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BAX Is Required for Neuronal Death after Trophic Factor Deprivation and during Development
Abstract
Members of the BCL2-related family of proteins either promote or repress programmed cell death. BAX, a death-promoting member, heterodimerizes with multiple death-repressing molecules, suggesting that it could prove critical to cell death. We tested whether Bax is required for neuronal death by trophic factor deprivation and during development. Neonatal sympathetic neurons and facial motor neurons from Bax-deficient mice survived nerve growth factor deprivation and disconnection from their targets by axotomy, respectively. These salvaged neurons displayed remarkable soma atrophy and reduced elaboration of neurities; yet they responded to readdition of trophic factor with soma hypertrophy and enhanced neurite outgrowth. Bax-deficient superior cervical ganglia and facial nuclei possessed increased numbers of neurons. Our observations demonstrate that trophic factor deprivation-induced death of sympathetic and motor neurons depends on Bax.
... BCL-2 is the direct antagonist of BAX and inhibits oligomerization directly interacting with BAX or by preventing BH3-only proteins, a subfamily of the pro-apoptotic group, from activating BAX [6,7]. The importance of the BCL-2 protein family for the proper development of the nervous system was demonstrated in different transgenic mouse studies [8][9][10][11][12][13]. Here, anti-apoptotic family members, like myeloid cell leukemia 1 (MCL-1) or B-cell lymphoma 2 like 1 (BCL-xL), are crucial for the survival of immature, newborn [9,13,14], and early postmitotic neurons [15]. ...
... Here, the increase in intracellular Ca 2+ initiates a multitude of signaling cascades [29,30], some of which are known to be beneficial for neuronal survival, repair, and maintenance in the peripheral and central nervous system [31][32][33]. In all these models, neuronal loss is prevented in mice lacking BAX [12,19,31,34]. Important regulators of neuronal survival, such as the AKT serine-threonine kinase 1 (AKT) and its counterpart phosphatase and tensin homolog (PTEN), are also impacted in expression and function by neuronal activity [19,35,36]. ...
... BAX and other members of the pro-apoptotic group of the BCL-2 family play critical or essential roles in developmental apoptosis [8][9][10][11][12]66]. Our data, corroborated by previous literature [47,67], show that developmental upregulation of BAX during the first postnatal days is accompanied by downregulation of BCL-2 protein expression, resulting in a BAX/BCL-2 ratio that is highest around P7 precisely when cortical cell death is high. ...
During early brain development, homeostatic removal of cortical neurons is crucial and requires multiple control mechanisms. We investigated in the cerebral cortex of mice whether the BAX/BCL-2 pathway, an important regulator of apoptosis, is part of this machinery and how electrical activity might serve as a set point of regulation. Activity is known to be a pro-survival factor; however, how this effect is translated into enhanced survival chances on a neuronal level is not fully understood. In this study, we show that caspase activity is highest at the neonatal stage, while developmental cell death peaks at the end of the first postnatal week. During the first postnatal week, upregulation of BAX is accompanied by downregulation of BCL-2 protein, resulting in a high BAX/BCL-2 ratio when neuronal death rates are high. In cultured neurons, pharmacological blockade of activity leads to an acute upregulation of Bax, while elevated activity results in a lasting increase of BCL-2 expression. Spontaneously active neurons not only exhibit lower Bax levels than inactive neurons but also show almost exclusively BCL-2 expression. Disinhibition of network activity prevents the death of neurons overexpressing activated CASP3. This neuroprotective effect is not the result of reduced caspase activity but is associated with a downregulation of the BAX/BCL-2 ratio. Notably, increasing neuronal activity has a similar, non-additive effect as the blockade of BAX. Conclusively, high electrical activity modulates BAX/BCL-2 expression and leads to higher tolerance to CASP3 activity, increases survival, and presumably promotes non-apoptotic CASP3 functions in developing neurons.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00018-023-04824-6.
... Apoptosis has been proposed as a mechanism leading to stress-related depression-like behaviour. Without appropriate trophic support or activity, newly generated neurons in adulthood die via apoptosis (Deckwerth et al., 1996). For instance, enhanced apoptosis was found in the brain structures of maternally separated rats (Lee et al., 2001). ...
... Consistent with a previous study , we found that the level of cleaved caspase-3 was significantly increased in the hippocampi of rats with depression-like behaviour, and this increase could be adjusted via GPT treatment. Bax is required for trophic factor deprivation-induced neuronal death (Deckwerth et al., 1996). The Bax/Bcl-2 ratio is important for cell survival because it critically controls apoptosis. ...
Ethnopharmacological relevance
Guipi Tang (GPT) is a widely used traditional Chinese medicine that is used to treat major depressive disorder. However, the molecular mechanisms of its effects remain unclear.
Aim of the study
This study aimed to investigate the antidepressant-like effects of GPT and explore its underlying molecular mechanisms.
Materials and methods
Male Sprague–Dawley rats were subjected to a chronic unpredictable mild stress (CUMS) procedure and treated with various doses of GPT, with fluoxetine treatment as a positive control. Behavioural tests (including sucrose preference test, novelty-suppressed feeding test, open-field test and forced swim test), Terminal deoxynucleotidyl transferase dUTP nick end labeling and enzyme-linked immunosorbent assay were conducted. The levels of Bax, Bcl-2, cleaved caspase-3, PI3K, p-PI3K, AKT, p-AKT, BDNF, TrkB and CREB or p-CREB were assessed at the protein level using western blotting or immunofluorescence.
Results
GPT consists of mainly known drugs, such as liquiritin and ginsenosides. It reversed depressive behaviours and decreased cell apoptosis in the hippocampi of CUMS rats. It significantly upregulated the levels of Bax, p-Akt, p-PI3K, BDNF, TrkB and p-CREB and downregulated the expression of cleaved caspase-3 and Bcl-2.
Conclusions
GPT had anti-depressive activity as indicated by the amelioration of depression-like behaviour and the inhibition of hippocampal neuronal apoptosis in CUMS rats. This inhibition was mediated partly by modulating the PI3K/Akt and/or BDNF/TrkB/CREB pathway, in which, glycosides, the main components of GPT, might be involved.
... Full loss of the Bax gene is not lethal and the mice develop without any major developmental abnormalities, although Bax À/À male mice are infertile (Knudson, Tung, Tourtellotte, Brown, & Korsmeyer, 1995). More subtle effects of Bax deletion have been observed, particularly in the brain and eye, including increased neuronal number (Deckwerth et al., 1996;White, Keller-Peck, Knudson, Korsmeyer, & Snider, 1998), absence of size differences in two sexually dimorphic nuclei (Forger & Peskin, 2005), cerebellar migration defects (Jung et al., 2008), increased retinal cell numbers, glial cell density, and altered microglial shape (Kawai et al., 2009;Mac Nair, Schlamp, Montgomery, Shestopalov, & Nickells, 2016;Mosinger Ogilvie, Deckwerth, Knudson, & Korsmeyer, 1998). Importantly, Bax deficiency reduces neuronal and retinal cell death resulting from various injuries (Deckwerth et al., 1996;Robinson, Conley, & Kern, 2003;Semaan, Li, & Nickells, 2010;W. ...
... More subtle effects of Bax deletion have been observed, particularly in the brain and eye, including increased neuronal number (Deckwerth et al., 1996;White, Keller-Peck, Knudson, Korsmeyer, & Snider, 1998), absence of size differences in two sexually dimorphic nuclei (Forger & Peskin, 2005), cerebellar migration defects (Jung et al., 2008), increased retinal cell numbers, glial cell density, and altered microglial shape (Kawai et al., 2009;Mac Nair, Schlamp, Montgomery, Shestopalov, & Nickells, 2016;Mosinger Ogilvie, Deckwerth, Knudson, & Korsmeyer, 1998). Importantly, Bax deficiency reduces neuronal and retinal cell death resulting from various injuries (Deckwerth et al., 1996;Robinson, Conley, & Kern, 2003;Semaan, Li, & Nickells, 2010;W. Sun & Oppenheim, 2003), including alcohol exposure. ...
Background:
During early development, alcohol exposure causes apoptotic cell death in discrete regions of the embryo which are associated with distinctive patterns of later-life abnormalities. In gastrulation, which occurs during the third week of human pregnancy, alcohol targets the ectoderm, the precursor of the eyes, face, and brain. This midline tissue loss leads to the craniofacial dysmorphologies, such as microphthalmia and a smooth philtrum, which define fetal alcohol syndrome (FAS). An important regulator of alcohol-induced cell death is the pro-apoptotic protein Bax. The current study determines if mice lacking the Bax gene are less susceptible to the pathogenic effects of gastrulation-stage alcohol exposure.
Methods:
Male and female Bax+/- mice mated to produce embryos with full (-/- ) or partial (+/- ) Bax deletions, or Bax+/+ wild-type controls. On Gestational Day 7 (GD 7), embryos received two alcohol (2.9 g/kg, 4 hr apart), or control exposures. A subset of embryos was collected 12 hr later and examined for the presence of apoptotic cell death, while others were examined on GD 17 for the presence of FAS-like facial features.
Results:
Full Bax deletion reduced embryonic apoptotic cell death and the incidence of fetal eye and face malformations, indicating that Bax normally facilitates the development of alcohol-induced defects. An RNA-seq analysis of GD 7 Bax+/+ and Bax-/- embryos revealed 63 differentially expressed genes, some of which may interact with the Bax deletion to further protect against apoptosis.
Conclusions:
Overall, these experiments identify that Bax is a primary teratogenic mechanism of gastrulation-stage alcohol exposure.
... Although Mcl-1 has a stronger affinity for Bak, the role of Bak in the developing nervous system is minimal and only observed when co-deleted with Bax (Lindsten et al., 2000). In contrast to Bak, Bax has a prominent role in nervous system development including regulating the size of the neuronal population (Knudson et al., 1995;Deckwerth et al., 1996). We therefore questioned whether Bax also has a pro-apoptotic role in the proliferating NPC population. ...
... As Mcl-1 has multiple pro-apoptotic targets, we focused on Bax, as it is the dominant pro-apoptotic protein expressed in the developing nervous system (Krajewski et al., 1994). Germline deletion of Bax rescues neurons from developmental death resulting in a 24-35% increase in neuronal populations (Deckwerth et al., 1996;White et al., 1998). In contrast, Bak null mice do not appear to have a CNS phenotype (Lindsten et al., 2000). ...
Myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic Bcl-2 protein, regulates neural precursor cell (NPC) survival in both the developing and adult mammalian nervous system. It is unclear when during the neurogenic period Mcl-1 becomes necessary for NPC survival and whether Bax is the sole pro-apoptotic target of Mcl-1. To address these questions, we used the nervous system-specific Nestin-Cre Mcl-1 conditional knockout mouse line (Mcl-1 CKO) to assess the anti-apoptotic role of Mcl-1 in developmental neurogenesis. Loss of Mcl-1 resulted in a wave of apoptosis beginning in the brainstem and cervical spinal cord at embryonic day 9.5 (E9.5) and in the forebrain at E10.5. Apoptosis was first observed ventrally in each region and spread dorsally over time. Within the spinal cord, apoptosis also spread in a rostral to caudal direction following the path of differentiation. Breeding the Mcl-1 CKO mouse with the Bax null mouse rescued the majority of NPC from apoptosis except in the dorsomedial brainstem and ventral thoracic spinal cord where only 50% were rescued. This demonstrates that Mcl-1 promotes NPC survival primarily by inhibiting the activation of Bax, but that Bax is not the sole pro-apoptotic target of Mcl-1 during embryonic neurogenesis. Interestingly, although co-deletion of Bax rescued the majority of NPC apoptosis, it resulted in embryonic lethality at E13, whereas conditional deletion of both Mcl-1 and Bax rescued embryonic lethality. In summary, this study demonstrates the widespread dependency on Mcl-1 during nervous system development.
... Knockout studies in rodents have solidified the importance of BCL-2 family proteins in the development of the nervous system. Selective deletion of the pro-apoptotic BCL-2 family protein BAX prevents the normal cell death that occurs in the cerebellum and retina, and these mice are reported to have increases in hippocampal and dorsal root ganglion (DRG) neurons [4][5][6][7]. Selective deletion of a BAX-activating, pro-apoptotic protein called p53 upregulated modulator of apoptosis (PUMA), also prevents apoptosis of DRG neurons in the peripheral nervous system (PNS). This results in an increased number of neurons and innervating branches during development [8,9], with no change in expected birth frequency for PUMA knockout mice [10,11]. ...
... One component may be the location of stress. Whole cell trophic-factor deprivation causes both PNS and CNS neurons to die in a PUMA-and BAXdependent manner [7,71,75], however, trophic-factor deprivation exclusively from axons results in degeneration [24]. Using microfluidic chambers that enable separate treatment of neuronal cell bodies and axons, trophic-factor deprivation from axons results in transcriptional upregulation of key genes required for axon degeneration such as bbc3, encoding PUMA [9,64] (Fig. 2). ...
Axonal degeneration and neuronal cell death are fundamental processes in development and contribute to the pathology of neurological disease in adults. Both processes are regulated by BCL-2 family proteins which orchestrate the permeabilization of the mitochondrial outer membrane (MOM). MOM permeabilization (MOMP) results in the activation of pro-apoptotic molecules that commit neurons to either die or degenerate. With the success of small-molecule inhibitors targeting anti-apoptotic BCL-2 proteins for the treatment of lymphoma, we can now envision the use of inhibitors of apoptosis with exquisite selectivity for BCL-2 family protein regulation of neuronal apoptosis in the treatment of nervous system disease. Critical to this development is deciphering which subset of proteins is required for neuronal apoptosis and axon degeneration, and how these two different outcomes are separately regulated. Moreover, noncanonical BCL-2 family protein functions unrelated to the regulation of MOMP, including impacting necroptosis and other modes of cell death may reveal additional potential targets and/or confounders. This review highlights our current understanding of BCL-2 family mediated neuronal cell death and axon degeneration, while identifying future research questions to be resolved to enable regulating neuronal survival pharmacologically.
... Studies have shown that endogenous BAX is important in programmed neuronal cell death. In bax Ϫ/Ϫ mice, sympathetic, peripheral nervous system (PNS), motor, and cerebellar granule neurons are all dramatically protected from apoptosis both in vitro and in vivo (15)(16)(17)(18)(19). Although BAX plays an important role under these circumstances and in these cells, few studies have examined a role for endogenous BAX in the cell death which occurs in the normal or injured CNS. ...
... The mutant allele generated a 507 basepair product which was amplified with a neo/pgk primer (0.16 M: 5'-CCGCTTCCATTGCTCAGCGG-3') and the same intron 5 reverse primer, which together generate a 507 basepair product. The cycling parameters for the reaction were 1 min at 94ЊC, 55ЊC, and 72ЊC each for a total of 30-35 cycles (16). ...
Background
Hypoxic-ischemic (H-I) injury to the neonatal brain has been shown to result in rapid cell death with features of acute excitotoxicity/necrosis as well as prominent delayed cell death with features of apoptosis such as marked caspase-3 activation. BAX, a pro-apoptotic molecule, has been shown to be required for apoptotic neuronal cell death during normal development but the contribution of endogenous BAX in cell death pathways following H-I injury to the developing or adult brain has not been studied. Materials and Methods
Bax +/+, +/−, and −/− mice at post-natal day 7 were subjected to unilateral carotid ligation followed by exposure to 45 minutes of 8% oxygen. At different timepoints following H-I, brain tissue was studied by conventional histology, immunohistochemistry, immunofluorescence, Western blotting, and enzymatic assay to determine the extent and type of cell injury as well as the amount of caspase activation. ResultsWe found that bax −/− mice had significantly less (38%) hippocampal tissue loss than mice expressing bax. Some of the remaining cell death in bax −/− mice, however, still had features of apoptosis including evidence of nuclear shrinkage and caspase-3 activation. Though bax −/− mice had significantly decreased caspase-3 activation as compared to bax expressing mice following H-I, the density of cells with activated caspase-8 in the CA3 region of the hippocampus did not differ between bax +/− and bax −/− mice. Conclusions
These findings demonstrate that endogenous BAX plays a role in regulating cell death in the central nervous system (CNS) following neonatal H-I, a model of cerebral palsy. In addition, while BAX appears to modulate the caspase-3 activation following neonatal H-I, caspase-8 which is linked to death receptor activation, may contribute to apoptotic-like neuronal death in a BAX-independent manner.
