RESUMEN
In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.
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Colágeno/metabolismo , Fibroblastos/citología , Lectinas Tipo C/metabolismo , Ganglios Linfáticos/citología , Glicoproteínas de Membrana/metabolismo , Amidas/farmacología , Animales , Supervivencia Celular/inmunología , Colágeno/inmunología , Citoesqueleto/inmunología , Citoesqueleto/ultraestructura , Inhibidores Enzimáticos/farmacología , Femenino , Fibroblastos/inmunología , Fibroblastos/ultraestructura , Lectinas Tipo C/inmunología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/ultraestructura , Masculino , Glicoproteínas de Membrana/inmunología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Fosforilación , Piridinas/farmacología , Organismos Libres de Patógenos EspecíficosRESUMEN
Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-ß (Aß) pathology (PS2APP) or combined Aß and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches.
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Enfermedad de Alzheimer , Glicoproteínas de Membrana , Microglía , Esclerosis Múltiple , Receptores Inmunológicos , Animales , Receptores Inmunológicos/agonistas , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Glicoproteínas de Membrana/agonistas , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Ratones , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Femenino , Masculino , Microglía/efectos de los fármacos , Microglía/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Ratones Transgénicos , Anticuerpos/farmacología , Humanos , Péptidos beta-Amiloides/metabolismo , Proteínas tau/metabolismoRESUMEN
Chronic kidney disease (CKD) is characterized by inflammation and fibrosis in the kidney. Renal biopsies and estimated glomerular filtration rate (eGFR) remain the standard of care, but these endpoints have limitations in detecting the stage, progression, and spatial distribution of fibrotic pathology in the kidney. MRI diffusion tensor imaging (DTI) has emerged as a promising noninvasive technology to evaluate renal fibrosis in vivo both in clinical and preclinical studies. However, these imaging studies have not systematically identified fibrosis particularly deeper in the kidney where biopsy sampling is limited, or completed an extensive analysis of whole organ histology, blood biomarkers, and gene expression to evaluate the relative strengths and weaknesses of MRI for evaluating renal fibrosis. In this study, we performed DTI in the sodium oxalate mouse model of CKD. The DTI parameters fractional anisotropy, apparent diffusion coefficient, and axial diffusivity were compared between the control and oxalate groups with region of interest (ROI) analysis to determine changes in the cortex and medulla. In addition, voxel-based analysis (VBA) was implemented to systematically identify local regions of injury over the whole kidney. DTI parameters were found to be significantly different in the medulla by both ROI analysis and VBA, which also spatially matched with collagen III immunohistochemistry (IHC). The DTI parameters in this medullary region exhibited moderate to strong correlations with histology, blood biomarkers, hydroxyproline, and gene expression. Our results thus highlight the sensitivity of DTI to the heterogeneity of renal fibrosis and importance of whole kidney noninvasive imaging.NEW & NOTEWORTHY Chronic kidney disease (CKD) can be characterized by inflammation and fibrosis of the kidney. Although standard of care methods have been limited in scope, safety, and spatial distribution, MRI diffusion tensor imaging (DTI) has emerged as a promising noninvasive technology to evaluate renal fibrosis in vivo. In this study, we performed DTI in an oxalate mouse model of CKD to systematically identify local kidney injury. DTI parameters strongly correlated with histology, blood biomarkers, hydroxyproline, and gene expression.
