Inter-species differences in regulation of the progranulin–sortilin axis in TDP-43 cell models of neurodegeneration

Cytoplasmic aggregates and nuclear depletion of the ubiquitous RNA-binding protein TDP-43 have been described in the autoptic brain tissues of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD) patients and both TDP-43 loss-of-function and gain-of-function mechanisms seem to contribute to the neurodegenerative process. Among the wide array of RNA targets, TDP-43 regulates progranulin (GRN) mRNA stability and sortilin (SORT1) splicing. Progranulin is a secreted neurotrophic and neuro-immunomodulatory factor whose endocytosis and delivery to the lysosomes are regulated by the neuronal receptor sortilin. Moreover, GRN loss-of-function mutations are causative of a subset of FTLD cases showing TDP-43 pathological aggregates. Here we show that TDP-43 loss-of-function differently affects the progranulin\u2013sortilin axis in murine and human neuronal cell models. We demonstrated that although TDP-43 binding to GRN mRNA occurs similarly in human and murine cells, upon TDP-43 depletion, a different control of sortilin splicing and protein content may determine changes in extracellular progranulin uptake that account for increased or unchanged secreted protein in murine and human cells, respectively. As targeting the progranulin\u2013sortilin axis has been proposed as a therapeutic approach for GRN-FTLD patients, the inter-species differences in TDP-43-mediated regulation of this pathway must be considered when translating studies from animal models to patients

Vascular endothelial growth factor-A and Poly(A) binding protein-interacting protein 2 expression in human head and neck carcinomas: correlation and prognostic significance

Vascular endothelial growth factor-A (VEGF-A) has been demonstrated to play an important role in tumour angiogenesis and to influence prognosis in many cancers. However its prognostic value in head and neck squamous cell carcinomas (HNSCCs) remains controversial. Therefore, we investigated the clinical relevance of VEGF-A expression in HNSCCs and analysed whether its expression was associated with PAIP2 protein levels, a VEGF-A mRNA-binding partner that strongly regulates VEGF-A expression in tissue culture. We determined the correlation of VEGF-A and PAIP2 protein levels, quantitatively evaluated in tumour tissue homogenates from 54 patients with HNSCC, to clinicopathological parameters. We showed that VEGF-A expression in HNSCC is correlated to the stage of tumour differentiation (P=0.050) and is an independent prognostic factor for progression-free survival (P=0.001) and overall survival (P=0.0004). In a pharynx carcinoma cell line, we demonstrated by RNA interference that VEGF-A expression is closely controlled by PAIP2. Moreover, in human HNSCCs, VEGF-A expression is significantly correlated to PAIP2 protein levels (P=0.0018). Nevertheless, PAIP2 expression is associated with neither clinicopathological factors nor patient's survival. Our data suggest that, in contrast to PAIP2 protein levels, which are unrelated to tumour prognosis, VEGF-A expression could serve as a prognostic marker in head and neck cancer and may be helpful for targeted therapies

Neurophysiology

Contains research objectives and reports on three research projects.U. S. Air Force Office of Scientific Research under Contract AF49(638)-398National Institutes of HealthTeagle Foundation, IncorporatedBell Telephone Laboratories, Incorporate

Neurophysiology

Contains research objectives and reports on nine research projects.The Teagle Foundation, Inc.U.S. Air Force (Aeronautical Systems Division) under Contract AF33(616)-7783Bell Telephone Laboratories, Inc.National Institutes of Health [Grant M-4235-(C1)]National Institutes of Health (Grant B-1865-(C3))National Institutes of Health (Grant MP-4737)National Institutes of Health (Grant B-2480(C1)

Superhydrophobic lab-on-chip measures secretome protonation state and provides a personalized risk assessment of sporadic tumour

Secretome of primary cultures is an accessible source of biological markers compared to more complex and less decipherable mixtures such as serum or plasma. The protonation state (PS) of secretome reflects the metabolism of cells and can be used for cancer early detection. Here, we demonstrate a superhydrophobic organic electrochemical device that measures PS in a drop of secretome derived from liquid biopsies. Using data from the sensor and principal component analysis (PCA), we developed algorithms able to efficiently discriminate tumour patients from non-tumour patients. We then validated the results using mass spectrometry and biochemical analysis of samples. For the 36 patients across three independent cohorts, the method identified tumour patients with high sensitivity and identification as high as 100% (no false positives) with declared subjects at-risk, for sporadic cancer onset, by intermediate values of PS. This assay could impact on cancer risk management, individual’s diagnosis and/or help clarify risk in healthy populations

Microenvironment Changes (in pH) Affect VEGF Alternative Splicing

Vascular endothelial growth factor-A (VEGF-A) has several isoforms, which differ in their capacity to bind extracellular matrix proteins and also in their affinity for VEGF receptors. Although the relative contribution of the VEGF isoforms has been studied in tumor angiogenesis, little is known about the mechanisms that regulate the alternative splicing process. Here, we tested microenvironment cues that might regulate VEGF alternative splicing. To test this, we used endometrial cancer cells that produce all VEGF isoforms as a model, and exposed them to varying pH levels, hormones, glucose and CoCl2 (to mimic hypoxia). Low pH had the most consistent effects in inducing variations in VEGF splicing pattern (VEGF121 increased significantly, p < 0.001, when compared to VEGF145, 165 or 189). This was accompanied by activation of the p38 stress pathway and SR proteins (splicing factors) expression and phosphorylation. SF2/ASF, SRp20 and SRp40 down-regulation by siRNA impaired the effects of pH stimulation, blocking the shift in VEGF isoforms production. Taken together, we show for the first time that acidosis (low pH) regulates VEGF-A alternative splicing, may be through p38 activation and suggest the possible SR proteins involved in this process

