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BACKGROUND: Infection with human immunodeficiency virus (HIV) influences the outcome and natural disease progression of hepatitis C virus (HCV) infection. While the majority of HCV mono-infected and HCV/HIV co-infected subjects develop chronic HCV infection, 20-46% of mono- and co-infected subjects spontaneously clear HCV infection. The mechanism underlying viral clearance is not clearly understood. Analysis of differential cellular gene expression (mRNA) between HIV-infected patients with persistent HCV infection or spontaneous clearance could provide a unique opportunity to decipher the mechanism of HCV clearance. METHODS: Plasma RNA from HIV/HCV co-infected subjects who cleared HCV and those who remained chronically infected with HCV was sequenced using Ion Torrent technology. The sequencing results were analyzed to identify transcripts that are associated with HCV clearance by measuring differential gene expression in HIV/HCV co-infected subjects who cleared HCV and those who remained chronically infected with HCV. RESULTS: We have identified plasma mRNA, the levels of which are significantly elevated (at least 5 fold, False Discovery Rate (FDR) <0.05) before HCV infection in subjects who cleared HCV compared to those who remained chronically infected. Upon further analysis of these differentially expressed genes, before and after HCV infection, we found that before HCV infection 12 genes were uniquely upregulated in the clearance group compared to the chronically infected group. Importantly, a number of these 12 genes and their upstream regulators (such as CCL3, IL17D, LBP, SOCS3, NFKBIL1, IRF) are associated with innate immune response functions. CONCLUSIONS: These results suggest that subjects who spontaneously clear HCV may express these unique genes associated with innate immune functions.
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Infecciones por VIH/virología , Hepatitis C/virología , ARN Viral/sangre , Coinfección/virología , Femenino , Regulación de la Expresión Génica , Hepatitis C Crónica/virología , Humanos , Inmunidad Innata/genética , Carga ViralAsunto(s)
Duodeno/patología , Ganglios Linfáticos/patología , Macroglobulinemia de Waldenström/patología , Anciano , Anemia/etiología , Axila , Médula Ósea/diagnóstico por imagen , Médula Ósea/patología , Endoscopía del Sistema Digestivo , Humanos , Cadenas Pesadas de Inmunoglobulina/sangre , Inmunoglobulina M/sangre , Cadenas kappa de Inmunoglobulina/sangre , Cadenas lambda de Inmunoglobulina/sangre , Ganglios Linfáticos/diagnóstico por imagen , Linfadenopatía/diagnóstico por imagen , Linfadenopatía/patología , Masculino , Mediastino , Tomografía de Emisión de Positrones , Esplenomegalia/diagnóstico por imagen , Macroglobulinemia de Waldenström/sangre , Macroglobulinemia de Waldenström/complicaciones , Macroglobulinemia de Waldenström/diagnósticoRESUMEN
Background: Ependymomas, rare glial brain tumors, account for <5% of all brain tumors. Interestingly, over 60% of ependymomas occur in the spinal cord of adults, including those originating from the filum terminale, while the rest are found within the brain. The World Health Organization (WHO) categorizes ependymomas into three grades: subependymomas and myxopapillary ependymomas ([MEPNs]; WHO grade I), classic ependymomas (WHO grade II), and anaplastic ependymomas (WHO grade III). Spinal ependymomas generally exhibit a more favorable prognosis compared to their intracranial counterparts and are primarily treated through gross total resection, which is considered the most effective surgical approach. As such, they are recognized as a distinct clinical entity that demands tailored management strategies. MEPNs, which constitute 13% of ependymomas, typically occur in the cauda equina and sometimes extend into the conus medullaris. Most other spinal ependymomas are of the classic type and predominantly arise in the cervical and thoracic regions of the spine. The mean age at diagnosis is 45 years of age. While prognosis varies based on molecular subtypes, complete resection is associated with improved survival. Case Description: Here, we demonstrate the technical nuances to safely achieve gross total resection of a giant spinal ependymoma in a 29-year-old female with a medical history notable for sept-optic dysplasia, and panhypopituitarism. The patient presented with progressive neck pain, upper and lower extremity weakness, and numbness for 1 year. On physical examination, she demonstrated mild weakness in her left arm. The preoperative magnetic resonance imaging revealed a cervicothoracic intramedullary mass extending from C4 to T2 with an associated syrinx at C4. Under intraoperative neural monitoring (somatosensory evoked potentials, motor-evoked potentials, and epidural direct wave recordings), the patient underwent a C4 - T2 laminectomy. In addition, spinal ultrasonography helped differentiate solid tumor mass from syrinx formation, thus guiding the focus and extent of the decompression . Conclusion: Gross total resection was achieved; at 18 postoperative months, the patient had mild residual motor deficit. The pathological evaluation revealed a WHO grade II ependymoma. Subsequent sequential enhanced MR studies at 3, 6, and 12 months confirmed no tumor recurrence.
