Human Molecular Genetics, 2001, Vol. 10, No. 15 1531-1538
© 2001 Oxford University Press
Double-stranded RNA-dependent protein kinase, PKR, binds preferentially to Huntington’s disease (HD) transcripts and is activated in HD tissue
The Buck Institute, 8001 Redwood Boulevard, Novato, CA 94945, USA and 1Center for Molecular Medicine and Therapeutics, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada
Received March 13, 2001; Revised and Accepted May 24, 2001.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Fourteen neurological diseases have been associated with the expansion of trinucleotide repeat regions. These diseases have been categorized into those that give rise to the translation of toxic polyglutamine proteins and those that are untranslated. Thus far, compelling evidence has not surfaced for the inclusion of a model in which a common mechanism may participate in the pathobiology of both translated and untranslated trinucleotide diseases. In these studies we show that a double-stranded RNA-binding protein, PKR, which has previously been linked to virally-induced and stress-mediated apoptosis, preferentially binds mutant huntingtin RNA transcripts immobilized on streptavidin columns that have been incubated with human brain extracts. These studies also show, by immunodetection in tissue slices, that PKR is present in its activated form in both human Huntington autopsy material and brain tissue derived from Huntington yeast artificial chromosome transgenic mice. The increased immunolocalization of the activated kinase is more pronounced in areas most affected by the disease and, coupled with the RNA binding results, suggests a role for PKR activation in the disease process.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Huntington’s disease (HD) is one of at least 14 neurological diseases associated with an expansion of unstable trinucleotide repeats (1). Eight of these diseases, including HD, involve translated polyglutamine products from CAG repeat sequences which are believed to be etiologic for disease pathogenesis. In tissue culture, overexpression of mutant huntingtin containing polyglutamine stretches is cytotoxic and may involve caspase-mediated cleavage and generation of a toxic N-terminal huntingtin fragment (2). Although it is a widely held hypothesis that translocation of the polyglutamine expansions to the nucleus and/or the neurites, and their subsequent aggregation, are important components of cellular pathology (3,4), evidence suggests that aggregation and/or nuclear localization may not be required for toxicity and may not be absolutely predictive of neuronal cell death in HD (5–7). One of the more puzzling features of HD is the cell type-specific vulnerability of projection neurons in the striatum, despite widespread expression of the protein in unaffected cell types within and outside of the CNS. Additionally, although protein expression is required for a disease phenotype in HD transgenic mouse lines (8), in the human disease there is an apparent lack of correlation between the sites of huntingtin protein expression and the cells that undergo selective neuronal loss (9).
The remainder of the trinucleotide neurological disorders are categorized as non-coding trinucleotide diseases. The trinucleotide repeat sequences that lead to these pathologies are generally longer than the translated expansions, ranging from a minimum of 50 repeats for myotonic dystrophy (10) to in excess of 230 repeats in fragile X syndrome, and include CCG, GCC, GAA and CTG (11). In contrast, the minimum number of translated CAG repeat sequences which give rise to pathological expression of the polyglutamine proteins range from 21 repeats for spinocerebellar ataxia type 6 (SCA6) to 61 for spinocerebellar ataxia type 3 (SCA3) (12,13). The molecular underpinnings of two untranslated trinucleotide repeat disorders have been identified. Both fragile X and fragile XE syndromes involve long, hypermethylated repeats in or near the promoter region which result in promoter silencing and loss of protein expression (14,15). Nonetheless, myotonic dystrophy (DM) and spinocerebellar ataxia type 8 (SCA8) involve 3' untranslated CTG repeats with unidentified mechanisms of pathology that are postulated to occur at the level of the RNA, i.e. trans-dominant RNA interference and/or RNA gain of function (16–19).
The reproducible toxicity of polyglutamine overexpression in tissue culture promotes a widely held belief that translated trinucleotide diseases are mechanistically distinct from their untranslated relatives. Without minimizing the importance of polyglutamine toxicity in HD, the data showing lack of correlation between the level of huntingtin expression and cell death in the disease suggest that polyglutamine expression alone may not be sufficient to explain the pattern and progression of the actual disease. Recently, a study of putative mechanisms underlying the untranslated DM trinucleotide pathology described a double-stranded RNA-binding protein, PKR, which binds CUG repeat RNAs in a length-dependent fashion (20). PKR is an interferon-induced protein kinase initially identified in viral response by virtue of its binding to and activation by the extensive secondary structure formed by viral RNA sequences. Upon binding to double-stranded RNA, PKR autophosphorylates and increases cellular sensitivity to apoptotic and pro-inflammatory stimuli through a number of putative pathways, including phosphorylation of its known substrate, eIF2
, which shuts down protein synthesis, release of NF-
B from its inhibitory complex and activation of the p38 MAPK pathway (21).
