- Email: [email protected]
Drug or poison: dosage is everything
Literature
MOLECULAR MEDICINE TODAY, APRIL 1999 (VOL. 5)
Background image reproduced from Ref. 1, with permission from Oxford University Press.
Huntingtin toxicity: nucleus or cytoplasm? CAG expansions encoding polyglutamine tracts have been identified as the causative mutations in eight neurodegenerative diseases, including Huntington’s disease (HD). These disorders are characterized by intracellular protein inclusions that contain the polyglutamine expansion. Cellular toxicity is determined by the number of glutamines but it is also influenced by fragment size. Inclusions are predominantly nuclear, however, cytoplasmic deposits have also been detected in HD brains. Hackam et al.1 have investigated the link between the subcellular localization of aggregates and toxicity in HD using constructs that express fragments of mutant huntingtin in vitro. Protein was localized using immunofluorescence microscopy and toxicity was assayed by measuring susceptibility to apoptotic stress. They found that the putative nuclear localization signal (NLS) of huntingtin is non-functional so they added a functional NLS to large fragments and a nuclear export sequence to shorter fragments to reverse
Drug or poison: dosage is everything Fabry disease is a lysosomal storage disorder, which is caused by a deficiency of a lysosomal enzyme, a-galactosidase A (a-Gal A) that results in the abnormal metabolism of glycosphingolipids. Fabry patients suffer from renal failure and premature cardiac arrest; no efficient treatment of this disease is currently available. In Fabry patients carrying the R301Q mutation in a-Gal A, the mutant enzyme retains an intact catalytic center but this is improperly folded. As a result, it gets ‘trapped’ in the endoplasmic reticulum and does not reach its final destination, the lysosome. When studying the effects of a potent inhibitor of a-Gal A, 1-deoxy-galactonojirimycin (DGJ), Fan et al.1 made a surprising discovery: when administered at lower doses, DGJ did not inhibit but enhanced a-Gal A activity in lymphoblasts from R301Q Fabry patients. Using reverse transcription (RT)-PCR and western-blot analyses, they found that in lymphoblasts cultured with DGJ, the amount of a-Gal A mRNA was unchanged but the amount of mature protein
146
their normal localizations. Irrespective of fragment size, and regardless of the length of the polyglutamine tract, they found no significant differences in the toxicity of nuclear or cytoplasmic aggregates. This work opens up the debate on the role of intraneuronal inclusions in disease pathogenesis as others have reported that cytotoxicity is exclusively nuclear2. These issues are of paramount importance if we are to target therapeutics effectively. 1 Hackam, A.S. et al. (1999) In vitro evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington’s disease, Hum. Mol. Genet. 8, 25–33 2 Saudou, F. et al. (1998) Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions, Cell 95, 55–66 Sarah Lloyd PhD [email protected]
was substantially increased and more of it was transported to the lysosomes. This suggests that at lower doses DGJ acts as a molecular chaperone and forces the mutant enzyme to adopt the proper configuration. DGJ was then administered to transgenic mice expressing the R301Q mutant enzyme. The consumption of the drug resulted in a severalfold increase of a-Gal A activity in the heart and kidney without any toxic effects to the treated animals. These results suggest that DGJ could be a long-awaited treatment of Fabry disease; because several DGJ derivatives are known potent inhibitors of other glycosidases, this type of drug could become a new paradigm to treat other metabolic disorders. These findings support an old adage: dosage is everything. 1 Fan, J-Q. et al. (1999) Accelerated transport and maturation of lysosomal a-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor, Nat. Med. 5, 112–115 Eugene Ivanov PhD [email protected]
