reduced neuronal expression of insulin-degrading enzyme in the dorsolateral prefrontal cortex of...

Journal of Psychiatric Research 43 (2009) 1095–1105

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Journal of Psychiatric Research

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Reduced neuronal expression of insulin-degrading enzyme in the dorsolateralprefrontal cortex of patients with haloperidol-treated, chronic schizophrenia

Hans-Gert Bernstein a,*, Theresia Ernst a, Uwe Lendeckel b,d, Alicja Bukowska b, Siegfried Ansorge c,Renate Stauch a, Sara Ten Have e, Johann Steiner a, Henrik Dobrowolny a, Bernhard Bogerts a

a Department of Psychiatry, University of Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germanyb Institute of Experimental Internal Medicine, University of Magdeburg, Germanyc IMTM Immune Technologies and Medicine GmbH, Leipziger Str. 44, D-39120 Magdeburg, Germanyd Department of Medical Biochemistry and Molecular Biology, University of Greifswald, Sauerbruchstraße, D-17487 Greifswald, Germanye The Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, Dundee University, Dundee, UK

a r t i c l e i n f o

Article history:Received 6 November 2008Received in revised form 20 February 2009Accepted 16 March 2009

Keywords:SchizophreniaInsulin-degrading enzymeNeocortexHypothalamusBasal nucleus of MeynertMorphometryWestern blotHaloperidolNeuropeptides

0022-3956/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.jpsychires.2009.03.006

* Corresponding author. Tel.: +49 391 67 14249; faE-mail address: [email protected]

a b s t r a c t

Insulin-degrading enzyme (IDE) is a neutral thiol metalloprotease, which cleaves insulin with high spec-ificity. Additionally, IDE hydrolyzes Ab, glucagon, IGF I and II, and b-endorphin. We studied the expressionof IDE protein in postmortem brains of patients with schizophrenia and controls because: (1) the geneencoding IDE is located on chromosome 10q23–q25, a gene locus linked to schizophrenia; (2) insulinresistance with brain insulin receptor deficits/receptor dysfunction was reported in schizophrenia; (3)the enzyme cleaves IGF-I and IGF-II which are implicated in the pathophysiology of the disease; and(4) brain c-endorphin levels, liberated from b-endorphin exclusively by IDE, have been reported to bealtered in schizophrenia. We counted the number of IDE immunoreactive neurons in the dorsolateral pre-frontal cortex, the hypothalamic paraventricular and supraoptic nuclei, and the basal nucleus of Meynertof 14 patients with schizophrenia and 14 matched control cases. Patients had long-term haloperidoltreatment. In addition, relative concentrations of IDE protein in the dorsolateral prefrontal cortex wereestimated by Western blot analysis. There was a significantly reduced number of IDE expressing neuronsand IDE protein content in the left and right dorsolateral prefrontal cortex in schizophrenia comparedwith controls, but not in other brain areas investigated. Results of our studies on the influence of halo-peridol on IDE mRNA expression in SHSY5Y neuroblastoma cells, as well as the effect of long-term treat-ment with haloperidol on the number of IDE immunoreactive neurons in rat brain, indicate thathaloperidol per se, is not responsible for the decreased neuronal expression of the enzyme in schizo-phrenics. Haloperidol however, might exert some effect on IDE, through changes of the expression levelsof its substrates IGF-I and II, insulin and b-endorphin. Reduced cortical IDE expression might be part ofthe disturbed insulin signaling cascades found in schizophrenia. Furthermore, it might contribute tothe altered metabolism of certain neuropeptides (IGF-I and IGF-II, b-endorphin), in schizophrenia.

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1. Introduction

Insulin-degrading enzyme (IDE, a.k.a insulinase and insulysin;EC 3.4.24.56) is a zinc-binding, neutral, thiol metalloproteasewhich belongs to the M16A pitrilysin subfamily of proteases. It re-quires both a free thiol and bivalent cations for its proteolyticactivity (Ansorge et al., 1984b). It is a single protein with amolecular weight of 110 kDa. IDE was co-isolated with the multi-catalytic proteinase, suggesting that IDE might be part of a proteincomplex (Bennett et al., 2000). Ubiquitin and an endogenous14 kDa inhibitor of IDE were shown to regulate its activity in cells

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x: +49 391 67 15223.(H.-G. Bernstein).

(Ogawa et al., 1992; Saric et al., 2003). In humans the geneencoding IDE is located on chromosome 10q23–q25, spanningabout 120 kb (Qiu and Folstein, 2006). IDE is widely expressedamong organs and tissues. In the brain its expression is highestduring development and decreases during aging (Runyan et al.,1979; Reiser et al., 1987; Baumeister et al., 1995). Since IDE iscapable of cleaving insulin with high specificity, the enzyme hasbeen regarded as a reliable marker for insulin catabolism (Dornet al., 1983; Ansorge et al., 1984a,b; Azam et al., 1990). Besidescleaving insulin, IDE is known to hydrolyze other natural sub-strates such as peptide Ab (Kurochkin and Goto, 1994), glucagon(Ansorge et al., 1984a,b; Authier et al., 1996), insulin-like growthfactors I and II (Roth et al., 2003), natriuretic peptide (Mülleret al., 1991), and b-endorphin (Safavi et al., 1996), as well as ATP(Del Carmen Camberos and Cresto, 2007). Due to its broad

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substrate spectrum IDE is prominently involved in a plethora ofcellular processes (Yfanti et al., 2008) and molecular variants ofthe gene encoding the enzyme may even have an influence on hu-man lifespan (Hong et al., 2008).

