SARS-CoV-2 infects human brain organoids causing cell death and loss of synapses that can be rescued by treatment with Sofosbuvir.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which was rapidly declared a pandemic by the World Health Organization (WHO). Early clinical symptomatology focused mainly on respiratory illnesses. However, a variety of neurological manifestations in both adults and newborns are now well-documented. To experimentally determine whether SARS-CoV-2 could replicate in and affect human brain cells, we infected iPSC-derived human brain organoids. Here, we show that SARS-CoV-2 can productively replicate and promote death of neural cells, including cortical neurons. This phenotype was accompanied by loss of excitatory synapses in neurons. Notably, we found that the U.S. Food and Drug Administration (FDA)-approved antiviral Sofosbuvir was able to inhibit SARS-CoV-2 replication and rescued these neuronal alterations in infected brain organoids. Given the urgent need for readily available antivirals, these results provide a cellular basis supporting repurposed antivirals as a strategic treatment to alleviate neurocytological defects that may underlie COVID-19- related neurological symptoms.

Impact of alcohol exposure on neural development and network formation in human cortical organoids.

Prenatal alcohol exposure is the foremost preventable etiology of intellectual disability and leads to a collection of diagnoses known as Fetal Alcohol Spectrum Disorders (FASD). Alcohol (EtOH) impacts diverse neural cell types and activity, but the precise functional pathophysiological effects on the human fetal cerebral cortex are unclear. Here, we used human cortical organoids to study the effects of EtOH on neurogenesis and validated our findings in primary human fetal neurons. EtOH exposure produced temporally dependent cellular effects on proliferation, cell cycle, and apoptosis. In addition, we identified EtOH-induced alterations in post-translational histone modifications and chromatin accessibility, leading to impairment of cAMP and calcium signaling, glutamatergic synaptic development, and astrocytic function. Proteomic spatial profiling of cortical organoids showed region-specific, EtOH-induced alterations linked to changes in cytoskeleton, gliogenesis, and impaired synaptogenesis. Finally, multi-electrode array electrophysiology recordings confirmed the deleterious impact of EtOH on neural network formation and activity in cortical organoids, which was validated in primary human fetal tissues. Our findings demonstrate progress in defining the human molecular and cellular phenotypic signatures of prenatal alcohol exposure on functional neurodevelopment, increasing our knowledge for potential therapeutic interventions targeting FASD symptoms.

Altered network and rescue of human neurons derived from individuals with early-onset genetic epilepsy.

Early-onset epileptic encephalopathies are severe disorders often associated with specific genetic mutations. In this context, the CDKL5 deficiency disorder (CDD) is a neurodevelopmental condition characterized by early-onset seizures, intellectual delay, and motor dysfunction. Although crucial for proper brain development, the precise targets of CDKL5 and its relation to patients' symptoms are still unknown. Here, induced pluripotent stem cells derived from individuals deficient in CDKL5 protein were used to generate neural cells. Proteomic and phosphoproteomic approaches revealed disruption of several pathways, including microtubule-based processes and cytoskeleton organization. While CDD-derived neural progenitor cells have proliferation defects, neurons showed morphological alterations and compromised glutamatergic synaptogenesis. Moreover, the electrical activity of CDD cortical neurons revealed hyperexcitability during development, leading to an overly synchronized network. Many parameters of this hyperactive network were rescued by lead compounds selected from a human high-throughput drug screening platform. Our results enlighten cellular, molecular, and neural network mechanisms of genetic epilepsy that could ultimately promote novel therapeutic opportunities for patients.

A human neurodevelopmental model for Williams syndrome.

