Newly identified disorder of copper metabolism caused by variants in CTR1, a high-affinity copper transporter.

The high-affinity copper transporter CTR1 is encoded by CTR1 (SLC31A1), a gene locus for which no detailed genotype-phenotype correlations have previously been reported. We describe identical twin male infants homozygous for a novel missense variant NM_001859.4:c.284 G > A (p.Arg95His) in CTR1 with a distinctive autosomal recessive syndrome of infantile seizures and neurodegeneration, consistent with profound central nervous system copper deficiency. We used clinical, biochemical and molecular methods to delineate the first recognized examples of human CTR1 deficiency. These included clinical phenotyping, brain imaging, assays for copper, cytochrome c oxidase (CCO), and mitochondrial respiration, western blotting, cell transfection experiments, confocal and electron microscopy, protein structure modeling and fetal brain and cerebral organoid CTR1 transcriptome analyses. Comparison with two other critical mediators of cellular copper homeostasis, ATP7A and ATP7B, genes associated with Menkes disease and Wilson disease, respectively, revealed that expression of CTR1 was highest. Transcriptome analyses identified excitatory neurons and radial glia as brain cell types particularly enriched for copper transporter transcripts. We also assessed the effects of Copper Histidinate in the patients' cultured cells and in the patients, under a formal clinical protocol. Treatment normalized CCO activity and enhanced mitochondrial respiration in vitro, and was associated with modest clinical improvements. In combination with present and prior studies, these infants' clinical, biochemical and molecular phenotypes establish the impact of this novel variant on copper metabolism and cellular homeostasis and illuminate a crucial role for CTR1 in human brain development. CTR1 deficiency represents a newly defined inherited disorder of brain copper metabolism.

Cerebral organoids containing an AUTS2 missense variant model microcephaly.

Variants in the AUTS2 gene are associated with a broad spectrum of neurological conditions characterized by intellectual disability, microcephaly, and congenital brain malformations. Here, we use a human cerebral organoid model to investigate the pathophysiology of a heterozygous de novo missense AUTS2 variant identified in a patient with multiple neurological impairments including primary microcephaly and profound intellectual disability. Proband cerebral organoids exhibit reduced growth, deficits in neural progenitor cell (NPC) proliferation and disrupted NPC polarity within ventricular zone-like regions compared to control cerebral organoids. We used CRISPR-Cas9-mediated gene editing to correct this variant and demonstrate rescue of impaired organoid growth and NPC proliferative deficits. Single-cell RNA sequencing revealed a marked reduction of G1/S transition gene expression and alterations in WNT-ß-catenin signalling within proband NPCs, uncovering a novel role for AUTS2 in NPCs during human cortical development. Collectively, these results underscore the value of cerebral organoids to investigate molecular mechanisms underlying AUTS2 syndrome.

PTEN somatic mutations contribute to spectrum of cerebral overgrowth.

Phosphatase and tensin homologue (PTEN) regulates cell growth and survival through inhibition of the mammalian target of rapamycin (MTOR) signalling pathway. Germline genetic variation of PTEN is associated with autism, macrocephaly and PTEN hamartoma tumour syndromes. The effect of developmental PTEN somatic mutations on nervous system phenotypes is not well understood, although brain somatic mosaicism of MTOR pathway genes is an emerging cause of cortical dysplasia and epilepsy in the paediatric population. Here we report two somatic variants of PTEN affecting a single patient presenting with intractable epilepsy and hemimegalencephaly that varied in clinical severity throughout the left cerebral hemisphere. High-throughput sequencing analysis of affected brain tissue identified two somatic variants in PTEN. The first variant was present in multiple cell lineages throughout the entire hemisphere and associated with mild cerebral overgrowth. The second variant was restricted to posterior brain regions and affected the opposite PTEN allele, resulting in a segmental region of more severe malformation, and the only neurons in which it was found by single-nuclei RNA-sequencing had a unique disease-related expression profile. This study reveals brain mosaicism of PTEN as a disease mechanism of hemimegalencephaly and furthermore demonstrates the varying effects of single- or bi-allelic disruption of PTEN on cortical phenotypes.

CCNA2 Ablation in Aged Mice Results in Abnormal rRNA Granule Accumulation in Hippocampus.