... She discovered that nerve growth factor was required to prevent death of sympathetic and sensory neurons in the peripheral nervous system [150]. Later work by many groups led to the discovery of apoptosis [151], and the recognition that growth-factor withdrawal dependent cell death was related to proapoptotic Bcl-2 family proteins such as Bax [152][153][154]. These proteins may also modulate mPTP formation through interactions at contact sites between the inner and outer mitochondrial membrane and changes in mitochondrial morphology [155]. ...
The mitochondrial permeability transition (mPT) describes a Ca 2+-dependent and cyclophilin D (CypD)-facilitated increase of inner mitochondrial membrane permeability that allows diffusion of molecules up to 1.5 kDa in size. It is mediated by a non-selective channel, the mitochondrial permeability transition pore (mPTP). Sustained mPTP opening causes mitochondrial swelling, which ruptures the outer mitochondrial membrane leading to subsequent apoptotic and necrotic cell death, and is implicated in a range of pathologies. However, transient mPTP opening at various sub-conductance states may contribute several physiological roles such as alterations in mitochondrial bioenergetics and rapid Ca 2+ efflux. Since its discovery decades ago, intensive efforts have been made to identify the exact pore-forming structure of the mPT. Both the adenine nucleotide translocase (ANT) and, more recently, the mitochondrial F 1 F O (F)-ATP synthase dimers, monomers or c-subunit ring alone have been implicated. Here we share the insights of several key investigators with different perspectives who have pioneered mPT research. We critically assess proposed models for the molecular identity of the mPTP and the mechanisms underlying its opposing roles in the life and death of cells. We provide in-depth insights into current controversies, seeking to achieve a degree of consensus that will stimulate future innovative research into the nature and role of the mPTP.
... 87 By contrast, proapoptotic Bcl-2-associated X-protein (Bax)knockout mice show Bax plays an essential role in neuronal death but not in axonal degeneration. [88][89][90][91] Although apoptosis in RGCs occurs primarily through the intrinsic pathway, continued RGC death in Bax-deficient mice treated with excitotoxins points toward the extrinsic apoptotic pathway as an alternative mechanism. 91 Glutamate-induced excitotoxicity, implicated in glaucoma pathogenesis, may be contributed by the extrinsic apoptotic pathway. ...
Optic neuropathies encompass a breadth of diseases that ultimately result in dysfunction and/or loss of retinal ganglion cells (RGCs). Although visual impairment from optic neuropathies is common, there is a lack of effective clinical treatments. Addressing a critical need for novel interventions, preclinical studies have been generating a growing body of evidence that identify promising new drug-based and cell-based therapies. Gene therapy is another emerging therapeutic field that offers the potential of specifically and robustly increasing long-term RGC survival in optic neuropathies. Gene therapy offers additional benefits of driving improvements following a single treatment administration, and it can be designed to target a variety of pathways that may be involved in individual optic neuropathies or across multiple etiologies. This review explores the history of gene therapy, the fundamentals of its application, and the emerging development of gene therapy technology as it relates to treatment of optic neuropathies.
... 35 Consistently, our study discovered that the expression of Bcl-2 was downregulated in ODG/R-induced neurons. In addition, BAX, a pro-apoptotic member of the Bcl-2 family, is required for neuronal death, 36 and PARP-1 activity is suppressed by Bcl-2 directly. 37 Previous study suggests that amantadine and topiramate can improve I/R injury by reducing apoptosis. ...
Background:
During ischemic stroke treatment, cerebral ischemia/reperfusion (I/R) injury results in neuronal cell death and neurological dysfunctions in brain. Previous studies indicate that basic helix-loop-helix family member e40 (BHLHE40) exerts protective effects on the pathology of neurogenic diseases. However, the protective function of BHLHE40 in I/R is unclear.
Objectives:
This study aimed to explore the expression, role and potential mechanism of BHLHE40 after ischemia.
Material and methods:
We established models of I/R injury in rats and of oxygen-glucose deprivation/reoxygenation (OGD/R) in primary hippocampal neurons. Nissl and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to detect neuronal injury and apoptosis. Immunofluorescence was used to detect BHLHE40 expression. Cell viability and cell damage measurements were conducted using Cell Counting Kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) assay. The regulation of BHLHE40 to pleckstrin homology-like domain family A, member 1 (PHLDA1) was assessed using the dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay.
Results:
Cerebral I/R rats exhibited severe neuronal loss and apoptosis in hippocampal cornu Ammonis 1 (CA1) region, accompanied by downregulated BHLHE40 expression at both mRNA and protein levels, indicating that BHLHE40 may regulate the apoptosis of hippocampal neurons. The function of BHLHE40 in neuronal apoptosis during cerebral I/R was further explored by establishing an OGD/R model in vitro. Low expression of BHLHE40 was also observed in neurons treated with OGD/R. The OGD/R administration inhibited cell viability and enhanced cell apoptosis in hippocampal neurons, whereas BHLHE40 overexpression reversed those changes. Mechanistically, we demonstrated that BHLHE40 could repress PHLDA1 transcription by binding to PHLDA1 promoter. The PHLDA1 is a facilitator of neuronal damage in brain I/R injury and its upregulation reversed the effects caused by BHLHE40 overexpression in vitro.
Conclusions:
The transcription factor BHLHE40 may protect against brain I/R injury through repressing cell damage via regulating PHLDA1 transcription. Thus, BHLHE40 may be a candidate gene for further study of molecular or therapeutic targets for I/R.
... In vitro models of cell death using embryonic sympathetic and DRG sensory neurons initially maintained and then deprived of NGF have identified several mechanisms that drive developmental neuronal degeneration (Unsain et al., 2013(Unsain et al., , 2014Geden et al., 2019). Among these are the role of the Bcl-2 family members, in particular BAX, to actively induce mitochondrial permeabilization, the importance of intracellular Ca 21 overload, particularly through TRPV1 channels, and the involvement of the caspase family of cysteine proteases as executors of cellular destruction (Deckwerth et al., 1996;Putcha et al., 2001;Simon et al., 2012;Unsain et al., 2013;Johnstone et al., 2019). Despite these findings, the precise molecular mechanisms driving the process of neuronal elimination during development are still not fully understood. ...
The activation of self-destructive cellular programs helps sculpt the nervous system during development, but the molecular mechanisms used are not fully understood. Prior studies have investigated the role of the APP in the developmental degeneration of sensory neurons with contradictory results. In this work, we sought to elucidate the impact of APP deletion in the development of the sensory nervous system in vivo and in vitro. Our in vivo data show an increase in the number of sciatic nerve axons in adult male and female APP-null mice, consistent with the hypothesis that APP plays a pro-degenerative role in the development of peripheral axons. In vitro, we show that genetic deletion of APP delays axonal degeneration triggered by nerve growth factor deprivation, indicating that APP does play a pro-degenerative role. Interestingly, APP depletion does not affect caspase-3 levels but significantly attenuates the rise of axoplasmic Ca2+ that occurs during degeneration. We examined intracellular Ca2+ mechanisms that could be involved and found that APP-null DRG neurons had increased Ca2+ levels within the endoplasmic reticulum and enhanced store-operated Ca2+ entry. We also observed that DRG axons lacking APP have more mitochondria than their WT counterparts, but these display a lower mitochondrial membrane potential. Finally, we present evidence that APP deficiency causes an increase in mitochondrial Ca2+ buffering capacity. Our results support the hypothesis that APP plays a pro-degenerative role in the developmental degeneration of DRG sensory neurons, and unveil the importance of APP in the regulation of calcium signaling in sensory neurons.Significance Statement:The nervous system goes through a phase of pruning and programmed neuronal cell death during development to reach maturity. In such context, the role played by the APP in the peripheral nervous system has been controversial, ranging from pro-survival to pro-degenerative. Here we present evidence in vivo and in vitro supporting the pro-degenerative role of APP, demonstrating the ability of APP to alter intracellular Ca2+ homeostasis and mitochondria, critical players of programmed cell death. This work provides a better understanding of the physiological function of APP and its implication in developmental neuronal death in the nervous system.
... Bax interacts with Bel-2 to form heterodimers, and its overexpression antagonises the protective effect ofBel-2 (Merry and Korsmeyer 1997). Bax has an important role in neuronal death (Deckwerth, Elliott et al. 1996), and this is highlighted by the consequences ofBax deficiency: in Bax knockout (Bax -/-) mice, developmental cell death is virtually eliminated in many populations of neurones (White, Keller-Peck et al. 1998). Despite this, Bax-/-mice show no other overt changes in the nervous system. ...
p>We raised polyclonal antibodies against components of sciatic nerves undergoing Wallerian degeneration. The antibodies have been screened against a range of naïve and injured tissues by immunohistochemistry and Western blotting. A proteomics approach has then been employed to deduce which antigen(s) the antibodies recognise. Immuno histochemistry on injured CNS tissues, using these novel antibodies, revealed staining of damaged axons from as early as 6 hours, which intensifies through to 7 days in the spinal cord and 28 days in the brain, the longest timepoints studied. Western blot analysis indicates a number of bands that are either upregulated or down regulated in response to injury in CNS tissue when compared to the appropriate naïve tissue. The bands identified by Western blot were subsequently analysed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF).
We identified changes in three proteins following CNS injury. Firstly, albumin appears to be taken up into damaged axons following injury prior to resealing. Secondly, kid-1, a transcription factor, was identified; Western blot and immunocytochemical analysis revealed no change in expression over the time course of injury investigated. And finally, focal adhesion kinase (FAK), a protein tyrosine kinase, was also identified.
Changes in FAK expression following injury were studied by Western blot and immunocytochemistry. Western blot analysis revealed a decrease in FAK expression following brain and spinal cord injury. Double immunofluorescence revealed that de novo FAK expression was present in oligodendrocytes as early as 6 hrs post injury. This is possibly the earliest event occurring in the oligodendrocyte in response to Wallerian degeneration of their associated axons. At later times FAK expression was detected, to a lesser extent, in astrocytes and possibly microglial cells.</p
... Therefore, limiting this process is a critical step in treating head trauma. In the present study, we evaluated whether treatment with bevacizumab was able to mitigate the apoptotic process induced by head trauma, evaluating the expression of two factors involved in the regulation of the apoptotic process such as Bax, responsible for the development of neuronal death [38], and Bcl-2 protective against neuronal cells [39,40]. Our results showed that after TBI, there is an increase in Bax expression and a significant decrease in Bcl-2, which highlights an imbalance in the apoptotic pathway. ...
Traumatic brain injury (TBI) disrupts the blood–brain barrier (BBB). Vascular endothelial growth factor (VEGF) is believed to play a key role in TBI and to be overexpressed in the absence of apolipoprotein E (ApoE). Bevacizumab, a VEGF inhibitor, demonstrated neuroprotective activity in several models of TBI. However, the effects of bevacizumab on Apo-E deficient mice are not well studied. The present study aimed to evaluate VEGF expression and the effects of bevacizumab on BBB and neuroinflammation in ApoE−/− mice undergoing TBI. Furthermore, for the first time, this study evaluates the effects of bevacizumab on the long-term consequences of TBI, such as atherosclerosis. The results showed that motor deficits induced by controlled cortical impact (CCI) were accompanied by increased brain edema and VEGF expression. Treatment with bevacizumab significantly improved motor deficits and significantly decreased VEGF levels, as well as brain edema compared to the control group. Furthermore, the results showed that bevacizumab preserves the integrity of the BBB and reduces the neuroinflammation induced by TBI. Regarding the effects of bevacizumab on atherosclerosis, it was observed for the first time that its ability to modulate VEGF in the acute phase of head injury prevents the acceleration of atherosclerosis. Therefore, the present study demonstrates not only the neuroprotective activity of bevacizumab but also its action on the vascular consequences related to TBI.
... transition and mitochondrial membrane potential resulting in the release of cytochrome c [29]. Mitochondrial initiated cytochrome c release is directly assisted by Bax [30]. ...
Context:
Consuming whole grain food has been motivated due to numerous health benefits arising from their bioactive components.
Aims:
This study aims to study whether the active compound extracted from Proso and Barnyard millets inhibits cell proliferation and induces apoptotic cell death in MCF-7 cell line.
Materials and methods:
Cell proliferative effect was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay using MCF-7 cell line. Cytotoxicity was determined by release of lactate dehydrogenase (LDH) enzyme from cells. Apoptotic morphological changes in MCF-7 cells were observe under fluorescence microscope using double staining of Hoeschst 33342/propidium iodide (PI). Induction of apoptosis was analyzed using Annexin V-fluorescein isothiocyanate/PI through flow cytometry.
Results:
In this study, cell proliferative effect of the bioactive compounds from proso millet (Compound 1) and barnyard millet (Compound 2) was evaluated using MCF-7 cell line. Both the compounds significantly inhibited the proliferation of MCF-7 cells after treated with 250 μg/ml and 1000 μg/ml concentration for 48 h. Cytotoxic activity of compounds was assessed by the release of LDH showed that these extracted compounds were not toxic to the cells. Apoptosis was confirmed by Hoechst 33,342/PI dual-staining, Annexin V-FTIC/PI staining, and flow cytometry results of cell cycle analysis shows that there was a significant cell arrest in the G0/G1 phase and increased the apoptotic cells in sub-G0 phase in a dose-dependent manner.
Conclusions:
This study suggests that the extracted vanillin compound from these millets have effectively induced apoptotic cell death in breast cancer cell line.
... instance, apoptosis in post-mitotic neurons is likely BAX-dependent as mature neurons have limited expression of full-length BAK that is competent to mediate MOMP (Deckwerth et al, 1996). Hence, deletion of BAX alone is often sufficient to confer protection to neurons in various animal models of both acute and chronic neurodegeneration (Table 1). ...
... In some knockout mice, the deletion of a molecule involved in apoptosis results in an initial protection from cell death, but the eventual elimination of these neurons, such as in p75 -/mice (Deppmann et al., 2008). In the BAX knockout mice, in contrast, all neurons are rescued from apoptosis permanently (Deckwerth et al., 1996), indicating that BAX is absolutely required for the apoptosis pathway in SCG neurons. To distinguish between these two possibilities, the number of neurons in the SCGs of P20 PlexA3 -/mice and PlexA3 +/+ mice were compared. ...