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Imagen de Difusión Tensora , Modelos Animales de Enfermedad , Fibrosis , Ratones Endogámicos C57BL , Insuficiencia Renal Crónica , Animales , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/diagnóstico por imagen , Masculino , Oxalatos/metabolismo , Riñón/patología , Riñón/diagnóstico por imagen , Riñón/metabolismo , RatonesRESUMEN
INTRODUCTION: Triggering receptor expressed on myeloid cells 2 (TREM2) agonists are being clinically evaluated as disease-modifying therapeutics for Alzheimer's disease. Clinically translatable pharmacodynamic (PD) biomarkers are needed to confirm drug activity and select the appropriate therapeutic dose in clinical trials. METHODS: We conducted multi-omic analyses on paired non-human primate brain and cerebrospinal fluid (CSF), and stimulation of human induced pluripotent stem cell-derived microglia cultures after TREM2 agonist treatment, followed by validation of candidate fluid PD biomarkers using immunoassays. We immunostained microglia to characterize proliferation and clustering. RESULTS: We report CSF soluble TREM2 (sTREM2) and CSF chitinase-3-like protein 1 (CHI3L1/YKL-40) as PD biomarkers for the TREM2 agonist hPara.09. The respective reduction of sTREM2 and elevation of CHI3L1 in brain and CSF after TREM2 agonist treatment correlated with transient microglia proliferation and clustering. DISCUSSION: CSF CHI3L1 and sTREM2 reflect microglial TREM2 agonism and can be used as clinical PD biomarkers to monitor TREM2 activity in the brain. HIGHLIGHTS: CSF soluble triggering receptor expressed on myeloid cells 2 (sTREM2) reflects brain target engagement for a novel TREM2 agonist, hPara.09. CSF chitinase-3-like protein 1 reflects microglial TREM2 agonism. Both can be used as clinical fluid biomarkers to monitor TREM2 activity in brain.
RESUMEN
Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.
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Endorribonucleasas/genética , Mieloma Múltiple/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Anciano , Animales , Bortezomib/farmacología , Estrés del Retículo Endoplásmico/genética , Endorribonucleasas/antagonistas & inhibidores , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lenalidomida/farmacología , Masculino , Ratones , Persona de Mediana Edad , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada/genética , Proteína 1 de Unión a la X-Box/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND AND PURPOSE: Develop a translational assay of Transient Receptor Potential Ankyrin 1 (TRPA1) activity for use as a preclinical and clinical biomarker. EXPERIMENTAL APPROACH: Allyl isothiocyanate (AITC), capsaicin or citric acid were applied to ears of wildtype and Trpa1-knock out (Trpa1 KO) rats, and changes in dermal blood flow (DBF) were measured by laser speckle contrast imaging. In humans, the DBF, pain and itch responses to 5-20% AITC applied to the forearm were measured and safety was evaluated. Reproducibility of the DBF, pain and itch responses to topically applied 10% and 15% AITC were assessed at two visits separated by 13-15 days. DBF changes were summarized at 5-minute intervals as areas under the curve (AUC) and maxima. Intraclass correlation coefficient (ICC) was calculated to assess arm-arm and period-period reproducibility. KEY RESULTS: AITC- and citric acid-induced DBF were significantly reduced in Trpa1 KO rats compared to wildtype (90 ± 2% and 65 ± 11% reduction, respectively), whereas capsaicin response did not differ. In humans, each AITC concentration significantly increased DBF compared to vehicle with the maximal increase occurring 5 minutes post application. Ten percent and 15% AITC were selected as safe and effective stimuli. AUC from 0 to 5 minutes was the most reproducible metric of AITC-induced DBF across arms (ICC = 0.92) and periods (ICC = 0.85). Subject-reported pain was more reproducible than itch across visits (ICC = 0.76 vs 0.17, respectively). CONCLUSION AND IMPLICATIONS: AITC-induced DBF is a suitable target engagement biomarker of TRPA1 activity for preclinical and clinical studies of TRPA1 antagonists.
