Sialin expression in the CNS implicates extralysosomal function in neurons

Aula, Nina; Kopra, Outi; Jalanko, Anu; Peltonen, Leena

doi:10.1016/j.nbd.2003.11.017

Cited by 34 publications

(24 citation statements)

References 32 publications

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“…Although sialin has been described as a lysosomal protein [4,9], our attempts to find another protein that consistently colocalizes with sialin have been unsuccessful. To characterize potential changes in subcellular localization associated with the mutations we therefore compared the expression patterns of the wild-type protein and the mutant proteins differentially tagged with the commonly used epitopes HA and myc.…”

Section: Resultsmentioning

confidence: 95%

G328E and G409E sialin missense mutations similarly impair transport activity, but differentially affect trafficking

Myall

Wreden

Wlizla

et al. 2007

Molecular Genetics and Metabolism

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Two disease-associated missense mutations in the sialin gene (G328E and G409E) have recently been identified in patients with lysosomal free sialic acid storage disease. We have assessed the effect of these mutations and find complete loss of measurable transport activity with both and impaired trafficking of the G409E protein. These results suggest that the two residues are important for proper function of sialin and confirm the association of loss of transport with disease causative mutations.

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Section: Resultsmentioning

confidence: 95%

G328E and G409E sialin missense mutations similarly impair transport activity, but differentially affect trafficking

Myall

Wreden

Wlizla

et al. 2007

Molecular Genetics and Metabolism

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“…This may explain the previous immunogold findings of glutamate and aspartate in the same nerve terminals [9,27]. There are so far no detailed electron microscopic localisation studies of VEAT in the brain, only previous light microscopic results showing that the sialin protein (see Introduction) is present at quite high concentrations throughout the forebrain, including in the hippocampus [46,47]. Whether vesicles containing VEAT is located in the same terminals as those containing VGLUT is one important question that must await further studies.…”

Section: Discussionmentioning

confidence: 95%

Vesicular Release of L- and D-Aspartate from Hippocampal Nerve Terminals: Immunogold Evidence~!2008-09-01~!2008-10-15~!2008-12-05~!

Holten¹,

Morland²,

Nordengen³

et al. 2008

TONEURJ

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Abstract:Glutamate is established as the most important excitatory transmitter in the brain. The transmitter status of aspartate is debated. There is evidence that aspartate is released from nerve terminals by exocytosis. However, release through excitatory amino acid transporters (EAATs) could be an alternative mechanism. We further investigated this by use of light and quantitative electron microscopic immunocytochemistry. The nerve terminal localisation of aspartate was compared to that of glutamate using antibodies specifically recognising the amino acids. Rat hippocampal slices were incubated under normal (3 mM) and depolarising (55 mM) concentrations of K + with and without the excitatory amino acid transporter inhibitor threo-beta-benzyloxyaspartate (TBOA). If aspartate is released either through reversal of the EAATs or through exchange with synaptically released glutamate, we would expect that TBOA would block the depolarisation induced release of aspartate. We found, however, that there was a substantial depletion of aspartate, as well as of glutamate, from hippocampal nerve terminals during K + induced depolarisation in the presence of TBOA. The possibility that aspartate is released through exocytosis from synaptic vesicles was further investigated by the use of a D-aspartate uptake assay, including exposure of the slices to exogenous D-aspartate and the use of D-aspartate immunogold cytochemistry to localise D-aspartate in the fixed tissue. We found that D-aspartate taken up into the terminals was concentrated in synaptic vesicles as opposed to in the cytoplasmic matrix. This is in line with the presence in synaptic vesicles of the recently identified vesicular transporter for aspartate.Keywords: Synaptic vesicles, immunocytochemistry, amino acids, electron microscopy, transport.The release of glutamate at synapses in the brain is well studied. Glutamate is released upon depolarisation of the presynaptic nerve terminal, leading to opening of voltage gated Ca 2+ channels and Ca 2+-dependent fusion of glutamate filled synaptic vesicles with the plasma membrane. The mechanism of release of L-aspartate at central synapses remains less defined. L-aspartate has been shown to be released in a Ca 2+ and clostridium toxin sensitive manner from various in vitro brain preparations (e.g. [1][2][3][4][5][6][7]). Strongly in favour of an exocytotic release mechanism are our previous immunocytochemical results showing that K + induced depolarisation elicited depletion of L-aspartate from nerve endings, which could be inhibited by low extracellular Ca 2+ -concentrations and tetanus toxin [8][9][10]. In addition, Daspartate has been shown to be subject to exocytotic release both from cultured neurons [11] There is ample evidence that excitatory nerve endings in the brain contain functional plasma membrane excitatory amino acid transporters (EAATs) [16][17][18][19][20]. The previous aspartate release data do not clearly distinguish between Laspartate release via exocytosis and via these EAATs, which could release as...

