Nonapoptotic cell death caused by the inhibition of RNA polymerase disrupts organelle distribution


      It is controversial whether the mode of cell death induced by CAG repeat diseases is apoptotic. One technical problem that affects this issue is that the very methods used for DNA injection may induce artificial apoptosis. A recent study demonstrated that the functions of RNA polymerase II are disrupted in spinocerebellar ataxia type 1 (SCA 1) pathology, one of the CAG repeat diseases, and that α-amanitin can inhibit the activity of RNA polymerase. To examine the cell death mechanisms involved in CAG repeat diseases, we treated cultured rat neurons with α-amanitin to avoid the artifacts caused by DNA transfection. Mature and immature rat neurons were treated with α-amanitin for 4–6 days and the effects of the treatment on the elongation of neurites, the distribution or morphology of organelles, and the nature of cell death were assessed by immunocytochemistry and quantitative analysis. Neurons exhibited a disruption of neurite elongation and eventually died by day 15 of the treatment. However, apoptosis was not detected. When the neurons survived well, but showed altered neurites, Golgi complexes and lysosomes exhibited changes in their normal intracellular distribution or morphology, but the endoplasmic reticulum and mitochondria did not. The distribution of phosphorylated Trk receptors was also disrupted in the neurites of treated neurons. The signal intensity of the dynein intermediate chain was markedly decreased in the treated neurons. Thus, organelle transport systems, particularly a minus-end-directed microtubule-dependent pathway, would be disrupted by the inhibition of RNA polymerase, and this change is likely to be an early event involved in SCA 1 pathology.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Journal of the Neurological Sciences
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Reddy P.H.
        • Williams M.
        • Charles V.
        • Garret L.
        • Pike-Buchanan L.
        • Whetsell Jr., W.O.
        Behavioral abnormalities and selective neuronal loss in HD transgenic mice expressing mutated full-length HD cDNA.
        Nat Genet. 1998; 20: 198-202
        • Wellington C.L.
        • Ellerby L.M.
        • Hackam A.S.
        • Margolis R.L.
        • Trifiro M.A.
        • Singaraja R.
        Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract.
        J Biol Chem. 1998; 273: 9158-9167
        • Hickey M.A.
        • Chesselet M.F.
        Apoptosis in Huntington's disease.
        Prog Neuro-Psychopharmacol Biol Psychiatry. 2003; 27: 255-265
        • Kim M.
        • Lee H.S.
        • Laforet G.
        • McIntyre C.
        • Martin E.J.
        • Chang P.
        • et al.
        Mutant huntingtin expression in clonal striatal cells: dissociation of inclusion formation and neuronal survival by caspase inhibition.
        J Neurosci. 1999; 19: 964-973
        • Okazawa H.
        • Rich T.
        • Chang A.
        • Lin X.
        • Waragai M.
        • Kajikawa M.
        • et al.
        Interaction between mutant ataxin-1 and PQBP-1 affects transcription and cell death.
        Neuron. 2002; 34: 701-713
        • Yamazaki T.
        • Selkoe D.J.
        • Koo E.H.
        Trafficking of cell surface β-amyloid precursor protein: retrograde and transcytotic transport in cultured neuron.
        J Cell Biol. 1995; 129: 431-442
        • Brewer G.J.
        • Torricelli J.R.
        • Evege E.K.
        • Price P.J.
        Optimized survival of hippocampal neurons in B27-supplemented neurobasal, a new serum-free medium combination.
        J Neurosci Res. 1993; 35: 567-576
        • Herkert M.
        • Shakman O.
        • Schweins E.
        • Becker C.M.
        β-Bungarotoxin is a potent inducer of apoptosis in cultured rat neurons by receptor-mediated internalization.
        Eur J Neurosci. 2001; 14: 821-828
        • Du J.
        • Feng L.
        • Zaitsev E.
        • Je H.S.
        • Liu X.W.
        • Lu B.
        Regulation of TrkB receptor tyrosine kinase and its internalization by neuronal activity and Ca2+ influx.
        J Cell Biol. 2003; 163: 385-395
        • Foud F.M.
        • Marshall W.D.
        • Farrell P.G.