Neurophysiology

Contains research objectives.Bell Telephone Laboratories, Inc.The Teagle Foundation, Inc.National Institutes of Health (Grant NB-01865-05)National Institutes of Health (Grant MH-04737-02)U.S. Air Force (Aeronautical Systems Division) under Contract AF33(616)-778

Pathogenic p62/SQSTM1 mutations impair energy metabolism through limitation of mitochondrial substrates

Abnormal mitochondrial function has been found in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Mutations in the p62 gene (also known as SQSTM1) which encodes the p62 protein have been reported in both disorders supporting the idea of an ALS/FTD continuum. In this work the role of p62 in energy metabolism was studied in fibroblasts from FTD patients carrying two independent pathogenic mutations in the p62 gene, and in a p62-knock-down (p62 KD) human dopaminergic neuroblastoma cell line (SH-SY5Y). We found that p62 deficiency is associated with inhibited complex I mitochondrial respiration due to lack of NADH for the electron transport chain. This deficiency was also associated with increased levels of NADPH reflecting a higher activation of pentose phosphate pathway as this is accompanied with higher cytosolic reduced glutathione (GSH) levels. Complex I inhibition resulted in lower mitochondrial membrane potential and higher cytosolic ROS production. Pharmacological activation of transcription factor Nrf2 increased mitochondrial NADH levels and restored mitochondrial membrane potential in p62-deficient cells. Our results suggest that the phenotype is caused by a loss-of-function effect, because similar alterations were found both in the mutant fibroblasts and the p62 KD model. These findings highlight the implication of energy metabolism in pathophysiological events associated with p62 deficiency

Lysophosphatidic acid and sphingosine-1-phosphate promote morphogenesis and block invasion of prostate cancer cells in three-dimensional organotypic models

Normal prostate and some malignant prostate cancer (PrCa) cell lines undergo acinar differentiation and form spheroids in three-dimensional (3-D) organotypic culture. Acini formed by PC-3 and PC-3M, less pronounced also in other PrCa cell lines, spontaneously undergo an invasive switch, leading to the disintegration of epithelial structures and the basal lamina, and formation of invadopodia. This demonstrates the highly dynamic nature of epithelial plasticity, balancing epithelial-to-mesenchymal transition against metastable acinar differentiation. This study assessed the role of lipid metabolites on epithelial maturation. PC-3 cells completely failed to form acinar structures in delipidated serum. Adding back lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) rescued acinar morphogenesis and repressed invasion effectively. Blocking LPA receptor 1 (LPAR1) functions by siRNA (small interference RNA) or the specific LPAR1 inhibitor Ki16425 promoted invasion, while silencing of other G-protein-coupled receptors responsive to LPA or S1P mainly caused growth arrest or had no effects. The G-proteins Gα12/13 and Gαi were identified as key mediators of LPA signalling via stimulation of RhoA and Rho kinases ROCK1 and 2, activating Rac1, while inhibition of adenylate cyclase and accumulation of cAMP may be secondary. Interfering with these pathways specifically impeded epithelial polarization in transformed cells. In contrast, blocking the same pathways in non-transformed, normal cells promoted differentiation. We conclude that LPA and LPAR1 effectively promote epithelial maturation and block invasion of PrCa cells in 3-D culture. The analysis of clinical transcriptome data confirmed reduced expression of LPAR1 in a subset of PrCa's. Our study demonstrates a metastasis-suppressor function for LPAR1 and Gα12/13 signalling, regulating cell motility and invasion versus epithelial maturation

C9orf72 expansion within astrocytes reduces metabolic flexibility in amyotrophic lateral sclerosis

It is important to understand how the disease process affects the metabolic pathways in amyotrophic lateral sclerosis and whether these pathways can be manipulated to ameliorate disease progression. To analyse the basis of the metabolic defect in amyotrophic lateral sclerosis we used a phenotypic metabolic profiling approach. Using fibroblasts and reprogrammed induced astrocytes from C9orf72 and sporadic amyotrophic lateral sclerosis cases we measured the production rate of reduced nicotinamide adenine dinucleotides (NADH) from 91 potential energy substrates simultaneously. Our screening approach identified that C9orf72 and sporadic amyotrophic lateral sclerosis induced astrocytes have distinct metabolic profiles compared to controls and displayed a loss of metabolic flexibility that was not observed in fibroblast models. This loss of metabolic flexibility, involving defects in adenosine, fructose and glycogen metabolism, as well as disruptions in the membrane transport of mitochondrial specific energy substrates, contributed to increased starvation induced toxicity in C9orf72 induced astrocytes. A reduction in glycogen metabolism was attributed to loss of glycogen phosphorylase and phosphoglucomutase at the protein level in both C9orf72 induced astrocytes and induced neurons. In addition, we found alterations in the levels of fructose metabolism enzymes and a reduction in the methylglyoxal removal enzyme GLO1 in both C9orf72 and sporadic models of disease. Our data show that metabolic flexibility is important in the CNS in times of bioenergetic stress