RESUMEN
JNJ-42491293 is a metabotropic glutamate 2 (mGlu2) positive allosteric modulator (PAM) that was radiolabelled with [11C]- to serve as a positron emission tomography (PET) ligand. Indeed, in vitro, the molecule displays high selectivity at mGlu2 receptors. However, PET experiments performed in rats, macaques and humans, have suggested that [11C]-JNJ-42491293 could interact with an unidentified, non-mGlu2 receptor binding site. The brain distribution of [11C]-JNJ-42491293 has not been determined in the brain of the common marmoset, a small non-human primate increasingly used in neuroscience research. Here, we investigated the distribution of [11C]-JNJ-42491293 in the marmoset brain. Three marmosets underwent brain magnetic resonance imaging (MRI) and 90-min dynamic PET scans with [11C]-JNJ-42491293 in combination with vehicle or the mGlu2 PAM AZD8529 (0.1, 1 and 10 mg/kg). In the scans in which [11C]-JNJ-42491293 was co-administered with vehicle, the brain areas with the highest standardised uptake values (SUVs) were the midbrain, cerebellum and thalamus, while the lowest SUVs were found in the pons. The addition of AZD8529 (0.1, 1 and 10 mg/kg) to [11C]-JNJ-42491293 did not modify the SUVs obtained with [11C]-JNJ-42491293 alone, and ex vivo blocking autoradiography with PAM AZD8529 (10, 100, 300 µM) on marmoset brain sections showed increased signals in the blocking conditions compared to vehicle, suggesting that no competition occurred between the 2 ligands. The results we obtained here do not suggest that [11C]-JNJ-42491293 interacts selectively, or even at all, with mGlu2 receptors in the marmoset, in agreement with findings previously reported in macaque and human.
Asunto(s)
Callithrix , Tomografía de Emisión de Positrones , Ratas , Animales , Tomografía de Emisión de Positrones/métodos , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Sitios de Unión , Unión ProteicaRESUMEN
In vivo biomarker abnormalities provide measures to monitor therapeutic interventions targeting amyloid-ß pathology as well as its effects on downstream processes associated with Alzheimer's disease pathophysiology. Here, we applied an in vivo longitudinal study design combined with imaging and cerebrospinal fluid biomarkers, mirroring those used in human clinical trials to assess the efficacy of a novel brain-penetrating anti-amyloid fusion protein treatment in the McGill-R-Thy1-APP transgenic rat model. The bi-functional fusion protein consisted of a blood-brain barrier crossing single domain antibody (FC5) fused to an amyloid-ß oligomer-binding peptide (ABP) via Fc fragment of mouse IgG (FC5-mFc2a-ABP). A five-week treatment with FC5-mFc2a-ABP (loading dose of 30 mg/Kg/iv followed by 15 mg/Kg/week/iv for four weeks) substantially reduced brain amyloid-ß levels as measured by positron emission tomography and increased the cerebrospinal fluid amyloid-ß42/40 ratio. In addition, the 5-week treatment rectified the cerebrospinal fluid neurofilament light chain concentrations, resting-state functional connectivity, and hippocampal atrophy measured using magnetic resonance imaging. Finally, FC5-mFc2a-ABP (referred to as KG207-M) treatment did not induce amyloid-related imaging abnormalities such as microhemorrhage. Together, this study demonstrates the translational values of the designed preclinical studies for the assessment of novel therapies based on the clinical biomarkers providing tangible metrics for designing early-stage clinical trials.
Asunto(s)
Enfermedad de Alzheimer , Amiloidosis , Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Biomarcadores , Estudios Longitudinales , Ratones , Tomografía de Emisión de Positrones , RatasRESUMEN
Neurofilament light chain (NFL) measurement has been gaining strong support as a clinically useful neuronal injury biomarker for various neurodegenerative conditions. However, in Alzheimer's disease (AD), its reflection on regional neuronal injury in the context of amyloid pathology remains unclear. This study included 83 cognitively normal (CN), 160 mild cognitive impairment (MCI), and 73 AD subjects who were further classified based on amyloid-beta (Aß) status as positive or negative (Aß+ vs Aß-). In addition, 13 rats (5 wild type and 8 McGill-R-Thy1-APP transgenic (Tg)) were examined. In the clinical study, reduced precuneus/posterior cingulate cortex and hippocampal grey matter density were significantly associated with increased NFL concentrations in cerebrospinal fluid (CSF) or plasma in MCI Aß+ and AD Aß+. Moreover, AD Aß+ showed a significant association between the reduced grey matter density in the AD-vulnerable regions and increased NFL concentrations in CSF or plasma. Congruently, Tg rats recapitulated and validated the association between CSF NFL and grey matter density in the parietotemporal cortex, entorhinal cortex, and hippocampus in the presence of amyloid pathology. In conclusion, reduced grey matter density and elevated NFL concentrations in CSF and plasma are associated in AD-vulnerable regions in the presence of amyloid positivity in the AD clinical spectrum and amyloid Tg rat model. These findings further support the NFL as a neuronal injury biomarker in the research framework of AD biomarker classification and for the evaluation of therapeutic efficacy in clinical trials.
