Browsing by Subject "Cell death"

Naturally-occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the central nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type – the astrocyte – has rarely been studied. It is presently unclear whether astrocytes are subject to significant amounts of developmental death, or how it occurs. Here we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia kill and engulf astrocytes to mediate their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally-occurring cell death, and demonstrate its importance for formation and integrity of the retinal gliovascular network.

Cellular senescence is a fundamental cell fate playing significant and complex roles during tumorigenesis and natural aging process. However, the molecular determinants distinguishing senescence from other temporary and permanent cell-cycle arrest states such as quiescence and post-mitotic state and the specified mechanisms underlying cell-fate decisions towards senescence versus cell death in response to cellular stress stimuli remain less understood. In our studies, we aimed to employ multi-omics approaches to deepen our understanding of cellular senescence, in particular, regarding the specific molecular determinants distinguishing cellular senescence from other non-dividing cell fates.

Notably, one of the most prominent features of cellular senescence differing from other non-dividing cell fates is the increased expression of senescence-associated beta-galactosidase. Because 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C12FDG) is known as the substrate catalyzed by beta-galactosidase for producing a green fluorescent product, we applied this compound to the cells undergoing G1 cell-cycle arrest (a mixture of senescent and quiescent cells). Employing fluorescence-activated cell sorting, we separated and collected senescent and quiescent cell populations based on green fluorescence intensity. As cellular senescence is more than just the non-dividing cell fate, we therefore systematically compared the gene expression between senescence and quiescence to provide insights into the specific features underlying senescence programming beyond cell cycle arrest. Following this strategy for the comparative gene expression analysis, we identified and characterized several genes critically involved in the program of cellular senescence, and one of the major findings was to identify IMMP2L, a nuclear-encoded mitochondrial intermembrane peptidase, can act as a molecular switch for determining the cell fates of healthy living, cell death, and senescence.

Inhibiting IMMP2L signaling through either the suicidal protease inhibitor SERPINB4 or transcriptional downregulation was sufficient to initiate cellular senescence by reprogramming the mitochondria functionality. Employing proteomics, we identified at least two mitochondrial target proteins processed by IMMP2L, including metabolic enzyme GPD2 and cell death regulator/electron transport chain complex I component AIF. Functional study suggests that, in healthy cells, the IMMP2L-GPD2 axis catalyzes redox reactions to produce phospholipid precursor Glycerol 3-phosphate; while under oxidative stress, IMMP2L cleaves AIF into its truncated pro-apoptotic form leading to cell death initiation to remove cells with irreparable damage. For cells programmed to senesce, the IMMP2L-GPD2 axis is switched off to block phospholipid biosynthesis leading to reduced availability of membrane building blocks for cell growth together with the disruption of mitochondrial localization of certain phospholipid-binding kinases, such as protein kinase C-δ (PKC-δ) and its downstream signaling. These alterations in mitochondria-associated metabolism and signaling network promote entry into a senescent state featuring high levels of reactive oxygen species (ROS). Simultaneously, blockage of pro-apoptotic AIF generation, which is due to the loss of IMMP2L, ensures the viability of senescent cells under ROS-mediated oxidative stress. Taken together, we have mechanistically uncovered IMMP2L-mediated signaling as a key regulatory pathway in the control of fates of healthy, apoptotic, and senescent cells.

In the physiological conditions, we observed that IMMP2L is downregulated in the muscle tissues and the blood samples of geriatric groups compared to that from young cohorts. Besides, centenarians display better genomic integrity at the IMMP2L locus when compared with the general population. Taken together, it suggests IMMP2L could also be an important player associated with the natural aging process.

Developmental cell death is essential for nervous system development, sculpting the developing tissue by controlling cell numbers. While developmental neuron death has been studied extensively, the most abundant cell type of the nervous system – the astrocyte – has often been overlooked. Our lab recently showed that astrocytes in the developing retina undergo an unusual non-apoptotic form of death that eliminates a vast proportion of the original population. Further, we found that microglia are the major effectors of astrocyte death. However, the mechanisms that induce microglia to kill astrocytes remain mysterious. It is important to understand these astrocyte death mechanisms because astrocytes play a crucial role in patterning the retinal blood vessel network. Developmental perturbations to astrocyte number have large effects on their patterning, and in turn cause severe vascular patterning defects – some of which resemble vasculopathies typical of human blinding disorders. Because death has such a major impact on astrocyte number, it presumably has an outsized impact on this critical patterning process. We therefore sought to identify the non-apoptotic mechanisms that drive astrocyte death. Previously, we showed that astrocyte numbers modulate microglial phagocytic activity – increasing this activity as astrocyte numbers rise and decreasing it as astrocyte numbers decline. This observation suggested that astrocytes themselves are the source of cues that drive their own death via recruitment of phagocytic microglia. Here we identify the membrane lipid phosphatidylserine (PtdSer) as one such astrocyte-derived “eat-me” cue. PtdSer is best known as an “eat-me” signal expressed on the surface of apoptotic cells. We show that PtdSer is also externalized on the cell surface of apparently normal astrocytes during the developmental death period. Moreover, using a genetic approach to increase cell-surface PtdSer, we show that it is sufficient to drive astrocyte death. For these studies, we used an astrocyte-specific mouse knockout of Tmem30a, an obligate subunit of the flippase enzymes that normally remove PtdSer from the cell surface. In these knockout animals, microglia are recruited to Tmem30a mutant astrocytes, engulf them, and cause a significant acceleration of cell number decline. This excess astrocyte loss has functional consequences for the development of the vasculature: The astrocytic template for angiogenesis is overly sparse, which leads to vascular patterning defects and delayed angiogenesis. Interestingly, these defects can be rescued by blocking the function of a phagocytic signaling pathway that can recognize PtdSer exposure, suggesting that the excess PtdSer exposure in the Tmem30a knockout animals is responsible for the increase in astrocyte death. Altogether our findings highlight the broad impact of dysregulated astrocyte death. Understanding how astrocyte population size is controlled will provide new insights into death mechanisms that are crucial for development not only in the retina but may also sculpt glial populations elsewhere in the central nervous system.

