Kerry L. Ferguson

Apoptosis-Inducing Factor Is a Key Factor in Neuronal Cell Death Propagated by BAX-Dependent and BAX-Independent Mechanisms

Mitochondria release proteins that propagate both caspase-dependent and caspase-independent cell death pathways. AIF (apoptosis-inducing factor) is an important caspase-independent death regulator in multiple neuronal injury pathways. Presently, there is considerable controversy as to whether AIF is neuroprotective or proapoptotic in neuronal injury, such as oxidative stress or excitotoxicity. To evaluate the role of AIF in BAX-dependent (DNA damage induced) and BAX-independent (excitotoxic) neuronal death, we used Harlequin (Hq) mice, which are hypomorphic for AIF. Neurons carrying double mutations for Hq/Apaf1-/- (apoptosis proteases-activating factor) are impaired in both caspase-dependent and AIF-mediated mitochondrial cell death pathways. These mutant cells exhibit extended neuroprotection against DNA damage, as well as glutamate-induced excitotoxicity. Specifically, AIF is involved in NMDA- and kainic acid- but not AMPA-induced excitotoxicity. In vivo excitotoxic studies using kainic acid-induced seizure showed that Hq mice had significantly less hippocampal damage than wild-type littermates. Our results demonstrate an important role for AIF in both BAX-dependent and BAX-independent mechanisms of neuronal injury.

Telencephalon-specific Rb knockouts reveal enhanced neurogenesis, survival and abnormal cortical development.

Correct cell cycle regulation and terminal mitosis are critical for nervous system development. The retinoblastoma (Rb) protein is a key regulator of these processes, as Rb−/− embryos die by E15.5, exhibiting gross hematopoietic and neurological defects. The extensive apoptosis in Rb−/− embryos has been attributed to aberrant S phase entry resulting in conflicting growth control signals in differentiating cells. To assess the role of Rb in cortical development in the absence of other embryonic defects, we examined mice with telencephalon‐specific Rb deletions. Animals carrying a floxed Rb allele were interbred with mice in which cre was knocked into the Foxg1 locus. Unlike germline knockouts, mice specifically deleted for Rb in the developing telencephalon survived until birth. In these mutants, Rb−/− progenitor cells divided ectopically, but were able to survive and differentiate. Mutant brains exhibited enhanced cellularity due to increased proliferation of neuroblasts. These studies demonstrate that: (i) cell cycle deregulation during differentiation does not necessitate apoptosis; (ii) Rb‐deficient mutants exhibit enhanced neuroblast proliferation; and (iii) terminal mitosis may not be required to initiate differentiation.

p107 regulates neural precursor cells in the mammalian brain

Here we show a novel function for Retinoblastoma family member, p107 in controlling stem cell expansion in the mammalian brain. Adult p107-null mice had elevated numbers of proliferating progenitor cells in their lateral ventricles. In vitro neurosphere assays revealed striking increases in the number of neurosphere forming cells from p107−/− brains that exhibited enhanced capacity for self-renewal. An expanded stem cell population in p107-deficient mice was shown in vivo by (a) increased numbers of slowly cycling cells in the lateral ventricles; and (b) accelerated rates of neural precursor repopulation after progenitor ablation. Notch1 was up-regulated in p107−/− neurospheres in vitro and brains in vivo. Chromatin immunoprecipitation and p107 overexpression suggest that p107 may modulate the Notch1 pathway. These results demonstrate a novel function for p107 that is distinct from Rb, which is to negatively regulate the number of neural stem cells in the developing and adult brain.

The Rb-CDK4/6 Signaling Pathway Is Critical in Neural Precursor Cell Cycle Regulation

The tumor suppressor, retinoblastoma (Rb), is involved in both terminal mitosis and neuronal differentiation. We hypothesized that activation of the Rb pathway would induce cell cycle arrest in primary neural precursor cells, independent of the proposed function of cyclin-dependent kinases 4/6 (CDK4/6) to sequester the CIP/KIP CDK inhibitors (CKIs) p21 and p27 from CDK2. We expressed dominant negative adenovirus mutants of CDKs 2, 4, and 6 (dnCDK2, dnCDK4, and dnCDK6) in neural progenitor cells derived from E12.5 wild type and Rb-deficient mouse embryos. In contrast to previous studies, our results demonstrate that in addition to dnCDK2, the dnCDK4/6 mutants can induce growth arrest. Moreover, the dnCDK4/6-mediated inhibition is Rb-dependent. The dnCDK2 partially inhibited cell growth in Rb-deficient cells, suggesting that CDK2 may have additional targets. A previously proposed function of CDK4/6 is CKI sequestration, thereby preventing the resulting inhibition of CDK2, believed to be the key regulator of cell cycle. However, our immunoprecipitations revealed that the dominant negative CDK mutants could arrest cell growth despite their interaction with p21 and p27. Taken together, our results demonstrate that both CDK2 and CDK4/6 are crucial for cell cycle regulation. Furthermore, our data underscore the importance of the Rb regulatory pathway in neuronal development and cell cycle regulation, independent of CKI sequestration.

