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The diagnosis of Alzheimer's disease (AD) was verified at post-mortem examination in 46 patients (9 male: 37 female; mean age 83.6 +/- 6.5 years) from a prospective clinical study. Compared to 10 age-matched controls, the total AD group showed a significant neuronal loss in the locus coeruleus, dorsal raphe nucleus, substantia nigra and in the basal nucleus of Meynert. Twelve patients (1 male: 11 female) had experienced symptoms of depression. These patients had significantly lower neuronal counts in the locus coeruleus, but less severe cell loss in the basal nucleus of Meynert compared to the AD patients without depression.

Down syndrome (trisomy 21) is a genetic disease with developmental brain abnormalities resulting in early mental retardation and precocious, age dependent Alzheimer-type neurodegeneration. We tried to discuss the role of neurodevelopmental abnormalities in connection with aberrant expression of genes on chromosome 21 including amyloid precursor protein (APP), CuZn superoxide dismutase (SOD1) and glial-derived S100 beta protein for neurodegeneration in DS. In this model, alterations in developmental pathways due to aberrant gene expression can impair cellular homeostasis and predispose to neurodegeneration of certain brain regions and types of nerve cells, involving cholinergic, serotonergic and catecholaminergic transmission, by shifting balance toward a pro-apoptotic state.

Information on gene expression in brain of patients with Down Syndrome (DS, trisomy 21) is limited and molecular biological research is focussing on mapping and sequencing chromosome 21. The information on gene expression in DS available follows the current concept of a gene dosage effect due to a third copy of chromosome 21 claiming overexpression of genes encoded on this chromosome. Based upon the availability of fetal brain and recent technology of gene hunting, we decided to use subtractive hybridization to evaluate differences in gene expression between DS and control brains. Subtractive hybridization was applied on two fetal brains with DS and two age and sex matched controls, 23rd week of gestation, and mRNA steady state levels were evaluated generating a subtractive library. Subtracted sequences were identified by gene bank and assigned by alignments to individual genes. We found a series of up- and downregulated sequences consisting of chromosomal transcripts, enzymes of intermediary metabolism, hormones, transporters/channels and transcription factors (TFs). We show that trisomy 21 or aneuploidy leads to the deterioration of gene expression and the derangement of transcripts describes the impairment of transport, carriers, channels, signaling, known metabolic and hormone imbalances. The dys-coordinated expression of transcription factors including homeobox genes, POU-domain TFs, helix-loop-helix-motifs, LIM domain containing TFs, leucine zippers, forkhead genes, maybe of pathophysiological significance for abnormal brain development and wiring found in patients with DS. This is the first description of the concomitant expression of a large series of sequences indicating disruption of the concerted action of genes in this disorder.

Overexpression of the transcription factor ETS2 and other genes localized in the socalled critical Down Syndrome region of chromosome 21 due to a gene dosage effect, is an attractive hypothesis for the explanation of the Down Syndrome phenotype. The overexpression of ETS2, however, has never been demonstrated in a human organ. We therefore challenged this hypothesis determining ETS2 levels in several brain regions of patients with Down Syndrome as compared to controls. We used a highly sensitive and quantitative RT-PCR method for the determination of ETS2 mRNA steady state levels in frontal, parietal, temporal, occipital lobe and cerebellum of 9 adult Down Syndrome patients and 9 adult controls. Significantly decreased ETS2 mRNA steady state levels (16.9 +/- 26.7 attogram mRNA ETS2/10 ng total RNA versus 87.7 +/- 92.9 in controls) in frontal lobe of Down Syndrome brain and decreased ETS2 mRNA steady state levels (6.99 +/- 6.4 attogram mRNA ETS2/100 pg beta-actin versus 19.8 +/- 15.7 in controls) in temporal lobe of Down Syndrome brain were found. In the other brain regions no statistically significant difference was detected. Our data provide evidence against the overexpression hypothesis for the development of the Down Syndrome phenotype. Decreased ETS2 transcripts found in temporal and frontal lobe of patients with Down Syndrome, however, may be involved in the pathogenesis of Down Syndrome including specific neurodegenerative processes and deteriorated plasticity of the brain taking place in Down Syndrome brain, as the concerted action of transcription factors may be seriously impaired.

