]>NBA5543S0197-4580(01)00218-410.1016/S0197-4580(01)00218-4Elsevier Science Inc.Fig. 1Fig. 1 represents the electrophoretic pattern of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) (SWISS-PROT number P09543). Frontal cortex, control sample.Fig. 2Fig. 2 represents the electrophoretic pattern of carbonic anhydrase II (CA II) (SWISS-PROT number P00918). Frontal cortex, control sample.Table 1Quantification of CNPase in human brain samples (arbitrary units)legend legend legendBrain regionControl mean ± SD (N)Down syndrome mean ± SD (N)Alzheimer’s disease mean ± SD (N)Frontal6.10 ± 2.35 (6)∗3.38 ± 1.64 (6)∗2.70 ± 0.95 (6)Temporal5.19 ± 2.02 (9)∗∗3.30 ± 1.28 (9)3.69 ± 1.94 (9)Occipital4.37 ± 0.98 (11)5.19 ± 1.85 (8)4.79 ± 1.86 (9)Parietal4.33 ± 2.30 (7)4.38 ± 2.78 (8)2.59 ± 1.53 (7)Cerebellum2.75 ± 0.47 (5)3.65 ± 1.24 (6)3.78 ± 1.84 (9)Thalamus5.41 ± 1.96 (7)5.06 ± 1.48 (7)4.74 ± 2.82 (5)Caudate nucleus4.02 ± 1.35 (6)6.04 ± 1.73 (7)5.53 ± 1.91 (8)legendData represent means ± SD (N = number of samples used from overall experimental group).legendMann-Whitney U-test was used to test differences between DS or AD and control group.legendStatistical significance∗p = 0.04,∗∗p = 0.02.Table 2Quantification of CA II in human brain samples (arbitrary units)legendBrain regionControl mean ± SD (N)Down syndrome mean ± SD (N)Alzheimer’s disease mean ± SD (N)Frontal11.16 ± 3.87 (9)16.70 ± 5.65 (5)9.74 ± 3.69 (7)Temporal10.54 ± 5.25 (10)13.73 ± 8.27 (8)13.55 ± 7.79 (10)Occipital11.16 ± 2.98 (9)16.25 ± 10.12 (8)12.03 ± 5.74 (10)Parietal13.86 ± 6.53 (7)10.66 ± 3.76 (8)14.14 ± 2.32 (5)Cerebellum8.87 ± 3.39 (11)7.15 ± 1.66 (7)9.50 ± 7.20 (9)Thalamus11.55 ± 2.93 (10)14.70 ± 3.41 (6)12.78 ± 3.38 (7)Caudate nucleus15.72 ± 8.73 (6)13.16 ± 3.35 (7)9.95 ± 2.41 (7)legendData represent mean ± SD (N = number of samples used from overall experimental group)Decreased brain levels of 2′,3′-cyclic nucleotide-3′-phosphodiesterase in Down syndrome and Alzheimer’s diseaseVlkolinskyRomanVlkolinskýa1NigelCairnsbMichaelFountoulakiscGertLubeca*Gert.Lubec@akh-wien.ac.ataUniversity of Vienna, Department of Pediatrics, Waehringer Guertel 18, A 1090 Vienna, AustriabInstitute of Psychiatry, Brain Bank, King’s College, De Crespigny Park London SE5 8AF, UKcPharmaceutical Research, Genomic Technologies, F. Hoffmann-La Roche, Ltd., CH-4070 Basel, Switzerland*Corresponding author. Tel.: +43-1-40400-32151The author is on leave from Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, Slovak Republic.AbstractIn Down syndrome (DS) as well as in Alzheimer’s disease (AD) oligodendroglial and myelin alterations have been reported. 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) and carbonic anhydrase II (CA II) are widely accepted as markers for oligodendroglia and myelin. However, only data on CNPase activity have been available in AD and DS brains so far. In our study we determined the protein levels of CNPase and CA II in DS, AD and in control post mortem brain samples in order to assess oligodendroglia and myelin alterations in both diseases. We used two dimensional electrophoresis to separate brain proteins that were subsequently identified by matrix assisted laser desorption and ionization mass-spectroscopy (MALDI-MS). Seven brain areas were investigated (frontal, temporal, occipital and parietal cortex, cerebellum, thalamus and caudate nucleus). In comparison to control brains we detected significantly decreased CNPase protein levels in frontal and temporal cortex of DS patients. The level of CA II protein in DS was unchanged in comparison to controls. In AD brains levels of CNPase were decreased in frontal cortex only. The level of CA II in all brain areas in AD group was comparable to controls. Changes of CNPase protein levels in DS and AD are in agreement with the previous finding of decreased CNPase activity in DS and AD brain. They probably reflect decreased oligodendroglial density and/or reduced myelination. These can be secondary to disturbances in axon/oligodendroglial communication due to neuronal loss present in both diseases. Alternatively, reduced CNPase levels in DS brains may be caused by impairment of glucose metabolism and/or alterations of thyroid functions.KeywordsOligodendrogliaNeurodegenerationMALDI-MSCarbonic anhydrase IIMyelin1IntroductionIn