,1 ,1

,2 , ,

*Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio,

USA

†College of Sciences, University of Texas at San Antonio, San Antonio, Texas, USA

‡Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky, USA

§Department of Neuroscience, Case Western Reserve University School of Medicine, Cleveland, Ohio,

USA

¶Department of Neurology, Case Western Reserve University School of Medicine, Cleveland, Ohio,

USA

AbstractLeptin signaling has received considerable attention in theAlzheimer disease (AD) field. Within the past decade, thepeptide hormone has been demonstrated to attenuate tauhyperphosphorylation in neuronal cells and to be modulatedby amyloid-b. Moreover, a role in neuroprotection and neuro-genesis within the hippocampus has been shown in animalmodels. To further characterize the association between leptinsignaling and vulnerable regions in AD, we assessed theprofile of leptin and the leptin receptor in AD and controlpatients. We analyzed leptin levels in CSF, and the concen-tration and localization of leptin and leptin receptor in thehippocampus. Significant elevations in leptin levels in bothCSF and hippocampal tissue of AD patients, compared with

age-matched control cases, indicate a physiological up-regu-lation of leptin in AD. However, the level of leptin receptormRNA decreased in AD brain and the leptin receptor proteinwas localized to neurofibrillary tangles, suggesting a severediscontinuity in the leptin signaling pathway. Collectively, ourresults suggest that leptin resistance in the hippocampus mayplay a role in the characteristic changes associated with thedisease. These findings are the first to demonstrate suchdysregulated leptin-signaling circuitry and provide novelinsights into the possible role of aberrant leptin signaling in AD.Keywords: Alzheimerdisease, leptin, leptin receptor,neurofibrillarytangles, tau.J. Neurochem. (2014) 128, 162–172.

The biological actions of leptin are typically associated withhypothalamic modulation of feeding behavior and energyexpenditure. Leptin is an anorexigenic peptide hormonesynthesized in and secreted from peripheral adipocytes; itsreceptor binding within the hypothalamus induces a bio-chemical cascade that ultimately reduces food intake andincreases energy usage (Morris and Rui 2009). While themolecular and biochemical mechanisms of leptin signaling inthe hypothalamus are relatively well-characterized, thoseinvolved in other brain regions such as hippocampus are lessclear. Interestingly, the long form of the leptin receptor(Ob-Rb), as well as the five alternatively spliced leptinreceptors (Ob-Ra, Ob-Rc, Ob-Rd, Ob-Re, and Ob-Rf), aredistributed throughout the hippocampus (Elmquist et al.

1998; Morash et al. 1999), and increasing evidence isestablishing the role of leptin in this region (Shanley et al.2002; Harvey 2003, 2007; Garza et al. 2008), particularly so

Received February 26, 2013; revised manuscript received July 22, 2013;accepted July 24, 2013.Address correspondence and reprint requests to Hyoung-gon Lee,

Department of Pathology, CaseWestern ReserveUniversity, 2103CornellRoad, Cleveland, OH 44106, USA. E-mail: hyoung-gon.lee@case.edu1These authors contributed equally to this work.2Deceased.Abbreviations used: AD, Alzheimer disease; Ab, amyloid-b; BBB,

blood–brain barrier; BMI, body mass index; CA, cornus ammonus; CSF,cerebrospinal fluid;MCI, mild cognitive impairment; NFT, neurofibrillarytangle; NGS, normal goat serum; Ob-Rb, long form of the leptin receptor.

162 © 2013 International Society for Neurochemistry, J. Neurochem. (2014) 128, 162--172

JOURNAL OF NEUROCHEMISTRY | 2014 | 128 | 162–172 doi: 10.1111/jnc.12380

in Alzheimer disease (AD) (Olsson et al. 1998; Power et al.2001; Greco et al. 2008, 2009b).AD is a pathologically complex disease characterized by

multiple etiological and biochemical aberrations includingoxidative stress, cell cycle aberration, transition metaldyshomeostasis, neurofibrillary tangle (NFT) formation,and amyloid-b (Ab) oligomerization/fibrillation (Castellaniet al. 2010). In longitudinal studies, several biomarkers arealso changed and predictive for the development of AD(Bateman et al. 2012). Notably, the serum levels of leptinhave been shown to be correlated with the development ofAD (Holden et al. 2009), suggesting a potential pathogenicrole of leptin. Indeed, leptin has previously been shown tomodulate tau hyperphosphorylation, the principal prerequi-site to the accumulation of NFTs in the brain (Greco et al.2008, 2009a,b), and to be affected by Ab-related signaling(Fewlass et al. 2004; Erol 2008). Moreover, the up-regula-tion of leptin has been proposed as a method of therapeuticintervention for AD (Tezapsidis et al. 2009). However, thestatus of the leptin signaling pathway in hippocampus andcerebral cortex, the vulnerable regions of AD, has not beendetermined. To this end, in this study, using multiplebiochemical and molecular biological methods, we analyzedthe levels of leptin, its major receptor Ob-Rb, and theactivated Ob-Rb (phosphorylated on tyrosine 985, pOb-RbT985) in the hippocampus of AD and control patients, aswell as in patients exhibiting mild cognitive impairment(MCI), a prodromal dementia associated with AD (Petersenet al. 1999).

