Cerebrospinal fluid biochemical studies in patients with Parkinson's disease: toward a potential search for biomarkers for this disease

The blood-brain barrier supplies brain tissues with nutrients and filters certain compounds from the brain back to the bloodstream. In several neurodegenerative diseases, including Parkinson's disease (PD), there are disruptions of the blood-brain barrier. Cerebrospinal fluid (CSF) has been widely investigated in PD and in other parkinsonian syndromes with the aim of establishing useful biomarkers for an accurate differential diagnosis among these syndromes. This review article summarizes the studies reported on CSF levels of many potential biomarkers of PD. The most consistent findings are: (a) the possible role of CSF urate on the progression of the disease; (b) the possible relations of CSF total tau and phosphotau protein with the progression of PD and with the preservation of cognitive function in PD patients; (c) the possible value of CSF beta-amyloid 1-42 as a useful marker of further cognitive decline in PD patients, and (d) the potential usefulness of CSF neurofilament (NFL) protein levels in the differential diagnosis between PD and other parkinsonian syndromes. Future multicentric, longitudinal, prospective studies with long-term follow-up and neuropathological confirmation would be useful in establishing appropriate biomarkers for PD.


INTRODUCTION
The diagnosis of Parkinson's disease (PD) in live patients is fundamentally clinical, and is based on the presence of its cardinal signs (rest tremor, rigidity, bradykinesia, and postural instability), and the absence of atypical data for idiopathic PD. The final confirmation of the diagnosis is made by post-mortem neuropathological analysis. To date, there are no definitive biomarkers to make an accurate differential diagnosis with other parkinsonian syndromes.
Because the cerebrospinal fluid (CSF) is in close contact with the extracellular space of the brain, it is believed that many of the biochemical modifications in the brain should be reflected in the CSF. Therefore, CSF has been widely investigated in PD and in other parkinsonian syndromes with the aim of acquiring knowledge on the pathogenesis of this disease. This article summarizes the data on analyses performed in the CSF of patients diagnosed with PD compared with controls, with regard to: (1) concentrations of neurotransmitters (mainly monoamines and their metabolites), neuromodulators, and related substances as possible biological markers of the disease itself or its complications; (2) concentrations of endogenous neurotoxins; (3) status of oxidative stress markers or substances which could be related with the induction of oxidative stress or with "neuroprotection" against it; (4) status of inflammation and immunological markers, neurotrophic and growth factors, and (5) concentrations of proteins related with the pathogenesis of PD or other compounds.
Friedman et al. (Friedman, 1985) reported an HVA/5-HIAA ratio in PD patients who developed levodopa-induced dyskinesias (LID) which was significantly higher than in PD patients under levodopa therapy and in controls, but Lunardi et al. (2009) found similar HVA/DA ratios in patients with and without LID. CSF DA, levodopa, and HVA levels were similar in PD patients treated with levodopa with wearing-off motor fluctuations to those without this complication of levodopa therapy, while CSF 3-ortho-methyldopa (3-OMD) levels were higher in the fluctuating patients (Tohgi et al., 1991a). CSF DOPAC and HVA were similar in PD patients with and without depression (Kuhn et al., 1996a), and in patients with major depression with PD than in those without PD (Pålhagen et al., 2010). CSF HVA levels were correlated with striatal uptake in PD patients measured with PET imaging with carbon-11labeled 2β-carbomethoxy-3β-(4-fluorophenyl)-tropane ( 11 C-FT) (Ishibashi et al., 2010). Tohgi et al. (1991bTohgi et al. ( , 1997 found a significant increase in tyrosine, and a significant decrease in CSF levodopa, DA, and 3-OMD in PD patients, which was related with levodopa dosage, and described an additional decrease in 3-OMD in subjects treated with tolcapone (Tohgi et al., 1995a). Other authors reported increased CSF 3-OMD related with levodopa therapy Krygowska-Wajs et al., 1997). On the other hand, Chia et al. (1993) found normal CSF 3-OMD concentrations. Moser et al. (1996) described increased CSF levodopa/3-OMD ratio in PD patients with hallucinations. Iacono et al. (1997) found similar HVA levels in PD patients with postural instability and gait disorders to PD patients without these symptoms.
Although many of the studies of DA metabolites were performed on patients with different types of parkinsonism, with different degrees of severity, and the fact that many of these studies were made using small sample sizes, there is a general consensus that CSF HVA levels are decreased in untreated PD patients and rise after levodopa therapy starts (decreased HVA may not be present in early stages of PD). It is to be expected that low CSF HVA levels should be a reflection of DA depletion in the nigroestriatal system. However, CSF DA metabolite levels are not useful to distinguish between different parkinsonian syndromes and could be normal in early stages of the disease. To our knowledge, no studies have been published regarding the correlation of CSF DA metabolite levels and brain DA levels, although the observation of a correlation between CSF HVA levels and striatal uptake of DA markers in PET imaging (Ishibashi et al., 2010), suggests this correlation.
Studies on the correlation of CSF 5-HT metabolite levels and brain 5-HT levels are lacking. The majority of studies report results on CSF 5-HIAA levels, with the controversial results based on short series of cohorts of patients with PD or other parkinsonian syndromes. Current data do not lend support to the role of CSF 5-HIAA as an unequivocal marker of depression linked to PD.
CSF activity of dopamine-β-hydroxylase (DBH), an enzyme involved in NE synthesis, has been found decreased in PD patients when compared with controls (Matsui et al., 1981;Hurst et al., 1985).
The normality of CSF MHPG levels found in nearly all studies with PD or other parkinsonian syndromes indicates that this is not a useful marker of PD. The correlation between CSF MHPG and brain NE is unknown.

