Human Molecular Genetics

Figure 3. Western blot analysis of in vitro binding of huntingtin to His-tagged CBS. (A) Rat brain S1 fraction (250 µg) was incubated with a His-CBS sample from E.coli (200 µg) and Ni²⁺-agarose or Ni²⁺-agarose alone. Following washing of the Ni²⁺-agarose, bound proteins were eluted with 50 µl 1× SDS-PAGE sample buffer and 10 µl loaded on 6% SDS-PAGE gels. Total protein attached to the Ni²⁺-agarose was undetectable by standard protein assay (59). Separated proteins were transferred onto nitrocellulose membrane and subjected to standard immunoblotting with anti-huntingtin antibody (see Materials and Methods). Lane 1, total rat S1 protein sample; lane 2, rat S1 bound to His-CBS; lane 3, rat S1 bound to Ni²⁺-agarose alone. (B) Binding of human huntingtin to His-CBS. Conditions as above except that 4% SDS-PAGE gels were used. Lane 1, human S1 bound to Ni²⁺-agarose alone; lane 2, human S1 bound to His-CBS; lane 3, blank lane; lane 4, total human S1 protein sample. (C) Coomassie stained 6% SDS-PAGE gel of human in vitro binding. Lane 1, total human S1 protein sample; lane 2, molecular mass standards; lane 3, human S1 bound to His-CBS; lane 4, human S1 bound to Ni²⁺-agarose.

CBS interacts with full-length huntingtin in vitro

To confirm the interaction between CBS and huntingtin by an independent method, 6× His-tagged CBS was produced in Escherichia coli. A soluble protein fraction (S1) was prepared from rat or human brain tissue and incubated with native recombinant His-CBS prepared from E.coli and Ni²⁺-agarose or with Ni²⁺-agarose alone. Following washing, proteins remaining bound to the Ni²⁺-agarose were separated by SDS-PAGE and subjected to western blot analysis using a well-characterized anti-peptide polyclonal antibody raised to amino acids 1-17 of huntingtin. Figure 3A shows that full-length rat huntingtin binds specifically to His-tagged CBS in vitro (lane 2). Figure 3B shows a similar, although weaker, result for full-length human huntingtin (lane 2). Figure 3C is a Coomassie blue stained 6% polyacrylamide gel of samples from an in vitro binding experiment, showing that nearly all the protein is washed off the Ni²⁺-agarose pellets. The inclusion of 0.5% Triton X-100 in the washing steps made no difference to binding of huntingtin to the NTA-agarose. These results indicate that recombinant CBS and full-length huntingtin do interact in vitro.

DISCUSSION

Both the yeast two-hybrid system and in vitro studies using His-tagged fusion proteins indicate that CBS binds specifically to rat and human huntingtin. Whilst only the N-terminus of huntingtin was used in the yeast two-hybrid screen, nearly full-length rat CBS was detected in the activation domain plasmid and this section of CBS with a His tag at its N-terminus was capable of specifically attaching full-length huntingtin from a clarified, but non-purified, brain homogenate from fresh rat brain and post-mortem frozen human brain samples. The Coomassie blue stained protein analytical gel (Fig. 3C) indicates that no detectable protein was isolated attached to the CBS His tag, but that huntingtin could be specifically detected on western blots (Fig. 3B). Even in partially purified huntingtin samples levels of huntingtin are too low to be detected on an analytical protein gel and must be western blotted for detection (A.L.Jones and J.D.Wood, unpublished results), so it is not surprising that a band corresponding to huntingtin is not seen on this analytical gel. The human huntingtin did not bind as strongly as the rat huntingtin to His-CBS. This could have been because the CBS protein was from rat, although the homology between rat and human CBS is high [>90% amino acid identity; (40)]. The human samples were frozen post-mortem brain tissue (post-mortem interval 24-48 h) and thus degradation will have occurred which may have reduced the specific binding. Nevertheless, both human and rat huntingtin specifically interacted with CBS. The interaction is dependent on more than just the polyglutamine repeat, as bait plasmids containing only the polyglutamine region with a few N-terminal amino acids did not interact with the activation domain plasmid carrying CBS (Table 2). Examination of SBMA and DRPLA bait plasmids carrying normal and expanded CAG repeats indicated that these proteins did not interact with CBS (Table 2). Therefore, it appears that areas of huntingtin outside the polyglutamine repeat tract are important for CBS interaction. The CBS-huntingtin interaction does not appear to be CAG repeat length dependent (Table 1).