Materials and methods

Tissue

Using an approved IRB protocol, human AD and non-dementedcontrolhippocampalorcortical tissuesampleswereobtainedatautopsy(AD: n = 33, age 63–95 years, mean 77.1 � 8.7 years; youngcontrols: n = 10, age 19–46 years, mean 29.5 � 11.7 years; agedcontrols: n = 16, age 62–86 years, mean 71.9 � 8.3 years) from theCase Medical Center at Case Western Reserve University. Brainsamples were fixed in either routine formalin or methacarn (metha-nol : chloroform : acetic acid; 6 : 3 : 1 v/v/v) at 4°C overnight.Following fixation, tissue was dehydrated through ascending concen-trations of ethanol, embedded in paraffin, and 5 lm sections wereplaced on coated slides.

Immunohistochemistry

Tissue sections were deparaffinized in xylene and hydrated throughdescending ethanol concentrations, and endogenous peroxidaseactivity was quenched by 30 min incubation in 3% hydrogenperoxide in methanol. For some experiments, antigen retrievalthrough pressure cooking was performed using manufacturer’srecommendations (Biocare Medical, Concord, MA, USA). Subse-quently, in all cases, non-specific binding sites were blocked with30 min incubation in 10% normal goat serum in Tris bufferedsaline, and then treated overnight at 4°C with primary antibodies.

Primary antibodies utilized for various detections included rabbitpolyclonal antibodies to leptin (Abcam 2125, Cambridge, MA,USA), leptin receptor (Pierce Thermo Scientific PA1-053. Rockford,IL, USA), phosphorylated leptin receptor at Tyr-985 (Millipore 07-097, Billerica, MA, USA), and mouse monoclonal antibody tohyperphosphorylated tau (Pierce-Endogen AT8, Rockford, IL,USA). Following incubation with species-specific secondary anti-bodies and peroxidase anti-peroxidase complexes, the antibodieswere detected with 3, 3′-diaminobenzidine, as the chromogen(Dako, Carpinteria, CA, USA). Verification of antibody specificityfor anti-Ob-Rb was done via absorption of the antibody with the Ob-Rb peptide (Thermo-Pierce PEP-014). Specifically, diluted antibodyin 1% normal goat serum was incubated with its specific peptideantigen and then applied onto an adjacent serial section stained withthe antibody alone.

Double label fluorescent microscopy was used to confirm thelocalization of Ob-Rb and AT8. Sections from three cases of ADwere rehydrated as above and after incubation with both mousemonoclonal AT8 and rabbit anti-sera to leptin receptor, AlexaFluor468 and 588 (Invitrogen, Grand Island, NY, USA) labeledsecondary antibodies were applied. Images were obtained usingZeiss Axiophot and Axiocam (Ziess, Thornwood, NY, USA).

Quantification of the levels of immunoreactivity in the sameneurons for both Ob-Rb and pOb-RbT985 was performed. Serialadjacent sections from three AD cases were immunostained. Threeof the exact same fields (209 magnification) were imaged for eachantibody using a Zeiss Axiocam (Ziess). Using landmark vessels,identical field orientation was determined and the same neuronslocated in each set. Using the Axiovision software (Zeiss) therelative staining intensity was measured with the backgroundsubtracted for neurons with and without NFT. Similar methodologywas applied to compare neurons stained with Ob-Rb and AT8.Images were obtained from the same fields from adjacent serialsections from eight cases of AD and the numbers of NFT stainedwith AT8 only, Ob-Rb only, or both AT8 and Ob-Rb were counted.

Leptin Radioimmunoassay (RIA)

To determine the leptin levels in CSF, samples obtained at autopsyfrom a series of well-characterized cases were obtained from theUniversity of Kentucky Sanders-Brown Center on Aging. For thesecases, age, postmortem interval, gender, Braak stage, Mini-MentalStatus Exam, body mass index (BMI), and protein levels wereavailable. All samples were collected with a low postmorteminterval (mean 3 h). AD (n = 21; age 57–95 years, mean78.5 � 10.2 years, Braak VI), MCI (n = 8; age 87–99 years, mean91.8 � 4.5 years, Braak III, IV,V), and control (n = 13; age 72–95 years, mean 83.7 � 8.1 years, Braak 0, I, II) cases were used.Table 1 details the case information. Samples were sent blinded forthe sensitive human leptin RIA (Linco, Millipore, St. Louis, MO,USA) and were performed in quadruplicates.

