Tissue morphology influences the temporal program of human brain organoid development.

Progression through fate decisions determines cellular composition and tissue architecture, but how that same architecture may impact cell fate is less clear. We took advantage of organoids as a tractable model to interrogate this interaction of form and fate. Screening methodological variations revealed that common protocol adjustments impacted various aspects of morphology, from macrostructure to tissue architecture. We examined the impact of morphological perturbations on cell fate through integrated single nuclear RNA sequencing (snRNA-seq) and spatial transcriptomics. Regardless of the specific protocol, organoids with more complex morphology better mimicked in vivo human fetal brain development. Organoids with perturbed tissue architecture displayed aberrant temporal progression, with cells being intermingled in both space and time. Finally, encapsulation to impart a simplified morphology led to disrupted tissue cytoarchitecture and a similar abnormal maturational timing. These data demonstrate that cells of the developing brain require proper spatial coordinates to undergo correct temporal progression.

Desensitization Protocols for Anti-SARS-CoV-2 Vaccines in Patients with High Risk of Allergic Reactions.

Vaccines for SAR-CoV-2 are the most effective preventive treatment able to reduce the risk of contracting the infection and experiencing worse outcomes whenever the infection is contracted. Despite their rarity, hypersensitivity reactions to the anti-SARS-CoV-2 vaccine have been described and could become the reason not to complete the vaccination. Desensitization protocols for other vaccines have been described and validated, while the use of this approach for anti-SARS-CoV-2 vaccines is still anecdotal. We herein describe our experience with 30 patients with previous allergic reactions to anti-SARS-CoV-2 vaccines or to any of their excipients, proving that they are effective and safe; only two patients experienced hypersensitivity reaction symptoms during the desensitization procedure. Moreover, in this article, we propose desensitization protocols for the most common anti-SARS-CoV-2 vaccines.

VSSP-activated macrophages mediate senescence and tumor inhibition in a preclinical model of advanced prostate cancer.

Androgen deprivation therapy (ADT) is a standard therapy for prostate cancer (PCa). Though disseminated disease is initially sensitive to ADT, an important fraction of the patients progresses to castration-resistant prostate cancer (CRPC). For this reason, the identification of novel effective therapies for treating CRPC is needed. Immunotherapeutic strategies focused on macrophages as antitumor effectors, directly enhancing their tumoricidal potential at the tumor microenvironment or their adoptive transfer after ex vivo activation, have arisen as promising therapies in several cancer types. Despite several approaches centered on the activation of tumor-associated macrophages (TAMs) in PCa are under investigation, to date there is no evidence of clinical benefit in patients. In addition, the evidence of the effectiveness of macrophage adoptive transfer on PCa is poor. Here we find that VSSP, an immunomodulator of the myeloid system, decreases TAMs and inhibits prostatic tumor growth when administered to castrated Pten-deficient prostate tumor-bearing mice. In mice bearing castration-resistant Ptenpc-/-; Trp53pc-/- tumors, VSSP administration showed no effect. Nevertheless, adoptive transfer of macrophages activated ex vivo with VSSP inhibited Ptenpc-/-; Trp53pc-/- tumor growth through reduction of angiogenesis and tumor cell proliferation and induction of senescence. Taken together, our results highlight the rationale of exploiting macrophage functional programming as a promising strategy for CRPC therapy, with particular emphasis on ex vivo-activated proinflammatory macrophage adoptive transfer. Video abstract.

BAP1 is a novel regulator of HIF-1α.