Huntington’s disease (HD) is a dominantly-inherited neurodegenerative disorder characterized by a constellation of motor, cognitive, and psychiatric features. Striatal medium spiny neurons, one of the most affected populations, are dependent on brain-derived neurotrophic factor (BDNF) anterogradely transported from the cortex for proper function and survival. Recent studies suggest both receptors for BDNF, TrkB and p75, are improperly regulated in striata of HD patients and mouse models of HD. While BDNF-TrkB signaling almost exclusively promotes survival and metabolic function, p75 signaling is able to induce survival or apoptosis depending on the available ligand and associated co-receptor. We investigated the role of p75 in the Q175 knock-in mouse model of HD by examining the levels and activation of downstream signaling molecules, and subsequently examining Q175WT/HD;p75-/- mice to determine if p75 represents a promising therapeutic target. In Q175WT/HD;p75+/+ mice, we observed enhanced survival signaling as evidenced by an increase in phosphorylation and activation of Akt and the p65 subunit of NFkB in the striatum at 5 months of age and an increase in XIAP expression compared to Q175WT/WT;p75+/+ mice; this increase was lost in Q175WT/HD;p75-/- mice. Q175WT/HD;p75-/- mice also showed a decrease in Bcl-XL expression by immunoblotting compared to Q175WT/HD;p75+/+ and Q175WT/WT;p75+/+ littermates. Consistent with diminished survival signaling, DARPP-32 expression decreased both by immunoblotting and by immunohistochemistry in Q175WT/HD;p75-/- mice compared to Q175WT/WT;p75+/+, Q175WT/HD;p75+/+, and Q175WT/WT;p75-/- littermates. Additionally, striatal volume declined to a greater extent in Q175WT/HD;p75-/- when compared to Q175WT/HD;p75+/+ littermates at 12 months, indicating a more aggressive onset of degeneration. These data suggest that p75 signaling plays an early role in augmenting pro-survival signaling in the striatum and that disruption of p75 signaling at a pre-symptomatic age may exacerbate pathologic changes in Q175WT/HD mice.
... It was established in a published paper that mice that lack caspase 3 expressions present excessive neuronal cells at birth and significantly reduced apoptosis [31]. The damaged mitochondrion in neurons initiates apoptosis with the help of proapoptotic protein Bax, belonging to the bcl-2 family, by activation of the mitochondrial membrane and release of cytochrome C and further caspase 9 activation [32]. This pathway mainly regulates apoptosis of the neurons even when the chief stimuli has not impaired the functioning of mitochondria. ...
The human body is highly complex and comprises of a variety of living cells 23 and extracellular material which forms tissues, organs, and organ systems. The human 24 cells tend to turn over readily to maintain homeostasis in tissues. However, the 25 postmitotic nerve cells exceptionally have an ability to regenerate and sustain for the en-26 tire life of an individual, to safeguard the physiological functioning of the central nervous 27 system. For efficient working of the CNS the neuronal death is essential, but extreme loss 28 of neurons declines the functioning of the nervous system and leads to onset of neuro-29 degenerative diseases. The neurodegenerative disease range from acute to chronic severe 30 life altering conditions like Parkinson's disease and Alzheimer disease. Over millions of 31 individuals worldwide are suffering from neurodegenerative disorders with less or neg-32 ligible treatment available thereby leading to decline in their quality of life. The neuropa-33 thological studies have identified a series of factors that explain the etiology of neuronal 34 degradation and its progression in neurodegenerative disease. The onset of neurological 35 diseases depends upon a combination of factors that causes disruption of neurons such 36 as environmental, biological, physiological, and genetic. The current review highlights 37 some of the major pathological factors responsible for neuronal degradation such as oxi-38 dative stress, cell death and neuroinflammation. All these factors have been described in 39 detail to enhance the understanding of their mechanisms and targeting them for disease 40 management. 41
... instance, apoptosis in post-mitotic neurons is likely BAX-dependent as mature neurons have limited expression of full-length BAK that is competent to mediate MOMP (Deckwerth et al, 1996). Hence, deletion of BAX alone is often sufficient to confer protection to neurons in various animal models of both acute and chronic neurodegeneration (Table 1). ...
Apoptotic cell death is implicated in both physiological and pathological processes. Since many types of cancerous cells intrinsically evade apoptotic elimination, induction of apoptosis has become an attractive and often necessary cancer therapeutic approach. Conversely, some cells are extremely sensitive to apoptotic stimuli leading to neurodegenerative disease and immune pathologies. However, due to several challenges, pharmacological inhibition of apoptosis is still only a recently emerging strategy to combat pathological cell loss. Here, we describe several key steps in the intrinsic (mitochondrial) apoptosis pathway that represent potential targets for inhibitors in disease contexts. We also discuss the mechanisms of action, advantages and limitations of small-molecule and peptide-based inhibitors that have been developed to date. These inhibitors serve as important research tools to dissect apoptotic signalling and may foster new treatments to reduce unwanted cell loss.
... This suggests that BH3-only activators play a significant role in regulating neuronal MOMP [103]. The central role of Bax in neuronal apoptosis is illustrated by the observation that Bax KO mice do not exhibit developmental programmed cell death of dorsal root ganglion sensory neurons, superior cervical ganglion sympathetic neurons, or motoneurons (MNs) [108][109][110]. Multiple studies have indicated that the deletion of the Bax gene protects neurons against excitotoxic apoptosis [97,111,112]. ...
The family of B-cell lymphoma-2 (Bcl-2) proteins exerts key functions in cellular health. Bcl-2 primarily acts in mitochondria where it controls the initiation of apoptosis. However, during the last decades, it has become clear that this family of proteins is also involved in controlling Ca²⁺ signaling in cells, a critical process for the function of most cell types, including neurons. Several anti- and pro-apoptotic Bcl-2 family members are expressed in neurons and impact neuronal function. Importantly, expression levels of neuronal Bcl-2 proteins are affected by age. In this review, we focus on the emerging roles of Bcl-2 proteins in neuronal cells. Specifically, we discuss how their dysregulation contributes to the onset, development, and progression of neurodegeneration in the context of Alzheimer's disease (AD). Aberrant Ca²⁺ signaling plays an important role in the pathogenesis of AD, and we propose that dysregulation of the Bcl-2-Ca²⁺ signaling axis may contribute to the progression of AD and that herein, Bcl-2 may constitute a potential therapeutic target for the treatment of AD.
... Indeed, in the CNS, errors in migration resulting in ectopic neurons are not uncommon as demonstrated by mice KO for Bcl-2-associated X protein-encoding, Bax (Jung et al, 2008). However, in sympathetic ganglia of Bax KO mice, increased neuronal survival in situ rather than gross ectopias are observed (Deckwerth et al, 1996). Kalirin suppression with an antisense RNA does not affect the survival of mature sympathetic neurons (May et al, 2002). ...
The migrational propensity of neuroblastoma is affected by cell identity, but the mechanisms behind the divergence remain unknown. Using RNAi and time-lapse imaging, we show that ADRN-type NB cells exhibit RAC1- and kalirin-dependent nucleokinetic (NUC) migration that relies on several integral components of neuronal migration. Inhibition of NUC migration by RAC1 and kalirin-GEF1 inhibitors occurs without hampering cell proliferation and ADRN identity. Using three clinically relevant expression dichotomies, we reveal that most of up-regulated mRNAs in RAC1- and kalirin–GEF1–suppressed ADRN-type NB cells are associated with low-risk characteristics. The computational analysis shows that, in a context of overall gene set poverty, the upregulomes in RAC1- and kalirin–GEF1–suppressed ADRN-type cells are a batch of AU-rich element–containing mRNAs, which suggests a link between NUC migration and mRNA stability. Gene set enrichment analysis–based search for vulnerabilities reveals prospective weak points in RAC1- and kalirin–GEF1–suppressed ADRN-type NB cells, including activities of H3K27- and DNA methyltransferases. Altogether, these data support the introduction of NUC inhibitors into cancer treatment research.
... Furthermore, the mRNA levels of Bax/Bcl2 were significantly upregulated in 10week-old βB1-CTGF mice. BAX is a proapoptotic member of the BCL2 family and plays a major role in mitochondrial-mediated apoptosis in different neuronal cells [23,24]. It was shown that deficiency of BAX prevented RGC degeneration in DBA/2J mice [25]. ...
To reveal the pathomechanisms of glaucoma, a common cause of blindness, suitable animal models are needed. As previously shown, retinal ganglion cell and optic nerve degeneration occur in βB1-CTGF mice. Here, we aimed to determine possible apoptotic mechanisms and degeneration of different retinal cells. Hence, retinae were processed for immunohistology (n = 5–9/group) and quantitative real-time PCR analysis (n = 5–7/group) in 5- and 10-week-old βB1-CTGF and wildtype controls. We noted significantly more cleaved caspase 3+ cells in βB1-CTGF retinae at 5 (p = 0.005) and 10 weeks (p = 0.02), and a significant upregulation of Casp3 and Bax/Bcl2 mRNA levels (p < 0.05). Furthermore, more terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL+) cells were detected in transgenic mice at 5 (p = 0.03) and 10 weeks (p = 0.02). Neurofilament H staining (p = 0.01) as well as Nefh (p = 0.02) and Tubb3 (p = 0.009) mRNA levels were significantly decreased at 10 weeks. GABAergic synapse intensity was lower at 5 weeks, while no alterations were noted at 10 weeks. The glutamatergic synapse intensity was decreased at 5 (p = 0.007) and 10 weeks (p = 0.01). No changes were observed for bipolar cells, photoreceptors, and macroglia. We conclude that apoptotic processes and synapse loss precede neuronal death in this model. This slow progression rate makes the βB1-CTGF mice a suitable model to study primary open-angle glaucoma.
... The proapoptotic Bcl-2 family member Bax is activated by signaling events in cells undergoing apoptotic cell death [11]. In neurons, genetic deletion of Bax is sufficient to block activation of the apoptotic pathway in response to multiple stimuli, including trophic factor withdrawal, excitotoxic insult, and ethanol exposure [10,12,13]. Blocking Bax is therefore a putative target to delineate neuron death occurring through activation of the apoptosis pathway from other mechanisms of injury. ...
Exposure to volatile anesthetics during the neonatal period results in acute neuron death. Prior work suggests that apoptosis is the dominant mechanism mediating neuron death. We show that Bax deficiency blocks neuronal death following exposure to isoflurane during the neonatal period. Blocking Bax-mediated neuron death attenuated the neuroinflammatory response of microglia following isoflurane exposure. We find that GABAergic interneurons are disproportionately overrepresented among dying neurons. Despite the increase in neuronal apoptosis induced by isoflurane exposure during the neonatal period, seizure susceptibility, spatial memory retention, and contextual fear memory were unaffected later in life. However, Bax deficiency alone led to mild deficiencies in spatial memory and contextual fear memory, suggesting that normal developmental apoptotic death is important for cognitive function. Collectively, these findings show that while GABAergic neurons in the neonatal brain undergo elevated Bax-dependent apoptotic cell death following exposure to isoflurane, this does not appear to have long-lasting consequences on overall neurological function later in life.
... The death of NGFdeprived neurons is triggered by insertion of the proapoptotic protein, BAX, into the outer mitochondrial membrane. This insertion causes the release of cytochrome c from the mitochondrial intermembrane space into the cytoplasm where it induces formation of the apoptosome and activation of caspase 3. The caspase 3 then cleaves critical protein substrates leading to cell death [11][12][13][14]. ...
Sympathetic neurons deprived of nerve growth factor (NGF) die by apoptosis. Chronic depolarization with elevated concentrations of extracellular potassium ([K+]E) supports long-term survival of these and other types of neurons in culture. While depolarization has long been used to support neuronal cultures, little is known about the mechanism. We explored how chronic depolarization of NGF-deprived mouse sympathetic neurons in culture blocks apoptotic death. First, we determined the effects of elevated [K+]E on proapoptotic BH3-only proteins reported to be upregulated in sympathetic neurons after NGF withdrawal. Upregulation of BIMEL was blocked by depolarization while upregulation of PUMA was not. BMF levels did not increase after NGF withdrawal, and elevated [K+]E had no effect on its expression. dp5/HRK was not detectable. A large increase in production of mitochondria-derived reactive species (RS), including reactive oxygen species (ROS), occurs in NGF-deprived sympathetic neurons. Suppressing these RS prevents cytochrome c release from mitochondria and apoptosis. The addition of high [K+]E to cultures rapidly blocked increased RS and cytochrome c release. Elevated [K+]E caused an increase of the cellular antioxidant glutathione (GSH). Preventing this increase prevented the elevated [K+]E from blocking cytochrome c release and death. While suppression of BIMEL upregulation by elevated [K+]E may contribute to high [K+]E pro-survival effects, we conclude that elevated [K+]E prevents apoptotic death of NGF-deprived sympathetic neurons primarily via an antioxidant mechanism.
... Further, BAX/BAK double knockout results in severe developmental defects that manifest in perinatal lethality in most cases, whereas the respective single gene knockout mice are viable with only mild phenotypes (Lindsten et al. 2000). Several studies have established a dominant role of BAX in neuronal apoptosis (Deckwerth et al. 1996;Steckley et al. 2007). Mice lacking only BAX show, among other defects, hyperplasia in the neuronal and lymphoid system (Knudson et al. 1995). ...
Mitochondria are key players of cellular metabolism, Ca2+ homeostasis, and apoptosis. The functionality of mitochondria is tightly regulated, and dysfunctional mitochondria are removed via mitophagy, a specialized form of autophagy that is compromised in hereditary forms of Parkinson's disease. Through mitophagy, cells are able to cope with mitochondrial stress until the damage becomes too great, which leads to the activation of pro-apoptotic BCL-2 family proteins located on the outer mitochondrial membrane. Active pro-apoptotic BCL-2 proteins facilitate the release of cytochrome c from the mitochondrial intermembrane space (IMS) into the cytosol, committing the cell to apoptosis by activating a cascade of cysteinyl-aspartate specific proteases (caspases). We are only beginning to understand how the choice between mitophagy and the activation of caspases is determined on the mitochondrial surface. Intriguingly in neurons, caspase activation also plays a non-apoptotic role in synaptic plasticity. Here we review the current knowledge on the interplay between mitophagy and caspase activation with a special focus on the central nervous system.
... In neurons, genetic deletion of Bax is sufficient to block the apoptotic pathway in 43 response to multiple stimuli, including trophic factor withdrawal, excitotoxic insult, and 44 ethanol exposure (10,12,13). Blocking Bax activation is therefore a putative target to 45 delineate neuronal death occurring through activation of the apoptotic pathway from 46 other mechanisms of injury. ...
Exposure to volatile anesthetics during the neonatal period results in acute neuronal death in rodent and non-human primate models, potentially leading to lasting cognitive deficits. We used Bax-/- mice to show that neuronal death following neonatal exposure to isoflurane is mediated by the apoptotic pathway, and that GABAergic interneurons are selectively vulnerable. Neonatal Bax-/- mice also showed attenuated microglial activation after exposure to isoflurane, indicating that neuroinflammatory response is secondary to neuronal apoptosis. Isoflurane-induced neuronal apoptosis in neonates appeared to have little effect on seizure threshold or cognitive function later in life. Collectively, these findings define the acute injury mechanism of volatile anesthetics during the neonatal period.
... NGF deprivationinduced apoptosis provides mechanistic support for dependence of developing sympathetic neurons on NGF (Huang and Reichardt, 2001). This idea is consolidated by the observation that developing peripheral neurons from Bax À/À mice are resistant to NGF withdrawal (Deckwerth et al., 1996). ...
Continuous neuronal survival is vital for mammals because mammalian brains have limited regeneration capability. After neurogenesis, suppression of apoptosis is needed to ensure a neuron’s long-term survival. Here we describe a robust genetic program that intrinsically attenuates apoptosis competence in neurons. Developmental downregulation of the splicing regulator PTBP1 in immature neurons allows neural-specific splicing of the evolutionarily conserved Bak1 microexon 5. Exon 5 inclusion triggers nonsense-mediated mRNA decay (NMD) and unproductive translation of Bak1 transcripts (N-Bak mRNA), leading to suppression of pro-apoptotic BAK1 proteins and allowing neurons to reduce apoptosis. Germline heterozygous ablation of exon 5 increases BAK1 proteins exclusively in the brain, inflates neuronal apoptosis, and leads to early postnatal mortality. Therefore, neural-specific exon 5 splicing and depletion of BAK1 proteins uniquely repress neuronal apoptosis. Although apoptosis is important for development, attenuation of apoptosis competence through neural-specific splicing of the Bak1 microexon is essential for neuronal and animal survival.
