ORIGINAL ARTICLE

Do CSF levels of t-Tau, p-Tau and β1-42 amyloid correlatewith dopaminergic system impairment in patients with a clinicaldiagnosis of Parkinson disease? A 123I-FP-CIT study in the earlystages of the disease

Agostino Chiaravalloti & Alessandro Stefani &Alessandro Fiorentini & Annamaria Lacanfora &

Paolo Stanzione & Orazio Schillaci

Received: 15 April 2014 /Accepted: 16 June 2014 /Published online: 10 July 2014# Springer-Verlag Berlin Heidelberg 2014

AbstractPurpose To investigate the relationships among cerebrospinalfluid (CSF) levels of t-Tau, p-Tau and Aβ1-42 amyloid peptideand 123I-FP-CIT uptake.Methods The study included 58 subjects (31 men and 27women, age 67 ± 9 years) with a clinical diagnosis ofParkinson disease diagnosed according to the UnitedKingdom Parkinson Disease Society Brain Bank criteria. Allsubjects underwent a CSF assay 28 ± 3 days before 123I-FP-CIT SPECT scanning. The relationships were evaluated bymeans of linear regression analysis and Pearson correlation.Results Striatal 123I-FP-CIT was positively related to both t-Tau and p-Tau CSF values with low levels of t-Tau and p-Taubeing related to a low uptake of 123I-FP-CIT. In particular,differences with higher statistical significance were found forthe striatum between the contralateral side and the side mainlyaffected on clinical examination (P<0.001). No significantrelationships were found between Aβ1-42 amyloid peptideand 123I-FP-CIT binding.Conclusion The results of our study suggest that the presyn-aptic dopaminergic system is more involved in Parkinsondisease patients with lower t-Tau and p-Tau CSF values while

values of Aβ1-42 amyloid peptide seems not to be related tonigrostriatal degeneration in our series.

Keywords Parkinson disease . DaTscan . Amyloid . p-Tau .

t-Tau

Introduction

In patients with a clinical diagnosis of Parkinson disease (PD)the relationships between clinical features and both the dopa-minergic and noradrenergic systems have been extensivelyinvestigated [1–3] and several studies have confirmed thecaudorostral ascending pattern of Lewy pathologies as in-ferred by Braak and colleagues, indicating that PD is a mul-tisystem disorder not confined to nigrostriatal dopamine defi-ciency [4]. In particular, it has been shown that impairment ofboth the dopaminergic and noradrenergic systems is related toseverity and disease duration [3]. The detection of dopami-nergic system impairment is actually considered one of themost accurate markers of disease showing a high sensitivity inthe diagnosis of Lewy pathologies [3, 5, 6] while evaluation ofthe noradrenergic system leads to a higher specificity [6].

To date the roles of Tau protein and the 42-amino acid formof beta-amyloid (Aβ1-42) in the neurodegenerative process ofPD are still a matter of debate. Assay of Tau protein, both astotal Tau (t-Tau) and phosphorylated Tau (p-Tau), and Aβ1-42

in cerebrospinal fluid (CSF) should reflect the central patho-genic processes in the brain and they have been proposed aspotential biochemical diagnostic markers of several neurode-generative disorders, becoming widely used in the evaluationof patients with Alzheimer disease (AD) [7]. While Aβ1-42 isthe core peptide that accumulates in senile plaques and is

A. Chiaravalloti (*) :A. Fiorentini :A. Lacanfora :O. SchillaciDepartment of Biomedicine and Prevention, University Tor Vergata,Viale Oxford 81, 00133 Rome, Italye-mail: agostino.chiaravalloti@gmail.com

A. Stefani : P. StanzioneDepartment of Neurosciences, University Tor Vergata, Rome, Italy

A. StefaniIRCCS Santa Lucia, Rome, Italy

O. SchillaciIRCCS Neuromed, Pozzilli, Italy

Eur J Nucl Med Mol Imaging (2014) 41:2137–2143DOI 10.1007/s00259-014-2841-4

considered a marker of ADwith relatively high specificity andis implicated in its pathogenesis [8], recently Tau protein hasbeen associated with PD [9]. Tau protein is a microtubule-associated protein that under physiological conditions regu-lates microtubules (assembly, dynamic behaviour, spatial or-ganization, and regulation of axonal transport of organelles,including mitochondria) [10]. Hyperphosphorylation of Tauleads to intracellular accumulation of Tau in the form ofneurofibrillary tangles, leading to subsequent inflamma-tion and neurodegenerative processes [9]. Hence, thepresence of Tau in the brain parenchyma or in theCSF is considered a consequence of abnormal Tau pro-tein accumulation. Moreover, it appears likely that t-Taulevels reflect neuronal degeneration. However, severalphysiological processes independent of cell death maybe responsible for Tau release from cell lines and neu-rons via multiple pathways (naked form or vesicle-associated) [10]. The detection of p-Tau protein in theCSF may therefore provide a useful biomarker of theneuropathological process (abnormal hyperphosphorylation)that is the key to the Tau misfolding process in diseased brainleading to abnormal conformations of Tau [11].

