Human iPSC-Derived Neuronal Model of Tau-A152T Frontotemporal Dementia Reveals Tau-Mediated Mechanisms of Neuronal Vulnerability
Frontotemporal dementia (FTD) and other tauopathies characterized by focal brain neurodegeneration and pathological accumulation of proteins are commonly associated with tau mutations. However, the mechanism of neuronal loss is not fully understood. To identify molecular events associated with tauopathy, we studied induced pluripotent stem cell (iPSC)-derived neurons from individuals carrying the tau-A152T variant. We highlight the potential of in-depth phenotyping of human neuronal cell models for pre-clinical studies and identification of modulators of endogenous tau toxicity. Through a panel of biochemical and cellular assays, A152T neurons showed accumulation, redistribution, and decreased solubility of tau. Upregulation of tau was coupled to enhanced stress-inducible markers and cell vulnerability to proteotoxic, excitotoxic, and mitochondrial stressors, which was rescued upon CRISPR/Cas9-mediated targeting of tau or by pharmacological activation of autophagy. Our findings unmask tau-mediated perturbations of specific pathways associated with neuronal vulnerability, revealing potential early disease biomarkers and therapeutic targets for FTD and other tauopathies.
MMP-9 and MMP-2 Contribute to Neuronal Cell Death in iPSC Models of Frontotemporal Dementia with MAPT Mutations
How mutations in the microtubule-associated protein tau (MAPT) gene cause frontotemporal dementia (FTD) remains poorly understood. We generated and characterized multiple induced pluripotent stem cell (iPSC) lines from patients with MAPT IVS10+16 and tau-A152T mutations and a control subject. In cortical neurons differentiated from these and other published iPSC lines, we found that MAPT mutations do not affect neuronal differentiation but increase the 4R/3R tau ratio. Patient neurons had significantly higher levels of MMP-9 and MMP-2 and were more sensitive to stress-induced cell death. Inhibitors of MMP-9/MMP-2 protected patient neurons from stress-induced cell death and recombinant MMP-9/MMP-2 were sufficient to decrease neuronal survival. In tau-A152T neurons, inhibition of the ERK pathway decreased MMP-9 expression. Moreover, ectopic expression of 4R but not 3R tau-A152T in HEK293 cells increased MMP-9 expression and ERK phosphorylation. These findings provide insights into the molecular pathogenesis of FTD and suggest a potential therapeutic target for FTD with MAPT mutations.
Chromatin is thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture occurring during genomic replication. Here, we measure a key aspect of chromatin structure dynamics during replication-how rapidly nucleosome positions are established on the newly replicated daughter genomes. By isolating newly synthesized DNA marked with 5-ethynyl-2'-deoxyuridine (EdU), we characterize nucleosome positions on both daughter genomes of S. cerevisiae during chromatin maturation. We find that nucleosomes rapidly adopt their mid-log positions at highly transcribed genes, which is consistent with a role for transcription in positioning nucleosomes in vivo. Additionally, experiments in hir1Delta mutants reveal a role for HIR in nucleosome spacing. We also characterized nucleosome positions on the leading and lagging strands, uncovering differences in chromatin maturation dynamics at hundreds of genes. Our data define the maturation dynamics of newly replicated chromatin and support a role for transcription in sculpting the chromatin template.
The impact of deliberate reflection with WISE-MDTM modules on critical thinking of nurse practitioner students: A prospective, randomized controlled pilot study
Objective: Nurse practitioner (NP) students at our graduate school of nursing use WISE-MDTM simulation modules in the curriculum. This prospective randomized controlled pilot study was undertaken to evaluate critical-thinking outcomes associated with adding metacognitive deliberate-reflection guidance to the learning strategy with WISE-MDTM simulation modules.
Methods: Of 33 NP students randomly assigned to intervention and control groups, 16 completed the study. The intervention group received WISE-MDTM learning modules with specific guidance or deliberate reflection. Controls used the modules with instructions for periodic free-thought reflections. Students’ tape-recorded reflections were categorized according to author-developed critical-thinking categories. Data were analyzed using NVIVOTM. Students’ feedback was collected by post-intervention anonymous survey.
Results: Critical thinking outcomes (student responses to exercises after free-thinking or deliberate-reflection guidance) did not differ between groups. However, the intervention group demonstrated a higher level of critical thought after deliberate-reflection guidance. Post-intervention quantitative and qualitative feedback from both groups endorsed the value of the WISE-MDTM modules for NP education.
