Lubov Nathanson

Transgenic Inhibition of Astroglial NF-κB Improves Functional Outcome in Experimental Autoimmune Encephalomyelitis by Suppressing Chronic Central Nervous System Inflammation

In the CNS, the transcription factor NF-κB is a key regulator of inflammation and secondary injury processes. Following trauma or disease, the expression of NF-κB-dependent genes is activated, leading to both protective and detrimental effects. In this study, we show that transgenic inactivation of astroglial NF-κB (glial fibrillary acidic protein-IκBα-dominant-negative mice) resulted in reduced disease severity and improved functional recovery following experimental autoimmune encephalomyelitis. At the chronic stage of the disease, transgenic mice exhibited an overall higher presence of leukocytes in spinal cord and brain, and a markedly higher percentage of CD8+CD122+ T regulatory cells compared with wild type, which correlated with the timing of clinical recovery. We also observed that expression of proinflammatory genes in both spinal cord and cerebellum was delayed and reduced, whereas the loss of neuronal-specific molecules essential for synaptic transmission was limited compared with wild-type mice. Furthermore, death of retinal ganglion cells in affected retinas was almost abolished, suggesting the activation of neuroprotective mechanisms. Our data indicate that inhibiting NF-κB in astrocytes results in neuroprotective effects following experimental autoimmune encephalomyelitis, directly implicating astrocytes in the pathophysiology of this disease.

Microarray analysis of gene expression in adult retinal ganglion cells

Retinal ganglion cells (RGCs) transfer visual information to the brain and are known to be susceptible to selective degeneration in various neuropathies such as glaucoma. This selective vulnerability suggests that these highly specialized neurons possess a distinct gene expression profile that becomes altered by neuropathy‐associated stresses, which lead to the RGC death. In this study, to identify genes expressed predominantly in adult RGCs, a global transcriptional profile of purified primary RGCs has been compared to that of the whole retina. To avoid alterations of the original gene expression profile by cell culture conditions, we isolated RNA directly from adult RGCs purified by immunopanning without prior sub‐cultivation. Genes expressed predominantly in RGCs included: Nrg1, Rgn, 14‐3‐3 family (Ywhah, Ywhaz, Ywhab), Nrn1, Gap43, Vsnl1, Rgs4. Some of these genes may serve as novel markers for these neurons. Our analysis revealed enrichment in genes controlling the pro‐survival pathways in RGCs as compared to other retinal cells.

Organization of mammalian cytoplasm.

ABSTRACT Although the role of macromolecular interactions in cell function has attracted considerable attention, important questions about the organization of cells remain. To help clarify this situation, we used a simple protocol that measures macromolecule release after gentle permeabilization for the examination of the status of endogenous macromolecules. Treatment of Chinese hamster ovary cells with saponin under carefully controlled conditions allowed entry of molecules of at least 800 kDa; however, there were minimal effects on internal cellular architecture and protein synthesis remained at levels comparable to those seen with intact cells. Most importantly, total cellular protein and RNA were released from these cells extremely slowly. The release of actin-binding proteins and a variety of individual cytoplasmic proteins mirrored that of total protein, while marker proteins from subcellular compartments were not released. In contrast, glycolytic enzymes leaked rapidly, indicating that cells contain at least two distinct populations of cytoplasmic proteins. Addition of microfilament-disrupting agents led to rapid and extensive release of cytoplasmic macromolecules and a dramatic reduction in protein synthesis. These observations support the conclusion that mammalian cells behave as highly organized, macromolecular assemblies (dependent on the actin cytoskeleton) in which endogenous macromolecules normally are not free to diffuse over large distances.

Active Aminoacyl-tRNA Synthetases Are Present in Nuclei as a High Molecular Weight Multienzyme Complex

Recent studies suggest that aminoacylation of tRNA may play an important role in the transport of these molecules from the nucleus to the cytoplasm. However, there is almost no information regarding the status of active aminoacyl-tRNA synthetases within the nuclei of eukaryotic cells. Here we show that at least 13 active aminoacyl-tRNA synthetases are present in purified nuclei of both Chinese hamster ovary and rabbit kidney cells, although their steady-state levels represent only a small percentage of those found in the cytoplasm. Most interestingly, all the nuclear aminoacyl-tRNA synthetases examined can be isolated as part of a multienzyme complex that is more stable, and consequently larger, than the comparable complex isolated from the cytoplasm. These data directly demonstrate the presence of active aminoacyl-tRNA synthetases in mammalian cell nuclei. Moreover, their unexpected structural organization raises important questions about the functional significance of these multienzyme complexes and whether they might play a more direct role in nuclear to cytoplasmic transport of tRNAs.