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Roedores , Animales , Biomarcadores , Humanos , Isotiocianatos , Ratas , Reproducibilidad de los Resultados , Canal Catiónico TRPA1RESUMEN
To date, there is no validated fluid biomarker for tau pathology in Alzheimer's disease, with contradictory results from studies evaluating the correlation between phosphorylated tau in CSF with tau PET imaging. Tau protein is subjected to proteolytic processing into fragments before being secreted to the CSF. A recent study suggested that tau cleavage after amino acid 368 by asparagine endopeptidase (AEP) is upregulated in Alzheimer's disease. We used immunoprecipitation followed by mass spectrometric analyses to evaluate the presence of tau368 species in CSF. A novel Simoa® assay for quantification of tau368 in CSF was developed, while total tau (t-tau) was measured by ELISA and the presence of tau368 in tangles was evaluated using immunohistochemistry. The diagnostic utility of tau368 was first evaluated in a pilot study (Alzheimer's disease = 20, control = 20), then in a second cohort where the IWG-2 biomarker criteria were applied (Alzheimer's disease = 37, control = 45), and finally in a third cohort where the correlation with 18F-GTP1 tau PET was evaluated (Alzheimer's disease = 38, control = 11). The tau368/t-tau ratio was significantly decreased in Alzheimer's disease (P < 0.001) in all cohorts. Immunohistochemical staining demonstrated that tau fragments ending at 368 are present in tangles. There was a strong negative correlation between the CSF tau368/t-tau ratio and 18F-GTP1 retention. Our data suggest that tau368 is a tangle-enriched fragment and that the CSF ratio tau368/t-tau reflects tangle pathology. This novel tau biomarker could be used to improve diagnosis of Alzheimer's disease and to facilitate the development of drug candidates targeting tau pathology. Furthermore, future longitudinal studies will increase our understanding of tau pathophysiology in Alzheimer's disease and other tauopathies.
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Enfermedad de Alzheimer/líquido cefalorraquídeo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Fragmentos de Péptidos/líquido cefalorraquídeo , Proteínas tau/líquido cefalorraquídeo , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Biomarcadores/líquido cefalorraquídeo , Estudios de Cohortes , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVE: Neurofibrillary tangles (NFTs), consisting of intracellular aggregates of the tau protein, are a pathological hallmark of Alzheimer's disease (AD). Here we report the identification and initial characterization of Genentech Tau Probe 1 ([18F]GTP1), a small-molecule PET probe for imaging tau pathology in AD patients. METHODS: Autoradiography using human brain tissues from AD donors and protein binding panels were used to determine [18F]GTP1 binding characteristics. Stability was evaluated in vitro and in vivo in mice and rhesus monkey. In the clinic, whole-body imaging was performed to assess biodistribution and dosimetry. Dynamic [18F]GTP1 brain imaging and input function measurement were performed on two separate days in 5 ß-amyloid plaque positive (Aß+) AD and 5 ß-amyloid plaque negative (Aß-) cognitive normal (CN) participants. Tracer kinetic modeling was applied and reproducibility was evaluated. SUVR was calculated and compared to [18F]GTP1-specific binding parameters derived from the kinetic modeling. [18F]GTP1 performance in a larger cross-sectional group of 60 Aß+ AD participants and ten (Aß- or Aß+) CN was evaluated with images acquired 60 to 90 min post tracer administration. RESULTS: [18F]GTP1 exhibited high affinity and selectivity for tau pathology with no measurable binding to ß-amyloid plaques or MAO-B in AD tissues, or binding to other tested proteins at an affinity predicted to impede image data interpretation. In human, [18F]GTP1 exhibited favorable dosimetry and brain kinetics, and no evidence of defluorination. [18F]GTP1-specific binding was observed in cortical regions of the brain predicted to contain tau pathology in AD and exhibited low (< 4%) test-retest variability. SUVR measured in the 60 to 90-min interval post injection correlated with tracer-specific binding (slope = 1.36, r2 = 0.98). Furthermore, in a cross-sectional population, the degree of [18F]GTP1-specific binding increased with AD severity and could differentiate diagnostic cohorts. CONCLUSIONS: [18F]GTP1 is a promising PET probe for the study of tau pathology in AD.
