CGG repeats in the human FMR1 gene regulate mRNA localization and cellular stress in developing neurons.

The human genome has many short tandem repeats, yet the normal functions of these repeats are unclear. The 5' untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene contains polymorphic CGG repeats, the length of which has differing effects on FMR1 expression and human health, including the neurodevelopmental disorder fragile X syndrome. We deleted the CGG repeats in the FMR1 gene (0CGG) in human stem cells and examined the effects on differentiated neurons. 0CGG neurons have altered subcellular localization of FMR1 mRNA and protein, and differential expression of cellular stress proteins compared with neurons with normal repeats (31CGG). In addition, 0CGG neurons have altered responses to glucocorticoid receptor (GR) activation, including FMR1 mRNA localization, GR chaperone HSP90α expression, GR localization, and cellular stress protein levels. Therefore, the CGG repeats in the FMR1 gene are important for the homeostatic responses of neurons to stress signals.

Rigor and reproducibility in human brain organoid research: Where we are and where we need to go.

Human brain organoid models have emerged as a promising tool for studying human brain development and function. These models preserve human genetics and recapitulate some aspects of human brain development, while facilitating manipulation in an in vitro setting. Despite their potential to transform biology and medicine, concerns persist about their fidelity. To fully harness their potential, it is imperative to establish reliable analytic methods, ensuring rigor and reproducibility. Here, we review current analytical platforms used to characterize human forebrain cortical organoids, highlight challenges, and propose recommendations for future studies to achieve greater precision and uniformity across laboratories.

3D bioprinting of human neural tissues with functional connectivity.

Probing how human neural networks operate is hindered by the lack of reliable human neural tissues amenable to the dynamic functional assessment of neural circuits. We developed a 3D bioprinting platform to assemble tissues with defined human neural cell types in a desired dimension using a commercial bioprinter. The printed neuronal progenitors differentiate into neurons and form functional neural circuits within and between tissue layers with specificity within weeks, evidenced by the cortical-to-striatal projection, spontaneous synaptic currents, and synaptic response to neuronal excitation. Printed astrocyte progenitors develop into mature astrocytes with elaborated processes and form functional neuron-astrocyte networks, indicated by calcium flux and glutamate uptake in response to neuronal excitation under physiological and pathological conditions. These designed human neural tissues will likely be useful for understanding the wiring of human neural networks, modeling pathological processes, and serving as platforms for drug testing.

3D Bioprinting of Human Neural Tissues with Functional Connectivity.

Probing how the human neural networks operate is hindered by the lack of reliable human neural tissues amenable for dynamic functional assessment of neural circuits. We developed a 3D bioprinting platform to assemble tissues with defined human neural cell types in a desired dimension using a commercial bioprinter. The printed neuronal progenitors differentiate to neurons and form functional neural circuits in and between tissue layers with specificity within weeks, evidenced by the cortical-to-striatal projection, spontaneous synaptic currents and synaptic response to neuronal excitation. Printed astrocyte progenitors develop into mature astrocytes with elaborated processes and form functional neuron-astrocyte networks, indicated by calcium flux and glutamate uptake in response to neuronal excitation under physiological and pathological conditions. These designed human neural tissues will likely be useful for understanding the wiring of human neural networks, modeling pathological processes, and serving as platforms for drug testing.

Transcriptional consequences of trisomy 21 on neural induction.

Introduction: Down syndrome, caused by trisomy 21, is a complex developmental disorder associated with intellectual disability and reduced growth of multiple organs. Structural pathologies are present at birth, reflecting embryonic origins. A fundamental unanswered question is how an extra copy of human chromosome 21 contributes to organ-specific pathologies that characterize individuals with Down syndrome, and, relevant to the hallmark intellectual disability in Down syndrome, how trisomy 21 affects neural development. We tested the hypothesis that trisomy 21 exerts effects on human neural development as early as neural induction. Methods: Bulk RNA sequencing was performed on isogenic trisomy 21 and euploid human induced pluripotent stem cells (iPSCs) at successive stages of neural induction: embryoid bodies at Day 6, early neuroectoderm at Day 10, and differentiated neuroectoderm at Day 17. Results: Gene expression analysis revealed over 1,300 differentially expressed genes in trisomy 21 cells along the differentiation pathway compared to euploid controls. Less than 5% of the gene expression changes included upregulated chromosome 21 encoded genes at every timepoint. Genes involved in specific growth factor signaling pathways (WNT and Notch), metabolism (including oxidative stress), and extracellular matrix were altered in trisomy 21 cells. Further analysis uncovered heterochronic expression of genes. Conclusion: Trisomy 21 impacts discrete developmental pathways at the earliest stages of neural development. The results suggest that metabolic dysfunction arises early in embryogenesis in trisomy 21 and may affect development and function more broadly.

