Cerebrospinal fluid biomarkers of neurodegeneration in chronic neurological diseases

Review

10.1586/14737159.8.4.479 © 2008 Expert Reviews Ltd ISSN 1473-7159 479www.expert-reviews.com

Cerebrospinal fluid biomarkers of neurodegeneration in chronic neurological diseasesExpert Rev. Mol. Diagn. 8(4), 479–494 (2008)

Hayrettin Tumani†, Charlotte Teunissen, Sigurd Süssmuth, Markus Otto, Albert C Ludolph and Johannes Brettschneider†Author for correspondenceDepartment of Neurology, University of Ulm, Oberer Eselsberg 45, 89081 Ulm, GermanyTel.: +49 731 177 5207Fax: +49 731 177 [email protected]

Chronic neurological diseases (CND) like amyotrophic lateral sclerosis (ALS), dementia ormultiple sclerosis (MS) share a chronic progressive course of disease that frequently leads tothe common pathological pathway of neurodegeneration, including neuroaxonal damage,apoptosis and gliosis. There is an ongoing search for biomarkers that could support earlydiagnosis of CND and help to identify responders to interventions in therapeutic treatmenttrials. Cerebrospinal fluid (CSF) is a promising source of biomarkers in CND, since the CSFcompartment is in close anatomical contact with the brain interstitial fluid, where biochemicalchanges related to CND are reflected. We review recent advances in CSF biomarkers researchin CND and thereby focus on markers associated with neurodegeneration.

KEYWORDS: amyotrophic lateral sclerosis • biomarker • cerebrospinal fluid • chronic neurological disease • dementia • multiple sclerosis • neurodegeneration

Chronic neurological diseases

Chronic neurological diseases (CND) like amyo-trophic lateral sclerosis (ALS), and different sub-types of dementia or multiple sclerosis (MS)form an important challenge to diagnostic andtherapeutical progress in neurology. Althoughclinically heterogeneous, these diseases share achronic progressive course of disease that is fre-quently accompanied by clinically severe, debili-tating symptoms. Furthermore, while those dis-eases demonstrate different pathophysiologicalorigins, they may all lead to a common patholog-ical pathway of neurodegeneration (FIGURE 1).These neurodegenerative pathways are character-ized by a slow continuous process of degenera-tion, leading to the apoptosis of a selective popu-lation of neurons, frequently accompanied by agliotic reaction [1–3].

Dementia occurs with a prevalence of approxi-mately 20% in 75–85-year olds [4]. Set against thebackground of progressive aging of Western socie-ties, dementia forms a huge socioeconomic burdento public health systems [5]. Alzheimer’s disease(AD) is the most common subtype of dementia.Pathological hallmarks are extensive amounts ofsenile plaques (amyloid-β is a major component)and neurofibrillary tangles (phosphorylated tau) as

well as a loss of cortical neurons and subcorticalprojections [6,7]. In vascular dementia (VD),damage to subcortical white matter is generallymore pronounced [6]. In frontotemporal demen-tia (FTD), frontal cortical neurons, frontal whitematter and in some cases the pyramidal tractsdegenerate as well [2].

ALS is the most frequent motor neuron dis-ease, occurring with an incidence of 1–2 casesper 100,000 [8]. It is characterized by selectivedegeneration of spinal and bulbar innervatingmotor neurons as well as pyramidal motor neu-rons, leading to death after a disease duration ofapproximately 3 years [9]. While several muta-tions such as in the gene encoding the cytosoliccopper-zinc superoxide dismutase have beenassociated with rare cases of familial ALS, thepathophysiological origins of the majority ofsporadic ALS remains unclear [9].

MS is a CND that most often initially presentsas an inflammatory demyelinating disease [10].However, the pathophysiological character maychange with neurodegenerative aspects thatdominate in later stages of disease [11,12]. Further-more, histopathological studies show neuro-axonal damage to be present in early stages of dis-ease [3,11] and to be the major morphologicalsubstrate of permanent clinical disability [13].

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Review Tumani, Teunissen, Süssmuth, Otto, Ludolph & Brettschneider

Biomarkers for CND

A biomarker is defined as a characteristic that is objectively meas-ured and evaluated as an indicator of normal biological processes,pathogenic processes or pharmacological responses to a therapeuticintervention [14]. The term ‘biomarker’ is often used interchangea-bly with the term ‘surrogate marker’. However, there is a clear hier-archical distinction between those two terms: The term ‘surrogate’indicates the ability of a biomarker to provide information aboutthe clinical prognosis or efficacy of a therapy. It implies a strongcorrelation with a clinical end point, about which a surrogate mustprovide information in a shorter time than would be needed byfollowing the clinical course of disease [14]. Prentice defined twoconditions that ensure the surrogacy of a biomarker [15]:

• The first requirement is a strong correlation between thebiomarker and the clinical end point;

• The second requirement is that a biomarker measures the neteffect of the treatment on the clinical end point;

While numerous biomarkers are able to reflect single clinical orpathophysiological aspects of CND, none to date have fulfilledthe criteria of a surrogate marker.

According to the review by Blennow and Hampel, biomark-ers of CND may function as either state markers or stage mark-ers [16]. While stage markers would give a measure of how farthe degenerative process has proceeded, state markers wouldreflect the occurrence and intensity of the disease process.

In CND, biomarkers reflecting neurodegeneration are indemand to support an early diagnosis, because upcoming treat-ment will be most effective if started early [17,18]. As an example,clinical diagnosis of early stages of AD may be difficult even forexperienced neurologists, while only an early treatment withantidementive agents, such as cholinesterase inhibitors, was

found to show noteworthy results [17]. Accordingly, there is ademand for biological markers to delineate potential earlystages of disease, characterized by mild cognitive impairment(MCI), but without overt dementia [19]. Biomarkers with prog-nostic value could identify patients that will benefit from a spe-cific treatment. Biomarkers could reflect specific pathophysio-logical aspects of CND and thereby help to identify respondersto interventions in therapeutic treatment trials [14].

