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1 JOURNAL OF NEUROCHEMISTRY (Suppl. 1) doi: /jnc.13390,,,, *Paracelsus-Elena-Klinik, Kassel, Germany University Medical Center (Department of Neuropathology), Georg-August University Goettingen, Goettingen, Germany Centro Disturbi della Memoria- Unita Valutativa Alzheimer, Clinica Neurologica, Universita di Perugia, Perugia, Italy Applied Neuroscience Group, CEITEC MU, Masaryk University, Brno, Czech Republic Department of Neurology, University Medical Center Ljubljana, Ljubljana, Slovenia **Division for Neurogeriatrics, Department of NVS, Karolinska Institutet, Center for Alzheimer Research, Stockholm, Sweden Centre for Age-Related Medicine, Stavanger University Hospital, Stavanger, Norway Institute of Clinical Medicine / Neurology, University of Eastern Finland, Kuopio, Finland Department for Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden Department of Neurology, Department of Laboratory Medicine, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, Nijmegen, The Netherlands ***Program in Neuroscience and Division of Neurology, The Ottawa Hospital, University of Ottawa Brain & Mind Research Institute, Ottawa, Ontario, Canada Abstract Cerebrospinal fluid (CSF) has been extensively studied to explore biochemical alterations in subjects with neurodegenerative disorders. In Alzheimer s disease, levels of increased CSF tau protein and decreased levels of b-amyloid 1 42 (Ab42) have been shown to correlate with brain plaque formation and tangle pathology. Intracellular Lewy inclusions containing aggregated a-synuclein (a-syn) represent a pathological hallmark of Parkinson s disease (PD). In most but not all studies published to date total CSF a-syn concentrations have been found to be decreased in disorders related to a-syn pathology, that is, PD, dementia with Lewy bodies and multiple system atrophy. However, these reports show extensive signal overlap among tested individuals, thereby diminishing its potential for routine use in clinical practice. To investigate potential biological (i.e., non-technical) confounders of reported CSF levels for a-syn, Ab42, and tau in PD and related disorders, we carried out a methodical review of Received February 17, 2015; revised manuscript received September 11, 2015; accepted September 21, Address correspondence and reprint requests to Brit Mollenhauer, Parsacelsus-Elena-Klinik, Klinikstrasse 16, Kassel, Germany. brit.mollenhauer@paracelsus-kliniken.de Abbreviations used: AD, Alzheimer s disease; APP, amyloid precursor protein; Ab42, b-amyloid 1 42; BBB, blood brain barrier; CBD, corticobasal degeneration; CJD, Creutzfeldt Jakob disease; CNS, central nervous system; CSF, cerebrospinal fluid; DLB, dementia with Lewy bodies; EDO, early disease onset; MMSE, mini mental status examination; MoCA, Montreal Cognitive Assessment; MSA, multiple system atrophy; NT-PD, non-tremor-dominant PD; PDD, Parkinson s disease with dementia; PD, Parkinson s disease; PPMI, Parkinson s Progression Marker Initiative; REM, rapid eye movement; SSRI, selective serotonine reuptake inhibitor; TD-PD, tremor-dominant Parkinson s disease; VP, vascular parkinsonism; a-syn, a-synuclein. 290

2 Biological confounders for the values of CSF proteins in PD 291 known factors that underlie signal variability and speculate on those that have not yet been tested. We discuss several biological factors, such as neuropathology, demographics, clinical phenotype, progression and duration of disease, concomitant illnesses and, last but not least, pharmacotherapy, which in isolation or combination can substantially alter values for CSF proteins of interest. Enhanced implementation of standardized clinical protocols, streamlined operating procedures, and further progress in the development of validated assays for CSF proteins have the potential to (i) inform us as to the pathogenesis of disease, (ii) support the laboratorybased diagnosis for symptomatic subjects in the future, and (iii) facilitate breakthrough therapies to alter the course of neurodegenerative disorders, such as PD and Alzheimer s disease. Keywords: cerebrospinal fluid biomarker, confounding factors, dementia, neuropathology, Parkinson s disease, a- synuclein, b-amyloid, tau-protein. J. Neurochem. (2016) 139 (Suppl. 1), This article is part of a special issue on Parkinson disease. Parkinson s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are neurodegenerative disorders that feature characteristic aggregates of insoluble a-synuclein (a-syn) within neurons (as in PD, DLB) or oligodendrocytes (as in MSA) (Spillantini et al. 1997; Gai et al. 1998; Jellinger 2003). Encoded by the SNCA gene, a-syn is an abundant intracellular protein. A rising number (currently five) of missense mutations and SNCA gene multiplication events have been linked to heritable forms of parkinsonism, dementia, and autonomic dysfunction (Polymeropoulos et al. 1997; Singleton et al. 2003) (reviewed in Farrer 2006; Schlossmacher 2007). The presence of a-syn in extracellular, biological fluids has been previously shown, including in plasma, conditioned cell media, and interstitial fluid of the brain as well as in cerebrospinal fluid (CSF) (Borghi et al. 2000; El-Agnaf et al. 2003, 2006; Lee et al. 2008; Mollenhauer et al. 2008; Tinsley et al. 2010). Because of high levels of a-syn in peripheral blood cells and the frequent contamination of CSF with blood products, the actual source of a-syn that is present in CSF has been carefully explored; recently it was shown to be primarily derived from neurons of the central nervous system (Hong et al. 2010; Mollenhauer et al. 2012). The quantification of total CSF a-syn has been proposed as a biomarker for a-syn-related disorders. Several studies have found decreased mean levels of total CSF a-syn in PD, DLB, and MSA compared to neurological and healthy controls in independent single- and multicenter cohort analyses (Mollenhauer et al. 2008, 2013b; Hong et al. 2010; Kang et al. 2013). Across studies the mean levels of CSF a-syn in untreated, de novo PD subjects show a net decrease of 10 15% in comparison to controls. If a reduction in extracellular a-syn species (CSF and interstitial) in the brain was to be widely confirmed, it would be tempting to speculate that it was as a result of a rise in intracellular a-syn deposits during the course of the disease, thereby diminishing the rate of its already small degree of physiological release from intracellular compartments (see neuropathology section below). However, studies have shown that increased SNCA gene transcription rates as well as the misprocessing of a-syn within cells actually promote more secretion of full-length a- syn (Emmanouilidou et al. 2010; Danzer et al. 2012). Alternatively, but currently considered less likely, a net reduction in CSF a-syn could also represent a general risk