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Enfermedad de Alzheimer , Disfunción Cognitiva , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides , Animales , Atrofia , Biomarcadores , Sustancia Gris/diagnóstico por imagen , Filamentos Intermedios , Proteínas de Neurofilamentos , Ratas , Proteínas tauRESUMEN
Clinical research has shown that chronic antipsychotic drug (APD) treatment further decreases cortical gray matter and hippocampus volume, and increases striatal and ventricular volume in patients with schizophrenia. D2-like receptor blockade is necessary for clinical efficacy of the drugs, and may be responsible for inducing these volume changes. However, the role of other D2-like receptors, such as D3, remains unclear. Following our previous work, we undertook a longitudinal study to examine the effects of chronic (9-week) typical (haloperidol (HAL)) and atypical (clozapine (CLZ)) APDs on the neuroanatomy of wild-type (WT) and dopamine D3-knockout (D3KO) mice using magnetic resonance imaging (MRI) and histological assessments in a sub-region of the anterior cingulate cortex (the prelimbic [PL] area) and striatum. D3KO mice had larger striatal volume prior to APD administration, coupled with increased glial and neuronal cell density. Chronic HAL administration increased striatal volume in both WT and D3KO mice, and reduced PL area volume in D3KO mice both at trend level. CLZ increased volume of the PL area of WT mice at trend level, but decreased D3KO PL area glial cell density. Both typical and atypical APD administration induced neuroanatomical remodeling of regions rich in D3 receptor expression, and typically altered in schizophrenia. Our findings provide novel insights on the role of D3 receptors in structural changes observed following APD administration in clinical populations.
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Antipsicóticos/farmacología , Cuerpo Estriado/efectos de los fármacos , Giro del Cíngulo/efectos de los fármacos , Receptores de Dopamina D3/metabolismo , Animales , Antipsicóticos/uso terapéutico , Recuento de Células , Clozapina/farmacología , Clozapina/uso terapéutico , Cuerpo Estriado/anatomía & histología , Cuerpo Estriado/diagnóstico por imagen , Femenino , Giro del Cíngulo/anatomía & histología , Giro del Cíngulo/diagnóstico por imagen , Haloperidol/farmacología , Haloperidol/uso terapéutico , Humanos , Inyecciones Intraperitoneales , Estudios Longitudinales , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Noqueados , Modelos Animales , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Trastornos Psicóticos/tratamiento farmacológico , Trastornos Psicóticos/patología , Receptores de Dopamina D3/genética , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/patologíaRESUMEN
Early-life stress (ELS) exposure has long-term consequences for both brain structure and function and impacts cognitive and emotional behavior. The basolateral amygdala (BLA) plays an important role in anxiety and fear conditioning through its extensive anatomical and functional connections, in particular to the medial prefrontal cortex (mPFC). However, how ELS affects amygdala function and connectivity in developing rats is unknown. We used the naturalistic limited bedding/nesting (LB) paradigm to induce chronic stress in the pups between postnatal day (PND) 1-10. Male normal bedding (NB, control) or LB offspring underwent structural and resting-state functional MRI (rs-fMRI) on PND18 and in adulthood (PND74-76). Adult male rats were tested for fear conditioning and extinction behavior prior to scanning. Seed-based functional connectivity maps were generated based on four BLA seeds (left, right, anterior and posterior). At both ages, LB induced different effects on anterior and posterior BLA networks, with significant reductions in rs-fMRI connectivity between the anterior BLA and mPFC in LB compared to NB offspring. BLA connectivity was lateralized by preweaning age, with the right hemisphere displaying more connectivity changes than the left. Weak negative volumetric correlations between the BLA and mPFC were also present, mostly in preweaning LB animals. rs-fMRI connectivity and volumetric changes were associated with enhanced fear behaviors in adult LB offspring. Activation of the LB-exposed neonatal amygdala described previously might accelerate the maturation of BLA-mPFC projections and/or modify the activity of reciprocal connections between these structures, leading to a net reduction in rs-fMRI connectivity and increased fear behavior.
