Supplementary Data for REVISED m/s # 200102577:
Dopamine transmission in DYT1 dystonia: a biochemical and autoradiographical study.
S. J. Augood PhD1, Z. Hollingsworth MPH1, D. S. Albers PhD3, L. Yang MD3, J-C. Leung BS2, B. Muller MD4, C. Klein MD4, X. O. Breakefield PhD2, and D. G. Standaert MD, PhD1
Author for correspondence:
S.J. Augood, Ph.D
Email:
Supplementary Data
(E) Introduction:
Evidence for the dopamine hypothesis of dystonia:
Mutations in genes involved in dopamine signaling 1can result in a dystonic phenotype (see 2 for recent review. Further, polymorphisms within the DA D5 receptor gene have been associated with cervical dystonia 3 and mechanical or ischemic lesions of the striatum, and administration of pharmacological agents that block DA D2 receptors in vivo, can result in a dystonic phenotype. Wild-type torsinA mRNA4 and protein are highly expressed within the nigro-striatal pathway in the normal post-mortem human brain and in pathologic cytoplasmic Lewy body inclusions within nigral DA neurons.5, 6. Together these findings provide compelling data in support of a dysfunction in striatal signaling in DYT1 dystonia, in particular that a perturbation in striatal DA transmission is an etiologic component of the phenotype.
(E) Methods:
Genetic screening for a mutation in the parkin gene:
Genomic DNA was isolated from fresh-frozen brain tissue from the one parkinsonism case bearing the GAG deletion in TOR1A, and was subjected to direct sequencing to detect point mutations and insertions/deltions in the parkin gene. Gene dosage analysis was also carried out to look for heterozygous exon deletions as described previously.7
Striatal content of dopamine and its metabolites.:
Fresh frozen striatal tissue (20-80 mg) was homogenized (1:10) in chilled 0.1 M perchloric acid and DA and its metabolites DOPAC and HVA were measured by high performance liquid chromatography (HPLC) with electrochemical detection.8 Concentrations of DA, DOPAC and HVA are standardized to protein content and expressed as nanograms per milligram of protein.
Statistical Analysis:
Statistical analyses were performed using a commercial software program (InStat 2.01) and significance levels were determined using an un-paired t-test.
Dopamine D1 and D2 receptor binding and dopamine uptake sites.
Quantitative receptor autoradiography was used to assay DA D1-like, D2-like, DA uptake and vesicular uptake binding sites. Levels of specific binding were determined by subtracting nonspecific binding from total binding. Striatal tissue sections from the control and the four DYT1 cases were processed for [3H]-SCH23390 (DA D1-like), [3H]-YM-08151-2 (DA D2-like), [3H]-mazindol (DA uptake) and [3H]-dihydrotetrabenazine (DHTB; VMAT2) ligand binding using standardized protocols.9 All [3H]-ligands were obtained from New England Nuclear (Boston, MA). In brief, assay buffer contained 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM MgCl2, 1M pargyline, and 0.001% ascorbate. For D1-like binding, slides were incubated in the dark with 1.54 nM [3H]-SCH-23390 (specific activity [s.a.] 75.5 Ci/mmol) for 2.5 h @ room temperature. Non-specific binding was defined in the presence of 1 M cis-flupentixol. For D2-like binding, slides were incubated in the dark with 187 pM [3H]-YM-09151-2 (s.a. 85 Ci/mmol) for 3 h @ room temperature. Non-specific binding was defined in the presence of 100 M DA. For DA uptake, slides were pre-washed in ice-cold binding buffer (50 mM Tris-HCl, 5 mM KCl and 300 mM NaCl, pH 7.9) for 5 mins. then incubated with 5.5 nM [3H]-mazindol (s.a. 23.5 Ci/mmol) in the presence of 300 nM desipramine for 1 h @ 4°. Non-specific binding was defined in the presence of 10 µM nomifensine. For VMAT2 binding, slides were warmed to room temperature, prewashed in buffer and then incubated with 5 nM [3H]-DHTB (s.a. 20 Ci/mmol) for 40 mins. @ room temperature. Non-specific binding was defined in the presence of 2 µM tetrabenazine. After incubation with [3H]-ligand, slides were rinsed, dried and apposed to tritium-sensitive film (Hyperfilm 3H, Amersham) with calibrated 14C-standards (ARC, Inc, St. Louis, MO). Films were analyzed using computer-assisted image analysis (M1, Imaging Research, St. Catherine’s, ON, Canada) and the density of the image converted to fmol/mg protein by automated extrapolation from the calibrated standards. Each case was assayed in duplicate and the specific binding was determined by subtraction of the non-specific value from the total binding value. All tissue sections were assayed in parallel.