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OBJECTIVE: Clear cell meningiomas (CCM) are an uncommon meningioma subtype marked by aggressive growth and high rates of recurrence despite initial resection. The present study evaluates the adjuvant benefit of stereotactic radiosurgery (SRS) for residual or recurrent tumors. METHODS: After review of our prospectively maintained database, we identified 6 patients (3 female) with histologically confirmed Grade 2 CCMs. The median age of the patients at the time of SRS was 45 years. Five patients had undergone prior gross total surgical resection and 1 patient had subtotal resection before SRS. The median SRS treatment volume was 4.7 cc and the median radiosurgical margin dose was 13 Gy (range: 10-15 Gy). RESULTS: The median follow-up time was 35.5 months (range 6-168 months). Three patients achieved tumor control after the first SRS procedure. Three patients experienced tumor progression at 4, 22, and 32 months after initial SRS. Tumor control was obtained in 2 of these patients after additional SRS. One patient with multiple SRS procedures had suspected adverse radiation effect that was successfully treated with corticosteroids followed by bevacizumab. CONCLUSIONS: Tumor control was maintained in 5 of 6 patients after one or more SRS procedures. SRS should be considered for early intervention after surgical resection of CCM. To maximize the tumor control rate, patients with diagnosed CCM should be treated more generously and higher margin dose should be prescribed. Patients with CCM should be counselled that more than one SRS may be necessary to provide sustained tumor control.
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Neoplasias Meníngeas , Meningioma , Radiocirugia , Humanos , Femenino , Persona de Mediana Edad , Meningioma/radioterapia , Meningioma/cirugía , Meningioma/etiología , Radiocirugia/métodos , Resultado del Tratamiento , Recurrencia Local de Neoplasia/cirugía , Recurrencia Local de Neoplasia/etiología , Neoplasias Meníngeas/radioterapia , Neoplasias Meníngeas/cirugía , Neoplasias Meníngeas/etiología , Estudios Retrospectivos , Estudios de SeguimientoRESUMEN
Glioblastoma (GBM) is fatal and the study of therapeutic resistance, disease progression, and drug discovery in GBM or glioma stem cells is often hindered by limited resources. This limitation slows down progress in both drug discovery and patient survival. Here we present a genetically engineered human cerebral organoid model with a cancer-like phenotype that could provide a basis for GBM-like models. Specifically, we engineered a doxycycline-inducible vector encoding shRNAs enabling depletion of the TP53, PTEN, and NF1 tumor suppressors in human cerebral organoids. Designated as inducible short hairpin-TP53-PTEN-NF1 (ish-TPN), doxycycline treatment resulted in human cancer-like cerebral organoids that effaced the entire organoid cytoarchitecture, while uninduced ish-TPN cerebral organoids recapitulated the normal cytoarchitecture of the brain. Transcriptomic analysis revealed a proneural GBM subtype. This proof-of-concept study offers a valuable resource for directly investigating the emergence and progression of gliomas within the context of specific genetic alterations in normal cerebral organoids.