Species-specific FMRP regulation of RACK1 is critical for prenatal cortical development.

Fragile X messenger ribonucleoprotein 1 protein (FMRP) deficiency leads to fragile X syndrome (FXS), an autism spectrum disorder. The role of FMRP in prenatal human brain development remains unclear. Here, we show that FMRP is important for human and macaque prenatal brain development. Both FMRP-deficient neurons in human fetal cortical slices and FXS patient stem cell-derived neurons exhibit mitochondrial dysfunctions and hyperexcitability. Using multiomics analyses, we have identified both FMRP-bound mRNAs and FMRP-interacting proteins in human neurons and unveiled a previously unknown role of FMRP in regulating essential genes during human prenatal development. We demonstrate that FMRP interaction with CNOT1 maintains the levels of receptor for activated C kinase 1 (RACK1), a species-specific FMRP target. Genetic reduction of RACK1 leads to both mitochondrial dysfunctions and hyperexcitability, resembling FXS neurons. Finally, enhancing mitochondrial functions rescues deficits of FMRP-deficient cortical neurons during prenatal development, demonstrating targeting mitochondrial dysfunction as a potential treatment.

ISSCR standards for the use of human stem cells in basic research.

The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.

Age-maintained human neurons demonstrate a developmental loss of intrinsic neurite growth ability.

Injury to adult mammalian central nervous system (CNS) axons results in limited regeneration. Rodent studies have revealed a developmental switch in CNS axon regenerative ability, yet whether this is conserved in humans is unknown. Using human fibroblasts from 8 gestational-weeks to 72 years-old, we performed direct reprogramming to transdifferentiate fibroblasts into induced neurons (Fib-iNs), avoiding pluripotency which restores cells to an embryonic state. We found that early gestational Fib-iNs grew longer neurites than all other ages, mirroring the developmental switch in regenerative ability in rodents. RNA-sequencing and screening revealed ARID1A as a developmentally-regulated modifier of neurite growth in human neurons. These data suggest that age-specific epigenetic changes may drive the intrinsic loss of neurite growth ability in human CNS neurons during development. One-Sentence Summary: Directly-reprogrammed human neurons demonstrate a developmental decrease in neurite growth ability.

Meta-Analysis of Down Syndrome Cortical Development Reveals Underdeveloped State of the Science.

Neurodevelopmental impairment contributes to the hallmark cognitive disability in individuals with Down syndrome (DS, trisomy 21, T21). The appearance of cognitive deficits in infancy suggests that alterations emerge during the earliest stages of neural development and continue throughout the lifespan in DS. Neural correlates of intellectual and language function include cortical structures, specifically temporal and frontal lobes that are smaller in DS. Yet, despite increased understanding of the DS cognitive-behavioral phenotype in childhood, there is very little structural and histological information to help explain the deficits. Consequently, attempts to effectively design therapeutic targets or interventions are limited. We present a systematic review of published research on cortical development in DS that reveals a paucity of studies that rigorously identify cellular features that may underlie the gross morphological deficits of the developing DS brain. We assessed 115 published reports retrieved through PubMed and other sources and found that only 23 reported histological and/or immunohistochemical data to define cell composition affected in DS post-mortem brain. Further, our analysis reveals that many reports have limited samples sizes and few DS samples, making it difficult to draw conclusions that are generally applicable to the DS population. Thus, the lack of replication and limited number of studies indicate that more developmentally focused research, ideally using equal numbers of age-matched samples in analyses, is needed to elucidate the cellular nature of smaller brain size in DS.

Altered patterning of trisomy 21 interneuron progenitors.

Individuals with Down syndrome (DS; Ts21), the most common genetic cause of intellectual disability, have smaller brains that reflect fewer neurons at pre- and post-natal stages, implicating impaired neurogenesis during development. Our stereological analysis of adult DS cortex indicates a reduction of calretinin-expressing interneurons. Using Ts21 human induced pluripotent stem cells (iPSCs) and isogenic controls, we find that Ts21 progenitors generate fewer COUP-TFII+ progenitors with reduced proliferation. Single-cell RNA sequencing of Ts21 progenitors confirms the altered specification of progenitor subpopulations and identifies reduced WNT signaling. Activation of WNT signaling partially restores the COUP-TFII+ progenitor population in Ts21, suggesting that altered WNT signaling contributes to the defective development of cortical interneurons in DS.

Cell models for Down syndrome-Alzheimer's disease research.