Unique Requirement for Rb/E2F3 in Neuronal Migration: Evidence for Cell Cycle-Independent Functions

ABSTRACT The cell cycle regulatory retinoblastoma (Rb) protein is a key regulator of neural precursor proliferation; however, its role has been expanded to include a novel cell-autonomous role in mediating neuronal migration. We sought to determine the Rb-interacting factors that mediate both the cell cycle and migration defects. E2F1 and E2F3 are likely Rb-interacting candidates that we have shown to be deregulated in the absence of Rb. Using mice with compound null mutations of Rb and E2F1 or E2F3, we asked to what extent either E2F1 or E2F3 interacts with Rb in neurogenesis. Here, we report that E2F1 and E2F3 are both functionally relevant targets in neural precursor proliferation, cell cycle exit, and laminar patterning. Each also partially mediates the Rb requirement for neuronal survival. Neuronal migration, however, is specifically mediated through E2F3, beyond its role in cell cycle regulation. This study not only outlines overlapping and distinct functions for E2Fs in neurogenesis but also is the first to establish a physiologically relevant role for the Rb/E2F pathway beyond cell cycle regulation in vivo.

A cell‐autonomous requirement for the cell cycle regulatory protein, Rb, in neuronal migration

Precise cell cycle regulation is critical for nervous system development. To assess the role of the cell cycle regulator, retinoblastoma (Rb) protein, in forebrain development, we studied mice with telencephalon‐specific Rb deletions. We examined the role of Rb in neuronal specification and migration of diverse neuronal populations. Although layer specification occurred at the appropriate time in Rb mutants, migration of early‐born cortical neurons was perturbed. Consistent with defects in radial migration, neuronal cell death in Rb mutants specifically affected Cajal–Retzius neurons. In the ventral telencephalon, although calbindin‐ and Lhx6‐expressing cortical neurons were generated at embryonic day 12.5, their tangential migration into the neocortex was dramatically and specifically reduced in the mutant marginal zone. Cell transplantation assays revealed that defects in tangential migration arose owing to a cell‐autonomous loss of Rb in migrating interneurons and not because of a defective cortical environment. These results revealed a cell‐autonomous role for Rb in regulating the tangential migration of cortical interneurons. Taken together, we reveal a novel requirement for the cell cycle protein, Rb, in the regulation of neuronal migration.

The Rb pathway in neurogenesis.

Cell division during embryogenesis plays a crucial role in the formation of the nervous system. During this developmental process, proliferating neural precursor cells commit to a neuronal fate and, as a consequence, undergo terminal mitosis and adopt a neuronal phenotype. A key cell cycle regulator, the tumor suppressor protein, retinoblastoma (Rb), is involved in both terminal mitosis and neuronal differentiation. Neural development is a complex process involving cell proliferation, cell fate determination and differentiation, as well as programmed cell death. In this review, we will examine each of these processes in turn, focussing on the role of the Rb family proteins to examine their many influences on these events.

The Conserved Ig Superfamily Member Turtle Mediates Axonal Tiling in Drosophila

Restriction of adjacent same-type axons/dendrites to separate single columns for specific neuronal connections is commonly observed in vertebrates and invertebrates, and is necessary for proper processing of sensory information. Columnar restriction is conceptually similar to tiling, a phenomenon referring to the avoidance of neurites from adjacent same-type neurons. The molecular mechanism underlying the establishment of columnar restriction or axonal/dendritic tiling remains largely undefined. Here, we identify Turtle (Tutl), a member of the conserved Tutl/Dasm1/IgSF9 subfamily of the Ig superfamily, as a key player in regulating the tiling pattern of R7 photoreceptor terminals in Drosophila. Tutl functions to prevent fusion between two adjacent R7 terminals, and acts in parallel to the Activin pathway. Tutl mediates homophilic cell–cell interactions. We propose that extrinsic terminal–terminal recognition mediated by Tutl, acts in concert with intrinsic Activin-dependent control of terminal growth, to restrict the connection made by each R7 axon to a single column.

Growth factors: can they promote neurogenesis?

Cortical development is a complex process in which extrinsic and intrinsic factors modulate the sequential generation of neurons and glia. Following successive rounds of division, precursors become determined along a neuronal or glial lineage prior to cell cycle exit and differentiation. Although the influence of growth factors in cell fate specification is not new, until recently little was known about the signaling pathways by which they regulate neuronal differentiation. Menard and colleagues have examined this issue and have demonstrated a role for the MEK-C/EBP (mitogen-activated-protein-kinase kinase and CCAAT/enhancer-binding protein) pathway in the promotion of growth factor-mediated neurogenesis.

Emerging Roles for the Retinoblastoma Gene Family

Research on the retinoblastoma protein has grown from studying its role as a tumour suppressor in cancer to identifying it as a key regulator of the cell cycle G1/S check point and today to exploring its function in numerous cellular processes. The recent development of conditional knockout mice has shed new light on the roles of Rb in embryonic development and has aided in the identification of the cell-of-origin in Retinoblastoma cancer. In this review, we will discuss the role of Rb as a tumour suppressor as well as its role in cell division, differentiation, apoptosis and cancer.