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The function, regulation and cellular distribution of GABA A receptor subunits have been extensively documented in the adult rodent brain and are linked to numerous neurological disorders. However, there is a surprising lack of knowledge on the cellular (sub-) distribution of GABA A receptor subunits and of their expressional regulation in developing healthy and diseased foetal human brains. To propose a role for GABA A receptor subunits in neurodevelopmental disorders, we studied the developing hippocampus of normal and Down syndrome foetuses. Among the α1-3 and γ2 subunits probed, we find significantly altered expression profiles of the α1, α3 and γ2 subunits in developing Down syndrome hippocampi, with the α3 subunit being most affected. α3 subunits were selectively down-regulated in all hippocampal subfields and developmental periods tested in Down syndrome foetuses, presenting a developmental mismatch by their adult-like distribution in early foetal development. We hypothesized that increased levels of the amyloid precursor protein (APP), and particularly its neurotoxic β-amyloid (1-42) fragment, could disrupt α3 gene expression, likely by facilitating premature neuronal differentiation. Indeed, we find increased APP content in the hippocampi of the Down foetuses. In a corresponding cellular model, soluble β-amyloid (1-42) administered to cultured SH-SY5Y neuroblastoma cells, augmented by retinoic acid-induced differentiation towards a neuronal phenotype, displayed a reduction in α3 subunit levels. In sum, this study charts a comprehensive regional and subcellular map of key GABA A receptor subunits in identified neuronal populations in the hippocampus of healthy and Down syndrome foetuses and associates increased β-amyloid load with discordant down-regulation of α3 subunits.

In this study, cortical receptor complex levels were determined in fetal Down syndrome (DS, trisomy 21) brain. Frontal cortices were obtained from individuals with DS (19th-22nd week of gestation) and controls. Membrane proteins were extracted, assayed on blue native gels and immunoblotted with brain receptor antibodies. Levels of a D1R-containing complex were markedly decreased in male and female cortices of DS individuals. Females with DS had significant reductions of nicotinic acetylcholine receptors α4 and α7, NMDA receptor GluN1 and AMPA receptor GluA1- and GluA3-containing receptor complexes. Levels of other brain receptor complexes (5-hydroxytryptamine 1A, GluA2 and GluR4 receptor-containing complexes) were comparable between the groups of females. Levels of GluA2- and GluA3-containing complexes were significantly increased in males. Decreased levels of D1R complexes in both sexes, along with the significant reduction of α4, α7-containing receptor complexes observed in females, may explain the brain deficits and impaired cognition observed in DS.

A large series of protein pathway components have been shown to be dysregulated in Down syndrome (DS) brain. No information about pathomechanisms linked to the trisomic state can be obtained from adult DS brain, however, as neurodegeneration occurs from the fourth decade. The aim of the study was to search for protein dysregulation in fetal DS brain before neurodegenerative changes are observed. Proteins were extracted from fetal DS and control frontal cortex, run on 2-DE, followed by quantification of protein spots with subsequent nano-ESI-LC-MS/MS analysis using an ion trap. Aberrant expression of proteins tropomodulin-2, tubulin alpha 1A chain, and alpha-internexin may indicate disturbed synaptic plasticity; fatty acid binding protein 7 suggests impaired maintenance of neuroepithelial cells; and creatine kinase B may reflect defective energy metabolism. RNA binding protein 4B derangement may represent impaired splicing, altered retrotransposon gag domain-containing protein 1 levels may be pointing to altered retrotransposition, and level changes of the potassium-chloride transporter solute carrier family 12 member 7 may lead to impaired ion fluxes with electrophysiological consequences. Taken together, aberrant protein levels from several pathways in fetal DS are challenging as well as fertilizing the area of research and providing the basis for additional neurochemical and functional studies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In a previous publication we observed aberrant levels of the human reduced folate carrier (hRFC) in cortex from fetal Down syndrome (DS) subjects. Immunoreactivity for hRFC was increased as the only chromosome 21 gene product studied. We, therefore, analyzed mice transgenic for hRFC (TghRFC1) and wild-type (WT) mice for cognitive functions, behavior and in an observational neurological battery (FOB). Cognitive functions were evaluated by the Morris water maze (MWM), the open field (OF) was used for exploratory behavior, locomotor activity and anxiety-related behavior. The elevated plus maze (EPM) was used to confirm findings in the OF testing anxiety-related behavior and the rota rod (RR) to evaluate motor function. In the MWM TghRFC1 mice performed significantly worse (P < 0.0003) on the probe trial than WT mice. In the FOB visual placing was significantly reduced inTghRFC1 mice. In the OF TghRFC1 mice crossed twice as often (P < 0.029) and in the EPM individuals from this group showed a reduced number of exits from the closed arm (P < 0.044) compared to WT mice. TghRFC1 mice showed impaired performance on the RR, spending one-fourth of the time of WT on the revolving rod (P < 0.0003). Cognitive impairment is an obligatory symptom of DS and this deficiency corresponds to findings in the MWM of mice transgenic for hRFC. Findings of visual placing and failure on the RR may reflect impaired motor performance including muscular hypotonia in DS subjects. Increased crossings in the OF may indicate modulated anxiety-related behavior observed in patients with DS.

Down syndrome (DS) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21. The phenotype of DS is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. Several reports have shown that the neuropathology of DS comprises developmental abnormalities and Alzheimer-like lesions such as senile plaques. A key component of senile plaques is amyloid beta-peptide which is generated from the amyloid precursor protein (APP) by sequential action of beta-secretases (BACE1 and BACE2) and gamma-secretase. While BACE1 maps to chromosome 11, APP and BACE2 are located on chromosome 21. To challenge the gene dosage effect and gain insight into the expressional relation between beta-secretases and APP in DS brain, we evaluated protein expression levels of BACE1, BACE2 and APP in fetal and adult DS brain compared to controls. In fetal brain, protein expression levels of BACE2 and APP were comparable between DS and controls. BACE1 was increased, but did not reach statistical significance. In adult brain, BACE1 and BACE2 were comparable between DS and controls, but APP was significantly increased. We conclude that APP overexpression seems to be absent during the development of DS brain up to 18-19 weeks of gestational age. However, its overexpression in adult DS brain could lead to disturbance of normal function of APP contributing to neurodegeneration. Comparable expression of BACE1 and BACE2 speaks against the hypothesis that increased beta-secretase results in (or even underlies) increased production of amyloidogenic A beta fragments. Furthermore, current data indicate that the DS phenotype cannot be fully explained by simple gene dosage effect.

