Upregulation of cholesterol synthesis by lysosomal defects requires a functional mitochondrial respiratory chain.

Mitochondria and lysosomes are two organelles that carry out both signaling and metabolic roles in cells. Recent evidence has shown that mitochondria and lysosomes are dependent on one another, as primary defects in one cause secondary defects in the other. Although there are functional impairments in both cases, the signaling consequences of primary mitochondrial dysfunction and lysosomal defects are dissimilar. Here, we used RNA sequencing to obtain transcriptomes from cells with primary mitochondrial or lysosomal defects to identify the global cellular consequences associated with mitochondrial or lysosomal dysfunction. We used these data to determine the pathways affected by defects in both organelles, which revealed a prominent role for the cholesterol synthesis pathway. We observed a transcriptional upregulation of this pathway in cellular and murine models of lysosomal defects, while it is transcriptionally downregulated in cellular and murine models of mitochondrial defects. We identified a role for the posttranscriptional regulation of transcription factor SREBF1, a master regulator of cholesterol and lipid biosynthesis, in models of mitochondrial respiratory chain deficiency. Furthermore, we found that retention of Ca2+ in lysosomes of cells with mitochondrial respiratory chain defects contributes to the differential regulation of the cholesterol synthesis pathway in the mitochondrial and lysosomal defects tested. Finally, we verified in vivo, using a model of mitochondria-associated disease in Caenorhabditis elegans that normalization of lysosomal Ca2+ levels results in partial rescue of the developmental delay induced by the respiratory chain deficiency.

Up-regulation of cholesterol synthesis by lysosomal defects requires a functional mitochondrial respiratory chain.

Mitochondria and lysosomes are two organelles that carry out both signaling and metabolic roles in the cells. Recent evidence has shown that mitochondria and lysosomes are dependent on one another, as primary defects in one cause secondary defects in the other. Nevertheless, the signaling consequences of primary mitochondrial malfunction and of primary lysosomal defects are not similar, despite in both cases there are impairments of mitochondria and of lysosomes. Here, we used RNA sequencing to obtain transcriptomes from cells with primary mitochondrial or lysosomal defects, to identify what are the global cellular consequences that are associated with malfunction of mitochondria or lysosomes. We used these data to determine what are the pathways that are affected by defects in both organelles, which revealed a prominent role for the cholesterol synthesis pathway. This pathway is transcriptionally up-regulated in cellular and mouse models of lysosomal defects and is transcriptionally down-regulated in cellular and mouse models of mitochondrial defects. We identified a role for post-transcriptional regulation of the transcription factor SREBF1, a master regulator of cholesterol and lipid biosynthesis, in models of mitochondrial respiratory chain deficiency. Furthermore, the retention of Ca 2+ in the lysosomes of cells with mitochondrial respiratory chain defects contributes to the differential regulation of the cholesterol synthesis pathway in the mitochondrial and lysosomal defects tested. Finally, we verified in vivo , using models of mitochondria-associated diseases in C. elegans , that normalization of lysosomal Ca 2+ levels results in partial rescue of the developmental arrest induced by the respiratory chain deficiency.

Multilevel spinal epidural empyema: series of clinical cases and bibliographic / Empiema epidural espinhal multinível: série de casos clínicos e revisão bibliográfica / Empiema epidural espinal multinivel: serie de casos clínicos y revisión bibliográfica

ABSTRACT: Multilevel spinal epidural empyema (SEE) is a rare and serious infection of the spine with a high rate of morbidity and mortality. Although abscesses or empyema of the spine sector are well studied, this pathology is surprising due to its rarity and diagnostic and therapeutic challenge. It stands out for being more common in adulthood and in males and is associated with predisposing pathologies. The bacteriological agent responsible in most cases is Staphylococcus aureus. Early treatment is essential and is based on two pillars: antibiotic therapy and decompressive surgery. We present two clinical cases with multilevel involvement that evolved favorably both infectiously and neurologically without causing spine instability and we carried out a bibliographic review of the subject. Level of Evidence IV; Case Report.

