PPP1R12A is a recycling endosomal phosphatase that facilitates YAP activation.

Yes-associated protein (YAP) is a transcriptional coactivator that is essential for the malignancy of various cancers. We have previously shown that YAP activity is positively regulated by phosphatidylserine (PS) in recycling endosomes (REs). However, the mechanism by which YAP is activated by PS in REs remains unknown. In the present study, we examined a group of protein phosphatases (11 phosphatases) that we had identified previously as PS-proximity protein candidates. Knockdown experiments of these phosphatases suggested that PPP1R12A, a regulatory subunit of the myosin phosphatase complex, was essential for YAP-dependent proliferation of triple-negative breast cancer MDA-MB-231 cells. Knockdown of PPP1R12A increased the level of phosphorylated YAP, reduced that of YAP in the nucleus, and suppressed the transcription of CTGF (a YAP-regulated gene), reinforcing the role of PPP1R12A in YAP activation. ATP8A1 is a PS-flippase that concentrates PS in the cytosolic leaflet of the RE membrane and positively regulates YAP signalling. In subcellular fractionation experiments using cell lysates, PPP1R12A in control cells was recovered exclusively in the microsomal fraction. In contrast, a fraction of PPP1R12A in ATP8A1-depleted cells was recovered in the cytosolic fraction. Cohort data available from the Cancer Genome Atlas showed that high expression of PPP1R12A, PP1B encoding the catalytic subunit of the myosin phosphatase complex, or ATP8A1 correlated with poor prognosis in breast cancer patients. These results suggest that the "ATP8A1-PS-YAP phosphatase" axis in REs facilitates YAP activation and thus cell proliferation.

Cellular senescence in white matter microglia is induced during ageing in mice and exacerbates the neuroinflammatory phenotype.

Cellular senescence, a state of irreversible cell-cycle arrest caused by a variety of cellular stresses, is critically involved in age-related tissue dysfunction in various organs. However, the features of cells in the central nervous system that undergo senescence and their role in neural impairment are not well understood as yet. Here, through comprehensive investigations utilising single-cell transcriptome analysis and various mouse models, we show that microglia, particularly in the white matter, undergo cellular senescence in the brain and spinal cord during ageing and in disease models involving demyelination. Microglial senescence is predominantly detected in disease-associated microglia, which appear in ageing and neurodegenerative diseases. We also find that commensal bacteria promote the accumulation of senescent microglia and disease-associated microglia during ageing. Furthermore, knockout of p16INK4a, a key senescence inducer, ameliorates the neuroinflammatory phenotype in damaged spinal cords in mice. These results advance our understanding of the role of cellular senescence in the central nervous system and open up possibilities for the treatment of age-related neural disorders.

Bacterial induction of B cell senescence promotes age-related changes in the gut microbiota.

The elucidation of the mechanisms of ageing and the identification of methods to control it have long been anticipated. Recently, two factors associated with ageing-the accumulation of senescent cells and the change in the composition of gut microbiota-have been shown to play key roles in ageing. However, little is known about how these phenomena occur and are related during ageing. Here we show that the persistent presence of commensal bacteria gradually induces cellular senescence in gut germinal centre B cells. Importantly, this reduces both the production and diversity of immunoglobulin A (IgA) antibodies that target gut bacteria, thereby changing the composition of gut microbiota in aged mice. These results have revealed the existence of IgA-mediated crosstalk between the gut microbiota and cellular senescence and thus extend our understanding of the mechanism of gut microbiota changes with age, opening up possibilities for their control.

Life and death of microglia: Mechanisms governing microglial states and fates.

Recent advances in genetic analysis and intensive research have revealed that microglial cells respond to external stimuli in various ways and adopt diverse states. Herein, we summarize the current knowledge on the possible microglial states during development, homeostasis, and perturbation. These states are characterized either by irreversible cellular changes, such as cell death and cellular senescence, or reversible alterations, triggered by temporary changes in the microenvironment. Subsequently, we discuss the modalities of the transcriptional and functional recovery of microglia from a molecular perspective. An understanding of (ir)reversible changes in their cellular fates or transcriptional states could provide novel options for the treatment of age-related and neurodegenerative disorders.

SARS-CoV-2 infection triggers paracrine senescence and leads to a sustained senescence-associated inflammatory response.

