Rebalancing of mitochondrial homeostasis through an NAD+-SIRT1 pathway preserves intestinal barrier function in severe malnutrition.

BACKGROUND: The intestine of children with severe malnutrition (SM) shows structural and functional changes that are linked to increased infection and mortality. SM dysregulates the tryptophan-kynurenine pathway, which may impact processes such as SIRT1- and mTORC1-mediated autophagy and mitochondrial homeostasis. Using a mouse and organoid model of SM, we studied the repercussions of these dysregulations on malnutrition enteropathy and the protective capacity of maintaining autophagy activity and mitochondrial health. METHODS: SM was induced through feeding male weanling C57BL/6 mice a low protein diet (LPD) for 14-days. Mice were either treated with the NAD +-precursor, nicotinamide; an mTORC1-inhibitor, rapamycin; a SIRT1-activator, resveratrol; or SIRT1-inhibitor, EX-527. Malnutrition enteropathy was induced in enteric organoids through amino-acid deprivation. Features of and pathways to malnutrition enteropathy were examined, including paracellular permeability, nutrient absorption, and autophagic, mitochondrial, and reactive-oxygen-species (ROS) abnormalities. FINDINGS: LPD-feeding and ensuing low-tryptophan availability led to villus atrophy, nutrient malabsorption, and intestinal barrier dysfunction. In LPD-fed mice, nicotinamide-supplementation was linked to SIRT1-mediated activation of mitophagy, which reduced damaged mitochondria, and improved intestinal barrier function. Inhibition of mTORC1 reduced intestinal barrier dysfunction and nutrient malabsorption. Findings were validated and extended using an organoid model, demonstrating that resolution of mitochondrial ROS resolved barrier dysfunction. INTERPRETATION: Malnutrition enteropathy arises from a dysregulation of the SIRT1 and mTORC1 pathways, leading to disrupted autophagy, mitochondrial homeostasis, and ROS. Whether nicotinamide-supplementation in children with SM could ameliorate malnutrition enteropathy should be explored in clinical trials. FUNDING: This work was supported by the Bill and Melinda Gates Foundation, the Sickkids Research Institute, the Canadian Institutes of Health Research, and the University Medical Center Groningen

The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells.

Phosphoinositide (PtdInsP) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes/late endosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological and enzyme-based assays dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized with Rab5-positive early endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes

Correction: The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells.

[This corrects the article DOI: 10.1371/journal.pone.0198454.]

The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells

Phosphoinositide (PtdInsP) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes/late endosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological and enzyme-based assays dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized with Rab5-positive early endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.  </p

Inhibition of Class III PtdIns 3-Kinase Vps34 displaces TgPH1 domain from membranes.

<p>A. PC3 and RPE cells expressing eGFP and treated with vehicle or 1 μM VPS34-IN1 for 1 h to block Vps34 activity. B. PC3, RPE, HeLa and RAW macrophages expressing GFP-2x-TgPH and treated with vehicle (control) or 1 μM VPS34-IN1 for 1 h. For A and B, cells were fixed and imaged by spinning disc confocal microscopy. C. Cos-7 cells expressing NES-iRFP-TgPH treated with vehicle or 1 μM Vps34-IN1 for 1 h. Cells were imaged by laser scanning confocal microscopy. For B and C, control cells displayed chimeric TgPH1 proteins on punctate structures, while cells treated with Vps34-IN1 exhibit mostly cytosolic TgPH1 distribution. D-G: The proportion of GFP-2x-TgPH1 associated with membranes relative to the cytosol was estimated by quantifying F^H/F_L fluorescence ratio as described in Methods using a 10-pixel wide and 30-40-pixel long line. H. The proportion of NES-iRFP-TgPH1 in the membrane versus cytosol was assessed by quantifying F_p/F_c as described in Methods. In all cases, these data quantitatively show that GFP-2x-TgPH1 and NES-iRFP-TgPH1 become cytosolic in VPS34-IN1-treated cells in a variety of cell lines. Error bars represent standard error of the mean derived from analyzing at least 20 cells per condition across N = 3 independent experiments. * p<0.05 against respective controls using Student’s t-test for F, G and H or using one-way ANOVA and Tukey’s test for D and E. Scale bars represent 10 μm.</p

Subcellular distribution of GFP-fused TgPH1 proteins in PIKfyve-inhibited RAW cells.

<p>RAW macrophages were transfected with GFP (A), GFP-TgPH1 (B), or GFP-2x-TgPH1 (C) and lysosomes were labelled with Alexa⁵⁴⁶-conjugated dextran as described in Methods. Cells were then left untreated (control) or exposed to 20 nM apilimod or 100 nM YM201636 for 1 h to inhibit PIKfyve and induce vacuolation. Cells were imaged live using spinning disc confocal microscopy. GFP-2x-TgPH1 displayed strong membrane association that was retained in PIKfyve-inhibited conditions, while GFP-TgPH1 displayed weaker membrane association and GFP was cytosolic under all treatments employed. Green line exemplifies line arrangements used to quantify F^H/F_L fluorescence ratio. D. Quantification of GFP, GFP-TgPH1 and GFP-2x-TgPH1 membrane association. Data shown are mean F^H/F_L ± SD from N = 3 from 25–40 cells per experiment per condition. Using a one-way ANOVA and Tukey's post-hoc test, there was significant difference between the F^H/F_L for GFP and GFP-TgPH1 for each respective condition (* p<0.05). In addition, there was a significant difference between the F^H/F_L for GFP-TgPH1 and GFP-2x-TgPH1 (** p<0.01). Scale bar represent 10 μm.</p

TgPH1 retains membrane association in genetically-impaired PIKfyve cells.

