Carlos R. Morales

From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus

Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes.

Qualitative and Quantitative Decline in Spermatogenesis of the Follicle-Stimulating Hormone Receptor Knockout (FORKO) Mouse

Abstract Sertoli cells express functional receptors for FSH, one of the two pituitary hormones that regulate spermatogenesis in mammals. We recently produced genetic mutant (FORKO) mice that lack FSH receptor, in order to examine the effects on testicular function and fertility. Mutant males exhibited weight loss of testis, epididymis, and seminal vesicle as well as low levels of testosterone. Except for reduced seminiferous tubular diameter, no gross changes were apparent upon histological examination. Analysis of testicular germ cells by flow cytometry revealed a significant increase in the percentage of 2C cells (spermatogonia and non-germ cells) and a significant decrease in the percentage of HC cells (elongated spermatids) of FORKO males. The absolute number of homogenization-resistant elongated spermatids was also significantly reduced in the mutant males. A 2-fold increase in c-kit-positive 2C cells was recorded in the mutant males. Elongated spermatids of FORKO males showed a dramatic increase in propidium iodide binding suggesting reduced nuclear compaction. The increase in size of the sperm head in mutants, as well as susceptibility to dithiothreitol-induced decondensation, suggests the inadequate condensation of sperm chromatin. Sperm chromatin structure assay, a technique that reflects DNA stability, revealed that sperm from FORKO males are susceptible to acid denaturation, indicating the poor quality of sperm. These data allow us to conclude that genetic disruption of FSH receptor signaling in the rodent induces major changes that might contribute to reduced fertility.

Secretion and endocytosis in the male reproductive tract: a role in sperm maturation

Publisher Summary The epithelial cells of the entire male reproductive duct system, from the testis to the vas deferens, contribute to a proper milieu for sperm maturation through two distinct activities: secretion and endocytosis. Examples are provided in the chapter of these activities by following the origin and fate of SGP-1, SGP-2, and immobilin in the excurrent duct system. These proteins typify the regional variations that exist for the secretion and endocytosis of proteins along the reproductive duct. The reasons for such regional variations in secretion and endocytosis of different proteins ultimately lies in the genetic regulatory factors for each protein, the type of association of each protein with the spermatozoa, if any, and the functional contributions that each protein plays in the final maturation of spermatozoa. An important concept discussed in this chapter is that the spermatozoon itself may contribute to its own maturation (i.e., glycosylation) providing that the appropriate conditions of its external milieu are met by the secretory and endocytic activities along the excurrent reproductive duct system.

Hepatic sortilin regulates both apolipoprotein B secretion and LDL catabolism

Genome-wide association studies (GWAS) have identified a genetic variant at a locus on chromosome 1p13 that is associated with reduced risk of myocardial infarction, reduced plasma levels of LDL cholesterol (LDL-C), and markedly increased expression of the gene sortilin-1 (SORT1) in liver. Sortilin is a lysosomal sorting protein that binds ligands both in the Golgi apparatus and at the plasma membrane and traffics them to the lysosome. We previously reported that increased hepatic sortilin expression in mice reduced plasma LDL-C levels. Here we show that increased hepatic sortilin not only reduced hepatic apolipoprotein B (APOB) secretion, but also increased LDL catabolism, and that both effects were dependent on intact lysosomal targeting. Loss-of-function studies demonstrated that sortilin serves as a bona fide receptor for LDL in vivo in mice. Our data are consistent with a model in which increased hepatic sortilin binds intracellular APOB-containing particles in the Golgi apparatus as well as extracellular LDL at the plasma membrane and traffics them to the lysosome for degradation. We thus provide functional evidence that genetically increased hepatic sortilin expression both reduces hepatic APOB secretion and increases LDL catabolism, providing dual mechanisms for the very strong association between increased hepatic sortilin expression and reduced plasma LDL-C levels in humans.

Function of vitamin A in normal and synchronized seminiferous tubules.