CSF fluid biomarkers

Biomarkers of CND include biochemical markers, which canbe drawn from the cerebrospinal fluid (CSF). While blood,urine, tears or saliva can easily be obtained, these liquids are vul-nerable to exogenous factors, such as inflammatory or metabolicinfluences, and results are consequently poorly reproducible inCNS diseases. By contrast, lumbar puncture of CSF is moreinvasive, although studies show complications to be rare if lum-bar puncture is performed by an experienced neurologist and ifnontraumatic needles with small gauge are used [20]. In general,CSF is a promising source of biomarkers in CND, since theCSF compartment is in close anatomical contact with the braininterstitial fluid, wherein biochemical changes related to the dis-ease may be reflected. Accordingly, alterations in protein expres-sion, post-translational modification or turnover within the tis-sue of the CNS associated with CND may be mirrored incorresponding changes in CSF protein content [16,21,22].

CSF physiology & its impact on CSF biomarker research

The majority of the protein content of the lumbar CSF is blood-derived (∼80%), while the remainder consists of brain-derived orintrathecally produced proteins (FIGURE 2). However, this recently

Figure 1. Common final pathway of neurodegeneration in chronic neurological diseases with different pathophysiological origins.

Alzheimer’s disease

Amyloid plaque formationNeurofibrillary tangles

Amyotrophic lateral sclerosis

ExcitotoxicityOxidative stressGlutamate toxicity

Disturbance ofaxonal transport

Multiple sclerosis

Inflammation (T-cell infiltration, macrophages, B-cell reaction)

RemyelinationDemyelination

Oligodendrocyte toxicity

Neuroaxonal damage

Neuronal apoptosis Glial reaction

Heterogeneouspathologicalonset of disease

Common final pathway ofneurodegeneration

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became a matter of debate because theCSF proteome appears to be different fromthe plasma proteome [23]. Under physio-logical conditions, blood-derived proteinsenter the CSF compartment via passivediffusion across the blood–CSF barrier.Depending on their molecular size andtheir blood concentration, CSF proteins ofblood origin show a specific CSF-to-bloodratio [24]. When molecular size and serumlevels are known, the CSF concentration ofa given protein can be calculated [25,26]. Ifthe concentration in the CSF exceeds thecalculated value, it indicates local produc-tion of the protein within the CNS (i.e.,intrathecal synthesis). Another importantfactor influencing the protein content isthe permeability of the blood–CSF barrier.The integrity of the blood–CSF barrier isbest characterized by the albumin CSF-to-serum ratio, since albumin is exclusivelyproduced in the liver and not by the ner-vous system. If the serum concentrationand the albumin CSF-to-serum ratio aretaken into account, the extent to whicheach protein in the CSF is producedwithin the CNS can be detirmined. Vari-ous formulae, such as the IgG-index, IgGsynthesis rate and IgG-loc, have been developed to discriminatebetween blood- and brain-derived fractions of immunoglobulins,which are used to confirm or rule out an inflammatory processwithin the CNS [27–29].

By contrast, CSF proteins that are predominantly synthesized inthe CNS (>95%), such as prostaglandin-D synthase, transthyretinor tau-protein, are not influenced by blood concentrations orchanges of barrier permeability and therefore do not have to berelated to their respective blood levels or albumin CSF/serum ratiowhen evaluated in CSF [30].

The sensitivity to detect intrathecally produced proteins inCSF also depends on the topographical relationship betweenthe site of the pathologic process and the CSF compartment.Certain areas of the brain, such as frontal, parietal or occipitalregions of the cortex, are considered CSF-distant. Pathologicalprocesses in these areas may not impact on the composition oflumbar CSF. Pathological processes localized in brain areasclose to ventricular and lumbar CSF space are more prone to bereflected by CSF changes [31]. Diseases of the meninges, peri-ventricular area, temporobasal region, spinal cord and rootsstrongly impact on the CSF parameters detected in lumbarCSF. Several additional factors, such as circadian variation, vol-ume of sampled CSF and rostrocaudal concentration gradient,may influence the concentration of CSF proteins and must beconsidered when studying CSF biomarkers [30,32,33]. Withrespect to the impact of the blood compartment on CSF, serum

should always be obtained at the time of the spinal tap and ana-lyzed in parallel. To ensure optimal performance and results,standardized protocols should be in place for the spinal tap andsample processing. Furthermore, it is important to analyze theCSF in a specialized laboratory that is routinely evaluated for itsperformance. If measuring proteins that potentially originatefrom both blood and brain compartments, CSF and serumsamples should be run in parallel in the same assay to minimizethe coefficient of variability.

Methods

In the following review, we give an update on CSF biomarkersin CND and thereby focus on markers associated with neuro-degeneration, apoptosis and gliosis, which are commonpathophysiological features in CND (FIGURE 1). Furthermore, wereview literature on recent developments in CSF biomarkerresearch using different methods for CSF proteome analysisand show future perspectives in biomarker research in CND.

Markers of neuroaxonal damage

Biomarkers of neuroaxonal damage (TABLES 1 & 2) are structuralproteins that are important for cytoskeletal stability and axonaltransport. Following neuroaxonal damage, these proteins canbe released into the intercellular space and from there into the

Figure 2. Compartments of the CNS and circulation of the CSF. Due to the close contact of the CSF with the brain and the spinal cord, the different steps in the pathogenesis of chronic neurological diseases might be detectable by changes of several markers. Between different compartments, there are distinct barriers to consider, such as the blood–CSF barrier, the blood–brain barrier and an albeit permeable CSF–brain barrier that basically comprises the cell membranes.CSF: Cerebrospinal fluid.

