Publications

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Salvioni, L;Zuppone, S;Andreata, F;Monieri, M;Mazzucchelli, S;Di Carlo, C;Morelli, L;Cordiglieri, C;Donnici, L;De Francesco, R;Corsi, F;Prosperi, D;Vago, R;Colombo, M.
Adv Therap,
3
(8)
, 2000007
(2020)
Systemic chemotherapy has not significantly reduced clinical demand for triple negative breast cancer (TNBC) treatments. To address the need for more effective therapy, the use of nonviral nanoparticles is explored to deliver suicide gene therapy as valuable alternative to protect nucleic acids in the bloodstream and improve their tumor uptake. Biocompatible cationic lipid nanoparticles are developed as a novel delivery system of a suicide plasmid gene encoding saporin. Active targeting is accomplished by taking advantage of nanoparticle functionalization with U11 peptide, designed to be directed toward urokinase plasminogen activator receptor, limiting off?target toxicity. The antitumor effect of U11?lipid?protamine?DNA (U11?LPD) nanoparticles are tested in TBNC cells, showing a strong prevalence of targeted versus nontargeted nanoparticles in terms of uptake kinetics and proliferation inhibition. Transfection of green fluorescent protein (GFP) plasmid in MDA?MB?231 cells is demonstrated. U11?LPD nanoparticles administered by retro bulbar injection exhibit excellent tumor tropism in TNBC orthotopic xenograft mice and effectively transfect TNBC cells with saporin plasmid resulting in tumor mass reduction. No systemic toxicity or organ damage is discovered after repeated treatments with nanoparticles. The findings suggest that systemic administration of targeted LPD nanoparticles to deliver saporin safely allows for active inhibition of cancer progression even in the absence of specific promoter gene sequences.
Holmgren, M;Ravicz, M;Hancock, K;Strelkova, O;Indzhykulian, A;Warchol, M;Sheets, L.
bioRxiv,
(2020)
Noise exposure damages sensory hair cells, resulting in loss of synaptic connections with auditory nerves and hair-cell death. The cellular mechanisms underlying noise-induced hair-cell damage and subsequent repair are not completely understood. Hair cells in neuromasts (NMs) of larval zebrafish are structurally and functionally comparable to mammalian hair cells but undergo robust regeneration following damage. We therefore developed a model for noise-induced hair-cell damage in this highly tractable system. Free swimming larvae exposed to strong water current for 2 hours displayed damage to NMs, including synapse loss, afferent neurite retraction, damaged hair bundles, and reduced mechanotransduction. Overstimulation also elicited an inflammatory response and macrophage recruitment. Remarkably, NM morphology and function appeared to fully recover within 2 days following exposure. Our results reveal morphological and functional changes in mechanically overstimulated lateral-line NMs that are analogous to changes observed in noise-exposed mammalian ear yet are rapidly and completely repaired.
Sun, Y;Chen, X;Fischer, S;Lu, S;Gillis, J;Zador, A.
bioRxiv,
(2020)
Functional circuits consist of neurons with diverse axonal projections and gene expression. Understanding the molecular signature of projections requires high-throughput interrogation of both gene expression and projections to multiple targets in the same cells at cellular resolution, which is difficult to achieve using current technology. Here, we introduce BARseq2, a technique that simultaneously maps projections and detects multiplexed gene expression by in situ sequencing. We determined the expression of cadherins and cell-type markers in 29,933 cells, and the projections of 3,164 cells in both the mouse motor cortex and auditory cortex. Associating gene expression and projections in 1,349 neurons revealed shared cadherin signatures of homologous projections across the two cortical areas. These cadherins were enriched across multiple branches of the transcriptomic taxonomy. By correlating multi-gene expression and projections to many targets in single neurons with high throughput, BARseq2 provides a path to uncovering the molecular logic underlying neuronal circuits.
Limatola, N;Chun, JT;Santella, L.