Vitamin A is clearly an important factor in spermatogenesis. Some of the new data on metabolism of retinoids in the testis has contributed to our understanding of the mechanism(s) involved in the action of vitamin A. It is probable that the requirement of the testis of vitamin A deficient rats for retinol but not retinoic acid involves access of the retinoids to various testicular compartments. Retinol may be required by germinal cells because of a requirement for esterification in order to be successfully transported by the Sertoli cells. Existing evidence suggests that both the Sertoli cells and the germinal cells have specific requirements for retinoids. In the vitamin A deficient rat there appears to be a developmental block at preleptotene spermatocyte and type Al spermatogonia stages. This block is removed by retinol and germinal cell development reinitiates in a synchronous manner. The synchronous testis model offers a number of advantages for the study of molecular events associated with the cycle of the seminiferous epithelium and the development of germinal cells as well as for investigations into the mechanism of action of the retinoids.

The turnover of amniotic fluid protein in the human conceptus

Abstract The amniotic fluid turnover of specific plasma proteins and protein hormones was studied in 19 pregnant women at 34 to 40 weeks of gestation. Seventeen of the women had normal pregnancies; of these, 6 were in labor during the study, and 11 were not. In the 2 remaining women, the fetuses were dead. Purified human serum albumin, serum γG, serum γA, chorionic gonadotropin, and growth hormone were labeled with either 131 I or 125 I, and the labeled proteins were then injected intra-amniotically singly or in pairs. Aliquots of amniotic fluid were obtained before the injection, 15 minutes after the injection, and at irregular intervals thereafter during the study period which lasted from 3½ hours to 13 days. Maternal and neonatal sera and urines were also obtained. All fluids were assayed for labeled protein as well as endogenous serum albumin, transferrin, γG, and γA. It was found that: (1) all 5 labeled proteins were cleared from amniotic fluid at similar rates, despite the marked differences in the molecular weights and metabolic functions of these proteins; (2) on the average, two thirds or more of the amniotic fluid volume was cleared of protein per day in the presence of a living fetus, over 80 per cent of this apparently by fetal swallowing, and the daily clearance of amniotic fluid averaged 342 ml. in the absence of labor and 554 ml. during labor, or 0.24 and 0.30 Gm. of amniotic fluid protein per kilogram of fetal weight, respectively; (3) fetal urine was the apparent source of a large fraction of the γG found in amniotic fluid, but fetal urine contributed less than 5 per cent of the albumin, less than 2 per cent of the transferrin, and little or none of the γA present in amniotic fluid; (4) amniotic fluid volume could change markedly in a matter of days—over a period of 5 days, it doubled in one normal patient and fell to half in another; (5) the volume of amniotic fluid swallowed by the fetus tended to vary directly with the volume of fluid in the amniotic cavity, a relation which, among other things, would serve to stabilize amniotic fluid volume.

ProNGF induces TNFα-dependent death of retinal ganglion cells through a p75NTR non-cell-autonomous signaling pathway

Neurotrophin binding to the p75 neurotrophin receptor (p75NTR) activates neuronal apoptosis following adult central nervous system injury, but the underlying cellular mechanisms remain poorly defined. In this study, we show that the proform of nerve growth factor (proNGF) induces death of retinal ganglion cells in adult rodents via a p75NTR-dependent signaling mechanism. Expression of p75NTR in the adult retina is confined to Müller glial cells; therefore we tested the hypothesis that proNGF activates a non-cell-autonomous signaling pathway to induce retinal ganglion cell (RGC) death. Consistent with this, we show that proNGF induced robust expression of tumor necrosis factor alpha (TNFα) in Müller cells and that genetic or biochemical ablation of TNFα blocked proNGF-induced death of retinal neurons. Mice rendered null for p75NTR, its coreceptor sortilin, or the adaptor protein NRAGE were defective in proNGF-induced glial TNFα production and did not undergo proNGF-induced retinal ganglion cell death. We conclude that proNGF activates a non-cell-autonomous signaling pathway that causes TNFα-dependent death of retinal neurons in vivo.