Interestingly, a recent study performed in a population of63 PD patients showed a negative correlation between CSFlevels of Tau proteins and motor symptoms of PD, low p-Taulevels being associatedwith increased severity of motor symp-toms in these patients [12]. The authors found that lower Aβ1-

42 and p-Tau levels were associated with a PD diagnosis andthat decreased CSF t-Tau and α-synuclein (α-syn) levels wereassociated with increased severity of motor symptoms [12].As a neuropathological correlate, Attems et al. found that in88.5 % of subjects with extrapyramidal symptoms (EPS) Taudeposition in the substantia nigra was a major pathologicalsubstrate, often associated with higher Braak stages [13].

The aim of this study was to investigate the relation-ships among the CSF biomarkers Aβ1-42, p-Tau and t-Tau and dopaminergic system impairment as detectableby means of N-(3-fluoropropyl)-2β-carbomethoxy-3β-(4-[123I]iodophenyl)nortropane (123I-FP-CIT) singlephoton emission computed tomography (SPECT).

Materials and methods

Patients

The study involved 58 patients with a clinical diagnosis of PD(31 men and 27 women, age 67 ± 9 years), 29 with Hoehn &Yahr (H&Y) stage 1, 11 with stage 1.5, 7 with stage 2 and 11with stage 3 disease, evaluated by 123I-FP-CIT SPECT, asshown in Table 1. CSF assay was performed 28 ± 3 daysbefore 123I-FP-CIT SPECT. A standard MR examination

without administration of contrast agent was performed7 ± 2 days before the SPECT examination.

A clinical examination was conducted by an experiencedneurologist (A.S.) and PD was diagnosed using the UnitedKingdom Parkinson’s Disease Society Brain Bank(UKPDSBB) criteria [14]. The motor part of the UKPDSBBwas used to assess the severity of disease [15]. The MiniMental State Examination did not reveal any cognitive defi-cits. The right side was the more affected side in all thesubjects examined (Table 1).

Informed consent was obtained from all patients in accor-dance with the principles of the Declaration of Helsinki.

CSF collection and analysis

CSF was collected following the procedure described in theConsensus Guidelines for CSF and Blood Biobanking forCNS Biomarker Studies [16]. The first 2 ml of CSF, obtainedby lumbar puncture, was analysed for cell count, proteinand glucose. An additional 10 ml of CSF was trans-ferred to polypropylene tubes, centrifuged at 2,000 g for10 min at room temperature and 0.5-ml aliquots weretransferred to polypropylene tubes which were immedi-ately frozen and stored at −80 °C until AD biomarkersware measured.

CSF levels of t-Tau, p-Tau and Aβ1-42 were measuredwithin 2 h of CSF collection by ELISA using a commerciallyavailable kit (Innotest; Innogenetics, Ghent, Belgium) and areexpressed as picograms per millilitre (Table 1).

MR imaging

MR imaging was performed using a 1.5-T superconductivesystem (OptimaTM MR450w; GE Medical Systems,Waukesha, WI) using a head coil. All patients were imagedwith the following sequences: fast spin echo (FSE) T2 in thecoronal plane, and FSE T1, T1, T2 and fluid attenuatedinversion recovery (FLAIR) in the axial plane with a slicethickness of 3 mm.

123I-FP-CIT SPECT

According to standard guidelines [17], each patient wasinjected intravenously with 185 MBq (5 mCi) of 123I-FP-CIT at the same time of day and under the same experimentalconditions. Perchlorate (1,000 mg) was administered at least30 min before injection of the radiopharmaceutical to blockthyroid uptake of free radioactive iodide [18]. Imaging wasalways performed 4 h after 123I-FP-CIT injection using a dual-head gamma camera (Millennium VG; GE Medical Systems,Milwaukee, WI) equipped with low-energy high-resolutioncollimators. SPECTstudies were acquired using the followingparameters: 128×128 matrix, 120 projections (rotation 360°),

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40 s per projection. The slice thickness was 4.42 mm.Reconstruction was performed using the ordered-subsets ex-pectation maximization algorithm (four iterations and 14subsets) with a Butterworth filter (cut-off frequency 0.5,order 10) to produce transaxial slices that wereattenuation-corrected. Attenuation correction was per-formed according to the method of Chang [19] using a coef-ficient μ of 0.11 cm−1 after manually drawing an ellipsearound the head contour.