BAP1 is a powerful tumor suppressor gene characterized by haplo insufficiency. Individuals carrying germline BAP1 mutations often develop mesothelioma, an aggressive malignancy of the serosal layers covering the lungs, pericardium, and abdominal cavity. Intriguingly, mesotheliomas developing in carriers of germline BAP1 mutations are less aggressive, and these patients have significantly improved survival. We investigated the apparent paradox of a tumor suppressor gene that, when mutated, causes less aggressive mesotheliomas. We discovered that mesothelioma biopsies with biallelic BAP1 mutations showed loss of nuclear HIF-1α staining. We demonstrated that during hypoxia, BAP1 binds, deubiquitylates, and stabilizes HIF-1α, the master regulator of the hypoxia response and tumor cell invasion. Moreover, primary cells from individuals carrying germline BAP1 mutations and primary cells in which BAP1 was silenced using siRNA had reduced HIF-1α protein levels in hypoxia. Computational modeling and co-immunoprecipitation experiments revealed that mutations of BAP1 residues I675, F678, I679, and L691 -encompassing the C-terminal domain-nuclear localization signal- to A, abolished the interaction with HIF-1α. We found that BAP1 binds to the N-terminal region of HIF-1α, where HIF-1α binds DNA and dimerizes with HIF-1ß forming the heterodimeric transactivating complex HIF. Our data identify BAP1 as a key positive regulator of HIF-1α in hypoxia. We propose that the significant reduction of HIF-1α activity in mesothelioma cells carrying biallelic BAP1 mutations, accompanied by the significant reduction of HIF-1α activity in hypoxic tissues containing germline BAP1 mutations, contributes to the reduced aggressiveness and improved survival of mesotheliomas developing in carriers of germline BAP1 mutations.

Pertuzumab study in the neoadjuvant setting for HER2-positive nonmetastatic breast cancer in Australia (PeRSIA).

The pertuzumab study in the neoadjuvant setting for HER2+ nonmetastatic breast cancer in Australia (PeRSIA-ML39622) is an analysis of safety and effectiveness data from the pertuzumab patient registry. Although the prognosis of patients with early stage HER2+ breast cancer has been greatly improved by advances in chemotherapy approximately 25% to 30% of patients develop recurrent disease. Our study aimed to examine the effectiveness of neoadjuvant pertuzumab on surgical outcomes, describe the medium-term effectiveness outcomes of patients treated with pertuzumab, and describe the planned and actual anticancer treatment regimens that patients received. Deidentified data were collected from the patients' medical records and entered into REDCap, between March 2018 and July 2019 (n = 95). The adverse events (AEs) reported most frequently were diarrhea (20; 21.1%), rash (4; 4.2%), and LVSD (4; 4.2%; two patients during neoadjuvant treatment and two patients during adjuvant treatment). AEs, ≥Grade 3 were diarrhea (2; 2.1%) and LVSD (1; 1.1%). Following surgery, a breast pathological complete response (bpCR) was achieved in 65 patients (70.7%; 95% CI: 60.2%-79.7%) and total pathological complete response (tpCR) in 59 patients (64.1%; 95% CI: 53.4%-73.9%). All patients who did not achieve a tpCR obtained a partial response (33/92, 35.9%). Our study is the first to capture real-world data on the use of pertuzumab in the neoadjuvant setting in Australia. The effectiveness and safety data are consistent with those reported in clinical trials of pertuzumab in patients with HER2+ breast cancer, with no new safety concerns.

Sirtuins and Hypoxia in EMT Control.

Epithelial-mesenchymal transition (EMT), a physiological process during embryogenesis, can become pathological in the presence of different driving forces. Reduced oxygen tension or hypoxia is one of these forces, triggering a large number of molecular pathways with aberrant EMT induction, resulting in cancer and fibrosis onset. Both hypoxia-induced factors, HIF-1α and HIF-2α, act as master transcription factors implicated in EMT. On the other hand, hypoxia-dependent HIF-independent EMT has also been described. Recently, a new class of seven proteins with deacylase activity, called sirtuins, have been implicated in the control of both hypoxia responses, HIF-1α and HIF-2α activation, as well as EMT induction. Intriguingly, different sirtuins have different effects on hypoxia and EMT, acting as either activators or inhibitors, depending on the tissue and cell type. Interestingly, sirtuins and HIF can be activated or inhibited with natural or synthetic molecules. Moreover, recent studies have shown that these natural or synthetic molecules can be better conveyed using nanoparticles, representing a valid strategy for EMT modulation. The following review, by detailing the aspects listed above, summarizes the interplay between hypoxia, sirtuins, and EMT, as well as the possible strategies to modulate them by using a nanoparticle-based approach.

Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

[This corrects the article DOI: 10.1371/journal.pbio.3001480.].

HMGB1-Mediated Activation of the Inflammatory-Reparative Response Following Myocardial Infarction.

