C. Michael Knudson

Bax-Deficient Mice with Lymphoid Hyperplasia and Male Germ Cell Death

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.

BAX is required for neuronal death after trophic factor deprivation and during development.

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.

The brain ryanodine receptor: a caffeine-sensitive calcium release channel.

The release of stored Ca2+ from intracellular pools triggers a variety of important neuronal processes. Physiological and pharmacological evidence has indicated the presence of caffeine-sensitive intracellular pools that are distinct from the well-characterized inositol 1,4,5,-trisphosphate (IP3)-gated pools. Here we report that the brain ryanodine receptor functions as a caffeine- and ryanodine-sensitive Ca2+ release channel that is distinct from the brain IP3 receptor. The brain ryanodine receptor has been purified 6700-fold with no change in [3H]ryanodine binding affinity and shown to be a homotetramer composed of an approximately 500 kd protein subunit, which is identified by anti-peptide antibodies against the skeletal and cardiac muscle ryanodine receptors. Our results demonstrate that the brain ryanodine receptor functions as a caffeine-sensitive Ca2+ release channel and thus is the likely gating mechanism for intracellular caffeine-sensitive Ca2+ pools in neurons.

Prolongation of ovarian lifespan into advanced chronological age by Bax -deficiency

Female mammals are endowed with a finite number of oocytes at birth, each enclosed by a single layer of somatic (granulosa) cells in a primordial follicle. The fate of most follicles is atretic degeneration, a process that culminates in near exhaustion of the oocyte reserve at approximately the fifth decade of life in women, leading to menopause. Apoptosis has a fundamental role in follicular atresia, and recent studies have shown that Bax, which is expressed in both granulosa cells and oocytes, may be central to ovarian cell death. Here we show that young adult female Bax–/– mice possess threefold more primordial follicles in their ovarian reserve than their wild–type sisters, and this surfeit of follicles is maintained in advanced chronological age, such that 20–22–month–old female Bax–/– mice possess hundreds of follicles at all developmental stages and exhibit ovarian steroid–driven uterine hypertrophy. These observations contrast with the ovarian and uterine atrophy seen in aged wild–type female mice. Aged female Bax–/– mice fail to become pregnant when housed with young adult males; however, metaphase II oocytes can be retrieved from, and corpora lutea form in, ovaries of aged Bax–/– females following superovulation with exogenous gonadotropins, and some oocytes are competent for in vitro fertilization and early embryogenesis. Therefore, ovarian lifespan can be extended by selectively disrupting Bax function, but other aspects of normal reproductive performance remain defective in aged Bax–/– female mice.

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

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

Neurotrophins support the development of diverse sensory axon morphologies

The initial outgrowth of peripheral axons in developing embryos is thought to occur independently of neurotrophins. However, the degree to which peripheral neurons can extend axons and elaborate axonal arborizations in the absence of these molecules has not been studied directly because of exquisite survival requirements for neurotrophins at early developmental stages. We show here that embryonic sensory neurons from BAX-deficient mice survived indefinitely in the absence of neurotrophins, even in highly dissociated cultures, allowing assessment of cell autonomous axon outgrowth. At embryonic day 11 (E11)–E13, stages of rapid axon growth toward targets in vivo, Bax−/− sensory neurons cultured without neurotrophins were almost invariably unipolar and extended only a rudimentary axon. Addition of neurotrophins caused outgrowth of a second axon and a marked, dose-dependent elongation of both processes. Surprisingly, morphological responses to individual neurotrophins differed substantially. Neurotrophin-3 (NT-3) supported striking terminal arborization of subsets of Bax−/− neurons, whereas NGF produced predominantly axon elongation in a different subset. We conclude that axon growth in vitro is neurotrophin dependent from the earliest stages of sensory neuron development. Furthermore, neurotrophins support the appearance of distinct axonal morphologies that characterize different sensory neuron subpopulations.