Cystathionine [beta]-synthase is a key enzyme in the generation of cysteine from methionine, catalysing formation of cystathionine by condensation of homocysteine and serine. Absence of cystathionine [beta]-synthase activity is associated with homocystinuria, a recessive disorder first recognized in the 1960s (41); the gene was isolated in 1990 (42) and a number of mutations have been detected in homocystinureic patients. A detailed account of this disease can be found in Mudd et al. (43). The initial metabolic consequence of CBS deficiency is intracellular accumulation of the enzyme's substrate, homocysteine, followed by its export from the cell and a rise in the level of homocysteine and its derivatives in plasma, interstitial fluid and urine (44). A number of other metabolic abnormalities also arise, such as rises in plasma Cu²⁺ and ornithine concentrations, but their relationship to the primary deficiency is unknown (45,46). CBS requires pyridoxal phosphate for activity and in some homocystinureics high doses of pyridoxine (vitamin B6) partially restore CBS acitvity (47).

Table 2. Co-transformation filter assays of various repeat-containing binding domain plasmids with the CBS activation domain plasmid in the yeast two-hybrid system

Binding domain construct	Activation domain construct	Interaction
pGBT9 only	Rat CBS amino acids 95-561	-
HD amino acids 1-171, 17 repeats	Rat CBS amino acids 95-561	+
HD amino acids 1-171, 50 repeats	Rat CBS amino acids 95-561	+
HD amino acids 864-1044	Rat CBS amino acids 95-561	-
HD amino acids 864-1215	Rat CBS amino acids 95-561	-
HD amino acids 1279-1695	Rat CBS amino acids 95-561	-
HD amino acids 1403-1695	Rat CBS amino acids 95-561	-
HD amino acids 1895-2063	Rat CBS amino acids 95-561	-
HD amino acids 2741-3144	Rat CBS amino acids 95-561	-
HD amino acids 2893-3144	Rat CBS amino acids 95-561	-
HD amino acids 7-16, 20 repeats	Rat CBS amino acids 95-561	-
HD amino acids 7-16, 57 repeats	Rat CBS amino acids 95-561	-
pAR2419	Rat CBS amino acids 95-561	-
pAR6619	Rat CBS amino acids 95-561	-
DRPLA amino acids 474-515*, 64 repeats	Rat CBS amino acids 95-561	-
pLAMc	Rat CBS amino acids 95-561	-
p53	pSV40	+

Mental retardation is often the first symptom that brings CBS deficiency to clinical attention, through developmental delay in the early years, and is the most frequent abnormality of the CNS (48). Around 20% of CBS-deficient patients have seizures and there is a high prevalence of psychiatric disorders, including depression, behavioural abnormalities and personality disorders (49). Pathology in the brain shows infarcts caused by cerebrovascular inclusions. The possibility that the neurological effects of CBS deficiency may be caused by excitotoxicity in the CNS has been suggested as the basis for the mental retardation and seizures seen in CBS-deficient patients (50). Two of the oxidation products of homocysteine, L-homocysteate and L-homocysteine sulphinate, are known to be potent agonists for NMDA receptors and thus to exert excitotoxic effects on neurons, which can be blocked by NMDA receptor antagonists (51). Both compounds can be detected in the urine of homocystinureic patients but not in normal controls (52).

One of the hypotheses for the selective neuronal death seen in HD is that it occurs through excitotoxic insult (31,32). There are a number of pieces of evidence that support this idea. HD initially affects the striatal area of the basal ganglia, which receives a major glutamatergic input from the cortex, thalamus and subthalamic nucleus (13). The neurochemical and neuropathological characteristics of HD can be mimicked in animals using glutamate receptor agonists such as kaininic and quinolinic acids, both excitotoxic amino acids. HD is characterized by a loss of striatal projection neurons and a sparing of striatal interneurons containing acetylcholine and somatostatin (13) and this pattern of selective vulnerability is also seen in rodents with quinolinic acid lesions of the striatum (31).

