Subcellular protein turnover in human neural progenitor cells revealed by correlative electron microscopy and nanoscale secondary ion mass spectrometry imaging.

Protein turnover is a critical process for accurate cellular function, in which damaged proteins in the cells are gradually replaced with newly synthesized ones. Many previous studies on cellular protein turnover have used stable isotopic labelling by amino acids in cell culture (SILAC), followed by proteomic bulk analysis. However, this approach does not take into account the heterogeneity observed at the single-cell and subcellular levels. To address this, we investigated the protein turnover of neural progenitor cells at the subcellular resolution, using correlative TEM and NanoSIMS imaging, relying on a pulse-chase analysis of isotopically-labelled protein precusors. Cellular protein turnover was found significantly heterogenous across individual organelles, which indicates a possible relation between protein turnover and subcellular activity. In addition, different isotopically-labelled amino acids provided different turnover patterns, in spite of all being protein precursors, suggesting that they undergo distinct protein synthesis and metabolic pathways at the subcellular level.

Lipid organization and turnover in the plasma membrane of human differentiating neural progenitor cells revealed by time-of-flight secondary ion mass spectrometry imaging.

Membrane lipids have been known to influence multiple signalling and cellular processes. Dysregulation of lipids at the neuronal membrane is connected to a significant alteration of the brain function and morphology, leading to brain diseases and neurodegeneration. Understanding the lipid composition and turnover of neuronal membrane will provide a significant insight into the molecular events underlying the regulatory effects of these biomolecules in a neuronal system. In this study, we aimed to characterize the composition and turnover of the plasma membrane lipids in human neural progenitor cells (NPCs) at an early differentiation stage into midbrain neurons using ToF-SIMS imaging. Lipid composition of the native plasma membrane was explored, followed by an examination of the lipid turnover using different isotopically labelled lipid precursors, including 13C-choline, 13C-lauric acid, 15N-linoleic, and 13C-stearic. Our results showed that differentiating NPCs contain a high abundance of ceramides, glycerophosphoserines, neutral glycosphingolipids, diradylglycerols, and glycerophosphocholines at the plasma membrane. In addition, different precursors were found to incorporate into different membrane lipids which are specific for the short- or long-carbon chains, and the unsaturation or saturation stage of the precursors. The lipid structure of neuronal membrane reflects the differentiation status of NPCs, and it can be altered significantly using a particular lipid precursor. Our study illustrates a potential of ToF-SIMS imaging to study native plasma membrane lipids and elucidate complex cellular processes by providing molecular -rich information at a single cell level.

FOXG1 targets BMP repressors and cell cycle inhibitors in human neural progenitor cells.

FOXG1 is a critical transcription factor in human brain where loss-of-function mutations cause a severe neurodevelopmental disorder, while increased FOXG1 expression is frequently observed in glioblastoma. FOXG1 is an inhibitor of cell patterning and an activator of cell proliferation in chordate model organisms but different mechanisms have been proposed as to how this occurs. To identify genomic targets of FOXG1 in human neural progenitor cells (NPCs), we engineered a cleavable reporter construct in endogenous FOXG1 and performed chromatin immunoprecipitation (ChIP) sequencing. We also performed deep RNA sequencing of NPCs from two females with loss-of-function mutations in FOXG1 and their healthy biological mothers. Integrative analyses of RNA and ChIP sequencing data showed that cell cycle regulation and Bone Morphogenic Protein (BMP) repression gene ontology categories were over-represented as FOXG1 targets. Using engineered brain cell lines, we show that FOXG1 specifically activates SMAD7 and represses CDKN1B. Activation of SMAD7 which inhibits BMP signaling may be one way that FOXG1 patterns the forebrain, while repression of cell cycle regulators such as CDKN1B may be one way that FOXG1 expands the NPC pool to ensure proper brain size. Our data reveal novel mechanisms on how FOXG1 may control forebrain patterning and cell proliferation in human brain development.

Characterization of Stress Granule Protein Turnover in Neuronal Progenitor Cells Using Correlative STED and NanoSIMS Imaging.

