cAMP/PKA signaling regulates TDP-43 aggregation and mislocalization.

Cytoplasmic mislocalization and aggregation of TDP-43 protein are hallmarks of amyotrophic lateral sclerosis (ALS) and are observed in the vast majority of both familial and sporadic cases. How these two interconnected processes are regulated on a molecular level, however, remains enigmatic. Genome-wide screens for modifiers of the ALS-associated genes TDP-43 and FUS have identified the phospholipase D (Pld) pathway as a key regulator of ALS-related phenotypes in the fruit fly Drosophila melanogaster [M. W. Kankel et al., Genetics 215, 747-766 (2020)]. Here, we report the results of our search for downstream targets of the enzymatic product of Pld, phosphatidic acid. We identify two conserved negative regulators of the cAMP/PKA signaling pathway, the phosphodiesterase dunce and the inhibitory subunit PKA-R2, as modifiers of pathogenic phenotypes resulting from overexpression of the Drosophila TDP-43 ortholog TBPH. We show that knockdown of either of these genes results in a mitigation of both TBPH aggregation and mislocalization in larval motor neuron cell bodies, as well as an amelioration of adult-onset motor defects and shortened lifespan induced by TBPH. We determine that PKA kinase activity is downstream of both TBPH and Pld and that overexpression of the PKA target CrebA can rescue TBPH mislocalization. These findings suggest a model whereby increasing cAMP/PKA signaling can ameliorate the molecular and functional effects of pathological TDP-43.

Amyotrophic Lateral Sclerosis Modifiers in Drosophila Reveal the Phospholipase D Pathway as a Potential Therapeutic Target.

Amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig's disease, is a devastating neurodegenerative disorder lacking effective treatments. ALS pathology is linked to mutations in >20 different genes indicating a complex underlying genetic architecture that is effectively unknown. Here, in an attempt to identify genes and pathways for potential therapeutic intervention and explore the genetic circuitry underlying Drosophila models of ALS, we carry out two independent genome-wide screens for modifiers of degenerative phenotypes associated with the expression of transgenic constructs carrying familial ALS-causing alleles of FUS (hFUSR521C) and TDP-43 (hTDP-43M337V). We uncover a complex array of genes affecting either or both of the two strains, and investigate their activities in additional ALS models. Our studies indicate the pathway that governs phospholipase D activity as a major modifier of ALS-related phenotypes, a notion supported by data we generated in mice and others collected in humans.

PINK1/Parkin Influences Cell Cycle by Sequestering TBK1 at Damaged Mitochondria, Inhibiting Mitosis.

PINK1 and Parkin are established mediators of mitophagy, the selective removal of damaged mitochondria by autophagy. PINK1 and Parkin have been proposed to act as tumor suppressors, as loss-of-function mutations are correlated with enhanced tumorigenesis. However, it is unclear how PINK1 and Parkin act in coordination during mitophagy to influence the cell cycle. Here we show that PINK1 and Parkin genetically interact with proteins involved in cell cycle regulation, and loss of PINK1 and Parkin accelerates cell growth. PINK1- and Parkin-mediated activation of TBK1 at the mitochondria during mitophagy leads to a block in mitosis due to the sequestration of TBK1 from its physiological role at centrosomes during mitosis. Our study supports a diverse role for the far-reaching, regulatory effects of mitochondrial quality control in cellular homeostasis and demonstrates that the PINK1/Parkin pathway genetically interacts with the cell cycle, providing a framework for understanding the molecular basis linking PINK1 and Parkin to mitosis.

The Notch Interactome: Complexity in Signaling Circuitry.

The Notch pathway controls a very broad spectrum of cell fates in metazoans during development, influencing proliferation, differentiation and cell death. Given its central role in normal development and homeostasis, misregulation of Notch signals can lead to various disorders including cancer. How the Notch pathway mediates such pleiotropic and differential effects is of fundamental importance. It is becoming increasingly clear through a number of large-scale genetic and proteomic studies that Notch interacts with a staggeringly large number of other genes and pathways in a context-dependent, complex, and highly regulated network, which determines the ultimate biological outcome. How best to interpret and analyze the continuously increasing wealth of data on Notch interactors remains a challenge. Here we review the current state of genetic and proteomic data related to the Notch interactome.

The Notch-Mediated Proliferation Circuitry.

