department of psychiatry & behavioral neurosciences, wayne ... · was from bio-rad (hercules,...

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*Department of Psychiatry & Behavioral Neurosciences, Wayne State University School of Medicine, Detroit, Michigan, USA

�Research & Development Service, John D. Dingell VA Medical Center, Detroit, Michigan, USA

�Behavioral Neuroscience Program, Department of Psychology, State University of New York at Binghamton, Binghamton, New York,

USA

Parkinson’s disease (PD) is a progressive neurodegenerativedisease of the dopamine (DA) neuronal system. Loss ofnigrostriatal DA neurons leads gradually to a severe move-ment disorder characterized by tremor, rigidity, bradykinesiaand impaired balance. The mechanisms underlying idiopathicPD are not fully understood but attention has focused onoxidative stress and inflammation (Glass et al. 2010),mitochondrial dysfunction (Yao and Wood 2009), environ-mental (Cicchetti et al. 2009) and genetic influences (Hatanoet al. 2009). It is not widely appreciated but the serotonin(5HT) neuronal system is severely degraded in PD.Postmortem PD brains have significant reductions in 5HT(Scatton et al. 1983), 5-hydroxyindole acetic acid (Mayeux

Received September 20, 2010; revised manuscript received November 2,2010; accepted November 15, 2010.Address correspondence and reprint requests to Donald M. Kuhn,

R&D Service (11R), John D. Dingell VA Medical Center, 4646 John R,Detroit, MI 48201, USA. E-mail: [email protected] used: 5HT, serotonin; CC (and CC-WT), catalytic core

domain of TPH2 (residues 150–488); CC-CL, cysteine-less CC; DA,dopamine; DTT, dithiothreitol; eGFP–TPH2, enhanced green fluorescentprotein–TPH2 fusion protein; FL-CD24, full-length TPH2 with C-ter-minal most 24 residues removed; FL-ND40, FL-TPH2 with N-terminalmost 40 residues removed; FL-RD-CL, FL-TPH2 with RD cysteines(C110,112) mutated to serines; FL-TPH2, FL form of TPH2; PAGE,polyacrylamide gel electrophoresis; RD (and RD-WT), regulatory do-main of TPH2 (residues 1–149); RD-C110S or C112S, RD with indi-cated cysteine-to-serine mutation; TPH, tryptophan hydroxylase.

Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative

disorder characterized by the loss of dopamine neurons of the

nigrostriatal system, resulting in severe motor disturbances.

Although much less appreciated, non-motor symptoms are

also very common in PD and many can be traced to serotonin

neuronal deficits. Tryptophan hydroxylase (TPH) 2, the rate-

limiting enzyme in the serotonin biosynthesis, is a phenotypic

marker for serotonin neurons and is known to be extremely

labile to oxidation. Therefore, the oxidative processes that

prevail in PD could cause TPH2 misfolding and modify sero-

tonin neuronal function much as is seen in dopamine neurons.

Oxidation of TPH2 inhibits enzyme activity and leads to the

formation of high molecular weight aggregates in a dith-

iothreitol-reversible manner. Cysteine-scanning mutagenesis

shows that as long as a single cysteine residue (out of a total

of 13 per monomer) remains in TPH2, it cross-links upon

oxidation and only cysteine-less mutants are resistant to this

effect. The effects of oxidants on TPH2 catalytic function and

cross-linking are also observed in intact TPH2-expressing

HEK293 cells. Oxidation shifts TPH2 from the soluble com-

partment into membrane fractions and large inclusion bodies.

Sequential non-reducing/reducing 2-dimensional sodium

dodecyl sulfate–polyacrylamide gel electrophoresis and

immunoblotting confirmed that TPH2 was one of a small

number of cytosolic proteins that form disulfide-bonded

aggregates. The propensity of TPH2 to misfold upon oxidation

of its cysteine residues is responsible for its catalytic lability

and may be related to loss of serotonin neuronal function in

PD and the emergence of non-motor (psychiatric) symptoms.

Keywords: disulfide cross-linking, non-motor symptoms,

Parkinson’s disease, protein aggregation, serotonin neurons,

tryptophan hydroxylase 2.

J. Neurochem. (2011) 116, 426–437.

JOURNAL OF NEUROCHEMISTRY | 2011 | 116 | 426–437 doi: 10.1111/j.1471-4159.2010.07123.x

426 Journal of Neurochemistry � 2010 International Society for Neurochemistry, J. Neurochem. (2011) 116, 426–437� 2010 The Authors

et al. 1984; Kostic et al. 1987), 5HT transporter content(Murai et al. 2001; Kish et al. 2008), tryptophan hydroxy-lase (TPH) 2 (Sawada et al. 1985) and 5HT neurons(Halliday et al. 1990a,b; Paulus and Jellinger 1991; Jellinger1999; Kovacs et al. 2003). Bilateral 6-hydroxydopaminelesions and subsequent L-DOPA treatment significantlyreduce midbrain TPH2 content (Eskow Jaunarajs et al.2010). Rotenone, a pesticide that causes PD-like symptomsin humans and animals (Cannon et al. 2009), selectively killscultured 5HT neurons (Ren and Feng 2007). The intensefocus on the motor symptomatology of PD is appropriate butincreased study of non-motor manifestations of PD is calledfor when considering that approximately 80% of PD patientssuffer from co-morbid neuropsychiatric conditions such asdepression, sleep disorders and anxiety (Chaudhuri et al.2006; Simuni and Sethi 2008; Obeso et al. 2010). Many ofthese conditions can be traced to dysfunctional 5HTneurochemistry (Ressler and Nemeroff 2000).

