Talin Gulesserian

Expression of apoptosis related proteins in brains of patients with Alzheimer's disease

An increasing number of proteins are implicated in apoptosis and several of them have been shown to be altered in Alzheimers disease (AD) brain. Because of this apoptosis is thought to be the underlying mechanism of neuronal cell loss in AD. To further substantiate this hypothesis we investigated the expression of a recently identified apoptosis related proteins and other apoptosis regulators in frontal cortex and cerebellum of AD by Western blot and enzyme-linked immunsorbent assay technique. Quantitative analysis revealed unaltered levels of Bax and RAIDD (Receptor interacting protein associated ICH-1 (caspase-2)/CED-3 (Caenorhabditis elegans death protease-3)-homologous protein with death domain) in both regions. ZIP (Zipper interacting protein) kinase, Bim/BOD (Bcl-2 interacting mediator of cell death/Bcl-2 related ovarian death gene) and p21 were significantly increased only in AD frontal cortex (P < 0.05, in all cases). Cerebellar Bcl-2 levels were significantly increased in AD (P < 0.01) while in AD frontal cortex, although the levels tended to increase did not reach significance level. The results indicate that apoptosis indeed account for the neuronal loss in AD. However, it does not seem to involve Bax and RAIDD.

Superoxide Dismutase SOD1, Encoded on Chromosome 21, but Not SOD2 Is Overexpressed in Brains of Patients With Down Syndrome

Abstract Background The antioxidant enzyme Cu/Zn-superoxide dismutase-1 (SOD1) gene is localized to chromosome 21q22.1 and catalyzes the dismutation of superoxide anions to hydrogen peroxide, which may lead to the increased production of active oxygen species in Down Syndrome (DS), trisomy 21. Although a number of studies have addressed this question, proposing the overexpression hypothesis, no specific protein-chemical data on SOD protein levels in the brains of patients with DS are available. Methods We therefore determined the protein SOD-1 and SOD-2 levels in the brains of controls (n=9) and adult patients with DS (n=9) and Alzheimer disease (AD; n=9). Two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization-mass spectroscopy detection and identification was used for the analyses. Results We found significantly increased SOD-1 levels in DS temporal, parietal, and occipital cortex, whereas SOD-1 was decreased in the AD temporal cortex and SOD-2 was comparable between all groups. Conclusions Increased SOD-1 levels in patients with DS may reflect the overexpression by the trisomic state, as a response to the oxidative stress, as has been proposed in DS by several authors. However, it well may be that glial proliferation, which is markedly increased in DS brain, may underlie the increased brain levels of this ubiquitous protein. The decrease of SOD1 in the temporal cortex of patients with AD may reflect an antiapoptotic mechanism or simply cell loss in the brain.

Aberrant expression of signaling‐related proteins 14‐3‐3 gamma and RACK1 in fetal Down Syndrome brain (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.

Antioxidant proteins in fetal brain: superoxide dismutase-1 (SOD-1) protein is not overexpressed in fetal Down syndrome

Exposure of living organisms to reactive oxygen species (ROS), notably oxygen free radicals and hydrogen peroxide is closely linked to the very fact of aerobic life. Oxidants, however, are not always detrimental for cell survival, indeed moderate concentrations of ROS serve as signaling molecules. To maintain this level, cells have evolved an antioxidant defense system. Disruption of this balance leads either to oxidative or reductive stress. Down syndrome (DS) is a genetic disorder associated with oxidative stress. Overexpression of superoxide dismutase-1 (SOD-1) as a result of gene loading is suggested to be responsible for this phenomenon. To examine this view, we investigated the expression of thirteen different proteins involved in the cellular antioxidant defense system in brains of control and DS fetuses by two-dimensional electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS). No detectable change was found in expression of SOD-1, catalase, phospholipid hydroperoxide glutathione peroxidase, glutathione reductase, antioxidant enzyme AOE372, thioredoxin-like protein and selenium binding protein between control and DS fetuses. By contrast, a significant reduction was observed in levels of glutathione synthetase (P < 0.01), glutathione-S-transferase mu2 (P < 0.01), glutathione-S-transferase p (P < 0.05), antioxidant protein 2 (P < 0.05), thioredoxin peroxidase-I (P < 0.05) and thioredoxin peroxidase-II (P < 0.01) in DS compared with controls. The data suggest that oxidative stress in fetal DS does not result from overexpression of SOD-1 protein, rather oxidative stress appears to be the consequence of low levels of reducing agents and enzymes involved in removal of hydrogen peroxide.

