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Cerebrospinal fluid in Creutzfeldt–Jakob disease
Handbook of Clinical Neurology, Vol. 146 (3rd series) Cerebrospinal Fluid in Neurologic Disorders F. Deisenhammer, C.E. Teunissen, and H. Tumani, Editors https://doi.org/10.1016/B978-0-12-804279-3.00008-3 Copyright © 2018 Elsevier B.V. All rights reserved
Chapter 8
Cerebrospinal fluid in Creutzfeldt–Jakob disease INGA ZERR*, SAIMA ZAFAR, MATTHIAS SCHMITZ, AND FRANC LLORENS Clinical Dementia Center, Department of Neurology, German Center for Neurodegenerative Diseases, University Medical Center G€ ottingen, G€ ottingen, Germany
Abstract Cerebrospinal fluid (CSF) contains a dynamic and complex mixture of proteins, which reflects physiologic or pathologic states of the central nervous system. Changes in CSF proteome have been described in various neurodegenerative disorders. Earliest publications came from the field of prion disease. Two major approaches have been followed aiming to detect the pathologic form of prion protein (PrPSc) in various peripheral tissues on one hand, but also looking for surrogate parameters as a consequence of the underlying neurodegenerative process. First observations were made using two-dimensional gel electrophoresis for proteins named p130/131, identified as belonging to the 14-3-3 protein family group. This protein became known as the first “wet” biomarker part of clinical diagnostic criteria. Other proteins were identified; most of the work in addition to 14-3-3 has been done on tau/p-tau. The development of PrPSc-based biomarkers was hampered by technical problems and detection limits. A novel technique which uses an amplification procedure followed by an aggregation step (real-time quaking-induced conversion: RT-QuIC) emerged and allows the detection of abnormally folded PrPSc in the CSF. This chapter summarizes the current knowledge of biomarker development in prion disease and discusses perspectives for new approaches.
ROUTINE PARAMETERS The routine tests of cerebrospinal fluid (CSF), such as cell count and blood–brain barrier function parameters, are generally normal in patients with Creutzfeldt–Jakob disease (CJD). A moderate increase in protein levels in the CSF can be observed in advanced disease stages. Although the disease is transmissible, there is no evidence for a typical inflammatory reaction and IgA, IgM, and IgG ratios are not altered. In exceptional cases, oligoclonal bands are detected in around 5% of cases, in a similar rate as in healthy controls (Jacobi et al., 2005).
BIOMARKER DEVELOPMENT 14-3-3 test The development of CSF-based biomarkers in CJD began in 1986, when a research group headed by M. Harrington identified two CSF proteins using two-dimensional gel
electrophoresis that he named p130/131 (Harrington et al., 1986). Almost 10 years later microsequencing showed that these two CSF proteins belong to the family of the 14-3-3 proteins (Hsich et al., 1996). The 14-3-3 protein family comprises several isoforms. Since anti-14-3-3 antibodies were available, their detection was facilitated by the use of Western blot assays. Other brain proteins, such as neuron-specific enolase, S100 protein, tau protein, and phosphorylated tau isoform p-tau (tau phosphorylated at threonine 181), were measured at high concentrations in the CSF of patients with CJD, to name only a few. Large numbers of studies on this subject have demonstrated that in the appropriate clinical circumstances a positive 14-3-3 protein detection is highly sensitive and specific for sporadic CJD (sCJD) diagnosis and correlates with clinical diagnosis in 85–94% of cases (Sanchez-Juan et al., 2006; Stoeck et al., 2012). Follow-up examinations revealed an increase in 14-3-3 protein levels with disease progression and a decrease in end-stage disease.
*Correspondence to: Inga Zerr, Clinical Dementia Center, Neurology, University Medical Center G€ ottingen, Robert-Koch-Str. 40, 37075 G€ottingen, Germany. Tel: +49-551-39-66636, Fax: +49-551-39-7020, E-mail: [email protected]
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Detection of elevated 14-3-3 protein levels became important in the differential diagnostic procedure. The most important differential diagnosis in suspected CJD is rapid progressive Alzheimer dementia, and in up to 30% potentially reversible dementia (Fig. 8.1) (Van Everbroeck et al., 2004; Heinemann et al., 2007; Kelley et al., 2009; Chitravas et al., 2011). Following the introduction of 14-3-3 tests in laboratories worldwide, although largely recognized as extremely useful, questions about the sensitivity, specificity, and predictive values of these tests have arisen. However, a multicenter European study demonstrated high specificity of the test of around 95% in the context of neurodegenerative disorders. Most “false positives” occurred in inflammatory diseases and in stroke patients as well as after epileptic fits – medical conditions which can be easily differentiated from CJD syndrome (Sanchez-Juan et al., 2006). Although indicative of acute neuronal damage or astrocyte activation, the detection of abnormal concentrations of the brain protein 14-3-3 alone is not sufficient to make the diagnosis of CJD and should always be used in the clinical context only. Therefore, clinical diagnostic criteria have been suggested and 14-3-3 detection in CSF is part of it (Zerr et al., 2000a, 2009). The identification of the 14-3-3 protein in CSF, in particular, has proved to be a useful method for discriminating CJD from other dementias (Stoeck et al., 2012). With respect to methodologic problems, the analysis of 14-3-3 protein is done using Western blotting and there is no generally accepted standard for which results should be compared or which isoform should be detected. Therefore, the evaluation of 14-3-3 test results can be subject to interpretative problems and requires experience from laboratory personnel. To overcome this difficulty, several quantitative methods, such as enzymelinked immunosorbent assay (ELISA) and protein capture assays were developed. Ring trials are extremely important to ensure high quality of the analyses (Schmitz et al., 2016).
Protein tau Another important marker involves the analysis of total tau protein and its phosphorylated isoforms. Tau levels in CSF are markedly elevated in patients with CJD and a pilot study indicates that detection in serum tau may be useful, too (Noguchi-Shinohara et al., 2011). While several p-tau test kits for detection of tau phosphorylated at different sites are commercially available, no comparative analysis has been performed to define the best detection methodology, optimal conditions, and optimal test variables. Another open question refers to the optimal detection range of tau in CSF, since the available commercial tests are optimized for the detection range 75–1200 pg/mL, which is suitable for classic Alzheimer disease (AD). Cut-off levels for CJD have been proposed, but vary between centers worldwide. Only limited information is available on tau/p-tau ratio and other biomarker patterns in CJD patients. Other problems include the comparative value between tau and 14-3-3 detection. Some research groups use tau as a single marker to confirm the diagnosis since the 14-3-3 test has been claimed to be less specific. On the other hand such studies have been biased by the selection of the samples analyzed.
