Connexin-43 (Cx43) is the most abundant gap junction protein in brain, where it is found primarily between astrocytes. Although the morphology of astrocytes from Cx43-null (knockout, KO) mice is similar to that of wild-type (WT) astrocytes, KO astrocytes exhibit reduced growth rate in culture. To evaluate the impact of deletion of Cx43 on other genes, including those encoding cell cycle proteins, we used DNA arrays to determine expression patterns in cultured astrocytes from sibling Cx43-null and WT mice. RNA samples extracted from astrocytes cultured from WT and Cx43-null neonatal mice were dye labeled and individually cohybridized with a reference of labeled cDNAs pooled from a variety of tissues on 8 gene arrays containing 8,975 mouse DNA sequences. Normal variability in expression of each gene was evaluated and incorporated into "expression scores" to statistically compare expression levels between WT and KO samples. In Cx43-null astrocytes, 4.1% of the 4,998 adequately quantifiable spots were found to have significantly (P < 0.05) decreased hybridization compared with controls, and 9.4% of the spots showed significantly higher hybridization. The significantly different spots corresponded to RNAs encoding 252 known proteins, many not previously linked to gap junctions, including transcription factors, channels and transporters, cell growth and death signals, enzymes and cell adhesion molecules. These data indicate a surprisingly high degree of impact of deletion of Cx43 on other astrocyte genes, implying that gap junction gene expression alters numerous processes in addition to intercellular communication.

Both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), its animal model, involve inflammatory attack on central nervous system (CNS) white matter, leading to demyelination and axonal damage. Changes in astrocytic morphology and function are also prominent features of MS and EAE. Resting astrocytes form a network that is interconnected through gap junctions, composed mainly of connexin43 (Cx43) protein. Although astrocytic gap junctional connectivity is known to be altered in many CNS pathologies, little is known about Cx43 expression in inflammatory demyelinating disease. Therefore, we evaluated the expression of Cx43 in spinal cords of EAE mice compared with healthy controls. Lumbar ventral white matter areas were heavily infiltrated with CD11β-immunoreactive monocytes, and within these infiltrated regions loss of Cx43 immunoreactivity was evident. These regions also showed axonal dystrophy, demonstrated by the abnormally dephosphorylated heavy-chain neurofilament proteins. Astrocytes in these Cx43-depleted lesions were strongly glial fibrillary acidic protein reactive. Significant loss (38%) of Cx43 protein in EAE mouse at the lumbar portion of spinal cords was confirmed by Western blot analysis. Decreased Cx43 transcript level was also observed on cDNA microarray analysis. In addition to changes in Cx43 expression, numerous other genes were altered, including those encoding adhesion and extracellular matrix proteins. Our data support the notion that, in addition to damage of myelinating glia, altered astrocyte connectivity is a prominent feature of inflammatory demyelination.Keywords white matter; inflammation; immunohistochemistry; microarray; experimental autoimmune encephalomyelitis; Cx43Multiple sclerosis (MS) is an immune-cell-mediated inflammatory demyelinating disease of the central nervous system (CNS;Miller and Karpus, 1994;Ewing and Bernard, 1998;Hohlfeld and Wekerle, 2001). MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS (Honegger and Langemann, 1989;Glabinski et al., 1997;Deloire-Grassin et al., 2000), are characterized by white matter (WM) inflammation accompanied by disturbed central nerve conductance and motor and sensory impairments (Juhler, 1988;Whitaker and Mitchell, 1997 Pathological findings in MS and EAE include infiltration of activated peripheral inflammatory cells into the CNS (Bauer et al., 1995;Owens et al., 1998;Imrich and Harzer, 2001), loss of myelin (Adams and Kubik, 1952;Raine et al., 1984;Whitaker and Mitchell, 1997), axonal damage (Ferguson et al., 1997;Trapp et al., 1998;Kornek et al., 2000;Bitsch et al., 2000;Deloire-Grassin et al., 2000), axonal loss (D'amelio et al., 1990;Landete et al., 2000;Bjartmar et al., 2001), edema (Claudio et al., 1990Levine et al., 1966), and astrocytic activation (Smith et al., 1983;Eng et al., 1992;Rafalowska et al., 1992). Notably, swelling of astrocytes is an early key event that precedes macrophage infiltration to the CNS in EAE (Smith et al., 1984;Eng et al., 1989Eng et al., , ...

Extensive studies on mice with total or partial disruption of either connexin43 (Cx43) or connexin32 (Cx32) have detected only subtle changes in central nervous system structure, growth, development, or function. We have used high density cDNA arrays to analyze the regulation, control, and coordination of the abundances of 7446 distinct transcripts in four brains, each of Cx43 null (K43), Cx43 heterozygous (H43), and Cx32 null (K32) mice as compared to the brains of wildtype (W) mice. The use of multiple samples allowed the determination of the statistical significance of gene regulation. Significantly regulated genes encoded proteins of all functional categories, extending beyond those that might be expected to depend on junctional communication. Moreover, we found a high degree of similarity between genes regulated in the K43 and H43 brains and a remarkable overlap between gene regulation in brains of K43 and K32. The regulated genes in both K43 and H43 brains showed an outstanding inverse coordination with the levels of expression of Cx43 in W brain, indicating that the regulated genes are largely predictable from their co-variance with Cx43 in the wildtype samples. These findings lead to the hypothesis that connexin expression may represent a central node in the regulation of gene expression patterns in brain.