ACETYLCHOLINE (Ach) AND RELATED SUBSTANCES
CSF levels of Ach (Duvoisin and Dettbarn, 1967;Welch et al., 1976;Yamada et al., 1996) and its precursor choline (Aquilonius et al., 1972;Welch et al., 1976;Nasr et al., 1993) have been reported to be similar in PD patients to controls with the exception of one study in which lower CSF choline levels were described in PD patients (Manyam et al., 1990).
CSF activity of butirylcholine-esterase (BchE) have been found to be similar in PD patients and controls (Ruberg et al., 1986;Sirviö et al., 1987), but increased in demented PD patients in a single study (Ruberg et al., 1986). Data on CSF Ach and related substances are scarce and based on short series of patients, and do not permit valid conclusions. CSF GABA levels in PD patients have been found to be decreased, when compared with controls, by many authors (Lakke and Teelken, 1976;Manyam et al., 1980Manyam et al., , 1988Kuroda et al., 1982;Manyam, 1982;Teychenné et al., 1982;Kuroda, 1983;de Jong et al., 1984;Araki et al., 1986;Tohgi et al., 1991c), while others have found this value to be normal (Enna et al., 1977;Abbott et al., 1982;Bonnet et al., 1987;Perschak et al., 1987;Mally et al., 1997;Engelborghs et al., 2003) or even increased . Manyam and Tremblay (1984) found reduced CSF free GABA levels and normality of conjugated levels. Abbot et al. (Perschak et al., 1987) found decreased CSF GABA levels in PD patients treated with levodopa, but not in "de novo" PD patients, while other authors found decreased CSF GABA in untreated PD patients (Manyam, 1982;de Jong et al., 1984), with CSF GABA normal (de Jong et al., 1984;Tohgi et al., 1991c) or slightly decreased (Manyam, 1982) in PD patients under levodopa therapy, suggesting that levodopa increases CSF levels. Teychenné et al. (1982) described low CSF GABA especially in PD patients with poor response to therapy or suffering from "on-off " motor fluctuations.
Data regarding other (non-neurotransmitter) amino acids are even more controversial. CSF levels of neutral and basic amino acids have been reported to be both increased (Van Sande et al., 1971;Lakke and Teelken, 1976;Lakke et al., 1987), and decreased (Molina et al., 1997a). Two groups reported decreased (Molina et al., 1997a;Engelborghs et al., 2003) and another increased CSF levels of taurine (Lakke and Teelken, 1976;Araki et al., 1986;Lakke et al., 1987). Ornithine, citruline, and arginine (implicated in the urea cycle, and the two latter in the synthesis of nitric oxide) have been found to be increased (Van Sande et al., 1971;Lakke and Teelken, 1976;Lakke et al., 1987), normal (Kuiper et al., 2000), or decreased (Molina et al., 1997a). Another group described increased CSF levels of total homocysteine but normal ones of free homocysteine in PD patients (Isobe et al., 2005), with an additional increase after treatment with levodopa, while total methionine levels decreased after this therapy (Isobe et al., 2010a).
In general, the results on CSF amino acid levels in PD patients are inconclusive, because they might be influenced by selection of study subjects, sample size, lack of adequate matching between cases and controls in many studies, differences in antiparkinsonian therapy, and differences in study techniques, storage and handling of the samples Molina et al., 1997a).