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Complejo Nuclear Basolateral/patología , Complejo Nuclear Basolateral/fisiopatología , Corteza Prefrontal/patología , Corteza Prefrontal/fisiopatología , Estrés Psicológico , Animales , Conducta Animal , Mapeo Encefálico , Condicionamiento Clásico , Extinción Psicológica , Miedo , Femenino , Imagen por Resonancia Magnética , Masculino , Vías Nerviosas/patología , Vías Nerviosas/fisiopatología , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Neuroanatomical alterations are well established in patients suffering from schizophrenia, however the extent to which these changes are attributable to illness, antipsychotic drugs (APDs), or their interaction is unclear. APDs have been extremely effective for treatment of positive symptoms in major psychotic disorders. Their therapeutic effects are mediated, in part, through blockade of D2-like dopamine (DA) receptors, i.e. the D2, D3 and D4 dopamine receptors. Furthermore, the dependency of neuroanatomical change on DA system function and D2-like receptors has yet to be explored. METHODS: We undertook a preclinical longitudinal study to examine the effects of typical (haloperidol (HAL)) and atypical (clozapine (CLZ)) APDs in wild type (WT) and dopamine D2 knockout (D2KO) mice over 9-weeks using structural magnetic resonance imaging (MRI). RESULTS: Chronic typical APD administration in WT mice was associated with reductions in total brain (pâ¯=â¯0.009) and prelimbic area (PL) (pâ¯=â¯0.02) volumes following 9-weeks, and an increase in striatal volume (pâ¯=â¯0.04) after six weeks. These APD-induced changes were not present in D2KOs, where, at baseline, we observed significantly smaller overall brain volume (pâ¯<â¯0.01), thinner cortices (qâ¯<â¯0.05), and enlarged striata (qâ¯<â¯0.05). Stereological assessment revealed increased glial density in PL area of HAL treated wild types. Interestingly, in WT and D2KO mice, chronic CLZ administration caused more limited changes in brain structure. CONCLUSIONS: Our results present evidence for the role of D2 DA receptors in structural alterations induced by the administration of the typical APD HAL and that chronic administration of CLZ has a limited influence on brain structure.
Asunto(s)
Antipsicóticos/administración & dosificación , Encéfalo/anatomía & histología , Encéfalo/efectos de los fármacos , Antagonistas de los Receptores de Dopamina D2/administración & dosificación , Receptores de Dopamina D2/fisiología , Animales , Clozapina/administración & dosificación , Femenino , Haloperidol/administración & dosificación , Estudios Longitudinales , Imagen por Resonancia Magnética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Dopamina D2/genéticaRESUMEN
PURPOSE: Indirect 13 C MRS by proton-observed carbon editing (POCE) is a powerful method to study brain metabolism. The sensitivity of POCE-MRS can be enhanced through the use of short TEs, which primarily minimizes homonuclear J-evolution related losses; previous POCE-MRS implementations use longer than optimal echo times due to sequence limitations, or short TE image selected in vivo spectroscopy-based multi-shot acquisitions for 3D localization. To that end, this paper presents a novel single-shot point resolved spectroscopy (PRESS)-localized POCE-MRS sequence that involves the application of simultaneous editing and localization pulses (SEAL)-PRESS, allowing the TE to be reduced to a theoretically optimal value of â¼ 1/JHC . METHODS: The optimized SEAL-PRESS sequence was first evaluated in simulation and in phantom; next, the sequence was validated with dynamic in vivo POCE-MRS performed in a rat preparation during a 1,6-13 C2 -Glc infusion, and on a microwave fixed rat brain following a 2-hour [1,6-13 C2 ]-Glc infusion. POCE spectra from the SEAL-PRESS sequence were compared against a previously described 12.6-ms PRESS-POCE sequence utilizing a classical carbon editing scheme. RESULTS: The SEAL-PRESS sequence provides > 95% editing efficiency, optimal sensitivity, and localization for POCE MRS with an overall sequence TE of 8.1 ms. Signal amplitude of 13 C-labeled metabolites Glu-H4, Gln-H4, Glx-H3, Glc-H6 +Glx-H2, and Asp-H2 were shown to be improved by >17% relative to a 12.6-ms PRESS-POCE sequence in vivo. CONCLUSION: We report for the first time, a single-shot PRESS-localized and edited 8.1-ms TE POCE-MRS sequence with optimal sensitivity, editing efficiency, and localization.