Down syndrome (DS), Alzheimer’s type of neurodegeneration develops invariably in later postnatal life, typically over the age 30. The neurodegenerative process in DS is sharing many pathological findings with Alzheimer’s disease (AD), the typical hallmarks being amyloid deposition, formation of neurofibrillary tangles, congophilic angiopathy and extensive neuronal cell loss [17,23]. In addition to neuronal involvement, glial cells also markedly participate in DS and AD pathology. For instance, persistent reactive astrogliosis has been clearly demonstrated in DS and AD brains [61,45].It was proposed that oligodendroglia, the principle myelin forming cells within CNS is also significantly affected in both diseases. In patients with DS and AD markers for oligodendroglial cells and myelin formation e.g. the activity of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), amount of myelin proteins and cerebroside were shown to be decreased [4,52]. Accordingly, myelination seems to be delayed in young DS patients as revealed by histology [65,66] and magnetic resonance imaging [28]. On the other hand, the proliferation of oligodendroglia by amyloid P component has been described in DS [1].Although evidence for the involvement of oligodendroglia in DS and AD pathology is accumulating, oligodendroglial markers such as CNPase and carbonic anhydrase II (CA II) activities but not their protein levels have been quantified so far [4,6,52]. Measuring enzyme activity may be misleading however, as this parameter can be influenced by different factors such as substrate availability/concentration, protein phosphorylation, etc. [49]. Moreover, the CNPase activity can be altered by the extent of neuro-glial communication therefore it need not to correspond to its protein levels [37]. This communication, in the case of DS and AD, may be considerably altered due to the extensive neuronal loss being present in both diseases.The limited data on white matter involvement in both diseases made us examine two oligodendroglial markers, CNPase and CA II in several brain regions of patients with DS and AD as compared to controls at the protein level. The use of 2-dimensional gel electrophoresis with subsequent unambiguous identification and characterization of the spots by matrix assisted laser desorption and ionization mass-spectroscopy (MALDI-MS) allowed the concomitant determination of two oligodendroglial markers on one gel.2′,3′-cyclic nucleotide 3′-phosphodiesterase (EC 3.1.4.37) is a protein accounting for approximately 4% of myelin protein content and for a long time it has been considered as an index of myelin formation [for review see 52,63]. There are two isoforms of this protein, CNP1ase (∼46 kDa) and CNP2ase (∼48 kDa), with CNP2ase differing from CNP1ase by a 20 amino acid extension at its N-terminus. Developmentally, it is one of the earliest myelination-specific polypeptides, synthesized by oligodendrocytes and its synthesis persists into adulthood, suggesting a role in the synthesis and maintaince of the myelin sheath [49]. It is synthesized on free polysomes in the oligodendrocyte cell bodies from where it reaches myelin. In myelin, CNPase is excluded from compact lamellar domains but it is present within the cytoplasmic channels that transverse the myelin sheath [15]. Therefore, it can be found at cytoplasmic sites of oligodendrocyte cell bodies, and at the periphery in the elongated oligodendroglial processes [43,47,67]. The physiological role of CNPase is not understood, but it is thought to be functionally involved in oligodendrocytes surface membrane expansion and migration during the earliest stages of axonal ensheathment [67].Carbonic anhydrases (carbonate hydrolase: EC 4.2.1.1) represent a group of isozymes the basic physiological function of which is to catalyze the reversible hydration of carbon dioxide to form H2CO3 which in turn decomposes to H+ and HCO3−. CA II is the main isozyme of the CA family in the human brain. It is located mainly in oligodendroglia and less in astrocytes [10,11,12,21,30]. CA II is a monomeric protein of theoretical relative molecular weight 29 kDa and theoretical pI value of 7.5 [3,35]. It has multiple functions in the brain and it’s deficiency is associated with pathological