The essential and highly conserved Notch signaling pathway controls a wide range of cell fate decisions during development, including cellular proliferation. Notch mediates both pro- and anti-proliferative effects in development, stem cells, and cancer depending on cellular context. Furthermore, it can induce proliferation in both cell-autonomous and non-cell-autonomous manners. Interacting genes and crosstalking signaling pathways play essential roles in regulating the proliferative response to Notch signals. A large number of genes that participate in the Notch network to influence proliferation have been identified, including several that activate the JNK signaling pathway, which interacts with Notch to induce both hyperplastic and invasive cellular behaviors. It is clear that dissecting the genetic circuitry surrounding Notch is essential to understanding the proliferative response to Notch in both development and cancer.

The Notch-mediated hyperplasia circuitry in Drosophila reveals a Src-JNK signaling axis.

Notch signaling controls a wide range of cell fate decisions during development and disease via synergistic interactions with other signaling pathways. Here, through a genome-wide genetic screen in Drosophila, we uncover a highly complex Notch-dependent genetic circuitry that profoundly affects proliferation and consequently hyperplasia. We report a novel synergistic relationship between Notch and either of the non-receptor tyrosine kinases Src42A and Src64B to promote hyperplasia and tissue disorganization, which results in cell cycle perturbation, JAK/STAT signal activation, and differential regulation of Notch targets. Significantly, the JNK pathway is responsible for the majority of the phenotypes and transcriptional changes downstream of Notch-Src synergy. We previously reported that Notch-Mef2 also activates JNK, indicating that there are commonalities within the Notch-dependent proliferation circuitry; however, the current data indicate that Notch-Src accesses JNK in a significantly different fashion than Notch-Mef2.

Notch signaling at a glance.

Cell-cell interactions define a quintessential aspect of multicellular development. Metazoan morphogenesis depends on a handful of fundamental, conserved cellular interaction mechanisms, one of which is defined by the Notch signaling pathway. Signals transmitted through the Notch surface receptor have a unique developmental role: Notch signaling links the fate of one cell with that of a cellular neighbor through physical interactions between the Notch receptor and the membrane-bound ligands that are expressed in an apposing cell. The developmental outcome of Notch signals is strictly dependent on the cellular context and can influence differentiation, proliferation and apoptotic cell fates. The Notch pathway is conserved across species (Artavanis-Tsakonas et al., 1999; Bray, 2006; Kopan and Ilagan, 2009). In humans, Notch malfunction has been associated with a diverse range of diseases linked to changes in cell fate and cell proliferation including cancer (Louvi and Artavanis-Tsakonas, 2012). In this Cell Science at a Glance article and the accompanying poster we summarize the molecular biology of Notch signaling, its role in development and its relevance to disease.

In vivo mapping of notch pathway activity in normal and stress hematopoiesis.

Accumulating evidence suggests that Notch signaling is active at multiple points during hematopoiesis. Until recently, the majority of such studies focused on Notch signaling in lymphocyte differentiation and knowledge of individual Notch receptor roles has been limited due to a paucity of genetic tools available. In this manuscript we generate and describe animal models to identify and fate-map stem and progenitor cells expressing each Notch receptor, delineate Notch pathway activation, and perform in vivo gain- and loss-of-function studies dissecting Notch signaling in early hematopoiesis. These models provide comprehensive genetic maps of lineage-specific Notch receptor expression and activation in hematopoietic stem and progenitor cells. Moreover, they establish a previously unknown role for Notch signaling in the commitment of blood progenitors toward the erythrocytic lineage and link Notch signaling to optimal organismal response to stress erythropoiesis.

Notch2 genetic fate mapping reveals two previously unrecognized mammary epithelial lineages.

Notch signalling is implicated in stem and progenitor cell fate control in numerous organs. Using conditional in vivo genetic labelling we traced the fate of cells expressing the Notch2 receptor paralogue and uncovered the existence of two previously unrecognized mammary epithelial cell lineages that we term S (Small) and L (Large). S cells appear in a bead-on-a-string formation and are embedded between the luminal and basal/myoepithelial layers in a unique reiterative pattern, whereas single or paired L cells appear among ductal and alveolar cells. Long-term lineage tracing and functional studies indicate that S and L cells regulate ipsi- and contralateral spatial placement of tertiary branches and formation of alveolar clusters. Our findings revise present models of mammary epithelial cell hierarchy, reveal a hitherto undescribed mechanism regulating branching morphogenesis and may have important implications for identification of the cell-of-origin of distinct breast cancer subtypes.

The Notch signalling system: recent insights into the complexity of a conserved pathway.