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Enfermedad de Alzheimer/diagnóstico por imagen , Radioisótopos de Flúor/farmacocinética , Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Proteínas tau/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Femenino , Radioisótopos de Flúor/administración & dosificación , Humanos , Cinética , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Ovillos Neurofibrilares/metabolismo , Tomografía de Emisión de Positrones/normas , Unión Proteica , Radiofármacos/administración & dosificación , Sensibilidad y EspecificidadRESUMEN
Extensive evidence implicates GluN2B-containing NMDA receptors (GluN2B-NMDARs) in excitotoxic-insult-induced neurodegeneration and amyloid ß (Aß)-induced synaptic dysfunction. Therefore, inhibiting GluN2B-NMDARs would appear to be a potential therapeutic strategy to provide neuroprotection and improve cognitive function in Alzheimer's disease (AD). However, there are no reports of long-term in vivo treatment of AD mouse models with GluN2B antagonists. We used piperidine18 (Pip18), a potent and selective GluN2B-NMDAR antagonist with favorable pharmacokinetic properties, for long-term dosing in AD mouse models. Reduced freezing behavior in Tg2576 mice during fear conditioning was partially reversed after subchronic (17 d) Pip18 treatment. However, analysis of freezing behavior in different contexts indicated that this increased freezing likely involves elevated anxiety or excessive memory generalization in both nontransgenic (NTG) and Tg2576 mice. In PS2APP mice chronically fed with medicated food containing Pip18 for 4 months, spatial learning and memory deficits were not rescued, plaque-associated spine loss was not affected, and synaptic function was not altered. At the same time, altered open field activity consistent with increased anxiety and degraded performance in an active avoidance task were observed in NTG after chronic treatment. These results indicate that long-term treatment with a GluN2B-NMDAR antagonist does not provide a disease-modifying benefit and could cause cognitive liabilities rather than symptomatic benefit in AD mouse models. Therefore, these results challenge the expectation of the therapeutic potential for GluN2B-NMDAR antagonists in AD.
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Enfermedad de Alzheimer/complicaciones , Déficit de la Atención y Trastornos de Conducta Disruptiva/inducido químicamente , Trastornos de la Memoria/etiología , Trastornos de la Memoria/patología , Sinapsis/patología , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Técnicas In Vitro , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Piperidinas/farmacologíaRESUMEN
Recent studies implicate death receptor 6 (DR6) in an amyloid precursor protein (APP)-dependent pathway regulating developmental axon pruning, and in a pruning pathway operating during plastic rearrangements in adult brain. DR6 has also been suggested to mediate toxicity in vitro of Aß peptides derived from APP. Given the link between APP, Aß, and Alzheimer's disease (AD), these findings have raised the possibility that DR6 contributes to aspects of neurodegeneration in AD. To test this possibility, we have used mouse models to characterize potential function(s) of DR6 in the adult CNS and in AD-related pathophysiology. We show that DR6 is broadly expressed within the adult CNS and regulates the density of excitatory synaptic connections onto pyramidal neurons in a genetic pathway with APP. DR6 knock-out also gives rise to behavioral abnormalities, some of which are similar to those previously documented in APP knock-out animals. However, in two distinct APP transgenic models of AD, we did not observe any alteration in the formation of amyloid plaques, gliosis, synaptic loss, or cognitive behavioral deficits with genetic deletion of DR6, though we did observe a transient reduction in the degree of microglial activation in one model. Our results support the view that DR6 functions with APP to modulate synaptic density in the adult CNS, but do not provide evidence for a role of DR6 in the pathophysiology of AD.
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Enfermedad de Alzheimer/fisiopatología , Precursor de Proteína beta-Amiloide/fisiología , Sistema Nervioso Central/citología , Receptores del Factor de Necrosis Tumoral/fisiología , Transducción de Señal/fisiología , Sinapsis/fisiología , Enfermedad de Alzheimer/patología , Animales , Reacción de Prevención/fisiología , Sistema Nervioso Central/crecimiento & desarrollo , Condicionamiento Operante/fisiología , Espinas Dendríticas/fisiología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Miedo/psicología , Gliosis/patología , Humanos , Hibridación in Situ , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Actividad Motora/fisiología , Vías Nerviosas/fisiología , Placa Amiloide/patologíaRESUMEN
In the developing brain, initial neuronal projections are formed through extensive growth and branching of developing axons, but many branches are later pruned to sculpt the mature pattern of connections. Despite its widespread occurrence, the mechanisms controlling pruning remain incompletely characterized. Based on pharmacological and biochemical analysis in vitro and initial genetic analysis in vivo, prior studies implicated a pathway involving binding of the Amyloid Precursor Protein (APP) to Death Receptor 6 (DR6) and activation of a downstream caspase cascade in axonal pruning. Here, we further test their involvement in pruning in vivo and their mechanism of action through extensive genetic and biochemical analysis. Genetic deletion of DR6 was previously shown to impair pruning of retinal axons in vivo. We show that genetic deletion of APP similarly impairs pruning of retinal axons in vivo and provide evidence that APP and DR6 act cell autonomously and in the same pathway to control pruning. Prior analysis had suggested that ß-secretase cleavage of APP and binding of an N-terminal fragment of APP to DR6 is required for their actions, but further genetic and biochemical analysis reveals that ß-secretase activity is not required and that high-affinity binding to DR6 requires a more C-terminal portion of the APP ectodomain. These results provide direct support for the model that APP and DR6 function cell autonomously and in the same pathway to control pruning in vivo and raise the possibility of alternate mechanisms for how APP and DR6 control pruning.