Background: In the immature developing fetal brain, amino acids (such as gamma-aminobutyric acid, and taurine) and monoamines (serotonin, noradrenaline, and dopamine) act as developmental signals or regulators. In subjects with Down syndrome, dysfunctional brain development is evident from birth as reduction in brain weight, as well as volume reductions in specific brain regions, and an altered number of neurons, dendrites, and dendritic branching is observed. However, mechanisms that underlie the observed dysfunctional brain development in Down syndrome are not clear., Objectives: Because diverse amino acids and monoamines are critical for normal brain development, we wanted to determine whether dysfunctional brain development observed in subjects with Down syndrome is associated with altered brain amino acid and/or monoamine levels., Design/methods: We quantified tissue concentrations of diverse amino acids, including gamma-aminobutyric acid and taurine, and the monoamines serotonin, noradrenaline, and dopamine in the frontal cortex of fetal Down syndrome tissue at a gestational age of approximately 20 weeks versus age-matched control aborted fetuses., Results: Fetal Down syndrome brains showed reductions in the levels of serotonin, gamma-aminobutyric acid, taurine, and dopamine in the frontal cortex. No alteration in the levels of arginine, aspartate, glutamine, glutamate, glycine, histidine, serine, or noradrenaline was observed., Conclusions: Serotonin, gamma-aminobutyric acid, taurine, and dopamine are critical for the acquisition of brain morphologic features, neuronal and glia proliferation, and synapse formation. The detected reductions in the levels of these neurotransmitters may indicate potential mechanisms for the observed dysfunctional neuronal development in the Down syndrome fetal brain.

Background: Down syndrome (DS, trisomy 21) is the most common genetic cause of mental retardation. A large series of biochemical defects have been observed in fetal and adult DS brain that help in unraveling the molecular mechanisms underlying mental retardation., Aims: As sialylation of glycoconjugates plays an important role in brain development, this study aimed to look at the sialic acid metabolism by measuring sialic acid synthase (SAS; N-acetylneuraminate synthase) in early second trimester fetal control and DS brain., Results: In this regard, protein profiling was performed by two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization mass-spectrometry followed by database search and subsequent quantification of spot using specific software. SAS, the enzyme catalyzing synthesis of N-acetyl-neuraminic acid (syn: sialic acid) was represented as a single spot and found to be significantly and manifold reduced (P < 0.01) in cortex of fetuses with DS (control vs. DS, 0.052 +/- 0.025 vs. 0.012 +/- 0.006)., Conclusion: The intriguing finding of the manifold decrease of SAS in DS fetal cerebral cortex as early as in the second trimester of pregnancy may help to explain the brain deficit observed in DS. Decreased SAS may well lead to altered sialic acid metabolism, required for brain development and, more specifically, for sialylation of key brain proteins, including neuronal cell adhesion molecule and myelin associated glycoprotein.

Premature aging and neuropathological features of Alzheimer's disease (AD) are commonly observed in Down syndrome (DS). Based on previous findings in a DS mouse model, the function of signaling pathways associated with adenylyl cyclase (AC) and phospholipase C (PLC) was assessed in cerebral cortex and cerebellum of age-matched adults with DS, AD, and controls. Basal production of cAMP was reduced in DS but not in AD cortex, and in both, DS and AD cerebellum. Responses to GTPgammaS, noradrenaline, SKF 38393 and forskolin were more depressed in DS than in AD cortex and cerebellum. Although no differences in PLC activity among control, DS and AD cortex were observed under basal and GTPgammaS- or Ca-stimulated conditions, the response of DS cortex to serotonergic and cholinergic stimulation was depressed, and that of AD was only impaired at cholinergic stimulation. No differences were documented in cerebellum. Our results demonstrate that PLC and AC were severely disturbed in the aged DS and AD brains, but the alterations in DS were more severe, and differed to some extent from those observed in AD.

Mice polytransgenic for chromosome 21 genes DSCR3, 5, 6, 9, and TTC3 within the Down Syndrome Critical Region-1 represent an animal model for Down Syndrome (DS). In a proteomic approach, we show a series of altered hippocampal protein levels that may be caused by overexpression of at least one of the five chromosome 21 genes and that fit fear-conditioned memory defects and were observed to be dysregulated in human fetal DS.

ADAMTS-1 is a disintegrin and metalloproteinase with thrombospondin 1 (TSP1)-like motifs with ubiquitous though variable expression. Natural substrates of this protease are proteoglycans as aggrecan and versican and null mutant mice propose a role for growth, fertility, organ structure and function. As the gene for this protein is encoded on chromosome 21 and maybe overexpressed due to the gene dosage hypothesis based upon the presence of a third chromosome in trisomy 21, we decided to study expression in Down syndrome (DS) brain and used brains of patients with Alzheimer's (AD) and Pick's disease (PD) as controls. Frontal cortex of controls, DS, AD and PD were homogenized and extracted proteins were used for immunoblotting using antibodies against ADAMTS-1 and ADAMTS-5. ADAMTS-1-immunoreactivity was manifold increased in brain with DS and neurodegeneration, whereas ADAMTS-5 levels were comparable. Overexpression of this metalloproteinase maybe specifically involved in proteoglycan degradation and handling in brain of patients with neurodegenerative disease which in turn may lead to or reflect pathological lesions in DS, AD and PD brain. The manifold overexpression of ADAMTS-1 may be used as marker protein for neurodegeneration.

Down syndrome (DS) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21 and characterized by somatic anomalies and mental retardation. The phenotype of DS is thought to result from overexpression of genes encoded on chromosome 21. Although several studies reported mRNA levels of genes localized on chromosome 21, mRNA data cannot be simply extrapolated to protein levels. Furthermore, most protein data have been generated using immunochemical methods. In this study we investigated expression of three proteins (cystathionine beta-synthase (CBS), pyridoxal kinase (PDXK), ES1 protein homolog, mitochondrial precursor (ES1)) whose genes are encoded on chromosome 21 in fetal DS (n = 8; mean gestational age of 19.8 +/- 2.0 weeks) and controls (n = 7; mean gestational age of 18.8 +/- 2.2 weeks) brains (cortex) using proteomic technologies. Two-dimensional electrophoresis (2-DE) with subsequent in-gel digestion of spots and matrix-assisted laser desorption ionization (MALDI) spectroscopic identification followed by quantification of spots with specific software was applied. Subsequent quantitative analysis of CBS and PDXK revealed levels comparable between DS and controls. By contrast, ES1 was two-fold elevated (P < 0.01) in fetal DS brain. This protein shows significant homology with the E. coli SCRP-27A/ELBB and zebrafish ES1 protein and contains a potential targeting sequence to mitochondria in its N-terminal region. Based on the assumption that structural similarities reflect functional relationship, it may be speculated that ES1 is serving a basic function in mitochondria. Although no function of the human ES1 protein is known yet, ES1 may be a candidate protein involved in the pathogenesis of the brain deficit in DS.

Down Syndrome (DS, trisomy 21) is the most common genetic cause of mental retardation. The completed sequencing of genes encoded on chromosome 21 provides excellent basic information, however the molecular mechanisms leading to the phenotype of DS remain to be elucidated. Although overexpression of chromosome 21 encoded genes has been documented information at the protein expression level is mandatory as it is the proteins that carry out function. We therefore decided to evaluated expression level of seven proteins whose genes are encoded on chromosome 21: DSCR4, DSCR5, DSCR6; KIR4.2, GIRK2, KCNE1 and KCNE2 in fetal cortex brain of DS and controls at the early second trimester of pregnancy by Western blotting. beta-actin and neuron specific enolase (NSE) were used to normalise cell loss and neuronal loss. DSCR5 (PIG-P), a component of glycosylphosphatidylinositol- N-acetylglucosaminyltransferase (GPI-GnT), was overexpressed about twofold, even when levels were normalised with NSE. DSCR6 was overexpressed in addition but when normalised versus NSE, levels were comparable to controls. DSCR4 was not detectable in fetal brain. Potassium channels KIR4.2 and GIRK2 were comparable between DS and controls, whereas KCNE1 and KCNE2 were not detectable. Quantification of these proteins encoded on chromosome 21 revealed that not all gene products of the DS critical region are overexpressed in DS brain early in life, indicating that the DS phenotype cannot be simply explained by the gene dosage effect hypothesis. Overexpression of PIG-P (DSCR5) may lead to or represent impaired glycosylphosphatidylinositol- N-acetylglucosaminyltransferase mediated posttranslational modifications and subsequent anchoring of proteins to the plasma membrane.

