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Evidence for a role for transglutaminase in Huntington's disease and the potential therapeutic implications
Neurochemistry International 40 (2002) 31 – 36 www.elsevier.com/locate/neuint
Evidence for a role for transglutaminase in Huntington’s disease and the potential therapeutic implications Marcela V. Karpuj a, Mark W. Becher b, Lawrence Steinman a,* a
Department of Neurological Sciences, Beckman Center for Molecular Medicine, B002, Stanford Uni6ersity, Stanford, CA 94305, USA b Department of Pathology, Uni6ersity of New Mexico Health Sciences Center, CRTC-B93, Albuquerque, NM 87131, USA
Abstract Transglutaminase (TGase) activity is increased in affected regions of brains from patients with Huntington’s disease (HD). TGase activity is particularly elevated in the nucleus compared with the cytoplasm from these brains. Gamma-glutaminyl–lysyl cross-links have been detected in nuclear inclusions in HD brain, indicating that TGase may play a prominent role in the aggregation of huntingtin (htt). Attempts to ameliorate experimental disease, via inhibition of TGase in transgenic models of HD in mice, are under investigation. © 2001 Elsevier Science Ltd. All rights reserved.
Huntingtin (htt) contains a stretch of glutamines near its N-terminus. When the length of the poly Q domain exceeds 36Q in htt, a lethal neurological disease, Huntington’s disease (HD), occurs. Aggregated htt in the nuclei of neurons, termed neuronal intranuclear inclusions (NII), and in dystrophic neurites in brain are pathologic hallmarks of HD and other polyglutamine diseases (DiFiglia et al., 1997). It remains unknown whether the aggregates are deleterious for neurons, or whether they represent an adaptive response to a desperate situation. Nuclear inclusions are also found in mice transgenic for the HD mutation, which have many of the neurologic features of patients with HD (Davies et al., 1997). Two hypotheses dominate our perception of how htt is aggregated. Perutz (1999) proposed that the polyglutamine domains on neighboring proteins, organize themselves into polar zippers from b-strands that can be assembled into sheets or barrels by hydrogen bonds formed between their main-chain and side-chain amides Perutz, 1999. Such polar zippered aggregations have the classic properties of amyloid, and have sufficient molecular order to intercalate the dye Congo Red, and show red – green birefrigence under polarized light (Scherzinger et al., 1997; Perutz, 1999). The second * Corresponding author. Tel.: + 1-650-725-6401; fax: +1-650-7250627. E-mail address: [email protected] (L. Steinman).
hypothesis was first proposed by Green (Green, 1993; Cooper et al., 1997; Kahlem et al., 1998), who suggested that the enzyme transglutaminase (TGase) covalently links htt into aggregations, which may be harmful. The two hypotheses are not mutually exclusive, and Perutz himself has argued that both mechanisms may occur in HD. Empirical evidence suggests that both mechanisms do occur in NII. Ubiquitinated NII that contain htt as well can be found in many neuronal populations, including striatal neurons of R6/2 mice by four weeks of age (Davies et al., 1997; DiFiglia et al., 1997; Becher et al., 1998). NII containing ubiquitinated aggregates of htt have subsequently been observed in the affected brain areas of patients with HD (DiFiglia et al., 1997; Becher et al., 1998). Isolated, concentrated aggregates of htt from HD brain, trapped on filters demonstrated red –green birefrigence with Congo Red staining, indicative of a b-sheet confirmation (Huang et al., 1998). However, tissue sections of NII from HD brain, show no evidence of red –green birefrigence with Congo Red, as was seen in comparable control sections from Alzheimer’s disease, where red –green birefrigence was present in amyloid plaques (Karpuj et al., 1999). McGowan et al. (2000) have reported that Congo Red birefrigence is seen in 1–2% of inclusions in HD brain that co-stained with an antibody against the N-terminus of htt. No amyloid-like inclusions were seen by McGowan et al. in R6/2 mice with an htt transgene. Recently evidence has
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been presented that gamma-glutaminyl– lysyl bonds can be co-localized with htt in NII in immunohistochemical studies of HD brain, suggesting that TGase plays a role in the formation of such inclusions in HD (Zainelli et al., 2000). The role of the NII in the pathogenesis of HD is unknown. There is debate about whether nuclear inclusions are involved in pathogenesis, or whether they may even have a protective function to sequester aggregated htt (Sisodia, 1998; Saudou et al., 1998). Recently Yamamoto showed in an inducible transgenic model of HD, that turning off the expression of htt was associated with improvement in symptoms and reduction in the extent of nuclear inclusions (Yamamoto et al., 2000; Orr and Zoghbi, 2000).
