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We investigate how matrix stiffness regulates chromatin reorganization and cell reprogramming and find that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, maximized at an intermediate stiffness of 20 kPa. ATAC sequencing analysis shows the same trend of chromatin accessibility to neuronal genes at these stiffness levels. Concurrently, we observe peak levels of histone acetylation and histone acetyltransferase (HAT) activity in the nucleus on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, the cotransporters shuttling HAT into the nucleus, rises with decreasing matrix stiffness; however, reduced importin-9 on soft matrices limits nuclear transport. These two factors result in a biphasic regulation of HAT transport into nucleus, which is directly demonstrated on matrices with dynamically tunable stiffness. Our findings unravel a mechanism of the mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications.

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

Abstract Ascl1 and Ngn2, closely related proneural transcription factors, are able to convert mouse embryonic stem cells into induced neurons. Despite their similarities, these factors elicit only partially overlapping transcriptional programs, and it remains unknown whether cells are converted via distinct mechanisms. Here we show that Ascl1 and Ngn2 induce mutually exclusive side populations by binding and activating distinct lineage drivers. Furthermore, Ascl1 rapidly dismantles the pluripotency network and installs neuronal and trophoblast cell fates, while Ngn2 generates a neural stem cell-like intermediate supported by incomplete shutdown of the pluripotency network. Using CRISPR-Cas9 knockout screening, we find that Ascl1 relies more on factors regulating pluripotency and the cell cycle, such as Tcf7l1. In the absence of Tcf7l1, Ascl1 still represses core pluripotency genes but fails to exit the cell cycle. However, overexpression of Cdkn1c induces cell cycle exit and restores the generation of neurons. These findings highlight that cell type conversion can occur through two distinct mechanistic paths, even when induced by closely related transcription factors.

Abstract The chromodomain helicase DNA-binding protein CHD8 is the most frequently mutated gene in autism spectrum disorder. Despite its prominent disease involvement, little is known about its molecular function in the human brain. CHD8 is a chromatin regulator which binds to the promoters of actively transcribed genes through genomic targeting mechanisms which have yet to be fully defined. By generating a conditional loss-of-function and an endogenously tagged allele in human pluripotent stem cells, we investigated the molecular function and the interaction of CHD8 with chromatin in human neurons. Chromatin accessibility analysis and transcriptional profiling revealed that CHD8 functions as a transcriptional activator at its target genes in human neurons. Furthermore, we found that CHD8 chromatin targeting is cell context-dependent. In human neurons, CHD8 preferentially binds at ETS motif-enriched promoters. This enrichment is particularly prominent on the promoters of genes whose expression significantly changes upon the loss of CHD8. Indeed, among the ETS transcription factors, we identified ELK1 as being most highly correlated with CHD8 expression in primary human fetal and adult cortical neurons and most highly expressed in our stem cell-derived neurons. Remarkably, ELK1 was necessary to recruit CHD8 specifically to ETS motif-containing sites. These findings imply that ELK1 and CHD8 functionally cooperate to regulate gene expression and chromatin states at MAPK/ERK target genes in human neurons. Our results suggest that the MAPK/ERK/ELK1 axis potentially contributes to the pathogenesis caused by CHD8 mutations in human neurodevelopmental disorders.

Pelizaeus-Merzbacher disease (PMD) is an X-linked leukodystrophy caused by mutations in Proteolipid Protein 1 (PLP1), encoding a major myelin protein, resulting in profound developmental delay and early lethality. Previous work showed involvement of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways, but poor PLP1 genotype-phenotype associations suggest additional pathogenetic mechanisms. Using induced pluripotent stem cell (iPSC) and gene-correction, we show that patient-derived oligodendrocytes can develop to the pre-myelinating stage, but subsequently undergo cell death. Mutant oligodendrocytes demonstrated key hallmarks of ferroptosis including lipid peroxidation, abnormal iron metabolism, and hypersensitivity to free iron. Iron chelation rescued mutant oligodendrocyte apoptosis, survival, and differentiationin vitro, and post-transplantation in vivo. Finally, systemic treatment of Plp1 mutant Jimpy mice with deferiprone, a small molecule iron chelator, reduced oligodendrocyte apoptosis and enabled myelin formation. Thus, oligodendrocyte iron-induced cell death and myelination is rescued by iron chelation in PMD pre-clinical models.

The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it is difficult to determine the function of single transcription factor binding sites within larger transcription networks. Here, we use deactivated Cas9 (dCas9) to disrupt binding to specific sites, a method we term CRISPRd. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting dCas9 to an Oct4 site in the Nanog promoter displaced Oct4 from this site, reduced Nanog expression, and slowed division. In contrast, disrupting the Oct4 binding site adjacent to Pax6 upregulated Pax6 transcription and disrupting Nanog binding its own promoter upregulated its transcription. Thus, we can easily distinguish between activating and repressing binding sites and examine autoregulation. Finally, multiple guide RNA expression allows simultaneous inhibition of multiple binding sites, and conditionally destabilized dCas9 allows rapid reversibility.

