16p13.11 deletion variants associated with neuropsychiatric disorders cause morphological and synaptic changes in induced pluripotent stem cell-derived neurons.

16p13.11 copy number variants (CNVs) have been associated with autism, schizophrenia, psychosis, intellectual disability, and epilepsy. The majority of 16p13.11 deletions or duplications occur within three well-defined intervals, and despite growing knowledge of the functions of individual genes within these intervals, the molecular mechanisms that underlie commonly observed clinical phenotypes remain largely unknown. Patient-derived, induced pluripotent stem cells (iPSCs) provide a platform for investigating the morphological, electrophysiological, and gene-expression changes that result from 16p13.11 CNVs in human-derived neurons. Patient derived iPSCs with varying sizes of 16p13.11 deletions and familial controls were differentiated into cortical neurons for phenotypic analysis. High-content imaging and morphological analysis of patient-derived neurons demonstrated an increase in neurite branching in patients compared with controls. Whole-transcriptome sequencing revealed expression level changes in neuron development and synaptic-related gene families, suggesting a defect in synapse formation. Subsequent quantification of synapse number demonstrated increased numbers of synapses on neurons derived from early-onset patients compared to controls. The identification of common phenotypes among neurons derived from patients with overlapping 16p13.11 deletions will further assist in ascertaining common pathways and targets that could be utilized for screening drug candidates. These studies can help to improve future treatment options and clinical outcomes for 16p13.11 deletion patients.

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.

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.

Generation and characterization of human induced pluripotent stem cells (iPSCs) from three male and three female patients with CDKL5 Deficiency Disorder (CDD).

CDKL5 Deficiency Disorder (CDD) is a rare X-linked monogenic developmental encephalopathy that is estimated to affect 1:42,000 live births. CDD is caused by pathogenic variants in the CDKL5 gene and is observed in both male and female patients. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from fibroblasts of six unrelated CDD patients-three males and three females. These patients are clinically diagnosed to present with classic CDD phenotypes, including refractory epilepsy and global developmental delay, and are being followed in a longitudinal clinical study.

Development of the Chinese Version of Medication Adherence Reasons Scale (ChMAR-Scale).

Medication non-adherence is a concern in chronic disease management. Currently, there is no scale that characterizes sufficient non-adherent reasons for practical use in the Chinese population. This study developed and validated the Chinese version of the Medication Adherence Reasons Scale (ChMAR-Scale) and described non-adherence reasons in adult patients taking blood pressure medicine in Taiwan. A forward-backward procedure was used to translate the original MAR-Scale, and new items pertinent to cultural differences were added. Patients aged above 20 years old who were taking blood pressure medicine were recruited from a regional hospital and eight community pharmacies in the Taipei metropolitan area. Data analyses were conducted with IBM SPSS 19 (Armonk, NY, USA). Exploratory factor analysis revealed six domains, including belief, self-perception, forgetfulness, management, availability, and miscellaneous issues, with Cronbach's alphas ranging from 0.649 to 0.852, item-total correlations ranging from 0.362 to 0.719, and factor loadings ranging from 0.365 to 0.775. Criterion-related validity with the visual analog scale and two global items were 0.525, 0.436, and 0.502. Forgetfulness, belief issues, and self-perception issues were the most common non-adherence reasons. In conclusion, the ChMAR-Scale showed good psychometric properties and identified more reasons for medication non-adherence than other existing scales. Healthcare providers should be vigilant of these problems while consulting patients.

A quantitative single-cell assay for retrograde membrane traffic enables rapid detection of defects in cellular organization.

Retrograde membrane trafficking from plasma membrane to Golgi and endoplasmic reticulum typifies one of the key sorting steps emerging from the early endosome that affects cell surface and intracellular protein dynamics underlying cell function. While some cell surface proteins and lipids are known to sort retrograde, there are few effective methods to quantitatively measure the extent or kinetics of these events. Here we took advantage of the well-known retrograde trafficking of cholera toxin and newly defined split fluorescent protein technology to develop a quantitative, sensitive, and effectively real-time single-cell flow cytometry assay for retrograde membrane transport. The approach can be applied in high throughput to elucidate the underlying biology of membrane traffic and how endosomes adapt to the physiologic needs of different cell types and cell states.

