Adult Neurogenesis in the Drosophila Brain: The Evidence and the Void.

Establishing the existence and extent of neurogenesis in the adult brain throughout the animals including humans, would transform our understanding of how the brain works, and how to tackle brain damage and disease. Obtaining convincing, indisputable experimental evidence has generally been challenging. Here, we revise the state of this question in the fruit-fly Drosophila. The developmental neuroblasts that make the central nervous system and brain are eliminated, either through apoptosis or cell cycle exit, before the adult fly ecloses. Despite this, there is growing evidence that cell proliferation can take place in the adult brain. This occurs preferentially at, but not restricted to, a critical period. Adult proliferating cells can give rise to both glial cells and neurons. Neuronal activity, injury and genetic manipulation in the adult can increase the incidence of both gliogenesis and neurogenesis, and cell number. Most likely, adult glio- and neuro-genesis promote structural brain plasticity and homeostasis. However, a definitive visualisation of mitosis in the adult brain is still lacking, and the elusive adult progenitor cells are yet to be identified. Resolving these voids is important for the fundamental understanding of any brain. Given its powerful genetics, Drosophila can expedite discovery into mammalian adult neurogenesis in the healthy and diseased brain.

Overexpression of Larp4B downregulates dMyc and reduces cell and organ sizes in Drosophila.

Regulation of cell and organ sizes is fundamental for all organisms, but its molecular basis is not fully understood. Here we performed a gain-of-function screen and identified larp4B whose overexpression reduces cell and organ sizes in Drosophila melanogaster. Larp4B is a member of La-related proteins (LARPs) containing an LA motif and an adjacent RNA recognition motif (RRM), and play diverse roles in RNA metabolism. However, the function of Larp4B has remained poorly characterized. We generated transgenic flies overexpressing wild-type Larp4B or a deletion variant lacking the LA and RRM domains, and demonstrated that the RNA-binding domains are essential for Larp4B to reduce cell and organ sizes. We found that the larp4B-induced phenotype was suppressed by dMyc overexpression, which promotes cell growth and survival. Furthermore, overexpression of larp4B decreased dMyc protein levels, whereas its loss-of-function mutation had an opposite effect. Our results suggest that Larp4B is a negative regulator of dMyc.

Targeted downregulation of dMyc restricts neurofibrillary tangles mediated pathogenesis of human neuronal tauopathies in Drosophila.

Formation of Neurofibrillary Tangles (NFTs) in neuronal tissues has been implicated as the hallmark of disease pathogenesis and tau mediated toxicity in human and mammalian models. However, previous studies had failed to correlate NFT formation with pathogenesis of human neuronal tauopathies in Drosophila disease models. Though, a recent report suggests formation of tau mediated NFTs like structures confined to dopaminergic neurons in Drosophila adult brain; by utilizing various approaches, we demonstrate distinct and recurrent formation of NFTs in Drosophila neuronal tissues upon expression of wild type or mutant isoforms of human tau protein, and this appears as the key mediator of the pathogenesis of human neuronal tauopathy in Drosophila. Further, we show that tissue specific downregulation of dMyc (Drosophila homolog of human c-myc proto-oncogene) alleviates h-tau mediated cellular and functional deficits by restricting the formation of NFTs in neuronal tissues. Therefore, our findings provide very critical and novel insights about pathogenesis of human neuronal tauopathies in Drosophila disease models.

Targeted upregulation of dMyc restricts JNK-mediated degeneration of dopaminergic neurons in the paraquat-induced Parkinson's disease model of Drosophila.

Parkinson's disease is the second most common neurodegenerative disease characterized by the loss of dopaminergic neurons in the brain. Parkinson's disease has both familial and sporadic cases of origin governed differentially by genetic and/or environmental factors. Different epidemiological studies have proposed an association between the pathogenesis of cancer and Parkinson's disease; however, a precise correlation between these two illnesses could not be established yet. In this study, we examined the disease-modifying property of dmyc (a Drosophila homolog of human cmyc proto-oncogene) in the paraquat-induced sporadic Parkinson's disease model of Drosophila. We report for the first time that targeted upregulation of dMyc significantly restricts paraquat-mediated neurotoxicity. We observed that paraquat feeding reduces the cellular level of dMyc. We further noted that targeted upregulation of dMyc in paraquat-exposed flies mitigates degeneration of dopaminergic neurons by reinstating the aberrantly activated JNK pathway, and this in turn improves the motor performance and survival rate of the flies. Our study provides the first evidence that improved cellular level of dMyc could efficiently minimize the neurotoxic effects of paraquat, which could be beneficial in designing novel therapeutic strategies against Parkinson's disease.

