Ronald W. Oppenheim

Naturally occurring and induced neuronal death in the chick embryo in vivo requires protein and RNA synthesis: Evidence for the role of cell death genes☆

Treatment of chick embryos in ovo for 10-12 hr with inhibitors of protein and RNA synthesis during the peak time of normal cell death (Embryonic Day 8) for motoneurons and dorsal root ganglion cells markedly reduces the number of degenerating neurons in these populations. The massive neuronal death induced by the early absence of the limbs was also blocked almost completely by these agents. Further, the death of neurons following peripheral axotomy at the end of the normal cell death period (Embryonic Day 10) was reduced significantly by treatment with inhibitors of biosynthetic reactions. These results indicate that, in vivo, naturally occurring neuronal death, neuronal death induced by the absence of peripheral targets, and axotomy-induced neuronal death later in development all require active gene expression and protein and RNA synthesis. Therefore, neuronal death in a variety of situations may reflect the expression of a developmental fate that can normally only be overridden or suppressed by specific environmental signals (e.g., neurotrophic molecules).

Complete Dissociation of Motor Neuron Death from Motor Dysfunction by Bax Deletion in a Mouse Model of ALS

The death of cranial and spinal motoneurons (MNs) is believed to be an essential component of the pathogenesis of amyotrophic lateral sclerosis (ALS). We tested this hypothesis by crossing Bax-deficient mice with mice expressing mutant superoxide dismutase 1 (SOD1), a transgenic model of familial ALS. Although Bax deletion failed to prevent neuromuscular denervation and mitochondrial vacuolization, MNs were completely rescued from mutant SOD1-mediated death. However, Bax deficiency extended lifespan and delayed the onset of motor dysfunction of SOD1 mutants, suggesting that Bax acts via a mechanism distinct from cell death activation. Consistent with this idea, Bax elimination delayed the onset of neuromuscular denervation, which began long before the activation of cell death proteins in SOD1 mutants. Additionally, we show that denervation preceded accumulation of mutant SOD1 within MNs and astrogliosis in the spinal cord, which are also both delayed in Bax-deficient SOD1 mutants. Interestingly, MNs exhibited mitochondrial abnormalities at the innervated neuromuscular junction at the onset of neuromuscular denervation. Additionally, both MN presynaptic terminals and terminal Schwann cells expressed high levels of mutant SOD1 before MNs withdrew their axons. Together, these data support the idea that clinical symptoms in the SOD1 G93A model of ALS result specifically from damage to the distal motor axon and not from activation of the death pathway, and cast doubt on the utility of anti-apoptotic therapies to combat ALS. Furthermore, they suggest a novel, cell death-independent role for Bax in facilitating mutant SOD1-mediated motor denervation.

Peptide inhibitors of the ice protease family arrest programmed cell death of motoneurons in vivo and in vitro

Members of the CED-3/interleukin-1 beta-converting enzyme (ICE) protease family have been implicated in cell death in both invertebrates and vertebrates. In this report, we show that peptide inhibitors of ICE arrest the programmed cell death of motoneurons in vitro as a result of trophic factor deprivation and in vivo during the period of naturally occurring cell death. In addition, interdigital cells that die during development are also rescued in animals treated with ICE inhibitors. Taken together, these results provide the first evidence that ICE or an ICE-like protease plays a regulatory role not only in vertebrate motoneuron death but also in the developmentally regulated deaths of other cells in vivo.

Neurotrophic Survival Molecules for Motoneurons: An Embarrassment of Riches

One obvious, albeit trivial, answer to this question is that the action of many of the factors in Table 1Table 1 reflect pharmacological rather than physiological effects. Other more interesting possibilities include the following: all or most motoneurons require multiple factors from diverse sources for optimal survival (Figure 1Figure 1); subpopulations of motoneurons, based on their function or connectivity, require different factors for survival; and motoneurons require distinct factors at different stages of development. At present, the available evidence does not appear to strongly favor any one of these alternatives over the others, and it remains possible that they are all correct. (It is nonetheless intriguing that no single putative motoneuron survival factor [including CT-1] has been shown to maintain more than 40%–50% of motoneurons either in vitro or in vivo [for references, see19xPennica, D, Arce, V, Swanson, T.A, Vejsada, R, Pollock, R.A, Armanini, M, Dudley, K, Phillips, H.S, Rosenthal, A, Kato, A.C, and Henderson, C.E. Neuron. 1996; 17: 63–74Abstract | Full Text | Full Text PDF | PubMed | Scopus (174)See all References, 16xOppenheim, R.W, Prevette, D, Haverkamp, L.J, Houenou, L, Qin-Wei, Y, and McManaman, J.L. J. Neurobiol. 1993; 24: 1065–1079Crossref | PubMedSee all References.) Perhaps by this “embarrassment of riches,” the embryo is trying to tell us something, but what? The hope, of course, is that future studies will reduce our ignorance on these matters and, in doing so, not only provide some final answers regarding the normal biology of motoneuron survival, but also suggest rational strategies as to how one might begin to use this information to treat human pathologies that involve the loss of motoneurons.Dedicated to Viktor Hamburger on the occasion of his 96th birthday.