A clear link exists between protein misfolding andneurodegeneration. Protein cysteine residues are cellularredox sensors and their modification can change proteinconformation as part of a controlled signaling process (Sitiaand Molteni 2004). Persistent oxidative stress can overwhelmcellular mechanisms (proteasomal and lysosomal proteolysis)that maintain the delicate balance between protein synthesisand degradation, leading eventually to neuronal damage anddeath (Chung et al. 2001; Glickman and Ciechanover 2002;Goldberg 2003). Examples of this link in PD are illustratedby parkin and a-synuclein (Schlehe et al. 2008; Winklhoferet al. 2008). TPH2, the rate limiting enzyme in 5HTsynthesis and a phenotypic marker for 5HT neurons, isremarkably unstable and prone to oxidative inactivation(Kuhn et al. 1980) perhaps as a result of its high cysteinecontent (13 cysteines/monomer or 52/tetramer). TPH2 cata-lytic function is determined by the redox status of its cysteineresidues such that losses in activity are highly correlated withthe number of cysteines that are oxidized (Kuhn and Arthur1997; Kuhn and Geddes 1999). Therefore, oxidative stressnot only lowers TPH2 activity and reduces 5HT synthesis, itmay also lead to misfolding and aggregation of the cysteine-rich protein. In light of strong precedence implicating altered5HT neuronal function in the non-motor symptoms (i.e.,psychiatric) of PD, we hypothesize that TPH2 is susceptibleto aggregation and misfolding upon oxidation, an effect thatcould contribute to 5HT neurochemical deficits and neuronaldamage in PD.

Materials and methods

MaterialsThe bacterial expression vector pGEX4T2 was obtained from

Amersham Biosciences (Piscataway, NJ, USA). Lipofectamine,

pcDNA3, Novex 4–12% gradient Tris-glycine gels and all cell

culture components were products of Invitrogen (Carlsbad, CA,

USA) and pEGFP was from Clontech (Mountain View, CA, USA).

Bacterial protease inhibitor cocktails, dithiothreitol, diamide, hydro-

gen peroxide, 5-hydroxytryptophan, tryptophan, catalase, EDTA,

ferrous ammonium sulfate, iodoacetamide, and all buffers and

detergents were purchased from Sigma-Aldrich (St. Louis, MO,

USA). HEK293 cells were provided by ATCC (Manassas, VA,

USA). Thrombin was acquired from GE Healthcare (Piscataway, NJ,

USA). Tetrahydrobiopterin was a product of Schircks Laboratories

(Jona, Switzerland). Imperial protein stain was obtained from Pierce

(Rockford, IL, USA) and the Bradford Protein Assay Dye Reagent

was from Bio-Rad (Hercules, CA, USA).

Expression of TPH2 in E. coli and protein purificationThe TPH2 cDNA from mouse brain was cloned into pGEX4T2 for

expression in E. coli as a glutathione S-transferase-fusion protein.

TPH2 was purified and cleaved from its glutathione S-transferasefusion tag with thrombin as previously reported (Sakowski et al.2006a). Mouse TPH1 was obtained in the same fashion (Sakowski

et al. 2006a). The resulting preparations of TPH2 (MW = 56 kDa)

and TPH1 (MW = 51 kDa) were judged to be approximately 95%

pure.

Site-directed and cysteine-scanning mutagenesis of TPH2Mutagenesis of TPH2 was carried out via splicing by overlap

extension (Horton et al. 1993) as previously employed (Kuhn and

Geddes 1999; Kuhn et al. 2002). TPH2 shares with the other

monooxygenase enzymes a domain structure (Vrana 1999; Carkaci-

Salli et al. 2006) comprised of an N-terminal regulatory domain

(RD; aa 1–149) and a catalytic core domain (CC; aa 150–488). Of

the 13 cysteines in TPH2, two are in the RD (C110, C112) and the

remaining 11 are in the CC (C162, C234, C248, C296, C315, C355,

C365, C394, C404, C408, C476). In an effort to assess the relative

contribution of the separate domains and selected cysteine residues

within them to oxidation-induced disulfide cross-linking, full-length

TPH2 and its RD and CC domains were studied independently. The

following mutants of wild-type, full-length (FL) TPH2 were

produced: FL-TPH2 (all 13 cysteines intact), FL-NDC6S (six

N-terminal-most cysteines mutated to serines), FL-CDC7S (seven

C-terminal-most cysteines mutated to serines), FL-TPH2-CL

(cysteine-less TPH2 with all 13 cysteines mutated to serines), FL-

RD-CL (cysteine-less RD with cysteines C110 and C112 mutated to

serines), FL-CC-CL (cysteine-less CC with all 11 CC cysteines

mutated to serines), FL-RD1CC1 (a mutant containing a single

cysteine residue within the RD [C112] and CC [C315], all remaining

cysteines mutated to serines) and FL-C476S [to mutate the only

cysteine residue in the tetramerization domain of TPH2 (Mockus

et al. 1997)]. Two truncation mutants of TPH2 were produced to

include FL-ND40 (N-terminal 40 amino acids removed to simulate

TPH1, MW = 51 kDa) and FL-CD24 (C-terminal 24 amino acids

truncated to remove the tetramerization domain of TPH2,

MW = 53 kDa). The following mutants of the RD-WT were made

as a stand-alone constructs (MW = 17 kDa): RD-C110S, RD-

C112S, and RD-CL (i.e., RD-C110,112S). The following mutants

of the CC-WT (MW = 39 kDa) were made as stand-alone con-

structs: CC + C315 (CC with all cysteines mutated to serine except

C315) and CC-CL (cysteine-less CC, all C-to-S mutants). All of the

above constructs were cloned into pGEX4T2 for expression and

purification as described above for FL-TPH2. All mutations of

� 2010 The AuthorsJournal of Neurochemistry � 2010 International Society for Neurochemistry, J. Neurochem. (2011) 116, 426–437

TPH2 misfolding and 5HT deficits in PD | 427

TPH2 cDNAs were confirmed by DNA sequencing. Each of the site

mutations and truncations made within FL-TPH2 or its separate

domains, as described above, is depicted in diagram form in

Figure S1 for reference.