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Ascitis/etiología , Diarrea/etiología , Esofagitis Eosinofílica/complicaciones , Síndrome Hipereosinofílico/complicaciones , Enfermedades del Íleon/complicaciones , Adulto , Ascitis/diagnóstico , Ascitis/tratamiento farmacológico , Biopsia , Budesonida/uso terapéutico , Colonoscopía , Diarrea/diagnóstico , Diarrea/tratamiento farmacológico , Esofagitis Eosinofílica/diagnóstico , Esofagitis Eosinofílica/tratamiento farmacológico , Esofagoscopía , Glucocorticoides/uso terapéutico , Humanos , Síndrome Hipereosinofílico/diagnóstico , Síndrome Hipereosinofílico/tratamiento farmacológico , Enfermedades del Íleon/diagnóstico , Enfermedades del Íleon/tratamiento farmacológico , Masculino , Inducción de Remisión , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
The group of CNS mesenchymal (non-meningothelial) and primary glial/neuronal tumors in association with EWSR1-non-ETS rearrangements comprises a growing spectrum of entities, mostly reported in isolation with incomplete molecular profiling. Archival files from three pediatric institutions were queried for unusual cases of pediatric (≤21 years) CNS EWSR1-rearranged tumors confirmed by at least one molecular technique. Extra-axial tumors and cases with a diagnosis of Ewing sarcoma (EWSR1-ETS family fusions) were excluded. Additional studies, including anchored multiplex-PCR with next-generation sequencing and DNA methylation profiling, were performed as needed to determine fusion partner status and brain tumor methylation class, respectively. Five cases (median 17 years) were identified (M:F of 3:2). Location was parenchymal (n = 3) and undetermined (n = 2) with topographic distributions including posterior fossa (n = 1), frontal (n = 1), temporal (n = 1), parietal (n = 1) and occipital (n = 1) lobes. Final designation with fusion findings included desmoplastic small round cell tumor (EWSR1-WT1; n = 1) and tumors of uncertain histogenesis (EWSR1-CREM, n = 1; EWSR1-CREB1, n = 1; EWSR1-PLAGL1, n = 1; and EWSR1-PATZ1, n = 1). Tumors showed a wide spectrum of morphology and biologic behavior. For EWSR1-CREM, EWSR1-PLAGL1 and EWSR1-PATZ1 tumors, no significant methylation scores were reached in the known brain tumor classes. Available outcome (4/5) was reported as favorable (n = 2) and unfavorable (n = 2) with a median follow-up of 30 months. In conclusion, we describe five primary EWSR1-non-ETS fused CNS tumors exhibiting morphologic and biologic heterogeneity and we highlight the clinical importance of determining specific fusion partners to improve diagnostic accuracy, treatment and monitoring. Larger prospective clinicopathological and molecular studies are needed to determine the prognostic implications of histotypes, anatomical location, fusion partners, breakpoints and methylation profiles in patients with these rare tumors.
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Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Proteína EWS de Unión a ARN/genética , Adolescente , Niño , Preescolar , Femenino , Humanos , Masculino , Fusión de Oncogenes , Proteínas de Fusión Oncogénica/genética , Adulto JovenRESUMEN
BACKGROUND: Spinal cavernous malformations are rare, accounting for approximately 5-12% of all spinal cord vascular lesions. Fortunately, improvements in imaging technologies have made it easier to establish the diagnosis of intramedullary spinal cavernomas (ISCs). CASE DESCRIPTION: Here, we report the case of a 63-year-old male with an >11-year history of left-sided radiculopathy, ataxia, and quadriparesis. Initially, radiographic findings were interpreted as consistent with spondylotic myelopathy with cord signal changes from the C3-C7 levels. The patient underwent a C3-C7 laminectomy/foraminotomy with instrumentation. It was only after several symptomatic recurrences and repeated magnetic resonance images (MRI) that the diagnosis of a ventrally-located intramedullary lesion, concerning for a cavernoma, at the level C6 was established. CONCLUSION: Early and repeated enhanced MR studies may be required to correctly establish the diagnosis and determine the optimal surgical management of ISCs.
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Neoplasias Meníngeas , Meningioma , Neoplasias de la Médula Espinal , Humanos , Meningioma/complicaciones , Meningioma/diagnóstico por imagen , Meningioma/cirugía , Neoplasias de la Médula Espinal/complicaciones , Neoplasias de la Médula Espinal/diagnóstico por imagen , Neoplasias de la Médula Espinal/cirugía , Neoplasias Meníngeas/diagnóstico por imagen , Neoplasias Meníngeas/cirugía , Neoplasias Meníngeas/patologíaRESUMEN
Although histology still plays a critical role in diagnosing diffuse gliomas, additional ancillary testing is an essential tool for VA pathology laboratories.
