Deconstructing cerebellar development cell by cell.

The cerebellum is a pivotal centre for the integration and processing of motor and sensory information. Its extended development into the postnatal period makes this structure vulnerable to a variety of pathologies, including neoplasia. These properties have prompted intensive investigations that reveal not only developmental mechanisms in common with other regions of the neuraxis but also unique strategies to generate neuronal diversity. How the phenotypically distinct cell types of the cerebellum emerge rests on understanding how gene expression differences arise in a spatially and temporally coordinated manner from initially homogeneous cell populations. Increasingly sophisticated fate mapping approaches, culminating in genetic-induced fate mapping, have furthered the understanding of lineage relationships between early- versus later-born cells. Tracing the developmental histories of cells in this way coupled with analysis of gene expression patterns has provided insight into the developmental genetic programmes that instruct cellular heterogeneity. A limitation to date has been the bulk analysis of cells, which blurs lineage relationships and obscures gene expression differences between cells that underpin the cellular taxonomy of the cerebellum. This review emphasises recent discoveries, focusing mainly on single-cell sequencing in mouse and parallel human studies that elucidate neural progenitor developmental trajectories with unprecedented resolution. Complementary functional studies of neural repair after cerebellar injury are challenging assumptions about the stability of postnatal cellular identities. The result is a wealth of new information about the developmental mechanisms that generate cerebellar neural diversity, with implications for human evolution.

PCARE and WASF3 regulate ciliary F-actin assembly that is required for the initiation of photoreceptor outer segment disk formation.

The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare-/- mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.

Functional and educational outcomes after treatment for intracranial arteriovenous malformations in children.

BACKGROUND: Arteriovenous malformations (AVMs) in the pediatric population are rare, yet they form the most frequent cause of hemorrhagic stroke in children. Compared to adults, children have been suggested to have beneficial neurological outcomes. However, few studies have focused on other variables than neurological outcomes. This study aims to assess the long-term functional and educational outcomes of children after multimodality approach of treatment for intracranial AVMs. METHODS: All children treated in our center between 1998 and 2016 for intracranial AVMs were reviewed. Patient characteristics, as well as AVM specifics, were collected. Functional outcomes were compared using the modified Rankin scale (mRs). Educational levels, using the International Standard Classification of Education (ISCED), were compared to the age-matched general population of the Netherlands. RESULTS: In total, 25 children were included at mean age of 10 years (range 2-16 years). Nineteen patients (76%) presented with intracranial bleeding. Mean follow-up was 11.5 ± 5.3 years (range 4.1-24.4). Four (16%) of patients were treated with embolization, three (12%) with microsurgery, and 18 patients (72%) received a combination of different treatment modalities. Altogether, 21 (84%) were embolized, 14 (56%) were treated with microsurgery, and eight (32%) received stereotactic radiosurgery. One child had a worse mRs at discharge compared to admission; all others improved (n = 11) or were stable (n = 13). At follow-up, all patients scored a stable or improved mRs compared to discharge, with 23 children (92%) scoring mRs 0 or 1. These 23 children followed regular education during follow-up without specialized or adapted schooling. No significant differences in educational level with the age-matched general population were found. CONCLUSION: This retrospective review shows positive long-term results of both functional and educational outcomes after multidisciplinary treatment of pediatric brain AVMs.

PTCH1-mutant human cerebellar organoids exhibit altered neural development and recapitulate early medulloblastoma tumorigenesis.

Patched 1 (PTCH1) is the primary receptor for the sonic hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma. As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we used cerebellar organoids differentiated from human induced pluripotent stem cells combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings demonstrate that developmental mechanisms in cerebellar organoids reflect in vivo processes of regionalisation and SHH signalling, and offer new insights into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models.

CRISPR-Cas Gene Perturbation and Editing in Human Induced Pluripotent Stem Cells.

Directing the fates of human pluripotent stem cells (hPSC) to generate a multitude of differentiated cell types allows the study of the genetic regulation of human development and disease. The translational potential of hPSC is maximized by exploiting CRISPR to silence or activate genes with spatial and temporal precision permanently or reversibly. Here, we summarize the increasingly refined and diverse CRISPR toolkit for the latter forms of gene perturbation in hPSC and their downstream applications. We discuss newer methods to install edits efficiently with single nucleotide resolution and describe pooled CRISPR screens as a powerful means of unbiased discovery of genes associated with a phenotype of interest. Last, we discuss the potential of these combined technologies in the treatment of hitherto intractable human diseases and the challenges to their implementation in the clinic.

A post-insertion strategy for surface functionalization of bacterial and mammalian cell-derived extracellular vesicles.

Extracellular vesicles (EVs) are nanoparticles which are released by cells from all three domains of life: Archaea, Bacteria and Eukarya. They can mediate cell-cell communication by transferring cargoes such as proteins and nucleic acids between cells. EVs receive great interest in both academia and industry as they have the potential to be natural drug carriers or vaccine candidates. However, limitations to their clinical translation exist as efficient isolation, loading, labelling and surface-engineering methods are lacking. In this article, we investigate a 'post-insertion' approach, which is commonly used in the functionalization of liposomes in the pharmaceutical field, on two different EV types: mammalian cell-derived EVs and bacteria-derived EVs. We aimed to find an easy and flexible approach to functionalize EVs, thereby improving the labelling, isolation, and surface-engineering.

Visual Outcomes after Endoscopic Endonasal Transsphenoidal Resection of Pituitary Adenomas: Our Institutional Experience.

