Syndrome Brain Research Articles

Rab5-stimulated Up-regulation of the Endocytic Pathway Increases Intracellular β-Cleaved Amyloid Precursor Protein Carboxyl-terminal Fragment Levels and Aβ Production

We previously identified abnormalities of the endocytic pathway in neurons as the earliest known pathology in sporadic Alzheimer's disease (AD) and Down's syndrome brain. In this study, we modeled aspects of these AD-related endocytic changes in murine L cells by overexpressing Rab5, a positive regulator of endocytosis. Rab5-transfected cells exhibited abnormally large endosomes immunoreactive for Rab5 and early endosomal antigen 1, resembling the endosome morphology seen in affected neurons from AD brain. The levels of both Abeta40 and Abeta42 in conditioned medium were increased more than 2.5-fold following Rab5 overexpression. In Rab5 overexpressing cells, the levels of beta-cleaved amyloid precursor protein (APP) carboxyl-terminal fragments (betaCTF), the rate-limiting proteolytic intermediate in Abeta generation, were increased up to 2-fold relative to APP holoprotein levels. An increase in beta-cleaved soluble APP relative to alpha-cleaved soluble APP was also observed following Rab5 overexpression. BetaCTFs were co-localized by immunolabeling to vesicular compartments, including the early endosome and the trans-Golgi network. These results demonstrate a relationship between endosomal pathway activity, betaCTF generation, and Abeta production. Our findings in this model system suggest that the endosomal pathology seen at the earliest stage of sporadic AD may contribute to APP proteolysis along a beta-amyloidogenic pathway.

AppGene Dosage Modulates Endosomal Abnormalities of Alzheimer's Disease in a Segmental Trisomy 16 Mouse Model of Down Syndrome

Altered neuronal endocytosis is the earliest known pathology in sporadic Alzheimer's disease (AD) and Down syndrome (DS) brain and has been linked to increased Abeta production. Here, we show that a genetic model of DS (trisomy 21), the segmental trisomy 16 mouse Ts65Dn, develops enlarged neuronal early endosomes, increased immunoreactivity for markers of endosome fusion (rab5, early endosomal antigen 1, and rabaptin5), and endosome recycling (rab4) similar to those in AD and DS individuals. These abnormalities are most prominent in neurons of the basal forebrain, which later develop aging-related atrophy and degenerative changes, as in AD and DS. We also show that App, one of the triplicated genes in Ts65Dn mice and human DS, is critical to the development of these endocytic abnormalities. Selectively deleting one copy of App or a small portion of the chromosome 16 segment containing App from Ts65Dn mice eliminated the endosomal phenotype. Overexpressing App at high levels in mice did not alter early endosomes, implying that one or more additional genes on the triplicated segment of chromosome 16 are also required for the Ts65Dn endosomal phenotype. These results identify an essential role for App gene triplication in causing AD-related endosomal abnormalities and further establish the pathogenic significance of endosomal dysfunction in AD.

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: Challenging the gene dosage effect hypothesis (Part IV).

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.

Understanding mental retardation in Down's syndrome using trisomy 16 mouse models.

Mental retardation in Down's syndrome, human trisomy 21, is characterized by developmental delays, language and memory deficits and other cognitive abnormalities. Neurophysiological and functional information is needed to understand the mechanisms of mental retardation in Down's syndrome. The trisomy mouse models provide windows into the molecular and developmental effects associated with abnormal chromosome numbers. The distal segment of mouse chromosome 16 is homologous to nearly the entire long arm of human chromosome 21. Therefore, mice with full or segmental trisomy 16 (Ts65Dn) are considered reliable animal models of Down's syndrome. Ts65Dn mice demonstrate impaired learning in spatial tests and abnormalities in hippocampal synaptic plasticity. We hypothesize that the physiological impairments in the Ts65Dn mouse hippocampus can model the suboptimal brain function occuring at various levels of Down's syndrome brain hierarchy, starting at a single neuron, and then affecting simple and complex neuronal networks. Once these elements create the gross brain structure, their dysfunctional activity cannot be overcome by extensive plasticity and redundancy, and therefore, at the end of the maturation period the mind inside this brain remains deficient and delayed in its capabilities. The complicated interactions that govern this aberrant developmental process cannot be rescued through existing compensatory mechanisms. In summary, overexpression of genes from chromosome 21 shifts biological homeostasis in the Down's syndrome brain to a new less functional state.