Conclusions: Despite no difference in unprompted outcomes between groups, the intervention group often verbalized more thoughtful clinical decision-making. We speculate that the deliberate-reflection guidance intervention utilized with students throughout only two modules was insufficient for them to internalize the critical-thinking process. We propose using free-thought reflections with one or two WISE-MDTM modules to identify struggling students’ clinical decision-making process. These students’ remediation plan could include recording their deliberate-reflection process while viewing WISE-MDTM modules. Students would be guided to verbalize and record their critical-thinking processes for faculty review until students sufficiently integrate the process into their clinical decision-making.
Skeletal muscle insulin resistance is a major characteristic of obesity and type 2 diabetes. Although obesity-mediated inflammation is causally associated with insulin resistance, the underlying mechanism is unclear. Our lab and others have shown that a chronic low-grade inflammation takes place in skeletal muscles during diet-induced obesity, as evidenced by increased macrophage markers and pro-inflammatory cytokine levels. Interleukin (IL)-10 is a Th2-type cytokine that inhibits the synthesis and activity of pro-inflammatory cytokines and counteracts the Toll-like receptor-mediated inflammation. Our lab has previously demonstrated the preventive role of IL-10 against insulin resistance. Here, I have analyzed the effects of IL-10 on the skeletal muscle glucose metabolism and myogenesis in three different insulin resistant states (high fat diet-induced, leptin-deficiency-induced and aging-induced). The first model involved long-term (16 weeks) high-fat diet (HFD) feeding that resulted in markedly obese and hyperglycemic mice, representative of obese type 2 diabetic subjects. In mice overexpressing IL-10 specifically in the skeletal muscle (MIL10), we observed improved whole-body and skeletal muscle insulin sensitivity as compared to wild-types after long-term high fat diet feeding. The improved insulin sensitivity in the skeletal muscle was due to increased Akt signaling and decreased muscle inflammation. Leptin is an important adipocyte-derived hormone that is elevated in obesity, and it regulates numerous physiological functions including the energy balance and inflammation. Thus, my second model examined the effects of muscle-specific overexpression of IL-10 on glucose metabolism in the hyperphagic, leptin-deficient ob/ob mice. We detected improved whole-body insulin sensitivity compared to the control mice. My third model examined the effects of increased IL-10 expression using MIL10 mice during aging-induced insulin resistance. In 18-month old MIL10 mice, we found enhanced whole-body and skeletal muscle insulin sensitivity due to improved insulin signaling and decreased muscle inflammation as compared to wild-type mice. Last, to test whether direct signaling of IL-10 on skeletal muscle is responsible for the beneficial effects of IL-10 on muscle glucose metabolism, I generated mice lacking IL-10 receptor 1 type chain selectively in skeletal muscle (M-IL10R-/-). We observed more prominent muscle inflammation and whole-body insulin resistance in HFD-fed M-IL10R-/- mice as compared to wild-type mice. Interestingly, when studying insulin resistance in the IL-10 transgenic mouse models, we identified a consistent increased lean mass phenotype, and conversely decreased lean mass in the HFD-fed M-IL10R-/- mice. Quantitative RT-PCR on HFD-fed MIL10 group muscles to measure myogenesis-related gene expression identified a correlation between lean mass and both IL-10 and MyoD mRNA expression levels. In support of this, I showed that IL-10 caused an increase in in vitro cultured myoblast proliferation rates. Together, these results highlight the potential benefits of IL-10 expression not only in muscle glucose metabolism but also in maintaining muscle mass during insulin resistant states. Overall, these results demonstrate that selective expression of IL-10 in skeletal muscle suppresses inflammation, improves glucose metabolism and muscle growth in obese and aging mice, and further establishes that these effects are at least partially mediated by direct activation of IL-10 signaling in skeletal muscle.
The human immunodeficiency virus type 1 (HIV-1) protease (PR) is a critical drug target as it is responsible for virion maturation. Mutations within the active site (1°) of the PR directly interfere with inhibitor binding while mutations distal to the active site (2°) to restore enzymatic fitness. Increasing mutation number is not directly proportional to the severity of resistance, suggesting that resistance is not simply additive but that it is interdependent. The interdependency of both primary and secondary mutations to drive protease inhibitor (PI) resistance is grossly understudied.
To structurally and dynamically characterize the direct role of secondary mutations in drug resistance, I selected a panel of single-site mutant protease crystal structures complexed with the PI darunavir (DRV). From these studies, I developed a network hypothesis that explains how mutations outside the active site are able to perpetuate changes to the active site of the protease to disrupt inhibitor binding.