NEUROPEPTIDES
Neuropeptides modulate neuronal communication by acting on cell surface receptors. Many of them are co-released with classical neurotransmitters. There have been reports of a number of changes in the concentrations of several neuropeptides in PD brain, which are mainly significant decreases in (Jiménez-Jiménez, 1994): (a) met-enkephalin (MET-ENK), substantia P (SP), and cholecystokinine 8 (CCK-8) in the substantia nigra; (b) MET-ENK and leu-enkephalin (LEU-ENK) in the putamen and globus pallidus; (c) MET-ENK in the ventral tegmental area; (d) SP, somatostatin and neurotensin in the neocortex, and (e) somatostatin and neurotensin in the hippocampus. It is likely that many of these changes are related with dopaminergic deficit, and the only clear relationship between a neuropeptide and a clinical feature of PD is that of somatostatin with the presence of cognitive impairment (Jiménez-Jiménez, 1994). Table 2 summarizes the findings of classical studies on CSF neuropeptide levels in PD patients. Most of these studies enrolled limited series of patients.
In recent years, there has been increased interest in the possible role of orexin-A/hypocretin-1, a neuropeptide hormone implicated in the pathogenesis of narcolepsia, on the development of excessive daytime sleepiness in PD patients. Since the first report by Drouot et al. (2003), who described decreased ventricular CSF orexin levels in PD patients, which were related with the severity of the disease, other authors have confirmed decreased CSF orexin in PD (Fronczek et al., 2007;Asai et al., 2009) and in other neurodegenerative parkinsonisms (Yasui et al., 2006), and the relation of CSF orexin with severity of PD (Asai et al., 2009), and with the presence of sleep attacks (Asai et al., 2009). In contrast, Compta et al. (2009a) found no significant differences in CSF orexin levels between demented PD patients, non-demented PD patients, and healthy controls, and found no relation between CSF orexine and Epworth sleepiness scale or Mini-Mental State Examination. Drouot et al. (2011) found a lack of association between low ventricular CSF orexin and sleepiness in PD, and a relation between high levels of orexin-A in PD associated with loss of REM muscle atonia (Bridoux et al., 2013), while Wienecke et al. (2012) reported association between low CSF orexin levels and sleepiness in PD. Finally, Pålhagen et al. (2010) described similar CSF orexin levels in patients with major depression with or without concomitant PD. The results regarding orexin A are controversial, and await confirmation. Pisani et al. (2005Pisani et al. ( , 2010 found increased CSF levels of the endogenous cannabinoid anandamide in untreated PD patients, which were unrelated to the severity of the disease (Pisani et al., 2005) and reversed by chronic dopaminergic replacement (Pisani et al., 2010). Zhou et al. (1997) found decreased CSF β-phenylethylamine (PEA) levels in PD patients which were correlated negatively with Hoehn & Yahr stage.

CYCLIC NUCLEOTIDES
These compounds act as intracellular second messengers of neurotransmitters or other compounds such as nitric oxide (NO). The most important are cyclic adenosine 3 5 monophosphate (cAMP) and cyclic guanosine 3 5 monophosphate (cGMP). Belmaker et al. (1978) reported a 40-50% decrease of CSF cAMP and an 80-90% decrease of CSF cGMP levels in PD patients who were not related with levodopa therapy. Decreased CSF cAMP levels in PD have also been reported in another study , while others found this value to be normal (Cramer et al., 1973(Cramer et al., , 1984Covicković-Sternić et al., 1987;Oeckl et al., 2012), both in PD patients with and without dementia (Oeckl et al., 2012). Four further studies described normal CSF cGMP levels Covicković-Sternić et al., 1987;Ikeda et al., Frontiers in Cellular Neuroscience www.frontiersin.org November 2014 | Volume 8 | Article 369 | 5  1995; Oeckl et al., 2012), while another found a non-significant trend toward higher CSF cGMP levels in PD patients when compared with controls and higher levels in levodopa-treated PD patients compared with those without levodopa treatment .