Alphaviruses to the rescue? The ability to genetically engineer animal viruses enables their application to medical research. The development of non-retroviral RNA viruses as vectors to deliver foreign genes for vaccination and gene therapy is particularly interesting, especially the domestication of the alphavirus group. Two papers in this field have recently been published1,2. Alphaviruses offer several advantages as expression vectors: they have a broad host range, high levels of expression and a small genome that is easy to manipulate. Vectors based on Semliki Forest virus (SFV), Sindbis virus and Venezuelan equine encephalitis have been constructed and deliver and express their cargo efficiently. However, the generation of wild-type virus, owing to recombination between the artificial genome and a helper genome, remains a problem in many recombinant viral systems. This biosafety issue is a concern, especially if vectors are to be used in vaccine delivery, and methods have been developed to reduce this risk. In the SFV system, Smerdou and Liljeström1 have prevented the production of wild-type virus by using two, independent helper RNAs; this system also circumvents the requirement of a viral protease and this portion of the genome has been mutated, conferring additional biosafety. Thus, at least two recombination events and one reversion are required to generate replication-competent virus particles. Extensive analysis has failed to demonstrate the presence of wild-type viruses, emphasizing the biosafety of the two-helper system. The expression strategy of alphaviruses imposes a severe burden on normal cellular biochemistry and infected cells die quickly, restricting the use of these vectors when long-term gene expression is required. However, Agapov et al.2 have developed noncytotoxic Sindbis virus vectors, which produce large amounts of foreign protein, by transfecting Sindbis virus replicons that express a puromycin N-acetyltransferase into cells grown in the presence of puromycin. Cells that survive in the presence of the drug carry replicons that are noncytotoxic and express the puromycin-inactivating enzyme. Such replicons can then be engineered to express other foreign genes for long periods in a noncytotoxic manner, which could be useful in both vaccinology and in the development of gene therapy. 1 Smerdou, C. and Liljeström, P. (1999) Two-helper RNA system for production of recombinant Semliki Forest virus particles, J. Virol. 73, 1092–1098 2 Agapov, E.V. et al. (1998) Noncytopathic Sindbis virus RNA vectors for heterologous gene expression, Proc. Natl. Acad. Sci. U. S. A. 95, 12689–12994 Roger Hewson [email protected]
1357-4310/99/$ - see front matter © 1999 Elsevier Science. All rights reserved.
MOLECULAR MEDICINE TODAY, APRIL 1999 (VOL. 5)
Background image reproduced from Ref. 1, with permission from Oxford University Press.
Huntingtin toxicity: nucleus or cytoplasm? CAG expansions encoding polyglutamine tracts have been identified as the causative mutations in eight neurodegenerative diseases, including Huntington’s disease (HD). These disorders are characterized by intracellular protein inclusions that contain the polyglutamine expansion. Cellular toxicity is determined by the number of glutamines but it is also influenced by fragment size. Inclusions are predominantly nuclear, however, cytoplasmic deposits have also been detected in HD brains. Hackam et al.1 have investigated the link between the subcellular localization of aggregates and toxicity in HD using constructs that express fragments of mutant huntingtin in vitro. Protein was localized using immunofluorescence microscopy and toxicity was assayed by measuring susceptibility to apoptotic stress. They found that the putative nuclear localization signal (NLS) of huntingtin is non-functional so they added a functional NLS to large fragments and a nuclear export sequence to shorter fragments to reverse
Drug or poison: dosage is everything Fabry disease is a lysosomal storage disorder, which is caused by a deficiency of a lysosomal enzyme, a-galactosidase A (a-Gal A) that results in the abnormal metabolism of glycosphingolipids. Fabry patients suffer from renal failure and premature cardiac arrest; no efficient treatment of this disease is currently available. In Fabry patients carrying the R301Q mutation in a-Gal A, the mutant enzyme retains an intact catalytic center but this is improperly folded. As a result, it gets ‘trapped’ in the endoplasmic reticulum and does not reach its final destination, the lysosome. When studying the effects of a potent inhibitor of a-Gal A, 1-deoxy-galactonojirimycin (DGJ), Fan et al.1 made a surprising discovery: when administered at lower doses, DGJ did not inhibit but enhanced a-Gal A activity in lymphoblasts from R301Q Fabry patients. Using reverse transcription (RT)-PCR and western-blot analyses, they found that in lymphoblasts cultured with DGJ, the amount of a-Gal A mRNA was unchanged but the amount of mature protein