I then expanded the panel to include highly mutated multi-drug resistant variants. To elucidate the interdependency between primary and secondary mutations I used statistical and machine-learning techniques to determine which specific mutations underlie the perturbations of key inter-molecular interactions. From these studies, I have determined that mutations distal to the active site are able to perturb the global PR hydrogen bonding patterns, while primary and secondary mutations cooperatively perturb hydrophobic contacts between the PR and DRV. Discerning and exploiting the mechanisms that underlie drug resistance in viral targets could proactively ameliorate both current treatment and inhibitor design for HIV-1 targets.
Drosophila IAP1-mediated ubiquitylation controls activation of the initiator caspase DRONC independent of protein degradation
Ubiquitylation targets proteins for proteasome-mediated degradation and plays important roles in many biological processes including apoptosis. However, non-proteolytic functions of ubiquitylation are also known. In Drosophila, the inhibitor of apoptosis protein 1 (DIAP1) is known to ubiquitylate the initiator caspase DRONC in vitro. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by caspase inhibition ("undead" cells), it is thought that DIAP1-mediated ubiquitylation causes proteasomal degradation of DRONC, protecting cells from apoptosis. However, contrary to this model, we show here that DIAP1-mediated ubiquitylation does not trigger proteasomal degradation of full-length DRONC, but serves a non-proteolytic function. Our data suggest that DIAP1-mediated ubiquitylation blocks processing and activation of DRONC. Interestingly, while full-length DRONC is not subject to DIAP1-induced degradation, once it is processed and activated it has reduced protein stability. Finally, we show that DRONC protein accumulates in "undead" cells due to increased transcription of dronc in these cells. These data refine current models of caspase regulation by IAPs.
In Drosophila imaginal epithelia, cells mutant for the endocytic neoplastic tumor suppressor gene vps25 stimulate nearby untransformed cells to express Drosophila Inhibitor-of-Apoptosis-Protein-1 (DIAP-1), conferring resistance to apoptosis non-cell autonomously. Here, we show that the non-cell autonomous induction of DIAP-1 is mediated by Yorkie, the conserved downstream effector of Hippo signaling. The non-cell autonomous induction of Yorkie is due to Notch signaling from vps25 mutant cells. Moreover, activated Notch in normal cells is sufficient to induce non-cell autonomous Yorkie activity in wing imaginal discs. Our data identify a novel mechanism by which Notch promotes cell survival non-cell autonomously and by which neoplastic tumor cells generate a supportive microenvironment for tumor growth.
Ligand-independent activation of the Hedgehog pathway displays non-cell autonomous proliferation during eye development in Drosophila
Deregulation of the Hedgehog (Hh) signaling pathway is associated with the development of human cancer including medullobastoma and basal cell carcinoma. Loss of Patched or activation of Smoothened in mouse models increases the occurrence of tumors. Likewise, in a Drosophila eye model, deregulated Hedgehog signaling causes overgrowth of eye and head tissues. Surprisingly, we show that cells with deregulated Hh signaling do not or only little contribute to the tissue overgrowth. Instead, they become more sensitive to apoptosis and may eventually be eliminated. Nevertheless, these mutant cells increase proliferation in the adjacent wild-type tissue, i.e., in a non-cell autonomous manner. This non-cell autonomous effect is position-dependent and restricted to mutant cells in the anterior portion of the eye. We also observe precocious non-cell autonomous differentiation in genetic mosaics with deregulated Hh signaling. Together, these non-cell autonomous growth and differentiation phenotypes in the Drosophila eye model reveal another strategy by which oncogenes may generate a supportive micro-environment for tumor growth.
Genes dedicated to killing cells must have evolved because of their positive effects on organismal survival. Positive functions of apoptotic genes have been well established in a large number of biological contexts, including their role in eliminating damaged and potentially cancerous cells. More recently, evidence has suggested that proapoptotic proteins-mostly caspases-can induce proliferation of neighboring surviving cells to replace dying cells. This process, that we will refer to as "apoptosis-induced proliferation," may be critical for stem cell activity and tissue regeneration. Depending on the caspases involved, at least two distinct types of apoptosis-induced proliferation can be distinguished. One of these types have been studied using a model in which cells have initiated cell death, but are prevented from executing it because of effector caspase inhibition, thereby generating "undead" cells that emit persistent mitogen signaling and overgrowth. Such conditions are likely to contribute to certain forms of cancer. In this review, we summarize the current knowledge of apoptosis-induced proliferation and discuss its relevance for tissue regeneration and cancer.