The past decade has witnessed growing interest in brain IDE,since it has been shown that IDE is the main soluble Ab-degradingenzyme at neutral pH in rat and human nervous tissue, in vitro(Kurochkin and Goto, 1994; McDermott and Gibson, 1997; Vekrel-lis et al., 2000; Hersh, 2006 and others). Its proven ability to cleavethe Leu16–Leu17 bond within the Ab region of the APP moleculeprecludes the formation of amyloidogenic fragments. Moreover,its dual role as an insulin- and Ab-cleaving enzyme in the CNS aswell as the reported genetic association between IDE and sporadicAlzheimer disease (AD; reviewed by Bernstein, 2005 and Qiu andFolstein, 2006) may link the pathology of this disease to that oftype II diabetes. This hypothesis (Henneberg and Hoyer, 1995; Frö-lich et al., 1998; Saric et al., 2003; Sigurdsson and Morelli, 2006)became one of the most debated topics with regard to the possibleorigin of sporadic AD (for recent considerations see De la Monteand Wands, 2005; Cole et al., 2007; Kim et al., 2007; Minerset al., 2008).

Table 1Demographical data of controls and cases with schizophrenia.

Case/sex (m/f) age (years) Postmortem interval (h) Storage tim

(a) Control cases for IDE immunohistochemistryControl cases31/m/40 96 63/m/50 72 332/m/64 35 826/f/33 72 919/f/64 24 2.558/f/64 26 1492/f/64 24 6153/m/29 60 3068/m/61 24 517/f/72 24 10

Established obese control cases (n = 4)95/f/38 24 828/f/50 72 47/m/47 24 695/f/38 24 8

(b) Individuals with schizophrenia for IDE immunohistochemistry72/m/34 5 11150/m/38 24 738/m/50 48 437/m/51 48 1040/m/57 72 518/f/65 66 744/f/38 12 –20/f/53 48 4106/f/54 24 584/f/59 48 915/f/60 48 5

Established obese cases with schizophrenia (n = 3)91/f/40 48 321/m/46 48 775/m/48 48 7

Case/sex (m/f) age Postmortem interv

(c) Control cases for IDE Western blotting (frozen material)m/51 16m/60 9f/55 12f/60 17f/62 15

(d) Cases with schizophrenia for IDE Western blotting (frozen material)m/49 16f/55 12f/58 14

Some recent findings, however, give reason to assume thatbrain IDE might be also involved in other neurological and neuro-psychiatric diseases (Morelli et al., 2004; Zhao et al., 2007; Chad-wick et al., 2007). Patients with schizophrenia often suffer fromthe so-called ‘‘metabolic syndrome” which is characterized by type2 diabetes, visceral obesity, elevated lipid levels and hypertension,and decreased sensitivity to insulin (for review see Sacks, 2003;Hägg et al., 2006). This co-morbidity significantly contributes toan increased risk of cardiovascular diseases in mentally ill patients(Casey, 2005; Hägg et al., 2006). In the case of schizophrenia, theincreased incidence of the metabolic syndrome/type 2 diabetes isnormally regarded a result of the treatment of the patients withatypical neuroleptics (for recent reviews see Holt et al., 2004; Zhaoet al., 2007 and De Hert et al., 2008), which is a major cause fornon-compliance of patients (Weiden et al., 2004). Recent workshows that treatment with typical neuroleptics may also increasethe risk of developing the metabolic syndrome (De Hert et al.,2008; Perez-Iglesias et al., 2009). The question, if drug-naïve pa-tients with schizophrenia may already show symptoms of themetabolic syndrome/diabetes has been controversially discussed(pro: Thakore et al., 2002; Kohen, 2004; Thakore, 2004; Huang

e (months) Cause of death BMI (kg/m2)

Not specified –Not specified –Ruptured aortic aneurysm 19.6Pulmonary embolism 20.7Peritonitis 14.3Myocardial infarction –Gastrointestinal hemorrhage –Not specified –Myocardial infarction 26.5Pneumonia, Ileus 21.0

Not specified 29.4Not specified 30.8Myocardial infarction 34.6Not specified 29.4

Suicide by hanging –Not specified –Pulmonary embolism 24.5Ileus 20.7Heart failure –Heart failure 14.4Not specified –Not specified –Not specified –Not specified –Heart failure 19.9

Ileus 36.0Pulmonary embolism 30.3Respiratory failure (aspiration) 29.8

al (h) Cause of death

Respiratory insufficiencyHeart failureHeart failureRuptured aortic aneurismLung embolism

Heart failureSuicide by hangingNot specified

H.-G. Bernstein et al. / Journal of Psychiatric Research 43 (2009) 1095–1105 1097

et al., 2006; Wang et al., 2007; contra: Perez-Iglesias et al., 2009;Mitchell, 2009). Indeed, there is yet little evidence that impairedglucose tolerance itself may increase the risk for schizophrenia(Perez-Iglesias et al., 2009; Mitchell, 2009). Observations thatnewly diagnosed and/or drug-naive schizophrenia patientsfrequently present with impaired glucose tolerance and/ordiabetes could well be due to current lifestyle factors (i.e. reducedphysical activity, impropriate nutrition and poorer health care)that predispose subjects to both disorders (Holt et al., 2004;Mitchell, 2009).

Interestingly, studies in schizophrenia identified robust de-creases in the expression levels of genes with metabolic functions,particularly in glycolysis and ATP generation. Many of these met-abolic genes are induced by insulin (Altar et al., 2009). Moreover,markedly reduced brain insulin receptor function and disturbedinsulin signaling pathways were recently found in patients withschizophrenia and in an animal model of the disease (Zhao et al.,2007). Thus, the observed changes in postmortem brains ofpatients with schizophrenia show certain similarities to what isfound in AD patients with regard to aberrant cerebral glucose-insulin metabolism (see above). Since IDE is a downstream targetof the insulin receptor signaling cascade (Zhao et al., 2004), wequeried whether this enzyme might also be affected inschizophrenia.

