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GI toxicity is a common dose-limiting adverse effect of platin chemotherapy treatment. Up to 50% of cancer survivors continue to experience symptoms of chronic constipation or diarrhea induced by their chemotherapy for many years after their treatment. This drug toxicity is largely attributed to damage to enteric neurons that innervate the GI tract and control GI motility. The mechanisms responsible for platin-induced enteric neurotoxicity and potential preventative strategies have remained unknown. Here, we use human pluripotent stem cell derived enteric neurons to establish a new model system capable of uncovering the mechanism of platin-induced enteric neuropathy. Utilizing this scalable system, we performed a high throughput screen and identified drug candidates and pathways involved in the disease. Our analyses revealed that excitotoxicity through muscarinic cholinergic signaling is a key driver of platin-induced enteric neuropathy. Using single nuclei transcriptomics and functional assays, we discovered that this disease mechanism leads to increased susceptibility of specific neuronal subtypes, including inhibitory nitrergic neurons, to platins. Histological assessment of the enteric nervous system in platin-treated patients confirmed the selective loss of nitrergic neurons. Finally, we demonstrated that pharmacological and genetic inhibition of muscarinic cholinergic signaling is sufficient to rescue enteric neurons from platin excitotoxicityin vitroand can prevent platin-induced constipation and degeneration of nitrergic neurons in mice. These studies define the mechanisms of platin-induced enteric neuropathy and serve as a framework for uncovering cell type-specific manifestations of cellular stress underlying numerous intractable peripheral neuropathies.

Summary/AbstractThe neural crest (NC) is highly multipotent and generates diverse lineages in the developing embryo. However, spatiotemporally distinct NC populations display differences in fate potential, such as increased gliogenic and parasympathetic potential from later migrating, nerve-associated Schwann cell precursors (SCPs). Interestingly, while melanogenic potential is shared by both early migrating NC and SCPs, differences in melanocyte identity resulting from differentiation through these temporally distinct progenitors have not been determined. Here, we leverage a human pluripotent stem cell (hPSC) model of NC temporal patterning to comprehensively characterize human NC heterogeneity, fate bias, and lineage development. We captured the transition of NC differentiation between temporally and transcriptionally distinct melanogenic progenitors and identified modules of candidate transcription factor and signaling activity associated with this transition. For the first time, we established a protocol for the directed differentiation of melanocytes from hPSCs through a SCP intermediate, termed trajectory 2 (T2) melanocytes. Leveraging an existing protocol for differentiating early NC-derived melanocytes, termed trajectory 1 (T1), we performed the first comprehensive comparison of transcriptional and functional differences between these distinct melanocyte populations, revealing differences in pigmentation and unique expression of transcription factors, ligands, receptors and surface markers. We found a significant link between the T2 melanocyte transcriptional signature and decreased survival in melanoma patients in the cancer genome atlas (TCGA). We performed anin vivoCRISPRi screen of T1 and T2 melanocyte signature genes in a human melanoma cell line and discovered several T2-specific markers that promote lung metastasis in mice. We further demonstrated that one of these factors, SNRPB, regulates the splicing of transcripts involved in metastasis relevant functions such as migration, cell adhesion and proliferation. Overall, this study identifies distinct developmental trajectories as a source of diversity in melanocytes and implicates the unique molecular signature of SCP-derived melanocytes in metastatic melanoma.

SUMMARYSchwann cells (SCs) are the myelinating and non-myelinating glia of the peripheral nervous system (PNS) and are essential for its function. Defects in SCs are associated with many PNS disorders including diabetic peripheral neuropathy (DPN), a condition affecting millions of patients. Here we present a strategy for deriving and purifying SCs from human pluripotent stem cells (hPSCs). The scalable cultures of SCs allow basic and translational applications such as high-resolution molecular and functional characterization, developmental studies, modeling and mechanistic understanding of SC diseases and drug discovery. Our hPSC-derived SCs recapitulate the key molecular features of primary SCs and are capable of engrafting efficiently and producing myelin in injured sciatic nerves in rats. We further established an hPSC-based in vitro model of DPN that revealed the selective vulnerability of human SCs to hyperglycemia-induced cytotoxicity. We established a high-throughput screening system that identified a candidate drug that counteracts glucose-mediated cytotoxicity in SCs and normalizes glucose-induced transcriptional and metabolic abnormalities in SCs. Treatment of hyperglycemic mice with this drug candidate rescues sensory function, prevents SC death, and counteracts myelin damage in sciatic nerves suggesting considerable potential as a novel treatment for DPN.

The enteric nervous system (ENS) plays a central role in gut physiology and mediating the crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS has remained elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here we map out the developmental and functional features of the human ENS, by establishing robust and scalable 2D ENS cultures and 3D enteric ganglioids from human pluripotent stem cells (hPSCs). These models recapitulate the remarkable neuronal and glial diversity found in primary tissue and enable comprehensive molecular analyses that uncover functional and developmental relationships within these lineages. As a salient example of the power of this system, we performed in-depth characterization of enteric nitrergic neurons (NO neurons) which are implicated in a wide range of GI motility disorders. We conducted an unbiased screen and identified drug candidates that modulate the activity of NO neurons and demonstrated their potential in promoting motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define the developmental programs involved in NO neuron specification and discovered that PDGFR inhibition boosts the induction of NO neurons in enteric ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice results in extensive tissue engraftment, providing a xenograft model for the study of human ENS in vivo and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathies.

Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulatinglevels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to a global health crisis, and yet our understanding of the disease pathophysiology and potential treatment options remains limited. SARS-CoV-2 infection occurs through binding and internalization of the viral spike protein to angiotensin converting enzyme 2 (ACE2) on the host cell membrane. Lethal complications are caused by damage and failure of vital organs that express high levels of ACE2, including the lungs, the heart and the kidneys. Here, we established a high-throughput drug screening strategy to identify therapeutic candidates that reduce ACE2 levels in human embryonic stem cell (hESC) derived cardiac cells. Drug target analysis of validated hit compounds, including 5 alpha reductase inhibitors, revealed androgen signaling as a key modulator of ACE2 levels. Treatment with the 5 alpha reductase inhibitor dutasteride reduced ACE2 levels and internalization of recombinant spike receptor binding domain (Spike-RBD) in hESC-derived cardiac cells and human alveolar epithelial cells. Finally, clinical data on coronavirus disease 2019 (COVID-19) patients demonstrated that abnormal androgen states are significantly associated with severe disease complications and cardiac injury as measured by blood troponin T levels. These findings provide important insights on the mechanism of increased disease susceptibility in male COVID-19 patients and identify androgen receptor inhibition as a potential therapeutic strategy.

Missense mutations of the human mitochondrial citrate carrier, encoded by the SLC25A1 gene, lead to an autosomal recessive neurometabolic disorder characterised by neonatal-onset encephalopathy with severe muscular weakness, intractable seizures, respiratory distress, and lack of psychomotor development, often resulting in early death. Here, we have measured the effect of all twelve known pathogenic mutations on the transport activity. The results show that nine mutations abolish transport of citrate completely, whereas the other three reduce the transport rate by >70%, indicating that impaired citrate transport is the most likely primary cause of the disease. Some mutations may be detrimental to the structure of the carrier, whereas others may impair key functional elements, such as the substrate binding site and the salt bridge network on the matrix side of the carrier. To understand the consequences of impaired citrate transport on metabolism, the substrate specificity was also determined, showing that the human citrate carrier predominantly transports citrate, isocitrate, cis-aconitate, phosphoenolpyruvate and malate. Although D-2- and L-2 hydroxyglutaric aciduria is a metabolic hallmark of the disease, it is unlikely that the citrate carrier plays a significant role in the removal of hydroxyglutarate from the cytosol for oxidation to oxoglutarate in the mitochondrial matrix. In contrast, computer simulations of central metabolism predict that the export of citrate from the mitochondrion cannot be fully compensated by other pathways, restricting the cytosolic production of acetyl-CoA that is required for the synthesis of lipids, sterols, dolichols and ubiquinone, which in turn explains the severe disease phenotypes.

SARS-CoV-2 infection has led to a global health crisis, and yet our understanding of the disease and potential treatment options remains limited. The infection occurs through binding of the virus with angiotensin converting enzyme 2 (ACE2) on the cell membrane. Here, we established a screening strategy to identify drugs that reduce ACE2 levels in human embryonic stem cell (hESC)-derived cardiac cells and lung organoids. Target analysis of hit compounds revealed androgen signaling as a key modulator of ACE2 levels. Treatment with antiandrogenic drugs reduced ACE2 expression and protected hESC-derived lung organoids against SARS-CoV-2 infection. Finally, clinical data on COVID-19 patients demonstrated that prostate diseases, which are linked to elevated androgen, are significant risk factors and that genetic variants that increase androgen levels are associated with higher disease severity. These findings offer insights on the mechanism of disproportionate disease susceptibility in men and identify antiandrogenic drugs as candidate therapeutics for COVID-19., Graphical Abstract, Highlights • Drug screens on hESC cardiac cells identify modulators of SARS-CoV-2 receptor ACE2 • Targets of drugs that reduce ACE2 converge on androgen signaling pathway • Androgen signaling inhibition reduces SARS-CoV-2 infection in hESC lung organoids • Elevated androgen increases COVID-19 susceptibility and severity in men, Men are more susceptibility to severe COVID-19 complications. Fattahi and colleagues leverage stem cell models, high-throughput drug screens, and patient records to demonstrate that male sex hormones regulate SARS-CoV-2 receptors and increase disease severity in men. They identify drug candidates that reduce viral infection by inhibiting these hormones.

The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

Highlights • Dicarboxylate or 2-oxoglutarate carriers are expressed in Lactococcus lactis. • Glutathione is not a competitive substrate of dicarboxylate or 2-oxoglutarate carriers. • Dicarboxylate and 2-oxoglutarate carriers do not transport glutathione. • The identity of the mitochondrial glutathione transporter is unknown., Glutathione carries out vital protective roles within mitochondria, but is synthesised in the cytosol. Previous studies have suggested that the mitochondrial dicarboxylate and 2-oxoglutarate carriers were responsible for glutathione uptake. We set out to characterise the putative glutathione transport by using fused membrane vesicles of Lactococcus lactis overexpressing the dicarboxylate and 2-oxoglutarate carriers. Although transport of the canonical substrates could be measured readily, an excess of glutathione did not compete for substrate uptake nor could transport of glutathione be measured directly. Thus these mitochondrial carriers do not transport glutathione and the identity of the mitochondrial glutathione transporter remains unknown.

Substrates of most transport proteins have not been identified, limiting our understanding of their role in physiology and disease. Traditional identification methods use transport assays with radioactive compounds, but they are technically challenging and many compounds are unavailable in radioactive form or are prohibitively expensive, precluding large-scale trials. Here, we present a high-throughput screening method that can identify candidate substrates from libraries of unlabeled compounds. The assay is based on the principle that transport proteins recognize substrates through specific interactions, which lead to enhanced stabilization of the transporter population in thermostability shift assays. Representatives of three different transporter (super)families were tested, which differ in structure as well as transport and ion coupling mechanisms. In each case, the substrates were identified correctly from a large set of chemically related compounds, including stereo-isoforms. In some cases, stabilization by substrate binding was enhanced further by ions, providing testable hypotheses on energy coupling mechanisms.