Blood compartment (5l)

Ventricle

Choroidplexus

CSF(150 ml)

Blood–CSFbarrier

Spinalnerveroots

Blood

Extracellular space

Glial activation

Apoptosis/degeneration

CSF–brainbarrier

Lumbar sac

Disturbedaxonaltransport

Glial proteins

Brain

Spinal cord

Arachnoidvilli

Neuroaxonalproteins

Blood–brain barrier

Ca2+

Expert Rev. Mol. Diagn. © Future Science Group Ltd (2008)



Table 1. Cerebrospinal fluid markers of neuroaxonal damage.

Marker Dementia Amyotrophic lateral sclerosis Multiple sclerosis

n Main findings n Main findings n Main findings

t-tau 75 ↑↑ in AD and VD vs controls [153] 69 ↑↑ in ALS vs controls [53] 36 ↑↑ in MS vs controls [55]

241 ↑↑ in AD vs controls [19] 20 ↑↑ in ALS vs controls [54] 60 ↑↑ in MS vs controls [60]

64 ↑↑ in AD vs FTD [43] 17 ↔ in ALS vs controls [55] 52 ↑↑ in MS vs controls; ↔ in RR-MS vs progressive MS [61]

77 ↑↑ in AD vs FTD, VD and controls [44]

18 ↔ in ALS vs controls [56] 52 ↑↑ in CIS vs controls [94]

78 ↑↑ in AD vs controls [85] 11 ↔ in ALS vs controls [57] 45 ↓ in MS vs controls [62]

407 ↑↑ in AD vs controls [45] 50 ↔ in MS vs controls [63]

72 ↑↑ in AD vs controls, VD and FTD [37]

32 ↑ in MS correlates with fast progression on the EDSS [65]

71 ↑↑ in AD vs controls [38] 114 ↑↑ in MS vs controls [59]

55 ↑↑ in AD vs controls [46] 20 ↔ in MS vs controls [64]

40 ↑↑ in AD and FTD vs controls [48] 17 ↑↑ in active MS vs controls [58]

33 ↑↑ in AD vs controls and DLB [49]

70 ↑↑ in AD vs controls [50]

p-tau 64 ↑↑ in AD vs controls [43] ↔ in ALS vs controls [57] 60 ↑↑ in MS vs controls [60]

41 ↑↑ in AD vs FTD, VD and controls [44]

41 ↑↑ in AD vs controls; in FTD vs controls [68]

72 ↑↑ in AD vs controls, VD, FTD [37]

154 ↑↑ in AD vs controls and DLB [154]

77 ↑ in MCI correlated with conversion to AD [155]

NfL 62 ↑↑ in AD, VD and FTD vs controls [82]

↑↑ in ALS vs controls [76] 99 ↑↑ in MS vs controls and relapse [88]

10 ↑↑ in AD, VD vs controls [81] ↑↑ in ALS vs controls; ↑↑ in sporadic vs SOD-1 familial ALS [77]

5 ↑↑ in RR-MS vs controls [81]

78 ↑↑ in AD and FTD vs controls [85] ↑↑ in ALS vs controls [81] 60 ↑↑ in RR-MS vs controls [89]

98 ↔ in AD vs VD, ↑↑ in AD vs controls [86]

66 ↑↑ in MS vs controls [90]

11 ↑↑ in AD vs controls [76] 35 ↑↑ in MS vs controls [91]

37 ↑↑ in AD vs controls [84]

NfH 190 ↑↑ in AD and VD vs controls [83] ↑↑ in ALS vs controls; ↑↑ in ALS with dominant UMN affection [53]

34 ↑↑ in progressive MS vs RR-MS [92]

37 ↑ in subgroup of FTD [84] 41 ↑↑ in ON vs controls [136]

98 ↑↑ in AD vs VD and controls [86] 52 ↑↑ in CIS vs controls [94]

34 ↑↑ in MS vs controls; ↑ in RR-MS indicates with worse prognosis [93]

↑: Increased; ↑↑: Significantly increased; ↓: Decreased; ↓↓: Significantly decreased; ↔: No alteration. AD: Alzheimer’s disease; ALS: Amyotrophic lateral sclerosis; DLB: Dementia with Lewy bodies; EDSS: Expanded Disability Status Scale; FTD: Frontotemporal dementia; MCI: Mild cognitive impairment; MS: Multiple sclerosis; Nfh: Heavy-chain neurofilament; Nfl: Light-chain neurofilament; ON: Optic neuritis; p-tau: Cerebrospinal fluid phosphorylated tau-protein; RR-MS: Relapsing-remitting multiple sclerosis; t-tau: Total cerebrospinal fluid tau; UMN: Upper motor neuron; VD: Vascular dementia.



CSF where they can be quantified. Candidate markers ofneuroaxonal damage include tau protein, different subtypes ofneurofilaments (Nf) as well as neuron-specific enolase (NSE)and 14-3-3 protein.

TauTau is a phosphoprotein that binds to tubulin and therebypromotes microtubule assembly and stability [34]. Because ofalternative splicing of tau mRNA, there are six isoforms withmolecular weights between 50 and 65 kDa [35]. The neuro-fibrillary tangles in AD are made up of an abnormally hyper-phosphorylated form of tau. Because of this hyper-phosphorylation, tau loses its ability to bind to themicrotubules and to stimulate their assembly [36]. To date, fourdifferent ELISA methods for quantification of t-tau in CSFhave been published [37–40].

CSF t-tauTotal CSF tau (t-tau) seems to reflect the extent of neuroaxonaldamage, with highest CSF concentrations found in conditionswith a rapid-progressive neuroaxonal degeneration [41,42]. Sev-eral studies consistently found a moderate-to-marked increaseof CSF t-tau in patients with AD [20,43,44]. The mean sensitivity

to discriminate AD from nondemented aged individuals wasapproximately 80%, with a specificity of about 90% [19]. InVD, elevated CSF t-tau has been found by some [38,45], but notall studies [39,46]. These inconsistent findings may be due to dis-crepancies between the studies with regard to patients includedand diagnostic criteria applied. Another factor influencing t-tauconcentrations may be concomitant AD pathology, which hasbeen frequently observed in VD [47]. Most other subtypes ofdementia, such as FTD or Lewy body dementia, show normal-to-mildly increased t-tau concentrations [38,43,48,49]. In patientswith MCI, high t-tau was found to discriminate patients thatdeveloped AD during clinical follow-up [50–52].