2. Material and methods

2.1. Human brain material

All brains were obtained from the New Magdeburg brain collec-tion. Case recruitment, acquisition of personal data, performance ofautopsy, and handling of autoptic material were conducted in strictaccordance with the Declaration of Helsinki, and have been ap-proved by the responsible Ethical Committee of Magdeburg. Brainsof 14 individuals without neurological or psychiatric disorders (sixmales, eight females; aged between 38 and 72 years) and 14 indi-viduals with schizophrenia (six males, six females, aged between34 and 65 years) were studied. All subjects with schizophreniahad histories of inpatient hospitalization. A lifetime psychiatricdiagnosis of schizophrenia was established according to DSM-III-R (American Psychiatric Association, 1987) by using the DiagnosticInstrument for brain studies (Dean et al., 2000), a structuredinstrument for the collection of clinical, pharmacological and otherrelevant information from case histories.

Only patients with well preserved and extensive clinical recordswere selected for the present study. Demographic and histologicaldata are summarized in Tables 1 and 2. Five brains of individualswithout a psychiatric disorder (two males, three females, aged

Table 2Clinical data.

Case/sex/age (years) Diagnosis, DSM-III-R Age of onset

72/m/34 Schizophrenia, chronic paranoid 32150/m/38 Schizophrenia, undifferentiated –21/m/46 Schizophrenia, undifferentiated 2875/m/48 Schizophrenia, undifferentiated 1638/m/50 Schizophrenia, undifferentiated 2537/m/51 Schizophrenia, chronic paranoid 2340/m/57 Schizophrenia, chronic paranoid 2818/m/65 Schizophrenia, chronic residual –44/f/38 Schizophrenia, chronic undifferentiated 3091/f/40 Schizophrenia, undifferentiated 2120/f/53 Schizophrenia, chronic undifferentiated 33106/f/54 Schizophrenia, chronic residual 3684/f/59 Schizophrenia, chronic paranoid 5215/f/60 Schizophrenia, chronic paranoid 44

51, 55, 60, 60 and 62 years) and three brains of patients withschizophrenia (one male, two females, aged 49, 55 and 58 years;all with long-term medication with haloperidol) were used forWestern blot analysis (see Table 1).

Brain volumes were calculated by the weight method usingfresh whole brain weight and brain density (Yamada et al.,1999). There were no significant differences (Students t -test, v2

analysis) between patients with schizophrenia and controls withregard to age, gender, and storage delay. Additionally, quantitativeneuropathological changes due to neurodegenerative disorderswere ruled out by an experienced neuropathologist as earlier de-scribed (Bernstein et al., 2001; Danos et al., 2003). In order to re-veal a possible influence of diagnosis-independent obesity on thecerebral expression of IDE individual body mass indices were cal-culated when possible. Knowing body weight and height of mostcases with schizophrenia and controls we were able to determineindividual body mass indices of a majority of cases (Tables 1aand b). Importantly, the average body mass index (BMI) of leancontrols (23.4 ± 2.1 kg/m2) did not significantly differ from that ofcases with schizophrenia (24.1 ± 2.3 kg/m2). The average BMI ofobese non-psychotic controls 34.3 ± 2.2 kg/m2. The individual BMIsof the four obese, non-psychotic patients were well beyond the re-cently calculated cut-off value of 28.7 kg/m2 for the metabolic syn-drome (Tirupati and Chua, 2007; Bernstein et al., 2008b). The sameholds true for three established obese cases with schizophrenia.

Brains destined for immunohistochemical studies were re-moved between 16 and 46 h after death. Tissue preparation wasperformed as described previously (Bernstein et al., 1999b). Briefly,brains were fixed in toto in 8% phosphate-buffered formaldehyde(pH 7.0) for 2 months. After embedding the brains in Paraplast�,serial coronal 20-lm-thick sections were cut on a microtome andmounted on slides. Every 50th section was stained for morpholog-ical orientation (combined cresyl violet and myelin stainingaccording to Nissl and Heidenhain-Woelcke). For Western blotanalysis samples of the left prefrontal cerebral cortex were used.These brains were removed between 9 and 17 h after death, dis-sected into small pieces, snap-frozen in liquid nitrogen and storedat �80�C.

2.2. Tissue processing, immunohistochemistry, and Western blotting

2.2.1. Tissue preparationThe brains for histological and immunohistochemical analysis

were processed in a standard manner, including immersion-fixa-tion in 8% phosphate-buffered formaldehyde for 2 months, embed-ded in Paraplast and cut with a microtome (whole-brain coronalsections, 20 lm thickness; Bernstein et al., 1998). Volume shrink-age was determined for each brain before and after dehydration

(years) Duration of illness (years) Medication before death (duration)

2 Haloperidol (2 years)– Haloperidol (at least 6 months)18 Haloperidol (duration unknown)32 Haloperidol (at least10 years)25 haloperidol (duration unknown)28 Haloperidol (at least 10 years)21 Haloperidol (at least 8 years)– Haloperidol (at least 1 year)8 Haloperidol (at least 3 years)19 Haloperidol (at least 4 years)20 Haloperidol (duration unknown)18 Haloperidol (at least 10 years)7 Haloperidol (duration unknown)16 Haloperidol (at least 10 years)


and embedding of tissue. Volume shrinkage factors were calculatedusing the formula: VF = (A1/A2)3/2 (VF = volume shrinkage factor;A1 = cross-sectional area before processing of tissue; A2 = cross sec-tional area after processing of tissue). Sections at intervals of400 lm (according to Cavalieris principle, Mayhew, 1992).

2.2.2. ImmunohistochemistryTwo different antibodies to IDE were employed in immunohis-

tochemical and Western blot studies. A polyclonal antiserumagainst the rat liver enzyme was raised in rabbits. For all proce-dures with rats and rabbits, ethical approval was sought accordingto the National Act of the Use of Experimental Animals of Germanyand the local Ethics Commission.