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Adenocarcinoma of the rete testis is a rare and aggressive malignancy arising from the epididymal epithelium. We present a case of a 66-year-old male who presented with left testis mass. Histopathological analysis of orchiectomy specimen was consistent with adenocarcinoma of the rete testis. Subsequent retroperitoneal lymph node dissection revealed metastatic disease not detected on preoperative PET-CT. Prior reports have suggested poor response rates to both systemic chemotherapy and radiation therapy. Aggressive surgical management of the retroperitoneum should be considered even in clinically node-negative patients given the paucity of other effective treatment regimens.
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Reactive oxygen species (ROS) are implicated in 6-hydroxydopamine (6-OHDA) injury to catecholaminergic neurons; however, the mechanism(s) are unclear. In addition to ROS generated during autoxidation, 6-OHDA may initiate secondary cellular sources of ROS that contribute to toxicity. Using a neuronal cell line, we found that catalytic metalloporphyrin antioxidants conferred protection if added 1 h after exposure to 6-OHDA, whereas the hydrogen peroxide scavenger catalase failed to protect if added more than 15 min after 6-OHDA. There was a temporal correspondence between loss of protection and loss of the ability of the antioxidant to inhibit 6-OHDA-induced ERK phosphorylation. Time course studies of aconitase inactivation, an indicator of intracellular superoxide, and MitoSOX red, a mitochondria targeted ROS indicator, demonstrate early intracellular ROS followed by a delayed phase of mitochondrial ROS production, associated with phosphorylation of a mitochondrial pool of ERK. Furthermore, on initiation of mitochondrial ROS and ERK activation, 6-OHDA-injured cells became refractory to rescue by metalloporphyrin antioxidants. Together with previous studies showing that inhibition of the ERK pathway confers protection from 6-OHDA toxicity, and that phosphorylated ERK accumulates in mitochondria of degenerating human Parkinson's disease neurons, these studies implicate mitochondrial ERK activation in Parkinsonian oxidative neuronal injury.
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Quinasas MAP Reguladas por Señal Extracelular/fisiología , Mitocondrias/efectos de los fármacos , Oxidopamina/farmacología , Animales , Butadienos/farmacología , Catalasa/farmacología , Células Cultivadas , Activación Enzimática , Flavonoides/farmacología , Metaloporfirinas/farmacología , Mitocondrias/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Nitrilos/farmacología , Oxidopamina/toxicidad , Ratas , Transducción de SeñalRESUMEN
INTRODUCTION: We examined mutational testing of the epidermal growth factor gene (EGFR) and erlotinib treatment among veterans diagnosed with non-small-cell lung cancer in the United States Department of Veterans Affairs (VA). Our objectives were to identify the prevalence of clinically actionable EGFR mutations, to determine whether testing and treatment were guideline concordant, to evaluate the impact of testing and treatment on survival, and to estimate the rate of testing. PATIENTS AND METHODS: Test results were linked to electronic health records from VA Corporate Data Warehouse and the VA Central Cancer Registry. We analyzed patient demographic and clinical characteristics, prevalence of EGFR mutations, and timing of EGFR mutational testing and erlotinib treatment based on pharmacy records. Overall survival was assessed by Kaplan-Meier analysis. RESULTS: Among 973 patients tested at 70 VA medical centers between 2011 and 2013, 64 (7%) had sensitizing EGFR mutations, 694 (71%) were EGFR wild type, and 168 (17%) had clinically insignificant polymorphisms or variants of unknown significance. Results were not documented in 47 tests (5%). Erlotinib administration was in agreement with test results in 843 cases (87%). CONCLUSION: Veterans have a much lower rate of sensitizing EGFR mutations than the reported average of 10% to 15%, which correlates with a high rate of smoking among veterans. This may partially explain clinicians' reluctance to prescribe EGFR testing, which results in underutilization. Although test results appear to have influenced erlotinib treatment decisions, we documented a substantial number of cases where treatment was not applied in accordance with clinical guidelines, potentially resulting in worse outcomes and unnecessary cost.
