Hypomyelination, hypodontia and craniofacial abnormalities in a Polr3b mouse model of leukodystrophy.

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.

Self-labelled encoder-decoder (SLED) for multi-echo gradient echo-based myelin water imaging.

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.

Emergence of deletions during treadmill locomotion as a function of supraspinal and sensory inputs.

During locomotion, alternating activity of flexor and extensor muscles is largely regulated by a spinal neuronal network, the central pattern generator, the activity of which is modulated by peripheral and supraspinal inputs. In the absence of these modulatory inputs, for example during fictive locomotion after spinalization and curarization, spontaneous failures of motor activation (deletions) in a muscle can occur without perturbing the rhythmic cycle structure of the antagonists on the same side or the contralateral side. This suggests that the central pattern generator can maintain the locomotor period when motoneuron discharges fail in a given pool of motoneurons. Here we first examined whether such deletions could occur during real locomotion on a treadmill and determined their consequences on the overt locomotor pattern. We also evaluated the role of supraspinal and sensory inputs in modulating the occurrence of failures of rhythmic activity by comparing the same cats in the intact state, then after a partial spinal cord injury (SCI), and finally after a complete SCI at different treadmill speeds. We showed that deletions: (1) are absent in intact animals and occur only after SCI; (2) affect only flexor muscle activity; (3) neither perturb the timing of rhythmic activity of these muscles in subsequent cycles nor interfere with the timing of the ipsilateral and contralateral agonists and antagonists; (4) do not affect significantly the locomotor pattern kinematics; and (5) are sensitive to treadmill speed and lesion severity, suggesting a role for sensory and supraspinal inputs in stabilizing rhythmic output activity.

Loss of symmetric cell division of apical neural progenitors drives DENND5A-related developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are a heterogenous group of epilepsies in which altered brain development leads to developmental delay and seizures, with the epileptic activity further negatively impacting neurodevelopment. Identifying the underlying cause of DEEs is essential for progress toward precision therapies. Here we describe a group of individuals with biallelic variants in DENND5A and determine that variant type is correlated with disease severity. We demonstrate that DENND5A interacts with MUPP1 and PALS1, components of the Crumbs apical polarity complex, which is required for both neural progenitor cell identity and the ability of these stem cells to divide symmetrically. Induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division during neural induction and have an inherent propensity to differentiate into neurons, and transgenic DENND5A mice, with phenotypes like the human syndrome, have an increased number of neurons in the adult subventricular zone. Disruption of symmetric cell division following loss of DENND5A results from misalignment of the mitotic spindle in apical neural progenitors. A subset of DENND5A is localized to centrosomes, which define the spindle poles during mitosis. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state and ultimately shortening the period of neurogenesis. This study provides a mechanism behind DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.

Radiosynthesis and In Vivo Evaluation of Four Positron Emission Tomography Tracer Candidates for Imaging of Melatonin Receptors.

Melatonin is a neurohormone that modulates several physiological functions in mammals through the activation of melatonin receptor type 1 and 2 (MT1 and MT2). The melatonergic system is an emerging therapeutic target for new pharmacological interventions in the treatment of sleep and mood disorders; thus, imaging tools to further investigate its role in the brain are highly sought-after. We aimed to develop selective radiotracers for in vivo imaging of both MT1 and MT2 by positron emission tomography (PET). We identified four previously reported MT ligands with picomolar affinities to the target based on different scaffolds which were also amenable for radiolabeling with either carbon-11 or fluorine-18. [11C]UCM765, [11C]UCM1014, [18F]3-fluoroagomelatine ([18F]3FAGM), and [18F]fluoroacetamidoagomelatine ([18F]FAAGM) have been synthesized in high radiochemical purity and evaluated in wild-type rats. All four tracers showed moderate to high brain permeability in rats with maximum standardized uptake values (SUVmax of 2.53, 1.75, 3.25, and 4.47, respectively) achieved 1-2 min after tracer administration, followed by a rapid washout from the brain. Several melatonin ligands failed to block the binding of any of the PET tracer candidates, while in some cases, homologous blocking surprisingly resulted in increased brain retention. Two 18F-labeled agomelatine derivatives were brought forward to PET scans in non-human primates and autoradiography on human brain tissues. No specific binding has been detected in blocking studies. To further investigate pharmacokinetic properties of the putative tracers, microsomal stability, plasma protein binding, log D, and membrane bidirectional permeability assays have been conducted. Based on the results, we conclude that the fast first pass metabolism by the enzymes in liver microsomes is the likely reason of the failure of our PET tracer candidates. Nevertheless, we showed that PET imaging can serve as a valuable tool to investigate the brain permeability of new therapeutic compounds targeting the melatonergic system.

Quantitation of Tissue Resection Using a Brain Tumor Model and 7-T Magnetic Resonance Imaging Technology.

BACKGROUND: Animal brain tumor models can be useful educational tools for the training of neurosurgical residents in risk-free environments. Magnetic resonance imaging (MRI) technologies have not used these models to quantitate tumor, normal gray and white matter, and total tissue removal during complex neurosurgical procedures. This pilot study was carried out as a proof of concept to show the feasibility of using brain tumor models combined with 7-T MRI technology to quantitatively assess tissue removal during subpial tumor resection. METHODS: Seven ex vivo calf brain hemispheres were used to develop the 7-T MRI segmentation methodology. Three brains were used to quantitate brain tissue removal using 7-T MRI segmentation methodology. Alginate artificial brain tumor was created in 4 calf brains to assess the ability of 7-T MRI segmentation methodology to quantitate tumor and gray and white matter along with total tissue volumes removal during a subpial tumor resection procedure. RESULTS: Quantitative studies showed a correlation between removed brain tissue weights and volumes determined from segmented 7-T MRIs. Analysis of baseline and postresection alginate brain tumor segmented 7-T MRIs allowed quantification of tumor and gray and white matter along with total tissue volumes removed and detection of alterations in surrounding gray and white matter. CONCLUSIONS: This pilot study showed that the use of animal tumor models in combination with 7-T MRI technology provides an opportunity to increase the granularity of data obtained from operative procedures and to improve the assessment and training of learners.