The success of the Human Genome Project (HGP) enables prediction of proteins by computer programs from nucleic acid sequences and for which there is no experimental evidence. Clues for function of hypothetical proteins are provided by sequence similarity with proteins of known function in model organisms. The availability of this bulk of new data is of immediate importance to Down's syndrome (DS) research. DS is the most common human chromosomal abnormality caused by an extra copy of chromosome 21 and is characterized by somatic anomalies and mental retardation. In addition, overexpression of chromosome 21 genes is directly or indirectly responsible for mental retardation and other phenotypic abnormalities of DS. To allow insight into how trisomy 21 represents the phenotype of DS, we constructed a two-dimensional protein map and investigated expression of 8 hypothetical proteins in fetal DS (n = 7) and control (n = 7) brains (cortex). Two-dimensional electrophoresis (2-DE) with subsequent in-gel digestion of spots and matrix-assisted laser desorption/ionization (MALDI) spectroscopic identification followed by quantification of spots with specific software was applied. Quantitative analysis of hypothetical protein FLJ10849, hypothetical protein FLJ20113, and activator of hsp90 ATPase homologue 1 (AHA1) revealed levels comparable between DS and controls. By contrast, expression levels of hypothetical protein KIAA1185, hypothetical protein 55.2 kDa, hypothetical protein 58.8 kDa, actin-related protein 3beta (ARP3beta), and putative GTP-binding protein PTD004 were significantly decreased (P < 0.05) in fetal DS brain, and domain analysis suggests involvement in cytoskeleton, signaling, and chaperone system abnormalities.

Amniocentesis is a valuable and standard procedure for prenatal diagnosis of genetic or inborn errors of metabolism. Amnion cells are cultivated and chromosomes or proteins can be examined to provide molecular diagnosis. Mainly individual proteins are searched for based upon pedigrees and/or anamnesis. As inborn errors of metabolism involve a vast diversity of metabolic enzymes, we aimed to find a screening method for a large series of metabolic enzymes. Amnion cells were obtained from amniocentesis and subjected to proteomic analysis. We used two-dimensional gel electrophoresis with in-gel digestion followed by matrix-assisted laser desorption/ionization-time of flight analysis, to identify metabolic enzymes. Furthermore, we compared metabolic proteins in amnion cells from controls with those from Down Syndrome (DS). Enzymes involved in carbohydrate handling, amino acid handling, -purine metabolism and intermediary metabolism as well as miscellaneous metabolic pathways were detected. Protein levels of several enzymes were significantly deranged in samples obtained from patients with DS. This approach, with the advantage of the concomitant determination of many enzyme proteins, may form the basis for future metabolic screens when amniocentesis is carried out.

Down syndrome (DS) is the most common genetic disorder with mental retardation and a host of deranged proteins has already been described. Protein hunting leads to rapid accumulation of aberrant proteins and proteomics methods not only allow unambiguous identification of proteins, they are also a powerful tools to identify new or predicted proteins. We applied two-dimensional gel electrophoresis with in-gel digestion of proteins and subsequent MALDI-TOF mass-spectrometrical identification and quantification of spots using specific software on cortical brain samples from 7 controls and 7 samples from fetal DS at the early second trimester. Nine hypothetical proteins were identified: three of them (4833418L03Rik protein Q9D614, mitochondrial inner membrane protein Q16891 and Nit protein 2 Q8WUF0) were significantly and about doublefold reduced in fetal DS brain. Hypothetical proteins CGI 99, FLJ10463, 70 kDa WD-repeat tumor rejection antigen homolog, KSRP, Hypothetical protein 49.6 kDa and Elongin A were comparable between groups. Domain analysis of deranged structures revealed a t&#95;SNARE domain for the Rik protein, indicating involvement of this protein in the exocytotic-synaptic machinery impaired in DS, a CN hydrolase domain for Nit protein 2, possibly reflecting aberrant nitrilase-related metabolism and handling and an inner mitochondrial protein, extending knowledge on the mitochondrial deficit in in fetal DS early in life.

Down syndrome (DS) is the most frequent genetic disorder with mental retardation and caused by trisomy 21. Although the molecular mechanisms of the various phenotypes of DS could be due to overexpression of gene(s) on chromosome 21, several groups have challenged this gene dosage effect hypothesis. The near completion of the sequencing of human chromosome 21 provides unprecedented opportunities to understand the molecular pathology of DS, however, functional information on gene products is limited so far. We therefore evaluated the levels of six proteins whose genes are encoded on chromosome 21 (trefoil factor 1, trefoil factor 2, trefoil factor 3, coxsackie virus and adenovirus receptor, carbonyl reductase 1 and interferon- alpha receptor) in fetal cerebral cortex from DS and controls at the early second trimester using Western blot analysis. None of the investigated proteins showed overexpression in DS compared to controls suggesting that these proteins are not involved in abnormal development of fetal DS brain and that DS phenotype can not be simply explained by the gene dosage effect hypothesis. We are systematically quantifying all proteins whose genes are encoded on chromosome 21 and these studies may provide a better understanding of genotype-phenotype correlation in DS.

RNA-binding proteins have important role in the post-transcriptional regulation of gene expression. They are involved in events such as mRNA processing, transport, stability and translation. Studies in different species indicate that mutants with defect in RNA-binding proteins are defective in cell growth and differentiation. Expression of various RNA-binding proteins in prenatal life was analyzed by the highly sensitive two-dimensional electrophoresis coupled to matrix-assisted laser desorption ionization mass spectroscopy. No apparent change was obtained in levels of heterogeneous nuclear ribonucleoproteins (A3, C1-C2, L and M), nucleolin, polyadenylate binding protein-1, nuclear factor associated with double stranded RNA-2 and RNA-binding motif protein-4 between control and Down syndrome fetuses. By contrast, U5 small nuclear ribonucleoprotein-specific 40 kDa protein (p < 0.05) and Lupus La protein (p < 0.01) were significantly elevated and reduced, respectively in fetal DS. As a conclusion we can say U5 small nuclear ribonucleoprotein-specific 40 kDa protein appears to play important role in spliceosome assembly and disassembly, whereas La protein is involved in small nuclear riboncleoprotein complex biogenesis and transfer RNA maturation. Aberrant expression of these proteins points to the fact that dysregulation of the splicing and translation processes is apparent early in prenatal life, and may contribute to the defective growth and differentiation in Down syndrome.

Major advances have been made in annotation of sequences of the human genome, although elucidating the functions of these newly discovered genes remains to be a strong challenge. In an effort to give insight into how triplication of chromosome 21 leads to mental retardation in Down syndrome, we have constructed a two-dimensional protein map from control and Down syndrome fetal brain and identified hypothetical proteins with no known functions. Subsequent quantitative analysis of these proteins revealed no apparent change in expression of hypothetical proteins DKZp564P0562.1 (fragment), 16.6, 21.4, 39.5, and 40kDa as well as putative 55kDa protein between controls and Down syndrome fetuses. By contrast, hypothetical protein 28.5kDa was significantly elevated (P<0.05) in fetal Down syndrome. This finding offers an important clue that a hypothetical protein might be involved in the pathomechanisms of brain abnormality in Down syndrome.

