The Fine Tuning of Drp1-Dependent Mitochondrial Remodeling and Autophagy Controls Neuronal Differentiation

Mitochondria play a critical role in neuronal function and neurodegenerative disorders, including Alzheimer's, Parkinson's and Huntington diseases and amyotrophic lateral sclerosis, that show mitochondrial dysfunctions associated with excessive fission and increased levels of the fission protein dynamin-related protein 1 (Drp1). Our data demonstrate that Drp1 regulates the transcriptional program induced by retinoic acid (RA), leading to neuronal differentiation. When Drp1 was overexpressed, mitochondria underwent remodeling but failed to elongate and this enhanced autophagy and apoptosis. When Drp1 was blocked during differentiation by overexpressing the dominant negative form or was silenced, mitochondria maintained the same elongated shape, without remodeling and this increased cell death. The enhanced apoptosis, observed with both fragmented or elongated mitochondria, was associated with increased induction of unfolded protein response (UPR) and ER-associated degradation (ERAD) processes that finally affect neuronal differentiation. These findings suggest that physiological fission and mitochondrial remodeling, associated with early autophagy induction are essential for neuronal differentiation. We thus reveal the importance of mitochondrial changes to generate viable neurons and highlight that, rather than multiple parallel events, mitochondrial changes, autophagy and apoptosis proceed in a stepwise fashion during neuronal differentiation affecting the nuclear transcriptional program

Drp1 overexpression induces desmin disassembling and drives kinesin-1 activation promoting mitochondrial trafficking in skeletal muscle

Mitochondria change distribution across cells following a variety of pathophysiological stimuli. The mechanisms presiding over this redistribution are yet undefined. In a murine model overexpressing Drp1 specifically in skeletal muscle, we find marked mitochondria repositioning in muscle fibres and we demonstrate that Drp1 is involved in this process. Drp1 binds KLC1 and enhances microtubule-dependent transport of mitochondria. Drp1-KLC1 coupling triggers the displacement of KIF5B from kinesin-1 complex increasing its binding to microtubule tracks and mitochondrial transport. High levels of Drp1 exacerbate this mechanism leading to the repositioning of mitochondria closer to nuclei. The reduction of Drp1 levels decreases kinesin-1 activation and induces the partial recovery of mitochondrial distribution. Drp1 overexpression is also associated with higher cyclin-dependent kinase-1 (Cdk-1) activation that promotes the persistent phosphorylation of desmin at Ser-31 and its disassembling. Fission inhibition has a positive effect on desmin Ser-31 phosphorylation, regardless of Cdk-1 activation, suggesting that induction of both fission and Cdk-1 are required for desmin collapse. This altered desmin architecture impairs mechanotransduction and compromises mitochondrial network stability priming mitochondria transport through microtubule-dependent trafficking with a mechanism that involves the Drp1-dependent regulation of kinesin-1 complex

Bioluminescence imaging of estrogen receptor activity during breast cancer progression

Estrogen receptors (ER) are known to play an important regulatory role in mammary gland development as well as in its neoplastic transformation. Although several studies highlighted the contribution of ER signaling in the breast transformation, little is known about the dynamics of ER state of activity during carcinogenesis due to the lack of appropriate models for measuring the extent of receptor signaling in time, in the same animal. To this aim, we have developed a reporter mouse model for the non-invasive in vivo imaging of ER activity: the ERE-Luc reporter mouse. ERE-Luc is a transgenic mouse generated with a firefly luciferase (Luc) reporter gene driven by a minimal promoter containing an estrogen responsive element (ERE). This model allows to measure receptor signaling in longitudinal studies by bioluminescence imaging (BLI). Here, we have induced sporadic mammary cancers by treating systemically ERE-Luc reporter mice with DMBA (9,10-dimethyl 1,2-benzanthracene) and measured receptor signaling by in vivo imaging in individual animals from early stage until a clinically palpable tumor appeared in the mouse breast. We showed that DMBA administration induces an increase of bioluminescence in the whole abdominal area 6 h after treatment, the signal rapidly disappears. Several weeks later, strong bioluminescence is observed in the area corresponding to the mammary glands. In vivo and ex vivo imaging analysis demonstrated that this bioluminescent signal is localized in the breast area undergoing neoplastic transformation. We conclude that this non-invasive assay is a novel relevant tool to identify the activation of the ER signaling prior the morphological detection of the neoplastic transformation