The finding that PKR binds a minimum of 15 CUG repeats, and that its activation increases in a repeat-dependent manner, suggests a potential role for PKR activation in CAG polyglutamine disease pathology. In an unrelated but relevant study, McLaughlin et al. (22) reported the association of an unidentified RNA-binding protein in rat and human brain tissue that specifically interacts with CAG repeat regions. Although the isolated protein was reported to migrate at 63 kDa on a polyacrylamide gel, this molecular weight is close to the molecular weight of PKR (69 kDa).
To assess whether PKR contributes to the pathogenesis of HD, we identified the presence of the activated kinase in Huntington autopsy material and investigated patterns of PKR phosphorylation in affected regions (e.g. striata), versus unaffected regions (e.g. hippocampi). Likewise, in preliminary studies, we noted strong PKR activation in the Huntington yeast artificial chromosome (YAC) transgenic mice, relative to controls, in areas affected by overexpression of the transgene. Additionally, expanding on data generated by the McLaughlin study described above (22), we used an RNA biotinylation protocol (23) to bind, isolate, and identify PKR as specifically associating with huntingtin exon 1 RNA fragments in human brain extracts, with binding and activation dependent on the length of the CAG repeat region. These findings suggest that activation of PKR and subsequent phosphorylation events may play a role in HD pathogenesis, the elucidation of which may lead to improved therapeutic intervention strategies.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Preferential binding of PKR to the expanded CAG region of mutant huntingtin RNA
To test the extent to which PKR from human tissue extracts binds CAG repeat sequences, truncated exon 1 fragments of wild-type and mutant huntingtin cDNAs, with 15 and 128 CAGs, respectively, were used to generate biotinylated RNA transcripts. Biotinylation was performed with a nucleotide that would modify the RNA outside of the CAG region, so as (i) not to interfere with the ability of the CAG region to form secondary structures and (ii) to favor equal incorporation of the biotin label and minimize the opportunity for preferential column binding of one RNA species over the other. The RNA was bound to immobilized streptavidin columns and incubated with supernatant from human brain extracts. Across several repetitions, using unique RNA preparations and three different non-diseased brain extracts, western blot analyses consistently identified clear PKR-immunopositive bands in the eluted fractions from columns with bound mutant huntingtin RNA fragments (Fig. 1D–F), with rare to very faint bands in the wild-type fractions (Fig. 1A–C). It was also of interest to determine whether the increased binding of PKR occurred simply as a function of the number of CAGs or whether PKR bound expanded CAG repeat sequences in excess of what would have been predicted simply by CAG counts. If the number of CAGs determined PKR binding and activation, a linear relationship would have existed between the number of CAGs present on the column and the amount of PKR that bound the column. Consequently, increasing the molarity of the wild-type RNAs to match the number of repeats in the mutant would have been expected to generate equivalent bands. If PKR binding and activation were dependent on secondary structure and the formation of double-stranded RNA regions, the longer CAG repeat region would have been expected to bind and activate PKR in excess of shorter RNAs with the same total amount of CAG repeats. To investigate this, an 8-fold molar excess of wild-type CAG RNA relative to the mutant was bound to the column and incubated with human tissue. This approximately equalized the molarity of CAG targets within the wild-type and mutant samples. Although a stronger band was detected by western blot analysis from these fractions (Fig. 2A and B) than from fractions using equimolar amounts of wild-type (Fig. 2E and F) and mutant (Fig. 2G and H) RNA, the intensity of the signal did not approach that achieved by incubation with the expanded huntingtin RNA (Fig. 2G and H).