Stress granules (SGs) are stress-induced biomolecular condensates which originate primarily from inactivated RNA translation machinery and translation initiation factors. SG formation is an important defensive mechanism for cell survival, while its dysfunction has been linked to neurodegenerative diseases. However, the molecular mechanisms of SG assembly and disassembly, as well as their impacts on cellular recovery, are not fully understood. More thorough investigations into the molecular dynamics of SG pathways are required to understand the pathophysiological roles of SGs in cellular systems. Here, we characterize the SG and cytoplasmic protein turnover in neuronal progenitor cells (NPCs) under stressed and non-stressed conditions using correlative STED and NanoSIMS imaging. We incubate NPCs with isotopically labelled (15N) leucine and stress them with the ER stressor thapsigargin (TG). A correlation of STED and NanoSIMS allows the localization of individual SGs (using STED), and their protein turnover can then be extracted based on the 15N/14N ratio (using NanoSIMS). We found that TG-induced SGs, which are highly dynamic domains, recruit their constituents predominantly from the cytoplasm. Moreover, ER stress impairs the total cellular protein turnover regimen, and this impairment is not restored after the commonly proceeded stress recovery period.

Putative Roles of SETBP1 Dosage on the SET Oncogene to Affect Brain Development.

Mutations in SET BINDING PROTEIN 1 (SETBP1) cause two different clinically distinguishable diseases called Schinzel-Giedion syndrome (SGS) or SETBP1 deficiency syndrome (SDD). Both disorders are disorders of protein dosage, where SGS is caused by decreased rate of protein breakdown due to mutations in a proteosome targeting domain, and SDD is caused by heterozygous loss-of-function mutations leading to haploinsufficiency. While phenotypes of affected individuals support a role for SETBP1 in brain development, little is known about the mechanisms that might underlie this. The binding partner which gave SETBP1 its name is SET and there is extensive literature on this important oncogene in non-neural tissues. Here we describe different molecular complexes in which SET is involved as well as the role of these complexes in brain development. Based on this information, we postulate how SETBP1 protein dosage might influence these SET-containing molecular pathways and affect brain development. We examine the roles of SET and SETBP1 in acetylation inhibition, phosphatase activity, DNA repair, and cell cycle control. This work provides testable hypotheses for how altered SETBP1 protein dosage affects brain development.

Kabuki syndrome stem cell models reveal locus specificity of histone methyltransferase 2D (KMT2D/MLL4).

Kabuki syndrome is frequently caused by loss-of-function mutations in one allele of histone 3 lysine 4 (H3K4) methyltransferase KMT2D and is associated with problems in neurological, immunological and skeletal system development. We generated heterozygous KMT2D knockout and Kabuki patient-derived cell models to investigate the role of reduced dosage of KMT2D in stem cells. We discovered chromosomal locus-specific alterations in gene expression, specifically a 110 Kb region containing Synaptotagmin 3 (SYT3), C-Type Lectin Domain Containing 11A (CLEC11A), Chromosome 19 Open Reading Frame 81 (C19ORF81) and SH3 And Multiple Ankyrin Repeat Domains 1 (SHANK1), suggesting locus-specific targeting of KMT2D. Using whole genome histone methylation mapping, we confirmed locus-specific changes in H3K4 methylation patterning coincident with regional decreases in gene expression in Kabuki cell models. Significantly reduced H3K4 peaks aligned with regions of stem cell maps of H3K27 and H3K4 methylation suggesting KMT2D haploinsufficiency impact bivalent enhancers in stem cells. Preparing the genome for subsequent differentiation cues may be of significant importance for Kabuki-related genes. This work provides a new insight into the mechanism of action of an important gene in bone and brain development and may increase our understanding of a specific function of a human disease-relevant H3K4 methyltransferase family member.

Evidence That Substantia Nigra Pars Compacta Dopaminergic Neurons Are Selectively Vulnerable to Oxidative Stress Because They Are Highly Metabolically Active.

There are 400-500 thousand dopaminergic cells within each side of the human substantia nigra pars compacta (SNpc) making them a minuscule portion of total brain mass. These tiny clusters of cells have an outsized impact on motor output and behavior as seen in disorders such as Parkinson's disease (PD). SNpc dopaminergic neurons are more vulnerable to oxidative stress compared to other brain cell types, but the reasons for this are not precisely known. Here we provide evidence to support the hypothesis that this selective vulnerability is because SNpc neurons sustain high metabolic rates compared to other neurons. A higher baseline requirement for ATP production may lead to a selective vulnerability to impairments in oxidative phosphorylation (OXPHOS) or genetic insults that impair Complex I of the electron transport chain. We suggest that the energy demands of the unique morphological and electrophysiological properties of SNpc neurons may be one reason these cells produce more ATP than other cells. We further provide evidence to support the hypothesis that transcription factors (TFs) required to drive induction, differentiation, and maintenance of midbrain dopaminergic neural progenitor cells which give rise to terminally differentiated SNpc neurons are uniquely involved in both developmental patterning and metabolism, a dual function unlike other TFs that program neurons in other brain regions. The use of these TFs during induction and differentiation may program ventral midbrain progenitor cells metabolically to higher ATP levels, allowing for the development of those specialized cell processes seen in terminally differentiated cells. This paper provides a cellular and developmental framework for understanding the selective vulnerability of SNpc dopaminergic cells to oxidative stress.