Lyssaviruses are the causative agents of rabies, a zoonotic, fatal disease that is thought to be ancestral to bats. In the last decade, the detection of bat associated lyssaviruses is increasing also in Europe. Within a retrospective bat associated lyssavirus surveillance study a total of 225 dead bats of 21 bat species were collected in Slovenia between 2012 and 2019 and tested by specific real-time RT-PCR method. The first lyssavirus positive sample in bats in Slovenia was detected using the real-time RT-PCR, the fluorescent antibody test, and next generation sequencing, while the rabies tissue culture inoculation test was unsuccessful due to sample degradation and storage conditions. The nearly complete genome of Divača bat lyssavirus from Slovenia consists of 11,871 nucleotides and reflects the characteristic gene organization known for lyssaviruses, encoding the five viral proteins. Phylogenetic analysis of Divača bat lyssavirus revealed that it belongs to phylogroup I lyssaviruses and is most closely related to Kotalahti bat lyssavirus (KBLV) with 87.20% nucleotide and 99.22% amino acid identity. Together with KBLV, Khujand virus, European bat lyssavirus 2, Bakeloh bat lyssavirus, and Aravan virus, Divača bat lyssavirus was detected in the genus Myotis suggesting its key role in the transmission and maintenance of certain lyssaviruses.

Abstract Background Neuroendocrine neoplasias (NENs) are a rare type of malignancy that arise from the cells of the neuroendocrine system. Most patients present with advanced, unresectable disease, typically with metastases to the liver. The presence of liver metastases dictates prognosis and there has been a number of studies investigating therapies that reduce the burden of liver disease. Selective Internal Radiation Therapy (SIRT) allows the delivery of targeted high dose radiation directly to tumours, with relative sparing of the surrounding liver tissue. Here, we describe the design and rationale of ArtTisaN, a phase II study to assess efficacy and tolerability of SIRT using TheraSpheres for the management of liver metastases secondary to NENs. Methods Twenty-four eligible participants will be recruited to receive SIRT with TheraSpheres. The primary objective is to determine the objective response rate to treatment, defined as the rate of best overall response in the treated liver volume. In addition, total hepatic response and overall response will be assessed according to RECIST 1.1. The second co-primary objective is to determine the incidence of adverse and serious adverse device events. The secondary objectives are progression free survival, overall survival and quality of life. Additional exploratory objectives include investigation of circulating biomarkers of response and identification of a radiomic signature of response. Discussion This trial will provide prospective evidence on the efficacy of SIRT using TheraSpheres for the management of liver metastases. Trial registration NCT04362436 .

Endogenous Cushing’s syndrome (CS) poses considerable diagnostic challenges. Although late-night salivary cortisol (LNSC) is recommended as a first-line screening investigation, it remains the least widely used test in many countries. The combined measurement of LNSC and late-night salivary cortisone (LNS cortisone) has shown to further improve diagnostic accuracy. We present a retrospective study in a tertiary referral centre comparing LNSC, LNS cortisone, overnight dexamethasone suppression test, low-dose dexamethasone suppression test and 24-h urinary free cortisol results of patients investigated for CS. Patients were categorised into those who had CS (21 patients) and those who did not (33 patients). LNSC had a sensitivity of 95% and a specificity of 91%. LNS cortisone had a specificity of 100% and a sensitivity of 86%. With an optimal cut-off for LNS cortisone of >14.5 nmol/L the sensitivity was 95.2%, and the specificity was 100% with an area under the curve of 0.997, for diagnosing CS. Saliva collection is non-invasive and can be carried out at home. We therefore advocate simultaneous measurement of LNSC and LNS cortisone as the first-line screening test to evaluate patients with suspected CS.

Abstract Background The zinc finger domain containing transcription factor Myt1l is tightly associated with neuronal identity and is the only transcription factor known that is both neuron-specific and expressed in all neuronal subtypes. We identified Myt1l as a powerful reprogramming factor that, in combination with the proneural bHLH factor Ascl1, could induce neuronal fate in fibroblasts. Molecularly, we found it to repress many non-neuronal gene programs, explaining its supportive role to induce and safeguard neuronal identity in combination with proneural bHLH transcriptional activators. Moreover, human genetics studies found MYT1L mutations to cause intellectual disability and autism spectrum disorder often coupled with obesity. Methods Here, we generated and characterized Myt1l-deficient mice. A comprehensive, longitudinal behavioral phenotyping approach was applied. Results Myt1l was necessary for survival beyond 24 h but not for overall histological brain organization. Myt1l heterozygous mice became increasingly overweight and exhibited multifaceted behavioral alterations. In mouse pups, Myt1l haploinsufficiency caused mild alterations in early socio-affective communication through ultrasonic vocalizations. In adulthood, Myt1l heterozygous mice displayed hyperactivity due to impaired habituation learning. Motor performance was reduced in Myt1l heterozygous mice despite intact motor learning, possibly due to muscular hypotonia. While anxiety-related behavior was reduced, acoustic startle reactivity was enhanced, in line with higher sensitivity to loud sound. Finally, Myt1l haploinsufficiency had a negative impact on contextual fear memory retrieval, while cued fear memory retrieval appeared to be intact. Limitations In future studies, additional phenotypes might be identified and a detailed characterization of direct reciprocal social interaction behavior might help to reveal effects of Myt1l haploinsufficiency on social behavior in juvenile and adult mice. Conclusions Behavioral alterations in Myt1l haploinsufficient mice recapitulate several clinical phenotypes observed in humans carrying heterozygous MYT1L mutations and thus serve as an informative model of the human MYT1L syndrome.