BIOPTERIN DERIVATIVES AND OTHER COFACTORS
Biopterins act as cofactors for aromatic amino acid hydroxylases, which produce a number of neurotransmitters including DA, NE, epinepherine, and 5-HT and are also required for the production of NO. CSF levels of neopterin and biopterin have been found decreased in PD patients by several groups, especially in those with early-onset PD (Fujishiro et al., 1990;Furukawa et al., 1992), and in carriers of the PARK8 mutation (Koshiba et al., 2011), which was negatively correlated with duration of illness in those patients with akinetic-rigid PD (Furukawa et al., 1991). In contrast, another group found increased CSF neopterin in PD (Widner et al., 2002). CSF concentration of hydroxylase cofactor, predominantly composed of tetrahydrobiopterin (BH 4 ), has also been found decreased (Williams et al., 1980a,b).
Thiamine is an essential cofactor for several important enzymes involved in brain oxidative metabolism. Our group found normal CSF levels of thiamine-diphosphate, thiaminemonophosphate, free thiamine, and total thiamine in PD patients (Jiménez-Jiménez et al., 1999).

ENDOGENOUS NEUROTOXINS
One of the classical etiological hypotheses of PD is related with the presence of endogenous substances which share structural similarities with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that induces a parkinsonism resembling PD.
Moser et al. (Moser and Kömpf, 1992;Moser et al., 1995) identified two tetrahydroisoquinolin (TIQ) derivatives in the CSF of PD patients, but not in healthy controls, 2-methyl and 1methyl-6,7-dihydroxy1,2,3,4-TIQ (2-MDTIQ and 1-MDTIQ or salsolinol). This group described a relation between high salsolinol levels and the presence of visual hallucinations (Moser et al., 1996), and reported an increased HVA/3OMD ratio in PD patients in which 2-MDTIQ was detected when compared with those PD in which it was not detectable.
CSF salsolinol levels have been reported to be increased in PD patients compared with controls by other groups (Maruyama et al., 1996;Antkiewicz-Michaluk et al., 1997;Krygowska-Wajs et al., 1997;Naoi and Maruyama, 1999), especially in demented PD patients , and in those patients with more severe parkinsonism , although other authors have described a trend toward decrease in CSF salsolinol levels with the progression of the disease . In contrast, another group reported similar CSF salsolinol (Müller et al., 1999a,b), but higher levels of harman and norharman β-carbolines (structural analogs of MPTP as well) in PD patients than in controls (Kuhn et al., 1996b). CSF levels of 1-benzyl-1,2,3,4-TIQ have also been found by another group to be increased (Kotake et al., 1995). Matsubara et al. (1995) measured β-carbolinium cations (BC+s) in the lumbar CSF of 22 PD patients and 11 age-matched controls, and found the 2,9-dimethylnorharmanium cation (2,9-Me2NH+) in 12 PD patients but not in controls. This group described decreased activity of nicotinamide N-methyltranserase (NNMT), an enzyme that catalyzes the N-methylation of nicotinamide and other pyridines in the CSF of younger PD patients compared with younger controls, and a trend toward decrease with aging in PD patients (Aoyama et al., 2001).
The results of studies on neurotoxins related with the risk for PD are based on small series and are not conclusive.

OXIDATIVE STRESS MARKERS
Because there is much evidence on the contribution of oxidative stress in the pathogenesis of PD (Figure 1) , the measurement of oxidative stress markers and substances related with oxidative and defense against oxidative phenomena in the CSF of PD patients is useful. Data regarding lipid peroxidation markers are controversial, while DNA oxidation markers have been found to be increased ( Table 3).
Transition metals such as iron, copper, and manganese, act as prooxidant agents, although copper is also essential for the antioxidant function of the protein ceruloplasmin, and copper and manganese are constituents of the cytosolic Cu +2 /Zn +2 and the mitochondrial Mn +2 -superoxide-dismutases (SOD, protective against oxidative processes). Zinc has antioxidant activity and is a constituent of Cu +2 /Zn +2 -SOD . The results of studies with CSF levels of iron and copper are controversial ( Table 3), but a recent meta-analysis showed similar values in PD patients to controls (Mariani et al., 2013), thus suggesting that these metals are not useful as markers of PD. Together with its role in glutamate excitotoxity, NO could contribute to oxidative stress mechanisms in the pathogenesis of PD by interacting with ferritin to release iron, inducing mitochondrial complex I damage , and by inducing nitrosylation of proteins (Fernández et al., 2013). However, studies on CSF levels of nitrates and nitrites have given controversial results ( Table 3).
Among other antioxidant enzymes and substances (Table 3), one study involving an important number of early PD patients showed the relationship between the presence of relatively higher levels of urate and the slower rates of clinical decline (Ascherio et al., 2009), despite the fact that CSF urate levels were found to be similar in PD patients and controls in the same study.
CSF levels of pros-methylimidazol acetic acid, an isomer of the histamine metabolite tele-methylimidazol acetic acid, have been found to be decreased in PD , and were highly positively correlated with the severity of the disease .
CSF complement 3 (C 3 ) and factor H (FH) levels were reported to be normal in one study (Wang et al., 2011), while another described a decrease in several isoforms of C 3b, C 4b , FH, and factor B (Finehout et al., 2005), and another normal C 4d (Yamada et al., 1994). CSF levels of heat shock proteins Hsp65 and Hsp70 have been found to be increased (Fiszer et al., 1996), and PD patients have shown higher HLA-DR expression in CSF monocytes in comparison with controls (Fiszer et al., 1994a).
Oligoclonal IgG bands have not been detected in the CSF of PD patients (Chu et al., 1983), but antibodies against DA neurons have been detected in 78% of PD patients and in only 3% of controls (Carvey et al., 1991), and the CSF of PD patients has shown a higher proportion of gamma-delta-T+ cells than in controls (Fiszer et al., 1994b).
The results of studies on inflammatory and immunological markers in PD have a low number of patients and controls enrolled, and are inconclusive.

