Outi Lahti

Publications (3)11.36 Total impact

• Source

  Available from: Marja Nissinen

  Article: Myelination in mouse dorsal root ganglion/Schwann cell cocultures.

  Satu Päiväläinen, Marja Nissinen, Henrika Honkanen, Outi Lahti, Salla M Kangas, Juha Peltonen, Sirkku Peltonen, Anthony M Heape
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  ABSTRACT: The established protocols for in vitro studies of peripheral nerve myelination with rat embryonic dorsal root ganglia (DRG) and postnatal Schwann cell cocultures do not work with mouse cells. Consequently, the full potential of this model, which would allow to perform cell type-specific, mixed genotype cocultures without cross-breeding the animals, cannot be exploited. We determined the conditions required to promote full myelination in cocultures of pre-purified mouse embryonic DRG and neonatal Schwann cells, and present a method which consistently yields 50-200 mature myelin sheaths/culture. Causes for the failure of the existing protocols to yield satisfactory results with mouse cells fell into three categories: the lack of adherent support provided by the substratum, growth factor and hormone deficiencies, and the high serum content of the media. For optimal results, mouse cocultures require a 3-dimensional substratum, a myelination-promoting culture medium containing pituitary extract, N2 supplement and forskolin, and a low serum concentration.
  Molecular and Cellular Neuroscience 04/2008; 37(3):568-78. · 3.66 Impact Factor
• Article: Isolation, purification and expansion of myelination-competent, neonatal mouse Schwann cells.

  Henrika Honkanen, Outi Lahti, Marja Nissinen, Riina M Myllylä, Salla Kangas, Satu Päiväläinen, Maria H Alanne, Sirkku Peltonen, Juha Peltonen, Anthony M Heape
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  ABSTRACT: Most studies of peripheral nerve myelination using culture models are performed with dorsal root ganglion neurons and Schwann cells pre-purified from the rat. The potential of this model is severely compromised by the lack of rat myelin mutants and the published protocols work poorly with mouse cells, for which numerous myelin mutants are available. This is partly due to difficulties in obtaining sufficient quantities of myelination-competent mouse Schwann cells. Here, we describe the isolation, purification and expansion of wild-type, myelination-competent Schwann cells from the sciatic nerves of 4-day-old mouse pups. The method consistently yields 1.9-3.3 x 10(6) of approximately 95% pure Schwann cells from the sciatic nerves of 12-15 4-day-old mouse pups, within 14-20 days. The Schwann cell proliferation rate ranges from 2.7- to 4.30-fold growth/week. Proliferation ceases within 4 weeks, when the cells become quiescent. Growth is reinduced by the presence of neurons; neuregulin is not sufficient for this effect. The Schwann cells isolated by this protocol are able to form compact myelin in culture, as judged by the segregated expression patterns of early (myelin-associated glycoprotein) and late (myelin basic protein) myelination markers in a three-dimensional neuron/Schwann cell coculture model. The Schwann cell batch yields are sufficient to perform 100-150 individual myelinating coculture assays. Employing mixed phenotype/genotype mouse neuron/Schwann cell cocultures, it will be possible to analyse the cell specificity of a mutation, and the cumulative effects of different mutations, without having to cross-breed the animals.
  European Journal of Neuroscience 09/2007; 26(4):953-64. · 3.63 Impact Factor
• Article: Degraded myelin-associated glycoprotein (dMAG) formation from pure human brain myelin-associated glycoprotein (MAG) is not mediated by calpain or cathepsin L-like activities.

  Satu Päiväläinen, Marko Suokas, Outi Lahti, Anthony M Heape
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  ABSTRACT: The myelin-associated glycoprotein (MAG) is a transmembrane cell adhesion molecule participating in myelin formation and maintenance. Calcium-activated/-dependent proteolysis of myelin-associated glycoprotein by calpain and cathepsin L-like activities has already been detected in purified myelin fractions, producing a soluble fragment, called degraded (d)MAG, characterized by the loss of the transmembrane and cytoplasmic domains. Here, we demonstrate and analyze dMAG formation from pure human brain myelin-associated glycoprotein. The activity never exhibited the high rate previously reported in human myelin fractions. Degradation is time-, temperature-, buffer- and structure-dependent, is inhibited at 4 degrees C and by denaturation of the sample, and is mediated by a trans-acting factor. There is no strict pH dependency of the proteolysis. Degradation was inhibited by excess aprotinin, but not by 1-10 micro g/mL aprotinin and was not eliminated by the use of an aprotinin-sepharose matrix during the purification. dMAG formation was not enhanced by calcium, nor inhibited by a wide variety of protease inhibitors, including specific calpain and cathepsin L inhibitors. Therefore, while cysteine proteases may be present in human myelin membrane fractions, they are not involved in dMAG formation from highly purified human brain myelin-associated glycoprotein preparations.
  Journal of Neurochemistry 03/2003; 84(3):533-45. · 4.06 Impact Factor