... For example, TERT protects against Bax-activating staurosporine in both 293T cells and MEFs 28 . Furthermore, the death of neonatal motor neurons after axotomy is mediated by Bax and the subsequent mitochondrial apoptotic cascades 29,30 . In addition to the prior reports, the present data provides additional evidence that hTERT could regulate mitochondrial function and its quality control processes by regulating PINK1. ...
Mutations in the phosphatase and tensin homologue-induced putative kinase 1 (PINK1) gene have been linked to an early-onset autosomal recessive form of familial Parkinson′s disease (PD). PINK1, a mitochondrial serine/threonine-protein kinase, plays an important role in clearing defective mitochondria by mitophagy – the selective removal of mitochondria through autophagy. Evidence suggests that alteration of the PINK1 pathway contributes to the pathogenesis of PD, but the mechanisms by which the PINK1 pathway regulates mitochondrial quality control through mitophagy remain unclear. Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase that functions in telomere maintenance as well as several non-telomeric activities. For example, hTERT has been associated with cellular immortalization, cell growth control, and mitochondrial regulation. We determined that hTERT negatively regulates the cleavage and cytosolic processing of PINK1 and enhances its mitochondrial localization by inhibiting mitochondrial processing peptidase β (MPPβ). Consequently, hTERT promotes mitophagy following carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced mitochondrial dysfunction and improves the function of damaged mitochondria by modulating PINK1. These findings suggest that hTERT positively regulates PINK1 function, leading to increased mitophagy following mitochondrial damage.
... Bax is a proapoptotic member of the bcl-2 family that enhances several forms of apoptosis and can directly induce cytochrome c release and apoptosis in transfected cells and in cell-free systems (16,17). Cells from animals lacking bax show variable resistance to apoptosis, although neurons from such animals are completely resistant to apoptosis induced by withdrawal of growth factors (18). The ability of p53 to induce the expression of bax in some cells is considered one of several ways that p53 triggers apoptosis in a transcriptionally dependent manner (8). ...
Objective
Studies were performed to determine if p53 mutations identified in rheumatoid arthritis (RA) synovial tissue are dominant negative.
Methods
Site‐directed mutagenesis was used to produce 2 RA‐derived mutants: asparagine→serine at codon 239 (N239S) and arginine→stop at codon 213 (R213*). HS68 dermal fibroblasts were transfected with either empty vector, wild‐type p53 cDNA (wt), or the N239S or R213* mutant p53 cDNA clones. Interleukin‐6 (IL‐6) and bax gene expression were determined by Northern blot analysis. Bax transcription was determined using a bax promoter/reporter gene construct (bax–luc).
Results
Transfection of HS68 cells with wt increased bax mRNA levels. This process was blocked by cotransfection with either mutant. The mutant p53 genes also increased IL‐6 gene expression. Low levels of bax promoter activity were detected in HS68 cells cotransfected with bax–luc and empty vector, N239S, or R213*, indicating that the RA mutants lacked transcriptional activity. Transfection with wt and bax–luc led to a 10‐fold increase in luciferase expression. When the wt gene was cotransfected with either of the mutants, there was a dose‐dependent inhibition of bax promoter activity.
Conclusion
These data indicate that at least 2 of the p53 mutants identified in RA joint samples are dominant negative and suppress endogenous wild‐type p53 function.
... We first established the neuroprotective effect of NeuroHeal treatment in a model of neonatal axotomy. It is known that neonatal axotomy of the sciatic nerve on or before postnatal day 4 triggers retrograde MN apoptosis [23][24][25]. We treated mothers of the pups with either vehicle or NeuroHeal through drinking water until sacrifice of pups according to workflow planning ( Figure 1A). ...
Rationale: Protective mechanisms allow healthy neurons to cope with diverse stresses. Excessive damage as well as aging can lead to defective functioning of these mechanisms. We recently designed NeuroHeal using artificial intelligence with the goal of bolstering endogenous neuroprotective mechanisms. Understanding the key nodes involved in neuroprotection will allow us to identify even more effective strategies for treatment of neurodegenerative diseases.
Methods: We used a model of peripheral nerve axotomy in rat pups, that induces retrograde apoptotic death of motoneurons. Nourishing mothers received treatment with vehicle, NeuroHeal or NeuroHeal plus nicotinamide, an inhibitor of sirtuins, and analysis of the pups were performed by immunohistochemistry, electron microscopy, and immunoblotting. In vitro, the post-translational status of proteins of interest was detailed using organotypic spinal cord cultures and genetic modifications in cell lines to unravel the neuroprotective mechanisms involved.
Results: We found that the concomitant activation of the NAD⁺-dependent deacetylase SIRT1 and the PI3K/AKT signaling pathway converge to increase the presence of deacetylated and phosphorylated FOXO3a, a transcription factor, in the nucleus. This favors the activation of autophagy, a pro-survival process, and prevents pro-apoptotic PARP1/2 cleavage.
Major conclusion: NeuroHeal is a neuroprotective agent for neonatal motoneurons that fine-tunes autophagy on by converging SIRT1/AKT/FOXO3a axis. NeuroHeal is a combo of repurposed drugs that allow its readiness for prospective pediatric use.
... These results contrasted with the phenotype observed in the Runx3 -/-;Bax -/embryos in which, in spite of the absence of sensory neuron cell death due to null mutation of the pro-apototic gene Bax (Deckwerth et al., 1996), their central afferents did not grow beyond the dorsal aspect of the spinal cord, as revealed by immunostaining for peripherin at E15.5 and VGLUT1 at E18.5 (Fig. 2G,H). Taken together, our results indicate that PSNs at limb levels require RUNX3 expression to project correctly into the ventral spinal cord, independently of its role on the outgrowth dynamic of their peripheral projections. ...
The control of all our motor outputs requires constant monitoring by proprioceptive sensory neurons (PSNs) that convey continuous muscle sensory inputs to the spinal motor network. Yet, the molecular programs that control the establishment of this sensorimotor circuit remain largely unknown. The transcription factor RUNX3 is essential for the early steps of PSNs differentiation, making it difficult to study its role during later aspects of PSNs specification. Here, we conditionally inactivate Runx3 in PSNs after peripheral innervation and identify that RUNX3 is necessary for maintenance of cell identity of only a subgroup of PSNs, without discernable cell death. RUNX3 controls also the sensorimotor connection between PSNs and motor neurons at limb level, with muscle-by-muscle variable sensitivities to the loss of Runx3 that correlate with levels of RUNX3 in PSNs. Finally, we find that muscles and neurotrophin-3 signaling are necessary for maintenance of RUNX3 expression in PSNs. Hence, a transcriptional regulator critical for specifying a generic PSN type identity after neurogenesis, is later regulated by target muscle-derived signal to contribute to the specialized aspects of the sensorimotor connection selectivity.
... This pathway functions to regulate neuronal apoptosis even in scenarios in which the primary insult does not involve mitochondrial damage, such as trophic factor withdrawal (Kristiansen and Ham 2014), suggesting that mitochondrial permeabilization broadly contributes to neuronal apoptosis. Neurons lacking Bax expression are strongly protected from degeneration and do not display caspase activation even after prolonged insult (Deckwerth et al. 1996;Vila et al. 2001;Libby et al. 2005). ...
Neurodegenerative disease is characterized by the progressive deterioration of neuronal function caused by the degeneration of synapses, axons, and ultimately the death of nerve cells. An increased understanding of the mechanisms underlying altered cellular homeostasis and neurodegeneration is critical to the development of effective treatments for disease. Here, we review what is known about neuronal cell death and how it relates to our understanding of neurodegenerative disease pathology. First, we discuss prominent molecular signaling pathways that drive neuronal loss, and highlight the upstream cell biology underlying their activation. We then address how neuronal death may occur during disease in response to neuron intrinsic and extrinsic stressors. An improved understanding of the molecular mechanisms underlying neuronal dysfunction and cell death will open up avenues for clinical intervention in a field lacking disease-modifying treatments.
... Reduction of apoptosis in the limbic system after MI may have important long-term repercussions on health, although this hypothesis still needs to be tested. It was proposed that neuron loss, which is associated with a reduction of neuronal activity of trophic support [20,21], could amplify the level of depression or contribute to the pathophysiology of dementia [22]. Our previous data pointed toward an effect of myocardial infarction-induced inflammation [1,23] underlying the increase in caspase-3 activity [4,5]. ...
Caspase-3 activation in the limbic system and depressive-like symptoms are observed after an acute myocardial infarction (MI) and studies suggest that inflammation may play a significant role. Combined treatment with the probiotic strains Bifidobacterium longum and Lactobacillus helveticus in rats has been shown to attenuate caspase-3 activation and depressive-like behaviour together with a reduction in pro-inflammatory cytokines. The present study was designed to determine the respective contribution of these two strains on caspase-3 activity in the limbic system and on depressive-like behaviour. Sprague-Dawley rats were assigned to one of four groups: Vehicle, L. helveticus R0052, B. longum R0175 and L. salivarius HA-118, administered orally for 14 days (10⁹CFU daily) before inducing MI by occlusion of the left anterior descending artery for 40 min followed by 14 days of reperfusion. Animals were then tested for socialisation, passive avoidance and forced swim test to assess depressive-like behaviour. At day 18 the animals were sacrificed; infarct size was estimated, plasma C-reactive protein concentration and brain caspase-3 activity were measured. Results indicated that infarct size did not vary across the different treatments. Rats treated with B. longum spent more time socializing, learned more rapidly the passive avoidance test and spent less time immobile in the forced swim test compared to the vehicle groups. Caspase-3 activity and plasma C-reactive protein concentrations were reduced in the lateral and medial amygdala as well as in the dentate gyrus of B. longum-supplemented animals. The only significant effect in the two groups receiving Lactobacilli compared to vehicle was that rats receiving L. salivarius learned more rapidly in the step-down passive avoidance test.
In conclusion, most of the beneficial effects that we previously reported with the combination of two probiotic strains in our experimentation regarding post-myocardial infarction depression are related to Bifidobacterium longum.
... For instance, perinatal brain development is associated with massive proliferation of neural progenitors 81 , followed by a wave of apoptosis that eliminates approximately half of cells that are either excessive or have not established appropriate synaptic connectivity 82 . The death of neuronal cells during this period is almost entirely BAX-dependent as demonstrated by the accumulation of excess neurons in Bax (but not Bak1) knockout animals 78,83 as well as BCL-2 overexpressing animals ( Fig. 3) 62 . Apart from BAX, also other pro-apoptotic proteins, including BIM and BID are highly expressed in the developing brain. ...
The loss of vital cells within healthy tissues contributes to the development, progression and treatment outcomes of many human disorders, including neurological and infectious diseases as well as environmental and medical toxicities. Conversely, the abnormal survival and accumulation of damaged or superfluous cells drive prominent human pathologies such as cancers and autoimmune diseases. Apoptosis is an evolutionarily conserved cell death pathway that is responsible for the programmed culling of cells during normal eukaryotic development and maintenance of organismal homeostasis. This pathway is controlled by the BCL-2 family of proteins, which contains both pro-apoptotic and pro-survival members that balance the decision between cellular life and death. Recent insights into the dynamic interactions between BCL-2 family proteins and how they control apoptotic cell death in healthy and diseased cells have uncovered novel opportunities for therapeutic intervention. Importantly, the development of both positive and negative small-molecule modulators of apoptosis is now enabling researchers to translate the discoveries that have been made in the laboratory into clinical practice to positively impact human health.
... Genetic and pharmacologic manipulation of Bcl-2 protein-protein interactions in vivo has demonstrated that processes which inhibit Bax oligomerization result in a substantial enhancement of neuronal survival following acute or chronic neural injury. 11,12 Consistent with this, peptides derived from two proteins known to interact with Bax (Ku70 and Bax inhibitor-1) have been shown to exhibit some ability to suppress Bax activation and in turn apoptotic cell death. 13,14 However, the administration of such agents exhibit significant clinical challenges due to their low bioavailability and stability in vivo. ...
Aberrant regulation of programmed cell death (PCD) has been tied to an array of human pathologies ranging from cancers to autoimmune disorders to diverse forms of neurodegeneration. Pharmacologic modulation of PCD signalling is therefore of central interest to a number of clinical and biomedical applications. A key component of PCD signalling involves the modulation of pro-and anti-apoptotic Bcl-2 family members. Among these, Bax translocation represents a critical regulatory phase in PCD. In the present study, we have employed a high-content high-throughput screen to identify small molecules which inhibit the cellular process of Bax re-distribution to the mitochondria following commitment of the cell to die. Screening of 6246 Generally Recognized As Safe compounds from four chemical libraries post-induction of cisplatin-mediated PCD resulted in the identification of 18 compounds which significantly reduced levels of Bax translocation. Further examination revealed protective effects via reduction of executioner caspase activity and enhanced mito-chondrial function. Consistent with their effects on Bax translocation, these compounds exhibited significant rescue against in vitro and in vivo cisplatin-induced apoptosis. Altogether, our findings identify a new set of clinically useful small molecules PCD inhibitors and highlight the role which cAMP plays in regulating Bax-mediated PCD.
... Other studies have shown that the first wave of apoptosis is often followed by a second wave [17]. This second wave may be attributed to loss of neuronal activity or trophic support for other neurons [6,18]. ...
Myocardial infarction (MI) in animal models induces cognitive deficits as well as the activation of caspase in the limbic system; both can be blocked by 2 weeks of treatment following MI using tricyclic antidepressants or selective serotonin uptake blockers. Here we used three different treatment schedules to test the short- and long-term effects of the combined serotonin-norepinephrine reuptake inhibitor desvenlafaxine on post-MI-associated cognitive deficits and caspase activation. MI was induced in 39 young adult rats, and 39 rats served as sham-operated controls. Desvenlafaxine (3 mg/kg/day, i.p.) or saline was administered according to one of three schedules: (1) for 2 weeks, starting right after surgery; (2) for 16 weeks, starting 2 weeks after surgery; (3) for 16 weeks, starting right after surgery. Behavior was tested 2 weeks (social interaction, passive avoidance) and 16 weeks (forced swimming, Morris water maze) after surgery. Caspase-3 and caspase-6 activities were measured 16 weeks after surgery. At 2 and 16 weeks post-surgery, saline-treated MI rats displayed performance deficits compared to desvenlafaxine-treated rats, regardless of the treatment schedule. Caspase-3 activity was higher in the amygdala (medial and lateral) and hippocampal CA3 region in untreated MI rats, whereas caspase-6 activity was higher in the CA1 region. Caspase-6 activity correlated positively with deficits in the Morris water maze. These results indicate that, independently of treatment schedules, various treatment schedules with desvenlafaxine can prevent MI-associated cognitive deficits and decrease caspase activities in the limbic system.
... BDNF levels are markedly reduced in the hippocampi of patients with depression [51]. Lower BDNF levels are associated with Bax-induced apoptosis [52]. Furthermore, an infusion of BDNF into the hippocampus produced antidepressant effects on rats [53]. ...