Down syndrome (DS) is the most common chromosomal abnormality and leads to intellectual disability, increased risk of cardiac defects, and an altered immune response. Individuals with DS have an extra full or partial copy of chromosome 21 (trisomy 21) and are more likely to develop early-onset Alzheimer's disease (AD) than the general population. Changes in expression of human chromosome 21 (Hsa21)-encoded genes, such as amyloid precursor protein (APP), play an important role in the pathogenesis of AD in DS (DS-AD). However, the mechanisms of DS-AD remain poorly understood. To date, several mouse models with an extra copy of genes syntenic to Hsa21 have been developed to characterise DS-AD-related phenotypes. Nonetheless, due to genetic and physiological differences between mouse and human, mouse models cannot faithfully recapitulate all features of DS-AD. Cells differentiated from human-induced pluripotent stem cells (iPSCs), isolated from individuals with genetic diseases, can be used to model disease-related cellular and molecular pathologies, including DS. In this review, we will discuss the limitations of mouse models of DS and how these can be addressed using recent advancements in modelling DS using human iPSCs and iPSC-mouse chimeras, and potential applications of iPSCs in preclinical studies for DS-AD.

ATase inhibition rescues age-associated proteotoxicity of the secretory pathway.

Malfunction of autophagy contributes to the progression of many chronic age-associated diseases. As such, improving normal proteostatic mechanisms is an active target for biomedical research and a key focal area for aging research. Endoplasmic reticulum (ER)-based acetylation has emerged as a mechanism that ensures proteostasis within the ER by regulating the induction of ER specific autophagy. ER acetylation is ensured by two ER-membrane bound acetyltransferases, ATase1 and ATase2. Here, we show that ATase inhibitors can rescue ongoing disease manifestations associated with the segmental progeria-like phenotype of AT-1 sTg mice. We also describe a pipeline to reliably identify ATase inhibitors with promising druggability properties. Finally, we show that successful ATase inhibitors can rescue the proteopathy of a mouse model of Alzheimer's disease. In conclusion, our study proposes that ATase-targeting approaches might offer a translational pathway for many age-associated proteopathies affecting the ER/secretory pathway.

Chemically induced senescence in human stem cell-derived neurons promotes phenotypic presentation of neurodegeneration.

Modeling age-related neurodegenerative disorders with human stem cells are difficult due to the embryonic nature of stem cell-derived neurons. We developed a chemical cocktail to induce senescence of iPSC-derived neurons to address this challenge. We first screened small molecules that induce embryonic fibroblasts to exhibit features characteristic of aged fibroblasts. We then optimized a cocktail of small molecules that induced senescence in fibroblasts and cortical neurons without causing DNA damage. The utility of the "senescence cocktail" was validated in motor neurons derived from ALS patient iPSCs which exhibited protein aggregation and axonal degeneration substantially earlier than those without cocktail treatment. Our "senescence cocktail" will likely enhance the manifestation of disease-related phenotypes in neurons derived from iPSCs, enabling the generation of reliable drug discovery platforms.

Building the Future Therapies for Down Syndrome: The Third International Conference of the T21 Research Society.

Research focused on Down syndrome has increased in the last several years to advance understanding of the consequences of trisomy 21 (T21) on molecular and cellular processes and, ultimately, on individuals with Down syndrome. The Trisomy 21 Research Society (T21RS) is the premier scientific organization for researchers and clinicians studying Down syndrome. The Third International Conference of T21RS, held June 6-9, 2019, in Barcelona, Spain, brought together 429 scientists, families, and industry representatives to share the latest discoveries on underlying cellular and molecular mechanisms of T21, define cognitive and behavioral challenges and better understand comorbidities associated with Down syndrome, including Alzheimer's disease and leukemia. Presentation of cutting-edge results in neuroscience, neurology, model systems, psychology, cancer, biomarkers and molecular and phar-ma-cological therapeutic approaches demonstrate the compelling interest and continuing advancement in all aspects of understanding and ameliorating conditions associated with T21.

Corrigendum: Basal Forebrain Cholinergic Neurons: Linking Down Syndrome and Alzheimer's Disease.

[This corrects the article DOI: 10.3389/fnagi.2021.703876.].

Basal Forebrain Cholinergic Neurons: Linking Down Syndrome and Alzheimer's Disease.

Down syndrome (DS, trisomy 21) is characterized by intellectual impairment at birth and Alzheimer's disease (AD) pathology in middle age. As individuals with DS age, their cognitive functions decline as they develop AD pathology. The susceptibility to degeneration of a subset of neurons, known as basal forebrain cholinergic neurons (BFCNs), in DS and AD is a critical link between cognitive impairment and neurodegeneration in both disorders. BFCNs are the primary source of cholinergic innervation to the cerebral cortex and hippocampus, as well as the amygdala. They play a critical role in the processing of information related to cognitive function and are directly engaged in regulating circuits of attention and memory throughout the lifespan. Given the importance of BFCNs in attention and memory, it is not surprising that these neurons contribute to dysfunctional neuronal circuitry in DS and are vulnerable in adults with DS and AD, where their degeneration leads to memory loss and disturbance in language. BFCNs are thus a relevant cell target for therapeutics for both DS and AD but, despite some success, efforts in this area have waned. There are gaps in our knowledge of BFCN vulnerability that preclude our ability to effectively design interventions. Here, we review the role of BFCN function and degeneration in AD and DS and identify under-studied aspects of BFCN biology. The current gaps in BFCN relevant imaging studies, therapeutics, and human models limit our insight into the mechanistic vulnerability of BFCNs in individuals with DS and AD.