No direct link between the possible excitotoxic death of specific subpopulations of striatal neurons and the initial genetic lesion, expansion of a CAG repeat, has been established in HD. However, as the oxidation products of homocysteine are known to be powerful excitotoxins, this could provide an explanation for some of the damage seen in HD brain. Huntingtin may, therefore, bind CBS and inhibit its activity, either directly or by preventing processing of the enzyme to its active form (53). Recent evidence showing specific accumulation of N-terminal huntingtin in the nuclei of neurons in brain areas vulnerable in HD (16) and in animals transgenic for the expanded HD gene (15), which appear to correlate well with disease progression, provides a basis for CBS involvement in the pathology, despite the lack of dependence of binding on repeat length. Such aggregations may provide binding sites for CBS, reducing its activity only in those cells that accumulate huntingtin, which are in the affected brain areas, i.e. any excitotoxic effect is secondary to huntingtin accumulation. Whilst the neuronal death in HD is initially specific, as the disease progresses virtually all neurons in the caudate and putamen are affected and other areas of the striatum and the cortex also atrophy; these are the cells that contain huntingtin-immunoreactive aggregates. Although the inclusions in patients are nuclear, huntingtin-positive dystrophic neurites are also seen in HD brain and in the transgenic mice aggregation appears to commence in the cytoplasm and be followed by translocation to the nucleus (15,16). Although there is strong circumstantial evidence that the aggregation observed in HD and other polyglutamine repeat diseases (54) is part of the pathological effect of the polyglutamine expansion, the actual primary pathological event remains unknown. Any huntingtin accumulation is likely to have a number of toxic effects and any pathological effect of other interacting proteins may also be mediated through such binding, for instance disruption of the neuronal cytoskeleton through interaction with HIP1.

CBS, like huntingtin, is ubiquitous, but the deficiency in CBS in homocystinureics causes defects in specific organs and systems. Heterozygotes, with only one functional CBS allele, possess only half the normal amount of protein and have <50% of normal CBS activity (55). Heterozygotes for homocystinuria are known to be at increased risk of peripheral vascular disease (55), so total CBS activity appears to be important in this respect and indicates that not all activity may need to be abolished for pathology to occur. However, HD patients do not have increased rates of peripheral vascular disease, so a partial reduction in CBS activity in peripheral systems in HD seems unlikely. Likewise, CBS deficiency in early life clearly has major consequences (43) and thus CBS must be active in presymptomatic carriers of the HD mutation. It would therefore seem likely that any effect on the activity of CBS caused through binding to huntingtin is likely to be temporally and spatially specific.

This finding indicates a possible mechanism contributing to the damage observed in HD caudate and subsequently elsewhere in the brain. Future work should examine the distribution of homocysteate and other homocysteine oxidation products in the brain tissue of HD patients and HD transgenic animal models. Assays for CBS activity can be undertaken, although conclusive results may be difficult to obtain as it seems likely that reduction in activity will be restricted to areas affected in HD brain. However, the observation of a possible link between huntingtin and the production of a known excitotoxin, homocysteate, allows a re-examination of the excitotoxic hypothesis in HD. It is also possible that in patients a deficit in CBS activity could be partially alleviated using high levels of vitamin B6 as a therapy.

MATERIALS AND METHODS

Yeast transformants growing on SD-Trp-Leu plates were used to innoculate 2 ml minimal medium and incubated with shaking at room temperature until an OD₆₀₀ of >0.1 was reached. To 100 µl of each culture in a 1.5 ml tube was added 700 ml Z buffer (0.1 M sodium phosphate, pH 7.0, 1 mM KCl, 1 mM MgSO₄) 0.3% [beta]-mercaptoethanol, 50 ml chloroform and 50 ml 0.1% SDS. Samples were vortexed for 30 s and 160 ml 4 mg/ml o-nitrophenylgalactoside in 0.1 M phosphate buffer, pH 7.0, was added. Tubes were incubated at 30°C for 1 h before reactions were quenched by addition of 400 ml 1 M Na₂CO₃. Cell debris was removed by centrifugation at 13 000 r.p.m. in a microfuge for 10 min and the absorbance of the samples was read at 420 nm.