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Secretasas de la Proteína Precursora del Amiloide/fisiología , Precursor de Proteína beta-Amiloide/genética , Axones/fisiología , Receptores del Factor de Necrosis Tumoral/genética , Transducción de Señal/fisiología , Animales , Animales Modificados Genéticamente , Recuento de Células , Células Cultivadas , Ganglios Espinales/citología , Ganglios Espinales/fisiología , Inmunohistoquímica , Inmunoprecipitación , Ratones , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Unión Proteica , ARN Interferente Pequeño/genética , Células Ganglionares de la Retina/fisiología , Células Receptoras Sensoriales/fisiologíaRESUMEN
Axon degeneration initiated by trophic factor withdrawal shares many features with programmed cell death, but many prior studies discounted a role for caspases in this process, particularly Caspase-3. Recently, Caspase-6 was implicated based on pharmacological and knockdown evidence, and we report here that genetic deletion of Caspase-6 indeed provides partial protection from degeneration. However, we find at a biochemical level that Caspase-6 is activated effectively only by Caspase-3 but not other "upstream" caspases, prompting us to revisit the role of Caspase-3. In vitro, we show that genetic deletion of Caspase-3 is fully protective against sensory axon degeneration initiated by trophic factor withdrawal, but not injury-induced Wallerian degeneration, and we define a biochemical cascade from prosurvival Bcl2 family regulators to Caspase-9, then Caspase-3, and then Caspase-6. Only low levels of active Caspase-3 appear to be required, helping explain why its critical role has been obscured in prior studies. In vivo, Caspase-3 and Caspase-6-knockout mice show a delay in developmental pruning of retinocollicular axons, thereby implicating both Caspase-3 and Caspase-6 in axon degeneration that occurs as a part of normal development.
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Axones/enzimología , Caspasa 3/fisiología , Caspasa 6/fisiología , Degeneración Nerviosa/enzimología , Colículos Superiores/crecimiento & desarrollo , Animales , Axones/patología , Axones/ultraestructura , Caspasa 3/genética , Caspasa 6/genética , Células Cultivadas , Activación Enzimática/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Imagen Molecular/métodos , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Factor de Crecimiento Nervioso/efectos adversos , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Células Receptoras Sensoriales/enzimología , Células Receptoras Sensoriales/patología , Transducción de Señal/genética , Transducción de Señal/fisiología , Colículos Superiores/enzimología , Degeneración Walleriana/enzimología , Degeneración Walleriana/genética , Degeneración Walleriana/patología , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/fisiologíaRESUMEN
In addition to being a hallmark of neurodegenerative disease, axon degeneration is used during development of the nervous system to prune unwanted connections. In development, axon degeneration is tightly regulated both temporally and spatially. Here, we provide evidence that degeneration cues are transduced through various kinase pathways functioning in spatially distinct compartments to regulate axon degeneration. Intriguingly, glycogen synthase kinase-3 (GSK3) acts centrally, likely modulating gene expression in the cell body to regulate distally restricted axon degeneration. Through a combination of genetic and pharmacological manipulations, including the generation of an analog-sensitive kinase allele mutant mouse for GSK3ß, we show that the ß isoform of GSK3, not the α isoform, is essential for developmental axon pruning in vitro and in vivo. Additionally, we identify the dleu2/mir15a/16-1 cluster, previously characterized as a regulator of B-cell proliferation, and the transcription factor tbx6, as likely downstream effectors of GSK3ß in axon degeneration.
