Immunocytochemical Characterization of the Mitochondrially Encoded ND1 Subunit of Complex I (NADH : Ubiquinone Oxidoreductase) in Rat Brain

Immunocytochemical Characterization of the MitochondriallyEncoded ND1 Subunit of Complex I (NADH:Ubiquinone

Oxidoreductase) in Rat Brain

*Edward H. Pettus, *Ranjita Betarbet, ‡Barbara Cottrell, ‡Douglas C. Wallace,*Vijay Madyastha, and *†§J. Timothy Greenamyre

Departments of*Neurology and†Pharmacology,‡Center for Molecular Medicine, and§Yerkes Regional PrimateResearch Center, Emory University, Atlanta, Georgia, U.S.A.

Abstract: In Parkinson’s disease, there is a selectivedefect in complex I of the electron transfer chain. Tobetter understand complex I and its involvement in neu-rodegenerative disease, we raised an antibody against aconserved epitope of the human mitochondrially en-coded subunit 1 of complex I (ND1). Antibodies wereaffinity purified and assessed by ELISA, immunoblotting,and immunocytochemistry. Immunoblots of brain ho-mogenates from mouse, rat, and monkey brain showed asingle 33-kDa band consistent with the predicted molec-ular mass of the protein. Subcellular fractionation showedthe protein to be enriched in mitochondria. Immunocyto-chemistry in rat brain revealed punctate labeling in cellbodies and processes of neurons. Immunoreactivity gen-erally co-localized with subunit IV of complex IV. In stri-atum, ND1 immunoreactivity was greatly enriched inlarge cholinergic neurons and neurons containing nitricoxide synthase, two cell populations that are resistant toexcitotoxic and metabolic insults. In substantia nigra,many dopaminergic neurons had little ND1 immunoreac-tivity, which may help to explain their sensitivity to com-plex I inhibitors. In spinal cord, ND1 immunoreactivitywas enriched in motor neurons. We conclude that com-plex I is differentially distributed across brain regions,between neurons and glia, and between types of neu-rons. This antibody should provide a valuable tool forassessing complex I in normal and pathological condi-tions. Key Words: Mitochondria—Complex I—Mitochon-drially encoded subunit I of NADH dehydrogenase (com-plex I)—Immunocytochemistry—Complex IV.J. Neurochem. 75, 383–392 (2000).

Mitochondrial oxidative phosphorylation (OXPHOS)accounts for;90% of the ATP production in neurons(Erecinska and Dagani, 1990). The OXPHOS systemoperates by the coordinated activity of the electron trans-fer chain (ETC) (complex I, NADH:ubiquinone oxi-doreductase; complex II, succinate ubiquinone oxi-doreductase; complex III, ubiquinol cytochromec oxi-doreductase; and complex IV, cytochromec oxidase),which uses the energy of reducing equivalents (NADH

and FADH2) to create a proton gradient that is used byATP synthase to generate ATP.

Complex I is the largest enzyme complex in theOXPHOS system. It comprises at least 41 subunits(Walker, 1995; Guenebaut et al., 1998), seven of whichare encoded by the mitochondrial genome (Chomynet al., 1986). The enzyme complex is composed of ahydrophobic transmembrane domain and a hydrophilicdomain that is exposed to the matrix side of the innermitochondrial membrane (Ragan, 1987; Patel et al.,1988). The NADH binding site, where electrons aretransferred from NADH to complex I, is in the hydro-philic domain (Deng et al., 1990; Pilkington et al., 1991;Fecke et al., 1994). Within this enzyme complex, elec-trons move through the various functional domains in aseries of reduction–oxidation reactions and are trans-ferred to ubiquinone in the hydrophobic domain(Friedrich et al., 1989).

The complex I subunits encoded by the mitochondrialgenome (ND1–6, ND4L) are all highly hydrophobic andcritical for enzyme activity (Friedrich et al., 1989;Tuschen et al., 1990; Wang et al., 1991). Mutations inthesesubunits are associated with a variety of neurode-generative disorders, most notably Leber’s hereditaryoptic neuropathy (Howell et al., 1991; Brown et al.,

Received December 22, 1999; revised manuscript received February29, 2000; accepted March 1, 2000.