Cell culture and transfectionHEK293 cells were transfected with the full-length cDNA of TPH2

using Lipofectamine (Sakowski et al. 2006a; Kuhn et al. 2007) inthe mammalian expression vectors pcDNA3 or pEGFP to produce a

eGFP–TPH2 fusion protein. Transformants were maintained in

neomycin selection until stable expression of TPH2 was confirmed

by measures of enzyme activity and immunoblotting using an

antibody selective for TPH2 (Sakowski et al. 2006b). Cells were

subsequently maintained in culture in Dulbecco’s modified Eagle’s

medium in 10% fetal bovine serum at 37�C in 5% CO2. When cells

reached approximately 75% confluence, they were harvested and

re-plated at a density of 5 · 105 cells/mL in preparation for

treatment.

Preparation of cell extractsTryptophan hydroxylase 2-expressing HEK293 cells were washed

free of media and harvested into 0.05 M Tris–HCl pH 7.4

containing a protease inhibitor cocktail. Cells were disrupted by

sonication and soluble and membrane fractions were isolated after

centrifugation of extracts at 40 000 g for 15 min at 4�C. Membrane

fractions were washed three times in 10 volumes homogenizing

buffer to remove any contaminating cytosolic proteins and sedi-

mented by centrifugation. Buffers used to prepare all TPH2

preparations specifically omitted dithiothreitol (DTT) to avoid

interference with protein disulfide cross-linking.

Exposure of TPH2 to oxidative conditionsTwo different preparations of TPH2 were used to study the response

of the enzyme to the cysteine oxidants diamide and H2O2: (i)

purified recombinant TPH2 and (ii) intact HEK293 cells expressing

TPH2. Two different oxidants were used as well. Diamide is a

highly specific cysteine reactant (Kosower and Kosower 1995) that

is used frequently to simulate oxidative stress in protein and intact

cell preparations. H2O2 is an endogenously occurring and physi-

ologically relevant oxidant also used frequently to promote

oxidative stress. Preliminary experiments tested each TPH2

preparation for its response to diamide and H2O2 over broad

concentration ranges and for different exposure times. TPH2 was

modified by oxidants in a concentration- and time-dependent

manner (data not shown) but single concentrations of each oxidant

and the times of exposure were selected for use in each preparation

(specified below). Recombinant TPH2 (2 lg) was exposed to

oxidants for 15 min at 30�C. Intact HEK293 cells expressing TPH2

cells (5 · 105) were exposed to oxidants in serum-free Dulbecco’s

modified Eagle’s medium for 15 min at 37�C. After treatment,

TPH2 was diluted in homogenizing buffer (1 : 10) ± DTT and

maintained at 30�C for 15 min and then and assayed for enzyme

activity and aggregation (see below). Intact cells were washed three

times with homogenizing buffer prior to isolation of soluble

fractions as described above. Iodoacetamide (40 mM) was added to

those samples intended for electrophoresis and immunoblotting to

prevent disulfide scrambling or adventitious air oxidation of any

unreacted cysteines.

TPH2 enzyme assayTryptophan hydroxylase 2 enzyme activity was determined by

measuring the accumulation of 5-hydroxytryptophan (i.e., the

product of tryptophan hydroxylation) by HPLC as previously

reported (Sakowski et al. 2006a; Kuhn et al. 2007). The TPH2

reaction mixture contained Tris pH 7.4 (50 mM), catalase (0.05 mg/

mL), tryptophan (200 lM), ferrous ammonium sulfate (100 lM)

and tetrahydrobiopterin (200 lM).

Electrophoresis and immunoblottingTryptophan hydroxylase 2 preparations were electrophoresed on

Novex 4–12% gradient Tris–glycine gels and exposed to immuno-

blotting as previously described (Sakowski et al. 2006b; Kuhn et al.2007) to determine the migration pattern of the protein on non-

reducing gels. The protein content of all preparations was

determined by the method of Bradford (1976) and samples were

diluted to the appropriate concentration and loaded onto gels after

heating to 95�C for 5 min. Duplicate samples were treated ± DTT to

determine the reversibility of the effects of oxidants on TPH2

assembly into high molecular weight aggregates. TPH2 migrates on

denaturing, non-reducing gels as a monomer (56 kDa). In those

instances where oxidized TPH2 aggregates were so large they did

not appear to enter denaturing gels, samples were applied to

cellulose acetate membranes as described by Niwa et al. (2007)because high molecular weight aggregates of many proteins are

retained on these filters (Scherzinger et al. 1997; Niwa et al. 2007).Filters were processed for TPH2 immunoreactivity in the same

manner as blots on nitrocellulose (see below).

Redox 2-dimensional sodium dodecyl sulfate–polyacrylamide gelelectrophoresisIntact HEK293 cells expressing TPH2 cells were treated with

diamide and after washing to remove excess oxidant, the soluble cell

fraction was prepared and subjected to redox 2-dimensional

polyacrylamide gel electrophoresis (PAGE) as previously described

(Brennan et al. 2004; Cumming et al. 2004). After electrophoresis,gels were stained with Imperial Protein Stain or they were

electroblotted to nitrocellulose for TPH2 immunoblotting.

Isolation of TPH2 aggregates in large inclusion bodiesIntact HEK293 cells (5 · 105) expressing either TPH2 or eGFP–

TPH2 were treated with diamide at 30�C for 15 min and large

inclusion bodies were isolated as described by Lee and Lee (2002)

as modified by Niwa et al. (2007). Cells were washed free of

diamide with cold phosphate buffered saline and detergent soluble

proteins were removed by treating cells for 5 min in 50 mM Tris–

HCl pH 7.4 containing 150 mM NaCl, 1% Nonidet P-40 (Sigma-

Aldrich, St. Louis, MO, USA) and 1 mM EDTA with a protease

inhibitor cocktail. After careful removal of the detergent soluble

material, detergent insoluble inclusion bodies were harvested by

scraping and incubated on ice for an additional 5 min. Extracts were

centrifuged at 80 g for 15 min and large inclusions were pipetted

onto a glass microscope slide, sealed with a coverslip and observed

for eGFP–TPH2 fluorescence on a BX51 fluorescence microscope

(Olympus, Center Valley, PA, USA). HEK293 cells transfected with

empty pEGFP vector were treated in an identical manner to

determine if diamide treatment caused eGFP to localize into large

cellular inclusions. In other experiments, the presence of wild-type

Journal of Neurochemistry � 2010 International Society for Neurochemistry, J. Neurochem. (2011) 116, 426–437� 2010 The Authors

428 | D. M. Kuhn et al.