marker for the development of PD. In contrast, it is widely agreed that significant neuronal (and synaptic) injury, such as occurs in prion disease and trauma, leads to a marked rise in CSF a-syn concentrations given its normally high intracellular concentration in mammalian brain (Mollenhauer et al. 2008). However, the published literature on CSF a-syn concentrations reveals great variability. Observed actual differences between patient groups are small (Tokuda et al. 2006; Mollenhauer et al. 2008; Hong et al. 2010) due to the profound overlap of individual values between diagnostic categories. Therefore, some authors have argued that a-syn is of little value as a biomarker for the (differential) diagnosis of parkinsonism (Aerts et al. 2012). In parallel, total and phosphorylated tau (t-tau and p-tau) and b-amyloid 1-42 (Ab42) are CSF biomarkers, which have been validated to reflect neurofibrillary tangle formation and amyloid plaque pathology in Alzheimer s disease (AD) brain, respectively. While both t-tau and a-syn release into the CSF and interstitial fluid are thought to at least partially reflect injury to the neuronal plasma membrane, several metabolites of the amyloid precursor protein (APP) including Ab42 are constitutively secreted under physiological conditions (Haass et al. 1992; Seubert et al. 1992). Thus, Ab42 s progressive reduction in CSF in patients is thought to reflect growing extracellular plaque deposition during the development and progression of AD (Bateman et al. 2012), and therefore, felt to be a process that is distinct from those mechanisms that promote the release of t-tau and a-syn (Mollenhauer et al. 2008; Toledo et al. 2013b). Nevertheless, these markers have also been investigated in PD. Several recent studies have suggested an early reduction in total CSF t-tau in PD, and a reduction in CSF Ab42 in those

3 292 B. Mollenhauer et al. PD cases that are associated with cognitive impairment (Siderowf et al. 2010; Alves et al. 2014). The reason for a decrease in CSF t-t-tau proteins in PD (vs. an increase in AD) remains unknown, but may be related to mechanisms other than neuronal injury (which is thought to underlie its rise in AD), and could be related to the reduction in CSF a-syn. For many of these CSF biomarkers, the overlap in values between healthy and diseased subjects may be attributable at least in part to a range of confounding factors. The following review addresses biological factors, summarizes our current understanding of the variability in CSF values for currently known, lead biomarker candidates, and speculates about which confounders need to be addressed in future studies. The lack of implementation of standardized operating procedures during the pre-analytical phase and technical confounders during the analytical phase, which can lead to CSF variability in select analytes, have been addressed elsewhere (Mollenhauer et al. 2010; del Campo et al. 2012). Known confounding variables Neuropathology to alter CSF a-syn levels Ideally, a disease-specific biomarker should reflect fundamental aspects of the underlying primary neuropathology or associated secondary changes. The major histopathological hallmarks of PD, PD with dementia (PDD) and DLB are a- syn positive intraneuronal inclusions known as a Lewy bodies (LB), Lewy neurites and albeit requiring more sensitive detection methods an abundance of small, presynaptic a-syn aggregates. However, a-syn aggregation disorders are neuropathologically heterogeneous. It has been estimated that at autopsy a-syn positive inclusions will be detected in more than 70% of patients with clinically assessed typical PD (Hughes et al. 2002). Furthermore, according to a recent study, pathological findings significant enough for the histologic diagnosis of AD were found coincidentally in 9% of PD cases at post mortem examination in subjects without any documentation of clinically recognized dementia (Irwin et al. 2012). Concurrent Ab pathology is increasingly recognized as an important factor of clinical variability in PD (Halliday et al. 2011). Detectable AD pathology is also a more common finding in PDD (Sabbagh et al. 2009; Irwin et al. 2012) and even more common in DLB brains compared to PD without dementia (Schoonenboom et al. 2012). On the other hand, a-syn positive inclusions have also been detected at autopsy in 8 17% of aged subjects (> 60 years) without any clear clinical symptoms of PD/DLB before death, which led to the creation of the term incidental Lewy body disease (Frigerio et al. 2011). Most biomarker studies so far have primarily used samples from clinically diagnosed patients based on neurological criteria rather than autopsy findings. The clinical diagnosis of PD is often imprecise, especially during early disease stages, as documented, for example, in recent clinicopathological studies, which demonstrated that 20 25% of PD patients eventually have a pathological diagnosis other than PD (Hughes et al. 1992; Jellinger 2008). Not surprisingly, diagnostic accuracy for PD intra vitam depends on the treating specialist and not surprisingly is consistently higher among movement disorder experts. Using neuropathologic findings of PD as the gold standard, Adler et al. (2014) reported only 26% accuracy for a clinical diagnosis of PD in untreated or not clearly responsive subjects, and only 53% accuracy in early PD responsive to medication (< 5 years duration). The presence of evidence for more than one coincidental neurodegenerative disease process is a common finding at post mortem examination (Jellinger 2008; Schneider et al. 2009; Tapiola et al. 2009; Jellinger and Attems 2010). The occurrence of mixed pathologies may, therefore, lead to clinical misdiagnosis, thus affecting CSF biomarker profiles in study participants (De Meyer et al. 2010). Similarly, Nelson and colleagues have shown that many DLB patients are falsely diagnosed as AD cases (Nelson et al. 2010). However, the specificity is high most of the subjects fulfilling clinical DLB criteria will indeed carry LB pathology in the brain. There is a high prevalence of AD-type pathology among subjects over 65 years old (Braak and Braak 1997). Compared to cortical LB pathology, where often few classical lesions are identified per field, AD-associated pathology may be microscopically more prevalent than routinely identifiable neuropathological hallmarks of DLB. Our ability to exclusively capture classical LB pathology in routine diagnosis carries with it the great risk of strongly underestimating the real amount of a-syn aggregation associated with the disorder. In cortical regions of DLB it has been shown that classical LB pathology accounts only for a small percentage of the total a-syn aggregate burden, with the vast majority of deposits being located pre-synaptically (Kramer and Schulz-Schaeffer 2007). Unfortunately, these pre-synaptic a-syn aggregates frequently evade recognition by conventional immunohistochemical methods, because most antibodies do not differentiate physiological a-syn, abundantly present in the nervous system, from its pathological aggregates. Pre-synaptic a-syn aggregates not only represent the most abundant type of a- syn pathology in DLB but also in cases of PD and PDD (Schulz-Schaeffer 2010). In addition, recent data strongly indicate that classical LB (and Lewy neurite) pathology seen in PD is not concordant with the degree of neuronal impairment (Milber et al. 2012; Schulz-Schaeffer 2012). Thus, pre-synaptic a-syn aggregates are considered by many to be a more sensitive and better correlation marker of functional deficits (Kramer and Schulz-Schaeffer 2007). However, their quantification in CSF specimens is still in the very early stage of assay development (Tokuda et al. 2010; Hansson et al. 2014);