FOXG1 dose tunes cell proliferation dynamics in human forebrain progenitor cells.

Heterozygous loss-of-function mutations in Forkhead box G1 (FOXG1), a uniquely brain-expressed gene, cause microcephaly, seizures, and severe intellectual disability, whereas increased FOXG1 expression is frequently observed in glioblastoma. To investigate the role of FOXG1 in forebrain cell proliferation, we modeled FOXG1 syndrome using cells from three clinically diagnosed cases with two sex-matched healthy parents and one unrelated sex-matched control. Cells with heterozygous FOXG1 loss showed significant reduction in cell proliferation, increased ratio of cells in G0/G1 stage of the cell cycle, and increased frequency of primary cilia. Engineered loss of FOXG1 recapitulated this effect, while isogenic repair of a patient mutation reverted output markers to wild type. An engineered inducible FOXG1 cell line derived from a FOXG1 syndrome case demonstrated that FOXG1 dose-dependently affects all cell proliferation outputs measured. These findings provide strong support for the critical importance of FOXG1 levels in controlling human brain cell growth in health and disease.

Growing as slow as a turtle: Unexpected maturational differences in a small, long-lived species.

Turtle body size is associated with demographic and other traits like mating success, reproductive output, maturity, and survival. As such, growth analyses are valuable for testing life history theory, demographic modeling, and conservation planning. Two important but unsettled research areas relate to growth after maturity and growth rate variation. If individuals exhibit indeterminate growth after maturity, older adults may have an advantage in fecundity, survival, or both over younger/smaller adults. Similarly, depending on how growth varies, a portion of the population may mature earlier, grow larger, or both. We used 23-years of capture-mark-recapture data to study growth and maturity in the Spotted Turtle (Clemmys guttata), a species suffering severe population declines and for which demographic data are needed for development of effective conservation and management strategies. There was strong support for models incorporating sex as a factor, with the interval growth model reparametrized for capture-mark-recapture data producing later mean maturation estimates than the age-based growth model. We found most individuals (94%) continued growing after maturity, but the instantaneous relative annual plastral growth rate was low. We recommend future studies examine the possible contribution of such slow, continued adult growth to fecundity and survival. Even seemingly negligible amounts of annual adult growth can have demographic consequences affecting the population vital rates for long-lived species.

Epigenetic priming in neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) affect about 1% of the population and can be caused by mutations in genes that affect the epigenetic code. There is limited functional understanding of most of these epigenetic modifiers, and we suggest that associated NDDs are caused, in part, by deficits in epigenetic priming, a prepatterning step that alters the genome in preparation to make cells competent to signaling cues. We provide evidence from high-resolution epigenetic and transcriptomic mapping studies to demonstrate how a failure to adequately prime the genome for neural induction could lead to impairment of terminally differentiated cells. This idea provides a framework for NDD pathogenesis and treatment.

Methylation of the tyrosine hydroxylase gene is dysregulated by cocaine dependence in the human striatum.

Cocaine dependence is a chronic, relapsing disorder caused by lasting changes in the brain. Animal studies have identified cocaine-related alterations in striatal DNA methylation; however, it is unclear how methylation is related to cocaine dependence in humans. We generated methylomic profiles of the nucleus accumbens using human postmortem brains from a cohort of individuals with cocaine dependence and healthy controls (n = 25 per group). We found hypermethylation in a cluster of CpGs within the gene body of tyrosine hydroxylase (TH), containing a putative binding site for the early growth response 1 (EGR1) transcription factor, which is hypermethylated in the caudate nucleus of cocaine-dependent individuals. We replicated this finding and found it to be specific to striatal neuronal nuclei. Furthermore, this locus demonstrates enhancer activity which is attenuated by methylation and enhanced by EGR1 overexpression. These results suggest that cocaine dependence alters the epigenetic regulation of dopaminergic signaling genes.

Lesch-Nyhan disease causes impaired energy metabolism and reduced developmental potential in midbrain dopaminergic cells.