<p>A. Live-cell imaging of Cos-7 cells expressing NES-iRFP-TgPH1 treated with vehicle alone or with 100 nM apilimod or 1 μM YM201636 for 1 h; the drugs cause a characteristic appearance of swollen vacuoles, some of which labelled with the TgPH1 probes. (B) Quantification (means ± SD of 38 ≤ n ≤ 40 cells) of TgPH localization on vesicular structures in cells expressed as F_p/F_c, the ratio of intensity in bright punctate objects to mean fluorescence of the whole cell; there is no decrease in membrane association of TgPH1 in COS-7 cells after PIKfyve inhibition. (C) COS-7 cells imaged as in A whilst co-expressing either GFP, GFP-PIKfyve or its dominant-negative K1831E mutant. The latter causes the appearance of swollen vacuoles due to defective PtdIns(3,5)P₂ synthesis, yet there is no effect on TgPH1 membrane association, as quantified in (D). D. Data shown is the mean ± SD of 90 cells per condition. Inset are zoomed areas (400 μm² delineated by the box in each panel. Corresponding differential interference contrast (DIC) are also shown.</p

GFP-2x-TgPH1 retains membrane association in several mammalian cells inhibited for PIKfyve.

<p>Human PC3 cells and RPE cells were transfected with GFP or GFP-2x-TgPH1, labelled with fluorescent dextran to identify lysosomes and then treated with vehicle or apilimod to inhibit PIKfyve for 1 h, followed by spinning disc confocal imaging. A, B. PC3 cells (A) and RPE (B) expressing GFP treated with vehicle or apilimod. In all cases, GFP appeared cytosolic. C, D: PC3 cells (C) and RPE cells (D) expressing GFP-2x-TgPH1 treated with vehicle or apilimod. In all cases, GFP-2x-TgPH1 retained membrane association. E, F. Quantification of GFP and GFP-2x-TgPH1 membrane association in PC3 (E) and RPE cells (F). Data shown are mean F^H/F_L ± STD from N = 3 from 25–40 cells per experiment per condition. Using a one-way ANOVA and Tukey's post-hoc test, there was a significance difference in the FH/FL for GFP versus GFP-2x-TgPH1 for the respective cells and treatments (* p<0.01) but there was no difference in the membrane distribution of GFP-2x-TgPH1 between vehicle and apilimod in PC3 or RPE cells. Scale bar represent 10 μm.</p

Depletion of PtdIns(3)P but not PtdIns(3,5)P₂ causes TgPH1 to dissociate from Rab5-positive membranes in COS-7 cells.

<p>A. Schematic of the rapamycin-induced dimerization system and the enzymatic activities of the FKBP-conjugated phosphatases. B, C. Time-lapse imaging of COS-7 cells expressing iRFP-FRB-Rab5 (magenta), mCherry-FKBP fused to the indicated enzyme (cyan) and eGFP^NES-TgPH1 (grayscale). B. The time-lapse shows the recruitment of the fused phosphatase (cyan) to Rab5-containing puncta (magenta) before and after the addition of 1 μM rapamycin, marked by time 0. C. A time-lapse series of the same cells showing the dynamics of eGFP^NES -TgPH1 before and after rapamycin. Images were acquired at 2 min intervals. The graphs at right show the normalized intensity at Rab5-positive membranes relative to the whole cell for FKBP-tagged enzymes (B) and eGFP^NES-TgPH1 (C). Data are means ±SEM of 41 (MTM1), 23 (C375S) or 27 (INPP5E) cells from 3 or 4 independent experiments. * p<0.05 against control using ANOVA and Tukey's test.</p

Growth Hormone Receptor (GHR) 6Ω Pseudoexon Activation: a Novel Cause of Severe Growth Hormone Insensitivity

Context: Severe forms of growth hormone insensitivity (GHI) are characterized by extreme short stature, dysmorphism, and metabolic anomalieObjective: This work aims to identify the genetic cause of growth failure in 3 "classical" GHI individuals.Methods: A novel intronic growth hormone receptor gene (GHR) variant was identified, and in vitro splicing assays confirmed aberrant splicing. A 6 Omega pseudoexon GHR vector and patient fibroblast analysis assessed the consequences of the novel pseudoexon inclusion and the impact on GHR function.Results: We identified a novel homozygous intronic GHR variant (g.5:42700940T &gt; G, c.618+836T &gt; G), 44 bp downstream of the previously recognized intronic 6. GHR pseudoexon mutation in the index patient. Two siblings also harbored the novel intronic 6 Omega pseudoexon GHR variant in compound heterozygosity with the known GHR c.181C &gt; T (R43X) mutation. In vitro splicing analysis confirmed inclusion of a 151-bp mutant 6 Omega pseudoexon not identified in wild-type constructs. Inclusion of the 6 Omega pseudoexon causes a frameshift resulting in a nonfunctional truncated GHR lacking the transmembrane and intracellular domains. The truncated 6 Omega pseudoexon protein demonstrated extracellular accumulation and diminished activation of STAT5B signaling following GH stimulation.Conclusion: Novel GHR 6 Omega pseudoexon inclusion results in loss of GHR function consistent with a severe GHI phenotype. This represents a novel mechanism of Laron syndrome and is the first deep intronic variant identified causing severe postnatal growth failure.The 2 kindreds originate from the same town in Campania, Southern Italy, implying common ancestry. Our findings highlight the importance of studying variation in deep intronic regions as a cause of monogenic disorders