Abeta The concentration of Ab peptides in CSF is thought to reflect disease-associated changes in AD and is widely applied as a diagnostic biomarker for this neurodegenerative disorder (Tamaoka et al., 1997; Shoji et al., 1998; Andreasen et al., 1999; Otto et al., 2000; Blennow and Zetterberg, 2009). A reduced level of Ab1-42 was also reported in other dementias, including CJD, without further analysis (with respect to biologic parameters). Also distinct patterns of Aß peptides in CJD, AD, and controls have been reported (Wiltfang et al., 2003). Since Aß1-40, Aß1-42 and their ratio are important biomarkers in AD
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Göttingen, Germany (1) Cleveland, USA (2)
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Mayo Clinic, Rochester, USA (3) Antwerp, Belgium (4)
40 30 20 10 0 AD
VD
LBD
FTD
other
treatable
Fig. 8.1. Differential diagnosis in suspected Creutzfeldt–Jakob disease. AD, Alzheimer disease; FTD, frontotemporal dementia; LBD, Lewy body disease; VD, vascular dementia.
CEREBROSPINAL FLUID IN CREUTZFELDT–JAKOB DISEASE diagnosis but are also clearly altered in CJD, further evaluation and standardization of these parameters with respect to the specific situation in prion diseases are needed.
MOLECULAR DISEASE PHENOTYPE, GENES, AND CSF ALTERATIONS IN CJD PRNP codon 129 genotype CSF biomarkers depend not only on the underlying disease, but also on several genetic determinants. The major well-known genetic factor influencing prion biomarker accuracy is the codon usage at position 129 of the prion protein gene (PRNP). In sCJD, combination of methionine–valine polymorphism at position 129 (MM, MV, VV), along with prion typing (type 1 or 2 depending on the electrophoretic mobility of the pathologic PrPSc protein) defines the molecular type of the disease. The different molecular subtypes present well-defined histopathologic features and differ by age at onset and disease course (Parchi et al., 2009, 2012). Several observations indicate that in neurodegenerative diseases CSF may mirror pathologic changes of the central nervous system (Tapiola et al., 2009; Sepp€al€a et al., 2012; Li et al., 2014) due to its unique relation with the peripheral blood system. As a consequence, the heterogeneity in the clinical features among different sCJD subtypes has a strong influence on the sensitivity to various diagnostic tests (Zerr et al., 2000b; Collins et al., 2006; Sanchez-Juan et al., 2007). Tau levels differed considerably between PRNP codon 129 genotypes in sCJD, showing better discriminatory ability only if codon129 genotype is known. Tau levels are considerably higher in PrP type 1 MM and MV patients but lower in those presenting with the VV genotype (Karch et al., 2015). Despite huge efforts in the establishment of disease-specific tau cut-off values in AD and sCJD (Sanchez-Juan et al., 2006; Humpel, 2011), sCJD subtypes with the lowest tau levels are not clearly distinguishable from AD cases (Karch et al., 2015; Gmitterová et al., 2016). Influence of sCJD molecular typing is not only restricted to tau but also to 14-3-3, neuron-specific enolase, and S100 proteins. For 14-3-3, differences among molecular subtypes appear to be related to the PrP type rather than to the codon 129 genotype (Castellani et al., 2004; Gmitterová et al., 2016). Indeed, a recent report using a quantitative 14-3-3 detection system has not been able to observe differences between homozygotes and heterozygotes at position 129 (Leitão et al., 2016). However, higher 14-3-3 protein levels are observed in the classic sCJD subtypes MM1 and MV1 compared to patients presenting atypical subtypes (MV2). Increased sensitivity was detected in PrP type 1 than in PrP type 2, whereas the lower levels were
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observed in the subtypes associated with longer disease duration (Castellani et al., 2004; Gmitterová et al., 2009). For S100 and neuron-specific enolase proteins, increased levels were detected in VV2 compared to VV1 subtype. When only codon 129 was significantly higher, values in homozygous group in comparison to heterozygous patients were detected (Gmitterová et al., 2016). Altogether the data indicate that PRNP codon 129 genotype alone or in combination with nongenetic factors such as PrPSc isotype defining the sCJD molecular subtype, and directly associated with the clinical and neuropathologic features of the disease, have a strong influence in the test sensitivity of prion CSF biomarkers currently used in the diagnostic routine. The impact of PRNP codon 129 genotypes has been also studied on the real-time quaking-induced conversion (RT-QuIC) signal (see below). Methionine homozygosity was associated with a higher signal maximum of RT-QuIC response, which was determined by increased signal in sCJD MM1 patients as compared to sCJD MV1 or VV1 patients. However, this effect was not observable for cases presenting with PrPSc type 2 (Cramm et al., 2015).
APOE genotype While PRNP codon 129 has not been demonstrated to influence the level of amyloid peptides in sCJD, APOE genotypes have been shown to modulate CSF Ab1-42 values in sCJD (Varges et al., 2011) in a similar manner as in AD (Galasko et al., 1998). The role of ApoE e4 has been investigated in the levels of the classic CSF prion biomarkers tau, neuron-specific enolase, S100 protein, Ab1-42, Ab1-40 peptides, and 14-3-3 (Varges et al., 2011; Leitão et al., 2016). In sCJD, ApoE e4 carriers with one ApoE e4 allele showed significantly reduced Ab1-42 values in comparison with noncarriers (Varges et al., 2011). Interestingly, ApoE e4 allele has no influence on sCJD disease duration or age at onset, and ApoE e4 allele has not been described as a risk factor for sCJD. These findings indicate that stratification by genetic factors might be a powerful tool for increasing the diagnostic performance of CSF biomarkers.
GENETIC PRION DISEASES Approximately 10–15% of all human prion disease cases present with a genetic etiology. In all cases development of hereditary prion diseases is associated with mutations in the gene encoding the prion protein (PRNP). Genetic CJD cases, fatal familial insomnia, and Gerstmann– Str€aussler–Scheinker syndrome represent the core phenotypes of genetic prion disease (Mead, 2006; Mastrianni, 2010). The CSF biomarker profile is highly
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dependent on the mutation type and associated with duration of illness, which in genetic cases is more heterogeneous than in sporadic cases, ranging from a few months to 13 years (Mastrianni, 2010). For 14-3-3, diagnostic accuracy in genetic CJD cases is similar to that in sCJD but only a low percentage of cases are 14-3-3-positive in Gerstmann–Str€aussler–Scheinker and fatal familial insomnia patients (Kovács et al., 2005; Ladogana et al., 2009; Gawinecka and Zerr, 2010). Similar observations are reported for tau (Ladogana et al., 2009; Llorens et al., 2016), S100, and neuron-specific enolase (Ladogana et al., 2009), in which genetic CJD cases presented similar levels as sCJD cases. Because of the generally slower rate of progression of fatal familial insomnia and Gerstmann–Str€aussler– Scheinker syndrome compared to genetic CJD and sCJD cases (Mead, 2006), the detection of 14-3-3, tau, and neuron-specific enolase, reflecting the neuropathologic lesions in the brain tissue as a consequence of rapid neuronal damage, may explain their low sensitivity in detecting cases with longer disease duration.
completely understood. Most probably it consists of a seeded induced assembly and conversion of PrPC directly within misfolded PrP aggregates (Bessen et al., 1995; Prusiner, 1998; Soto et al., 2002). Minuscule amounts of PrPSc act as a seed in the reaction. Single recPrP substrate is recruited by the PrP seed which induces the conversion and a conformational change of the substrate molecule by integrating them into an amyloid-like aggregate (Fig. 8.2). In the RT-QuIC assay, samples are subjected to shaking which breaks PrP aggregates into new reactive seeds for conversion and incubation. With each cycle the amyloid reaction product can increase exponentially (Fig. 8.2). Aggregated PrP can be easily monitored by the use of a fluorescence dye, thioflavin-T, in a fluorescent plate reader. In contrast to PMCA, the amyloid reaction product of RT-QuIC is considered to be noninfectious.
