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Stanislav L Karsten

Publications and source records attributed to Stanislav L Karsten.

11 recordsLinked to original sources
PubMed2006

Degradation of tau protein by puromycin-sensitive aminopeptidase in vitro.

Tau, a microtubule associated protein, aggregates into intracellular paired helical filaments (PHFs) by an unknown mechanism in Alzheimer's disease (AD) and other tauopathies. A contributing factor may be a failure to metabolize free cytosolic tau within the neuron. The buildup of tau may then drive the aggregation process through mass action. Therefore, proteases that normally degrade tau are of great interest. A recent genetic screen identified puromycin-sensitive aminopeptidase (PSA) as a potent modifier of tau-induced pathology and suggested PSA as a possible tau-degrading enzyme. Here we have extended these observations using human recombinant PSA purified from Escherichia coli. The enzymatic activity and characteristics of the purified PSA were verified using chromogenic substrates, metal ions, and several specific and nonspecific protease inhibitors, including puromycin. PSA was shown to digest recombinant human full-length tau in vitro, and this activity was hindered by puromycin. The mechanism of amino terminal degradation of tau was confirmed using a novel N-terminal cleavage-specific tau antibody (Tau-C6g, specific for cleavage between residues 13-14) and a C-terminal cleavage-specific tau antibody (Tau-C3). Additionally, PSA was able to digest soluble tau purified from normal human brain to a greater extent than either soluble or PHF tau purified from AD brain, indicating that post-translational modifications and/or polymerization of tau may affect its digestion by PSA. These results are consistent with observations that PSA modulates tau levels in vivo and suggest that this enzyme may be involved in tau degradation in human brain.

Alzheimer Disease↗
PubMed2006

Genetic analysis of anterior posterior expression gradients in the developing mammalian forebrain.

Intrinsic regulatory factors play critical roles in early cortical patterning, including the development of the anteroposterior (A-P) axis. To identify genes that are differentially expressed along the A-P axis of the developing cerebral cortex, we analyzed gene expression in presumptive frontal, parietal, and occipital cerebral walls of E12.5 mouse using complementary DNA microarrays. We identified 106 genes, including expressed sequence tags (ESTs), expressed in an A-P gradient in the embryonic brain and screened 88 by in situ hybridization for confirmation. Central nervous system (CNS) expression patterns of many of these genes were previously unknown. Others, such as Sfrp1, CoupTF1, and FABP7, were expressed in a manner consistent with previous studies, providing independent confirmation. Two related transcription factors, previously not implicated in CNS development, Fhl1 and Fhl2, were observed to be enriched in posterior and anterior telencephalon, respectively. We studied patterning gradients in Fhl1 knockout mice but observed no changes in gene expression related to A-P regionalization in the Fhl1 knockout mice. These data provide an important set of new candidates for studies of cortical patterning and maturation.

Animals↗
PubMed2006

A genomic screen for modifiers of tauopathy identifies puromycin-sensitive aminopeptidase as an inhibitor of tau-induced neurodegeneration.

Neurofibrillary tangles (NFT) containing tau are a hallmark of neurodegenerative diseases, including Alzheimer's disease (AD). NFT burden correlates with cognitive decline and neurodegeneration in AD. However, little is known about mechanisms that protect against tau-induced neurodegeneration. We used a cross species functional genomic approach to analyze gene expression in multiple brain regions in mouse, in parallel with validation in Drosophila, to identify tau modifiers, including the highly conserved protein puromycin-sensitive aminopeptidase (PSA/Npepps). PSA protected against tau-induced neurodegeneration in vivo, whereas PSA loss of function exacerbated neurodegeneration. We further show that human PSA directly proteolyzes tau in vitro. These data highlight the utility of using both evolutionarily distant species for genetic screening and functional assessment to identify modifiers of neurodegeneration. Further investigation is warranted in defining the role of PSA and other genes identified here as potential therapeutic targets in tauopathy.

Aminopeptidases↗
PubMed2006

FACS-array profiling of striatal projection neuron subtypes in juvenile and adult mouse brains.

A major challenge in systems neuroscience is to perform precise molecular genetic analyses of a single neuronal population in the context of the complex mammalian brain. Existing technologies for profiling cell type-specific gene expression are largely limited to immature or morphologically identifiable neurons. In this study, we developed a simple method using fluorescent activated cell sorting (FACS) to purify genetically labeled neurons from juvenile and adult mouse brains for gene expression profiling. We identify and verify a new set of differentially expressed genes in the striatonigral and striatopallidal neurons, two functionally and clinically important projection neuron subtypes in the basal ganglia. We further demonstrate that Ebf1 is a lineage-specific transcription factor essential to the differentiation of striatonigral neurons. Our study provides a general approach for profiling cell type-specific gene expression in the mature mammalian brain and identifies a set of genes critical to the function and dysfunction of the striatal projection neuron circuit.

Aging↗
PubMed2005

Exercise your amyloid.

The interplay of genetics and the environment in the etiology of Alzheimer's disease (AD) is not well understood. Now, Lazarov and coworkers show that a simple paradigm of environmental enrichment alleviates amyloid burden and alters disease-associated gene expression changes in a double transgenic mouse model of AD.

Alzheimer Disease↗
PubMed2004

Large-scale microarray gene expression analysis in discrete electrophysiologically identified neuronal clusters.