Mutations in HPRT1, a gene encoding a rate-limiting enzyme for purine salvage, cause Lesch-Nyhan disease which is characterized by self-injury and motor impairments. We leveraged stem cell and genetic engineering technologies to model the disease in isogenic and patient-derived forebrain and midbrain cell types. Dopaminergic progenitor cells deficient in HPRT showed decreased intensity of all developmental cell-fate markers measured. Metabolic analyses revealed significant loss of all purine derivatives, except hypoxanthine, and impaired glycolysis and oxidative phosphorylation. real-time glucose tracing demonstrated increased shunting to the pentose phosphate pathway for de novo purine synthesis at the expense of ATP production. Purine depletion in dopaminergic progenitor cells resulted in loss of RHEB, impairing mTORC1 activation. These data demonstrate dopaminergic-specific effects of purine salvage deficiency and unexpectedly reveal that dopaminergic progenitor cells are programmed to a high-energy state prior to higher energy demands of terminally differentiated cells.

Non-CG methylation and multiple histone profiles associate child abuse with immune and small GTPase dysregulation.

Early-life adversity (ELA) is a major predictor of psychopathology, and is thought to increase lifetime risk by epigenetically regulating the genome. Here, focusing on the lateral amygdala, a major brain site for emotional homeostasis, we describe molecular cross-talk among multiple mechanisms of genomic regulation, including 6 histone marks and DNA methylation, and the transcriptome, in subjects with a history of ELA and controls. In the healthy brain tissue, we first uncover interactions between different histone marks and non-CG methylation in the CAC context. Additionally, we find that ELA associates with methylomic changes that are as frequent in the CAC as in the canonical CG context, while these two forms of plasticity occur in sharply distinct genomic regions, features, and chromatin states. Combining these multiple data indicates that immune-related and small GTPase signaling pathways are most consistently impaired in the amygdala of ELA individuals. Overall, this work provides insights into genomic brain regulation as a function of early-life experience.

Cocaine-related DNA methylation in caudate neurons alters 3D chromatin structure of the IRXA gene cluster.

Epigenetic mechanisms, like those involving DNA methylation, are thought to mediate the relationship between chronic cocaine dependence and molecular changes in addiction-related neurocircuitry, but have been understudied in human brain. We initially used reduced representation bisulfite sequencing (RRBS) to generate a methylome-wide profile of cocaine dependence in human post-mortem caudate tissue. We focused on the Iroquois Homeobox A (IRXA) gene cluster, where hypomethylation in exon 3 of IRX2 in neuronal nuclei was associated with cocaine dependence. We replicated this finding in an independent cohort and found similar results in the dorsal striatum from cocaine self-administering mice. Using epigenome editing and 3C assays, we demonstrated a causal relationship between methylation within the IRX2 gene body, CTCF protein binding, three-dimensional (3D) chromatin interaction, and gene expression. Together, these findings suggest that cocaine-related hypomethylation of IRX2 contributes to the development and maintenance of cocaine dependence through alterations in 3D chromatin structure in the caudate nucleus.

Stimulation of L-type calcium channels increases tyrosine hydroxylase and dopamine in ventral midbrain cells induced from somatic cells.

Making high-quality dopamine (DA)-producing cells for basic biological or small molecule screening studies is critical for the development of novel therapeutics for disorders of the ventral midbrain. Currently, many ventral midbrain assays have low signal-to-noise ratio due to low levels of cellular DA and the rate-limiting enzyme of DA synthesis, tyrosine hydroxylase (TH), hampering discovery efforts. Using intensively characterized ventral midbrain cells derived from human skin, which demonstrate calcium pacemaking activity and classical electrophysiological properties, we show that an L-type calcium agonist can significantly increase TH protein levels and DA content and release. Live calcium imaging suggests that it is the immediate influx of calcium occurring simultaneously in all cells that drives this effect. Genome-wide expression profiling suggests that L-type calcium channel stimulation has a significant effect on specific genes related to DA synthesis and affects expression of L-type calcium receptor subunits from the CACNA1 and CACNA2D families. Together, our findings provide an advance in the ability to increase DA and TH levels to improve the accuracy of disease modeling and small molecule screening for disorders of the ventral midbrain, including Parkinson's disease.

A roadmap for neurodevelopmental disease modeling for non-stem cell biologists.