ROLE OF IN VITRO PROTEIN MISFOLDING AMPLIFICATION ASSAYS IN PRION DISEASE DIAGNOSTICS The self-propagating replication of the abnormally folded host-derived prion protein (PrPC) is characteristic for human prion diseases. In its pathologic conformation, PrPSc is prone to aggregation. The pathogenic conversion mechanism of PrP is the basis for a number of different in vitro protein misfolded amplification assays which enable the study of the conversion processes of PrPSc in vitro. Currently, several in vitro conversion systems, e.g., protein misfolding cyclic amplification (PMCA) (first described by Saborio et al., 2001), amyloid seeding assay (reported by Colby et al., 2007), QuIC (Atarashi et al., 2008), and RT-QuIC (Wilham et al., 2010; Atarashi et al., 2011) have been developed, mainly adapted to a brain seed. The next step in the development of in vitro amplification systems was the adaptation for the measurement of aggregated PrPSc in human CSF. This was an innovation for premortem diagnostics. Currently, the most common in vitro amplification assays are PMCA and RT-QuIC, which are comparable to “polymerase chain reaction for misfolded proteins.” PMCA, based on sonication, usually employs normal mice brain homogenate as substrate while RT-QuIC uses recombinant synthesized prion protein (recPrP), derived from several sources, such as hamster, chimeric hamster-sheep, bank vole, or human. The mechanism of prion conversion process in this assay is not
Fig. 8.2. Schematic diagram of the real-time quaking-induced conversion (RT-QuIC) assay. A pathologic form of prion protein (PrPSc) seed and recombinant synthesized prion protein (recPrPC) substrate are mixed and incubated, inducing the conversion and incorporation of the substrate molecules producing misfolded b-strand-enriched PrP polymers. Cycles of shaking and incubation of b-strand-enriched PrP polymers breaks and generates more seeds, inducing an exponential amplification reaction.
CEREBROSPINAL FLUID IN CREUTZFELDT–JAKOB DISEASE Meanwhile, RT-QuIC has already been applied to human brain tissue, CSF, and olfactory neuroepithelium. In prion disease diagnostics, the CSF RT-QuIC has been standardized and validated thoroughly. Its high reproducibility (in ring trial studies) and stability under defined CSF storage conditions (short- and long-term up to 5 years) have been demonstrated (Cramm et al., 2016), suggesting the RT-QuIC as an innovative and robust diagnostic test method. In prion disease diagnosis this assay exhibited a specificity of almost 100% and a sensitivity of 85% (Atarashi et al., 2011; McGuire et al., 2012; Sano et al., 2013; Cramm et al., 2016). Genetic CJD cases showed a sensitivity of 100%, while analysis of sCJD and fatal familial insomnia CSF samples revealed a sensitivity of 80% and 57%, respectively (Cramm et al., 2016). Another application of the RT-QuIC assay is the detection of PrPSc in the olfactory neuroepithelium of CJD patients (Orrú et al., 2014). RT-QuIC reactions, seeded with a PrPSc seed from nasal brushing, revealed a sensitivity of 97% and specificity of 100% (Orrú et al., 2014; Zanusso et al., 2014). Compared to the CSF RT-QuIC, testing of nasal brushings provoked a faster and stronger RT-QuIC seeding response. Even though, the nasal brushing RT-QuIC is less standardized, the sampling provides an alternative and is less invasive than lumbar puncture. The concept of protein misfolding was previously thought to be related to prion diseases, solely. However, previous reports described similar characteristics also for other misfolded proteins, such as Ab or tau protein in Alzheimer patients or alpha-synuclein in Parkinson’s disease, indicating “prion-like” protein propagation. Thus, the RT-QuIC methodology has considerable diagnostic potential that may become relevant also for other misfolded proteins/diseases and will result in an increase of the application spectrum of this test (Salvadores et al., 2014; Stancu et al., 2015).
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increased a-synuclein levels in sCJD cases compared to controls (Kasai et al., 2014; Llorens et al., 2015b). However, these studies could not validate the suspected excellent clinical accuracy of synuclein detection in discrimination of sCJD cases reported in the first study. Recently, the use of an electrochemiluminescencebased platform for the quantification of CSF a-synuclein has been demonstrated to improve the range and sensitivity of the test over colorimetric assays. Using this assay in a small cohort of control, AD, and sCJD cases the authors concluded that CSF a-synuclein levels are highly increased in sCJD compared to control and AD cases (Llorens et al., 2015a). Further studies in independent and large cohorts of sCJD cases and other neurodegenerative diseases will determine the accurate clinical parameters of this promising sCJD biomarker. The physiopathologic mechanisms leading to an increase of a-synuclein in the CSF of CJD cases are unknown, but no synuclein-related pathology has been reported so far in sCJD brain tissue. The enrichment of a-synuclein in the presynaptic terminals (Maroteaux et al., 1988), coupled with massive synaptic damage to sCJD brain tissue (Ferrer et al., 1999), suggests that release of synaptic proteins into the interstitial fluid and clearance into the CSF are a potential source of elevated a-synuclein in the CSF of sCJD. Of interest, the decreased levels of CSF a-synuclein in a-synuclein aggregation disorders, especially in dementia with Lewy bodies, combined with the elevated levels in sCJD, may help to distinguish both diseases with higher clinical accuracy. sCJD and dementia with Lewy bodies presents a partial overlap in the CSF biomarker panel, with decreased Ab42 and increased tau levels (Parnetti et al., 2008; Andersson et al., 2011; Llorens et al., 2016). In addition, sCJD and dementia with Lewy bodies have overlapping clinical symptoms that can lead to their misdiagnosis (Tartaglia et al., 2012). Thus inclusion on a-synuclein measurement, either as single or composite biomarker, may help disease discrimination in the differential diagnostic context.
