High fat / high cholesterol diet does not provoke atherosclerosis in the ω3-and ω6-polyunsaturated fatty acid synthesis-inactivated Δ6-fatty acid desaturase-deficient mouse.

Objective: An increased ω6/ω3-polyunsaturated fatty acid ratio in the current Western diet is regarded as a critical epigenetic nutritional factor in the pathogenesis of several human lifestyle diseases, metabolic syndrome, cardiovascular disease, the central nervous system and the female and male reproductive systems. The impact of nutrient ω3-and ω6-PUFAs in the pathogenesis of dyslipoproteinemia and atherosclerosis has been a topic of intense efforts for several decades. Cellular homeostasis of the ω3-and ω6- PUFA pool is maintained by the synthesis of ω3-and ω6-PUFAs from essential fatty acids (EFA) (linoleic and α-linolenic acid) and their dietary supply.

Dietary ω3-and ω6-Polyunsaturated fatty acids reconstitute fertility of Juvenile and adult Fads2-Deficient mice.

Objective: Polyunsaturated fatty acids (PUFAs), including essential fatty acids linoleic and α-linolenic acid and derived long chain and very long chain ω3-and ω6-polyunsaturated fatty acids, are vital structures in mammalian membrane systems and signaling molecules, pivotal in brain development, lipid, and energy metabolism and in female and male fertility during human evolution. Numerous nutritional studies suggest imbalance of PUFA metabolism as a critical factor in the pathogenesis of several human lifestyle diseases: dyslipoproteinemia, obesity, cardiovascular and neurodegenerative diseases, and infertility. The lack of unbiased animal models impedes molecular interpretation of the role of synthesized and dietary supplied PUFAs in these conditions.

Neutral Sphingomyelinase 2 (SMPD3) Deficiency in Mice Causes Chondrodysplasia with Unimpaired Skeletal Mineralization.

SMPD3 deficiency in the neutral sphingomyelinase (Smpd3) mouse results in a novel form of juvenile dwarfism, suggesting smpd3 is a polygenetic determinant of body height. SMPD3 controls homeostasis of the sphingomyelin cycle in the Golgi compartment, essential for membrane remodeling, initiating multiform vesicle formation and transport in the Golgi secretory pathway. Using the unbiased Smpd3 genetic model, this study shows that the perturbed Golgi secretory pathway of chondrocytes of the epiphyseal growth zone leads to dysproteostasis, skeletal growth inhibition, malformation, and chondrodysplasia, but showed unimpaired mineralization in primary and secondary enchondral ossification centers.

Novel CB1-ligands maintain homeostasis of the endocannabinoid system in ω3- and ω6-long-chain-PUFA deficiency.

Mammalian ω3- and ω6-PUFAs are synthesized from essential fatty acids (EFAs) or supplied by the diet. PUFAs are constitutive elements of membrane architecture and precursors of lipid signaling molecules. EFAs and long-chain (LC)-PUFAs are precursors in the synthesis of endocannabinoid ligands of G protein-coupled cannabinoid receptor (CB)1 and CB2 in the endocannabinoid system, which critically regulate energy homeostasis as the metabolic signaling system in hypothalamic neuronal circuits and behavioral parameters.

SMPD3 deficiency perturbs neuronal proteostasis and causes progressive cognitive impairment.

Neutral sphingomyelinase smpd3 is most abundantly expressed in neurons of brain. The function of SMPD3 has remained elusive. Here, we report a pathogenetic nexus between absence of SMPD3 in the Golgi compartment (GC) of neurons of the smpd3-/- mouse brain, inhibition of Golgi vesicular protein transport and progressive cognitive impairment.

Hair Growth Cycle Is Arrested in SCD1 Deficiency by Impaired Wnt3a-Palmitoleoylation and Retrieved by the Artificial Lipid Barrier.

Stearoyl-CoA desaturase 1 (SCD1) is the dominant member of the SCD-isozyme family, regarded as a major regulator of lipid and energy metabolism in liver and adipose tissue. SCD1 deficiency impairs the desaturation of de novo-synthesized palmitoyl- and stearoyl-CoA to palmitoleoyl- and oleoyl-CoA. Scd1 mice develop metabolic waste syndrome and skin lesions: epidermal barrier disruption, alopecia, and degeneration of sebaceous glands.

Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition.

Systemic loss of neutral sphingomyelinase (SMPD3) in mice leads to a novel form of systemic, juvenile hypoplasia (dwarfism). SMPD3 deficiency in mainly two growth regulating cell types contributes to the phenotype, in chondrocytes of skeletal growth zones to skeletal malformation and chondrodysplasia, and in hypothalamic neurosecretory neurons to systemic hypothalamus-pituitary-somatotropic hypoplasia. The unbiased smpd3-/- mouse mutant and derived smpd3-/- primary chondrocytes were instrumental in defining the enigmatic role underlying the systemic and cell autonomous role of SMPD3 in the Golgi compartment.

Obesity resistance and deregulation of lipogenesis in Δ6-fatty acid desaturase (FADS2) deficiency.