Quantitative RT-PCR

Frozen hippocampal tissues from clinically and pathologicallyconfirmed cases of AD (n = 16, ages 65–85, mean 78.1) and non-AD age-matched controls (n = 11, ages 61–91, mean 79.8) withNational Institute of Aging and Consortium to Establish a Registryfor Alzheimer’s Disease criteria were obtained at autopsy by theCase Western Reserve University Brain Bank under an IRB-

© 2013 International Society for Neurochemistry, J. Neurochem. (2014) 128, 162--172

Disruption of leptin signaling in AD 163

approved protocol. Frozen white adipose tissue was used forverification of PCR technique (provided by the Human TissueProcurement Facility of Case Western Reserve University and UHCase Medical Center).

Frozen tissue was homogenized in TRIzol reagent (Invitrogen,Carlsbad, CA, USA) following chloroform extraction. RNA wasfurther purified with the RNeasy Mini Kit (Qiagen, Germantown,MD, USA), following the instructions of the manufacturer. RNAwas treated with DNase (Turbo DNase; Ambion) to remove traceDNA. RNA was converted to cDNA using the High Capacity cDNAReverse Transcription Kit (Applied Biosystems, Carlsbad, CA,

USA) according to the manufacturer’s instructions. Briefly, thepurified total RNA (100 ng) was reverse transcribed with clonedMoloney murine leukemia virus reverse transcriptase (5 U) byincubating at 37°C for 120 min, followed by heating at 85°C for5 min. Real-time RT-PCR was used to quantify relative leptin andOb-Rb expression. TaqMan PCR assay for leptin (Hs00174877_m1)and Ob-Rb (Hs00174497_m1) was performed on cDNA samples in96-well optical plates on an ABI StepOne Plus Thermocycler withglyceraldehyde 3-phosphate dehydrogenase used as an endogenouscontrol (Taqman; Applied Biosystems). For each 20 lL TaqManreaction, 20 ng cDNA was mixed with 10 lL 2 9 TaqMan FastUniversal PCR Master Mix (Applied Biosystems) and 1 lL of20 9 TaqMan assay mix. Standard fast PCR parameters were used.Data are expressed as relative quantity (RQ value).

Western blot

To assess if leptin is degraded over time, as a potential result ofincreased postmortem interval (PMI), in vitro analysis wasperformed by incubating 1 lg purified leptin in either Tris-bufferedsaline or human CSF (n = 2) for 4 h on ice or at 37°C to mimic thelongest PMI of the samples used in the RIA analysis, and thenresolved on 15% sodium dodecyl sulfate–polyacrylamide gelelectrophoresis and analyzed by western blot.

Frozen hippocampus samples were homogenized in lysis buffer(Cell signaling, Danvers, MA, USA) with protease inhibitors added(Roche, Indianapolis, IN, USA). After determining protein concen-tration with bicinchoninic acid BCA assay (Pierce), 30 lg of proteinwas resolved using sodium dodecyl sulfate–polyacrylamide gelelectrophoresis. For leptin analysis, 15% gels were used, for Ob-Rb10% gels were used. Proteins were transferred to Immobilonpolyvinylidene difluoride membrane (Millipore), blocked with 10%milk or 5% bovine serum albumin. Primary antibodies wereincubated overnight, then the blots rinsed in Tris-buffered saline-Tween (TBST) and horseradish peroxidase-labeled secondaryantibodies applied. After rinsing in TBST, blots were developedusing Millipore ECL detection reagent and blots were exposed toX-ray film.

Statistical analysis

Data are expressed as means � SEM. Differences between groupswere determined by ANOVA followed by Bonferroni’s post hoc testusing Origin 8 program (OriginLab, Northampton, MA, USA). ForFig. 1a, a two-way ANOVA was used for the analysis. p < 0.05 wasconsidered statistically significant.

Results

Leptin levels in CSF and brain

Leptin concentration in CSF obtained at the time of autopsywas significantly higher in AD compared to both MCI(*p<0.05) and control (*p<0.05) cases (Fig. 1a). CSF leptinwas higher in females compared with males only within theAD group (*p < 0.05) (Fig. 1a), in agreement with previousfindings demonstrating a gender difference in circulatingleptin levels (Lieb et al. 2009; Fulda et al. 2010). Leptinlevel was lower in MCI than controls; however, this trend didnot reach statistical significance (Fig. 1a). Moreover, CSF

Table 1 Information on the cases used for CSF leptin analysis

Diagnosis Age (years) Braak Stage PMI (h) BMI Gender

Control 90 0 3.5 24.4 Male

Control 92 0 3.25 25.8 MaleControl 95 0 3.5 23.0 FemaleControl 72 1 3.75 28.7 Female

