels in chick but at thoracic levels in rat. At a given rostrocaudal level, medial motoneurons tend to die before lateral populations, but here too there are exceptions. Motoneuron cell death is thus regulated in a highly stereotyped manner during development of vertebrate spinal cord. Our technique will provide a basis for the monitoring even localized changes in this pattern.

Role of neurotrophic factors in motoneuron development.

More than 10 factors from different gene families are now known to enhance motoneuron survival, and to be expressed in a manner consistent with a role in regulating motoneuron numbers during development. We provide evidence that: a) different factors may act on different sub-populations of motoneurons; b) different factors may act in synergy on a given motoneuron. Thus, the functional diversity of motoneurons, and the cellular complexity of their environment, may be reflected in the mechanisms that have evolved to keep them alive.

Stability of immunoglobulin E (IgE) in stored obstetric sera.

OBJECTIVE: To determine the stability of immunoglobulin E levels in obstetric sera. METHODS: AlaSTAT(R) and AlaTOP(R) (Diagnostic Products) were used to assay total and specific IgE levels in obstetric sera collected in Memphis, TN and Portland, OR. The samples were collected from the Collaborative Perinatal Project (CPP) between 1959 and 1965 and stored at -20 degrees C. The assay results were compared with IgE levels found in sera collected at the same locations for the Calcium for Pre-eclampsia Prevention Study (CPEP) and stored since 1992 at -70 degrees C. The samples were also assayed for cockroach (CR) and mouse urine specific IgE using the AlaSTAT(R) assay (Diagnostic Products). RESULTS: Total IgE and specific IgE to CR and mouse urine were detectable in older and recent samples. The median total IgE for the recent and older Portland samples was 26 IU/ml and 65 IU/ml, respectively. The median total IgE was identical (40 IU/ml) in the recent and older Memphis samples. CONCLUSION: Long-term storage does not diminish the ability to measure serum IgE. Levels of IgE in sera stored 32-37 years were equal to or greater than levels in sera stored for 5 years. reserved.

Calpain inhibitors, but not caspase inhibitors, prevent actin proteolysis and DNA fragmentation during apoptosis.

Apoptosis, or programmed cell death, involves a cascade of regulatory events leading to the activation of specific proteases. However, the key substrates for these proteases remain to be identified. We previously demonstrated that levels of five unidentified polypeptides were specifically increased in neurons from embryonic chicken ciliary ganglia undergoing apoptosis by trophic deprivation. Here we show by microsequencing of two of these polypeptides that they are fragments of actin. One of them represents cleavage of actin at the site of interaction with DNase I. The same actin fragments are also found at early stages of apoptosis in chicken and rat dorsal root ganglion neurons, chicken spinal motoneurons and rat thymocytes. Actin fragmentation may play a role in the apoptotic process, since calpain inhibitors I and II both inhibit neuronal death and suppress actin fragmentation. In contrast, caspase (ICE family) inhibitors, though effective in delaying neuronal death, do not prevent actin cleavage or DNA fragmentation. These results indicate a key role for calpain-like proteases in neuronal programmed cell death and suggest that actin fragmentation in the cell is correlated with subsequent DNA fragmentation.

Custodial grandparenting and the impact of grandchildren with problems on role satisfaction and role meaning.

This study compared three groups of grandparents, attempting to disentangle grandparental role demands from child-specific problems as sources of role-specific and grandchild-relationship distress. Those grandparents raising grandchildren reported to demonstrate neurological, physical, emotional, or behavioral problems exhibited the most personal distress, the least role satisfaction and role meaning, and the most deteriorated grandparent-grandchild relationships. Custodial grandparents raising grandchildren reported to have few difficulties also differed in the ways listed above from those grandparents not raising their grandchildren and from those raising grandchildren displaying problems. For men, but not women, more positive grandparent meaning was associated with raising a grandchild. Significantly, custodial grandparents were more likely to be raising boys, suggesting that boys may be either more difficult for grandparents to raise or that boys react more negatively to the adverse circumstances under which grandparents assume care.

Synergistic effects of schwann- and muscle-derived factors on motoneuron survival involve GDNF and cardiotrophin-1 (CT-1).

The survival of central neurons depends on multiple neurotrophic factors produced by different cell types. We demonstrate that media conditioned by muscle and Schwann cell lines show strong synergistic effects on survival of purified embryonic day 14.5 rat motoneurons in culture. Different lines of evidence implicate glial cell line-derived neurotrophic factor (GDNF) and cardiotrophin-1 (CT-1) in this synergy. Their expression in the environment of the motoneuron is compartmentalized: gdnf transcripts are expressed principally in Schwann cell lines, whereas ct-1 mRNA is present in myotubes. Blocking antibodies to GDNF inhibit the trophic activity of Schwann cell line-conditioned media by 75%, whereas CT-1 antibodies diminish the myotube-derived activity by 46%. CT-1 and GDNF act synergistically to enhance motoneuron survival in vitro. In vivo, individual motoneurons coexpress both GDNF and CT-1 receptor components. GDNF and CT-1, therefore, are major components of the trophic support provided by the Schwann and muscle cells, respectively. The possibility that they act together on individual motoneurons suggests that the motoneuron must integrate distinct signals from different cellular partners when deciding whether to die or to survive.