Based on the MR scan, regions of interest (ROI) werecreated manually for each patient including detailedregions for both putamina, caudate nuclei and occipitalcortices (Fig. 1). The MR scan was coregistered with123I-FP-CIT SPECT using Mimvista software (MIMvistaCorp., Cleveland, OH) [20] and then reoriented so as tobe parallel to the canthomeatal line. For analysis of123I-FP-CIT striatal uptake, three adjacent transverseslices that showed the most intense striatal uptake werechosen and the mean of the ratios of specific to nonspe-cific binding was calculated. The ratios of specific tononspecific binding were calculated according to thefollowing expression: 123I-FP-CIT binding = (ROI−Occ)/Occ, where ROI is the mean count in the ROI (putamen orcaudate nucleus) and Occ is the mean count in the occipitalcortex [21], as shown in Fig. 1.

Statistical analysis

We calculated the means and standard deviations of age,UPDRS III score, H&Y grade, disease duration and analysedthe semiquantitative 123I-FP-CIT SPECT data (Table 2). Two

way ANOVA was used to evaluate the differences amonggender and CSFmarker levels and striatal 123I-FP-CIT uptake.To determine if the 123I-FP-CIT uptake data showed aGaussian distribution, D’Agostino’s K-squared normality testwas applied (where the null hypothesis is that the data arenormally distributed).

CSF levels of Aβ1-42, p-Tau ant t-Tau and striatal123I-FP-CIT uptake, UPDRS, H&Y stage, disease dura-tion and age were correlated by linear regression andPearson correlation.

Since reporting effect sizes is considered good practicewhen presenting empirical research finding and is able toenhance the statistical power of a test regardless the P value(especially in the context of an F-test for ANOVA or multipleregression) [22], here we report a standardized measure ofeffect size (Cohen’s f2) which allows an evaluation of localeffect size (i.e. telling us exactly how large the relationshipreally is between the variables we’ve studied and is indepen-dent of howmany people were tested) [23]. By convention, ƒ2

effect sizes of 0.02, 0.15, and 0.35 are termed small, medium,and large, respectively [24].

A hypothesis was considered valid when P value was≤0.05.

Results

On visual examination, the 123I-FP-CIT SPECT data werepositive for dopaminergic presynaptic impairment in all thepatients. A general overview of the population studied is

Table 1 General overview of the58 PD patients (27 men, 31women)

There were no significant differ-ences between the men andwomen

All patients (n=58) Men Women

Age (years), mean±SD 67±9 66.2±7 67.8±10

Disease duration (months), mean±SD 21.76±11.75 – –

Hoehn & Yahr stage, n

1 29 11 18

1.5 11 8 3

2 7 4 3

3 11 5 6

UPRDS III score, mean±SD 26.27±6.37 27.2±7 25.34±5

Tau (pg/ml), mean±SD 273.04±233.70 273.03±233.7 273.05±232.2

p-Tau (pg/ml), mean±SD 40.64±25.08 40.6±25 40.6±24

Aβ1-42 (pg/ml), mean±SD 514.17±289.15 514±289.1 513.83±288.1

23I-FP-CIT binding ratios, mean±SD

Striatum total 2.37±0.59 2.38±0.6 2.36±0.6

Right caudate 2.63±0.65 2.65±0.7 2.64±0.3

Left caudate 2.49±0.66 2.48±0.8 2.49±0.9

Right putamen 2.16±0.63 2.17±0.6 2.13±0.9

Left putamen 2.02±0.61 2.02±0.5 2.01±0.6

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shown in Table 1. CSF biomarker levels and 123I-FP-CIT uptake were not significantly different between genders

(P>0.05). All the 123I-FP-CIT uptake data were normallydistributed.