Control 74 1 4 25.8 MaleControl 75 1 3.5 26.5 FemaleControl 76 1 2 20.9 Female

Control 86 1 1.75 21.8 FemaleControl 79 2 2.25 28.5 MaleControl 79 2 1.75 27.5 Male

Control 87 2 2 25.8 MaleControl 90 2 4 24.0 FemaleControl 93 2 2.75 NA FemaleMCI 87 3 2.25 26.6 Male

MCI 91 3 5 NA FemaleMCI 93 3 2.75 20.0 FemaleMCI 97 3 2.75 25.7 Female

MCI 87 4 3.5 24.0 MaleMCI 92 4 2 26.4 MaleMCI 88 5 2.25 34.1 Female

MCI 99 5 2 24.9 FemaleLate AD 57 6 3 NA FemaleLate AD 65 6 4 18.7 MaleLate AD 67 6 2.25 NA Female

Late AD 68 6 3.25 31.8 FemaleLate AD 69 6 4 NA MaleLate AD 70 6 3.25 21.1 Female

Late AD 72 6 2.25 20.7 FemaleLate AD 73 6 2 30.4 MaleLate AD 75 6 2.33 21.8 Female

Late AD 78 6 3.75 21.2 MaleLate AD 78 6 3.5 25.0 MaleLate AD 84 6 4.5 21.6 Male

Late AD 84 6 2.75 25.4 MaleLate AD 85 6 2.75 21.8 MaleLate AD 86 6 4.25 31.7 FemaleLate AD 86 6 3.25 NA Female

Late AD 86 6 2 NA FemaleLate AD 90 6 2.8 21.0 FemaleLate AD 90 6 2.75 21.6 Female

Late AD 90 6 3.25 23.0 MaleLate AD 95 6 4 25.8 Male

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164 D. J. Bonda et al.

leptin concentration was significantly higher in late severestage AD cases than early mild stage AD and MCI caseswhen correlated with Braak staging of pathological NFT

burden. Specifically, statistical comparison of Braak stage VIcases and Braak stage 0–V cases found the mean CSF leptinin stage VI was more than six times greater (p < 0.01).However, CSF leptin concentration did not correlate with agein control (r = 0.43), MCI (r = �0.02), or AD groups(r = �0.31) (Fig. 1b), indicating the observed trend ofhigher CSF leptin in late stage AD is more likely adisease-related rather than an age-related phenomenon.There were no significant differences in BMI among

experimental groups, ages, and genders (data not shown).Furthermore, no correlation was found between CSF leptinlevels and BMI in the control (r = 0.10), MCI (r = �0.02),or even the AD group (r = 0.05) (Fig. 1c). Regarding thestability of leptin in our CSF samples, we found nocorrelation between CSF leptin levels and PMI in any ofthe groups, or even across all samples together. To furtherconfirm the stability of leptin over time, in vitro analysis wasperformed. Purified leptin was incubated in either Tris-buffered saline or in aliquots of CSF and kept on ice orplaced at 37°C to mimic the in vivo postmortem environ-ment. No degradation of leptin was found in either conditioneven after 4 h, the longest PMI of the CSF samples used inthe RIA analysis (Figure S1a). Consistent with this finding,in multiple previous studies, leptin was shown to berelatively stable in biological samples (Ma et al. 1996;Flower et al. 2000; Miller et al. 2006).Immunohistochemical analysis of hippocampal tissue

sections revealed leptin localized to pyramidal neurons inboth control (Fig. 2a) and AD cases (Fig. 2b); a subpopu-lation of neurons in the AD cases demonstrated relativelyhigher levels of leptin. Quantitative RT-PCR was performedon leptin mRNA purified from hippocampus from cases ofAD and control to confirm the source of leptin. No leptinmRNA was found in any case, confirming leptin is notsynthesized in the hippocampus, while the positive controladipose tissue showed high levels of leptin mRNA (Fig. 2c).

Leptin receptor levels in the brainIn quantitative RT-PCR analysis with total RNA isolatedfrom AD and control brain, the expression level of Ob-RbmRNA was significantly low in AD hippocampal tissue(p < 0.05) (Fig. 2c) compared with control. Interestingly,there was no significant change in Ob-Rb mRNA levels as afunction of age in either AD or control group (data notshown).Immunohistochemical staining further confirmed the pres-

ence of Ob-Rb in hippocampal and cortical neurons. In age-matched controls, a weak cytoplasmic localization wasprominent with occasional nuclear staining (Fig. 3a). Nota-bly, AD cases showed a robust association of Ob-Rb withboth intracellular and extracellular NFTs (Fig. 3b, c) as wellas neuritic plaques (Fig. 3d). In older controls, age-relatedneurofibrillary pathology was also labeled by Ob-Rb anti-body (Fig. 3a, arrow). The specificity of the antibody for

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n = 6Control AlzheimerMCI

n = 7 n = 3 n = 5 n = 10 n = 11FemaleMale FemaleMale FemaleMale

Fig. 1 CSF leptin levels in control, mild cognitive impairment (MCI),

and Alzheimer disease (AD). (a) CSF leptin is significantly higher in ADthan MCI and control cases (*p < 0.05). CSF leptin was higher infemales compared with males within the AD group (*p < 0.05). (b)

CSF leptin level did not correlate with age in control MCI or ADsuggesting higher level of CSF leptin in AD cases is disease relatedrather than age relate. (c) Nor did the leptin levels correlate with body

mass index (BMI) in any of the groups.