TPH2 in detergent-insoluble inclusion bodies was determined by

immunoblotting. No attempt was made to characterize these

inclusion bodies morphologically and these studies followed

published protocols used with other proteins showing misfolding

and aggregation (Lee and Lee 2002; Niwa et al. 2007).

Measures of turbidityAggregation of diamide-treated recombinant TPH2 was monitored

by measuring the absorbance at 405 nm. TPH2 (1 mg/mL) was

placed into flat-bottomed microtiter plates in a volume of 100 lL.Diamide (1 mM) was added and absorbance was monitored at 1-min

intervals using a Molecular Devices (Sunnyvale, CA, USA)

Versamax tunable microplate reader. TPH2 samples omitting

diamide treatment served as controls. Plates were agitated by orbital

shaking every 30 s between readings to re-suspend any aggregates.

After the final absorbance reading, samples of control and diamide-

treated TPH2 were subjected to electrophoresis and immunoblotting

as described above.

Distribution of TPH2 between soluble and membranecompartmentsHEK293 cells expressing TPH2 were treated with diamide as

described above after which cells were washed to remove excess

oxidant. Cells treated with phosphate-buffered saline served as

controls. Cells were harvested, disrupted by sonication, and

centrifuged at 20 000 g at 4�C. The supernatant was removed and

the membrane fraction was washed three times and resuspended

finally in phosphate-buffered saline. The soluble and membrane

fractions were immunoblotted to determine the distribution of TPH2

between compartments.

Statistical analysisData for TPH2 activity is expressed as the mean ± SEM of the

number of experiments indicated in the respective figure legends.

The main treatment effects on enzyme activity were tested by one-

way ANOVA using GraphPad Prism 5 for Windows (GraphPad

Software, San Diego, CA, USA). All of the main effects were

significant (p < 0.001) and are not indicated hereafter. Comparisons

of individual groups to their respective controls were carried out

with Tukey’s multiple comparison test using GraphPad Prism 5 and

the level of significance for each comparison is indicated on each

figure and in the figure legends.

Results

Oxidants inactivate TPH2 and cause the formation of highmolecular weight aggregatesTryptophan hydroxylase 2 was treated with H2O2 or diamideand the effects on enzyme activity are presented in Fig. 1(a). Itcan be seen that 500 lM H2O2 or 100 lM diamide reducedTPH2 activity to 30% or 5% of control, respectively. Theseeffects of H2O2 and diamide could be reversed partially butsignificantly by DTT. Analysis of oxidant-treated TPH2 byimmunoblotting revealed that both H2O2 and diamide treat-ment led to the formation of high molecular weight aggre-gates. Figure 1(b) shows that H2O2 diminished the amount ofTPH2 migrating as the monomer (56 kDa) on denaturing,

non-reducing gels and increased the amount of immunoreac-tive TPH2 species at approximately 130 kDa. Another majorband was observed at� 250 kDa and even larger species wereobserved as a smear that extended to the top of the resolvinggel. The pattern of effects of diamide on TPH2 was essentiallythe same as H2O2 but were much greater. In fact, the TPH2monomer was no longer observed after diamide treatment andthe majority of the protein appeared in a major band of�250 kDa with only minor smearing. The very high molec-ular weight TPH2 aggregates formed upon treatment withdiamide did not enter the gel but could be trapped and detectedon cellulose acetate membranes (data not shown). Treatmentof oxidant-exposed TPH2 with DTT led to a near-totalremoval of the high molecular weight aggregates such that themajor species on gels was the 56 kDa monomer. If excessDTT was added to enzyme preparations prior to oxidanttreatment, the effects on activity and aggregation wereprevented (data not shown). It can even be seen in Fig. 1(b)

Fig. 1 Inhibition of recombinant TPH2 and aggregation by cysteine

oxidants. Purified recombinant TPH2 was treated with 500 lM H2O2 or

100 lM diamide as described under ‘Experimental Procedures’. (a)

The effects of oxidants on TPH2 catalytic activity under substrate-

saturating conditions. The data represents means ± SEM of four

independent experiments carried out in duplicate and results are ex-

pressed as percent control TPH2 activity. The inhibitory effects of

H2O2 and diamide were significantly different from control (*p < 0.001)

and the DTT-mediated reversal of TPH2 inhibition was significantly

different for each oxidant by comparison to no-DTT conditions

(#p < 0.05 for H2O2; +p < 0.01 for diamide). (b) Aggregation of re-

combinant TPH2 by disulfide-bonding. TPH2 was treated with 500 lM

H2O2 or 100 lM diamide and aggregation of the protein was deter-

mined on immunoblots under non-reducing ()DTT) or reducing

(+DTT) conditions. Fully reduced TPH2 appears as a 56 kDa mono-

mer on denaturing, non-reducing gels.



that freshly prepared, untreated TPH2 shows a minor amountof cross-linking (130 kDa species) that is removed by DTT aswell, demonstrating its propensity to undergo cysteineoxidation and aggregation during purification in bufferscontaining oxygen (i.e., room air).

As an adjunct to immunoblotting to document misfoldingof TPH2 after cysteine oxidation, the turbidity of diamide-treated TPH2 preparations was determined by absorbancemeasures at 405 nm. Figure 2 shows the time-dependentincrease in turbidity upon oxidation of the protein. After a lagperiod of approximately 5 min, diamide-treated TPH2showed a steady increase in turbidity that reached maximumwithin 20–30 min. These solutions were visibly cloudy.Control preparations did not show increased turbidity overthe same time period. Samples of each TPH2 preparationwere subjected to immunoblotting and the results areincluded in the inset to Fig. 2. These data agree very wellwith results shown in Fig. 1(b) and indicate that diamidecaused extensive cross-linking of TPH2 into high molecularweight aggregates. DTT removed most of these highmolecular weight species. It is also evident in Fig. 2 (inset)that untreated preparations of TPH2 developed high molec-ular weight aggregates during turbidity measures, but notnearly to the same extent as seen during diamide treatment.We attribute this response to the need to use higherconcentrations of protein and diamide in this experiment toallow detection of aggregates at 405 nm.