Williams syndrome is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with Williams syndrome lack precisely the same set of genes, with breakpoints in chromosome band 7q11.23 (refs 1-5). The contribution of specific genes to the neuroanatomical and functional alterations, leading to behavioural pathologies in humans, remains largely unexplored. Here we investigate neural progenitor cells and cortical neurons derived from Williams syndrome and typically developing induced pluripotent stem cells. Neural progenitor cells in Williams syndrome have an increased doubling time and apoptosis compared with typically developing neural progenitor cells. Using an individual with atypical Williams syndrome, we narrowed this cellular phenotype to a single gene candidate, frizzled 9 (FZD9). At the neuronal stage, layer V/VI cortical neurons derived from Williams syndrome were characterized by longer total dendrites, increased numbers of spines and synapses, aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in neurons from Williams syndrome were validated after Golgi staining of post-mortem layer V/VI cortical neurons. This model of human induced pluripotent stem cells fills the current knowledge gap in the cellular biology of Williams syndrome and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain.

Leaf extracts of Campomanesia xanthocarpa positively regulates atherosclerotic-related protein expression.

Atherosclerosis is caused by a monocyte-mediated inflammatory process that, in turn, is stimulated by cytokines and adhesion molecules. Monocytes are then differentiated into macrophages, leading to the formation of arterial atherosclerotic plaques. Recently, guavirova leaf extracts from Campomanesia xanthocarpa (EG) have shown potential effects on the treatment of plaque formation by reducing cholesterol, LDL levels and serum oxidative stress. We evaluated the effect of EG on the viability of human monocytic and endothelial cell lines at three time points (24, 48 and 72 hours) and whether it can modulate the migration and in vitro expression of CD14, PECAM-1, ICAM-1, HLA-DR and CD105. Cell viability was affected only at higher concentrations and times. We observed decreased ICAM-1 expression in cells treated with 50 µg/ml EG and CD14 expression with IFN-γ and without IFN-γ. CD14 also decreased endothelial cell expression in the presence of IFN-γ and GE. We also found decreased expression of PECAM-1 when treated with EG and IFN-γ. In addition, EG-treated endothelial cells showed higher migration than the control group. Reduced expression of these markers and increased migration may lead to decreased cytokines, which may be contributing to decreased chronic inflammatory response during atherosclerosis and protecting endothelial integrity.

The Expression Profile of Dental Pulp-Derived Stromal Cells Supports Their Limited Capacity to Differentiate into Adipogenic Cells.

Mesenchymal stromal cells (MSCs) can self-renew, differentiate into specialised cells and have different embryonic origins-ectodermal for dental pulp-derived MSCs (DPSCs) and mesodermal for adipose tissue-derived MSCs (ADSCs). Data on DPSCs adipogenic differentiation potential and timing vary, and the lack of molecular and genetic information prompted us to gain a better understanding of DPSCs adipogenic differentiation potential and gene expression profile. While DPSCs differentiated readily along osteogenic and chondrogenic pathways, after 21 days in two different types of adipogenic induction media, DPSCs cultures did not contain lipid vacuoles and had low expression levels of the adipogenic genes proliferator-activated receptor gamma (PPARG), lipoprotein lipase (LPL) and CCAAT/enhancer-binding protein alpha (CEBPA). To better understand this limitation in adipogenesis, transcriptome analysis in undifferentiated DPSCs was carried out, with the ADSC transcriptome used as a positive control. In total, 14,871 transcripts were common to DPSCs and ADSCs, some were unique (DPSCs: 471, ADSCs: 1032), and 510 were differentially expressed genes. Detailed analyses of overrepresented transcripts showed that DPSCs express genes that inhibit adipogenic differentiation, revealing the possible mechanism for their limited adipogenesis.

Evidence of nuclei-encoded spliceosome mediating splicing of mitochondrial RNA.

Mitochondria are thought to have originated as free-living prokaryotes. Mitochondria organelles have small circular genomes with substantial structural and genetic similarity to bacteria. Contrary to the prevailing concept of intronless mitochondria, here we present evidence that mitochondrial RNA transcripts (mtRNA) are not limited to policystronic molecules, but also processed as nuclei-like transcripts that are differentially spliced and expressed in a cell-type specific manner. The presence of canonical splice sites in the mtRNA introns and of core components of the nuclei-encoded spliceosome machinery within the mitochondrial organelle suggest that nuclei-encoded spliceosome can mediate splicing of mtRNA.

