Expression of human‐specific ARHGAP11B in mice leads to neocortex expansion and increased memory flexibility

Neocortex expansion during human evolution provides a basis for our enhanced cognitive abilities. Yet, which genes implicated in neocortex expansion are actually responsible for higher cognitive abilities is unknown. The expression of human‐specific ARHGAP11B in embryonic/foetal mouse, ferret and marmoset neocortex was previously found to promote basal progenitor proliferation, upper‐layer neuron generation and neocortex expansion during development, features commonly thought to contribute to increased cognitive abilities. However, a key question is whether this phenotype persists into adulthood and if so, whether cognitive abilities are indeed increased. Here, we generated a transgenic mouse line with physiological ARHGAP11B expression that exhibits increased neocortical size and upper‐layer neuron numbers persisting into adulthood. Adult ARHGAP11B‐transgenic mice showed altered neurobehaviour, notably increased memory flexibility and a reduced anxiety level. Our data are consistent with the notion that neocortex expansion by ARHGAP11B, a gene implicated in human evolution, underlies some of the altered neurobehavioural features observed in the transgenic mice, such as the increased memory flexibility, a neocortex‐associated trait, with implications for the increase in cognitive abilities during human evolution.     

Human cerebral organoids reveal progenitor pathology in EML1‐linked cortical malformation

Malformations of human cortical development (MCD) can cause severe disabilities. The lack of human‐specific models hampers our understanding of the molecular underpinnings of the intricate processes leading to MCD. Here, we use cerebral organoids derived from patients and genome edited‐induced pluripotent stem cells to address pathophysiological changes associated with a complex MCD caused by mutations in the echinoderm microtubule‐associated protein‐like 1 (EML1) gene. EML1‐deficient organoids display ectopic neural rosettes at the basal side of the ventricular zone areas and clusters of heterotopic neurons. Single‐cell RNA sequencing shows an upregulation of basal radial glial (RG) markers and human‐specific extracellular matrix components in the ectopic cell population. Gene ontology and molecular analyses suggest that ectopic progenitor cells originate from perturbed apical RG cell behavior and yes‐associated protein 1 (YAP1)‐triggered expansion. Our data highlight a progenitor origin of EML1 mutation‐induced MCD and provide new mechanistic insight into the human disease pathology.     

RAB6 and dynein drive post‐Golgi apical transport to prevent neuronal progenitor delamination

Radial glial (RG) cells are the neural stem cells of the developing neocortex. Apical RG (aRG) cells can delaminate to generate basal RG (bRG) cells, a cell type associated with human brain expansion. Here, we report that aRG delamination is regulated by the post‐Golgi secretory pathway. Using in situ subcellular live imaging, we show that post‐Golgi transport of RAB6+ vesicles occurs toward the minus ends of microtubules and depends on dynein. We demonstrate that the apical determinant Crumbs3 (CRB3) is also transported by dynein. Double knockout of RAB6A/A'' and RAB6B impairs apical localization of CRB3 and induces a retraction of aRG cell apical process, leading to delamination and ectopic division. These defects are phenocopied by knockout of the dynein activator LIS1. Overall, our results identify a RAB6‐dynein‐LIS1 complex for Golgi to apical surface transport in aRG cells, and highlights the role of this pathway in the maintenance of neuroepithelial integrity.     

ARHGAP45 controls naïve T‐ and B‐cell entry into lymph nodes and T‐cell progenitor thymus seeding

T and B cells continually recirculate between blood and secondary lymphoid organs. To promote their trans‐endothelial migration (TEM), chemokine receptors control the activity of RHO family small GTPases in part via GTPase‐activating proteins (GAPs). T and B cells express several RHO‐GAPs, the function of most of which remains unknown. The ARHGAP45 GAP is predominantly expressed in hematopoietic cells. To define its in vivo function, we describe two mouse models where ARHGAP45 is ablated systemically or selectively in T cells. We combine their analysis with affinity purification coupled to mass spectrometry to determine the ARHGAP45 interactome in T cells and with time‐lapse and reflection interference contrast microscopy to assess the role of ARGHAP45 in T‐cell polarization and motility. We demonstrate that ARHGAP45 regulates naïve T‐cell deformability and motility. Under physiological conditions, ARHGAP45 controls the entry of naïve T and B cells into lymph nodes whereas under competitive repopulation it further regulates hematopoietic progenitor cell engraftment in the bone marrow, and T‐cell progenitor thymus seeding. Therefore, the ARGHAP45 GAP controls multiple key steps in the life of T and B cells.     