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Axones/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/patología , Neuronas/patología , Fosfotransferasas/metabolismo , Transducción de Señal/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Electroporación , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Femenino , Ganglios Espinales/citología , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Genotipo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Proteínas Fluorescentes Verdes/genética , Hipocampo/citología , Humanos , Inmunoprecipitación , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/prevención & control , Factor de Crecimiento Nervioso/deficiencia , Proteínas del Tejido Nervioso/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Técnicas de Cultivo de Órganos , Fosforilación/fisiología , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Células Ganglionares de la Retina/metabolismo , Transducción de Señal/efectos de los fármacos , Transfección , Proteína Fluorescente RojaRESUMEN
Passive immunization against ß-amyloid (Aß) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aß monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aß, protected against Aß1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aß oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aß, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aß antibody that takes advantage of a unique Aß binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.
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Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/inmunología , Inmunoglobulina G/farmacología , Microglía/efectos de los fármacos , Microglía/metabolismo , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Animales Recién Nacidos , Receptor 1 de Quimiocinas CX3C , Células Cultivadas , Corteza Cerebral/citología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta Inmunológica , Método Doble Ciego , Ensayo de Inmunoadsorción Enzimática , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Proteínas Fluorescentes Verdes/genética , Hipocampo/citología , Humanos , Inmunoglobulina G/metabolismo , Masculino , Ratones , Ratones Transgénicos , Microscopía Confocal , Persona de Mediana Edad , Mutación/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Placa Amiloide/inmunología , Placa Amiloide/metabolismo , Placa Amiloide/patología , Presenilina-1/genética , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de Quimiocina/genética , Estadísticas no Paramétricas , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: To quantitatively compare aortic curvature and motion with resulting aneurysm location, direction of expansion, and pathophysiological features in experimental abdominal aortic aneurysms (AAAs). METHODS AND RESULTS: MRI was performed at 4.7 T with the following parameters: (1) 3D acquisition for vessel geometry and (2) 2D cardiac-gated acquisition to quantify luminal motion. Male 24-week-old mice were imaged before and after AAA formation induced by angiotensin II (AngII)-filled osmotic pump implantation or infusion of elastase. AngII-induced AAAs formed near the location of maximum abdominal aortic curvature, and the leftward direction of expansion was correlated with the direction of suprarenal aortic motion. Elastase-induced AAAs formed in a region of low vessel curvature and had no repeatable direction of expansion. AngII significantly increased mean blood pressure (22.7 mm Hg, P<0.05), whereas both models showed a significant 2-fold decrease in aortic cyclic strain (P<0.05). Differences in patterns of elastin degradation and localization of fluorescent signal from protease-activated probes were also observed. CONCLUSIONS: The direction of AngII aneurysm expansion correlated with the direction of motion, medial elastin dissection, and adventitial remodeling. Anterior infrarenal aortic motion correlated with medial elastin degradation in elastase-induced aneurysms. Results from both models suggest a relationship between aneurysm pathological features and aortic geometry and motion.
