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Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.
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Virus de la Hepatitis B , Hepatitis B Crónica , Hepatocitos , Humanos , Hepatocitos/virología , Hepatocitos/efectos de los fármacos , Animales , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/fisiología , Ratones , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Proteínas del Núcleo Viral/metabolismo , Antivirales/farmacología , Antivirales/uso terapéutico , Antígenos del Núcleo de la Hepatitis B/metabolismo , Cápside/metabolismo , Cápside/efectos de los fármacos , Hígado/virología , Hígado/efectos de los fármacos , Hígado/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Ensamble de Virus/efectos de los fármacos , Apoptosis/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). SIGNIFICANCE: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.
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Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Animales , Ratones , Línea Celular Tumoral , Proteínas Proto-Oncogénicas p21(ras)/genética , Femenino , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Guanosina Trifosfato/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutación , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , MasculinoRESUMEN
Broad-spectrum RAS inhibition has the potential to benefit roughly a quarter of human patients with cancer whose tumours are driven by RAS mutations1,2. RMC-7977 is a highly selective inhibitor of the active GTP-bound forms of KRAS, HRAS and NRAS, with affinity for both mutant and wild-type variants3. More than 90% of cases of human pancreatic ductal adenocarcinoma (PDAC) are driven by activating mutations in KRAS4. Here we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models. We observed broad and pronounced anti-tumour activity across models following direct RAS inhibition at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumour versus normal tissues. Treated tumours exhibited waves of apoptosis along with sustained proliferative arrest, whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC mouse model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumours identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identify a promising candidate combination therapeutic regimen to overcome monotherapy resistance.
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Antineoplásicos , Carcinoma Ductal Pancreático , Guanosina Trifosfato , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Animales , Femenino , Humanos , Ratones , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Variaciones en el Número de Copia de ADN , Resistencia a Antineoplásicos/efectos de los fármacos , Genes myc , Guanosina Trifosfato/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto , MutaciónRESUMEN
Background & Aims: Patterns of liver HBV antigen expression have been described but not quantified at single-cell resolution. We applied quantitative techniques to liver biopsies from individuals with chronic hepatitis B and evaluated sampling heterogeneity, effects of disease stage, and nucleos(t)ide (NUC) treatment, and correlations between liver and peripheral viral biomarkers. Methods: Hepatocytes positive for HBV core and HBsAg were quantified using a novel four-plex immunofluorescence assay and image analysis. Biopsies were analysed from HBeAg-positive (n = 39) and HBeAg-negative (n = 75) participants before and after NUC treatment. To evaluate sampling effects, duplicate biopsies collected at the same time point were compared. Serum or plasma samples were evaluated for levels of HBV DNA, HBsAg, hepatitis B core-related antigen (HBcrAg), and HBV RNA. Results: Diffusely distributed individual HBV core+ cells and foci of HBsAg+ cells were the most common staining patterns. Hepatocytes positive for both HBV core and HBsAg were rare. Paired biopsies revealed large local variation in HBV staining within participants, which was confirmed in a large liver resection. NUC treatment was associated with a >100-fold lower median frequency of HBV core+ cells in HBeAg-positive and HBeAg-negative participants, whereas reductions in HBsAg+ cells were not statistically significant. The frequency of HBV core+ hepatocytes was lower in HBeAg-negative participants than in HBeAg-positive participants at all time points evaluated. Total HBV+ hepatocyte burden correlated with HBcrAg, HBV DNA, and HBV RNA only in baseline HBeAg-positive samples. Conclusions: Reductions in HBV core+ hepatocytes were associated with HBeAg-negative status and NUC treatment. Variation in HBV positivity within individual livers was extensive. Correlations between the liver and the periphery were found only between biomarkers likely indicative of cccDNA (HBV core+ and HBcrAg, HBV DNA, and RNA). Impact and Implications: HBV infects liver hepatocyte cells, and its genome can exist in two forms that express different sets of viral proteins: a circular genome called cccDNA that can express all viral proteins, including the HBV core and HBsAg proteins, or a linear fragment that inserts into the host genome typically to express HBsAg, but not HBV core. We used new techniques to determine the percentage of hepatocytes expressing the HBV core and HBsAg proteins in a large set of liver biopsies. We find that abundance and patterns of expression differ across patient groups and even within a single liver and that NUC treatment greatly reduces the number of core-expressing hepatocytes.
