👤 Diego Peretti

Increasing levels of the cold-shock protein, RNA-binding motif 3 (RBM3), either through cooling or by ectopic over-expression, prevents synapse and neuronal loss in mouse models of neurodegeneration. To exploit this process therapeutically requires an understanding of mechanisms controlling cold-induced RBM3 expression. Here, we show that cooling increases RBM3 through activation of TrkB via PLCγ1 and pCREB signaling. RBM3, in turn, has a hitherto unrecognized negative feedback on TrkB-induced ERK activation through induction of its specific phosphatase, DUSP6. Thus, RBM3 mediates structural plasticity through a distinct, non-canonical activation of TrkB signaling, which is abolished in RBM3-null neurons. Both genetic reduction and pharmacological antagonism of TrkB and its downstream mediators abrogate cooling-induced RBM3 induction and prevent structural plasticity, whereas TrkB inhibition similarly prevents RBM3 induction and the neuroprotective effects of cooling in prion-diseased mice. Conversely, TrkB agonism induces RBM3 without cooling, preventing synapse loss and neurodegeneration. TrkB signaling is, therefore, necessary for the induction of RBM3 and related neuroprotective effects and provides a target by which RBM3-mediated synapse-regenerative therapies in neurodegenerative disorders can be used therapeutically without the need for inducing hypothermia. Show less

The LMF1 (lipase maturation factor 1) gene encodes a protein involved in lipoprotein lipase and hepatic lipase maturation. Homozygous mutations in LMF1 leading to severe hypertriglyceridemia (SHTG) are rare in the literature. A few additional rare LMF1 variants have been described with poor functional studies. The aim of this study was to assess the frequency of LMF1 variants in a cohort of 385 patients with SHTG, without homozygous or compound heterozygous deleterious mutations identified in lipoprotein lipase (LPL), apolipoprotein A5 (APOA5), apolipoprotein C2 (APOC2), glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) genes, and to determine their functionality. LMF1 coding variants were screened using denaturing high-performance liquid chromatography followed by direct sequencing. In silico studies of LMF1 variants were performed, followed by in vitro functional studies using human embryonic kidney 293T (HEK-293T) cells cotransfected with vectors encoding human LPL and LMF1 cDNA. LPL activity was measured in cell culture medium after heparin addition using human VLDL-TG as substrate. Nineteen nonsynonymous coding LMF1 variants were identified in 65 patients; 10 variants were newly described in SHTG. In vitro, p.Gly172Arg, p.Arg354Trp, p.Arg364Gln, and p.Arg537Trp LMF1 variants decreased LPL activity, and the p.Trp464Ter variant completely abolished LPL activity. We identified a young girl heterozygote for the p.Trp464Ter variant and a homozygote carrier of the p.Gly172Arg variant with a near 50% decreased LPL activity in vitro and in vivo. The study confirms the rarity of LMF1 variants in a large cohort of patients with SHTG. LMF1 variants are likely to be involved in multifactorial hyperchylomicronemia. Partial LMF1 defects could be associated with intermittent phenotype as described for p.Gly172Arg homozygous and p.Trp464Ter heterozygous carriers. Show less

Optimal molecular diagnosis of primary dyslipidemia is challenging to confirm the diagnosis, test and identify at risk relatives. The aim of this study was to test the application of a single targeted next-generation sequencing (NGS) panel for hypercholesterolemia, hypocholesterolemia, and hypertriglyceridemia molecular diagnosis. NGS workflow based on a custom AmpliSeq panel was designed for sequencing the most prevalent dyslipidemia-causing genes (ANGPTL3, APOA5, APOC2, APOB, GPIHBP1, LDLR, LMF1, LPL, PCSK9) on the Ion PGM Sequencer. One hundred and forty patients without molecular diagnosis were studied. In silico analyses were performed using the NextGENe software and homemade tools for detection of copy number variations (CNV). All mutations were confirmed using appropriate tools. Eighty seven variations and 4 CNV were identified, allowing a molecular diagnosis for 40/116 hypercholesterolemic patients, 5/13 hypocholesterolemic patients, and 2/11, hypertriglyceridemic patients respectively. This workflow allowed the detection of CNV contrary to our previous strategy. Some variations were found in previously unexplored regions providing an added value for genotype-phenotype correlation and familial screening. In conclusion, this new NGS process is an effective mutation detection method and allows better understanding of phenotype. Consequently this assay meets the medical need for individualized diagnosis of dyslipidemia. Show less

GPIHBP1 is a new endothelial binding site for lipoprotein lipase (LPL), the key enzyme for intravascular lipolysis of triglyceride-rich lipoproteins (TGRL). We have identified two new missense mutations of the GPIHBP1 gene, C89F and G175R, by systematic sequencing in a cohort of 376 hyperchylomicronemic patients without mutations on the LPL, APOC2, or APOA5 gene. Phenotypic expression and functional consequences of these two mutations were studied. We performed clinical and genotypic studies of probands and their families. GPIHBP1 functional alterations were studied in CHO pgsA-745 transfected cells. Probands are an adult with a homozygous G175R mutation and a child with a hemizygous C89F neomutation and a deletion of the second allele. C89F mutation was associated with a C14F signal peptide polymorphism on the same haplotype. Both patients had resistant hyperchylomicronemia, low LPL activity, and history of acute pancreatitis. In CHO pgsA-745 cells, both G175R and C14F variants reduce the expression of GPIHBP1 at the cell surface. C89F mutation is responsible for a drastic LPL-binding defect to GPIHBP1. C14F may further potentiate C89F effect. The emergence of hyperchylomicronemia in the generation after a neomutation further establishes a critical role for GPIHBP1 in TGRL physiopathology in humans. Our results highlight the crucial role of C65-C89 disulfide bond in LPL binding by GPIHBP1 Ly6 domain. Furthermore, we first report a mutation of the hydrophobic C-terminal domain that impairs GPIHBP1 membrane targeting. Show less