Address correspondence and reprint requests to Dr. J. T. Greenamyreat Department of Neurology, Emory University, WMRB 6000, 1639Pierce Dr., Atlanta, GA 30322, U.S.A. E-mail: [email protected]

Drs. E. H. Pettus and R. Betarbet contributed equally to the article.Abbreviations used:BSA, bovine serum albumin; ChAT, choline

acetyltransferase; COXIV, nuclear encoded subunit IV of cytochromeoxidase (complex IV); ETC, electron transfer chain; FITC, fluoresceinisothiocyanate; ICC, immunocytochemistry; ND1, mitochondrially en-coded subunit 1 of NADH dehydrogenase (complex I); ND1-ir, ND1immunoreactivity; nNOS, neuronal nitric oxide synthase; OXPHOS,oxidative phosphorylation; PBS, phosphate-buffered saline; PVDF,polyvinylidene difluoride; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; TH, tyrosine hydroxylase; TSA, tyramide signal am-plification.

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Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 2000 International Society for Neurochemistry

1992a,b). In addition to defined mitochondrial mutationsof complex I subunits, impaired complex I activity hasbeen associated with Parkinson’s disease (Schapira et al.,1989, 1990; Shoffner et al., 1991; Cardellach et al., 1993;Reichmann et al., 1993; Janetzky et al., 1994), Hunting-ton’s disease (Arenas et al., 1998), and lathyrism (Paiand Ravindranath, 1993; Sriram et al., 1998).

As noted, genetic and biochemical studies support arole for complex I impairment in the pathogenesis ofneurodegeneration; however, with the exception of[3H]dihydrorotenone binding (Greenamyre et al., 1992;Higgins and Greenamyre, 1996), available techniques toassay complex I have been unable to distinguish betweenneuronal and glial components or to provide a compre-hensive anatomical map of complex I distribution. Tounderstand a specific role for complex I in neurodegen-erative disorders, it will be important to define the re-gional and cellular distribution of the enzyme and deter-mine how this relates to pathological changes. The cur-rent communication reports the generation of anantibody specific to the mitochondrially encoded ND1subunit of complex I. This antibody has enabled us tocharacterize the regional and cellular distribution ofcomplex I in the central nervous system.

MATERIALS AND METHODS

Peptide and antibody productionA polyclonal antibody was raised against a synthetic peptide

conjugated to a carrier protein, bovine serum albumin (BSA).The amino acid sequence chosen for peptide synthesis relied onthe published human sequence for ND1 (Arnason et al., 1996).The specific sequence used for the peptide construct, ND133–43

(NH2-NQLRKGPNVVGPY-COOH; MW 5 1,441.56), wasselected based on its predicted antigenic characteristics(MacVector; Oxford Molecular Group, San Jose, CA, U.S.A.)and assessed for sequence homology in the NCBI Entrez databank (National Center for Biotechnology Information, Be-thesda, MD, U.S.A.). The ND133–43 peptide was synthesizedwith tert-butyloxycarbonyl, purified by HPLC, and character-ized by microbore protein analysis and mass spectroscopy atthe Emory University Microchemistry Facility (Dr. Jan Pohl).Twelve milligrams of the peptide was conjugated to 25 mg ofBSA via a bis-diazobenzidine-tyrosine coupling reaction, asdescribed elsewhere (Doolittle, 1986). Following dialysis, theconcentration of peptide conjugated to BSA was assessed byEdman degradation amino-terminus sequence analysis.

The antibody host animal was a New Zealand rabbit (Co-vance, Denver, PA, U.S.A.). Conjugated peptide was adminis-tered subcutaneously to the host every 21 days (500-mg initialdose, 250-mg second, third, and fourth dose, and 150-mg main-tenance dose), and serum was drawn 10 days following antigenadministration. Antibodies specific for ND133–43 (ND1) wereisolated from serum by affinity column chromatography. First,serum was passed twice over a BSA-agarose column (Sigma,St. Louis, MO, U.S.A.) to eliminate anti-BSA antibodies fromthe effluent. The collected effluent from the BSA column wasthen passed over an ND133–43-Sepharose column. The boundantibodies were eluted with 0.2M glycine (pH 2.4), collected in0.8-ml fractions, brought to pH 7.4 with 1.0M Tris (pH 10),and then assayed for protein concentration using a Bio-Rad DCprotein assay kit (Bio-Rad, Hercules, CA, U.S.A.). Antibodieswere frozen and stored at220°C.

ELISASerial dilutions of the antigens, ND133–43 and BSA, were

prepared in 20 mM Tris (pH 7.4), and 100ml of each dilutionwas added to individual wells of a 96-well flat-bottom ELISAplate and incubated at 37°C for 1 h. The antibody was seriallydiluted (KPL diluent; KPL, Gaithersburg, MD, U.S.A.) at1:200, 1:400, 1:600, 1:800, 1:1,200, 1:6,400, and 1:12,800,applied to the wells, and incubated at 37°C for 1 h. Wells werewashed three times in 250ml of phosphate-buffered saline(PBS)/0.05% Tween-20, and 100ml of horseradish peroxidase-conjugated goat anti-rabbit antibody (1:2,000; KPL) was ap-plied to all wells. Wells were washed twice with 250ml ofPBS/0.05% Tween-20 and then three times with 14 mM Tris(pH 7.4). 2,29-Azinodi(3-ethylbenzdiazoline sulfonate) (100ml) was applied to each well and allowed to react for;10 minuntil the color was adequate for densitometry. The chromo-genic reaction was halted by addition of 5% sodium dodecylsulfate (SDS) in water. Densitometry of the microtiter plateswas conducted on a Thermomax plate reader (Molecular De-vices, Sunnyvale, CA, U.S.A.).