© 2013 International Society for Neurochemistry, J. Neurochem. (2014) 128, 162--172

Disruption of leptin signaling in AD 165

Ob-Rb was confirmed by the absorption assay with itsspecific peptide which greatly reduced the antibody (Fig. 3e,f) and no difference in immunoreactivity was observedbetween methacarn and formalin fixed sections (data notshown). Western blot analysis of soluble fraction of hippo-campal homogenates from both AD and control casesrevealed the antibody used recognized the full length leptinreceptor (approximately 125 kDa) in both AD and controlpatients (Figure S1b). It is not unexpected that no strikingincrease in Ob-Rb is seen in the AD cases by western blot,since it is likely that the Ob-Rb in NFT could be insolubleand difficult to detect with this method.To confirm the co-localization of Ob-Rb with NFTs, we

performed a comparative analysis of adjacent serial sectionsfrom eight cases of AD and found that on average 40% ofneurons stained with Ob-Rb (Fig. 4a) also contained thepathological form of tau, labeled with the monoclonalantibody AT8 (Fig. 4b). Another 40% of NFT containedonly AT8, and about 20% of NFT were only positive for Ob-Rb (Fig. 4c). To substantiate the quantification obtainedusing serial adjacent sections stained using anti-Ob-Rb andAT8, double fluorescent microscopy on a single section wasalso used to confirm that many of the neurons stronglystained for Ob-Rb (red) also contained AT8 (green), a markerfor NFT (Fig. 4d–f). Indeed, some NFT only demonstratedOb-Rb (Fig. 4g–i) and some only displayed AT8 (Fig. 4j–l),yet Ob-Rb was still seen as a weak nuclear localization, butnot accumulated in the cytoplasmic NFT formation in theseinstances.When leptin binds to its receptor, Ob-Rb becomes

phosphorylated at T985 for its activation. Using an antibodyspecific for the activated form, we found that pOb-RbT985

localized to neuronal cytoplasm at qualitatively higher levelsin the hippocampus in AD (Fig. 5b) compared with controls(Fig. 5a). However, importantly, the neurons positive forpOb-RbT985 did not appear to be associated with NFTs inany cases tested in this study. To confirm this finding further,we carefully examined the cornus ammonus (CA) regions ofthe hippocampus within individual AD cases. Neuronswithin the CA3 region, which contains relatively little NFTpathology, exhibited strong pOb-RbT985 staining (Fig. 5b)and Ob-Rb was also present in many of these same neuronscontaining the activated form (Fig. 5b, c, black arrows).Conversely, neurons in the CA1 region on the same sectionsfrom the same patients visually had less pOb-RbT985

immunoreactivity (Fig. 5e). Compared to the CA3 region,many NFT showed robust Ob-Rb staining (Fig. 5f), yet mostof these cells had little or no pOb-RbT985 immunoreactivity(black arrows in Fig. 5e, f). Many of the non-NFT bearingneurons in the CA1 region contained both Ob-Rb and pOb-RbT985 (red arrows in Fig. 5e, f).Quantification of the pOb-RbT985 immunoreactivity in the

population of neurons with and without Ob-Rb-positive NFTrevealed a significant decrease in the activated form in NFT.

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C (n = 11) (n = 2)Ob-Rb Leptin Ob-Rb Leptin

Fig. 2 Immunohistochemical staining finds the antibody to leptinstains neuronal cytoplasm in the hippocampus from both control (a)and Alzheimer disease (AD) cases (b). Many neurons in the AD casesdemonstrated relatively higher levels of leptin. Scale bar = 50 lm.

Quantitative RT-PCR found leptin mRNA isolated from brain tissue ofAD and control absent in all cases, confirming leptin is not synthesizedin these brain tissues, while the positive control adipose tissue showed

high levels of leptin mRNA (c). qRT-PCR analysis found the level oflong form of the leptin receptor (Ob-Rb) mRNA was significantly low inAD hippocampal tissue (*p < 0.05, c) compared with control.

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166 D. J. Bonda et al.

Using adjacent serial sections from three cases of AD,neurons that were present in both sections stained for eitherOb-Rb or pOb-RbT985 were densitometrically analyzed.pOb-RbT985 immunoreactivity was found to be lower inNFT-bearing neurons in all three cases analyzed (p < 0.05and p < 0.001, Fig. 5d).