4 Biological confounders for the values of CSF proteins in PD 293 nevertheless, these species could potentially impact the preclinical assessment of subjects, as has been shown in asymptomatic LRRK2 mutation carriers (Aasly et al. 2014). Efforts are also underway to test them in patients with idiopathic rapid eye movement sleep behavior disorder (Compta et al. 2015). At this time, and without the benefit of routine intra vitam or post mortem brain examination in subjects clinically diagnosed with PD, the absence or presence of concomitant a-syn pathology cannot be definitely confirmed, and thus the possibility of misdiagnosis cannot be ruled out. It is therefore of great importance that the clinical value of any new biomarker candidate (and the method for its quantification) is carefully validated in neuropathologically confirmed cases. To our knowledge, the publication of multiple studies evaluating CSF biomarkers in patients with neuropathologically confirmed PD is still lacking. Only two studies have included confirmed PDD patients (Engelborghs et al. 2008; Tapiola et al. 2009). Moreover, most of these studies encompass only a limited number of subjects ranging from 1 to 7; only 4 of these studies have more than 10 subjects (Koopman et al. 2009; Mollenhauer et al. 2010; Brunnstrom et al. 2013; Slaets et al. 2013). To date, two reports have assessed total CSF a-syn levels in autopsy-confirmed DLB patients (Mollenhauer et al. 2008, 2011b). It is important to note that neuropathological examination is conducted more often when patients present with an atypical clinical course. Thus, published studies of DLB cases have to be interpreted with caution, because they may also contain a sampling bias as a potential confounder. Of note, and as is to be expected, in the case of ongoing and widespread cell death CSF a-syn will rise dramatically, as was shown in eight Creutzfeldt Jakob disease autopsies to date (Mollenhauer et al. 2008). What is also currently missing is a detailed analysis of CSF a- syn values for overall rare heritable forms of PD, with either early or late onset, particularly in confirmed carriers of a mutation in one of the two a-syn-encoding SNCA alleles. In dominantly inherited AD, detailed laboratory studies have greatly informed us as to the course of blood and CSF biomarker behaviors from the pre-symptomatic stage, to mild cognitive impairment, to fully penetrant dementia (Bateman et al. 2012). A summary of studies assessing classical AD biomarkers in the CSF of PD patients can be found in Tables 1 and 2. A summary of published studies comparing neuropathological findings and CSF biomarkers in PD, PDD, DLB, and AD can be found in Table 3. Effects of demographics (age and gender) A sex effect on CSF proteins? Too early to tell There is a known predominance in the overall prevalence of PD and DLB in men, although for some of its subtypes, such as mutant LRRK2-linked cases, women appear to be more affected (at least in some ethnicities). Only a few studies have addressed any associations between sex and CSF a-syn levels in patients with a-syn-related disorders. In these studies, no significant effect by gender was found when healthy aged controls were compared with PD subjects (Tokuda et al. 2006; Hong et al. 2010), when DLB patients were compared to AD cases (Noguchi-Shinohara et al. 2009; Kasuga et al. 2010), and when DLB patients (n = 16) were compared to both AD (n = 18) and healthy control subjects (n = 22) (Kapaki et al. 2013). Conversely, the results were influenced by sex in two studies. In the first one it was reported that only female (but not male) DLB patients displayed lower levels of CSF a-syn compared to AD patients and controls(wennstrom et al. 2012). Women with DLB demonstrated 40% less CSF a-syn levels than both female controls and female AD patients, where levels were highly similar. Results of another study by the same group showed that sex appeared to be linked to a-syn levels in patients with PD/PDD (Wennstrom et al. 2013). The authors investigated CSFlevels for total a-syn and neurosin(suggested to be an a-syn cleaving enzyme) in patients diagnosed with DLB (n = 33),PD(n = 38),andPDD(n = 22)versusthosewithAD (n = 46) and 52 non-demented controls. There, lower CSF levels of a-syn in patients with a-syn aggregation disease and higher levels in AD were found, which may reflect different pathological events, such as intracellular aggregation (and less release into the extracellular environment) in the former and increased cell death in the latter (Mollenhauer et al. 2008, 2011b;Honget al.2010;toledoet al.2013a).inthepdgroup, female patients demonstrated higher a-syn levels than male patients ( vs pg/ml, p = 0.029). In the PDD group the results were reversed: higher levels were shown in male versus female patients ( vs pg/ ml, p = 0.043). Furthermore, when comparing females and males, across the diagnostic groups female AD patients (n = 35; pg/ml) significantly differed from female DLB patients (n = 18; pg/ml, p = 0.004) and from PDD patients (n = 6, pg/ml, p = 0.005). When comparing male subjects across the groups, AD patients (n = 11; pg/ml) differed from PD patients (n = 20; pg/ml, p = 0.001) and subjects with PDD (n = 16; pg/ml, p = 0.033) but not from subjects with DLB (n = 15; pg/ml, p = 0.253). Taken together, there is currently not enough evidence to suggest that sex functions as a confounder when assessing total CSF a-syn levels in patients with a-syn aggregation disorders; if present, any sex effect has yet to be determined in larger cohort-studies with comparable numbers of women and men enrolled. Age as a possible confounder of CSF a-syn levels The published results regarding an age dependence of CSF total a-syn levels in patients with PD and controls have yet to be reconciled (Mollenhauer et al. 2010): A decrease in CSF a-syn with progression in age was first reported in 33 PD patients and 38 control subjects (Tokuda et al. 2006).