Biol Bull,
239
(1)
, 13-23
(2020)
Fertilization and early development are usually the most vulnerable stages in the life of marine animals, and the biological processes during this period are highly sensitive to the environment. In nature, sea urchin gametes are shed in seawater, where they undergo external fertilization and embryonic development. In a laboratory, it is possible to follow the exact morphological and biochemical changes taking place in the fertilized eggs and the developing embryos. Thus, observation of successful fertilization and the subsequent embryonic development of sea urchin eggs can be used as a convenient biosensor to assess the quality of the marine environment. In this paper, we have examined how salinity and pH changes affect the normal fertilization process and the following development of Paracentrotus lividus. The results of our studies using confocal microscopy, scanning and transmission electron microscopy, and time-lapse Ca2+ image recording indicated that both dilution and acidification of seawater have subtle but detrimental effects on many aspects of the fertilization process. They include Ca2+ signaling and coordinated actin cytoskeletal changes, leading to a significantly reduced rate of successful fertilization and, eventually, to abnormal or delayed embryonic development.
Hudson, A;Loughran, G;Szabo, N;Wills, N;Atkins, J;Cooley, L.
bioRxiv,
(2020)
Stop codon readthrough during translation occurs in many eukaryotes, including Drosophila, yeast, and humans. Recoding of UGA, UAG or UAA to specify an amino acid allows the ribosome to synthesize C-terminally extended proteins. We previously found evidence for tissue-specific regulation of stop codon readthrough in decoding the Drosophila kelch gene, whose first open reading frame (ORF1) encodes a subunit of a Cullin3-RING ubiquitin ligase. Here, we show that the efficiency of kelch readthrough varies markedly by tissue. Immunoblotting for Kelch ORF1 protein revealed high levels of the readthrough product in lysates of larval and adult central nervous system (CNS) tissue and larval imaginal discs. A sensitive reporter of kelch readthrough inserted after the second kelch open reading frame (ORF2) directly detected synthesis of Kelch readthrough product in these tissues. To analyze the role of cis-acting sequences in regulating kelch readthrough, we used cDNA reporters to measure readthrough in both transfected human cells and transgenic Drosophila. Results from a truncation series suggest that a predicted mRNA stem-loop 3’ of the ORF1 stop codon stimulates high-efficiency readthrough. Expression of cDNA reporters using cell type-specific Gal4 drivers revealed that CNS readthrough is restricted to neurons. Finally, we show that high-effficiency readthrough in the CNS is common in Drosophila, raising the possibility that the neuronal proteome includes many proteins with conserved C-terminal extensions. This work provides new evidence for a remarkable degree of tissue- and cell-specific dynamic stop codon redefinition in Drosophila.
Moreno-Andrés, D;Yokoyama, H;Scheufen, A;Holzer, G;Lue, H;Schellhaus, AK;Weberruss, M;Takagi, M;Antonin, W.
Cells,
9
(7)
, 1702
(2020)
The eukaryotic nucleus remodels extensively during mitosis. Upon mitotic entry, the nuclear envelope breaks down and chromosomes condense into rod-shaped bodies, which are captured by the spindle apparatus and segregated during anaphase. Through telophase, chromosomes decondense and the nuclear envelope reassembles, leading to a functional interphase nucleus. While the molecular processes occurring in early mitosis are intensively investigated, our knowledge about molecular mechanisms of nuclear reassembly is rather limited. Using cell free and cellular assays, we identify the histone variant H2A.Z and its chaperone VPS72/YL1 as important factors for reassembly of a functional nucleus after mitosis. Live-cell imaging shows that siRNA-mediated downregulation of VPS72 extends the telophase in HeLa cells. In vitro, depletion of VPS72 or H2A.Z results in malformed and nonfunctional nuclei. VPS72 is part of two chromatin-remodeling complexes, SRCAP and EP400. Dissecting the mechanism of nuclear reformation using cell-free assays, we, however, show that VPS72 functions outside of the SRCAP and EP400 remodeling complexes to deposit H2A.Z, which in turn is crucial for formation of a functional nucleus.
Chandrakar, P;Varghese, M;Aghvami, S;Baskaran, A;Dogic, Z;Duclos, G.
Physical Review Letters,
125
(25)
(2020)
Spontaneous growth of long-wavelength deformations is a defining feature of active liquid crystals. We investigate the effect of confinement on the instability of 3D active liquid crystals in the isotropic phase composed of extensile microtubule bundles and kinesin molecular motors. When shear aligned, such fluids exhibit finite-wavelength self-amplifying bend deformations. By systematically changing the channel size we elucidate how the instability wavelength and its growth rate depend on the channel dimensions. Experimental findings are qualitatively consistent with a minimal hydrodynamic model, where the fastest growing deformation is set by a balance of active driving and elastic relaxation. Our results demonstrate that confinement determines the structure and dynamics of active fluids on all experimentally accessible length scales.