In ALS, some studies reported elevated CSF t-tau concentra-tions [53,54], while others observed them to be normal [55–57].Generally, t-tau appears to be inferior to Nf in monitoringneuroaxonal damage in ALS [53].

In MS, CSF t-tau levels were found to be increased by most[55,58–62], though not by all studies [63,64]. CSF t-tau concentra-tions correlated positively with gadolinium-enhancing lesionsand relapse activity. On the other hand, an inverse correlationwas seen between t-tau concentrations and disease duration,with highest values found in clinically isolated syndrome (CIS)patients and lowest in secondary progressive MS (SPMS)

Table 2. Cerebrospinal fluid markers of neuroaxonal damage.

Marker Dementia Multiple sclerosis

n Main findings n Main findings

Tubulin 35 ↑↑ in MS vs controls [91]

Actin 20 ↑↑ in AD homozygous for ApoE ε4-allele [156] 35 ↑↑ in MS vs controls [91]

NSE 30 ↓ in AD and VD vs controls [99] 66 66 ↔ in MS vs controls [90]

64 ↑↑ in AD and VD vs controls; ↔ in AD vs VD [95] 91 91 ↔ in MS vs controls [104]

43 ↑↑ in dementia vs controls [96] 34 34 ↔ in MS vs controls [105]

159 ↔ in dementia vs controls (↑↑ in CJD) [98]

44 ↔ in AD vs controls, ↓↓ in VD vs controls and AD [97]

58 ↑↑ in CJD vs controls [100]

16 ↑ increased persistent in CJD [102]



14-3-3 129 Positive in CJD vs controls [103] 63 14-3-3 detectable in 38% of MS [109]

31 Positive in CJD vs controls [101] 38 14-3-3 detectable in 13.2% of MS [108]

10 10β, γ, ε, η positive in CJD vs other dementia [107] 47 14-3-3 detectable in 8.2% of MS; associated with higher relapse rate [65]

37 14-3-3 detectable in 8% of MS [110]

NAA 46 ↑↑ in RR-MS vs SPMS [112]

↑: Increased; ↑↑: Significantly increased; ↓: Decreased; ↓↓: Significantly decreased; ↔: No alteration. AD: Alzheimer’s disease; CJD: Creutzfeldt–Jakob disease; MS: Multiple sclerosis; NAA: N-acetyl aspartic acid; NSE: Neuron-specific enolase; RR-MS: Relapsing-remitting multiple sclerosis; SPMS: Secondary progressive multiple sclerosis; VD: Vascular dementia.



patients [60,61]. One study suggested a prognostic relevance oft-tau in reporting a correlation of higher levels with faster clinicaldeterioration on the expanded disability-severity scale (EDSS) [65].The results of the CSF tau studies support the fact that theintensity of the neuronal damage is most prominent in the earlyphase of MS. This finding is in accordance with histopathologi-cal findings from brain biopsies and magnetic resonance (MR)spectroscopy studies of MS patients [3,66].

CSF phosphorylated tau-proteinHyperphosphorylation of tau has been observed in severalneurological diseases, including AD [34,36]. CSF phosphorylatedtau-protein (p-tau) levels were found to be normal or only mildlyincreased in neurological diseases such as Creutzfeldt–Jakob dis-ease (CJD) that show a marked increase in CSF t-tau [51,67].Consequently, p-tau does not appear to be a general marker ofneuroaxonal damage, but to reflect specifically the phosphoryla-tion state of tau. Comparable to t-tau, the sensitivity to discrimi-nate AD from nondemented controls was about 80%, with aspecificity of approximately 90% [16]. However, the sensitivity ofp-tau to differentiate AD from controls was higher than for t-tauor amyloid-β1-42, but not necessarily more sensitive to distin-guish AD from other subtypes of dementia [43,44,68,69]. A markedincrease in CSF p-tau was found in MCI cases that progressed toAD during follow-up [67,70,71]. Normal levels for p-tau werereported in non-AD dementias such as VD, Lewy body demen-tia or FTD, as well as in other CND, such as ALS [43,44,57,68].Differences between the studies may be due to ELISA methodsspecific for different phosphorylated tau epitopes [19]. Recentlythe combination of p-tau, t-tau and amyloid-β1-42 was seen asindicative for the development of AD in MCI patients [52]. Thecomparison between different p-tau epitopes gave only aminimal additional diagnostic improvement [72].

NeurofilamentsNeurofilaments belong to the class IV intermediate filaments.Nfs form an important component of the axoskeleton and areparticularly abundant in large myelinated axons. Nfs are het-eropolymers that are composed of four subunits: a light (NfL),medium (NfM) and heavy (NfH) chain and α-internexin [73].The length of the C-terminal tail domain differs considerablybetween these subunits and accounts for the differences inmolecular mass [73,74].

In ALS, an increased staining for phosphorylated NfH wasobserved in axons and neuronal cell bodies in ALS [75]. In CSFof ALS patients, elevation of NfL as well as of NfH wasobserved using ELISA [53,76,77]. NfL tended to be unstable inCSF due to a marked susceptibility to proteases [78], which con-siderably reduced their reliability as a biochemical marker. Bycontrast, the more heavily phosphorylated NfH proved to bemore stable [78]. Like the aforementioned tau protein, bothNfH and NfL were observed to be higher in patients with dam-age to predominantly the upper motor neurons [53,76]. As a pos-sible explanation, it was suggested that upper motor neuron

damage would result in the degeneration of large caliber axonsalong the entire length of their spinal pathway, leading to therelease of large quantities of NfH into the CSF. By contrast,degeneration of the lower motor neuron would only releaseNfH into the CSF from proximal axons adjacent to the ante-rior horn cell, which would be proportionally less important.Very high concentrations of CSF NfH were observed inpatients with a rapidly progressive course of disease and mayaccordingly be associated with a poorer prognosis [53].