1. TGase cross-links a fragment of translated htt containing the polyQ domain We examined whether soluble htt constructs could be cross-linked by TGase in vitro. We used a rabbit reticulocyte–lysate system to translate transcripts containing portions of exon 1 of htt with polyQ23, polyQ4l, or polyQ67. We first constructed a 310 amino acid fragment, beginning with the N-terminal methionine of htt, corresponding to a predicted 40 kDa protein. We chose this length because a 40 kDa band, recognized by anti-htt antibodies, in total protein homogenates and in nuclear extracts is detected in HD cortex, but not in control brain (Karpuj et al., 1999). Thus httQ23 is a 310 amino acid fragment of the N-terminus of htt, and httQ4l is a 330 amino acid fragment. We also constructed a 90 amino acid fragment from the N-terminus of htt, httQ23 a 110 amino acid fragment from httQ4l and a 135 amino acid fragment, httQ67. We intrinsically labeled httQ23, httQ41 or httQ67 with S35 methionine. Upon addition of TGase, both httQ23 (310aa) and httQ41 (330aa), aggregate into a high molecular weight polymer within 45 min. There is no increase in aggregation with the longer httQ41 compared with the httQ23 (Karpuj et al., 1999). Similarly we showed that on the addition of TGase, httQ23 (90aa), httQ41 (110aa), and httQ67 (135aa), all aggregate into a high molecular weight polymer within 45 min. There was an increased aggregation with the constructs httQ41 (110aa) and httQ67 (135 aa), containing polyQ domains exceeding the pathologic threshold of Q36, compared with httQ23 (90aa) (PB 0.002 httQ67 vs. httQ23 at 0.1 U Tgase/ml; P B 0.01 httQ67 vs. httQ23 at 0.05 U TGase/ml; P B 0.02 httQ67 vs. httQ41 at 0.1 U TGase/ml, published in Karpuj et al., 1999). Interestingly, there is a direct correlation in the amount of aggregation and the size of the polyQ domain. This phenomenon indicates that the protein context is important in the correlation between the
length of the polyQ segment and the activity of TGase. It is interesting to note that shorter fragments are present after caspase mediated proteolysis of htt; a process, which may be vital in the pathogenesis of HD. Inhibition of caspases, was shown to ameliorate experimental HD (Ona et al., 1999). The ordered structure of amyloid allows binding of the dye Congo Red at regular intervals, producing a characteristic red–green birefringence under polarized light (Prusiner et al., 1983; Karpuj et al., 1999). This red–green birefringence was noted in htt aggregates in vitro after the enzymatic cleavage of the fusion protein GST (Becher et al., 1998). The TGase mediated aggregates of htt in vitro, did not show green birefringence after staining with Congo Red and thus cannot be considered amyloid, based on a widely accepted definition of amyloid (Karpuj et al., 1999; Prusiner et al., 1983). The aggregation of htt is dependent on the TGase activity. Using a monoclonal antibody against TGase that is known to block its activity, CUB 7402 (Neomarkers, Union City, CA), the aggregation of htt is inhibited (Karpuj et al., 1999). TGase itself can be detected in the aggregates of httQ23, httQ41, or httQ67, using monoclonal anti-TGase antibodies. On Western blots with anti-TGase antibodies, products are seen which co-migrate with the S35 labeled httQ23, S35 labeled httQ41, or S35 labeled httQ67 aggregation. It is noteworthy that httQ23, httQ41 and httQ67 are all soluble. In contrast, GST fusion constructs of htt with Q\ 30 are insoluble, after the GST protein is enzymatically cleaved (Scherzinger et al., 1997). The in vitro translated material, in our experiments, is soluble and does not form any aggregates, when analyzed by SDSPAGE after up to 125 h at room temperature (data not shown).