Discussion

Our findings indicate a marked alteration in leptin signalingin the hippocampus of AD patients. We found elevations inleptin concentrations in CSF samples (Fig. 1a) from ADpatients when compared with controls. These differenceswere not correlated with variations in age or BMI within orbetween groups (Fig. 1b, c); they are therefore likelyassociated with the disease itself. While the women ADend stage patients were found to have higher leptin levels inthe CSF, women have previously been shown to have higherciruculating levels of leptin in the serum than men (Bennettet al. 1997). This study may have implications for under-standing the gender differences associated with ADincidence. Interestingly, our analyses of leptin CSF levelsalong the Braak staging continuum demonstrated a strikingleptin increase in the most severe cases. Specifically, Braak

VI patients exhibited a six-fold increase in leptin CSFconcentrations compared to Braak 0–V patients. The Braakscoring system provides an assessment of neuritic pathologyin postmortem brain tissue that is independent of, thoughcomplementary to, antemortem diagnosis, with Braak 0 or Irepresenting minimal tau pathology and Braak VI represent-ing severe NFT pathology (Braak and Braak 1994). Braakstages I–II are typically manifest as clinically silent, stagesIII–IV as incipient, mild AD, and stages V–VI as fullydeveloped, severe AD (Braak and Braak 1995). Theseresults, taken together with prospective studies in whichincreased levels of baseline plasma leptin concentrationscorrelated with a reduced risk of dementia or AD onset(Holden et al. 2009; Lieb et al. 2009), strongly suggest acorrelation between the leptin level in brains and CSF withthe progression of AD. These studies did not monitor leptinlevels following the initial measurement and, thus did notcollect end-stage data for patients in the Braak VI category ofAD, for which we found striking increases of leptin.Nonetheless, our study also found a trend toward decreasedlevels of CSF leptin in MCI compared with control, whichsupports the attributed benefits associated with leptin signal-ing in the brain, and may be important in elucidating theetiological occurrence of AD.

(a) (b)

(c) (d)

(e) (f)

Fig. 3 Immunohistochemistry reveals weak

long form of the leptin receptor (Ob-Rb)localization to neuronal cytoplasm inpyramidal neurons of control individuals

(a) as well as the occasional age-relatedneurofibrillary tangles (a, arrow). In theAlzheimer disease (AD) cases examined,however, neurofibrillary tangle (NFT) are

prominently labeled (b) both intracellullarand extracellular NFT (marked by * in b).The NFT exhibits typical fibrillar morphology

(c). Ob-Rb is also colocalized with theneuritic pathology associated with senileplaques (d). Adsorption with the peptide

antigen nearly abolishes the NFT andneuritic plaque localization (f) compared tothe non-absorbed adjacent sections (e).

Scale bars = 50 lm (a, b, d–f) and 20 lm(c).

© 2013 International Society for Neurochemistry, J. Neurochem. (2014) 128, 162--172

Disruption of leptin signaling in AD 167

Leptin is synthesized in the periphery and activelytransported across the blood–brain barrier (BBB) or blood–CSF barrier (Kurrimbux et al. 2004), thus our finding thatleptin mRNA is not present in the brain while its protein islocalized in neurons is consistent with previous reports. Theprecise dynamics of leptin transport are incompletely under-stood; it is not yet clear whether CSF leptin is exiting thebrain via the choroid plexus epithelial cells (Uotani et al.1999) or whether it is entering the brain in a mannerindependent of leptin BBB entry. Furthermore, leptintransport across the BBB is believed to be a saturablesystem whereby brain entry is strictly limited by transportdynamics (Banks et al. 1996). Given that BBB has beendemonstrated to be compromised in AD brains (Deane andZlokovic 2007), increased level of leptin in severe AD casesmay be reflective of this disease-associated pathology. Alongwith the marked increase in leptin in AD CSF, we found asignificant decrease in Ob-Rb mRNA levels in AD patientscompared with controls. The expression of Ob-Rb isregulated by a number of factors, including circulating leptin

levels, insulin levels, and diet, and the precise dynamics andconditions of leptin receptor regulation are incompletelyunderstood (Hikita et al. 2000; Mitchell et al. 2009).Therefore, the alteration found in our study (i.e., theincreased levels of CSF leptin, the high levels of Ob-Rbsequestered in NFT, the up-regulation of pOb-RbT985 in non-tangle-bearing neurons) may all contribute to the negativeregulation of Ob-Rb mRNA expression and this mechanismneeds to be determined in future study.Leptin plays a major role in the regulation of appetite and

body energy metabolism. It has been shown that biomarkersfor AD such as Ab and tau are associated with lower BMI inboth MCI and cognitively normal individuals (Vidoni et al.2011). For example, patients with moderate AD were shownto have lower lean body mass, and the male patients energyhomeostasis, measured by serum ghrelin and leptin levels, isnot maintained (Theodoropoulou et al. 2012). Other appetiteand body energy metabolism associated neuropeptides, suchas galanin and melanocyte stimulating hormone in the CSF,are also correlated with AD progression and specifically NFT