In AD plaques, increased staining for phosphorylated NfH isevident in the proximal axon and in the perikaryon [79,80]. NfHhyperphosphorylation in axons elongating from AD plaques hasbeen interpreted as evidence of early axonal injury caused bytoxic properties of the plaque components [80]. Several studiesshowed CSF NfH and NfL levels to be higher in patients withAD, FTD and VD when compared with age-matched non-demented controls [81–86]. The strongest difference was observedfor the comparison of FTD patients with nondemented controls,both for NfL and NfH [84,87]. According to one study, increasedCSF NfL levels were also able to separate patients with FTDfrom those with AD [85], while NfH showed no marked capacityto differentiate between subtypes of dementia [83,84]. Generally,CSF NfH and NfL levels do not appear to be useful as a screen-ing test in the diagnosis of dementia. However, both Nf proteinsmay be of value for investigation of disease progression in somepatients with FTD, VD and AD [87].

In MS, increased levels of NfL have been consistentlyreported by several studies [88–91]. NfL correlated with thedegree of disability and relapse rate, indicating continuousaxonal damage during the entire course of the disease with themost profound damage associated with acute relapses [89,90]. Inanother study, a significant higher proportion of patients withprogressive MS showed an increase in NfH levels when com-pared with those with a relapsing-remitting (RR) course of dis-ease [92]. Furthermore, NfH correlated with disability scales,indicating that cumulative neuroaxonal loss is associated withsustained disability and that elevated NfH levels may suggest apoor prognosis [92,93]. Together with t-tau, the NfH phospho-form NfHSMI35 could improve predicting conversion from CISto clinically definite MS [94].

Together with Nf, microtubules are the main axoskeletal pro-teins. Tubulin-α and -β subunits form the major structural com-ponent of the microtubule. Actin is an important constituent ofthe microfilaments, which are part of presynaptic terminals anddendritic spines. One study found elevated CSF actin and tubu-lin concentrations in progressive MS as compared with RR-MSas well as controls, and also observed a correlation with severityof disease as measured by EDSS [91]. To date, confirmatorystudies and data on CSF levels in other CND are missing.

Neuron-specific enolaseEnolase is one of many glycolytic enzymes and consists of threesubunits (α, β and γ). In the CNS, the α-γ and γ-γ isoforms aremainly localized within the neurons and therefore called NSE.



NSE converts 2-phospho-glycerate tophosphoenolpyruvate. Studies on CSFNSE in dementia are controversial. Whilesome studies reported CSF NSE to beelevated in AD and VD as compared withnondemented controls [95,96], othersobserved no such difference [97,98] andone study even reported CSF NSE to bedecreased in AD [99]. In contrast to theseconflicting data, very high concentrationsof CSF NSE are well established as amarker for diagnosis of CJD [100–103]. InMS, CSF NSE were within normal rangecompared with normal controls as well asbetween subtypes of MS [104,105].

14-3-3 proteinsThe 14-3-3 proteins (∼30 kDa) arehomologous dimeric proteins, expressedin almost all eukaryotic cells. In humans,seven isoforms (β, γ, ε, η, σ, τ, ξ) areknown. Five major isoforms are found inthe CNS. 14-3-3 are mainly detected bynonquantitative western blots. To date,only one study described detection of14-3-3 by an ELISA (awaiting furtherconfirmation) [106].

Similar to t-tau, S100β (S100b), NSEand 14-3-3 are well-known as diagnosticmarkers for CJD, which can be deline-ated by determination of specific 14-3-3 isoforms (β, γ, ε, η)from other subtypes of dementia [42,101,103,107]. In patientswith CIS, it was detected in five of 38 cases and its presencepredicted short-term evolution of RR-MS. These results wereconfirmed in larger cohorts of patients [65,108,109]. Anothergroup failed to confirm these results [110]. The reason for thismight be that the 14-3-3 antibodies which were used in thesestudies cross-react with the light chain of immunoglobulin,which is elevated in MS patients [111]. However, further evalu-ation of 14-3-3 in a larger cohort of MS patients with animproved protocol might be useful.

N-acetyl aspartic acidN-acetyl aspartic acid (NAA) is a neuron-specific marker con-stantly identified in MR-spectroscopy studies of the normaland MS brain. One first study investigating CSF NAA in MSusing gas chromatography mass spectrometry observed higherconcentrations in RR-MS as compared with SPMS as well as acorrelation with severity of disease and MRI parameters [112].

In summary, the CSF concentration of markers of neuro-degeneration (e.g., t-tau), may be elevated in various diseasesassociated with neuroaxonal damage regardless of underlyingetiology [58]. As illustrated in FIGURE 3, CSF t-tau concentrationsappear to be mainly influenced by the intensity of the neuronal

damage. Accordingly, t-tau may function as a state markeraccording to Blennow and Hampel [16]. In addition, within asingle disease entity the CSF t-tau concentration may be ofprognostic value, in other words, it may indicate a faster pro-gression or conversion to definite disease (e.g., from MCI toAD, from CIS to RR-MS, or fast vs slow progressing ALS).

Biomarkers of neuroaxonal damage represent a category ofparameters with high surrogate potential in CND. Sincemost CNDs are heterogeneous with regard to the furtherintraindividual disease course, the baseline CSF concentra-tion of CSF biomarkers may become a relevant predictivediagnostic tool. While biomarkers of neurodegeneration arewell established in patients with AD (tau-protein in combina-tion with amyloid-β) and in patients with CJD (14-3-3 pro-tein), thorough evaluation of these biomarkers in larger stud-ies using standardized assays warrant further efforts inpatients with ALS and MS [113].