RESUMO: O empiema epidural espinhal multinível (EEE) e uma infecção rara e grave da coluna vertebral, com alta taxa de morbidade e mortalidade. Embora os abscessos ou empiemas de um setor da coluna vertebral sejam bem estudados, esta patologia surpreende pela sua raridade e desafio diagnóstico e terapêutico. Destaca-se por ser mais comum na idade adulta, no sexo masculino, e estar associada a patologias predisponentes. O agente bacteriológico responsável na maioria dos casos e o Staphylococcus aureus. O tratamento precoce e essencial e baseia-se em dois pilares: antibioticoterapia e cirurgia descompressiva. Apresentamos dois casos clínicos com envolvimento multinível que evoluíram favoravelmente tanto infecciosa quanto neurologicamente sem causar instabilidade da coluna vertebral e realizamos uma revisão bibliográfica do assunto. Nível de Evidencia IV; Estudo de Caso-controle.

RESUMEN: El empiema epidural espinal (EEE) multinivel es una infección rara y grave de la columna vertebral con alta tasa de morbimortalidad. Si bien los abscesos o empiemas de un sector de la columna están bien estudiados, esta patología sorprende por su rareza, reto diagnóstico y terapeutico. Se destaca por ser más frecuente en la edad adulta, en el sexo masculino y se ve asociada a patologías predisponentes. El agente bacteriológico responsable en la mayoría de los casos es el Staphylococcus aureus. El tratamiento precoz es fundamental y está basado en dos pilares: antibioticoterapia y quirúrgico descompresivo Presentamos dos casos clínicos con afectación multinivel que evolucionaron favorablemente tanto en lo infeccioso como en lo neurológico sin provocar una inestabilidad del raquis y realizamos revisión bibliográfica del tema. Nivel de Evidencia IV; Estudio de Caso-control.

Mitochondrial respiratory chain deficiency inhibits lysosomal hydrolysis.

Mitochondria are key organelles for cellular metabolism, and regulate several processes including cell death and macroautophagy/autophagy. Here, we show that mitochondrial respiratory chain (RC) deficiency deactivates AMP-activated protein kinase (AMPK, a key regulator of energy homeostasis) signaling in tissue and in cultured cells. The deactivation of AMPK in RC-deficiency is due to increased expression of the AMPK-inhibiting protein FLCN (folliculin). AMPK is found to be necessary for basal lysosomal function, and AMPK deactivation in RC-deficiency inhibits lysosomal function by decreasing the activity of the lysosomal Ca2+ channel MCOLN1 (mucolipin 1). MCOLN1 is regulated by phosphoinositide kinase PIKFYVE and its product PtdIns(3,5)P2, which is also decreased in RC-deficiency. Notably, reactivation of AMPK, in a PIKFYVE-dependent manner, or of MCOLN1 in RC-deficient cells, restores lysosomal hydrolytic capacity. Building on these data and the literature, we propose that downregulation of the AMPK-PIKFYVE-PtdIns(3,5)P2-MCOLN1 pathway causes lysosomal Ca2+ accumulation and impaired lysosomal catabolism. Besides unveiling a novel role of AMPK in lysosomal function, this study points to the mechanism that links mitochondrial malfunction to impaired lysosomal catabolism, underscoring the importance of AMPK and the complexity of organelle cross-talk in the regulation of cellular homeostasis. Abbreviation: ΔΨm: mitochondrial transmembrane potential; AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; ATP: adenosine triphosphate; ATP6V0A1: ATPase, H+ transporting, lysosomal, V0 subbunit A1; ATP6V1A: ATPase, H+ transporting, lysosomal, V0 subbunit A; BSA: bovine serum albumin; CCCP: carbonyl cyanide-m-chlorophenylhydrazone; CREB1: cAMP response element binding protein 1; CTSD: cathepsin D; CTSF: cathepsin F; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; EBSS: Earl's balanced salt solution; ER: endoplasmic reticulum; FBS: fetal bovine serum; FCCP: carbonyl cyanide-p-trifluoromethoxyphenolhydrazone; GFP: green fluorescent protein; GPN: glycyl-L-phenylalanine 2-naphthylamide; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCOLN1/TRPML1: mucolipin 1; MEF: mouse embryonic fibroblast; MITF: melanocyte inducing transcription factor; ML1N*2-GFP: probe used to detect PtdIns(3,5)P2 based on the transmembrane domain of MCOLN1; MTORC1: mechanistic target of rapamycin kinase complex 1; NDUFS4: NADH:ubiquinone oxidoreductase subunit S4; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; pcDNA: plasmid cytomegalovirus promoter DNA; PCR: polymerase chain reaction; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol-3,5-bisphosphate; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; P/S: penicillin-streptomycin; PVDF: polyvinylidene fluoride; qPCR: quantitative real time polymerase chain reaction; RFP: red fluorescent protein; RNA: ribonucleic acid; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; shRNA: short hairpin RNA; siRNA: small interfering RNA; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3; TMRM: tetramethylrhodamine, methyl ester, perchlorate; ULK1: unc-51 like autophagy activating kinase 1; ULK2: unc-51 like autophagy activating kinase 2; UQCRC1: ubiquinol-cytochrome c reductase core protein 1; v-ATPase: vacuolar-type H+-translocating ATPase; WT: wild-type.