Effects of freezingmethods on brain catecholamine content:
Striatal DA tissue content was measured in samples from control cases which had been either passive-frozen or quick-frozen in liquid nitrogen vapor, as previous reports have suggested that the method of tissue processing can impact significantly on the content of catecholamines measured 10, 11. We measured a marked and significant increase in tissue DA content in slow, passive-frozen striatal tissue (34.37 ± 3.19 ng/mg protein) compared to rapid, quick frozen (6.35 ±1.20 ng/mg protein) blocks. DOPAC and HVA values (ng/mg protein) were more consistent between the quick frozen (DOPAC, 2.29 ± 1.13; HVA 37.21 ± 3.44) and passive frozen (DOPAC, 2.26 ± 0.27; HVA, 60.02 ± 6.85) samples. The striatal tissue content of DA and its metabolites, DOPAC and HVA, measured here for control passive frozen tissue are consistent with the reports of others9, 12 and underscore the importance of matching postmortem variables when using human tissue, as recently reviewed by Hornykiewicz.12
Inherent problems of human postmortem studies:
When interpreting human post-mortem biochemical studies there are several factors that must be taken into account. Firstly, the method of tissue preparation can significantly influence tissue integrity.13 Our biochemical data comparing striatal tissue DA content in passive-frozen versus quick-frozen tissue, clearly demonstrate that method of freezing must be considered. Secondly, it is now well documented that both pre- and post-mortem variables can impact significantly on catecholamine content, and post-mortem delay has been reported to negatively impact on striatal DA content.12 In this study the post-mortem interval of our passive-frozen control group (26.77 ± 2.3 hrs.) was significantly longer than the DYT1 dystonia group (12.37 ± 1.01 hrs.), suggesting that the actual difference in striatal DA content premortem may have been greater than is represented by the data here. Further, DA ligand binding has been shown to be stable post mortem9 suggesting that this is not a confounding variable. Thirdly, small group sizes can be problematic due to the inherent variability between human cases. In this study we report on three DYT1 dystonia cases which, to our knowledge, is the total number of cases available in U.S. federally-funded brain banks. Thus, additional fresh-frozen post-mortem brain tissue from symptomatic DYT1 dystonia cases is not currently available.
(E) Table 1
Demographics of the postmortem human cases used in this study.
Case: / Dx: / Freezing method / GAG deletionin DYT1 / Age (yrs), Sex / PMI (hrs)
B3229 / Control / Liquid N2 / No / 81, M / 19.2
B4571 / Control / Liquid N2 / No / 71, M / 24.0
B3956 / Control / Liquid N2 / No / 69, M / 14.0
B3423 / Control / Unknown / No / 79, M / 23.3
m96017 / Control / Liquid N2 / No / 66, F / unknown
B4635 / Control / Passive / No / 62, M / 29.2
B4646 / Control / Passive / No / 68, F / 21.5
B4596 / Control / Passive / No / 49, M / 24.6
1 / Dystonia / Passive / Yes / 67, F / 13.5
6 / Dystonia / Passive / Yes / 83, F / 12.5
8 / Dystonia / Passive / Yes / 86, M / 11.1
7 / Parkinsonism / passive / Yes / 79, F / 16.2
Dx; diagnosis, PMI = postmortem interval.
Three of the control cases have been shown previously to express torsinA mRNA.4
(E) References:
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