Apoptosis-related markersTransglutaminaseTransglutaminase belongs to a family of enzymes that catalyzethe formation of a covalent bond between a free amine group(e.g., protein-bound lysine) and the γ-carboxamide group of

Figure 3. Correlation of a biomarker with the dynamics of the natural course of chronic neurological diseases adapted from data on t-tau. In MS and ALS, neuronal damage appears to predominate in a very early phase of the disease and in progressive subtypes of disease. In dementia, higher levels of tau occur in later stages of diseases, presumably indicating the cumulative involvement of neuronal loss.AD: Alzheimer’s disease; ALS: Amyotrophic lateral sclerosis; CJD: Creutzfeldt–Jakob disease; FTD: Frontotemporal dementia; MS: Multiple sclerosis; t-tau: Total cerebrospinal fluid tau; VD: Vascular dementia.

Tau

leve

l

Rat

e o

f d

isea

se p

rog

ress

ion

Very high

Elevated

Normal

Early stages Late stages

High

Low

Clinicalmanifestation

CJD

AD

ALS, MS

VD, FT

Disease duration



protein-bound glutamine (TABLE 3). Tissue transglutaminase(tTG) is activated during the apoptotic cell death cascade andplays a key role in the formation of apoptotic bodies [114]. InAD and VD (but also in other neurological diseases such asParkinson’s disease), elevated CSF tTG levels have beenreported, indicating increased neural cell apoptosis [115,116]. Nocapacity to differentiate between subtypes of dementia or corre-lation with clinical parameters was observed [115]. In ALS, lowenzyme levels of CSF tTG have been found, suggesting thatenzymatic activity becomes depleted at the terminal stages ofthe disease when most of the spinal motor neuronal perikaryahave been destroyed [114]. A candidate marker related to trans-glutaminase activity is N-ε-γ-glutamyl-lysine isodipeptide,which is released from the breakdown of proteins crosslinked

by transglutaminase enzymes [117]. Similar to tTG, N-lysine iso-peptide levels were observed to be elevated in AD and VD com-pared with nondemented controls, while no potential to differ-entiate between subtypes of dementia was observed [117]. Again,the specificity appears to be low because elevated CSF levels ofN-ε-γ-glutamyl-lysine isodipeptide were also reported in otherneurological diseases (e.g., Huntington’s disease) [118].

Ceramide was demonstrated to accumulate in neurons dur-ing acute or chronic neurodegeneration and to induce neuronalapoptosis by upregulation of proapoptotic mitochondrial pro-teins [119–121]. Ceramide was shown to be elevated in a pre-senilin-1 mouse model of AD and to induce apoptosis in neu-rons and glial cells in AD [119,122]. One study reported elevatedCSF ceramide levels in AD as compared with ALS and other

Table 3. Cerebropsinal fluid markers of apoptosis and glial response.

Biomarker Dementia Amyotrophic lateral sclerosis Multiple sclerosis

n Main findings n Main findings n Main findings

Apoptosis-related cerebrospinal fluid markers

tTG 51 ↑↑ in AD vs controls; ↔ in VD vs controls [115]

17 ↑↑ in early stage ALS vs controls, significantly decreased in late-stage ALS vs controls [114]

N-lysine isopeptide

25 ↑↑ in AD and VD vs controls; ↔ in VD vs AD [117]

Ceramide 16 ↑↑ in AD vs ALS and controls [123]

Gliosis-related cerebrospinal fluid markers

GFAP 27 ↑↑ in AD vs controls [131] 99 ↑↑ in MS vs controls [88]

65 ↑↑ in dementia vs controls [132] 13 ↑↑ in MS vs controls; correlation with clinical deficits [137]

23 ↑↑ in NPH vs dementia and controls [133]

66 ↑↑ in SPMS vs controls; correlation with EDSS [90]

51 ↑ in MS with poor ambulation [135]

S100b 43 ↑↑ in dementia vs controls [96] 20 ↓ with ongoing disease [54] 66 ↔ in MS vs controls [90]

119 ↑↑ in dementia vs controls and MS [157]

34 ↑↑ in MS vs controls [93]

159 ↔ in dementia vs controls (↑↑ in CJD) [98]

91 ↔ in MS vs controls [104]

129 ↑↑ in CJD vs controls [103] 10 Highest in third week after onset of relapse [134]

67 ↑↑ in AD and FTD vs controls [128] 41 ↔ in ON vs controls [136]

68 ↔ in AD vs controls; ↑↑ in early AD vs controls [129]

51 ↑↑ in RR-MS vs controls [135]

40 ↑↑ in FTD vs controls [130]

↑: Increased; ↑↑: Significantly increased; ↓: Decreased; ↓↓: Significantly decreased; ↔: No alteration. AD: Alzheimer’s disease; ALS: Amyotrophic lateral sclerosis; CJD: Creutzfeldt–Jakob disease; EDSS: Expanded Disability Status Scale; FTD: Frontotemporal dementia; GFAP: Glial fibrillary acidic protein; MS: Multiple sclerosis; ON: Optic neuritis; RR-MS: Relapsing-remitting multiple sclerosis; NPH: Normal pressure hydrocephalus; SPMS: Secondary progressive multiple sclerosis; tTG: Tissue transglutaminse; VD: Vascular dementia.



neurological diseases, although no correlation with clinicalparameters was observed [123]. In vitro, ceramide was found toinduce apoptosis in oligodendrocytes [124], indicating a possibleinvolvement in MS pathology, although no study on ceramideCSF levels in MS exists at present.

Markers of glial activation

Glial activation generally occurs very early in the cascade ofneurodegeneration (TABLE 3) [125]. S100b and glial fibrillary acidicprotein (GFAP) are candidate markers for the measurement ofglial activation. S100b is an acidic calcium-binding proteinfound mostly in specific glial cells, such as astrocytes andSchwann cells [126]. GFAP is the major structural protein of theintermediate filament of astrocytes [127].