The normal processes of learning and memory as well as the pathological progress of various neurological diseases may result in changes in gene expression in small, local populations of neurons in any given brain area, leading to the occurrence of specific patterns of electrical activity without easily detectable changes in the morphology of this brain area. One way of identifying these changes might be the comparison of gene expression of areas which generate and areas which do not generate specific patterns of electrical activity. A method for microbiopsy of limited (0.5-1.0 mm3) tissue samples from electrophysiologically identified areas of neurons generating epileptiform activity in the rat brain is described. Here we demonstrate that total RNA isolated from individual microbiopsy samples might be successfully used for microarray based gene expression analysis of any discretely localized neuronal group which can be identified electrophysiologically, including neurons in cortical columns, cell assemblies or other functional units.

Animals↗
PubMed2004

Identification of a Hoxd10-regulated transcriptional network and combinatorial interactions with Hoxa10 during spinal cord development.

Hoxd10 is expressed in the posterior spinal cord and hindlimbs of the mouse. Hoxd10, along with other Hox transcription factors, is thought to regulate the activity of genes involved in nervous system patterning and motor neuron development, but little is known about the downstream targets regulated by this gene. cDNA microarrays were used to investigate the transcriptional network regulated by Hoxd10 in homozygous knockout animals. Sixty-nine genes were identified with altered expression levels in mutant spinal cords. Among these were genes involved in such diverse cellular events as cellular communication, cell cycle control, development and differentiation, and neuronal survival. The expression of some of these genes was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. Nine genes showed changes in expression of the same sign and similar magnitude using RT-PCR in Hoxd10 single mutant animals, with additional changes in expression seen in Hoxa10/Hoxd10 double mutant animals. In situ hybridization studies also demonstrated changes in expression consistent with microarray results. Analysis of putative promoter regions for Hox protein binding sites suggested that some genes may be direct Hoxd10 targets, whereas others likely are regulated through intermediate steps. Using cDNA microarrays to study a single gene knockout during critical developmental stages has identified a large number of genes regulated by Hoxd10, many of which would not have been approached as candidates for Hox gene regulation based on function or expression.

Animals↗
PubMed2003

Global analysis of gene expression in neural progenitors reveals specific cell-cycle, signaling, and metabolic networks.

The genetic programs underlying neural stem cell (NSC) proliferation and pluripotentiality have only been partially elucidated. We compared the gene expression profile of proliferating neural stem cell cultures (NS) with cultures differentiated for 24 h (DC) to identify functionally coordinated alterations in gene expression associated with neural progenitor proliferation. The majority of differentially expressed genes (65%) were upregulated in NS relative to DC. Microarray analysis of this in vitro system was followed by high throughput screening in situ hybridization to identify genes enriched in the germinal neuroepithelium, so as to distinguish those expressed in neural progenitors from those expressed in more differentiated cells in vivo. NS cultures were characterized by the coordinate upregulation of genes involved in cell cycle progression, DNA synthesis, and metabolism, not simply related to general features of cell proliferation, since many of the genes identified were highly enriched in the CNS ventricular zones and not widely expressed in other proliferating tissues. Components of specific metabolic and signal transduction pathways, and several transcription factors, including Sox3, FoxM1, and PTTG1, were also enriched in neural progenitor cultures. We propose a putative network of gene expression linking cell cycle control to cell fate pathways, providing a framework for further investigations of neural stem cell proliferation and differentiation.

Animals↗
PubMed2002

An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis.

Archival formalin-fixed, paraffin-embedded and ethanol-fixed tissues represent a potentially invaluable resource for gene expression analysis, as they are the most widely available material for studies of human disease. Little data are available evaluating whether RNA obtained from fixed (archival) tissues could produce reliable and reproducible microarray expression data. Here we compare the use of RNA isolated from human archival tissues fixed in ethanol and formalin to frozen tissue in cDNA microarray experiments. Since an additional factor that can limit the utility of archival tissue is the often small quantities available, we also evaluate the use of the tyramide signal amplification method (TSA), which allows the use of small amounts of RNA. Detailed analysis indicates that TSA provides a consistent and reproducible signal amplification method for cDNA microarray analysis, across both arrays and the genes tested. Analysis of this method also highlights the importance of performing non-linear channel normalization and dye switching. Furthermore, archived, fixed specimens can perform well, but not surprisingly, produce more variable results than frozen tissues. Consistent results are more easily obtainable using ethanol-fixed tissues, whereas formalin-fixed tissue does not typically provide a useful substrate for cDNA synthesis and labeling.

Animals↗
PubMed2002

Thresholding rules for recovering a sparse signal from microarray experiments.

We consider array experiments that compare expression levels of a high number of genes in two cell lines with few repetitions and with no subject effect. We develop a statistical model that illustrates under which assumptions thresholding is optimal in the analysis of such microarray data. The results of our model explain the success of the empirical rule of two-fold change. We illustrate a thresholding procedure that is adaptive to the noise level of the experiment, the amount of genes analyzed, and the amount of genes that truly change expression level. This procedure, in a world of perfect knowledge on noise distribution, would allow reconstruction of a sparse signal, minimizing the false discovery rate. Given the amount of information actually available, the thresholding rule described provides a reasonable estimator for the change in expression of any gene in two compared cell lines.

Animals↗