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Cellular Distribution of Torsin a and Torsin B in Normal Human Brain

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Cellular Distribution of Torsin a and Torsin B in Normal Human Brain ORIGINAL CONTRIBUTION Cellular Distribution of Torsin A and Torsin B in Normal Human Brain Marina Konakova, PhD; Duong P. Huynh, PhD; William Yong, MD; Stefan M. Pulst, MD Background: Early-onset torsion dystonia is a hyper- teins. In Western blot analysis of normal human brain kinetic movement disorder caused by a deletion of 1 glu- homogenates, the antibodies specifically recognized 38-kd tamic acid residue in torsin A protein, a novel member endogenous torsin A and 62-kd endogenous torsin B. Ab- of the AAA family of adenosine triphosphatases. No mu- sorption controls showed that labeling was blocked by tation has been found so far in the closely related torsin cognate peptide used for immunization. Immunolocal- B protein. Little is known about the molecular basis of ization studies revealed that torsin A and torsin B were the disease, and the cellular functions of torsin proteins widely expressed throughout the human central ner- remain to be investigated. vous system. Both proteins displayed cytoplasmic dis- tribution, although torsin B localization in some neu- Objective: To study the regional, cellular, and subcel- rons was perinuclear. Strong labeling of neuronal lular distribution of the torsin A and torsin B proteins. processes was detected for both proteins. Methods: Expression of torsin proteins in the central Conclusions: Torsin A and torsin B have similar distri- nervous system was analyzed by Western blot analysis bution in the central nervous system, although their sub- and immunohistochemistry in human postmortem brain cellular localization is not identical. Strong expression tissues. in neuronal processes points to a potential role for tor- sin proteins in synaptic functioning. Results: We generated polyclonal antipeptide antibod- ies directed against human torsin A and torsin B pro- Arch Neurol. 2001;58:921-927 RIMARY EARLY-ONSET torsion Despite significant progress in un- dystonia is an autosomal derstanding the genetic basis of early- dominant inherited disorder onset torsion dystonia, the molecular that has been linked to the ge- mechanism by which this mutation re- netic locus DYT1 on human sults in the disease phenotype is not well chromosomeP 9q34.1,2 Two genes from this understood. The process is thought to in- locus (TOR1A and TOR1B) have been re- volve a deficiency in dopamine release in cently identified through positional clon- the substantia nigra.5,6 To address the mo- ing.3,4 The TOR1A gene encodes a 37-kd lecular mechanisms of early-onset dysto- protein called torsin A. The adjacentTOR1B nia, several studies have examined the ana- gene encodes a closely related protein called tomical distribution of torsin proteins in torsin B of unknown molecular weight. A the central nervous system (CNS). One of GAG deletion at codon 302 in torsin A re- these studies focused on the expression of sults in loss of 1 glutamic acid and causes torsin A and torsin B messenger RNA disease, although penetrance of this mu- (mRNA) expression in the normal hu- tant allele is only 30%. man brain7—torsin A mRNA was found Sequence analysis of the complemen- in many brain regions, including the do- From the Rose Moss Laboratory tary DNA encoding torsin A and torsin B pamine neurons of the substantia nigra; for Parkinson’s and indicated that these proteins belong to the however, no expression of torsin B mRNA Neurodegenerative Diseases, AAA family of adenosine triphosphatases was detected in the same regions. One Burns and Allen Research (ATPases), associated with diverse cellu- study examined the expression of torsin Institute (Drs Konakova, lar activities. This family of ATPases is de- A protein in the frontal cortex, cerebel- Huynh, and Pulst), the fined by a conserved ATPase domain that lum, and substantia nigra of rat and hu- Department of Surgical 8 Pathology (Dr Yong), and the contains Walker homology sequences. man brains. Localization was nuclear and Division of Neurology Since AAA-type ATPases are implicated in cytoplasmic in neurons; the distribution (Dr Pulst), Cedars-Sinai a wide variety of functions, it is difficult of torsin B protein was not analyzed. Medical Center, UCLA School to predict what cellular processes in- Nuclear localization of torsin A in this of Medicine, Los Angeles, Calif. volve the torsin proteins. study did not correspond to cytoplasmic (REPRINTED) ARCH NEUROL / VOL 58, JUNE 2001 WWW.ARCHNEUROL.COM 921 Downloaded from www.archneurol.com at University of Utah, on March 29, 2012 ©2001 American Medical Association. All rights reserved. METHODS GEL ELECTROPHORESIS AND WESTERN BLOTTING Cell lysates were mixed with an equal volume of 2X load- PREPARATION OF ing buffer (135-mmol/L Tris