Approaches to differentiating pluripotent stem cells (PSCs) into neurons currently face two major challenges-(i) generated cells are immature, with limited functional properties; and (ii) cultures exhibit heterogeneous neuronal subtypes and maturation stages. Using lineage-determining transcription factors, we previously developed a single-step method to generate glutamatergic neurons from human PSCs. Here, we show that transient expression of the transcription factors Ascl1 and Dlx2 (AD) induces the generation of exclusively GABAergic neurons from human PSCs with a high degree of synaptic maturation. These AD-induced neuronal (iN) cells represent largely nonoverlapping populations of GABAergic neurons that express various subtype-specific markers. We further used AD-iN cells to establish that human collybistin, the loss of gene function of which causes severe encephalopathy, is required for inhibitory synaptic function. The generation of defined populations of functionally mature human GABAergic neurons represents an important step toward enabling the study of diseases affecting inhibitory synaptic transmission.

Normal differentiation and induced reprogramming require the activation of target cell programs and silencing of donor cell programs. In reprogramming, the same factors are often used to reprogram many different donor cell types. As most developmental repressors, such as RE1-silencing transcription factor (REST) and Groucho (also known as TLE), are considered lineage-specific repressors, it remains unclear how identical combinations of transcription factors can silence so many different donor programs. Distinct lineage repressors would have to be induced in different donor cell types. Here, by studying the reprogramming of mouse fibroblasts to neurons, we found that the pan neuron-specific transcription factor Myt1-like (Myt1l) exerts its pro-neuronal function by direct repression of many different somatic lineage programs except the neuronal program. The repressive function of Myt1l is mediated via recruitment of a complex containing Sin3b by binding to a previously uncharacterized N-terminal domain. In agreement with its repressive function, the genomic binding sites of Myt1l are similar in neurons and fibroblasts and are preferentially in an open chromatin configuration. The Notch signalling pathway is repressed by Myt1l through silencing of several members, including Hes1. Acute knockdown of Myt1l in the developing mouse brain mimicked a Notch gain-of-function phenotype, suggesting that Myt1l allows newborn neurons to escape Notch activation during normal development. Depletion of Myt1l in primary postmitotic neurons de-repressed non-neuronal programs and impaired neuronal gene expression and function, indicating that many somatic lineage programs are actively and persistently repressed by Myt1l to maintain neuronal identity. It is now tempting to speculate that similar many-but-one lineage repressors exist for other cell fates; such repressors, in combination with lineage-specific activators, would be prime candidates for use in reprogramming additional cell types.

Abstract Exposure to early life stress (ELS) during childhood or prenatally increases the risk of future psychiatric disorders. The effect of stress exposure during the neonatal period is less well understood. In preterm infants, exposure to invasive procedures is associated with altered brain development and future stress responses suggesting that the neonatal period could be a key time for the programming of mental health. Previous studies suggest that ELS affects the hypothalamic epigenome, making it a good candidate to mediate these effects. In this study, we used a mouse model of early life stress (modified maternal separation; MMS). We hypothesised MMS would affect the hypothalamic transcriptome and DNA methylome, and impact on adult behaviour. MMS involved repeated stimulation of pups for 1.5 h/day, whilst separated from their mother, from postnatal day (P) 4–6. 3’mRNA sequencing and DNA methylation immunoprecipitation (meDIP) sequencing were performed on hypothalamic tissue at P6. Behaviour was assessed with the elevated plus, open field mazes and in-cage monitoring at 3–4 months of age. MMS was only associated with subtle changes in gene expression, but there were widespread alterations in DNA methylation. Notably, differentially methylated regions were enriched for synapse-associated loci. MMS resulted in hyperactivity in the elevated plus and open field mazes, but in-cage monitoring revealed that this was not representative of habitual hyperactivity. ELS has marked effects on DNA methylation in the hypothalamus in early life and results in stress-specific hyperactivity in young adulthood. These results have implications for the understanding of ELS-mediated effects on brain development.