Cysteine-scanning mutagenesis establishes that TPH2aggregates upon oxidation via disulfide cross-linkingOxidant-induced cross-linking of TPH2 into higher molec-ular weight species did not appear to reflect an orderedassembly into non-sodium dodecyl sulfate-dissociable mul-timers of 56 kDa monomers (see Fig. 1b). This resultsuggests the possibility that any of the cysteines throughoutthe TPH2 sequence could be cross-linking to others in aninter- and/or intra-molecular arrangement. Therefore, wetested a large series of TPH2 cysteine mutants for theirresponse to oxidation. Results for the regulatory domain(RD-WT) construct were interesting for two reasons. First,the predominant form of untreated cysteine-containing RDspecies on gels (Fig. 3a) has a MWof about 40 kDa (slightlylarger than two cross-linked RDs) and another band at theexpected MW of 17 kDa. This higher MW immunoreactiveband in untreated samples reverted to 17 kDa upon treatmentwith DTT (data not shown), indicating that two RDs weredisulfide cross-linked. Second, treatment of the RD-WT withdiamide caused cross-linking as evidenced by a reduction inthe amount of protein at the native MW of 17 kDa and theformation of two higher molecular weight species (� 40 and60 kDa). Mutants of the RD that contained only a singlecysteine (i.e., RD-C110S or RD-C112S) cross-linked into asingle major species of 40 kDa but the 60 kDa form was notformed. The cysteine-less RD construct (i.e., RD-CL; C110,C112S) did not cross-link when treated with diamide and didnot form the 40 kDa dimer under control conditions(Fig. 3a). Selected cysteine mutants of the TPH2 catalyticcore (CC; 39 kDa) were also treated with diamide and theresults in Fig. 3(b) show that the CC readily cross-links intohigh molecular weight species, seen predominantly as areduction in the 39 kDa species and extensive streaking. Aslong as the CC retains a single cysteine (i.e., CC + C315)diamide causes cross-linking, but to a smaller extent. TheCC-CL construct did not cross-link when treated withdiamide. Selected cysteine mutants of FL-TPH2 were treatedwith diamide and the results are presented in Fig. 3(c). FL-TPH2 cross-links as shown above (Fig. 1b) when treatedwith diamide. Truncation mutants of FL-TPH2 lacking thesix N-terminal most (i.e., FL-NDC6S) or the seven C-terminal most cysteine residues (i.e., FL-CDC7S) also cross-linked upon oxidation, seen as a reduction in the monomericspecies of each and streaking to the higher molecular weightregions of the gel. TPH2 lacking all cysteine residues(FL-TPH2-CL) did not cross-link as shown in Fig. 3(c).

Cysteine residues within the RD or the CC of FL-TPH2were mutated and responses of these mutants to diamideare presented in Fig. 3(d). The FL-TPH2 lacking cysteineswithin the RD (i.e., FL-RD-CL) showed considerablecross-linking whereas a mutant lacking cysteines in theCC (i.e., FL-CC-CL) mutant showed substantially lesscross-linking by comparison to either FL-TPH2 or FL-RD-CL much as seen in the CC mutant containing only one

Fig. 2 Increased turbidity of recombinant TPH2 after treatment with

diamide. Recombinant TPH2 was treated with 1 mM diamide and

turbidity was assessed through absorbance measures at 405 nm.

Turbidity was monitored at 1-min intervals with orbital shaking be-

tween each reading to re-suspend any aggregates. Untreated TPH2

preparations or preparations treated with diamide in the presence of

excess DTT did not show increases in absorbance at 405 nm. The

data in the graph represents the mean of five independent experi-

ments. Inset, aggregation of TPH2 into high molecular weight species

on western blots of gels run under non-reducing ()DTT) and reducing

(+DTT) conditions. Samples in the inset are taken from proteins used

in turbidity experiments.



cysteine. If a single cysteine residue remained in the RD(i.e., C112) and the CC (i.e., C315), referred to as FL-RD1CC1, this species also showed cross-linking whenexposed to diamide. Finally, TPH1 and two truncationmutants of TPH2, FL-ND40 and FL-CD24 were treatedwith diamide and the results are presented in Fig. 3(e). FL-ND40 lacks the N-terminal most 40 residues that set itapart from TPH1 and it can be seen that this mutant, aswell as TPH1, show considerable cross-linking uponoxidation. FL-C476S lacks the only cysteine residue withinthe leucine zipper domain and FL-CD24 omits the leucinezipper altogether, yet both constructs show substantialoxidant-induced cross-linking. When all mutants shown inFig. 3(a–e) above were treated with DTT (after diamide)and electrophoresed under reducing conditions, diamide-induced cross-linking was reversed (data not shown).Taken together, these results point to cysteine modification,and not other amino acid residues, as the mechanism bywhich oxidants cause cross-linking of TPH2 into highermolecular weight aggregates.