NLRP3 activation contributes to endothelin‐1‐induced erectile dysfunction

In the present study, we hypothesized that endothelin (ET) receptors (ET_A and ET_B) stimulation, through increased calcium and ROS formation, leads to Nucleotide Oligomerization Domain‐Like Receptor Family, Pyrin Domain Containing 3 (NLRP3) activation. Intracavernosal pressure (ICP/MAP) was measured in C57BL/6 (WT) mice. Functional and immunoblotting assays were performed in corpora cavernosa (CC) strips from WT, NLRP3^−/− and caspase^−/− mice in the presence of ET‐1 (100 nM) and vehicle, MCC950, tiron, BAPTA AM, BQ123, or BQ788. ET‐1 reduced the ICP/MAP in WT mice, and MCC950 prevented the ET‐1 effect. ET‐1 decreased CC ACh‐, sodium nitroprusside (SNP)‐induced relaxation, and increased caspase‐1 expression. BQ123 an ET_A receptor antagonist reversed the effect. The ET_B receptor antagonist BQ788 also reversed ET‐1 inhibition of ACh and SNP relaxation. Additionally, tiron, BAPTA AM, and NLRP3 genetic deletion prevented the ET‐1‐induced loss of ACh and SNP relaxation. Moreover, BQ123 diminished CC caspase‐1 expression, while BQ788 increased caspase‐1 and IL‐1β levels in a concentration‐dependent manner (100 nM–10 μM). Furthermore, tiron and BAPTA AM prevented ET‐1‐induced increase in caspase‐1. In addition, BAPTA AM blocked ET‐1‐induced ROS generation. In conclusion, ET‐1‐induced erectile dysfunction depends on ET_A‐ and ET_B‐mediated activation of NLRP3 in mouse CC via Ca²⁺‐dependent ROS generation.     

Trimethylamine modulates dauer formation,neurodegeneration, and lifespan through tyra‐3/daf‐11 signaling in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, signals derived from bacteria in the diet, the animal''s major nutrient source, can modulate both behavior and healthspan. Here we describe a dual role for trimethylamine (TMA), a human gut flora metabolite, which acts as a nutrient signal and a neurotoxin. TMA and its associated metabolites are produced by the human gut microbiome and have been suggested to serve as risk biomarkers for diabetes and cardiovascular diseases. We demonstrate that the tyramine receptor TYRA‐3, a conserved G protein‐coupled receptor (GPCR), is required to sense TMA and mediate its responses. TMA activates guanylyl cyclase DAF‐11 signaling through TYRA‐3 in amphid neurons (ASK) and ciliated neurons (BAG) to mediate food‐sensing behavior. Bacterial mutants deficient in TMA production enhance dauer formation, extend lifespan, and are less preferred as a food source. Increased levels of TMA lead to neural damage in models of Parkinson''s disease and shorten lifespan. Our results reveal conserved signaling pathways modulated by TMA in C. elegans that are likely to be relevant for its effects in mammalian systems.     

Delta‐like ligand‐4 regulates Notch‐mediated maturation of second heart field progenitor‐derived pharyngeal arterial endothelial cells

Mesodermal progenitors in the second heart field (SHF) express Delta‐like‐ligand 4 (Dll4) that regulates Notch‐mediated proliferation. As cells of SHF lineage mature to assume endocardial and myocardial cell fates, we have shown that Dll4 expression is lost, and the subsequent expression of another Notch ligand Jagged1 regulates Notch‐mediated maturation events in the developing heart. A subset of SHF progenitors also matures to form the pharyngeal arch artery (PAA) endothelium. Dll4 was originally identified as an arterial endothelial‐specific Notch ligand that plays an important role in blood vessel maturation, but its role in aortic arch maturation has not been studied to date secondary to the early lethality observed in Dll4 knockout mice. We show that, unlike in SHF‐derived endocardium and myocardium, Dll4 expression persists in SHF‐derived arterial endothelial cells. Using SHF‐specific conditional deletion of Dll4, we demonstrate that as SHF cells transition from their progenitor state to an endothelial fate, Dll4‐mediated Notch signalling switches from providing proliferative to maturation cues. Dll4 expression maintains arterial identity in the PAAs and plays a critical role in the maturation and re‐organization of the 4th pharyngeal arch artery, in particular. Haploinsufficiency of Dll4 in SHF leads to highly penetrant aortic arch artery abnormalities, similar to those observed in the clinic, primarily resulting from aberrant reorganization of bilateral 4th pharyngeal arch arteries. Hence, we show that cells of SHF lineage that assume an arterial endothelial fate continue to express Dll4 and the resulting Dll4‐mediated Notch signalling transitions from an early proliferative to a later maturation role during aortic arch development.     

