Fig. 1. Transgene detection by PCR. Products correspond to the following templates: lane 1, negative control, no DNA added; lane 2, pGemHDEL plasmid; lane 3, DNA from a patient; lanes 4 and 5, DNA from nontransgenic mice; lanes 6 and 7, DNA from transgenic mice. Arrow indicates 100 bp.

the phenotype earlier, and their motor activity has been characterized in greater detail than that of the other lines.9,21,22 The progressive neurological phenotype of the R6/2 mice includes weight loss, resting tremor, abrupt movements, seizures, progressive hypoactivity, and the presence of dyskinesia of the limbs, when held by the tail, that develops to clasping feet.9,21,22 We use the R6/1 mice, because we consider that the longer time that elapses to the manifestation of the phenotype may more closely resemble HD, whereas the R6/2 line may be more similar to juvenile HD, in which it is common to observe seizures.15 Because the behavior of the R6/1 mice has not been studied so thoroughly as that of the R6/2 line, and we are interested in determining the relationship between oxidative damage and the expression of the neurological phenotype, we have examined the development of motor behavior of R6/1 mice starting at 11 weeks of age (a time at which R6/1

21 R. J. Carter, L. A. Lione, L. Humby, L. Mangiarini, A. Mahal, G. P. Bates, S. B. Dunnett, and J. A. Morton, J. Neurosci. 19, 3248 (1999).

22 L. A. Lione, R. J. Carter, M. J. Hunt, G. P. Bates, J. A. Morton, and S. B. Dunnett, J. Neurosci. 19, 10428 (1999).

mice are undistinguishabie from wild-type mice by simple observation), up to 35 weeks of age.

To determine the neurological phenotype, both transgenic and nontransgenic littermates are tested for motor activity and for feet clasping. Mice at various ages, ranging from 11 to 35 weeks of age, are tested for motor behavior by an automated system (electronic motility meter 40Fc; Motron Products, Stockholm, Sweden). Mice are placed in a clear plastic box, and both horizontal and vertical movements (rearing up onto their back legs) are automatically recorded for a 10-min session for each animal. Each mouse is subjected to only one motor activity test. Feet clasping is determined by holding the mice by the tail. If a mouse does not clasp its feet within a maximum period of 2 min, it is considered as not presenting the behavior. Mice are tested only once for feet clasping. We also record mouse body weight every week. These simple observations allow us to relate the onset of the neurological phenotype with striatal oxidative damage.

The difference in body weight between R6/1 and wild-type mice becomes evident by 24 weeks of age, and by 35 weeks the weight of transgenic mice is 63% of that of age-matched nontransgenic controls. Decreased horizontal motor activity becomes manifest by 19 weeks of age in transgenic mice. At that age R6/1 mice show a 45% reduction in horizontal motor activity when compared with nontransgenic littermates. By 35 weeks of age, motor behavior of transgenic mice is only 18% compared with age-matched controls. With respect to vertical motor behavior, transgenic mice show a 43% reduction, compared with controls at 24 weeks of age, and by 35 weeks they had only 21% of the activity of wild-type mice. Nontransgenic control mice never present feet clasping behavior, whereas R6/1 mice show the behavior starting at 19 weeks of age. Transgenic mice also present resting tremor.16 It is worth pointing out that all mice that presented the phenotype have been identified as transgenics; behavioral evaluations are relevant to validate the use of this model.

Oxidative Damage Lipid Peroxidation

We determine lipid peroxidation (LP) as a measure of oxidative cell damage induced by the formation of free radicals. In pathological conditions, free radicals cause different cellular toxic effects. One of the most relevant is LP, the most commonly used index to measure the biological effect of free radicals.23 LP is oxidative damage of the polyunsaturated membrane lipids, which are susceptible to oxidation by molecular oxygen through a free radical chain process.24 The final result of LP is a change in the lipid composition of the cell membrane that

