Mature neurons dynamically restrict apoptosis via redundant premitochondrial brakes

Apoptotic cell death is critical for the early development of the nervous system, but once the nervous system is established, the apoptotic pathway becomes highly restricted in mature neurons. However, the mechanisms underlying this increased resistance to apoptosis in these mature neurons are not completely understood. We have previously found that members of the miR-29 family of microRNAs (miRNAs) are induced with neuronal maturation and that overexpression of miR-29 was sufficient to restrict apoptosis in neurons. To determine whether endogenous miR-29 alone was responsible for the inhibition of cytochrome c release in mature neurons, we examined the status of the apoptotic pathway in sympathetic neurons deficient for all three miR-29 family members. Unexpectedly, we found that the apoptotic pathway remained largely restricted in miR-29-deficient mature neurons. We therefore probed for additional mechanisms by which mature neurons resist apoptosis. We identify miR-24 as another miRNA that is upregulated in the maturing cerebellum and sympathetic neurons that can act redundantly with miR-29 by targeting a similar repertoire of pro-death BH3-only genes. These results reveal that mature neurons engage redundant brakes to restrict the apoptotic pathway and ensure their long-term survival

Bcl-xL Is Essential for the Survival and Function of Differentiated Neurons in the Cortex That Control Complex Behaviors

Apoptosis plays an essential role during brain development, yet the precise mechanism by which this pathway is regulated in the brain remains unknown. In particular, mammalian cells are known to express multiple anti-apoptotic Bcl-2 family proteins. However, the cells of the developing brain could also exist in a primed state in which the loss of a single anti-apoptotic Bcl-2 family protein is sufficient to trigger apoptosis. Here, we examined the critical role of Bcl-xL, an anti-apoptotic protein, during brain development. Using conditional knock-out mice in which Bcl-xL is deleted in neural progenitor cells (Bcl-xLEmx1–Cre), we show that the loss of Bcl-xL is not sufficient to trigger apoptosis in these proliferating progenitors. In contrast, specific populations of postmitotic neurons derived from these progenitors, including upper layer cortical neurons and the CA1–CA3 regions of the hippocampus, were acutely dependent on Bcl-xL. Consistent with this finding, deletion of Bcl-xL selectively in the postmitotic neurons in the brain (Bcl-xLNex–Cre) also resulted in similar patterns of apoptosis. This Bcl-xL deficiency-induced neuronal death was a consequence of activation of the apoptotic pathway, because the cell death was rescued with codeletion of the proapoptotic proteins Bax and Bak. Importantly, the loss of these Bcl-xL-dependent neurons led to severe neurobehavioral abnormalities, including deficits in motor learning, hyperactivity, and increased risk-taking and self-injurious behaviors. Together, our results identify a population of neurons in the developing brain that are acutely dependent on Bcl-xL during the peak period of synaptic connectivity that are important for the establishment of higher-order complex behaviors

Distinct pathways mediate axon degeneration during apoptosis and axon-specific pruning

Neurons can activate pathways that destroy the whole cell via apoptosis or selectively degenerate only the axon (pruning). Both apoptosis and axon degeneration require Bax and caspases. Here we demonstrate that despite this overlap, the pathways mediating axon degeneration during apoptosis versus axon pruning are distinct. While caspase-6 is activated in axons following nerve growth factor (NGF) deprivation, microfluidic chamber experiments reveal that caspase-6 deficiency only protects axons during axon-specific but not whole-cell (apoptotic) NGF deprivation. Strikingly, axon-selective degeneration requires the apoptotic proteins Caspase-9 and Caspase-3 but, in contrast to apoptosis, not Apaf-1. Additionally, cell bodies of degenerating axons are protected from caspase activation by protea some activity and XIAP. Also, mature neurons restrict apoptosis but remain permissive for axon degeneration, further demonstrating the independent regulation of these two pathways. These results reveal insight into how neurons allow for precise control over apoptosis and axon-selective degeneration pathways, thereby permitting long-term plasticity without risking neurodegeneration

Essential Function of Dicer in Resolving DNA Damage in the Rapidly Dividing Cells of the Developing and Malignant Cerebellum

