RESUMEN
Myelin Basic Protein (MBP) is essential for both elaboration and maintenance of CNS myelin, and its reduced accumulation results in hypomyelination. How different Mbp mRNA levels affect myelin dimensions across the lifespan and how resident glial cells may respond to such changes are unknown. Here, to investigate these questions, we used enhancer-edited mouse lines that accumulate Mbp mRNA levels ranging from 8% to 160% of wild type. In young mice, reduced Mbp mRNA levels resulted in corresponding decreases in Mbp protein accumulation and myelin sheath thickness, confirming the previously demonstrated rate-limiting role of Mbp transcription in the control of initial myelin synthesis. However, despite maintaining lower line specific Mbp mRNA levels into old age, both MBP protein levels and myelin thickness improved or fully normalized at rates defined by the relative Mbp mRNA level. Sheath length, in contrast, was affected only when mRNA levels were very low, demonstrating that sheath thickness and length are not equally coupled to Mbp mRNA level. Striking abnormalities in sheath structure also emerged with reduced mRNA levels. Unexpectedly, an increase in the density of all glial cell types arose in response to reduced Mbp mRNA levels. This investigation extends understanding of the role MBP plays in myelin sheath elaboration, architecture, and plasticity across the mouse lifespan and illuminates a novel axis of glial cell crosstalk.
Asunto(s)
Proteína Básica de Mielina , Vaina de Mielina , Neuroglía , ARN Mensajero , Animales , Proteína Básica de Mielina/metabolismo , Proteína Básica de Mielina/genética , Vaina de Mielina/metabolismo , Vaina de Mielina/genética , ARN Mensajero/metabolismo , Neuroglía/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MasculinoRESUMEN
RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit. Here, we determined the impact of POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic Pdgfrα-Cre/ERT; R26-Stopfl-EYFP; Polr3bfl mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed. Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis. In summary, we characterized the Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions.
Asunto(s)
Anodoncia , Anomalías Craneofaciales , Enfermedades Desmielinizantes , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias , Enfermedades Neurodegenerativas , Humanos , Animales , Ratones , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Mutación/genéticaRESUMEN
PURPOSE: Reconstruction of high quality myelin water imaging (MWI) maps is challenging, particularly for data acquired using multi-echo gradient echo (mGRE) sequences. A non-linear least squares fitting (NLLS) approach has often been applied for MWI. However, this approach may produce maps with limited detail and, in some cases, sub-optimal signal to noise ratio (SNR), due to the nature of the voxel-wise fitting. In this study, we developed a novel, unsupervised learning method called self-labelled encoder-decoder (SLED) to improve gradient echo-based MWI data fitting. METHODS: Ultra-high resolution, MWI data was collected from five mouse brains with variable levels of myelination, using a mGRE sequence. Imaging data was acquired using a 7T preclinical MRI system. A self-labelled, encoder-decoder network was implemented in TensorFlow for calculation of myelin water fraction (MWF) based on the mGRE signal decay. A simulated MWI phantom was also created to evaluate the performance of MWF estimation. RESULTS: Compared to NLLS, SLED demonstrated improved MWF estimation, in terms of both stability and accuracy in phantom tests. In addition, SLED produced less noisy MWF maps from high resolution MR microscopy images of mouse brain tissue. It specifically resulted in lower noise amplification for all mouse genotypes that were imaged and yielded mean MWF values in white matter ROIs that were highly correlated with those derived from standard NLLS fitting. Lastly, SLED also exhibited higher tolerance to low SNR data. CONCLUSION: Due to its unsupervised and self-labeling nature, SLED offers a unique alternative to analyze gradient echo-based MWI data, providing accurate and stable MWF estimations.