Notch signalling links the fate of one cell to that of an immediate neighbour and consequently controls differentiation, proliferation and apoptotic events in multiple metazoan tissues. Perturbations in this pathway activity have been linked to several human genetic disorders and cancers. Recent genome-scale studies in Drosophila melanogaster have revealed an extraordinarily complex network of genes that can affect Notch activity. This highly interconnected network contrasts our traditional view of the Notch pathway as a simple linear sequence of events. Although we now have an unprecedented insight into the way in which such a fundamental signalling mechanism is controlled by the genome, we are faced with serious challenges in analysing the underlying molecular mechanisms of Notch signal control.

Notch and Mef2 synergize to promote proliferation and metastasis through JNK signal activation in Drosophila.

Genetic analyses in Drosophila revealed a synergy between Notch and the pleiotropic transcription factor Mef2 (myocyte enhancer factor 2), which profoundly influences proliferation and metastasis. We show that these hyperproliferative and invasive Drosophila phenotypes are attributed to upregulation of eiger, a member of the tumour necrosis factor superfamily of ligands, and the consequent activation of Jun N-terminal kinase signalling, which in turn triggers the expression of the invasive marker MMP1. Expression studies in human breast tumour samples demonstrate correlation between Notch and Mef2 paralogues and support the notion that Notch-MEF2 synergy may be significant for modulating human mammary oncogenesis.

Notch and disease: a growing field.

Signals through the Notch receptors are used throughout development to control cellular fate choices. Our intention here is to provide an overview of the involvement of Notch signaling in human disease, which, keeping pace with the known biology of the pathway, manifests itself in a pleiotropic fashion. A pathway with such broad action in normal development, a profound involvement in the biology of adult stem cells and intricate and complex controls governing its activity, poses numerous challenges. We provide an overview of Notch related pathologies identified thus far and emphasize aspects that have been modeled in experimental systems in order to understand the underlying pathobiology and, hopefully, help the definition of rational therapeutic avenues.

Trp53 regulates Notch 4 signaling through Mdm2.

Notch receptors and their ligands have crucial roles in development and tumorigenesis. We present evidence demonstrating the existence of an antagonistic relationship between Notch 4 and Trp53, which is controlled by the Mdm2-dependent ubiquitylation and degradation of the Notch receptor. We show that this signal-controlling mechanism is mediated by physical interactions between Mdm2 and Notch 4 and suggest the existence of a trimeric complex between Trp53, Notch 4 and Mdm2, which ultimately regulates Notch activity. Functional studies indicate that Trp53 can suppress NICD4-induced anchorage-independent growth in mammary epithelial cells and present evidence showing that Trp53 has a pivotal role in the suppression of Notch-associated tumorigenesis in the mammary gland.

Notch: the past, the present, and the future.

Proliferating investigations of the Notch pathway have given rise to the Notch "field," which has grown exponentially over the past 30 years. This field, founded by investigations of embryology and genetics in Drosophila, now encompasses many metazoa, including humans. The increasingly diverse scope of the field has engendered an expanding understanding that normal Notch pathway function is central to most developmental decision-making in animals, and that pathway dysfunction is implicated in many diseases, including cancer. We provide a personal view of the foundations and rapid evolution of the Notch field; and we discuss a variety of outstanding conundrums and questions regarding Notch biology, for which answers will be found and refined during the next 30 years.

Dose-dependent induction of distinct phenotypic responses to Notch pathway activation in mammary epithelial cells.

Aberrant activation of Notch receptors has been implicated in breast cancer; however, the mechanisms contributing to Notch-dependent transformation remain elusive because Notch displays dichotomous functional activities, promoting both proliferation and growth arrest. We investigated the cellular basis for the heterogeneous responses to Notch pathway activation in 3D cultures of MCF-10A mammary epithelial cells. Expression of a constitutively active Notch-1 intracellular domain (NICD) was found to induce two distinct types of 3D structures: large, hyperproliferative structures and small, growth-arrested structures with reduced cell-to-matrix adhesion. Interestingly, we found that these heterogeneous phenotypes reflect differences in Notch pathway activation levels; high Notch activity caused down-regulation of multiple matrix-adhesion genes and inhibition of proliferation, whereas low Notch activity maintained matrix adhesion and provoked a strong hyperproliferative response. Moreover, microarray analyses implicated NICD-induced p63 down-regulation in loss of matrix adhesion. In addition, a reverse-phase protein array-based analysis and subsequent loss-of-function studies identified STAT3 as a dominant downstream mediator of the NICD-induced outgrowth. These results indicate that the phenotypic responses to Notch are determined by the dose of pathway activation; and this dose affects the balance between growth-stimulative and growth-suppressive effects. This unique feature of Notch signaling provides insights into mechanisms that contribute to the dichotomous effects of Notch during development and tumorigenesis.

