Cdk8/CDK19 promotes mitochondrial fission through Drp1 phosphorylation and can phenotypically suppress pink1 deficiency in Drosophila.

Cdk8 in Drosophila is the orthologue of vertebrate CDK8 and CDK19. These proteins have been shown to modulate transcriptional control by RNA polymerase II. We found that neuronal loss of Cdk8 severely reduces fly lifespan and causes bang sensitivity. Remarkably, these defects can be rescued by expression of human CDK19, found in the cytoplasm of neurons, suggesting a non-nuclear function of CDK19/Cdk8. Here we show that Cdk8 plays a critical role in the cytoplasm, with its loss causing elongated mitochondria in both muscles and neurons. We find that endogenous GFP-tagged Cdk8 can be found in both the cytoplasm and nucleus. We show that Cdk8 promotes the phosphorylation of Drp1 at S616, a protein required for mitochondrial fission. Interestingly, Pink1, a mitochondrial kinase implicated in Parkinson's disease, also phosphorylates Drp1 at the same residue. Indeed, overexpression of Cdk8 significantly suppresses the phenotypes observed in flies with low levels of Pink1, including elevated levels of ROS, mitochondrial dysmorphology, and behavioral defects. In summary, we propose that Pink1 and Cdk8 perform similar functions to promote Drp1-mediated fission.

Loss of the endoplasmic reticulum protein Tmem208 affects cell polarity, development, and viability.

Nascent proteins destined for the cell membrane and the secretory pathway are targeted to the endoplasmic reticulum (ER) either posttranslationally or cotranslationally. The signal-independent pathway, containing the protein TMEM208, is one of three pathways that facilitates the translocation of nascent proteins into the ER. The in vivo function of this protein is ill characterized in multicellular organisms. Here, we generated a CRISPR-induced null allele of the fruit fly ortholog CG8320/Tmem208 by replacing the gene with the Kozak-GAL4 sequence. We show that Tmem208 is broadly expressed in flies and that its loss causes lethality, although a few short-lived flies eclose. These animals exhibit wing and eye developmental defects consistent with impaired cell polarity and display mild ER stress. Tmem208 physically interacts with Frizzled (Fz), a planar cell polarity (PCP) receptor, and is required to maintain proper levels of Fz. Moreover, we identified a child with compound heterozygous variants in TMEM208 who presents with developmental delay, skeletal abnormalities, multiple hair whorls, cardiac, and neurological issues, symptoms that are associated with PCP defects in mice and humans. Additionally, fibroblasts of the proband display mild ER stress. Expression of the reference human TMEM208 in flies fully rescues the loss of Tmem208, and the two proband-specific variants fail to rescue, suggesting that they are loss-of-function alleles. In summary, our study uncovers a role of TMEM208 in development, shedding light on its significance in ER homeostasis and cell polarity.

De novo variants in PLCG1 are associated with hearing impairment, ocular pathology, and cardiac defects.

Phospholipase C isozymes (PLCs) hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol, important signaling molecules involved in many cellular processes. PLCG1 encodes the PLCγ1 isozyme that is broadly expressed. Hyperactive somatic mutations of PLCG1 are observed in multiple cancers, but only one germline variant has been reported. Here we describe three unrelated individuals with de novo heterozygous missense variants in PLCG1 (p.Asp1019Gly, p.His380Arg, and p.Asp1165Gly) who exhibit variable phenotypes including hearing loss, ocular pathology and cardiac septal defects. To model these variants in vivo, we generated the analogous variants in the Drosophila ortholog, small wing (sl). We created a null allele slT2A and assessed the expression pattern. sl is broadly expressed, including in wing discs, eye discs, and a subset of neurons and glia. Loss of sl causes wing size reductions, ectopic wing veins and supernumerary photoreceptors. We document that mutant flies exhibit a reduced lifespan and age-dependent locomotor defects. Expressing wild-type sl in slT2A mutant rescues the loss-of-function phenotypes whereas expressing the variants causes lethality. Ubiquitous overexpression of the variants also reduces viability, suggesting that the variants are toxic. Ectopic expression of an established hyperactive PLCG1 variant (p.Asp1165His) in the wing pouch causes severe wing phenotypes, resembling those observed with overexpression of the p.Asp1019Gly or p.Asp1165Gly variants, further arguing that these two are gain-of-function variants. However, the wing phenotypes associated with p.His380Arg overexpression are mild. Our data suggest that the PLCG1 de novo heterozygous missense variants are pathogenic and contribute to the features observed in the probands.