Differential L1 regulation in pluripotent stem cells of humans and apes.

Identifying cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic understanding of the evolution and diversity of our own species. Until now, preserved tissues have been the main source for most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). However, these tissue samples do not fairly represent the distinctive traits of live cell behaviour and are not amenable to genetic manipulation. We propose that induced pluripotent stem (iPS) cells could be a unique biological resource to determine relevant phenotypical differences between human and NHPs, and that those differences could have potential adaptation and speciation value. Here we describe the generation and initial characterization of iPS cells from chimpanzees and bonobos as new tools to explore factors that may have contributed to great ape evolution. Comparative gene expression analysis of human and NHP iPS cells revealed differences in the regulation of long interspersed element-1 (L1, also known as LINE-1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution. Decreased levels of L1-restricting factors APOBEC3B (also known as A3B) and PIWIL2 (ref. 7) in NHP iPS cells correlated with increased L1 mobility and endogenous L1 messenger RNA levels. Moreover, results from the manipulation of A3B and PIWIL2 levels in iPS cells supported a causal inverse relationship between levels of these proteins and L1 retrotransposition. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPS cells in culture and may have also occurred in the germ line or embryonic cells developmentally upstream to germline specification during primate evolution. We propose that differences in L1 mobility may have differentially shaped the genomes of humans and NHPs and could have continuing adaptive significance.

Layered hydrogels accelerate iPSC-derived neuronal maturation and reveal migration defects caused by MeCP2 dysfunction.

Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays, as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here, we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders, such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. Using this platform, we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus, this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types.

Cockayne syndrome-derived neurons display reduced synapse density and altered neural network synchrony.

Cockayne syndrome (CS) is a rare genetic disorder in which 80% of cases are caused by mutations in the Excision Repair Cross-Complementation group 6 gene (ERCC6). The encoded ERCC6 protein is more commonly referred to as Cockayne Syndrome B protein (CSB). Classical symptoms of CS patients include failure to thrive and a severe neuropathology characterized by microcephaly, hypomyelination, calcification and neuronal loss. Modeling the neurological aspect of this disease has proven difficult since murine models fail to mirror classical neurological symptoms. Therefore, a robust human in vitro cellular model would advance our fundamental understanding of the disease and reveal potential therapeutic targets. Herein, we successfully derived functional CS neural networks from human CS induced pluripotent stem cells (iPSCs) providing a new tool to facilitate studying this devastating disease. We identified dysregulation of the Growth Hormone/Insulin-like Growth Factor-1 (GH/IGF-1) pathway as well as pathways related to synapse formation, maintenance and neuronal differentiation in CSB neurons using unbiased RNA-seq gene expression analyses. Moreover, when compared to unaffected controls, CSB-deficient neural networks displayed altered electrophysiological activity, including decreased synchrony, and reduced synapse density. Collectively, our work reveals that CSB is required for normal neuronal function and we have established an alternative to previously available models to further study neural-specific aspects of CS.

De novo transcriptome assembly and analysis to identify potential gene targets for RNAi-mediated control of the tomato leafminer (Tuta absoluta).