Enzymatic Activity of Lysosomal Carboxypeptidase (Cathepsin) A Is Required for Proper Elastic Fiber Formation and Inactivation of Endothelin-1

Background— Lysosomal carboxypeptidase, cathepsin A (protective protein, CathA), is a component of the lysosomal multienzyme complex along with &bgr;-galactosidase (GAL) and sialidase Neu1, where it activates Neu1 and protects GAL and Neu1 against the rapid proteolytic degradation. On the cell surface, CathA, Neu1, and the enzymatically inactive splice variant of GAL form the elastin-binding protein complex. In humans, genetic defects of CathA cause galactosialidosis, a metabolic disease characterized by combined deficiency of CathA, GAL, and Neu1 and a lysosomal storage of sialylated glycoconjugates. However, several phenotypic features of galactosialidosis patients, including hypertension and cardiomyopathies, cannot be explained by the lysosomal storage. These observations suggest that CathA may be involved in hemodynamic functions that go beyond its protective activity in the lysosome. Methods and Results— We generated a gene-targeted mouse in which the active CathA was replaced with a mutant enzyme carrying a Ser190Ala substitution in the active site. These animals expressed physiological amounts of catalytically inactive CathA protein, capable of forming lysosomal multienzyme complex, and did not develop secondary deficiency of Neu1 and GAL. Conversely, the mice showed a reduced degradation rate of the vasoconstrictor peptide, endothelin-1, and significantly increased arterial blood pressure. CathA-deficient mice also displayed scarcity of elastic fibers in lungs, aortic adventitia, and skin. Conclusions— Our results provide the first evidence that CathA acts in vivo as an endothelin-1–inactivating enzyme and strongly confirm a crucial role of this enzyme in effective elastic fiber formation.

Hamster sperm antigen P26h is a phosphatidylinositol-anchored protein

We have previously identified a hamster sperm protein, P26h, proposed to be involved in the interaction between spermatozoa and the eggs zona pellucida. In this study we investigated the mechanism of P26h accumulation on hamster spermatozoa during epididymal maturation. Immunocytochemical studies showed an accumulation of P26h on the acrosomal cap of hamster spermatozoa during epididymal transit. To document the anchoring mechanism of P26h, cauda epididymal spermatozoa were exposed to different treatments. High‐salt buffered solutions were unable to remove P26h from the surface of intact spermatozoa. P26h was released in a dose‐dependent manner when live spermatozoa were treated with a solution of phospholipase C specific to phosphatidylinositol. In contrast, the P26h remained associated to the sperm surface following treatment with trypsin. To document the transfer mechanisms of P26h on the maturing spermatozoa, prostasomes were isolated from the epididymal fluid and subjected to immunodetection. Western blots and immunogold studies showed that P26h was associated to epididymal prostasomes. Phospholipase C treatment performed on epididymal prostasomes, indicated that P26h also is anchored to these vesicles via a phosphatidylinositol. These results suggest that epididymal sperm maturation involves a cell to cell transfer of a phosphaditylinositol‐anchored protein and that prostasomes may be implicated in this process. Mol. Reprod. Dev. 52:225–233, 1999.

The lysosomal trafficking of acid sphingomyelinase is mediated by sortilin and mannose 6-phosphate receptor.

Acid sphingomyelinase (ASM), a member of the saposin‐like protein (SAPLIP) family, is a lysosomal hydrolase that converts sphingomyelin to ceramide. Deficiency of ASM causes a variant form of Niemann‐Pick disease. The mechanism of lysosomal targeting of ASM is poorly known. Previous studies suggest that ASM could use in part the mannose 6‐phosphate receptor (M6P‐Rc). Sortilin, a type I transmembrane glycoprotein that belongs to a novel family of receptor proteins, presents structural features of receptors involved in lysosomal targeting. In this study we examined the hypothesis that sortilin may be implicated in the trafficking of ASM to the lysosomes. Using a dominant‐negative sortilin construct lacking the cytoplasmic tail, which is essential to recruit adaptor proteins and clathrin, we demonstrated that sortilin is also involved in the lysosomal targeting of ASM. Confocal microscopy revealed that truncated sortilin partially inhibited the lysosomal trafficking of ASM in COS‐7 cells and abolished the lysosomal targeting of ASM in I‐cells. Pulse‐chase experiments corroborated that sortilin is involved in normal sorting of newly synthesized ASM. Furthermore, over‐expression of truncated sortilin accelerated and enhanced the secretion of ASM from COS‐7 cells and I‐cells. Co‐immunoprecipitation assays confirmed the interaction between sortilin and ASM. In conclusion, ASM uses sortilin as an alternative receptor to be targeted to the lysosomes.