Asunto(s)
Vaina de Mielina , Sustancia Blanca , Animales , Ratones , Agua , Sustancia Blanca/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Encéfalo/diagnóstico por imagenRESUMEN
One of the limiting factors in cone-beam CT (CBCT) image quality is system blur, caused by detector response, x-ray source focal spot size, azimuthal blurring, and reconstruction algorithm. In this work, we develop a novel iterative reconstruction algorithm that improves spatial resolution by explicitly accounting for image unsharpness caused by different factors in the reconstruction formulation. While the model-based iterative reconstruction techniques use prior information about the detector response and x-ray source, our proposed technique uses a simple measurable blurring model. In our reconstruction algorithm, denoted as simultaneous deblurring and iterative reconstruction (SDIR), the blur kernel can be estimated using the modulation transfer function (MTF) slice of the CatPhan phantom or any other MTF phantom, such as wire phantoms. The proposed image reconstruction formulation includes two regularization terms: (1) total variation (TV) and (2) nonlocal regularization, solved with a split Bregman augmented Lagrangian iterative method. The SDIR formulation preserves edges, eases the parameter adjustments to achieve both high spatial resolution and low noise variances, and reduces the staircase effect caused by regular TV-penalized iterative algorithms. The proposed algorithm is optimized for a point-of-care head CBCT unit for image-guided radiosurgery and is tested with CatPhan phantom, an anthropomorphic head phantom, and 6 clinical brain stereotactic radiosurgery cases. Our experiments indicate that SDIR outperforms the conventional filtered back projection and TV penalized simultaneous algebraic reconstruction technique methods (represented by adaptive steepest-descent POCS algorithm, ASD-POCS) in terms of MTF and line pair resolution, and retains the favorable properties of the standard TV-based iterative reconstruction algorithms in improving the contrast and reducing the reconstruction artifacts. It improves the visibility of the high contrast details in bony areas and the brain soft-tissue. For example, the results show the ventricles and some brain folds become visible in SDIR reconstructed images and the contrast of the visible lesions is effectively improved. The line-pair resolution was improved from 12 line-pair/cm in FBP to 14 line-pair/cm in SDIR. Adjusting the parameters of the ASD-POCS to achieve 14 line-pair/cm caused the noise variance to be higher than the SDIR. Using these parameters for ASD-POCS, the MTF of FBP and ASD-POCS were very close and equal to 0.7 mm-1 which was increased to 1.2 mm-1 by SDIR, at half maximum.
Asunto(s)
Encéfalo/diagnóstico por imagen , Tomografía Computarizada de Haz Cónico/métodos , Cabeza/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/métodos , Fantasmas de Imagen , Radiocirugia/métodos , Cirugía Asistida por Computador/métodos , Tomografía Computarizada por Rayos X/métodos , Algoritmos , Artefactos , Humanos , Modelos TeóricosRESUMEN
Neuromodulation approaches to treating lower urinary tract dysfunction could be substantially improved by a sensor able to detect when the bladder is full. A number of approaches to this problem have been proposed, but none has been found entirely satisfactory. Electrical plethysmography approaches attempt to relate the electrical impedance of the bladder to its volume, but have previously focused only on the amplitudes of the measured signals. We investigated whether the phase relationships between sinusoidal currents applied through a pair of stimulating electrodes and measured through a pair of recording electrodes could provide information about bladder volume. Acute experiments in a rabbit model were used to investigate how phase-to-volume or amplitude-to-volume regression models could be used to predict bladder volumes in future recordings, with and without changes to the saline conductivity. Volume prediction errors were found to be 6.63 ± 1.12 mL using the phase information and 8.32 ± 3.88 mL using the amplitude information (p = 0.44 when comparing the phase and amplitude results, n = 6), where the volume of the filled bladder was about 25 mL. When a full/empty binary decision rule was applied based on the regression model, the difference between the actual threshold that would result from this rule and the desired threshold was found to be 4.24 ± 0.65 mL using the phase information and 106.92 ± 189.82 mL using the amplitude information (p = 0.03, n = 6). Our results suggest that phase information can form the basis for more effective and robust electrical plethysmography approaches to bladder volume measurement.
Asunto(s)
Vejiga Urinaria/anatomía & histología , Vejiga Urinaria/fisiología , Animales , Impedancia Eléctrica , Electrodos , Masculino , Pletismografía , ConejosRESUMEN
OBJECTIVE: Dimethyl fumarate (DMF), a therapy for relapsing-remitting multiple sclerosis (RRMS), is implicated as acting on inflammatory and antioxidant responses within both systemic immune and/or central nervous system (CNS) compartments. Orally administered DMF is rapidly metabolized to monomethyl fumarate (MMF). Our aim was to analyze the impact of fumarates on antiinflammatory and antioxidant profiles of human myeloid cells found in the systemic compartment (monocytes) and in the inflamed CNS (blood-derived macrophages and brain-derived microglia). METHODS: We analyzed cytokine and antioxidant expression in monocytes from untreated or DMF-treated RRMS patients and controls, and in monocyte-derived macrophages (MDMs) and microglia isolated from adult and fetal human brain tissue. RESULTS: Monocytes from multiple sclerosis (MS) patients receiving DMF had reduced expression of the proinflammatory micro-RNA miR-155 and of antioxidant genes HMOX1 and OSGIN1 compared to untreated MS patients; similar changes were observed in patients receiving FTY720 and/or natalizumab. In vitro addition of DMF but not MMF to MDMs and microglia inhibited lipopolysaccharide-induced production of inflammatory cytokines and increased expression of the antioxidant gene HMOX1 in the absence of significant cytotoxicity. INTERPRETATION: Our in vivo-based observations that effects of DMF therapy on systemic myeloid cell gene expression are also observed with FTY720 and natalizumab therapy suggests that the effect may be indirect, reflecting reduced overall disease activity. Our in vitro results demonstrate significant effects of DMF but not MMF on inflammation and antioxidant responses by MDMs and microglia, questioning the mechanisms whereby DMF therapy would modulate myeloid cell properties within the CNS.