GROWTH AND NEUROTROPHIC FACTORS
CSF Brain Derived Neurotrophic Factor (BDNF) levels have been found to be similar in PD patients with major depression to those in patients with major depression without PD in one study (Pålhagen et al., 2010), while another described this value as increased in PD patients compared with controls (Salehi and Mashayekhi, 2009). CSF Transforming Growth Factor α (TGF-α) has been found to be increased in juvenile parkinsonism (Mogi and Nagatsu, 1999). TGF-β1 has been found to be increased (Mogi et al., 1995(Mogi et al., , 1996aVawter et al., 1996;Mogi and Nagatsu, 1999) or normal (Rota et al., 2006), and TGF-β2 increased (Vawter et al., 1996). CSF insulin-like growth factor-1 (IGF-1) and IGF binding proteins (IGFBPs) expression is increased in PD patients (Mashayekhi et al., 2010). Finally, a single study found a non-significant trend toward increased CSF levels of neuroregulins (which belong to the Epidermal Growth Factor or EGF family) in PD patients (Pankonin et al., 2009). The results of studies on growth and neurotrophic factors in PD, involving a low number of patients and controls, do not permit definitive conclusions.

PROTEINS INVOLVED IN THE PATHOGENESIS OF PARKINSON'S DISEASE MICROTUBULAR ASSOCIATED PROTEIN Tau (MAPT)
Because MAPT gene is one of the main genes involved in the risk for PD (Alonso-Navarro et al., 2014), the measurement of CSF protein tau levels are hypothetically useful as a marker of this disease. Tau protein is important for maintaining the stability of axonal microtubules involved in the mediation of fast axonal transport of synaptic constituents. Hyperphosphorylation of tau causes reduces binding affinity for microtubules, leading to their malfunction. Following neuronal damage, tau is released into extracellular space and may be increased in the CSF. Tau is an important component of the neurofibrillary tangles (pairwise, helical protein filaments which are found in the cytoskeleton or neuronal cells in Alzheimer's disease (AD) brains. CSF tau protein levels are increased in AD patients, and so are a useful marker for this disease. The high risk of PD patients of developing cognitive impairment or dementia patients makes measurement of CSF tau reasonable as a possible marker of this disease.
Many studies have shown similar CSF total tau and phosphorylated tau (phosphotau) in PD patients to controls (Blennow et al., 1995;Molina et al., 1997c;Jansen Steur et al., 1998;Sjögren et (Mollenhauer et al., 2006;Compta et al., 2009b). The 33 KDa/55 KDa tau isoforms ratio have also been found to be normal in PD (Borroni et al., 2008(Borroni et al., , 2009), but decreased in progressive supranuclear palsy (PSP), and normal in patients with diffuse Lewy body disease (DLBD), demented PD patients (PDD), AD, and frontotemporal dementia (FTD) (Borroni et al., 2008(Borroni et al., , 2009). Some authors have found decreased CSF total tau and phosphotau levels when compared with controls (Mollenhauer et al., 2011;Shi et al., 2011;Kang et al., 2013) and similar levels in PD to PSP, DLBD, and MSA (Mollenhauer et al., 2011), while others found higher CSF tau in DLBD compared with PDD patients (Andersson et al., 2011), and still others higher CSF total tau in MSA than in PD patients (Herbert et al., 2014). Hall et al. (2012) reported decreased CSF total tau and normal phosphotau both in PD and PDD, while total tau was increased in CBD and normal in PSP, DLBD, and MSA, and phosphotau was decreased in PSP and MSA in comparison with controls.
Přikrylová Vranová et al. (2010) found increased CSF tau levels in PD patients with less than 2 years of evolution, and increased CSF tau levels which were higher in patients with PDD than in PD, and in PD than in controls, and similar CSF tau in DLDB than in controls (Vranová et al., 2014). This group and others found increased CSF total tau levels in patients with non-tremor variants of PD as compared to tremor-dominant PD and controls (Jellinger, 2012;Přikrylová Vranová et al., 2012). Compta et al. (2011) described increased CSF tau levels in PD patients carrying the allele rs242557A. Siderowf et al. (2010) showed a lack of association between baseline CSF tau levels and cognitive decline in PD patients. Patients with corticobasal degeneration (CBD) and PSP have shown higher CSF total and phospotau levels (Aerts et al., 2011), and patients with DLBD showed similar CSF tau levels to PD patients in one study (Ohrfelt et al., 2009), while other authors found higher CSF tau levels in AD than in DLBD, in DLDB higher than in PDD, and in PDD higher than in PD .
Baseline CSF levels of total and phosphotau in the DATATOP study, involving 403 early PD patients, were negatively correlated with disease progression assessed with the Unified PD Rating Scale (UPDRS) (Zhang et al., 2013). Beyer et al. (2013) reported a lack of correlation between CSF levels of total and phosphotau, and ventricular size in 73 nondemented PD patients and 18 PD patients with mild cognitive impairment.
The results of the studies reported on CSF tau levels in PD are summarized in Table 4. Although these results are not conclusive, CSF tau levels could be related to the progression of the disease (Zhang et al., 2013), and to the preservation of cognitive function in PD patients (Stewart et al., 2014).