Mounting evidence in the literature suggests that RNA-RNA binding protein aggregations can disturb neuronal homeostasis and lead to symptoms associated with normal aging as well as dementia. The specific ablation of cyclin A2 in adult neurons results in neuronal polyribosome aggregations and learning and memory deficits. Detailed histologic and ultrastructural assays of aged mice revealed that post-mitotic hippocampal pyramidal neurons maintain cyclin A2 expression and that proliferative cells in the dentate subgranular zone express cyclin A2. Cyclin A2 loss early during neural development inhibited hippocampal development through canonical/cell-cycle mechanisms, including prolonged cell cycle timing in embryonic hippocampal progenitor cells. However, in mature neurons, cyclin A2 colocalized with dendritic rRNA. Cyclin A2 ablation in adult hippocampus resulted in decreased synaptic density in the hippocampus as well as in accumulation of rRNA granules in dendrite shafts. We conclude that cyclin A2 functions in a noncanonical/non-cell cycle regulatory role to maintain adult pyramidal neuron ribostasis.

The role of PHOX2B-derived astrocytes in chemosensory control of breathing and sleep homeostasis.

KEY POINTS: The embryonic PHOX2B-progenitor domain generates neuronal and glial cells which together are involved in chemosensory control of breathing and sleep homeostasis. Ablating PHOX2B-derived astrocytes significantly contributes to secondary hypoxic respiratory depression as well as abnormalities in sleep homeostasis. PHOX2B-derived astrocyte ablation results in axonal pathologies in the retrotrapezoid nucleus. ABSTRACT: We identify in mice a population of ∼800 retrotrapezoid nucleus (RTN) astrocytes derived from PHOX2B-positive, OLIG3-negative progenitor cells, that interact with PHOX2B-expressing RTN chemosensory neurons. PHOX2B-derived astrocyte ablation during early life results in adult-onset O2 chemoreflex deficiency. These animals also display changes in sleep homeostasis, including fragmented sleep and disturbances in delta power after sleep deprivation, all without observable changes in anxiety or social behaviours. Ultrastructural evaluation of the RTN demonstrates that PHOX2B-derived astrocyte ablation results in features characteristic of degenerative neuro-axonal dystrophy, including abnormally dilated axon terminals and increased amounts of synapses containing autophagic vacuoles/phagosomes. We conclude that PHOX2B-derived astrocytes are necessary for maintaining a functional O2 chemosensory reflex in the adult, modulate sleep homeostasis, and are key regulators of synaptic integrity in the RTN region, which is necessary for the chemosensory control of breathing. These data also highlight how defects in embryonic development may manifest as neurodegenerative pathology in an adult.

Modeling neurodegenerative diseases with cerebral organoids and other three-dimensional culture systems: focus on Alzheimer's disease.

Many neurodegenerative diseases (NDs) such as Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis and Huntington's disease, are characterized by the progressive accumulation of abnormal proteinaceous assemblies in specific cell types and regions of the brain, leading to cellular dysfunction and brain damage. Although animal- and in vitro-based studies of NDs have provided the field with an extensive understanding of some of the mechanisms underlying these diseases, findings from these studies have not yielded substantial progress in identifying treatment options for patient populations. This necessitates the development of complementary model systems that are better suited to recapitulate human-specific features of ND pathogenesis. Three-dimensional (3D) culture systems, such as cerebral organoids generated from human induced pluripotent stem cells, hold significant potential to model NDs in a complex, tissue-like environment. In this review, we discuss the advantages of 3D culture systems and 3D modeling of NDs, especially AD and FTD. We also provide an overview of the challenges and limitations of the current 3D culture systems. Finally, we propose a few potential future directions in applying state-of-the-art technologies in 3D culture systems to understand the mechanisms of NDs and to accelerate drug discovery. Graphical abstract.

AUTS2 Syndrome: Molecular Mechanisms and Model Systems.

AUTS2 syndrome is a genetic disorder that causes intellectual disability, microcephaly, and other phenotypes. Syndrome severity is worse when mutations involve 3' regions (exons 9-19) of the AUTS2 gene. Human AUTS2 protein has two major isoforms, full-length (1259 aa) and C-terminal (711 aa), the latter produced from an alternative transcription start site in exon 9. Structurally, AUTS2 contains the putative "AUTS2 domain" (∼200 aa) conserved among AUTS2 and its ohnologs, fibrosin, and fibrosin-like-1. Also, AUTS2 contains extensive low-complexity sequences and intrinsically disordered regions, features typical of RNA-binding proteins. During development, AUTS2 is expressed by specific progenitor cell and neuron types, including pyramidal neurons and Purkinje cells. AUTS2 localizes mainly in cell nuclei, where it regulates transcription and RNA metabolism. Some studies have detected AUTS2 in neurites, where it may regulate cytoskeletal dynamics. Neurodevelopmental functions of AUTS2 have been studied in diverse model systems. In zebrafish, auts2a morphants displayed microcephaly. In mice, excision of different Auts2 exons (7, 8, or 15) caused distinct phenotypes, variously including neonatal breathing abnormalities, cerebellar hypoplasia, dentate gyrus hypoplasia, EEG abnormalities, and behavioral changes. In mouse embryonic stem cells, AUTS2 could promote or delay neuronal differentiation. Cerebral organoids, derived from an AUTS2 syndrome patient containing a pathogenic missense variant in exon 9, exhibited neocortical growth defects. Emerging technologies for analysis of human cerebral organoids will be increasingly useful for understanding mechanisms underlying AUTS2 syndrome. Questions for future research include whether AUTS2 binds RNA directly, how AUTS2 regulates neurogenesis, and how AUTS2 modulates neural circuit formation.