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The validity and relevance of histologic disease activity in Crohn's disease (CD) is unclear, owing to disconnects with endoscopic pathology. Here, we explore relationships between endoscopic, histologic, and molecular activity. This post hoc analysis of the Phase 2 FITZROY trial (NCT02048618) assessed baseline and week 10 (W10) inflammation across matched ileal and colonic segments in CD patients receiving filgotinib 200âmg (n = 42) vs placebo (n = 18). Macroscopic and microscopic disease were assessed by Simple Endoscopic Score for CD ulceration subscore (uSES-CD) and Global Histologic Activity Score activity subscore (aGHAS), respectively. Molecular activity was quantified by phosphorylated signal transducer and activator of transcription (pSTAT)1 and pSTAT3 in epithelium and nonepithelium. Segments were classified as "low" or "high" activity; correlations and concordance were calculated. Logistic regression identified W10 outcome predictors. Overall, 300 segments in 60 patients were assessed. Baseline uSES-CD and aGHAS correlations were 0.72 and 0.53 in colon and ileum, respectively. pSTAT levels had poor-to-moderate concordance with uSES-CD (κârange, 0.11-0.49) but moderate-to-good concordance with aGHAS (0.43-0.77). With filgotinib vs placebo, uSES-CD and aGHAS decreased in significantly more segments with high baseline uSES-CD and aGHAS, and significantly more segments with high baseline pSTAT improved at W10. pSTAT1 was more sensitive to change than uSES-CD and aGHAS. Low baseline pSTAT3 in colon nonepithelium predicted W10 low uSES-CD (P = .044). There was better concordance between histologic and molecular disease activity associated with higher sensitivity to change vs endoscopic severity in ileocolonic CD. Our results suggest histologic activity be included in the assessment of CD inflammatory burden.
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Enfermedad de Crohn , Humanos , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/patología , Endoscopía Gastrointestinal/métodos , Mucosa Intestinal , Piridinas/uso terapéutico , Factor de Transcripción STAT1RESUMEN
INTRODUCTION: Although diabetic kidney disease (DKD) is responsible for more than half of all chronic and end-stage kidney disease (ESKD), the association of light (LM) and electron microscopic (EM) structural changes with clinical parameters and prognosis in DKD is incompletely understood. METHODS: This is an interim analysis of 62 patients diagnosed with biopsy-confirmed DKD from the multicenter TRIDENT (Transformative Research in Diabetic Nephropathy) study. Twelve LM and 8 EM descriptors, representing changes in glomeruli, tubulointerstitium, and vasculature were analyzed for their relationship with clinical measures of renal function. Patients were followed every 6 months. RESULTS: Multivariable linear regression analysis revealed that estimated glomerular filtration rate (eGFR) upon enrollment correlated the best with interstitial fibrosis. On the other hand, the rate of kidney function decline (eGFR slope) correlated the most with glomerular lesions including global glomerulosclerosis and mesangiolysis. Unbiased clustering analysis based on histopathologic data identified 3 subgroups. The first cluster, encompassing subjects with the mildest histologic lesions, had the most preserved kidney function. The second and third clusters had similar degrees of kidney dysfunction and structural damage, but differed in the degree of glomerular epithelial cell and podocyte injury (podocytopathy DKD subtype). Cox proportional hazard analysis showed that subjects in cluster 2 had the highest risk to reach ESKD (hazard ratio: 17.89; 95% confidence interval: 2.13-149.79). Glomerular epithelial hyperplasia and interstitial fibrosis were significant predictors of ESKD in the multivariate model. CONCLUSION: The study highlights the association between fibrosis and kidney function and identifies the role of glomerular epithelial changes and kidney function decline.