Fig. 1 Sample axial T1 MR and 123I-FP-CIT SPECT images selectedfrom a patient with t-Tau and p-Tau CSF levels of 155 and 33 pg/ml (a, b)and from a patient with t-Tau and p-Tau CSF levels of 1,200 and 186 pg/ml (d, e). On the T1 images (a, d), ROIs were traced on the right and leftcaudate (RC and LC), right and left putamen (RP and LP) and occipital

region (OCC), and then placed on the coregistered 123I-FP-CIT SPECTimages (b, e). The Striatal/Occ ratio is more affected in b than in e, beingequal to 1.88 and 3.06, respectively. c, f Axial T2 SPECT/MR imagesshowing the findings in b and e, respectively

Table 2 Statistical analysis of 123I-FP-CIT uptake in relation to Tau, Aβ1-42 and P-Tau CSF levels

Region t-Tau p-Tau Aβ1-42

Linear regression Pearson correlation Linear regression Pearson correlation Linear regression Pearson correlation

r2 P value r P value r2 P value r P value r2 P value r P value

Striatum total 0.1676 0.0014 0.4094 0.0014 0.09519 0.0185 0.3085 0.0185 0.02334 ns −0.1528 ns

Left caudate 0.1273 0.0060 0.3569 0.0060 0.07293 0.0404 0.2701 0.0404 0.02933 ns −0.1713 ns

Right caudate 0.7298 0.0403 0.2702 0.0403 0.0576 ns 0.2361 ns 0.03216 ns −0.1522 ns

Left putamen 0.1999 0.0004 0.4471 0.0004 0.09093 0.0214 0.3016 0.0214 0.02112 ns −0.1453 ns

Right putamen 0.1964 0.0005 0.4432 0.0005 0.1255 0.0064 0.3543 0.0064 0.01727 ns −0.1314 ns

ns not significant

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CSF levels of Aβ1-42, p-Tau, t-Tau and striatal 123I-FP-CITuptake

As shown in Table 2 and Fig. 2, t-Tau levels weresignificantly related to striatal 123I-FP-CIT uptake. Similarresults were obtained for p-Tau with the exception of theright caudate. On the other hand no significant relation-ships were found between the CSF Aβ1-42 peptide valuesand the SPECT data.

With few exceptions, the size effect on linear regressionanalysis was small in our study cohort. In particular, forregression analysis of t-Tau levels and 123I-FP-CIT uptake, f2

was 0.2013 for both the right and left striatum, 0.1458 for theleft caudate, 0.07298 for the right caudate, 0.2498 for the leftputamen and 0.2444 for the right putamen. For p-Tau levelsand 123I-FP-CIT uptake, f2 was 0.1052 for both the right andleft striatum, 0.0786 for the left caudate, 0.1000 for the leftputamen, 0.1435 for the right putamen and 0.0611 for the rightcaudate. For Aβ1-42 levels and 123I-FP-CIT uptake, f2 was0.0238 for both the right and left striatum, 0.0302 for the leftcaudate, 0.0215 for the left putamen and 0.0175 for the rightputamen.

Discussion

The main finding of this study was the positive relationshipsamong CSF t-Tau and p-Tau levels and 123I-FP-CIT uptake, as

shown in Fig. 2. These finding are in agreement with those ofseveral clinical studies showing decreased concentrations ofCSF t-Tau, p-Tau or both, in patients with PD as comparedwith controls, the most including 50 or more patients [12,25–27].

To the best of our knowledge, the relationships among 123I-FP-CIT and CSF t-Tau, p-Tau and Aβ1-42 peptide have notpreviously been investigated. Shi et al. compared CSF t-Tau,p-Tau and Aβ1-42 in subjects with a clinical diagnosis of AD,PD or multiple system atrophy (MSA) and a control group.They found that the levels of t-Tau in subjects with PD andMSAwere significantly lower than in controls (P=0.026, PDvs. controls; P=0.004, MSA vs. controls) and significantlylower than in subjects with AD. Alterations in p-Taumirrored those of t-Tau, but again significantly lower p-Tau levels were found in subjects with PD than in con-trols [27]. Kang et al. found lower levels of t-Tau, p-Tauand α-syn in subjects with PD than in healthy controlsand, while p-Tau levels were associated with a diagnosisof PD diagnosis, lower CSF t-Tau and α-syn levels wereassociated with more severe motor symptoms [12]. Ourstudy using 123I-FP-CIT SPECT confirmed that dopami-nergic system involvement was correlated with low levelsof p-Tau and t-Tau proteins in the CSF. Most of ourpatients had a relatively short disease duration (about21 months) and early-stage disease (69 % of our patients,40/58, were H&Y stage 1 or 1.5; Table 1). This suggests thata relationship between CSF Tau and nigrostriatal degenerationis detectable during the early stages of PD.