Maintenance of genomic integrity is critical during neurodevelopment, particularly in rapidly dividing cerebellar granule neuronal precursors that experience constitutive replication-associated DNA damage. As Dicer was recently recognized to have an unexpected function in the DNA damage response, we examined whether Dicer was important for preserving genomic integrity in the developing brain. We report that deletion of Dicer in the developing mouse cerebellum resulted in the accumulation of DNA damage leading to cerebellar progenitor degeneration, which was rescued with p53 deficiency; deletion of DGCR8 also resulted in similar DNA damage and cerebellar degeneration. Dicer deficiency also resulted in DNA damage and death in other rapidly dividing cells including embryonic stem cells and the malignant cerebellar progenitors in a mouse model of medulloblastoma. Together, these results identify an essential function of Dicer in resolving the spontaneous DNA damage that occurs during the rapid proliferation of developmental progenitors and malignant cells

Human Embryonic Stem Cells Have Constitutively Active Bax at the Golgi and Are Primed to Undergo Rapid Apoptosis

Human embryonic stem (hES) cells activate a rapid apoptotic response after DNA damage but the underlying mechanisms are unknown. A critical mediator of apoptosis is Bax, which is reported to become active and translocate to the mitochondria only after apoptotic stimuli. Here we show that undifferentiated hES cells constitutively maintain Bax in its active conformation. Surprisingly, active Bax was maintained at the Golgi rather than at the mitochondria, thus allowing hES cells to effectively minimize the risks associated with having pre-activated Bax. After DNA damage, active Bax rapidly translocated to the mitochondria by a p53-dependent mechanism. Interestingly, upon differentiation, Bax was no longer active and cells were not acutely sensitive to DNA damage. Thus, maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death, likely to prevent the propagation of mutations during the early critical stages of embryonic development

miR-29b is activated during neuronal maturation and targets BH3-only genes to restrict apoptosis

The execution of apoptosis is critical for proper development of the nervous system. However, it is equally important that neurons strictly inhibit apoptosis after development to ensure their survival throughout the lifetime of the organism. Here we show that a microRNA, miR-29b, is markedly induced with neuronal maturation and functions as a novel inhibitor of neuronal apoptosis. The prosurvival function of miR-29b is mediated by targeting genes in the proapoptotic BH3-only family. Our results identify a unique strategy evolved by maturing neurons that uses a single microRNA to inhibit the multiple, redundant BH3-only proteins that are key initiators of apoptosis

Characterization of a Cul9–Parkin double knockout mouse model for Parkinson’s disease

Acknowledgements We thank Viktoriya Nikolova at the UNC Mouse Behavioural Phenotyping Laboratory for her technical assistance. This work was supported by a Rapid Response Innovation Award (ID 9056) from the Michael J Fox Foundation for Parkinson’s Research and by the NIH Grant GM118331 to M.D. V.J. was supported by Training in translational Research in Neurology NIH Fellowship 2T32NS007480. The Tansey laboratory is supported by NIH/NIA 1R01 AG057247, NIH/NINDS 5R01 NS092122, NIH/NIA 3RF1 AG051514-01, and the Norman Fixel Institute for Neurological Diseases to M.G.T. The UNC Mouse Behavioural Phenotyping Laboratory is supported by a grant from the National Institute of Child Health and Human Development (NICHD), U54-HD079124. The Neuroscience Microscopy Core Facility is supported, in part, by funding from the NIH-NINDS Neuroscience Center Support Grant P30 NS045892 and the NIH-NICHD Intellectual and Developmental Disabilities Research Center Support Grant U54 HD079124. Contributions E.H. conducted the in vitro experiments and prepared the mice cohorts. V.S. and A.N. helped with generating the Cul9, Parkin double KO mice. V.J and M.J.T. performed the stereological analysis and quantification of dopaminergic neurons. S.M. conducted and analysed the neurobehavioral assessments. M.D. outlined and supervised the project. E.H. and M.D. produced the final version of the manuscript.Peer reviewedPublisher PD

Bcl-xL Is Essential for the Survival and Function of Differentiated Neurons in the Cortex That Control Complex Behaviors.

