The effects of dietary fish oil on hepatic high density and low density lipoprotein receptor activities in the rat

AbstractRats were fed either a standard ration diet or that diet supplemented with 8% by wt of a marine fish oil or safflower oil. After 10 days, plasma triacylglycerols, total cholesterol, high density lipoprotein (HDL) cholesterol, hepatic cholesterol and fatty acid synthesis and hepatic low density lipoprotein (LDL) receptor activity were significantly depressed while HDL receptor activity was significantly increased in rats fed fish oil. Fish oil-induced effects on cholesterol metabolism in the rat therefore include reciprocal changes in the activities of hepatic LDL and HDL receptors

Mutations in the facilitative glucose transporter GLUT10 alter angiogenesis and cause arterial tortuosity syndrome

Arterial tortuosity syndrome (ATS) is an autosomal recessive disorder characterized by tortuosity, elongation, stenosis and aneurysm formation in the major arteries owing to disruption of elastic fibers in the medial layer of the arterial wall1. Previously, we used homozygosity mapping to map a candidate locus in a 4.1-Mb region on chromosome 20q13.1 (ref. 2). Here, we narrowed the candidate region to 1.2 Mb containing seven genes. Mutations in one of these genes, SLC2A10, encoding the facilitative glucose transporter GLUT10, were identified in six ATS families. GLUT10 deficiency is associated with upregulation of the TGFb pathway in the arterial wall, a finding also observed in Loeys-Dietz syndrome, in which aortic aneurysms associate with arterial tortuosity3. The identification of a glucose transporter gene responsible for altered arterial morphogenesis is notable in light of the previously suggested link between GLUT10 and type 2 diabetes4,5. Our data could provide new insight on the mechanisms causing microangiopathic changes associated with diabetes and suggest that therapeutic compounds intervening with TGFb signaling represent a new treatment strategy

CLASP2 links Reelin to the cytoskeleton during neocortical development

Published in final edited form as: Neuron. 2017 March 22; 93(6): 1344–1358.e5. doi:10.1016/j.neuron.2017.02.039.INTRODUCTION The complex architecture of the brain requires precise control over the timing of neurogenesis, neuron migration, and differentiation. These three developmental processes are exquisitely controlled during the expansion of the mammalian neocortex. The six morphologically distinct layers of the neocortex form in an “inside-out” pattern with early-born neurons forming deeper layers and later-born neurons migrating past them to form superficial layers of the cortical plate (Rakic, 1974). The Reelin signaling pathway plays a crucial role in cortical lamination. Reelin is a secreted glycoprotein that exerts its function by binding to the lipoprotein receptors ApoER2 and VLDLR and inducing tyrosine phosphorylation of the intracellular adaptor protein Disabled (Dab1) (Howell et al., 2000, Bock and Herz, 2003). Phosphorylated Dab1 then recruits downstream signaling molecules to promote cytoskeletal changes necessary for neuronal migration, final positioning, and morphology (D’Arcangelo, 2005). Mutations of Reelin, the dual ApoER2/VLDLR receptor, or Dab1 lead to an inversion of the normal inside-out pattern of cortex development (D’Arcangelo et al., 1995, Howell et al., 1997, Trommsdorff et al., 1999). In addition, a number of mutations in cytoskeletal-encoded genes produce deficits in neuron migration and cortical lamination phenotypically similar to Reelin mutants, firmly establishing a mechanistic and developmentally critical connection between Reelin and the cytoskeleton. For example, human mutations in lissencephaly-1, doublecortin, and tubulin, integral components of the microtubule cytoskeleton, cause severe cortical lamination defects with later-born neurons failing to migrate past previously born neurons (Reiner et al., 1993, Gleeson et al., 1998, Romaniello et al., 2015). The culmination of these genetic studies indicates that several signaling pathways, including the Reelin pathway, converge on downstream cytoskeletal proteins to affect proper neuronal migration and brain development. However, the molecular effectors of these pathways have not been fully characterized. CLASPs (cytoplasmic linker associated proteins) belong to a heterogeneous family of plus-end tracking proteins (+TIPs) that specifically accumulate at the growth cone. This localization strategically places them in a position to control neurite growth, directionality, and the crosstalk between microtubules and the actin cytoskeleton (Akhmanova and Hoogenraad, 2005, Basu and Chang, 2007, Akhmanova and Steinmetz, 2008). Previous evidence showed that CLASPs accumulate asymmetrically toward the leading edge of migrating fibroblasts, indicating a role for CLASPs in cell polarity and movement (Akhmanova et al., 2001, Wittmann and Waterman-Storer, 2005). We found that CLASP2 protein levels steadily increase throughout neuronal development and are specifically enriched at the growth cones of extending neurites. In particular, short-hairpin RNA (shRNA)-mediated knockdown of CLASP2 in primary mouse neurons decreases neurite length, whereas overexpression of human CLASP2 causes the formation of multiple axons, enhanced dendritic branching, and Golgi condensation (Beffert et al., 2012). These results implicate a role for CLASP2 in neuronal morphogenesis and polarization; however, the function of CLASP2 during brain development is unknown. Here we demonstrate that CLASP2 is a modifier of the Reelin signaling pathway during cortical development. In vivo knockdown experiments demonstrate that CLASP2 plays significant roles in neural precursor proliferation, neuronal migration, and morphogenesis. In addition, we show that GSK3β-mediated phosphorylation of CLASP2 controls its binding to the Reelin adaptor protein Dab1, a required molecular step governing CLASP2’s regulatory effects on neuron morphology and movement. RESULTS CLASP2 Expression Is Functionally Associated with the Reelin Signaling Pathway To identify novel genes downstream of Reelin signaling, we examined the expression of mRNA transcripts by microarray between adult brain cortices from mice deficient in either Reelin, the double ApoER2/VLDLR receptor mutant, or Dab1 and compared Affymetrix gene expression profiles against age-matched, wild-type mice. Importantly, each of these mutant mouse models present a similar phenotype that includes severe neuronal migration defects (D’Arcangelo et al., 1995, Howell et al., 1997, Trommsdorff et al., 1999). We defined a large network of genes perturbed above a threshold of 1.5-fold in response to deficient Reelin signaling, identifying 832 upregulated and 628 downregulated genes that were common to all three mouse models (Figure 1A). Ingenuity Pathway Analysis revealed a network of genes that is functionally related to cytoskeleton organization, microtubule dynamics, neurogenesis, and migration of cells (Figure 1B). Of the few cytoskeletal candidate genes identified, CLASP2 was the only microtubule +TIP. Specifically, CLASP2 mRNA expression was increased in all three Reelin mutant phenotypes, while CLASP1 mRNA expression remained unchanged (Figure 1B). Consistent with the microarray data, CLASP2 protein levels were ∼2.8-fold higher in Dab1 knockout mice (Figure 1C). These findings suggest that Reelin signaling controls CLASP2 expression and establishes the first molecular link between a plus-end, microtubule binding protein downstream of extracellular Reelin signaling.We thank Drs. Thomas C. Sudhof, Joachim Herz, Santos Franco, and Torsten Wittmann for plasmids and antibodies. We thank Alicia Dupre, Elias Fong, and Christine Learned for technical support. This work was supported by grants from the National Institutes of Health (R21 MH100581 to T.F.H., U.B., and A.H.). (R21 MH100581 - National Institutes of Health)Accepted manuscrip