Oxidative stress inactivates TPH2 and causes the formationof high molecular weight aggregates in intact cellsIntact HEK293 cells expressing TPH2 were exposed toH2O2 or diamide and the effects on TPH2 activity areshown in Fig. 4(a). H2O2 (2 mM) lowered enzyme activityby 60% whereas diamide (1 mM) inhibited TPH2 functionby 75%. Figure 4(a) also shows that DTT reversed theinhibition of TPH2 caused by either oxidant. Immunoblot-ting of cells after oxidant treatment revealed that bothH2O2 and diamide resulted in the formation of highmolecular weight aggregates of TPH2, as shown inFig. 4(b). TPH2 from untreated HEK293 cells migratedentirely as the monomeric species. H2O2 caused a minorshift in TPH2 migration in gels from the monomericspecies toward the formation of immunoreactive bands of� 110–140 kDa. Diamide led to much more extensiveTPH2 cross-linking as evidenced by a near total loss of themonomeric species accompanied by the formation of aband at � 130 kDa and smearing of TPH2 immunoreac-tivity from � 170 kDa to the top of the resolving gel. DTTcompletely removed these high molecular weight aggre-gates and returned TPH2 to the monomeric form. If intactHEK293 cells were treated with H2O2 or diamide in thepresence of excess DTT, TPH2 inhibition and aggregationwas prevented (data not shown).

(a)

(b)

(c)

(d)

(e)

Fig. 3 Aggregation of TPH2 cysteine mutants by diamide-induced

oxidation. Cysteine-to-serine mutants within the RD (a), CC (b) or FL-

TPH2 (c,d) and truncation mutants of TPH2 (e) were expressed,

purified and treated under control conditions (C) or with diamide (D;

100 lM) and aggregation of the respective TPH2 constructs was

determined on immunoblots under denaturing and non-reducing gels.



Oxidative stress shifts TPH2 from the soluble compartmentof intact cells into membranes and large inclusion bodiesThe effect of diamide (1 mM) on TPH2 distribution inHEK293 cells was examined and Fig. 5(a) shows that TPH2is located primarily in the soluble fraction (72% of total) ofthe untreated cells. Diamide causes a substantial increase inthe amount of TPH2 that is recovered in the membranefraction, from 28% to 45% of total, based on semi-quantitative scans of the blot in Fig. 5(a). No attempt wasmade in these experiments to recover TPH2 as highmolecular weight aggregates after fractionation of cells sogels were run under reducing conditions to allow detection oftotal monomeric TPH2. Figure 5(b–e) shows results fromexperiments that exposed HEK293 cells expressing eGFPalone or an eGFP–TPH2 fusion construct to diamide

(1 mM). Cells were washed and large inclusion bodies wereisolated as detergent-insoluble complexes. Diamide (Fig. 5c)did not change the amount of eGFP found in the inclusionsby comparison to control (Fig. 5b). However, it is clear fromFig. 5(e) that diamide caused a substantial increase in theamount of eGFP–TPH2 found in large cellular inclusions.Untreated controls did show minor amounts of eGFP–TPH2

Fig. 4 Inhibition of TPH2 and aggregation after treatment of intact

cells with oxidants. (a) Intact HEK293 cells stably expressing TPH2

were treated with 2 mM H2O2 or 1 mM diamide and the effects on

catalytic activity were determined. Data are means ± SEM of four

independent experiments carried out in duplicate and results are ex-

pressed as percent control TPH2 activity. The inhibitory effects of

H2O2 and diamide were significantly different from control (*p < 0.001)

and the DTT-mediated reversal of TPH2 inhibition was significantly

different for each oxidant by comparison to no-DTT conditions

(#p < 0.05). (b) Intact HEK293 cells expressing TPH2 were treated

with 2 mM H2O2 or 1 mM diamide and aggregation of the protein was

assessed on western blots of gels run under non-reducing ()DTT) or

reducing (+DTT) conditions.

Fig. 5 TPH2 associates with membrane fractions and large inclusion

bodies after treatment of intact cells with diamide. Intact HEK293 cells

stably expressing TPH2 were treated with 1 mM diamide and the

distribution of TPH2 in membrane and soluble cellular compartments

as well as in detergent insoluble inclusion bodies was determined by

western blotting. (a) Intact TPH2-expressing HEK293 cells were

fractionated into soluble and membrane fractions after treatment with

diamide and the distribution of TPH2 was determined under reducing

conditions by western blotting. (b–d) Intact HEK293 cells expressing

an EGFP–TPH2 fusion protein were treated with diamide and deter-

gent insoluble inclusion bodies were isolated and probed for EGFP-

TPH2 using fluorescence microscopy. Images were captured from

untreated HEK293 cells stably expressing EGFP (panel b) or from

cells treated with diamide (panel c) and from untreated cells

expressing EGFP-TPH2 (panel d) or from cells treated with diamide

(panel e). (f) Intact HEK293 cells expressing wild-type TPH2 were

treated with the indicated concentrations of diamide after which large

detergent-soluble fractions and detergent-insoluble inclusion bodies

were isolated and probed for TPH2 using western blotting.



in the inclusions (Fig. 5d) but the magnitude of this effectwas small by comparison to diamide-treated cells. The resultspresented in Fig. 5(b–e) did not indicate a role for eGFP inenhancing aggregation for its TPH2 fusion partner. To ruleout this possibility fully, these experiments were repeated inHEK293 cells expressing wild-type TPH2 and detergent-insoluble inclusion bodies were immunoblotted under reduc-ing conditions to detect total monomeric TPH2. Figure 5(f)shows that diamide caused a progressive dose-effect onmovement of TPH2 into large inclusions. TPH2 is foundalmost exclusively in the detergent-soluble compartment ofuntreated controls but is shifted to a large extent to the largeinclusion bodies after treatment with increasing concentra-tions of diamide.