5 294 B. Mollenhauer et al. Table 1 Studies assessing CSF AD classical biomarkers in PD Study Patients CSF Ab42 CSF total tau CSF phospho-tau Comments Sjogren et al. 15 PD, 19 AD, 14 (2002) FTD, 11 ALS, 17 HC Bibl et al. (2006) 23 AD, 21 DLB, 21 PDD, 23HC Mollenhauer et al. (2006b) Mollenhauer et al. (2007) Parnetti et al. (2008) Zhang et al. (2008) Compta et al. (2009) Siderowf et al. (2010) Alves et al. (2010) Montine et al. (2010) Prikrylova Vranova et al. (2010) Leverenz et al. (2011) 23 PD, 73 PDD, 41 HC 15 AD, 20 PSP, 12 CBD, 20 DLB, 11 PD, 18 MSA, 19 HC 20 PD, 18 PDD, 19 DLB, 20 HC 40 PD, 48 AD, 95 HC 20 PD, 20 PDD, 30 HC Longitudinal study (1 year f/u): 45 PD, no HC 109 de novo untreated PD, 36 HC 35 HC 50 year, 115 HC > 50 year, 24 amci, 49 AD, 49 PD, 69 PDCIND, 11 PDD in AD & ALS, in FTD & PD Ab42 /37 in AD versus HC Ab1 40* in DLB versus PDD in PDD versus PD in AD, PSP, CBD, and DLB in DLB > PDD > PD > HC in AD N/A in PDD versus PD in AD Slight in PD and CBD versus HC DLB > PDD > PD No differences between PD and HC in AD N/A N/A N/A in PD in AD N/A No difference No correlations with age, disease duration, H&Y, MMSE Positive correlation with MMSE No correlation with age or disease duration Ab42 associated with performance at Word Fluency and Prose Memory tests in PD in PDD > PD > HC in PDD versus PD & HC in PDD versus PD & HC Ab42 associated with Verbal Fluency; tau associated with worse memory, visuospatial functions, naming, and advanced age Unchanged Unchanged Unchanged baseline Ab42 associated with faster cognitive decline ( Ab42 independent predictor of cognitive decline) in PD No difference No difference Ab42 associated with memory impairment in PDCIND & PDD versus PD &HC No difference No difference 32 PD, 30HC PD = HC in PD Not measured clusterin levels; t-tau, t-tau/ab42 and clusterin associated with disease duration 22 PD, no HC Unchanged Unchanged Not measured Ab42 and/or Ab42/t-tau associated with performance at Digit Symbol and Category Fluency tests

6 Biological confounders for the values of CSF proteins in PD 295 Table 1 (continued) Study Patients CSF Ab42 CSF total tau CSF phospho-tau Comments Parnetti et al. (2011) Shi et al. (2011) Andersson et al. (2011) Prikrylova Vranova et al. (2012) Bech et al. (2012) 38 PD, 32 DLB, 48 AD, 31 FTD, 32 HC 126 PD, 32 MSA, 50 AD, 137 HC PD-HC PD = HC PD = HC T-tau & p-tau associated with age & disease duration; Ab42 associated with higher age slightly in PD& MSA versus HC 17 PDD, 47 DLB in DLB versus PDD 48 PD without dementia (17 EDO, 15 TD, 16 NT), 18 AD, 19 HC 10 MSA, 10 PSP, 11 DLB, 22 PD (2 PDD), No HC Nutu et al. (2013) 90 PD, 32 PDD, 68 DLB, 48 AD, 45 PSP, 46 MSA, 12 CBD Compta et al. (2013) Slight in NT PD in DLB versus other groups 27 PD in dementia converters Alves et al. (2013) 99 de novo PD (39 PIGD, 60 TD), 46 HC Alves et al. (2014) 104 PD with 5 year follow-up Stav et al. (2015) 21 early PD, 28 MCI non-pd, 34 NC Kang et al. (2013) 63 de novo PD 39 HC in PD versus HC in PD versus HC Fractalkine /Ab42 ratio associated with H&Y & UPDRS. t-tau & p-tau associated with advanced age in DLB PDD No difference total tau/ab42 in PD NT No difference N/A No difference Tau-protein associated with disease severity // // // Lower levels of Ab15/ 16 detected in PD, PDD, PSP and MSA groups in PIGD-PD versus TD-PD and HC CSF levels at PD diagnosis in PD compared to NC, but above cut-off for AD No significant differences between dementia converters and not converters No significant differences between dementia converters and not converters // // PIGD phenotype also showed Ab38, Ab42/ 40 and Ab38/40 levels versus TD-PD and HC No significant change from normal levels in PD than in NC and in MCI non- PD No significant change from normal levels in PD than in NC and in MCI non- PD Ab42 predicted a substantially risk for subsequent dementia at high sensitivity (85%), with hazard ratios of 9.9 (95% confidence interval) Lower Ab42 was associated with reduced cognitive functions in early PD Subgroup of multicenter PPMI study PD, Parkinson s disease; PDD, Parkinson s disease dementia; DLB, dementia with Lewy bodies; HC, healthy controls; MSA, multiple system atrophy; PSP, progressive supranuclear palsy; CBD, corticobasal degeneration; TD, tremor dominant; PIGD, postural instability and gait disorder; EDO, early disease onset; NT, non-tremor dominant; AD, Alzheimer s disease, ALS, amyotrophic lateral sclerosis; FTD, frontotemporal dementia; MMSE, mini mental state examination; H&Y, Hoehn Yahr scale; UPDRS, unified Parkinson s disease rating scale; a MCI, amnestic mild cognitive impairment; Ab42, b-amyloid 1 42; Abxy, b-amyloid 1-xy; t-tau, total tau-protein; p-tau, phosphorylated tau-protein; MCI, mild cognitive impairment; PPMI, Parkinson Progression Marker Initiative.

