Wilhelm Stoffel

Glutamate transporter EAAC‐1‐deficient mice develop dicarboxylic aminoaciduria and behavioral abnormalities but no neurodegeneration

Four L‐glutamate neurotransmitter transporters, the three Na+‐dependent GLAST‐1, GLT‐1 and EAAC‐1, and the Cl−‐dependent EAAT‐4, form a new family of structurally related integral plasma membrane proteins with different distribution in the central nervous system. They may have pivotal functions in the regulation of synaptic L‐glutamate concentration during neurotransmission and are believed to prevent glutamate neurotoxicity. To investigate the specific physiological and pathophysiological role of the neuronal EAAC‐1, which is also expressed in kidney and small intestine, we have generated two independent mouse lines lacking EAAC‐1. eaac‐1−/− mice develop dicarboxylic aminoaciduria. No neurodegeneration has been observed during a period of >12 months, but homozygous mutants display a significantly reduced spontaneous locomotor activity.

APPLICATION OF SPECIFIC EXTRACORPOREAL REMOVAL OF LOW DENSITY LIPOPROTEIN IN FAMILIAL HYPERCHOLESTEROLAEMIA

A highly selective method for the removal of apolipoprotein-B-containing serum lipoproteins (low density lipoproteins [LDL] and very-low-density lipoproteins [VLDL]) from hypercholesterolaemic patients by immunoadsorption in an extracorporeal system consists of separation of plasma from the blood cells by a blood-separation centrifuge, followed by the selective adsorption of LDL from the plasma effluent, from which high-density lipoproteins (HDL) and other plasma proteins are not removed, is returned to the patient with the blood cells. Three patients with familial hypercholesterolaemia, one homozygous and two heterozygous, were treated repeatedly during a period of 9 months. No undesirable side-effects or changes in clinical, chemical, haematological, or immunological parameters have yet been observed. The new procedure has several advantages over treatments currently used; it is non-invasive, more specific, and less costly and lowers LDL to a greater degree.

Acid sphingomyelinase-deficient mice mimic the neurovisceral form of human lysosomal storage disease (Niemann-Pick disease)

We have generated an acid sphingomyelinase (aSMase)-deficient mouse line by gene targeting. This novel strain of mutant mouse mimics the lethal, neurovisceral form of the human sphingomyelin storage disease, known as Niemann-Pick disease. Homozygous mice accumulate sphingomyelin extensively in the reticuloendothelial system of liver, spleen, bone marrow, and lung, and in the brain. Most strikingly, the ganglionic cell layer of Purkinje cells of the cerebellum degenerates completely, leading to severe impairment of neuromotor coordination. The Niemann-Pick mouse might facilitate studies on the function of aSMase in the generation of ceramide as proposed second messenger in the intracellular signaling pathways and across the plasma membrane. Furthermore, it provides a suitable model for the development of strategies for somatic gene therapy.

Inhibition of the High‐Affinity Brain Glutamate Transporter GLAST‐1 via Direct Phosphorylation

Abstract: Neurotransmission at excitatory glutamatergic synapses is terminated by the reuptake of the neurotransmitter by high‐affinity transporters, which keep the extracellular glutamate concentration below excitotoxic levels. The amino acid sequence of the recently isolated and cloned brain‐specific glutamate/aspartate transporter (GLAST‐1) of the rat reveals three consensus sequences of putative phosphorylation sites for protein kinase C (PKC). The PKC activator phorbol 12‐myristate 13‐acetate (PMA) decreased glutamate transport activity in Xenopus oocytes and human embryonic kidney cells (HEK293) expressing the cloned GLAST‐1 cDNA, within 20 min, to 25% of the initial transport activity. This down‐regulation was blocked by the PKC inhibitor staurosporine. GLAST‐1 transport activity remains unimpaired by phorbol 12‐monomyristate. Removal of all putative PKC sites of wild‐type GLAST‐1 by site‐directed mutagenesis did not abolish inhibition of glutamate transport. [32P]Phosphate‐labeled wild‐type and mutant transport proteins devoid of all predicted PKC sites were detected by immunoprecipitation after stimulation with PMA. Immunoprecipitation of [35S]methionine‐labeled transporter molecules indicates a similar stability of phosphorylated and nonphosphorylated GLAST‐1 protein. Immunofluorescence staining did not differentiate surface staining of HEK293 cells expressing GLAST‐1 with and without PMA treatment. These data suggest that the neurotransmitter transporter activity of GLAST‐1 is inhibited by phosphorylation at a non‐PKC consensus site.

Δ6-Desaturase (FADS2) deficiency unveils the role of ω3- and ω6-polyunsaturated fatty acids

Mammalian cell viability is dependent on the supply of the essential fatty acids (EFAs) linoleic and α‐linolenic acid. EFAs are converted into ω3‐ and ω6‐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. Here, we show that deletion of Δ6‐fatty acid desaturase (FADS2) gene expression in the mouse abolishes the initial step in the enzymatic cascade of PUFA synthesis. The lack of PUFAs and eicosanoids does not impair the normal viability and lifespan of male and female fads2−/− mice, but causes sterility. We further provide the molecular evidence for a pivotal role of PUFA‐substituted membrane phospholipids in Sertoli cell polarity and blood–testis barrier, and the gap junction network between granulosa cells of ovarian follicles. The fads2−/− mouse is an auxotrophic mutant. It is anticipated that FADS2 will become a major focus in membrane, haemostasis, inflammation and atherosclerosis research.