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Angiotensina II/efectos adversos , Aorta Abdominal/patología , Aorta Abdominal/fisiopatología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/fisiopatología , Animales , Aorta Abdominal/diagnóstico por imagen , Aneurisma de la Aorta Abdominal/patología , Fenómenos Biomecánicos , Presión Sanguínea/fisiología , Progresión de la Enfermedad , Elastina/metabolismo , Hipertensión/fisiopatología , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Modelos Cardiovasculares , UltrasonografíaRESUMEN
Clathrin is a coat protein involved in vesicle budding from several membrane-bound compartments within the cell. Here we present an analysis of a temperature-sensitive (ts) mutant of clathrin heavy chain (CHC) in a multicellular animal. As expected Caenorhabditis elegans chc-1(b1025ts) mutant animals are defective in receptor-mediated endocytosis and arrest development soon after being shifted to the restrictive temperature. Steady-state clathrin levels in these mutants are reduced by more than 95% at all temperatures. Hub interactions and membrane associations are lost at the restrictive temperature. chc-1(b1025ts) animals become paralyzed within minutes of exposure to the restrictive temperature because of a defect in the nervous system. Surprisingly synaptic vesicle number is not reduced in chc-1(b1025ts) animals. Consistent with the normal number of vesicles, postsynaptic miniature currents occur at normal frequencies. Taken together, these results indicate that a high level of CHC activity is required for receptor-mediated endocytosis in nonneuronal cells but is largely dispensable for maintenance of synaptic vesicle pools.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Cadenas Pesadas de Clatrina/metabolismo , Endocitosis , Receptores de Superficie Celular/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Cadenas Pesadas de Clatrina/genética , Mutación/genética , Unión Neuromuscular/citología , Unión Neuromuscular/metabolismo , Unión Neuromuscular/ultraestructura , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Transmisión Sináptica , Vesículas Sinápticas/ultraestructuraRESUMEN
BACKGROUND: Understanding patterns of association between CSF phosphorylated tau (p-tau) species and clinical disease severity will aid Alzheimer's disease (AD) diagnosis and treatment. OBJECTIVE: To evaluate changes in tau phosphorylation ratios to brain imaging (amyloid PET, [18F]GTP1 PET, and MRI) and cognition across clinical stages of AD in two different cohorts. METHODS: A mass spectrometry (MS)-based method was used to evaluate the relationship between p-tau/tau phosphorylation ratios on 11 sites in CSF and AD pathology measured by tau PET ([18F]GTP1) and amyloid PET ([18F]florbetapir or [18F]florbetaben). Cohort A included cognitively normal amyloid negative (nâ=â6) and positive (nâ=â5) individuals, and amyloid positive prodromal (nâ=â13), mild (nâ=â12), and moderate AD patients (nâ=â10); and Cohort B included amyloid positive prodromal (nâ=â24) and mild (nâ=â40) AD patients. RESULTS: In this cross-sectional analysis, we identified clusters of phosphosites with different profiles of phosphorylation ratios across stages of disease. Eight of 11 investigated sites were hyperphosphorylated and associated with SUVR measures from [18F]GTP1 and amyloid PET. Novel sites 111, 153, and 208 may be relevant biomarkers for AD diagnosis to complement tau hyperphosphorylation measures on previously established sites 181, 205, 217, and 231. Hypophosphorylation was detected on residues 175, 199, and 202, and was inversely associated with [18F]GTP1 and amyloid PET. CONCLUSION: Hyperphosphorylated and hypophosphorylated forms of tau are associated with AD pathologies, and due to their different site-specific profiles, they may be used in combination to assist with staging of disease.
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Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/patología , Encéfalo/patología , Tomografía de Emisión de Positrones , Proteínas tau/líquido cefalorraquídeo , Anciano , Enfermedad de Alzheimer/diagnóstico por imagen , Biomarcadores/líquido cefalorraquídeo , Encéfalo/diagnóstico por imagen , Cognición , Estudios Transversales , Femenino , Radioisótopos de Flúor/metabolismo , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Radiofármacos/metabolismoRESUMEN