7 296 B. Mollenhauer et al. Table 2 Other large cross-sectional cohorts Study Cases Protein Method Remarks Age Mattsson et al. (2012) 529 AD dementia (median age 71, range years), 304 HC (67, years), and a longitudinal cohort of 750 MCI (69, years) followed for at least 2 years, or until dementia diagnosis Ab42, total tau, p-tau at 181 ELISA (Innotest â, Innogenetics, Ghent, Belgium) or Luminex xmap â (Inno-Bia AlzBio3 â, Innogenetics) Diagnostic accuracies for AD with age, but the predictive values for a combination of biomarkers remained essentially stable Age correlated with t-tau (r = 0.22, p < 0.001) and p-tau (r = 0.23, p < 0.001) in HC, and with Ab42 SMCI (r = 0.23, p < 0.001). The main source of variation was diagnosis rather than age AD, Alzheimer s disease, HC, healthy controls, MCI, mild cognitive impairment, SMCI, stable mild cognitive impairment; Ab42, b-amyloid 1 42; t-tau, total tau-protein; p-tau, phosphorylated tau-protein. Similarly, an inverse relation between CSF a-syn levels and age was reported in another study (PD/DLB, n = 57) (Mollenhauer et al. 2008). In contrast, two studies demonstrated a positive correlation between total a-syn levels and age comprising 132 healthy controls and 117 patients with PD in one report, and 107 controls and 90 PD subjects in another study (Hong et al. 2010; Hall et al. 2012). According to Hong et al. (2010), CSF a-syn levels were significantly increased as a function of age in normal controls. However, the age dependence became weaker in patients with PD. In addition, an age-dependent increase in CSF levels of phosphorylated a-syn was recently reported in 126 healthy controls (aged years; p < 0.05; r = 0.22) (Wang et al. 2012). However, the observed age dependence disappeared when subjects younger than 50 years (n = 51) were excluded from the analysis. To complicate things further, subsequent studies have revealed that there was no change in CSF a-syn with age in neurologically healthy controls and PD patients (van Geel et al. 2008; Ohrfelt et al. 2009; Reesink et al. 2010; Parnetti et al. 2011; Kapaki et al. 2013). Thus, either age plays an insignificant role in the concentration of CSF a-syn levels, or other confounding factors such as cohort size and variables not yet accounted for in these studies are likely to underlie these inconsistencies. Age as a confounder of CSF b-amyloid and tau-levels in PD and DLB The majority of published reports found no correlation between the levels of CSF Ab42 and age in PD patients (Mollenhauer et al. 2006b, 2007; Compta et al. 2009; Prikrylova Vranova et al. 2010; Reesink et al. 2010; Beyer et al. 2013) whereas an inverse correlation with age was reported by Parnetti and colleagues (Parnetti et al. 2011). The published literature also reveals conflicting findings regarding an age dependence for other classical CSF biomarkers, that is, total tau protein and phosphorylated tau, in PD patients (Mollenhauer et al. 2006b; Prikrylova Vranova et al. 2010; Reesink et al. 2010). Significant associations between CSF total tau and phosphorylated tau with age were reported by some investigators (Compta et al. 2009; Parnetti et al. 2011; Beyer et al. 2013) although similar to the issue of CSF a-syn no consistent pattern has yet emerged (Mollenhauer et al. 2006b; Prikrylova Vranova et al. 2010; Reesink et al. 2010). Concerning patients with DLB, in most of the published investigations no correlation was found between age and CSF Ab42 levels (Gomez-Tortosa et al. 2003; Mollenhauer et al. 2005b, 2007; Bibl et al. 2007a, 2010; Wada-Isoe et al. 2007; Mulugeta et al. 2011; Brunnstrom et al. 2013). However, according to one study (Schoonenboom et al. 2012), CSF Ab42 levels decrease with increasing age in DLB patients (as they do in subjects complaining about subjective memory dysfunction). How does one explain these rather variable results regarding the association in DLB between CSF Ab42 and t-tau levels and the age of subjects? Indeed, decreased CSF levels of Ab42 and increased levels of total tau and phosphorylated tau have been validated and as of today serve as widely accepted biomarkers of AD (for review, see e.g., Mattsson et al. 2009; Blennow et al. 2010). A correlation between reduced CSF Ab42 and cognitive impairment has also been proposed for PD (Alves et al. 2010; Montine et al. 2010; Siderowf et al see also below). Thus, while AD-like CSF pathology is seen in PD patients with cognitive impairment and older age at disease onset (Halliday and McCann 2010; Irwin et al. 2012), it seems to be even more prevalent in DLB. As outlined above (in the section on neuropathology), DLB frequently represents a mixed type of dementia with both a-syn inclusion-positive and AD-like pathology (Armstrong et al. 2000; Edison et al. 2008) that typically presents in the 8 th decade of life (James et al. 2012). In accordance, results of inverse correlations between age and Ab42 in some PD/PDD cases and

8 Biological confounders for the values of CSF proteins in PD 297 Table 3 Summary of published studies comparing neuropathological findings and CSF biomarkers in PD, PPD, DLB, and AD Study Cases Protein measurements Method Neuropathology Remarks Beyer et al. (2013) Slaets et al. (2013) Le Bastard et al. (2013) Toledo et al. (2012) Schoonenboom et al. (2012) 91 PD Ab38, Ab40, Ab42, t-tau and p-tau 18 autopsy-confirmed DLB 66 autopsy-confirmed dementia patients (51 AD and 15 non-ad) and 95 C 142 autopsy-confirmed dementia (71 AD, 29 FTD, 3 ALS, 7 DLB, 32 of which cases also had coincident diagnoses) 512 AD, 272 OD, 135 PSY, 275 SMC 52 DLB, 34 VaD), 16 CBD, 20, PSP Autopsy obtained in a subgroup of 17 patients Ab42, t-tau, p-tau181p Ab42, t-tau, p-tau181p Ab42, t-tau, p-tau181p Ab42, t-tau, p-tau181p Abeta triplex assay (Human Abeta peptide Ultra-Sensitive Kit; Meso Scale): Innotest htau-ag and Innotest p-tau (181P), Innogenetics ELISA (Innotest, Innogenetics) A multi-analyte Luminex assay (INNO-BIA AlzBio3), a single-analyte ELISA tests (Innotest) 96% (ELISA) and 77% had Luminex, with 43 and 46 C for comparison, respectively ELISA (Innogenetics) Postmortem pathologic examinations of the ParkWest cohort are planned In 14 DLB + senile plaques: significantly CSF Ab42 concentrations versus 5 DLB patients 1 senile plaques but not compared to AD No differences in CSF t-tau or p-tau181p concentrations in 9 DLB + NFT versus 9 DLB-NFT Ab42 + p-tau181p: optimal discrimination of AD and non-ad patients (diagnostic accuracy = 0.86) No pathologically confirmed controls available Clinical and NPDG diagnoses: 81.4% overall agreement. NPDG AD plus another pathology: 26.8% of the sample, 69.4% (ELISA), 96.4% (Luminex) were classified as AD according to biomarker profiles. Clinical instead of NPDG: 14 17% underestimation of the biomarker accuracy CSF Ab42 + p-tau: a correct classification in AD (92%) and in OD (66%). Concordance between clinical and NPDG: 85%. CSF markers reflected neuropathology in 94% Association between CSF Abeta measures and structural brain changes in newly diagnosed drugna ıve PD patients Concomitant amyloid pathology in DLB limits the use of CSF Ab42 for the differential diagnosis of AD versus DLB Age should be considered as a possible confounding factor CSF Ab and tau assays are useful for diagnosis of AD even at MCI stages DLB and FTD must be established based on NPDG rather than clinical diagnoses, diagnostic CSF panels needed Slightly CSF levels Ab42 and p-tau levels in DLB Limited number of NPDG available

