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Sulf1/Sulf2 genes are highly expressed during early fetal cardiovascular development but down-regulated during later stages correlating with a number of cell signalling pathways in a positive or a negative manner. Immunocytochemical analysis confirmed SULF1/SULF2 expression not only in endothelial cell lining of blood vessels but also in the developing cardiomyocytes but not in the adult cardiomyocytes despite persisting at reduced levels in the adult endothelial cells. The levels of both SULFs in adult ischemic human hearts and in murine hearts following coronary occlusion increased in endothelial lining of some regional blood vessels but with little or no detection in the cardiomyocytes. Unlike the normal adult heart, the levels of SULF1 and SULF2 were markedly increased in the adult canine right-atrial haemangiosarcoma correlating with increased TGFβ cell signalling. Cell signalling relationship to ischaemia was further confirmed by in vitro hypoxia of HMec1 endothelial cells demonstrating dynamic changes in not only vegf and its receptors but also sulfotransferases and Sulf1 & Sulf2 levels. In vitro hypoxia of HMec1 cells also confirmed earlier up-regulation of TGFβ cell signalling revealed by Smad2, Smad3, ALK5 and TGFβ1 changes and later down-regulation correlating with Sulf1 but not Sulf2 highlighting Sulf1/Sulf2 differences in endothelial cells under hypoxia.

SummaryAutophagy is essential for neuronal development and its deregulation contributes to neurodegenerative diseases. NDR1 and NDR2 are highly conserved kinases implicated in neuronal development, mitochondrial health and autophagy, but how they affect mammalian brain development in vivo is not known. Using single and double Ndr1/2 knockout mouse models we show that, dual, but not individual loss of Ndr1/2 in neurons causes neurodegeneration during brain development, but also in adult mice. Proteomic and phosphoproteomic comparisons between Ndr1/2 knockout and control brains revealed novel kinase substrates and indicated that endocytosis is significantly affected in the absence of NDR1/2. We validated the endocytic protein, Raph1/Lpd1 as a novel NDR1/2 substrate and showed that both NDR1/2 and Raph1 are critical for endocytosis and membrane recycling. In NDR1/2 knockout brains, we observed prominent accumulation of transferrin receptor, p62 and ubiquitinated proteins, indicative of a major impairment of protein homeostasis. Furthermore, the levels of LC3-positive autophagosomes were reduced in knockout neurons, implying that reduced autophagy efficiency mediates p62 accumulation and neurotoxicity. Mechanistically, pronounced mislocalisation of the transmembrane autophagy protein ATG9A at the neuronal periphery, impaired axonal ATG9A trafficking and increased ATG9A surface levels further confirm defects in membrane trafficking and could underlie the impairment in autophagy. We provide novel insight into the roles of NDR1/2 kinases in maintaining neuronal health.HighlightsDual neuronal Ndr1 and Ndr2 knockout during development or in adult mice causes neurodegeneration.Phosphoproteomics comparison of Ndr1/2 knockouts with control littermates shows endocytosis and membrane trafficking to be affected and reveals novel substrates.Raph1/Lamellipodin is a novel NDR1/2 substrate that is required for TfR endocytosis.Ndr1/2 knockout brains exhibit a severe defect in ubiquitinated protein clearance and reduced autophagy.NDR1/2 and Raph1 are required for the trafficking of the only transmembrane autophagy protein, ATG9A.

Autophagy is essential for neuronal development and its deregulation contributes to neurodegenerative diseases. NDR1 and NDR2 are highly conserved kinases, implicated in neuronal development, mitochondrial health and autophagy, but how they affect mammalian brain development in vivo is not known. Using single and doubleNdr1/2knockout mouse models, we show that only dual loss ofNdr1/2in neurons causes neurodegeneration. This phenotype was present when NDR kinases were deleted both during embryonic development, as well as in adult mice. Proteomic and phosphoproteomic comparisons betweenNdr1/2knockout and control brains revealed novel kinase substrates and indicated that endocytosis is significantly affected in the absence of NDR1/2. We validated the endocytic protein Raph1/Lpd1, as a novel NDR1/2 substrate, and showed that both NDR1/2 and Raph1 are critical for endocytosis and membrane recycling. In NDR1/2 knockout brains, we observed prominent accumulation of transferrin receptor, p62 and ubiquitinated proteins, indicative of a major impairment of protein homeostasis. Furthermore, the levels of LC3-positive autophagosomes were reduced in knockout neurons, implying that reduced autophagy efficiency mediates p62 accumulation and neurotoxicity. Mechanistically, pronounced mislocalisation of the transmembrane autophagy protein ATG9A at the neuronal periphery, impaired axonal ATG9A trafficking and increased ATG9A surface levels further confirm defects in membrane trafficking, and could underlie the impairment in autophagy. We provide novel insight into the roles of NDR1/2 kinases in maintaining neuronal health.

NDR kinases NDR1 and NDR2 are highly conserved regulators of neuronal differentiation that are also implicated in mitochondrial health and autophagy. The roles of NDR1/2 in mammalian brain homeostasis in vivo have not been explored. Using constitutive Ndr1 and/or neuron-specific Ndr2 knockout mouse models we show that, dual, but not individual loss of Ndr1/2 causes neurodegeneration with progressive accumulation of p62 and ubiquitinated proteins. Consistent with impaired autophagy, we found reduced LC3-positive autophagosomes in the brain and lower autophagosome formation rates and axonal LC3 trafficking in primary neurons expressing NDR1/2 shRNA. Quantitative proteomics revealed altered regulation of membrane trafficking and identified the endocytic protein Raph1/Lamellipodin (Lpd) as a novel substrate of NDR kinases. Pronounced mislocalisation of the transmembrane autophagy protein ATG9A at the neuronal periphery, partially at synapses, impaired axonal ATG9A trafficking and increased ATG9A surface levels reflect defective endocytosis, as shown by reduced transferrin uptake. We provide mechanistic understanding of how NDR kinases maintain neuronal autophagy and health.

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Despite widespread use of behavioral observations to evaluate child feeding behaviors in research and clinical practice, few studies have comprehensively characterized mealtimes or identified features that differentiate children with and without disordered feeding; these were the aims of the current study. Mealtime observations were conducted for 18 children with avoidant restrictive food intake disorder (ARFID) and 21 typically developing children. Observations were coded inductively, and associations between disorder and observed mealtime actions were examined. Most behaviors were observed across both clinical and nonclinical mealtimes, and many did not differ in frequency between children with and without ARFID. However, significant group differences were observed in the frequencies of behaviors relating to food intake, visual and physical engagement with feeding, and movement during mealtimes. The comparability of behaviors across clinical and nonclinical groups suggests that eating behaviors exist on a continuum from "normal" to "abnormal," with group differences relating to frequency rather than type of behavior. The behavioral differences observed in this study suggest that identification of children with ARFID should focus on child engagement with food and restlessness during mealtimes. Reliance on emotional and escape-maintained behaviors will lead to underrecognition of families in need of clinical support.