Δ-6-fatty acid desaturase (FADS2) is the key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs), the essential structural determinants of mammalian membrane lipid-bilayers. We developed the auxotrophic fads2(-/-) mouse mutant to assess the enigmatic role of ω3- and ω6-PUFAs in lipid homeostasis, membrane structure and function. Obesity resistance is another major phenotype of the fads2(-/-) mutant, the molecular basis of which is unknown.

Functional redundancy of mitochondrial enoyl-CoA isomerases in the oxidation of unsaturated fatty acids.

Mitochondrial enoyl-CoA isomerase (ECI1) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild-type (WT) and Eci1-deficient knockout (KO) mice to explore a potential presentation of human ECI1 deficiency.

Isolation and characterization of living primary astroglial cells using the new GLAST-specific monoclonal antibody ACSA-1.

Astrocytes show large morphological and functional heterogeneity and are involved in many aspects of neural function. Progress in defining astrocyte subpopulations has been hampered by the lack of a suitable antibody for their direct detection and isolation. Here, we describe a new monoclonal antibody, ACSA-1, which was generated by immunization of GLAST1 knockout mice.

Altered phenotype of the vestibular organ in GLAST-1 null mice.

Various studies point to a crucial role of the high-affinity sodium-coupled glutamate aspartate transporter GLAST-1 for modulation of excitatory transmission as shown in the retina and the CNS. While 2-4-month-old GLAST-1 null mice did not show any functional vestibular abnormality, we observed profound circling behavior in older (7 months) animals lacking GLAST-1. An unchanged total number of otoferlin-positive vestibular hair cells (VHCs), similar ribbon numbers in VHCs, and an unchanged VGLUT3 expression in type II VHCs were detected in GLAST-1 null compared to wild-type mice.

Versican V2 assembles the extracellular matrix surrounding the nodes of ranvier in the CNS.

The CNS-restricted versican splice-variant V2 is a large chondroitin sulfate proteoglycan incorporated in the extracellular matrix surrounding myelinated fibers and particularly accumulating at nodes of Ranvier. In vitro, it is a potent inhibitor of axonal growth and therefore considered to participate in the reduction of structural plasticity connected to myelination. To study the role of versican V2 during postnatal development, we designed a novel isoform-specific gene inactivation approach circumventing early embryonic lethality of the complete knock-out and preventing compensation by the remaining versican splice variants.

Delta6-desaturase (FADS2) deficiency unveils the role of omega3- and omega6-polyunsaturated fatty acids.

Mammalian cell viability is dependent on the supply of the essential fatty acids (EFAs) linoleic and alpha-linolenic acid. EFAs are converted into omega3- and omega6-polyunsaturated fatty acids (PUFAs), which are essential constituents of membrane phospholipids and precursors of eicosanoids, anandamide and docosanoids. Whether EFAs, PUFAs and eicosanoids are essential for cell viability has remained elusive.

Neutral sphingomyelinase (SMPD3) deficiency causes a novel form of chondrodysplasia and dwarfism that is rescued by Col2A1-driven smpd3 transgene expression.

Neutral sphingomyelinase SMPD3 (nSMase2), a sphingomyelin phosphodiesterase, resides in the Golgi apparatus and is ubiquitously expressed. Gene ablation of smpd3 causes a generalized prolongation of the cell cycle that leads to late embryonic and juvenile hypoplasia because of the SMPD3 deficiency in hypothalamic neurosecretory neurons. We show here that this novel form of combined pituitary hormone deficiency is characterized by the perturbation of the hypothalamus-pituitary growth axis, associated with retarded chondrocyte development and enchondral ossification in the epiphyseal growth plate.

Obesity resistance of the stearoyl-CoA desaturase-deficient (scd1-/-) mouse results from disruption of the epidermal lipid barrier and adaptive thermoregulation.

Targeted deletion of the stearoyl-CoA desaturase 1 gene (scd1) in mouse causes obesity resistance and a severe skin phenotype. Here, we demonstrate that SCD1 deficiency disrupts the epidermal lipid barrier and leads to uncontrolled transepidermal water loss, breakdown of adaptive thermoregulation and cold resistance, as well as a metabolic wasting syndrome. The loss of omega-hydroxylated very long-chain fatty acids (VLCFA) and ceramides substituted with omega-hydroxylated VLCFA covalently linked to corneocyte surface proteins leads to the disruption of the epidermal lipid barrier in scd1-/- mutants.

Neutral sphingomyelinase 2 (smpd3) in the control of postnatal growth and development.

Neutral sphingomyelinases sphingomyelin phosphodiesterase (SMPD)2 and -3 hydrolyze sphingomyelin to phosphocholine and ceramide. smpd2 is expressed ubiquitously, and smpd3 is expressed predominantly in neurons of the CNS. Their activation and the functions of the released ceramides have been associated with signaling pathways in cell growth, differentiation, and apoptosis.

Functional analysis of glutamate transporters in excitatory synaptic transmission of GLAST1 and GLAST1/EAAC1 deficient mice.