consequences such as mental retardation and brain calcification [57,62]. CA II is providing HCO3−, which regulates membrane transport of Na+/water and contributes to cerebrospinal fluid formation. CA II has been proposed to participate in the processes of myelination. Controlling extracellular ion concentration, CA II activity is probably involved also in regulation of neuronal excitability [44] and susceptibility to seizures [54].In this report we describe decreased levels of CNPase protein in frontal and temporal cortex of DS brain samples along with unchanged levels of CA II. Similarly, we detected decreased protein levels of CNPase in frontal lobe of AD brains while CA II levels were not altered.2Methods2.1Brain samples for determination of protein levelsThe brain samples from frontal, temporal, parietal, occipital cortex, cerebellum, thalamus and caudate nucleus of caryotyped patients with DS (n = 9; 3 females, 6 males; 55.7 ± 7.48 years old), AD (n = 11, 4 females, 7 males; 59.55 ± 6.1 years old) and controls (n = 14, 4 females, 10 males 56.1 ± 9.87 years old) were used for the studies at the protein level. Post mortem brain samples were obtained from the MRC London Brain Bank of Neurodegerative Diseases, Institute of Psychiatry). The controls were brains from individuals with no history of neurological or psychiatric disorders. The major cause of death was bronchopneumonia in DS and AD and heart disease in controls. Post mortem interval of brain dissection in DS, AD and controls was 31.44 ± 19.56, 32.45 ± 27.83 and 37.67 ± 21.8 h, respectively). Tissue samples were stored at −70°C and the freezing chain was never interrupted.2.2Two-dimensional electrophoresis of brain proteinsBrain tissue was suspended in 0.5 ml of sample buffer consisting of 40 mM Tris, 5 M urea (Merck, Darmstadt, Germany), 2 M thiourea (Sigma, St Louis, MO, USA), 4% CHAPS (Sigma), 10 mM 1,4 dithioerythritol (Merck), 1 mM EDTA (Merck) and a mixture of protease inhibitors, 1 mM PMSF and 1 μg of each pepstatin A, chymostatin, leupeptin and antipain. The suspension was sonicated for approximately 30 s and centrifuged at 10000 × g for 10 min and the supernatant was centrifuged further at 150000 × g for 45 min. The protein content in the supernatant was determined by the Coomassie blue method [7].The 2-D gel electrophoresis was performed essentially as reported [36]. Samples of approximately 1.5 mg were applied on immobilized pH 3-10 nonlinear gradient strips (IPG, Amersham Pharmacia Biotechnology Uppsala, Sweden), at both, the basic and acidic ends of the strips. The proteins were focused at 300 V for 1 h, after which the voltage was gradually increased to 3500 V within 6 h. Focusing was continued at 5000 V for 48 h. The second-dimensional separation was performed on 9–16% linear gradient polyacrylamide gel (Serva, Heidelberg, Germany and Bio-Rad, Hercules, CA, USA). The gels were stained with colloidal Coomassie blue (Novex, San Diego, CA, USA) for 48 h, destained with water and scanned in a Molecular Dynamics Personal densitometer. The images were processed using PhotoShop (Adobe) and PowerPoint (Microsoft) software. Protein spots were quantified using the ImageMaster 2D Elite software (Amersham Pharmacia Biotechnology). During the quantification the volumes of the spots were calculated as a summation of pixel intensities making up the spots. The individual spot volume was expressed as a percentage of the total volume of the spots present in the gel part considered.2.3Matrix assisted laser desorption ioization—mass spectroscopy (MALDI-MS)MALDI-MS analysis was performed as described with minor modifications [20]. Briefly, spots were excised, destained with 50% acetonitril in 0.1 M ammonium bicarbonate and dried in a speedvac evaporator. The dried gel pieces were reswollen with 3 μl of 3 mM Tris-HCL, pH 8.8, containing 50 ng trypsin (Promega, Madison, WI, USA) and after 15 min, 3 μl of water were added. One μl was applied onto the dried matrix spot. The matrix consisted of 15 mg nitrocellulose (Bio-Rad) and 20 mg (α-cyano-4-hydroxycynnamic acid (Sigma) dissolved in 1 ml acetone:isopropanol (1:1, v/v). 0.5 μl of the matrix solution was applied on the sample target. Specimen were analyzed in time-of-flight PerSeptive Biosystems mass spectrometer (Voyager Elite, Cambridge, MA, USA) equipped with a reflectron. An accelerating voltage of 20 kV was used. Calibration was internal to the samples. We used standards to correct the measured peptide masses, reducing, thus, the window of mass tolerance and increasing the confidence of identification. The peptide masses were matched with the theoretical peptide masses of all proteins from all species of the SWISS-PROT database. For protein search, monoisotopic masses were used and a mass tolerance of 0.0075% was allowed.2.4Statistical analysisResults are expressed as means ± standard deviation (SD). Following Bartlett’s test for homogeneity of variances, Mann-Whitney U-test was used to evaluate differences between control vs. DS and AD study groups. The exact p values were calculated for those comparisons where the statistical significance reached p < 0.05 (∗p = 0.04, ∗∗p = 0.02).3ResultsHuman brain protein extracts from seven brain regions were separated by 2-D gel electrophoresis and visualized using colloidal Coomassie blue staining. The pattern of separated protein spots was mostly identical to that previously published [38]. Protein spots were analyzed by MALDI-MS, following in-gel digestion as described above. The 2-D gels with proteins from corresponding brain regions of patients with DS and AD were compared with each other, in order to detect differences at the protein level.CNPase was represented by a well-defined single spot (Fig. 1).  By the technique used we could not distinguish between the two known isoforms of the protein, but the protein was unambiguously identified by MALDI-MS (SWISS-PROT accession number: P09543).We detected a significant decrease of CNPase protein levels in frontal and temporal cortex of brains of DS patients. In other brain regions levels of CNPase were not significantly different from controls. In brain samples from patients with AD we detected significant decrease of this protein in frontal cortex only (Table 1). CA II (SWISS-PROT accession number: P00918) was represented by two spots that could not be clearly separated from each other in most gels (Fig. 2).  The sum of both CA II spots was therefore used for statistical evaluation.Compared to control group we could not detect any significant changes in CA II protein level neither in DS nor in AD brain samples (Table 2). 4DiscussionUsing 2-dimensional electrophoresis with MALDI-MS identification we have clearly shown that CNPase and CA II are regularly expressed in brains of controls. In our study CNPase was significantly reduced in frontal and temporal cortex of DS patients and in frontal cortex in AD patients.4.1CNPase and CAII protein levels in DS brain samplesIn DS patients the reduced CNPase activity (along with other parameters of myelin such as the amount of proteolipid protein and myelin basic protein) was already reported by Banik et al. [4] suggesting reduced oligodendroglia and possibly myelination in DS. This is in agreement with our observation of decreased protein level of CNPase. Impaired myelination in DS brain starting from early childhood was confirmed histologically [9,65,66] and has been proposed by magnetic resonance imaging [28].CA II protein was not altered in any of DS brain regions, which is not a contradiction to the observed decrease of CNPase activity. Unchanged levels of CA II in DS brains of our cohort may have been resulting from the (low abundance) expression of CA II in astrocytes [26,48,50,58], proliferating in DS [45,61] thus masking a potential decrease of CA II. In a previous study, increased levels of an astroglial marker, glial fibrillary acidic protein (GFAP) have been found also in DS brains of our cohort [22].A number of pathological mechanisms may explain oligodendroglia/myelination deficits in DS. The evidence is growing that myelination and oligodendroglial maturation in CNS is strongly dependent on their close communication with neurons. This communication involves e.g. cell adhesion molecules that could convey the axonal signal to oligodendroglial cells and/or electrical activity of neurons itself that may regulate the myelination process [14,16,40]. In DS brains, however, a massive neuronal loss, starting early in postnatal life, has been reported due to either β-amyloid toxicity to