De-regulation of JNK and JAK/STAT signaling in ESCRT-II mutant tissues cooperatively contributes to neoplastic tumorigenesis
Multiple genes involved in endocytosis and endosomal protein trafficking in Drosophila have been shown to function as neoplastic tumor suppressor genes (nTSGs), including Endosomal Sorting Complex Required for Transport-II (ESCRT-II) components vacuolar protein sorting 22 (vps22), vps25, and vps36. However, most studies of endocytic nTSGs have been done in mosaic tissues containing both mutant and non-mutant populations of cells, and interactions among mutant and non-mutant cells greatly influence the final phenotype. Thus, the true autonomous phenotype of tissues mutant for endocytic nTSGs remains unclear. Here, we show that tissues predominantly mutant for ESCRT-II components display characteristics of neoplastic transformation and then undergo apoptosis. These neoplastic tissues show upregulation of c-Jun N-terminal Kinase (JNK), Notch, and Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signaling. Significantly, while inhibition of JNK signaling in mutant tissues partially inhibits proliferation, inhibition of JAK/STAT signaling rescues other aspects of the neoplastic phenotype. This is the first rigorous study of tissues predominantly mutant for endocytic nTSGs and provides clear evidence for cooperation among de-regulated signaling pathways leading to tumorigenesis.
Correct spatial and temporal induction of numerous cell type-specific genes during development requires regulated removal of the repressive histone H3 lysine 27 trimethylation (H3K27me3) modification. Here we show that the H3K27me3 demethylase dUTX is required for hormone-mediated transcriptional regulation of apoptosis and autophagy genes during ecdysone-regulated programmed cell death of Drosophila salivary glands. We demonstrate that dUTX binds to the nuclear hormone receptor complex Ecdysone Receptor/Ultraspiracle, and is recruited to the promoters of key apoptosis and autophagy genes. Salivary gland cell death is delayed in dUTX mutants, with reduced caspase activity and autophagy that coincides with decreased apoptosis and autophagy gene transcripts. We further show that salivary gland degradation requires dUTX catalytic activity. Our findings provide evidence for an unanticipated role for UTX demethylase activity in regulating hormone-dependent cell death and demonstrate how a single transcriptional regulator can modulate a specific complex functional outcome during animal development.
Caspases are a highly specialized class of cell death proteases. Since they are synthesized as inactive full-length zymogens, activation--at least of effector caspases and to some extent also of initiator caspases-requires a proteolytic cleavage event, generating a large and a small subunit, two of each forming the active caspase. The proteolytic cleavage event generates neo-epitopes at both the C-terminus of the large subunit and the N-terminus of the small subunit. The cleaved Caspase-3 (CC3) antibody was raised against the neo-epitope of the large subunit and thus detects only cleaved, but not full-length, Caspase-3. Although raised against human cleaved Caspase-3, the CC3 antibody cross-reacts in other species and detects cleaved caspases, most notably DrICE and Dcp-1, in Drosophila. This protocol describes the procedure for use of the CC3 antibody to detect caspase activity in larval imaginal discs in Drosophila.
Multiple mechanisms modulate distinct cellular susceptibilities toward apoptosis in the developing Drosophila eye
Although apoptosis is mechanistically well understood, a comprehensive understanding of how cells modulate their susceptibility toward apoptosis in a developing tissue is lacking. Here, we reveal striking dynamics in the apoptotic susceptibilities of different cell types in the Drosophila retina over a period of only 24 hr. Mitotic cells are extremely susceptible to apoptotic signals, while postmitotic cells have developed several strategies to promote survival. For example, photoreceptor neurons accumulate the inhibitor of apoptosis, Diap1. In unspecified cells, Cullin-3-mediated degradation keeps Diap1 levels low. These cells depend on EGFR signaling for survival. As development proceeds, developmentally older photoreceptors degrade Diap1, resulting in increased apoptosis susceptibility. Finally, R8 photoreceptors have very efficient survival mechanisms independent of EGFR or Diap1. These examples illustrate how complex cellular susceptibility toward apoptosis is regulated in a developing organ. Similar complexities may regulate apoptosis susceptibilities in mammalian development, and tumor cells may take advantage of it.