ImmunoblotWhole-tissue homogenates or subcellular fractions were

used for immunoblotting. Subcellular fractions, including crudemitochondrial fractions, were prepared according to Gray andWhittaker (1962). Fresh tissue samples were homogenized inbuffer H (210 mM mannitol, 70 mM sucrose, 1 mM EGTA, 5mM HEPES) with protease inhibitors (1mg/ml apoprotinin,leupeptin, and pepstatin A) and assayed for protein concentra-tion as described above. Laemmli SDS sample buffer wasadded to aliquots of homogenate, which were incubated over-night at 37°C in microcentrifuge tubes. Samples were thenloaded on a 12% acrylamide minigel and run at 130 V inrunning buffer (0.1% SDS, 125 mM Tris base, 1M glycine) ona Bio-Rad minigel apparatus. Gels were rinsed in transferbuffer (10% methanol, 25 mM Tris, 192 mM glycine) for 10min, apposed to polyvinylidene difluoride (PVDF) membrane(Millipore, Bedford, MD, U.S.A.), and transferred overnight at35 V under cooling conditions. PVDF membranes were incu-bated in KPL blocker for.3 h on a shaker at room temperatureand then incubated in blocker containing primary antibody(1:500) and overnight at 4°C on a shaker. Membranes werethoroughly rinsed in PBS/Tween, incubated with horseradishperoxidase-conjugated secondary antibody for 1 h at roomtemperature, and rinsed thoroughly in PBS/Tween. Secondaryantibodies were detected by chemiluminescence (AmershamECL, U.K., or Pierce CL or Ultra CL, Rockford, IL, U.S.A.).

Immunocytochemistry (ICC)Six adult Sprague–Dawley rats were used for this study. The

rats were anesthetized with Equithesin and transcardially per-fused with chilled isotonic saline, followed by 3% paraformal-dehyde in phosphate buffer. Brains were harvested and post-fixed in 3% paraformaldehyde for 1 h. They were then cryo-protected in 30% sucrose in 0.1M phosphate buffer. Brainswere frozen on dry ice and cut at 40mm on a sliding micro-tome. The brain sections were washed in Tris-buffered saline(TBS; 4 3 30 min) and incubated in endogenous peroxidaseinhibitor for 10 min (3% H2O2 in TBS). Following a 33 10-min wash in TBS, tissue was incubated in 10% normal goatserum in TBS/0.1 % Triton X-100 for 1 h. Sections were thenincubated for 48 h in the same solution containing either aprimary antibody to ND1 (polyclonal rabbit 1:1,000) or to thenuclear encoded subunit IV of cytochrome oxidase (complexIV) (COXIV; monoclonal mouse, 1:500; Molecular Probes,