Swoger, M;Gupta, S;Charrier, E;Bates, M;Hehnly, H;Patteson, A.
bioRxiv,
(2020)
The ability of cells to take and change shape is a fundamental feature underlying development, wound repair, and tissue maintenance. Central to this process is physical and signaling interactions between the three cytoskeletal polymeric networks: F-actin, microtubules, and intermediate filaments (IFs). Vimentin is an IF protein that is essential to the mechanical resilience of cells and regulates cross-talk amongst the cytoskeleton, but its role in how cells sense and respond to the surrounding extracellular matrix is largely unclear. To investigate vimentin’s role in substrate sensing, we designed polyacrylamide hydrogels that mimic the elastic and viscoelastic nature of in vivo tissues. Using wild-type and vimentin-null mouse embryonic fibroblasts, we show that vimentin enhances cell spreading on viscoelastic substrates, even though it has little effect in the limit of purely elastic substrates. Our results provide compelling evidence that the vimentin cytoskeletal network is a physical modulator of how cells sense and respond to mechanical properties of their extracellular environment.
Keith, S;Bishop, C;Fallacaro, S;McCartney, B.
bioRxiv,
(2020)
Perturbations to animal-associated microbial communities (the microbiota) have deleterious effects on various aspects of host fitness, including dysregulated energy metabolism. However, the molecular processes underlying these microbial impacts on the host are poorly understood. In this study, we identify a novel connection between the microbiota and the neuronal factor Arc1 that affects metabolism and development in Drosophila. We find that Arc1 exhibits tissue-specific microbiota-dependent expression changes, and that flies bearing a null mutation of Arc1 complete larval development at a dramatically slowed rate compared to wild-type animals. In contrast, monoassociation with a single Acetobacter sp. isolate was sufficient to enable Arc1 mutants to develop at a wild-type rate. These developmental phenotypes are highly sensitive to composition of the larval diet, suggesting the growth rate defects of GF flies lacking Arc1 reflect metabolic dysregulation. Additionally, we show that pre-conditioning the larval diet with Acetobacter sp. partially accelerates Arc1 mutant development, but live bacteria are required for the full growth rate promoting effect. Finally, GF Arc1 mutants display multiple traits consistent with reduced insulin signaling activity that are reverted by association with Acetobacter sp., suggesting a potential mechanism underlying the microbe-dependent developmental phenotypes. Our results reveal a novel role for Arc1 in modulating insulin signaling, metabolic homeostasis, and growth rate that is specific to the host’s microbial and nutritional environment.SUMMARYDrosophila Arc1 exhibits microbiota-dependent, tissue-specific differential expression, mitigates the impacts of germ-free rearing on insulin signaling and growth rate, but is dispensable for metabolic homeostasis in Acetobacter-colonized flies.
González, G;Baruffaldi, D;Martinengo, C;Angelini, A;Chiappone, A;Roppolo, I;Pirri, CF;Frascella, F.
Nanomaterials (Basel),
10
(9)
, 1788
(2020)
Light-based 3D printing techniques could be a valuable instrument in the development of customized and affordable biomedical devices, basically for high precision and high flexibility in terms of materials of these technologies. However, more studies related to the biocompatibility of the printed objects are required to expand the use of these techniques in the health sector. In this work, 3D printed polymeric parts are produced in lab conditions using a commercial Digital Light Processing (DLP) 3D printer and then successfully tested to fabricate components suitable for biological studies. For this purpose, different 3D printable formulations based on commercially available resins are compared. The biocompatibility of the 3D printed objects toward A549 cell line is investigated by adjusting the composition of the resins and optimizing post-printing protocols; those include washing in common solvents and UV post-curing treatments for removing unreacted and cytotoxic products. It is noteworthy that not only the selection of suitable materials but also the development of an adequate post-printing protocol is necessary for the development of biocompatible devices.
Seabright, AP;Fine, NHF;Barlow, JP;Lord, SO;Musa, I;Gray, A;Bryant, JA;Banzhaf, M;Lavery, GG;Hardie, DG;Hodson, DJ;Philp, A;Lai, YC.
FASEB J,
34
(5)
, 6284-6301
(2020)
Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.