neurons, altered regulation of apoptotic processes or free radical damage (for review see 46,25]. Thus, the neuro-oligodendroglial communication may be profoundly disrupted with the consequence of altered myelination.Additionally, there are several other pathological changes in DS brain metabolism that may explain our results:In DS impaired brain glucose metabolisms may be responsible for oligodendroglial loss and consequently myelin deficits. Oligodendroglial cells are more susceptible to glucose deprivation than astrocytes [41] and thus, glucose metabolism disturbances in DS would preferably affect this cell population and the oligodendrocyte-dependent myelination. In temporal and frontal lobe of DS brains of our cohort we have recently shown down-regulated expression and decreased activity of phosphoglucose isomerase, a key glucose handling enzyme [32]. This change was not found in AD, however. Moreover, the expression and the activities of phosphoglycerate kinase and glyceraldehyde 3-phosphate dehydrogenase levels, important enzymes of glucose metabolism, were found increased in DS brains but not in AD [34,39]. These findings indicate that disturbances of glucose metabolism that possibly explain oligodendroglial pathology, are linked to DS rather than to AD.Thyroid hormone abnormalities are strongly associated with DS, the most consistent finding being elevated levels of thyroid stimulating hormone [18,24]. In our cohort we showed elevated thyroid stimulating hormone receptor protein levels in DS brains [33]. It is well established that myelin protein synthesis [2] and oligodendroglial development [28] are strongly dependent on tissue thyroid hormone levels. Early in development thyroid hormones function as an instructive signal for the generation of oligodendrocytes and enhance the proliferation of the committed precursor oligodendrocyte cells. Thyroid hormones regulate the number of oligodendrocytes generated by directly promoting their differentiation and they are required to achieve adequate oligodendrocyte numbers [53]. Finally, thyroid hormone increases morphological and functional maturation of postmitotic oligodendrocytes by stimulation of the expression of various myelin genes. The direct stimulatory influence of triiodothyronine on the activity of CNPase and its mRNA expression has also been demonstrated [5,42]. Therefore, alterations of thyroid hormones commonly encountered in DS, may have led not only to altered CNPase activity in DS patient found by Banik et al. [4] but also to the decrease on CNPase protein levels in our samples. Additionally, to show another possible link between CNPase decrease and thyroid hormone disturbances, it has been suggested that sustained increase of thyroid hormone levels in DS may promote oligodendroglial cell death by apoptosis [42]. Indeed, in DS brain samples of our cohort we detected increased levels of DNAse I transcripts, an enzyme that is most probably involved in apoptotic processes [56]. In addition, by quantitative ELISA, we detected significant increase of p53 protein in frontal and temporal cortex along with increase of CD95 protein levels in temporal cortex only [55]. The upregulation of both proteins usually precedes apoptosis in many cell types and is probably involved also in apoptosis-induced cell loss in DS. Interestingly, CD95 (APO-1/Fas) receptor that mediate receptor-dependent programmed cell death, is expressed in adult human brain mainly by oligodendroglial cells. The alteration of apoptotic markers determined in these studies may well be reflecting, apart from apoptosis of neurons, the enhanced apoptosis of oligodendroglial cells with a consequence of reduced myelination. Hence, we assume that decreased CNPase protein levels detected in DS brains of our cohort might be the consequence of enhanced apoptosis either in oligodendroglial cells, or be secondarily to neuronal loss.4.2CNPase and CAII protein levels in AD brainsThe involvement of white matter in AD pathology has been described. Alterations of myelin constituents such as myelin lipids were shown to be significantly decreased in AD brains, including semioval center [64], temporal and frontal cortices, caudate nucleus and hippocampus [60]. Additionally, imaging techniques were used to reveal