2. TGase activity is increased in nuclei in HD brains Although the effect of guinea pig liver TGase on polyglutamine substrates including htt, has been studied in vitro (Kahlem et al., 1996; Cooper et al., 1997; Kahlem et al., 1998), we explored whether TGase activity is present in HD brain, and whether there is TGase activity in HD brain nuclei. We asked whether an extract from HD brain contains TGase activity. First, we ascertained on Western blots, that TGase is seen in nuclei isolated from the brain of an HD patient (httQ63/Q26) (and from control brain), as well as from cytoplasm isolated from HD brains (httQ44/Q16; htt Q63/Q26), (and from control brain) (Karpuj et al., 1999). Nuclear TGase has been described in neuroblastoma cells in vitro (Lesort et al., 1998), but not heretofore in HD brain.
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In Table 1 (reprinted from Karpuj et al., 1999), we showed that a cytosolic extract from the HD brain and control brain provides enzymatic activity for the incorporation of radiolabeled putrescine, as an amine donor, into dimethylated casein, which serves as an amine acceptor (Lorand and Conrad, 1984; Folk, 1980; Lorand et al., 1979). Enzymatic activity is completely inhibited by MDC (monodansylcadaverine), which competes with radiolabeled putrescine as a substrate for TGase (Table 1). In the HD brain, neuronal loss is prevalent in cortex, while areas like cerebellum, are occasionally affected in adult HD, and routinely affected in juvenile HD. TGase activity was greater in HD brain, than in corresponding areas of control brain (HD cortex 14 88892863 cpm vs. normal cortex 669791410 cpm, mean91SEM, P B 0.009 for HD cortex vs. normal cortex; HD cerebellum 11 22192426 cpm vs. control cerebellum 26069719 cpm, mean9 1SEM PB 0.001 for HD cerebellum vs. control cerebellum). TGase activity was also greater in extracts of nuclei from brains of HD patients than controls (HD brain cortical nuclear extract 63689764 cpm vs. control brain cortical nuclear extract 23579226 cpm, mean9 1SEM PB 0.0001) (Table 1). Our experiment demonstrates that TGase activity is present in compartments of the cell where nuclear inclusions are found in HD patient (cytosol and nuclei). Moreover, it is noteworthy that even in the cortex of HD patients, damaged from the extensive degeneration seen in the disease, that TGase activity is actually increased, compared with
Table 1 HD brain a Acti6ity of TGase in HD and control brain Cortex 14 888 9 2863 b (n= 3) Cerebellum 11 221 9 2426 c (n= 3) Nuclear extract 6368 9764 d (n= 3) Cortex+MDC 94 918 (n=3)
Control brain
66979 1468 b (n= 5) 2606 9 798 c (n= 3) 23579240 d (n= 4) 639 10 (n= 5)
Inhibition of TGase acti6ity with monodansylcada6erine Casein+brain 100 889 9 14866 e lysate +putrescine Casein+brain 6349346 e Lysath +MDC +putrescine From Karpuj et al. (1999). a Mean CPM+1SD, expressed as incorporation of [3H]putrescine NH(CH2)4NH2 into N,N dimethylated casein. b PB0.03. c PB0.013. d PB0.001. e PB0.0001.
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controls. These results were confirmed subsequently by Lesort et al. (1999). Using an immunoblot analysis, they showed that in the striatum the levels of tissue TGase are significantly increased in all HD cases compared with controls. Immunohistochemical staining of the HD striatum revealed that tissue TGase immunoreactivity is markedly increased in HD brains with various degrees of disease, as compared with controls. In the superior frontal cortex, tissue TGase activity is significantly higher in all HD cases as compared with controls. Quantitative analysis of immunoblots demonstrated that tissue TGase levels are elevated in HD grades 2 and 3 cases. Finally, Tissue TGase immunoreactivity within the superior frontal neocortex was shown to be greater in all the HD cases compared with controls.