Joshi, K;Matlack, T;Pyonteck, S;Menzel, R;Rongo, C.
bioRxiv,
(2020)
Multicellular organisms use multiple pathways to restore protein homeostasis (proteostasis) in response to adverse physiological conditions, changing environment, and developmental aging. The nervous system can regulate proteostasis in different tissues, but it is unclear how it mobilizes proteostasis pathways to offset physiological decline. Here we show that C. elegans employs the humoral biogenic amine neurotransmitters dopamine, serotonin, and tyramine to regulate proteostasis and the activity of the Ubiquitin Proteasome System (UPS) in epithelial tissues. Mutants for biogenic amine synthesis show decreased poly-ubiquitination and turnover of a GFP-based UPS substrate. Using RNA-seq, we determined the expression profile of genes regulated by biogenic amine signaling. We find that biogenic amines promote the expression of a subset of cytochrome P450 monooxygenases involved in eicosanoid production from polyunsaturated fatty acids (PUFAs). Mutants for these P450s share the same UPS phenotype observed in biogenic amine mutants. The production of n-3 PUFAs is required for UPS substrate turnover, whereas mutants that accumulate n-3 PUFAs show accelerated turnover of this GFP-based substrate. Our results suggest that neurosecretory sensory neurons release biogenic amines to modulate the lipid signaling profile, which in turn activates stress response pathways to maintain proteostasis.
Horspool, AM;Wang, T;Scaringella, YS;Taub, ME;Chan, TS.
Drug Metab Dispos,
48
(8)
, 645-654
(2020)
Human liver microsomes (HLM) are a commonly used tool to study drug metabolism in vitro. Typical experiments conducted using suspensions of HLM can be challenging to separate from the incubation solution without lengthy ultracentrifugation steps. Magnetizable beads coated with silica (MGBS) were found to bind strongly to HLM, which could then be isolated and purified using a magnet. Binding of HLM to the MGBS (HLM-MGBS) was demonstrated to be mediated by strong interactions between microsomal phospholipids and MGBS, as artificially prepared phosphatidylcholine (PC) liposomes could be more efficiently captured by the MGBS. HLM-MGBS complexes retained functional cytochrome P450 and uridine-diphosphate-glucuronosyltransferase (UGT) activity as indicated by CYP2C8-mediated amodiaquine de-ethylation, CYP3A4-mediated midazolam 1’hydroxylation, UGT1A1-mediated glucuronidation of estradiol, UGT1A9-mediated glucuronidation of propofol, and UGT2B7-mediated glucuronidation of zidovudine. When comparing suspension HLM alone with HLM-MGBS complexes containing equivalent amounts of HLM, the intrinsic clearance (CLint) of CYP450 substrates was comparable; however, CLint of UGT1A1, UGT1A9, and UGT2B7 was increased in the HLM-MGBS system between 1.5- and 6-fold. HLM-MGBS used in an incubation could also be readily replaced with fresh HLM-MGBS to maintain the presence of active enzymes. Thus, HLM-MGBS demonstrate increased in vitro metabolic efficiency and manipulability, providing a new platform for determination of accurate metabolic parameters. SIGNIFICANCE STATEMENT: The following work describes the strong binding of HLM to magnetizable beads. In addition, the preservation of enzyme activity on the bound HLM provides a novel means to conduct preclinical metabolism studies.
Arnold, ML;Cooper, J;Grant, BD;Driscoll, M.
J Vis Exp,
(163)
(2020)
Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease. C. elegans adult neurons that express aggregating proteins can extrude large (~4 µm) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called “exophers” and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases. While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons. Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.
De, A;Beligala, DH;Sharma, VP;Burgos, CA;Lee, AM;Geusz, ME.
Clin Exp Metastasis,
37
(5)
, 617-635
(2020)
Epithelial-mesenchymal transition (EMT) is a key event preceding tumor cell metastasis that increases cell invasiveness and cancer stem cell (CSC) populations. Studies suggest that genes used in generating circadian rhythms also serve in regulating EMT. To test the role of circadian clocks in cellular EMT events two cancer cell lines were compared, one that has a well-established circadian clock, C6 from rat glioma, and one that does not, MCF-7 from human breast tumor. MCF-7 tumorsphere cultures were tested for evidence of circadian rhythms because of previously reported circadian rhythm enhancement in C6 tumorspheres shown by elevated rhythm amplitude and increased expression of circadian clock gene Per2. Bioluminescence imaging of Per2 gene expression in MCF-7 tumorspheres revealed a previously unconfirmed circadian clock in this important cancer research model. Inducing CSC generation through EMT in C6 and MCF-7 monolayer cultures revealed circadian oscillations in the size of the post-EMT CSC population, confirming that circadian rhythms are additional processes controlling this stage of cancer progression. EMT was verified by distinct cellular morphological changes and expression of stem cell proteins OCT4, nestin, MSI1, and CD133 along with EMT-related proteins ZEB1, vimentin, and TWIST. Quantifying single-cell events and behaviors through time-lapse imaging indicated the post-EMT population size was determined largely by circadian rhythms in epithelial-like cancer cells undergoing EMT. We then identified a specific phase of the circadian rhythm in Per2 gene activation as a potential target for therapeutic treatments that may suppress EMT, minimize CSCs, and limit metastasis.
