Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain

Science

Volumn 277, Issue 5334, 1997, Pages 1990-1993

  DiFiglia, Marian ^a   Sapp, Ellen ^a   Chase, Kathryn O ^d   Davies, Stephen W ^b   Bates, Gillian P ^c   Vonsattel, J P ^a   Aronin, Neil ^d  

^a MASSACHUSETTS GENERAL HOSPITAL   (United States)

^b UNIVERSITY COLLEGE LONDON   (United Kingdom)

^c KING'S COLLEGE LONDON   (United Kingdom)

^d UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BRAIN PROTEIN; UBIQUITIN;

ARTICLE; BRAIN CORTEX; CONTROLLED STUDY; CORPUS STRIATUM; DYSTROPHY; HUMAN; HUMAN TISSUE; HUNTINGTON CHOREA; NEURITE; NEUROPATHOLOGY; PATHOGENESIS; PRIORITY JOURNAL; PROTEIN AGGREGATION;

ADOLESCENT; ADULT; AGED; BLOTTING, WESTERN; BRAIN CHEMISTRY; CELL NUCLEUS; CEREBRAL CORTEX; CORPUS STRIATUM; FLUORESCENT ANTIBODY TECHNIQUE; HUMANS; HUNTINGTON DISEASE; IMMUNOENZYME TECHNIQUES; MIDDLE AGED; MUTATION; NERVE TISSUE PROTEINS; NEURITES; NEURONS; NUCLEAR PROTEINS; UBIQUITINS;

EID: 0030752709     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5334.1990     Document Type: Article

References (44)