ALPHA-SYNUCLEIN
Alpha-synuclein (α-synuclein) is a 140 amino acid-long presynaptic protein, which is the major component of the Lewy bodies (the neuropatologic hallmark of PD), and has been implicated in the pathogenesis of PD and in synucleinopathies such as MSA and DLBD. Mutations of the α-synuclein (SNCA) gene are related with early-onset monogenic familial PD and are associated with increased risk for sporadic PD (Alonso- Navarro et al., 2014). Although early studies failed to detect the native form of α-synuclein in the CSF of PD and control patients (Jakowec et al., 1998), later studies have detected monomeric SNC in the CSF, with similar levels in PD patients and controls (Borghi et al., 2000). Several studies have found similar CSF total αsynuclein levels in PD patients and in controls (Woulfe et al., 2002;Ohrfelt et al., 2009;Park et al., 2011;Parnetti et al., 2011;Tateno et al., 2012) and others decreased CSF α-synuclein in PD (Tokuda et al., 2006;Hong et al., 2010;Mollenhauer et al., 2011Mollenhauer et al., , 2013Hall et al., 2012;Wang et al., 2012;Kang et al., 2013;Wennström et al., 2013;Parnetti et al., 2014a,b;Mondello et al., 2014;van Dijk et al., 2014), DLBD (Parnetti et al., 2011;Wennström et al., 2013), MSA Mondello et al., 2014), and PSP . Four studies have reported increased CSF oligomeric α-synuclein levels in PD compared with controls (Tokuda et al., 2010;Park et al., 2011;        et al., 2014a,b), and one of them showed increased CSF α-Syn in PD patients compared with patients with PSP and AD (Tokuda et al., 2010). Wang et al. (2012) found increased CSF levels of the phosphorylated α-synuclein phospho-Ser129 (PS-129) in PD patients when compared with controls, but lower levels in MSA and PSP of this protein than in PD patients and controls. Aerts et al. (2012) found similar CSF α-synuclein levels in PD patients to DLBD, PSP, and MSA. Hall et al. (2012) found higher CSF α-synuclein in PSP than in PD, PDD, DLBD, and MSA. Tateno et al. (2012) reported similar CSF α-synuclein levels in PD, MSA, DLBD, and controls but higher CSF αsynuclein levels in AD patients, while Ohrfelt et al. (2009) found higher CSF α-Syn levels in AD than in DLDB and PD, and in DLBD than in PD patients. Foulds et al. (2012) found similar post-mortem CSF total α-synuclein levels in PD, MSA, DLBD, and PSP, but increased CSF levels of phosforylated oligomers in MSA.
van Dijk et al. (2014) reported a lack of relation between CSF α-synuclein levels and striatal dopaminergic deficit measured by dopamine transporter binding and single photon emission computed tomography. In addition, a recent study by Shi et al. (2012) described a lack of relation between the loss of striatal dopaminergic function, assessed by positron emission tomography (PET), and CSF α-synuclein levels, in asymptomatic carriers of mutations in the LRRK2 gene. CSF neurosin (a protease that degrades α-synuclein) levels have been found to be decreased (Wennström et al., 2013).
Lower baseline CSF α-synuclein levels in the DATATOP study predicted a better preservation of cognitive function in early PD patients with up to 8 years of follow-up (Stewart et al., 2014).
The results of the studies reported on CSF α-synuclein levels in PD are summarized in Table 5. The majority of recent studies have shown decreased CSF α-synuclein levels both in PD and in other synucleopathies. Therefore, this should be a useful marker to distinguish this disease from controls, but not to distinguish among synucleopathies.
Baseline CSF Aβ levels in the DATATOP study, were negatively correlated with disease progression assessed with UPDRS (Zhang et al., 2013). Baseline CSF levels of Aβ1-42 in two studies (Siderowf et al., 2010;Parnetti et al., 2014b); and the combination of lower baseline CSF Aβ, worse verbal learning, semantic fluency and visuoperceptual scores, and thinner superior-frontal/anterior cingulated in precentral regions by 3Tbrain-Magnetic Resonance Imaging in another (Compta et al., 2013) have been associated with further cognitive decline in PD patients.