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“…Cell Culture-Primary astrocyte cultures from E16.5 embryos were prepared and cultured essentially as described (16). Briefly, astrocytes prepared from the striata of the embryos were cultured in Dulbecco's modified Eagle's medium supplemented with 100 IU/ml penicillin, 100 g/ml streptomycin, 0.5 mM L-glutamine, 25 mM Hepes, pH 7.4, 1ϫ G-5 supplement, and 1% characterized fetal bovine serum on poly-D-lysine (50 g/ml)-coated culture dishes (Nunc).…”

Section: Methodsmentioning

confidence: 99%

Secretion of Sterols and the NPC2 Protein from Primary Astrocytes

Mutka

Lusa

Linder

et al. 2004

Journal of Biological Chemistry

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Astrocytes secrete cholesterol in lipoprotein particles.Here we show that primary murine embryonic astrocytes secrete endogenously synthesized cholesterol but also the cholesterol precursors desmosterol and lathosterol. In astrocyte membranes, desmosterol and cholesterol were the predominant sterols. Astrocytes derived from Niemann-Pick type C lipidosis (NPC1 ؊/؊ ) mice displayed late endosomal cholesterol deposits, but the secretion of biosynthetic sterols from the cells was not inhibited. Both wild-type and NPC1 ؊/؊ astrocytes secreted the NPC2 protein. Size-exclusion chromatography combined with electron microscopy showed that the majority of sterols were secreted separately from NPC2 in heterogeneous spherical particles with an average diameter of 20 nm. These data suggest that NPC2 and the majority of sterols secreted from astrocytes are not released together and that the secretion of neither sterols nor NPC2 requires NPC1 function. In addition, the findings reveal a complexity of sterol species in astrocytes and bring up the possibility that some of the effects assigned to astrocyte cholesterol may be attributed to its penultimate precursors. The brain is the most cholesterol-rich organ in the body, containing roughly 25% of the unesterified cholesterol present in the whole individual. The input of cholesterol into the brain comes entirely, or almost entirely, from in situ synthesis because blood lipoproteins do not cross the blood-brain-barrier (1). Glial cells are thought to be responsible for a large part of this biosynthetic activity. Most of the sterol in the brain is acquired during myelination in the early stages of development and is produced by oligodendrocytes (2).Astrocytes, the most abundant glial cells, are intimately associated with neuronal synapses and secrete cholesterol in lipoprotein particles. Astrocytes have been proposed to provide cholesterol for synapse formation (3) as well as to participate in the recycling of cholesterol after injury (4). Nascent lipoproteins isolated from neonatal mouse astrocytes are a heterogeneous population of particles in the size range of plasma high density lipoproteins and appear to be composed of two separate classes that contain either apolipoprotein E or J (5).Niemann-Pick type C disease (NPC) 1 is an inherited, fatal neurodegenerative disorder in which large amounts of cholesterol and sphingolipids accumulate intracellularly within late endocytic organelles. The disease is caused by mutations in either the NPC1 or NPC2 gene (6). The NPC1 protein is a late endosomal membrane protein harboring a sterol-sensing domain, whereas NPC2 is a secretory protein that has cholesterol binding properties and uses the mannose 6-phosphate marker for late endosomal targeting (7). The exact functions of the proteins remain unknown, but genetic evidence suggests that they function in concert to facilitate lysosomal lipid egress (8).In the NPC1 Ϫ/Ϫ mouse astrocytes are considered to contribute to neurodegeneration (9, 10). Interestingly, NPC1 Ϫ/Ϫ astrocytes were shown t...

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Sialin expression in the CNS implicates extralysosomal function in neurons

Cited by 34 publications

References 32 publications

G328E and G409E sialin missense mutations similarly impair transport activity, but differentially affect trafficking

G328E and G409E sialin missense mutations similarly impair transport activity, but differentially affect trafficking

Vesicular Release of L- and D-Aspartate from Hippocampal Nerve Terminals: Immunogold Evidence~!2008-09-01~!2008-10-15~!2008-12-05~!

Secretion of Sterols and the NPC2 Protein from Primary Astrocytes

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