Sujkowski, A;Gretzinger, A;Soave, N;Todi, SV;Wessells, R.
PLoS Genet,
16
(6)
, e1008778
(2020)
Endurance exercise has broadly protective effects across organisms, increasing metabolic fitness and reducing incidence of several age-related diseases. Drosophila has emerged as a useful model for studying changes induced by chronic endurance exercise, as exercising flies experience improvements to various aspects of fitness at the cellular, organ and organismal level. The activity of octopaminergic neurons is sufficient to induce the conserved cellular and physiological changes seen following endurance training. All 4 octopamine receptors are required in at least one target tissue, but only one, Oct?1R, is required for all of them. Here, we perform tissue- and adult-specific knockdown of alpha- and beta-adrenergic octopamine receptors in several target tissues. We find that reduced expression of Oct?1R in adult muscles abolishes exercise-induced improvements in endurance, climbing speed, flight, cardiac performance and fat-body catabolism in male Drosophila. Importantly, Oct?1R and OAMB expression in the heart is also required cell-nonautonomously for adaptations in other tissues, such as skeletal muscles in legs and adult fat body. These findings indicate that activation of distinct octopamine receptors in skeletal and cardiac muscle are required for Drosophila exercise adaptations, and suggest that cell non-autonomous factors downstream of octopaminergic activation play a key role.
Angelini, G;Castagneto-Gissey, L;Casella-Mariolo, J;Caristo, ME;Russo, MF;Lembo, E;Verrastro, O;Stefanizzi, G;Marini, PL;Casella, G;Bornstein, SR;Rubino, F;Mingrone, G.
Am J Physiol Gastrointest Liver Physiol,
319
(4)
, G502-G511
(2020)
Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver-related mortality. NAFLD is associated with obesity, hepatic fat accumulation, and insulin resistance, all of which contribute to its pathophysiology. Weight-loss is the main therapy for NAFLD, and metabolic surgery is the most effective treatment for morbid obesity and its metabolic comorbidities. Although has been reported that Roux-en-Y gastric bypass can reverse NAFLD, it is unclear whether such effects result from reduced weight, from a lower calorie-intake, or from the direct influence of surgery on mechanisms contributing to NAFLD. We aimed to investigate whether gastrointestinal (GI) bypass surgery could induce direct effects on hepatic fat accumulation and insulin resistance, independently of weight reduction. Twenty Wistar rats on a high-fat diet underwent duodenal-jejunal-bypass (DJB) or sham operation and were pair fed (PF) for 15 wk after surgery to obtain a matched weight. Outcome measures include ectopic fat deposition, expression of genes and proteins involved in fat metabolism, insulin-signaling, and gluconeogenesis in liver and muscle. Despite no differences in body weight and calorie intake, DJB showed lower ectopic fat accumulation, improved peripheral and hepatic insulin sensitivity, and enhanced lipid droplet degradation. In both tissues, DJB increased insulin signaling, whereas hepatic key enzymes involved in gluconeogenesis and de novo lipogenesis were decreased. These findings suggest that DJB can reverse, independently of weight loss, ectopic fat deposition and insulin resistance, two features of NAFLD that share a mutual pathway, in which perilipin-2 (PLIN2) seems to be the main player, supporting further investigation into strategies that target the gut to treat metabolic liver diseases.NEW & NOTEWORTHY Our findings suggest that duodenal-jejunal bypass can reverse, independently of weight loss, ectopic fat deposition and insulin resistance, two features of nonalcoholic fatty liver disease that share a mutual pathway, in which perilipin-2 seems to be the main player. Our study supports further investigation into the role of proximal small intestine exclusion in the pathophysiology of nonalcoholic fatty liver disease to uncover less invasive treatments that mimic the effects of metabolic surgery and aims to prevent and treat metabolic liver disease.