Naturally occurring cell death during neural development

Abstract In many populations of developing neurons, massive numbers of cells die prior to the completion of differentiation. This naturally occurring cell loss is not a pathological process but, as the name implies, it is a normal feature of neurogenesis. Although in recent years considerable attention has been focused on this seemingly implausible developmental phenomenon, many fundamental issues concerning both mechanisms and the biological function of neuron death remain unresolved. Some progress has been made, however, and in the present brief review I consider evidence pertaining to a few of the critical questions about this phenomenon in vertebrates.

PROGRAMMED CELL DEATH IN THE DEVELOPING NERVOUS SYSTEM

Virtually all cell populations in the vertebrate nervous system undergo massive “naturally‐occurring” or “programmed” cell death (PCD) early in development. Initially neurons and glia are overproduced followed by the demise of approximately one‐half of the original cell population. In this review we highlight current hypotheses regarding how large‐scale PCD contributes to the construction of the developing nervous system. More germane to the theme of this symposium, we emphasize that the survival of cells during PCD depends critically on their ability to access “trophic” molecular signals derived primarily from interactions with other cells. Here we review the cell‐cell interactions and molecular mechanisms that control neuronal and glial cell survival during PCD, and how the inability of such signals to suppress PCD may contribute to cell death in some diseases such as spinal muscular atrophy. Finally, by using neurotrophic factors (e.g. CNTF, GDNF) and genes that control the cell death cascade (e.g. Bcl‐2) as examples, we underscore the importance of studying the mechanisms that control neuronal and glial cell survival during normal development as a means of identifying molecules that prevent pathology‐induced cell death. Ultimately this line of investigation could reveal effective strategies for arresting neuronal and glial cell death induced by injury, disease, and/or aging in humans.

Cell death of motoneurons in the chick embryo spinal cord: VIII. Motoneurons prevented from dying in the embryo persist after hatching

A chronic neuromuscular blockade during those embryonic stages when naturally occurring spinal motoneuron death occurs, results in the prevention of this cell loss. The excess motoneurons are maintained as long as the neuromuscular blockade is continued; once embryonic neuromuscular activity resumes, however, the excess motoneurons undergo a delayed period of cell death. By contrast, the resumption of neuromuscular activity in these same preparations after hatching did not result in a delayed cell death. The excess motoneurons, prevented from dying in the embryo, persisted for as long as 4 days postnatally despite the presence of considerable limb motility. The maintenance of motoneurons may be regulated differently before and after hatching.

Chapter 13 Neuron Death in Vertebrate Development: In Vivo Methods

Publisher Summary The study of neuronal death involves demonstrating that it occurs in a given situation, estimating the magnitude and timing of the loss, evaluating which particular neurons die, analyzing why and how they die, and understanding the role or purpose of the loss. A wide range of methods is available for achieving these ends; most involve the use of histological sections, although biochemical analysis of homogenized tissue can also provide useful information. Counting healthy neurons in histological sections is the most direct and widely used method for estimating the number of neurons that die, and the timing of their loss. Because most cases of neuronal death occur in postmitotic populations, there is rarely any need to consider complex tissue kinetics; the number of neurons lost is simply the initial number minus the final number in a defined population. However, the fact that subtraction is used makes the final estimation of neuronal death highly sensitive to errors in the estimations of total neuronal number. To meet the high standards of accuracy that are required in counting healthy neurons, it is essential to avoid two main sources of error: those because of inadequate definition of the population to be counted and those in counting.

Programmed Cell Death of Adult-Generated Hippocampal Neurons Is Mediated by the Proapoptotic Gene Bax

In the dentate gyrus (DG) of the adult mouse hippocampus, a substantial number of new cells are generated daily, but only a subset of these survive and differentiate into mature neurons, whereas the majority undergo programmed cell death (PCD). However, neither the intracellular machinery required for adult stem cell-derived neuronal death nor the biological implications of the significant loss of these newly generated cells have been examined. Several markers for apoptosis failed to reveal cell death in Bax-deficient mice, and this, together with a progressive increase in neuron number in the DG of the Bax knock-out, indicates that Bax is critical for the PCD of adult-generated hippocampal neurons. Whereas the proliferation of neural progenitor cells was not altered in the Bax-knock-out, there was an accumulation of doublecortin, calretinin+, and neuronal-specific nuclear protein+ postmitotic neurons, suggesting that Bax-mediated PCD of adult-generated neurons takes place during an early phase of differentiation. The absence of PCD in the adult also influenced the migration and maturation of adult-generated DG neurons. These results suggest that PCD in the adult brain plays a significant role in the regulation of multiple aspects of adult neurogenesis.

Reduction of endogenous transforming growth factors |[beta]| prevents ontogeneticneuron death

We show that following immunoneutralization of endogenous transforming growth factors β (TGF-β) in the chick embryo, ontogenetic neuron death of ciliary, dorsal root and spinal motor neurons was largely prevented, and neuron losses following limb bud ablation were greatly reduced. Likewise, preventing TGF-β signaling by treatment with a TβR-II fusion protein during the period of ontogenetic cell death in the ciliary ganglion rescued all neurons that normally die. TUNEL staining revealed decreased numbers of apoptotic cells following antibody treatment. Exogenous TGF-β rescued the TGF-β-deprived phenotype. We conclude that TGF-β is critical in regulating ontogenetic neuron death as well as cell death following neuronal target deprivation.