References Cases/Controls
The results of the studies reported on CSF Aβ levels in PD are summarized in Table 6. Many of these studies suggest the potential usefulness of CSF Aβ1-42 levels to predict cognitive impairment in PD patients.
CSF neuronal thread protein (NTP) levels have been found increased when compared with controls and decreased when compared with AD patients in one study (de la Monte et al., 1992), and similar to those of controls in another (Yamada et al., 1993). CSF annexine V has been found to be decreased in PD (Vermes et al., 1999). Glial fibrilar acidic protein (GFAP) has been found to be normal in the CSF of PD, MSA, PSP, and CBD patients (Constantinescu et al., 2010). CSF levels of the glial activation marker YKL-40 have been found to be decreased in PD, MS, PSP, and CBD (Olsson et al., 2013).

OTHER PROTEINS
Defects in the gene encoding DJ-1 protein cause an autosomal recessive early-onset PD, PARK7 (Alonso- Navarro et al., 2014). This protein is also a marker of oxidative stress. CSF levels of DJ-1 protein have been found to be increased in PD in 2 studies (Waragai et al., 2006;Herbert et al., 2014) and decreased in another 2 (Shi et al., 2011;Hong et al., 2010). One of these studies described decreased CSF DJ-1 in MSA as well (Shi et al., 2011), and other increased DJ-1 in MSA compared with PD and with controls (Herbert et al., 2014). Shi et al. (2012) described a lack of relation between the loss of striatal dopaminergic function and CSF DJ-1 levels in asymptomatic carriers of mutations in the LRRK2 gene (PARK8). The results on DJ-1 are, therefore, inconsistent and should not be considered as a marker of PD.   Defects in the gene encoding ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) cause familial PD, PARK5. A recent study found decreased CSF UCH-L1 levels in PD, MSA, and PSP compared with controls (Mondello et al., 2014).

OTHER COMPOUNDS
In patients with PD there have been reports of normal CSF levels of the proteoglycan N-acetyl neuraminic acid (Lipman and Papadopoulos, 1973), and CSF insulin levels (Jiménez-Jiménez et al., 2000) have been found normal in PD patients.
The CSF levels of corticosterone (Pålhagen et al., 2010) and neuroactive steroids such as allopregnanolone (THP) and 5 αdihydroprogesterone (DHP) (di Michele et al., 2003) have been found to be decreased in PD. Björkhem et al. (2013) reported that 10% of the PD patients were found to have increased CSF levels of 24S-hydroxycholesterol, and that there was a significant correlation between this value and duration of the disease. Lee et al. (2008) described a significant increase in the CSF levels of the polyunsaturated fatty acid eicosapentanoic acid (EPA) in patients with PD and MSA. Paik et al. (2010) measured several polyamines in the CSF of patients with PD, MSA and controls. These substances are important for cell growth, and act as important modulators of a variety of ion channels, including glutamate NMDA and AMPA receptors. CSF total polyamine, N 1 acetyl-cadaverine, and cadaverine levels were increased both in PD and MSA, but PD patients showed higher CSF putrescine and lower CSF spermidine levels than MSA and controls, and MSA patients showed lower CSF N 1 acetylputrescine than PD and controls. CSF N 8acetylspermidine levels were higher in PD patients than in controls, and in MSA than in PD patients and controls.