Gene regulatory networks underlie the long-term changes in cell specification, growth of synaptic connections, and adaptation that occur throughout neonatal and postnatal life. Here we show that the transcriptional response in neurons is exquisitely sensitive to the temporal nature of action potential firing patterns. Neurons were electrically stimulated with the same number of action potentials, but with different inter-burst intervals. We found that these subtle alterations in the timing of action potential firing differentially regulates hundreds of genes, across many functional categories, through the activation or repression of distinct transcriptional networks. Our results demonstrate that the transcriptional response in neurons to environmental stimuli, coded in the pattern of action potential firing, can be very sensitive to the temporal nature of action potential delivery rather than the intensity of stimulation or the total number of action potentials delivered. These data identify temporal kinetics of action potential firing as critical components regulating intracellular signalling pathways and gene expression in neurons to extracellular cues during early development and throughout life.

Chronic chagasic cardiomyopathy is a leading cause of heart failure in Latin American countries. About 30% of Trypanosoma cruzi-infected individuals develop this severe symptomatic form of the disease, characterized by intense inflammatory response accompanied by fibrosis in the heart. We performed an extensive microarray analysis of hearts from a mouse model of this disease and determined significant alterations in expression of ∼12% of the sampled genes. Extensive upregulations were associated with immune-inflammatory responses (chemokines, adhesion molecules, cathepsins and MHC molecules) and fibrosis (extracellular matrix components, lysyl oxidase and Timp-1). Our results indicate potentially relevant factors involved in the pathogenesis of the disease that may provide new therapeutic targets in chronic Chagas' disease.

We hypothesize that distinct cell phenotypes are governed by different sets of gene master regulators (GMRs) whose strongly protected (by the homeostatic mechanisms) abundance modulates most cell processes by coordinating the expression of numerous genes from the corresponding functional pathways. Gene Commanding Height (GCH), a composite measure of gene expression control and coordination, is introduced to establish the gene hierarchy in each phenotype. If the hypothesis is true, than one can selectively destroy cancer nodules from a heterogeneous tissue by altering the expression of genes whose GCHs are high in cancer but low in normal cell phenotype. Here, we test the hypothesis and show its utility for the thyroid cancer (TC) gene therapy. First, we prove that malignant and cancer free surrounding areas of a surgically removed papillary TC (PTC) tumor are governed by different GMRs. Second, we show that stable transfection of a gene induces larger transcriptomic alterations in the cells where it has higher GCH than in other cells. For this, we profiled the transcriptomes of the papillary BCPAP and anaplastic 8505C TC cell lines before and after stable transfection with NEMP1, DDX19B, PANK2 or UBALD1. The four genes were selected to have similar expression levels but significantly different GCH scores in the two cell lines before transfection. Indeed, each of the four genes triggered larger alterations in the cells where they had larger GCH. Our results prove the feasibility of a personalized gene therapy approach that selectively targets the cancer cells from a tissue.

The dynamic and never exactly repeatable tumor transcriptomic profile of people affected by the same form of cancer requires a personalized and time-sensitive approach of the gene therapy. The Gene Master Regulators (GMRs) were defined as genes whose highly controlled expression by the homeostatic mechanisms commands the cell phenotype by modulating major functional pathways through expression correlation with their genes. The Gene Commanding Height (GCH), a measure that combines the expression control and expression correlation with all other genes, is used to establish the gene hierarchy in each cell phenotype. We developed the experimental protocol, the mathematical algorithm and the computer software to identify the GMRs from transcriptomic data in surgically removed tumors, biopsies or blood from cancer patients. The GMR approach is illustrated with applications to our microarray data on human kidney, thyroid and prostate cancer samples, and on thyroid, prostate and blood cancer cell lines. We proved experimentally that each patient has his/her own GMRs, that cancer nuclei and surrounding normal tissue are governed by different GMRs, and that manipulating the expression has larger consequences for genes with higher GCH. Therefore, we launch the hypothesis that silencing the GMR may selectively kill the cancer cells from a tissue.

Microarray experiments have generally focused on magnitude of gene expression changes in pathological conditions, thereby using the method as a high throughput screen to identify candidate marker genes and/or to validate phenotypic differences. We have used novel strategies to extract additional information from array studies, including expression variability and coordination, from which organizational principles of transcriptomes are emerging. We have reported that the expression level, variability and coordination of numerous genes are regulated in brains of connexin43 null (Gja1(-/-)) mouse with respect to wildtype. Moreover, expression coordination with Gja1 in wildtype largely predicted the expression regulation in Gja1(-/-) tissues. We now report a remarkable overlap between regulations in Gja1(-/-) and connexin32 null (Gjb1(-/-)) brains, and that both differ markedly from those in connexin36 null (Gja9(-/-)) brain. Since in brain these three connexins are expressed in different cell types (Cx43 in astrocytes, ependymal and vascular cells, Gjb1 in oligodendrocytes, and Cx36 in neurons and microglia), and because astrocytes and oligodendrocytes (and possibly neurons and microglia) may form syncytia coupled by gap junction channels, these observations suggest the existence of distinct connexin-dependent panglial and neuronal transcriptomic networks. Such networks, where linkage partners are rearranged and strengths modified in brains of knockouts, may explain downstream and parallel "ripples" of phenotypic change resulting from single gene manipulations as illustrated by alterations in transcription factor networks resulting from deletion of Gja1 or Gjb1. The transcription factors also formed network hubs with genes from other functional categories, thus allowing regulation of one functional pathway through manipulation of another.