9 298 B. Mollenhauer et al. Table 3 (continued) Study Cases Protein measurements Method Neuropathology Remarks Brunnstrom et al. (2013) To investigate correlations between CSF biomarker levels and histopathological findings, with a focus on concomitant AD pathology, in NPDG verified DLB cases Mollenhauer et al. (2011b) To assess whether reduction of a-syn in CSF was a marker for a-syn deposition in the brain, and therefore diagnostic of synucleinopathies Mollenhauer et al. (2011a) To compare absolute and relative quantities of CSF Abpeptides with a larger cohort of clinically diagnosed patients with probable AD, probable DLB, and non-demented controls 16 DLB Ab42, t-tau, p-tau181p CSF, in vivo 41 autopsy-confirmed DLB (n = 13), AD, or OND Neuropathologically defined patients with 11 definite AD; dad and 12 definite; ddlb A larger cohort of clinically diagnosed patients with 71 probable AD; pad; 32 probable DLB; pdlb; and 71 NDC Total a-syn and total tau Ab-peptides (Abxy%) T-tau: ELISA constructed to measure both normal and hyperphosphorylated tau P-tau: ELISA constructed to specifically measure tau phosphorylated at Thr181. Ab42: ELISA constructed to specifically measure b-amyloid ELISA The quantitative Ab-SDS-PAGE/ immunoblot Both Braak and PPAD9 stages inversely correlated with Ab42 levels CERAD stage: no significant correlations. Cerebral amyloid angiopathy correlated positively with t-tau and t-tau/ Ab42 ratio, and inversely with Ab42 levels but a very heterogeneous extent of cerebral amyloid angiopathy present CSF a-syn levels in all definite DLB versus AD and OND. No correlation between CSF a-syn values in DLB and frequency of LB in the cortex. In 41 autopsy-confirmed cases CSF a-syn discriminated between DLB and AD (p = ; AUC = 0.687) CSF Ab-peptides (Ab x%): similar relative distribution in NPDG and clinically defined groups. Ab42% in dad versus NDC (p = ), but no difference in dad versus pad. Ab40 ox% in ddlb versus NDC (p = ), but no difference in ddlb versus pdlb absolute value of each Ab peptide in ddlb versus pdlb; most pronounced for Ab42 (p = ), non-significant for the other peptides Significantly levels of Ab42% in ddlb versus pdlb The burden of concomitant Alzheimer pathology correlates with CSF Ab42 but not with t-tau levels in cases with neuropathologically defined DLB Significantly mean CSF a-syn concentrations as measured by ELISA in PD, DLB, and MSA than in OND The CSF Ab42% and Ab40ox%: useful as diagnostic biomarkers for AD and DLB, respectively. Confirmed Ab peptide patterns in NPDG patients with AD and DLB

10 Biological confounders for the values of CSF proteins in PD 299 Table 3 (continued) Study Cases Protein measurements Method Neuropathology Remarks Andersson et al. (2011) To investigate CSF biomarkers and the associated cognitive profile in DLB and PDD Le Bastard et al. (2010) To study whether CSF biomarkers could have helped the clinician in differential dementia diagnosis in case of clinically ambiguous diagnoses, as compared to autopsy-confirmed dementia diagnosis as gold standard Brunnstrom et al. (2010) Tapiola et al. (2009) To study the relationship between antemortem CSF biomarker levels and AD neuropathological changes in the brain Koopman et al. (2009) Clinically diagnosed 47 DLB (one DLB with autopsy) and 17 PDD patients 22 autopsy-confirmed dementias (15 AD, 1 MXD, 2 VAD, 1 FTD, 2 DLB and 1 CJD in population totaling 157 patients with an ambiguous clinical diagnosis at CSF sampling 43 patients with a clinical dementia disorder underwent autopsy (8 AD, 7 DLB, 12 FTD, 5 VaD and OD) 79 with clinically diagnosed AD, (71 probable AD, 8 possible AD)? neuropathologically 1 DLB (among 29 clinical other dementia; 3 DLB, 2 PDD)? neuropathologically 1 DLB, 1 PDD and 15 OND) 95 definite AD and 50 non-ad dementias (18 DLB, 10 FTD, 6CJD, 16 VaD Ab42, t-tau, p-tau181p t-tau and Ab42 Ab42, t-tau, p-tau181p Ab42, t-tau, p-tau181p Antemortem lumbar CSF samples Neuropathologic evaluations included the classic silver impregnation method and IHC for Ab, hyperphosphorylated tau, and a-syn Single-parameter ELISA kits Ab42 and t-tau concentrations: AD = NON-AD. P-tau181P concentrations: significantly in AD versus NON-AD The biomarker-based diagnostic model correctly classified 18 of 22 (82%) patients with clinically ambiguous diagnoses In 8 patients (5 with AD had t-tau and Ab42), while both values for the other three patients were normal Slightly t-tau and/or Ab42 were also seen in several patients with OD such as DLB, VaD and FTD Inverse correlation of CSF Ab42 levels with total Ab load in the brain. Correlation of CSF tau levels with results HP-tau and with the presence of neocortical NFT The number of NP in he brain remained a significant predictor of CSF Ab42 level and of CSF tau level. The ratio of ptau/ab42 showed S = 91.6%, Sp = 85.7%, with an overall accuracy of 90.2% for the presence of pathologic NP in the brain No neuropathological confirmations of diagnosis have been obtained (except for one DLB patient where the diagnose was confirmed) A correct diagnosis would have been established in the majority of autopsyconfirmed AD and NON-AD cases, indicating that biomarkers have an added diagnostic value in cases with ambiguous clinical diagnoses There is no perfect concordance between CSF levels and pathological findings, which should be taken into account in the clinical diagnostic setting CSF Ab42 and tau protein levels are related to amyloid load and the presence of NFT in the brain. Limited number of patients: definite conclusions about the behavior of CSF biomarkers in association with other pathologic changes in the brain, not possible Limited statistical validity: the sample size of the pooled non-ad group (+ limited individual non-ad categories) was about half of the size of the AD group