a-synuclein With the aim of completing the analyses of core dementia CSF biomarkers in prion disease, the quantification of a-synuclein levels in sCJD has been recently addressed. A pioneer cross-sectional study suggested elevated CSF a-synuclein levels in a small cohort of sCJD cases, compared to controls. In the same study, the authors reported increased synuclein levels in AD and decreased levels in aggregation disorders (Parkinson disease, dementia with Lewy bodies, and multiple-system atrophy) (Mollenhauer et al., 2008). Further studies validated
Immunologic markers in prion diseases In neurodegenerative disease, immunologic responses can be involved in disease progression and clinical heterogeneity, e.g., AD or sCJD. There is abundant evidence that cytokine-mediated interactions between neurons and glia cells contribute to cognitive impairment, suggesting that the brain’s innate immune system has a major influence on the pathogenesis of neurodegenerative diseases. Understanding inflammatory mechanisms is pivotal for achieving better
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insights into changes in brain metabolism. Previous studies had already demonstrated that the formation of plaques in AD or in CJD can be influenced at an early disease stage by the immune system through an involvement of major histocompatibiltity complex II and complement activation which may result in a microglia recruitment and secretion of proinflammatory cytokines (Sharief et al., 1999; Van Everbroeck et al., 2002). To investigate the function of the immune system as a modifier of disease pathogenesis in CJD, cytokine levels in CJD patients were determined by different detection systems (e.g., multiplex analysis based on the Luminex technology or ELISA). A number of different cytokines have been identified as being regulated in CJD (Stoeck et al., 2005, 2006; Fujita et al., 2013; Llorens et al., 2014) highlighting the potential importance for the course of the disease. However, CSF findings in sCJD patients are controversial. Several studies observed an increase in proinflammatory cytokines, such as tumor necrosis factor-a and interleukin-1b (IL-1b) (Sharief et al., 1999; Van Everbroeck et al., 2002), whereas others noted elevated levels of anti-inflammatory cytokines such as IL-4 and IL-10 (Stoeck et al., 2005, 2006). In addition, levels of IL-8, IL-1 receptor antagonist, and IL-17 (which induces inflammation) were reported to be elevated in sCJD patients, while transforming growth factor-b2 has been observed to be decreased compared to a control group (Stoeck et al., 2006, 2014; Fujita et al., 2013). Variable findings may be explained by different detection assays, a relatively low number of cases, and the fact that the level of single cytokine is variable during the course of the disease, as shown in infected mice brains (Llorens et al., 2014). Consequently, the usefulness of immunologic markers in prion disease diagnostic is limited because of the instability, a lack of repetitious accuracy across different laboratories, and relatively low sensitivity and specificity. For example, IL-8 exhibited a sensitivity of 70% and a specificity of 82% for sCJD (Stoeck et al., 2006), which is relatively low compared to 14-3-3 (approximately 90% sensitivity and specificity) (Zerr et al., 1998; Schmitz et al., 2016) or RT-QuIC (85% and 99%) (Cramm et al., 2016).
Proteomics: global alterations of CSF Proteomics is the study of the entire protein content translated by the genomewide machinery in a cell. Therefore, any alteration in this vicinity shows up the distinct geographies of gene byproducts, i.e., modifications at posttranslational level, different protein isoforms, localization at a subcellular level, and interactive association between other proteins (Pandey and Mann, 2000;
Aebersold and Mann, 2003; Lamond et al., 2012). For instance, proteins are the molecules that arefunctional in the cell and may indicate disease-specific alterations in tissues or organs. CSF interconnects with the brain tissue and comprises many proteins derived from brain and consequently characterizes a promising strategy for the discovery of biomarkers in neurologic diseases. For the analysis of protein contents in CSF, many recent techniques have emerged in the field of proteomics. The most commonly used methods encompass global differential proteome analysis likening dualistic or more experimental conditions. Quantitative approaches are able to measure protein expression (both absolute and relative amount) and in detail depth of posttranslational modifications (van Gool and Hendrickson, 2012; Craft et al., 2013). Global proteome alteration analysis includes: (1) classic gel based system (two-dimensional electrophoresis or two-dimensional differential fluorescence gel electrophoresis); and (2) gel-free system. Gelbased proteome analysis is still largely used with some limitations, including the isolation of specific types and numbers of proteins coupled with mass spectrometry (Fig. 8.3). In CSF the amount of protein is comparatively low and largely masked by the high amount of salts (> 150 mM) (Sickmann et al., 2002) and highly abundant proteins, but features a highly diverse proteome. In human CSF, the proteomic alterations typically comprise many protein fractionation and concentration steps in association with the depletion of highly abundant proteins, i.e., serum albumin. In general, from few milliliters of human CSF approximately 2600 proteins can be identified (Righetti et al., 2005). Despite all these limitations, cumulative characterization strategy of the CSF proteome field is growing with different advancements of three major MS modules: ion source, mass analyzer, and detection unit. Based on the different ion sources used, most mass spectrometers used in the field of proteomics can be generally categorized into two types: electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) instruments. In neurologic disorders, especially in prion diseases, early detection of potential biomarkers is highly desirable in association with molecular pathogenesis (Vitzthum et al., 2005). Under pathologic conditions, the altered proteins are released from the brain cells into CSF and thus are easily accessible for analysis (Hu et al., 2005). Different studies to date have demonstrated a wide range of subsets of altered proteome potential biomarkers. Apart from this, the high amount of immunoglobulins in many neurologic diseases also indicates the need for specific isolation and fractionation methods (Aebersold and Mann, 2003; Qian et al., 2006). Using
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Fig. 8.3. Proteomics approaches in combination with validation parameters. CSF, cerebrospinal fluid; MALDI-FTMS, matrixassisted laser desorption/ionization Fourier transform mass spectrometry; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; NanoLC-ESI-MS, nano-scale liquid chromatographic electrospray ionization mass spectrometry.
multiple affinity purification columns, most of the abundant proteins from CSF can be depleted before the proteomics analysis (Sitnikov et al., 2006). In prion diseases, only a handful of research in the field of CSF has been performed and showed potential differential proteome alteration. In humans, mass spectrometry and gel-based systems revealed differential proteome-wide alterations in the sporadic form of CJD and demonstrated the role of codon 129 genotype (MM, MV, and VV) and the protease-resistant form of prion protein (type 1 and type 2) at translational level (Piubelli et al., 2006; Gawinecka et al., 2010; Gmitterová et al., 2016). In the prion-infected hamster model, MALDI Fourier transform mass spectrometry (MALDI-FTMS), and support vector machines have demonstrated peptide profiles characteristic of disease state in CSF and used for the further development of prion disease multiple diagnostics markers (Herbst et al., 2009). Some recent studies also demonstrated the comprehensive analysis of extracellular vesicles from human CSF using tandem mass spectrometry in conjunction with bioinformatics (Sanchez et al., 2004; Chiasserini et al., 2014). This proteome-wide screening showed that human CSF extracellular vesicles contain prionogenic proteins such as the amyloid precursor protein and prion protein and showed the involvement of extracellular vesicles in the spread of neurodegeneration to different brain areas (Chiasserini et al., 2014). The proteomic shotgun-based approach of tandem mass tag labeling has established growing consideration for neuroproteomics analysis and proves to be an effective tool for the quantitative identification of 437 potential proteins; with 95% confidence interval 49 proteins were verified from the CSF of CJD patients (Chen et al., 2014). Recent proteome-based data from CSF of
sCJD patients highlighted the differentially regulated levels of complement components (Chen et al., 2016). In conclusion, the proteomic approaches are appropriate for the identification of early diagnostic markers and to get insight into the proteome-wide alteration during the disease course. This could also be useful for the increment of scientific signs for the alterations of CSF homeostasis throughout the pathogenesis of prion diseases.