Asunto(s)
Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Espectroscopía de Resonancia Magnética con Carbono-13/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Animales , Química Encefálica/fisiología , Glucosa/administración & dosificación , Glucosa/química , Glucosa/farmacocinética , Masculino , Fantasmas de Imagen , Ratas , Ratas Long-EvansRESUMEN
PURPOSE: In vivo carbon-13 (13 C) MR spectroscopy (MRS) is capable of measuring energy metabolism and neuroenergetics, noninvasively in the brain. Indirect (1 H-[13 C]) MRS provides sensitivity benefits compared with direct 13 C methods, and normally includes a 1 H surface coil for both localization and signal reception. The aim was to develop a coil platform with homogenous B1+ and use short conventional pulses for short echo time proton observed carbon edited (POCE) MRS. METHODS: A 1 H-[13 C] MRS coil platform was designed with a volumetric resonator for 1 H transmit, and surface coils for 1 H reception and 13 C transmission. The Rx-only 1 H surface coil nullifies the requirement for a T/R switch before the 1 H preamplifier; the highpass filter and preamplifier can be placed proximal to the coil, thus minimizing sensitivity losses inherent with POCE-MRS systems described in the literature. The coil platform was evaluated with a PRESS-POCE sequence (TE = 12.6 ms) on a rat model. RESULTS: The coil provided excellent localization, uniform spin nutation, and sensitivity. 13 C labeling of Glu-H4 and Glx-H3 peaks, and the Glx-H2 peaks were observed approximately 13 and 21 min following the infusion of 1-13 C glucose, respectively. CONCLUSION: A convenient and sensitive platform to study energy metabolism and neurotransmitter cycling is presented. Magn Reson Med 79:628-635, 2018. © 2017 International Society for Magnetic Resonance in Medicine.
Asunto(s)
Encéfalo/diagnóstico por imagen , Isótopos de Carbono/química , Espectroscopía de Protones por Resonancia Magnética/métodos , Animales , Fantasmas de Imagen , Protones , Ratas , Ratas Long-EvansRESUMEN
Imaging biomarkers are frequently proposed as endpoints for clinical trials targeting brain amyloidosis in Alzheimer's disease (AD); however, the specific impact of amyloid-ß (Aß) aggregation on biomarker abnormalities remains elusive in AD. Using the McGill-R-Thy1-APP transgenic rat as a model of selective Aß pathology, we characterized the longitudinal progression of abnormalities in biomarkers commonly used in AD research. Middle-aged (9-11 months) transgenic animals (both male and female) displayed mild spatial memory impairments and disrupted cingulate network connectivity measured by resting-state fMRI, even in the absence of hypometabolism (measured with PET [18F]FDG) or detectable fibrillary amyloidosis (measured with PET [18F]NAV4694). At more advanced ages (16-19 months), cognitive deficits progressed in conjunction with resting connectivity abnormalities; furthermore, hypometabolism, Aß plaque accumulation, reduction of CSF Aß1-42 concentrations, and hippocampal atrophy (structural MRI) were detectable at this stage. The present results emphasize the early impact of Aß on brain connectivity and support a framework in which persistent Aß aggregation itself is sufficient to impose memory circuits dysfunction, which propagates to adjacent brain networks at later stages.SIGNIFICANCE STATEMENT The present study proposes a "back translation" of the Alzheimer pathological cascade concept from human to animals. We used the same set of Alzheimer imaging biomarkers typically used in large human cohorts and assessed their progression over time in a transgenic rat model, which allows for a finer spatial resolution not attainable with mice. Using this translational platform, we demonstrated that amyloid-ß pathology recapitulates an Alzheimer-like profile of biomarker abnormalities even in the absence of other hallmarks of the disease such as neurofibrillary tangles and widespread neuronal losses.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/análisis , Encéfalo/patología , Modelos Animales de Enfermedad , Imagen por Resonancia Magnética/métodos , Trastornos de la Memoria/patología , Imagen Multimodal/métodos , Neuroimagen/métodos , Placa Amiloide/patología , Tomografía de Emisión de Positrones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Amiloidosis/patología , Animales , Animales Modificados Genéticamente , Biomarcadores , Química Encefálica , Disfunción Cognitiva/patología , Femenino , Radioisótopos de Flúor , Fluorodesoxiglucosa F18 , Masculino , Trastornos de la Memoria/metabolismo , Mutación , Placa Amiloide/química , Agregación Patológica de Proteínas , Radiofármacos , Ratas , Ratas Transgénicas , Ratas WistarRESUMEN
BACKGROUND: Preclinical neuroimaging allows for the assessment of brain anatomy, connectivity and function in laboratory animals, such as mice and rats. Most of these studies are performed under anesthesia to avoid movement during the scanning sessions. METHOD: Due to the limitations associated with anesthetized imaging, recent efforts have been made to conduct rodent imaging studies in awake animals, habituated to the restraint systems used in these instances. As of now, only one such system is commercially available for mouse scanning (Animal Imaging Research, Boston, MA, USA) integrating the radiofrequency coil electronics with the restraining element, an approach which, although effective in reducing head motion during awake imaging, has some limitations. In the current report, we present a novel mouse restraining system that addresses some of these limitations. RESULTS/COMPARISON TO OTHER METHODS: The effectiveness of the restraining system was evaluated in terms of three-dimensional linear head movement across two consecutive functional MRI scans (total 20min) in 33 awake mice. Head movement was minimal, recorded in roughly 12% of the time-series. Respiration rate during the acclimation procedure dropped while the bolus count remained unchanged. Body movement during functional acquisitions did not have a significant effect on magnetic field (B0) homogeneity. CONCLUSION/NOVELTY: Compared to the commercially available system, the benefit of the current design is two-fold: 1) it is compatible with a range of commercially-available coils, and 2) it allows for the pairing of neuroimaging with other established techniques involving intracranial cannulation (i.e. microinfusion and optogenetics).