23 J. M. Gutteridge and B. Halliwell, Trends Biochem. Sci. 15,129 (1990).

24 B. Halliwell and J. M. Gutteridge, Mol. Aspects Med. 8, 89 (1985).

induces alterations of its physicochemical properties, including increased membrane rigidity, which may eventually lead to cell death due to energy and structural changes.25,26 At different ages both R6/1 mice and nontransgenic littermates are killed by decapitation. Brains are obtained and the striata and other regions (cortices and cerebella) are rapidly dissected out on an ice-cold surface. The formation of lipid-soluble fluorescence is determined on the basis of the method of Triggs and Willmore,27 with modifications.16 Tissue (i.e., one striatum) is homogenized in 3 ml of distilled water, and 1-ml aliquots of the homogenates are added to 4 ml of a chloroform-methanol [high-performance liquid chromatography (HPLC) grade] mixture (2:1, v/v). After stirring, mixtures are ice-cooled for 30 min, and the top phase is gently removed by aspiration; the phase must be completely removed so that it will not interfere with the fluorescence determination. Fluorescence of the chloroform phase is measured at 350-nm excitation and 430-nm emission wavelengths. To calibrate the sensitivity of the assay, the spectrophotometer is adjusted to 140 fluorescence units with a 0.001-mg/ml concentration of a quinine standard prepared in 0.05 M H2SO4. Protein content is determined by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Results are expressed as relative fluorescence units per milligram of protein.

Nitric Oxide Synthase Activity

Nitric oxide is synthesized by the oxidation of a terminal guanidino nitrogen atom of L-arginine by the enzyme nitric oxide synthase (NOS).28 Two major forms of NOS, a constitutive and an inducible form, have been described.29"34 The constitutive form requires calcium, calmodulin, NADPH, and tetrahydrobiopterin (BH4) for full activity,30-35 whereas the inducible form (iNOS) is calcium and calmodulin independent.36-38 The method described to measure NOS activity is

25 P. H. Chan, M. Yurko, and R. A. Fishman, J. Neurochem. 38, 531 (1982).

26 K. Kogure, B. D. Watson, R. Busto, and K. Abe, Neurochem. Res. 7, 1405 (1982).

27 W. P. Triggs and L. J. Willmore, J. Neurochem. 42,976 (1984).

28 R. M. J. Palmer, D. S. Ashton, and S. Moncada, Nature (London) 333, 664 (1988).

29 D. S. Bredt and S. H. Snyder, Proc. Natl. Acad. Sei. U.S.A. 87, 682 (1990).

30 B. Mayer, M. John, and E. Böhme, FEBS Lett. 277, 215 (1990).

31 R. Busse and A. Mulsch, FEBS Lett. 265, 133 (1990).

32 U. Forstermann, J. S. Pollock, H. H. H. W. Schmidt, M. Heller, and F. Murad, Proc. Natl. Acad. Sei. U.S.A. 88, 1788 (1991).

33 R. G. Knowles, M. Merrett, M. Salter, and S. Moncada, Biochem. J. 270, 833 (1990).

34 S. Hauschlidt, A. Lackhoff, A. Mulsch, J. Kohler, W. Bessler, and R. Busse, Biochem. J. 270, 354 (1990).

35 J. Giovannelli, K. L. Campos, and S. Kaufman, Proc. Natl. Acad. Sei. U.S.A. 88, 7091 (1991).

36 S. S. Gross, E. A. Jaffe, R. Levi, and G. Kilbourn, Biochem. Biophys. Res. Commun. 178,823 (1991).

37 R. Busse and A. Mulsch, FEBS Lett. 275, 87 (1990).

38 D. J. Stuehr, H. J. Cho, N. S. Kwon, M. F. Weise, and C. F. Nathan, Proc. Natl. Acad. Sei. U.S.A. 88,7773 (1991).

based on the stoichiometric conversion of L-arginine to L-citrulline, a stable byproduct of NO production from L-arginine.29,39