Although gene hunting has been carried out in Down Syndrome (DS) cells, information on expressional differences in DS brain is limited. We have recently described expressional differences in fetal DS brain but cannot assign these findings to "DS per" se or simply to "neurodegeneration". We therefore performed gene hunting in cerebellum of adult patients with DS and Alzheimer's disease (AD) neuropathology, AD and controls. The gene hunting method used was differential display and pools of the individual groups were examined to rule out allelic differences. Differential display revealed the absence of a band, identified by sequencing and gene bank work as matching the NADH3 gene (99.1% identity) in cerebellum of DS patients. Dot blots showed the presence of NADH3 signals in only two out of 7 DS patients. We show at the transcriptional level that a mitochondrial enzyme, the complex I, NADH3, is significantly downregulated in DS cerebellum. This extends previous work on deficiencies of the electron transport chain in platelets of patients with DS.

Human DNAse I (EC 3.1.21.1) is an enzyme most probably involved in apoptotic processes. Splicing of the DNAse I primary transcript in normal and apoptotic cells into up to 20 splicing forms and the recent description of a different family of caspase-activated DNAses, hampered studies on the role of DNAse I in apoptosis research. Performing gene hunting in fetal brain of patients with DS we found a sequence with 100% homology to DNAse I and this formed the Rationale for studies in adult DS brain. It was therefore the aim of the study to evaluate DNAse I-mRNA steady state levels in DS brain using adult brain without brain pathologies and Alzheimer's Disease (AD) brain as control, in order to rule out that DNAse I--overexpression may not be specific for DS but rather reflecting apoptosis per se, a hallmark of both disorders. Determination of DNAse I-mRNA steady state levels was carried out by a blotting method in frontal, parietal, temporal occipital lobe and cerebellum. We found significantly increased DNAse I transcripts in brain of DS and AD both, when normalized versus the house-keeping gene beta actin or total RNA. We demonstrate the significant increase of DNAse I--transcript in the pathogenesis of DS and AD suggesting a role for this enzyme in the apoptotic process known to occur in both disorders. We are now going to carry out protein and enzyme activity levels in our laboratory to confirm our findings at the transcriptional level.

Apoptosis has been implicated in the selective neuronal loss of Down syndrome (DS). Apoptosis activates a family of cysteine proteases with specificity for aspartic acid residues referred to as caspases that play a key role in dismantling a cell committed to die. Caspase activity is regulated by a variety of proteins that possess a domain resembling the prodomains of caspases. Little is known, however, about the changes of caspases and their regulatory proteins in DS. Here, we investigated levels of nine such different proteins by western blot technique in frontal cortex and cerebellum of control and DS subjects. The protein levels of DFF45 (DNA fragmentation factor 45), and FLIP (FADD like interleukin-1beta-converting enzyme inhibitory proteins) were significantly decreased whereas that of RICK (RIP-like interacting CLARP kinase) increased in both regions of DS. In contrast, cytochrome c, Apaf-1 (apoptosis protease activating factor-1), procaspase-9 and ARC (apoptosis repressor with caspase recruitment domain) were unchanged. Procaspase-3 and -8 were significantly decreased in frontal cortex but no significant change was observed in cerebellum. Regression analysis revealed no correlation between postmortem interval and levels of the investigated proteins. However, inconsistent correlation was found between age and levels of proteins as well as amongst the density of individual proteins. These findings demonstrate that dysregulation of apoptotic proteins does exist in DS brain and may underlie the neuropathology of DS. The study further suggests that apoptosis in DS may occur via the death receptor pathway independent of cytochrome c. Hence, therapeutic strategies that target caspase activation may prove useful in combating neuronal loss in this disorder.