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Neuroimagen/instrumentación , Restricción Física/instrumentación , Adaptación Psicológica , Animales , Encéfalo/diagnóstico por imagen , Diseño de Equipo , Cabeza , Campos Magnéticos , Imagen por Resonancia Magnética/instrumentación , Masculino , Ratones Endogámicos C57BL , Movimiento (Física) , Impresión Tridimensional , Estrés Psicológico/fisiopatología , VigiliaRESUMEN
BACKGROUND: We describe the fabrication and performance of a chronic in situ coil system designed to allow focal brain stimulation in rats while acquiring functional MRI data. NEW METHOD: An implantable receive-only surface radiofrequency coil (iCoil) was designed to be fitted subcutaneously, directly onto to the rat skull surface during the intracerebral cannulation procedure. The coil is fixed in place using acrylic dental cement anchored to four screws threaded into the skull. To demonstrate the use of this coil system in situ, whole-brain functional MRI scans were acquired during various stimuli, including intracranial microinfusions of bicuculline and morphine in the prefrontal cortex and ventral tegmental area, respectively. RESULTS/COMPARISON TO OTHER METHODS: SNR performance of the iCoil was superior to three commercially-available coils, in some instances by a factor of two. Widespread BOLD activation was observed in response to bicuculline and morphine microinfusions. CONCLUSION: A new approach was demonstrated for high-SNR MR imaging of the brain in rats with intracranial implants using an implantable surface coil. This approach enables mapping the functional response to highly targeted stimuli such as intracranial microinfusions.
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Encéfalo/diagnóstico por imagen , Estimulación Encefálica Profunda/instrumentación , Estimulación Encefálica Profunda/veterinaria , Bombas de Infusión Implantables , Imagen por Resonancia Magnética/instrumentación , Imagen por Resonancia Magnética/veterinaria , Microinyecciones/veterinaria , Animales , Diseño de Equipo , Análisis de Falla de Equipo , Masculino , Microinyecciones/instrumentación , Prótesis e Implantes , Ratas , Ratas Long-Evans , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Transductores/veterinariaRESUMEN
BACKGROUND CONTEXT: Chronic low back pain is debilitating and difficult to treat. Depending on the etiology, responses to treatments vary widely. Although chronic low back pain is frequently related to intervertebral disc degeneration, the relationship between disc degeneration severity and clinical symptoms are still poorly understood. In humans, studies investigating the relationship between disc degeneration severity and low back pain are limited by the difficulty of obtaining disc samples from well-characterized patients and pain-free controls. We have previously described the secreted protein, acidic, rich in cysteine (SPARC)-null mouse model of chronic low back pain. SPARC is a matricellular protein involved in regulating the assembly and composition of extracellular matrix. The SPARC-null mice develop age-dependent disc degeneration of increasing severity accompanied by behavioral signs suggestive of axial low back pain, radiating leg pain, and motor impairment. The existence of this model allows for examination of the relationships between clinical symptoms in vivo and pathological signs of disc degeneration ex vivo. PURPOSE: The goal of this study was to explore the relationship between behavioral signs of pain and the severity of lumbar disc degeneration using the SPARC-null mouse model of disc degeneration-related low back pain. STUDY DESIGN: This study used a cross-sectional, multiple-cohort behavioral and histological study of disc degeneration and behavioral symptoms in a mouse model of low back pain associated with disc degeneration. METHODS: SPARC-null and wild-type control mice ranging from 6 to 78 weeks of age were used in this study. The severity of disc degeneration was determined by ex vivo analysis of the lumbar spine using colorimetric histological staining and a scoring system adapted from the Pfirrmann scale. Behavioral signs of axial low back pain, radiating leg pain, and motor impairment were quantified as tolerance to axial stretching in the grip force assay, hypersensitivity to cold or mechanical stimuli on the hindpaw (acetone and von Frey tests), and latency to fall in the rotarod assay, respectively. RESULTS: The SPARC-null mice exhibited decreased tolerance to axial stretching, hindpaw cold hypersensitivity, and motor impairment compared with age-matched control mice. The severity of disc degeneration increased with age in both SPARC-null and control mice and by 78 weeks of age, the same proportion of lumbar discs were abnormal in SPARC-null and control mice. However, the degree of degeneration was more severe in the SPARC-null mice. In both SPARC-null and control mice, tolerance to axial stretching but not hindpaw cold sensitivity correlated with disc degeneration severity. Motor impairment correlated with degeneration severity in the SPARC-null mice only. CONCLUSIONS: These data suggest that internal disc disruption contributes to axial low back pain and motor impairment but not to radiating leg pain. These results have implications for the optimization of mechanism-based treatments strategies.