BACKGROUND: Providing double-stranded RNA (dsRNA) to insects has been proven to silence target genes, and this approach has emerged as a potential method to control agricultural pests by engineering plants to express insect dsRNAs. A critical step of this technology is the screening of effective target genes essential for insect development and/or survival. The tomato leafminer (Tuta absoluta Meyrick) is a major Solanum lycopersicum (tomato) pest that causes significant yield losses and has recently invaded Europe, from where it is spreading at an alarming rate. To explore RNA interference (RNAi) against T. absoluta, sequence information on potential target genes is necessary, but only a few sequences are available in public databases. RESULTS: We sequenced six libraries from RNA samples from eggs, adults, and larvae at four stages, obtaining an overall total of around 245 million reads. The assembled T. absoluta transcriptome contained 93,477 contigs with an average size of 1,574 bp, 59.8 % of which presented positive Blast hits, with 19,995 (21.4 %) annotated by gene ontology. From the transcriptome, most of the core genes of the RNAi mechanism of Lepidoptera were identified indicating the potential suitability of T. absoluta for gene silencing. No contigs displayed significant similarity with a RNA-dependent RNA Polymerase. Genes from the juvenile hormone and ecdysteroid biosynthetic pathways were identified, representing potential target genes for systemic silencing. Comparisons of transcript profiles among stages revealed 1,577 genes differentially expressed at earlier larval stages, from which potential gene targets were identified. Five of these genes were evaluated using in vitro transcribed dsRNA absorbed by tomato leaflets, which were fed to 1(st) instar T. absoluta larvae, resulting in significant reduction of larval body weight while exhibiting significant knockdown for three of the genes. CONCLUSIONS: The transcriptome we generated represents a valuable genomic resource for screening potential gene targets that affect the development or survival of T. absoluta larvae. Five novel genes that showed greater expression at the 1(st) larval stage were demonstrated to be effective potential RNAi targets by reducing larval weight and can be considered good candidates for use in RNAi-mediated crop protection.

Genetic network analysis indicate that individuals affected by neurodevelopmental conditions have genetic variations associated with ophthalmologic alterations: A critical review of literature.

Changes in the nervous system are related to a wide range of mental disorders, which include neurodevelopmental disorders (NDD) that are characterized by early onset mental conditions, such as schizophrenia and autism spectrum disorders and correlated conditions (ASD). Previous studies have shown distinct genetic components associated with diverse schizophrenia and ASD phenotypes, with mostly focused on rescuing neural phenotypes and brain activity, but alterations related to vision are overlooked. Thus, as the vision is composed by the eyes that itself represents a part of the brain, with the retina being formed by neurons and cells originating from the glia, genetic variations affecting the brain can also affect the vision. Here, we performed a critical systematic literature review to screen for all genetic variations in individuals presenting NDD with reported alterations in vision. Using these restricting criteria, we found 20 genes with distinct types of genetic variations, inherited or de novo, that includes SNP, SNV, deletion, insertion, duplication or indel. The variations occurring within protein coding regions have different impact on protein formation, such as missense, nonsense or frameshift. Moreover, a molecular analysis of the 20 genes found revealed that 17 shared a common protein-protein or genetic interaction network. Moreover, gene expression analysis in samples from the brain and other tissues indicates that 18 of the genes found are highly expressed in the brain and retina, indicating their potential role in adult vision phenotype. Finally, we only found 3 genes from our study described in standard public databanks of ophthalmogenetics, suggesting that the other 17 genes could be novel target for vision diseases.

Loss of GTF2I promotes neuronal apoptosis and synaptic reduction in human cellular models of neurodevelopment.

Individuals with Williams syndrome (WS), a neurodevelopmental disorder caused by hemizygous loss of 26-28 genes at 7q11.23, characteristically portray a hypersocial phenotype. Copy-number variations and mutations in one of these genes, GTF2I, are associated with altered sociality and are proposed to underlie hypersociality in WS. However, the contribution of GTF2I to human neurodevelopment remains poorly understood. Here, human cellular models of neurodevelopment, including neural progenitors, neurons, and three-dimensional cortical organoids, are differentiated from CRISPR-Cas9-edited GTF2I-knockout (GTF2I-KO) pluripotent stem cells to investigate the role of GTF2I in human neurodevelopment. GTF2I-KO progenitors exhibit increased proliferation and cell-cycle alterations. Cortical organoids and neurons demonstrate increased cell death and synaptic dysregulation, including synaptic structural dysfunction and decreased electrophysiological activity on a multielectrode array. Our findings suggest that changes in synaptic circuit integrity may be a prominent mediator of the link between alterations in GTF2I and variation in the phenotypic expression of human sociality.