Generation of Neurosphere-Derived Organoid-Like-Aggregates (NEDAS) from Neural Stem Cells.

Neurosphere cultures have been used to propagate and study the intrinsic properties of neural stem cells (NSCs) for more than two decades but this method has many limitations. It is well known that neurospheres fuse in culture, but the long-term biological consequences of this phenomena are not well characterized. We leveraged the fusion behavior of human neurospheres to improve upon this technique with our Neurosphere-derived organoid-like aggregates (NEDAS) culture method, allowing the fusion of human NSCs at high density, which were maintained in orbital shaker conditions for 8-12 weeks without passing leading to the formation of 3D organoid-like aggregates without the use of Matrigel. NEDAS organoids proliferate and self-organize into neural rosettes, expressing PAX6 and SOX2 in ventricular zone (VZ)-like proliferative areas. Outside these rosettes, we identified corridors of migratory radial glial progenitors expressing Phospo-vimentin, CRYAB. In addition to DLX2, CXCR4 + progenitors. Further, we found immature neurons within cortical-like areas highly enriched for DCX and TUJ1, in addition to GABA+ and excitatory VGLUT1+ neurons. Here, we provide a protocol to generate NEDAS, additionally, we present a protocol for immunostaining of NEDAS organoids for confocal imaging. This protocol may be useful to dissect the self-organization and morphogenetic programs of populations of human NSCs offering an advantageous alternative to the conventional neurospheres method, generating more cell type diversity, within tissue-like aggregates over extended periods of time without dissociation or passing. NEDAS may be a complementary method to cerebral organoids protocols from IPSCs. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation and expansion of cultures of human neural stem cells in reduced growth factor basement matrix Basic Protocol 2: Formation and fusion of neurospheres derived matrigel-free organoid-like aggregates (NEDAS) Basic Protocol 3: Harvest, cryosection, and imaging protocol for NEDAS.

Electrophysiological Maturation of Cerebral Organoids Correlates with Dynamic Morphological and Cellular Development.

Cerebral organoids (COs) are rapidly accelerating the rate of translational neuroscience based on their potential to model complex features of the developing human brain. Several studies have examined the electrophysiological and neural network features of COs; however, no study has comprehensively investigated the developmental trajectory of electrophysiological properties in whole-brain COs and correlated these properties with developmentally linked morphological and cellular features. Here, we profiled the neuroelectrical activities of COs over the span of 5 months with a multi-electrode array platform and observed the emergence and maturation of several electrophysiologic properties, including rapid firing rates and network bursting events. To complement these analyses, we characterized the complex molecular and cellular development that gives rise to these mature neuroelectrical properties with immunohistochemical and single-cell transcriptomic analyses. This integrated approach highlights the value of COs as an emerging model system of human brain development and neurological disease.

Development of a Novel FIJI-Based Method to Investigate Neuronal Circuitry in Neonatal Mice.

The emergence of systems neuroscience tools requires parallel generation of objective analytical workflows for experimental neuropathology. We developed an objective analytical workflow that we used to determine how specific autonomic neural lineages change during postnatal development. While a wealth of knowledge exists regarding postnatal alterations in respiratory neural function, how these neural circuits change and develop in the weeks following birth remains less clear. In this study, we developed our workflow by combining genetic mouse modeling and quantitative immunofluorescent confocal microscopy and used this to examine the postnatal development of neural circuits derived from the transcription factors NKX2.2 and OLIG3 into three medullary respiratory nuclei. Our automated FIJI-based image analysis workflow rapidly and objectively quantified synaptic puncta in user-defined anatomic regions. Using our objective workflow, we found that the density and estimated total number of Nkx2.2-derived afferents into the pre-Bötzinger Complex significantly decreased with postnatal age during the first three weeks of postnatal life. These data indicate that Nkx2.2-derived structures differentially influence pre-Bötzinger Complex respiratory oscillations at different stages of postnatal development.