Rabbits received the primary s.c. injections of 2 ml of homoge-nate of IDE containing polyacrylamide gel slices (in 0.15 mol/l NaCland 0.5 mmol/l dithiothreitol) emulsified in Freunds completeadjuvant into the inguinal region. Booster injections (2 ml) weregiven after 3 months at weekly intervals. Each injection containedapproximately 0.1 mg highly purified proteinase protein. A total of3–4 injections were necessary to get reasonable titers. The rabbitswere bled one week after the last inoculation. Immunoinhibitoryproperties of the IDE antibody (i.e. inhibition of 125I-insulin and125I-glucagon degradation by IDE) were studied and an almost totalinhibition of peptide degradation was revealed [over 90% for eachpeptide; Ansorge et al. (1984a)]. The purity of the preparation wasfurther tested by two-dimensional electrophoresis, immunodiffu-sion, and crossed immunoelectrophoresis as described earlier(Dorn et al., 1983; Ansorge et al., 1984a,b). In all immunologicalspecificity tests a single precipitation line was observed.

The second antibody used, was a commercially available, well-characterized monoclonal antibody to IDE from Abcam (chargenumber 25733, Abcam Cambridge, UK). Immunohistochemicaluse of this antibody was described in sufficient detail by Cookand colleagues (Cook et al., 2003). In pilot studies we comparedour polyclonal anti-IDE antiserum and a commercially availablemonoclonal antibody against the enzyme with regard to theirimmunostaining properties. Both antibodies labeled identical cellpopulations in different brain areas. However, the signal-to-noise-ratio was better with the polyclonal antiserum. Hence, weused this antiserum for all further studies (for details, see Bernsteinet al., 2008a).

Immunohistochemical control experiments involved omissionof the primary antiserum, its substitution by rabbit normal serumor buffer.

2.2.3. Immunoblot assayFrozen tissue samples from the prefrontal cortex were pulver-

ized in liquid nitrogen and subsequently homogenized in lysis buf-fer (50 mM Tris–HCl, pH 7.5, 5 mM EDTA, 100 mM NaCl, 0.5%Triton X-100, 10% glycerol, 10 mM K2HPO4, 0.5% NP-40), contain-ing a protease inhibitor cocktail (Roche Diagnostics, Heidelberg,Germany), 1 mM sodium vanadate, 0.5% deoxycholate, 0.1 mMPMSF, 20 mM NaF, and 20 mM glycerol 2-phosphate (all from Sig-ma, Heidelberg, Germany). Tissue homogenates were centrifugedat 15,000 rpm for 15 min and the resulting supernatant was storedat �20 �C until further use. Aliquots representing 30 lg proteinwere subjected to SDS–PAGE, followed by transfer to a polyvinyli-dene fluoride membrane (NEN Life Science, Boston, MA, USA).Membranes were blocked with Roti-Block (Roth, Karlsruhe, Ger-many) in TBS and then incubated with either the polyclonal antise-rum (diluted in Roti-Block/TBS 1:200) or a mouse monoclonalantibody against IDE (9B12.225, Abcam; diluted in PBS/0.05%Tween20 1:300). Anti-rabbit or anti-mouse horseradish peroxidaseconjugated antibodies (Cell Signaling, Frankfurt/Main, Germany)diluted 1:3000 in Roti-Block/TBS were applied after washing theblots three times in TBS. For chemoluminescence detection, the

SuperSignal West Dura substrate (Pierce, Bonn, Germany) wasused. To compare the different groups, densitometric quantifica-tion was performed only on equally processed blots and exposedon the same X-ray film. The expression of glyceraldehyde-3-phos-phate dehydrogenase (GADPH), a house keeping enzyme (Guptaet al., 1999), was used as internal standard.

2.3. Stereology and statistical analysis

IDE expressing neurons were counted in different brain areas(layers of the left and right dorsolateral prefrontal cortex, thehypothalamic paraventricular (PVN) and supraoptic (SON) nucleiand basal nucleus of Meynert). The section thickness after the his-tological procedures was 18.9 ± 1.0 lm (mean ± SD). A countinggrid was used to define a three-dimensional box within the thick-ness of the section as described earlier (Bernstein et al., 1998)allowing at least 4-lm guard zones at the top and bottom of thesection, and to apply a direct, three-dimensional counting method.IDE immunopositive neurons were counted separately in a linearprobe of stacked counting boxes extending from the pial surfaceto the underlying white matter (Selemon et al., 2003). Laminarboundaries, which were most clearly visible at low power (6.3�),were marked on the linear probes by switching back and forth be-tween 6.3� and 40� objectives during the analysis so that cell den-sity for each of the six layers could be calculated. Total length ofthe linear probe provided a measurement of cortical thickness. Val-ues from the five linear probes were averaged to obtain a mean va-lue of neuronal density. Fifteen boxes per cortical layer werecounted. We examined the densities of all (Nissl stained) neuronsin the selected area as published (Bernstein et al., 1998, and Bern-stein et al., 2007; Spiechowicz et al., 2006). To estimate the numberof IDE-immunopositive PVN and SON neurons we counted at high-er magnification the number of cell profiles per slice using the opti-cal disector method as previously described for nitric oxidesynthase-containing hypothalamic neurons (Bernstein et al.,1998). Left and right hemispheres were counted separately. Sincethe actual thickness of the sections was between 18 and 20 lm,two well-defined optical planes within the section were used (dis-tance 16 lm) and immunostained cells were counted that comeinto focus as one passes from the upper to the lower optical plane.The number of objects was related to the square of the PVN andSON at the counting levels and to the number of neuronal profilesof Nissl-stained neurons at adjacent sections. Knowing the numberof cell profiles within this ‘‘counting box” (i.e. between the twoplanes of the dissector) and the square of the counting area (asdetermined by the square of the PVN and SON at the adjacent sec-tion) it was possible to calculate the amount of IDE-like-immuno-reactive cells within a given tissue volume (cell density). Whenneglecting minimal fluctuations of the number of immunostainedneuronal profiles between the counted sections, it was possibleto estimate the total number of IDE-containing nerve cells withinthe whole PVN and SON by multiplying the average numerical celldensity by the volume of the population (Purba et al., 1996). Therostral starting level for cell counting in the PVN was neuroana-tomically defined by the appearance of the central part of the sup-rachiasmatic nucleus on the section (guiding structure). Cellcounting was performed under blind conditions by two indepen-dent raters. The inter-rater correlation (reliability) was 0.91. Vol-umes of the PVN and SON were determined by integrating areameasurements from the most rostral to the most caudal Nissl-stained sections of each population. Numbers of IDE-immunoreac-tive neurons in the basal nucleus of Meynert were estimatedaccording to Chui et al. (1984). Neurons were counted at the siteof maximal neuronal density after reviewing serial sections aroundthe decussation of the anterior commissure (Chui et al., 1984). Thedata were statistically analysed by the nonparametric, two-tailed