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Degeneración del Disco Intervertebral/fisiopatología , Locomoción , Dolor de la Región Lumbar/fisiopatología , Anciano , Animales , Femenino , Humanos , Degeneración del Disco Intervertebral/patología , Dolor de la Región Lumbar/patología , Ratones , Ratones Endogámicos C57BLRESUMEN
Hippocampal shrinkage is a commonly found neuroanatomical change in stress-related mood disorders such as depression and post-traumatic stress disorders (PTSD). Since the onset and severity of these disorders have been found to be closely related to stressful life events, and as stress alone has been shown to reduce hippocampal volume in animal studies, vulnerability to mood disorders may be related to a susceptibility to stress-induced hippocampal shrinkage. However, a smaller hippocampal volume before stress exposure has also been suggested to confer vulnerability of stressed individuals to PTSD or depression. In this study, we examined the contribution of either innate hippocampal volume differences or hippocampal susceptibility to stress-induced shrinkage to the formation of stress-related psychopathology using longitudinal MRI measurements of hippocampal volume in inbred C57 mice before and after chronic social defeat stress. We found that only half of the stressed C57 mice were susceptible to stress and developed psychopathological behaviors such as social avoidance. The other half was resilient to stress and exhibited no social avoidance. Before exposure to stress, we observed a positive correlation between hippocampal volume and social avoidance. After chronic social defeat stress, we found significant increases in left hippocampal volume in resilient and nonstressed control mice. Intriguingly, this increase in hippocampal volume was not found in susceptible mice, suggesting an arrestment of hippocampal growth in these mice. Our findings suggest that both a susceptibility to stress-induced hippocampal volume changes and a larger hippocampus before stress exposure confer vulnerability to psychopathology after chronic stress.
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Hipocampo/patología , Resiliencia Psicológica , Estrés Psicológico/patología , Animales , Reacción de Prevención , Peso Corporal , Enfermedad Crónica , Modelos Animales de Enfermedad , Dominación-Subordinación , Lateralidad Funcional , Estudios Longitudinales , Imagen por Resonancia Magnética , Masculino , Ratones Endogámicos C57BL , Tamaño de los Órganos , Conducta Social , Estrés Psicológico/psicologíaRESUMEN
High-throughput screening of Tranzyme Pharma's proprietary macrocycle library using the aequorin Ca2+-bioluminescence assay against the human ghrelin receptor (GRLN) led to the discovery of novel agonists against this G-protein coupled receptor. Early hits such as 1 (Ki=86 nM, EC50=134 nM) though potent in vitro displayed poor pharmacokinetic properties that required optimization. While such macrocycles are not fully rule-of-five compliant, principally due to their molecular weight and clogP, optimization of their pharmacokinetic properties proved feasible largely through conformational rigidification. Extensive SAR led to the identification of 2 (Ki=16 nM, EC50=29 nM), also known as ulimorelin or TZP-101, which has progressed to phase III human clinical trials for the treatment of postoperative ileus. X-ray structure and detailed NMR studies indicated a rigid peptidomimetic portion in 2 that is best defined as a nonideal type-I' ß-turn. Compound 2 is 24% orally bioavailable in both rats and monkeys. Despite its potency, in vitro and in gastric emptying studies, 2 did not induce growth hormone (GH) release in rats, thus demarcating the GH versus GI pharmacology of GRLN.
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Compuestos Macrocíclicos/síntesis química , Peptidomiméticos/síntesis química , Receptores de Ghrelina/agonistas , Administración Oral , Animales , Disponibilidad Biológica , Células CACO-2 , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Hormona del Crecimiento/metabolismo , Humanos , Técnicas In Vitro , Macaca fascicularis , Compuestos Macrocíclicos/farmacocinética , Compuestos Macrocíclicos/farmacología , Espectroscopía de Resonancia Magnética , Masculino , Microsomas Hepáticos/metabolismo , Oligopéptidos/síntesis química , Oligopéptidos/farmacocinética , Oligopéptidos/farmacología , Peptidomiméticos/farmacocinética , Peptidomiméticos/farmacología , Conformación Proteica , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A new method for solid phase parallel synthesis of chemically and conformationally diverse macrocyclic peptidomimetics is reported. A key feature of the method is access to broad chemical and conformational diversity. Synthesis and mechanistic studies on the macrocyclization step are reported.