|
|
Immunodetection of activated PKR in Huntington and control brains and in Huntington YAC transgenic mice
Tissue sections from the striatum, globus pallidus and hippocampus of three HD brains and three age-matched control brains were processed for activated PKR immunoreactivity using an antibody developed against the phosphorylated, activated form of PKR, anti-PKR [pT451]. When PKR binds to double-stranded RNA, it undergoes a conformational change that exposes a phosphorylation site at Thr451. Subsequent auto-phosphorylation at this site leads to activation and kinase activity. In the control tissue samples, light to moderate phospho-PKR immunoreactivity was detected in the gray matter in cell bodies, predominantly nuclear (Fig. 3A, C and E). Intense staining was found in the Huntington striatum and globus pallidus, predominantly in cells with a rounded morphology (Fig. 3B, D and F). In both control and Huntington tissue, activated PKR immunoreactivity could be found in the white matter tracts. This staining was markedly increased in the Huntington brains, particularly in the striatum (Fig. 4A and B). Increased immunoreactivity was also found throughout the neuropil of Huntington striata (Fig. 5). Conversely, staining in the hippocampal regions of Huntington tissue was remarkably similar to control. Punctate staining was localized to neuronal nuclei, with some diffuse cytoplasmic labeling, as well as punctate immunoreactivity in the neuropil (data not shown). Immunoreactivity was also detected in the white matter tracts in the hippocampus. Interestingly, occasionally strong and predominantly cytoplasmic immunoreactivity was detected in large neuronal cells in the hippocampus that differed from the pattern of staining noted elsewhere.
|
|
|
Although we are presently undertaking more comprehensive immunocytochemical analyses of Huntington YAC transgenic mice, preliminary results of PKR immunodetection parallel what has been seen in analyses of the human tissue. At 3 months of age, no differences can be seen between control and Huntington mice, with only punctate staining in the striatum and a moderate amount of immunoreactivity in the white matter tracts of the cerebellum (data not shown). In contrast, at 9 months, dense immunoreactivity was found in the brains of the Huntington transgenic mice in the anterior caudate, cingulum, external capsule, internal capsule and in the tracts penetrating the striatum (Fig. 6B and D). Only moderate staining in these areas was detected in the controls at 9 months (Fig. 6A and C). In the hippocampus no differences were detected between Huntington and control mice, with very little immunoreactivity evident.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
These results support a role for PKR in the pathogenesis of HD and suggest that a common mechanism may exist at the level of the RNA for both translated and non-translated trinucleotide disorders. We have shown that CAG repeat regions in huntingtin mRNA are capable of binding PKR in human brain tissue extracts and that the binding of PKR is increased with longer trinucleotide stretches. It is possible that, for untranslated triplet repeat disease, longer stretches and greater secondary structure may be required to activate sufficient PKR to mobilize cell death pathways. The translation of toxic polyglutamine proteins, in contrast, may render the cell more vulnerable and, consequently, the requirement for RNA-activated PKR would be lowered. In addition, previous studies have shown that molecules such as dextran and chondroitin sulfate, heparin and polyglutamines can also activate PKR, as can environmental stress (21).
Compelling evidence exists for the requirement of protein synthesis to generate demonstrable phenotypes in transgenic models of neurological CAG repeat diseases. In one study, for example, no behavioral phenotype was seen for mice expressing high levels of untranslated huntingtin mRNA carrying 44 CAG expansions. It is worth noting, however, that even expression of expanded polyglutamine proteins in that range has not always been sufficient to generate a phenotype for transgenic models of HD, spinal and bulbar muscular atrophy or Machado–Joseph disease (24). Consequently, the absence of phenotype in transgenic models is not always readily interpretable. The most convincing argument against an RNA mechanism in CAG repeat disease is found in a recent study of SCA1 showing that, not only is protein synthesis absolutely required for a phenotype in the transgenic model, but that elimination of a nuclear localization signal quite some distance from the expanded trinucleotide region was sufficient for reversal of phenotype, suggesting that even the expression of expanded polyglutamines was not sufficient to produce a disease pathology (25). Interestingly, the initial study generating this SCA1 transgenic line describes the production of five lines in which high levels (50–100 times endogenous) of mutant mRNA were transcribed with no detectable protein expression from the mutant transgene. All five lines exhibited a significant neurological phenotype relative to transgene controls with normal length repeats (26). Although mRNA levels were not correlated with severity of phenotype and the suggestion was made that the mutant protein was probably quickly degraded and, thus, undetectable, these data certainly support the possibility that expanded RNA trinucleotide repeats can contribute to neurological disease. Finally, the recent identification of SCA8, a human spinocerebellar ataxia mediated by an untranslated CAG expansion, provides the best support that untranslated trinucleotide repeats are capable of mediating CNS disease pathology (19).