Biallelic Mutations in TSC2 Lead to Abnormalities Associated with Cortical Tubers in Human iPSC-Derived Neurons.

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in TSC1 or TSC2 Patients frequently have epilepsy, autism spectrum disorder, and/or intellectual disability, as well as other systemic manifestations. In this study, we differentiated human induced pluripotent stem cells (iPSCs) from a female patient with TSC with one or two mutations in TSC2 into neurons using induced expression of NGN2 to examine neuronal dysregulation associated with the neurological symptoms in TSC. Using this method, neuronal differentiation was comparable between the three genotypes of iPSCs. We observed that TSC2+/- neurons show mTOR complex 1 (mTORC1) hyperactivation and associated increased cell body size and process outgrowth, as well as exacerbation of the abnormalities by loss of the second allele of TSC2 in TSC2-/- neurons. Interestingly, iPSC-derived neurons with either a single or biallelic mutation in TSC2 demonstrated hypersynchrony and downregulation of FMRP targets. However, only neurons with biallelic mutations of TSC2 demonstrated hyperactivity and transcriptional dysregulation observed in cortical tubers. These data demonstrate that loss of one allele of TSC2 is sufficient to cause some morphological and physiological changes in human neurons but that biallelic mutations in TSC2 are necessary to induce gene expression dysregulation present in cortical tubers. Finally, we found that treatment of iPSC-derived neurons with rapamycin reduced neuronal activity and partially reversed gene expression abnormalities, demonstrating that mTOR dysregulation contributes to both phenotypes. Therefore, biallelic mutations in TSC2 and associated molecular dysfunction, including mTOR hyperactivation, may play a role in the development of cortical tubers.SIGNIFICANCE STATEMENT In this study, we examined neurons derived from induced pluripotent stem cells with two, one, or no functional TSC2 (tuberous sclerosis complex 2) alleles and found that loss of one or both alleles of TSC2 results in mTORC1 hyperactivation and specific neuronal abnormalities. However, only biallelic mutations in TSC2 resulted in elevated neuronal activity and upregulation of cell adhesion genes that is also observed in cortical tubers. These data suggest that loss of heterozygosity of TSC1 or TSC2 may play an important role in the development of cortical tubers, and potentially epilepsy, in patients with TSC.

A High-Content Screen Identifies TPP1 and Aurora B as Regulators of Axonal Mitochondrial Transport.

Dysregulated axonal trafficking of mitochondria is linked to neurodegenerative disorders. We report a high-content screen for small-molecule regulators of the axonal transport of mitochondria. Six compounds enhanced mitochondrial transport in the sub-micromolar range, acting via three cellular targets: F-actin, Tripeptidyl peptidase 1 (TPP1), or Aurora Kinase B (AurKB). Pharmacological inhibition or small hairpin RNA (shRNA) knockdown of each target promotes mitochondrial axonal transport in rat hippocampal neurons and induced pluripotent stem cell (iPSC)-derived human cortical neurons and enhances mitochondrial transport in iPSC-derived motor neurons from an amyotrophic lateral sclerosis (ALS) patient bearing one copy of SOD1A4V mutation. Our work identifies druggable regulators of axonal transport of mitochondria, provides broadly applicable methods for similar image-based screens, and suggests that restoration of proper axonal trafficking of mitochondria can be achieved in human ALS neurons.

RBFOX1 and RBFOX2 are dispensable in iPSCs and iPSC-derived neurons and do not contribute to neural-specific paternal UBE3A silencing.