Cell cycle dependent oscillatory expression of estrogen receptor-α links Pol II elongation to neoplastic transformation

Decades of studies provided a detailed view of the mechanism of estrogen receptor-\u3b1 (ER\u3b1) regulated gene transcription and the physio-pathological relevance of the genetic programs controlled by this receptor in a variety of tissues. However, still limited is our knowledge on the regulation of ER\u3b1 synthesis. Preliminary observations showed that the expression of ER\u3b1 is cell cycle regulated. Here, we have demonstrated that a well described polymorphic sequence in the first intron of ER\u3b1 (PvuII and XbaI) has a key role in regulating the ER\u3b1 content in cycling cells. We have shown that the RNA Pol II (Pol II) elongation is blocked at the polymorphic site and that the proto-oncogene c-MYB modulates the release of the pausing polymerase. It is well known that the two SNPs are associated to an increased risk, progression, survival and mortality of endocrine-related cancers, here we have demonstrated that the c-MYB-dependent release of Pol II at a specific phase of the cell cycle is facilitated by the px haplotype, thus leading to a higher ER\u3b1 mitogenic signal. In breast cancer, this mechanism is disrupted when the hormone refractory phenotype is established; therefore, we propose this oscillator as a novel target for the development of therapies aimed at sensitizing breast cancer resistant to hormonal treatments. Because PvuII and XbaI were associated to a broad range physio-pathological conditions beside neoplastic transformation, we expect that the ER\u3b1 oscillator contributes to the regulation of the estrogen signal in several tissues

Essential role for acid sphingomyelinase-inhibited autophagy in melanoma response to cisplatin

The sphingolipid metabolising enzyme Acid Sphingomyelinase (A-SMase) has been recently shown to inhibit melanoma progression and correlate inversely to tumour grade. In this study we have investigated the role of A-SMase in the chemo-resistance to anticancer treatmentusing mice with melanoma allografts and melanoma cells differing in terms of expression/activity of A-SMase. Since autophagy is emerging as a key mechanism in tumour growth and chemo-resistance, we have also investigated whether an action of A-SMase in autophagy can explain its role. Melanoma sensitivity to chemotherapeutic agent cisplatin in terms of cell viability/apoptosis, tumour growth, and animal survival depended directly on the A-SMase levels in tumoural cells. A-SMase action was due to inhibition of autophagy through activation of Akt/mammalian target of rapamycin (mTOR) pathway. Treatment of melanoma-bearing mice with the autophagy inhibitor chloroquine restored sensitivity to cisplatin of tumours expressing low levels of A-SMase while no additive effects were observed in tumours characterised by sustained A-SMase levels. The fact that A-SMase in melanomas affects mTOR-regulated autophagy and plays a central role in cisplatin efficacy encourages pre-clinical testing on the modulation of A-SMase levels/activity as possible novel anti-neoplastic strategy

Local Knockdown of ERK2 in the Adult Mouse Brain Via Adeno-Associated Virus-Mediated RNA Interference

In recent years RNA interference (RNAi) has become a useful genetic tool to downregulate candidate disease genes for which pharmaceutical inhibitors are not available. In combination with viral vectors to trigger RNAi in the mammalian body, it allows the localized and specific manipulation of the expression of single or multiple genes in vivo. The MAP kinases ERK1 and ERK2 are involved in the transduction of extracellular signals to nuclear effectors. A role for ERKs has been proposed in the adult brain in mediating neuronal functions, as for fear learning in the lateral amygdala. To study the role of ERK in anxiety disorders characterized by disturbed fear learning processes we developed Erk-specific RNAi tools and tested the efficacy of a viral Erk2 vector in the adult mouse brain. We found shRNAs that showed silencing of either both ERK1/2 or only ERK2. In particular, our analysis showed that an Erk2-specific shRNA reduced the activity of this gene at comparable efficiency both in vitro and in vivo. This reagent provides a useful tool to study the role of ERK2, for which small molecule inhibitors are not available, in the development of anxiety and other psychiatric disorders