A defect in mitochondrial fatty acid synthesis impairs iron metabolism and causes elevated ceramide levels.

In most eukaryotic cells, fatty acid synthesis (FAS) occurs in the cytoplasm and in mitochondria. However, the relative contribution of mitochondrial FAS (mtFAS) to the cellular lipidome is not well defined. Here we show that loss of function of Drosophila mitochondrial enoyl coenzyme A reductase (Mecr), which is the enzyme required for the last step of mtFAS, causes lethality, while neuronal loss of Mecr leads to progressive neurodegeneration. We observe a defect in Fe-S cluster biogenesis and increased iron levels in flies lacking mecr, leading to elevated ceramide levels. Reducing the levels of either iron or ceramide suppresses the neurodegenerative phenotypes, indicating an interplay between ceramide and iron metabolism. Mutations in human MECR cause pediatric-onset neurodegeneration, and we show that human-derived fibroblasts display similar elevated ceramide levels and impaired iron homeostasis. In summary, this study identifies a role of mecr/MECR in ceramide and iron metabolism, providing a mechanistic link between mtFAS and neurodegeneration.

Sphingolipids in neurodegenerative diseases.

Neurodegenerative Diseases (NDDs) are a group of disorders that cause progressive deficits of neuronal function. Recent evidence argues that sphingolipid metabolism is affected in a surprisingly broad set of NDDs. These include some lysosomal storage diseases (LSDs), hereditary sensory and autonomous neuropathy (HSAN), hereditary spastic paraplegia (HSP), infantile neuroaxonal dystrophy (INAD), Friedreich's ataxia (FRDA), as well as some forms of amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Many of these diseases have been modeled in Drosophila melanogaster and are associated with elevated levels of ceramides. Similar changes have also been reported in vertebrate cells and mouse models. Here, we summarize studies using fly models and/or patient samples which demonstrate the nature of the defects in sphingolipid metabolism, the organelles that are implicated, the cell types that are initially affected, and potential therapeutics for these diseases.

Mechanisms of IRF2BPL-related disorders and identification of a potential therapeutic strategy.

The recently discovered neurological disorder NEDAMSS is caused by heterozygous truncations in the transcriptional regulator IRF2BPL. Here, we reprogram patient skin fibroblasts to astrocytes and neurons to study mechanisms of this newly described disease. While full-length IRF2BPL primarily localizes to the nucleus, truncated patient variants sequester the wild-type protein to the cytoplasm and cause aggregation. Moreover, patient astrocytes fail to support neuronal survival in coculture and exhibit aberrant mitochondria and respiratory dysfunction. Treatment with the small molecule copper ATSM (CuATSM) rescues neuronal survival and restores mitochondrial function. Importantly, the in vitro findings are recapitulated in vivo, where co-expression of full-length and truncated IRF2BPL in Drosophila results in cytoplasmic accumulation of full-length IRF2BPL. Moreover, flies harboring heterozygous truncations of the IRF2BPL ortholog (Pits) display progressive motor defects that are ameliorated by CuATSM treatment. Our findings provide insights into mechanisms involved in NEDAMSS and reveal a promising treatment for this severe disorder.

An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination.

Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.

Loss of IRF2BPL impairs neuronal maintenance through excess Wnt signaling.

De novo truncations in Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) lead to severe childhood-onset neurodegenerative disorders. To determine how loss of IRF2BPL causes neural dysfunction, we examined its function in Drosophila and zebrafish. Overexpression of either IRF2BPL or Pits, the Drosophila ortholog, represses Wnt transcription in flies. In contrast, neuronal depletion of Pits leads to increased wingless (wg) levels in the brain and is associated with axonal loss, whereas inhibition of Wg signaling is neuroprotective. Moreover, increased neuronal expression of wg in flies is sufficient to cause age-dependent axonal loss, similar to reduction of Pits. Loss of irf2bpl in zebrafish also causes neurological defects with an associated increase in wnt1 transcription and downstream signaling. WNT1 is also increased in patient-derived astrocytes, and pharmacological inhibition of Wnt suppresses the neurological phenotypes. Last, IRF2BPL and the Wnt antagonist, CKIα, physically and genetically interact, showing that IRF2BPL and CkIα antagonize Wnt transcription and signaling.