J. Neurochem., Vol. 75, No. 1, 2000

384 E. H. PETTUS ET AL.

Eugene, OR, U.S.A.) or a combination of primary antibodiesincluding antibodies to ND1 and antibodies to choline acetyl-transferase (ChAT; monoclonal mouse, 1:100; Dr. BruceWainer, Emory University, Atlanta, GA, U.S.A.) (Levey et al.,1983), tyrosine hydroxylase (TH; monoclonal mouse, 1:2,000;Chemicon, Temecula, CA, U.S.A.), or neuronal nitric oxidesynthase (nNOS; polyclonal rabbit, 2mg/ml; Upstate Biotech-nology, Lake Placid, NY, U.S.A.). ND1 and COXIV werevisualized using the tyramide signal amplification (TSA) pro-cedure (Shindler and Roth, 1996; Betarbet and Greenamyre,1999). Following primary antibody incubation, sections wereincubated in appropriate biotinylated secondary antibody (bio-tinylated goat anti-rabbit or anti-mouse IgG, 1:200; JacksonImmunoResearch Laboratories, West Grove, PA, U.S.A.). TSAkits from NEN (Boston, MA, U.S.A.) were then used to am-plify the antigen signal. Sections were incubated in streptavidinconjugated to horseradish peroxidase (1:100) for 1 h and bio-tinylated tyramide (1:50) for 30 min. For single-labeling stud-ies, the avidin–biotin complex method was used to detect theantigen signal (ABC Elite Kit; Vector Laboratories, Burlin-game, CA, U.S.A.) and 3,39-diaminobenzidine tetrachloridewas used to visualize the final product. For double-labelingstudies, the TSA procedure with fluorescein isothiocyanate(FITC)-conjugated tyramide was used to visualize ND1,whereas ChAT, TH, and nNOS were visualized using theappropriate Texas Red-conjugated secondary antibodies. Toavoid cross-reactivity, labeling for ND1 and ChAT or ND1 andTH was performed sequentially, even though the antibodieswere raised in different hosts. For ND1 and nNOS immunola-beling, because the antibodies were raised in the same host,protocol changes were made to maintain the specificity of theantibodies. Sections were incubated with nNOS antibody fol-lowed by goat anti-rabbit Fab fragments conjugated to TexasRed fluorophore (1:200; Jackson ImmunoResearch Laborato-ries). Before incubating the sections with the second primaryantibody (ND1), monovalent unconjugated Fab fragments(Jackson ImmunoResearch Laboratories) were used to occupyany remaining free “goat anti-rabbit” binding sites on the firstprimary antibody (nNOS). Then the second secondary antibodywas used to detect the second primary antibody. As mentionedpreviously, the ND1 signal was amplified using the TSA pro-cedure, and in the double-labeling experiments, ND1 was vi-sualized using FITC conjugated to tyramide (1:50, TSA kit;NEN). For controls, one or both primary antibodies were omit-ted.

Immunostained sections were examined using bright-fieldmicroscopy or conventional fluorescence microscopy. Imageswere captured on a Leitz microscope (Leica) linked to an imageanalysis system (Imaging Research, St. Catharines, Ontario,Canada) with selective filter sets to visualize FITC and TexasRed separately as well as simultaneously. For final output,images were processed using Adobe Photoshop 5.0 software.

Antibody preadsorptionIn addition to omission of primary antibody, both immuno-

blotting and ICC experiments employed controls in which theprimary antibody was preadsorbed against anchored ND133–43

peptide. Five hundred microliters of concentrated ND1 anti-body in PBS (1:50) was added to 500ml of ND133–43-Sepha-rose in a microcentrifuge tube and agitated at room temperaturefor 1 h. This tube was centrifuged at 100g for 3 min, and thesupernatant was brought to 5 ml (KPL diluent), a 1:500 dilutionof the original antibody titer. Simultaneously, an equal titer ofantibody was subjected to the same procedure in PBS, omittingthe ND133–43-Sepharose (1:500). Both of these antibody prep-

arations were used in immunoblotting experiments against40-mg rat brain samples and in ICC of rat brain sectionsprepared and processed as described above.

RESULTS

ELISAControl ELISA experiments run against blank wells or

omitting the primary antibody or using preimmune se-rum revealed no reaction product. Unpurified serumshowed a strong affinity for both the peptide and BSAthat was dependent on both serum titer and antigenconcentration (not shown). The affinity-purified ND1antibody showed titer- and antigen concentration-depen-dent immunoreactivity but no reactivity against the BSAcarrier (not shown). These results indicate that the serumcontained antibodies reactive against the conjugate andthat affinity column chromatography effectively isolatedantibodies against the peptide.

ImmunoblotsConsistent with the predicted molecular mass of the

subunit (Cooper and Clark, 1994), the ND1 antibodyspecifically marked a single;33-kDa band in homoge-nates of rat brain (Fig. 1A). No bands were seen when theprimary antibody was omitted or when the ND1 antibodywas preadsorbed against the Sepharose-anchoredND133–43peptide (Fig. 1A). The 33-kDa band was alsofound in rat liver, mouse brain, monkey brain, and PC12cells (Fig. 1B). Relative to whole rat brain homogenates,ND1-immunoreactive protein was enriched in mitochon-drial fractions (Fig. 1B). Additionally, the ND1 bandwas identified in homogenates of human lymphoblasts(Fig. 1C).

ND1 immunoreactivity (ND1-ir) in rat brainThe ND1 antibody was used to characterize the ana-

tomical distribution of the ND1 subunit protein in rat

FIG. 1. Immunoblot analysis of the ND1 antibody in varioustissue homogenates. A: In homogenates of rat brain (40 mg ofprotein/lane), a single band of ;33 kDa is seen. No immunore-action product is detected when the primary antibody is omittedor when the antibody is preadsorbed against the ND133–43 pep-tide. B: ND1-ir is enriched in rat brain compared to liver and inmitochondrial fractions relative to whole-brain homogenates. Itis also found in mouse and monkey brain (temporal cortex) andin PC-12 cells. Each lane was loaded with 40 mg of protein. C:ND1-ir in homogenates from two different human lymphoblas-toid cell lines is shown. Note the different molecular mass mark-ers in C.