Different cell types belonging to the innate and adaptive immune system play mutually non-exclusive roles during the different phases of the inflammatory-reparative response that occurs following myocardial infarction. A timely and finely regulation of their action is fundamental for the process to properly proceed. The high-mobility group box 1 (HMGB1), a highly conserved nuclear protein that in the extracellular space can act as a damage-associated molecular pattern (DAMP) involved in a large variety of different processes, such as inflammation, migration, invasion, proliferation, differentiation, and tissue regeneration, has recently emerged as a possible regulator of the activity of different immune cell types in the distinct phases of the inflammatory reparative process. Moreover, by activating endogenous stem cells, inducing endothelial cells, and by modulating cardiac fibroblast activity, HMGB1 could represent a master regulator of the inflammatory and reparative responses following MI. In this review, we will provide an overview of cellular effectors involved in these processes and how HMGB1 intervenes in regulating each of them. Moreover, we will summarize HMGB1 roles in regulating other cell types that are involved in the different phases of the inflammatory-reparative response, discussing how its redox status could affect its activity.

Androgens increase excitatory neurogenic potential in human brain organoids.

The biological basis of male-female brain differences has been difficult to elucidate in humans. The most notable morphological difference is size, with male individuals having on average a larger brain than female individuals1,2, but a mechanistic understanding of how this difference arises remains unknown. Here we use brain organoids3 to show that although sex chromosomal complement has no observable effect on neurogenesis, sex steroids-namely androgens-lead to increased proliferation of cortical progenitors and an increased neurogenic pool. Transcriptomic analysis and functional studies demonstrate downstream effects on histone deacetylase activity and the mTOR pathway. Finally, we show that androgens specifically increase the neurogenic output of excitatory neuronal progenitors, whereas inhibitory neuronal progenitors are not increased. These findings reveal a role for androgens in regulating the number of excitatory neurons and represent a step towards understanding the origin of sex-related brain differences in humans.

Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

Polythionic Acids in the Wackenroder Reaction.

Polythionic acids, whose general formula is H2S n O6, with n greater than 2, were discovered in the aqueous solution of SO2 and H2S, known as the Wackenroder liquid. Their reactions with each other and with other reagents are, mostly, difficult to characterize, since such compounds readily decompose and interconvert, especially in solution. Nevertheless, they play an important role in technical applications (e.g., gold leaching, magnesium milling, cooling in metal processing) and in reactions of inorganic chemistry of sulfur. A few years ago, Shell-Paques/Paqell patented the first industrial process for the biological conversion of H2S into a colloidal mixture of sulfur and polythionates. Such hydrophilic sulfur can be used as a fertilizer and soil improver in agriculture in all but alkaline soils. Recently, Eni S.p.A. has developed to bench plant scale a new process, the HydroClaus process for the conversion of H2S into an acidic hydrophilic slurry of sulfur and polythionate ions. Such a slurry can be used as a soil improver where the very alkaline soil pH hinders the cultivation. The aim of this work is to study the laboratory-scale production of polythionates in view of the novel HydroClaus process scale-up at the industrial level. After the literature related to polythionates and their characterization has been revised, the sulfur-based mixture has been synthesized and the polythionate ions concentration has been determined. Also, the effect of the reaction operating conditions has been investigated to assess how they can influence the nature and the distribution of products in solution.

Modeling neurodegeneration with mutant-tau organoids.

Greater understanding of the events preceding neurodegeneration is needed to design effective preventive and therapeutic strategies. In this issue of Cell, Bowles et al. (2021) report cerebral organoids that reveal early events in frontotemporal dementia pathogenesis due to mutations in microtubule-associated protein tau (MAPT), shedding light on a novel mechanism involving abnormal splicing and glutamate signaling.

miR-200c-3p Regulates Epitelial-to-Mesenchymal Transition in Epicardial Mesothelial Cells by Targeting Epicardial Follistatin-Related Protein 1.