Viloria, K;Nasteska, D;Briant, LJB;Heising, S;Larner, DP;Fine, NHF;Ashford, FB;da Silva Xavier, G;Ramos, MJ;Hasib, A;Cuozzo, F;Manning Fox, JE;MacDonald, PE;Akerman, I;Lavery, GG;Flaxman, C;Morgan, NG;Richardson, SJ;Hewison, M;Hodson, DJ.
Cell Rep,
31
(11)
, 107761
(2020)
Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly localized to the liver and pancreatic ? cells. Although DBP serum levels, gene polymorphisms, and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates ? cell morphology, ? cell function, and glucagon secretion. Deletion of DBP leads to smaller and hyperplastic ? cells, altered Na+ channel conductance, impaired ? cell activation by low glucose, and reduced rates of glucagon secretion both in vivo and in vitro. Mechanistically, this involves reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects are also seen in ? cell and ? cell function. Immunostaining of human pancreata reveals generalized loss of DBP expression as a feature of late-onset and long-standing, but not early-onset, type 1 diabetes. Thus, DBP regulates ? cell phenotype, with implications for diabetes pathogenesis.
Wahis, J;Kerspern, D;Althammer, F;Baudon, A;Goyon, S;Hagiwara, D;Lefèvre, A;Boury-Jamot, B;Bellanger, B;Abatis, M;da Gouveia, M;Benusiglio, D;Eliava, M;Rozov, A;Weinsanto, I;Knobloch-Bollmann, H;Wang, H;Pertin, M;Inquimbert, P;Pitzer, C;Siemens, J;Goumon, Y;Boutrel, B;Darbon, P;Lamy, C;Stern, J;Décosterd, I;Chatton, J;Young, W;Stoop, R;Poisbeau, P;Grinevich, V;Charlet, A.
bioRxiv,
(2020)
Oxytocin orchestrates social and emotional behaviors through modulation of neural circuits in brain structures such as the central amygdala (CeA). The long-standing dogma is that oxytocin signaling in the central nervous system occurs exclusively via direct actions on neurons. However, several findings over the last decades showed that astrocytes actively participate in the modulation of neuronal circuits. Here, we investigate the degree of astrocytes’ involvement in oxytocin functions. Using astrocyte’ specific gain and loss of function approaches, we demonstrate that CeA astrocytes not only directly respond to oxytocin, but are actually necessary for its effects on neuronal circuits and ultimately behavior. Our work identifies astrocytes as a crucial cellular substrate underlying the promotion of a positive emotional state by oxytocin. These results further corroborate that astrocytes are key regulators of neuronal circuits activity by responding to specific neuropeptidergic inputs, and opens up new perspectives to understand how neuromodulators gate brain functions.
Fujise, K;Okubo, M;Abe, T;Yamada, H;Nishino, I;Noguchi, S;Takei, K;Takeda, T.
The Journal of biological chemistry,
(2020)
Membrane remodeling is required for dynamic cellular processes such as cell division, polarization and motility. BAR domain proteins and dynamins are key molecules in membrane remodeling that work together for membrane deformation and fission. In striated muscles, sarcolemmal invaginations termed T-tubules are required for excitation-contraction coupling. BIN1 and DNM2, which encode a BAR domain protein BIN1 and dynamin 2, respectively, have been reported to be causative genes of centronuclear myopathy (CNM), a hereditary degenerative disease of skeletal muscle, and deformation of T-tubules is often observed in the CNM patients. However, it remains unclear how BIN1 and dynamin 2 are implicated in T-tubule biogenesis, and how mutations in these molecules cause CNM to develop.Here, using an in cellulo reconstitution assay, we demonstrate that dynamin 2 is required for stabilization of membranous structures equivalent to T-tubules. GTPase activity of wild type dynamin 2 is suppressed through interaction with BIN1, whereas that of the disease-associated mutant dynamin 2 remains active due to lack of the BIN1-mediated regulation thus causing aberrant membrane remodeling. Finally, we show that in cellulo aberrant membrane remodeling by mutant dynamin 2 variants is correlated with their enhanced membrane fission activities, and the results can explain severity of the symptoms in patients. Thus, this study provides molecular insights into dysregulated membrane remodeling triggering the pathogenesis of DNM2-related centronuclear myopathy. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.