hydrochloride, pH 6.8; 20% ANTIPEPTIDE ANTIBODIES glycerol; 4% sodium dodecyl sulfate; 5% 2-mercaptoetha- nol; and 0.0025% bromphenol blue) and subjected to one- Antisera against human torsin A and B were generated by dimensional sodium dodecly sulfate–polyacrylamide gel immunizing rabbits with synthetic peptides conjugated to electrophoresis (4%-15% acrylamide gels) according to the keyhole limpet hemocyanin. These peptides were synthe- method of Laemmli.11 sized with a COOH-terminal cysteine. Synthetic peptides After electrophoresis, proteins were transferred to a ni- used for immunization were as follows: peptide 1 trocellulosemembrane(AmershamPharmaciaBiotech)intrans- (GQKRSLSREALQK [residues 51-65]) and peptide 2 fer buffer (25-mmol/L Tris, 192-mmol/L glycine, and 20% (SGKQREDIKLKDIE [residues 224-237]) of human tor- methanol)usingatankblotapparatus.Blotswerestainedwith sin A and peptide 3 (HEQKIKLYQDQLQK [residues 77- PonceauStoverifyequalproteinloadingperlane.After1hour 90]) of human torsin B. of blocking using 5% nonfat milk powder in Tris-buffered salinewithTween(20-mmol/LTris-hydrochloride,pH7.6;137- AFFINITY PURIFICATION mmol/L sodium chloride; and 0.05% Tween 20), blots were OF ANTIBODIES probedwithantibodies(1or2µg/mLdilutedinTris-buffered salinewithTweencontaining5%milkpowder)overnight.The Individual peptides were coupled to cyanogen bromide– primaryantibodiesweredetectedwithhorseradishperoxidase– activated Sepharose 4B (Amersham Pharmacia Biotech, conjugated antirabbit antibodies (1:5000 dilution in Tris- Piscataway, NJ) in 0.1-mol/L carbonate-bicarbonate buffered saline with Tween containing 5% milk powder) and buffer (pH 11) overnight. Antiserum samples were passed an ECL detection system (Amersham Pharmacia Biotech). through the columns in a slow, regulated flow (0.5 mL/min). The columns were then washed with phosphate IMMUNOHISTOCHEMISTRY buffer and peptide-specific antibodies were eluted with Allimmunohistologicalstainingwasperformedon7-µmsec- 0.5-mol/L glycine hydrochloride buffer (pH 2.5). The elu- tions.Tissuesectionsweredewaxedinxyleneandrehydrated ants were immediately neutralized with 10% (vol/vol) throughwashesindecreasingconcentrationsofethanol(100%, Tris base buffer (pH 8.0), desalted, and concentrated with 95%,and70%).Sectionsweresubsequentlytreatedwithapro- Centriprep 30 columns (Amicon Pharmaceuticals Inc, teasecocktail(BiomedaCorp,Hayward,Calif)andavidin-biotin Beverly, Mass). and blocked with 3% normal goat serum. Sections were then incubatedwithtorsinAandBantibodies,20µg/mL,overnight PROTEIN EXTRACTION at4°C.PrimaryantibodiesweredetectedusingaVectoravidin- biotincomplexeliteperoxidasekit(VectorLaboratories,Bur- Nondenaturing Triton X-100 lysates of human trabecular lingame,Calif),enhancedbydiaminobenzidineenhancer(Bio- bone (HTB) cells were made by resuspending cell pellets medaCorp),andvisualizedwithdiaminobenzidine.Sections in ice-cold lysis buffer (100-mmol/L Tris hydrochloride, were counterstained with aqueous hematoxylin (Xymed Co, pH 7.5; 150-mmol/L sodium chloride; 5-mmol/L EDTA; 1% SanFrancisco,Calif).Alldilutionsandwasheswereperformed Triton X-100; and a cocktail of protease inhibitors). Ly- in0.1-mol/Lphosphate-bufferedsalinecontaining0.01%Tri- sates were clarified by centrifuging in a microcentrifuge at ton X-100. The specificity of immunostaining was confirmed full speed for 15 minutes. Brain homogenates were pre- by preabsorption of the primary antibodies with the specific pared as described previously.10 peptides used for immunization, 100 µmol/L. localization of normal and mutant torsin A in cultured dicted amino acid sequences of torsin proteins. Pep- cells.9 tides were chosen based on the analysis of hydrophilicity To address the discrepancies in previous studies and (Kyte-Doolittle scale), antigenic index (Jameson-Wolf to compare torsin A and torsin B protein distribution, method), and surface probability (Emini formula). The we generated and characterized torsin A– and torsin chosen peptide epitopes did not show significant homol- B–specific antibodies. Using these antibodies, we per- ogy to known proteins. Two torsin A–specific antibod- formed a comprehensive comparative analysis of the cel- ies (A1 and A2) and 1 torsin B–specific (B1) antibody lular expression of torsin A and torsin B in the human were prepared. The A1 and A2 antibodies were gener- CNS. We also made the first estimate of the molecular ated using an N-terminal (residues 51-65) and a C- weight of torsin B. terminal (residues 224-237) torsin A peptide, respec- tively. The B1 antibody was raised against an N-terminal RESULTS torsin B peptide (putative residues 77-90). Expression of torsin A and B proteins in the brain PRODUCTION AND EVALUATION was examined by Western blot analysis of whole human OF POLYCLONAL ANTIBODIES brain homogenates (Figure 1A). Analysis using A1and A2 antibodies revealed
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