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19. 2-terminal peptide. Staining was absent under these conditions. For double-label immunofluorescence microscopy, tissues were treated with antiserum to huntingtin in combination with either monoclonal antibody to ubiquitin (Chemicon) or monoclonal antiserum to neurofilament (SMI312; Sternberger Monoclonals, Inc). Secondary antisera were rabbit BODIPY fluorescein (Molecular Probes, Inc.) and mouse Cy 5 (Jackson ImmunoResearch, Inc.). The double-stained sections were examined in a Bio-Rad 1024 laser confocal microscope. For analysis of ultrastructure, some immunoperoxidase-labeled sections were embedded in Epon and thin sections were cut on an ultramicrotome, mounted on formvar-coated slot grids, and examined in a JEOL 100 CX electron microscope. An antibody to ubiquitin (Dako; dilution 1:500) was also used to label some sections. A series of slides, from the same control and HD patients, stained with Ab 585 (9) was also available.
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22. To determine the frequency of neurons with hNlls, we viewed noncounterstained tissue sections from control and HD cortex in a Zeiss light microscope (LM) at × 640 magnification and with differential interference filtering (Nomarski optics). Neurons with and without hNlls were recorded in successive microscopic fields that spanned the dorsoventral extent of the cortical gray matter. The size of hNlls, nuclei, and nucleoli was determined with the assistance of a × 100 oil immersion objective lens and a drawing tube attached to the microscope. Drawings were scanned into a computer and the cross-sectional area and major and minor axis for each structure were determined by using NIH Image software. To determine the frequency of DNs with huntingtin and ubiquitin labeling, we examined adjacent sections labeled for these antigens in the LM at × 160. We scanned successive microscopic fields throughout the dorsoventral extent of the gray matter and recorded all neuntes in each field. We estimated the shape of nuclear inclusions (spherical, ovoid, elliptical) from the ratio of the major and minor axes. Total neurons examined in all HD patients was 8055 for analysis of hNlls, 3415 for analysis of ubiquitin-positive Nils, 4373 for hDNs, and 1983 for ubiquitin-positive DNs. Data analysis was performed by using Microsoft Excel and t -tests were done with Graphpad Instat.
23. 2-terminal-directed antisera to huntingtin label nuclear inclusions (9). Antibody 1C2, which preferentially recognizes polyglutamine domains in mutant huntingtin [Y. Trottier et al., Nature 378, 403 (1995)], recognizes the transgene protein in the HD mouse by Western blot but does not label nuclear inclusions by immunohistochemistry.
24. Brain extracts enriched for nuclei were prepared by a modification [J. L. Sonnenberg, P. F. Macgregor-Leon, T. Curran, J. I. Morgan, Neuron 3, 359 (1989)] of a procedure reported by our laboratories [N. Aronin, K. Chase, S. M. Sagar, F. R. Sharp, M. DiFiglia, Neuroscience 44, 409 (1991)]. Protein separation and Western blot analysis for huntingtin with Ab 1 were performed as described (4, 11).
25. Brain extracts enriched for nuclei were prepared by a modification [J. L. Sonnenberg, P. F. Macgregor-Leon, T. Curran, J. I. Morgan, Neuron 3, 359 (1989)] of a procedure reported by our laboratories [N. Aronin, K. Chase, S. M. Sagar, F. R. Sharp, M. DiFiglia, Neuroscience 44, 409 (1991)]. Protein separation and Western blot analysis for huntingtin with Ab 1 were performed as described (4, 11).
26. The soluble form of the mutant huntingtin fragment detected in the soluble nuclear fraction by Western blot analysis most likely contributes to formation of the hNlls. Recent evidence in HD transgenic mice shows that nuclear protein fractions isolated from brain contained huntingtin-immunoreactive low molecular weight proteins and a high molecular weight product resistant to conventional protein separation [E. Scherzinger ef al., Cell 90, 549 (1997)].
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31. Little or no ubiquitin staining of Nlls was obtained in juveniles J12 and J13.
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33. Although a nucleolus distinct from the hNll was identified routinely in individual HD neurons, we cannot rule out the possibility that the hNlls are of nucleolar origin because multiple nucleoli may exist in cells.
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35. 2-terminal products of huntingtin are increased in axons after blockade of axonal transport in rat peripheral nerve [J. Block-Galarza et al., Neuroreport 8, 2247 (1997)]. Some dystrophic neurites may be dendrites, because dendrites are enriched in huntingtin in normal brain and dendritic changes in HD cortical neurons have been identified. Also, we cannot exclude the possibility that some of the structures identified as DNs are in fact hNlls retained in the brain after degeneration and dissolution of the affected neurons. Further analysis in HD brain and in transgenic mice may help resolve this issue.
36. 2-terminal products of huntingtin are increased in axons after blockade of axonal transport in rat peripheral nerve [J. Block-Galarza et al., Neuroreport 8, 2247 (1997)]. Some dystrophic neurites may be dendrites, because dendrites are enriched in huntingtin in normal brain and dendritic changes in HD cortical neurons have been identified. Also, we cannot exclude the possibility that some of the structures identified as DNs are in fact hNlls retained in the brain after degeneration and dissolution of the affected neurons. Further analysis in HD brain and in transgenic mice may help resolve this issue.
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39. A striking example of a disorder involving the development of nuclear inclusions is neuronal intranuclear inclusion disease, which is characterized by neurodegeneration within the central and peripheral nervous systems and may include extrapyramidal dysfunction [M. Haltia, H. Somer, J. Palo, W. G. Johnson, Ann. Neurol. 15, 316 (1984); N. Funata et al., Clin. Neuropathol. 9, 89 (1990)]. Some types of nuclear inclusions may be involved in the storage, degradation, or transport of pre-mRNA and pre-rRNA [K. Brasch and R. L. Ochs, Exp. Cell Res. 202, 211 (1992)].
40. A striking example of a disorder involving the development of nuclear inclusions is neuronal intranuclear inclusion disease, which is characterized by neurodegeneration within the central and peripheral nervous systems and may include extrapyramidal dysfunction [M. Haltia, H. Somer, J. Palo, W. G. Johnson, Ann. Neurol. 15, 316 (1984); N. Funata et al., Clin. Neuropathol. 9, 89 (1990)]. Some types of nuclear inclusions may be involved in the storage, degradation, or transport of pre-mRNA and pre-rRNA [K. Brasch and R. L. Ochs, Exp. Cell Res. 202, 211 (1992)].
41. A striking example of a disorder involving the development of nuclear inclusions is neuronal intranuclear inclusion disease, which is characterized by neurodegeneration within the central and peripheral nervous systems and may include extrapyramidal dysfunction [M. Haltia, H. Somer, J. Palo, W. G. Johnson, Ann. Neurol. 15, 316 (1984); N. Funata et al., Clin. Neuropathol. 9, 89 (1990)]. Some types of nuclear inclusions may be involved in the storage, degradation, or transport of pre-mRNA and pre-rRNA [K. Brasch and R. L. Ochs, Exp. Cell Res. 202, 211 (1992)].
42. Ubiquitin attaches to misfolded or abnormal proteins to be degraded in the proteosome, a large multiprotein complex that is found in both nucleoplasm and cytoplasm [M. Peters. W. W. Franke, J. A. Kleinschmidt, J. Biol. Chem. 269, 7709 (1994)].
43. In the HD mouse ubiquitin immunoreactivity develops in nuclear inclusions several weeks after detection of the transgene protein (9), further supporting the idea that ubiquitin-dependent proteolysis of mutant huntingtin is delayed. The presence of fewer ubiquitin-positive Nils and DNs compared with those with mutant huntingtin could also be due to a greater instability of ubiquitin in postmortem tissue. The latter may also explain previous failures to detect ubiquitinated mutant huntingtin in the HD brain in biochemical assays (4, 16).
44. Supported by grants NS 16367 to M.D. and J.P.V. and NS 31579 to M.D. and N.A. and by grants from the Hereditary Disease Foundation to M.D. and N.A. We appreciate the technical assistance of L. Cherkas; the helpful suggestions of Drs. J. Lawrence, T. Smith, G. Stein, P. Bhide, J. Francis, B. Hyman, M. Irizarry, and M. Kim; and the contribution of postmortem tissue of patient A4 provided by Drs. A. Young and J. Penney.