BACKGROUND: The role and implementation of tau PET imaging for predicting subsequent cognitive decline in Alzheimer's disease (AD) remains uncertain. This study was designed to evaluate the relationship between baseline [18F]GTP1 tau PET and subsequent longitudinal change across multiple cognitive measures over 18 months. METHODS: Our analyses incorporated data from 67 participants, including cognitively normal controls (n = 10) and ß-amyloid (Aß)-positive individuals ([18F] florbetapir Aß PET) with prodromal (n = 26), mild (n = 16), or moderate (n = 15) AD. Baseline measurements included cortical volume (MRI), tau burden ([18F]GTP1 tau PET), and cognitive assessments [Mini-Mental State Examination (MMSE), Clinical Dementia Rating (CDR), 13-item version of the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog13), and Repeatable Battery for the Assessment of Neuropsychological Status (RBANS)]. Cognitive assessments were repeated at 6-month intervals over an 18-month period. Associations between baseline [18F]GTP1 tau PET indices and longitudinal cognitive performance were assessed via univariate (Spearman correlations) and multivariate (linear mixed effects models) approaches. The utility of potential prognostic tau PET cut points was assessed with ROC curves. RESULTS: Univariate analyses indicated that greater baseline [18F]GTP1 tau PET signal was associated with faster rates of subsequent decline on the MMSE, CDR, and ADAS-Cog13 across regions of interest (ROIs). In multivariate analyses adjusted for baseline age, cognitive performance, cortical volume, and Aß PET SUVR, the prognostic performance of [18F]GTP1 SUVR was most robust in the whole cortical gray ROI. When AD participants were dichotomized into low versus high tau subgroups based on baseline [18F]GTP1 PET standardized uptake value ratios (SUVR) in the temporal (cutoff = 1.325) or whole cortical gray (cutoff = 1.245) ROIs, high tau subgroups demonstrated significantly more decline on the MMSE, CDR, and ADAS-Cog13. CONCLUSIONS: Our results suggest that [18F]GTP1 tau PET represents a prognostic biomarker in AD and are consistent with data from other tau PET tracers. Tau PET imaging may have utility for identifying AD patients at risk for more rapid cognitive decline and for stratification and/or enrichment of participant selection in AD clinical trials. Trial registration ClinicalTrials.gov NCT02640092 . Registered on December 28, 2015.
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Enfermedad de Alzheimer , Disfunción Cognitiva , Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/psicología , Péptidos beta-Amiloides , Disfunción Cognitiva/diagnóstico por imagen , Disfunción Cognitiva/psicología , Humanos , Tomografía de Emisión de Positrones/métodos , Proteínas tauRESUMEN
Vascular leakage, or edema, is a serious complication of acute allergic reactions. Vascular leakage is triggered by the release of histamine and serotonin from granules within tissue-resident mast cells. Here, we show that expression of Neutrophil Serine Protease 4 (NSP4) during the early stages of mast cell development regulates mast cell-mediated vascular leakage. In myeloid precursors, the granulocyte-macrophage progenitors (GMPs), loss of NSP4 results in the decrease of cellular levels of histamine, serotonin and heparin/heparan sulfate. Mast cells that are derived from NSP4-deficient GMPs have abnormal secretory granule morphology and a sustained reduction in histamine and serotonin levels. Consequently, in passive cutaneous anaphylaxis and acute arthritis models, mast cell-mediated vascular leakage in the skin and joints is substantially reduced in NSP4-deficient mice. Our findings reveal that NSP4 is required for the proper storage of vasoactive amines in mast cell granules, which impacts mast cell-dependent vascular leakage in mouse models of immune complex-mediated diseases.
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Mastocitos/enzimología , Serina Proteasas/metabolismo , Traslado Adoptivo , Animales , Complejo Antígeno-Anticuerpo , Regulación Enzimológica de la Expresión Génica , Histamina/metabolismo , Ratones , Ratones Noqueados , Neutrófilos , Serina Proteasas/genética , Serotonina/metabolismoRESUMEN
NMDA receptors (NMDARs) play subunit-specific roles in synaptic function and are implicated in neuropsychiatric and neurodegenerative disorders. However, the in vivo consequences and therapeutic potential of pharmacologically enhancing NMDAR function via allosteric modulation are largely unknown. We examine the in vivo effects of GNE-0723, a positive allosteric modulator of GluN2A-subunit-containing NMDARs, on brain network and cognitive functions in mouse models of Dravet syndrome (DS) and Alzheimer's disease (AD). GNE-0723 use dependently potentiates synaptic NMDA receptor currents and reduces brain oscillation power with a predominant effect on low-frequency (12-20 Hz) oscillations. Interestingly, DS and AD mouse models display aberrant low-frequency oscillatory power that is tightly correlated with network hypersynchrony. GNE-0723 treatment reduces aberrant low-frequency oscillations and epileptiform discharges and improves cognitive functions in DS and AD mouse models. GluN2A-subunit-containing NMDAR enhancers may have therapeutic benefits in brain disorders with network hypersynchrony and cognitive impairments.