U-test (Mann and Whitney) and t-test with Tukey–HSD correction.The critical level for statistical significance was P 6 0.05. To assesschanges secondary to hospitalization and autolysis, IDE cell num-bers found in post mortem dorsolateral prefrontal cortex and hypo-thalamic nuclei of neuropsychiatric patients were correlated to theduration of the illness and the post mortem delay (Spearman’s non-parametric ranking test).

2.4. Neuronal expression of IDE immunoreactivity in haloperidol-treated rats

2.4.1. AnimalsFifteen adult male Wistar rats, weighing between 190 and 230 g

were divided into three groups, each containing five animals, to in-clude low-dose (group a), high-dose (group b), and controls (groupc). Animals were housed separately in standard plastic cages in anair-conditioned room, at 22�C, under a 12:12-h light/dark cycle,and fed standard laboratory chow and tap water during the exper-iment. The experiment was performed in accordance with the na-tional guidelines for the use and care of laboratory animals. Allprocedures were approved by the local animal care committee ofthe University of Magdeburg.

2.4.2. Drug administrationHaloperidol (Sigma St. Louis, MO, USA.) dissolved in normal sal-

ine (0.9% NaCl) to give a dose of either 0.4 (group a, low dose) or 1.0(group b, high dose) mg/kg, was given intraperitoneally once a dayfor 6 weeks. Haloperidol at a dose of 1.0 mg/kg was chosen asbeing equivalent to the highest dosage used in human therapies(Chang et al., 1989). The same volume of normal saline (0.9% NaCl)was administered to the control group (group c). At the end of thesixth week, animals were anesthetized with chloral hydrate andtranscardially perfused with 8% formalin.

2.4.3. Immunohistochemistry, morphometry, and statisticsBrains were removed from the cranium and fixed in formalin

and then paraffin-embedded as described earlier (Keilhoff et al.,2001). Serial 6 lm thick sections were cut from the brains. Everyfifth section was stained for hematoxylin–eosin. The immuno-chemical protocol (including specificity controls) for IDE was thesame as for human brain material. To estimate the amount ofIDE-immunopositive neurons in the prefrontal cortex and in hypo-thalamic areas (PVN) we counted stained cell profiles at highermagnification using the optical disector method as described else-where (Bernstein et al., 1998). A counting grid was used to obtain areference square. At least three sections per region and case werecounted. Since the thickness of the paraffin sections was only6 lm, no infrasectional counting box was defined (Bernsteinet al., 2008b). Tissue shrinkage was calculated and taken into ac-count. The data were statistically analysed by the non-parametricU-test (Mann and Whitney).

2.5. Expression of IDE mRNA in haloperidol-treated SHSY5Yneuroblastoma cells

2.5.1. Cell cultureThe human SH-SY5Y neuroblastoma cell line was purchased

from DSMZ (Braunschweig, Germany; ACC 209) and grown to80% confluency in Dulbeccos modified Eagles medium (DMEM;PAA Laboratories, Linz, Austria), supplemented with 15% heat-inac-tivated fetal calf serum (PAA). To study the effect of different con-centrations of haloperidol, cells were seeded into six well plates ata density of 2 � 106 cells/3 ml and cultured in the absence or pres-ence of haloperidol as indicated in the figure for 24 h. Cells werethen washed twice in PBS and RNA preparation was started by di-rectly adding the lysis buffer (see RNA preparation).

2.5.2. RNA preparation and RT-PCRSamples for RT-PCR were rapidly frozen in liquid nitrogen and

stored until further analysis. As described recently (Bukowskaet al., 2006) by applying the method of Chomczynski and Sacchi(1987). A 1 lg quantity of total RNA was transcribed into cDNA.A 25 ll reaction mixture consisted of 1� SensiMix (Quantace,UK), 0.5 ll of SYBR-Green I (Quantace), 1 ll cDNA, and 0.3 lmol/lof the specific primers for IDE (Hs_IDE_1 SG QuantiTect, Qiagen,Hilden, Germany) or b-actin (forward: AAgATgACCCAgATCATgTTT-gAg; reverse: AggAggCAATgATCTTgATCTT, BioTeZ, Berlin, Ger-many). PCR was performed in an iCycler (BioRad, Munich,Germany). Initial denaturationat 95 �C for 30 s, annealing at 62 �Cfor 30 s, and elongation at 72 �C for 60 s.

3. Results

3.1. Immunolocalization of IDE in human brain regions – qualitativeobservations

3.1.1. Human brain cortexConsistent with previously published findings (Bernstein et al.,

2008a) IDE was found to be widely but unevenly distributed in hu-man cortical neurons. IDE immunoreactive neurons were locatedin all cortical areas investigated. Almost all layer III and V pyrami-dal neurons showed an intense immunolabeling. Additionally,numerous interneurons were also found to express the protein.Intracellularly, the immunoreaction was located in the perikaryaand the dendrites of the nerve cells (Fig. 1A). Typically, cell nucleiwere free of reaction product. Sometimes, the neuropil showed aweak to moderate immunostaining of IDE. Remarkably, a consider-able portion of brain sections showed a markedly reduced neuro-nal immunostaining for IDE compared to the other ones. Afterunblinding for the diagnoses it was revealed that among these lessintense stained sections all but one were obtained from patientswith schizophrenia (an example is given in Fig. 1B). On the otherhand were two cases with ‘‘normal” immunostaining for IDE sub-sequently identified as being from individuals with schizophrenia.

