RESUMEN
Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons from the substantia nigra (SN) that project to the dorsal striatum (caudate-putamen). To better understand the molecular mechanisms underlying PD, we performed combined lipid profiling and RNA sequencing of SN and putamen samples from PD patients and age-matched controls. SN lipid analysis pointed to a neuroinflammatory component and included elevated levels of the endosomal lipid Bis (Monoacylglycero)Phosphate 42:8, while two of the three depleted putamen lipids were saturated sphingomyelin species. Remarkably, we observed gender-related differences in the SN and putamen lipid profiles. Transcriptome analysis revealed that the top-enriched pathways among the 354 differentially expressed genes (DEGs) in the SN were "protein folding" and "neurotransmitter transport", and among the 261 DEGs from putamen "synapse organization". Furthermore, we identified pathways, e.g., "glutamate signaling", and genes, encoding, e.g., an angiotensin receptor subtype or a proprotein convertase, that have not been previously linked to PD. The identification of 33 genes that were common among the SN and putamen DEGs, which included the α-synuclein paralog ß-synuclein, may contribute to the understanding of general PD mechanisms. Thus, our proof-of-concept data highlights new genes, pathways and lipids that have not been explored before in the context of PD.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Lípidos/sangre , Enfermedad de Parkinson/fisiopatología , Putamen/fisiopatología , Sustancia Negra/fisiopatología , Anciano , Anciano de 80 o más Años , Femenino , Humanos , MasculinoRESUMEN
Parkinson's disease (PD) is a highly prevalent neurodegenerative disease for which no disease-modifying treatments are available, mainly because knowledge about its pathogenic mechanism is still incomplete. Recently, a key role for lipids emerged, but lipid profiling of brain samples from human subjects is demanding. Here, we used an unbiased approach, lipidomics, to determine PD-linked changes in the lipid profile of a well-established cell model for PD, the catecholaminergic neuronal cell line SH-SY5Y treated with the neurotoxin 6-hydroxydopamine (6-OHDA). We observed changes in multiple lipid classes, including phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), and total cholesterol, in 6-OHDA-treated SH-SY5Y cells. Furthermore, we found differences in the length and degree of unsaturation of the fatty acyl chains, indicating changes in their metabolism. Except for the observed decreased PS levels, the alterations in PC, PG, PI, and cholesterol levels are in agreement with the results of previous studies on PD-patient material. Opposite to what has been previously described, the cholesterol-lowering drug statins did not have a protective effect, while low doses of cholesterol supplementation partially protected SH-SY5Y cells from 6-OHDA toxicity. However, cholesterol supplementation triggered neuronal differentiation, which could have confounded the results of cholesterol modulation. Taken together, our results show that 6-OHDA-treated SH-SY5Y cells display many lipid changes also found in PD patient and animal model brains, although the SH-SY5Y cell model seems less suitable to study the involvement of cholesterol in PD initiation and progression.
Asunto(s)
Lípidos/análisis , Modelos Biológicos , Oxidopamina/toxicidad , Enfermedad de Parkinson/metabolismo , Línea Celular Tumoral , Colesterol/metabolismo , Humanos , Lipidómica , Simvastatina/farmacologíaRESUMEN
The congenital form of myotonic dystrophy type 1 (cDM) is caused by the large-scale expansion of a (CTGâ¢CAG)n repeat in DMPK and DM1-AS. The production of toxic transcripts with long trinucleotide tracts from these genes results in impairment of the myogenic differentiation capacity as cDM's most prominent morpho-phenotypic hallmark. In the current in vitro study, we compared the early differentiation programs of isogenic cDM myoblasts with and without a (CTG)2600 repeat obtained by gene editing. We found that excision of the repeat restored the ability of cDM myoblasts to engage in myogenic fusion, preventing the ensuing myotubes from remaining immature. Although the cDM-typical epigenetic status of the DM1 locus and the expression of genes therein were not altered upon removal of the repeat, analyses at the transcriptome and proteome level revealed that early abnormalities in the temporal expression of differentiation regulators, myogenic progression markers, and alternative splicing patterns before and immediately after the onset of differentiation became normalized. Our observation that molecular and cellular features of cDM are reversible in vitro and can be corrected by repeat-directed genome editing in muscle progenitors, when already committed and poised for myogenic differentiation, is important information for the future development of gene therapy for different forms of myotonic dystrophy type 1 (DM1).