Trisomy of human chromosome 21 is a major cause of mental retardation and other phenotypic abnormalities collectively known as Down syndrome. Down syndrome is associated with developmental failure followed by processes of neurodegeneration that are known to supervene later in life. Despite a widespread interest in Down syndrome, the cause of developmental failure is unclear. The brain of a child with Down syndrome develops differently from that of a normal one, although characteristic morphological differences have not been noted in prenatal life. On the other hand, a review of the existing literature indicates that there are a series of biochemical alterations occurring in fetal Down syndrome brain that could serve as substrate for morphological changes. We propose that these biochemical alterations represent and/or precede morphological changes. This review attempts to dissect these molecular changes and to explain how they may lead to mental retardation.

The synaptosomal associated protein of 25kDa (SNAP-25) is widely distributed in the brain and reduced in neurodegenerative diseases. In a previous paper we have shown reduced amounts of SNAP-25 protein in adult Down Syndrome (DS) brain. Neuronal cell death and downregulation at the transcriptional level may be responsible for the decrease. Therefore SNAP-25 mRNA levels were determined in frontal cortex and cerebellum of adult DS by a competitive reverse transcription-polymerase chain reaction. We found significantly increased mRNA levels in DS either related to 10 ng total RNA (P < 0.05 level in cerebellum: DS 2622 +/- 1081 attogr mean +/- SEM and controls 154 +/- 37 attogr. mean +/- SEM) or normalized versus the house keeping gene beta-actin (P < 0.05 level in frontal cortex: DS 1324 +/- 504 attogr. mean +/- SEM and control 131 +/- 32 attogr. mean +/- SEM; P<0.01 in cerebellum: DS 632 +/- 189 attogr. mean +/- SEM and control 21 +/- 2 attogr. mean +/- SEM). The main finding of this study shows elevated mRNA levels of SNAP-25 in adult DS brain whereas histological and protein-chemical evidence for decreased synaptosomal structures including SNAP-25 in a comparable cohort has been reported. We suggest compensatory mechanisms for the upregulation at the transcriptional level. We propose that SNAP-25 as many other brain proteins are regulated by protein stability rather than at the mRNA level., (Copyright 2002 Elsevier Science Ireland Ltd.)

Brain histopathology of 32 fetuses with Down syndrome was compared to that of 25 age-matched normal controls and 9 brains of fetuses of HIV positive mothers. Four cases of Down syndrome and 1 HIV case showed microdysgenesia of the cerebral cortex. As the pathogenetic background of cortical irregularities is presently not known, we analyzed the neuronal expression of drebrin, an actin-binding protein of neuronal dendritic spines. This protein is thought to play a role in synaptic formation and was recently shown to be manifold reduced in brains of fetuses with Down syndrome. However, immunocytochemistry revealed no differences in drebrin expression pattern between Down patients and controls. We conclude that cerebral cortical microdysgenesia is an infrequent non-specific pathology in fetal Down syndrome.

Down syndrome is the most common birth defect associated with mental retardation. Identifying proteins that are aberrantly expressed therefore helps to understand how chromosomal imbalance leads to subnormal intelligence in Down syndrome. In the present study, we generated a fetal brain map with the use of an analytical method based on two-dimensional electrophoresis coupled with mass spectrometry and searched the proteome for differential protein expression. Among 49 proteins analyzed in seven control and nine Down syndrome fetuses, we found 11 proteins that have been deregulated in cerebral cortex of fetal Down syndrome. While double-strand break repair protein rad 21 homologue, eukaryotic translation initiation factor 3 subunit 5, mixed lineage leukemia septin-like fusion protein-B and heat shock protein 75 were increased; beta-amyloid precursor-like protein 1, tropomyosin 4-anaplastic lymphoma kinase fusion oncoprotein type 2, Nck adaptor protein 2, Src homology domain growth factor receptor bound 2-like endophilin B2, beta tubulin, septin 7 and hematopoietic stem/progenitor cells 140 were decreased. The current data suggest that misexpression of proteins that have functions ranging from signaling to cellular structural organization could contribute to or reflect brain dysgenesis in Down syndrome.

Trisomy 21, Down Syndrome, is the most common genetic cause of human mental retardation and results from non-disjunction of chromosome 21. Several reports have been linking folate metabolism to DS and indeed, chromosome 21 even encodes for a specific folate carrier. The availability of brain tissue along with the advent of proteomics enabled us to identify and quantify C1-tetrahydrofolate synthase (THF-S), a key element in folate metabolism in brain along with other enzymes involved in C1-metabolism. Brains of controls and DS subjects at the 18th-19th week of gestation were homogenised and separated on 2 dimensional gel electrophoresis with subsequent in-gel digestion and mass spectrometrical identification and quantification with specific software. THF-S was represented by three spots, possibly representing isoforms or posttranslational modifications. Two spots were significantly, about twofold, increased in fetal DS brain: Controls [means +/- SD: (spot 1) 2.55 +/- 0.69; (spot 3) 1.39 +/- 0.86] vs. Down syndrome [means +/- SD: (spot 1) 4.25 +/- 1.63; (spot 3) 4.43 +/- 2.13]. These results were reproducible when THF-S levels were normalised versus the housekeeping protein actin and neuron specific enolase to compensate cell or neuronal loss. C1-metabolism related enzymes ribose-phosphate pyrophosphokinase I, inositol monophosphate dehydrogenase, guanidine monophosphate synthease and S-adenosylmethionine synthase, gamma form, were comparable between groups. Overexpression of this key enzyme in fetal DS brain at the early second trimester may indicate abnormal folate metabolism and may reflect folate deficiency. This may be of pathomechanistic relevance and thus extends and confirms the involvement of folate metabolism in trisomy 21.

Trisomy 21 (Down syndrome, DS) is the most common genetic cause of mental retardation, resulting from triplication of the whole or distal part of human chromosome 21. Overexpression of genes located on chromosome 21, as a result of extra gene load, has been considered a central hypothesis for the explanation of the DS phenotype. This gene dosage hypothesis has been challenged, however. We have therefore decided to study proteins whose genes are encoded on chromosome 21 in brain of patients with DS and Alzheimer's disease (AD), as all patients with DS from the fourth decade show Alzheimer-related neuropathology. Using immunoblotting we determined Coxsackievirus and adenovirus receptor (CAR), Claudin-8, C21orf2, Chromatin assembly factor 1 p60 subunit (CAF-1 p60) in frontal cortex from DS, AD and control patients. Significant reduction of C21orf2 and CAF-1 p60, but comparable expression of CAR and claudin-8 was observed in DS but all proteins were comparable to controls in AD, even when related to NSE levels to rule out neuronal cell loss or actin to normalise versus a housekeeping protein. Reduced CAF-1 p60 may reflect impaired DNA repair most probably due to oxidative stress found as early as in fetal life continuing into adulthood. The decrease of C21orf2 may represent mitochondrial dysfunction that has been reported repeatedly and also data on CAR and claudin-8 are not supporting the gene-dosage hypothesis at the protein level. As aberrant expression of the four proteins was not found in brains of patients with AD, decreased CAF and C21orf2 can be considered specific for DS.