Negative regulation of diminutive cancer regulator through differentiation and microRNA pathway components in Drosophila cells.

Drosophila model is intensively studied for the development of cancer. The diminutive (dMyc), a homolog of the human MYC gene, is responsible for cell- apoptosis and its upregulation is responsible for determining the fate of cancerous growth in humans and Drosophila model. This work implores the requirement of dMyc and its expression as one of the major regulator of cancer with other proteins and repression of dMyc mRNA in Drosophila S2 cells. Here we report protein complex of Argonaute 1 (AGO1), Bag of marbles (Bam), and Brain tumor (Brat) proteins and not the individual factor of this complex repression of dMyc mRNA in Drosophila Schneider 2 cells and promote differentiation in cystoblast of Drosophila ovary. These results exhibit the significant role of this complex, including master differentiation factor Bam with other various differentiation factor Brat and microRNA pathway component AGO1, which may negatively regulate dMyc mRNA and so the dMyc protein.

The transcription factor Spalt and human homologue SALL4 induce cell invasion via the dMyc-JNK pathway in Drosophila.

Cancer cell metastasis is a leading cause of mortality in cancer patients. Therefore, revealing the molecular mechanism of cancer cell invasion is of great significance for the treatment of cancer. In human patients, the hyperactivity of transcription factor Spalt-like 4 (SALL4) is sufficient to induce malignant tumorigenesis and metastasis. Here, we found that when ectopically expressing the Drosophila homologue spalt (sal) or human SALL4 in Drosophila, epithelial cells delaminated basally with penetration of the basal lamina and degradation of the extracellular matrix, which are essential properties of cell invasion. Further assay found that sal/SALL4 promoted cell invasion via dMyc-JNK signaling. Inhibition of the c-Jun N-terminal kinase (JNK) signaling pathway through suppressing matrix metalloprotease 1, or basket can achieve suppression of cell invasion. Moreover, expression of dMyc, a suppressor of JNK signaling, dramatically blocked cell invasion induced by sal/SALL4 in the wing disc. These findings reveal a conserved role of sal/SALL4 in invasive cell movement and link the crucial mediator of tumor invasion, the JNK pathway, to SALL4-mediated cancer progression.This article has an associated First Person interview with the first author of the paper.

Drosophila HUWE1 Ubiquitin Ligase Regulates Endoreplication and Antagonizes JNK Signaling During Salivary Gland Development.

The HECT-type ubiquitin ligase HECT, UBA and WWE Domain Containing 1, (HUWE1) regulates key cancer-related pathways, including the Myc oncogene. It affects cell proliferation, stress and immune signaling, mitochondria homeostasis, and cell death. HUWE1 is evolutionarily conserved from Caenorhabditis elegance to Drosophila melanogaster and Humans. Here, we report that the Drosophila ortholog, dHUWE1 (CG8184), is an essential gene whose loss results in embryonic lethality and whose tissue-specific disruption establishes its regulatory role in larval salivary gland development. dHUWE1 is essential for endoreplication of salivary gland cells and its knockdown results in the inability of these cells to replicate DNA. Remarkably, dHUWE1 is a survival factor that prevents premature activation of JNK signaling, thus preventing the disintegration of the salivary gland, which occurs physiologically during pupal stages. This function of dHUWE1 is general, as its inhibitory effect is observed also during eye development and at the organismal level. Epistatic studies revealed that the loss of dHUWE1 is compensated by dMyc proeitn expression or the loss of dmP53. dHUWE1 is therefore a conserved survival factor that regulates organ formation during Drosophila development.