CONCLUSIONS
(A) The majority of classical biochemical studies on neurotransmitter and related substances have described decreased CSF HVA, and normal NE, MHPG, ACh, AChE, glutamate, aspartate, and glycine levels in patients with PD. Results on CSF GABA and 5-HIAA levels are controversial. Many of these classical studies included patients with different types of Parkinsonism and had a limited number of patients and controls. (B) Studies on the possible value of endogenous neurotoxins, oxidative stress markers, inflammatory and immunological markers, and growth and neurotrophic factors as biological markers of PD should be considered as inconclusive. The most consistent finding related with these issues is the possible role of CSF urate on the progression of the disease (Ascherio et al., 2009). (C) Data regarding the role of CSF total tau and phosphotau as biological markers for PD are inconsistent. The most interesting findings are the possible relations of these markers with the progression of the disease (Zhang et al., 2013), and with the preservation of cognitive function in PD patients (Stewart et al., 2014). (D) CSF α-synuclein levels have been found to be decreased in most, but not all, studies in PD patients compared with controls. This marker should be useful for the differential diagnosis between synucleopathies and other parkinsonian syndromes, but its usefulness to differentiate among synucleopathies (PD, PDD, DLBD, and MSA), remains to be elucidated. (E) CSF Aβ1-42 levels could be considered as a useful marker of the presence of further cognitive decline in PD patients. (F) CSF NFL protein levels should be useful for the differential diagnosis of PSP, MSA, CBD, and PDD from PD, but not to discriminate between PD and healthy controls.

FUTURE APPROACHES
While possible biomarkers for PD in classical studies have been hypothesis-driven, attempts to develop effective procedures for the differential diagnosis of PD in its early stages have led to the performance of CSF multianalyte methods including systematic measurements of patterns of variation in proteins (proteomics) or small molecules (metabolomics). These methods have led to the identification of possible unexpected biomarkers of diseases involved in neurodegenerative processes. However, the results of these types of studies, which are briefly described below, are not clearly established and await replication. Guo et al. (2009), in a proteomic analysis of the CSF of PD patients and controls, found significantly higher CSF levels of apolipoprotein E, autotoxin, and some SOD1 isoforms, and lower levels of complement C 4 when compared with controls, while Pigment epithelium-derived factor (PEDF or serpin F1) was not significantly increased, and complement C 3 and haptoglobin were similar in PD patients and controls. Zhang et al. (2008) performed a proteomics-discovered multianalyte profile (MAP) in CSF on 95 control subjects, 48 patients with probable AD, and 40 patients with probable PD, and concluded that the optimal MAP leading to the correct diagnosis was composed of the following proteins in order of contribution: tau, BDNF, IL-8, Aβ42, β2-microglobulin, vitamin D binding protein, apoA2, and apoE. Maarouf et al. (2012) analyzed ventricular CSF from PD and controls obtained in the immediate post-mortem period using a two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrophotometry protein identification, and found differences between the 2 groups in 6 molecules: fibrinogen, transthyretin, apoE, clusterin, apoA1, and glutathione-Stransferase-Pi (GSTP). Trupp et al. (2014) reported a generally lower level of metabolites in PD as compared to controls, with a specific decrease in 3-hydroxyisovaleric acid, tryptophan, and creatinine, a significant decrease in the levels of Aβ-38 and Aβ-42, and an increase in soluble amyloid peptide precursor α (APPα) in CSF of patients.
Ideally, future studies should fulfill the following conditions: (a) a multicenter and prospective design; (b) inclusion of patients diagnosed with PD and other types of parkinsonism according to standardized criteria; (c) measurement of multiple potential biological markers in the CSF; (d) a very long-term follow-up period (till death as end-point), with assessment of both clinical features and serial determinations of the biological markers; and (e) final neuropathological confirmation by examination of the brains of patients at death (this is lacking in most of the studies published).

ACKNOWLEDGMENTS
English grammar was reviewed by Professor James McCue. Natalia Gutiérrez Casado (Librarian of Hospital Universitario del Sureste) contributed in getting many of the classical references. Research at authors' laboratories is financed by grants PI12/00241, PI12/00324, and RETICS RD12/0013/0002 from Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Spain, Innovation and GR10068 from Junta de Extremadura, Spain. Financed in part with FEDER funds from the European Union.