A brain derived peptide preparation reduces the translation dependent loss of a cytoskeletal protein in primary cultured chicken neurons

  • Robert Wronski
  • S. Kronawetter
  • B. Hutter-Paier
  • K. Crailsheim
  • M. Windisch
Conference paper


Neuronal cytoskeletal proteins like the microtubule associated protein 2 (MAP2) are objected to pathological proteolysis in case of Alzheimer’s disease and brain ischemia. The neurotrophic peptidergic drug Cerebrolysin® (EBEWE Arzneimittel, Austria, Europe) is produced by a standardized enzymatic break-down of lipid free porcine brain proteins. Cerebolysin® protected MAP2 in primary neuronal cultures after a brief histotoxic hypoxia and in a rat model of acute brain ischemia. Furthermore the drug was shown to inhibit the proteases μ- and m-calpain dose dependency in several cell free protease activity assays.

The question if the higher MAP2 levels are due to an alleviation of proteolysis, to a higher synthesis rate or both is addressed in the current investigation: Monitoring the MAP2 content of primary neuronal cell cultures over a period of eight days revealed MAP2 to reach a peak level on day six in vitro followed by a degradation phase. In other experiments the protein synthesis of Cerebrolysin® treated and untreated cells was blocked with cycloheximide at that moment when all cells exhibited the same MAP2 content. After the following MAP2 degradation phase — i. e. after eight days in vitro — the MAP2 contents were determined by western blotting. Cerebrolysin® treated cells contained more MAP2 than untreated controls proving that the drug protects MAP2 independently from de novo synthesis, although further work is in progress to investigate if the drug supplementary boosts this effect by increasing MAP2 synthesis.