Human microglial cells as a therapeutic target in a neurodevelopmental disease model.

Although microglia are macrophages of the central nervous system, their involvement is not limited to immune functions. The roles of microglia during development in humans remain poorly understood due to limited access to fetal tissue. To understand how microglia can impact human neurodevelopment, the methyl-CpG binding protein 2 (MECP2) gene was knocked out in human microglia-like cells (MGLs). Disruption of the MECP2 in MGLs led to transcriptional and functional perturbations, including impaired phagocytosis. The co-culture of healthy MGLs with MECP2-knockout (KO) neurons rescued synaptogenesis defects, suggesting a microglial role in synapse formation. A targeted drug screening identified ADH-503, a CD11b agonist, restored phagocytosis and synapse formation in spheroid-MGL co-cultures, significantly improved disease progression, and increased survival in MeCP2-null mice. These results unveil a MECP2-specific regulation of human microglial phagocytosis and identify a novel therapeutic treatment for MECP2-related conditions.

Comment on "Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neanderthals".

Pinson et al. (1) concluded that the modern human TKTL1 gene is responsible for an increased number of cortical neurons. We show that the "putative Neanderthal variant" of TKTL1 is present in modern human backgrounds. We dispute their argument that this genetic variant is responsible for brain differences in modern humans as opposed to Neanderthals.

IARA: a complete and curated atlas of the biogenesis of spliceosome machinery during RNA splicing.

Splicing is one of the most important post-transcriptional processing systems and is responsible for the generation of transcriptome diversity in all living eukaryotes. Splicing is regulated by the spliceosome machinery, which is responsible for each step of primary RNA processing. However, current molecules and stages involved in RNA splicing are still spread over different studies. Thus, a curated atlas of spliceosome-related molecules and all involved stages during RNA processing can provide all researchers with a reliable resource to better investigate this important mechanism. Here, we present IARA (website access: https://pucpr-bioinformatics.github.io/atlas/), an extensively curated and constantly updated catalog of molecules involved in spliceosome machinery. IARA has a map of the steps involved in the human splicing mechanism, and it allows a detailed overview of the molecules involved throughout the distinct steps of splicing.

Response to Comment on "Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment".

Maricic et al. performed an undisclosed in silico­only whole-exome sequencing analysis of our data and found genomic alterations previously undetected in some clones. Some of the predicted alterations, if true, could change the original genotype of the clones. We failed to experimentally validate all but one of these genomic alterations, which did not affect our previous results or data interpretation.

Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment.

The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 (NOVA1) plays a key role in neural development and function. NOVA1 also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of NOVA1 Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in NOVA1, which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species' evolution.

Vertical lossless genomic data compression tools for assembled genomes: A systematic literature review.

The recent decrease in cost and time to sequence and assemble of complete genomes created an increased demand for data storage. As a consequence, several strategies for assembled biological data compression were created. Vertical compression tools implement strategies that take advantage of the high level of similarity between multiple assembled genomic sequences for better compression results. However, current reviews on vertical compression do not compare the execution flow of each tool, which is constituted by phases of preprocessing, transformation, and data encoding. We performed a systematic literature review to identify and compare existing tools for vertical compression of assembled genomic sequences. The review was centered on PubMed and Scopus, in which 45726 distinct papers were considered. Next, 32 papers were selected according to the following criteria: to present a lossless vertical compression tool; to use the information contained in other sequences for the compression; to be able to manipulate genomic sequences in FASTA format; and no need prior knowledge. Although we extracted performance compression results, they were not compared as the tools did not use a standardized evaluation protocol. Thus, we conclude that there's a lack of definition of an evaluation protocol that must be applied by each tool.