Asunto(s)
Mioblastos/patología , Distrofia Miotónica/genética , Repeticiones de Trinucleótidos , Línea Celular , Epigénesis Genética , Edición Génica , Terapia Genética , Humanos , Desarrollo de Músculos , Mioblastos/citología , Mioblastos/metabolismo , Distrofia Miotónica/patología , Distrofia Miotónica/terapia , Proteína Quinasa de Distrofia Miotónica/genéticaRESUMEN
BACKGROUND: Study motivation and knowledge retention benefit from regular student self-assessments. Inclusion of certainty-based learning (CBL) in computer-assisted formative tests may further enhance this by enabling students to identify whether they are uninformed or misinformed regarding the topics tested, which may trigger future study actions including instructor consultation. METHODS: Using a cross-over study design involving two out of thirteen computer-assisted formative assessments (CAFAs) of a first-year cell biology course, we compared student-instructor interactions, student learning experiences and final exam scores between two (bio)medical science student cohorts who worked with different CBL-containing CAFAs. RESULTS: A total of 389 students participated in the study. After completion 159 (41%) filled in a questionnaire on their experience with CBL during supervised CAFAs. In the control group the median duration of student-instructor interactions was 90 s (range 60-140 s), and this increased with 20 s to 110 s (range 60-150 s) in the group working with a CBL-based CAFA. The number of interactions was similar in both groups (0.22 per student per hour, regardless of CBL inclusion). Forty percent of the students expected that CBL would positively influence their study behavior, and 23% also anticipated a positive effect on examination scores. Student examination scores, however, were not affected by CBL. Almost half of the students (43%) were in favor of CBL inclusion in future computer-assisted learning modules, whereas 33% did not see merit in including CBL in CAFAs. CONCLUSIONS: Incorporation of CBL in a single formative assessment led to a slight increase in student-instructor interaction times, but had effect neither on the number of student-instructor interactions nor on exam scores. CBL inclusion positively influenced student's appreciation of the coursework, presumably by helping students to evaluate their mastery level and identify misconceptions. A more extensive enrollment of CBL beyond an individual formative assessment, throughout a course or a curriculum, may possibly reveal positive effects on study efficacy.
Asunto(s)
Evaluación Educacional/métodos , Estudiantes de Medicina/psicología , Adolescente , Instrucción por Computador/métodos , Estudios Cruzados , Femenino , Retroalimentación Formativa , Humanos , Masculino , Adulto JovenRESUMEN
Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disorder caused by expansion of a CTG repeat in the 3'-untranslated region (UTR) of the DMPK gene. Mutant DMPK transcripts form aberrant structures and anomalously associate with RNA-binding proteins (RBPs). As a first step toward better understanding of the involvement of abnormal DMPK mRNA folding in DM1 manifestation, we used SHAPE, DMS, CMCT, and RNase T1 structure probing in vitro for modeling of the topology of the DMPK 3'-UTR with normal and pathogenic repeat lengths of up to 197 CUG triplets. The resulting structural information was validated by disruption of base-pairing with LNA antisense oligonucleotides (AONs) and used for prediction of therapeutic AON accessibility and verification of DMPK knockdown efficacy in cells. Our model for DMPK RNA structure demonstrates that the hairpin formed by the CUG repeat has length-dependent conformational plasticity, with a structure that is guided by and embedded in an otherwise rigid architecture of flanking regions in the DMPK 3'-UTR. Evidence is provided that long CUG repeats may form not only single asymmetrical hairpins but also exist as branched structures. These newly identified structures have implications for DM1 pathogenic mechanisms, like sequestration of RBPs and repeat-associated non-AUG (RAN) translation.
Asunto(s)
Regiones no Traducidas 3' , Secuencias Invertidas Repetidas , Proteína Quinasa de Distrofia Miotónica/genética , Repeticiones de Trinucleótidos , Emparejamiento Base , Expresión Génica , Humanos , Modelos Genéticos , Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , Distrofia Miotónica/patología , Proteína Quinasa de Distrofia Miotónica/metabolismo , Conformación de Ácido Nucleico , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Parkinson's disease (PD) is a neurodegenerative disease characterized by a progressive loss of dopaminergic neurons from the nigrostriatal pathway, formation of Lewy bodies, and microgliosis. During the past decades multiple cellular pathways have been associated with PD pathology (i.e., oxidative stress, endosomal-lysosomal dysfunction, endoplasmic reticulum stress, and immune response), yet disease-modifying treatments are not available. We have recently used genetic data from familial and sporadic cases in an unbiased approach to build a molecular landscape for PD, revealing lipids as central players in this disease. Here we extensively review the current knowledge concerning the involvement of various subclasses of fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and lipoproteins in PD pathogenesis. Our review corroborates a central role for most lipid classes, but the available information is fragmented, not always reproducible, and sometimes differs by sex, age or PD etiology of the patients. This hinders drawing firm conclusions about causal or associative effects of dietary lipids or defects in specific steps of lipid metabolism in PD. Future technological advances in lipidomics and additional systematic studies on lipid species from PD patient material may improve this situation and lead to a better appreciation of the significance of lipids for this devastating disease.