We have shown strong immunoreactivity of activated PKR by immunodetection of its phosphorylated form in autopsy material from diseased Huntington brain. Although a striking increase in immunoreactivity was found in areas of the Huntington brain that are affected by the disease relative to areas in Huntington brains that are spared, and likewise an increase in staining from diseased areas relative to similar areas from age-matched control tissue, without serial sections throughout each complete brain region it is impossible to compare one area of the brain to another statistically and unequivocally. Consequently we can say only that this pattern of PKR immunoreactivity in the Huntington postmortem tissue would support a role for the involvement of activated PKR in the disease process. Because PKR functions as a part of the stress response pathway, the relative contributions of polyglutamine toxicity and secondary RNA structure to PKR activation cannot be determined conclusively and both are likely to participate in activation of the kinase. The moderate level of phosphorylated PKR in normal brain, particularly in neurons, is not unanticipated. PKR plays a role in cell cycle regulation and its expression is increased in growth-arrested cells (27). Of note, the levels of total PKR protein are not coupled with its activation and are less predictive of cell cycle stage than is its phosphorylated or activated state. This may be an important distinction when investigating a potential mechanism for PKR in neuronal tissue relative to cell lines and in using overexpression paradigms without consideration of the phosphorylation state of the kinase.
Although unphosphorylated PKR is more highly expressed in the cytoplasm than in the nucleus, upon activation by double-stranded RNA phosphorylated PKR translocates to the nucleus (28). This would explain the predominance of phosphorylated PKR staining in the nucleus relative to the cytoplasm in brain tissue. The strong immunoreactivity of phosphorylated PKR in diseased white matter tracts and in the Huntington striatal neuropil, however, was not expected and has not been described previously. Even the non-diseased white matter tracts had easily detectable levels of phospho-specific PKR immunoreactivity, although much lower than the diseased brain. The PKR neuropil staining in Huntington brain samples bears a striking similarity to N-terminal huntingtin aggregates found in the neuropil of human postmortem tissue, reported by Gutekunst et al. (7), and in huntingtin N-terminal fragments in mice transgenic for the Huntington mutation (29). Additionally, other reports have found strong N-terminal huntingtin immunoreactivity in white matter tracts in HD autopsy material (30). The presence of a basal level of phosphorylated PKR that we note in neurites and in axon tracts of normal brain tissue may render these structures more vulnerable to increased activation by CAG repeat mRNA and by disease-mediated toxic stimuli.
These data do not support, nor are we proposing, a causative mechanism at the RNA level for HD pathogenesis. However, identifying the presence of a well characterized pro-apoptotic kinase, which can be activated by both CAG mRNA secondary structure as well as by polyglutamine toxicity, may be significant in appreciating the mechanisms underlying the specific cellular vulnerability in this disease. It will be important to determine whether the activation of PKR occurs late in disease progression, secondary to polyglutamine toxicity, or whether phosphorylation of the kinase is an early mediator of cell death programs. Additionally, it will be necessary to examine the presence of activated PKR in other trinucleotide repeat disorders before postulating a role for PKR as a common mechanism in translated and untranslated triplet repeat disorders. Nonetheless, the presence of activated PKR in HD brain tissue, coupled with the propensity for long CAG repeats to bind PKR preferentially in human brain supernatants, argue for the inclusion of RNA-mediated mechanisms in HD pathogenesis that act in parallel with polyglutamine toxicity to promote cell death.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Immunohistochemistry
Formalin-fixed human tissue was provided by the Harvard Brain Tissue Resource Center and paraffin-embedded by the Histology Core Facility at the Buck Institute. Three different grade 3 Huntington cases, aged 42, 67 and 72 years, and three different age-matched controls were examined. Tissue sections from 3- and 9-month-old Huntington YAC transgenic mice (31) were derived from transgenic lines generated at the Center for Molecular Medicine and Therapeutics, University of British Columbia, and also processed by the Histology Core Facility at the Buck Institute. Non-transgenic littermates were used as control tissue at the same time periods. Sections were deparaffinized in xylenes (2 x 10 min) followed by 5 min incubations each in 100, 95 and 75% ethanol. Sections were rinsed in water and washed (2 x 5 min) in 0.5x PBS. To unmask antigens, the slides were immersed for 30 min in a copeland jar filled with citric acid buffer (pH 6.0) in an 80°C water bath. The copeland jar was subsequently removed from the water bath and allowed to come to room temperature, rinsed in PBS for 5 min, and incubated in 10% methanol and 3% H2O2 in water to quench endogenous peroxidase activity. The sections were then processed for antigens using a highly sensitive method for immunodetection of proteins which involves a biotinylated tyramine amplification step described by Adams (32). Briefly, sections were rinsed in PBS, blocked with 10% goat serum/PBS/0.1% Triton X 100 for 1 h at room temperature, and incubated in primary antibody, a phospho-specific antibody against activated PKR (Biosource), at 1:1000 dilution overnight at 4°C. Sections were rinsed (3 x 10 min) in 0.1x PBS followed by incubation in a biotinylated donkey anti-rabbit secondary antibody (Jackson Immunochemicals, 1:500) for 2 h at room temperature, rinsed (3 x 10 min in 0.1x PBS), and immersed in an ABC solution (Vector, 1:1000) for 1 h. Sections were again rinsed in PBS, placed in an 0.5% biotinylated tyramine/0.01% H2O2 solution for 10 min, rinsed (3 x 10 min in 0.1x PBS), and placed back in the ABC solution for 30 min. After final rinses, the tissue was incubated in diaminobenzidine (DAB) substrate (Vector) for detection of horseradish peroxidase (HRP), mounted and coverslipped.