Regulation of Notch signaling by E3 ubiquitin ligases.

Notch signaling is an evolutionarily conserved pathway that is widely used for multiple cellular events during development. Activation of the Notch pathway occurs when the ligand from a neighboring cell binds to the Notch receptor and induces cleavage of the intracellular domain of Notch, which further translocates into the nucleus to activate its downstream genes. The involvement of the Notch pathway in diverse biological events is possible due to the complexity in its regulation. In order to maintain tight spatiotemporal regulation, the Notch receptor, as well as its ligand, undergoes a series of physical and biochemical modifications that, in turn, helps in proper maintenance and fine-tuning of the signaling outcome. Ubiquitination is the post-translational addition of a ubiquitin molecule to a substrate protein, and the process is regulated by E3 ubiquitin ligases. The present review describes the involvement of different E3 ubiquitin ligases that play an important role in the regulation and maintenance of proper Notch signaling and how perturbation in ubiquitination results in abnormal Notch signaling leading to a number of human diseases.

TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.

De novo mutations in TOMM70, a receptor of the mitochondrial import translocase, cause neurological impairment.

The translocase of outer mitochondrial membrane (TOMM) complex is the entry gate for virtually all mitochondrial proteins and is essential to build the mitochondrial proteome. TOMM70 is a receptor that assists mainly in mitochondrial protein import. Here, we report two individuals with de novo variants in the C-terminal region of TOMM70. While both individuals exhibited shared symptoms including hypotonia, hyper-reflexia, ataxia, dystonia and significant white matter abnormalities, there were differences between the two individuals, most prominently the age of symptom onset. Both individuals were undiagnosed despite extensive genetics workups. Individual 1 was found to have a p.Thr607Ile variant while Individual 2 was found to have a p.Ile554Phe variant in TOMM70. To functionally assess both TOMM70 variants, we replaced the Drosophila Tom70 coding region with a Kozak-mini-GAL4 transgene using CRISPR-Cas9. Homozygous mutant animals die as pupae, but lethality is rescued by the mini-GAL4-driven expression of human UAS-TOMM70 cDNA. Both modeled variants lead to significantly less rescue indicating that they are loss-of-function alleles. Similarly, RNAi-mediated knockdown of Tom70 in the developing eye causes roughening and synaptic transmission defect, common findings in neurodegenerative and mitochondrial disorders. These phenotypes were rescued by the reference, but not the variants, of TOMM70. Altogether, our data indicate that de novo loss-of-function variants in TOMM70 result in variable white matter disease and neurological phenotypes in affected individuals.

Regulation of Notch Signaling in Drosophila melanogaster: The Role of the Heterogeneous Nuclear Ribonucleoprotein Hrp48 and Deltex.

Notch signaling is an evolutionarily conserved pathway that plays a central role in a number of cellular events during metazoan development. Due to its involvement in numerous developmental events, Notch signaling requires tight spatial and temporal regulation. Deltex is a cytoplasmic protein that physically binds to the Notch and regulates its signaling activity in a context-dependent manner. However, the biology of Deltex in regulation of Notch signaling is not well explored. For a better understanding of Deltex activity in the regulatory circuit of Notch pathway, a co-IP-based screening was performed. Hrp48, an RNA-binding protein, was identified as an interacting partner of Deltex in that screening. Interaction of these two proteins seemed to regulate the Notch signaling outcome in the epithelial tissue. Additionally, it was found that coexpression of Deltex and Hrp48 can lead to cell death as well as JNK activation. Considering the fact of well conserved nature of Notch as well as both of these two proteins, namely, Hrp48 and Deltex, this interaction can be helpful to understand the regulation of Notch signaling both in development and disease condition.

Regulation of Notch signaling by the chromatin-modeling protein Hat-trick.