385IMMUNOCHEMICAL CHARACTERIZATION OF COMPLEX I

brain. The antibody stained the brain differentially asseen in a representative low-magnification sectionthrough the anterior striatum (Fig. 2A). The cortex, ol-factory tubercle, and septal regions were intenselystained. The striatum showed patchy staining and thecorpus callosum was faintly labeled. Control experi-ments omitting the ND1 antibody or using antibodypreadsorbed against ND133–43-Sepharose showed no im-munolabeling (Fig. 2B and C). As described below,neuronal cell staining was observed throughout the brain,but minimal labeling was observed in glial cells andwhite matter.

Cerebral cortex.A laminated pattern of ND1-ir wasobserved in the cortex (Fig. 2A), and ND1-ir was mostnotable in the pyramidal cells of layer V. Reaction prod-uct was intense in the soma and the apical dendrites,whereas the nuclei appeared devoid of immunoreactivity(Figs. 3A and 4A). Inspection at higher magnificationrevealed a punctate pattern of immunolabeling in the

soma and dendrites (Fig. 4B). Layer III pyramidal neu-rons and large multipolar cells in layer VI also demon-strated ND1-ir. In addition to variable ND1 expressionbetween the cortical neuronal populations, intercellularvariability was also noted within the layer V pyramidalneurons. Intensely labeled cells were occasionally neigh-bored by lightly labeled neurons of the same cell popu-lation (Fig. 4B). ND1-ir was not found to distinguishglial cells; however, light punctate background reactiv-ity, possibly indicative of glial ND1, was observable inselect micrographs (Fig. 4B). The pattern of COXIVimmunolabeling in the cerebral cortex was similar to thatof ND-1, with distinct labeling observed in the layer Vpyramidal somata and dendrites (Fig. 3B). Layer IIIpyramidal cells and the large multipolar cells of layer VIwere also immunolabeled.

Striatum.In striatum, two populations of cells showedintense ND1-ir (Fig. 5). Large stellate neurons andsmaller ovoid neurons both showed heavy labeling in thecell bodies and dendritic processes. These populations ofcells, like those of layer V pyramidal neurons in thecortex, also showed punctate labeling and clear nuclei.The vast majority of other cell types in the striatum,including medium spiny neurons, displayed low levels of

FIG. 3. ND1 and COXIV ICC in the parietal cortex. A: PunctateND1-ir is observed in layer V pyramidal neurons, in the cell soma,as well as in the apical dendrites. B: COXIV immunolabeling wassimilar to that of ND1. Intense COXIV immunolabeling is ob-served in the soma and dendrites of layer V neurons. Bar 5 50mm.

FIG. 2. Specificity of ND1 ICC in rat brain. Adjacent coronalsections through the anterior striatum were processed for im-munohistochemistry with ND1 antibody (A), blocking serum only(B), and ND1 antibody preadsorbed with ND133–43 peptide (C).ND1 immunolabeling was concentrated in cellular regions andless conspicuous in the white matter. Sections incubated withthe blocking serum only or with preadsorbed antibody showedno immunolabeling. CC, corpus callosum; CT, cerebral cortex;OT, olfactory tubercle; S, septal regions; ST, striatum.



ND1-ir and were detectable following cresyl violet coun-terstaining (Fig. 5). Cell morphology implied that thelarge ND1-positive neurons were the cholinergic inter-neurons and the medium-sized neurons with fusiformcell bodies were interneurons that stain for NOS orsomatostatin. To confirm that the cholinergic and nNOScells did stain intensely for ND1, immunofluorescent

double labeling was performed. Among the cells withintense ND1-ir, the large neurons were immunoreactivefor ChAT, whereas medium-sized neurons showed noChAT signal (Fig. 6). Double labeling with ND1 andnNOS showed medium-sized ND1-immunoreactive neu-rons to be positive for nNOS, whereas the large ND1-immunoreactive neurons did not express nNOS (Fig. 7).These experiments indicate that ND1 is differentiallyexpressed in the striatum, with the large cholinergic cellsand the medium-sized nNOS-positive cells expressinghigher levels of ND1 than the other neurons of thestriatum.

FIG. 4. Differential distribution of ND1-ir in the parietal cortex. A:Punctate ND1-ir is evident in the cell bodies and apical dendritesof layer V neurons. Cell nuclei are usually clear of ND1-ir. Bar5 50 mm. B: A representative photomicrograph shows differen-tial ND1 labeling in the cortex at a higher magnification. Thepyramidal neuron on the right displays intense punctate immu-nolabeling, and the neighboring neuron on the left is lightlylabeled. Sections were counterstained with cresyl violet. Bar5 20 mm.