Reduced estradiol levels are associated with depression in women during the transition to and after menopause. A considerable number of studies focusing on the theme of treating depression through the activation of erythropoietin (EPO)-induced signaling pathways have been published. Venlafaxine is an approved antidepressant drug that inhibits both serotonin and norepinephrine transporters. The aim of the present study was to investigate the effects of venlafaxine on the depressive-like behaviors and serum estradiol levels in female rats following ovariectomy (OVX) and the possible roles of EPO-induced signaling pathways. Venlafaxine (10 mg/kg/day) was orally administered to OVX rats over a period of 4 weeks using two different treatment regimens: either starting 24 h or 2 weeks after OVX. Venlafaxine showed a superior efficacy in inducing antidepressant-like effects after an acute treatment (24 h post-OVX) than after the delayed treatment (2 weeks post-OVX) and was characterized by a decreased immobility time in the forced swimming test. In parallel, venlafaxine induced EPO and EPO receptor mRNA expression and increased levels of phospho-Janus kinase 2 (p-JAK2), phospho-signal transducer and activator of transcription 5, and phospho-extracellular signal-regulated kinase 1/2 in the hippocampus of OVX rats. Meanwhile, rats exhibited a marked reduction in the hippocampal Bax/Bcl2 ratio, caspase-3 activity, and tumor necrosis factor alpha levels after venlafaxine treatment. Venlafaxine also increased the hippocampal brain-derived neurotrophic factor and serum estradiol levels. Based on these findings, venlafaxine exerts a neuroprotective effect on OVX rats that is at least partially attributed to the activation of EPO/EPOR/JAK2 signaling pathways, anti-apoptotic activities, anti-inflammatory activities, and neurotrophic activities, as well as an increase in serum estradiol level.
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... Previous studies have demonstrated that germline deletion of Bak does not impact nervous system development [27]. In contrast, germline deletion of Bax (Bax Null) affects the development of several neuronal populations including motor neurons resulting in supernumerary cells [28]. Furthermore, Bax protein expression has been shown to increase in differentiating neurons [20]. ...
During neurogenesis, proliferating neural precursor cells (NPC) exit the cell cycle and differentiate into postmitotic neurons. The proteins that regulate cell survival through the stages of differentiation, however, are still poorly understood. Here, we examined the roles of the anti-apoptotic Bcl-2 proteins, Mcl-1 and Bcl-xL, in promoting survival as cells progress through the stages of neurogenesis in the mouse embryonic central nervous system. We used Nestin-mediated, nervous system-specific conditional deletion of mcl-1, bcl-x or both to identify their distinct and overlapping roles. Individual conditional deletion of mcl-1 (MKO) and bcl-x (BKO) suggested sequential roles in promoting cell survival during developmental neurogenesis. In the MKO embryo, apoptosis begins at embryonic day 10 (E10) in the proliferating NPC population throughout the entire developing nervous system. In the BKO embryo, apoptosis begins later at E11 within the postmitotic neuron populations. In the double (mcl-1 and bcl-x) conditional knockout (DKO), cell death extended throughout both proliferating and non-proliferating cell populations resulting in embryonic lethality at E12, earlier than in either the MKO or BKO. Apoptotic cell death of the entire central nervous system in the DKO demonstrates that both genes are necessary for cell survival during developmental neurogenesis. To determine whether Mcl-1 and Bcl-xL have overlapping anti-apoptotic roles during neurogenesis, we examined the impact of gene dosage. Loss of a single bcl-x allele in the MKO embryo exasperated apoptotic cell death within the NPC population revealing a novel anti-apoptotic role for Bcl-xL in proliferating NPCs. Cells were rescued from apoptosis in both the MKO and BKO embryos by breeding with the Bax null mouse line indicating that Mcl-1 and Bcl-xL have a common pro-apoptotic target during developmental neurogenesis. Taken together, these findings demonstrate that Mcl-1 and Bcl-xL are the two essential anti-apoptotic Bcl-2 proteins required for the survival of the developing mammalian nervous system.
In multicellular organisms, sexed gonads have evolved that facilitate release of sperm versus eggs, and bilaterian animals purposefully combine their gametes via mating behaviors. Distinct neural circuits have evolved that control these physically different mating events for animals producing eggs from ovaries versus sperm from testis. In this review, we will describe the developmental mechanisms that sexually differentiate neural circuits across three major clades of bilaterian animals–Ecdysozoa, Deuterosomia, and Lophotrochozoa. While many of the mechanisms inducing somatic and neuronal sex differentiation across these diverse organisms are clade‐specific rather than evolutionarily conserved, we develop a common framework for considering the developmental logic of these events and the types of neuronal differences that produce sex‐differentiated behaviors.
This article is categorized under: Congenital Diseases > Stem Cells and Development
Neurological Diseases > Stem Cells and Development
Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.
The post-ganglionic sympathetic neurons play an important role in modulating visceral functions and maintaining homeostasis through complex and reproducible axonal and dendritic connections between individual neurons and with their target tissues. Disruptions in these connections and in sympathetic nervous system function are observed in several neurological, cardiac and immune-related disorders, which underscores the need for understanding the mechanisms underlying neuronal polarity, axonal growth and dendritic growth in these neurons. The goals of this chapter are to explore our current understanding of the various growth factors, their signaling pathways, downstream effectors and interplay between these pathways to regulate different stages of axonal and dendritic growth in sympathetic neurons.
Like other herpesviruses, varicella-zoster virus (VZV) evolved a wide range of functions to modulate a broad array of host defences, presumably as a means to provide a survival advantage to the virus during infection. In addition to control of components of the adaptive immune response, VZV also modulates a range of innate responses. In this context, it has become increasingly apparent that VZV encodes specific functions that interfere with programmed cell death (PCD) pathways. This review will overview the current understanding of VZV-mediated control of PCD pathways, focussing on the three most well-defined PCD pathways: apoptosis, necroptosis and pyroptosis. We will also discuss future directions about these PCD pathways that are yet to be explored in the context of VZV infection.KeywordsVaricella-zoster virusApoptosisProgrammed cell death
In the central nervous system (CNS), execution of programmed cell death (PCD) is crucial for proper neurodevelopment. However, aberrant activation of these pathways in adult CNS leads to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). How a cell dies is critical, as it can drive local immune activation and tissue damage. Classical apoptosis engages several mechanisms to evoke “immunologically silent” responses, whereas other forms of programmed death such as pyroptosis, necroptosis, and ferroptosis release molecules that can potentiate immune responses and inflammation. In ALS, a fatal neuromuscular disorder marked by progressive death of lower and upper motor neurons, several cell types in the CNS express machinery for multiple PCD pathways. The specific cell types engaging PCD, and ultimate mechanisms by which neuronal death occurs in ALS are not well defined. Here, we provide an overview of different PCD pathways implicated in ALS. We also examine immune activation in ALS and differentiate apoptosis from necrotic mechanisms based on downstream immunological consequences. Lastly, we highlight therapeutic strategies that target cell death pathways in the treatment of neurodegeneration and inflammation in ALS.
Apoptosis is implicated in both developmental and disease related neuronal loss. The characteristic morphological changes that define apoptosis appear to result from the activation of members of a class of cysteine proteases known as caspases. While identification of the relevant caspases and their means of regulation have been investigated in many cell types, the corresponding pathways in neurones are still unclear. One activating mechanism in non neuronal cell types depends upon caspase-8 autocatalytic activation upon aggregation induced by interactions with protein domains referred to as death domains (DD) and death effector domains (DED). The aim of this study was the identification of neuronal caspases and potential DD/DED upstream regulators. These were studied in a model of developmental neuronal apoptosis, rat P1 superior cervical ganglion (SCG) neurones which are dependent upon nerve growth factor (NGF) for survival. The screening of a rat brain library for DD/DED encoding cDNAs resulted in the cloning of the rat homologue of FADD. Wild type (WT) FADD and a dominant negative (DN) FADD mutant were overexpressed by microinjection in SCG neurones. Overexpression of FADD WT did not induce cell death in neurones in the presence of NGF, whereas both the WT and DN rescued neurones from NGF withdrawal induced apoptosis, suggesting that DDs are involved in neuronal apoptosis. The expression of several caspases and DD-related proteins was assessed in SCG neurones with an optimised RT-PCR protocol. While caspases-2, -3, -6, -7 and -9 were found to be normally expressed, caspase-8, a known partner of FADD in non neuronal systems, was absent. Furthermore, the expression of caspase-8 and DD-related mRNAs such as FADD, Fas and Fas ligand were not induced after NGF withdrawal. These findings suggest a regulatory role for DD proteins in SCG neuronal apoptosis, which is different from the Fas-FADD-caspase-8 pathway described for non neuronal cells.
Tau is a microtubule-associated protein, normally binding to tubulin, in an interaction mediated by chaperones. Unwanted tau is degraded by proteasomes. The tauopathies are neurodegenerative disorders characterized by a tau-predominant neuropathology. Tauopathies include Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP-17, which can be caused by mutations in the tau gene). Pathological tau is hyperphosphorylated, and has reduced microtubule binding capacity. Tau mutations in the microtubule-binding region have similar effects. In some tauopathies, tau deposition is isoform-specific, and pathogenic mutations can cause isoform imbalances. Oxidative stress may also be a factor in tauopathies. In addition, tau pathology has been associated with an ubiquitin variant, which causes proteasome inhibition. There is evidence of aberrations in the chaperone system in diseased cells, reducing tau-microtubule binding, and degradation. To investigate the role of UBB+1 in tauopathies, a model system was developed with SH-SY5Y neuroblastoma cells, which showed that UBB+1 inhibits the proteasome. UBB+1 expression did not affect tau, but was coupled with a chaperone expression response. Subsequently, contrary to expectations, cells with induced UBB+1 expression coped better with oxidative insults. These proteins were studied in sections from PSP brains; UBB+1 and abnormal tau were only present in the diseased tissue. Chaperones and UBB+1 did not co-localise, indicating a failure in the chaperone response. In cells, mutant-tau localized with microtubules as effectively as wild-type. Through neuronal differentiation, tau concentrations were greatly increased, and phosphorylation was decreased. Proteasome inhibition did not increase tau concentration or phosphorylation, or affect isoform balance. A failure in the chaperone response in the brains of tauopathies could explain an increase in unbound cytosolic-tau, tau aggregation, decreased proteasome activity, and susceptibility to oxidative stress.
The ability of the small heat shock protein 27 (Hsp27) to prevent apoptotic neuronal death has been examined in primary sensory and motor neurons. The first part of the thesis investigates whether there is a correlation between the expression of Hsp27 and the survival of adult and neonatal sensory and motor neurons. Peripheral nerve injury is well known to induce a substantial death of neonatally injured, but not of adult injured sensory and motor neurons. In the adult, sciatic nerve axotomy results in a rapid upregulation of Hsp27 expression in all injured sensory and motor neurons. In contrast, sciatic nerve injury performed at birth induces Hsp27 upregulation only in a very small proportion (~5%) of injured neurons. Those neonatal neurons that express Hsp27 after nerve injury survive, and those that do not undergo apoptosis. The hypothesis that Hsp27 functions as an intrinsic neuronal survival factor is directly tested. Employing viral vectors to induce human Hsp27 expression in neonatal sensory neurons in vitro and neonatal motor neurons in vivo, Hsp27 is demonstrated to rescue both sensory and motor neurons from nerve injury induced cell death. Delivery of an antisense human Hsp27 construct with a Herpes-Simplex viral vector to knockdown endogenous Hsp27 is shown to induce apoptotic cell death in adult primary sensory neurons. These findings suggest that Hsp27 is both necessary and sufficient for cell survival in these neurons. Immunohistochemical analysis of the apoptotic pathway reveals that Hsp27 inhibits apoptosis in sensory neurons by acting downstream of cytochrome-c release from the mitochondria but upstream of caspase-3 activation. The contribution of post-translational changes in conferring a survival role for Hsp27 is studied by examining the ability of a non-phosphorylatable mutant of Hsp27 to protect neonatally injured motor neurons from death in vivo, and by investigating changes in the phosphorylation of Hsp27 on Seri5 after nerve injury. These findings suggest phosphorylation is important in the neuroprotective role of Hsp27. These findings that all injured adult sensory and motor neurons upregulate Hsp27 and survive and that only a minority of neonatal sensory and motor neurons upregulate Hsp27 and most die, together with the demonstration that Hsp27 sense expression can rescue neonatal cells while antisense constructs induce adult neuronal death, indicate that Hsp27 is indeed an intrinsic neuronal survival factor. That the Hsp27 in all adult injured sensory and motor neurons is phosphorylated, while constitutively expressed Hsp27 is not, as well as the failure of a non-phosphorylatable mutant Hsp27 to rescue cells, suggests that post-translational as well as transcriptional regulation of Hsp27 is important for promoting neuronal survival. The survival of sensory and motor neurons depends on the presence of intrinsic factors that include Hsp27, preventing full activation the apoptotic pathway.
Multiple members of the tumour necrosis factor superfamily (TNFSF) regulate the growth and branching of neural processes late in development, when neurons are establishing and refining connections. Here, we present the first evidence that a TNFSF member acts much earlier in development, when axons are growing to their targets. CD40L transiently enhanced axon growth from embryonic mouse DRG neurons cultured at this early stage. Early spinal nerves of embryos lacking the CD40L receptor (Cd40-/- mice) were significantly shorter in vivo than those of Cd40+/+ littermates. CD40L was synthesized in early DRG targets and was co-expressed with CD40 in early DRG neurons. Whereas CD40L enhanced early axon growth independently of neurotrophins, disruption of a CD40L/CD40 autocrine loop impaired early neurotrophin-promoted axon growth. In marked contrast to the widespread regulation of axon and dendrite growth by CD40L reverse signalling later in development, CD40-Fc, which activates reverse signalling, had no effect on early sensory axon growth. These results suggest that CD40 forward signalling is a novel physiological regulator of early axon growth that acts by target-derived and autocrine mechanisms.
Purpose of Review
This article summarizes the current studies on molecular biomarkers with potential implications in diagnosis, prognosis, and response to treatment in patients with glaucoma.
Recent Findings
Important advances have occurred in the understanding of the pathogenesis of glaucomatous neurodegeneration. Protein biomarkers associated with inflammatory, neurodegenerative, and other molecular pathways have been described in glaucoma patients in tear film, aqueous fluid, vitreous fluid, and serum, however, we are still far from having a clear picture of the whole molecular network that relates to the disease and its implications in clinical use.
Summary
Although more studies are needed, current and emerging molecular biomarkers candidates in glaucoma may eventually transition into clinical use and contribute to outline the concept of precision medicine and precision health in glaucoma.
Apoptosis plays a major role in shaping the developing nervous system during embryogenesis as neuronal precursors differentiate to become post‐mitotic neurons. However, once neurons are incorporated into functional circuits and become mature, they greatly restrict their capacity to die via apoptosis, thus allowing the mature nervous system to persist in a healthy and functional state throughout life. This robust restriction of the apoptotic pathway during neuronal differentiation and maturation is defined by multiple unique mechanisms that function to more precisely control and restrict the intrinsic apoptotic pathway. However, while these mechanisms are necessary for neuronal survival, mature neurons are still capable of activating the apoptotic pathway in certain pathological contexts. In this review, we highlight key mechanisms governing the survival of post‐mitotic neurons, while also detailing the physiological and pathological contexts in which neurons are capable of overcoming this high apoptotic threshold.
Naturally occurring cell death (NOCD) is a prominent feature of the developing nervous system. During this process, neurons express bcl-2, a major regulator of cell death whose expression may determine whether a neuron dies or survives. To gain insight into the possible role of bcl-2 during NOCD in vivo, we generated lines of transgenic mice in which neurons overexpress the human BCL-2 protein under the control of the neuron-specific enolase (NSE) or phosphoglycerate kinase (PGK) promoters. BCL-2 overexpression reduced neuronal loss during the NOCD period, which led to hypertrophy of the nervous system. For instance, the facial nucleus and the ganglion cell layer of the retina had, respectively, 40% and 50% more neurons than normal. Consistent with this finding, more axons than normal were found in the facial and optic nerves. We also tested whether neurons overexpressing BCL-2 were more resistant to permanent ischemia induced by middle cerebral artery occlusion; in transgenic mice, the volume of the brain infarction was reduced by 50% as compared with wild-type mice. These animals represent an invaluable tool for studying the effects of increased neuronal numbers on brain function as well as the mechanisms that control the survival of neurons during development and adulthood.