Inhibition of SIRT1 deacetylase and p53 activation uncouples the anti-inflammatory and chemopreventive actions of NSAIDs

Background: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed as chemopreventive agents for many tumours; however, the mechanism responsible for their anti-neoplastic activity remains elusive and the side effects due to cyclooxygenase (COX) inhibition prevent this clinical application. Methods: Molecular biology, in silico, cellular and in vivo tools, including innovative in vivo imaging and classical biochemical assays, were applied to identify and characterise the COX-independent anti-cancer mechanism of NSAIDs. Results: Here, we show that tumour-protective functions of NSAIDs and exisulind (a sulindac metabolite lacking anti-inflammatory activity) occur through a COX-independent mechanism. We demonstrate these NSAIDs counteract carcinogen-induced proliferation by inhibiting the sirtuin 1 (SIRT1) deacetylase activity, augmenting acetylation and activity of the tumour suppressor p53 and increasing the expression of the antiproliferative gene p21. These properties are shared by all NSAIDs except for ketoprofen lacking anti-cancer properties. The clinical interest of the mechanism identified is underlined by our finding that p53 is activated in mastectomy patients undergoing intraoperative ketorolac, a treatment associated with decreased relapse risk and increased survival. Conclusion: Our study, for the first-time, links NSAID chemopreventive activity with direct SIRT1 inhibition and activation of the p53/p21 anti-oncogenic pathway, suggesting a novel strategy for the design of tumour-protective drugs

P19 H-Ras Induces G1/S Phase Delay Maintaining Cells in a Reversible Quiescence State

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: Three functional c-ras genes, known as c-H-ras, c-K-ras, and c-N-ras, have been largely studied in mammalian cells with important insights into normal and tumorigenic cellular signal transduction events. Two K-Ras mRNAs are obtained from the same pre-mRNA by alternative splicing. H-Ras pre-mRNA can also be alternatively spliced in the IDX and 4A terminal exons, yielding the p19 and p21 proteins, respectively. However, despite the Ras gene family’s established role in tumorigenic cellular signal transduction events, little is known about p19 function. Previous results showed that p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multi-protein complexes in different signaling pathways (Cancer Res 2003, 63 p5178). This observation suggests that p19 and p21 play differential and complementary roles in the cell.[Principal Findings]: We found that p19 regulates telomerase activity through its interaction with p73a/b proteins. We also found that p19 overexpression induces G1/S phase delay; an observation that correlates with hypophosphorylation of both Akt and p70SK6. Similarly, we also observed that FOXO1 is upregulated when p19 is overexpressed. The three observations of (1) hypophosphorylation of Akt, (2) G1/S phase delay and (3) upregulation of FOXO1 lead us to conclude that p19 induces G1/S phase delay, thereby maintaining cells in a reversible quiescence state and preventing entry into apoptosis. We then assessed the effect of p19 RNAi on HeLa cell growth and found that p19 RNAi increases cell growth, thereby having the opposite effect of arrest of the G1/S phase or producing a cellular quiescence state.[Significance]: Interestingly, p19 induces FOXO1 that in combination with the G1/S phase delay and hypophosphorylation of both Akt and p70SK6 leads to maintenance of a reversible cellular quiescence state, thereby preventing entry into apoptosis.This work was supported by Fundacion de Investigacion Medica Mutua Madrileña Automovilista (Fundacion MMA), the Plan Nacional (MEC) BFU2005-00701 and the Fundacion Eugenio Rodriguez Pascual. M.C. was a recipient of a Fmed MMA fellowship.Peer reviewe