Primary Neuronal Culture Nootropic Drug Acute Brain Ischemia Primary Neuronal Cell Culture Daily Harvesting 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. Akai F, Hiruma S, Sato T, Iwamotu N, Fujimotu N, Iohu M, Hashimotu S (1992) Neurotrophic factor-like effect of FPF 1070 on septal cholinergic neurons after transections of fimbria-fornix in the rat brain. Histol Histopath 7: 213 – 221Google Scholar
  2. Alexa A, Tompa P, Baki A, Vereb G, Friedrich P (1996) Mutual protection of microtubule associated protein 2 (MAP2) and cyclic AMP-dependent protein kinase II against μ-calpain. J Neurosci Res 44: 438 – 445PubMedCrossRefGoogle Scholar
  3. Avila J, Brandt R, Kosik KS (1997) Preface of: Brain microtubule associated proteins — modifications in disease, Harwood Academic Publ., Amsterdam, p viiGoogle Scholar
  4. Blomgren K, McRae A, Bona E, Saido TC, Karlsson JO, Hagberg H (1995) Degradation of fodrin and MAP 2 after neonatal cerebral hypoxic-ischemia. Brain Res 684: 136 – 142PubMedCrossRefGoogle Scholar
  5. Boado RJ (1998) Brain-derived peptides increase blood-brain barrier GLUT1 glucose transporter gene expression via mRNA stabilization. Neurosci Lett 255: 147 – 150PubMedCrossRefGoogle Scholar
  6. Caceres A, Mautino J, Kosik KS (1992) Suppression of MAP2 in cultured cerebellar macroneurons inhibits minor neurite formation. Neuron 9: 607 – 618PubMedCrossRefGoogle Scholar
  7. Charrière-Bertrand C, Garner C, Tardy M, Nunez J (1991) Expression of various microtubule-associated protein 2 forms in the developing mouse brain and in cultured neurons and astrocytes. J Neurochem 56: 385 – 391PubMedCrossRefGoogle Scholar
  8. Dammerman M, Yen SH, Shafit-Zagardo B (1989) Sequence of a human MAP-2 region sharing epitopes with Alzheimer neurofibrillary tangles. J Neurosci Res 24: 487 – 495PubMedCrossRefGoogle Scholar
  9. Francis-Turner L, Valouskova V (1996) Nerve growth factor and nootropic drug Cerebrolysin but not fibroblast growth factor can reduce spatial memory impairment elicited by fimbria-fornix transection: Short term study. Neurosci Lett 202: 1 – 4CrossRefGoogle Scholar
  10. Friedrich P, Aszodi A (1991) MAP2: a sensitive cross-linker and adjustable spacer in dendritic architecture. FEBS Lett 295: 5 – 9PubMedCrossRefGoogle Scholar
  11. Gschanes A, Valouskova V, Windisch M (1997) Ameliorative influence of a nootropic drug on motor activity of rats after bilateral carotid artery occlusion. J Neural Transm 104: 1319 – 1327PubMedCrossRefGoogle Scholar
  12. Hutter-Paier B, Friiwirth M, Grygar E, Windisch M (1996) Cerebrolysin protects neurons from ischemia-induced loss of microtubule-associated protein 2. J Neural Transm [Suppl 47]: 276Google Scholar
  13. Hutter-Paier B, Steiner E, Windisch M (1998) Cerebrolysin protects isolated cortical neurons from neurodegeneration after brief histotoxic hypoxia. J Neural Transm [Suppl 54]: 343 – 349Google Scholar
  14. Iqbal K, Grundke-Iqbal I, Wisniewski HM (1987) Alterations of the neuronal cytoskeleton in Alzheimer’s disease and related conditions. In: Alterations in the neuronal cytoskeleton in Alzheimer’s disease, Plenum Publ., New York, pp 109 – 136Google Scholar
  15. Iqbal K, Zaidi T, Bancher C, Grundke-Iqbal I (1994) Alzheimer paired helical filaments, restoration of the biological activity by dephosphorylation. FEBS Lett 349: 104 – 108PubMedCrossRefGoogle Scholar
  16. Johnson GVW, Jope RS (1992) The role of microtubule-associated protein 2 (MAP-2) in neuronal growth, plasticity, and degeneration. J Neurosci Res 33: 505 – 512PubMedCrossRefGoogle Scholar
  17. Johnson GVW, Guttman RP (1997) Calpains intact and active? Bioessays 19: 1011 – 1018PubMedCrossRefGoogle Scholar
  18. Kaech S, Matus A (1997) Possible roles for MAP2 in neuronal pathology. In: Brain microtubule associated proteins — modifications in disease, Harwood Academic Publishers, Amsterdam, p 33Google Scholar
  19. Khatoon S, Grundke-Iqbal I, Iqbal K (1992) Brain levels of microtubule associated protein x are elevated in Alzheimer’s disease: A radioimmuno-slot-blot assay for nanograms of protein. J Neurochem 59: 750 – 753PubMedCrossRefGoogle Scholar
  20. Kindler S, Schulz B, Goedert M, Garner CC (1990) Molecular structure of microtubule- associated protein 2b and 2c from rat brain. J Biol Chem 265: 19679 – 19684PubMedGoogle Scholar
  21. Koppi ST, Barolin GS (1996) Hämodilutionstherapie mit nervenzellstoffwechsel-aktiver Therapie beim ischämischen Insult — ermutigende Result ate einer Vergleichsstudie. Wien Med Wochenschr 146 /3: 1 – 8Google Scholar
  22. Kosik KS, Conlogue L (1993) Microtubule associated protein function: Lessons from expression in spodoptera frugiperdacells. Cell Motil Cytoskeleton 28: 195 – 198CrossRefGoogle Scholar
  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680 – 685PubMedCrossRefGoogle Scholar
  24. Leclerc N, Baas PW, Garner CC, Kosik KS (1996) Juvenile and mature MAP2 isoforms induce distinct patterns of process outgrowth. Mol Biol Cell 7: 443 – 455PubMedGoogle Scholar
  25. Lee G, Rook SL (1992) Expression of tau protein in non-neuronal cells: microtubule binding and stabilization. J Cell Sci 102: 227 – 237PubMedGoogle Scholar
  26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265 – 275PubMedGoogle Scholar
  27. Mandelkow E, Song YH, Schweers O, Marx A, Mandelkow EM (1995) On the structure of microtubules, tau, and paired helical filaments. Neurobiol Aging 16: 347 – 354PubMedCrossRefGoogle Scholar