Opportunities, barriers, and recommendations in down syndrome research.

BACKGROUND: Recent advances in medical care have increased life expectancy and improved the quality of life for people with Down syndrome (DS). These advances are the result of both pre-clinical and clinical research but much about DS is still poorly understood. In 2020, the NIH announced their plan to update their DS research plan and requested input from the scientific and advocacy community. OBJECTIVE: The National Down Syndrome Society (NDSS) and the LuMind IDSC Foundation worked together with scientific and medical experts to develop recommendations for the NIH research plan. METHODS: NDSS and LuMind IDSC assembled over 50 experts across multiple disciplines and organized them in eleven working groups focused on specific issues for people with DS. RESULTS: This review article summarizes the research gaps and recommendations that have the potential to improve the health and quality of life for people with DS within the next decade. CONCLUSIONS: This review highlights many of the scientific gaps that exist in DS research. Based on these gaps, a multidisciplinary group of DS experts has made recommendations to advance DS research. This paper may also aid policymakers and the DS community to build a comprehensive national DS research strategy.

Physiological Characterization and Transcriptomic Properties of GnRH Neurons Derived From Human Stem Cells.

Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus play a key role in the regulation of reproductive function. In this study, we sought an efficient method for generating GnRH neurons from human embryonic and induced pluripotent stem cells (hESC and hiPSC, respectively). First, we found that exposure of primitive neuroepithelial cells, rather than neuroprogenitor cells, to fibroblast growth factor 8 (FGF8), was more effective in generating GnRH neurons. Second, addition of kisspeptin to FGF8 further increased the efficiency rates of GnRH neurogeneration. Third, we generated a fluorescent marker mCherry labeled human embryonic GnRH cell line (mCh-hESC) using a CRISPR-Cas9 targeting approach. Fourth, we examined physiological characteristics of GnRH (mCh-hESC) neurons: similar to GnRH neurons in vivo, they released the GnRH peptide in a pulsatile manner at ~60 min intervals; GnRH release increased in response to high potassium, kisspeptin, estradiol, and neurokinin B challenges; and injection of depolarizing current induced action potentials. Finally, we characterized developmental changes in transcriptomes of GnRH neurons using hESC, hiPSC, and mCh-hESC. The developmental pattern of transcriptomes was remarkably similar among the 3 cell lines. Collectively, human stem cell-derived GnRH neurons will be an important tool for establishing disease models to understand diseases, such as idiopathic hypothalamic hypogonadism, and testing contraceptive drugs.

DSCAM/PAK1 pathway suppression reverses neurogenesis deficits in iPSC-derived cerebral organoids from patients with Down syndrome.

Down syndrome (DS), caused by trisomy of chromosome 21, occurs in 1 of every 800 live births. Early defects in cortical development likely account for the cognitive impairments in DS, although the underlying molecular mechanism remains elusive. Here, we performed histological assays and unbiased single-cell RNA-Seq (scRNA-Seq) analysis on cerebral organoids derived from 4 euploid cell lines and from induced pluripotent stem cells (iPSCs) from 3 individuals with trisomy 21 to explore cell-type-specific abnormalities associated with DS during early brain development. We found that neurogenesis was significantly affected, given the diminished proliferation and decreased expression of layer II and IV markers in cortical neurons in the subcortical regions; this may have been responsible for the reduced size of the organoids. Furthermore, suppression of the DSCAM/PAK1 pathway, which showed enhanced activity in DS, using CRISPR/Cas9, CRISPR interference (CRISPRi), or small-molecule inhibitor treatment reversed abnormal neurogenesis, thereby increasing the size of organoids derived from DS iPSCs. Our study demonstrates that 3D cortical organoids developed in vitro are a valuable model of DS and provide a direct link between dysregulation of the DSCAM/PAK1 pathway and developmental brain defects in DS.

Balancing serendipity and reproducibility: Pluripotent stem cells as experimental systems for intellectual and developmental disorders.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) and their differentiation into neural lineages is a revolutionary experimental system for studying neurological disorders, including intellectual and developmental disabilities (IDDs). However, issues related to variability and reproducibility have hindered translating preclinical findings into drug discovery. Here, we identify areas for improvement by conducting a comprehensive review of 58 research articles that utilized iPSC-derived neural cells to investigate genetically defined IDDs. Based upon these findings, we propose recommendations for best practices that can be adopted by research scientists as well as journal editors.