RNA synthesis
Huntingtin plasmids containing the huntingtin coding sequence with either 15 or 128 CAGs were digested with HindIII to generate transcripts ending 
352 nucleotides 3' to the CAG repeat segment. Preparation of biotinylated transcripts was performed using the Ambion MEGAscript in vitro Transcription Kit combined with biotin-16-dUTP (Boehringer Mannheim). One microgram of digested template plasmid DNA was used for each reaction and the ratio of biotinylated dUTP to unlabeled dUTP was 1:2 according to instructions provided. The reaction was completed by treatment with DNase (2 U/reaction) at 37°C for 15 min followed by lithium chloride precipitation to remove the unincorporated nucleotides. The labeled wild-type huntingtin transcript was 449 nucleotides long and the mutant transcript was 788 nucleotides. RNA concentration was assessed by measuring absorbance at 260 nm and was confirmed by visualization on a 1% agarose gel. Incorporation of biotin was confirmed by spotting a nylon membrane with dilutions of the synthesized RNA followed by UV crosslinking for 3 min. The membrane was washed (3 x 5 min) with detergent buffer and incubated with streptavidin conjugated to HRP (1:1000, NEN). After three final washes, the membrane was incubated for 1 min with ECL chemiluminescence reagent and exposed to film (Kodak BIOMAX).
Preparation of cytoplasmic extracts
Cytosolic extracts from human, non-disease autopsy material were generously provided by Dr Simon Melov’s laboratory at the Buck Institute as well as the Harvard Tissue Bank and were prepared according to standard lysis and homogenization protocols. In brief, tissue was homogenized in 2x volumes of ice-cold extraction buffer (10 mM Tris–Cl pH 8.0, with Roche Complete Mini Cocktail protease inhibitors) and spun at 16 000 g for 10 min at 4°C. The supernatant was transferred to fresh microfuge tubes and used in the binding experiments. Control brain samples were derived from subjects between 65 and 74 years of age at postmortem intervals ranging from 12.5 to 23.9 h.
Biotinylated RNA affinity column
One milliliter syringes were packed with a small amount of glass wool and loaded with 125 µl of ImmunoPure immobilized streptavidin (Pierce). The columns were equilibrated with 2 ml HEPES buffer including 25 mM HEPES (pH 7.5), 10 mM NaCl, 5% glycerol, 5 mM DTT, and 0.1 mM EDTA. Labeled RNA was diluted in binding buffer comprised of 25 mM HEPES (pH 7.5), 10 mM NaCl, 5% glycerol, 5 mM DTT, 0.1 mM EDTA, 3% Superasin (Ambion) and 25 µg/ml tRNA (Sigma), pipetted onto the column, and incubated for 5 min. The columns were rinsed with 4 ml of HEPES buffer. Supernatant from human tissue extracts were diluted 1:1 v/v with binding buffer and pipetted onto the column. The columns were washed with 5 ml of HEPES buffer. The bound proteins were eluted with 300 µl of high-salt buffer (1x PBS, 1 mM MgCl2, 2.5 mM KCl, 1 M NaCl). After allowing the first two drops to pass through, the eluate was collected in 100 µl fractions. The column protocol was reproduced on five separate occasions with three unique preparations of biotinylated RNA and three different human tissue extracts with the same results. Aliquots from eluted fractions were electrophoresed on a 10% NuPAGE Bis-Tris gel for 50 min in MOPS buffer at 200 V constant. Proteins were transferred to PVDF membranes for western blotting applications.