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

Fig. 4 Staining of adjacent serial sectionsreveals many of the neurons containing long

form of the leptin receptor (Ob-Rb) (a) alsocontain phosphorylated tau, labeled withthe monoclonal antibody AT8 (b), marked

by arrows. * denotes landmark vessel.Quantification of eight cases of Alzheimerdisease (AD) found that on average 40% of

neurons stained with Ob-Rb also containedthe pathological form of tau, while another40%of neurofibrillary tangle (NFT) containedonly AT8, and about 20% of NFT was only

positive for Ob-Rb (c). Double fluorescentmicroscopy on a single section was alsoused to confirm that many of the neurons

strongly stained for Ob-Rb (left panels, red,d, g, j) also contained AT8, a marker for NFT(middle panels, green, e, h, k). Right panels

show themerged images. Indeed, someNFTonly demonstrated Ob-Rb (g–i) and someonly displayed AT8 (j–l), yet Ob-Rb was still

seen as a weak nuclear localization, but notaccumulated in the cytoplasmic NFTformation in these instances.

© 2013 International Society for Neurochemistry, J. Neurochem. (2014) 128, 162--172

168 D. J. Bonda et al.

related biomarkers tau (Costa et al. 2011). Many studieshave found relationships to midlife obesity and increased riskof AD and dementia. However, weight loss is also a factor inAD patients, though it may not be directly related to loss offat and instead may be a result of loss of muscle (Lee 2011).Other AD related factors may also play a role in thedysregulation of leptin signaling reported here. Mice lackingapolipoprotein E (APOE) show decreased circulating leptin,linking appetite, leptin, cholesterol levels with APOE, ofwhich the APOE4 allele is less effective at clearing awaycholesterol, and which poses the strongest genetic risk factorfor AD (Raber et al. 2000). In any case, there seems to be asevere disconnect in the leptin signaling pathway in AD.Our data demonstrate a striking colocalization of the leptin

receptor to NFTs in the hippocampus (Fig. 4) with a weakassociation between the active form of Ob-Rb (pOb-RbT985)and NFTs (Fig. 5) in AD. Within the same tissue sections ofthe same AD cases, we noted discrepancies in the regionalimmunoreactivity of pOb-RbT985 and resident NFT, such thatthe NFT-positive region (i.e., CA1) contained drasticallyreduced pOb-RbT985 stainingwhile the pathology-free regions(CA3/4) contained elevated pOb-RbT985 staining (Fig. 5).

These incongruities continue despite the elevated presence ofleptin in the AD brains and CSF mentioned above. Thus, thereseems to be an NFT-induced blockade of Ob-Rb accessibilityand an effective leptin resistance. That is, because ofpathological sequestration of Ob-Rb by NFT, regions con-taining high concentrations of NFT lose the ability tocommunicate leptin signal transmission and therefore do notdemonstrate immunohistochemical presence of pOb-RbT985

and fail to receive the aforementioned benefits of leptinsignaling in the hippocampus by effectively preventing leptinfrom gaining access to its receptor in the affected region.Consequently, these pathological alterations in leptin signal-ing result in leptin resistance in the vulnerable neurons in AD(Fig. 6) and this mechanism may be critical to the cognitivedwindling phenotype characteristic of AD. Supporting thishypothesis, it is interesting to note that, throughout the cyclefrom expression to degradation, Ob-Rb follows an elaboratetrafficking pathway that relies chiefly onmicrotubules (Wilckeand Walum 2000). Since tau is a microtubule binding protein(Castellani et al. 2008) and becomes hyperphosphorylatedand aggregates into fibrillized NFTs in AD (Mondragon-Rodriguez et al. 2008), it is likely that Ob-Rb becomes

(a) (b) (c)

(d) (e) (f)

Fig. 5 Theactivated formof the leptin receptor, pOb-RbT985, is detected

at higher levels in the hippocampus of theAlzheimer disease (AD) cases(b) compared with controls (a). However, within individual AD cases, thepOb-RbT985 immunoreactivity is strongest in the CA4/CA3 regions, andis much reduced in the CA2/CA1 regions. In the CA3 region of an AD

case, many of the neurons with high levels of pOb-RbT985 (b), alsodemonstrate a diffuse cytoplasmic localization of long form of the leptinreceptor (Ob-Rb) (c; same neurons denoted by black arrows). On the

same section, pyramidal neurons in the CA1 region contain lower levels

of pOb-RbT985 (e). Red arrows mark those cells containing pOb-RbT985

and diffuse cytoplasmic Ob-Rb pattern (d). Conversely, the neurofibril-lary tangle (NFT)-bearing neurons immunostained for Ob-Rb (blackarrows, f), often do not contain pOb-RbT985 (e).* denotes landmark

vessels. Scale bar = 50 lm. Quantification (d) reveals a significantlylower relative intensity of pOb-RbT985 in those cells with NFT comparedto cells without NFT (*p < 0.001, **p < 0.05).