3. Intranuclear inclusions in human HD neurons cannot be detected with polarized light when stained with Congo Red In order to explore whether the intranuclear protein aggregates found in human postmortem HD cases are capable of incorporating Congo Red to produce applegreen birefringence on polarized light, we developed a double staining method. Given that these inclusions can be demonstrated only by immunocytochemical (or electron microscopic) techniques, it would be difficult to interpret a negative Congo Red stain either by itself not knowing where the inclusions are, or as a double label on a previously reacted section which contains a large antigen–antibody –chromagen complex. Thus, we chose to stain with Congo Red first, photograph many neuronal nuclei with both bright field and polarized light, and then reveal the intranuclear aggregates via immunocytochemistry using a single section, and thus study the identical cells. This technique definitively demonstrates that the nuclei visualized with polarized light, regardless of whether they were positive or negative, definitely contain protein aggregates with the known typical staining characteristics of HD inclusions. The viability of this method was established through the simultaneous staining of similarly prepared neocortical tissue from a case of Alzheimer’s disease with congophilic angiopathy (vascular amyloid) and senile plaques. This positive control for both amyloid and ubiquitin had abundant polarizable amyloid in both blood vessels and senile plaques and ubiquitin immunoreactivity in neurites of senile plaques. With this method, no Congo Red stained material was identified in neuronal nuclei, or other neural components in the HD case, by either bright field or polarized light (Fig. 1, reprinted from Karpuj et al., 1999). This included the numerous nuclei that con-
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Fig. 1. Congo Red staining of intranuclear inclusions in human HD neurons. Congo Red stained intranuclear inclusions in cortical neurons (A, B) from a juvenile HD (genetically confirmed) patient do not show red staining when viewed by bright field (left) or display apple-green birefringence with polarized light (middle). Sequential staining of the identical section with ubiquitin (right) confirms the presence of a typical HD inclusion (arrows) which is distinct from the nucleolus (arrowheads) and is not visualized in the previous images. Amyloid in a blood vessel with congophilic angiopathy and a senile plaque from a patient with Alzheimer’s disease (C) serves as a positive control for both the Congo Red (C, left and middle) and ubiquitin (C, right) stains. Bars: A, B = 10 mm; C =50 mm. Hematoxylin counterstain (from Karpuj et al., 1999).
tained ubiquitin immunoreactive protein aggregates in the same plane of focus (Fig. 1). Although some inclusion-bearing nuclei had chromatin clearing in the area of the protein aggregate, it was not possible in this directed search to predict which nuclei contained inclusions based on the hematoxylin stain only. Thus, in the configuration found in human HD neurons, the protein aggregates of HD do not incorporate Congo Red to yield polarized light birefringence. It appears via immunocytochemistry that the protein aggregates of HD are well defined and large enough to be detected by a dye-based method such as Congo Red. Therefore, this negative result is not simply because of the lack of protein that stains. Rather, it suggests, within the constraints of this postmortem experiment,
that the proteins in these polyglutamine-rich aggregates do not have a classic amyloid-like configuration. Inclusion bodies in DRPLA and in SCA-3, other polyglutamine diseases, have been described. In COS cells, DRPLA aggregates do not stain with Congo Red (Igarashi et al., 1998), though no mention was made of the staining properties in tissue sections from the human brain. In SCA-3, the inclusions (Paulson et al., 1997) do not stain with Congo Red (John Trojanowski, personal communication, ‘the aggregations in SCA-3 are not Congo Red positive’). The in vitro aggregates of htt cross-linked with TGase in our study cannot be considered amyloid either (Prusiner et al., 1983). Thus, neither the TGase catalyzed polymers of htt, nor the polymers that appear in HD tissue have the biophysical
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characteristics of amyloid. The covalent linkages of htt polymerized with TGase probably does not give enough order for the periodic binding of Congo Red, necessary for the red– green birefringence. However, it should be noted that Gusella and colleagues showed that after the purification of aggregates on filters some degree of Congo Red staining is visible (Huang et al., 1998). Thus, it is highly probable that both TGase mediated, and polar zippered aggregations are present in NII in HD. These results may lead to the development of a novel therapy for HD and other diseases involving polyglutamine expansions. We have shown that MDC, a competitor for the substrate of TGase, can block cross-linking of radiolabeled putrescine with casein, in vitro (Table 1). Similarly, the TGase catalyzed polymerization of htt was shown by Green and colleagues to be prevented by competitors of substrate for TGase (Kahlem et al., 1998). Recently, Igarashi et al. (1998) showed the suppression of aggregate formation and apoptosis in COS cells by competitors of TGase mediated cross-linking, including cystamine and MDC. Both these studies open an extremely genuine possibility of attempting to treat diseases mediated by proteins with polyglutamine domains, by inhibiting TGase in brain (Lorand, 1996, 1998; Kahlem et al., 1998). Such investigations are underway, with positive results reported in an abstract form using the TGase inhibitor cystamine (Karpuj et al., 2000). Other novel ways of treating HD including inhibition of caspases (Ona et al., 1999), minocycline (Chen et al., 2000), and carnitine (Ferrante et al., 2000), all modulate alternative pathways in the pathogenesis of HD, and may synergize with the inhibition of TGase. In combination, these various approaches may offer hope for a potential therapy of HD and other polyglutamine disorders.