Asunto(s)
Metabolismo de los Lípidos , Enfermedad de Parkinson/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Femenino , Humanos , MasculinoRESUMEN
Myotonic dystrophy type 1 (DM1) and 2 (DM2) are autosomal dominant degenerative neuromuscular disorders characterized by progressive skeletal muscle weakness, atrophy, and myotonia with progeroid features. Although both DM1 and DM2 are characterized by skeletal muscle dysfunction and also share other clinical features, the diseases differ in the muscle groups that are affected. In DM1, distal muscles are mainly affected, whereas in DM2 problems are mostly found in proximal muscles. In addition, manifestation in DM1 is generally more severe, with possible congenital or childhood-onset of disease and prominent CNS involvement. DM1 and DM2 are caused by expansion of (CTGâ¢CAG)n and (CCTGâ¢CAGG)n repeats in the 3' non-coding region of DMPK and in intron 1 of CNBP, respectively, and in overlapping antisense genes. This critical review will focus on the pleiotropic problems that occur during development, growth, regeneration, and aging of skeletal muscle in patients who inherited these expansions. The current best-accepted idea is that most muscle symptoms can be explained by pathomechanistic effects of repeat expansion on RNA-mediated pathways. However, aberrations in DNA replication and transcription of the DM loci or in protein translation and proteome homeostasis could also affect the control of proliferation and differentiation of muscle progenitor cells or the maintenance and physiological integrity of muscle fibers during a patient's lifetime. Here, we will discuss these molecular and cellular processes and summarize current knowledge about the role of embryonic and adult muscle-resident stem cells in growth, homeostasis, regeneration, and premature aging of healthy and diseased muscle tissue. Of particular interest is that also progenitor cells from extramuscular sources, such as pericytes and mesoangioblasts, can participate in myogenic differentiation. We will examine the potential of all these types of cells in the application of regenerative medicine for muscular dystrophies and evaluate new possibilities for their use in future therapy of DM.
RESUMEN
Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of AONs with highest in vitro efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood. Here, we report on the unassisted uptake of naked AONs, so-called gymnosis, in muscle cells in culture. We found that gymnosis works similarly well for proliferating myoblasts as for terminally differentiated myotubes. Cell biological analyses combined with microscopy imaging showed that a phosphorothioate backbone promotes efficient gymnosis, that uptake is clathrin mediated and mainly results in endosomal-lysosomal accumulation. Nuclear localization occurred at a low level, but the gymnotically delivered AONs effectively modulated the expression of their nuclear RNA targets. Chloroquine treatment after gymnotic delivery helped increase nuclear AON levels. In sum, we demonstrate that gymnosis is feasible in proliferating and non-proliferating muscle cells and we confirm the relevance of AON chemistry for uptake and intracellular trafficking with this method, which provides a useful means for bio-activity screening of AONs in vitro.