Global up-regulation of chromosome 21 gene expression in the developing down syndrome brain

Down syndrome (DS) results from complete or partial triplication of human chromosome 21. It is assumed that the neurological and other symptoms are caused by the overexpression of genes on chromosome 21, but this hypothesis has not yet been assessed on a chromosome-wide basis. Here we show that expression of genes localized to chromosome 21 is globally up-regulated in human fetal trisomy 21 cases, both in cerebral cortex extracts and in astrocytic cell lines cultured from cerebral cortex. This abnormal regulation of gene expression is specific to chromosome 21. Our data describe transcriptional changes that are specific to many genes assigned to chromosome 21 and do not directly measure the clinical phenotype of DS. However, it is possible that these gene expression changes ultimately relate to the phenotypic variability of DS.

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part III).

Down syndrome (DS) is the most frequent genetic disorder with mental retardation and caused by trisomy 21. Although the gene dosage effect hypothesis has been proposed to explain the impact of extra chromosome 21 on the pathology of DS, a series of evidence that challenge this hypothesis has been reported. The availability of the complete sequences of genes on chromosome 21 serves now as starting point to find functional information of the gene products, but information on gene products is limited so far. We therefore evaluated expression levels of six proteins whose genes are encoded on chromosome 21 (synaptojanin-1, chromosome 21 open reading frame 2, oligomycin sensitivity confering protein, peptide 19, cystatin B and adenosine deaminase RNA-specific 2) in fetal cerebral cortex from DS and controls at 18-19 weeks of gestational age using Western blot analysis. Synaptojanin-1 and C21orf2 were increased in DS, but others were comparable between DS and controls, suggesting that the DS phenotype cannot be simply explained by gene dosage effects. We are systematically quantifying all proteins whose genes are encoded on chromosome 21 in order to provide a better understanding of the pathobiochemistry of DS at the protein level. These studies are of significance as they show for the first time protein levels that are carrying out specific function in human fetal brain with DS.

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part II).

Down syndrome (DS) is the most common genetic cause of mental retardation. To explain the impact of extra chromosome 21 in the pathology of DS, gene dosage effect hypothesis has been proposed, but several investigators including our group have challenged this hypothesis. Although analysis of the sequence of chromosome 21 has been essentially completed, the molecular and biochemical mechanisms underlying the pathology are still unknown. We therefore investigated expression levels of six proteins encoded on chromosome 21 (HACS1, DYRK1A, alphaA-crystallin, FTCD, GARS-AIRS-GART, and CBS) in fetal cerebral cortex from DS and controls at 18-19 weeks of gestational age using Western blot analysis. Protein expression of HACS1 was significantly and remarkably decreased in DS, and the expression levels of five proteins were comparable between DS and controls suggesting that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are continuing to quantify proteins whose genes are encoded on chromosome 21 in order to provide a better understanding of the pathobiochemistry of DS at the protein level.

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part I).

Down syndrome (DS) is the most significant genetic disorder with mental retardation and is caused by trisomy 21. The phenotype of DS is thought to result from overexpression of a gene(s) located on the triplicated chromosome (region). An increasing body of evidence that challenge this "gene dosage effect" hypothesis, however, has been reported indicating that this hypothesis still remains to be elucidated. The availability of the complete sequence of genes on chromosome 21 could have an immediate impact on DS research, but no conclusions can be drawn from nucleic acid levels. This made us evaluate protein levels of six proteins, gene products, encoded on chromosome 21 (T-cell lymphoma invasion and metastasis inducing Tiam1 protein, holocarboxylase synthetase, human interferon-regulated resistance GTP-binding protein MxA, Pbx regulating protein 1, autoimmune regulator, and pericentrin) in fetal cortex from DS and controls at 18-19 weeks of gestational age using Western blot technique. None of the investigated proteins showed overexpression in DS compared to controls. Our present data showing unaltered expression of six proteins on chromosome 21 in fetal DS brain suggest that the existence of the trisomic state is not involved in abnormal development of fetal DS brain and that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are in the process of quantifying all gene products of chromosome 21 and our first results do not support the gene dosage hypothesis.

Molecular changes in fetal Down syndrome brain

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.

Increased RNA levels of the 25 kDa synaptosomal associated protein in brain samples of adult patients with Down Syndrome

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.

Manifold decreased protein levels of matrin 3, reduced motor protein HMP and hlark in fetal Down's syndrome brain.

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.

Balanced X chromosome inactivation patterns in the Rett syndrome brain.