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Evidence for a role for transglutaminase in Huntington’s disease and the potential therapeutic implications Marcela V. Karpuj a, Mark W. Becher b, Lawrence Steinman a,* a
Department of Neurological Sciences, Beckman Center for Molecular Medicine, B002, Stanford Uni6ersity, Stanford, CA 94305, USA b Department of Pathology, Uni6ersity of New Mexico Health Sciences Center, CRTC-B93, Albuquerque, NM 87131, USA
Abstract Transglutaminase (TGase) activity is increased in affected regions of brains from patients with Huntington’s disease (HD). TGase activity is particularly elevated in the nucleus compared with the cytoplasm from these brains. Gamma-glutaminyl–lysyl cross-links have been detected in nuclear inclusions in HD brain, indicating that TGase may play a prominent role in the aggregation of huntingtin (htt). Attempts to ameliorate experimental disease, via inhibition of TGase in transgenic models of HD in mice, are under investigation. © 2001 Elsevier Science Ltd. All rights reserved.
Huntingtin (htt) contains a stretch of glutamines near its N-terminus. When the length of the poly Q domain exceeds 36Q in htt, a lethal neurological disease, Huntington’s disease (HD), occurs. Aggregated htt in the nuclei of neurons, termed neuronal intranuclear inclusions (NII), and in dystrophic neurites in brain are pathologic hallmarks of HD and other polyglutamine diseases (DiFiglia et al., 1997). It remains unknown whether the aggregates are deleterious for neurons, or whether they represent an adaptive response to a desperate situation. Nuclear inclusions are also found in mice transgenic for the HD mutation, which have many of the neurologic features of patients with HD (Davies et al., 1997). Two hypotheses dominate our perception of how htt is aggregated. Perutz (1999) proposed that the polyglutamine domains on neighboring proteins, organize themselves into polar zippers from b-strands that can be assembled into sheets or barrels by hydrogen bonds formed between their main-chain and side-chain amides Perutz, 1999. Such polar zippered aggregations have the classic properties of amyloid, and have sufficient molecular order to intercalate the dye Congo Red, and show red – green birefrigence under polarized light (Scherzinger et al., 1997; Perutz, 1999). The second * Corresponding author. Tel.: + 1-650-725-6401; fax: +1-650-7250627. E-mail address: [email protected] (L. Steinman).
hypothesis was first proposed by Green (Green, 1993; Cooper et al., 1997; Kahlem et al., 1998), who suggested that the enzyme transglutaminase (TGase) covalently links htt into aggregations, which may be harmful. The two hypotheses are not mutually exclusive, and Perutz himself has argued that both mechanisms may occur in HD. Empirical evidence suggests that both mechanisms do occur in NII. Ubiquitinated NII that contain htt as well can be found in many neuronal populations, including striatal neurons of R6/2 mice by four weeks of age (Davies et al., 1997; DiFiglia et al., 1997; Becher et al., 1998). NII containing ubiquitinated aggregates of htt have subsequently been observed in the affected brain areas of patients with HD (DiFiglia et al., 1997; Becher et al., 1998). Isolated, concentrated aggregates of htt from HD brain, trapped on filters demonstrated red –green birefrigence with Congo Red staining, indicative of a b-sheet confirmation (Huang et al., 1998). However, tissue sections of NII from HD brain, show no evidence of red –green birefrigence with Congo Red, as was seen in comparable control sections from Alzheimer’s disease, where red –green birefrigence was present in amyloid plaques (Karpuj et al., 1999). McGowan et al. (2000) have reported that Congo Red birefrigence is seen in 1–2% of inclusions in HD brain that co-stained with an antibody against the N-terminus of htt. No amyloid-like inclusions were seen by McGowan et al. in R6/2 mice with an htt transgene. Recently evidence has
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been presented that gamma-glutaminyl– lysyl bonds can be co-localized with htt in NII in immunohistochemical studies of HD brain, suggesting that TGase plays a role in the formation of such inclusions in HD (Zainelli et al., 2000). The role of the NII in the pathogenesis of HD is unknown. There is debate about whether nuclear inclusions are involved in pathogenesis, or whether they may even have a protective function to sequester aggregated htt (Sisodia, 1998; Saudou et al., 1998). Recently Yamamoto showed in an inducible transgenic model of HD, that turning off the expression of htt was associated with improvement in symptoms and reduction in the extent of nuclear inclusions (Yamamoto et al., 2000; Orr and Zoghbi, 2000).