Western blotting
Membranes were washed briefly in Tris-buffered saline with Tween (TBST) and blocked in TBST/5% non-fat dried milk (NFDM) for a minimum of 1 h. Membranes were incubated overnight at 4°C in a primary monoclonal antibody generated against PKR (Research Genetics), diluted 1:1000 in 5% NFDM. Membranes were rinsed (3 x 10 min) in TBST and incubated in anti-mouse HRP-conjugated secondary antibody (Amersham) at 1:1000 dilution in TBST. After three final washes, the membrane was incubated for 1 min with ECL chemiluminescence reagent and exposed to film (Kodak BIOMAX).
|
ACKNOWLEDGEMENTS |
|---|
We would like to acknowledge the Hereditary Disease Foundation for their generous support of this work and for their tireless efforts to seek a cause and cure for HD. We would also like to thank Drs Eberwine, Mascotti, Beal and Spanggord for their valuable insight and expertise. The human tissue in these studies was provided by the Harvard Brain Tissue Resource Center, which is supported in part by PHS grant number MH/NS 31862.
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +1 415 209 2274; Fax: +1 415 209 2230; Email: apeel@buckinstitute.org
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Cummings, C.J. and Zoghbi, H.Y. (2000) Fourteen and counting: unraveling trinucleotide repeat diseases. Hum. Mol. Genet., 9, 909–916.
2 Wellington, C.L., Singaraja, R., Ellerby, L., Savill, J., Roy, S., Leavitt, B., Cattaneo, E., Hackam, A., Sharp, A., Thornberry, N. et al. (2000) Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and non-neuronal cells. J. Biol. Chem., 275, 19831–19838.
3 Li, H., Li, S.H., Johnston, H., Shelbourne, P.F. and Li, X.J. (2000) Amino-terminal fragments of mutant huntingtin show selective accumulation in striatal neurons and synaptic toxicity. Nat. Genet., 25, 385–389.[Web of Science][Medline]
4 Sapp, E., Penney, J., Young, A., Aronin, N., Vonsattel, J.P. and DiFiglia, M. (1999) Axonal transport of N-terminal huntingtin suggests early pathology of corticostriatal projections in Huntington disease. J. Neuropathol. Exp. Neurol., 58, 165–173.[Web of Science][Medline]
5 Kuemmerle, S, Gutekunst, C.A., Klein, A.M., Li, X.J., Li, S.H., Beal, M.F., Hersch, S.M. and Ferrante, R.J. (1999) Huntington aggregates may not predict neuronal death in Huntington’s disease. Ann. Neurol., 46, 842–849.[Web of Science][Medline]
6 Saudou, F., Finkbeiner, S., Devys, D. and Greenberg, M.E. (1998) Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell, 95, 55–66.[Web of Science][Medline]
7 Gutekunst, C.A., Li, S.H., Yi, H., Mulroy, J.S., Kuemmerle, S., Jones, R., Rye, D., Ferrante, R.J., Hersch, S.M. and Li, X.J. (1999) Nuclear and neuropil aggregates in Huntington’s disease: relationship to neuropathology. J. Neurosci., 19, 2522–2534.
8 Goldberg, Y.P., Kalchman, M.A., Metzler, M., Nasir, J., Zeisler, J., Graham, R., Koide, H.B., O’Kusky, J., Sharp, A.H., Ross, C.A. et al. (1996) Absence of disease phenotype and intergenerational stability of the CAG repeat in transgenic mice expressing the human Huntington’s disease transcript. Hum. Mol. Genet., 5, 177–185.
9 Fusco, F.R., Chen, Q., Lamoreaux, W.J., Figueredo-Cardenas, G., Jiao, Y., Coffman, J.A., Surmeier, D.J., Honig, M.G., Carlock, L.R., Reiner, A. et al. (1999) Cellular localization of huntingtin in striatal and cortical neurons in rats: lack of correlation with neuronal vulnerability in Huntington’s disease. J. Neurosci., 15, 1189–1202.
10 Brook, J.D., McCurrach, M.E., Harley, H.G., Buckler, A.J., Church, D., Aburatani, H., Hunter, K., Stanton, V.P., Thirion, J.P., Hudson, T. et al. (1992) Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell, 21, 799–808.
11 Warren, S.T. and Nelson, D.L. (1994) Advances in molecular analysis of fragile X syndrome. J. Am. Med. Assoc., 16, 536–542.
