mTORC2 Loss in Oligodendrocyte Progenitor Cells Results in Regional Hypomyelination in the Central Nervous System.

In the CNS, oligodendrocyte progenitor cells (OPCs) differentiate into mature oligodendrocytes to generate myelin, an essential component for normal nervous system function. OPC differentiation is driven by signaling pathways, such as mTOR, which functions in two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), containing Raptor or Rictor, respectively. In the current studies, mTORC2 signaling was selectively deleted from OPCs in PDGFRα-Cre X Rictorfl/fl mice. This study examined developmental myelination in male and female mice, comparing the impact of mTORC2 deletion in the corpus callosum and spinal cord. In both regions, Rictor loss in OPCs resulted in early reduction in myelin RNAs and proteins. However, these deficits rapidly recovered in spinal cord, where normal myelin was noted at P21 and P45. By contrast, the losses in corpus callosum resulted in severe hypomyelination and increased unmyelinated axons. The hypomyelination may result from decreased oligodendrocytes in the corpus callosum, which persisted in animals as old as postnatal day 350. The current studies focus on uniquely altered signaling pathways following mTORC2 loss in developing oligodendrocytes. A major mTORC2 substrate is phospho-Akt-S473, which was significantly reduced throughout development in both corpus callosum and spinal cord at all ages measured, yet this had little impact in spinal cord. Loss of mTORC2 signaling resulted in decreased expression of actin regulators, such as gelsolin in corpus callosum, but only minimal loss in spinal cord. The current study establishes a regionally specific role for mTORC2 signaling in OPCs, particularly in the corpus callosum.SIGNIFICANCE STATEMENT mTORC1 and mTORC2 signaling has differential impact on myelination in the CNS. Numerous studies identify a role for mTORC1, but deletion of Rictor (mTORC2 signaling) in late-stage oligodendrocytes had little impact on myelination in the CNS. However, the current studies establish that deletion of mTORC2 signaling from oligodendrocyte progenitor cells results in reduced myelination of brain axons. These studies also establish a regional impact of mTORC2, with little change in spinal cord in these conditional Rictor deletion mice. Importantly, in both brain and spinal cord, mTORC2 downstream signaling targets were impacted by Rictor deletion. Yet, these signaling changes had little impact on myelination in spinal cord, while they resulted in long-term alterations in myelination in brain.

Intrinsic and extrinsic regulators of oligodendrocyte progenitor proliferation and differentiation.

Oligodendrocytes are highly specialized glial cells, responsible for producing myelin in the central nervous system (CNS). The multi-stage process of oligodendrocyte development is tightly regulated to ensure proper lineage progression of oligodendrocyte progenitor cells (OPCs) to mature myelin producing oligodendrocytes. This developmental process involves complex interactions between several intrinsic signaling pathways that are modulated by an array of extrinsic factors. Understanding these regulatory processes is of crucial importance, as it may help to identify specific molecular targets both to enhance plasticity in the normal CNS and to promote endogenous recovery following injury or disease. This review describes two major regulators that play important functional roles in distinct phases of oligodendrocyte development: OPC proliferation and differentiation. Specifically, we highlight the roles of the extracellular astrocyte/radial glia-derived protein Endothelin-1 in OPC proliferation and the intracellular Akt/mTOR pathway in OPC differentiation. Lastly, we reflect on how recent advances in neuroscience and scientific technology will enable greater understanding into how intrinsic and extrinsic regulators interact to generate oligodendrocyte diversity.

Spatiotemporal control of necroptotic cell death and plasma membrane recruitment using engineered MLKL domains.

Necroptosis is a form of programmed necrotic cell death in which a signaling cascade induces oligomerization of mixed lineage kinase domain-like (MLKL) protein, leading to plasma membrane rupture. Necroptotic cell death is recognized as important for protection against viral infection and has roles in a variety of diseases, including cancer and diabetes. Despite its relevance to health and disease states, many questions remain about the precise mechanism of necroptotic cell death, cellular factors that can protect cells from necroptosis, and the role of necroptosis in disease models. In this study, we engineered a light-activated version of MLKL that rapidly oligomerizes and is recruited to the plasma membrane in cells exposed to light, inducing rapid cell death. We demonstrate this tool can be controlled spatially and temporally, used in a chemical genetic screen to identify chemicals and pathways that protect cells from MLKL-induced cell death, and used to study signaling responses of non-dying bystander cells. In additional studies, we re-engineered MLKL to block its cell-killing capacity but retain light-mediated membrane recruitment, developing a new single-component optogenetic tool that allows modulation of protein function at the plasma membrane.

A Short CEP135 Splice Isoform Controls Centriole Duplication.

Centriole duplication is coordinated such that a single round of duplication occurs during each cell cycle. Disruption of this synchrony causes defects including supernumerary centrosomes in cancer and perturbed ciliary signaling [1-5]. To preserve the normal number of centrioles, the level, localization, and post-translational modification of centriole proteins is regulated so that, when centriole protein expression and/or activity are increased, centrioles self-assemble. Assembly is initiated by the formation of the cartwheel structure that comprises the base of centrioles [6-11]. SAS-6 constitutes the cartwheel, and SAS-6 levels remain low until centriole assembly is initiated at S phase onset [3, 12, 13]. CEP135 physically links to SAS-6 near the site of microtubule nucleation and binds to CPAP for triplet microtubule formation [13, 14]. We identify two distinct protein isoforms of CEP135 that antagonize each other to modulate centriole duplication: full-length CEP135 (CEP135(full)) promotes new assembly, whereas a short isoform, CEP135(mini), represses it. CEP135(mini) represses centriole duplication by limiting the centriolar localization of CEP135(full) binding proteins (SAS-6 and CPAP) and the pericentriolar localization of γ-tubulin. The CEP135 isoforms exhibit distinct and complementary centrosomal localization during the cell cycle. CEP135(mini) protein decreases from centrosomes upon anaphase onset. We suggest that the decrease in CEP135(mini) from centrosomes promotes centriole assembly. The repression of centriole duplication by a splice isoform of a protein that normally promotes it serves as a novel mechanism to limit centriole duplication.

Extracellular vesicles secreted from cancer cell lines stimulate secretion of MMP-9, IL-6, TGF-ß1 and EMMPRIN.

Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-ß1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles.