RESUMO
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.
Assuntos
Colágeno/metabolismo , Fibroblastos/citologia , Lectinas Tipo C/metabolismo , Linfonodos/citologia , Glicoproteínas de Membrana/metabolismo , Amidas/farmacologia , Animais , Sobrevivência Celular/imunologia , Colágeno/imunologia , Citoesqueleto/imunologia , Citoesqueleto/ultraestrutura , Inibidores Enzimáticos/farmacologia , Feminino , Fibroblastos/imunologia , Fibroblastos/ultraestrutura , Lectinas Tipo C/imunologia , Linfonodos/imunologia , Linfonodos/ultraestrutura , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Fosforilação , Piridinas/farmacologia , Organismos Livres de Patógenos EspecíficosRESUMO
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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Doença de Alzheimer , Glicoproteínas de Membrana , Microglia , Esclerose Múltipla , Receptores Imunológicos , Animais , Receptores Imunológicos/agonistas , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Glicoproteínas de Membrana/agonistas , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Camundongos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Feminino , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Anticorpos/farmacologia , Humanos , Peptídeos beta-Amiloides/metabolismo , Proteínas tau/metabolismoRESUMO
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.
Assuntos
Imagem de Tensor de Difusão , Modelos Animais de Doenças , Fibrose , Camundongos Endogâmicos C57BL , Insuficiência Renal Crônica , Animais , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/diagnóstico por imagem , Masculino , Oxalatos/metabolismo , Rim/patologia , Rim/diagnóstico por imagem , Rim/metabolismo , CamundongosRESUMO
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.
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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.
Assuntos
Endorribonucleases/genética , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Idoso , Animais , Bortezomib/farmacologia , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/antagonistas & inibidores , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lenalidomida/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-Box/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Roedores , Animais , Biomarcadores , Humanos , Isotiocianatos , Ratos , Reprodutibilidade dos Testes , Canal de Cátion TRPA1RESUMO
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.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Biomarcadores/líquido cefalorraquidiano , Estudos de Coortes , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Doença de Alzheimer/diagnóstico por imagem , Radioisótopos de Flúor/farmacocinética , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/farmacocinética , Proteínas tau/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Feminino , Radioisótopos de Flúor/administração & dosagem , Humanos , Cinética , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/metabolismo , Tomografia por Emissão de Pósitrons/normas , Ligação Proteica , Compostos Radiofarmacêuticos/administração & dosagem , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Doença de Alzheimer/complicações , Transtornos de Deficit da Atenção e do Comportamento Disruptivo/induzido quimicamente , Transtornos da Memória/etiologia , Transtornos da Memória/patologia , Sinapses/patologia , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Técnicas In Vitro , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Piperidinas/farmacologiaRESUMO
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.
Assuntos
Doença de Alzheimer/fisiopatologia , Precursor de Proteína beta-Amiloide/fisiologia , Sistema Nervoso Central/citologia , Receptores do Fator de Necrose Tumoral/fisiologia , Transdução de Sinais/fisiologia , Sinapses/fisiologia , Doença de Alzheimer/patologia , Animais , Aprendizagem da Esquiva/fisiologia , Sistema Nervoso Central/crescimento & desenvolvimento , Condicionamento Operante/fisiologia , Espinhas Dendríticas/fisiologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Medo/psicologia , Gliose/patologia , Humanos , Hibridização In Situ , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/fisiologia , Vias Neurais/fisiologia , Placa Amiloide/patologiaRESUMO
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.
Assuntos
Secretases da Proteína Precursora do Amiloide/fisiologia , Precursor de Proteína beta-Amiloide/genética , Axônios/fisiologia , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Contagem de Células , Células Cultivadas , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Degeneração Neural/genética , Degeneração Neural/patologia , Ligação Proteica , RNA Interferente Pequeno/genética , Células Ganglionares da Retina/fisiologia , Células Receptoras Sensoriais/fisiologiaRESUMO
Purpose: We evaluated the impact of partial volume correction (PVC) methods on the quantification of longitudinal [18F]GTP1 tau positron-emission tomography (PET) in Alzheimer's disease and the suitability of describing the tau pathology burden temporal trajectories using linear mixed-effects models (LMEM). Methods: We applied van Cittert iterative deconvolution (VC), 2-compartment, and 3-compartment, and the geometric transfer matrix plus region-based voxelwise methods to data acquired in an Alzheimer's disease natural history study over 18 months at a single imaging site. We determined the optimal PVC method by comparing the standardized uptake value ratio change (%ΔSUVR) between diagnostic and tau burden-level groups and the longitudinal repeatability derived from the LMEM. The performance of LMEM analysis for calculating %ΔSUVR was evaluated in a natural history study and in a multisite clinical trial of semorinemab in prodromal to mild Alzheimer's disease by comparing results to traditional per-visit estimates. Results: The VC, 2-compartment, and 3-compartment PVC methods had similar performance, whereas region-based voxelwise overcorrected regions with a higher tau burden. The lowest within-subject variability and acceptable group separation scores were observed without PVC. The LMEM-derived %ΔSUVR values were similar to the per-visit estimates with lower variability. Conclusion: The results indicate that the tested PVC methods do not offer a clear advantage or improvement over non-PVC images for the quantification of longitudinal [18F]GTP1 PET data. LMEM offers a robust framework for the longitudinal tau PET quantification with low longitudinal test-retest variability. Clinical trial registration: NCT02640092 and NCT03289143.
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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.
Assuntos
Axônios/enzimologia , Caspase 3/fisiologia , Caspase 6/fisiologia , Degeneração Neural/enzimologia , Colículos Superiores/crescimento & desenvolvimento , Animais , Axônios/patologia , Axônios/ultraestrutura , Caspase 3/genética , Caspase 6/genética , Células Cultivadas , Ativação Enzimática/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Imagem Molecular/métodos , Degeneração Neural/genética , Degeneração Neural/patologia , Fator de Crescimento Neural/efeitos adversos , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Células Receptoras Sensoriais/enzimologia , Células Receptoras Sensoriais/patologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Colículos Superiores/enzimologia , Degeneração Walleriana/enzimologia , Degeneração Walleriana/genética , Degeneração Walleriana/patologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/fisiologiaRESUMO
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.
Assuntos
Axônios/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Degeneração Neural/enzimologia , Degeneração Neural/patologia , Neurônios/patologia , Fosfotransferases/metabolismo , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Eletroporação , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Feminino , Gânglios Espinais/citologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genótipo , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Humanos , Imunoprecipitação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Degeneração Neural/tratamento farmacológico , Degeneração Neural/prevenção & controle , Fator de Crescimento Neural/deficiência , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Cultura de Órgãos , Fosforilação/fisiologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Células Ganglionares da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Proteína Vermelha FluorescenteRESUMO
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.
Assuntos
Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/imunologia , Imunoglobulina G/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Animais Recém-Nascidos , Receptor 1 de Quimiocina CX3C , Células Cultivadas , Córtex Cerebral/citologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Relação Dose-Resposta Imunológica , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Humanos , Imunoglobulina G/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Pessoa de Meia-Idade , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Placa Amiloide/imunologia , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Presenilina-1/genética , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Quimiocinas/genética , Estatísticas não Paramétricas , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Priming of the organ-specific premetastatic sites is thought to be an important yet incompletely understood step during metastasis. In this study, we show that the metastatic tumors we examined overexpress granulocyte-colony stimulating factor (G-CSF), which expands and mobilizes Ly6G+Ly6C+ granulocytes and facilitates their subsequent homing at distant organs even before the arrival of tumor cells. Moreover, G-CSF-mobilized Ly6G+Ly6C+ cells produce the Bv8 protein, which has been implicated in angiogenesis and mobilization of myeloid cells. Anti-G-CSF or anti-Bv8 antibodies significantly reduced lung metastasis. Transplantation of Bv8 null fetal liver cells into lethally irradiated hosts also reduced metastasis. We identified an unexpected role for Bv8: the ability to stimulate tumor cell migration through activation of one of the Bv8 receptors, prokineticin receptor (PKR)-1. Finally, we show that administration of recombinant G-CSF is sufficient to increase the numbers of Ly6G+Ly6C+ cells in organ-specific metastatic sites and results in enhanced metastatic ability of several tumors.
Assuntos
Antígenos Ly/imunologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Granulócitos/efeitos dos fármacos , Granulócitos/imunologia , Neoplasias Pulmonares/patologia , Metástase Neoplásica , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Feminino , Perfilação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/genética , Granulócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos SCID , Análise em Microsséries , Transplante de Neoplasias , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
INTRODUCTION: Recently, an increasing number of tau tracers have become available. There is a need to standardize quantitative tau measures across tracers, supporting a universal scale. We developed several cortical tau masks and applied them to generate a tau imaging universal scale. METHOD: One thousand forty-five participants underwent tau scans with either 18F-flortaucipir, 18F-MK6240, 18F-PI2620, 18F-PM-PBB3, 18F-GTP1, or 18F-RO948. The universal mask was generated from cognitively unimpaired amyloid beta (Aß)- subjects and Alzheimer's disease (AD) patients with Aß+. Four additional regional cortical masks were defined within the constraints of the universal mask. A universal scale, the CenTauRz, was constructed. RESULTS: None of the regions known to display off-target signal were included in the masks. The CenTauRz allows robust discrimination between low and high levels of tau deposits. DISCUSSION: We constructed several tau-specific cortical masks for the AD continuum and a universal standard scale designed to capture the location and degree of abnormality that can be applied across tracers and across centers. The masks are freely available at https://www.gaain.org/centaur-project.
RESUMO
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.
Assuntos
Angiotensina II/efeitos adversos , Aorta Abdominal/patologia , Aorta Abdominal/fisiopatologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/fisiopatologia , Animais , Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/patologia , Fenômenos Biomecânicos , Pressão Sanguínea/fisiologia , Progressão da Doença , Elastina/metabolismo , Hipertensão/fisiopatologia , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Modelos Cardiovasculares , UltrassonografiaRESUMO
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.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Cadeias Pesadas de Clatrina/metabolismo , Endocitose , Receptores de Superfície Celular/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Cadeias Pesadas de Clatrina/genética , Mutação/genética , Junção Neuromuscular/citologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Transmissão Sináptica , Vesículas Sinápticas/ultraestruturaRESUMO
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.