Aberrant protein expression in cerebral cortex of fetus with 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.

Brain t-complex polypeptide 1 (TCP-1) related to its natural substrate β1 tubulin is decreased in Alzheimer's disease

The t-complex polypeptide 1 is a selective molecular chaperone in tubulin biogenesis, by that nascent tubulin subunits are bound to t-complex polypeptide 1 and released in assembly competent forms. In neurodegenerative diseases with Alzheimer pathology cytoskeletal proteins are deficient and aggregated. Therefore we examined t-complex polypeptide 1 as represented by the zeta subunit and its specific substrate beta 1 tubulin represented by a truncated product in six brain regions of nine patients with Alzheimers disease, nine patients with Down syndrome and nine controls. We used 2 dimensional electrophoresis with in-gel-digestion and matrix-assisted laser desorption/ ionization- mass spectrometry for the separation and identification of human brain t-complex polypeptide 1 and beta 1 tubulin. When t-complex polypeptide I was related to its natural and specific substrate beta 1 tubulin, the ratio was significantly decreased in the temporal, frontal, parietal cortex and in thalamus of patients with Alzheimers disease. In Down syndrome the t-complex polypeptide 1/beta 1 tubulin ratio was significantly increased in frontal and parietal cortex suggesting a different mechanism for aggregation of microfilament proteins e.g. beta 1 tubulin. Relatively decreased molecular chaperoning of beta 1 tubulin by t-complex polypeptide 1 may lead to misfolded tubulin aggregating and accumulating in plaques and tangles, a hallmark of Alzheimers disease. Our contribution provides first clues for a mechanism of microtubular accumulation in Alzheimers disease and challenges further studies on different chaperones and chaperonins in the brain of patients with neurodegenerative diseases.

Effect of a Rapeseed Oil Substituting Diet on Serum Lipids and Lipoproteins in Children and Adolescents with Familial Hypercholesterolemia

Objective:Familial hypercholesterolemia (FH) is a predominantly inherited disorder, which contributes to a defect of the LDL-cholesterol receptor. For adults with familial hypercholesterolemia (FH), it is known that a supplementary diet of monounsaturated fatty acids reduces elevated levels of total cholesterol and LDL-cholesterol and may further increase HDL-cholesterol. In particular the reduced intake of dietary fat reduces total serum cholesterol and LDL-cholesterol in the range of 10% to 15% and inhibits LDL-oxidation. Once the diagnosis of familial hypercholesterolemia is made in early childhood a supplementary diet with rapeseed oil should be started as early as possible to prevent development of atherosclerosis and subsequent complications. So far there are no reports of a lipid lowering diet enriched with rapeseed oil in children and adolescents. Methods:Seventeen children and young adolescents (male = 6, female = 11, ages 4 to 19 years) diagnosed with FH were enrolled in this study. They received dietary training and a classical low fat/low cholesterol diet enriched with rapeseed oil over five months. In the first two months they received orally mean 15 g/day (8–23 g/day), for the remaining three months mean 22 g/day (15–30 g/day) rapeseed oil. The calculation of the three-days dietary protocols showed the following characteristics: 29.5% calories from fat, 14.3% calories from protein and 54.6% calories from carbohydrates. The subjects had six sessions of dietary counseling, and serum lipids levels and lipoprotein(a) were estimated; each month’s diet adherence was controlled by a dietitian and discussed with the patients and their families during this five-month study. Result:During five months of rapeseed oil diet serum triglycerides decreased by 29% (119.2 ± 62.8 mg/dL vs. 84.9 mean ± 39.7 mg/dL), VLDL-cholesterol by 27% (23 ± 12 mg/dL vs. 17 ± 8 mg/dL), total cholesterol by 10% (233 ± 35 mg/dL vs. 213 ± 36 mg/dL), LDL-cholesterol by 7% (151 ± 31 mg/dL vs. 142 ± 31 mg/dL). HDL-cholesterol (59 ± 15 mg/dL vs. 57 ± 11 mg/dL) and Lp(a) (29.8 ± 36.3 mg/dL vs. 32.6 ± 40.7 mg/dL) were not changed significantly. The diet was well accepted; in most families a sustained change was reported. Conclusions:Our results indicate that in children and adolescents with FH a lipid-lowering diet with rapeseed oil has a similar effect on total serum cholesterol and LDL-cholesterol compared to classical cholesterol reduction diets (step I). However, an additional pronounced effect on lowering of triglycerides and VLDL-cholesterol can be observed.