In AD, mildly to moderately elevated levels of CSF S100b havebeen reported by various studies [98,128,129], with one study sug-gesting that levels may be highest in early stages of disease [129].Generally, no correlation with severity of dementia wasobserved, although one study reported a correlation with brainatrophy [128]. Elevation of S100b was also observed in othersubtypes of dementia, such as FTD, indicating that S100b hasno potential as a marker for differential diagnosis in dementia[98,128,130]. Similar to S100b, CSF GFAP was observed to be ele-vated in AD [131,132], but also in other subtypes of dementia(e.g., FTD or normal pressure hydrocephalus) [132,133]. As forS100b, no consistent correlation of GFAP with clinical severityof dementia could be observed [131,132].

In ALS, S100b levels were found to be within the normal ref-erence range with a weak correlation with duration of the dis-ease [54]. However, studies of CSF S100b in a larger cohort ofALS patients are still missing.

In MS, increased CSF S100b was reported particularly duringan acute relapse [93,104,134], which is in line with observations onpathology that described S100b and GFAP in acute inflammatoryplaques associated with activation or destruction of astrocytes[12,127]. One author reported significantly higher S100b levels inRR-MS as compared with chronic progressive subtypes [135],although this could not be confirmed by another study [136].

In RR-MS, CSF GFAP levels were observed to correlate withclinical deficits [90,135,137] and to increase over a 24-month follow-up period [135,137]. Therefore, GFAP may serve as a biomarker inMS for disease progression, probably reflecting the increasingrate of astrogliosis. In summary, gliosis-related markers appear tohave only low potential for differential diagnosis of CND. How-ever, further evaluation of their surrogacy potential is needed inlarger cohorts and with standardized assays.

Identification of new biomarkers using proteome analysis

Research with unbiased approaches, such as proteomics, belongsto the more recent omics technologies that have been applied todiscover new candidate biomarkers in CSF [138]. To analyze the

CSF proteome (e.g., the total protein content) in patients withCND, different technical approaches have been used. SELDI-TOF mass spectrometry has been successfully applied in demen-tia as well as ALS [139–141]. Alternative approaches are 2D gel elec-trophoresis followed by MALDI-TOF mass spectrometry as wellas liquid chromatography (LC) mass spectrometry in conjunc-tion with ICAT. TABLE 4 provides an overview of different CSFproteomics studies in CND.

Each of the available methods has specific inherent advantagesas well as limitations. SELDI-TOF mass spectrometry does nothave a high sensitivity, which makes protein identification diffi-cult, and mainly detects proteins with a lower molecular weight,and thus may miss potentially interesting markers [140,141]. 2D gelelectrophoresis followed by MALDI-TOF mass spectrometry islabor-intensive because it requires isolating each protein spotbefore identification of the protein [142]. The most abundant pro-teins (e.g., albumin or immunoglobulins) limit the total amountof protein that can be loaded on the 2D gel [143]. To increase thedetection of less abundant proteins, CSF must be preprocessedby extracting the bulk of extracerebral proteins and salts. To ena-ble preprocessing steps, large quantities of CSF are necessary.This demand can be met by pooling of CSF samples; however,pooling may impede the detection of protein alterations found inindividual patients [142]. A recent study performed an integratedanalysis of the CSF peptidome and proteome by combination ofSDS-PAGE with nano-LC mass spectrometry, and identified798 proteins in the whole CSF proteome [23]. Using a bead-basedmethod for MALDI tandem mass spectrometry, approximately150 mass-intensity peaks of peptides and proteins of less than 30kDa could be identified in a volume of 100 µl of CSF [144]. In LCmass spectrometry labeled with ICAT, only cysteine-containingpeptides are labeled (∼90% of the CSF proteome containscysteine residues). Another setback is that during each run, only asubset of peptides separated by LC gains access to the mass spec-trometer, accounting for marked differences between successiveruns of identical samples, which limit the reproducibility of thisapproach [145,146]. Generally, the different methodical approachesto proteome analysis are semiquantitative methods [147,148].Accordingly, results must be validated using easier, quantitativeassays, such as multiparameter immunoassays.

Expert commentary

Although several studies have been conducted to date, only a fewmarkers are used in clinical practice. While biomarkers of neuro-degeneration are well established in patients with AD (tau-proteinin combination with amyloid-β) and in patients with CJD (14-3-3 protein), in patients with ALS and MS, thorough evaluation ofbiomarkers in larger studies using standardized assays are war-ranted. The great majority of biochemical–biological studies,although of paramount importance (as discussed above), generallysuffer from several drawbacks:

• The studies are monocentric and samples sizes are too small,resulting in underpowered studies;



• Sample collection and storage conditions are not standardized;

• Clinical/biological data collection is not standardized andincomplete and clinical characteristics are often missing;

• Biochemical–biological methods have not been validatedinternally and externally;

• The studies are mainly cross-sectional and no appropriatecontrol groups are included (healthy instead of neurologicalcontrols, which are seen in clinical practice);

• Study goals are poorly defined and sample size estimationsare usually missing;

Table 4. Candidate cerebrospinal fluid biomarkers from proteome analysis.