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Chapter 8
Cerebrospinal fluid in Creutzfeldt–Jakob disease INGA ZERR*, SAIMA ZAFAR, MATTHIAS SCHMITZ, AND FRANC LLORENS Clinical Dementia Center, Department of Neurology, German Center for Neurodegenerative Diseases, University Medical Center G€ ottingen, G€ ottingen, Germany
Abstract Cerebrospinal fluid (CSF) contains a dynamic and complex mixture of proteins, which reflects physiologic or pathologic states of the central nervous system. Changes in CSF proteome have been described in various neurodegenerative disorders. Earliest publications came from the field of prion disease. Two major approaches have been followed aiming to detect the pathologic form of prion protein (PrPSc) in various peripheral tissues on one hand, but also looking for surrogate parameters as a consequence of the underlying neurodegenerative process. First observations were made using two-dimensional gel electrophoresis for proteins named p130/131, identified as belonging to the 14-3-3 protein family group. This protein became known as the first “wet” biomarker part of clinical diagnostic criteria. Other proteins were identified; most of the work in addition to 14-3-3 has been done on tau/p-tau. The development of PrPSc-based biomarkers was hampered by technical problems and detection limits. A novel technique which uses an amplification procedure followed by an aggregation step (real-time quaking-induced conversion: RT-QuIC) emerged and allows the detection of abnormally folded PrPSc in the CSF. This chapter summarizes the current knowledge of biomarker development in prion disease and discusses perspectives for new approaches.
ROUTINE PARAMETERS The routine tests of cerebrospinal fluid (CSF), such as cell count and blood–brain barrier function parameters, are generally normal in patients with Creutzfeldt–Jakob disease (CJD). A moderate increase in protein levels in the CSF can be observed in advanced disease stages. Although the disease is transmissible, there is no evidence for a typical inflammatory reaction and IgA, IgM, and IgG ratios are not altered. In exceptional cases, oligoclonal bands are detected in around 5% of cases, in a similar rate as in healthy controls (Jacobi et al., 2005).
BIOMARKER DEVELOPMENT 14-3-3 test The development of CSF-based biomarkers in CJD began in 1986, when a research group headed by M. Harrington identified two CSF proteins using two-dimensional gel
electrophoresis that he named p130/131 (Harrington et al., 1986). Almost 10 years later microsequencing showed that these two CSF proteins belong to the family of the 14-3-3 proteins (Hsich et al., 1996). The 14-3-3 protein family comprises several isoforms. Since anti-14-3-3 antibodies were available, their detection was facilitated by the use of Western blot assays. Other brain proteins, such as neuron-specific enolase, S100 protein, tau protein, and phosphorylated tau isoform p-tau (tau phosphorylated at threonine 181), were measured at high concentrations in the CSF of patients with CJD, to name only a few. Large numbers of studies on this subject have demonstrated that in the appropriate clinical circumstances a positive 14-3-3 protein detection is highly sensitive and specific for sporadic CJD (sCJD) diagnosis and correlates with clinical diagnosis in 85–94% of cases (Sanchez-Juan et al., 2006; Stoeck et al., 2012). Follow-up examinations revealed an increase in 14-3-3 protein levels with disease progression and a decrease in end-stage disease.
*Correspondence to: Inga Zerr, Clinical Dementia Center, Neurology, University Medical Center G€ ottingen, Robert-Koch-Str. 40, 37075 G€ottingen, Germany. Tel: +49-551-39-66636, Fax: +49-551-39-7020, E-mail: [email protected]
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Detection of elevated 14-3-3 protein levels became important in the differential diagnostic procedure. The most important differential diagnosis in suspected CJD is rapid progressive Alzheimer dementia, and in up to 30% potentially reversible dementia (Fig. 8.1) (Van Everbroeck et al., 2004; Heinemann et al., 2007; Kelley et al., 2009; Chitravas et al., 2011). Following the introduction of 14-3-3 tests in laboratories worldwide, although largely recognized as extremely useful, questions about the sensitivity, specificity, and predictive values of these tests have arisen. However, a multicenter European study demonstrated high specificity of the test of around 95% in the context of neurodegenerative disorders. Most “false positives” occurred in inflammatory diseases and in stroke patients as well as after epileptic fits – medical conditions which can be easily differentiated from CJD syndrome (Sanchez-Juan et al., 2006). Although indicative of acute neuronal damage or astrocyte activation, the detection of abnormal concentrations of the brain protein 14-3-3 alone is not sufficient to make the diagnosis of CJD and should always be used in the clinical context only. Therefore, clinical diagnostic criteria have been suggested and 14-3-3 detection in CSF is part of it (Zerr et al., 2000a, 2009). The identification of the 14-3-3 protein in CSF, in particular, has proved to be a useful method for discriminating CJD from other dementias (Stoeck et al., 2012). With respect to methodologic problems, the analysis of 14-3-3 protein is done using Western blotting and there is no generally accepted standard for which results should be compared or which isoform should be detected. Therefore, the evaluation of 14-3-3 test results can be subject to interpretative problems and requires experience from laboratory personnel. To overcome this difficulty, several quantitative methods, such as enzymelinked immunosorbent assay (ELISA) and protein capture assays were developed. Ring trials are extremely important to ensure high quality of the analyses (Schmitz et al., 2016).
Protein tau Another important marker involves the analysis of total tau protein and its phosphorylated isoforms. Tau levels in CSF are markedly elevated in patients with CJD and a pilot study indicates that detection in serum tau may be useful, too (Noguchi-Shinohara et al., 2011). While several p-tau test kits for detection of tau phosphorylated at different sites are commercially available, no comparative analysis has been performed to define the best detection methodology, optimal conditions, and optimal test variables. Another open question refers to the optimal detection range of tau in CSF, since the available commercial tests are optimized for the detection range 75–1200 pg/mL, which is suitable for classic Alzheimer disease (AD). Cut-off levels for CJD have been proposed, but vary between centers worldwide. Only limited information is available on tau/p-tau ratio and other biomarker patterns in CJD patients. Other problems include the comparative value between tau and 14-3-3 detection. Some research groups use tau as a single marker to confirm the diagnosis since the 14-3-3 test has been claimed to be less specific. On the other hand such studies have been biased by the selection of the samples analyzed.
Abeta The concentration of Ab peptides in CSF is thought to reflect disease-associated changes in AD and is widely applied as a diagnostic biomarker for this neurodegenerative disorder (Tamaoka et al., 1997; Shoji et al., 1998; Andreasen et al., 1999; Otto et al., 2000; Blennow and Zetterberg, 2009). A reduced level of Ab1-42 was also reported in other dementias, including CJD, without further analysis (with respect to biologic parameters). Also distinct patterns of Aß peptides in CJD, AD, and controls have been reported (Wiltfang et al., 2003). Since Aß1-40, Aß1-42 and their ratio are important biomarkers in AD
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Göttingen, Germany (1) Cleveland, USA (2)
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Mayo Clinic, Rochester, USA (3) Antwerp, Belgium (4)
40 30 20 10 0 AD
VD
LBD
FTD
other
treatable
Fig. 8.1. Differential diagnosis in suspected Creutzfeldt–Jakob disease. AD, Alzheimer disease; FTD, frontotemporal dementia; LBD, Lewy body disease; VD, vascular dementia.