The eukaryotic cell cycle is characterized by a series of tightly orchestrated events during which cellular components are replicated and subsequently divided. In mammalian cells, the precise spatiotemporal control of cell cycle progression is achieved through multiple cyclin-dependent-kinases (Cdks), which are pivotal for both the proper timing of the cell cycle and the maintenance of genomic integrity. The cell progresses through a series of different phases before division, including the pre-mitotic G2 phase and the mitotic M phase. As the interval between DNA replication and mitosis, G2 phase is the stage where protein synthesis occurs in preparation for mitosis. On the other hand, mitosis is characterized by the most dramatic events of the cell cycle, featuring chromosome condensation and segregation into two daughter cells. Transition from G2 to M phase is driven by the activation of Cdk1, which forms a heterodimer with its obligate allosteric activator Cyclin B1. This heterodimer phosphorylates downstream effectors, thereby inducing entry into mitosis. Entry into mitosis is halted by activation of the G2/M cell cycle checkpoint, which stimulates the Myt1 and Wee1 kinases; these activated kinases phosphorylate Cdk1 at Thr14 and Tyr15, resulting in Cdk1 inactivation. Failure of G2/M cell cycle arrest leads to premature mitosis and triggers a form of cell death marked by multiple micronuclei and decondensed chromatin known as "mitotic catastrophe". Mitotic catastrophe can be stimulated by the presence of incompletely replicated DNA or DNA damage, which leads to the unscheduled activation of the Cdk1/CyclinB complex.

Recent studies in the brain tumor glioblastoma (GBM) have suggested that abrogation of the G2/M checkpoint with a Wee1 inhibitor enhances the efficacy of chemotherapy and ionizing radiation[1]. Interestingly, I have found that Wee1 protein levels vary dramatically between different types of brain tumors including glioblastoma and medulloblastoma. Also, Wee1 protein abundance determines tumor sensitivity to DNA-damaging chemotherapies. Furthermore, similar to findings in the Xenopus egg extract, in a mammalian cell culture system I have found that Wee1 protein abundance is subject to regulation by the arginine methyltransferase PRMT5. Taken together, these findings suggest that Wee1 or PRMT5 may be used as effective targets for therapy in a subset of glioblastoma and medulloblastoma patients.

1. Mir, S.E., et al., In Silico Analysis of Kinase Expression Identifies WEE1 as a Gatekeeper against Mitotic Catastrophe in Glioblastoma. Cancer Cell, 2010. 18(3): p. 244-257.

T cells are a critical arm of the adaptive immune system which function to coordinate and orchestrate complex immune reactions, as well as, kill damaged and infected cells. Production of a diverse peripheral T cell compartment requires massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment at the DN2 stage and following successful rearrangement and selection for functional TCRβ chains in DN3 thymocytes, which promotes development of DN4 cells to the DP stage. Signals driving expansion of DN2 thymocytes are well studied, however, factors regulating the proliferation and survival of DN4 cells remain poorly understood.

E proteins are transcription factors which have been shown to play essential non-redundant roles throughout T cell development. The functions of E proteins in T cell development include, enforcing T lineage commitment, promoting proper TCR rearrangements, regulating developmental progression and functional checkpoints, and coordinating complex transcriptional networks underpinning developmental progression. Due to the large number of genome-wide binding sites and massive number of genes regulated by E proteins, their numerous functions are poorly understood. The goal of this dissertation is to determine the role of the E protein-regulated transcription factor Zfp335 in T cell development.

We utilized conditional deletion models to determine the role of Zfp335 in early and late stages of conventional and unconventional T cell development. Through these efforts we uncovered we uncover an unexpected link between the transcription factor Zfp335 and control of the cGAS/STING pathway for sensing cytosolic DNA in post-β-selection DN4 thymocytes. The absence of Zfp335 drives cGAS/STING-dependent death of DN4 cells. Zfp335 controls survival by sustaining expression of Ankle2, which in turn regulates the activity of Baf to suppress cGAS/STING-dependent cell death. Additionally, genetic ablation of Zfp335 precludes the development of unconventional iNKT cells due to STING-independent cell death following lineage commitment along with preventing effector differentiation of surviving cells.

Our studies also uncovered an additional cGAS/STING-independent role in the terminal maturation of conventional αβ T cells. The absence of Zfp335 prevents the establishment of a naïve T cell compartment, inhibits differentiation of CD4 T cells and promotes the developmental acquisition of an effector program in CD8 T cells. Using in vivo and ex vivo genetic manipulation combined with detailed bioinformatic analyses we show that Zfp335 functions to promote T cell development, maturation, and effector differentiation through the regulation of a small but essential set of genes.

To our knowledge, these studies detail the first described role for cGAS/STING in T cell development and strongly suggest a transcriptional mechanism downstream of Zfp335 which coordinates genome-wide alterations to chromatin compaction required for proper establishment of conventional and unconventional T cell pools. Together, these studies will provide a novel framework for understanding the life and death of developing T cells and may uncover novel pathways for enhancing the efficacy of T cell-based therapeutics.