3.1.2. HypothalamusThe hypothalamus was found to be the brain region with the

most prominent immunolocalization of IDE. The protein was re-vealed in a vast majority of neurons located in different hypotha-lamic nuclei. The most pronounced immunostaining appeared inthe PVN, where almost all neurons expressed IDE immunoreactiv-ity. A similar scenario was seen in the supraoptic (Fig. 1C and D),the suprachiasmatic, the lateral hypothalamic, and the arcuate nu-clei. Some IDE immunopositive neurons were detected in themammillary bodies.

Qualitative inspection of the sections did not reveal obviousstaining differences between the cases.

3.1.3. Basal nucleus of MeynertWe could replicate our previous observation (Bernstein et al.,

1999a), in that most (íf not all), neurons of this nucleus showeda moderate immunostaining for IDE. Qualitative inspection of thesections did not reveal obvious staining differences between thecases.

3.2. Immunolocalization of IDE in human brain regions inschizophrenia and controls – quantitative estimation

3.2.1. Dorsolateral prefrontal cortexAs previously reported, we have examined the densities of all

IDE expressing neurons in left and right dorsolateral prefrontal cor-tex, DLPFC (Bernstein et al., 2007), in order to clarify if possible

Fig. 1. Immunohistochemical demonstration of IDE in human brain areas in haloperidol-treated patients with schizophrenia and control cases. (A) Localization of IDEimmunoreactivity in DLPFC cortex neurons of an non-psychotic individual. Multiple pyramidal and interneurons are immunopositive ofr IDE. Bar = 100 lm. (B) Localization ofIDE immunoreactivity in DLPFC cortex neurons of an individual with schizophrenia. Please notice the reduced density of immunostained cells in schizophrenia. Bar = 120 lm.(C) Immunodetection of IDE in hypothalamic SON neurons of an non-psychotic individual. A majority of neurons express the antigen. Bar = 120 lm. (D) Immunodetection ofIDE in hypothalamic SON neurons of an individual with schizophrenia. Bar = 120 lm.


changes in the number of IDE immunoreactive neurons are possi-bly due to alterations in the overall number/density of neurons. Nosignificant differences with regard to the overall number of neu-rons and neuronal density were found between controls and caseswith schizophrenia.

Compared to controls, in schizophrenia cases, the overall den-sity of IDE-immunopositive cells located in the left DLPFC was re-duced by more than 40% (7883 ± 1491 cells/mm3 vs. 5405 ± 2660cells/mm3). In the right DLPFC the reduction was less than 25%(7578 cells/mm3 ± 1160 vs. 6708 ± 2183 cells/mm3). Hence, thereduction of the overall density of IDE-immunoreactive neuronswas significant only on the left side (p = 0.018), whereas on theright side only a tendency was seen (p = 0.074). The data is givenin Fig. 2. Layer-specific cell counting revealed significant reduc-tions for left DLPFC layers I (p = 0.015), II (p = 0.012), IV (p =0.0350) and V (p = 0.027), but not for layers III (p = 0.090) and VI(p = 0.058). On the right side, a significant reduction was seen forlayers V (p = 0.044) and VI (p = 0.040). There was no significantinfluence of BMI on IDE cell numbers.

3.2.2. Hypothalamic nuclei PVN and SONPreviously we have shown that there are only minor and insig-

nificant differences between the volumes of hypothalamic nuclei ofpatients with schizophrenia and controls (for details, see Bernsteinet al., 2008b). Estimation of the numerical density of IDE-express-ing neurons revealed only insignificant reductions of cell density inPVN and SON in schizophrenia (Fig. 3). No significant differenceswith regard to IDE cell density were found between lean and obeseindividuals.

3.2.3. Basal nucleus of MeynertNo difference was found between the cell densities in the basal

nucleus of Meynert of patients with schizophrenia and controls(see Fig. 3).

3.3. Western immunoblot analysis

Western blotting of human postmortem prefrontal cortex tissueobtained from five individual brains from non-psychotic subjects(n1–n5) and three from patients with schizophrenia (s1–s3)showed the characteristic enzyme species possessing a molecularweight of about 110 kDa as described earlier with this antibody(Bernstein et al., 1999a, 2008a). Densitometric analysis of thebands revealed that compared to controls, there is a significantreduction in expression of IDE protein in DLPFC tissue of patientswith schizophrenia (p = 0.049; Fig. 4).

3.4. Neuronal expression of IDE in cortical and hypothalamic areas ofhaloperidol-treated and control rats

As in the human brain IDE immunoreactivity was widespread inrat brain neurons of haloperidol-and saline-treated rats, withhypothalamic neurons showing the most dense and intense stain-ing. Cell counts (number of rats n = 5 for each of the three groups)in the prefrontal cortex did not reveal statistically significant dif-ferences between the groups with regard to IDE-immunoreactiveprefrontal cortex neurons (saline group 16400 ± 800 cells/mm3,low dose group 17400 ± 900 cell/mm3 and high dose group17600 ± 1000 cells/mm3). Estimation of cell numbers in the PVNalso showed insignificant differences with regard to the numberof immunostained neurons between the groups (saline-treated rats96.7 ± 6.0 neurons; low-dose-treated rats 94.9 ± 10.1 neurons;high-dose-treated rats 98.2 ± 8.2 neurons per left PVN/section.