Angelman Syndrome (AS) is a rare neurodevelopmental disorder caused by loss of function of the maternally inherited copy of UBE3A, an imprinted gene expressed biallelically in most tissues, but expressed exclusively from the maternal allele in neurons. Active transcription of the neuron-specific long non-coding RNA (lncRNA), UBE3A-ATS, has been shown to silence paternal UBE3A. We hypothesized that alternative splicing factors RBFOX2 and RBFOX1 might mediate splicing changes and result in the transcription of UBE3A-ATS in neurons. We found that RBFOX2 and RBFOX1 both bind to UBE3A-ATS transcript in neurons, but are not required for gene expression and/or neuron-specific processing in the SNURF/SNRPN-UBE3A region. However, we found that depletion of RBFOX2 causes a proliferation phenotype in immature neural cultures, suggesting that RBFOX2 is involved in division versus differentiation decisions in iPSC-derived neural progenitors. Absence of RBFOX2 also altered the expression of some genes that are important for glutamatergic neocortical development and Wnt-Frizzled signalling in mature neuronal cultures. Our data show that while RBFOX1 and RBFOX2 do not mediate neuron-specific processing of UBE3A-ATS, these proteins play important roles in developing neurons and are not completely functionally redundant.

Modeling Genomic Imprinting Disorders Using Induced Pluripotent Stem Cells.

Induced pluripotent stem cell (iPSC) technology has allowed for the invaluable modeling of many genetic disorders including disorders associated with genomic imprinting. Genomic imprinting involves differential DNA and histone methylation and results in allele-specific gene expression. Most of the epigenetic marks in somatic cells are erased and reestablished during the process of reprogramming into iPSCs. Therefore, in generating models of disorders associated with genomic imprinting, it is important to verify that the imprinting status and allele-specific gene expression patterns of the parental somatic cells are maintained in their derivative iPSCs. Here, we describe three techniques: DNA methylation analysis, allele-specific PCR, and RNA FISH, which we use to analyze genomic imprinting in iPSC models of neurogenetic disorders involving copy number variations of the chromosome 15q11-q13 region.

Colchicine-induced degeneration of the micronucleus during conjugation in Tetrahymena.

One of the most dramatic examples of nuclear morphogenesis occurs during conjugation in Tetrahymena when the micronucleus elongates to a size longer than the cell itself. After contraction to a spherical shape, the nucleus moves directly to chromosome separation in the first meiotic division. Here we investigate the consequences of interrupting the elongation process. Colchicine, a microtubule inhibitor, caused retraction of elongated structures. With time, cells began to lose their micronuclei, and by five hours more than half of the paired cells had at least one cell missing a micronucleus. After reversing the colchicine block, existing micronuclei did not undergo elongation again, nor did meiosis occur. These observations indicate that micronuclear elongation is critical to subsequent meiotic division. Further, nuclear elimination occurs, which could be due to meiotic failure or possibly a problem downstream from meiosis. An analysis of the process of colchicine-induced micronuclear degeneration indicated that it was regulated by a caspase-dependent mechanism, characteristic of apoptosis, and then resorbed by a lysosome-dependent autophagic mechanism. Amicronucleate cells failed to grow when returned to nutrient medium, likely because of a lesion in the post-conjugation reconstruction of a functioning oral apparatus. The ease by which a large number of nuclei are induced to "self-destruct" may make this system useful in investigating the link between colchicine treatment and nuclear death in Tetrahymena, and in investigating how nuclear death could be regulated in living cells more generally. Finally, we note that this phenomenon might relate to the evolution of amicronucleate species of Tetrahymena.