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Drug-related sinusoidal dilatation (SD) is a common form of hepatotoxicity associated with oxaliplatin-based chemotherapy used prior to resection of colorectal liver metastases (CRLM). Recently, hepatic SD has also been associated with anti-delta like 4 (DLL4) cancer therapies targeting the NOTCH pathway. To investigate the hypothesis that NOTCH signaling plays an important role in drug-induced SD, gene expression changes were examined in livers from anti-DLL4 and oxaliplatin-induced SD in non-human primate (NHP) and patients, respectively. Putative mechanistic biomarkers of bevacizumab (bev)-mediated protection against oxaliplatin-induced SD were also investigated. RNA was extracted from whole liver sections or centrilobular regions by laser-capture microdissection (LCM) obtained from NHP administered anti-DLL4 fragment antigen-binding (F(ab')2 or patients with CRLM receiving oxaliplatin-based chemotherapy with or without bev. mRNA expression was quantified using high-throughput real-time quantitative PCR. Significance analysis was used to identify genes with differential expression patterns (false discovery rate (FDR) < 0.05). Eleven (CCL2, CCND1, EFNB2, ERG, ICAM1, IL16, LFNG, NOTCH1, NOTCH4, PRDX1, and TGFB1) and six (CDH5, EFNB2, HES1, IL16, MIK67, HES1 and VWF) candidate genes were differentially expressed in the liver of anti-DLL4- and oxaliplatin-induced SD, respectively. Addition of bev to oxaliplatin-based chemotherapy resulted in differential changes in hepatic CDH5, HEY1, IL16, JAG1, MMP9, NOTCH4 and TIMP1 expression. This work implicates NOTCH and IL16 pathways in the pathogenesis of drug-induced SD and further explains the hepato-protective effect of bev in oxaliplatin-induced SD observed in CRLM patients.
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Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Neoplasias Colorrectales/tratamiento farmacológico , Hígado/efectos de los fármacos , Hígado/patología , Oxaliplatino/efectos adversos , Transcriptoma , Anciano , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biopsia , Capilares/efectos de los fármacos , Capilares/metabolismo , Capilares/patología , Neoplasias Colorrectales/patología , Dilatación Patológica/inducido químicamente , Dilatación Patológica/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Hígado/irrigación sanguínea , Hígado/metabolismo , Neoplasias Hepáticas/secundario , Macaca fascicularis , Masculino , Persona de Mediana Edad , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/genética , Oxaliplatino/administración & dosificación , Transcriptoma/efectos de los fármacosRESUMEN
Inhibition of the kinase activity of leucine-rich repeat kinase 2 (LRRK2) is under investigation as a possible treatment for Parkinson's disease. However, there is no clinical validation as yet, and the safety implications of targeting LRRK2 kinase activity are not well understood. We evaluated the potential safety risks by comparing human and mouse LRRK2 mRNA tissue expression, by analyzing a Lrrk2 knockout mouse model, and by testing selective brain-penetrating LRRK2 kinase inhibitors in multiple species. LRRK2 mRNA tissue expression was comparable between species. Phenotypic analysis of Lrrk2 knockout mice revealed morphologic changes in lungs and kidneys, similar to those reported previously. However, in preclinical toxicity assessments in rodents, no pulmonary or renal changes were induced by two distinct LRRK2 kinase inhibitors. Both of these kinase inhibitors induced abnormal cytoplasmic accumulation of secretory lysosome-related organelles known as lamellar bodies in type II pneumocytes of the lung in nonhuman primates, but no lysosomal abnormality was observed in the kidney. The pulmonary change resembled the phenotype of Lrrk2 knockout mice, suggesting that this was LRRK2-mediated rather than a nonspecific or off-target effect. A biomarker of lysosomal dysregulation, di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP), was also decreased in the urine of Lrrk2 knockout mice and nonhuman primates treated with LRRK2 kinase inhibitors. Our results suggest a role for LRRK2 in regulating lysosome-related lamellar bodies and that pulmonary toxicity may be a critical safety liability for LRRK2 kinase inhibitors in patients.
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Pulmón/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/patología , Animales , Biomarcadores/sangre , Biomarcadores/orina , Relación Dosis-Respuesta a Droga , Femenino , Células HEK293 , Humanos , Riñón/anomalías , Riñón/efectos de los fármacos , Riñón/patología , Riñón/ultraestructura , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Pulmón/anomalías , Pulmón/patología , Pulmón/ultraestructura , Macaca fascicularis , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Morfolinas/química , Morfolinas/farmacología , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Pirazoles/química , Pirazoles/farmacología , Pirimidinas/química , Pirimidinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-DawleyRESUMEN
The ability to distinguish between similar experiences is a critical feature of episodic memory and is primarily regulated by the dentate gyrus (DG) region of the hippocampus. However, the molecular mechanisms underlying such pattern separation tasks are poorly understood. We report a novel role for the small GTPase ADP ribosylation factor 4 (Arf4) in controlling pattern separation by regulating dendritic spine development. Arf4(+/-) mice at 4-5 months of age display severe impairments in a pattern separation task, as well as significant dendritic spine loss and smaller miniature excitatory post-synaptic currents (mEPSCs) in granule cells of the DG. Arf4 knockdown also decreases spine density in primary neurons, whereas Arf4 overexpression promotes spine development. A constitutively active form of Arf4, Arf4-Q71L, promotes spine density to an even greater extent than wildtype Arf4, whereas the inactive Arf4-T31N mutant does not increase spine density relative to controls. Arf4's effects on spine development are regulated by ASAP1, a GTPase-activating protein that modulates Arf4 GTPase activity. ASAP1 overexpression decreases spine density, and this effect is partially rescued by concomitant overexpression of wildtype Arf4 or Arf4-Q71L. In addition, Arf4 overexpression rescues spine loss in primary neurons from an Alzheimer's disease-related apolipoprotein (apo) E4 mouse model. Our findings suggest that Arf4 is a critical modulator of DG-mediated pattern separation by regulating dendritic spine development.
