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Purpose Clinically evaluate the noninferiority of a custom virtual reality (VR) perimetry system when compared to a clinically and routinely used perimeter on both healthy subjects and glaucoma patients. Methods We use a custom-designed VR perimetry system tailored for visual field testing. The system uses Oculus Quest VR headset (Facebook Technologies, LLC, Bern, Switzerland), that includes a clicker for participant response feedback. A prospective, single center, study was conducted at the Department of Ophthalmology of the Bern University Hospital (Bern, Switzerland) for 12 months. Of the 114 participants recruited 70 subjects (36 healthy and 34 glaucoma patients with early to moderate visual field loss) were included in the study. Participants underwent perimetry tests on an Octopus 900 (Haag-Streit, K��niz, Switzerland) as well as on the custom VR perimeter. In both cases, standard dynamic strategy (DS) was used in conjunction with the G testing pattern. Collected visual fields (VFs) from both devices were then analyzed and compared. Results High mean defect (MD) correlations between the two systems (Spearman, �� ��� 0.75) were obtained. The VR system was found to slightly underestimate VF defects in glaucoma subjects (1.4 dB). No significant bias was found with respect to eccentricity or subject age. On average, a similar number of stimuli presentations per VF was necessary when measuring glaucoma patients and healthy subjects. Conclusions This study demonstrates that a clinically used perimeter and the proposed VR perimetry system have comparable performances with respect to a number of perimetry parameters in healthy and glaucoma patients with early to moderate visual field loss. Translational Relevance This suggests that VR perimeters have the potential to assess VFs with high enough confidence, whereby alleviating challenges in current perimetry practices by providing a portable and more accessible visual field test.

INTRODUCTION: The cerebral cortex is responsible for higher-order functions in the nervous system and has undergone substantial expansion in size in primates. The development of the forebrain, including the assembly of the expanded human cerebral cortex, is a lengthy process that involves the diversification and expansion of neural progenitors, the generation and positioning of layer-specific glutamatergic neurons, the cellular migration of γ-aminobutyric acid (GABA)-ergic neurons, and the formation and maturation of glial cells. Disruption of these cellular events by either genetic or environmental factors can lead to neurodevelopmental disease, including autism spectrum disorders and intellectual disability. RATIONALE: Human forebrain development is, to a large extent, inaccessible for cellular-level study, direct functional investigation, or manipulation. The lack of availability of primary brain tissue samples—in particular, at later stages—as well as the limitations of conventional in vitro cellular models have precluded a detailed mechanistic understanding of corticogenesis in healthy and disease states. Therefore, tracking epigenetic changes in specific forebrain cell lineages over long time periods, has the potential to unravel the molecular programs that underlie cell specification in the human cerebral cortex and, by temporally mapping disease risk onto these changes, to identify cell types and periods of increased disease susceptibility. RESULTS: We used three-dimensional (3D) directed differentiation of human pluripotent stem cells into dorsal and ventral forebrain domains and applied the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) in combination with RNA-sequencing (RNA-seq) to map the epigenetic and gene expression signatures of neuronal and glial cell lineages over 20 months in vitro. We show, through direct comparison with primary brain tissue from our study and several epigenetic datasets, that human stem cell-derived 3D forebrain organoids recapitulated in vivo chromatin accessibility patterns over time. We then integrated these data to discover putative enhancer-gene linkages and lineage-specific transcription factor regulators, including a diverse repertoire of factors that may control cortical specification. We validated protein expression of some of these transcription factors using immunofluorescence, confirming cellular and temporal dynamics in both primary tissue and forebrain organoids. Next, we used this resource to map genes and genetic variants associated with schizophrenia and autism spectrum disorders to distinct accessibility patterns to reveal cell types and periods of susceptibility. Last, we identified a wave of chromatin remodeling during cortical neurogenesis, during which a quarter of regulatory regions are active, and then highlighted transcription factors that may drive these developmental changes. CONCLUSION: Using long-term 3D neural differentiation of stem cells as well as primary brain tissue samples, we found that organoids intrinsically undergo chromatin state transitions in vitro that are closely related to human forebrain development in vivo. Leveraging this platform, we identified epigenetic alterations putatively driven by specific transcription factors and discovered a dynamic period of chromatin remodeling during human cortical neurogenesis. Finally, we nominated several key transcription factors that may coordinate over time to drive these changes and mapped cell type-specific disease-associated variation over time and in specific cell types.

In the developing brain, neurons expressing VEGF-A and blood vessels grow in close apposition, but many of the molecular pathways regulating neuronal VEGF-A and neurovascular system development remain to be deciphered. Here, we show that miR-9 links neurogenesis and angiogenesis through the formation of neurons expressing VEGF-A. We found that miR-9 directly targets the transcription factors TLX and ONECUTs to regulate VEGF-A expression. miR-9 inhibition leads to increased TLX and ONECUT expression, resulting in VEGF-A overexpression. This untimely increase of neuronal VEGF-A signal leads to the thickening of blood vessels at the expense of the normal formation of the neurovascular network in the brain and retina. Thus, this conserved transcriptional cascade is critical for proper brain development in vertebrates. Because of this dual role on neural stem cell proliferation and angiogenesis, miR-9 and its downstream targets are promising factors for cellular regenerative therapy following stroke and for brain tumor treatment.

Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics. Here we describe a capsid selection method, called Cre recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications.

Objective: Ganglioside-induced differentiation associated-protein 1 ( GDAP1 ) mutations are commonly associated with autosomal recessive Charcot-Marie-Tooth (ARCMT) neuropathy; however, in rare instances, they also lead to autosomal dominant Charcot-Marie-Tooth (ADCMT). We aimed to investigate the frequency of disease-causing heterozygous GDAP1 mutations in ADCMT and their associated phenotype. Methods: We performed mutation analysis in a large cohort of ADCMT patients by means of bidirectional sequencing of coding regions and exon-intron boundaries of GDAP1 . Intragenic GDAP1 deletions were excluded using an allele quantification assay. We confirmed the pathogenic character of one sequence variant by in vitro experiments assaying mitochondrial morphology and function. Results: In 8 Charcot-Marie-Tooth disease (CMT) families we identified 4 pathogenic heterozygous GDAP1 mutations, 3 of which are novel. Three of the mutations displayed reduced disease penetrance. Disease onset in the affected individuals was variable, ranging from early childhood to adulthood. Disease progression was slow in most patients and overall severity milder than typically seen in autosomal recessive GDAP1 mutations. Electrophysiologic changes are heterogeneous but compatible with axonal neuropathy in the majority of patients. Conclusions: With this study, we broaden the phenotypic and genetic spectrum of autosomal dominant GDAP1 -associated neuropathies. We show that patients with dominant GDAP1 mutations may display clear axonal CMT, but may also have only minimal clinical and electrophysiologic abnormalities. We demonstrate that cell-based functional assays can be reliably used to test the pathogenicity of unknown variants. We discuss the implications of phenotypic variability and the reduced penetrance of autosomal dominant GDAP1 mutations for CMT diagnostic testing and counseling.

The human cerebral cortex develops through an elaborate succession of cellular events that, when disrupted, can lead to neuropsychiatric disease. The ability to reprogram somatic cells into pluripotent cells that can be differentiated in vitro provides a unique opportunity to study normal and abnormal corticogenesis. Here, we present a simple and reproducible 3D culture approach for generating a laminated cerebral cortex-like structure, named human cortical spheroids (hCSs), from pluripotent stem cells. hCSs contain neurons from both deep and superficial cortical layers and map transcriptionally to in vivo fetal development. These neurons are electrophysiologically mature, display spontaneous activity, are surrounded by nonreactive astrocytes and form functional synapses. Experiments in acute hCS slices demonstrate that cortical neurons participate in network activity and produce complex synaptic events. These 3D cultures should allow a detailed interrogation of human cortical development, function and disease, and may prove a versatile platform for generating other neuronal and glial subtypes in vitro.

Summary There is significant need to develop physiologically relevant models for investigating human astrocytes in health and disease. Here, we present an approach for generating astrocyte lineage cells in a three-dimensional (3D) cytoarchitecture using human cerebral cortical spheroids (hCSs) derived from pluripotent stem cells. We acutely purified astrocyte-lineage cells from hCSs at varying stages up to 20 months in vitro using immunopanning and cell sorting and performed high-depth bulk and single-cell RNA sequencing to directly compare them to purified primary human brain cells. We found that hCS-derived glia closely resemble primary human fetal astrocytes and that, over time in vitro , they transition from a predominantly fetal to an increasingly mature astrocyte state. Transcriptional changes in astrocytes are accompanied by alterations in phagocytic capacity and effects on neuronal calcium signaling. These findings suggest that hCS-derived astrocytes closely resemble primary human astrocytes and can be used for studying development and modeling disease.

Mutations in the ganglioside-induced differentiation associated protein 1 (GDAP1) cause severe peripheral motor and sensory neuropathies called Charcot-Marie-Tooth disease. GDAP1 expression induces fission of mitochondria and peroxisomes by a currently elusive mechanism, while disease causing mutations in GDAP1 impede the protein’s role in mitochondrial dynamics. In silico analysis reveals sequence similarities of GDAP1 to glutathione S-transferases (GSTs). However, a proof of GST activity and its possible impact on membrane dynamics are lacking to date. Using recombinant protein, we demonstrate for the first time theta-class-like GST activity for GDAP1, and it’s activity being regulated by the C-terminal hydrophobic domain 1 (HD1) of GDAP1 in an autoinhibitory manner. Moreover, we show that the HD1 amphipathic pattern is required to induce membrane dynamics by GDAP1. As both, fission and GST activities of GDAP1, are critically dependent on HD1, we propose that GDAP1 undergoes a molecular switch, turning from a pro-fission active to an auto-inhibited inactive conformation.