FIG. 5. Differential distribution of ND1-ir in the striatum. Themost intense ND1-ir is observed in the cell bodies and dendriticprocesses of the large stellate (arrowhead) and medium-sizedovoid (large arrow) neurons. Notable in the same field are nu-merous cells, probably the medium-sized projection neurons,with low levels of punctate perinuclear labeling (small arrows).The sections are counterstained with cresyl violet. Bar 5 50 mm.

FIG. 6. Differential distribution of ND1-ir in the striatum. Thelarge stellate cells immunolabeled most intensely for ND1 arecholinergic, as confirmed by double-immunofluorescent stainingof ND1 and ChAT. A: Green ND1-ir is present in two cell popu-lations, large (arrows) and medium-sized (arrowheads) neurons.B: Red ChAT immunoreactivity is observed only in the largeneurons. C: Yellow fluorescence indicates co-localization of ND1and ChAT. Bar 5 30 mm.



Red nucleus.Punctate ND1-ir was concentrated in thecell bodies and dendrites of the magnocellular neurons ofthe red nucleus (Fig. 8). Punctate labeling was observedin the surrounding neuropil.

Cerebellum.In the cerebellum, punctate ND1-ir wasobserved in Purkinje cells and their dendritic arbors inthe molecular layer (Fig. 9). The neuropil of the molec-ular layer had punctate ND1-ir, whereas, in contrast, thegranule cell layer showed clear somata and little or nolabeling (Fig. 9B and C).

Globus pallidus.ND1-ir was concentrated in the so-mata and dendrites of pallidal neurons (Fig. 10A). Inaddition to the somatodendritic labeling, punctate immu-noreaction product was observed in the surrounding neu-ropil. Immunolabeling with the COXIV antibody wasalso observed in the somatodendritic compartment but, incontrast to the pattern of ND1-ir, was minimal in theneuropil (Fig. 10B).

Substantia nigra.In the pars compacta, there werelarge neurons with relatively intense ND1-ir in their cellbodies and proximal processes; lightly staining neuronswere also seen (Fig. 11A). To determine if these ND1-

positive cells of the pars compacta were the dopaminer-gic cells of the region, double-immunofluorescent label-ing with TH and ND1 antibodies was performed (Fig.12). Although the large neurons in this region were, infact, dopaminergic, it was found that a sizable fraction ofthe TH-immunoreactive cells had little or no ND1-ir. Inthe pars reticulata, smaller ovoid neurons, presumablyGABAergic projection neurons, were rich in ND1-ir(Fig. 11B). In contrast to the pars compacta cells, punc-tate ND1-ir was apparent in small-caliber distal pro-cesses of these neurons. As a result, there was moreneuropil ND1-ir in the pars reticulata.

Hippocampus.Throughout the hippocampus, ND1-irwas observed in the pyramidal cells, with the most in-

FIG. 7. Differential distribution of ND1-ir in the striatum. Thesmall subset of medium-sized cells that immunolabel intenselyfor ND1 contain nNOS, as confirmed by double-immunofluores-cent staining of ND1 and nNOS. A: Green ND1-ir is present inthree cell populations, large (large arrowhead) and medium-sized (arrows) neurons that are intensely stained for ND1 andmedium-sized neurons that are lightly stained for ND1 (smallarrowheads). B: Red nNOS immunoreactivity is observed only inthe subset of medium-sized neurons (arrows). C: Yellow fluores-cence indicates co-localization of ND1 and nNOS. The threepopulations of striatal cells immunolabeled for ND1 are clearlyapparent in this photomicrograph. Bar 5 30 mm.

FIG. 8. Neurons in the red nucleus have intense punctate ND1labeling. Punctate staining can be observed in the surroundingneuropil also. Sections were counterstained with cresyl violet.Bar 5 50 mm.

FIG. 9. Differential distribution of ND1-ir in the cerebellum. A:Coronal sections through the cerebellum demonstrate ND1 im-munolabeling in the Purkinje cell layer (PCL) and the molecularlayer (ML), with very little labeling in the granule cell layer (GCL).Bar 5 100 mm. B: Higher magnification shows punctate immu-noreactivity in Purkinje cells and dendrites. This is in stark con-trast to the light background reactivity of the molecular layer andthe lack of reactivity in the granule cell layer. Bar 5 25 mm. C:Sagittal section through the cerebellum demonstrates ND1-ir ina Purkinje cell and its dendritic arbor. Bar 5 25 mm.



tense labeling seen in the CA3 region (Fig. 13A). Apicaldendrites could be followed well into the stratum radia-tum and stratum lucidum, and the punctate nature of thedendritic labeling gave the dendrites a beaded appear-ance (Fig. 13B). Aside from the somatodendritic label-ing, the stratum lucidum and stratum radiatum of theCA3 region consistently showed a darker backgroundlabeling than CA1 (Fig. 13A). COXIV distribution in thehippocampus was similar to that of ND1. Punctate den-dritic labeling could be followed into the stratum radia-tum and stratum lucidum and yielded the same beadedlabeling pattern seen in ND1-stained sections (Fig. 13C).