brain white matter changes in AD [8,19,59].In AD information on CNPase and CA II is poor and in quantitative terms only Reinikainen et al. determined activity of CNPase reflecting oligodendroglial density and possibly myelin composition of AD brains [52]. The authors found CNPase activities to be slightly increased in temporal cortex, parietal cortex and gyrus parahippocampalis, but decreased in the hippocampus and putamen, pointing to decreased oligodendroglial density or myelination deficits in the later two regions. The relatively higher CNPase activities in former three regions were ascribed to excessive loss of neurons without substantial change in oligodendroglia. However, as stated above, results from CNPase activity and protein levels may well be divergent. Hence, measuring the CNPase protein levels may be a more reliable parameter of oligodendroglial density.We revealed significantly decreased CNPase protein level in frontal cortex of AD. Although a tendency of decreased CNPase protein level could be detected also in temporal and parietal cortex of AD patients, the changes did not reach statistical significance. Nevertheless, the finding confirms the already detected pathological myelin alterations commonly found in AD [8,19,59]. We assume, however, that the myelin changes in AD are rather secondary to neuronal/axonal degeneration common in AD than the consequence of above mentioned glucose metabolism disturbances or imbalance of thyroid functions present in DS.Unaltered levels of CA II in AD brains may be explained similarly to the finding observed in DS brains by reactive astrogliosis which may mask potential decrease of CA II [51]. Indeed, using 2-D electrophoresis technique an elevated level of GFAP was found in AD brains of our cohort pointing to astroglial proliferation [22].4.3Regional differences of CNPase protein levels in DS and ADThe reasons for the uneven distribution of white matter damage, noticeable in DS in frontal and temporal lobes and in AD in frontal lobe only, are presently not clear. The regional differences might be simply explained by well known selective vulnerability of different neuronal populations to various noxious stimuli being present in both diseases and selective neuronal death with the consequence of decreased myelination in corresponding areas. The frontal and temporal lobes, where the decrease of CNPase protein reached statistical significance, are typically underdeveloped in young DS patients [9,13] and these are the brain regions, where thyroid-receptor derangement was detected [33]. Similarly, in AD the frontal lobe is usually among most affected regions based on measurements by volumetric neuroimaging [31].The differential vulnerability of individual neurons in different brain regions is well known. Studies on apoptosis in a comparable cohort of DS brain brains showed that temporal but not frontal lobes presented with deranged CD95, a hallmark of apoptosis [55]. In a comparable panel we found synaptosomal loss in temporal cortex of AD and DS patients but not in frontal cortex [68]. Similarly, regional and disease-specific changes of neuroendocrine-specific protein (NSP-C), a marker of neuronal differentiation, were decreased in frontal and temporal lobes in DS but sparing temporal cortex in AD [68]. These findings may help to explain different CNPase levels in individual brain regions but also indicate the problem to find a primary cause for myelination deficits.4.4Concluding remarksIn conclusion, we report decreased CNPase in frontal cortex of patients with DS and AD and in temporal cortex of patients with DS as compared to controls with the tentative biological meaning of decreased oligodendroglia. The methodology used permitted unambiguous identification of this marker protein for oligodendroglia and most probably for myelination. This finding is in agreement with decreased myelination in DS and AD as revealed by histological and imaging methods. Although in agreement with and confirming previous findings of decreased CNPase activity in DS brain [4], decreased CNPase protein determined by 2-dimensional electrophoresis with MALDI-MS identification may be more reliable and suitable than evaluating enzyme activity. 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