The N-Terminal Domain of ERK1 Accounts for the Functional Differences with ERK2

The Extracellular Regulated Kinase 1 and 2 transduce a variety of extracellular stimuli regulating processes as diverse as proliferation, differentiation and synaptic plasticity. Once activated in the cytoplasm, ERK1 and ERK2 translocate into the nucleus and interact with nuclear substrates to induce specific programs of gene expression. ERK1/2 share 85% of aminoacid identity and all known functional domains and thence they have been considered functionally equivalent until recent studies found that the ablation of either ERK1 or ERK2 causes dramatically different phenotypes. To search a molecular justification of this dichotomy we investigated whether the different functions of ERK1 and 2 might depend on the properties of their cytoplasmic-nuclear trafficking. Since in the nucleus ERK1/2 is predominantly inactivated, the maintenance of a constant level of nuclear activity requires continuous shuttling of activated protein from the cytoplasm. For this reason, different nuclear-cytoplasmic trafficking of ERK1 and 2 would cause a differential signalling capability. We have characterised the trafficking of fluorescently tagged ERK1 and ERK2 by means of time-lapse imaging in living cells. Surprisingly, we found that ERK1 shuttles between the nucleus and cytoplasm at a much slower rate than ERK2. This difference is caused by a domain of ERK1 located at its N-terminus since the progressive deletion of these residues converted the shuttling features of ERK1 into those of ERK2. Conversely, the fusion of this ERK1 sequence at the N-terminus of ERK2 slowed down its shuttling to a similar value found for ERK1. Finally, computational, biochemical and cellular studies indicated that the reduced nuclear shuttling of ERK1 causes a strong reduction of its nuclear phosphorylation compared to ERK2, leading to a reduced capability of ERK1 to carry proliferative signals to the nucleus. This mechanism significantly contributes to the differential ability of ERK1 and 2 to generate an overall signalling output

Effects of estrogens and bladder inflammation on mitogen-activated protein kinases in lumbosacral dorsal root ganglia from adult female rats

BACKGROUND: Interstitial cystitis is a chronic condition associated with bladder inflammation and, like a number of other chronic pain states, symptoms associated with interstitial cystitis are more common in females and fluctuate during the menstrual cycle. The aim of this study was to determine if estrogens could directly modulate signalling pathways within bladder sensory neurons, such as extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases. These signalling pathways have been implicated in neuronal plasticity underlying development of inflammatory somatic pain but have not been as extensively investigated in visceral nociceptors. We have focused on lumbosacral dorsal root ganglion (DRG) neurons projecting to pelvic viscera (L1, L2, L6, S1) of adult female Sprague-Dawley rats and performed both in vitro and in vivo manipulations to compare the effects of short- and long-term changes in estrogen levels on MAPK expression and activation. We have also investigated if prolonged estrogen deprivation influences the effects of lower urinary tract inflammation on MAPK signalling. RESULTS: In studies of isolated DRG neurons in short-term (overnight) culture, we found that estradiol and estrogen receptor (ER) agonists rapidly stimulated ER-dependent p38 phosphorylation relative to total p38. Examination of DRGs following chronic estrogen deprivation in vivo (ovariectomy) showed a parallel increase in total and phosphorylated p38 (relative to beta-tubulin). We also observed an increase in ERK1 phosphorylation (relative to total ERK1), but no change in ERK1 expression (relative to beta-tubulin). We observed no change in ERK2 expression or phosphorylation. Although ovariectomy increased the level of phosphorylated ERK1 (vs. total ERK1), cyclophosphamide-induced lower urinary tract inflammation did not cause a net increase of either ERK1 or ERK2, or their phosphorylation. Inflammation did, however, cause an increase in p38 protein levels, relative to beta-tubulin. Prior ovariectomy did not alter the response to inflammation. CONCLUSIONS: These results provide new insights into the complex effects of estrogens on bladder nociceptor signalling. The diversity of estrogen actions in these ganglia raises the possibility of developing new ways to modulate their function in pelvic hyperactivity or pain states