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Factores de Ribosilacion-ADP/metabolismo , Espinas Dendríticas/metabolismo , Giro Dentado/metabolismo , Factores de Ribosilacion-ADP/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células Cultivadas , Electrofisiología , Inmunohistoquímica , Ratones , Ratones MutantesRESUMEN
Apolipoprotein E4 (apoE4), the major genetic risk factor for late onset Alzheimer disease, assumes a pathological conformation, intramolecular domain interaction. ApoE4 domain interaction mediates the detrimental effects of apoE4, including decreased mitochondrial cytochrome c oxidase subunit 1 levels, reduced mitochondrial motility, and reduced neurite outgrowth in vitro. Mutant apoE4 (apoE4-R61T) lacks domain interaction, behaves like apoE3, and does not cause detrimental effects. To identify small molecules that inhibit domain interaction (i.e. structure correctors) and reverse the apoE4 detrimental effects, we established a high throughput cell-based FRET primary assay that determines apoE4 domain interaction and secondary cell- and function-based assays. Screening a ChemBridge library with the FRET assay identified CB9032258 (a phthalazinone derivative), which inhibits domain interaction in neuronal cells. In secondary functional assays, CB9032258 restored mitochondrial cytochrome c oxidase subunit 1 levels and rescued impairments of mitochondrial motility and neurite outgrowth in apoE4-expressing neuronal cells. These benefits were apoE4-specific and dose-dependent. Modifying CB9032258 yielded well defined structure-activity relationships and more active compounds with enhanced potencies in the FRET assay (IC(50) of 23 and 116 nm, respectively). These compounds efficiently restored functional activities of apoE4-expressing cells in secondary assays. An EPR binding assay showed that the apoE4 structure correction resulted from direct interaction of a phthalazinone. With these data, a six-feature pharmacophore model was constructed for future drug design. Our results serve as a proof of concept that pharmacological intervention with apoE4 structure correctors negates apoE4 detrimental effects in neuronal cells and could be further developed as an Alzheimer disease therapeutic.
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Apolipoproteína E4/antagonistas & inhibidores , Apolipoproteína E4/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Apolipoproteína E4/química , Línea Celular , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Neuronas/metabolismo , Ftalazinas/química , Ftalazinas/farmacología , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.
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Apolipoproteína E4/metabolismo , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Transporte Biológico , Línea Celular , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Aparato de Golgi/metabolismo , Hipocampo/citología , Humanos , Ratones , Modelos Biológicos , Mutación , Neuronas/metabolismoRESUMEN
Amyloid-ß (Aß) peptides, derived from the amyloid precursor protein, and the microtubule-associated protein tau are key pathogenic factors in Alzheimer's disease (AD). How exactly they impair cognitive functions is unknown. We assessed the effects of Aß and tau on axonal transport of mitochondria and the neurotrophin receptor TrkA, cargoes that are critical for neuronal function and survival and whose distributions are altered in AD. Aß oligomers rapidly inhibited axonal transport of these cargoes in wild-type neurons. Lowering tau levels prevented these defects without affecting baseline axonal transport. Thus, Aß requires tau to impair axonal transport, and tau reduction protects against Aß-induced axonal transport defects.