1. TGase cross-links a fragment of translated htt containing the polyQ domain We examined whether soluble htt constructs could be cross-linked by TGase in vitro. We used a rabbit reticulocyte–lysate system to translate transcripts containing portions of exon 1 of htt with polyQ23, polyQ4l, or polyQ67. We first constructed a 310 amino acid fragment, beginning with the N-terminal methionine of htt, corresponding to a predicted 40 kDa protein. We chose this length because a 40 kDa band, recognized by anti-htt antibodies, in total protein homogenates and in nuclear extracts is detected in HD cortex, but not in control brain (Karpuj et al., 1999). Thus httQ23 is a 310 amino acid fragment of the N-terminus of htt, and httQ4l is a 330 amino acid fragment. We also constructed a 90 amino acid fragment from the N-terminus of htt, httQ23 a 110 amino acid fragment from httQ4l and a 135 amino acid fragment, httQ67. We intrinsically labeled httQ23, httQ41 or httQ67 with S35 methionine. Upon addition of TGase, both httQ23 (310aa) and httQ41 (330aa), aggregate into a high molecular weight polymer within 45 min. There is no increase in aggregation with the longer httQ41 compared with the httQ23 (Karpuj et al., 1999). Similarly we showed that on the addition of TGase, httQ23 (90aa), httQ41 (110aa), and httQ67 (135aa), all aggregate into a high molecular weight polymer within 45 min. There was an increased aggregation with the constructs httQ41 (110aa) and httQ67 (135 aa), containing polyQ domains exceeding the pathologic threshold of Q36, compared with httQ23 (90aa) (PB 0.002 httQ67 vs. httQ23 at 0.1 U Tgase/ml; P B 0.01 httQ67 vs. httQ23 at 0.05 U TGase/ml; P B 0.02 httQ67 vs. httQ41 at 0.1 U TGase/ml, published in Karpuj et al., 1999). Interestingly, there is a direct correlation in the amount of aggregation and the size of the polyQ domain. This phenomenon indicates that the protein context is important in the correlation between the
length of the polyQ segment and the activity of TGase. It is interesting to note that shorter fragments are present after caspase mediated proteolysis of htt; a process, which may be vital in the pathogenesis of HD. Inhibition of caspases, was shown to ameliorate experimental HD (Ona et al., 1999). The ordered structure of amyloid allows binding of the dye Congo Red at regular intervals, producing a characteristic red–green birefringence under polarized light (Prusiner et al., 1983; Karpuj et al., 1999). This red–green birefringence was noted in htt aggregates in vitro after the enzymatic cleavage of the fusion protein GST (Becher et al., 1998). The TGase mediated aggregates of htt in vitro, did not show green birefringence after staining with Congo Red and thus cannot be considered amyloid, based on a widely accepted definition of amyloid (Karpuj et al., 1999; Prusiner et al., 1983). The aggregation of htt is dependent on the TGase activity. Using a monoclonal antibody against TGase that is known to block its activity, CUB 7402 (Neomarkers, Union City, CA), the aggregation of htt is inhibited (Karpuj et al., 1999). TGase itself can be detected in the aggregates of httQ23, httQ41, or httQ67, using monoclonal anti-TGase antibodies. On Western blots with anti-TGase antibodies, products are seen which co-migrate with the S35 labeled httQ23, S35 labeled httQ41, or S35 labeled httQ67 aggregation. It is noteworthy that httQ23, httQ41 and httQ67 are all soluble. In contrast, GST fusion constructs of htt with Q\ 30 are insoluble, after the GST protein is enzymatically cleaved (Scherzinger et al., 1997). The in vitro translated material, in our experiments, is soluble and does not form any aggregates, when analyzed by SDSPAGE after up to 125 h at room temperature (data not shown).
2. TGase activity is increased in nuclei in HD brains Although the effect of guinea pig liver TGase on polyglutamine substrates including htt, has been studied in vitro (Kahlem et al., 1996; Cooper et al., 1997; Kahlem et al., 1998), we explored whether TGase activity is present in HD brain, and whether there is TGase activity in HD brain nuclei. We asked whether an extract from HD brain contains TGase activity. First, we ascertained on Western blots, that TGase is seen in nuclei isolated from the brain of an HD patient (httQ63/Q26) (and from control brain), as well as from cytoplasm isolated from HD brains (httQ44/Q16; htt Q63/Q26), (and from control brain) (Karpuj et al., 1999). Nuclear TGase has been described in neuroblastoma cells in vitro (Lesort et al., 1998), but not heretofore in HD brain.