We have shown that overexpression of Bcl-x can rescue sympathetic neurones from nerve growth factor deprivation in vitro. We have also examined the distribution and expression of Bcl-x mRNA in the developing and adult nervous system using Northern blot and in situ hybridization. Bcl-x mRNA is widespread during development of the nervous system. In embryonic spinal cord, mRNA levels increase at the beginning of the naturally occurring cell death period, suggesting that Bcl-x may be involved in the selection of neurones during this period. In the brain, Bcl-x expression increases after birth to reach a high level in the adult brain. Neurones from the cortex, olfactory bulb, and Purkinje cells are among those expressing the highest levels of Bcl-x mRNA. The widespread expression of Bcl-x during development and in adult brain suggests of a role for Bcl-x beyond simply protecting neurones from developmental cell death.
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Fibroblast growth factor receptor (FGF-R) mRNA expression was examined in the adult rat CNS. Northern blot analysis showed a distinct 4.3 kb transcript in various CNS regions. In situ hybridization revealed widely distributed, but specific, populations of cells that express FGF-R mRNA. The most intense hybridization signals were observed in the hippocampus and in the pontine cholinergic neurons. The limbic system and brainstem nuclei, including motor nuclei, showed robust labeling. Cerebellar granule cells and spinal cord neurons were positive for FGF-R mRNA. The distribution of FGF-R mRNA differed significantly from that of NGF receptor mRNA; particularly, no hybridization signal was detected in basal forebrain cholinergic neurons. These results strongly suggest that FGF or FGF-like molecules may exert effects on specific neuronal populations in the mature CNS.
The period of natural cell death in the development of rodent motor neurons is followed by a period of sensitivity to axonal injury. In the rat this early postnatal period of vulnerability coincides with that of very low ciliary neurotrophic factor (CNTF) levels in the sciatic nerve before CNTF increases to the high, adult levels. The developmental time course of CNTF expression, its regional tissue distribution and its cytosolic localization (as suggested by its primary structure) favour a role for CNTF as a lesion factor rather than a target-derived neurotrophic molecule like nerve growth factor. Nevertheless CNTF exhibits neurotrophic activity in vitro on different populations of embryonic neurons. To determine whether the vulnerability of motor neurons to axotomy in the early postnatal phase is due to insufficient availability of CNTF, we transected the axons of newborn rat motor neurons and demonstrated that local application of CNTF prevents the degeneration of the corresponding cell bodies.
The patterns of expression of the bcl-2, bax, and bci-X genes were examined immunohistochemically in neurons of the adult rat brain before and after 10 min of global ischemia induced by transient cardiac arrest. High levels of the cell death promoting protein Bax and concomitant low levels of the apoptosis-blocking protein Bcl-2 were found in some populations of neurons that are particularly sensitive to cell death induced by transient global ischemia, such as the CA1 sector of the hippocampus and the Purkinje cells of the cerebellum. Moreover, within 0.5 to 3 hr after an ischemic episode, immunostaining for Bax was markedly increased within neurons with morphological features of degeneration in many regions of the brain. Use of a two-color staining method for simultaneous analysis of Bax protein and in situ detection of DNA-strand breaks revealed high levels of Bax immunoreactivity in many neurons undergoing apoptosis. Postischemic elevations in Bax protein levels in the hippocampus, cortex, and cerebellum were also demonstrated by immunoblotting. At early times after transient ischemia, regulation of Bcl-2 and Bcl-x protein levels varied among neuronal subpopulations, but from 3 hr on, those neurons with morphological evidence of degeneration uniformly contained reduced levels of Bci-2 and particularly Bci-X immunoreactivity. The findings suggest that differential expression of some members of the bcl-2 gene family may play an important role in determining the relative sensitivity of neuronal subpopulations to ischemia and that postischemic alterations in the expression of bax, bcl-2, and bcl-x may contribute to the delayed neuronal cell death that occurs during the repurfusion phase after a transient ischemic episode.
Bcl-2 and its analogs protect different classes of neurons from apoptosis in several experimental situations. These proteins may therefore provide a means for treatment of neurodegenerative diseases. We examined the effects of Bcl-2 overexpression in a genetic mouse model with motor neuron disease (progressive motor neuronopathy/pmn). Pmn/pmn mice lose motoneurons and myelinated axons, and die at 6 weeks of age. When these mice were crossed with transgenic mice that overexpress human Bcl-2, there was a rescue of the facial motoneurons with a concomitant restoration of their normal soma size and expression of choline acetyltransferase. However, Bcl-2 overexpression did not prevent degeneration of myelinated axons in the facial and phrenic motor nerves and it did not increase the life span of the animals. Since Bcl-2 acts strictly on neuronal cell body survival without compensating for nerve degeneration in pmn/pmn/bcl-2 mice, this proto-oncogene would not in itself be sufficient for treatment of neurodegenerative diseases where axonal impairment is a major component.
The time course of molecular events that accompany degeneration and death after nerve growth factor (NGF) deprivation and neuroprotection by NGF and other agents was examined in cultures of NGF-dependent neonatal rat sympathetic neurons and compared to death by apoptosis. Within 12 h after onset of NGF deprivation, glucose uptake, protein synthesis, and RNA synthesis fell precipitously followed by a moderate decrease of mitochondrial function. The molecular mechanisms underlying the NGF deprivation-induced decrease of protein synthesis and neuronal death were compared and found to be different, demonstrating that this decrease of protein synthesis is insufficient to cause death subsequently. After these early changes and during the onset of neuronal atrophy, inhibition of protein synthesis ceased to halt neuronal degeneration while readdition of NGF or a cAMP analogue remained neuroprotective for 6 h. This suggests a model in which a putative killer protein reaches lethal levels several hours before the neurons cease to respond to readdition of NGF with survival and become committed to die. Preceding loss of viability by 5 h and concurrent with commitment to die, the neuronal DNA fragmented into oligonucleosomes. The temporal and pharmacological characteristics of DNA fragmentation is consistent with DNA fragmentation being part of the mechanism that commits the neuron to die. The antimitotic and neurotoxin cytosine arabinoside induced DNA fragmentation in the presence of NGF, supporting previous evidence that it mimicked NGF deprivation-induced death closely. Thus trophic factor deprivation-induced death occurs by apoptosis and is an example of programmed cell death.
Most examples of cell death in animals are controlled by a genetic program that is activated within the dying cell. The apoptotic process is further regulated by a set of genes that act as repressors of cell death. Of these, bcl-2 is expressed in a variety of embryonic and postnatal tissues which suggests a critical role for bcl-2 in organogenesis and tissue homeostasis. Surprisingly, mutant mice with targeted disruption of bcl-2 appear normal at birth and complete maturation of lymphoid tissues before succumbing to fulminant lymphopenia and polycystic renal disease by 2-5 weeks of age. This suggests that there may be genes other than bcl-2 that can regulate apoptosis during development. To begin to investigate this possibility, we have cloned and characterized the murine bcl-x gene, whose human counterpart displays striking homology to bcl-2. The predicted murine bcl-xL gene product exhibits a high level of amino acid identity (97%) to its human counterpart. Just like Bcl-2, the murine bcl-xL gene product can act as a dominant inhibitor of cell death upon growth factor withdrawal. In addition, the bulk of the bcl-xL product localizes to the periphery of mitochondria as assessed by a bcl-xL-tag expression system, suggesting that both Bcl-2 and Bcl-xL proteins prevent cell death by a similar mechanism. bcl-xL is the most abundant bcl-x mRNA species expressed in embryonic and adult tissues. The levels of bcl-xL mRNA appear higher than those of bcl-2 during embryonal development and in several adult organs including bone marrow, brain, kidney and thymus. In addition to bcl-xL, we have identified another form of bcl-x mRNA, bcl-x beta, that results from an unspliced bcl-x transcript. bcl-x beta mRNA is expressed in various embryonic and postnatal tissues. Surprisingly, the expression of bcl-xS (a negative regulator of programmed cell death) was undetectable by a sensitive S1-nuclease assay and polymerase chain reaction analysis of mouse tissues. Based on its tissue and developmental patterns of expression, it appears that bcl-x may play an important role in the regulation of cell death during development and tissue homeostasis.
Serum-free PC12 cell cultures have been used to study the mechanisms of neuronal death after neurotrophic factor deprivation. We previously reported that PC12 cells undergo "apoptotic" internucleosomal DNA cleavage after withdrawal of trophic support. Here, we have used a sensitive method to detect PC12 cell DNA fragmentation within three hrs of serum removal and have exploited this assay to examine several aspects regarding the mechanisms of neuronal survival/death. Major advantages of this assay are that it permits acute experiments to be performed well before other manifest signs of cell death and under conditions that cannot be applied chronically. We find that this apopotic DNA fragmentation is distinct from the random DNA degradation that occurs during necrotic death. Major observations include the following: (a) There is a good correlation between the ability of trophic substances to promote PC12 cell survival and to inhibit early DNA fragmentation. (b) Phorbol ester, an activator of PKC, acutely suppresses DNA fragmentation, but does not promote long-term survival or inhibition of endonuclease activity when applied chronically due to its downregulation of PKC. (c) Cells undergoing apoptosis within 3 h of serum withdrawal have a "commitment point" of only 1.0-1.5 h beyond which they can no longer be rescued by NGF. (d) Aurin, a non-carboxylic analog of the endonuclease inhibitor ATA, also inhibits DNA fragmentation and promotes short-term survival of PC12 cells. (e) Macromolecular synthesis is not required for DNA fragmentation or for NGF to prevent this event. (f) Extracellular Ca2+ is not required for internucleosomal DNA cleavage caused by serum withdrawal or for suppression of this by NGF. (g) DNA fragmentation can also be detected in cultures of rat sympathetic neurons as early as 10 h after removal of NGF. As in PC12 cell cultures, this precedes morphological signs of cell death.
Members of the Bcl-2 family of proteins are characterized by their ability to modulate cell death. Bcl-2 and some of its homologues inhibit apoptosis, whereas other family members, such as Bax, will accelerate apoptosis under certain conditions. Here we describe the identification and characterization of a complementary DNA that encodes a previously unknown Bcl-2 homologue designated Bak. Like Bax, the bak gene product primarily enhances apoptotic cell death following an appropriate stimulus. Unlike Bax, however, Bak can inhibit cell death in an Epstein-Barr-virus-transformed cell line. The widespread tissue distribution of Bak messenger RNA, including those containing long-lived, terminally differentiated cell types, suggests that cell-death-inducing activity is broadly distributed, and that tissue-specific modulation of apoptosis is controlled primarily by regulation of molecules that inhibit apoptosis.
The bcl-2 protooncogene, which protects various cell types from apoptotic cell death, is expressed in the developing and adult nervous system. To explore its role in regulation of neuronal cell death, we generated transgenic mice expressing Bcl-2 under the control of the neuron-specific enolase promoter, which forced expression uniquely in neurons. Sensory neurons isolated from dorsal root ganglia of newborn mice normally require nerve growth factor for their survival in culture, but those from the bcl-2 transgenic mice showed enhanced survival in its absence. Furthermore, apoptotic death of motor neurons after axotomy of the sciatic nerve was inhibited in these mice. The number of neurons in two neuronal populations from the central and peripheral nervous system was increased by 30%, indicating that Bcl-2 expression can protect neurons from cell death during development. The generation of these transgenic mice suggests that Bcl-2 may play an important role in survival of neurons both during development and throughout adult life.
We have examined the hypothesis that neuronal programmed cell death requires a genetic program; we used a model wherein rat sympathetic neurons maintained in vitro are deprived of NGF and subsequently undergo apoptosis. To evaluate gene expression potentially necessary for this process, we used a PCR-based technique and in situ hybridization; patterns of general gene repression and selective gene induction were identified in NGF-deprived neurons. A temporal cascade of induced genes included "immediate early genes," which were remarkable in that their induction occurred hours after the initial stimulus of NGF removal and the synthesis of some required ongoing protein synthesis. The cascade also included the cell cycle gene c-myb and the genes encoding the extracellular matrix proteases transin and collagenase. Concurrent in situ hybridization and nuclear staining revealed that while c-jun was induced in most neurons, c-fos induction was restricted to neurons undergoing chromatin condensation, a hallmark of apoptosis. To evaluate the functional role of the proteins encoded by these genes, neutralizing antibodies were injected into neurons. Antibodies specific for either c-Jun or the Fos family (c-Fos, Fos B, Fra-1, and Fra-2) protected NGF-deprived neurons from apoptosis, whereas antibodies specific for Jun B, Jun D, or three nonimmune antibody preparations had no protective effect. Because these induced genes encode proteins ranging from a transcription factor necessary for death to proteases likely involved in tissue remodeling concurrent with death, these data may outline a genetic program responsible for neuronal programmed cell death.
To study molecular mechanisms underlying neuronal cell death, we have used sympathetic neurons from superior cervical ganglia which undergo programmed cell death when deprived of nerve growth factor. These neurons have been microinjected with expression vectors containing cDNAs encoding selected proteins to test their regulatory influence over cell death. Using this procedure, we have shown previously that sympathetic neurons can be protected from NGF deprivation by the protooncogene Bcl-2. We now report that the E1B19K protein from adenovirus and the p35 protein from baculovirus also rescue neurons. Other adenoviral proteins, E1A and E1B55K, have no effect on neuronal survival. E1B55K, known to block apoptosis mediated by p53 in proliferative cells, failed to rescue sympathetic neurons suggesting that p53 is not involved in neuronal death induced by NGF deprivation. E1B19K and p35 were also coinjected with Bcl-Xs which blocks Bcl-2 function in lymphoid cells. Although Bcl-Xs blocked the ability of Bcl-2 to rescue neurons, it had no effect on survival that was dependent upon expression of E1B19K or p35.
Interactions of the Bcl-2 protein with itself and other members of the Bcl-2 family, including Bcl-X-L, Bcl-X-S, Mcl-1, and Bax, were explored with a yeast two-hybrid system. Fusion proteins were created by linking Bcl-2 family proteins to a LexA DNA-binding domain or a B42 trans-activation domain. Protein-protein interactions were examined by expression of these fusion proteins in Saccharomyces cerevisiae having a lacZ (beta-galactosidase) gene under control of a LexA-dependent operator. This approach gave evidence for Bcl-2 protein homodimerization. Bcl-2 also interacted with Bcl-X-L and Mcl-1 and with the dominant inhibitors Bax and Bcl-X-S. Bcl-X-L displayed the same pattern of combinatorial interactions with Bcl-2 family proteins as Bcl-2. Use of deletion mutants of Bcl-2 suggested that Bcl-2 homodimerization involves interactions between two distinct regions within the Bcl-2 protein, since a LexA protein containing Bcl-2 amino acids 83-218 mediated functional interactions with a B42 fusion protein containing Bcl-2 amino acids 1-81 but did not complement a B42 fusion protein containing Bcl-2 amino acids 83-218. In contrast to LexA/Bcl-2 fusion proteins, expression of a LexA/Bax protein was lethal to yeast. This cytotoxicity could be abrogated by B42 fusion proteins containing Bcl-2, Bcl-X-L, or Mcl-1 but not those containing Bcl-X-S (an alternatively spliced form of Bcl-X that lacks a well-conserved 63-amino acid region). The findings suggest a model whereby Bax and Bcl-X-S differentially regulate Bcl-2 function, and indicate that requirements for Bcl-2/Bax heterodimerization may be different from those for Bcl-2/Bcl-2 homodimerization.