  28. Matesic DF, Lin RCS (1994) Microtubule-associated protein 2 as an early indicator of ischemia-induced neurodegeneration in the gerbil forebrain. J Neurochem 63: 1012– 1020Google Scholar
  29. Metcalfe JC, Smith GA (1991) NMR measurement of cytoplasmic free calcium concentration by fluorine labelled indicators in: Cellular calcium — a practical approach, Oxford University Press, Oxford, p 124Google Scholar
  30. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxic assays. J Immunol Methods 65: 55 – 63PubMedCrossRefGoogle Scholar
  31. Okabe S, Hirokawa N (1989) Rapid turnover of microtubule-associated protein MAP2 in the axon revealed by microinjection of biotinylated MAP2 into cultured neurons. PNAS 86: 4127 – 4131PubMedCrossRefGoogle Scholar
  32. Pontremoli S, Viotti PL, Michetti M, Sparatore P, Salamino F, Melloni E (1990) Identification of an endogenous activator of calpain in rat skeletal muscle. Biochem Biophys Res Comm 171: 569 – 574PubMedCrossRefGoogle Scholar
  33. Rüther E, Ritter R, Apecechea M, Freytag S, Windisch M (1994) Efficacy of the peptidergic nootropic drug cerebrolysin in patients with senile dementia of the Alzheimer type (SDAT). Pharmacopsychiatry 27: 32 – 40PubMedCrossRefGoogle Scholar
  34. Ruben GC, Iqbal K, Grundke-Iqbal I, Wisniewski HM, Ciardelli TL, Johnson JE Jr. (1991) The microtubule-associated protein tau forms a triple-stranded left hand polymer. J Biol Chem 266: 22019 – 22027PubMedGoogle Scholar
  35. Saido TC, Sorimachi H, Suzuki K (1994) Calpain: new perspectives in molecular diversity and physiological-pathological involvement. FASEB J 8: 814 – 822PubMedGoogle Scholar
  36. Salamino F, De Tullio R, Mengotti P, Viotti PL, Melloni E, Pontremoli S (1993) Site directed activation of calpain is promoted by a membrane associated natural activator protein. Biochem J 290: 191 – 197PubMedGoogle Scholar
  37. Satou T, Imano M, Akai F, Hashimoto S, Itoh T, Fujimoto M (1993) Morphological observation of effects of Cerebrolysin on cultured neural cells. Adv Biosci 87: 195– 196Google Scholar
  38. Satou T, Itoh T, Fujimoto M, Hashimoto S (1994) Neurotrophic-like effects of FPF-1070 on cultured neurons from chick embryonic dorsal root ganglia. Jpn Pharmacol Ther 22 /4: 205 – 212Google Scholar
  39. Schwab M, Antonow-Schlorke I, Zwiener U, Bauer R (1998) Brain derived peptides reduce the size of cerebral infarction and loss of MAP2 immunoreactivity after focal ischemia in rats. J Neural Transm [Suppl 54]: 299 – 311Google Scholar
  40. Sharma N, Kress Y, Shaft-Zagardo B (1994) Antisense MAP2-oligonucleotides induce changes in microtubule assembly and neuritic elongation in pre-existing neurites of rat cortical neurons. Cell Motil Cytoskeleton 27: 234 – 247PubMedCrossRefGoogle Scholar
  41. Shea TB (1997) Restriction of μM-Calcium-requiring calpain activation to the plasma membrane in human neuroblastoma cells: Evidence for regionalized influence of a calpain activator protein. J Neurosci Res 48: 543 – 550PubMedCrossRefGoogle Scholar
  42. Siman R, Noszek JC (1988) Excitatory amino acids activate calpain I and induce structural protein breakdown in vivo. Neuron 1: 279 – 287PubMedCrossRefGoogle Scholar
  43. Tompa P, Schád E, Baki A, Alexa A, Batke J (1995) An ultrasensitive continuous fluorimetric assay for calpain activity. Anal Biochem 228: 287 – 293PubMedCrossRefGoogle Scholar
  44. Trojanowski JQ, Schmidt ML, Shin RW, Bramblett GT, Goedert M, Lee VMY (1993) PHFτ (A68): From pathological marker to potential mediator of neuronal dysfunction and degeneration in Alzheimer’s disease. Clin Neurosci 1: 184 – 191Google Scholar
  45. Ulloa L, Dombrádi V, Díaz-Nido J, Szücs K, Gergely P, Friedrich P, Avila J (1993) Dephosphorylation of distinct sites on microtubule-associated protein MAP1B by protein phosphatases 1, 2A and 2B. FEBS Lett 330: 85 – 89Google Scholar
  46. Valouskova V, Francis-Turner L (1998) Can Cerebrolysin influence chronic deterioration of spatial learning and memory? J Neural Transm [Suppl 53]: 343 – 349Google Scholar
  47. Wiche G, Oberkanins C, Himmler A (1991) Molecular structure and function of microtubule-associated proteins. Int Rev Cytol 124: 217 – 273PubMedCrossRefGoogle Scholar
  48. Windisch M, Frtihwirth M, Grygar E, Hutter-Paier B (1997) Cerebrolysin normalizes MAP2 homeostasis after glutamate induced neuronal cell death. J Neurol Sci 150: 200 – 201CrossRefGoogle Scholar
  49. Windisch M, Gschanes A, Hutter-Paier B (1998) Neurotrophic activities and therapeutic experience with a brain derived peptide preparation. J Neural Transm [Suppl 54]: 289 – 298Google Scholar
  50. Wronski R, Tompa P, Hutter-Paier B, Crailsheim K, Friedrich P, Windisch (2000) Inhibitory effect of a brain derived peptide preparation on the Ca++-dependent protease, calpain. J Neural Transm 107 (2): 145 – 157PubMedCrossRefGoogle Scholar
  51. Zhang W, Lane RD, Mellgren RL (1996) The major calpain isozymes are long lived proteins. J Biol Chem 271: 18825 – 18830PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag/Wien 2000

Authors and Affiliations

  • Robert Wronski
    • 1
    • 2
    • 3
  • S. Kronawetter
    • 1
  • B. Hutter-Paier
    • 1
  • K. Crailsheim
    • 2
  • M. Windisch
    • 1
  1. 1.JSW Research ForschungslaborGrazAustria
  2. 2.Institute of ZoologyKarl-Franzens-UniversityGrazAustria
  3. 3.JSW Research Forschungslabor Ges. m. b. H.GrazAustria

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