11 300 B. Mollenhauer et al. Table 3 (continued) Study Cases Protein measurements Method Neuropathology Remarks Engelborghs et al. (2008) Mollenhauer et al. (2008) Mukaetova- Ladinska et al. (2008) Buerger et al. (2006) To study whether CSF levels of p-tau reflect neurofibrillary changes within the brain 100 autopsy-confirmed dementia (46 AD, 9 MXD (n = 9), 3 FTD, 1DLB, 6 PDD, 7 CJD, 1 CJD (Heidenhain variant), 5 VaD, 1 NPH) and 100 C; without autopsy) 8 PD, 38 DLB (one autopsy), 13 AD, 8 CJD, 13 NCO (a group of nonneurodegenerative disease controls) 25 CSF postmortem ventricular samples (8 NDC, 3 VaD, 9 AD, 2 DLB, 2 PD, 1 PDD) 26 AD patients, with intra vitam CSF as well as postmortem neuropathological data Ab42, t-tau, p-tau181p as a-syn P-tau phosphorylated at threonine 231 (p-tau231p) Commercially available singleparameter ELISA kits (respectively, Innotest â _- Amyloid (1 42), Innotest â htau Ag, Innotest â Phosphotau (181P); Innogenetics) A built, validated and optimized sandwich-type, enzyme-linked immunoadsorbent assay (ELISA) to measure total as levels in unconcentrated CSF Dementia discriminated from C with S = 86%, Sp = 89%. T-tau and Ab42, optimally discriminated AD from NON-AD and C (S = 90%, Sp = 89%). AD discriminated from NON- AD using p-tau181p and Ab42 (S = 80%, Sp = 93%) Diagnostic accuracy of the latter model (82.7%) was comparable to clinical diagnostic accuracy (81.6%) levels of a-syn in CSF in AD, DLB and VaD versus NDC. Syn more in DLB and VaD. a-syn from Braak stage III onwards and stable until Braak stage VI. These results were not influenced by age at death or postmortem delay. All DLB patients had neocortical LB present, and cortical LB score, modified for syn ranged from 9 to 16 CSF levels of p-tau231p associated with NFT count and HP-tau load (significant correlations between CSF p-tau231p concentrations and scores of NFTs and HP-tau load) within all neocortical regions, such as frontal, temporal and parietal lobe, but not in the allocortical CA1 region of the HP Correlation of the scores of NPs with CSF p-tau231p only within the frontal cortex Using this model, in cases with clinically doubtful diagnoses, a correct diagnosis would have been established in 4/6 autopsy-confirmed AD and 3/3 autopsyconfirmed Non-AD cases. The value of biomarkers in differential dementia diagnosis was shown, using pathological diagnosis as a reference A first feasibility study employing a novel ELISA. CSF as concentrations in subjects with parkinsonism linked to synucleinopathy, PD, and DLB in a-syn in the ventricular CSF of individuals with dementia is a novel finding. Further studies needed to investigate whether using these markers may help to discriminate DLB from OD The findings for early stages still remain to be studied CSF p-tau231p may serve as an in vivo surrogate biomarker of neurofibrillary pathology in AD

12 Biological confounders for the values of CSF proteins in PD 301 Table 3 (continued) Study Cases Protein measurements Method Neuropathology Remarks Mollenhauer et al. (2005a) Mollenhauer et al. (2005b) Clark et al. (2003) To correlate antemortem CSF tau and Ab levels with definitive dementia diagnosis Strozyk et al. (2003) To investigate the relationship of amyloid neuropathology to postmortem CSF Ab42 levels in an autopsy sample Tschampa et al. (2001) 21 DLB, 19 AD in 2 AD cases confirmation by autopsy 71 DLB, 67 AD, 41 COND, in 2 DLB cases clinical diagnosis confirmed by autopsy 103 patients with dementia, 3 DLB, 73 COND CSF obtained from the ventricles at death in dementia patients Autopsy-confirmed 11 AD, 5 DLB, 17 COND Ab42, Ab40, t-tau and p-tau 181P t-tau and Ab42 t-tau and Ab42 A set of commercially available assays A set of commercially available assays Innotest htau- Ag; Innogenetics NV, ELISA developed by Suzuki et al., 1994 Only a minor comment about the autopsy Patients, fulfilling all three-core criteria (DLB- III), have tau levels average tau value for the LB + AD than the value for patients with AD-LB. The AD + LB patients have NFT and more amyloid plaques than patients with AD-LB number of NP in the neocortex (p = 0.001) and in the hippocampus (p = 0.003) was strongly associated with levels of Ab42. Pathologically confirmed AD had Ab 42 levels (coefficient 0.74; 95% CI 1.4, 0.1) compared to clinical dementia CSF tau levels: AD = DLB, but than in HC. No correlation of the NFT density with tau levels CSF tau levels are associated with AD pathology and can help discriminate AD from other dementias. However, some patients with AD have a level less than the mean 2 SDs of the HC cohort There is a wide range of tau pathology in DLB a-syn, alpha synuclein; AD, Alzheimer s disease; ALS, amyotrophic lateral sclerosis; C, Controls; CJD, Creutzfeldt Jakob s disease; CBD, corticobasal degeneration; COND, controls with other neurological diseases; DLB, dementia with Lewy bodies; FTD, frontotemporal dementia; HC, healthy controls; HD, Huntington s disease; HP, hippocampus; LB, Lewy body; MSA, multiple system atrophy; MXD, mixed dementia; NDC, non-demented controls; NFT, neurofibrillary tangles; NP, neuritic plaques; NPDG, neuropathological diagnosis; NPH, normal pressure hydrocephalus; OD, other dementias; PD, Parkinson s disease; PDD, Parkinson s disease dementia; PFP, peripheral facial palsy; PND, peripheral neurological disease; PPAD9, poly-pathology Alzheimer s disease assessment; nine areas; PSP, progressive supranuclear palsy; PSY, patient with psychiatric disorder; S, sensitivity; Sp, specificity; SC, patients with subjective complaints; SMC, subjective memory complaint; SCA, spinocerebellar ataxia; SSP, spastic spinal palsy; VaD, vascular dementia; Ab42, b-amyloid 1 42; CSF, cerebrospinal fluid, ddlb, definite DLB, pdlb, probable DLB; t-tau, total tau-protein; p-tau, phosphorylated tau-protein; OND, other neurological diseases; SD, standard deviation. particularly in DLB appear to be the result of concomitant AD pathology (Mollenhauer et al. 2011b), as does the positive correlation between age and CSF t-tau/phosphorylated tau levels in some of these patients. A summary of studies on age and sex in relation to CSF markers in PD, PDD, and DLB is given in Table 4. Clinical variants of PD based on cognitive changes differential diagnosis Cognitive impairment is more common in PD (> 30%) than in the general population even during early stages of the disease (Aarsland et al. 2009; Aarsland and Kurz 2010). The risk of dementia is approximately 80% for those PD patients with more than 20 years of disease duration (Aarsland et al. 2003; Hely et al. 2008). Increased levels of total and phosphorylated tau, and decreased levels of Ab42 characterize the CSF AD profile (Hansson et al. 2006). Siderowf et al. (2010) showed that, in addition to these clinical changes, reduced CSF Ab42 represents an independent predictor of cognitive decline in patients with PD, as was recently confirmed by Alves et al. (2014). The link between CSF Ab42 levels and cognitive decline in PD has been analyzed in follow-up studies by others