Intra‐epithelial non‐canonical Activin A signaling safeguards prostate progenitor quiescence

The healthy prostate is a relatively quiescent tissue. Yet, prostate epithelium overgrowth is a common condition during aging, associated with urinary dysfunction and tumorigenesis. For over thirty years, TGF‐β ligands have been known to induce cytostasis in a variety of epithelia, but the intracellular pathway mediating this signal in the prostate, and its relevance for quiescence, have remained elusive. Here, using mouse prostate organoids to model epithelial progenitors, we find that intra‐epithelial non‐canonical Activin A signaling inhibits cell proliferation in a Smad‐independent manner. Mechanistically, Activin A triggers Tak1 and p38 ΜAPK activity, leading to p16 and p21 nuclear import. Spontaneous evasion from this quiescent state occurs upon prolonged culture, due to reduced Activin A secretion, a condition associated with DNA replication stress and aneuploidy. Organoids capable to escape quiescence in vitro are also able to implant with increased frequency into immunocompetent mice. This study demonstrates that non‐canonical Activin A signaling safeguards epithelial quiescence in the healthy prostate, with potential implications for the understanding of cancer initiation, and the development of therapies targeting quiescent tumor progenitors.     

Therapeutic stem cell‐derived alveolar‐like macrophages display bactericidal effects and resolve Pseudomonas aeruginosa‐induced lung injury

Bacterial lung infections lead to greater than 4 million deaths per year with antibiotic treatments driving an increase in antibiotic resistance and a need to establish new therapeutic approaches. Recently, we have generated mouse and rat stem cell‐derived alveolar‐like macrophages (ALMs), which like primary alveolar macrophages (1''AMs), phagocytose bacteria and promote airway repair. Our aim was to further characterize ALMs and determine their bactericidal capabilities. The characterization of ALMs showed that they share known 1''AM cell surface markers, but unlike 1''AMs are highly proliferative in vitro. ALMs effectively phagocytose and kill laboratory strains of P. aeruginosa (P.A.), E. coli (E.C.) and S. aureus, and clinical strains of P.A. In vivo, ALMs remain viable, adapt additional features of native 1''AMs, but proliferation is reduced. Mouse ALMs phagocytose P.A. and E.C. and rat ALMs phagocytose and kill P.A. within the lung 24 h post‐instillation. In a pre‐clinical model of P.A.‐induced lung injury, rat ALM administration mitigated weight loss and resolved lung injury observed seven days post‐instillation. Collectively, ALMs attenuate pulmonary bacterial infections and promote airway repair. ALMs could be utilized as an alternative or adjuvant therapy where current treatments are ineffective against antibiotic‐resistant bacteria or to enhance routine antibiotic delivery.     

Mito‐nuclear discordance at a mimicry color transition zone in bumble bee Bombus melanopygus

As hybrid zones exhibit selective patterns of gene flow between otherwise distinct lineages, they can be especially valuable for informing processes of microevolution and speciation. The bumble bee, Bombus melanopygus, displays two distinct color forms generated by Müllerian mimicry: a northern “Rocky Mountain''’ color form with ferruginous mid‐abdominal segments (B. m. melanopygus) and a southern “Pacific''’ form with black mid‐abdominal segments (B. m. edwardsii). These morphs meet in a mimetic transition zone in northern California and southern Oregon that is more narrow and transitions further west than comimetic bumble bee species. To understand the historical formation of this mimicry zone, we assessed color distribution data for B. melanopygus from the last 100 years. We then examined gene flow among the color forms in the transition zone by comparing sequences from mitochondrial COI barcode sequences, color‐controlling loci, and the rest of the nuclear genome. These data support two geographically distinct mitochondrial haplogroups aligned to the ancestrally ferruginous and black forms that meet within the color transition zone. This clustering is also supported by the nuclear genome, which, while showing strong admixture across individuals, distinguishes individuals most by their mitochondrial haplotype, followed by geography. These data suggest the two lineages most likely were historically isolated, acquired fixed color differences, and then came into secondary contact with ongoing gene flow. The transition zone, however, exhibits asymmetries: mitochondrial haplotypes transition further south than color pattern, and both transition over shorter distances in the south. This system thus demonstrates alternative patterns of gene flow that occur in contact zones, presenting another example of mito‐nuclear discordance. Discordant gene flow is inferred to most likely be driven by a combination of mimetic selection, dominance effects, and assortative mating.     