Objectives Visual dysfunction in patients with pituitary adenomas is a clear indication for endoscopic endonasal transsphenoidal surgery (EETS). However, the visual outcomes vary greatly among patients and it remains unclear what tumor, patient, and surgical characteristics contribute to postoperative visual outcomes. Methods One hundred patients with pituitary adenomas who underwent EETS between January 2011 and June 2015 in a single institution were retrospectively reviewed. General patient characteristics, pre- and postoperative visual status, clinical presentation, tumor characteristics, hormone production, radiological features, and procedural characteristics were evaluated for association with presenting visual signs and visual outcomes postoperatively. Suprasellar tumor extension (SSE) was graded 0 to 4 following a grading system as formulated by Fujimoto et al. Results Sixty-six (66/100) of all patients showed visual field defects (VFD) at the time of surgery, of whom 18% (12/66) were asymptomatic. VFD improved in 35 (35%) patients and worsened in 4 (4%) patients postoperatively. Mean visual acuity (VA) improved from 0.67 preoperatively to 0.84 postoperatively ( p = 0.04). Nonfunctioning pituitary adenomas (NFPAs) and Fujimoto grade were independent predictors of preoperative VFD in the entire cohort ( p = 0.02 and p < 0.01 respectively). A higher grade of SSE was the only factor independently associated with postoperative improvement of VFD ( p = 0.03). NFPA and Fujimoto grade 3 were independent predictors of VA improvement (both p = 0.04). Conclusion EETS significantly improved both VA and VFD for most patients, although a few patients showed deterioration of visual deficits postoperatively. Higher degrees of SSE and NFPA were independent predictors of favorable visual outcomes.

Bacterial membrane vesicles as promising vaccine candidates.

Both Gram-positive and Gram-negative bacteria can release nano-sized lipid bilayered structures, known as membrane vesicles (MVs). These MVs play an important role in bacterial survival by orchestrating interactions between bacteria and between bacteria and host. The major constituents of MVs are proteins, lipids and nucleic acids. Due to the immunogenicity of the membrane lipids and/or proteins of the MVs, in combination with adjuvant danger signals and the repeating patterns on the nanosized surface, MVs can effectively stimulate the innate and adaptive immune system. Since they are non-replicating, they are safer than attenuated vaccines. In addition, by genetic engineering of the donor cells, further improvements to their safety profile, immunogenicity and yield can be achieved. To date, one MV-based vaccine against Neisseria meningitidis (N. meningitidis) serogroup B was approved. Other (engineered) MVs in the pipeline study are mostly in the preclinical phase.

Length of Thromboprophylaxis in Patients Operated on for a High-Grade Glioma: A Retrospective Study.

OBJECTIVE: High-grade gliomas are associated with venous thromboembolism (VTE). This retrospective study with a parallel cohort design investigated influence of continuing prophylactic anticoagulation after discharge on rate of VTE and intracranial hemorrhage (ICH) in patients operated on for high-grade glioma. METHODS: Consecutive adult patients who underwent subtotal or gross total resection for high-grade glioma at a single institution were included. Multivariable logistic regression analysis was used to investigate the association between duration of thromboprophylaxis (dalteparin administered 21 days vs. 0-7 days) and occurrence of VTE and ICH within 21 or 90 days after surgery, corrected for known risk factors. RESULTS: Of 301 included patients, 166 received short-term thromboprophylaxis, and 135 received prolonged thromboprophylaxis. In multivariable analysis, prolonged thromboprophylaxis was not significantly associated with occurrence of VTE within 21 days (3.0% vs. 1.2%; P = 0.24) or 90 days (8.9% vs. 4.8%; P = 0.09) after surgery; however, prolonged prophylaxis was associated with occurrence of ICH (5.9% vs. 0.6%; P = 0.03). Additionally, immobility (P = 0.03) and high body mass index (P = 0.02) were associated with occurrence of VTE. CONCLUSIONS: Prophylactic anticoagulation for 21 days postoperatively was not associated with a decreased rate of VTE compared with thromboprophylaxis until discharge. ICH was more common with prolonged thromboprophylaxis. These results provide insufficient evidence to extend duration of prophylaxis beyond hospitalization. Large-scale randomized prospective studies are needed to clarify safety, efficacy, and optimal timing of postoperative thromboprophylaxis in patients with high-grade glioma.

International practice variation in postoperative imaging of chronic subdural hematoma patients.

OBJECTIVE: The value of CT scanning after burr hole surgery in chronic subdural hematoma (CSDH) patients is unclear, and practice differs between countries. At the Brigham and Women's Hospital (BWH) in Boston, Massachusetts, neurosurgeons frequently order routine postoperative CT scans, while the University Medical Center Utrecht (UMCU) in the Netherlands does not have this policy. The aim of this study was to compare the use of postoperative CT scans in CSDH patients between these hospitals and to evaluate whether there are differences in clinical outcomes. METHODS: The authors collected data from both centers for 391 age- and sex-matched CSDH patients treated with burr hole surgery between January 1, 2002, and July 1, 2016, and compared the number of postoperative scans up to 6 weeks after surgery, the need for re-intervention, and postoperative neurological condition. RESULTS: BWH patients were postoperatively scanned a median of 4 times (interquartile range [IQR] 2-5), whereas UMCU patients underwent a median of 0 scans (IQR 0-1, p < 0.001). There was no significant difference in the number of re-operations (20 in the BWH vs 27 in the UMCU, p = 0.34). All re-interventions were preceded by clinical decline and no recurrences were detected on scans performed on asymptomatic patients. Patients' neurological condition was not worse in the UMCU than in the BWH (p = 0.43). CONCLUSIONS: While BWH patients underwent more scans than UMCU patients, there were no differences in clinical outcomes. The results of this study suggest that there is little benefit to routine scanning in asymptomatic patients who have undergone surgical treatment of uncomplicated CSDH and highlight opportunities to make practice more efficient.