        • Goldberg M.
        • Ruhenstroth-Bauer G.
        Inhibition by the mushroom toxins α-amanitin and phalloidin of hepatoprotein-induced 3H-thymidine incorporation into rat liver DNA and of plasma protein production in hepatocyte cultures.
        Toxicon. 1987; 12: 1265-1271
        • Kolodziej P.A.
        • Woychik N.
        • Liao S.M.
        • Young R.A.
        RNA polymerase II subunit composition, stoichiometry, and phosphorylation.
        Moll Cell Biol. 1990; 10: 1915-1920
        • Nguyen V.T.
        • Giannoni F.
        • Dubois M.F.
        • Seo S.J.
        • Vigneron M.
        • Kedinger C.
        In vivo degradation of RNA polymerase II largest subunit triggered by α-amanitin.
        Nucleic Acid Res. 1996; 24: 2924-2929
        • Bhattacharyya A.
        • Watson F.L.
        • Pomeroy S.L.
        • Zhang Y.J.
        • Stiles C.D.
        • Segal R.A.
        High-resolution imaging demonstrates dynein-based vesicular transport of activated Trk receptors.
        J Neurobiol. 2002; 51: 302-312
        • Turmaine M.
        • Raza A.
        • Mahal A.
        • Mangiarini L.
        • Bates G.P.
        • Davies S.W.
        Nonapoptotic neurodegeneration in a transgenic mouse model of Huntington's disease.
        Proc Natl Acad Sci USA. 2000; 97: 8093-8097
        • Burkhardt J.K.
        • Echeverri C.J.
        • Nilsson T.
        • Valee R.B.
        Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution.
        J Cell Biol. 1997; 139: 469-484
        • Morris R.L.
        • Hollenbeck P.J.
        Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons.
        J Cell Biol. 1995; 131: 1315-1326
        • Terasaki M.
        • Reese T.S.
        Characterization of endoplasmic reticulum by co-localization of BiP and dicarbocyanine dyes.
        J Cell Sci. 1992; 101: 315-322
        • Kreis T.E.
        Role of microtubules in the organization of the Golgi apparatus.
        Cell Motil Cytoskeleton. 1990; 15: 67-70
        • Matteoni R.
        • Kreis T.E.
        Translocation and clustering of endosomes and lysosomes depends on microtubules.
        J Cell Biol. 1987; 105: 1253-1265
        • Hirokawa N.
        • Sato-Yoshitake R.
        • Yoshida T.
        • Kawashima T.
        Brain dynein (MAP1C) localizes on both retrogradely transported membranous organelles in vivo.
        J Cell Biol. 1990; 111: 1027-1037
        • Julien J.P.
        Neurofilaments and motor neuron disease.
        Trends Cell Biol. 1997; 7: 243-249
        • Gunawardena S.
        • Goldstein L.S.B.
        Polyglutamine disease and transport problems.
        Arch Neurol. 2005; 62: 46-51
        • Difiglia M.
        • Sapp E.
        • Chase K.O.
        • Davies S.W.
        • Bates G.P.
        • Vonsattel J.P.
        Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain.
        Science. 1997; 277: 1990-1993
        • Li H.
        • Li S.H.
        • Yu Z.X.
        • Li X.J.
        Huntingtin aggregate-associated axonal degeneration is an early pathological event in Huntington's disease mice.
        J Neurosci. 2001; 21: 8473-8481
        • Yang Q.
        • Hashizume Y.
        • Yoshida M.
        • Wang Y.
        • Goto Y.
        • Mitsuma N.
        • et al.
        Morphological Purkinje cell changes in spinocerebellar ataxia type 6.
        Acta Neuropathol. 2000; 100: 371-376
        • Gavin R.H.
        Microtubule-microfilament synergy in the cytoskeleton.
        Int Rev Cytol. 1997; 173: 207-242
        • Hafezparast M.
        • Klocke R.
        • Ruhrberg C.
        • Marquardt A.
        • Ahmad-Annuar A.
        • Bowen S.
        • et al.
        Mutations in dynein link motor neuron degeneration to defects in retrograde transport.
        Science. 2003; 300: 808-812