Targeted Downregulation of dMyc Suppresses Pathogenesis of Human Neuronal Tauopathies in Drosophila by Limiting Heterochromatin Relaxation and Tau Hyperphosphorylation.

Human tauopathies such as Alzheimer's Disease (AD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), Pick's disease etc., are a group of neurodegenerative diseases which are characterized by abnormal hyperphosphorylation of tau that leads to formation of neurofibrillary tangles. Recapitulating several features of human neurodegenerative disorders, the Drosophila tauopathy model displays compromised lifespan, locomotor function impairment, and brain vacuolization in adult brain which is progressive and age dependent. Here, we demonstrate that tissue-specific downregulation of the Drosophila homolog of human c-myc proto-oncogene (dMyc) suppresses tau-mediated morphological and functional deficits by reducing abnormal tau hyperphosphorylation and restoring the heterochromatin loss. Our studies show for the first time that the inherent chromatin remodeling ability of myc proto-oncogenes could be exploited to limit the pathogenesis of human neuronal tauopathies in the Drosophila disease model. Interestingly, recent reports on successful uses of some anti-cancer drugs against Alzheimer's and Parkinson's diseases in clinical trials and animal models strongly support our findings and proposed possibility.

Investigation of protein synthesis in Drosophila larvae using puromycin labelling.

Translational control of gene expression is an important regulator of growth, homeostasis and aging in Drosophila The ability to measure changes in protein synthesis in response to genetic and environmental cues is therefore important in studying these processes. Here we describe a simple and cost-effective approach to assay protein synthesis in Drosophila larval cells and tissues. The method is based on the incorporation of puromycin into nascent peptide chains. Using an ex vivo approach, we label newly synthesized peptides in larvae with puromycin and then measure levels of new protein synthesis using an anti-puromycin antibody. We show that this method can detect changes in protein synthesis in specific cells and tissues in the larvae, either by immunostaining or western blotting. We find that the assay reliably detects changes in protein synthesis induced by two known stimulators of mRNA translation - the nutrient/TORC1 kinase pathway and the transcription factor dMyc. We also use the assay to describe how protein synthesis changes through larval development and in response to two environmental stressors - hypoxia and heat shock. We propose that this puromycin-labelling assay is a simple but robust method to detect protein synthesis changes at the levels of cells, tissues or whole body in Drosophila.

Controlling the Master: Chromatin Dynamics at the MYC Promoter Integrate Developmental Signaling.

The transcription factor and cell growth regulator MYC is potently oncogenic and estimated to contribute to most cancers. Decades of attempts to therapeutically target MYC directly have not resulted in feasible clinical applications, and efforts have moved toward indirectly targeting MYC expression, function and/or activity to treat MYC-driven cancer. A multitude of developmental and growth signaling pathways converge on the MYC promoter to modulate transcription through their downstream effectors. Critically, even small increases in MYC abundance (<2 fold) are sufficient to drive overproliferation; however, the details of how oncogenic/growth signaling networks regulate MYC at the level of transcription remain nebulous even during normal development. It is therefore essential to first decipher mechanisms of growth signal-stimulated MYC transcription using in vivo models, with intact signaling environments, to determine exactly how these networks are dysregulated in human cancer. This in turn will provide new modalities and approaches to treat MYC-driven malignancy. Drosophila genetic studies have shed much light on how complex networks signal to transcription factors and enhancers to orchestrate Drosophila MYC (dMYC) transcription, and thus growth and patterning of complex multicellular tissue and organs. This review will discuss the many pathways implicated in patterning MYC transcription during development and the molecular events at the MYC promoter that link signaling to expression. Attention will also be drawn to parallels between mammalian and fly regulation of MYC at the level of transcription.

CNBP regulates wing development in Drosophila melanogaster by promoting IRES-dependent translation of dMyc.