Microarray analysis of fiber cell maturation in the lens

The mammalian lens consists of an aged core of quiescent cells enveloped by layers of mature fully elongated cells and younger, continuously elongating transcriptionally active cells. The fiber cell maturation is initiated when fiber cells cease to elongate. The process of maturation represents a radical switch from active elongation to a life‐long quiescence and has not been studied previously. It may also include critical stages of preparation for the organelle removal and denucleation. In the present study, we used laser capture microdisection (LCM) microdissection and RNA amplification to compare global gene expression profiles of young elongating and mature, non‐elongating fiber cells. Analysis of microarray data from three independent dye‐swap experiments identified 65 differentially expressed genes (FDR < 0.1) with greater than 2‐fold change in expression levels. Microarray array results for a group of randomly selected genes were confirmed by quantitative RT‐PCR. These microarray results provide clues to understanding the molecular pathways underlying lens development. The identified changes in the profile of gene expression reflected a shift in cell physiology characterizing the lens fiber maturation.

Inhibition of astroglial NF-kappaB enhances oligodendrogenesis following spinal cord injury

BackgroundAstrocytes are taking the center stage in neurotrauma and neurological diseases as they appear to play a dominant role in the inflammatory processes associated with these conditions. Previously, we reported that inhibiting NF-κB activation in astrocytes, using a transgenic mouse model (GFAP-IκBα-dn mice), results in improved functional recovery, increased white matter preservation and axonal sparing following spinal cord injury (SCI). In the present study, we sought to determine whether this improvement, due to inhibiting NF-κB activation in astrocytes, could be the result of enhanced oligodendrogenesis in our transgenic mice.MethodsTo assess oligodendrogenesis in GFAP-IκBα-dn compared to wild-type (WT) littermate mice following SCI, we used bromodeoxyuridine labeling along with cell-specific immuno-histochemistry, confocal microscopy and quantitative cell counts. To further gain insight into the underlying molecular mechanisms leading to increased white matter, we performed a microarray analysis in naïve and 3 days, 3 and 6 weeks following SCI in GFAP-IκBα-dn and WT littermate mice.ResultsInhibition of astroglial NF-κB in GFAP-IκBα-dn mice resulted in enhanced oligodendrogenesis 6 weeks following SCI and was associated with increased levels of myelin proteolipid protein compared to spinal cord injured WT mice. The microarray data showed a large number of differentially expressed genes involved in inflammatory and immune response between WT and transgenic mice. We did not find any difference in the number of microglia/leukocytes infiltrating the spinal cord but did find differences in their level of expression of toll-like receptor 4. We also found increased expression of the chemokine receptor CXCR4 on oligodendrocyte progenitor cells and mature oligodendrocytes in the transgenic mice. Finally TNF receptor 2 levels were significantly higher in the transgenic mice compared to WT following injury.ConclusionsThese studies suggest that one of the beneficial roles of blocking NF-κB in astrocytes is to promote oligodendrogenesis through alteration of the inflammatory environment.

Evidence of novel fine-scale structural variation at autism spectrum disorder candidate loci

BackgroundAutism spectrum disorders (ASD) represent a group of neurodevelopmental disorders characterized by a core set of social-communicative and behavioral impairments. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, acting primarily via the GABA receptors (GABR). Multiple lines of evidence, including altered GABA and GABA receptor expression in autistic patients, indicate that the GABAergic system may be involved in the etiology of autism.MethodsAs copy number variations (CNVs), particularly rare and de novo CNVs, have now been implicated in ASD risk, we examined the GABA receptors and genes in related pathways for structural variation that may be associated with autism. We further extended our candidate gene set to include 19 genes and regions that had either been directly implicated in the autism literature or were directly related (via function or ancestry) to these primary candidates. For the high resolution CNV screen we employed custom-designed 244 k comparative genomic hybridization (CGH) arrays. Collectively, our probes spanned a total of 11 Mb of GABA-related and additional candidate regions with a density of approximately one probe every 200 nucleotides, allowing a theoretical resolution for detection of CNVs of approximately 1 kb or greater on average. One hundred and sixty-eight autism cases and 149 control individuals were screened for structural variants. Prioritized CNV events were confirmed using quantitative PCR, and confirmed loci were evaluated on an additional set of 170 cases and 170 control individuals that were not included in the original discovery set. Loci that remained interesting were subsequently screened via quantitative PCR on an additional set of 755 cases and 1,809 unaffected family members.ResultsResults include rare deletions in autistic individuals at JAKMIP1, NRXN1, Neuroligin4Y, OXTR, and ABAT. Common insertion/deletion polymorphisms were detected at several loci, including GABBR2 and NRXN3. Overall, statistically significant enrichment in affected vs. unaffected individuals was observed for NRXN1 deletions.ConclusionsThese results provide additional support for the role of rare structural variation in ASD.