Characterizing Vocal Fold Injury Recovery in a Rabbit Model With Three-Dimensional Virtual Histology.

OBJECTIVES/HYPOTHESIS: In animal studies of vocal fold scarring and treatment, imaging-based evaluation is most often conducted by tissue slicing and histological staining. Given variation in anatomy, injury type, severity, and sacrifice timepoints, planar histological sections provide limited spatiotemporal details of tissue repair. Three-dimensional (3D) virtual histology may provide additional contextual spatial information, enhancing objective interpretation. The study's aim was to evaluate the suitability of magnetic resonance imaging (MRI), microscale computed tomography (CT), and nonlinear laser-scanning microscopy (NM) as virtual histology approaches for rabbit studies of vocal fold scarring. METHODS: A unilateral injury was created using microcup forceps in the left vocal fold of three New Zealand White rabbits. Animals were sacrificed at 3, 10, and 39 days postinjury. ex vivo imaging of excised larynges was performed with MRI, CT, and NM modalities. RESULTS: The MRI modality allowed visualization of injury location and morphological internal features with 100-µm spatial resolution. The CT modality provided a view of the injury defect surface with 12-µm spatial resolution. The NM modality with optical clearing resolved second-harmonic generation signal of collagen fibers and two-photon autofluorescence in vocal fold lamina propria, muscle, and surrounding cartilage structures at submicrometer spatial scales. CONCLUSIONS: Features of vocal fold injury and wound healing were observed with MRI, CT, and NM. The MRI and CT modalities provided contextual spatial information and dissection guidance, whereas NM resolved extracellular matrix structure. The results serve as a proof of concept to motivate incorporation of 3D virtual histology techniques in future vocal fold injury animal studies. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1578-1587, 2021.

Evaluation of Lanthanide-Doped Upconverting Nanoparticles for in Vitro and in Vivo Applications.

Because of their unique physicochemical properties, lanthanide-doped upconverting nanoparticles (Ln-UCNPs) have exceptional potential for biological applications. However, the use in biological systems is hampered by the limited understanding of their bionano interactions. Our multidisciplinary study has generated these insights through in-depth and quantitative analyses. The Ln-UCNPs examined here are spherical, monodisperse, and stable in aqueous environments. We show that Ln-UCNPs were associated with HeLa (cervical cancer) and LLC-PK1 (renal proximal tubule) cells and were nontoxic over a wide concentration range. Multiple biomarkers were assessed to monitor the cellular homeostasis in Ln-UCNP-treated cells. To this end, we evaluated the nuclear lamina, nucleoli, and nuclear transport factors. Single-cell analyses quantified the impact on Nrf2 and NF-κB, two transcription factors that control stress and immune responses. Moreover, we measured Ln-UCNP-induced changes in the abundance of molecular chaperones. Collectively, in vitro studies confirmed that Ln-UCNPs are nontoxic and trigger minor cellular stress responses. This lack of toxicity was verified in vivo, using the model organism Caenorhabditis elegans. The compatibility with biological systems prompted us to assess Ln-UCNPs as potential contrast agents for magnetic resonance imaging. We demonstrated that the Ln-UCNPs examined here were especially suitable as T2 contrast agents; they clearly outperformed the clinically used Gadovist. Taken together, our interdisciplinary work provides robust evidence for the nontoxicity of Ln-UCNPs. This sets the stage for the translation of Ln-UCNP for use in complex biological systems.

Creating a Comprehensive Research Platform for Surgical Technique and Operative Outcome in Primary Brain Tumor Neurosurgery.

BACKGROUND: The operative environment poses many challenges to studying the relationship between surgical acts and patient outcomes in intracranial oncological neurosurgery. We sought to develop a framework in which neurosurgical performance and extent of resection could be precisely quantified in a controlled setting. METHODS: The stiffness of an alginate hydrogel-based tumor was modified with differing concentrations of the cross-linking agent calcium sulfate until biomechanical properties similar to those of human primary brain tumors measured at resection were achieved. The artificial tumor was subsequently incorporated into an ex-vivo animal brain as a final model. Magnetic resonance imaging enhancement and ultraviolet fluorescence was achieved by incorporating gadolinium and fluorescein solution, respectively. Video recordings from the operative microscope, ceiling cameras, and instrument-mounted fiducial markers within a surgical suite environment captured operative performance. RESULTS: A total of 24 rheometer measurements were conducted on alginate hydrogels containing 10-, 11-, and 12-mM concentrations of calcium sulfate. Sixty-eight stiffness measurements were conducted on eight patient tumor samples. No differences were found between the alginate and brain tumor stiffness values [Kruskal-Wallis χ2(4) = 9.187; P = 0.057]. Tumor was identified using ultraviolet fluorescence and ultrasonography. The volume and location of the resected white and gray matter and residual tumor could be quantified in 0.003-mm3 increments using a 7T magnetic resonance imaging coil. Ultrasonic aspirator and bipolar electrocautery movement data were successfully transformed into performance metrics. CONCLUSION: The developed framework can offer clinicians, learners, and researchers the ability to perform operative rehearsal, teaching, and studies involving brain tumor surgery in a controlled laboratory environment and represents a crucial step in the understanding and training of expertise in neurosurgery.