Asunto(s)
Núcleo Celular/química , Endocitosis , Endosomas/química , Fibras Musculares Esqueléticas/química , Distrofia Muscular de Duchenne/terapia , Oligonucleótidos Antisentido/química , Análisis de Varianza , Animales , Diferenciación Celular , Línea Celular , Proliferación Celular , Cloroquina/farmacología , Clatrina/metabolismo , Humanos , Hidrazonas/farmacología , Ratones , Ratones Transgénicos , Microscopía Confocal , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , ARN/efectos de los fármacos , ARN/genética , ARN/metabolismoRESUMEN
In yeast and higher eukaryotes nuclear retention of transcripts may serve in control over RNA decay, nucleocytoplasmic transport and premature cytoplasmic appearance of mRNAs. Hyperadenylation of RNA is known to be associated with nuclear retention, but the cause-consequence relationship between hyperadenylation and regulation of RNA nuclear export is still unclear. We compared polyadenylation status between normal and expanded DMPK transcripts in muscle cells and tissues derived from unaffected individuals and patients with myotonic dystrophy type 1 (DM1). DM1 is an autosomal dominant disorder caused by (CTG)n repeat expansion in the DMPK gene. DM1 etiology is characterized by an almost complete block of nuclear export of DMPK transcripts carrying a long (CUG)n repeat, including aberrant sequestration of RNA-binding proteins. We show here by use of cell fractionation, RNA size separation and analysis of poly(A) tail length that a considerable fraction of transcripts from the normal DMPK allele is also retained in the nucleus (~30%). They carry poly(A) tails with an unusually broad length distribution, ranging between a few dozen to >500 adenosine residues. Remarkably, expanded DMPK (CUG)n transcripts from the mutant allele, almost exclusively nuclear, carry equally long poly(A) tails. Our findings thus suggest that nuclear retention may be a common feature of regulation of DMPK RNA expression. The typical forced nuclear residence of expanded DMPK transcripts affects this regulation in tissues of DM1 patients, but not through hyperadenylation.
Asunto(s)
Núcleo Celular/metabolismo , Regulación Enzimológica de la Expresión Génica , Proteína Quinasa de Distrofia Miotónica/biosíntesis , Poli A , Expansión de Repetición de Trinucleótido , Alelos , Línea Celular Transformada , Núcleo Celular/genética , Núcleo Celular/patología , Humanos , Distrofia Miotónica/enzimología , Distrofia Miotónica/genética , Distrofia Miotónica/patología , Proteína Quinasa de Distrofia Miotónica/genéticaRESUMEN
Parkinson's disease (PD) is a devastating and highly prevalent neurodegenerative disease for which only symptomatic treatment is available. In order to develop a truly effective disease-modifying therapy, improvement of our current understanding of the molecular and cellular mechanisms underlying PD pathogenesis and progression is crucial. For this purpose, standardization of research protocols and disease models is necessary. As human dopaminergic neurons, the cells mainly affected in PD, are difficult to obtain and maintain as primary cells, current PD research is mostly performed with permanently established neuronal cell models, in particular the neuroblastoma SH-SY5Y lineage. This cell line is frequently chosen because of its human origin, catecholaminergic (though not strictly dopaminergic) neuronal properties, and ease of maintenance. However, there is no consensus on many fundamental aspects that are associated with its use, such as the effects of culture media composition and of variations in differentiation protocols. Here we present the outcome of a systematic review of scientific articles that have used SH-SY5Y cells to explore PD. We describe the cell source, culture conditions, differentiation protocols, methods/approaches used to mimic PD and the preclinical validation of the SH-SY5Y findings by employing alternative cellular and animal models. Thus, this overview may help to standardize the use of the SH-SY5Y cell line in PD research and serve as a future user's guide.
Asunto(s)
Línea Celular Tumoral , Neuronas , Enfermedad de Parkinson , HumanosRESUMEN
The unstable (CTG·CAG)n trinucleotide repeat in the myotonic dystrophy type 1 (DM1) locus is bidirectionally transcribed from genes with terminal overlap. By transcription in the sense direction, the DMPK gene produces various alternatively spliced mRNAs with a (CUG)n repeat in their 3' UTR. Expression in opposite orientation reportedly yields (CAG)n-repeat containing RNA, but both structure and biologic significance of this antisense gene (DM1-AS) are largely unknown. Via a combinatorial approach of computational and experimental analyses of RNA from unaffected individuals and DM1 patients we discovered that DM1-AS spans >6 kb, contains alternative transcription start sites and uses alternative polyadenylation sites up- and downstream of the (CAG)n repeat. Moreover, its primary transcripts undergo alternative splicing, whereby the (CAG)n segment is removed as part of an intron. Thus, in patients a mixture of DM1-AS RNAs with and without expanded (CAG)n repeat are produced. DM1-AS expression appears upregulated in patients, but transcript abundance remains very low in all tissues analyzed. Our data suggest that DM1-AS transcripts belong to the class of long non-coding RNAs. These and other biologically relevant implications for how (CAG)n-expanded transcripts may contribute to DM1 pathology can now be explored experimentally.