Spinal cord.ND1 distribution was examined in thecervical, thoracic, and lumbar spinal cord. Immunoreac-tivity was darker in the central gray matter, but scatteredcells in white matter were stained intensely. In the ven-tral horns of the cervical and lumbar regions, large motorneurons had more ND1-ir than the surrounding neuropil(Fig. 14).

DISCUSSION

To analyze the regional, cellular, and subcellular dis-tributions of the ND1 protein in brain and hence thedistribution of complex I, a polyclonal antibody wasraised against a unique epitope of the mitochondriallyencoded ND1 protein. The antibody was affinity purified,characterized by immunoblotting and ELISA, and thenused for ICC studies of rat brain.

Antibody specificityAfter affinity purification, ELISA showed a specific

reaction product that was dependent on both antibodyand peptide (ND133–43) concentration. Western blotting

FIG. 10. ND1 and COXIV ICC in the globus pallidus. A: ND1labeling is observed in the cell bodies and dendrites of globuspallidus neurons. In addition, punctate ND1 labeling is observedthroughout the neuropil. B: COXIV ICC yielded somatic anddendritic labeling similar to that of ND1; however, the neuropillabeling associated with ND1 ICC was not present. Bar 5 50 mm.

FIG. 11. Distribution of ND1-ir in the substantia nigra. A: In thepars compacta, a dense population of multipolar neurons islabeled with ND1. Immunolabeling is present in the cell bodiesand proximal dendrites, but it is absent in the nucleus. Some ofthe neurons are stained quite lightly. Bar 5 100 mm. B: In thepars reticulata, neurons are intensely labeled for ND1, and, incontrast to the pars compacta, immunoreactivity is easily ob-served in small-caliber distal dendrites. Bar 5 100 mm.

FIG. 12. Heterogeneity of ND1 expression in the dopaminergiccells of the substantia nigra. A brain section through the sub-stantia nigra was double-labeled for ND1 (A) and TH (B). Notethat many dopaminergic nigral neurons have very little ND1-ir(arrows). Bar 5 50 mm.



consistently labeled a single band with an estimatedmolecular mass of;33 kDa, consistent with the pre-dicted mass of the ND1 gene product (Cooper and Clark,1994). Preadsorption of the antibody with the ND133–43peptide eliminated all reaction product in immunoblotand ICC studies. We therefore conclude that the antibodyrecognizes specifically the ND1 subunit of complex I.

Mitochondrial localization of ND1-ir in brainAlthough mitochondria are essential components of all

brain cells, many studies have found that mitochondrialmarkers are not distributed homogeneously (Hevner andWong-Riley, 1989; Porter et al., 1994). Consistent withthese other studies, we have found striking heterogeneityin the distribution of ND1 in the brain, as discussedbelow. Our ICC results are generally consistent with ourprevious studies that mapped complex I with an autora-diographic assay of [3H]dihydrorotenone binding (Hig-gins and Greenamyre, 1996). For example, both assaysshow that there is relatively little complex I in whitematter, and distinctive laminar distributions in cortex,cerebellum, and hippocampus. Although the autoradio-

graphic assay permits a more quantitative assessment ofthe regional distribution of complex I, ICC with the ND1antibody provides a much higher level of anatomicalresolution. Thus, with light microscopy, it is readilyapparent that ND1-ir has a punctate distribution, consis-tent with its presumed mitochondrial localization. Otherstudies have shown that ND1-ir co-localizes with Mito-Tracker dyes (authors’ unpublished data), further con-firming that it is a mitochondrial marker. Moreover, thepunctate somatodendritic pattern of staining with theND1 antibody is generally very similar to that of theCOXIV subunit of cytochrome oxidase. Together withthe ELISA and immunoblot data, the ICC data indicatethat the antibody is specific for the ND1 subunit ofmitochondrial complex I.