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Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Análisis Mutacional de ADN/métodos , Receptores ErbB/genética , Neoplasias Pulmonares/diagnóstico , Mutación/genética , Veteranos , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Clorhidrato de Erlotinib/uso terapéutico , Femenino , Adhesión a Directriz , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , Análisis de Supervivencia , Estados Unidos , United States Department of Veterans Affairs , Adulto JovenRESUMEN
ANO1 is a calcium-activated chloride channel that is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and other cancers. While ANO1 expression negatively correlates with survival in several cancers, its epigenetic regulation is poorly understood. We analyzed HNSCC samples from TCGA and a separate dataset of HPV+ oropharyngeal squamous cell carcinoma (OPSCC) samples to identify differentially methylated regions. E6 and E7 transfected normal oral keratinocytes (NOK) were used to induce hypermethylation of the ANO1 promoter. We found three CpG islands that correlated with ANO1 expression, including two positively correlated with expression. Using two HNSCC datasets with differential expression of ANO1, we showed hypermethylation of positively correlated CpG islands potentiates ANO1 expression. E7 but not E6 transfection of NOK cells led to hypermethylation of a positively correlated CpG island without a change in ANO1 expression. ANO1 promoter methylation was also correlated with patient survival. Our results are the first to show the contribution of positively correlated CpG's for regulating gene expression in HNSCC. Hypermethylation of the ANO1 promoter was strongly correlated with but not sufficient to increase ANO1 expression, suggesting methylation of positively correlated CpG's likely serves as an adjunct to other mechanisms of ANO1 activation.
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Anoctamina-1/biosíntesis , Carcinoma de Células Escamosas/patología , Islas de CpG , Metilación de ADN , Epigénesis Genética , Neoplasias de Cabeza y Cuello/patología , Proteínas de Neoplasias/biosíntesis , Humanos , Regiones Promotoras GenéticasRESUMEN
Purpose: TMEM16A is a calcium-activated chloride channel that is amplified in a variety of cancers, including 30% of head and neck squamous cell carcinomas (HNSCCs), raising the possibility of an anti-apoptotic role in malignant cells. This study investigated this using a multimodal, translational investigation.Experimental Design: Combination of (i) in vitro HNSCC cell culture experiments assessing cell viability, apoptotic activation, and protein expression (ii) in vivo studies assessing similar outcomes, and (iii) molecular and staining analysis of human HNSCC samples.Results: TMEM16A expression was found to correlate with greater tumor size, increased Erk 1/2 activity, less Bim expression, and less apoptotic activity overall in human HNSCC. These findings were corroborated in subsequent in vitro and in vivo studies and expanded to include a cisplatin-resistant phenotype with TMEM16A overexpression. A cohort of 41 patients with laryngeal cancer demonstrated that cases that recurred after chemoradiation failure were associated with a greater TMEM16A overexpression rate than HNSCC that did not recur.Conclusions: Ultimately, this study implicates TMEM16A as a contributor to tumor progression by limiting apoptosis and as a potential biomarker of more aggressive disease. Clin Cancer Res; 23(23); 7324-32. ©2017 AACR.
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Anoctamina-1/genética , Apoptosis/genética , Proteína 11 Similar a Bcl2/genética , Carcinoma de Células Escamosas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Proteínas de Neoplasias/genética , Animales , Anoctamina-1/metabolismo , Antineoplásicos/farmacología , Proteína 11 Similar a Bcl2/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Cisplatino/farmacología , Regulación hacia Abajo , Femenino , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Masculino , Ratones Desnudos , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Head and neck squamous cell carcinoma (HNSCC) has a variety of causes. Recently, the human papilloma virus (HPV) has been implicated in the rising incidence of oropharyngeal cancer and has led to variety of studies exploring the differences between HPV-positive and HPV-negative HNSCC. The calcium-activated chloride channel TMEM16A is overexpressed in a variety of cancers, including HNSCC, but whether or not it plays different roles in HPV-positive and HPV-negative HNSCC is unknown. Here, we demonstrate that TMEM16A is preferentially overexpressed in HPV-negative HNSCC and that this overexpression of TMEM16A is associated with decreased patient survival. We also show that TMEM16A expression is decreased in HPV-positive HNSCC at the DNA, RNA, and protein levels in patient samples as well as cell lines. We demonstrate that the lower levels of TMEM16A expression in HPV-positive tumors can be attributed to both a combination of copy number alteration and promoter methylation at the DNA level. Additionally, our cellular data show that HPV-negative cell lines are more dependent on TMEM16A for survival than HPV-positive cell lines. Therefore, we suspect that the down-regulation of TMEM16A in HPV-positive HNSCC makes TMEM16A a poor therapeutic target in HPV-positive HNSCC, but a potentially useful target in HPV-negative HNSCC.
