Expression of heterologous proteins in cultured rat hippocampal neurons using the semliki forest virus vector

Olkkonen, Vesa M.; Liljeström, Peter; Garoff, Henrik; Simons, Kai; Dotti, Carlos G.

doi:10.1002/jnr.490350412

Cited by 78 publications

(66 citation statements)

References 31 publications

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“…SP, signal peptide, ␣, ␤, ␥, ␦; APP secretases. scription mixture of both pSFV1/APP and pSFV1/C99 or pSFV1/C111 and pSFVhelper was co-transfected into baby hamster kidney cells using electroporation (77). The baby hamster cells were cultured in Glasgow's modified Eagle's medium supplemented with 5% fetal bovine serum, 2 mM glutamate, and 10% tryptose phosphate broth.…”

Section: Methodsmentioning

confidence: 99%

Independent Inhibition of Alzheimer Disease β- and γ-Secretase Cleavage by Lowered Cholesterol Levels

Grimm

Tomic

et al. 2008

Journal of Biological Chemistry

120

107

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The major molecular risk factor for Alzheimer disease so far identified is the amyloidogenic peptide A␤ 42 . In addition, growing evidence suggests a role of cholesterol in Alzheimer disease pathology and A␤ generation. However, the cellular mechanism of lipid-dependent A␤ production remains unclear. Here we describe that the two enzymatic activities responsible for A␤ production, ␤-secretase and ␥-secretase, are inhibited in parallel by cholesterol reduction. Importantly, our data indicate that cholesterol depletion within the cellular context inhibits both secretases additively and independently from each other. This is unexpected because the ␤-secretase ␤-site amyloid precursor protein cleaving enzyme and the presenilin-containing ␥-secretase complex are structurally different from each other, and these enzymes are apparently located in different subcellular compartments. The parallel and additive inhibition has obvious consequences for therapeutic research and may indicate an intrinsic cross-talk between Alzheimer disease-related amyloid precursor protein processing, amyloid precursor protein function, and lipid biology.A␤ peptides are the main proteinaceous component of Alzheimer disease amyloid plaques. A␤ is derived from posttranslational cleavage of the amyloid precursor protein (APP). Cleavage of APP by BACE I (1) at the N terminus of the A␤ sequence generates a C-terminal fragment (C99) that includes the entire A␤ sequence. In mouse cortical neurons BACE I is essential for APP ␤-cleavage (2). A second proteolytic activity termed ␥-secretase cleaves APP at the C-terminal end of the A␤ sequence, releasing A␤ 40 and A␤ 42 during normal cellular metabolism of APP (3, 4). A fraction of APP is processed by the ␣-secretase pathway in which APP is cleaved within the A␤ region thus precluding A␤ formation. However, neurons predominately use the ␤-secretory pathway at the expense of the ␣-secretory pathway to process APP (5). Moreover, neurons produce significant amounts of intracellular A␤ in vivo and in vitro (6 -8). A specific feature of ␥-secretase is that it is capable of cleaving APP only after a major part of the APP luminal domain is removed. Under normal circumstances it is therefore not possible to assay ␥-secretase activity directly.Analyses of APP-FAD mutations (9) as well as of PS-FAD mutations (10) have corroborated the assumption that a small increase in A␤ 42 levels causes AD (11). The subcellular activities of both ␤-and ␥-secretase have been extensively studied. Processing of APP to A␤ differs for different intracellular compartments (12) and depends among others on the interaction of membrane composition and the APP transmembrane domain (13). Variable amounts of ␤-secretase activity were found along the secretory pathway starting in the ER/intermediate compartment, post-Golgi vesicles, TGN, and endosomes (14, 15). In contrast, ␥-secretase activity was found to be prominent in the ER, TGN, and plasma membrane and to produce different A␤ isoforms in different compartments (reviewed by Hartmann (...

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Section: Methodsmentioning

confidence: 99%

Independent Inhibition of Alzheimer Disease β- and γ-Secretase Cleavage by Lowered Cholesterol Levels

Grimm

Tomic

et al. 2008

Journal of Biological Chemistry

120

107

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“…pSFV-LacZ, pSFV-NPC2, and pSFV-helper 1 were linearized, and runoff transcription was performed according to Liljeström and Garoff (21). The NPC2 transcription mix was cotransfected with the helper transcription mix into baby hamster kidney cells using electroporation as described (22). The culture supernatant was collected after 24 h of incubation.…”

Section: Methodsmentioning

confidence: 99%

“…The culture supernatant was collected after 24 h of incubation. Titration of the viral stocks on baby hamster kidney cells was performed as described (22). Recombinant SFV was added on near-confluent dishes of astrocytes, and viral entry was allowed for 60 min at 37°C, after which the incubation was continued in serum-free astrocyte medium at 37°C for 6 or 14 h (latter for size-exclusion chromatography).…”

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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“…Replication-deficient vectors have been shown to infect primary cultures of hippocampal and cortical neurons efficiently. 11 Interestingly, cultures on a feeder layer of glial cells suggest that the expression is highly neuron-specific and very few glial cells show any transgene expression. Similarly, the b-galactosidase expression pattern was highly neuron-specific for both SIN and SFV vectors injected into hippocampal slices cultured in vivo.…”

Section: Neurosciencementioning

confidence: 99%

“…The first step towards gene therapy application was the demonstration of high transduction rates of various Large-scale production Drug screening, structural biology 9,10 Primary neurons Localization, electrophysiology 11 Hippocampal slices Localization, electrophysiology 12,13 In vivo, rat brain Localization, duration 16 Gene therapy Cancer vaccines Tumor protection, tumor regression 20 Tumor cell lines Transduction, cell killing 17,18 Intratumoral injection Tumor regression 23,24 Liposome-encapsulation Tumor-targeted gene delivery, therapy 28 18 Clearly, the infection rate was modest, although a bystander effect was observed for the therapeutic TK (thymidine kinase) after ganciclovir treatment. Another application of alphaviruses for cancer therapy has been vaccination with tumor antigens.…”

Section: Gene Therapymentioning

confidence: 99%