Fusion protein expression

The CBS insert was released from the HybriZAP vector by digestion with EcoRI and XhoI and blunt end cloned into the SmaI site of pQE31 (Qiagen), creating pQECBS. This plasmid was transformed into XLI-Blue and transformants selected on LB plates containing carbenicillin. Individual colonies were grown at 37°C overnight in 5 ml LB containing carbenicillin and this culture used to inoculate 100 ml LB. This was grown to an OD₆₀₀ of 0.7-0.9 before expression was induced for 3 h with 2 mM IPTG. Cells were harvested by centrifugation at 4000 g for 20 min at 4°C and stored overnight at -20°C in 10 ml sonication buffer with protease inhibitors [50 mM NaH₂PO₄, 300 mM NaCl, pH 7.8, Complete protease inhibitors (Boehringer)]. After thawing in cold water, cells were sonicated and centrifuged at >10 000 g for 20 min and the supernatant containing soluble proteins removed.

Preparation of huntingtin samples

For in vitro binding studies whole cell extracts were prepared by homogenizing tissue samples in 5 vol. homogenization buffer with protease inhibitors [50 mM NaH₂PO₄, 300 mM NaCl, 0.5% Triton X-100, pH 7.8, Complete protease inhibitors (Boehringer)], using 20 strokes of a glass homogenizer. Homogenates were centrifuged at 1000 g for 5 min to produce a P1 pellet, which was washed by resuspension in 5 vol. homogenization buffer and recentrifuged for 5 min at 1000 g. The two supernatants were combined to give an S1 fraction and stirred on ice for 20 min. The S1 fraction was clarified by centrifugation at 15 000 r.p.m. for 20 min and filtration through a 0.2 µm filter.

In vitro binding experiments

Five hundred microlitres of pQECBS protein sample were incubated with 500 µl rat or human brain S1 fraction and 50 µl 50% Ni²⁺-agarose slurry (Qiagen) with shaking at room temperature for 1 h. Bound proteins were collected by centrifugation for 10 s and the Ni²⁺-agarose pellet washed with 3 × 1 ml sonication buffer. The addition of 0.5% Triton X-100 to the sonication buffer did not elute any detectable protein from the column. Bound proteins were eluted with SDS sample buffer, separated by 4 or 6% SDS-PAGE and transferred onto nitrocellulose membrane. Filters were incubated with anti-huntingtin serum 675 (1:1000) followed by an anti-rabbit secondary antibody conjugated to horseradish peroxidase.

ACKNOWLEDGEMENTS

We are grateful to the MRC for a Centre Initiative in Neuropsychiatric Genetics and for a studentship (J.M.B.). The SBMA bait plasmids pAR24:15 and pAR66:16 were a kind gift from Drs K.Fischbeck and D.Merry (University of Pennsylvania, PA) and pDR64 a kind gift from Dr S.Tsuji (Niigata University, Japan).

REFERENCES

2. Macdonald,M.E., Ambrose,C.M., Duyao,M.P., Myers,R.H., Lin,C., Srinidhi,L., Barnes,G., Taylor,S.A., James,M., Groot,N., Macfarlane,H., Jenkins,B., Anderson,M.A., Wexler,N.S., Gusella,J.F., Bates,G.P., Baxendale,S., Hummerich,H., Kirby,S., North,M., Youngman,S., Mott,R., Zehetner,G., Sedlacek,Z., Poustka,A., Frischauf,A.M., Lehrach,H., Buckler,A.J., Church,D., Doucettestamm,L., Odonovan,M.C., Ribaramirez,L., Shah,M., Stanton,V.P., Strobel,S.A., Draths,K.M., Wales,J.L., Dervan,P., Housman,D.E., Altherr,M., Shiang,R., Thompson,L., Fielder,T., Wasmuth,J.J., Tagle,D., Valdes,J., Elmer,L., Allard,M., Castilla,L., Swaroop,M., Blanchard,K., Collins,F.S., Snell,R., Holloway,T., Gillespie,K., Datson,N., Shaw,D. and Harper,P.S. (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntingtons disease chromosomes. Cell, 72, 971-983.