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In Table 1 (reprinted from Karpuj et al., 1999), we showed that a cytosolic extract from the HD brain and control brain provides enzymatic activity for the incorporation of radiolabeled putrescine, as an amine donor, into dimethylated casein, which serves as an amine acceptor (Lorand and Conrad, 1984; Folk, 1980; Lorand et al., 1979). Enzymatic activity is completely inhibited by MDC (monodansylcadaverine), which competes with radiolabeled putrescine as a substrate for TGase (Table 1). In the HD brain, neuronal loss is prevalent in cortex, while areas like cerebellum, are occasionally affected in adult HD, and routinely affected in juvenile HD. TGase activity was greater in HD brain, than in corresponding areas of control brain (HD cortex 14 88892863 cpm vs. normal cortex 669791410 cpm, mean91SEM, P B 0.009 for HD cortex vs. normal cortex; HD cerebellum 11 22192426 cpm vs. control cerebellum 26069719 cpm, mean9 1SEM PB 0.001 for HD cerebellum vs. control cerebellum). TGase activity was also greater in extracts of nuclei from brains of HD patients than controls (HD brain cortical nuclear extract 63689764 cpm vs. control brain cortical nuclear extract 23579226 cpm, mean9 1SEM PB 0.0001) (Table 1). Our experiment demonstrates that TGase activity is present in compartments of the cell where nuclear inclusions are found in HD patient (cytosol and nuclei). Moreover, it is noteworthy that even in the cortex of HD patients, damaged from the extensive degeneration seen in the disease, that TGase activity is actually increased, compared with
Table 1 HD brain a Acti6ity of TGase in HD and control brain Cortex 14 888 9 2863 b (n= 3) Cerebellum 11 221 9 2426 c (n= 3) Nuclear extract 6368 9764 d (n= 3) Cortex+MDC 94 918 (n=3)
Control brain
66979 1468 b (n= 5) 2606 9 798 c (n= 3) 23579240 d (n= 4) 639 10 (n= 5)
Inhibition of TGase acti6ity with monodansylcada6erine Casein+brain 100 889 9 14866 e lysate +putrescine Casein+brain 6349346 e Lysath +MDC +putrescine From Karpuj et al. (1999). a Mean CPM+1SD, expressed as incorporation of [3H]putrescine NH(CH2)4NH2 into N,N dimethylated casein. b PB0.03. c PB0.013. d PB0.001. e PB0.0001.
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controls. These results were confirmed subsequently by Lesort et al. (1999). Using an immunoblot analysis, they showed that in the striatum the levels of tissue TGase are significantly increased in all HD cases compared with controls. Immunohistochemical staining of the HD striatum revealed that tissue TGase immunoreactivity is markedly increased in HD brains with various degrees of disease, as compared with controls. In the superior frontal cortex, tissue TGase activity is significantly higher in all HD cases as compared with controls. Quantitative analysis of immunoblots demonstrated that tissue TGase levels are elevated in HD grades 2 and 3 cases. Finally, Tissue TGase immunoreactivity within the superior frontal neocortex was shown to be greater in all the HD cases compared with controls.