Asunto(s)
Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica/genética , ARN sin Sentido/genética , ARN Mensajero/química , Expansión de Repetición de Trinucleótido , Regiones no Traducidas 3' , Adolescente , Empalme Alternativo , Estudios de Casos y Controles , Línea Celular , Biología Computacional/métodos , Humanos , Masculino , Proteína Quinasa de Distrofia Miotónica/química , Sistemas de Lectura Abierta , Poliadenilación , ARN sin Sentido/química , ARN Largo no Codificante/genética , ARN Mensajero/genética , Sitio de Iniciación de la Transcripción , Regulación hacia ArribaRESUMEN
Myotonic dystrophy type 1 (DM1) is caused by (CTGâ CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5' or 3' unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTGâ CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTGâ CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Inestabilidad Genómica , Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica/genética , Expansión de Repetición de Trinucleótido , Repeticiones de Trinucleótidos , Animales , Proteínas Bacterianas/genética , Secuencia de Bases , Proteína 9 Asociada a CRISPR , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Codón , Modelos Animales de Enfermedad , Endonucleasas/genética , Fibroblastos/metabolismo , Expresión Génica , Orden Génico , Sitios Genéticos , Humanos , Ratones , ARN Guía de Kinetoplastida , ARN Mensajero/genética , ARN Mensajero/metabolismo , Eliminación de SecuenciaRESUMEN
Muscular manifestation of myotonic dystrophy type 1 (DM1), a common inheritable degenerative multisystem disorder, is mainly caused by expression of RNA from a (CTG·CAG)n-expanded DM1 locus. Here, we report on comparative profiling of expression of normal and expanded endogenous or transgenic transcripts in skeletal muscle cells and biopsies from DM1 mouse models and patients in order to help us in understanding the role of this RNA-mediated toxicity. In tissue of HSA(LR) mice, the most intensely used 'muscle-only' model in the DM1 field, RNA from the α-actin (CTG)250 transgene was at least 1000-fold more abundant than that from the Dmpk gene, or the DMPK gene in humans. Conversely, the DMPK transgene in another line, DM500/DMSXL mice, was expressed â¼10-fold lower than the endogenous gene. Temporal regulation of expanded RNA expression differed between models. Onset of expression occurred remarkably late in HSA(LR) myoblasts during in vitro myogenesis whereas Dmpk or DMPK (trans)genes were expressed throughout proliferation and differentiation phases. Importantly, quantification of absolute transcript numbers revealed that normal and expanded Dmpk/DMPK transcripts in mouse models and DM1 patients are low-abundance RNA species. Northern blotting, reverse transcriptase-quantitative polymerase chain reaction, RNA-sequencing and fluorescent in situ hybridization analyses showed that they occur at an absolute number between one and a few dozen molecules per cell. Our findings refine the current RNA dominance theory for DM1 pathophysiology, as anomalous factor binding to expanded transcripts and formation of soluble or insoluble ribonucleoprotein aggregates must be nucleated by only few expanded DMPK transcripts and therefore be a small numbers game.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Músculo Esquelético/citología , Distrofia Miotónica/genética , ARN Mensajero/genética , Expansión de Repetición de Trinucleótido , Actinas/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Desarrollo de Músculos , Músculo Esquelético/patología , Distrofia Miotónica/patología , Proteína Quinasa de Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica/metabolismoRESUMEN
Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTGâ¢CAG)n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and target cell membranes. It has been reported that DM1 patients show elevated levels of markers of muscle damage or loss of sarcolemmal integrity in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface expression of dystrophin was normal and calcium-positive fibers, indicating elevated intracellular calcium levels, were only rarely seen. Combined, our findings indicate that cells in DM1 tissues do not display compromised membrane integrity. Hence, the cell membrane is a barrier that must be overcome in future work towards effective drug delivery in DM1 therapy.