The high affinity, Na(+)-dependent, electrogenic glial L-glutamate transporters GLAST1 and GLT1, and two neuronal EAAC1 and EAAT4, regulate the neurotransmitter concentration in excitatory synapses of the central nervous system. We dissected the function of the individual transporters in the monogenic null allelic mouse lines, glast1(-/-) and eaac1(-/-), and the derived double mutant glast(-/-)eaac1(-/-). Unexpectedly, the biochemical analysis and the behavioral phenotypes of these null allelic mouse lines were inconspicuous.

Nerve conduction velocity measurements reveal the functional deficit in ceramide galactosyl transferase-deficient (cgt-/-) mice.

Biochemical and ultrastructural studies of ceramide galactosyltransferase (CGT) in a CGT-deficient mouse line (cgt-/-) were complemented by nerve conduction velocity (NCV) measurements in motor nerves (sciatic nerve in the hind limbs) of wild type (wt) and cgt-/- mice. Stimulation and recording electrodes were adapted to the small size of developing mice during their myelination period. Motor NCVs in wt mice ranged between 16 and 26 m/s but in cgt-/- mice between 6 and 13 m/s, which corresponds to the conductance of unmyelinated peripheral nerves.

Neutral sphingomyelinase 1 deficiency in the mouse causes no lipid storage disease.

Sphingomyelin is a major lipid in the bilayer of subcellular membranes of eukaryotic cells. Different sphingomyelinases catalyze the initial step in the catabolism of sphingomyelin, the hydrolysis to phosphocholine and ceramide. Sphingomyelinases have been postulated to generate ceramide as a lipophilic second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis.

Tolerability and safety of two new preservative-free tear film substitutes.

Purpose: The tolerability, safety, and visual comfort of two new tear film substitutes were studied in a phase I clinical study.

Methods: Two amphipathic lipids (phosphatidylcholine, cholesterol, sphingomyelin, and gangliosides) containing solutions (I and II) of well-defined stoichiometry, free of preservatives, were studied in 20 eyes of 20 healthy volunteers (age range, 26-32 years) in a randomized, double-blind, crossover study. Randomization was achieved by treating one eye of each volunteer four times daily for 7 days with composite solution I.

Disruption of mitochondrial beta -oxidation of unsaturated fatty acids in the 3,2-trans-enoyl-CoA isomerase-deficient mouse.

Cellular energy metabolism is largely sustained by mitochondrial beta-oxidation of saturated and unsaturated fatty acids. To study the role of unsaturated fatty acids in cellular lipid and energy metabolism we generated a null allelic mouse, deficient in 3,2-trans-enoyl-CoA isomerase (ECI) (eci(-/-) mouse). ECI is the link in mitochondrial beta-oxidation of unsaturated and saturated fatty acids and essential for the complete degradation and for maximal energy yield.

Oligodendrocytes expressing exclusively the DM20 isoform of the proteolipid protein gene: myelination and development.

Oligodendroglia and Schwann cells synthesize myelin-specific proteins and lipids for the assembly of the highly organized myelin membrane of the motor-sensory axons in the central (CNS) and peripheral nervous system (PNS), respectively, allowing rapid saltatory conduction. The isoforms of the main myelin proteins, the peripheral myelin basic isoproteins (MBP) and the integral proteolipid proteins, PLP and DM20, arise from alternative splicing. Activation of a cryptic splice site in exon III of plp leads to the deletion of 105 bp encoding the PLP-specific 35 amino acid residues within the cytosolic loop 3 of the four-transmembrane domain (TMD) integral membrane protein.

Rumpshaker-like proteolipid protein (PLP) ratio in a mouse model with unperturbed structural and functional integrity of the myelin sheath and axons in the central nervous system.

The gene plp on the X chromosome encodes the isoforms proteolipid protein (PLP) and DM(20), two dominant integral membrane proteins of central nervous system (CNS) myelin. DM(20) results from the activation of the cryptic splice site in exon III of the PLP gene. We inserted a sense-orientated loxP flanked neomycin-gene into intron III of the plp sequence, using homologous recombination in embryonic stem cells and generated the homozygous neoS mouse line.

Assembly of myelin by association of proteolipid protein with cholesterol- and galactosylceramide-rich membrane domains.

Myelin is a specialized membrane enriched in glycosphingolipids and cholesterol that contains a limited spectrum of proteins. We investigated the assembly of myelin components by oligodendrocytes and analyzed the role of lipid-protein interactions in this process. Proteolipid protein (PLP), the major myelin protein, was recovered from cultured oligodendrocytes from a low-density CHAPS-insoluble membrane fraction (CIMF) enriched in myelin lipids.

Functional analysis of acid and neutral sphingomyelinases in vitro and in vivo.

The molecular cloning and the elucidation of the gene structures of the acid (aSMase) and a neutral sphingomyelinases (nSMase) of mouse and human facilitated the structural and functional analysis of these enzymes responsible for the catabolism of sphingomyelin present ubiquitously in the membrane lipid bilayer of mammalian cells. The protein and enzymic properties of the glycoprotein aSMase and of a non-glycosylated nSMase residing in the membranes of the endoplasmic reticulum have been analysed in the native as well as in the recombinant shingomyelinases. Important insight was gained from gene targeting experiments in which an aSMase deficient mouse line was generated which mimics the neurovisceral form of the human Niemann-Pick disease.