Recent findings suggest that epithelial to mesenchymal transition (EMT), a key step during heart development, is involved in cardiac tissue repair following myocardial infarction (MI). MicroRNAs (miRNAs) act as key regulators in EMT processes; however, the mechanisms by which miRNAs target epicardial EMT remain largely unknown. Here, by using an in vitro model of epicardial EMT, we investigated the role of miRNAs as regulators of this process and their potential targets. EMT was induced in murine epicardial-mesothelial cells (EMCs) through TGF ß1 treatment for 48, 72, and 96 h as indicated by the expression of EMT-related genes by qRT-PCR, WB, and immunofluorescence. Further, enhanced expression of stemness genes was also detected. Among several EMT-related miRNAs, miR-200c-3p expression resulted as the most strongly suppressed. Interestingly, we also found a significant upregulation of Follistatin-related protein 1 (FSTL1), a miR-200c predicted target already identified as a potent cardiogenic factor produced by epicardial cells that promotes regeneration following MI. Dual-luciferase reporter assay demonstrated that miR-200c-3p directly targeted the 3'-untranslated region of FSTL1 in EMCs. Consistently, WB analysis showed that knockdown of miR-200c-3p significantly increased FSTL1 expression, whereas overexpression of miR-200c-3p counteracted TGF ß1-mediated FSTL1 upregulation. Importantly, FSTL1 silencing maintained epithelial features in EMCs, despite EMT induction by TGF ß1, and attenuated EMT-associated traits, including migration and stemness. In conclusion, epicardial FSTL1, an important cardiogenic factor in its secreted form, induces EMT, stemness, and migration of EMCs in a miR-200c-3p dependent pathway.

Breaking the barrier: In vitro models to study choroid plexus development.

The choroid plexus is central to normal brain function by secreting cerebrospinal fluid and dynamically regulating its composition throughout development and homoeostasis. Much of our current understanding of this region of the brain comes from studies in animal models. These fundamental studies have shed light on choroid plexus mechanisms of secretion, barrier function and homoeostatic regulation. However, how these specific mechanisms are regulated in the human choroid plexus is much less understood, due to ethical and technical limitations. A number of recent breakthroughs have enabled a new range of techniques and tools for functional characterisation of choroid plexus development and physiology. With the advance of new technologies such as in vivo imaging, single-cell transcriptomics and in vitro three-dimensional cultures we are now able to address a number of outstanding questions in choroid plexus biology. Here, we discuss some of these recent breakthroughs and we focus in particular on how in vitro models can be a powerful tool to study human cerebrospinal fluid secretion and barrier function.

SARS-CoV-2 Infects the Brain Choroid Plexus and Disrupts the Blood-CSF Barrier in Human Brain Organoids.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, leads to respiratory symptoms that can be fatal. However, neurological symptoms have also been observed in some patients. The cause of these complications is currently unknown. Here, we use human-pluripotent-stem-cell-derived brain organoids to examine SARS-CoV-2 neurotropism. We find expression of viral receptor ACE2 in mature choroid plexus cells expressing abundant lipoproteins, but not in neurons or other cell types. We challenge organoids with SARS-CoV-2 spike pseudovirus and live virus to demonstrate viral tropism for choroid plexus epithelial cells but little to no infection of neurons or glia. We find that infected cells are apolipoprotein- and ACE2-expressing cells of the choroid plexus epithelial barrier. Finally, we show that infection with SARS-CoV-2 damages the choroid plexus epithelium, leading to leakage across this important barrier that normally prevents entry of pathogens, immune cells, and cytokines into cerebrospinal fluid and the brain.

Human CNS barrier-forming organoids with cerebrospinal fluid production.

Cerebrospinal fluid (CSF) is a vital liquid, providing nutrients and signaling molecules and clearing out toxic by-products from the brain. The CSF is produced by the choroid plexus (ChP), a protective epithelial barrier that also prevents free entry of toxic molecules or drugs from the blood. Here, we establish human ChP organoids with a selective barrier and CSF-like fluid secretion in self-contained compartments. We show that this in vitro barrier exhibits the same selectivity to small molecules as the ChP in vivo and that ChP-CSF organoids can predict central nervous system (CNS) permeability of new compounds. The transcriptomic and proteomic signatures of ChP-CSF organoids reveal a high degree of similarity to the ChP in vivo. Finally, the intersection of single-cell transcriptomics and proteomic analysis uncovers key human CSF components produced by previously unidentified specialized epithelial subtypes.