Heterogeneous ND1 distributionAlthough ND1-ir is present throughout the brain, its

differential staining pattern is clear. As noted, there is amarked difference between neurons and glia. ICC withthe ND1 antibody fails to clearly identify glial cells, andsimilarly no definitive glial labeling was observed withCOXIV antibody. That neither ETC antibody labels glialcells well is consistent with the hypothesis that most glialmetabolism occurs independently of mitochondrial OX-

FIG. 14. Distribution of ND1-ir in the spinal cord. A: Coronalsection of the spinal cord at the cervical level demonstrates ND1immunolabeling in the gray and white matter. Bar 5 500 mm. B:Higher magnification of the boxed area in A shows large motorneurons with intense ND1 labeling in the ventral horn. Bar 5 100mm.

FIG. 13. Distribution of ND1-ir in the hippocampus. A: Photomi-crograph at low magnification illustrates that the ND1-ir in thehippocampus is concentrated in the pyramidal cell layer. Bar5 200 mm. B: At higher magnification, the punctate somatoden-dritic ND1-ir in the CA1 pyramidal cells can be appreciated. C:Like ND1 labeling, punctate COXIV immunoreactivity can beobserved in the CA1 pyramidal cell dendrites. Bar 5 100 mm. st.l, stratum lucidum; st. o, stratum oriens; st. p, stratum pyrami-dale; st. r, stratum radiatum.



PHOS (Tsacopoulos and Magistretti, 1996). There is alsoheterogeneity in staining between neuronal types. Forexample, all cortical neurons express ND1-ir, but layer Vneurons are, in general, stained most intensely with theND1 antibody. Even within this layer, some neuronsstain much more intensely than do others, as shown inFig. 4B. Within the cerebellum, the Purkinje cells displayintense, punctate ND1-ir in their cell bodies and den-drites, but the adjacent granule cells are seemingly de-void of ND1-ir (Fig. 9). The different levels of ND1expression by various cell populations likely reflect dif-ferent levels of complex I activity, as expression ofcytochrome oxidase subunits has been shown to correlatewith levels of complex IV activity in neurons (Hevnerand Wong-Riley, 1989). It should be noted, however,that the relationship between ND1 concentration andOXPHOS activity is as yet unknown.

Some of these differences in expression levels mayhave important functional consequences. For example, inthe striatum, large cholinergic neurons and nNOS-posi-tive neurons have much more ND1-ir than the projectionneurons. These two classes of neurons are selectivelyresistant to excitotoxic insults and mitochondrial toxins(Beal et al., 1993; Brouillet et al., 1993; Wu¨llner et al.,1994; Schulz et al., 1996; Greene et al., 1998), and inHuntington’s disease, despite extensive striatal pathol-ogy, the cholinergic and nNOS-positive neurons arespared (Ferrante et al., 1987). It is tempting to speculatethat the higher level of ND1 (and, presumably, complexI activity) in these cells helps to confer resistance againstsuch metabolic insults.

Conversely, the low levels of ND1-ir seen in many ofthe dopaminergic neurons of the substantia nigra mayindicate that they have less capacity to withstand defectsin complex I activity. Consistent with this interpretation,nigrostriatal dopamine neurons show a selective vulner-ability to systemic inhibition of complex I by rotenone(MacKenzie and Greenamyre, 1998). Moreover, an ap-parent systemic defect in complex I activity has beensuggested to play a pathogenic role in Parkinson’s dis-ease (Schapira et al., 1989; 1990; Shoffner et al., 1991;Cardellach et al., 1993; Reichmann et al., 1993; Janetzkyet al., 1994).

Although the distributions of ND1 and COXIV weregenerally very similar, exceptions were found in theglobus pallidus (Fig. 10) and substantia nigra (Fig. 11).In these regions, there was much more ND1-ir thanCOXIV immunoreactivity in fine processes and neuropil.Preliminary subcellular fractionation studies of synapticversus nonsynaptic mitochondria suggest that ND1 ispreferentially enriched in synaptic mitochondria relativeto COXIV (Pettus et al., 1998). Thus, the ND1-ir seen infine processes and neuropil may reflect synaptic mito-chondria.

ConclusionIn summary, we have used a polyclonal antibody

raised against the ND1 subunit of complex I to examinethe distribution of this key enzyme system in brain. Our

results indicate that components of the ETC are distrib-uted differentially in brain and further suggest that theirrelative density may help to explain some patterns ofselective vulnerability and resistance in neurological dis-orders. This antibody should be a useful tool for assess-ing the involvement of complex I in neurodegeneration.

Acknowledgment: This work was supported by NIH grantNS33779 and the Emory Alzheimer’s Disease Center(AG10130). We thank Craig Heilman for performing immuno-blots of lymphoblast samples.

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Immunocytochemical Characterization of the Mitochondrially Encoded ND1 Subunit of Complex I (NADH : Ubiquinone Oxidoreductase) in Rat Brain