Notch and Wnt signals cooperatively control cell proliferation and tumorigenesis in the intestine.

Notch and Wnt signals play essential roles in intestinal development and homeostasis, yet how they integrate their action to affect intestinal morphogenesis is not understood. We examined the interplay between these two signaling pathways in vivo, by modulating Notch activity in mice carrying either a loss- or a gain-of-function mutation of Wnt signaling. We find that the dramatic proliferative effect that Notch signals have on early intestinal precursors requires normal Wnt signaling, whereas its influence on intestinal differentiation appears independent of Wnt. Analogous experiments in Drosophila demonstrate that the synergistic effects of Notch and Wnt are valid across species. We also demonstrate a striking synergy between Notch and Wnt signals that results in inducing the formation of intestinal adenomas, particularly in the colon, a region rarely affected in available mouse tumor models, but the primary target organ in human patients. These studies thus reveal a previously unknown oncogenic potential of Notch signaling in colorectal tumorigenesis that, significantly, is supported by the analysis of human tumors. Importantly, our experimental evidence raises the possibility that Notch activation might be an essential initial event triggering colorectal cancer.

Crossing paths with Notch in the hyper-network.

The development of complex and diverse metazoan morphologies is coordinated by a surprisingly small number of evolutionarily conserved signaling mechanisms. These signals can act in parallel but often appear to function as an integrated hyper-network. The nodes defining this complex molecular circuitry are poorly understood, but the biological significance of pathway cross-talk is profound. The importance of such large-scale signal integration is exemplified by Notch and its ability to cross-talk with all the major pathways to influence cell differentiation, proliferation, survival and migration. The Notch pathway is, thus, a useful paradigm to illustrate the complexity of pathway cross-talk: its pervasiveness, context dependency, and importance in development and disease.

Myc is a Notch1 transcriptional target and a requisite for Notch1-induced mammary tumorigenesis in mice.

To explore the potential involvement of aberrant Notch1 signaling in breast cancer pathogenesis, we have used a transgenic mouse model. In these animals, mouse mammary tumor virus LTR-driven expression of the constitutively active intracellular domain of the Notch1 receptor (N1(IC)) causes development of lactation-dependent mammary tumors that regress upon gland involution but progress to nonregressing, invasive adenocarcinomas in subsequent pregnancies. Up-regulation of Myc in these tumors prompted a genetic investigation of a potential Notch1/Myc functional relationship in breast carcinogenesis. Conditional ablation of Myc in the mammary epithelium prevented the induction of regressing N1(IC) neoplasms and also reduced the incidence of nonregressing carcinomas, which developed with significantly increased latency. Molecular analyses revealed that both the mouse and human Myc genes are direct transcriptional targets of N1(IC) acting through its downstream Cbf1 transcriptional effector. Consistent with this mechanistic link, Notch1 and Myc expression is positively correlated by immunostaining in 38% of examined human breast carcinomas.

Notch signals control the fate of immature progenitor cells in the intestine.

The Notch signalling pathway plays a crucial role in specifying cellular fates in metazoan development by regulating communication between adjacent cells. Correlative studies suggested an involvement of Notch in intestinal development. Here, by modulating Notch activity in the mouse intestine, we directly implicate Notch signals in intestinal cell lineage specification. We also show that Notch activation is capable of amplifying the intestinal progenitor pool while inhibiting cell differentiation. We conclude that Notch activity is required for the maintenance of proliferating crypt cells in the intestinal epithelium.

Modulation of notch signaling elicits signature tumors and inhibits hras1-induced oncogenesis in the mouse mammary epithelium.

Deregulation of Notch signaling, which normally affects a broad spectrum of cell fates, has been implicated in various neoplastic conditions. Here we describe a transgenic mouse model, which demonstrates that expression of a constitutively active form of the Notch1 receptor in the mammary epithelium induces the rapid development of pregnancy/lactation-dependent neoplasms that consistently exhibit a characteristic histopathological pattern. These signature tumors retain the ability to respond to apoptotic stimuli and regress on initiation of mammary gland involution, but eventually appear to progress in subsequent pregnancies to nonregressing malignant adenocarcinomas. Additionally, we present evidence indicating that cyclin D1 is an in vivo target of Notch signals in the mammary glands and demonstrate that we can effectively inhibit Hras1-driven, cyclin D1-dependent mammary oncogenesis by transgenic expression of the Notch antagonist Deltex.