Long-term follow-up of ais treated surgically with pedicular screws / Seguimiento a largo plazo de eia intervenida quirúrgicamente con tornillos pediculares / Acompanhamento a longo prazo de eia tratada cirurgicamente com parafusos peduculares

ABSTRACT Objective: In the last three decades, there have been great advances in the surgical treatment of adolescent idiopathic scoliosis. There are few studies that focus on the long-term clinical and radiographic results of AIS operated on with pedicle screws that also consider psychological repercussions. Methods: We conducted an observational longitudinal study. We reviewed the AIS cases that were operated on with pedicle screws in our center between January 2009 and December 2010. We conducted follow-up until July 2019. A short questionnaire was administered to assess patient satisfaction and the long-term impact from both psychological and functional points of view. Results: A total of 19 patients met the inclusion criteria. The mean preoperative Cobb was 58°, the postoperative was 23° and at the end of follow-up it was 26°. No major complications were reported. Ninety percent were very satisfied with the overall results of the surgery. Ninety-five percent had no limitation for sports or daily activities and 90% were satisfied with the cosmetic results. Conclusion: The short- and long-term radiographic evolution in patients treated with third generation material presented good clinical results. In 3 cases (16%) loss of correction greater than 10% was reported. The overall satisfaction index and cosmetic results were very good at the end of follow-up despite the low correction rate (60%). There were no major complications and the incidence of functional limitation and pain at the end of the follow-up was very low. Level of evidence IV; Review article.

RESUMO Objetivo: Nas últimas três décadas houve grandes avanços no tratamento cirúrgico da escoliose idiopática do adolescente. Existem poucos trabalhos que estudam os resultados clínicos e radiográficos a longo prazo de EIA, tratados cirurgicamente com parafusos pediculares que também considerem a repercussão psicológica. Métodos: Realizamos um estudo longitudinal observacional. Analisamos a EIA de pacientes operados entre janeiro de 2009 e dezembro de 2010 que receberam parafusos pediculares em nosso centro. Realizamos o acompanhamento até julho de 2019. Foi realizado um breve questionário para avaliar a satisfação dos pacientes e a repercussão a longo prazo do ponto de vista psicológico e funcional. Resultados: Um total de 19 pacientes satisfizeram os critérios de inclusão. A média de Cobb pré-operatório foi de 58° e o pós-operatório foi de 23° e ao final do acompanhamento, de 26°. Não foram registradas complicações relevantes. Quanto à satisfação, 90% estão muito satisfeitos com o resultado geral da cirurgia, 95% não têm limitações para esportes ou atividades diárias e 90% estão satisfeitos com os resultados estéticos. Conclusões: A evolução radiográfica a curto e longo prazo nos pacientes tratados com material de terceira geração apresentou bons resultados clínicos. Em 3 casos (16%), registrou-se perda de correção superior a 10%. O índice geral de satisfação e resultado estético é muito bom no final do acompanhamento, apesar da baixa taxa de correção (60%). Não se constataram complicações importantes e a incidência de limitação funcional e dor ao final do acompanhamento foi muito baixa. Nível de evidência IV; Artigo de revisão.