3. Rubinsztein,D.C., Leggo,J., Coles,R., Almqvist,E., Biancalana,V., Cassiman,J.J., Chotai,K., Connarty,M., Craufurd,D., Curtis,A., Curtis,D., Davidson,M.J., Differ,A.M., Dode,C., Dodge,A., Frontali,M., Ranen,N.G., Stine,O.C., Sherr,M., Abbott,M.H., Franz,M.L., Graham,C.A., Harper,P.S., Hedreen,J.C., Jackson,A., Kaplan,J.C., Losekoot,M., Macmillan,J.C., Morrison,P., Trottier,Y., Novelletto,A., Simpson,S.A., Theilmann,J., Whittaker,J.L., Folstein,S.E., Ross,C.A. and Hayden,M.R. (1996) Phenotypic characterization of individuals with 30-40 CAG repeats in the Huntington disease (HD) gene reveals hd cases with 36 repeats and apparently normal elderly individuals with 36-39 repeats. Am. J. Hum. Genet., 59, 16-22. MEDLINE Abstract

4. Macmillan,J.C., Snell,R.G., Tyler,A., Houlihan,G.D., Fenton,I., Cheadle,J.P., Lazarou,L.P., Shaw,D.J. and Harper,P.S. (1993) Molecular analysis and clinical correlations of the Huntingtons disease mutation. Lancet, 342, 954-958. MEDLINE Abstract

5. Duyao,M.P., Ambrose,C.M., Myers,R.H., Novelletto,A., Persichetti,E., Barnes,G., Srinidhi,J., Bird,E., Vonsattel,J.P., Macdonald,M.E. and Gusella,J.F. (1993) Trinucleotide repeat length instability and age-of-onset in Huntingtons disease. Am. J. Hum. Genet., 53, 1152.

6. Andrew,S.E., Goldberg,Y.P., Kremer,B., Telenius,H., Theilmann,J., Adam,S., Starr,E., Squitieri,F., Lin,B.Y., Kalchman,M.A., Graham,R.K. and Hayden,M.R. (1993) The relationship between trinucleotide (CAG) repeat length and clinical features of Huntingtons disease. Nature Genet., 4, 398-403. MEDLINE Abstract

7. Difiglia,M., Sapp,E., Chase,K., Schwarz,C., Meloni,A., Young,C., Martin,E., Vonsattel,J.P., Carraway,R., Reeves,S.A., Boyce,F.M. and Aronin,N. (1995) Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons. Neuron, 14, 1075-1081. MEDLINE Abstract

8. Ross,C.A., Li,S.H., Young,W.S., Schilling,G., Li,X.J., Stine,O.C., Margolis,R.L., Wagster,M.V., Ranen,N.G., Hedreen,J.C. and Folstein,S.E. (1993) Expression of IT-15 and lack of reduction in Huntingtons disease (HD). Am. J. Hum. Genet., 53, 85.

9. Trottier,Y., Devys,D., Imbert,G., Saudou,F., An,I., Lutz,Y., Weber,C., Agid,Y., Hirsch,E.C. and Mandel,J.L. (1995) Cellular-localization of the Huntingtons disease protein and discrimination of the normal and mutated form. Nature Genet., 10, 104-110. MEDLINE Abstract

10. Kosinski,C.M., Cha,J.H., Young,A.B., Persichetti,F., Macdonald,M., Gusella,J.F., Penney,J.B. and Standaert,D.G. (1997) Huntingtin immunoreactivity in the rat neostriatum: differential accumulation in projection and interneurons. Exp. Neurol., 144 239-247. MEDLINE Abstract

11. Jou,Y.S. and Myers,R.M. (1995) Evidence from antibody studies that the CAG repeat in the Huntington disease gene is expressed in the protein. Hum. Mol. Genet., 4, 465-469. MEDLINE Abstract

12. Aronin,N., Chase,K., Young,C., Sapp,E., Schwarz,C., Matta,N., Kornreich,R., Landwehrmeyer,B., Bird,E., Beal,M.F., Vonsattel,J.P., Smith,T., Carraway,R., Boyce,F.M., Young,A.B., Penney,J.B. and Difiglia,M. (1995) CAG expansion affects the expression of mutant huntingtin in the Huntingtons disease brain. Neuron, 15, 1193-1201. MEDLINE Abstract

13. Hedreen,J.C. and Folstein,S.E. (1995) Early loss of neostriatal striosome neurons in Huntington's disease. J. Neuropathol. Exp. Neurol., 54, 105-120. MEDLINE Abstract

14. Macmillan,J.C. and Quarrell,O.W.J. (1996) The neurobiology of Huntington's disease. In Harper,P.S. (ed.), Huntington's Disease, 2nd Edn. WB Saunders, London, UK, pp. 317-357.