12 Zhuchenko, O., Bailey, J., Bonnen, P., Ashizawa, T., Stockton, D.W., Amos, C., Dobyns, W.B., Subramony, S.H., Zoghbi, H.Y. and Lee C.C. (1997) Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel. Nat. Genet., 15, 62–69.[Web of Science][Medline]
13 Maruyama, H., Nakamura, S., Matsuyama, Z., Sakai, T., Doyu, M., Sobue, G., Seto, M., Tsujihata, M., Oh-i, T., Nishio, T. et al. (1995) Molecular features of the CAG repeats and clinical manifestation of Machado–Joseph disease. Hum. Mol. Genet., 4, 807–812.
14 Pieretti, M., Zhang, F.P., Fu, Y.H., Warren, S.T., Oostra, B.A., Caskey, C.T. and Nelson, D.L. (1991) Absence of expression of the FMR-1 gene in fragile X syndrome. Cell, 66, 817–822.[Web of Science][Medline]
15 Gecz, J., Gedeon, A.K., Sutherland, G.R. and Mulley, J.C. (1996) Identification of the gene FMR2, associated with FRAXE mental retardation. Nat. Genet., 13, 105–108.[Web of Science][Medline]
16 Philips, A.V., Timchenko, L.T. and Cooper, T.A. (1998) Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy. Science, 280, 737–741.
17 Miller, J.W., Urbinati, C.R., Teng-Umnuay, P., Stenberg, M.G., Byrne, B.J., Thornton, C.A. and Swanson, M.S. (2000) Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J., 19, 4439–4448.[Web of Science][Medline]
18 Wang, J., Pegoraro, E., Menegazzo, E., Gennarelli, M., Hoop, R.C., Angelini, C. and Hoffman, E.P. (1995) Myotonic dystrophy: evidence for a possible dominant-negative RNA mutation. Hum. Mol. Genet., 4, 599–606.
19 Koob, M.D., Moseley, M.L., Schut, L.J., Benzow, K.A., Bird, T.D., Day, J.W. and Ranum, L.P. (1999) An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8). Nat. Genet., 21, 379–384.[Web of Science][Medline]
20 Tian, B., White, R.J., Xia, T., Welle, S., Turner, D.H., Mathews, M.B. and Thornton, C.A. (2000) Expanded CUG repeat RNAs form hairpins that activate the double-stranded RNA-dependent protein kinase PKR. RNA, 6, 79–87.[Abstract]
21 Williams, B.R. (1999) PKR; a sentinel kinase for cellular stress. Oncogene, 18, 6112–6120.[Web of Science][Medline]
22 McLaughlin, B.A., Spencer, C. and Eberwine, J. (1996) CAG trinucleotide RNA repeats interact with RNA-binding proteins. Am. J. Hum. Genet., 59, 561–569.[Web of Science][Medline]
23 Rodgers, J.T., Patel, P., Hennes, J.L., Bolognia, S.L. and Mascotti, D.P. (2000) Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays. Anal. Biochem., 15, 254–259.
24 Bates, G.P., Mangiarini, L., Mahal, A. and Davies, S.W. (1997) Transgenic models of Huntington’s disease. Hum. Mol. Genet., 6, 1633–1637.
25 Klement, I.A., Skinner, P.J., Kaytor, M.D., Yi, H., Hersch, S.M., Clark, H.B., Zoghbi, H.Y. and Orr, H.T. (1998) Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice. Cell, 95, 41–53.[Web of Science][Medline]
26 Burright, E.N., Clark, H.B., Servadio, A., Matilla, T., Feddersen, R.M., Yunis, W.S., Duvick, L.A., Zoghbi, H.Y. and Orr, H.T. (1995) SCA1 transgenic mice: a model for neurodegeneration caused by an expanded CAG trinucleotide repeat. Cell, 82, 937–948.[Web of Science][Medline]
27 Samuel, C.E., Kuhen, K.L., George, C.X., Ortega, L.G., Rende-Fournier, R. and Tanaka, H. (1997) The PKR protein kinase–an interferon-inducible regulator of cell growth and differentiation. Int. J. Hematol., 65, 227–237.[Web of Science][Medline]
28 Takizawa, T., Tatematsu, C., Watanabe, M., Yoshida, M. and Nakajima, K. (2000) Three leucine-rich sequences and the N-terminal region of double-stranded RNA-activated protein kinase (PKR) are responsible for its cytoplasmic localization. J. Biochem., 128, 471–476.
29 Li, H., Li, S.H., Cheng, A.L., Mangiarini, L., Bates, G.P. and Li, X.J. (1999) Ultrastructural localization and progressive formation of neuropil aggregates in Huntington’s disease transgenic mice. Hum. Mol. Genet., 8, 1227–1236.