Dementia Amyotrophic lateral sclerosis Multiple sclerosis

SELDI-TOF mass spectometryCystatin C ↑ β2-microglobulin ↑VGF precursor protein ↓ [139]

↓ transthyretin, ↓ cystatin C, c-terminal fragment of neuroendocrine protein 7B2 [140]

↓ cystatin C, ↓ fragment of VGF, ↓ 7.6 kDa protein that could not be identified [141]

2D GE followed by MALDI-TOF mass spetrometryIn AD, significantly altered (increased or decreased) vs controls: ↑ transthyretin, ↑ retinol-binding protein, ↑ β2-microglobulin, ↓ ApoA1, ↓ ApoE [158]

↓ granin-like neuroendocrine precursor, ↓ Apo E, ↓ pigment epithelium-derived factor, ↓ retinol-binding protein, ↓ haptoglobin [159]

RR-MS vs controls:Igκ chain precursor, transferrin, serine proteinase inhibitor, α2-HS-glycoprotein, apoE, transthyretin CIS vs controls:IgGκ chain, pro-ApoA1, serum albumin precursor, complement factor-3, serine proteinase inhibitor, vitamin D-binding protein, translation-initiation factor elF-4-γ, apoE, transthyretin [142]

LC mass spectrometry163 different proteins AD vs controls; of these, 39 had ACR-ASAP ≥1.2 and 46 had ACR-ASAP ≤0.8 [145]

Total of 430 spots in the MS CSF proteome representing 61 distinct proteins: 103 spots were not seen on control gels; all but four of these 103 were proteins known to be present in normal human CSF [147]: • CRTAC-1B (cartilage acidic protein)• Tetranectin (a plasminogen-binding protein)• SPARC-like protein (a calcium-binding cell signaling glycoprotein) • Autotaxin T (phosphodiesterase) 65 different proteins were identified from 300 spots; 18 of these proteins have not been reported previously on 2D gel in human CSF [148]:• Aldolase A• Annexin 1• Calmodulin-related protein NB-1• Cystatin A• EWI-2 (CD81 partner 3)• Glutathione peroxidase• N-acetyllactosaminide β-1,3,-N-acetyl glucosaminyltransferase• Niemann-Pick disease type C2 protein• Procollagen C-proteinase enhancer protein• Psoriasin (s100A7)• Semenogelin 1 and 2• Superoxide dismutase• Tetranectin• Complement factor H-related protein 1• Dermcidin• Galectin-7• Hornerin

ACR-ASAP: Alzheimer/controls ratio-automated statistical analysis of proteine abundance; AD: Alzheimer’s disease; CSF: Cerebrospinal fluid; LC: Liquid chromatography; MS: Multiple sclerosis; RR-MS: Relapsing-remitting multiple sclerosis; SPARC: Secreted protein acidic and rich in cysteine.



• Confirmation studies with an independent set of samplesare missing;

• Important covariates and confounders are not considered;

• The statistical methods are not appropriate and rarely pre-defined;

• Such small single center projects are just able to create primaryexploratory data and technical validation of assays;

• In addition, for most markers tested in the CSF, the blood–CSFbarrier function has not been considered sufficiently, in order todiscriminate whether the biomarker of interest originates fromthe systemic circulation or from intrathecal synthesis, whichwould indicate the CNS specificity.

Five-year view

Standardization of sampling protocols as well as ethical proto-cols is necessary in future biomarker research studies in CND.This would allow combining sample sets from differentresearch groups within multicenter studies. Such protocols

would also allow investigators to replicate the studies with sam-ples that match the initial pilot study. In addition to standard-ized sampling protocols, there is a growing need for standard-ized guidelines for other steps of biomarker research, includingexperimental design, criteria for data analysis and representa-tion and proof-of-principle research. Several guidelines forreporting research in different areas (REMARK, STARD,MIAME and PROTEOMICS) already exist [149–152]. Similarapproaches are on the way for CSF biomarker research in MSand in other neurological diseases [201].

Financial & competing interests disclosure

This work has been supported in part by fundings of the DeutscheForschungsgemeinschaft to ACL (DFG LU 336/12-1-KFO142-TP P4).The authors have no other relevant affiliations or financial involvementwith any organization or entity with a financial interest in or financialconflict with the subject matter or materials discussed in the manuscriptapart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Key issues

• Chronic neurological diseases (CND) such as amyotrophic lateral sclerosis (ALS), different subtypes of dementia or multiple sclerosis (MS), form an important challenge to diagnostic and therapeutical progress in neurology.

• CND may all lead to the common pathological pathway of neurodegeneration, including neuroaxonal damage, neuronal apoptosis and glial reaction.

• Biomarkers could support early diagnosis of CND and help to identify responders to interventions in therapeutic treatment trials.

• Cerebrospinal fluid (CSF) is a promising source of biomarkers in CND, since the CSF compartment is in close anatomical contact with the brain interstitial fluid, where biochemical changes related to CND are reflected.

• Different approaches of CSF proteome analysis have recently been applied to identify new candidate biomarkers in CND.

• To increase validity and power of future biomarker studies in CND, a multicenter approach based on standardization of sampling protocols, experimental design and data analysis is necessary.

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Website

201 European BioMS Societywww.BioMS.eu

Affiliations

• Hayrettin Tumani, MDDepartment of Neurology, University of Ulm, Oberer Eselsberg 45, 89081 Ulm, GermanyTel.: +49 731 177 5207Fax: +49 731 177 [email protected]

• Charlotte Teunissen, PhDDepartment of Molecular Cell Biology & Immunology, VU University Medical Center, 1007 MB Amsterdam, The NetherlandsTel.: +31 20 444 8061Fax: +31 20 444 [email protected]

• Sigurd Suessmuth, MDDepartment of Neurology, University of Ulm, Oberer Eselsberg 45, 89081 Ulm, GermanyTel.: +49 731 177 1206Fax: +49 731 177 [email protected]

• Markus OttoDepartment of Neurology, University of Ulm, Oberer Eselsberg 45, 89081 Ulm, GermanyTel.: +49 731 500 63019Fax: +49 731 500 [email protected]

• Albert C Ludolph, MDDepartment of Neurology, University of Ulm, Oberer Eselsberg 45, 89081 Ulm, GermanyTel.: +49 731 177 1201Fax: +49 731 177 [email protected]

• Johannes Brettschneider, MDDepartment of Neurology, University of Ulm, Oberer Eselsberg 45, 89081 Ulm, GermanyTel.: +49 731 177 1206Fax: +49 731 177 [email protected]

Cerebrospinal fluid biomarkers of neurodegeneration in chronic neurological diseases

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