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Disruption of leptin signaling in AD 169

sequestered within the pathological NFTs during its process-ing and movement along the cytoskeleton. Indeed, aberrationin cytosolic transport machinery has been previously identifiedin AD, affecting the functioning of signaling cascadesassociated with Smad2 (Lee et al. 2006b) and importin a1(Lee et al. 2006a), and the data presented here correspondsnicely to these observations. In addition, reduction in down-stream signaling molecules of the leptin pathway in AD hasbeen described, indicating a lack of effective leptin-basedcommunication in the disease (Chiba et al. 2009).These data raise important questions as to the nature of the

impact that these interactions have on the neurophysiology/cognitive phenotype of the affected patients. The disruption in

leptin signaling in AD is clear; whether or not this is amanifestation of leptin resistance because of NFT develop-ment, or the result of some other neurological defects,however, is not clear. In relation to the reported benefits ofleptin signaling in the hippocampus (Harvey 2003; Garzaet al. 2008), the ultimate effect may be partly responsible forthe dwindling cognitive capacity that characterizes AD. Thatis, perhaps the disjointed intracellular processing of Ob-Rbimpairs leptin signaling in the affected regions (i.e., thehippocampal formation of the mediotemporal lobe), creating aleptin resistance that drastically reduces the benefits of leptinsignaling (Garza et al. 2008; Perez-Gonzalez et al. 2011) inan already decaying brain state. The data listed here, inconjunction with the findings of Chiba et al. (2009), in whichthe JAK2/STAT3 signaling molecules of the leptin cascadewere found to be altered in AD, support this postulation.The goal of AD research is the generation of translational

therapeutics that can prevent and reverse disease symptoms.Leptin has been previously demonstrated to exert neuropro-tective effects in the hippocampus (Garza et al. 2008; Perez-Gonzalez et al. 2011) as well as an amelioration of thehyperphosphorylation of tau in these regions (Greco et al.2008, 2009a,b). Our findings seem to similarly point to theleptin pathway as a target for disease control. It may bepossible to simultaneously reduce tau neuropathology andincrease cognitive restoration through an increase in leptinsignaling and appropriate leptin receptor trafficking.

Acknowledgments

This study was supported by the National Institutes of Health(AG028679 to HGL and AG031852 to XWZ) and P30 AG028383(UK-ADC database). The funding sources had no involvement indata collection, analysis or interpretation of the study.

Conflict of interest

Dr Xiongwei Zhu was a consultant for and received grant supportfrom Medivation. Dr. Mark A. Smith was a consultant for AnavexLife Sciences Corporation, Eisai, Medivation, Neurotez, and TakedaPharmaceuticals; owned stock options in Aria Neurosciences,Neurotez, Panacea and Voyager, and received lecture fees fromGSK, Medivation and Pfizer.

Supporting information

Additional supporting information may be found in the onlineversion of this article at the publisher's web-site:

Figure S1. Western blot analysis of purified leptin incubated inhuman CSF collected from two individuals, case A and case B,shows no degradation of either the monomer or dimer band evenafter 4 h incubation at 37°C, mimicking the longest post-morteminterval of the subjects used for the leptin RIA analysis (a). Theleptin receptor antibody, which detects NFT in AD brain byimmunocytochemistry, detects the full length Ob-Rb at approxi-mately 125kDa.

AD onset

Dendrite

Free circulating Leptin

Sequestered Ob-Rb

Reduced Leptin signal transduction

Neuronal weakening/cognitive decline

Increased leptinproduction

NFT deposition

Fig. 6 Schematic depicting the proposed mechanism underlying leptinresistance in Alzheimer disease (AD). Following onset of the disease,

hyperphosphorylation of tau leads to the formation of neurofibrillarytangle (NFT) and corresponding impairment of intracellular traffickingnetworks. The long form of the leptin receptor (Ob-Rb) becomes

unable to reach the cell membrane and thus becomes unable to gainaccess to circulating free leptin disrupting leptin signaling, and thecognitive/neuronal benefits associated with its activity do not manifest.

Concurrently, the lack of leptin signal transduction within affectedneurons creates the illusion of inadequate circulating leptin levels,ultimately communicating a physiological need for more leptin secre-

tion. Ultimately, this cascade may cause a gradual weakening ofaffected neurons and cognitive decline even with increased level ofleptin.

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170 D. J. Bonda et al.

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