Biallelic loss of function variants in PPP1R21 cause a neurodevelopmental syndrome with impaired endocytic function

Next‐generation sequencing (NGS) has been instrumental in solving the genetic basis of rare inherited diseases, especially neurodevelopmental syndromes. However, functional workup is essential for precise phenotype definition and to understand the underlying disease mechanisms. Using whole exome (WES) and whole genome sequencing (WGS) in four independent families with hypotonia, neurodevelopmental delay, facial dysmorphism, loss of white matter, and thinning of the corpus callosum, we identified four previously unreported homozygous truncating PPP1R21 alleles: c.347delT p.(Ile116Lysfs*25), c.2170_2171insGGTA p.(Ile724Argfs*8), c.1607dupT p.(Leu536Phefs*7), c.2063delA p.(Lys688Serfs*26) and found that PPP1R21 was absent in fibroblasts of an affected individual, supporting the allele's loss of function effect. PPP1R21 function had not been studied except that a large scale affinity proteomics approach suggested an interaction with PIBF1 defective in Joubert syndrome. Our co‐immunoprecipitation studies did not confirm this but in contrast defined the localization of PPP1R21 to the early endosome. Consistent with the subcellular expression pattern and the clinical phenotype exhibiting features of storage diseases, we found patient fibroblasts exhibited a delay in clearance of transferrin‐488 while uptake was normal. In summary, we delineate a novel neurodevelopmental syndrome caused by biallelic PPP1R21 loss of function variants, and suggest a role of PPP1R21 within the endosomal sorting process or endosome maturation pathway

A Novel and Lethal De Novo LQT-3 Mutation in a Newborn with Distinct Molecular Pharmacology and Therapeutic Response

SCN5A encodes the alpha-subunit (Na(v)1.5) of the principle Na(+) channel in the human heart. Genetic lesions in SCN5A can cause congenital long QT syndrome (LQTS) variant 3 (LQT-3) in adults by disrupting inactivation of the Na(v)1.5 channel. Pharmacological targeting of mutation-altered Na(+) channels has proven promising in developing a gene-specific therapeutic strategy to manage specifically this LQTS variant. SCN5A mutations that cause similar channel dysfunction may also contribute to sudden infant death syndrome (SIDS) and other arrhythmias in newborns, but the prevalence, impact, and therapeutic management of SCN5A mutations may be distinct in infants compared with adults.Here, in a multidisciplinary approach, we report a de novo SCN5A mutation (F1473C) discovered in a newborn presenting with extreme QT prolongation and differential responses to the Na(+) channel blockers flecainide and mexiletine. Our goal was to determine the Na(+) channel phenotype caused by this severe mutation and to determine whether distinct effects of different Na(+) channel blockers on mutant channel activity provide a mechanistic understanding of the distinct therapeutic responsiveness of the mutation carrier. Sequence analysis of the proband revealed the novel missense SCN5A mutation (F1473C) and a common variant in KCNH2 (K897T). Patch clamp analysis of HEK 293 cells transiently transfected with wild-type or mutant Na(+) channels revealed significant changes in channel biophysics, all contributing to the proband's phenotype as predicted by in silico modeling. Furthermore, subtle differences in drug action were detected in correcting mutant channel activity that, together with both the known genetic background and age of the patient, contribute to the distinct therapeutic responses observed clinically.The results of our study provide further evidence of the grave vulnerability of newborns to Na(+) channel defects and suggest that both genetic background and age are particularly important in developing a mutation-specific therapeutic personalized approach to manage disorders in the young