146
their normal localizations. Irrespective of fragment size, and regardless of the length of the polyglutamine tract, they found no significant differences in the toxicity of nuclear or cytoplasmic aggregates. This work opens up the debate on the role of intraneuronal inclusions in disease pathogenesis as others have reported that cytotoxicity is exclusively nuclear2. These issues are of paramount importance if we are to target therapeutics effectively. 1 Hackam, A.S. et al. (1999) In vitro evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington’s disease, Hum. Mol. Genet. 8, 25–33 2 Saudou, F. et al. (1998) Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions, Cell 95, 55–66 Sarah Lloyd PhD [email protected]
was substantially increased and more of it was transported to the lysosomes. This suggests that at lower doses DGJ acts as a molecular chaperone and forces the mutant enzyme to adopt the proper configuration. DGJ was then administered to transgenic mice expressing the R301Q mutant enzyme. The consumption of the drug resulted in a severalfold increase of a-Gal A activity in the heart and kidney without any toxic effects to the treated animals. These results suggest that DGJ could be a long-awaited treatment of Fabry disease; because several DGJ derivatives are known potent inhibitors of other glycosidases, this type of drug could become a new paradigm to treat other metabolic disorders. These findings support an old adage: dosage is everything. 1 Fan, J-Q. et al. (1999) Accelerated transport and maturation of lysosomal a-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor, Nat. Med. 5, 112–115 Eugene Ivanov PhD [email protected]
Alphaviruses to the rescue? The ability to genetically engineer animal viruses enables their application to medical research. The development of non-retroviral RNA viruses as vectors to deliver foreign genes for vaccination and gene therapy is particularly interesting, especially the domestication of the alphavirus group. Two papers in this field have recently been published1,2. Alphaviruses offer several advantages as expression vectors: they have a broad host range, high levels of expression and a small genome that is easy to manipulate. Vectors based on Semliki Forest virus (SFV), Sindbis virus and Venezuelan equine encephalitis have been constructed and deliver and express their cargo efficiently. However, the generation of wild-type virus, owing to recombination between the artificial genome and a helper genome, remains a problem in many recombinant viral systems. This biosafety issue is a concern, especially if vectors are to be used in vaccine delivery, and methods have been developed to reduce this risk. In the SFV system, Smerdou and Liljeström1 have prevented the production of wild-type virus by using two, independent helper RNAs; this system also circumvents the requirement of a viral protease and this portion of the genome has been mutated, conferring additional biosafety. Thus, at least two recombination events and one reversion are required to generate replication-competent virus particles. Extensive analysis has failed to demonstrate the presence of wild-type viruses, emphasizing the biosafety of the two-helper system. The expression strategy of alphaviruses imposes a severe burden on normal cellular biochemistry and infected cells die quickly, restricting the use of these vectors when long-term gene expression is required. However, Agapov et al.2 have developed noncytotoxic Sindbis virus vectors, which produce large amounts of foreign protein, by transfecting Sindbis virus replicons that express a puromycin N-acetyltransferase into cells grown in the presence of puromycin. Cells that survive in the presence of the drug carry replicons that are noncytotoxic and express the puromycin-inactivating enzyme. Such replicons can then be engineered to express other foreign genes for long periods in a noncytotoxic manner, which could be useful in both vaccinology and in the development of gene therapy. 1 Smerdou, C. and Liljeström, P. (1999) Two-helper RNA system for production of recombinant Semliki Forest virus particles, J. Virol. 73, 1092–1098 2 Agapov, E.V. et al. (1998) Noncytopathic Sindbis virus RNA vectors for heterologous gene expression, Proc. Natl. Acad. Sci. U. S. A. 95, 12689–12994 Roger Hewson [email protected]
1357-4310/99/$ - see front matter © 1999 Elsevier Science. All rights reserved.