There is a series of about 12 transcription factors expressed on chromosome 21. These transcription factors (TFs) are major candidates for playing a pathogenetic role for the abnormal wiring of the brain in fetal Down Syndrome (DS) as approximately 5,000 TFs are developmentally involved in the complex architecture of the human brain. TF derangement in DS has been already reported and we decided to contribute to the problem by studying four TFs encoded on chromosome 21 in fetal DS brain. We used fetal cortex of 8 DS fetuses and 6 controls (females) from the 18-19th week of gestation. Brain homogenates were subject to immunoblotting using goat-anti-BACH1, rabbit anti-heme oxygenase 1 (HO1), rabbit anti-ERG, rabbit anti-RUNX1 and goat anti-SIM2 l. Antibodies against beta-actin were used to normalise cell loss and antibodies against neuron-specific enolase were used to compensate neuronal loss. BACH1 was significantly overexpressed in fetal DS (p < 0.008) as compared to controls whereas RUNX1 and ERG proteins were comparable between groups, and SIM2 l was not detectable in any specimen. BACH1 was even significantly increased in the DS panel when normalised versus the housekeeping protein beta-actin (p < 0.01) or the neuron specific enolase (p < 0.01). HO-1 was found comparable between groups. BACH1, a member of the family of BTB-basic leucine zipper transcription factors, regulates gene expression through the NF-E2 site. More specifically, BACH1 suppresses expression of HO1. Increased BACH1, however, did not lead to decreased HO1, which would have explained oxidative stress observed in fetal DS.

Although cytoskeleton derangement has been reported in brain of patients with neurodegenerative disorders, basic information on integral constituents forming this network including stoichiometric composition is missing. It was therefore the aim of the study to qualitatively and quantitatively evaluate individual proteins of the three major classes representing the cytoskeleton of human brain. Cytoskeleton proteins beta-actin (betaA), alpha-actinin (Act), tubulin beta-III (betaIII), microtubule associated protein 1 (MAP1), neurofilaments NF-L, NF-M and NF-H and neuron specific enolase (NSE), a marker for neuronal density, were determined by immunoblotting. Brain samples (frontal cortex) of controls (CO), patients with Down Syndrome (DS), Alzheimer's disease (AD) and Pick's disease (PD) were used for the study. In DS brain betaIII, NF-H and NF-M, in AD brain NF-M and NF-H and in PD brain NF-L, NF-M and NF-H were significantly reduced. Stoichiometry of cytoskeleton proteins in control brain revealed the following relations: betaA:Act:betaIII:MAP1:NF-L:NF-M:NF-H = 1.0:0.8:3.8:2.4:3.2:2.2. This stoichiometrical ratios were aberrant in DS, AD and PD with the main outcome that ratios of members of the neurocytoskeleton (betaIII, NF's) in relation to betaA were remarkably decreased. This finding confirms data of decreased neuronal density using NSE in DS and AD. We propose stoichiometry of cytoskeleton elements in normal brain and confirm and extend knowledge on cytoskeleton defects in neurodegenerative diseases. The finding of significantly decreased individual elements may well lead to or represent disassembly of the neurocytoskeleton observed in neurodegenerative diseases.

Impairment of the RE-1-silencing transcription factor (REST) and REST-dependent genes in Down Syndrome (DS) neuronal progenitor cells and neurospheres has been published recently. As dysregulation of this system has been shown at the RNA level and considering the long and unpredictable way from RNA to proteins, and as it is the proteins that do the function in brain, we decided to test this hypothesis at the protein level. Cortex of brains of patients with Down Syndrome at the early second trimester were used. REST-dependent structures as synapsin I, brain derived neurotrophic factor BDNF and neuronal growth-associated protein SCG10 were determined at the protein level using immunoblotting. Proteins were comparably expressed in fetal Down syndrome and control brains. Even when normalized versus housekeeping genes (glyceraldehyde-6-phosphate-dehydrogenease) and a marker for neuronal density (neuron-specific enolase) DS results were resembling controls. Therefore, we cannot confirm the REST-hypothesis by our studies in the 18/19th week of gestation at the protein level in brain and taking into account that the hypothesis was based upon studies in progenitor cells.

Down syndrome (trisomy of chromosome 21) (DS) is the most common genetic cause of mental retardation. In our study we employed immunoblotting to evaluate protein expression of reduced folate carrier (hRFC), encoded by a gene localised on chromosome 21, in fetal DS brain. We observed increased expression of hRFC-immunoreactive band with an apparent MW of approximately 150 kDa, whereas the other bands (MWs approximately 60 and 50 kDa), were comparable to control. In conclusion, we suggest that aberrant hRFC expression may well have a role in the already observed deterioration of folate metabolism in DS. Moreover, no alterations of expression level of p53 and Sp1, supposed to play a role in the regulation of hRFC, suggest that regulation of hRFC expression in fetal life by these proteins is highly unlikely, at least by changes in their protein level.

In comparison to most other groups with intellectual disability individuals with Down syndrome are at lower risk for significant psychopathology, although relative to their typically developing peers they have higher rates of behavioural and emotional problems. A total of 43 Down syndrome patients (21 females and 22 males), who ranged in age from 5.33 to 30.58 years, were examined for the presence of age-related changes in the spectrum of externalizing and internalizing problems. Intelligence tests included Hamburg-Wechsler-Intelligenz Test für Kinder III (HAWIK-III), Hamburg-Wechsler-Intelligenz Test für Erwachsene (HAWIE-R) and Kaufman-Assessment-Battery for Children, German Version (K-ABC). Behavioural and emotional problems were assessed by the the Strengths and Difficulties Questionnaire for Parents, German Version (SDQ) and the Clinical Assessment Scale for Child and Adolescent Psychopathology (CASCAP). IQ was significantly inversly related to the age of patients. Externalizing behaviours (dominant, opposing/refusing, impulsiveness, inattention and increased motor activity) were significantly higher in the 5-10 years old group, whereas internalizing behaviours (shy/insecure, low self confidence, decreased motor activity) where more prevalent in adolescents and adults (10-30 years). Possible relationships between this age-related changes and increased risks of later-onset psychopathology (depression and dementia) are discussed.

A couple of transporters and channels has been proposed as candidate genes involved in the pathomechanisms leading to the neurodevelopmental abnormalities and the phenotype of Down Syndrome (DS, trisomy 21). No systematic study, however, has been carried out showing the concomitant expression of several candidate RNAs during fetal life. It was therefore the aim of the study to apply an array of 96 brain RNAs mainly consisting of channels and transporters to show their expressional levels in fetal DS brain at the early second trimester. Brain RNA was extracted from fetal cortex of the 18-19th week of gestation of controls and DS individuals and used for the GEArray Q Series Human Neuroscience-1/Ion Channels & Transporters analysis. 15 out of 96 RNAs of the array were observed on the films in both groups during this gestational period consisting of genes for potassium, sodium, calcium channels and transporters (ASIC3, ATP1B1, CACNA1B, KCNB2, KCNC1, KCND2, KCNF1, KCNN1, KCNN3, hKCa4, KCNQ2, lipid transfer protein II, SCN2B, acetyl choline transporter, glutamate transporter3). There was no statistically significant difference between the control and the DS group. We provide information on the developmental expression of the aforementioned 15 RNAs and the absence of the residual examined 81 RNAs at the 18th/19th week of gestation in fetal cortex that was never reported before and show that channels and transporters present with unchanged expression in fetal DS brain.