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Química Farmacéutica/métodos , Péptidos Cíclicos/química , Técnicas Químicas Combinatorias , Dimerización , Dipéptidos , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Imitación Molecular , Estructura Molecular , Péptidos/química , Plata/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
In order to study the effect of phosphorylation on the function of the steroidogenic acute regulatory protein (StAR), 10 putative phosphorylation sites were mutated in the hamster StAR. In pcDNA3.1-StAR transfected COS-1 cells, decreases in basal activity were found for the mutants S55A, S185A and S194A. Substitution of S185 by D or E to mimic phosphorylation resulted in decreased activity for all mutants; we concluded that S185 was not a phosphorylation site and we hypothesized that mutations on S185 created StAR conformational changes resulting in a decrease in its binding affinity for cholesterol. In contrast, the mutation S194D resulted in an increase in StAR activity. We have calculated the relative rate of pregnenolone formation (App. V(max)) in transfected COS-1 cells with wild type (WT) and mutant StAR-pcDNA3.1 under control and (Bu)(2)-cAMP stimulation. The App. V(max) values refer to the rate of cholesterol transported and metabolized by the cytochrome P450scc enzyme present in the inner mitochondrial membrane. The App. V(max) was 1.61 +/- 0.28 for control (Ctr) WT StAR and this value was significantly increased to 4.72 +/- 0.09 for (Bu)(2)-cAMP stimulated preparations. App. V(max) of 5.53 (Ctr) and 4.82 ((Bu)(2)-cAMP) found for S194D StAR preparations were similar to that of the WT StAR stimulated preparations. At equal StAR quantity, an anti-phospho-(S/T) PKA substrate antibody revealed four times more phospho-(S/T) in (Bu)(2)-cAMP than in control preparations. The intensity of phosphorylated bands was decreased for the S55A, S56A and S194A mutants and it was completely abolished for the S55A/S56A/S194A mutant. StAR activity of control and stimulated preparations were diminished by 73 and 72% for the mutant S194A compared to 77 and 83% for the mutant S55A/S56A/S194A. The remaining activity appears to be independent of phosphorylation at PKA sites and could be due to the intrinsic activity of non-phosphorylated StAR or to an artefact due to the pharmacological quantity of StAR expressed in COS-1. In conclusion we have shown that (Bu)(2)-cAMP provokes an augmentation of both the quantity and activity of StAR, and that an enhancement in StAR phosphorylation increases its activity. The increased quantity of StAR upon (Bu)(2)-cAMP stimulation could be due to an augmentation of its mRNA or protein synthesis stability, or both; this is yet to be determined.
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Glándulas Suprarrenales/metabolismo , Mutación/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Animales , Sitios de Unión , Bucladesina/farmacología , Células COS , Chlorocebus aethiops , Colesterol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol , Cricetinae , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias , Mutagénesis Sitio-Dirigida , Fosforilación , Pregnenolona/metabolismo , Unión Proteica , Conformación Proteica/efectos de los fármacos , TransfecciónRESUMEN
We studied the effect of the adrenocorticotropin hormone (ACTH) on the expression of the steroidogenic acute regulatory protein (StAR) in vivo in rat and hamster adrenals and also in transfection experiments using COS-1 cells. In vivo, ACTH increased the level of StAR mRNA within 30-60 minutes and also increased the quantity of StAR, but with a 2-3-hour delay. ACTH induced the formation of many acidic StAR species as analyzed by two-dimensional gel electrophoresis and immunoblotting. In the transfection experiments, (Bu)(2)-cAMP also induced the formation of many acidic species for the hamster StAR; in COS-1 cells, StAR is phosphorylated mainly on serine (S) residue(s). When alanine (A) was substituted for serine, S13A, S185A, and S194A mutants had decreased StAR activity compared to wildtype, thus determining the importance of these amino acid residues in StAR action. The full-length WT, N46-truncated StAR lacking its mitochondrial import sequence, and N46-S194A had similar activities, whereas N46-S185A had completely lost its activity. Our results suggest that S194, but not S185, functions in association with the mitochondrial import sequence for the initiation of StAR activation. Further studies showed that S185 is implicated in salt bridge stability, not in StAR phosphorylation, suggesting its importance for StAR folding. Thermodynamic calculations of the hamster StAR homology model based on MLN64 show that StAR can partially unfold to bind cholesterol and serve as a rapid transfer mechanism for eventual translocation into mitochondria. This is supportive of a StAR functioning either outside the mitochondria or in the mitochondrial intermembrane space.