3. Intranuclear inclusions in human HD neurons cannot be detected with polarized light when stained with Congo Red In order to explore whether the intranuclear protein aggregates found in human postmortem HD cases are capable of incorporating Congo Red to produce applegreen birefringence on polarized light, we developed a double staining method. Given that these inclusions can be demonstrated only by immunocytochemical (or electron microscopic) techniques, it would be difficult to interpret a negative Congo Red stain either by itself not knowing where the inclusions are, or as a double label on a previously reacted section which contains a large antigen–antibody –chromagen complex. Thus, we chose to stain with Congo Red first, photograph many neuronal nuclei with both bright field and polarized light, and then reveal the intranuclear aggregates via immunocytochemistry using a single section, and thus study the identical cells. This technique definitively demonstrates that the nuclei visualized with polarized light, regardless of whether they were positive or negative, definitely contain protein aggregates with the known typical staining characteristics of HD inclusions. The viability of this method was established through the simultaneous staining of similarly prepared neocortical tissue from a case of Alzheimer’s disease with congophilic angiopathy (vascular amyloid) and senile plaques. This positive control for both amyloid and ubiquitin had abundant polarizable amyloid in both blood vessels and senile plaques and ubiquitin immunoreactivity in neurites of senile plaques. With this method, no Congo Red stained material was identified in neuronal nuclei, or other neural components in the HD case, by either bright field or polarized light (Fig. 1, reprinted from Karpuj et al., 1999). This included the numerous nuclei that con-
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Fig. 1. Congo Red staining of intranuclear inclusions in human HD neurons. Congo Red stained intranuclear inclusions in cortical neurons (A, B) from a juvenile HD (genetically confirmed) patient do not show red staining when viewed by bright field (left) or display apple-green birefringence with polarized light (middle). Sequential staining of the identical section with ubiquitin (right) confirms the presence of a typical HD inclusion (arrows) which is distinct from the nucleolus (arrowheads) and is not visualized in the previous images. Amyloid in a blood vessel with congophilic angiopathy and a senile plaque from a patient with Alzheimer’s disease (C) serves as a positive control for both the Congo Red (C, left and middle) and ubiquitin (C, right) stains. Bars: A, B = 10 mm; C =50 mm. Hematoxylin counterstain (from Karpuj et al., 1999).
tained ubiquitin immunoreactive protein aggregates in the same plane of focus (Fig. 1). Although some inclusion-bearing nuclei had chromatin clearing in the area of the protein aggregate, it was not possible in this directed search to predict which nuclei contained inclusions based on the hematoxylin stain only. Thus, in the configuration found in human HD neurons, the protein aggregates of HD do not incorporate Congo Red to yield polarized light birefringence. It appears via immunocytochemistry that the protein aggregates of HD are well defined and large enough to be detected by a dye-based method such as Congo Red. Therefore, this negative result is not simply because of the lack of protein that stains. Rather, it suggests, within the constraints of this postmortem experiment,
that the proteins in these polyglutamine-rich aggregates do not have a classic amyloid-like configuration. Inclusion bodies in DRPLA and in SCA-3, other polyglutamine diseases, have been described. In COS cells, DRPLA aggregates do not stain with Congo Red (Igarashi et al., 1998), though no mention was made of the staining properties in tissue sections from the human brain. In SCA-3, the inclusions (Paulson et al., 1997) do not stain with Congo Red (John Trojanowski, personal communication, ‘the aggregations in SCA-3 are not Congo Red positive’). The in vitro aggregates of htt cross-linked with TGase in our study cannot be considered amyloid either (Prusiner et al., 1983). Thus, neither the TGase catalyzed polymers of htt, nor the polymers that appear in HD tissue have the biophysical
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characteristics of amyloid. The covalent linkages of htt polymerized with TGase probably does not give enough order for the periodic binding of Congo Red, necessary for the red– green birefringence. However, it should be noted that Gusella and colleagues showed that after the purification of aggregates on filters some degree of Congo Red staining is visible (Huang et al., 1998). Thus, it is highly probable that both TGase mediated, and polar zippered aggregations are present in NII in HD. These results may lead to the development of a novel therapy for HD and other diseases involving polyglutamine expansions. We have shown that MDC, a competitor for the substrate of TGase, can block cross-linking of radiolabeled putrescine with casein, in vitro (Table 1). Similarly, the TGase catalyzed polymerization of htt was shown by Green and colleagues to be prevented by competitors of substrate for TGase (Kahlem et al., 1998). Recently, Igarashi et al. (1998) showed the suppression of aggregate formation and apoptosis in COS cells by competitors of TGase mediated cross-linking, including cystamine and MDC. Both these studies open an extremely genuine possibility of attempting to treat diseases mediated by proteins with polyglutamine domains, by inhibiting TGase in brain (Lorand, 1996, 1998; Kahlem et al., 1998). Such investigations are underway, with positive results reported in an abstract form using the TGase inhibitor cystamine (Karpuj et al., 2000). Other novel ways of treating HD including inhibition of caspases (Ona et al., 1999), minocycline (Chen et al., 2000), and carnitine (Ferrante et al., 2000), all modulate alternative pathways in the pathogenesis of HD, and may synergize with the inhibition of TGase. In combination, these various approaches may offer hope for a potential therapy of HD and other polyglutamine disorders.
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