Approximately half of the neurons produced during embryogenesis normally die before adulthood. Although target-derived neurotrophic factors are known to be major determinants of programmed cell death--apoptosis--the molecular mechanisms by which trophic factors interfere with cell death regulation are largely unknown. Overexpression of the bcl-2 proto-oncogene in cultured sympathetic neurons has now been shown to prevent apoptosis normally induced by deprivation of nerve growth factor. This finding, together with the previous demonstration of bcl-2 expression in the nervous system, suggests that the Bcl-2 protein may be a major mediator of the effects of neurotrophic factors on neuronal survival.
We have examined the hypothesis that neuronal programmed cell death requires a genetic program; we used a model wherein rat sympathetic neurons maintained in vitro are deprived of NGF and subsequently undergo apoptosis. To evaluate gene expression potentially necessary for this process, we used a PCR-based technique and in situ hybridization; patterns of general gene repression and selective gene induction were identified in NGF-deprived neurons. A temporal cascade of induced genes included "immediate early genes," which were remarkable in that their induction occurred hours after the initial stimulus of NGF removal and the synthesis of some required ongoing protein synthesis. The cascade also included the cell cycle gene c-myb and the genes encoding the extracellular matrix proteases transin and collagenase. Concurrent in situ hybridization and nuclear staining revealed that while c-jun was induced in most neurons, c-fos induction was restricted to neurons undergoing chromatin condensation, a hallmark of apoptosis. To evaluate the functional role of the proteins encoded by these genes, neutralizing antibodies were injected into neurons. Antibodies specific for either c-Jun or the Fos family (c-Fos, Fos B, Fra-1, and Fra-2) protected NGF-deprived neurons from apoptosis, whereas antibodies specific for Jun B, Jun D, or three nonimmune antibody preparations had no protective effect. Because these induced genes encode proteins ranging from a transcription factor necessary for death to proteases likely involved in tissue remodeling concurrent with death, these data may outline a genetic program responsible for neuronal programmed cell death.
Bcl-2 is a mitochondrial- and perinuclear-associated protein that prolongs the lifespan of a variety of cell types by interfering with programmed cell death (apoptosis). Bcl-2 seems to function in an antioxidant pathway, and it is believed that membrane attachment mediated by a COOH-terminal hydrophobic tail is required for its full activity. To identify critical regions in bcl-2 alpha for subcellular localization, activity, and/or interaction with other proteins, we created, by site-directed mutagenesis, various deletion, truncation, and point mutations. We show here that membrane attachment is not required for the survival activity of bcl-2 alpha. A truncation mutant of bcl-2 alpha lacking the last 33 amino acids (T3.1) including the hydrophobic COOH terminus shows full activity in blocking apoptosis of nerve growth factor-deprived sympathetic neurons or TNF-alpha-treated L929 fibroblasts. Confocal microscopy reveals that the T3 mutant departs into the extremities of neurites in neurons and filopodias in fibroblasts. Consistently, T3 is predominantly detected in the soluble fraction by Western blotting, and is not inserted into microsomes after in vitro transcription/translation. We further provide evidence for motifs (S-N and S-II) at the NH2 and COOH terminus of bcl-2, which are crucial for its activity.
Sympathetic neurons require NGF for survival, but it is not known when these cells first become dependent on neurotrophic factors. We have examined in vitro mitotically active sympathetic neuroblasts immuno-isolated from different embryonic stages, and have correlated this functional data with the expression of neurotrophin receptor mRNAs in vivo. Cells from E14.5 ganglia are supported by neurotrophin-3 (NT-3) in a serum-free medium, but not by NGF; NT-3 acts as a bona fide survival factor for these cells and not simply as a mitogen. By birth, sympathetic neurons are well-supported by NGF, whereas NT-3 supports survival only weakly and at very high doses. This change in neurotrophin-responsiveness is correlated with a reciprocal switch in the expression of trkC and trkA mRNAs by sympathetic neuroblasts in vivo. These data suggest that neurotrophic factors may control neuronal number at earlier stages of development than previously anticipated. They also suggest that the acquisition of NGF-dependence may occur, at least in part, through the loss of receptors for these interim survival factors.
Fibroblast growth factor receptor (FGF-R) mRNA expression was examined in the adult rat CNS. Northern blot analysis showed a distinct 4.3 kb transcript in various CNS regions. In situ hybridization revealed widely distributed, but specific, populations of cells that express FGF-R mRNA. The most intense hybridization signals were observed in the hippocampus and in the pontine cholinergic neurons. The limbic system and brainstem nuclei, including motor nuclei, showed robust labeling. Cerebellar granule cells and spinal cord neurons were positive for FGF-R mRNA. The distribution of FGF-R mRNA differed significantly from that of NGF receptor mRNA; particularly, no hybridization signal was detected in basal forebrain cholinergic neurons. These results strongly suggest that FGF or FGF-like molecules may exert effects on specific neuronal populations in the mature CNS.
An experimental autoimmune model of nerve growth factor (NGF) deprivation has been used to assess the role of NGF in the development of various cell types in the nervous system. Adult rats immunized with 2.5S mouse NGF in complete Freund's adjuvant produced antibodies that crossreacted with their own NGF and that were transferred in utero to the fetus and in milk to the neonate. Cross-fostering experiments were carried out to separate the effects of exposure to anti-NGF in utero from those due to exposure through the milk. Anti-NGF transferred in utero and in milk resulted in the destruction of peripheral sympathetic neurons assessed by morphological methods (light microscopy) and biochemical methods (tyrosine hydroxylase activity, choline acetyltransferase activity, and protein content). No effects were observed on the adrenal medulla. Offspring of NGF-immunized females exposed to anti-NGF in utero had a decreased protein content in the dorsal root ganglia and were unable to transport (125)I-labeled NGF injected in the forepaw to the dorsal root ganglia. These results suggest that a subpopulation of sensory neurons is dependent on NGF for survival during some period of fetal development. This model offers the potential for determining the degree and time of dependence of various cell types on NGF.
Current ideas about the dependence of neurons on target-derived growth factors were formulated on the basis of experiments involving neurons with projections to the periphery. Nerve growth factor (NGF) and recently identified members of the NGF family of neuronal growth factors, known as neurotrophins, are thought to regulate survival of sympathetic and certain populations of sensory ganglion cells during development. Far less is known about factors that regulate the survival of spinal and cranial motor neurons, which also project to peripheral targets. NGF has not been shown to influence motor neuron survival, and whether the newly identified neurotrophins promote motor neuron survival is unknown. We show here that brain-derived neurotrophic factor (BDNF) is retrogradely transported by motor neurons in neonatal rats and that local application of BDNF to transected sciatic nerve prevents the massive death of motor neurons that normally follows axotomy in the neonatal period. These results show that BDNF has survival-promoting effects on motor neurons in vivo and suggest that BDNF may influence motor neuron survival during development.
Approximately half of the neurons produced during embryogenesis normally die before adulthood. Although target-derived neurotrophic factors are known to be major determinants of programmed cell death--apoptosis--the molecular mechanisms by which trophic factors interfere with cell death regulation are largely unknown. Overexpression of the bcl-2 proto-oncogene in cultured sympathetic neurons has now been shown to prevent apoptosis normally induced by deprivation of nerve growth factor. This finding, together with the previous demonstration of bcl-2 expression in the nervous system, suggests that the Bcl-2 protein may be a major mediator of the effects of neurotrophic factors on neuronal survival.
During normal vertebrate development, about half of spinal motoneurons are lost by a process of naturally occurring or programmed cell death. Additional developing motoneurons degenerate after the removal of targets or afferents. Naturally occurring motoneuron death as well as motoneuron death after loss of targets or after axotomy can be prevented by in vivo treatment with putative target (muscle) derived or other neurotrophic agents. Motoneurons can also be prevented from dying in vitro and in vivo (Y.Q.-W., R.W., D.P., J. Johnson and L. Van Eldik, unpublished data and refs 7, 13, 14) by treatment with central nervous system extracts (brain or spinal cord) and purified central nervous system and glia-derived proteins. Here we report that in vivo treatment of chick embryos with brain-derived neurotrophic factor rescues motoneurons from naturally occurring cell death. Furthermore, in vivo treatment with brain-derived neurotrophic factor (and nerve growth factor) also prevents the induced death of motoneurons that occurs following the removal of descending afferent input (deafferentation). These data indicate that members of the neurotrophin family can promote the survival of developing avian motoneurons.
The t(14;18)(q32;21) chromosomal translocation characteristic of follicular lymphomas is the most common cytogenetic abnormality known to be associated with any specific type of hematolymphoid malignancy. A fragment of DNA containing the crossover point between chromosomes 14 and 18 was cloned from the tumor cells of a patient with a follicular lymphoma carrying this translocation. Nucleotide sequence analysis of the breakpoint DNA revealed that the break in chromosome 14 occurred in joining region 4(J4) of the nonfunctional immunoglobulin heavy chain allele. This finding and other structural similarities of the breakpoint with the functional diversity region-joining region (D-J) joint in this lymphoma suggest that D-J recombination enzymes played a role in the mechanism of the t(14;18) translocation. Hybridization analysis of DNA from 40 follicular lymphomas showed that the majority of t(14;18) translocations occur on chromosome 18 DNA within 4.2 kilobases of the cloned breakpoint. A DNA probe from this breakpoint-cluster region detects transcription products in the tumor cells from which it was cloned and in a B-lymphoma cell line containing a t(14;18) translocation.
Specific chromosomal translocations found in distinct neoplasms suggest that genes that flank such breakpoints play a critical role in transformation. We have characterized the t(14;18)(q32;q21) chromosomal translocation present in over 60% of human follicular lymphomas. We exploited an unexpected rearrangement of an Ig heavy-chain gene to clone the chromosomal breakpoint. An element isolated from 18q21 mediated translocations in all four t(14;18) bearing cell lines and in six of 11 follicular lymphomas, but did not normally rearrange in other B or non-B cells. The breakpoints clustered within a small 4.3 kb region on chromosome 18. The breakpoints on chromosome 14 were focused within or immediately 5' to JH. These breakpoints retained the Ig enhancer region close to a new transcriptional unit identified on chromosome segment 18q21. Since none of the cellular oncogenes are known to map to 18q21, cloning this element provides an opportunity to characterize a potentially new transforming gene.
In this study, the joining sequences between chromosomes 14 and 18 on the 14q+ chromosomes of a patient with pre-B-cell leukemia and four patients with follicular lymphoma carrying a t(14;18) chromosome translocation were analyzed. In each case, the involved segment of chromosome 18 has recombined with the immunoglobulin heavy-chain joining segment (JH) on chromosome 14. The sites of the recombination on chromosome 14 are located close to the 5' end of the involved JH segment, where the diversity (D) regions are rearranged with the JH segments in the production of active heavy-chain genes. As extraneous nucleotides (N regions) were observed at joining sites and specific signal-like sequences were detected on chromosome 18 in close proximity to the breakpoints, it is concluded that the t(14;18) chromosome translocation is the result of a mistake during the process of VDJ joining at the pre-B-cell stage of differentiation. The putative recombinase joins separated DNA segments on two different chromosomes instead of joining separated segments on the same chromosome, causing a t(14;18) chromosome translocation in the involved B cells.
The wild-type functions of the genes ced-3 and ced-4 are required for the initiation of programmed cell deaths in the nematode Caenorhabditis elegans. The reduction or loss of ced-3 or ced-4 function results in a transformation in the fates of cells that normally die; in ced-3 or ced-4 mutants, such cells instead survive and differentiate, adopting fates that in the wild type and associated with other cells. ced-3 and ced-4 mutants appear grossly normal in morphology and behavior, indicating that programmed cell death is not an essential aspect of nematode development. The genes ced-3 and ced-4 define the first known step of a developmental pathway for programmed cell death, suggesting that these genes may be involved in determining which cells die during C. elegans development.
Although ganglia from neonatal mouse sympathetic ganglia require nerve growth factor (NGF) for survival in culture, explanted sympathetic ganglia from early embryonic stages do not require added NGF for survival and growth. To determine whether the change in growth factor requirement is due to changes in the neurons themselves, to variations in neuronal populations, or to changes in nonneuronal cells, we examined the response to growth factors by dissociated sympathetic neurons at various stages of development. Results indicate that neurons from the 14-day gestational (E14) superior cervical ganglion (SCG) do not require NGF for initial survival and neurite extension, but do require the conditioned medium neurite extension factor, CMF. By 2 to 3 days thereafter, whether in vivo or in culture, most neurons have developed a requirement for NGF for survival in culture. During the same period, there is a concomitant increase in responsiveness to NGF alone as a trophic agent. Changes in response to NGF are not due to changes in NGF content of ganglia, to interactions in culture with nonneuronal cells, or to age-related differences in NGF requirements for maximum survival. The changes in growth factor requirements may be related to mechanisms regulating specificity of nerve-target connections.
Motoneuron cell death in the lumbar lateral motor column of the mouse embryo and neonate was examined to determine the timing and position of cell death with respect to events occurring in the limb. Counts of motoneurons in histological sections of the entire lumbar lateral motor column were made in mice ranging in age from 12 1/2 days of embryonic development to 20 days of neonatal life. Between 13 and 18 days of embryonic development, 67% of the motoneurons initially present in the motor column die, or approximately 3350 out of 5000 cells. Peak motoneuron cell death occurs at 14 days. No cell death occurs during the first neonatal weeks when polyneuronal muscle fiber innervation is lost. Counts of the total number of cells present at 18 days were similar to those previously reported for adult mice, suggesting that all motoneuron cell death has occurred by the end of prenatal development. In a few 13- to 14-day mouse embryos, the hindlimbs were totally filled with horseradish peroxidase (HRP) to define the boundaries and size of the lateral motor population which projects to the limb at early stages. Counts of HRP labeled motoneurons in selected lumbar cord segments were close to the total number of lateral motoneurons in the same segments. As the HRP injections were made prior to or at the onset of cell death, these observations indicate that many cells which die have sent axons into the limb.
Sympathetic neurons, when placed in culture during the period of naturally occurring cell death, will die by apoptosis when deprived of nerve growth factor (NGF). In this system, the mRNA levels of the BCL-2 family members decrease after NGF deprivation and during apoptosis. Sympathetic neurons from BCL-2-deficient mice died more rapidly after NGF deprivation than neurons from wild-type littermates. Sympathetic neurons of adult animals are relatively independent of NGF for survival. If sympathetic neurons are maintained in vitro for several weeks, loss of acute trophic factor dependence develops with a time course similar to that seen in the intact animal. Examination of neurons from BCL-2-deficient mice showed that BCL-2 expression is not required for the development of trophic factor independence. Therefore, BCL-2 is an important regulator of the survival of sympathetic neurons after NGF deprivation during the period of naturally occurring programmed neuronal death, but BCL-2 is not involved in the development of trophic factor independence in mature sympathetic neurons.
BAX, a heterodimeric partner of BCL2, counters BCL2 and promotes apoptosis in gain-of-function experiments. A Bax knockout mouse was generated that proved viable but displayed lineage-specific aberrations in cell death. Thymocytes and
B cells in this mouse displayed hyperplasia, and Bax-deficient ovaries contained unusual atretic follicles with excess granulosa cells. In contrast, Bax-deficient males were infertile as a result of disordered seminiferous tubules with an accumulation of atypical premeiotic
germ cells, but no mature haploid sperm. Multinucleated giant cells and dysplastic cells accompanied massive cell death. Thus,
the loss of Bax results in hyperplasia or hypoplasia, depending on the cellular context.