CEREBROSPINAL FLUID IN CREUTZFELDT–JAKOB DISEASE diagnosis but are also clearly altered in CJD, further evaluation and standardization of these parameters with respect to the specific situation in prion diseases are needed.
MOLECULAR DISEASE PHENOTYPE, GENES, AND CSF ALTERATIONS IN CJD PRNP codon 129 genotype CSF biomarkers depend not only on the underlying disease, but also on several genetic determinants. The major well-known genetic factor influencing prion biomarker accuracy is the codon usage at position 129 of the prion protein gene (PRNP). In sCJD, combination of methionine–valine polymorphism at position 129 (MM, MV, VV), along with prion typing (type 1 or 2 depending on the electrophoretic mobility of the pathologic PrPSc protein) defines the molecular type of the disease. The different molecular subtypes present well-defined histopathologic features and differ by age at onset and disease course (Parchi et al., 2009, 2012). Several observations indicate that in neurodegenerative diseases CSF may mirror pathologic changes of the central nervous system (Tapiola et al., 2009; Sepp€al€a et al., 2012; Li et al., 2014) due to its unique relation with the peripheral blood system. As a consequence, the heterogeneity in the clinical features among different sCJD subtypes has a strong influence on the sensitivity to various diagnostic tests (Zerr et al., 2000b; Collins et al., 2006; Sanchez-Juan et al., 2007). Tau levels differed considerably between PRNP codon 129 genotypes in sCJD, showing better discriminatory ability only if codon129 genotype is known. Tau levels are considerably higher in PrP type 1 MM and MV patients but lower in those presenting with the VV genotype (Karch et al., 2015). Despite huge efforts in the establishment of disease-specific tau cut-off values in AD and sCJD (Sanchez-Juan et al., 2006; Humpel, 2011), sCJD subtypes with the lowest tau levels are not clearly distinguishable from AD cases (Karch et al., 2015; Gmitterová et al., 2016). Influence of sCJD molecular typing is not only restricted to tau but also to 14-3-3, neuron-specific enolase, and S100 proteins. For 14-3-3, differences among molecular subtypes appear to be related to the PrP type rather than to the codon 129 genotype (Castellani et al., 2004; Gmitterová et al., 2016). Indeed, a recent report using a quantitative 14-3-3 detection system has not been able to observe differences between homozygotes and heterozygotes at position 129 (Leitão et al., 2016). However, higher 14-3-3 protein levels are observed in the classic sCJD subtypes MM1 and MV1 compared to patients presenting atypical subtypes (MV2). Increased sensitivity was detected in PrP type 1 than in PrP type 2, whereas the lower levels were
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observed in the subtypes associated with longer disease duration (Castellani et al., 2004; Gmitterová et al., 2009). For S100 and neuron-specific enolase proteins, increased levels were detected in VV2 compared to VV1 subtype. When only codon 129 was significantly higher, values in homozygous group in comparison to heterozygous patients were detected (Gmitterová et al., 2016). Altogether the data indicate that PRNP codon 129 genotype alone or in combination with nongenetic factors such as PrPSc isotype defining the sCJD molecular subtype, and directly associated with the clinical and neuropathologic features of the disease, have a strong influence in the test sensitivity of prion CSF biomarkers currently used in the diagnostic routine. The impact of PRNP codon 129 genotypes has been also studied on the real-time quaking-induced conversion (RT-QuIC) signal (see below). Methionine homozygosity was associated with a higher signal maximum of RT-QuIC response, which was determined by increased signal in sCJD MM1 patients as compared to sCJD MV1 or VV1 patients. However, this effect was not observable for cases presenting with PrPSc type 2 (Cramm et al., 2015).
APOE genotype While PRNP codon 129 has not been demonstrated to influence the level of amyloid peptides in sCJD, APOE genotypes have been shown to modulate CSF Ab1-42 values in sCJD (Varges et al., 2011) in a similar manner as in AD (Galasko et al., 1998). The role of ApoE e4 has been investigated in the levels of the classic CSF prion biomarkers tau, neuron-specific enolase, S100 protein, Ab1-42, Ab1-40 peptides, and 14-3-3 (Varges et al., 2011; Leitão et al., 2016). In sCJD, ApoE e4 carriers with one ApoE e4 allele showed significantly reduced Ab1-42 values in comparison with noncarriers (Varges et al., 2011). Interestingly, ApoE e4 allele has no influence on sCJD disease duration or age at onset, and ApoE e4 allele has not been described as a risk factor for sCJD. These findings indicate that stratification by genetic factors might be a powerful tool for increasing the diagnostic performance of CSF biomarkers.
GENETIC PRION DISEASES Approximately 10–15% of all human prion disease cases present with a genetic etiology. In all cases development of hereditary prion diseases is associated with mutations in the gene encoding the prion protein (PRNP). Genetic CJD cases, fatal familial insomnia, and Gerstmann– Str€aussler–Scheinker syndrome represent the core phenotypes of genetic prion disease (Mead, 2006; Mastrianni, 2010). The CSF biomarker profile is highly
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dependent on the mutation type and associated with duration of illness, which in genetic cases is more heterogeneous than in sporadic cases, ranging from a few months to 13 years (Mastrianni, 2010). For 14-3-3, diagnostic accuracy in genetic CJD cases is similar to that in sCJD but only a low percentage of cases are 14-3-3-positive in Gerstmann–Str€aussler–Scheinker and fatal familial insomnia patients (Kovács et al., 2005; Ladogana et al., 2009; Gawinecka and Zerr, 2010). Similar observations are reported for tau (Ladogana et al., 2009; Llorens et al., 2016), S100, and neuron-specific enolase (Ladogana et al., 2009), in which genetic CJD cases presented similar levels as sCJD cases. Because of the generally slower rate of progression of fatal familial insomnia and Gerstmann–Str€aussler– Scheinker syndrome compared to genetic CJD and sCJD cases (Mead, 2006), the detection of 14-3-3, tau, and neuron-specific enolase, reflecting the neuropathologic lesions in the brain tissue as a consequence of rapid neuronal damage, may explain their low sensitivity in detecting cases with longer disease duration.
completely understood. Most probably it consists of a seeded induced assembly and conversion of PrPC directly within misfolded PrP aggregates (Bessen et al., 1995; Prusiner, 1998; Soto et al., 2002). Minuscule amounts of PrPSc act as a seed in the reaction. Single recPrP substrate is recruited by the PrP seed which induces the conversion and a conformational change of the substrate molecule by integrating them into an amyloid-like aggregate (Fig. 8.2). In the RT-QuIC assay, samples are subjected to shaking which breaks PrP aggregates into new reactive seeds for conversion and incubation. With each cycle the amyloid reaction product can increase exponentially (Fig. 8.2). Aggregated PrP can be easily monitored by the use of a fluorescence dye, thioflavin-T, in a fluorescent plate reader. In contrast to PMCA, the amyloid reaction product of RT-QuIC is considered to be noninfectious.