Apoptosis has essential roles in a variety of cellular and developmental processes. Although the pathway is well studied, how the activities of individual components in the pathway are regulated is less understood. In Drosophila, a key component in apoptosis is Drosophila inhibitor of apoptosis protein 1 (DIAP1), which is required to prevent caspase activation. Here, we demonstrate that Drosophila CG42593 (ubr3), encoding the homolog of mammalian UBR3, has an essential role in regulating the apoptosis pathway. We show that loss of ubr3 activity causes caspase-dependent apoptosis in Drosophila eye and wing discs. Our genetic epistasis analyses show that the apoptosis induced by loss of ubr3 can be suppressed by loss of initiator caspase Drosophila Nedd2-like caspase (Dronc), or by ectopic expression of the apoptosis inhibitor p35, but cannot be rescued by overexpression of DIAP1. Importantly, we show that the activity of Ubr3 in the apoptosis pathway is not dependent on its Ring-domain, which is required for its E3 ligase activity. Furthermore, we find that through the UBR-box domain, Ubr3 physically interacts with the neo-epitope of DIAP1 that is exposed after caspase-mediated cleavage. This interaction promotes the recruitment and ubiquitination of substrate caspases by DIAP1. Together, our data indicate that Ubr3 interacts with DIAP1 and positively regulates DIAP1 activity, possibly by maintaining its active conformation in the apoptosis pathway.
Extracellular Reactive Oxygen Species Drive Apoptosis-Induced Proliferation via Drosophila Macrophages
Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. Here we show that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. We propose that in an immortalized ("undead") model of AiP, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer.
The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles
Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-beta to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events.
Genetic characterization of two gain-of-function alleles of the effector caspase DrICE in Drosophila
Caspases are the executioners of apoptosis. Although much is known about their physiological roles and structures, detailed analyses of missense mutations of caspases are lacking. As mutations within caspases are identified in various human diseases, the study of caspase mutants will help to elucidate how caspases interact with other components of the apoptosis pathway and how they may contribute to disease. DrICE is the major effector caspase in Drosophila required for developmental and stress-induced cell death. Here, we report the isolation and characterization of six de novo drICE mutants, all of which carry point mutations affecting amino acids conserved among caspases in various species. These six mutants behave as recessive loss-of-function mutants in a homozygous condition. Surprisingly, however, two of the newly isolated drICE alleles are gain-of-function mutants in a heterozygous condition, although they are loss-of-function mutants homozygously. Interestingly, they only behave as gain-of-function mutants in the presence of an apoptotic signal. These two alleles carry missense mutations affecting conserved amino acids in close proximity to the catalytic cysteine residue. This is the first time that viable gain-of-function alleles of caspases are described in any intact organism and provides a significant exception to the expectation that mutations of conserved amino acids always abolish the pro-apoptotic activity of caspases. We discuss models about how these mutations cause the gain-of-function character of these alleles.
The beneficial role of extracellular reactive oxygen species in apoptosis-induced compensatory proliferation
Apoptosis-induced proliferation (AiP) maintains tissue homeostasis following massive stress-induced cell death. During this phenomenon, dying cells induce proliferation of the surviving cells to compensate for the tissue loss, and thus restore organ size. Along with wound healing and tissue regeneration, AiP also contributes to tumor repopulation following radiation or chemotherapy. There are several models of AiP. Using an "undead" AiP model that causes hyperplastic overgrowth of Drosophila epithelial tissue, we recently demonstrated that extracellular reactive oxygen species (eROS) are produced by undead epithelial cells, and are necessary for inducing AiP and overgrowth. Furthermore, hemocytes, the Drosophila blood cells, are seen adjacent to the undead epithelial tissue, and may secrete the TNF ortholog Eiger that signals through the TNF receptor to active Jun-N-terminal kinase (JNK) in the undead tissue and induce proliferation. We propose that undead epithelial tissue triggers an inflammatory response that resembles recruitment of macrophages to human epithelial tumors, and that these tumor-associated macrophages release signals for proliferation and tumor growth of the epithelium. This Extra View article summarizes these recent findings with a focus on the role of eROS for promoting regeneration and inflammation-induced tumorigenesis.
BACKGROUND: ATG1 belongs to the Uncoordinated-51-like kinase protein family. Members of this family are best characterized for roles in macroautophagy and neuronal development. Apoptosis-induced proliferation (AiP) is a caspase-directed and JNK-dependent process which is involved in tissue repair and regeneration after massive stress-induced apoptotic cell loss. Under certain conditions, AiP can cause tissue overgrowth with implications for cancer.
RESULTS: Here, we show that Atg1 in Drosophila (dAtg1) has a previously unrecognized function for both regenerative and overgrowth-promoting AiP in eye and wing imaginal discs. dAtg1 acts genetically downstream of and is transcriptionally induced by JNK activity, and it is required for JNK-dependent production of mitogens such as Wingless for AiP. Interestingly, this function of dAtg1 in AiP is independent of its roles in autophagy and in neuronal development.
CONCLUSION: In addition to a role of dAtg1 in autophagy and neuronal development, we report a third function of dAtg1 for AiP.