15. Davies,S.W., Turmaine,M., Cozens,B.A., DiFiglia,M., Sharp,A.H., Ross,C.A., Scherzinger,E., Wanker,E.E., Mangiarini,L. and Bates,G.P. (1997) Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell, 90, 537-548. MEDLINE Abstract

16. DiFiglia,M., Sapp,E., Chase,K.O., Davies,S.W., Bates,G.P., Vonsattel,J.P. and Aronin,N. (1997) Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science, 277, 1990-1993. MEDLINE Abstract

17. Li,X.J., Li,S.H., Sharp,A.H., Nucifora,F.C., Schilling,G., Lanahan,A., Worley,P., Snyder,S.H. and Ross,C.A. (1995) A huntingtin-associated protein enriched in brain with implications for pathology. Nature, 378, 398-402. MEDLINE Abstract

18. Li,X.J., Sharp,A.H., Li,S.H., Dawson,T.M., Snyder,S.H. and Ross,C.A. (1996) Huntingtin-associated protein (HAP1)-discrete neuronal localizations in the brain resemble those of neuronal nitric-oxide synthase. Proc. Natl. Acad. Sci. USA, 93, 4839-4844. MEDLINE Abstract

19. Burke,J.R., Enghild,J.J., Martin,M.E., Jou,Y.S., Myers,R.M., Roses,A.D., Vance,J.M. and Strittmatter,W.J. (1996) Huntington and DRPLA proteins selectively interact with the enzyme gapdh. Nature Med., 2, 347-350.

20. Bao,J., Sharp,A.H., Wagster,M.V., Becher,M., Schilling,G., Ross,C.A., Dawson,V.L. and Dawson,T.M. (1996) Expansion of polyglutamine repeat in huntingtin leads to abnormal protein interactions involving calmodulin. Proc. Natl. Acad. Sci. USA, 93, 5037-5042. MEDLINE Abstract

21. Koshy,B., Matilla,T., Burright,E.N., Merry,D.E., Fischbeck,K.H., Orr,H.T. and Zoghbi,H.Y. (1996) Spinocerebellar ataxia type-1 and spinobulbar muscular-atrophy gene products interact with glyceraldehyde-3-phosphate dehydrogenase. Hum. Mol. Genet., 5, 1311-1318. MEDLINE Abstract

22. Brennan,W.A., Bird,E.D. and Aprille,J.R. (1985) Regional mitochondrial respiratory activity in Huntington's disease brain. J. Neurochem., 44, 1948-1950. MEDLINE Abstract

23. Parker,W.D., Boyson,S.J., Luder,A.S. and Parks,J.K. (1990) Evidence for a defect in NADH:ubiquinone oxidoreductase (complex I) in Huntington's disease. Neurology, 40, 1231-1233. MEDLINE Abstract

24. Gu,M., Gash,M.T., Mann,V.M., Javoyagid,F., Cooper,J.M. and Schapira,A.H.V. (1996) Mitochondrial defect in Huntingtons disease on caudate-nucleus. Ann. Neurol., 39, 385-389. MEDLINE Abstract

25. Koroshetz,W.J., Jenkins,B.G., Rosen,B.R. and Beal,M.F. (1997) Energy metabolism defects in Huntington's disease and effects of coenzyme Q(10). Ann. Neurol., 41, 160-165. MEDLINE Abstract

26. Kalchman,M.A., Graham,R.K., Xia,G., Koide,H.B., Hodgson,J.G., Graham,K.C., Goldberg,Y.P., Gietz,R.D., Pickart,C.M. and Hayden,M.R. (1996) Huntingtin is ubiquitinated and interacts with a specific ubiquitin-conjugating enzyme. J. Biol. Chem., 271, 19385-19394. MEDLINE Abstract

27. Wanker,E.E., Rovira,C., Scherzinger,E., Hasenbank,R., Walter,S., Tait,D., Colicelli,J. and Lehrach,H. (1997) HIP-1: a huntingtin interacting protein isolated by the yeast two-hybrid system. Hum. Mol. Genet., 6, 487-495. MEDLINE Abstract