CCHC-type zinc finger nucleic acid binding protein (CNBP) is a small conserved protein, which plays a key role in development and disease. Studies in animal models have shown that the absence of CNBP results in severe developmental defects that have been mostly attributed to its ability to regulate c-myc mRNA expression. Functionally, CNBP binds single-stranded nucleic acids and acts as a molecular chaperone, thus regulating both transcription and translation.   In this work we report that in Drosophila melanogaster, CNBP is an essential gene, whose absence causes early embryonic lethality. In contrast to what observed in other species, ablation of CNBP does not affect dMyc mRNA expression, whereas the protein levels are markedly reduced. We demonstrate for the first time that dCNBP regulates dMyc translation through an IRES-dependent mechanism, and that knockdown of dCNBP in the wing territory causes a general reduction of wing size, in keeping with the reported role of dMyc in this region. Consistently, reintroduction of dMyc in CNBP-deficient wing imaginal discs rescues the wing size, further supporting a key role of the CNBP-Myc axis in this context. Collectively, these data show a previously uncharacterized mechanism, whereby, by regulating dMyc IRES-dependent translation, CNBP controls Drosophila wing development. These results may have relevant implications in other species and in pathophysiological conditions.

MicroRNA miR-308 regulates dMyc through a negative feedback loop in Drosophila.

The abundance of Myc protein must be exquisitely controlled to avoid growth abnormalities caused by too much or too little Myc. An intriguing mode of regulation exists in which Myc protein itself leads to reduction in its abundance. We show here that dMyc binds to the miR-308 locus and increases its expression. Using our gain-of-function approach, we show that an increase in miR-308 causes a destabilization of dMyc mRNA and reduced dMyc protein levels. In vivo knockdown of miR-308 confirmed the regulation of dMyc levels in embryos. This regulatory loop is crucial for maintaining appropriate dMyc levels and normal development. Perturbation of the loop, either by elevated miR-308 or elevated dMyc, caused lethality. Combining elevated levels of both, therefore restoring balance between miR-308 and dMyc levels, resulted in lower apoptotic activity and suppression of lethality. These results reveal a sensitive feedback mechanism that is crucial to prevent the pathologies caused by abnormal levels of dMyc.

Investigation of dmyc Promoter and Regulatory Regions.

Products of the myc gene family integrate extracellular signals by modulating a wide range of their targets involved in cellular biogenesis and metabolism; the purpose of this integration is to regulate cell death, proliferation, and differentiation. However, understanding the regulation of myc at the transcription level remains a challenge. We performed rapid amplification of dmyc cDNA ends (5' RACE) and mapped the transcription start site at P1 promoter, 18 base pairs upstream of the start of the known EST GM01143 and within the 5' UTR. Our data show that the first TATA box, previously computationally predicted, is utilized to generate dmyc full length mRNA. The largest transcript contains all three exons, generated after the removal of the introns by constitutively regulated splicing events. Further investigation of Downstream Promoter Element (DPE) was achieved by studying lacZ reporter activity; investigation revealed that this element and its upstream cluster of binding sites are required for the dmyc intron 2 activity. These findings may provide valuable tools for further analysis of dmyc cis-elements.

Identification of cis-Regulatory Elements in the dmyc Gene of Drosophila Melanogaster.

Myc is a crucial regulator of growth and proliferation during animal development. Many signals and transcription factors lead to changes in the expression levels of Drosophila myc, yet no clear model exists to explain the complexity of its regulation at the level of transcription. In this study we used Drosophila genetic tools to track the dmyc cis-regulatory elements. Bioinformatics analyses identified conserved sequence blocks in the noncoding regions of the dmyc gene. Investigation of lacZ reporter activity driven by upstream, downstream, and intronic sequences of the dmyc gene in embryonic, larval imaginal discs, larval brain, and adult ovaries, revealed that it is likely to be transcribed from multiple transcription initiation units including a far upstream regulatory region, a TATA box containing proximal complex and a TATA-less downstream promoter element in conjunction with an initiator within the intron 2 region. Our data provide evidence for a modular organization of dmyc regulatory sequences; these modules will most likely be required to generate the tissue-specific patterns of dmyc transcripts. The far upstream region is active in late embryogenesis, while activity of other cis elements is evident during embryogenesis, in specific larval imaginal tissues and during oogenesis. These data provide a framework for further investigation of the transcriptional regulatory mechanisms of dmyc.