Rachel Weitzdoerfer

Fetal life in Down Syndrome starts with normal neuronal density but impaired dendritic spines and synaptosomal structure

Information on fetal brain in Down Syndrome (DS) is limited and there are only few histological, mainly anecdotal reports and no systematic study on the wiring of the brain in early prenatal life exists. Histological methods are also hampered by inherent problems of morphometry of neuronal structures. It was therefore the aim of the study to evaluate neuronal loss, synaptic structures and dendritic spines in the fetus with Down Syndrome as compared to controls by biochemical measurements. 2 dimensional electrophoresis with subsequent mass spectroscopical identification of spots and their quantification with specific software was selected. This technique identifies proteins unambiguously and concomitantly on the same gel. Fetal cortex samples were taken at autopsy with low post-mortem time, homogenized and neuron specific enolase (NSE) determined as a marker for neuronal density, the synaptosomal associated proteins alpha SNAP [soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein], beta SNAP, SNAP 25 and the channel associated protein of synapse 110 (chapsyn 110) as markers for synaptosomal structures and drebrin (DRB) as marker for dendritic spines. NSE, chapsyn 110 and beta SNAP were comparable in the control fetus panel and in Down Syndrome fetuses. Drebrin was significantly and remarkably reduced and not even detectable in several Down Syndrome brain samples. Quantification of SNAP 25 revealed significantly reduced values in DS cortex and alpha SNAP was only present in half of the DS individuals. We conclude that at the time point of about 19 weeks of gestation (early second trimester) no neuronal loss can be detected but drebrin, a marker for dendritic spines and synaptosomal associated proteins alpha SNAP and SNAP 25 were significantly reduced indicating impaired synaptogenesis. Early dendritic deterioration maybe leading to the degeneration of the dendritic tree and arborization, which is a hallmark of Down Syndrome from infancy.

Reduction of actin-related protein complex 2/3 in fetal Down syndrome brain

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.

Aberrant expression of dihydropyrimidinase related proteins-2,-3 and -4 in fetal Down Syndrome brain

Pathfinding of growing axons to reach their target during brain development is a subtle process needed to build up contacts between neurons. Abnormalities in brain development in Down Syndrome (DS) are described in a couple of morphological reports but the molecular mechanisms underlying abnormal wiring in fetal DS brain are not yet elucidated. We therefore performed a study using the proteomic approach to show differences in protein levels involved in the guidance of axons between control and DS brain in early prenatal life. Proteins obtained from autopsy of human fetal abortus were applied on 2-dimensional gel, identified and quantified. We quantified 5 members of the semaphorin/collapsin family, the dihydropyrimidinase related proteins 1-4 and the collapsin response mediator protein-5 (CRMP-5) in 8 DS and 7 control cortex samples. DRP-1 and CRMP-5 levels were comparable in the control and DS samples. Evaluation of DRP-2, DRP-3 and DRP-4 revealed significantly decreased levels of 2 of the 15 spots assigned to DRP-2 and increased levels of one spot assigned to DRP-3 and increased DRP-4 in DS brain. We conclude that as early as from the 19th week of gestation pathfinding cues of the outgrowing axons are impaired in DS. These findings may help to elucidate mechanisms leading to abnormalities in neural migration of DS 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.

Expression of cystathionine β-synthase, pyridoxal kinase, and ES1 protein homolog (mitochondrial precursor) in fetal Down syndrome brain

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.

Long-Term Influence of Perinatal Asphyxia on the Social Behavior in Aging Rats

Background: Various groups have been addressing the question of whether perinatal asphyxia (PA) affects the behavior of young animals, but no information is available on long-term effects of PA on the behavior in aged rats, although it has been postulated that PA may lead to neurological and psychiatric deficits in adult life. Objective: We, therefore, decided to study the effects of PA on social and anxiety-related behaviors in 2-year-old rats, using a noninvasive animal model resembling the clinical situation. Methods: For the behavioral studies, the open-field test, the elevated plus-maze test, and a social interaction test in pairs were performed. Magnetic resonance imaging of the brain was selected to rule out neuropathological changes due to the aging process per se, as well as asphyxia-induced pathologies in the brain areas known to play an important role in the modulation of behavior. Results: The social interaction test revealed a statistically significant increase in the number of social grooming episodes and the time spent running alone, whereas the numbers of social sniffing and fighting episodes and the time spent running together were decreased in the asphyxiated group. The elevated plus- maze test revealed a higher presence of entries into the closed arm. Furthermore, sniffing and self-grooming episodes were significantly increased in the asphyxiated group. Conclusions: We found a significantly decreased social aggressiveness and an increased social contact behavior as well as increased anxiety levels in the asphyxiated animals. The present findings may provide important information on the long-term behavioral sequelae of PA in the aged individual.

Long-term sequelae of perinatal asphyxia in the aging rat

Abstract. Information on the consequences of perinatal asphyxia (PA) on brain morphology and function in the aging rat is missing although several groups have hypothesized that PA may be responsible for neurological and psychiatric deficits in the adult. We therefore decided to study the effects of PA on the central nervous system (CNS) in terms of morphology, immunohistochemistry, neurology and behavior in the aging animal. Hippocampus and cerebellum were evaluated morphologically by histological, immunohistochemical and magnetic resonance imaging and cerebellum also by stereological tests. Neurological function was tested by an observational test battery and rota rod test. Cognitive functions were examined by multiple-T-maze and the Morris water maze (MWM). Increased serotonin transporter (SERT) immunoreactivity in the CA2 region of the hippocampus and a significant difference in the escape latency, when the platform of the MWM was moved to a new location, were observed in asphyxiated rats. We showed that deteriorated cognitive functions accompanied by aberrant expression of hippocampal SERT and impaired relearning are long-term sequelae of perinatal asphyxia, a finding that may form the basis for understanding CNS pathology in the aging subject, animal or human.

Derangement of hypothetical proteins in fetal Down's syndrome brain.

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 Downs 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 3β (ARP3β), 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.

Life-long effects of perinatal asphyxia on stress-induced proteins and dynamin 1 in rat brain.

In previous work, we have shown that perinatal asphyxia (PA) in the rat leads to life-long neurotransmitter deficits and impairment of cognitive functions and behavior. This observation made us examine protein expression in hippocampus of rats with PA at the end of the life span. We applied a well-documented and characterized animal model of PA. Pups, normoxic and asphyxiated for 20 min, were brought up until the age of 24 months and then were sacrificed. Hippocampal tissue was dissected from the brains, and proteins were run on two-dimensional gel electrophoresis with in-gel digestion and subsequent identification of proteins by MALDI-TOF followed by quantification of protein spots by specific software. In hippocampus of rats with PA, the stress proteins protein disulfide isomerase A3 precursor and stress-induced phosphoprotein-1 were significantly increased, whereas the microtubule-associated protein dynamin-1 was significantly reduced. Increased stress protein levels may represent long-term effects of PA or, alternatively, could reflect conditioning of the stress protein machinery known to occur as a neuroprotective principle following hypoxic-ischemic conditions. Decreased dynamin-1 levels may be considered as a long-term effect on the exocytotic system possibly reflecting or leading to impaired neuronal transport and vesicle-trafficking in PA of the rat of advanced age.

Transcription factor REST dependent proteins are comparable between Down Syndrome and control brains: challenging a hypothesis

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.