Reports of post-acute COVID-19 syndrome, in which the inflammatory response persists even after SARS-CoV-2 has disappeared, are increasing1, but the underlying mechanisms of post-acute COVID-19 syndrome remain unknown. Here, we show that SARS-CoV-2-infected cells trigger senescence-like cell-cycle arrest2,3 in neighboring uninfected cells in a paracrine manner via virus-induced cytokine production. In cultured human cells or bronchial organoids, these SASR-CoV-2 infection-induced senescent cells express high levels of a series of inflammatory factors known as senescence-associated secretory phenotypes (SASPs)4 in a sustained manner, even after SARS-CoV-2 is no longer detectable. We also show that the expression of the senescence marker CDKN2A (refs. 5,6) and various SASP factor4 genes is increased in the pulmonary cells of patients with severe post-acute COVID-19 syndrome. Furthermore, we find that mice exposed to a mouse-adapted strain of SARS-CoV-2 exhibit prolonged signs of cellular senescence and SASP in the lung at 14 days after infection when the virus was undetectable, which could be substantially reduced by the administration of senolytic drugs7. The sustained infection-induced paracrine senescence described here may be involved in the long-term inflammation caused by SARS-CoV-2 infection.

Gut bacteria identified in colorectal cancer patients promote tumourigenesis via butyrate secretion.

Emerging evidence is revealing that alterations in gut microbiota are associated with colorectal cancer (CRC). However, very little is currently known about whether and how gut microbiota alterations are causally associated with CRC development. Here we show that 12 faecal bacterial taxa are enriched in CRC patients in two independent cohort studies. Among them, 2 Porphyromonas species are capable of inducing cellular senescence, an oncogenic stress response, through the secretion of the bacterial metabolite, butyrate. Notably, the invasion of these bacteria is observed in the CRC tissues, coinciding with the elevation of butyrate levels and signs of senescence-associated inflammatory phenotypes. Moreover, although the administration of these bacteria into ApcΔ14/+ mice accelerate the onset of colorectal tumours, this is not the case when bacterial butyrate-synthesis genes are disrupted. These results suggest a causal relationship between Porphyromonas species overgrowth and colorectal tumourigenesis which may be due to butyrate-induced senescence.

A Role of Phosphatidylserine in the Function of Recycling Endosomes.

Cells internalize proteins and lipids in the plasma membrane (PM) and solutes in the extracellular space by endocytosis. The removal of PM by endocytosis is constantly balanced by the replenishment of proteins and lipids to PM through recycling pathway. Recycling endosomes (REs) are specific subsets of endosomes. Besides the established role of REs in recycling pathway, recent studies have revealed unanticipated roles of REs in membrane traffic and cell signalling. In this review, we highlight these emerging issues, with a particular focus on phosphatidylserine (PS), a phospholipid that is highly enriched in the cytosolic leaflet of RE membranes. We also discuss the pathogenesis of Hermansky Pudlak syndrome type 2 (HPS2) that arises from mutations in the AP3B1 gene, from the point of view of dysregulated RE functions.

A BET family protein degrader provokes senolysis by targeting NHEJ and autophagy in senescent cells.

Although cellular senescence acts primarily as a tumour suppression mechanism, the accumulation of senescent cells in vivo eventually exerts deleterious side effects through inflammatory/tumour-promoting factor secretion. Thus, the development of new drugs that cause the specific elimination of senescent cells, termed senolysis, is anticipated. Here, by an unbiased high-throughput screening of chemical compounds and a bio-functional analysis, we identify BET family protein degrader (BETd) as a promising senolytic drug. BETd provokes senolysis through two independent but integrated pathways; the attenuation of non-homologous end joining (NHEJ), and the up-regulation of autophagic gene expression. BETd treatment eliminates senescent hepatic stellate cells in obese mouse livers, accompanied by the reduction of liver cancer development. Furthermore, the elimination of chemotherapy-induced senescent cells by BETd increases the efficacy of chemotherapy against xenograft tumours in immunocompromised mice. These results reveal the vulnerability of senescent cells and open up possibilities for its control.

Endosomal phosphatidylserine is critical for the YAP signalling pathway in proliferating cells.