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Carcinoma de Células Escamosas/genética , Canales de Cloruro/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/genética , Anoctamina-1 , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virología , Línea Celular Tumoral , Canales de Cloruro/metabolismo , Perfilación de la Expresión Génica/métodos , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/virología , Interacciones Huésped-Patógeno , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Proteínas de Neoplasias/metabolismo , Papillomaviridae/fisiología , Interferencia de ARN , Análisis de SupervivenciaRESUMEN
A number of reports indicate the potential for redox signalling via extracellular signal-regulated protein kinases (ERK) during neuronal injury. We have previously found that sustained ERK activation contributes to toxicity elicited by 6-hydroxydopamine (6-OHDA) in the B65 neuronal cell line. To determine whether reactive oxygen species (ROS) play a role in mediating ERK activation and 6-OHDA toxicity, we examined the effects of catalase, superoxide dismutase (SOD1), and metalloporphyrin antioxidants ('SOD mimetics') on 6-OHDA-treated cells. We found that catalase and metalloporphyrin antioxidants not only conferred protection against 6-OHDA but also inhibited development of sustained ERK phosphorylation in both differentiated and undifferentiated B65 cells. However, exogenously added SOD1 and heat-inactivated catalase had no effect on either toxicity or sustained ERK phosphorylation. This correlation between antioxidant protection and inhibition of 6-OHDA-induced sustained ERK phosphorylation suggests that redox regulation of ERK signalling cascades may contribute to neuronal toxicity.
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Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/efectos de los fármacos , Oxidopamina/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Catalasa/metabolismo , Catalasa/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Activación Enzimática , Metaloporfirinas/metabolismo , Metaloporfirinas/farmacología , Fosforilación , Ratas , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/farmacologíaRESUMEN
Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP(+)) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease-associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl-cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity.
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Autofagia/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Bucladesina/metabolismo , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Neuronas/citología , Neuronas/metabolismo , Fagosomas/metabolismo , Fosforilación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Sirolimus/farmacologíaRESUMEN
Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.
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Autofagia/fisiología , Homeostasis/fisiología , Mitocondrias/metabolismo , Estrés Oxidativo/fisiología , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína 7 Relacionada con la Autofagia , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Proteínas Quinasas/genética , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Degenerating neurons of Parkinson's disease (PD) patient brains exhibit granules of phosphorylated extracellular signal-regulated protein kinase 1/2 (ERK1/2) that localize to autophagocytosed mitochondria. Here we show that 6-hydroxydopamine (6-OHDA) elicits activity-related localization of ERK1/2 in mitochondria of SH-SY5Y cells, and these events coincide with induction of autophagy and precede mitochondrial degradation. Transient transfection of wildtype (WT) ERK2 or constitutively active MAPK/ERK Kinase 2 (MEK2-CA) was sufficient to induce mitophagy to a degree comparable with that elicited by 6-OHDA, while constitutively active ERK2 (ERK2-CA) had a greater effect. We developed green fluorescent protein (GFP) fusion constructs of WT, CA, and kinase-deficient (KD) ERK2 to study the role of ERK2 localization in regulating mitophagy and cell death. Under basal conditions, cells transfected with GFP-ERK2-WT or GFP-ERK2-CA, but not GFP-ERK2-KD, displayed discrete cytoplasmic ERK2 granules of which a significant fraction colocalized with mitochondria and markers of autophagolysosomal maturation. The colocalizing GFP-ERK2/mitochondria granules are further increased by 6-OHDA and undergo autophagic degradation, as bafilomycin-A, an inhibitor of autolysosomal degradation, robustly increased their detection. Interestingly, increasing ERK2-WT or ERK2-CA expression was sufficient to promote comparable levels of macroautophagy as assessed by analysis of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3). In contrast, the level of mitophagy was more tightly correlated with ERK activity levels, potentially explained by the greater localization of ERK2-CA to mitochondria compared to ERK2-WT. These data indicate that mitochondrial localization of ERK2 activity is sufficient to recapitulate the effects of 6-OHDA on mitophagy and autophagic cell death.