30 Jones, A.L. (1999) The localization and interactions of huntingtin. Philos. Trans. R. Soc. Lond. B. Biol. Sci., 354, 1021–1027.
31 Hodgson, J.G., Agopyan, N., Gutekunst, C.A., Leavitt, B.R., LePiane, F., Singaraja, R., Smith, D.J., Bissada, N., McCutcheon, K., Nasir, J. et al. (1999) A YAC mouse model for Huntington’s disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration. Neuron, 23, 181–192.[Web of Science][Medline]
32 Adams, J.C. (1992) Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J. Histochem. Cytochem., 40, 1457–1463.[Abstract]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Alirezaei, D. D. Watry, C. F. Flynn, W. B. Kiosses, E. Masliah, B. R. G. Williams, M. Kaul, S. A. Lipton, and H. S. Fox Human Immunodeficiency Virus-1/Surface Glycoprotein 120 Induces Apoptosis through RNA-Activated Protein Kinase Signaling in Neurons J. Neurosci., October 10, 2007; 27(41): 11047 - 11055. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Shimazawa, Y. Ito, Y. Inokuchi, and H. Hara Involvement of Double-Stranded RNA-Dependent Protein Kinase in ER Stress-Induced Retinal Neuron Damage Invest. Ophthalmol. Vis. Sci., August 1, 2007; 48(8): 3729 - 3736. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Osborne and C. A. Thornton RNA-dominant diseases Hum. Mol. Genet., October 15, 2006; 15(suppl_2): R162 - R169. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Chen, C. Ma, K. A. Bower, Z. Ke, and J. Luo Interaction between RAX and PKR Modulates the Effect of Ethanol on Protein Synthesis and Survival of Neurons J. Biol. Chem., June 9, 2006; 281(23): 15909 - 15915. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. McLeod, L. V. O'Keefe, and R. I. Richards The pathogenic agent in Drosophila models of 'polyglutamine' diseases Hum. Mol. Genet., April 15, 2005; 14(8): 1041 - 1048. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Sobczak and W. J. Krzyzosiak CAG Repeats Containing CAA Interruptions Form Branched Hairpin Structures in Spinocerebellar Ataxia Type 2 Transcripts J. Biol. Chem., February 4, 2005; 280(5): 3898 - 3910. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Everett and N. W. Wood Trinucleotide repeats and neurodegenerative disease Brain, November 1, 2004; 127(11): 2385 - 2405. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhang, J. Li, D. P. Sejas, K. R. Rathbun, G. C. Bagby, and Q. Pang The Fanconi Anemia Proteins Functionally Interact with the Protein Kinase Regulated by RNA (PKR) J. Biol. Chem., October 15, 2004; 279(42): 43910 - 43919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Sobczak and W. J. Krzyzosiak Imperfect CAG Repeats Form Diverse Structures in SCA1 Transcripts J. Biol. Chem., October 1, 2004; 279(40): 41563 - 41572. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Kino, D. Mori, Y. Oma, Y. Takeshita, N. Sasagawa, and S. Ishiura Muscleblind protein, MBNL1/EXP, binds specifically to CHHG repeats Hum. Mol. Genet., March 1, 2004; 13(5): 495 - 507. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Pang, T. A. Christianson, T. Koretsky, H. Carlson, L. David, W. Keeble, G. R. Faulkner, A. Speckhart, and G. C. Bagby Nucleophosmin Interacts with and Inhibits the Catalytic Function of Eukaryotic Initiation Factor 2 Kinase PKR J. Biol. Chem., October 24, 2003; 278(43): 41709 - 41717. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Jasinska, G. Michlewski, M. de Mezer, K. Sobczak, P. Kozlowski, M. Napierala, and W. J. Krzyzosiak Structures of trinucleotide repeats in human transcripts and their functional implications Nucleic Acids Res., October 1, 2003; 31(19): 5463 - 5468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Sobczak, M. de Mezer, G. Michlewski, J. Krol, and W. J. Krzyzosiak RNA structure of trinucleotide repeats associated with human neurological diseases Nucleic Acids Res., October 1, 2003; 31(19): 5469 - 5482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Nussbaum, S. Gunnery, and M. B. Mathews The 3'-untranslated regions of cytoskeletal muscle mRNAs inhibit translation by activating the double-stranded RNA-dependent protein kinase PKR Nucleic Acids Res., March 1, 2002; 30(5): 1205 - 1212. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||