Imprinted expression of UBE3A in non-neuronal cells from a Prader-Willi syndrome patient with an atypical deletion.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two neurodevelopmental disorders most often caused by deletions of the same region of paternally inherited and maternally inherited human chromosome 15q, respectively. AS is a single gene disorder, caused by the loss of function of the ubiquitin ligase E3A (UBE3A) gene, while PWS is still considered a contiguous gene disorder. Rare individuals with PWS who carry atypical microdeletions on chromosome 15q have narrowed the critical region for this disorder to a 108 kb region that includes the SNORD116 snoRNA cluster and the Imprinted in Prader-Willi (IPW) non-coding RNA. Here we report the derivation of induced pluripotent stem cells (iPSCs) from a PWS patient with an atypical microdeletion that spans the PWS critical region. We show that these iPSCs express brain-specific portions of the transcripts driven by the PWS imprinting center, including the UBE3A antisense transcript (UBE3A-ATS). Furthermore, UBE3A expression is imprinted in most of these iPSCs. These data suggest that UBE3A imprinting in neurons only requires UBE3A-ATS expression, and no other neuron-specific factors. These data also suggest that a boundary element lying within the PWS critical region prevents UBE3A-ATS expression in non-neural tissues.

Gene expression analysis of human induced pluripotent stem cell-derived neurons carrying copy number variants of chromosome 15q11-q13.1.

BACKGROUND: Duplications of the chromosome 15q11-q13.1 region are associated with an estimated 1 to 3% of all autism cases, making this copy number variation (CNV) one of the most frequent chromosome abnormalities associated with autism spectrum disorder (ASD). Several genes located within the 15q11-q13.1 duplication region including ubiquitin protein ligase E3A (UBE3A), the gene disrupted in Angelman syndrome (AS), are involved in neural function and may play important roles in the neurobehavioral phenotypes associated with chromosome 15q11-q13.1 duplication (Dup15q) syndrome. METHODS: We have generated induced pluripotent stem cell (iPSC) lines from five different individuals containing CNVs of 15q11-q13.1. The iPSC lines were differentiated into mature, functional neurons. Gene expression across the 15q11-q13.1 locus was compared among the five iPSC lines and corresponding iPSC-derived neurons using quantitative reverse transcription PCR (qRT-PCR). Genome-wide gene expression was compared between neurons derived from three iPSC lines using mRNA-Seq. RESULTS: Analysis of 15q11-q13.1 gene expression in neurons derived from Dup15q iPSCs reveals that gene copy number does not consistently predict expression levels in cells with interstitial duplications of 15q11-q13.1. mRNA-Seq experiments show that there is substantial overlap in the genes differentially expressed between 15q11-q13.1 deletion and duplication neurons, Finally, we demonstrate that UBE3A transcripts can be pharmacologically rescued to normal levels in iPSC-derived neurons with a 15q11-q13.1 duplication. CONCLUSIONS: Chromatin structure may influence gene expression across the 15q11-q13.1 region in neurons. Genome-wide analyses suggest that common neuronal pathways may be disrupted in both the Angelman and Dup15q syndromes. These data demonstrate that our disease-specific stem cell models provide a new tool to decipher the underlying cellular and genetic disease mechanisms of ASD and may also offer a pathway to novel therapeutic intervention in Dup15q syndrome.

Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader-Willi syndromes.

Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are neurodevelopmental disorders of genomic imprinting. AS results from loss of function of the ubiquitin protein ligase E3A (UBE3A) gene, whereas the genetic defect in PWS is unknown. Although induced pluripotent stem cells (iPSCs) provide invaluable models of human disease, nuclear reprogramming could limit the usefulness of iPSCs from patients who have AS and PWS should the genomic imprint marks be disturbed by the epigenetic reprogramming process. Our iPSCs derived from patients with AS and PWS show no evidence of DNA methylation imprint erasure at the cis-acting PSW imprinting center. Importantly, we find that, as in normal brain, imprinting of UBE3A is established during neuronal differentiation of AS iPSCs, with the paternal UBE3A allele repressed concomitant with up-regulation of the UBE3A antisense transcript. These iPSC models of genomic imprinting disorders will facilitate investigation of the AS and PWS disease processes and allow study of the developmental timing and mechanism of UBE3A repression in human neurons.