A family of Bcl-2-related proteins regulates cell death and shares highly conserved BH1 and BH2 domains. BH1 and BH2 domains of Bcl-2 were required for it to heterodimerize with Bax and to repress apoptosis. A yeast two-hybrid assay accurately reproduced this interaction and defined a selectivity and hierarchy of further dimerizations. Bax also heterodimerizes with Bcl-xL, Mcl-1, and A1. A Gly-159-->Ala substitution in BH1 of Bcl-xL disrupted its heterodimerization with Bax and abrogated its inhibition of apoptosis in mammalian cells. This suggests that the susceptibility to apoptosis is determined by multiple competing dimerizations in which Bax may be a common partner.
During their lifespan, immature cells normally pass through sequential transitions to a differentiated state and eventually undergo cell death. This progression is aberrant in cancer, although the transition to differentiation can be reestablished in inducible leukemia cell lines. This report describes a gene, MCL1, that we isolated from the ML-1 human myeloid leukemia cell line during phorbol ester-induced differentiation along the monocyte/macrophage pathway. Our results demonstrate that expression of MCL1 increases early in the induction, or "programming," of differentiation in ML-1 (at 1-3 hr), before the appearance of differentiation markers and mature morphology (at 1-3 days). They further show that MCL1 has sequence similarity to BCL2, a gene involved in normal lymphoid development and in lymphomas with the t(14;18) chromosome translocation. MCL1 and BCL2 do not fall into previously known gene families. BCL2 differs from many oncogenes in that it inhibits programmed cell death, promoting viability rather than proliferation; this parallels the association of MCL1 with the programming of differentiation and concomitant maintenance of viability but not proliferation. Thus, in contrast to proliferation-associated genes, expression of MCL1 and BCL2 relates to the programming of differentiation and cell viability/death. The discovery of MCL1 broadens our perspective on an emerging MCL1/BCL2 gene family and will allow further comparison with oncogene families.
Cells are eliminated in a variety of physiological settings by apoptosis, a genetically encoded process of cellular suicide. Apoptosis comprises an intrinsic cellular defence against tumorigenesis, which, when suppressed, may contribute to the development of malignancies. The bcl-2 oncogene, which is activated in follicular lymphomas, functions as a potent suppressor of apoptosis under diverse conditions. Here we describe the complementary DNA cloning and functional analysis of a new Bcl-2 homologue, Bak, which promotes cell death and counteracts the protection from apoptosis provided by Bcl-2. Moreover, enforced expression of Bak induces rapid and extensive apoptosis of serum-deprived fibroblasts. This raises the possibility that Bak is directly involved in activating the cell death machinery.
A number of DNA viruses carry apoptosis-inhibiting genes which enable the virus to escape from the host response. The adenovirus E1B 19K protein can inhibit apoptosis induced by E1A, tumour-necrosis factor-alpha, FAS antigen and nerve growth factor deprivation. The molecular basis of this inhibition remains poorly understood, but the fact that protection is seen in the absence of other viral proteins suggests that E1B 19K targets cellular proteins. We report here the identification of three cellular proteins that bind E1B 19K. One of these is a new member of the bcl-2 family, which we have called bak (for bcl-2 homologous antagonist/killer). This protein, which is expressed in a wide variety of cell types, binds to E1B 19K and to the Bcl-2 homologue Bcl-XL (ref. 17) in yeast. In addition, overexpression of bak in sympathetic neurons deprived of nerve growth factor accelerates apoptosis and blocks the protective effect of co-injected E1B 19K.
Neuronal cell death is an important regressive event during the normal development of the peripheral and central nervous systems of many vertebrate and invertebrate species. Furthermore, when neurons are deprived of their target following axonal injury (axotomy) during embryonic, fetal, or early postnatal development, they undergo massive cell death. Both naturally occurring and axotomy-induced neuronal cell death can be prevented by treatment with growth factors or neurotrophic agents. Naturally occurring cell death of spinal MNs has been extensively studied in both avians and mammals. However, compared with mammals, there is little information on the effects of axotomy in avian species and it is not known whether trophic agents can modify axotomy-induced death in avian MNs. It is also not known whether trophic/growth factors can promote the in vivo survival of mammalian MNs during the period of naturally occurring cell death. We have examined (1) the time course of axotomy-induced death of lumbar spinal MNs in chick and mouse, and (2) the survival-promoting activity of a number of previously characterized growth and trophic factors on both programmed and axotomy-induced MN death in these two species. We show that axotomy performed on, or prior to, E12 in the chick results in a rapid decrease (i.e. 50%) in MN numbers within 3-4 days postsurgery, whereas these cells were able to survive for up to 1 week following axotomy on E14. By contrast, mouse MNs remained vulnerable to axotomy for at least 5 days after birth.(ABSTRACT TRUNCATED AT 250 WORDS)
Transgenic mice homozygously lacking in the bcl-2 gene were generated using homologous recombination in embryonal stem cells. The complete absence of Bcl-2 alpha and -beta proteins did not interfere with normal embryonic development. Abnormalities became evident after birth, although the severity varied among homozygous null mice, bcl-2-/- mice displayed pleiotropic abnormalities similar to those in the previously described bcl-2-/- mice, including growth retardation, smaller ears, short lives, polycystic kidney, atrophic thymus and spleen with accelerated apoptotic cell death of lymphocytes, and hair hypopigmentation in the second hair follicle cycle. Our bcl-2-/- mice also revealed novel defects in the small intestine, characterized by retarded development, accelerated exfoliation of epithelial cells, and very few mitotic progenitor cells.
During normal development of the vertebrate nervous system, large numbers of neurons in the central and peripheral nervous system undergo naturally occurring cell death. For example, about half of all spinal motor neurons die over a period of a few days in developing avian, rat and mouse embryos. Previous studies have shown that extracts from muscle and brain, secreted factors from glia, as well as several growth factors and neurotrophic agents, including muscle-derived factors, can promote the survival of developing motor neurons in vitro and in vivo. But because neurotrophins and other known trophic agents administered alone or in combination are insufficient to rescue all developing motor neurons from cell death, other neurotrophic molecules are probably essential for the survival and differentiation of motor neurons. Here we report that glial-cell-line-derived neurotrophic factor (GDNF), a potent neurotrophic factor that enhances survival of mammalian midbrain dopaminergic neurons, rescues developing avian motor neurons from natural programmed cell death in vivo and promotes the survival of enriched populations of cultured motor neurons. Furthermore, treatment with this agent in vivo also prevents the induced death and atrophy of both avian and mouse spinal motor neurons following peripheral axotomy.
Motor neurons require neurotrophic factor(s) for their survival during development and for maintenance of function in adulthood. In vivo studies have shown that motor neurons respond to a variety of molecules, including ciliary neurotrophic factor, members of the neurotrophin family, and the insulin growth factor IGF-1 (refs 3-13). Here we investigate the potential motor neuron neurotrophic effects of glial-cell-line-derived neurotrophic factor (GDNF), initially identified as a neurotrophic factor for substantia nigra dopaminergic neurons. We find that GDNF is retrogradely transported, in a receptor-mediated fashion, by spinal cord motor neurons in neonatal rats. Local application of GDNF to the transected facial nerve prevents the massive motor neuron cell death and atrophy that normally follows axotomy in the neonatal period. In adult rats, GDNF administered locally or systemically can markedly attenuate the lesion-induced decrease of choline acetyltransferase immunoreactivity in the facial nucleus. Our data indicate that GDNF has very profound neurotrophic effects in vivo on developing as well as on adult motor neurons, and is the most potent motor neuron trophic factor found so far.
To extend the mammalian cell death pathway, we screened for further Bcl-2 interacting proteins. Both yeast two-hybrid screening and lambda expression cloning identified a novel interacting protein, Bad, whose homology to Bcl-2 is limited to the BH1 and BH2 domains. Bad selectively dimerized with Bcl-xL as well as Bcl-2, but not with Bax, Bcl-xs, Mcl-1, A1, or itself. Bad binds more strongly to Bcl-xL than Bcl-2 in mammalian cells, and it reversed the death repressor activity of Bcl-xL, but not that of Bcl-2. When Bad dimerized with Bcl-xL, Bax was displaced and apoptosis was restored. When approximately half of Bax was heterodimerized, death was inhibited. The susceptibility of a cell to a death signal is determined by these competing dimerizations in which levels of Bad influence the effectiveness of Bcl-2 versus Bcl-xL in repressing death.
Sympathetic neurons in culture die by apoptosis when deprived of nerve growth factor (NGF). We used this model of programmed cell death to study the mechanisms that mediate neuronal apoptosis. Cultured sympathetic neurons were injected with copper/zinc superoxide dismutase protein (SOD) or with an expression vector containing an SOD cDNA. In both cases apoptosis was delayed when the neurons were deprived of NGF. The delay was similar to that seen when a bcl-2 expression vector was injected. SOD, injected 8 hr after NGF deprivation, provided no protection, indicating that superoxide production may occur early in response to trophic factor deprivation. We have demonstrated, with a redox-sensitive dye, an increase in reactive oxygen species (ROS) that peaked at 3 hr after sympathetic neurons were deprived of NGF. If NGF was added back to the culture medium after the period of peak ROS generation, apoptosis was completely prevented, suggesting that ROS production serves as an early signal, rather than a toxic agent, to mediate apoptosis.
bcl-x is a member of the bcl-2 gene family, which may regulate programmed cell death. Mice were generated that lacked Bcl-x.
The Bcl-x-deficient mice died around embryonic day 13. Extensive apoptotic cell death was evident in postmitotic immature
neurons of the developing brain, spinal cord, and dorsal root ganglia. Hematopoietic cells in the liver were also apoptotic.
Analyses of bcl-x double-knockout chimeric mice showed that the maturation of Bcl-x-deficient lymphocytes was diminished.
The life-span of immature lymphocytes, but not mature lymphocytes, was shortened. Thus, Bcl-x functions to support the viability
of immature cells during the development of the nervous and hematopoietic systems.
In vivo thymocyte maturation models were used to investigate the differentiation role of Bcl-2. In alpha/beta T cell receptor (TCR) class II-restricted transgenic mice, Bcl-2 was upregulated at the CD4+ CD8+ stage during positive selection. The lckpr-bcl2 transgene was bred onto MHC classes I-I- and II-I-, MHC-I-, and alpha/beta TCR backgrounds to determine whether Bcl-2 promoted thymocyte maturation in the absence of coreceptor-MHC interaction. Bcl-2 rescued CD8+ thymocytes in class I-I- and alpha/beta TCR in mice; however, they were not exported to the periphery. Bcl-2 had no effect on CD4 lineage maturation in class II-I- mice. No single-positive thymocytes accumulate in MHC-I- mice despite overexpressed Bcl-2. Thus, Bcl-2 enables selection of certain TCRs on class II molecules and their differentiation along the CD8 pathway; however, Bcl-2 did not substitute for positive selection. In RAG-1-I- mice, Bcl-2 promoted differentiation to the CD4+ CD8+ stage. Bcl-2 can promote thymocyte maturation at several control points.
Although proliferation is fundamental to the generation of neuronal populations, little is known about the function of trophic mechanisms during neurogenesis. We now describe a novel role for neurotrophin-3 (NT-3): the neurotrophin stimulates proliferation of sympathetic neuroblasts through trophic mechanisms. NT-3 promotes survival of the dividing precursors, but does not directly stimulate mitosis. NT-3 trophic effects differ markedly from those of the sympathetic mitogen, insulin. Furthermore, whereas NT-3 exhibits trophic activity for dividing neuroblasts, nerve growth factor characteristically promotes survival of postnatal sympathetic neurons. The stage-specific activity of NT-3 and nerve growth factor in culture parallels the sequence of trkC and trkA receptor gene expression detected in vivo. Thus, neurotrophins apparently serve as trophic factors during ontogeny, acting sequentially during establishment of individual populations.
The activity of the C. elegans gene ced-9 is required to protect cells that normally survive from undergoing programmed cell death. Here we describe the cloning and molecular characterization of this gene. ced-9 is an element of a polycistronic locus that also contains the gene cyt-1, which encodes a protein similar to cytochrome b560 of complex II of the mitochondrial respiratory chain. ced-9 encodes a 280 amino acid protein showing sequence and structural similarities to the mammalian proto-oncogene bcl-2. Overexpression of bcl-2 can mimic the protective effect of ced-9 on C. elegans cell death and can prevent the ectopic cell deaths that occur in ced-9 loss-of-function mutants. These results suggest that ced-9 and bcl-2 are homologs and that the molecular mechanism of programmed cell death has been conserved from nematodes to mammals.
Naturally occurring cell death (NOCD) is a prominent feature of the developing nervous system. During this process, neurons express bcl-2, a major regulator of cell death whose expression may determine whether a neuron dies or survives. To gain insight into the possible role of bcl-2 during NOCD in vivo, we generated lines of transgenic mice in which neurons overexpress the human BCL-2 protein under the control of the neuron-specific enolase (NSE) or phosphoglycerate kinase (PGK) promoters. BCL-2 overexpression reduced neuronal loss during the NOCD period, which led to hypertrophy of the nervous system. For instance, the facial nucleus and the ganglion cell layer of the retina had, respectively, 40% and 50% more neurons than normal. Consistent with this finding, more axons than normal were found in the facial and optic nerves. We also tested whether neurons overexpressing BCL-2 were more resistant to permanent ischemia induced by middle cerebral artery occlusion; in transgenic mice, the volume of the brain infarction was reduced by 50% as compared with wild-type mice. These animals represent an invaluable tool for studying the effects of increased neuronal numbers on brain function as well as the mechanisms that control the survival of neurons during development and adulthood.
Zoltan N. Oitvai* and Stanley 3. Korsmeyefl *Department of Pathology Northwestern University School of Medicine Chicago, Illinois 60610 tHoward Hughes Medical institute Division of Molecular Oncology Departments of Medicine and Pathology Washington University School of Medicine St. Louis, Missouri 63110 Recent evidence has emphasized the indispensibie role of programmed cell death in the development and mainte- nance of homeostasis within ail multicellular organisms. Genetic and molecular analysis from nematodes to hu- mans has indicated that cellular suicide is highly con- sewed (Hengartner and Horvitz, 1994, and references therein). Thus, it appears that the evolutionary switch from a unicellular to a multicellular existence hinged in part upon the creation of a regulated cell suicide response. A Cell-Autonomous Susceptibility to Dying From an individual cell’ s point of view, signals received from its environment can prompt the ultimate sacrifice: its own demise. The capacity to carry out apoptosis appears to be inherent to most (if not ail) ceils that depend upon an extracellular milieu of survival factors or cell-ceil contact molecules for their viability (Raff, 1992). Yet it is increasingly evident that the decision to die is not solely determined by extraceiluiar signals. in a number of biological systems, the degree of sensitivity to a given death stimulus is cell-type specific. For example, CD4+CD6+ cortical thymocytes are exquisitely sensitive to a wide vari- ety of apoptotic stimuli, while the more mature meduliary thymocytes are resistant. Similarly, in the Drosophila eye, a given signal can effectively rescue cells from apoptosis at one developmental stage, but not at another (Bonini et al., 1993). These observations imply the existence of an autonomous regulation of the apoptotic program. intracellular Checkpoints: The f3cM/Bax Rheostat The resistance of mature meduliary thymocytes to apop- totic signals correlates with a high expression level of the Bcl-2 protein. Bcl-2, originally identified at the t(l4;16) breakpoint in foilicular B cell lymphoma, can counter some but not ail stimuli known to induce cell death (Vaux et al., 1968). Bcl-2 is the founding member of an expanding family of proteins, whose principle homology is clustered within two conserved motifs called the Bcl-2 homology 1 (BHl) and homology 2 (BH2) domains (reviewed by Wii- liams and Smith, 1993). This family includes Bax, a homo- log that dimerizes with itself or with Bcl-2 and that, when overproduced, promotes apoptosis (Korsmeyer et al., 1993). Of note, Bax itself cannot trigger cell death, but requires a cell death signal (Figure 1). Selected mutations