Abnormal cell sorting and altered early neurogenesis in a human cortical organoid model of Protocadherin-19 clustering epilepsy.

Introduction: Protocadherin-19 (PCDH19)-Clustering Epilepsy (PCE) is a developmental and epileptic encephalopathy caused by loss-of-function variants of the PCDH19 gene on the X-chromosome. PCE affects females and mosaic males while male carriers are largely spared. Mosaic expression of the cell adhesion molecule PCDH19 due to random X-chromosome inactivation is thought to impair cell-cell interactions between mutant and wild type PCDH19-expressing cells to produce the disease. Progress has been made in understanding PCE using rodent models or patient induced pluripotent stem cells (iPSCs). However, rodents do not faithfully model key aspects of human brain development, and patient iPSC models are limited by issues with random X-chromosome inactivation. Methods: To overcome these challenges and model mosaic PCDH19 expression in vitro, we generated isogenic female human embryonic stem cells with either HA-FLAG-tagged PCDH19 (WT) or homozygous PCDH19 knockout (KO) using genome editing. We then mixed GFP-labeled WT and RFP-labeled KO cells and generated human cortical organoids (hCOs). Results: We found that PCDH19 is highly expressed in early (days 20-35) WT neural rosettes where it co-localizes with N-Cadherin in ventricular zone (VZ)-like regions. Mosaic PCE hCOs displayed abnormal cell sorting in the VZ with KO and WT cells completely segregated. This segregation remained robust when WT:KO cells were mixed at 2:1 or 1:2 ratios. PCE hCOs also exhibited altered expression of PCDH19 (in WT cells) and N-Cadherin, and abnormal deep layer neurogenesis. None of these abnormalities were observed in hCOs generated by mixing only WT or only KO (modeling male carrier) cells. Discussion: Our results using the mosaic PCE hCO model suggest that PCDH19 plays a critical role in human VZ radial glial organization and early cortical development. This model should offer a key platform for exploring mechanisms underlying PCE-related cortical hyperexcitability and testing of potential precision therapies.

Protocol for selecting single human pluripotent stem cells using a modified micropipetter.

Single-cell clonal selection is a critical procedure for generating a homogeneous population of human pluripotent stem cells. Here, we present a protocol that repurposes the STRIPPER Micropipetter, normally used for in vitro fertilization, to pick single stem cells. We describe steps for tool and reagent preparation, single-cell picking, and colony passaging. We then detail procedures for amplification and analysis. Our protocol does not require cell sorting and produces homogenous clonal cultures with more than 50% survival rate. For complete details on the use and execution of this protocol, please refer to Deng et al.1.

BMAL1 Regulates the Daily Timing of Colitis.

Many physiological functions exhibit circadian rhythms: oscillations in biological processes that occur in a 24-hour period. These daily rhythms are maintained through a highly conserved molecular pacemaker known as the circadian clock. Circadian disruption has been proposed to cause increased risk of Inflammatory Bowel Disease (IBD) but the underlying mechanisms remain unclear. Patients with IBD experience chronic inflammation and impaired regeneration of intestinal epithelial cells. Previous animal-based studies have revealed that colitis models of IBD are more severe in mice without a circadian clock but the timing of colitis, and whether its inflammatory and regenerative processes have daily rhythms, remains poorly characterized. We tested circadian disruption using Bmal1-/- mutant mice that have a non-functional circadian clock and thus no circadian rhythms. Dextran Sulfate Sodium (DSS) was used to induce colitis. The disease activity of colitis was found to exhibit time-dependent variation in Bmal1+/+ control mice but is constant and elevated in Bmal1-/- mutants, who exhibit poor recovery. Histological analyses indicate worsened colitis severity in Bmal1-/- mutant colon, and colon infiltration of immune system cells shows a daily rhythm that is lost in the Bmal1-/- mutant. Similarly, epithelial proliferation in the colon has a daily rhythm in Bmal1+/+ controls but not in Bmal1-/- mutants. Our results support a critical role of a functional circadian clock in the colon which drives 24-hour rhythms in inflammation and healing, and whose disruption impairs colitis recovery. This indicates that weakening circadian rhythms not only worsens colitis, but delays healing and should be taken into account in the management of IBD. Recognition of this is important in the management of IBD patients required to do shift work.