A CRMP4‐dependent retrograde axon‐to‐soma death signal in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal non‐cell‐autonomous neurodegenerative disease characterized by the loss of motor neurons (MNs). Mutations in CRMP4 are associated with ALS in patients, and elevated levels of CRMP4 are suggested to affect MN health in the SOD1^G93A‐ALS mouse model. However, the mechanism by which CRMP4 mediates toxicity in ALS MNs is poorly understood. Here, by using tissue from human patients with sporadic ALS, MNs derived from C9orf72‐mutant patients, and the SOD1^G93A‐ALS mouse model, we demonstrate that subcellular changes in CRMP4 levels promote MN loss in ALS. First, we show that while expression of CRMP4 protein is increased in cell bodies of ALS‐affected MN, CRMP4 levels are decreased in the distal axons. Cellular mislocalization of CRMP4 is caused by increased interaction with the retrograde motor protein, dynein, which mediates CRMP4 transport from distal axons to the soma and thereby promotes MN loss. Blocking the CRMP4‐dynein interaction reduces MN loss in human‐derived MNs (C9orf72) and in ALS model mice. Thus, we demonstrate a novel CRMP4‐dependent retrograde death signal that underlies MN loss in ALS.     

Parkin drives pS65‐Ub turnover independently of canonical autophagy in Drosophila

Parkinson''s disease‐related proteins, PINK1 and Parkin, act in a common pathway to maintain mitochondrial quality control. While the PINK1‐Parkin pathway can promote autophagic mitochondrial turnover (mitophagy) following mitochondrial toxification in cell culture, alternative quality control pathways are suggested. To analyse the mechanisms by which the PINK1–Parkin pathway operates in vivo, we developed methods to detect Ser65‐phosphorylated ubiquitin (pS65‐Ub) in Drosophila. Exposure to the oxidant paraquat led to robust, Pink1‐dependent pS65‐Ub production, while pS65‐Ub accumulates in unstimulated parkin‐null flies, consistent with blocked degradation. Additionally, we show that pS65‐Ub specifically accumulates on disrupted mitochondria in vivo. Depletion of the core autophagy proteins Atg1, Atg5 and Atg8a did not cause pS65‐Ub accumulation to the same extent as loss of parkin, and overexpression of parkin promoted turnover of both basal and paraquat‐induced pS65‐Ub in an Atg5‐null background. Thus, we have established that pS65‐Ub immunodetection can be used to analyse Pink1‐Parkin function in vivo as an alternative to reporter constructs. Moreover, our findings suggest that the Pink1‐Parkin pathway can promote mitochondrial turnover independently of canonical autophagy in vivo.     

Modelling of primary ciliary dyskinesia using patient‐derived airway organoids

Patient‐derived human organoids can be used to model a variety of diseases. Recently, we described conditions for long‐term expansion of human airway organoids (AOs) directly from healthy individuals and patients. Here, we first optimize differentiation of AOs towards ciliated cells. After differentiation of the AOs towards ciliated cells, these can be studied for weeks. When returned to expansion conditions, the organoids readily resume their growth. We apply this condition to AOs established from nasal inferior turbinate brush samples of patients suffering from primary ciliary dyskinesia (PCD), a pulmonary disease caused by dysfunction of the motile cilia in the airways. Patient‐specific differences in ciliary beating are observed and are in agreement with the patients'' genetic mutations. More detailed organoid ciliary phenotypes can thus be documented in addition to the standard diagnostic procedure. Additionally, using genetic editing tools, we show that a patient‐specific mutation can be repaired. This study demonstrates the utility of organoid technology for investigating hereditary airway diseases such as PCD.     

Quantification of noradrenergic‐, dopaminergic‐, and tectal‐neurons during aging in the short‐lived killifish Nothobranchius furzeri

Parkinson''s disease (PD) is characterized by phosphorylation and aggregation of the protein α‐Synuclein and ensuing neuronal death progressing from the noradrenergic locus coeruleus to midbrain dopaminergic neurons. In 2019, Matsui and colleagues reported a spontaneous age‐dependent degeneration of dopaminergic neurons and an even greater neurodegeneration of the noradrenergic neurons in the short‐lived killifish Nothobranchius furzeri. Given the great possible relevance of a spontaneous model for PD, we assessed neurodegeneration of noradrenergic and dopaminergic neurons in two further laboratory strains of N. furzeri. We implemented, for the first time in N. furzeri, a whole‐brain clarification technique and proceeded to entire 3D nuclei reconstruction to quantify total cell numbers in two different stains of N. furzeri. In both strains, we observed that age‐dependent neurodegeneration is limited to the locus coeruleus and does not involve the posterior tuberculum. We also applied 3D counting to the optic tectum, an area of active adult neurogenesis, and detected an increase of neurons with age. Our results confirm age‐dependent neurodegeneration of noradrenergic neurons, a condition reminiscent of the presymptomatic stage of PD indicating that N. furzeri could be used in the future to identify modifying factors for age‐dependent neurodegeneration and open the intriguing possibility that natural genetic variation may influence the susceptibility of dopaminergic neurons.     

Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.