Fatty acid binding proteins (FABPs) are thought to play a role in the binding, targeting and transport of long-chain fatty acids, and at least three types of FABPs are found in human brain; heart type (H)-FABP, brain type (B)-FABP and epidermal type (E)-FABP. Although all three FABPs could be involved in normal brain function in prenatal and postnatal life, a neurobiological role of FABPs in neurodegenerative diseases has not been reported yet. These made us evaluate the protein levels of FABPs in brains from patients with Down syndrome (DS) and Alzheimer's disease (AD) and fetal cerebral cortex with DS using two-dimensional (2-D) gel electrophoresis with subsequent matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) identification and specific software for quantification of proteins. In adult brain, B-FABP was significantly increased in occipital cortex of DS, and H-FABP was significantly decreased in DS (frontal, occipital and parietal cortices) and AD (frontal, temporal, occipital and parietal cortices). In fetal brain, B-FABP and epidermal E-FABP levels were comparable in controls and DS. We conclude that aberrant expression of FABPs, especially H-FABP may alter membrane fluidity and signal transduction, and consequently could be involved in cellular dysfunction in neurodegenerative disorders.

Down syndrome (DS; trisomy 21) is a genetic disorder associated with early mental retardation and patients inevitably develop Alzheimer's disease (AD)-like neuropathological changes. The molecular defects underlying the DS-phenotype may be due to overexpression of genes encoded on chromosome 21. This so-called gene dosage hypothesis is still controversial and demands systematic work on protein expression. A series of transcription factors (TF) are encoded on chromosome 21 and are considered to play a pathogenetic role in DS. We therefore decided to study brain expression of TF encoded on chromosome 21 in patients with DS and AD compared to controls: Frontal cortex of 6 male DS patients, 6 male patients with AD and 6 male controls were used for the experiments. Immunoblotting was used to determine protein levels of TF BACH1, ERG, SIM2 and RUNX1. SIM2 and RUNX1 were comparable between groups, while BACH1 was significantly reduced in DS, and ERG was increased in DS and AD as compared to controls. These findings may indicate that DS pathogenesis cannot be simply explained by the gene dosage effect hypothesis and that results of ERG expression in DS were paralleling those in AD probably reflecting a common pathogenetic mechanism possibly explaining why all DS patients develop AD like neuropathology from the fourth decade. We conclude that TF derangement is not only due to the process of neurodegeneration and propose that TFs BACH1 and ERG play a role for the development of AD-like neuropathology in DS and pathogenesis of AD per se and the manifold increase of ERG in both disorders may form a pivotal pathogenetic link.

Down's syndrome (DS) is the most frequent genetic cause of mental retardation and although known for more than a hundred years, the underlying pathomechanisms for the phenotype and abnormal brain functions remain elusive. Performing protein hunting in fetal Down's syndrome (trisomy 21) brain we detected aberrant expression of several proteins. Fetal brain cortex from controls and Down's syndrome at the early second trimester of gestation was used for expressional analysis. Two-dimensional electrophoresis with subsequent in-gel digestion of spots and matrix-assisted laser desorption/ionization spectroscopical identification followed by quantification of spots with specific software was applied. The nuclear matrix protein matrin 3, cytoskeletal motor protein HMP, and the circadian clock protein hlark were significantly decreased in fetal DS brain. C8ORF2 protein was reduced but did not reach statistical significance. Prox1 and predicted protein Dj149A16.6 were comparable between groups. Reduction of brain proteins may represent or cause deficient cytoskeletal structure, transcription machinery and exocytosis, functions already known to be deteriorated in DS brain. Some of the described proteins were only predicted and we here show the existence of the corresponding proteins in fetal human brain.

Trisomy 21 (Down's syndrome) is the most common genetic cause of human mental retardation. In Down's syndrome (DS) patients, deteriorated glucose, lipid, purine, folate and methionine/homocysteine metabolism has been reported. In our study, we used a proteomic approach to evaluate protein expression of enzyme proteins of intermediary metabolism in the brain of Down's syndrome fetuses. In fetal DS brain, we detected increased protein levels of mitochondrial aconitase as well as NADP-linked isocitrate dehydrogenase, decreased protein expression of citrate synthase and cytosolic aspartate aminotransferase. From two spots that corresponded to either pyruvate kinase M1 or M2 isozymes, significant elevation was observed only in one, while the second spot as well as the sum of the spots showed no differences between DS and controls. These results suggest derangement of intermediary metabolism during prenatal development of DS individuals.

Down syndrome (DS) is characterized by abnormal brain morphology and neurological and behavioral functions. The pivotal role of helicases in brain development, growth, and differentiation made us evaluate three DEAD BOX proteins, DEAD-box protein 1 (DBP-RB), DEAD-box protein 3 (HLP2), DEAD-box protein 72 (P72), and the RuvB-like DNA helicase (TIP49b), in fetal brain of controls and DS subjects, using two-dimensional electrophoresis with subsequent mass spectroscopic (MALDI-MS) identification. HLP2 and TIP49b brain levels were comparable between DS and controls, and protein levels of p72 and DBP-RB were significantly reduced in DS fetal cortex (p72: 2.04+/-1.90 vs. 5.57+/-2.56 in controls, p < 0.01; DBP-RB: 0.58+/-0.94 vs. 1.90+/-0.97 in controls, p < 0.01). Impairment of the helicases p72 and DBP-RB may reflect or lead to deficient growth and differentiation of brain development early in life and can be considered pathogenetic factors along with the reported deficits of transcription, splicing, and elongation factors already described in fetal DS brains.

Down syndrome (DS) is the most common genetic birth defect associated with mental retardation. The mechanism(s) underlying the neuropathology of DS is not completely understood. Different hypotheses have been advanced to explain this mystery, including the gene dosage effect, the amplified developmental instability, and the molecular misreading concept. Overexpression of genes residing in chromosome 21 has been assumed to be a central point in the neuropathology of DS, although reports disagreeing with this notion have also been published. In addition, an accumulating body of evidence indicates that genes located on other chromosomes are also involved in the process. DS thus appears to be a disease process involving numerous gene products and this interaction and interplay in the final analysis determines the outcome of the disease. In this regard, transcription factors, reactive oxygen species and apoptosis related proteins are viewed as potential candidates that play a significant role in the disease process. Therapeutic modalities that target these factors including antioxidants and caspase inhibitors might have some benefit in alleviating the symptoms of DS.