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Péptidos beta-Amiloides/metabolismo , Transporte Axonal , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas tau/metabolismo , Péptidos beta-Amiloides/farmacología , Animales , Células Cultivadas , Hipocampo/citología , Ratones , Mitocondrias/metabolismo , Fragmentos de Péptidos/farmacología , Receptor trkA/metabolismoRESUMEN
Neuronal expression of apolipoprotein (apo) E4 may contribute to the pathogenesis of Alzheimer's disease (AD). In studying how apoE expression is regulated in neurons, we identified a splicing variant of apoE mRNA with intron-3 retention (apoE-I3). ApoE-I3 mRNA was detected in neuronal cell lines and primary neurons, but not in astrocytic cell lines or primary astrocytes, from humans and mice by reverse transcription (RT)-PCR. In both wild-type and human apoE knock-in mice, apoE-I3 was found predominantly in cortical and hippocampal neurons by in situ hybridization. Cell fractionation and quantitative RT-PCR revealed that over 98% of the apoE-I3 mRNA was retained in the nucleus without protein translation. In transfected primary neurons, apoE expression increased dramatically when intron-3 was deleted from a genomic DNA construct and decreased markedly when intron-3 was inserted into a cDNA construct, suggesting that intron-3 retention/splicing controls apoE expression in neurons. In response to excitotoxic challenge, the apoE-I3 mRNA was markedly increased in morphologically normal hippocampal neurons but reduced in degenerating hippocampal neurons in mice; apoE mRNA showed the opposite pattern. This apparent precursor-product relationship between apoE-I3 and apoE mRNA was supported by a transcriptional inhibition study. Thus, neuronal expression of apoE is controlled by transcription of apoE-I3 under normal conditions and by processing of apoE-I3 into mature apoE mRNA in response to injury.
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Apolipoproteínas E/biosíntesis , Apolipoproteínas E/genética , Corteza Cerebral/metabolismo , Regulación de la Expresión Génica/fisiología , Hipocampo/metabolismo , Intrones/fisiología , Neuronas/metabolismo , Empalme del ARN/fisiología , Animales , Apolipoproteínas E/metabolismo , Línea Celular , Corteza Cerebral/citología , Hipocampo/citología , Humanos , Hibridación in Situ , Ratones , Ratones Mutantes , Procesamiento Postranscripcional del ARN/fisiologíaRESUMEN
Convergent evidence has revealed an association between insulin resistance and Alzheimer's disease (AD), and the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, rosiglitazone, an insulin sensitizer and mitochondrial activator, improves cognition in patients with early or mild-to-moderate AD. Apolipoprotein (apo) E4, a major genetic risk factor for AD, exerts neuropathological effects through multiple pathways, including impairment of dendritic spine structure and mitochondrial function. Here we show that rosiglitazone significantly increased dendritic spine density in a dose-dependent manner in cultured primary cortical rat neurons. This effect was abolished by the PPAR-gamma-specific antagonist, GW9662, suggesting that rosiglitazone exerts this effect by activating the PPAR-gamma pathway. Furthermore, the C-terminal-truncated fragment of apoE4 significantly decreased dendritic spine density. Rosiglitazone rescued this detrimental effect. Thus, rosiglitazone might improve cognition in AD patients by increasing dendritic spine density.
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Apolipoproteína E4/fisiología , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Dendritas/efectos de los fármacos , Espinas Dendríticas/efectos de los fármacos , Neuronas/metabolismo , Tiazolidinedionas/farmacología , Animales , Animales Recién Nacidos , Apolipoproteína E4/biosíntesis , Recuento de Células , Células Cultivadas , Corteza Cerebral/patología , Neuronas/patología , Ratas , RosiglitazonaRESUMEN
The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and cerebellar ataxia. A mutation in Cacna2d2, the gene encoding the alpha 2 delta-2 voltage-dependent calcium channel accessory subunit, has been found to underlie the ducky phenotype. The alpha 2 delta-2 mRNA is strongly expressed in cerebellar Purkinje cells. We show that du/du mice have abnormalities in their Purkinje cell dendritic tree. The mutation in alpha 2 delta-2 results in the introduction of a premature stop codon and predicts the expression of a truncated protein encoded by the first three exons of Cacna2d2, followed by 8 novel amino acids. We show that both mRNA and protein corresponding to this predicted transcript are expressed in du/du cerebellum and present in Purkinje cells. Whereas the alpha 2 delta-2 subunit increased the peak current density of the Ca(V)2.1/beta(4) channel combination when co-expressed in vitro, co-expression with the truncated mutant alpha 2 delta-2 protein reduced current density, indicating that it may contribute to the du phenotype.