Yes-associated protein (YAP) is a recently discovered growth-promoting transcription coactivator that has been shown to regulate the malignancy of various cancers. How YAP is regulated is not fully understood. Here, we show that one of the factors regulating YAP is phosphatidylserine (PS) in recycling endosomes (REs). We use proximity biotinylation to find proteins proximal to PS. Among these proteins are YAP and multiple proteins related to YAP signalling. Knockdown of ATP8A1 (an RE PS-flippase) or evectin-2 (an RE-resident protein) and masking of PS in the cytoplasmic leaflet of membranes, all suppress nuclear localization of YAP and YAP-dependent transcription. ATP8A1 knockdown increases the phosphorylated (activated) form of Lats1 that phosphorylates and inactivates YAP, whereas evectin-2 knockdown reduces the ubiquitination and increased the level of Lats1. The proliferation of YAP-dependent metastatic cancer cells is suppressed by knockdown of ATP8A1 or evectin-2. These results suggest a link between a membrane phospholipid and cell proliferation.

Transport of the cholera toxin B-subunit from recycling endosomes to the Golgi requires clathrin and AP-1.

The retrograde pathway is defined by the transport of proteins and lipids from the plasma membrane through endosomes to the Golgi complex, and is essential for a variety of cellular activities. Recycling endosomes are important sorting stations for some retrograde cargo. SMAP2, a GTPase-activating protein (GAP) for Arf1 with a putative clathrin-binding domain, has previously been shown to participate in the retrograde transport of the cholera toxin B-subunit (CTxB) from recycling endosomes. Here, we found that clathrin, a vesicle coat protein, and clathrin adaptor protein complex 1 (AP-1) were present at recycling endosomes and were needed for the retrograde transport of CTxB from recycling endosomes to the Golgi, but not from the plasma membrane to recycling endosomes. SMAP2 immunoprecipitated clathrin and AP-1 through a putative clathrin-binding domain and a CALM-binding domain, and SMAP2 mutants that did not interact with clathrin or AP-1 could not localize to recycling endosomes. Moreover, knockdown of Arf1 suppressed the retrograde transport of CTxB from recycling endosomes to the Golgi. These findings suggest that retrograde transport is mediated by clathrin-coated vesicles from recycling endosomes and that the role of the coat proteins is in the recruitment of Arf GAP to transport vesicles.

Transport through recycling endosomes requires EHD1 recruitment by a phosphatidylserine translocase.

P4-ATPases translocate aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of membranes. PS is highly enriched in recycling endosomes (REs) and is essential for endosomal membrane traffic. Here, we show that PS flipping by an RE-localized P4-ATPase is required for the recruitment of the membrane fission protein EHD1. Depletion of ATP8A1 impaired the asymmetric transbilayer distribution of PS in REs, dissociated EHD1 from REs, and generated aberrant endosomal tubules that appear resistant to fission. EHD1 did not show membrane localization in cells defective in PS synthesis. ATP8A2, a tissue-specific ATP8A1 paralogue, is associated with a neurodegenerative disease (CAMRQ). ATP8A2, but not the disease-causative ATP8A2 mutant, rescued the endosomal defects in ATP8A1-depleted cells. Primary neurons from Atp8a2-/- mice showed a reduced level of transferrin receptors at the cell surface compared to Atp8a2+/+ mice. These findings demonstrate the role of P4-ATPase in membrane fission and give insight into the molecular basis of CAMRQ.

SMAP2 regulates retrograde transport from recycling endosomes to the Golgi.

Retrograde transport is where proteins and lipids are transported back from the plasma membrane (PM) and endosomes to the Golgi, and crucial for a diverse range of cellular functions. Recycling endosomes (REs) serve as a sorting station for the retrograde transport and we recently identified evection-2, an RE protein with a pleckstrin homology (PH) domain, as an essential factor of this pathway. How evection-2 regulates retrograde transport from REs to the Golgi is not well understood. Here, we report that evection-2 binds to SMAP2, an Arf GTPase-activating protein. Endogenous SMAP2 localized mostly in REs and to a lesser extent, the trans-Golgi network (TGN). SMAP2 binds evection-2, and the RE localization of SMAP2 was abolished in cells depleted of evection-2. Knockdown of SMAP2, like that of evection-2, impaired the retrograde transport of cholera toxin B subunit (CTxB) from REs. These findings suggest that evection-2 recruits SMAP2 to REs, thereby regulating the retrograde transport of CTxB from REs to the Golgi.