3.5. Influence of haloperidol on IDE mRNA expression in SHSY5Yneuroblastoma cells

We tested the influence of different concentrations of haloper-idol on the expression of IDE mRNA in neuroblastoma cells and

Fig. 2. Numerical density of IDE expressing neurons for each of the six cortical layers for left (L) and right (R) dorsolateral prefrontal cortex is shown. In schizophrenia thedensity of IDE-immunopositive cells located in the left DLPFC is significantly reduced by more than 40%.

Fig. 3. Numerical density of IDE expressing neurons for left and right supraoptic, paraventricular nuclei as well for left and right basal nucleus of Meynert is shown. There areno significant differences between cases with schizophrenia and controls.


found a bell-shaped curve: under the influence of 1 lg/ml of thedrug IDE mRNA is considerably elevated. Higher concentrations(2.5 lg/ ml and more) of haloperidol reduce IDE mRNA levels tothe normal state (Fig. 5).

4. Discussion

The main finding of this study is that the number of IDE proteinexpressing neurons is reduced in the post-mortem DLPFC of pa-tients with haloperidol-treated, chronic schizophrenia. In addition,the IDE protein concentration was found to be significantly re-

duced in brain tissue obtained from patients with schizophrenia.Patients with schizophrenia show a high prevalence of the meta-bolic syndrome/type 2 diabetes, which is frequently associatedwith peripheral hyperinsulinism, insulin resistance, and abnormal-ities in glucose metabolism (reviewed in Hägg et al., 2006). Brainimaging of patients with schizophrenia revealed decreased brainglucose utilization (Tamminga et al., 1992; Benes and Berretta,2000; Henderson et al., in press and others) and reduced neuronalmetabolism (Weinberger, 1999; Fannon et al., 2003). It is notknown whether schizophrenia is accompanied by increased cere-bral insulin levels. However, there is evidence that treatment of

Fig. 4. Expression of IDE in human left DLPFC of normal control cases (n1–n5) andindividuals with schizophrenia (s1–s3) as revealed by Western blot analysis.Compared with controls a significant reduction of neocortical IDE immunoreactivityis seen in cases with schizophrenia.

Fig. 5. Influence of haloperidol on IDE mRNA expression in human neuroblastomacells. A single dose of 1 lg/ml of the drug elevate the amount of IDE mRNA. Higherconcentrations (2.5 lg/ml and more) of haloperidol slightly reduce IDE mRNA levelsto the normal state.


rats with olanzapine increases the concentration of insulin 2 (ananalogue of human insulin) in the frontal cortex (Fatemi et al.,2006). Recently, an insulin receptor dysfunction in the dorsolateralprefrontal cortex has been demonstrated in schizophrenia (Zhaoet al., 2007). These investigators found a 50% decrease in theamounts and the extent of autophosphorylation of insulin receptorsubunit b, associated with a strong decrease in PKB/Akt expressionand activity. This inhibition of insulin receptor signaling wasaccompanied by elevated amounts of glycogen synthase kinase(GSK)-3a and GSK-3b. Elevated activity of GSK-3 has also been de-scribed in AD (Hooper et al., 2008). The fact that IDE is a down-stream target of the cerebral insulin receptor (Zhao et al., 2004)might explain the reduced cortical expression of IDE in schizophre-nia. Ho and colleagues (2004) were able to show that insulin resis-tance (as reported for brain tissue in schizophrenia by Zhao et al.(2007)) is associated with reduced IDE levels in an animal modelof AD. The possible influence of medication on cerebral IDE expres-sion requires further consideration. Out of the 12 individuals withschizophrenia studied by Zhao and co-workers (2007), only two re-

ceived atypical neuroleptics, whereas nine received typical neuro-leptics and one individual was drug-free. Consequently, theobserved changes in cerebral insulin metabolism in schizophreniacannot solely be attributed to atypical antipsychotics. Here, we de-scribe a significant reduction of the cellular expression of IDE, adownstream target of insulin receptor signaling (Zhao et al.,2004; Van der Heide et al., 2006), in the same brain region as stud-ied by Zhao et al. (2007). Patients in our study had only treatmentwith haloperidol, thus, the findings fit to those of Zhao et al. (2007)in that disturbed cerebral insulin/IGF signaling may occur in pa-tients with schizophrenia with typical neuroleptics during lifetime.It has recently been hypothesized that therapeutic and adversemetabolic effects of neuroleptics might be related to a commonpharmacologic mechanism, and that some abnormalities in insulinsignaling in schizophrenia might be ameliorated by antipsychotics(probably through their interaction with Akt and/or GSK; Girgiset al., 2008). Since Akt and GSK are upstream to IDE in the insulinsignaling cascade, and Akt has been indeed shown to influence IDEexpression through insulin (Zhao et al., 2007). So, reduced corticalIDE immunoreactivity in schizophrenia might be an indirect conse-quence of antipsychotic treatment. To learn more about the rela-tionship between haloperidol and reduced IDE expression, westudied the influence of different concentrations of the drug on en-zyme mRNA in a cell culture system as well as on neuronal IDEimmunoreactivity in chronically treated rats. At a concentrationof haloperidol by far exceeding the maximal brain tissue concen-tration measured in human post-mortem brains after long-termdrug treatment (Kornhuber et al., 2006), haloperidol increases(not decreases) IDE expression. At clearly pharmacologicdoses the drug slightly reduces IDE mRNA. Furthermore, we foundthat the number of IDE immunopositive neurons in the medial pre-frontal cortex and in the PVN of rats after long-term administrationof the drug did not significantly differ from that in saline-treatedanimals. Hence, it is unlikely that the reduction of IDE expressionseen in cortical areas of patients with schizophrenia is due toneuroleptic drug treatment per se. An indirect effect of haloperidolthrough Akt or GSK cannot be excluded, however (Girgis et al.,2008). Another point to be considered is the influence of themetabolic state of the individuals on cerebral IDE expression. Wefailed to find a significant effect of BMI on IDE expression in oursamples.