RESUMEN Objetivo: En las últimas tres décadas han habido grandes avances en el tratamiento quirúrgico de las escoliosis idiopática del adolescente. Existen pocos trabajos que estudien el resultado clínico y radiográfico a largo plazo en las EIA intervenidas con tornillos pediculares que además contemplen repercusión psicológica. Métodos: Realizamos un estudio longitudinal observacional. Revisamos las EIA intervenidas entre enero de 2009 y diciembre de 2010 con tornillos pediculares en nuestro centro. Realizamos seguimiento hasta julio de 2019. Se realizó un breve cuestionario para evaluar satisfacción de los pacientes y la repercusión a largo plazo de punto de vista psicológico y funcional. Resultados: Un total de 19 pacientes cumplieron los criterios de inclusión. La media del Cobb pre operatorio fue de 58° y el post operatorio de 23° y al final del seguimiento, de 26°. No se registraron complicaciones mayores. El 90% está muy satisfecho con el resultado global de la cirugía. El 95% no presenta limitación para el deporte o actividad cotidiana y el 90% está conforme con resultado cosmético. Conclusiones: La evolución radiográfica a corto y largo plazo en pacientes tratados con material de tercera generación presentó buenos resultados clínicos. En 3 casos (16%) se registró perdida de corrección mayor a 10%. El índice de satisfacción global y resultado cosmético es muy bueno al final del seguimiento a pesar de la baja tasa de corrección (60%). No se constataron complicaciones mayores y la incidencia de limitación funcional y dolor al final del seguimiento fue muy baja. Nivel de evidencia IV; Estudio de revisión.

Síndrome de cola de caballo secundario a hernia discal / Cauda equina syndrome secondary to herniated disc / Síndrome de cauda eqüina secundária a hérnia discal

Resumen: Introducción: el síndrome de cola de caballo (SCC) es una entidad poco frecuente, provocado por la compresión de las raíces nerviosas en el canal a nivel de la cola de caballo. Puede dejar graves secuelas si no es diagnosticado y tratado de forma precoz. Únicamente 2% a 6% de las hernias discales lumbares van a provocar un SCC. El diagnóstico de esta patología se basa en criterios clínicos, siendo éstos objeto de controversia dada la variabilidad de presentación del cuadro clínico. El objetivo de este trabajo es analizar la presentación clínica y evolución posoperatoria de los pacientes intervenidos por SCC secundaria a hernia de disco por equipo del Centro de Deformidades de Columna (CE.DEF.CO), entre enero de 2009 y diciembre de 2018. Material y método: realizamos un análisis retrospectivo. La población objetivo son los pacientes intervenidos por SCC secundario a hernia discal entre enero de 2009 y diciembre de 2018 por equipo del CE.DEF.CO. Analizamos 20 pacientes intervenidos quirúrgicamente, de ellos 17 (85%) casos presentaron síntomas urinarios, 18 (90%) casos dolor o elementos deficitarios en miembros inferiores, 13 casos anestesia/hipoestesia en silla de montar, 6 casos síntomas intestinales y 3 casos presentaron disfunciones sexuales. En 19 casos se realizó procedimiento quirúrgico antes de las 48 de iniciados los síntomas. Resultados: la remisión de síntomas esfinterianos al mes es de 83% y al año posoperatorio de casi 87%. Se constató un caso de disfunción sexual al año posoperatorio. Conclusión: el diagnóstico precoz por el médico emergencista es fundamental, por lo que el conocimiento de esta patología es imprescindible. Nuestros resultados en pacientes intervenidos de forma precoz, antes de las 48 horas, fueron similares a los publicados en la bibliografía internacional con bajo porcentaje de secuelas.