Differential gene expression profiling of large and small retinal ganglion cells

Different sub-populations of retinal ganglion cells (RGCs) vary in their sensitivity to pathological conditions such as retinal ischemia, diabetic retinopathy and glaucoma. Comparative transcriptomic analysis of such groups will likely reveal molecular determinants of differential sensitivity to stress. However, gene expression profiling of primary neuronal sub-populations represent a challenge due to the cellular heterogeneity of retinal tissue. In this manuscript, we report the use of a fluorescent neural tracer to specifically label and selectively isolate RGCs with different soma sizes by fluorescence-activated cell sorting (FACS) for the purpose of differential gene expression profiling. We identified 145 genes that were more active in the large RGCs and 312 genes in the small RGCs. Differential data were validated by quantitative RT-PCR, several corresponding proteins were confirmed by immunohistochemistry. Functional characterization revealed differential activity of genes implicated in synaptic transmission, neurotransmitter secretion, axon guidance, chemotaxis, ion transport and tolerance to stress. An in silico reconstruction of cellular networks suggested that differences in pathway activity between the two sub-populations of RGCs are controlled by networks interconnected by SP-1, Erk2 (MAPK1), Egr1, Egr2 and, potentially, regulated via transcription factors C/EBPbeta, HSF1, STAT1- and c-Myc. The results show that FACS-aided purification of retrogradely labeled cells can be effectively utilized for transcriptional profiling of adult retinal neurons.

Myocardial Alternative RNA Splicing and Gene Expression Profiling in Early Stage Hypoplastic Left Heart Syndrome

Hypoplastic Left Heart Syndrome (HLHS) is a congenital defect characterized by underdevelopment of the left ventricle and pathological compensation of the right ventricle. If untreated, HLHS is invariably lethal due to the extensive increase in right ventricular workload and eventual failure. Despite the clinical significance, little is known about the molecular pathobiological state of HLHS. Splicing of mRNA transcripts is an important regulatory mechanism of gene expression. Tissue specific alterations of this process have been associated with several cardiac diseases, however, transcriptional signature profiles related to HLHS are unknown. In this study, we performed genome-wide exon array analysis to determine differentially expressed genes and alternatively spliced transcripts in the right ventricle (RV) of six neonates with HLHS, compared to the RV and left ventricle (LV) from non-diseased control subjects. In HLHS, over 180 genes were differentially expressed and 1800 were differentially spliced, leading to changes in a variety of biological processes involving cell metabolism, cytoskeleton, and cell adherence. Additional hierarchical clustering analysis revealed that differential gene expression and mRNA splicing patterns identified in HLHS are unique compared to non-diseased tissue. Our findings suggest that gene expression and mRNA splicing are broadly dysregulated in the RV myocardium of HLHS neonates. In addition, our analysis identified transcriptome profiles representative of molecular biomarkers of HLHS that could be used in the future for diagnostic and prognostic stratification to improve patient outcome.

Regional and temporal differences in gene expression of LH(BETA)T(AG) retinoblastoma tumors.

PURPOSE The purpose of this study was to evaluate by microarray the hypothesis that LH(BETA)T(AG) retinoblastoma tumors exhibit regional and temporal variations in gene expression. METHODS LH(BETA)T(AG) mice aged 12, 16, and 20 weeks were euthanatized (n = 9). Specimens were taken from five tumor areas (apex, anterior lateral, center, base, and posterior lateral). Samples were hybridized to gene microarrays. The data were preprocessed and analyzed, and genes with a P < 0.01, according to the ANOVA models, and a log(2)-fold change >2.5 were considered to be differentially expressed. Differentially expressed genes were analyzed for overlap with known networks by using pathway analysis tools. RESULTS There were significant temporal (P < 10(-8)) and regional differences in gene expression for LH(BETA)T(AG) retinoblastoma tumors. At P < 0.01 and log(2)-fold change >2.5, there were significant changes in gene expression of 190 genes apically, 84 genes anterolaterally, 126 genes posteriorly, 56 genes centrally, and 134 genes at the base. Differentially expressed genes overlapped with known networks, with significant involvement in regulation of cellular proliferation and growth, response to oxygen levels and hypoxia, regulation of cellular processes, cellular signaling cascades, and angiogenesis. CONCLUSIONS There are significant temporal and regional variations in the LH(BETA)T(AG) retinoblastoma model. Differentially expressed genes overlap with key pathways that may play pivotal roles in murine retinoblastoma development. These findings suggest the mechanisms involved in tumor growth and progression in murine retinoblastoma tumors and identify pathways for analysis at a functional level, to determine significance in human retinoblastoma. Microarray analysis of LH(BETA)T(AG) retinal tumors showed significant regional and temporal variations in gene expression, including dysregulation of genes involved in hypoxic responses and angiogenesis.