Notch signaling plays a pleiotropic role in a variety of cellular processes, including cell fate determination, differentiation, proliferation and apoptosis. The increasingly complex regulatory mechanisms of Notch signaling account for the many functions of Notch during development. Using a yeast two-hybrid screen, we identified the Drosophila DNA-binding protein Hat-trick (Htk) to be an interacting partner of Notch-intracellular domain (Notch-ICD); their physical interaction was further validated by co-immunoprecipitation experiments. htk genetically interacts with Notch pathway components in trans-heterozygous combinations. Loss of htk function in htk mutant somatic clones resulted in the downregulation of Notch targets, whereas its overexpression caused ectopic expression of Notch targets, without affecting the level of the Notch protein. In the present study, immunocytochemical analyses demonstrate that Htk and overexpressed Notch-ICD colocalize in the same nuclear compartment. Here, we also show that Htk cooperates with Notch-ICD and Suppressor of Hairless to form an activation complex and binds to the regulatory sequences of Notch downstream targets such as Enhancer of Split complex genes, to direct their expression. Together, our results suggest a novel mode of regulation of Notch signaling by the chromatin-modeling protein Htk.

Synergistic interaction of Deltex and Hrp48 leads to JNK activation.

The communication among the cells plays a seminal role in metazoan development by coordinating multiple cellular processes that, in turn, helps in the maintenance of biological homeostasis. Our previous study demonstrated that Dx and Hrp48 together downregulate Notch signaling and induce cell death in Drosophila. To understand the signaling events behind the Dx and Hrp48-induced cell death in a greater detail, we performed a set of genetic experiments followed by immunocytochemical analyses. Our data revealed that Dx along with Hrp48 induced JNK activation and consequently cell death in the eye tissue. Additionally, using genetic and molecular approaches, we identified the domain of Dx protein responsible for its synergistic activity with Hrp48. Altogether, our analyses suggest that coexpression of Dx and Hrp48 activates JNK pathway to induce cell death in eye disc of Drosophila melanogaster.

Regulation of Notch signaling in the developing Drosophila eye by a T-box containing transcription factor, Dorsocross.

Owing to a multitude of functions, there is barely a tissue or a cellular process that is not being regulated by Notch signaling. To allow the Notch signal to be deployed in numerous contexts, many different mechanisms have evolved to regulate the level, duration and spatial distribution of Notch activity. To identify novel effectors of Notch signaling in Drosophila melanogaster, we analyzed the whole transcriptome of the wing and eye imaginal discs in which an activated form of Notch was overexpressed. Selected candidate genes from the transcriptome analysis were subjected to genetic interaction experiments with Notch pathway components. Among the candidate genes, T-box encoding gene, Dorsocross (Doc) showed strong genetic interaction with Notch ligand, Delta. Genetic interaction between them resulted in reduction of eye size, loss of cone cells, and cell death, which represent prominent Notch loss of function phenotypes. Immunocytochemical analysis in Df(3L)DocA/Dl 5f trans-heterozygous eye discs showed accumulation of Notch at the membrane. This accumulation led to decreased Notch signaling activity as we found downregulation of Atonal, a Notch target and reduction in the rate of Notch-mediated cell proliferation. Doc mutant clones generated by FLP-FRT system showed depletion in the expression of Delta and subsequent reduction in the Notch signaling activity. Similarly, Doc overexpression in the eye discs led to modification of Delta expression, loss of Atonal expression and absence of eye structure in pharate adults. Taken together, our results suggest that Doc regulates the expression of Delta and influence the outcome of Notch signaling in the eye discs.

Deltex interacts with Eiger and consequently influences the cell death in Drosophila melanogaster.

TNF-JNK signaling is one of the highly conserved signaling pathways that regulate a broad spectrum of cellular processes including proliferation and apoptosis. Eiger, the sole homologue of TNF in Drosophila, initiates the TNF-JNK pathway to induce cell death. Previously, Deltex (Dx) has been identified as a Notch signaling component that regulates vesicular trafficking of Notch. In the present study, we have investigated the interaction between these two proteins in order to identify how Dx influences the activity of Eiger. Dx is found to act as a novel modulator of JNK-mediated cell death inducing activity of Eiger. Additionally, we observe that dx genetically interacts with eiger during wing development, and these two proteins, Dx and Eiger, colocalize in the cytoplasm. Our analysis reveals that Dx is involved in the cytoplasmic relocalization of Eiger from the cell membrane, thereby influencing Eiger-mediated JNK-activation process. Moreover, we demonstrate that Dx potentiates the activity of Eiger to downregulate Notch signaling pathway by retaining the Notch protein in the cytoplasm. Together, our findings reveal a novel role of Dx to modulate the signaling activity of Eiger and subsequent JNK-mediated cell death.