TPH2 is particularly sensitive to oxidative stress in intactcells as revealed by redox 2-dimensional sodium dodecylsulfate–PAGEThe disulfide-mediated cross-linking of TPH2 into highmolecular aggregates is somewhat difficult to place incontext, even in intact cells exposed to oxidizing conditions,because it is possible that H2O2 or diamide causes non-selective aggregation of the majority of cytoplasmic proteins.To gain a fuller appreciation of the extent to which TPH2 ismodified by oxidation in intact cells in relation to othersoluble proteins, HEK293 cells expressing TPH2 weresubjected to redox 2-dimensional PAGE and immunoblottingafter diamide (1 mM) treatment. The stained gels in Fig. 6(a– control cells and b – diamide-treated cells) show apredominant diagonal band of proteins that are not modified

by disulfide cross-linking after either treatment, in excellentagreement with previous, related studies (Brennan et al.2004; Cumming et al. 2004). However, proteins that cross-link into DTT-sensitive high molecular weight aggregatesafter diamide treatment (Fig. 6b) can be seen below and tothe right of the major diagonal band. These proteins are farmore prominent in the diamide-treated cells (Fig. 6b) than incontrols (Fig. 6a). Gels from identically treated cell prepara-tions were subjected to immunoblotting for TPH2, and it canbe seen in Fig. 6(c) that TPH2 is found almost entirely withinthe major diagonal band migrating as the 56 kDa monomer.However, diamide treatment results in the appearance of anextended TPH2 immunoreactive band at 56 kDa below andto the right of the diagonal band. The immunoreactivityrunning horizontally toward the right side of the blot at56 kDa represents aggregates that appear as higher molecularweight bands and smears on one-dimensional bands.

Discussion

Parkinson’s disease is the prototypical neurodegenerativedisorder of the DA neuronal system. Destruction of nigro-striatal pathways with attendant loss of DA neurotrans-mission leads gradually to severe motor impairment. Thepredominant therapeutic approach to treatment of the motorproblems associated with PD is L-DOPA, reflecting theemphasis on restoration of DA function (Ahlskog 2007).However, it is becoming clear that PD is a multicentricneurodegenerative process and loss of non-DA cells is likelyresponsible for the numerous non-motor symptoms that are

Fig. 6 Disulfide cross-linking of TPH2 in cytosol of diamide-treated

HEK293 cells. Intact HEK293 cells expressing TPH2 were treated with

1 mM diamide and the cytosolic proteins were fractionated on

sequential non-reducing/reducing gels. (a) Stained diagonal gel of

cytosolic proteins from untreated cells; (b) stained diagonal gel of

cytosolic proteins from diamide-treated cells; (c) western blot of diag-

onal gel of cytosolic proteins probed with antibodies against TPH2;

(d) western blot of diagonal gel of cytosolic proteins probed with

antibodies against TPH2. Arrows point to the TPH2 monomer at

56 kDa.



associated with PD. The non-motor symptoms of PD areserious and contribute to worsened disability, impairedquality of life and shortened life expectancy (Schrag 2006;McKinlay et al. 2008). Affective disorders in PD are also notsimply a consequence of psychological distress because ofthe development of a chronic debilitating disease. The co-morbidity of depression in PD (� 1 in 2) far exceeds thatfound in the general population (� 1 in 50; Beekman et al.1999) and in other neurodegenerative conditions (Wragg andJeste 1989; Chwastiak et al. 2002). Neither depression noranxiety is correlated with motor disability and affectivesymptomatology remains in PD patients after therapeuticimprovement in motor status (Kostic et al. 1987; Wang et al.2009). The fact that depression is frequently the presentingsymptom before the emergence of clinically significant motorsymptoms suggests that depression could even be considereda risk factor for PD (Nilsson et al. 2001; Schuurman et al.2002; Aarsland et al. 2009). As further recognition of theimportance of non-motor symptoms in PD, the developmentof an animal model that recapitulates many of theseprogressive deficits is revealing the involvement of dysfunc-tions in 5HT neurochemistry (Taylor et al. 2009).

How might the 5HT neuronal system be targeted fordestruction in PD? Those risk factors that target the DAneuronal system in PD (i.e., oxidative stress, mitochondrialdysfunction, and environmental factors) may well exert thesame general influence on 5HT neurons. However, thepossibility exists that a factor specific to 5HT neurons may beinvolved, as occurs with Parkin and a-synuclein in the caseof DA neurons (Schlehe et al. 2008; Winklhofer et al. 2008).One such factor could be TPH2. Apart from its role inregulating 5HT synthesis and function, TPH2 is concentratedselectively in 5HT neurons of the midbrain raphe nuclei.Previous work in our lab (Kuhn et al. 1980; Kuhn and Arthur1996, 1997; Kuhn and Geddes 1999) and others (Carkaci-Salli et al. 2006; Tenner et al. 2007) has established thatTPH2 is extremely unstable and quickly loses catalyticfunction upon oxidative attack. In light of its relatively highcysteine content (52 cysteine residues per functional tetra-mer) and selective localization in 5HT neurons, we hypoth-esized that TPH2 is highly susceptible to misfolding andaggregation under conditions simulating PD-induced oxida-tive stress. Therefore, the present studies represent anessential first step in the characterization of TPH2 as apotential endogenous neurotoxic protein for 5HT neuronsmuch as occurs with superoxide dismutase in amyotrophiclateral sclerosis (Cozzolino et al. 2008) and glyceraldehyde3-phosphate dehydrogenase and b-amyloid in Alzheimer’sdisease (Cumming and Schubert 2005). In these latter cases,protein misfolding via disulfide cross-linking is thought toplay a role in neurodegeneration.

Tryptophan hydroxylase 2 is shown presently to be highlysensitive to inactivation by cysteine oxidants. TPH2 misfoldsand forms high molecular weight aggregates upon oxidation.

