Assembly of functionally integrated human forebrain spheroids.

The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.

Role of DNA repair in Bacillus subtilis spore resistance to high energy and low energy electron beam treatments.

Bacillus subtilis spore inactivation mechanisms under low energy electron beam (LEEB) and high energy electron beam (HEEB) treatment were investigated using seven mutants lacking specific DNA repair mechanisms. The results showed that most of the DNA repair-deficient mutants, including ΔrecA, ΔKu ΔligD, Δexo Δnfo, ΔuvrAB and ΔsbcDC, had reduced resistances towards electron beam (EB) treatments at all investigated energy levels (80 keV, 200 keV and 10 MeV) compared to their wild type. This result suggested DNA damage was induced during EB treatments. The mutant lacking recA showed the lowest resistance, followed by the mutant lacking Ku and ligD. These findings indicated that recA, Ku and ligD and their associated DNA repair mechanisms, namely, homologous recombination and non-homologous end joining, play important roles in spore survival under EB treatment. Furthermore, exoA, nfo, uvrAB, splB, polY1 and polY2, which are involved in nucleotide damage repair/removal, showed different levels of effects on spore resistance under EB treatment. Finally, the results suggested that HEEB and LEEB inactivate B. subtilis spores through similar mechanisms. This research will provide a better understanding of how EB technologies inactivate B. subtilis spores and will contribute to the application of these technologies as a non-thermal, gentle spore control approach.

Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture.

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.

Charcot-Marie-Tooth disease-associated mutants of GDAP1 dissociate its roles in peroxisomal and mitochondrial fission.

Mitochondria and peroxisomes can be fragmented by the process of fission. The fission machineries of both organelles share a set of proteins. GDAP1 is a tail-anchored protein of mitochondria and induces mitochondrial fragmentation. Mutations in GDAP1 lead to Charcot-Marie-Tooth disease (CMT), an inherited peripheral neuropathy, and affect mitochondrial dynamics. Here, we show that GDAP1 is also targeted to peroxisomes mediated by the import receptor Pex19. Knockdown of GDAP1 leads to peroxisomal elongation that can be rescued by re-expressing GDAP1 and by missense mutated forms found in CMT patients. GDAP1-induced peroxisomal fission is dependent on the integrity of its hydrophobic domain 1, and on Drp1 and Mff, as is mitochondrial fission. Thus, GDAP1 regulates mitochondrial and peroxisomal fission by a similar mechanism. However, our results reveal also a more critical role of the amino-terminal GDAP1 domains, carrying most CMT-causing mutations, in the regulation of mitochondrial compared to peroxisomal fission.

The Gdap1 knockout mouse mechanistically links redox control to Charcot-Marie-Tooth disease.

The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial fission factor and mutations in GDAP1 cause Charcot-Marie-Tooth disease. We found that Gdap1 knockout mice (Gdap1(-/-)), mimicking genetic alterations of patients suffering from severe forms of Charcot-Marie-Tooth disease, develop an age-related, hypomyelinating peripheral neuropathy. Ablation of Gdap1 expression in Schwann cells recapitulates this phenotype. Additionally, intra-axonal mitochondria of peripheral neurons are larger in Gdap1(-/-) mice and mitochondrial transport is impaired in cultured sensory neurons of Gdap1(-/-) mice compared with controls. These changes in mitochondrial morphology and dynamics also influence mitochondrial biogenesis. We demonstrate that mitochondrial DNA biogenesis and content is increased in the peripheral nervous system but not in the central nervous system of Gdap1(-/-) mice compared with control littermates. In search for a molecular mechanism we turned to the paralogue of GDAP1, GDAP1L1, which is mainly expressed in the unaffected central nervous system. GDAP1L1 responds to elevated levels of oxidized glutathione by translocating from the cytosol to mitochondria, where it inserts into the mitochondrial outer membrane. This translocation is necessary to substitute for loss of GDAP1 expression. Accordingly, more GDAP1L1 was associated with mitochondria in the spinal cord of aged Gdap1(-/-) mice compared with controls. Our findings demonstrate that Charcot-Marie-Tooth disease caused by mutations in GDAP1 leads to mild, persistent oxidative stress in the peripheral nervous system, which can be compensated by GDAP1L1 in the unaffected central nervous system. We conclude that members of the GDAP1 family are responsive and protective against stress associated with increased levels of oxidized glutathione.

Spatial transcriptomics using multiplexed deterministic barcoding in tissue.

Spatially resolved transcriptomics of tissue sections enables advances in fundamental and applied biomedical research. Here, we present Multiplexed Deterministic Barcoding in Tissue (xDBiT) to acquire spatially resolved transcriptomes of nine tissue sections in parallel. New microfluidic chips were developed to spatially encode mRNAs over a total tissue area of 1.17 cm2 with a 50 µm resolution. Optimization of the biochemical protocol increased read and gene counts per spot by one order of magnitude compared to previous reports. Furthermore, the introduction of alignment markers allowed seamless registration of images and spatial transcriptomic spots. Together with technological advances, we provide an open-source computational pipeline to prepare raw sequencing data for downstream analysis. The functionality of xDBiT was demonstrated by acquiring 16 spatially resolved transcriptomic datasets from five different murine organs, including the cerebellum, liver, kidney, spleen, and heart. Factor analysis and deconvolution of spatial transcriptomes allowed for in-depth characterization of the murine kidney.