UNLABELLED: Apoptosis plays an essential role during brain development, yet the precise mechanism by which this pathway is regulated in the brain remains unknown. In particular, mammalian cells are known to express multiple anti-apoptotic Bcl-2 family proteins. However, the cells of the developing brain could also exist in a primed state in which the loss of a single anti-apoptotic Bcl-2 family protein is sufficient to trigger apoptosis. Here, we examined the critical role of Bcl-xL, an anti-apoptotic protein, during brain development. Using conditional knock-out mice in which Bcl-xL is deleted in neural progenitor cells (Bcl-xL(Emx1-Cre)), we show that the loss of Bcl-xL is not sufficient to trigger apoptosis in these proliferating progenitors. In contrast, specific populations of postmitotic neurons derived from these progenitors, including upper layer cortical neurons and the CA1-CA3 regions of the hippocampus, were acutely dependent on Bcl-xL. Consistent with this finding, deletion of Bcl-xL selectively in the postmitotic neurons in the brain (Bcl-xL(Nex-Cre)) also resulted in similar patterns of apoptosis. This Bcl-xL deficiency-induced neuronal death was a consequence of activation of the apoptotic pathway, because the cell death was rescued with codeletion of the proapoptotic proteins Bax and Bak. Importantly, the loss of these Bcl-xL-dependent neurons led to severe neurobehavioral abnormalities, including deficits in motor learning, hyperactivity, and increased risk-taking and self-injurious behaviors. Together, our results identify a population of neurons in the developing brain that are acutely dependent on Bcl-xL during the peak period of synaptic connectivity that are important for the establishment of higher-order complex behaviors. SIGNIFICANCE STATEMENT: Although Bcl-xL is known to inhibit apoptosis, exactly which cells in the brain are dependent on Bcl-xL has remained unclear because of the embryonic lethality of mice globally deleted for Bcl-xL. Here, we conditionally deleted Bcl-xL in the brain and found that this did not result in widespread apoptosis in the proliferating progenitors. Instead, Bcl-xL deficiency induced apoptosis in a select population of differentiated neurons predominantly in the early postnatal stages. Importantly, these Bcl-xL-dependent neurons are not essential for survival of the organism but instead regulate complex behaviors. Our results show that the selective loss of these Bcl-xL-dependent neurons results in mice exhibiting severe neurobehavioral abnormalities, including self-injurious and risk-taking behaviors, hyperactivity, and learning and memory defects

Bcl-xL Is Essential for the Survival and Function of Differentiated Neurons in the Cortex That Control Complex Behaviors.

UnlabelledApoptosis plays an essential role during brain development, yet the precise mechanism by which this pathway is regulated in the brain remains unknown. In particular, mammalian cells are known to express multiple anti-apoptotic Bcl-2 family proteins. However, the cells of the developing brain could also exist in a primed state in which the loss of a single anti-apoptotic Bcl-2 family protein is sufficient to trigger apoptosis. Here, we examined the critical role of Bcl-xL, an anti-apoptotic protein, during brain development. Using conditional knock-out mice in which Bcl-xL is deleted in neural progenitor cells (Bcl-xL(Emx1-Cre)), we show that the loss of Bcl-xL is not sufficient to trigger apoptosis in these proliferating progenitors. In contrast, specific populations of postmitotic neurons derived from these progenitors, including upper layer cortical neurons and the CA1-CA3 regions of the hippocampus, were acutely dependent on Bcl-xL. Consistent with this finding, deletion of Bcl-xL selectively in the postmitotic neurons in the brain (Bcl-xL(Nex-Cre)) also resulted in similar patterns of apoptosis. This Bcl-xL deficiency-induced neuronal death was a consequence of activation of the apoptotic pathway, because the cell death was rescued with codeletion of the proapoptotic proteins Bax and Bak. Importantly, the loss of these Bcl-xL-dependent neurons led to severe neurobehavioral abnormalities, including deficits in motor learning, hyperactivity, and increased risk-taking and self-injurious behaviors. Together, our results identify a population of neurons in the developing brain that are acutely dependent on Bcl-xL during the peak period of synaptic connectivity that are important for the establishment of higher-order complex behaviors.Significance statementAlthough Bcl-xL is known to inhibit apoptosis, exactly which cells in the brain are dependent on Bcl-xL has remained unclear because of the embryonic lethality of mice globally deleted for Bcl-xL. Here, we conditionally deleted Bcl-xL in the brain and found that this did not result in widespread apoptosis in the proliferating progenitors. Instead, Bcl-xL deficiency induced apoptosis in a select population of differentiated neurons predominantly in the early postnatal stages. Importantly, these Bcl-xL-dependent neurons are not essential for survival of the organism but instead regulate complex behaviors. Our results show that the selective loss of these Bcl-xL-dependent neurons results in mice exhibiting severe neurobehavioral abnormalities, including self-injurious and risk-taking behaviors, hyperactivity, and learning and memory defects