28. Kalchman,M.A., Koide,H.B., Mccutcheon,K., Graham,R.K., Nichol,K., Nishiyama,K., Kazemiesfarjani,P., Lynn,F.C., Wellington,C., Metzler,M., Goldberg,Y.P., Kanazawa,I., Gietz,R.D. and Hayden,M.R. (1997) HIP1,a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain. Nature Genet., 16, 44-53. MEDLINE Abstract

29. Gutekunst,C.A., Levey,A.I., Heilman,C.J., Whaley,W.L., Yi,H., Nash,N.R., Rees,H.D., Madden,J.J. and Hersch,S.M. (1995) Identification and localization of huntingtin in brain and human lymphoblastoid cell-lines with anti-fusion protein antibodies. Proc. Natl. Acad. Sci. USA, 92, 8710-8714. MEDLINE Abstract

30. Wood,J.D., Macmillan,J.C., Harper,P.S., Lowenstein,P.R. and Jones,A.L. (1996) Partial characterization of murine huntingtin and apparent variations in the subcellular-localization of huntingtin in human, mouse and rat-brain. Hum. Mol. Genet., 5, 481-487. MEDLINE Abstract

31. Beal,M.F., Ferrante,R.J., Swartz,K.J. and Kowall,N.W. (1991) Chronic quinolinic acid lesions in rats closely resemble Huntington's disease. J. Neurosci., 11, 1649-1659. MEDLINE Abstract

32. Difiglia,M. (1990) Excitotoxic injury in the neostriatum: a model for Huntington's disease. Trends Neurosci., 13, 286-289. MEDLINE Abstract

33. Beal,M.F., Hyman,B.T. and Koroshetz,W. (1993) Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases. Trends Neurosci., 16, 125-131. MEDLINE Abstract

34. Beal,M.F., Brouillet,E., Jenkins,B.G., Ferrante,R.J., Kowall,N.W., Miller,J.M., Storey,E., Srivastava,R., Rosen,B.R. and Hyman,B.T. (1993) Neurochemical and histologic characterization of striatal excitotoxic lesions produced by the mitochondrial toxin 3-nitropropionic acid. J. Neurosci., 13, 4181-4192. MEDLINE Abstract

35. Beal,M.F. (1994) Neurochemistry and toxin models in Huntingtons disease. Curr. Opin. Neurol., 7, 542-547. MEDLINE Abstract

36. Mangiarini,L., Sathasivam,K., Seller,M., Cozens,B., Harper,A., Hetherington,C., Lawton,M., Trottier,Y., Lehrach,H., Davies,S.W. and Bates,G.P. (1996) Exon-1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell, 87, 493-506. MEDLINE Abstract

37. Choi,D.W. (1988) Glutamate neurotoxicity and diseases of the nervous system. Neuron, 1, 623-634. MEDLINE Abstract

38. Ankarcrona,M., Dypbukt,J.M., Bonfoco,E., Zhivotovsky,B., Orrenius,S., Lipton,S.A. and Nicotera,P. (1995) Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron, 15, 961-973. MEDLINE Abstract

39. Fields,S. and Song,O.K. (1989) A novel genetic system to detect protein-protein interactions. Nature, 340, 245-246. MEDLINE Abstract

40. Kraus,J.P., Le,K., Swaroop,M., Ohura,T., Tahara,T., Rosenberg,L.E., Roper,M.D. and Kozich,V. (1993) Human cystathionine [beta]-synthase cDNA-sequence, alternative splicing and expression in cultured-cells. Hum. Mol. Genet., 2, 1633-1638. MEDLINE Abstract

41. Mudd,S.H., Finkelstein,J.D., Irreverre,F. and Laster,L. (1964) Homocystinuria: an enzymatic defect. Science, 143, 1443.

42. Kraus,J.P. (1990) Molecular analysis of cystathionine [beta]-synthase-a gene on chromosome 21. Prog. Clin. Biol. Res., 360, 201. MEDLINE Abstract

43. Mudd,S.H., Levy,H.L. and Skovby,F. (1995) Disorders of transsulfuration. In Scriver,C.R., Beaudet,A.L., Sly,W.S. and Valle,D. (eds), The Metabolic and Molecular Bases of Inherited Disease, 7th Edn. McGraw-Hill, New York. pp. 1279-1327.