Increased protein levels of serotonin transporter in frontal cortex of patients with Down syndrome.

Serotonin transporters (SERTs) are presynaptic proteins specialized for the clearance of serotonin from the synaptic cleft. A large body of evidence exists on altered platelet serotonin uptake and metabolism in Down syndrome (DS). Besides, dysregulation of SERTs expression have been reported in various complex behavioural traits and disorders including, neurodegenerative disorders. This prompted us to investigate SERT protein levels in adult brain specimens. Western blot analyses were performed in frontal cortex and cerebellum of aged controls and patients with DS and Alzheimers disease (AD). The result revealed that frontal cortex SERT was significantly increased (P<0.05) in DS, whereas in AD it was comparable to controls. In cerebellum, no significant difference was observed among the study populations. A remarkable difference was noted when SERT was normalized vs. neuron specific enolase (NSE), a neuronal marker. The increase in SERT/NSE was highly significant (P<0.01) in DS frontal cortex compared to controls. Neither AD frontal cortex nor DS and AD cerebellum did show any significant difference. These findings indicate that a region specific alteration in SERT expression may exist in DS with AD-like pathology. As little is known about the status of serotenergic synaptic markers in DS brain, the findings may contribute to an effort made to delineate the underlying causes of serotonergic dysfunction in DS and the quest for therapeutic strategies. The study also suggest caution should be taken in applying data obtained from DS to model AD biochemical defects.

Changes in nicotinic acetylcholine receptor subunits expression in brain of patients with Down syndrome and Alzheimer's disease

Cholinergic deficit associated with loss of nicotinic acetylcholine receptors (nAChRs) has been described in Alzheimers 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%) (P < 0.05) and AD (93%) (P < 0.05), whereas the 26kDa, an isoform/truncated form of alpha3, displayed a reversed pattern. It was significantly decreased in DS (75%) (P < 0.001) and AD (52.6%) (P < 0.05). Alpha4 was comparable in all groups by contrast, alpha7 was significantly decreased in AD (64%) (P < 0.05). 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%) (P < 0.01). In cerebellum, no significant alteration was observed in any of the subunits except beta2. It exhibited a significant increase (161%) (P < 0.01) 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.

Deterioration of the transcriptional, splicing and elongation machinery in brain of fetal Down Syndrome

Perturbation of brain development i.e. regulation of gene expression, differentiation, growth and migration in Down Syndrome (DS) has been reported to occur early in life pointing to impairment of the complex system of transcription and or translation and indeed, altered expression of transcription factors has been reported in adult DS brain. We therefore decided to compare the transcriptional and translational machinery in cortex of brains of controls and fetuses with Down syndrome in the second trimenon of gestation. We determined a series of transcription/translation factors by 2 D-electrophoresis followed by MALDI--identification and quantification with specific software. The protooncogene C-CRK, CRK-like protein, elongation factor 1-alpha 1, elongation factor 2, elongation factor tu and two out of four spots representing PTB-associated splicing factor PSF were significantly downregulated in brain of fetal DS fetuses as compared to controls. The finding of reduced transcription and translation factors may indicate deranged protein synthesis. The underlying cause for individual reduced transcription, splicing and translation factors may be explained by chromosomal imbalance or by posttranslational modifications as e.g. phosphorylation, known to be aberrant in DS. Reduced expression of transcription factors in fetal DS during early life may be responsible or reflecting impaired brain development and deficient wiring of the brain in DS.