Sequential screening nominates the Parkinson's disease associated kinase LRRK2 as a regulator of Clathrin-mediated endocytosis.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are an established cause of inherited Parkinson's disease (PD). LRRK2 is expressed in both neurons and glia in the central nervous system, but its physiological function(s) in each of these cell types is uncertain. Through sequential screens, we report a functional interaction between LRRK2 and Clathrin adaptor protein complex 2 (AP2). Analysis of LRRK2 KO tissue revealed a significant dysregulation of AP2 complex components, suggesting LRRK2 may act upstream of AP2. In line with this hypothesis, expression of LRRK2 was found to modify recruitment and phosphorylation of AP2. Furthermore, expression of LRRK2 containing the R1441C pathogenic mutation resulted in impaired clathrin-mediated endocytosis (CME). A decrease in activity-dependent synaptic vesicle endocytosis was also observed in neurons harboring an endogenous R1441C LRRK2 mutation. Alongside LRRK2, several PD-associated genes intersect with membrane-trafficking pathways. To investigate the genetic association between Clathrin-trafficking and PD, we used polygenetic risk profiling from IPDGC genome wide association studies (GWAS) datasets. Clathrin-dependent endocytosis genes were found to be associated with PD across multiple cohorts, suggesting common variants at these loci represent a cumulative risk factor for disease. Taken together, these findings suggest CME is a LRRK2-mediated, PD relevant pathway.

The Parkinson's Disease Protein LRRK2 Interacts with the GARP Complex to Promote Retrograde Transport to the trans-Golgi Network.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease (PD). However, the precise function of LRRK2 remains unclear. We report an interaction between LRRK2 and VPS52, a subunit of the Golgi-associated retrograde protein (GARP) complex that identifies a function of LRRK2 in regulating membrane fusion at the trans-Golgi network (TGN). At the TGN, LRRK2 further interacts with the Golgi SNAREs VAMP4 and Syntaxin-6 and acts as a scaffolding platform that stabilizes the GARP-SNAREs complex formation. Therefore, LRRK2 influences both retrograde and post-Golgi trafficking pathways in a manner dependent on its GTP binding and kinase activity. This action is exaggerated by mutations associated with Parkinson's disease and can be blocked by kinase inhibitors. Disruption of GARP sensitizes dopamine neurons to mutant LRRK2 toxicity in C. elegans, showing that these pathways are interlinked in vivo and suggesting a link in PD.

CDCP1 overexpression drives prostate cancer progression and can be targeted in vivo.

The mechanisms by which prostate cancer shifts from an indolent castration-sensitive phenotype to lethal castration-resistant prostate cancer (CRPC) are poorly understood. Identification of clinically relevant genetic alterations leading to CRPC may reveal potential vulnerabilities for cancer therapy. Here we find that CUB domain-containing protein 1 (CDCP1), a transmembrane protein that acts as a substrate for SRC family kinases (SFKs), is overexpressed in a subset of CRPC. Notably, CDCP1 cooperates with the loss of the tumor suppressor gene PTEN to promote the emergence of metastatic prostate cancer. Mechanistically, we find that androgens suppress CDCP1 expression and that androgen deprivation in combination with loss of PTEN promotes the upregulation of CDCP1 and the subsequent activation of the SRC/MAPK pathway. Moreover, we demonstrate that anti-CDCP1 immunoliposomes (anti-CDCP1 ILs) loaded with chemotherapy suppress prostate cancer growth when administered in combination with enzalutamide. Thus, our study identifies CDCP1 as a powerful driver of prostate cancer progression and uncovers different potential therapeutic strategies for the treatment of metastatic prostate tumors.

MicroRNAs in Cancer Treatment-Induced Cardiotoxicity.

Cancer treatment has made significant progress in the cure of different types of tumors. Nevertheless, its clinical use is limited by unwanted cardiotoxicity. Aside from the conventional chemotherapy approaches, even the most newly developed, i.e., molecularly targeted therapy and immunotherapy, exhibit a similar frequency and severity of toxicities that range from subclinical ventricular dysfunction to severe cardiomyopathy and, ultimately, congestive heart failure. Specific mechanisms leading to cardiotoxicity still remain to be elucidated. For instance, oxidative stress and DNA damage are considered key players in mediating cardiotoxicity in different treatments. microRNAs (miRNAs) act as key regulators in cell proliferation, cell death, apoptosis, and cell differentiation. Their dysregulation has been associated with adverse cardiac remodeling and toxicity. This review provides an overview of the cardiotoxicity induced by different oncologic treatments and potential miRNAs involved in this effect that could be used as possible therapeutic targets.