For each assay we use one striatum. Tissue from R6/1 mice and from nontrans-genic littermates is dissected out. Tissue is homogenized on ice in 250 /il of a buffer containing a cocktail of protease inhibitors. A 50 mMTris-HCl-0.1 mMEDTA-0.1 mM EGTA-0.1% (v/v) 2-mercaptoethanol solution (pH 7.5) is prepared and kept in the refrigerator. Before homogenizing, the protease inhibitors are mixed to the following final concentrations: 100/xMleupeptin, 1 mMphenylmethylsulfonyl fluoride (PMSF) from a 100 mM stock dissolved in ethanol (ethanol may cloud the homogenizing buffer, which must be vortexed frequently), aprotinin (2 /Ltg/ml), soybean trypsin inhibitor (SBTI; 10 /ig/ml), and 0.1% (v/v) Tergitol type NP-40 (Sigma, St. Louis, MO). Protein content is measured by the BCA assay. After the tissue has been homogenized in the presence of the protease inhibitor cocktail, volumes containing 500 ng of protein are taken for each reaction, and the following reagents are added to the mix (all final concentrations): 1 mM NADPH (100 mM stock is 100 mg/1.2 ml of homogenizing buffer without protease inhibitors; prepare fresh); 100 nM calmodulin, from a 10 ¡tM stock in buffer without protease inhibitors kept at —20°; 30 ¡jlM tetrahydrobiopterin (stock solution is 100 mM in sterile water; prepare fresh); and 2.5 mM CaCl2 from a 25 mM solution in Tris buffer. Reaction mix also contains 10 \jlM L-arginine-HCl, from a 10 mM stock (2.1 mg/ml in water; prepare fresh), and 0.2 /¿Ci of L-[3H]arginine (approximately 66 Ci/mmol, 1 /iCi//il; Amersham, Arlington Heights, IL) is added per 100-/xl reaction (labeled arginine is diluted 1:5 in the original Tris buffer, and the necessary amount is added to the master mix). Master mix must be prepared according to the number of samples that will be assayed. Total arginine is 10 ¡iM unlabeled plus 3 pmol labeled for each reaction. Final reaction volumes are adjusted to 100 fi\ with homogenizing buffer. Starting with a 250-/xl volume should be sufficient to run a duplicate of each reaction both in the presence and in the absence of calcium. This final mixture is incubated for 30 min at 37°. Reactions are stopped by adding 1 ml of ice-cold stop buffer (2 mM EGTA, 2 mM EDTA, 20 mM HEPES, pH 5.5). The reaction mixture, now 1.1 ml, is applied to a 1-ml column of Dowex-50W resin that had been previously equilibrated with stop buffer. The cation-exchange resin retains labeled arginine and allows L-[3H]citrulline to flow through the column. Wash the incubation tube with 1 ml of distilled water, vortex, and pass it over the column to wash through the sample. Samples are measured by liquid scintillation spectroscopy [5 ml of Aquasol-2 (New England Nuclear, Boston, MA) per approximately 2 ml of eluate]. Check, by duplicate, total counts of tube containing 0.2 fiCi [3H]arginine of master mix. To determine background radioactivity, tubes are prepared without tissue, and the procedure described is followed. Background should not exceed about 4% of total counts added. Subtract background

39 A. Rengasamy and R. A. Johns, Neuroprotocols Companion Methods Neurosci. 1, 159 (1992).

counts and calculate nanomoles of citrulline generated, knowing the specific activity of the labeled L-arginine added (cpm/nmol). We have expressed results as nanomoles of L-[3H]citrulline per milligram of protein per 30 min. To test for calcium dependency of NOS activity, and thus differentiate NOS and iNOS activities, enzyme activity is measured, both in the presence (2.5 mM) and in the absence of CaCl2, with 1.0 mM EGTA (from a 10 mM stock prepared in water; a drop or two of 1 N NaOH may be needed to dissolve) to chelate residual calcium in the incubation mixture. To prepare the ion-exchange column 100-200 g of Dowex-50W (50X8-200; Sigma) resin is placed in distilled water. The mixture is swirled into a slurry. After the gel settles, the water is removed and the resin is washed twice with 50-100 ml of I N NaOH to convert the acid form of the resin to a salt. NaOH is removed, and the resin is washed with abundant water until the supernatant reaches a pH lower than pH 8.0 (we have observed best and most consistent results when the pH is between pH 7.5 and 7.9). A few minutes before stopping the incubation of the tissue 1 ml of preequilibrated resin is placed in filtration columns (S/P screening columns P 5194; Scientific Products, McGraw Park, IL). Alternatively, the preequilibrated resin can be stored in stop buffer at 4°.

Concluding Remarks

Mice transgenic for the HD mutation represent interesting systems for studying the cellular and molecular mechanisms that underlie the onset of the human disease. Moreover, they may provide relevant models to test therapeutic treatments, including treatments aimed at protecting cells from damage induced by free radicals. This chapter has described the manner in which this laboratory handles R6/1 transgenic mice, and a reliable PCR method to genotype them. Simple behavioral analyses and observations were also discussed that indicate the progression of the neurological phenotype and that can be related to the degree of oxidative damage sustained by the striata of transgenic mice. Finally, two biochemical assays were described that provide information regarding the oxidative status of the brains of transgenic mice. These assays indicate the degree of oxidative damage (LP and NOS activity). These data provide an overview of the relationships between the expression of the HD mutation, the neurological phenotype, and the degree of oxidative damage sustained by the brain.


This work was supported by a CONACYT grant (33042-N).

0 0

Post a comment