The Circadian Clock Gene, Bmal1, Regulates Intestinal Stem Cell Signaling and Represses Tumor Initiation.

BACKGROUND & AIMS: Circadian rhythms are daily physiological oscillations driven by the circadian clock: a 24-hour transcriptional timekeeper that regulates hormones, inflammation, and metabolism. Circadian rhythms are known to be important for health, but whether their loss contributes to colorectal cancer is not known. We tested the nonredundant clock gene Bmal1 in intestinal homeostasis and tumorigenesis, using the Apcmin model of colorectal cancer. METHODS: Bmal1 mutant, epithelium-conditional Bmal1 mutant, and photoperiod (day/night cycle) disrupted mice bearing the Apcmin allele were assessed for tumorigenesis. Tumors and normal nontransformed tissue were characterized. Intestinal organoids were assessed for circadian transcription rhythms by RNA sequencing, and in vivo and organoid assays were used to test Bmal1-dependent proliferation and self-renewal. RESULTS: Loss of Bmal1 or circadian photoperiod increases tumor initiation. In the intestinal epithelium the clock regulates transcripts involved in regeneration and intestinal stem cell signaling. Tumors have no self-autonomous clock function and only weak clock function in vivo. Apcmin clock-disrupted tumors show high Yes-associated protein 1 (Hippo signaling) activity but show low Wnt (Wingless and Int-1) activity. Intestinal organoid assays show that loss of Bmal1 increases self-renewal in a Yes-associated protein 1-dependent manner. CONCLUSIONS: Bmal1 regulates intestinal stem cell pathways, including Hippo signaling, and the loss of circadian rhythms potentiates tumor initiation. Transcript profiling: GEO accession number: GSE157357.

Impaired ECM Remodeling and Macrophage Activity Define Necrosis and Regeneration Following Damage in Aged Skeletal Muscle.

Regenerative capacity of skeletal muscle declines with age, the cause of which remains largely unknown. We investigated extracellular matrix (ECM) proteins and their regulators during early regeneration timepoints to define a link between aberrant ECM remodeling, and impaired aged muscle regeneration. The regeneration process was compared in young (three month old) and aged (18 month old) C56BL/6J mice at 3, 5, and 7 days following cardiotoxin-induced damage to the tibialis anterior muscle. Immunohistochemical analyses were performed to assess regenerative capacity, ECM remodeling, and the macrophage response in relation to plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-9 (MMP-9), and ECM protein expression. The regeneration process was impaired in aged muscle. Greater intracellular and extramyocellular PAI-1 expression was found in aged muscle. Collagen I was found to accumulate in necrotic regions, while macrophage infiltration was delayed in regenerating regions of aged muscle. Young muscle expressed higher levels of MMP-9 early in the regeneration process that primarily colocalized with macrophages, but this expression was reduced in aged muscle. Our results indicate that ECM remodeling is impaired at early time points following muscle damage, likely a result of elevated expression of the major inhibitor of ECM breakdown, PAI-1, and consequent suppression of the macrophage, MMP-9, and myogenic responses.

A Bird's-Eye View of the Multiple Biochemical Mechanisms that Propel Pathology of Alzheimer's Disease: Recent Advances and Mechanistic Perspectives on How to Halt the Disease Progression Targeting Multiple Pathways.

Neurons consume the highest amount of oxygen, depend on oxidative metabolism for energy, and survive for the lifetime of an individual. Therefore, neurons are vulnerable to death caused by oxidative-stress, accumulation of damaged and dysfunctional proteins and organelles. There is an exponential increase in the number of patients diagnosed with neurodegenerative diseases such as Alzheimer's (AD) as the number of elderly increases exponentially. Development of AD pathology is a complex phenomenon characterized by neuronal death, accumulation of extracellular amyloid-ß plaques and neurofibrillary tangles, and most importantly loss of memory and cognition. These pathologies are most likely caused by mechanisms including oxidative stress, mitochondrial dysfunction/stress, accumulation of misfolded proteins, and defective organelles due to impaired proteasome and autophagy mechanisms. Currently, there are no effective treatments to halt the progression of this disease. In order to treat this complex disease with multiple biochemical pathways involved, a complex treatment regimen targeting different mechanisms should be investigated. Furthermore, as AD is a progressive disease-causing morbidity over many years, any chemo-modulator for treatment must be used over long period of time. Therefore, treatments must be safe and non-interfering with other processes. Ideally, a treatment like medicinal food or a supplement that can be taken regularly without any side effect capable of reducing oxidative stress, stabilizing mitochondria, activating autophagy or proteasome, and increasing energy levels of neurons would be the best solution. This review summarizes progress in research on different mechanisms of AD development and some of the potential therapeutic development strategies targeting the aforementioned pathologies.