ROLE OF IN VITRO PROTEIN MISFOLDING AMPLIFICATION ASSAYS IN PRION DISEASE DIAGNOSTICS The self-propagating replication of the abnormally folded host-derived prion protein (PrPC) is characteristic for human prion diseases. In its pathologic conformation, PrPSc is prone to aggregation. The pathogenic conversion mechanism of PrP is the basis for a number of different in vitro protein misfolded amplification assays which enable the study of the conversion processes of PrPSc in vitro. Currently, several in vitro conversion systems, e.g., protein misfolding cyclic amplification (PMCA) (first described by Saborio et al., 2001), amyloid seeding assay (reported by Colby et al., 2007), QuIC (Atarashi et al., 2008), and RT-QuIC (Wilham et al., 2010; Atarashi et al., 2011) have been developed, mainly adapted to a brain seed. The next step in the development of in vitro amplification systems was the adaptation for the measurement of aggregated PrPSc in human CSF. This was an innovation for premortem diagnostics. Currently, the most common in vitro amplification assays are PMCA and RT-QuIC, which are comparable to “polymerase chain reaction for misfolded proteins.” PMCA, based on sonication, usually employs normal mice brain homogenate as substrate while RT-QuIC uses recombinant synthesized prion protein (recPrP), derived from several sources, such as hamster, chimeric hamster-sheep, bank vole, or human. The mechanism of prion conversion process in this assay is not
Fig. 8.2. Schematic diagram of the real-time quaking-induced conversion (RT-QuIC) assay. A pathologic form of prion protein (PrPSc) seed and recombinant synthesized prion protein (recPrPC) substrate are mixed and incubated, inducing the conversion and incorporation of the substrate molecules producing misfolded b-strand-enriched PrP polymers. Cycles of shaking and incubation of b-strand-enriched PrP polymers breaks and generates more seeds, inducing an exponential amplification reaction.
CEREBROSPINAL FLUID IN CREUTZFELDT–JAKOB DISEASE Meanwhile, RT-QuIC has already been applied to human brain tissue, CSF, and olfactory neuroepithelium. In prion disease diagnostics, the CSF RT-QuIC has been standardized and validated thoroughly. Its high reproducibility (in ring trial studies) and stability under defined CSF storage conditions (short- and long-term up to 5 years) have been demonstrated (Cramm et al., 2016), suggesting the RT-QuIC as an innovative and robust diagnostic test method. In prion disease diagnosis this assay exhibited a specificity of almost 100% and a sensitivity of 85% (Atarashi et al., 2011; McGuire et al., 2012; Sano et al., 2013; Cramm et al., 2016). Genetic CJD cases showed a sensitivity of 100%, while analysis of sCJD and fatal familial insomnia CSF samples revealed a sensitivity of 80% and 57%, respectively (Cramm et al., 2016). Another application of the RT-QuIC assay is the detection of PrPSc in the olfactory neuroepithelium of CJD patients (Orrú et al., 2014). RT-QuIC reactions, seeded with a PrPSc seed from nasal brushing, revealed a sensitivity of 97% and specificity of 100% (Orrú et al., 2014; Zanusso et al., 2014). Compared to the CSF RT-QuIC, testing of nasal brushings provoked a faster and stronger RT-QuIC seeding response. Even though, the nasal brushing RT-QuIC is less standardized, the sampling provides an alternative and is less invasive than lumbar puncture. The concept of protein misfolding was previously thought to be related to prion diseases, solely. However, previous reports described similar characteristics also for other misfolded proteins, such as Ab or tau protein in Alzheimer patients or alpha-synuclein in Parkinson’s disease, indicating “prion-like” protein propagation. Thus, the RT-QuIC methodology has considerable diagnostic potential that may become relevant also for other misfolded proteins/diseases and will result in an increase of the application spectrum of this test (Salvadores et al., 2014; Stancu et al., 2015).
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increased a-synuclein levels in sCJD cases compared to controls (Kasai et al., 2014; Llorens et al., 2015b). However, these studies could not validate the suspected excellent clinical accuracy of synuclein detection in discrimination of sCJD cases reported in the first study. Recently, the use of an electrochemiluminescencebased platform for the quantification of CSF a-synuclein has been demonstrated to improve the range and sensitivity of the test over colorimetric assays. Using this assay in a small cohort of control, AD, and sCJD cases the authors concluded that CSF a-synuclein levels are highly increased in sCJD compared to control and AD cases (Llorens et al., 2015a). Further studies in independent and large cohorts of sCJD cases and other neurodegenerative diseases will determine the accurate clinical parameters of this promising sCJD biomarker. The physiopathologic mechanisms leading to an increase of a-synuclein in the CSF of CJD cases are unknown, but no synuclein-related pathology has been reported so far in sCJD brain tissue. The enrichment of a-synuclein in the presynaptic terminals (Maroteaux et al., 1988), coupled with massive synaptic damage to sCJD brain tissue (Ferrer et al., 1999), suggests that release of synaptic proteins into the interstitial fluid and clearance into the CSF are a potential source of elevated a-synuclein in the CSF of sCJD. Of interest, the decreased levels of CSF a-synuclein in a-synuclein aggregation disorders, especially in dementia with Lewy bodies, combined with the elevated levels in sCJD, may help to distinguish both diseases with higher clinical accuracy. sCJD and dementia with Lewy bodies presents a partial overlap in the CSF biomarker panel, with decreased Ab42 and increased tau levels (Parnetti et al., 2008; Andersson et al., 2011; Llorens et al., 2016). In addition, sCJD and dementia with Lewy bodies have overlapping clinical symptoms that can lead to their misdiagnosis (Tartaglia et al., 2012). Thus inclusion on a-synuclein measurement, either as single or composite biomarker, may help disease discrimination in the differential diagnostic context.