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Hormona Adrenocorticotrópica/farmacología , Fosfoproteínas/fisiología , Animales , Colesterol/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Fosfoproteínas/análisis , Fosfoproteínas/genética , Fosforilación , ARN Mensajero/análisis , Relación Estructura-Actividad , TermodinámicaRESUMEN
The cytochrome P450c17 isoforms from various animal species have different substrate selectivity, especially for 17,20-lyase activity. In particular, the human P450c17 selectively produces dehydroepiandrosterone with little androstenedione (AD). Hamster P450c17, on the other hand, produces both of these steroids at comparable rates. We thus investigated if computational analysis could explain the difference in activity profiles. Therefore, we inserted the four P450c17 substrates-pregnenolone, progesterone, and their 17alpha-hydroxylated forms-inside our hamster P450c17 model, which we derived from our human P450c17 model based on the crystal structure of P450BMP. We performed molecular dynamics (MD) simulations on the complexes and analyzed the resultant trajectories to identify amino acids that interact with substrates. Starting with substrates in two different orientations, we obtained two sets of binding trajectories in each case. The first set of trajectories reveal structural rearrangements that occur during binding, whereas the second set of trajectories reflects substrate orientations during catalysis. Our modeling suggests that three distinct steps are required for substrate selectivity and binding to the hamster P450c17: (1) recognition of the substrate at the putative substrate entrance, characterized by a pocket at the surface of the hamster P450c17 containing charged residues R96 and D116; (2) entry of the substrate into the active site, in an intermediate position directed by possible hydrogen bonding of the substrates with the heme D-ring propionate group, R96, R440, and T306; followed by (3) 90 degrees counterclockwise rotation of the substrates, positioning them in optimal position for reactivity, a process that may be directed by hydrogen bonding to the 110-112 region of the hamster P450c17. With some substrates, we obtained trajectories which suggest that major distortions in the I-helix and opening of the H-I loop occur during substrate binding. In conclusion, these modeling exercises provide insight to possible structural reorganizations that occur during substrate binding and suggest that amino acids that participate in three distinct steps of this process may all contribute to substrate binding and activity.
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Esteroide 17-alfa-Hidroxilasa/química , 17-alfa-Hidroxipregnenolona/química , 17-alfa-Hidroxiprogesterona/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Simulación por Computador , Cricetinae , Isoenzimas/química , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Pregnenolona/química , Progesterona/química , Esteroide 17-alfa-Hidroxilasa/metabolismo , Especificidad por SustratoRESUMEN
In order to understand the activity specificity of the hamster cytochrome P450 17 alpha-hydroxylase/17,20-lyase (P450c17), we have studied its structure/activity using three hamster P450c17 recombinant mutants (T202N/D240N/D407H). In transiently transfected COS-1 cells, the mutation T202N reduced 17 alpha-hydroxylation of pregnenolone and progesterone to 24 and 44% of wild type (WT), respectively, followed by reduced 17,20-cleavage to 71 and 67%, respectively. On the other hand, the mutation D240N decreased specifically 17,20-lyase activity to 61% of WT when incubated with pregnenolone while the mutation D407H only decreased 17 alpha-hydroxylation to 46% when incubated with progesterone.To comprehend the altered activity profiles of these hamster P450c17 mutants, we have elaborated a 3D model of the hamster P450c17 and compared it to our preceding model of the human P450c17. Analysis of the mutants with this model showed that, without direct contact to the substrates, these mutations transmit structural changes to the active site. By analogy, these results support the concept that any cellular changes modifying the external structure of P450c17, such as phosphorylation, could have influence on its active site and enzymatic activities.
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Glándulas Suprarrenales/enzimología , Modelos Moleculares , Esteroide 17-alfa-Hidroxilasa/química , Esteroide 17-alfa-Hidroxilasa/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Cricetinae , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Esteroide 17-alfa-Hidroxilasa/genética , Relación Estructura-ActividadRESUMEN
Although much progress has been achieved in the study of the steroidogenic acute regulatory protein (StAR) dependent cholesterol transfer inside mitochondria, not one mechanism can account for all experimental data obtained to date. We have thus investigated the possibility that molecular modeling and structure-based thermodynamic calculations (STC) could enlighten these discrepancies. Starting from the crystallographic data of the human MLN64, a StAR homology model was generated and subjected to STC to verify the importance of StAR structural alterations for proper function. As expected, the model resembled the MLN64 crystal, although no binding site "tunnel" was obtained. Instead, a closed cavity was discovered, approximately the size and shape of cholesterol. This suggests that StAR does indeed require structural alterations to allow cholesterol binding, most evidently by the C-terminal alpha-helix above the U-shaped beta-barrel. Through STC, it is shown that unfolding of this helix is probable and leads to a 2% subpopulation of partially unfolded StAR, supportive of both the intermembrane shuttle and the molten globule hypotheses.