Corrigendum: Cancer Cell Mitochondria Targeting by Pancratistatin Analogs is Dependent on Functional Complex II and III.

This corrects the article DOI: 10.1038/srep42957.

The Circadian Clock Gene BMAL1 Coordinates Intestinal Regeneration.

BACKGROUND & AIMS: The gastrointestinal syndrome is an illness of the intestine caused by high levels of radiation. It is characterized by extensive loss of epithelial tissue integrity, which initiates a regenerative response by intestinal stem and precursor cells. The intestine has 24-hour rhythms in many physiological functions that are believed to be outputs of the circadian clock: a molecular system that produces 24-hour rhythms in transcription/translation. Certain gastrointestinal illnesses are worsened when the circadian rhythms are disrupted, but the role of the circadian clock in gastrointestinal regeneration has not been studied. METHODS: We tested the timing of regeneration in the mouse intestine during the gastrointestinal syndrome. The role of the circadian clock was tested genetically using the BMAL1 loss of function mouse mutant in vivo, and in vitro using intestinal organoid culture. RESULTS: The proliferation of the intestinal epithelium follows a 24-hour rhythm during the gastrointestinal syndrome. The circadian clock runs in the intestinal epithelium during this pathologic state, and the loss of the core clock gene, BMAL1, disrupts both the circadian clock and rhythmic proliferation. Circadian activity in the intestine involves a rhythmic production of inflammatory cytokines and subsequent rhythmic activation of the JNK stress response pathway. CONCLUSIONS: Our results show that a circadian rhythm in inflammation and regeneration occurs during the gastrointestinal syndrome. The study and treatment of radiation-induced illnesses, and other gastrointestinal illnesses, should consider 24-hour timing in physiology and pathology.

Cancer Cell Mitochondria Targeting by Pancratistatin Analogs is Dependent on Functional Complex II and III.

Enhanced mitochondrial stability and decreased dependence on oxidative phosphorylation confer an acquired resistance to apoptosis in cancer cells, but may present opportunities for therapeutic intervention. The compound pancratistatin (PST) has been shown to selectively induce apoptosis in cancer cells. However, its low availability in nature has hindered its clinical advancement. We synthesized PST analogs and a medium-throughput screen was completed. Analogs SVTH-7, -6, and -5 demonstrated potent anti-cancer activity greater than PST and several standard chemotherapeutics. They disrupted mitochondrial function, activated the intrinsic apoptotic pathway, and reduced growth of tumor xenografts in vivo. Interestingly, the pro-apoptotic effects of SVTH-7 on cancer cells and mitochondria were abrogated with the inhibition of mitochondrial complex II and III, suggesting mitochondrial or metabolic vulnerabilities may be exploited by this analog. This work provides a scaffold for characterizing distinct mitochondrial and metabolic features of cancer cells and reveals several lead compounds with high therapeutic potential.

Inhibition of stress induced premature senescence in presenilin-1 mutated cells with water soluble Coenzyme Q10.

A water-soluble formulation of CoQ10 (WS-CoQ10) was shown to stabilize mitochondria and prevent oxidative stress-induced neuronal death. Presenilin-1 (PS-1)-mutated Alzheimer's Disease (AD) fibroblasts (PSAF) were used for studying the effects of PS-1 mutation. PS-1 mutation correlated to increased reactive oxygen species (ROS) production and stress induced premature senescence (SIPS) in PSAF; WS-CoQ10 treatment decreased ROS generation, increased population doublings, and postponed SIPS. Treated PSAF had higher PCNA expression, and lower levels of MnSOD, p21, p16Ink4A, and Rb. WS-CoQ10 caused the resumption of autophagy in PSAF. Thus, WS-CoQ10 as inhibitor of SIPS and ameliorator of autophagy could be an effective prophylactic/therapeutic agent for AD.