Myelin glycolipids and their functions

During myelination, oligodendrocytes in the CNS and Schwann cells in the PNS synthesise myelin-specific proteins and lipids for the assembly of the axon myelin sheath. A dominant class of lipids in the myelin bilayer are the glycolipids, which include galactocerebroside (GalC), galactosulfatide (sGalC) and galactodiglyceride (GalDG). A promising approach for unravelling the roles played by various lipids in the myelin membrane involves knocking out the genes encoding important enzymes in lipid biosynthesis. The recent ablation of the ceramide galactosyltransferase ( cgt) gene in mice is the first example. The cgt gene encodes a key enzyme in glycolipid biosynthesis. Its absence causes glycolipid deficiency in the lipid bilayer, breakdown of axon insulation and loss of saltatory conduction. Additional knock-out studies should provide important insights into the various functions of glycolipids in myelinogenesis and myelin structure.

Myelin-deficient rat: a point mutation in exon III (A----C, Thr75----Pro) of the myelin proteolipid protein causes dysmyelination and oligodendrocyte death.

The expression of the proteolipid protein (PLP) gene of the myelin deficient (md) and normal rat was studied during the myelination period. The sizes of the PLP transcripts (1.6 and 3.2 kb) in the md and normal rat were identical although the md PLP messenger RNA level was extremely reduced as shown by in situ hybridization and Northern blot hybridization analysis. The structure of the md proteolipid protein gene was analyzed on the cDNA and genomic level. The molecular basis of the myelin deficiency phenotype has been elucidated: a point mutation in exon III (A‐‐‐‐C transversion) verified by cDNA and genomic DNA sequencing causes a mutation of Thr75 to Pro and creates an additional AvaII restriction site in exon III of the md rat. The threonine to proline mutation located within the second transmembranal alpha‐helix might induce a conformational change and thereby prohibit the integration of PLP into the membrane with the clinical manifestation of dysmyelination leading to premature death within 3‐6 weeks.

[50] Chemical synthesis of choline-labeled lecithins and sphingomyelins

Publisher Summary Synthetic or naturally occurring phosphatidylcholines or sphingomyelins are demethylated to the corresponding phosphatidyl-N, N moiety. The reaction scheme given in this chapter summarizes the two steps. The procedure allows the convenient chemical preparation of choline labeled lecithins and sphingomyelins of high specific activity regardless of the degree of unsaturation. The procedure is simple, rapid, and inexpensive as compared to the biosynthetic preparation of choline-labeled lecithins and sphingomyelins. The latter can hardly be obtained in high specific activity by biosynthetic procedures.

GALACTOSPHINGOLIPIDS AND AXONO-GLIAL INTERACTION IN MYELIN OF THE CENTRAL NERVOUS SYSTEM

Abstract The myelin of central and peripheral nervous system of UDP-galactose-ceramide galactosyltransferase deficient mice (cgt-/-) is completely depleted of its major lipid constituents, galactocerebrosides and sulfatides. The deficiency of these glycolipids affects the biophysical properties of the myelin sheath and causes the loss of the rapid saltatory conduction velocity of myelinated axons. With the onset of myelination, null mutant cgt-/- mice develop fatal neurological defects. CNS and PNS analysis of cgt-/- mice revealed (1) hypomyelination of axons of the spinal cord and optic nerves, but no apoptosis of oligodendrocytes, (2) redundant myelin in younger mice leading to vacuolated nerve fibers in cgt-/- mice, (3) the occurrence of multiple myelinated CNS axons, and (4) severely distorted lateral loops in CNS paranodes. The loss of saltatory conduction is not associated with a randomization of voltage-gated sodium channels in the axolemma of PNS fibers. We conclude that cerebrosides (GalC) and sulfatides (sGalC) play a major role in CNS axono-glial interaction. A close axono-glial contact is not a prerequisite for the spiraling and compaction process of myelin. Axonal sodium channels remain clustered at the nodes of Ranvier independent of the change in the physical properties of myelin membrane devoid of galactosphingolipids. Increased intracellular concentrations of free ceramides do not trigger apoptosis of oligodendrocytes.

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

Abstract 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 ω-hydroxylated very long-chain fatty acids (VLCFA) and ceramides substituted with ω-hydroxylated VLCFA covalently linked to corneocyte surface proteins leads to the disruption of the epidermal lipid barrier in scd1 -/- mutants. Artificial occlusion of the skin by topical lipid application largely reconstituted the epidermal barrier and also reversed dysregulation of thermogenesis and cold resistance, as well as the metabolic disturbances. Interestingly, SCD1 deficiency abolished expression of the key transcription factor Lef1, which is essential for interfollicular epidermis, sebaceous glands, and hair follicle development. Finally, the occurrence of SCD1 and a newly described hSCD5 (ACOD4) gene in humans suggests that the scd1 -/- mouse mutant might be a valuable animal model for the study of human skin diseases associated with epidermal barrier defects.