Stathmin, distributed in neurons with high abundance, acts as an intracellular relay, integrating various transduction pathways triggered by extracellular signals and it is involved in physiological regulation of microtubule destabilization. Stathmin has been also shown to be a critical molecule in pathology of neurodegeneration such as Alzheimer's disease (AD), particularly, in neurofibrillary tangle (NFT) formation. Here we evaluated protein levels of stathmin in adult brain from patients with AD and Down syndrome (DS) showing AD-like pathology by applying proteomic technologies with two-dimensional (2-D) gel electrophoresis, matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) identification and specific software for quantification of proteins. Significantly decreased protein levels of stathmin were observed in frontal (2.12+/-1.17, n = 6) and temporal (3.05+/-2.81, n = 10) cortices of AD compared to controls (frontal cortex: 4.41+/-1.70, n = 8; temporal cortex: 5.26+/-2.26, n = 13). Stathmin was also significantly decreased in frontal (2.47+/-1.11, n = 7) and temporal (2.02+/-1.18, n = 9) cortices of DS. We also investigated stathmin levels in fetal brain. Stathmin was not significantly changed between fetal DS brain and controls. We suggest that the decreased protein level of stathmin in brains is associated with tangle formation and microtubule instability in DS as well as AD, but stathmin is not involved in the abnormal development of fetal DS brain.

A series of impaired metabolic functions in Down Syndrome (DS) including glucose handling has been described. Recent information from positron emission tomography studies in DS patients and our finding of downregulated phosphoglucose isomerase (PGI) in fetal brain with DS by gene hunting using subtractive hybridization, made us investigate PGI, a key enzyme of glucose metabolism, in brain of patients with DS, Alzheimer's disease (AD) and controls. PGI and phosphofructokinase (PFK) activities were determined in frontal, parietal, temporal, occipital lobe and cerebellum of 9 controls, 9 patients with DS and 9 patients with AD. PGI activity in DS brain was significantly decreased in frontal, temporal lobe and cerebellum, comparable to controls in parietal lobe and elevated in occipital lobe. Brain PGI activity of patients with AD was comparable to controls in all regions tested, PFK, a rate limiting enzyme of glucose metabolism, was comparable between all brain regions of all three groups. Data of this study confirm impaired glucose metabolism in DS proposed in literature and found by positron emission tomography (PET) studies. We show that changes in glucose handling in patients with AD as evaluated by PET studies are not supported by our data, although not contradictory, as determinants other than glucose metabolizing enzymes as e.g. vascular factors and glucose transport may account for these findings. Changes of downregulated PGI found by subtractive hybridization at the transcriptional level in fetal DS brain along with our findings in DS brain regions suggest a strong specific link between glucose metabolism and DS rather than AD.

Down syndrome (DS) is a genetic disease that exhibits significant neuropathological parallels with Alzheimer's disease (AD). One of the features of DS, neuronal loss, has been hypothesized to occur as a result of apoptosis. An increasing number of proteins are implicated in apoptosis and several of them were shown to be altered in AD, however, the knowledge in DS is far from complete. To further substantiate the hypothesis that apoptosis is the underlying mechanism for neuronal loss and contribute towards the current knowledge of apoptosis in DS, we analyzed the expression of apoptosis related proteins in frontal cortex and cerebellum of DS by western blot and ELISA techniques. Quantitative analysis revealed a significant increase in DS frontal (P0.0001) and cerebellar (P0.05) Bim/BOD (Bcl-2 interacting mediator of cell death/Bcl-2 related ovarian death gene), cerebellar Bcl-2 (P0.01) as well as p21 (P0.05) levels compared to controls. No significant change was detected in Bax, RAIDD (receptor interacting protein (RIP)-associated ICH-1/CED-3-homologus protein with death domain), ZIP (Zipper interacting protein) kinase and NF-kappaB p65 levels in both regions, although frontal cortex levels of RAIDD, Bcl-2 and p21 levels tended to increase. In addition, a 45 kDa truncated form of NF-kappaB p65 displayed a significant elevation (P0.05) in DS cerebellum. No significant correlation had been obtained between postmortem interval and level of the proteins analyzed. With regard to age, it was only NF-kappaB p65 that showed significant correlation (r = -0.8964, P = 0.0155, n = 9) in frontal cortex of controls. These findings provide further evidence that apoptosis indeed accounts for the neuronal loss in DS but Bax and RAIDD do not appear to take part in this process.