Asunto(s)
Permeabilidad de la Membrana Celular , Membrana Celular/metabolismo , Distrofia Miotónica/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Calcio/metabolismo , Niño , Distrofina/genética , Distrofina/metabolismo , Azul de Evans/farmacocinética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Distrofia Miotónica/tratamiento farmacológicoRESUMEN
Subcellular partitioning of creatine kinase contributes to the formation of patterns in intracellular ATP distribution and the fuelling of cellular processes with a high and sudden energy demand. We have previously shown that brain-type creatine kinase (CK-B) accumulates at the phagocytic cup in macrophages where it is involved in the compartmentalized generation of ATP for actin remodeling. Here, we report that CK-B catalytic activity also helps in the formation of protrusive ruffle structures which are actin-dependent and abundant on the surface of both unstimulated and LPS-activated macrophages. Recruitment of CK-B to these structures occurred transiently and inhibition of the enzyme's catalytic activity with cyclocreatine led to a general smoothening of surface morphology as visualized by scanning electron microscopy. Comparison of the dynamics of distribution of YFP-tagged CK-mutants and isoforms by live imaging revealed that amino acid residues in the C-terminal segment (aa positions 323-330) that forms one of the protein's two mobile loops are involved in partitioning over inner regions of the cytosol and nearby sites where membrane protrusions occur during induction of phagocytic cup formation. Although wt CK-B, muscle-type CK (CK-M), and a catalytically dead CK-B-E232Q mutant with intact loop region were normally recruited from the cytosolic pool, no dynamic transition to the phagocytic cup area was seen for the CK-homologue arginine kinase and a CK-B-D326A mutant protein. Bioinformatics analysis helped us to predict that conformational flexibility of the C-terminal loop, independent of conformational changes induced by substrate binding or catalytic activity, is likely involved in exposing the enzyme for binding at or near the sites of membrane protrusion formation.
Asunto(s)
Membrana Celular/metabolismo , Extensiones de la Superficie Celular/metabolismo , Forma BB de la Creatina-Quinasa/metabolismo , Macrófagos/metabolismo , Actinas/metabolismo , Animales , Línea Celular , Extensiones de la Superficie Celular/efectos de los fármacos , Biología Computacional , Creatinina/análogos & derivados , Creatinina/farmacología , Drosophila melanogaster , Inhibidores Enzimáticos/farmacología , Humanos , Macrófagos/ultraestructura , Ratones , Estructura Terciaria de ProteínaRESUMEN
Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H) (i.e. NAD+ and NADH) and NADP(H) (i.e. NADP+ and NADPH) play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD+-supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT), found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD+ in macrophages. Inhibition of NAD+ salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD+ levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD+ induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD+ levels by NAD+, NMN, or NADP+ supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD+'s cytosolic role in the regulation of morphofunctional characteristics of macrophages.
Asunto(s)
Acrilamidas/farmacología , Citoesqueleto de Actina/fisiología , Adenosina Trifosfato/metabolismo , Citocinas/metabolismo , Macrófagos/citología , NAD/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Piperidinas/farmacología , Western Blotting , Línea Celular , Citocinas/antagonistas & inhibidores , Cartilla de ADN/genética , Fluorescencia , Humanos , Macrófagos/fisiología , Microscopía Electrónica de Rastreo , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Oxidación-Reducción , Consumo de Oxígeno/fisiología , Fagocitosis/efectos de los fármacos , TransfecciónRESUMEN
Macrophages constantly undergo morphological changes when quiescently surveying the tissue milieu for signs of microbial infection or damage, or after activation when they are phagocytosing cellular debris or foreign material. These morphofunctional alterations require active actin cytoskeleton remodeling and metabolic adaptation. Here we analyzed RAW 264.7 and Maf-DKO macrophages as models to study whether there is a specific association between aspects of carbohydrate metabolism and actin-based processes in LPS-stimulated macrophages. We demonstrate that the capacity to undergo LPS-induced cell shape changes and to phagocytose complement-opsonized zymosan (COZ) particles does not depend on oxidative phosphorylation activity but is fueled by glycolysis. Different macrophage activities like spreading, formation of cell protrusions, as well as phagocytosis of COZ, were thereby strongly reliant on the presence of low levels of extracellular glucose. Since global ATP production was not affected by rewiring of glucose catabolism and inhibition of glycolysis by 2-deoxy-D-glucose and glucose deprivation had differential effects, our observations suggest a non-metabolic role for glucose in actin cytoskeletal remodeling in macrophages, e.g. via posttranslational modification of receptors or signaling molecules, or other effects on the machinery that drives actin cytoskeletal changes. Our findings impute a decisive role for the nutrient state of the tissue microenvironment in macrophage morphodynamics.