Fig. 2 Linear regression analysis of CSF levels of Aβ1-42, p-Tau, t-Tau inrelation to striatal 123I-FP-CIT uptake. The higher the t-Tau CSF levels,the higher the 123I-FP-CIT uptake in the striatum (a), left putamen (b),right putamen (c), left caudate (d) and right caudate (e). Similar results are

shown for p-Tau (f–i) with the exception of right caudate (l) (see text). Nosignificant relationships have been found for the relationships betweenAβ1-42 CSF levels and 123I-FP-CIT uptake (m–q)

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In PD, Lewy bodies (that are detectable in the substantianigra pars compacta, SNPc) can occasionally contain Tau,usually incorporated into filaments [28], and increased Tauphosphorylation has also been discovered in synapse-enrichedfractions from PD brains [29]. On the basis or our results, itcou ld be specula ted tha t reduced c learance ofhyperphosphorylated Tau may be responsible for Tau accu-mulation in the brain (in turn responsible for neurodegenera-tive processes in particular in the SNPc) and for reduced Tauconcentrations in the CSF (with a mechanism similar to that ofamyloid in AD) [30].

The usefulness of molecular imaging using 123I-FP-CITSPECT in the evaluation of patients with movement disordersis well known [31]. Considering DATas a surrogate marker ofdopaminergic nigrostriatal neurons, 123I-FP-CIT imaging ofDAT sites can identify nigrostriatal dopaminergic deficits inPD patients in vivo, and several SPECT studies have demon-strated high accuracy in the various clinical subtypes of PD [1]and in differentiating patients with PD and parkinsonian syn-dromes from patients with essential tremor and healthy con-trols [1].

A few studies have failed to identify a relationship betweenTau levels and EPS. In particular, in a study of the impact ofnigral Tau lesions in a population of AD subjects with EPS,Attems et al. found that the density of nigral Tau lesions,which were present in 88.5 % of 35 EPS-positive patients,was not correlated with the intensity of EPS [13], ruling out apossible direct influence of these proteins on the nigrostriataldegeneration process [13]. Since these findings were obtainedin patients with a clinical diagnosis of AD, future studiescould investigate the relationship between 123I-FP-CIT striataluptake and CSF Tau levels in these patients.

One of the main limitations of this study was the lack ofCSF samples from a control group of healthy subjects (that isof the utmost importance in neuroimaging studies). ROCanalysis of CSF t-Tau and p-Tau levels in healthy subjectscompared to the levels in our PD patients would have allowedthe identification of t-Tau and p-Tau CSF cut-off values relat-ed to the presence/absence of a dopaminergic deficit. Thedecision not to use a sample of healthy individuals was dueto the extremely high cost of building up such a cohort ofsubjects and of the difficulty in preventing them from beingexposed to radiation and to invasive procedures. Future stud-ies, possibly including a larger cohort of PD patients, arenecessary in this regard.

Several CSF biomarkers including α-syn and DJ-1 (twoproteins intimately involved in the pathogenesis of familialand sporadic PD) have been proposed as additional markers ofPD in recent years, and in a recent study involving a largecohort of subjects a decrease in CSF DJ-1 and/or α-synprovided a sensitivity of 90 – 92 % and a specificity of58 – 70% in distinguishing PD subjects from healthy controlsand patients with AD [32]. The authors did not find significant

associations between DJ-1 or α-syn levels and the severity ofPD [32]. Future studies should include other CSF functionalparameters for investigating possible relationships with 123I-FP-CIT striatal uptake and, again, comparison with a controlgroup of healthy subjects could further support the data.

The value of Aβ1-42 CSF levels in our patients (514.17±289.15 pg/ml, Table 1) is similar to the levels found by others(e.g. 510.6±27.8 pg/ml found by Zhang et al. in 40 PDpatients [33]). The regression analyses (Fig. 2. m–q) showeda mild trend to decreased values that is consistent with anegative relationship between Aβ1-42 CSF levels and 123I-FP-CIT uptake. Nevertheless, we did not find any statisticallysignificant relationship between CSF levels of Aβ1-42 and123I-FP-CIT uptake, as shown in Table 2. These results arenot surprising since many studies have not shown any signif-icant reduction in Aβ1-42 CSF concentrations in PD patientswithout cognitive impairment [25–27, 33] (as in our patients,in whom the main neuropsychological tests did not reveal anycognitive impairment), suggesting that progressive cognitiveimpairment in patients with PD may be associated with in-creased deposition of fibrillar Aβ in the cerebrum [26].

Conclusion

The results of this study suggest that lower levels of CSF t-Tauand p-Tau are related to greater dopaminergic impairment inpatients with a clinical diagnosis of PD. If available, theseparameters could help in the interpretation of 123I-FP-CITSPECT results.

Conflicts of interest None.

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