In brain, nucleoside diphosphate kinase (NDPK) and its coding gene, nm23, have been implicated to modulate neuronal cell proliferation, differentiation, and neurite outgrowth. However, a role of NDPK in neurodegenerative diseases has not been reported yet. Using proteomics techniques, we evaluated the protein levels of NDPK-A in seven brain regions from patients with Alzheimer's disease (AD) and Down syndrome (DS) showing AD-like neuropathology. NDPK-A was significantly decreased in brain regions (frontal, occipital, and parietal cortices) of both disorders. Due to the limitation of brain samples, the activity of NDPK was measured in three brain regions (frontal cortex, temporal cortex, and cerebellum). The specific activity of NDPK was significantly decreased in AD (frontal cortex) and DS (frontal and temporal cortices). Since NDPK-B could also drive the activity of NDPK, protein expression levels of both NDPK-A and NDPK-B were studied in frontal cortex by Western blot analysis. NDPK-A was significantly decreased in AD, which was consistent with the results of proteomics. However, NDPK-A was slightly decreased in DS and protein expression levels of NDPK-B in both DS and AD were moderately decreased, without reaching statistical significance. We propose that oxidative modification of NDPK could lead to the decreased activity of NDPK and, subsequently, influence several neuronal functions in neurodegenerative diseases as multifunctional enzyme through several mechanisms.

Nascent polypeptide-associated complex (NAC) protein, a heterodimeric complex of alpha- and beta-subunits, prevents mistargeting of nascent polypeptide chains to the endoplasmic reticulum membranes. alpha-NAC has sequence similarities with transcription-regulating proteins and has been reported to function as a transcriptional coactivator potentiating c-Jun-mediated transcription. Performing gene hunting using differential display-polymerase chain reaction, a downregulated sequence in the frontal cortex of patients with Alzheimer's disease (AD) and Down syndrome (DS) with AD-like neuropathology was identified as a-NAC with 100% homology. The significant decrease in alpha-NAC mRNA was shown by semiquantitative reverse transcription-polymerase chain reaction, and in parallel, the significant decrease of alpha-NAC protein, which was even more pronounced when related to either actin or neuron-specific enolase levels, was also observed in both disorders. Linear regression analysis revealed a strong, significant correlation between alpha-NAC protein and mRNA expression. In fetal DS brain, however, mRNA levels of alpha-NAC were comparable between DS and controls, suggesting that the decrease in alpha-NAC might be involved in the pathology of neurodegenerative diseases. The decrease in alpha-NAC as a transcriptional coactivator could contribute to the characteristic decline of the c-Jun-mediated transcriptional machinery and could function as the complementary mechanism in c-Jun-mediated apoptosis. Decreased alpha-NAC may result in the mistargeting, mistranslation, and proteolysis of proteins by affecting overall NAC function.

Drebrin, located in the dendritic spines of the neuron, plays a role in the synaptic plasticity together with actin filaments. Although drebrin regulates the morphological changes of spines in neurodegenerative disease such as Alzheimer's disease (AD), drebrin in Down syndrome (DS) showing AD-like neuropathology has not been studied. We used Western blotting to determine protein levels of drebrin and F-actin in frontal, temporal cortex and cerebellum from patients with DS and AD as compared to controls. A monoclonal antibody against drebrin and F-actin was used. Drebrin levels were significantly decreased in frontal (means +/- standard deviation; DS 0.24 +/- 0.52; AD 0.16 +/- 0.14; controls 2.56 +/- 3.48) and temporal cortex (DS 0.07 +/- 0.11; AD 0.07 +/- 0.15; controls 1.71 +/- 1.51) and drebrin was also decreased when normalized with F-actin. No changes were observed in cerebellum. Decreased drebrin could not simply be due to cell loss (F-actin) or neuronal loss (comparable neuron-specific enolase between groups). Reduced drebrin could be responsible for or representing the loss of spine plasticity in DS and may be a useful indicator for the impaired arborization in neurodegenerative disorders.

Down syndrome (DS) patients present with morphological abnormalities in brain development, leading to mental retardation. Given the importance of actin cytoskeleton to form the basis of various cell functions, the regulation of actin system is crucial during brain development. We therefore aimed to study the expression levels of actin binding proteins in fetal DS and control cortex. We evaluated the levels of eight actin binding proteins using the proteomic approach of two-dimensional gel electrophoresis with subsequent mass spectroscopical identification of protein spots. In fetal DS brain we found a significant reduction of the actin-related protein complex 2/3 (Arp2/3) 20 kDa subunit and the coronin-like protein p57, which are involved in actin filament cross-linking and nucleation and capping of actin filaments. We conclude that deficient levels of these proteins may, at least partially, be involved in the dysgenesis of the brain in DS., (Copyright 2002 Elsevier Science (USA).)

Histamine-N-methyltransferase (HMT) inactivates the neurotransmitter histamine. Central histaminergic deficits may contribute to the cognitive impairment of neurodegenerative disorders including Alzheimer's disease (AD) and Down syndrome (DS). However, there is no evidence for histaminergic deficits in Pick's disease (PiD). HMT levels were measured in the frontal cortex and cerebellum of brains of patients with AD, DS, and PiD, and normal aged subjects using proteomics techniques. In frontal cortex, HMT was significantly decreased in DS, but significantly increased in PiD compared with controls. HMT levels were comparable in cerebellum of all groups. Elevated HMT in PiD could lead to increased histamine degradation that in turn would be in agreement with impaired cognitive functions of PiD. Decreased HMT in DS would be compatible with findings of decreased histamine synthesis, thus reflecting a compensation mechanism to antagonize reduced synthesis by decreased degradation.

Down syndrome (DS, trisomy 21) is the most frequent genetic cause of mental retardation. Although known for more than a hundred years the underlying pathomechanisms for the phenotype and impaired brain functions remain elusive. Performing protein hunting in fetal DS brain, we detected a series of cytoskeleton proteins with aberrant expression in fetal DS cortex. Fetal brain cortex samples of controls and DS of the early second trimenon of gestation were used for the experiments. We applied two-dimensional electrophoresis with in-gel digestion of protein spots, subsequent mass spectroscopical (MALDI) identification, and quantification of spots using specific software. Centractin alpha, F-actin capping protein alpha-1, alpha-2 and beta subunits were significantly reduced in fetal DS cortex, whereas dynein intermediate clear 2, dynein intermediate chain 2, and kinesin light chain protein levels were unchanged. Centractins and F-actin capping proteins are major determinants of the cytoskeleton and are involved in pivotal functions including cellular, organelle, and nuclear motility. Deranged centractins and F-actin capping proteins may represent or induce deficient axonal transport and may well contribute to deterioration of the cytoskeleton's mitotic functions in trisomy 21.

Although Down Syndrome (DS, trisomy 21) is the most frequent isolated cause of mental retardation, information on brain protein expression and in particular protein expression of signaling-related proteins is limited. Impaired signaling in DS involving different signaling systems has been proposed and the availability of fetal brain along with recent proteome technologies unambiguously identifying individual brain proteins made us study individual signaling factors in the brain. We studied fetal brain cortex of controls (n = 7) and DS (n = 9) from early second trimester of gestation by two-dimensional gel electrophoresis with subsequent matrix-assisted laser/desorption ionization (MALDI) identification followed by quantification with specific software. Four 14-3-3 protein isoforms, mitogen-activated protein kinase 1, receptor for activited kinase 1 (RACK1), constitutive photomorphogenesis (COP9) complex subunit 4 and cAMP-dependent protein kinase type II have been identified. Quantification showed that protein 14-3-3 gamma (means +/- standard deviation of controls: 10.18+/-2.30 and of DS 4.20+/-1.19) and two spots assigned to RACK1 (controls spot 1: 4.15+/-2.45 and DS 1.95+/-0.93; controls spot 2: 5.08+/-2.4 vs. DS: 2.56+/-1.19) were significantly decreased in DS cortex. Reduced 14-3-3 gamma may represent impaired neuronal differentiation, synaptic plasticity and impaired signaling by PKC and Raf while decreased RACK1 (anchoring protein receptor for activated C-kinase) may reflect or generate deranged beta-II- protein kinease C (PKC) function with the putative biological meaning of aberrant migration and neuritic outgrowth in DS early in life.