Asunto(s)
Glucosa/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Línea Celular , Proliferación Celular , Forma de la Célula/efectos de los fármacos , Supervivencia Celular , Proteínas del Sistema Complemento/metabolismo , Citoesqueleto/metabolismo , Desoxiglucosa/metabolismo , Glucólisis , Lactatos/metabolismo , Ratones , Fosforilación Oxidativa , Fagocitosis , Seudópodos/metabolismo , Zimosan/metabolismoRESUMEN
Myotonic dystrophy type 1 (DM1) is caused by DM protein kinase (DMPK) transcripts containing an expanded (CUG)n repeat. Antisense oligonucleotide (AON)-mediated suppression of these mutant RNAs is considered a promising therapeutic strategy for this severe disorder. Earlier, we identified a 2'-O-methyl (2'-OMe) phosphorothioate (PT)-modified (CAG)7 oligo (PS58), which selectively silences mutant DMPK transcripts through recognition of the abnormally long (CUG)n tract. We present here a comprehensive collection of triplet repeat AONs and found that oligo length and nucleotide chemistry are important determinants for activity. For significant reduction of expanded DMPK mRNAs, a minimal length of five triplets was required. 2'-O,4'-C-ethylene-bridged nucleic acid (ENA)-modified AONs appeared not effective, probably due to lack of nuclear internalization. Selectivity for products from the expanded DMPK allele in patient myoblasts, an important requirement to minimize unwanted side effects, appeared also dependent on AON chemistry. In particular, RNase-H-dependent (CAG)n AONs did not show (CUG)n length specificity. We provide evidence that degradation of long DMPK transcripts induced by PS58-type AONs is an RNase-H independent process, does not involve oligo-intrinsic RNase activity nor does it interfere with splicing of DMPK transcripts. Our collection of triplet repeat AONs forms an important resource for further development of a safe therapy for DM1 and other unstable microsatellite diseases.Molecular Therapy-Nucleic Acids (2013) 2, e81; doi:10.1038/mtna.2013.9; published online 19 March 2013.
RESUMEN
Oncogenic transformation involves reprogramming of cell metabolism, whereby steady-state levels of intracellular NAD(+) and NADH can undergo dramatic changes while ATP concentration is generally well maintained. Altered expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD(+)-salvage, accompanies the changes in NAD(H) during tumorigenesis. Here, we show by genetic and pharmacological inhibition of NAMPT in glioma cells that fluctuation in intracellular [NAD(H)] differentially affects cell growth and morphodynamics, with motility/invasion capacity showing the highest sensitivity to [NAD(H)] decrease. Extracellular supplementation of NAD(+) or re-expression of NAMPT abolished the effects. The effects of NAD(H) decrease on cell motility appeared parallel coupled with diminished pyruvate-lactate conversion by lactate dehydrogenase (LDH) and with changes in intracellular and extracellular pH. The addition of lactic acid rescued and knockdown of LDH-A replicated the effects of [NAD(H)] on motility. Combined, our observations demonstrate that [NAD(H)] is an important metabolic component of cancer cell motility. Nutrient or drug-mediated modulation of NAD(H) levels may therefore represent a new option for blocking the invasive behavior of tumors.
Asunto(s)
Movimiento Celular/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Glioma/fisiopatología , NAD/metabolismo , Invasividad Neoplásica/fisiopatología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Northern Blotting , Western Blotting , Glioma/metabolismo , Humanos , Concentración de Iones de Hidrógeno , L-Lactato Deshidrogenasa/metabolismo , Ácido Láctico/farmacología , Nicotinamida Fosforribosiltransferasa/genética , Nicotinamida Fosforribosiltransferasa/metabolismo , Imagen de Lapso de Tiempo , Células Tumorales CultivadasRESUMEN
Transformed cancer cells have an altered metabolism, characterized by a shift towards aerobic glycolysis, referred to as 'the Warburg phenotype'. A change in flux through mitochondrial OXPHOS and cytosolic pathways for ATP production and a gain of capacity for biomass production in order to sustain the needs for altered growth and morphodynamics are typically involved in this global rewiring of cancer cell metabolism. Characteristically, these changes in metabolism are accompanied by enhanced uptake of nutrients like glucose and glutamine. Here we focus on the relationship between cell metabolism and cell dynamics, in particular the formation and function of invadopodia, specialized structures for focal degradation of the extracellular matrix. Since we recently found presence of enzymes that are active in glycolysis and associated pathways in invadopodia, we hypothesize that metabolic adaptation and invadopodia formation are linked processes. We give an overview on the background for this idea and show for the first time that extracellular matrix degradation by invadopodia can be differentially manipulated, without effects on cell proliferation, by use of metabolic inhibitors or changes in nutrient composition of cell culture media. We conclude that cell metabolism and carbohydrate availability, especially pyruvate, are involved in fuelling of invadopodia formation and activity.