Bax-Dependent Spermatogonia Apoptosis Is Required for Testicular Development and Spermatogenesis

Abstract Bax is a multidomain, proapoptotic member of the Bcl-2 family that is required for normal spermatogenesis in mice. Despite its proapoptotic function, previous results found that Bax-deficient mature male mice demonstrate increased cell death and dramatic testicular atrophy. The present study examined the role of Bax during the normal development of the testis to determine whether the increased cell death in mature mice could be explained by decreased apoptosis earlier in development. Consistent with this hypothesis, testicular atrophy is preceded by increased testicular weight and hypercellular tubules in immature Bax-deficient mice. TUNEL staining at Postnatal Day (P) 7 and morphological quantitation between P5 and P15 demonstrates decreased germ cell apoptosis in Bax-deficient mice. By P15, increased numbers of type A spermatogonia, and at P12 and P15, an increase in intermediate type spermatogonia were noted in Bax-deficient animals. By P25, the number of basal compartment cells was greatly increased in Bax-deficient animals compared with controls such that four or five layers of preleptotene spermatocytes were routinely present within the basal compartment of the testis. Although the Sertoli cell barrier was significantly removed from the basement membrane, it appeared intact as judged by the hypertonic fixation test. During late pubertal development, massive degeneration of germ cells took place, including many of those cell types that previously survived in the first wave of spermatogenesis. The data indicate that Bax is required for normal developmental germ cell death in the type A spermatogonia, specifically dividing (A2, A3, and A4) spermatogonia, at a time at which the number of spermatogonia is regulated in a density-dependent manner. The massive hyperplasia that occurs in Bax-deficient mice subsequently results in Bax independent cell death that may be triggered by overcrowding of the seminiferous epithelium.

Bcl-xL/Bcl-2 coordinately regulates apoptosis, cell cycle arrest and cell cycle entry.

Bcl‐xL and Bcl‐2 inhibit both apoptosis and proliferation. In investigating the relationship between these two functions of Bcl‐xL and Bcl‐2, an analysis of 24 Bcl‐xL and Bcl‐2 mutant alleles, including substitutions at residue Y28 previously reported to selectively abolish the cell cycle activity, showed that cell cycle delay and anti‐apoptosis co‐segregated in all cases. In determining whether Bcl‐2 and Bcl‐xL act in G0 or G1, forward scatter and pyronin Y fluorescence measurements indicated that Bcl‐2 and Bcl‐xL cells arrested more effectively in G0 than controls, and were delayed in G0–G1 transition. The cell cycle effects of Bcl‐2 and Bcl‐xL were reversed by Bad, a molecule that counters the survival function of Bcl‐2 and Bcl‐xL. When control and Bcl‐xL cells of equivalent size and pyronin Y fluorescence were compared, the kinetics of cell cycle entry were similar, demonstrating that the ability of Bcl‐xL and Bcl‐2 cells to enhance G0 arrest contributes significantly to cell cycle delay. Our data suggest that cell cycle effects and increased survival both result from intrinsic functions of Bcl‐2 and Bcl‐xL.

The pro-apoptotic gene Bax is required for the death of ectopic primordial germ cells during their migration in the mouse embryo

In the mouse embryo, significant numbers of primordial germ cells (PGCs) fail to migrate correctly to the genital ridges early in organogenesis. These usually die in ectopic locations. In humans, 50% of pediatric germ line tumors arise outside the gonads, and these are thought to arise from PGCs that fail to die in ectopic locations. We show that the pro-apoptotic gene Bax, previously shown to be required for germ cell death during later stages of their differentiation in the gonads, is also expressed during germ cell migration, and is required for the normal death of germ cells left in ectopic locations during and after germ cell migration. In addition, we show that Bax is downstream of the known cell survival signaling interaction mediated by the Steel factor/Kit ligand/receptor interaction. Together, these observations identify the major mechanism that removes ectopic germ cells from the embryo at early stages.

The TCF-1 and LEF-1 transcription factors have cooperative and opposing roles in T-cell development and malignancy

The TCF-1 and LEF-1 transcription factors are known to play critical roles in normal thymocyte development. Unexpectedly, we found that TCF-1-deficient (Tcf7(-/-)) mice developed aggressive T cell malignancy, resembling human T cell acute lymphoblastic leukemia (T-ALL). LEF-1 was aberrantly upregulated in premalignant Tcf7(-/-) early thymocytes and lymphoma cells. We further demonstrated that TCF-1 directly repressed LEF-1 expression in early thymocytes and that conditional inactivation of Lef1 greatly delayed or prevented T cell malignancy in Tcf7(-/-) mice. In human T-ALLs, an early thymic progenitor (ETP) subtype was associated with diminished TCF7 expression, and two of the ETP-ALL cases harbored TCF7 gene deletions. We also showed that TCF-1 and LEF-1 were dispensable for T cell lineage commitment but instead were required for early thymocytes to mature beyond the CD4(-)CD8(-) stage. TCF-1 thus has dual roles, i.e., acting cooperatively with LEF-1 to promote thymocyte maturation while restraining LEF-1 expression to prevent malignant transformation of developing thymocytes.