Stem and derivative cells induced from somatic tissues are a critical tool for disease modeling but significant technical hurdles hamper their use. The purpose of this review is to provide an overview of pitfalls and mitigation strategies for the nonstem cell biologist using induced pluripotent stem cells and investigating neurodevelopmental disorders. What sample sizes are reasonable? What derivation and purification protocols should be used to make human neurons? In what way should gene editing technologies be used to support discoveries? What kinds of preclinical studies are the most feasible? It is hoped that this roadmap will provide the necessary details for experimental planning and execution for those less familiar in the area of stem cell disease modeling. High-quality human preclinical models will allow for the discovery of molecular and cellular phenotypes specific to different neurodevelopmental disorders, and may provide the assays to advance translational medicine for unmet medical needs.

Human iPSC-derived Down syndrome astrocytes display genome-wide perturbations in gene expression, an altered adhesion profile, and increased cellular dynamics.

Down syndrome (DS), caused by the triplication of human chromosome 21, leads to significant alterations in brain development and is a major genetic cause of intellectual disability. While much is known about changes to neurons in DS, the effects of trisomy 21 on non-neuronal cells such as astrocytes are poorly understood. Astrocytes are critical for brain development and function, and their alteration may contribute to DS pathophysiology. To better understand the impact of trisomy 21 on astrocytes, we performed RNA-sequencing on astrocytes from newly produced DS human induced pluripotent stem cells (hiPSCs). While chromosome 21 genes were upregulated in DS astrocytes, we found consistent up- and down-regulation of genes across the genome with a strong dysregulation of neurodevelopmental, cell adhesion and extracellular matrix molecules. ATAC (assay for transposase-accessible chromatin)-seq also revealed a global alteration in chromatin state in DS astrocytes, showing modified chromatin accessibility at promoters of cell adhesion and extracellular matrix genes. Along with these transcriptomic and epigenomic changes, DS astrocytes displayed perturbations in cell size and cell spreading as well as modifications to cell-cell and cell-substrate recognition/adhesion, and increases in cellular motility and dynamics. Thus, triplication of chromosome 21 is associated with genome-wide transcriptional, epigenomic and functional alterations in astrocytes that may contribute to altered brain development and function in DS.

FOXG1 Dose in Brain Development.

Brain development is a highly regulated process that involves the precise spatio-temporal activation of cell signaling cues. Transcription factors play an integral role in this process by relaying information from external signaling cues to the genome. The transcription factor Forkhead box G1 (FOXG1) is expressed in the developing nervous system with a critical role in forebrain development. Altered dosage of FOXG1 due to deletions, duplications, or functional gain- or loss-of-function mutations, leads to a complex array of cellular effects with important consequences for human disease including neurodevelopmental disorders. Here, we review studies in multiple species and cell models where FOXG1 dose is altered. We argue against a linear, symmetrical relationship between FOXG1 dosage states, although FOXG1 levels at the right time and place need to be carefully regulated. Neurodevelopmental disease states caused by mutations in FOXG1 may therefore be regulated through different mechanisms.

Correction: A new microdeletion syndrome involving TBC1D24, ATP6V0C, and PDPK1 causes epilepsy, microcephaly, and developmental delay.

The original version of this Article contained an error in the spelling of the author Siddharth Banka, which was incorrectly given as Siddhart Banka. This has now been corrected in both the PDF and HTML versions of the Article.

A new microdeletion syndrome involving TBC1D24, ATP6V0C, and PDPK1 causes epilepsy, microcephaly, and developmental delay.

PURPOSE: Contiguous gene deletions are known to cause several neurodevelopmental syndromes, many of which are caused by recurrent events on chromosome 16. However, chromosomal microarray studies (CMA) still yield copy-number variants (CNVs) of unknown clinical significance. We sought to characterize eight individuals with overlapping 205-kb to 504-kb 16p13.3 microdeletions that are distinct from previously published deletion syndromes. METHODS: Clinical information on the patients and bioinformatic scores for the deleted genes were analyzed. RESULTS: All individuals in our cohort displayed developmental delay, intellectual disability, and various forms of seizures. Six individuals were microcephalic and two had strabismus. The deletion was absent in all 13 parents who were available for testing. The area of overlap encompasses seven genes including TBC1D24, ATP6V0C, and PDPK1 (also known as PDK1). Bi-allelic TBC1D24 pathogenic variants are known to cause nonsyndromic deafness, epileptic disorders, or DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, seizures). Sanger sequencing of the nondeleted TBC1D24 allele did not yield any additional pathogenic variants. CONCLUSIONS: We propose that 16p13.3 microdeletions resulting in simultaneous haploinsufficiencies of TBC1D24, ATP6V0C, and PDPK1 cause a novel rare contiguous gene deletion syndrome of microcephaly, developmental delay, intellectual disability, and epilepsy.