Cholinergic deficit associated with loss of nicotinic acetylcholine receptors (nAChRs) has been described in Alzheimer's disease (AD) by receptor binding assays, positron emission tomography and immunoblotting. However, little is known about the alteration of these receptors in a related disease, Down syndrome (DS) which might be of importance for therapeutic strategies. The protein levels of neuronal nAChR alpha and beta subunits in human postmortem brain samples (frontal cortex and cerebellum) of control, adult DS, and AD were investigated by making use of western blot analysis. Two major bands at 26 and 45 kDa for alpha3, one at 50 kDa for alpha4 and beta2, and one at 45 kDa for alpha7 were detected by the respective antibodies. Specific alteration in individual subunits was also apparent in DS and AD. In frontal cortex, the 45kDa alpha3 subunit was significantly increased in DS (121%) (P0.05) and AD (93%) (P0.05), whereas the 26kDa, an isoform/truncated form of alpha3, displayed a reversed pattern. It was significantly decreased in DS (75%) (P0.001) and AD (52.6%) (P0.05). Alpha4 was comparable in all groups by contrast, alpha7 was significantly decreased in AD (64%) (P0.05). In DS, however, although the levels tended to be lower (17.3%) the reduction was not significant. Beta2 was unchanged in AD but showed a significant increase in DS frontal cortex (98.1%) (P0.01). In cerebellum, no significant alteration was observed in any of the subunits except beta2. It exhibited a significant increase (161%) (P0.01) in DS. Derangement in expression of nAChRs is apparent in DS, as in AD that may have some relevance to DS neuropathology. Furthermore, the increase in beta2 expression indicate that these subunits may have more than a structural role. Hence, therapeutic strategies tailored towards these end might be of some benefit for cognitive enhancement in these disorders.

The semantic variant of primary progressive aphasia (svPPA) is a clinical syndrome characterized by neurodegeneration and progressive loss of semantic knowledge. Unlike many other forms of frontotemporal lobar degeneration (FTLD), svPPA has a highly consistent underlying pathology composed of TDP-43 (a regulator of RNA and DNA transcription metabolism). Previous genetic studies of svPPA are limited by small sample sizes and a paucity of common risk variants. Despite this, svPPA’s relatively homogenous clinicopathologic phenotype makes it an ideal investigative model to examine genetic processes that may drive neurodegenerative disease. In this study, we used GWAS metadata, tissue samples from pathologically confirmed frontotemporal lobar degeneration, and in silico techniques to identify and characterize protein interaction networks associated with svPPA risk. We identified 64 svPPA risk genes that interact at the protein level. The protein pathways represented in this svPPA gene network are critical regulators of RNA metabolism and cell death, such as SMAD proteins and NOTCH1. Many of the genes in this network are involved in TDP-43 metabolism. Contrary to the conventional notion that svPPA is a clinical syndrome with few genetic risk factors, our analyses show that svPPA risk is complex and polygenic in nature. Risk for svPPA is likely driven by multiple common variants in genes interacting with TDP-43, along with cell death,x` working in combination to promote neurodegeneration.

ARPP-19 (cAMP-regulated phosphoprotein of Mr = 19,000) is a substrate for cAMP-dependent protein kinase (PKA). ARPP-19 is found in all brain regions but the function of ARPP-19 is not fully elucidated yet. We detected a downregulated sequence with 100% homology with ARPP-19 in temporal cortex of patients with Down syndrome (DS) as compared to controls, but not in Alzheimer's disease (AD) using differential displaypolymerase chain reaction (DD-PCR). We subsequently determined protein levels of ARPP-19 in temporal cortex and cerebellum by immunoblotting and observed significant reduction of ARPP-19 in DS (temporal cortex) and AD (cerebellum). We also observed decreased activities of PKA in DS (temporal cortex and cerebellum) and AD (temporal cortex). These findings suggest that decreased ARPP-19 along with decreased activities of PKA is involved in pathomechanisms of both neurodegenerative disorders. Furthermore, these findings provide first evidence for an impaired mechanism of cAMP-related signal transduction and phosphorylation in both dementing disorders.