What could be the possible pathophysiological consequences ofreduced neocortical IDE expression in schizophrenia? Due to somesimilarities between Alzheimers disease and schizophrenia withregard to compromised cerebral insulin signalling, and the reducedneuronal expression of the Ab-clearing enzyme IDE found in bothdiseases (AD: Perez et al., 2000; Miners et al., 2008 and others;schizophrenia: this study), one might expect an increased inci-dence of Alzheimer-like pathology in elder patients with schizo-phrenia. This is due to insufficient removal of amyloid-formingAb molecules over time. This is, however, not the case (Higakiet al., 1997; Religa et al., 2003). The reasons for this are unknown,but might have to do with: (1) compensation of reduced IDE by in-creased activities of other Ab-clearing enzymes (Miners et al.,2008); (2) a protective effect of haloperidol against amyloid forma-tion and its toxic effects (Higaki et al., 1997; Palotas et al., 2004);and (3), smoking, which has been shown to attenuate Ab deposi-tion in the cortical areas of normal eldery individuals (Courtet al., 2005). Since many individuals with schizophrenia are heavysmokers, smoking may contribute to reduced Ab pathology inschizophrenia. Secondly, decremented IDE expression in schizo-phrenia might affect the concentrations of neuropeptides andgrowth factors, which are natural substrates of the enzyme. IDEis known to generate c-endorphin from b-endorphin (Safaviet al., 1996), and to play a role in the general turnover of b-endor-phin in the CNS (Reed et al., 2008). Wiegant et al. (1988) have


shown that c-endorphin is increased in post-mortem brains of pa-tients with schizophrenia, which cannot be explained by decreasedlevels of the c-endorphin generating enzyme, IDE. Conflicting datahave been reported on the b-endorphin levels in brains of patientswith schizophrenia. Several authors (including our group) reporteddecreased expression of the peptide in brains of individuals withschizophrenia, while others found unchanged or even increasedlevels of the peptide (for detailed information, see Bernsteinet al., 2002).

Especially intriguing in this context, is the emerging role of twoother naturally occurring substrates of the enzyme, IGF-I and IGF-II, in schizophrenia. Significantly lower fasting plasma IGF-I levels(but increased insulin concentrations) were measured in drug-naïve schizophrenia patients compared to non-psychotic controls(Venkatasubramanian et al., 2007). These authors suppose thatIGF-I deficit might be responsible for, or at least significantly con-tribute to, insulin resistance in schizophrenia. Genome-wide scanshave identified regions on chromosome 12p13–p24, close to thosefor the IGF-I gene, to be linked to schizophrenia (DeLisi et al.,2002). Further, abnormalities of foetal growth are consistently re-lated to risk of neuro-developmental disorder and schizophrenia.Restricted foetal growth which may be associated with early-onsetillness in males, has been linked to abnormal parental IGF imprint-ing (Abel, 2004). So, low-birth weight infants, who are at increasedrisk of developing schizophrenia, often have reduced IGF levels atbirth (reviewed in Gunnell and Holly, 2004). Since there are noknown gene polymorphisms of IGF-I linked to schizophrenia (Gun-nell et al., 2007), an altered metabolism (by IDE or other proteases)might be a reason for the reduced blood plasma levels. Unlike IGF-I,IGF-II is significantly increased in blood samples of haloperidol-medicated, male schizophrenia patients (Akanji et al., 2007). Thisindeed might also a consequence of reduced proteolytic degrada-tion by reduced IDE (or any other peptidase/protease involved inIGF-II biotransformation). IGF-II (Lin et al., 2006), but not IGF-I(Mehler-Wex et al., 2006), has shown expression alteration dueto haloperidol. Hence, it cannot be excluded that long-term medi-cation with haloperidol alters the expression of immunoreactiveIDE through changes in the expression of its substrate, IGF-II. Morestudies are warranted in regard to IGF-I and IGF-II concentration inbrains of individuals with schizophrenia, in order to judge a possi-ble role of IDE in mediating the levels of these growth factors inschizophrenia. Lastly, there might be a genetic association betweenIDE and schizophrenia. Several linkage analyses point to an associ-ation of schizophrenia with chromosome loci between 10q23 and25 (Mowry et al., 2000; Lerer et al., 2003; Williams et al.,2003; Devlin et al., 2007; Fanous et al., 2008), where the IDE geneis encoded. However, unlike for a possible genetic association ofIDE with AD (Vepsäläinen et al., 2007) none of the aforementionedstudies has really tried to reveal if SNPs in the IDE gene areassociated with an increased risk for developing schizophrenia.Moreover, even if certain IDE gene polymorphisms were found tobe associated with schizophrenia, this help explain only a smallpercentage of the cases, not the general decrease in the expressionof the gene product, as found here for brain cortical areas inschizophrenia. Besides the already discussed aspects, manyyet unknown factors (for example, dysregulated micro RNAscontroling IDE protein levels, Beveridge et al., 2008) mayhave an influence on disease-related cerebral IDE proteinexpression. Thus, more studies are needed to better define theplace of reduced IDE in the puzzle of metabolic changes seen inschizophrenia.

Funding sources

The study was financed in part by BMBF of Germany.

Contributors

H.-G. Bernstein: design of the study, participation in cellcountings, writing of the article, T. Ernst: cell coutings, staistics,U. Lendeckel: biochemical and molecular work, A. Bukowska: bio-chemical and molecular work, S. Ansorge: production and charac-terization of IDE antisera, R. Stauch: immunostainings for IDE, S.Ten Have: experiments with rats, language, J. Steiner: involved instudy design, H. Dobrowolny: bio-Statistics, B. Bogerts: involvedin study design.

Conflict of interest statement

None declared.

Acknowledgements

This study was supported by Stanley Consortium and BMBF ofGermany. We thank R. Stauch, K. Matzke and K. Paelchen for skilledtechnical assistance.

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reduced neuronal expression of insulin-degrading enzyme in the dorsolateral prefrontal cortex of...

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