13 302 B. Mollenhauer et al. (Compta et al. 2009; Alves et al. 2010; Montine et al. 2010; Siderowf et al. 2010; Leverenz et al. 2011; Parnetti et al. 2014). Compta and co-workers found lower levels of CSF Ab42 and increased total and phosphorylated tau levels in PDD compared to controls and compared to PD subjects without dementia. The same study demonstrated that higher concentrations of CSF total tau and phosphorylated tau in PDD patients were associated with impaired memory along with naming and visuospatial deficits, whereas reduced Ab42 levels were correlated with impaired phonetic and semantic verbal fluency (Compta et al. 2009). A significant correlation between decreased Ab peptide CSF levels (Ab42, Ab40 and Ab38) and memory impairment was observed in a recent cohort study of de novo subjects with PD without detectable dementia [109 patients; mean mini mental status examination (MMSE) of 27.8]; it included 36 age-matched controls (mean MMSE of 29) and 20 patients with mild AD (mean MMSE of 24.4). No association with executive and visuospatial functions was found in the PD subjects (Alves et al. 2010). In another study (Leverenz et al. 2011), an association between lower CSF Ab42 levels and Ab42/t-tau ratio with worse performance on the Digit Symbol test was observed in 22 non-demented PD patients. These results reinforce the role of incipient, concomitant AD pathology in the development of dementia in PD patients. Recent reports of altered in vivo amyloid imaging in PDD patients are consistent with this interpretation (Gomperts et al. 2013). A prospective study of 45 PD patients with a 1-year follow-up period demonstrated that lower levels of Ab42 at baseline were associated with a more rapid cognitive decline, particularly in attention, ability to conceptualize and memory performance (Siderowf et al. 2010). Importantly, this study provided evidence for a role of low Ab42 in the prediction of cognitive decline in PD patients. Similarly, a prospective study carried out in 44 PD patients and 25 neurological controls showed that low CSF Ab42 levels were associated with a higher rate of decline in MMSE and Montreal Cognitive Assessment scores, confirming its role as independent predictive factor for cognitive decline in PD (Parnetti et al. 2014). Under these circumstances, a reduction in CSF a-syn probably does not add any value to the issue of cognitive change in these patients. However, a marked rise in it would point to an alternate diagnosis, such as a fulminant neurodegenerative process. It has also been shown that not only CSF Ab42 reduction, but also a rise in a-syn, might predict future cognitive and motor impairment in PD (Stewart et al. 2014; Hall et al. 2015); it indicates that a-syn levels may not only be controlled by its dysregulation inside neurons (e.g., Lewy bodies), therefore possibly contributing to lower extracellular CSF levels, but could also reflect ongoing synaptic degeneration. Of note, it has been shown that CSF a-syn is increased in, for example, Creutzfeldt-Jakob disease and AD, as well as in PD subjects with progressive cognitive decline (Hall et al., 2015) and worsening motor performance (Kang et al., 2013). Therefore, the diagnostic utility of total CSF a- syn (as a single marker) is limited. When it is reduced below the mean it is currently thought to reflect the chronic disease process that underlies PD, DLB and MSA. When it is markedly increased above the mean it is believed to occur as a result of severe synaptic injury. Progression, disease duration, and endophenotypes as confounders Several studies have explored CSF levels of a-syn and AD biomarkers in PD with regard to correlation between analyte levels and disease severity (Mollenhauer et al. 2006a, 2008, 2011b; Tokuda et al. 2006, 2010; Ohrfelt et al. 2009; Hong et al. 2010; Shi and Zhang 2011; Prikrylova Vranova et al. 2012; Wang et al. 2012) as well as disease duration (Prikrylova Vranova et al. 2010; Tokuda et al. 2010; Parnetti et al. 2011; Wang et al. 2012). Prikrylova reported higher CSF t-tau in non-tremordominant PD patients (n = 32) versus controls (n = 30), suggesting that the most significant neurodegenerative changes in PD may occur in the initial stage of the disease (Prikrylova Vranova et al. 2012). Alternatively, and currently less debated, is the possibility that an early dysregulation of total CSF tau reflects a risk marker for the development of PD. It is important to note, that the strong association of single nucleotide polymorphisms at the microtubule-associated protein tau locus (which encodes human tau isoforms) in genome-wide association studies of idiopathic PD (Simon-Sanchez et al. 2009) has not yet been adequately explained in pathogenetic concepts of the disorder. On the other hand, Parnetti et al. (2011) reported an association of total CSF tau and phosphorylated tau with longer disease duration. But there were no differences between the levels of CSF Ab1 42, CSF t-tau, and CSF phosphorylated tau in PD (n = 38) versus controls (n = 32). In their study, the levels of CSF total a-syn were found to be reduced in PD, but did not correlate with disease severity, as measured by Hoehn and Yahr staging. Hong et al. (2010) suggested that there might be a floor effect and CSF biomarkers may be altered more significantly during early stages of the disease; that is, correlating better with early PD and its progression. Prikrylova Vranova et al. (2012) reported a distinct profile of CSF neurodegenerative markers (other than CSF a-syn) for various endophenotypes of PD in a cohort of 48 subjects. Non-tremor-dominant PD patients showed significantly more elevated levels of CSF total tau and of the index t-tau/ab42 than other parkinsonian subgroups (early disease onset and tremor-dominant forms) and controls. These results corroborate the concept that CSF levels of t-tau protein and the index total tau/ab42 may be regarded as laboratory markers for the presence and severity of neurodegeneration (Brunn-