Current studies establish clearly that these effects aremediated by disulfide cross-linking. First, the effects ofoxidants are prevented by the thiol reducing agent DTT, andDTT reverses oxidant-induced aggregation of TPH2 andpartially restores catalytic function. Failure to achievecomplete restoration of enzyme activity with DTT is relatedto the somewhat harsh conditions required to reverseaggregation (i.e., DTT with heating) but which lead tofurther inactivation of the enzyme. Second, a broad series ofTPH2 cysteine mutants reveal that virtually any cysteineresidue within TPH2 can participate in cross-linking. TPH2does not appear to cross-link in an ordered fashion intospecies that are multiples of the 56 kDa monomer, but formshigher molecular aggregates indicative of intra- and inter-molecular cross-linking (Cumming et al. 2004). Removal ofthe C-terminal lysine zipper of TPH2 (or the single cysteineresidue therein), which mediates its assembly into functionaltetramers (Mockus et al. 1997), does not prevent disulfidecross-linking indicating further that oxidation is not formingtetramers that are resistant to disassembly. Neither theregulatory nor catalytic core domain of TPH2 confersspecificity on cross-linking, and as long as 1–2 cysteinesremain in TPH2, it misfolds upon oxidation. Only thecysteine-less mutant of TPH2 is totally resistant to oxidant-induced cross-linking. These results are important mecha-nistically because they establish that cysteines alone mediatethe responses of TPH2 to oxidation. TPH2 is also verysensitive to oxidant stress-induced modification in intactcells. Both diamide and H2O2 inhibit TPH2 catalytic functionand lead to the formation of high molecular weightaggregates in HEK293 cells. TPH2 is a cytoplasmic proteinbut in reaction to oxidative stress shifts into membranefractions and large cellular inclusion bodies. We did notpresently characterize these subcellular compartments but itis clear nonetheless that oxidants cause a dramatic reductionin TPH2 catalytic function and lead to misfolding andaggregation. Studies using redox 2D electrophoresis alsoshow that TPH2 undergoes oxidative cross-linking in intactcells.

L-DOPA remains the standard pharmacotherapy for PDand the majority of patients will receive it at some point intheir treatment. L-DOPA is converted to DA in brain by L-aromatic amino acid decarboxylase. This treatment increasesDA in all cells expressing the decarboxylase to include DAneurons, as desired, as well as in 5HT and other neurons. Theinappropriate deposition of DA within 5HT neurons candramatically alter their neurochemical function. L-DOPAincreases DA at the expense of decreased 5HT release innormal (Borah and Mohanakumar 2007) or DA-depleted rats(Maruyama et al. 1992; Navailles et al. 2010a). It has alsobeen shown recently (Navailles et al. 2010b) that 5HTneurons mediate ectopic release of DA after treatment ofhemi-parkinsonian rats with L-DOPA. The release of DA as a‘false transmitter’ from 5HT neurons has been implicated in



the L-DOPA-induced dyskinesia seen in PD rats (Carta et al.2007, 2008) and could conceivably play a role in theexacerbation of affective symptoms seen in more advancedstages of PD, when the efficacy of L-DOPA therapy iscompromised. L-DOPA may also accentuate damage to the5HT system. DA- and L-DOPA-derived quinones reduce5HT synthesis by inactivating TPH2 (Kuhn and Arthur 1998,1999). We have also shown recently that bilateral 6-hy-droxydopamine lesions in rats reduce TPH2 content inmidbrain raphe nuclei, an effect that is accentuated byL-DOPA treatment (Eskow Jaunarajs et al. 2010).

It is recognized by now that the majority of the disabilityassociated with late-stage PD arises from dysfunction withinnon-DA systems (Ahlskog 2007). The emergence of numer-ous psychiatric disorders including depression, apathy andanxiety could well be related to 5HT neurochemical deficits.Unfortunately, investigations into non-DA mechanisms ofPD lag far behind those for DA to the point of being virtuallyabsent (Taylor et al. 2009). In light of the importance ofprotein misfolding (i.e., proteinopathy) in neurodegenerativediseases in general (Glickman and Ciechanover 2002;Goldberg 2003) and in PD specifically (Chung et al. 2001;Braak et al. 2004; Schlehe et al. 2008; Winklhofer et al.2008; Tan et al. 2009), studies specific for the 5HT neuronalsystem are long overdue. Considering existing evidence, amechanistic framework by which misfolded TPH2 coulddamage 5HT neurons can be proposed. TPH2 is a cytoplas-mic protein that is extensively associated with microtubulesin axons and dendrites (Joh et al. 1975; Pickel et al. 1976).We have also noted that TPH2 associates with tubulin underphosphorylating conditions insofar as immunoprecipitationof TPH2 co-precipitates tubulin (Johansen et al. 1995, 1996).Disruption of axonal transport may be a causative event inneurodegenerative diseases including PD (De Vos et al.2008) and the rotenone-induced damage to 5HT neuronsoccurs through a microtubule-dependent mechanism (Renand Feng 2007). Evidence for decreased transport andaccumulation of TPH2 has been presented in Alzheimer’sdisease (Burke et al. 1990). Therefore, misfolding of TPH2and cross-linking to microtubules could exert deleteriouseffects on 5HT neurons.

The present work represents an essential, initial charac-terization of TPH2 as a misfolding protein that couldinfluence 5HT neuronal function in advancing PD. TPH2satisfies several criteria in playing such a role to includeselective expression in 5HT neurons, extreme sensitivity toinactivation and misfolding by oxidative stress, and abnor-mal sorting from cytoplasm into membranes and inclusionbodies. The current approach depends by necessity onoxidants that model conditions prevalent in PD because thespecific oxidant(s) that play roles in the neurodegeneration ofPD are not known. Similarly, the present studies used TPH2-expressing HEK293 cells to model intact human cells andbecause other 5HT neuronal-like cells such as the mouse

teratocarcinoma (Buc-Caron et al. 1990) and RN46A cells(White and Whittemore 1992) express too little TPH2 todetect using immunoblotting and enzymatic assays. Thepresent results are also limited in that they have not yetestablished that oxidation and misfolding of TPH2 specifi-cally leads to cellular damage. Unfortunately, the selectiveoxidation of TPH2 in intact cells is not possible. Nonethe-less, the results are highly relevant in that acute oxidativestress results in reductions in 5HT synthesis and dramaticchanges in TPH2 cellular status, conditions that exist in PD.

Acknowledgements

This work was supported, in whole or in part, by National Institutes

of Health grants DA010756, DA017327, and a VA Merit Award.

The authors declare that they have no conflicts of interest.

Supporting information

Additional supporting information may be found in the online

version of this article:

Figure S1. Mutagenesis of TPH2.

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peer-reviewed and may be re-organized for online delivery, but are

not copy-edited or typeset. Technical support issues arising from

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department of psychiatry & behavioral neurosciences, wayne ... · was from bio-rad (hercules,...

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