Exclusion of mutations in the PRNP, JPH3, TBP, ATN1, CREBBP, POU3F2 and FTL genes as a cause of disease in Portuguese patients with a Huntington-like phenotype

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder characterised by chorea, cognitive impairment, dementia and personality changes, caused by the expansion of a CAG repeat in the HD gene. Often, patients with a similar clinical presentation do not carry expansions of the CAG repeat in this gene [Huntington disease-like (HDL) patients]. We report the genetic analysis of 107 Portuguese patients with an HDL phenotype. The HDL genes PRNP and JPH3, encoding the prion protein and junctophilin-3, respectively, were screened for repeat expansions in these patients. Given the partial clinical overlap of SCA17, DRPLA and neuroferritinopathy with HD, their causative genes (TBP, ATN1, and FTL, respectively) were also analysed. Finally, repeat expansions in two candidate genes, CREBBP and POU3F2, which encode the nuclear transcriptional coactivator CREB-binding protein and the CNS-specific transcription factor N-Oct-3, respectively, were also studied. Expansions of the repetitive tracts of the PRNP, JPH3, TBP, ATN1, CREBBP and POU3F2 genes were excluded in all patients, as were sequence alterations in the FTL gene. Since none of the genes already included in the differential diagnosis of HD was responsible for the disease in our sample, the genetic heterogeneity of the HDL phenotype is still open for investigation.Fundação para a Ciência e a Tecnologia (FCT) and FEDER (grant CBO/33485/99). BIC included in grant CBO/33485/99, respectivel

Predictive value of pathological and immunohistochemical parameters for axillary lymph node metastasis in breast carcinoma

<p>Abstract</p> <p>Background/Objective</p> <p>While several prognostic factors have been identified in breast carcinoma, the clinical outcome remains hard to predict for individual patients. Better predictive markers are needed to help guide difficult treatment decisions. Axillary lymph node metastasis (ALNM) is one of the most important prognostic determinants in breast carcinoma; however, the reasons why tumors vary in their capability to result in axillary metastasis remain unclear. Identifying breast carcinoma patients at risk for ALNM would improve treatment planning. This study aimed to identify the factors associated with ALNM in breast carcinoma, with particular emphasis on basal-like phenotype.</p> <p>Methods</p> <p>Breast carcinoma patients (n = 210) who underwent breast conserving surgery and axillary lymph node dissection (ALND) (level I and II) or modified radical mastectomy were included in this study. Pathological and immunohistochemical data including individual receptor/gene status was collected for analysis. The basal phenotype status was ascertained using the basal cytokeratin markers CK5, CK14, CK17 and EGFR.</p> <p>Results</p> <p>ALNM was found in 55% (n = 116) of the patients. On univariate analysis, multicentric disease, large tumor size (>2 cm), vascular and lymphatic invasion, epithelial hyperplasia, necrosis, in situ carcinoma and perineural invasion were associated with higher risk for ALNM, whereas CK5, CK14, EGFR positivity and basal-like tumor type were associated with lower risk. On multivariate analysis, CK5 positivity (OR 0.003, 95%CI 0.000-0.23, p = 0.009) and lymphatic/vascular invasion (OR 17.94, 95%CI 4.78-67.30, p < 0.001) were found to be independent predictors.</p> <p>Conclusions</p> <p>Although the value of complete ALND has been questioned in invasive breast cancer patients, treatment decisions for breast carcinoma have been influenced by many parameters, including lymph node status. Since histopathologic characteristics and expression of biological markers varies among the same histologic subtypes of breast carcinoma, specific clinical and histopathologic features of the primary tumor and ALN status like sentinel node might be used to tailor the loco-regional and systemic treatment in different clinical settings.</p

Surgical Management of Multicentricity, Nipple-Areola Involvement, and Bilaterality in Breast Carcinoma

During a five-year period, 457 women who underwent partial, segmental, or total mastectomy were assessed for multicentricity (31%), nipple-areola involvement (17%), and bilateral breast carcinoma (9%). These factors determined the extent of surgery performed and the possibilities for later breast reconstruction. The frequency of nipple-areola involvement in breast cancer strongly indicates the need for alternatives to their preservation for breast reconstruction. The study concludes that total mastectomy for invasive carcinoma is the preferred method of treatment for early breast cancer until further clinical trials have assessed the value of segmentectomy