In Rett syndrome (RTT), an X-linked disorder essentially limited to females, neurological development goes awry. Causing this disarray in neuronal function is a mutated form of a protein known as methyl-CpG-binding protein 2 (MeCP2). Because the MECP2 gene is subject to X chromosome inactivation (XCI) in females, a number of studies have addressed whether the percentage of cells inactivating the normal vs. mutant chromosome in heterozygous females influences the phenotypic outcome of MECP2 mutations. Because most of these studies measured XCI in peripheral blood, however, interpretation of the results requires the assumption that XCI patterns in blood are representative of those in the brain, the primarily affected tissue. Here, we have analyzed the MECP2 sequence and XCI status in 13 brains of RTT patients. Mutations were identified in nine of the cases, with eight of these representing C to T transitions at CpG dinucleotides, and one being a novel frameshift mutation (765delA). Patterns of XCI were balanced in 10 of 10 cases for which the assay was informative. As previous studies have shown that a majority of RTT patients have balanced XCI patterns in peripheral blood, our results suggest that the pattern in blood is an accurate indicator of XCI patterns in the brain for a majority of cases, but there are some notable exceptions that this study may help explain. Given the correlation between balanced XCI and classic RTT, these results suggest that a certain percentage of neurons expressing the mutant MECP2 gene may be required for RTT to become manifest.

Caspase-cleaved amyloid precursor protein and activated caspase-3 are co-localized in the granules of granulovacuolar degeneration in Alzheimer's disease and Down's syndrome brain.

Granulovacuolar degeneration (GVD) is a diagnostic neuropathological feature of Alzheimer's disease (AD). In some neurons, apoptosis has been hypothesized to be a primary mechanism causing neuronal cell death in AD. In this study we investigated CA1 neurons with GVD in AD and Down's syndrome (DS) brain. We demonstrated that activated caspase-3 and a caspase-cleaved cleavage product of the amyloid precursor protein (cAPP) are co-localized in GVD granules, and that these same cells often show nuclear DNA damage. In contrast, activated caspase-8 is present in the cytoplasm but not within the granules of GVD neurons. A caspase-cleavage product of fodrin that accumulates in many AD and DS neurons is not present in GVD granules. These data support a role for the activation of apoptotic mechanisms in selective compartments exhibiting GVD.

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 Centractin and Capping Proteins, Integral Constituents of the Dynactin Complex, in Fetal Down Syndrome Brain

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 α, F-actin capping protein α-1, α-2 and β 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.

Intraneuronal Aβ42 accumulation in Down syndrome brain

Alzheimer's disease (AD) brains display A beta (Aβ) plaques, inflammatory changes and neurofibrillary tangles (NFTs). Converging evidence suggests a neuronal origin of Aβ. We performed a temporal study of intraneuronal Aβ accumulation in Down syndrome (DS) brains. Sections from temporal cortex of 70 DS cases aged 3 to 73 years were examined immunohistochemically for immunoreactivity (IR) for the Aβ N-terminal, the Aβ40 C-terminus and the Aβ42 C-terminus. N-terminal antibodies did not detect intracellular Aβ Aβ40 antibodies did not detect significant intracellular Aβ, but older cases showed Aβ40 IR in mature plaques. in contrast, Aβ42 antibodies revealed clear-cut intraneuronal IR. All Aβ42 antibodies tested showed strong intraneuronal Aβ42 IR in very young DS patients, especially in the youngest cases studied (e.g., 3 or 4 yr. old), but this IR declined as extracellular Applaques gradually accumulated and matured. No inflammatory changes were associated with intraneuronal Aβ. We also studied the temporal development of gliosis and NFT formation, revealing that in DS temporal cortex, inflammation and NFT follow A β deposition. We conclude that Aβ42 accumulates intracellulary prior to extracellular Aβ deposition in Down syndrome, and that subsequent maturation of extracellular Aβ deposits elicits inflammatory responses and precedes NFTs.

Reduced levels of DEAD-box proteins DBP-RB and p72 in fetal Down syndrome brains.

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.

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.

Protein expression in Down syndrome brain.

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.

Gene Expression Profiling in Postmortem Rett Syndrome Brain: Differential Gene Expression and Patient Classification

The identification of mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MECP2) gene in Rett Syndrome (RTT) suggests that an inappropriate release of transcriptional silencing may give rise to RTT neuropathology. Despite this progress, the molecular basis of RTT neuropathogenesis remains unclear. Using multiple cDNA microarray technologies, subtractive hybridization, and conventional biochemistry, we generated comprehensive gene expression profiles of postmortem brain tissue from RTT patients and matched controls. Many glial transcripts involved in known neuropathological mechanisms were found to have increased expression in RTT brain, while decreases were observed in the expression of multiple neuron-specific mRNAs. Dramatic and consistent decreases in transcripts encoding presynaptic markers indicated a specific deficit in presynaptic development. Employing multiple clustering algorithms, it was possible to accurately segregate RTT from control brain tissue samples based solely on gene expression profile. Although previously achieved in cancers, our results constitute the first report of human disease classification using gene expression profiling in a complex tissue source such as brain.