Summary: Introduction: cauda equina syndrome (CES) is a rare entity, caused by compression of the nerve roots in the spinal canal at the cauda equina level. It can leave serious sequelae if it is not diagnosed and treated early. Only 2-6% of lumbar disc herniation will cause CES. The diagnosis of this pathology is based on clinical criteria, these being the subject of controversy given the variability of presentation of the clinical picture. Materials and methods: we conducted a retrospective study. The target population are the patients operated on for CES secondary to herniated disc between January 2009 and December 2018 by a team from CE.DEF.CO. (center for spinal deformities). The objective is to carry out a set-up regarding the clinical presentation of this entity and to evaluate the correlation between surgical time and neurological improvement. Out of 20 patients who were operated, 17 (85%) presented urinary symptoms, 18 (90%) were patients in pain or presenting deficit elements in the lower limbs, 13 were cases of anesthesia / hypoesthesia in the saddle, 6 cases evidenced intestinal symptoms and 3 cases presented sexual dysfunction. In 19 cases, a surgical procedure was performed before 48 hours after symptoms started. Results: the remission of sphincter symptoms after one month is 83% and it rises to almost 87% one year after the surgery. One case of sexual dysfunction persisted one year after surgery. Conclusion: early diagnosis by the emergency physician is essential, so knowledge of this pathology is essential. Our experience and good results allow us to conclude that early surgical treatment is associated with symptomatic improvement and fewer neurological sequelae in the short and long term.

Resumo: Introdução: a síndrome da cauda eqüina (SCE) é uma entidade rara causada pela compressão das raízes nervosas no canal no nível da cauda eqüina. Pode deixar seqüelas graves se não for diagnosticada e tratada precocemente. Apenas 2-6% das hérnias do disco lombar causarão a síndrome da cauda eqüina. O diagnóstico desta patologia é baseado em critérios clínicos, sendo este motivo de controvérsia, dada a variabilidade da apresentação do quadro clínico. Materiais e métodos: realizamos um estudo retrospectivo. A população-alvo estava composta por pacientes operados por SCE secundária a hérnia discal entre janeiro de 2009 e dezembro de 2018 pela equipe do CE. DEF.CO (Centro de Defeitos da Coluna Vertebral). Foram analisados 20 pacientes operados, dos quais 17 (85%) apresentaram sintomas urinários, 18 (90%) dor ou elementos de deficiência em membros inferiores, 13 anestesia / hipoestesia em sela, 6 sintomas intestinais e 3 disfunções sexuais. Em 19 casos, um procedimento cirúrgico foi realizado antes de 48 horas após o início dos sintomas. Resultado: a remissão dos sintomas esfincterianos em um mês é de 83% e em um ano pós-operatório é de quase 87%. Um caso de disfunções sexuais foi encontrado um ano após a cirurgia. Conclusão: o diagnóstico precoce pelo médico de emergência é essencial, portanto o conhecimento desta patologia é fundamental. Nossos resultados em pacientes operados precocemente, antes de 48 horas, foram semelhantes aos publicados na literatura internacional com baixo percentual de seqüelas.

Cardiac mitochondrial biogenesis in endotoxemia is not accompanied by mitochondrial function recovery.

Mitochondrial biogenesis emerges as a compensatory mechanism involved in the recovery process in endotoxemia and sepsis. The aim of this work was to analyze the time course of the cardiac mitochondrial biogenesis process occurring during endotoxemia, with emphasis on the quantitative analysis of mitochondrial function. Female Sprague-Dawley rats (45 days old) were ip injected with LPS (10 mg/kg). Measurements were performed at 0-24 h after LPS administration. PGC-1α and mtTFA expression for biogenesis and p62 and LC3 expression for autophagy were analyzed by Western blot; mitochondrial DNA levels by qPCR, and mitochondrial morphology by transmission electron microscopy. Mitochondrial function was evaluated as oxygen consumption and respiratory chain complex activity. PGC-1α and mtTFA expression significantly increased in every time point analyzed, and mitochondrial mass was increased by 20% (P<0.05) at 24 h. p62 expression was significantly decreased in a time-dependent manner. LC3-II expression was significantly increased at all time points analyzed. Ultrastructurally, mitochondria displayed several abnormalities (internal vesicles, cristae disruption, and swelling) at 6 and 18 h. Structures compatible with fusion/fission processes were observed at 24 h. A significant decrease in state 3 respiration was observed in every time point analyzed (LPS 6h: 20%, P<0.05). Mitochondrial complex I activity was found decreased by 30% in LPS-treated animals at 6 and 24h. Complex II and complex IV showed decreased activity only at 24 h. The present results show that partial restoration of cardiac mitochondrial architecture is not accompanied by improvement of mitochondrial function in acute endotoxemia. The key implication of our study is that cardiac failure due to bioenergetic dysfunction will be overcome by therapeutic interventions aimed to restore cardiac mitochondrial function.