A new missense GDAP1 mutation disturbing targeting to the mitochondrial membrane causes a severe form of AR-CMT2C disease.

Charcot-Marie-Tooth disease (CMT) caused by mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene is characterized by a spectrum of phenotypes. Recurrent nonsense mutations (Q163X and S194X) showing regional distribution segregate with an early onset, severe course of recessive CMT disease with early loss of ambulancy. Missense mutations in GDAP1 have been reported in sporadic CMT cases with variable course of disease, among them the recurrent L239F missense GDAP1 mutation occurring in the European population. Finally, some GDAP1 mutations are associated with a mild form of CMT inherited as an autosomal dominant trait. In this study, we characterize the CMT phenotype in one Polish family with recessive trait of inheritance at the clinical, electrophysiological, morphological, cellular, and genetic level associated with a new Gly327Asp mutation in the GDAP1 gene. In spite of the nature of Gly327Asp mutation (missense), the CMT phenotype associated with this variant may be characterized as an early onset, severe axonal neuropathy, with severe skeletal deformities. The mutation lies within the transmembrane domain of GDAP1 and interferes with the mitochondrial targeting of the protein, similar to the loss of the domain in the previously reported Q163X and S194X mutations. We conclude that the loss of mitochondrial targeting is associated with a severe course of disease. Our study shows that clinical outcome of CMT disease caused by mutations in the GDAP1 gene cannot be predicted solely on the basis of genetic results (missense/nonsense mutations).

Virtual Reality-Based and Conventional Visual Field Examination Comparison in Healthy and Glaucoma Patients.

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, Köniz, 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, ρ ≥ 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.

Chromatin accessibility dynamics in a model of human forebrain development.

Forebrain development is characterized by highly synchronized cellular processes, which, if perturbed, can cause disease. To chart the regulatory activity underlying these events, we generated a map of accessible chromatin in human three-dimensional forebrain organoids. To capture corticogenesis, we sampled glial and neuronal lineages from dorsal or ventral forebrain organoids over 20 months in vitro. Active chromatin regions identified in human primary brain tissue were observed in organoids at different developmental stages. We used this resource to map genetic risk for disease and to explore evolutionary conservation. Moreover, we integrated chromatin accessibility with transcriptomics to identify putative enhancer-gene linkages and transcription factors that regulate human corticogenesis. Overall, this platform brings insights into gene-regulatory dynamics at previously inaccessible stages of human forebrain development, including signatures of neuropsychiatric disorders.

Human Astrocyte Maturation Captured in 3D Cerebral Cortical Spheroids Derived from Pluripotent Stem Cells.

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.

MicroRNA-9 Couples Brain Neurogenesis and Angiogenesis.

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.

Glutathione-conjugating and membrane-remodeling activity of GDAP1 relies on amphipathic C-terminal domain.

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.

Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.

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.

Confident non-invasive diagnosis of pseudolesions of the liver using diffusion-weighted imaging at 3T MRI.

PURPOSE: Pseudolesions of the liver including focal steatosis or non-steatosis and THID (transient hepatic intensity differences) are often challenging, especially when imaging patients with underlying malignant disease. We evaluated the efficacy of diffusion-weighted imaging (DWI) in the diagnostic work-up of pseudolesions. MATERIALS AND METHODS: Forty-eight patients with pseudolesions of the liver were consecutively examined and the images were retrospectively analyzed. MRI was performed on a clinical 3T scanner using T1-GRE in-phase and opposed phase images, T2-TSE-FS, diffusion-weighted sequences (b-value 50, 300, 600), ADC mapping, and dynamic post-contrast T1-VIBE-FS sequences (32 patients received Gd-EB-DTPA and 16 patients received gadolinium chelates). All images were analyzed by two experienced radiologists in consensus. As a standard of reference, we used the T1-w GRE, in-phase and out of phase, and the contrast enhanced series, as well as long-term follow-up. RESULTS: In the 48 patients, a total of 116 liver lesions were found. Of these, 40 were benign and eleven were malignant focal lesions. Benign lesions included one FNH, 26 simple cysts, and twelve hemangiomas. In addition, 65 pseudolesions (20 focal steatosis, 13 focal non-steatosis, and 32 THIDs) were found. All pseudolesions could be identified either on the T1-GRE in-phase and opposed phase images or on the contrast-enhanced series, or on both. However, none of them were visible on the diffusion-weighted images. CONCLUSION: Pseudolesions are invisible on DWI (negative predictive value = 1); therefore, DWI can be used as an additional sequence to significantly increase diagnostic confidence in the differentiation between pseudolesions and other focal liver lesions.