Notch signals modulate lgl mediated tumorigenesis by the activation of JNK signaling.

OBJECTIVES: Oncogenic potential of Notch signaling and its cooperation with other factors to affect proliferation are widely established. Notch exhibits a cooperative effect with loss of a cell polarity gene, scribble to induce neoplastic overgrowth. Oncogenic Ras also show cooperative effect with loss of cell polarity genes such as scribble (scrib), lethal giant larvae (lgl) and discs large to induce neoplastic overgrowth and invasion. Our study aims at assessing the cooperation of activated Notch with loss of function of lgl in tumor overgrowth, and the mode of JNK signaling activation in this context. RESULTS: In the present study, we use Drosophila as an in vivo model to show the synergy between activated Notch (N act ) and loss of function of lgl (lgl-IR) in tumor progression. Coexpression of N act and lgl-IR results in massive tumor overgrowth and displays hallmarks of cancer, such as MMP1 upregulation and loss of epithelial integrity. We further show activation of JNK signaling and upregulation of its receptor, Grindelwald in N act /lgl-IR tumor. In contrast to previously described Notch act /scrib-/- tumor, our experiments in N act /lgl-IR tumor showed the presence of dying cells along with tumorous overgrowth.

Pleiotropic Functions of the Chromodomain-Containing Protein Hat-trick During Oogenesis in Drosophila melanogaster.

Chromatin-remodeling proteins have a profound role in the transcriptional regulation of gene expression during development. Here, we have shown that the chromodomain-containing protein Hat-trick is predominantly expressed within the oocyte nucleus, specifically within the heterochromatinized karyosome, and that a mild expression is observed in follicle cells. Colocalization of Hat-trick with Heterochromatin Protein 1 and synaptonemal complex component C(3)G along with the diffused karyosome after hat-trick downregulation shows the role of this protein in heterochromatin clustering and karyosome maintenance. Germline mosaic analysis reveals that hat-trick is required for maintaining the dorso-ventral patterning of eggs by regulating the expression of Gurken. The increased incidence of double-strand breaks (DSBs), delayed DSB repair, defects in karyosome formation, altered Vasa mobility, and, consequently, misexpression and altered localization of Gurken in hat-trick mutant egg chambers clearly suggest a putative involvement of Hat-trick in the early stages of oogenesis. In addition, based on phenotypic observations in hat-trick mutant egg chambers, we speculate a substantial role of hat-trick in cystoblast proliferation, oocyte determination, nurse cell endoreplication, germ cell positioning, cyst encapsulation, and nurse cell migration. Our results demonstrate that hat-trick has profound pleiotropic functions during oogenesis in Drosophila melanogaster.

Regulation of Notch Signaling by the Heterogeneous Nuclear Ribonucleoprotein Hrp48 and Deltex in Drosophila melanogaster.

Notch signaling is an evolutionarily conserved pathway that is found to be involved in a number of cellular events throughout development. The deployment of the Notch signaling pathway in numerous cellular contexts is possible due to its regulation at multiple levels. In an effort to identify the novel components integrated into the molecular circuitry affecting Notch signaling, we carried out a protein-protein interaction screen based on the identification of cellular protein complexes using co-immunoprecipitation followed by mass-spectrometry. We identified Hrp48, a heterogeneous nuclear ribonucleoprotein in Drosophila, as a novel interacting partner of Deltex (Dx), a cytoplasmic modulator of Notch signaling. Immunocytochemical analysis revealed that Dx and Hrp48 colocalize in cytoplasmic vesicles. The dx mutant also showed strong genetic interactions with hrp48 mutant alleles. The coexpression of Dx and Hrp48 resulted in the depletion of cytoplasmic Notch in larval wing imaginal discs and downregulation of Notch targets cut and wingless Previously, it has been shown that Sex-lethal (Sxl), on binding with Notch mRNA, negatively regulates Notch signaling. The overexpression of Hrp48 was found to inhibit Sxl expression and consequently rescued Notch signaling activity. In the present study, we observed that Dx together with Hrp48 can regulate Notch signaling in an Sxl-independent manner. In addition, Dx and Hrp48 displayed a synergistic effect on caspase-mediated cell death. Our results suggest that Dx and Hrp48 together negatively regulate Notch signaling in Drosophila melanogaster.