44. Refsum,H., Helland,S. and Ueland,P.M. (1985) Radioenzymic determination of homocysteine in plasma and urine. Clin. Chem., 31, 624. MEDLINE Abstract

45. Perry,T.L. (1981) Mild elevations of plasma ornithine in homocystinuria. Clin. Chim. Acta, 117, 97.

46. Dudman,N.P.B. and Wilcken,D.E.L. (1983) Increased plasma copper in patients with homocystinuria due to cystathionine [beta]-synthase deficiency. Clin. Chim. Acta, 127, 105. MEDLINE Abstract

47. Barber,G.W. and Spaeth,G.L. (1967) Pyridoxine therapy in homocystinuria. Lancet, i, 337.

48. Mudd,S.H., Skovby,F., Levy,H.L., Pettigrew,K.D., Wilcken,B., Pyeritz,R.E., Andria,G., Boers,G.H.J., Bromberg,I.L., Cerone,R., Fowler,B., Grobe,H., Schmidt,H. and Schweitzer,L. (1985) The natural history of homocystinuria due to cystathionine [beta]-synthase deficiency. Am. J. Hum. Genet., 37, 1-31. MEDLINE Abstract

49. Abbott,M.H., Folstein,S.E., Abbey,H. and Pyeritz,R.E. (1987) Psychiatric manifestations of homocystinuria due to cystathionine [beta]-synthase deficiency: prevalence, natural history, and relationship to neurologic impairment and vitamin B6-responsiveness. Am. J. Med. Genet., 26, 959-969. MEDLINE Abstract

50. Schwarz,S. and Zhou,G.Z. (1991) N-Methyl-D-aspartate receptors and CNS symptoms of homocystinuria. Lancet, 337, 1226-1227. MEDLINE Abstract

51. Schwarz,S., Zhou,G.Z., Katki,A.G. and Rodbard,D. (1990) L-Homocysteate stimulates [³H]MK-801 binding to the phencyclidine recognition site and thus is an agonist for the N-methyl-D-aspartate operated cation channel. Neuroscience, 37, 193-200. MEDLINE Abstract

52. Ohmori,S., Kodama,H., Ikegami,T., Mizuhara,S., Oura,T., Isshiki,G. and Uemera,I. (1972) Unusual sulfur-containing amino acids in the urine of homocystinuric patients: III. Homocysteic acid, homocysteine sulfinic acid, S-(carboxymethylthio)homocysteine, and S-(3-hydroxy-3-carboxy-n-propyl) homocysteine. Physiol. Chem. Phys., 4, 286.

53. Skovby,F., Kraus,J.P. and Rosenberg,L.E. (1984) Biosynthesis and proteolytic activation of cystathionine [beta]-synthase in rat liver. J. Biol. Chem., 259, 588.

54. Paulson,H.L., Perez,M.K., Trottier,Y., Trojanowski,J.Q., Subramony,S.H., Das,S.S., Vig,P., Mandel,J.-L., Fischbeck,K.H. and Pittman,R.N. (1997) Intranuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3. Neuron, 19, 333-344. MEDLINE Abstract

55. Boers,G.H.J., Smals,A.G.H. and Trijbels,F.J.M. (1985) Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease. New Engl. J. Med., 313, 709-715.

56. Nagafuchi,S., Yanagisawa,H., Ohsaki,E., Shirayama,T., Tadokoro,K., Inoue,T. and Yamada,M. (1994) Structure and expression of the gene responsible for the triplet repeat disorder, dentatorubral and pallidoluysian atrophy (DRPLA). Nature Genet., 8, 177-182. MEDLINE Abstract

57. Schiestl,R.H. and Gietz,R.D. (1989) High efficiency transformation of intact cells using single stranded nucleic acids as a carrier. Curr. Genet., 16, 339-346. MEDLINE Abstract

58. Hoffman,C.S. and Winston,F. (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of E. coli. Gene, 57, 267-272. MEDLINE Abstract

59. Peterson,G.L. (1977) A simplification of the protein assay method of Lowry et al.) which is more generally applicable. Anal. Biochem., 83, 346-356. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +44 1222 745175; Fax: +44 1222 747603; Email: wmgalj@cardiff.ac.uk

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