a-synuclein With the aim of completing the analyses of core dementia CSF biomarkers in prion disease, the quantification of a-synuclein levels in sCJD has been recently addressed. A pioneer cross-sectional study suggested elevated CSF a-synuclein levels in a small cohort of sCJD cases, compared to controls. In the same study, the authors reported increased synuclein levels in AD and decreased levels in aggregation disorders (Parkinson disease, dementia with Lewy bodies, and multiple-system atrophy) (Mollenhauer et al., 2008). Further studies validated
Immunologic markers in prion diseases In neurodegenerative disease, immunologic responses can be involved in disease progression and clinical heterogeneity, e.g., AD or sCJD. There is abundant evidence that cytokine-mediated interactions between neurons and glia cells contribute to cognitive impairment, suggesting that the brain’s innate immune system has a major influence on the pathogenesis of neurodegenerative diseases. Understanding inflammatory mechanisms is pivotal for achieving better
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insights into changes in brain metabolism. Previous studies had already demonstrated that the formation of plaques in AD or in CJD can be influenced at an early disease stage by the immune system through an involvement of major histocompatibiltity complex II and complement activation which may result in a microglia recruitment and secretion of proinflammatory cytokines (Sharief et al., 1999; Van Everbroeck et al., 2002). To investigate the function of the immune system as a modifier of disease pathogenesis in CJD, cytokine levels in CJD patients were determined by different detection systems (e.g., multiplex analysis based on the Luminex technology or ELISA). A number of different cytokines have been identified as being regulated in CJD (Stoeck et al., 2005, 2006; Fujita et al., 2013; Llorens et al., 2014) highlighting the potential importance for the course of the disease. However, CSF findings in sCJD patients are controversial. Several studies observed an increase in proinflammatory cytokines, such as tumor necrosis factor-a and interleukin-1b (IL-1b) (Sharief et al., 1999; Van Everbroeck et al., 2002), whereas others noted elevated levels of anti-inflammatory cytokines such as IL-4 and IL-10 (Stoeck et al., 2005, 2006). In addition, levels of IL-8, IL-1 receptor antagonist, and IL-17 (which induces inflammation) were reported to be elevated in sCJD patients, while transforming growth factor-b2 has been observed to be decreased compared to a control group (Stoeck et al., 2006, 2014; Fujita et al., 2013). Variable findings may be explained by different detection assays, a relatively low number of cases, and the fact that the level of single cytokine is variable during the course of the disease, as shown in infected mice brains (Llorens et al., 2014). Consequently, the usefulness of immunologic markers in prion disease diagnostic is limited because of the instability, a lack of repetitious accuracy across different laboratories, and relatively low sensitivity and specificity. For example, IL-8 exhibited a sensitivity of 70% and a specificity of 82% for sCJD (Stoeck et al., 2006), which is relatively low compared to 14-3-3 (approximately 90% sensitivity and specificity) (Zerr et al., 1998; Schmitz et al., 2016) or RT-QuIC (85% and 99%) (Cramm et al., 2016).
Proteomics: global alterations of CSF Proteomics is the study of the entire protein content translated by the genomewide machinery in a cell. Therefore, any alteration in this vicinity shows up the distinct geographies of gene byproducts, i.e., modifications at posttranslational level, different protein isoforms, localization at a subcellular level, and interactive association between other proteins (Pandey and Mann, 2000;
Aebersold and Mann, 2003; Lamond et al., 2012). For instance, proteins are the molecules that arefunctional in the cell and may indicate disease-specific alterations in tissues or organs. CSF interconnects with the brain tissue and comprises many proteins derived from brain and consequently characterizes a promising strategy for the discovery of biomarkers in neurologic diseases. For the analysis of protein contents in CSF, many recent techniques have emerged in the field of proteomics. The most commonly used methods encompass global differential proteome analysis likening dualistic or more experimental conditions. Quantitative approaches are able to measure protein expression (both absolute and relative amount) and in detail depth of posttranslational modifications (van Gool and Hendrickson, 2012; Craft et al., 2013). Global proteome alteration analysis includes: (1) classic gel based system (two-dimensional electrophoresis or two-dimensional differential fluorescence gel electrophoresis); and (2) gel-free system. Gelbased proteome analysis is still largely used with some limitations, including the isolation of specific types and numbers of proteins coupled with mass spectrometry (Fig. 8.3). In CSF the amount of protein is comparatively low and largely masked by the high amount of salts (> 150 mM) (Sickmann et al., 2002) and highly abundant proteins, but features a highly diverse proteome. In human CSF, the proteomic alterations typically comprise many protein fractionation and concentration steps in association with the depletion of highly abundant proteins, i.e., serum albumin. In general, from few milliliters of human CSF approximately 2600 proteins can be identified (Righetti et al., 2005). Despite all these limitations, cumulative characterization strategy of the CSF proteome field is growing with different advancements of three major MS modules: ion source, mass analyzer, and detection unit. Based on the different ion sources used, most mass spectrometers used in the field of proteomics can be generally categorized into two types: electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) instruments. In neurologic disorders, especially in prion diseases, early detection of potential biomarkers is highly desirable in association with molecular pathogenesis (Vitzthum et al., 2005). Under pathologic conditions, the altered proteins are released from the brain cells into CSF and thus are easily accessible for analysis (Hu et al., 2005). Different studies to date have demonstrated a wide range of subsets of altered proteome potential biomarkers. Apart from this, the high amount of immunoglobulins in many neurologic diseases also indicates the need for specific isolation and fractionation methods (Aebersold and Mann, 2003; Qian et al., 2006). Using
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Fig. 8.3. Proteomics approaches in combination with validation parameters. CSF, cerebrospinal fluid; MALDI-FTMS, matrixassisted laser desorption/ionization Fourier transform mass spectrometry; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; NanoLC-ESI-MS, nano-scale liquid chromatographic electrospray ionization mass spectrometry.
multiple affinity purification columns, most of the abundant proteins from CSF can be depleted before the proteomics analysis (Sitnikov et al., 2006). In prion diseases, only a handful of research in the field of CSF has been performed and showed potential differential proteome alteration. In humans, mass spectrometry and gel-based systems revealed differential proteome-wide alterations in the sporadic form of CJD and demonstrated the role of codon 129 genotype (MM, MV, and VV) and the protease-resistant form of prion protein (type 1 and type 2) at translational level (Piubelli et al., 2006; Gawinecka et al., 2010; Gmitterová et al., 2016). In the prion-infected hamster model, MALDI Fourier transform mass spectrometry (MALDI-FTMS), and support vector machines have demonstrated peptide profiles characteristic of disease state in CSF and used for the further development of prion disease multiple diagnostics markers (Herbst et al., 2009). Some recent studies also demonstrated the comprehensive analysis of extracellular vesicles from human CSF using tandem mass spectrometry in conjunction with bioinformatics (Sanchez et al., 2004; Chiasserini et al., 2014). This proteome-wide screening showed that human CSF extracellular vesicles contain prionogenic proteins such as the amyloid precursor protein and prion protein and showed the involvement of extracellular vesicles in the spread of neurodegeneration to different brain areas (Chiasserini et al., 2014). The proteomic shotgun-based approach of tandem mass tag labeling has established growing consideration for neuroproteomics analysis and proves to be an effective tool for the quantitative identification of 437 potential proteins; with 95% confidence interval 49 proteins were verified from the CSF of CJD patients (Chen et al., 2014). Recent proteome-based data from CSF of
sCJD patients highlighted the differentially regulated levels of complement components (Chen et al., 2016). In conclusion, the proteomic approaches are appropriate for the identification of early diagnostic markers and to get insight into the proteome-wide alteration during the disease course. This could also be useful for the increment of scientific signs for the alterations of CSF homeostasis throughout the pathogenesis of prion diseases.
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