Down syndrome (DS) is the most common chromosomal abnormality associated with early mental retardation and neurological abnormalities followed by precocious age dependent Alzheimer-type neurode generation later in life. Knowledge of the pathological mechanisms involved in DS is far from complete, but overexpression of genes residing in chromosome 21 was considered to be the central point for the DS phenotype. In this regard, beta amyloid precursor protein (APP), CuZn superoxide dismutase (SOD1) and S100beta have been implicated in causing apoptosis, a mechanism thought to be responsible for neuronal loss in DS, in one way or another. The gene dosage hypothesis has been challenged, however, and dysregulation of expression of genes located on other chromosomes has been described, which may well be secondary to chromosomal imbalance or a direct consequence of the disease process. The present review focuses on the protein expression profile in DS and we postulate that abnormalities in the coordinated expression, as well as interaction of proteins may be responsible for the neuropathology of DS. A series of candidate proteins are discussed that may be directly causing or reflecting the DS phenotype, in particular the brain abnormalities in DS.

In 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.

In Down syndrome, enhanced apoptosis (programmed cell death) may play a role in the pathogenesis of characteristic early mental retardation and precocious neurodegeneration of Alzheimer type. Various apoptosis-associated proteins (Bax, Bcl-2, Fas, p53, Hsp70, neuronal apoptosis inhibitory protein-like immunoreactivity) were investigated in four different cortical regions and the cerebellum of one fetal Down syndrome (35 weeks' gestation) postmortem brain sample compared with a control brain sample. The most impressive finding was an at least fivefold elevation of Bax protein together with decreased Bcl-2 values in all Down syndrome cerebral regions investigated. In addition, antiapoptotic, presumably caspase-inhibitory, principles like heat shock protein 70 and neuronal apoptosis inhibitory protein were also reduced. Whereas Fas protein, an important member of receptor-mediated apoptosis, was inconsistently altered, a rather surprising finding was reduced proapoptotic, regulatory protein p53 in four of five regions. The findings are in good agreement with the proposed role of the Bcl-2 protein family in regulating developmental (naturally occurring) apoptotic neuronal death and further suggest that developmental apoptosis may be inappropriately commandeered by so far undefined pathologic processes in Down syndrome.

NADH: ubiquinone oxidoreductase (complex I), one of the most complicated multi-protein enzyme complexes, is important for energy metabolism because it is the initial enzyme of the mitochondrial respiratory chain. Deficiency of complex I is frequently found in various tissues of patients with neurodegenerative disease. Here we studied the protein levels of complex I 24- and 75-kDa subunits in several brain regions from patients with Down syndrome (DS) and Alzheimer's disease (AD). We determined protein levels of complex I 24-, 75-kDa subunits and mitochondrial marker proteins mitochondrial matrix protein P1 (hsp60) and aconitate hydratase from seven brain regions of patients with DS, AD and controls. Proteins were separated by two-dimensional (2-D) gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Complex I 24-kDa subunit was significantly reduced in occipital cortex and thalamus in patients with DS and temporal and occipital cortices in patients with AD. Complex I 75-kDa subunit was significantly reduced in brain regions from patients with DS (temporal, occipital and caudate nucleus) and AD (parietal cortex). Reductions of two subunits of complex I may lead to the impairment of energy metabolism and result in neuronal cell death (apoptosis), a hallmark of both neurodegenerative disorders.

Although it is well-known that synaptosomal proteins are deranged in neurodegenerative disorders, no information is available at the protein-chemical level as mainly immunochemical or immunohistochemical data were reported previously. We therefore investigated synaptosomal proteins in brain specimens from patients with Down syndrome (DS) and Alzheimer's disease (AD) to challenge the DS synaptic pathology as well as the relevance of DS to AD in synaptic pathology. For the aim of this study, we employed two-dimensional electrophoresis and matrix-associated laser desorption ionization mass spectroscopy and determined beta-soluble N-ethylmaleimide-sensitive factor attachment protein (beta-SNAP), gamma-SNAP and synaptotagmin I (SYT I) in 7 individual brain regions of controls and patients with DS and AD. In DS brain, beta-SNAP was significantly reduced in temporal cortex (p < 0.01). SYT I (p65) and SYT I (pI 7.0) were significantly reduced in thalamus (p < 0.01 and p < 0.05, respectively). In AD brain, beta-SNAP was significantly decreased in temporal cortex (p < 0.05). SYT I (p65) was significantly reduced in cerebellum (p < 0.05), and temporal (p < 0.001) and parietal cortex (p < 0.01). SYT I (pI 7.0) was significantly reduced in temporal (p < 0.001) and parietal cortex (p < 0.01) and thalamus (p < 0.01). gamma-SNAP did not show any change in both DS and AD. The findings may explain impaired synaptogenesis in DS and AD brain, which is well documented in DS brain already early in life, and/or synaptosomal loss secondary to neuronal loss observed in both neurodegenerative disorders. It may also represent, reflect or account for the impaired neuronal transmission in DS and AD, caused by deterioration of the exocytic machinery. Here, we provide evidence for several deranged synaptosomal proteins in several brain regions at the protein level indicating deficient synaptosomal wiring of the brain in DS and AD., (Copyright 2001 S. Karger AG, Basel.)

Heat shock proteins (HSPs) in their molecular capacity as chaperones have been reported to regulate the apoptotic pathway and also play a critical role in protein conformational diseases such as Alzheimer's disease (AD). As all Down syndrome (DS) brains display AD-like neuropathology, neuronal loss in DS was shown to be mediated by apoptosis. We decided to investigate the expression patterns of HSPs in seven brain regions of adults with DS using two-dimensional polyacrylamide gel electrophoresis (2-DE). Following 2-DE, approximately 120 protein spots were successfully identified by matrix-assisted laser desorption/ionization--mass spectrometry (MALDI-MS) followed by quantification of the identified proteins. We unambiguously identified and quantified nine different chaperone proteins. Accordingly, all but three chaperone proteins did exhibit a significant change in expression. HSP 70 RY, heat shock cognate (HSC) 71 and glucose-regulated protein (GRP) 75 showed a significant decrease (P < 0.05) in DS temporal cortex whereas HSP 70.1 and GRP 78 were significantly increased (P<0.05) in cerebellum. Whilst T-complex 1 (TCP-1) epsilon subunit showed a significant decrease (P< 0.05) in parietal cortex, a similar extent of increase (P<0.05) as that observed in cerebellum was obtained in parietal levels of GRP 78. Alpha-crystallin B, HSP 60 and GRP 94 did not show any detectable changes in expression patterns. This report presents the first approach to quantify nine different chaperones simultaneously at the protein level in different brain regions and provides evidence for aberrant chaperone expression patterns in DS. The relevance of this aberrant expression patterns are discussed in relation to the biochemical and neuropathological abnormalities in DS brain.