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Recent studies indicate that regulation of cellular oxidative capacity through enhancing mitochondrial biogenesis may be beneficial for neuronal recovery and survival in human neurodegenerative disorders. The peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) has been shown to be a master regulator of mitochondrial biogenesis and cellular energy metabolism in muscle and liver. The aim of our study was to establish whether PGC-1alpha and PGC-1beta control mitochondrial density also in neurons and if these coactivators could be up-regulated by deacetylation. The results demonstrate that PGC-1alpha and PGC-1beta control mitochondrial capacity in an additive and independent manner. This effect was observed in all studied subtypes of neurons, in cortical, midbrain, and cerebellar granule neurons. We also observed that endogenous neuronal PGC-1alpha but not PGC-1beta could be activated through its repressor domain by suppressing it. Results demonstrate also that overexpression of SIRT1 deacetylase or suppression of GCN5 acetyltransferase activates transcriptional activity of PGC-1alpha in neurons and increases mitochondrial density. These effects were mediated exclusively via PGC-1alpha, since overexpression of SIRT1 or suppression of GCN5 was ineffective where PGC-1alpha was suppressed by short hairpin RNA. Moreover, the results demonstrate that overexpression of PGC-1beta or PGC-1alpha or activation of the latter by SIRT1 protected neurons from mutant alpha-synuclein- or mutant huntingtin-induced mitochondrial loss. These evidences demonstrate that activation or overexpression of the PGC-1 family of coactivators could be used to compensate for neuronal mitochondrial loss and suggest that therapeutic agents activating PGC-1 would be valuable for treating neurodegenerative diseases in which mitochondrial dysfunction and oxidative damage play an important pathogenic role.

Organelle transport in neuronal processes is central to the organization, developmental fate, and functions of neurons. Organelles must be transported through the slender, highly branched neuronal processes, making the axonal transport vulnerable to any perturbation. However, some intracellular structures like mitochondria are able to considerably modify their volume. We therefore hypothesized that swollen mitochondria could impair the traffic of other organelles in neurite shafts. To test this hypothesis, we have investigated the effects of mitochondrial swellers on the organelle traffic. Our data demonstrate that treatment of neurons with potassium ionophore valinomycin led to the fast time-dependent inhibition of organelle movement in cerebellar granule neurons. Similar inhibition was observed in neurons treated with the inhibitors of the mitochondrial respiratory chain, sodium azide and antimycin, which also induced swelling. No decrease in the motility of organelles was observed in cultures treated with inhibitors of ATP production or transport, oligomycin or bongkrekic acid, suggesting that inhibition of the ATP-generating activity itself without swelling does not affect the motility of organelles. The effect of swellers on the traffic was more important in thin processes, thus indicating the role of steric hindrance of swollen mitochondria. We propose that the size and morphology of the transported cargo is also relevant for seamless axonal transport and speculate that mitochondrial swelling could be one of the reasons for impaired organelle transport in neuronal processes.

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease. In human SLE patients, as well as in mouse models of SLE, the development of disease is associated with increased levels of pro-inflammatory cytokines, such as interleukin-6 (IL-6). However, IL-6 target genes contributing to the development of disease remain to be identified. Our previous studies of one mouse model of SLE identified an interferon-inducible gene, Ifi202, as a major contributor to the disease. We now report that IL-6 induces expression of the Ifi202 gene. We found that IL-6 treatment of mouse splenocytes increased levels of Ifi202 mRNA and p202 protein. Furthermore, IL-6 treatment of NIH 3T3 cells or expression of a constitutively active form of STAT3, a known mediator of IL-6 signaling, stimulated the activity of a 202-luc-reporter through a potential STAT3 DNA-binding site (the 202-SBS) present in the 5'-regulatory region of the Ifi202 gene. Moreover, treatment of cells with IL-6 stimulated binding of the transcription factor STAT3 to an oligonucleotide containing the 202-SBS in gel-mobility shift assays and to the 5'-regulatory region of the Ifi202 gene in chromatin immunoprecipitation assays. Importantly, site-directed mutagenesis of 202-SBS or expression of a dominant negative form of STAT3 significantly reduced constitutive as well as IL-6-stimulated activity of the 202-luc-reporter. Together, our observations support the idea that IL-6 stimulates transcription of the Ifi202 gene through STAT3 activation and predict that increased levels of IL-6 in lupus contribute to up-regulation of p202.

p38 MAPK pathway signaling is known to participate in cell proliferation, apoptosis, and differentiation, in a manner dependent on the cellular context. The factors that determine the specific biological response in a given cell type, however, remain largely unknown. We report opposite effects of the p38 isoforms on regulation of AP-1-dependent activities by p38 activators MAPK kinase 6 (MKK6) and/or arsenite in human breast cancer cells. The p38β isoform increases the activation of AP-1 transcriptional activities by MKK6 and/or arsenite, whereas p38γ/p38δ inhibits or has no effect on the stimulation. The p38β does so by increasing the levels of phosphorylated c-Jun, whereas the p38γ and -δ isoforms may act by regulating the c-jun transcription. AP-1-dependent processes such as vitamin D receptor gene promoter activation and cellular proliferation were similarly activated by the p38β or inhibited by the p38γ and/or -δ isoforms. Whereas the human breast cancer cells express all four isoforms, mouse NIH 3T3 and EMT-6 cells express only some of the p38 family members, with p38β higher in 3T3 cells but p38δ only detected in the EMT-6 line. Consistent with the positive and negative roles of p38β and p38δ in AP-1 regulation, MKK6 stimulates AP-1-dependent transcription in NIH 3T3 but not EMT-6 cells. In support of a role of c-Jun regulation by p38 isoforms in determining AP-1 activity, the levels of endogenous c-Jun and its phosphorylated form on p38 activation are higher in NIH 3T3 cells. These results demonstrate the contrasting activities of the different p38 isoforms in transmitting the upstream signal to AP-1 and show that the expression profile of p38 isoforms determines whether the p38 signal pathway activates or inhibits AP-1-dependent processes.

The p53 tumor suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. We reported previously that overexpression of p202, an interferon-inducible negative regulator of cell growth, negatively regulates the transcriptional activity of p53. Now we identify the gene encoding p202 as one whose mRNA and protein expression decrease in cells following the expression of wild-type, but not mutant, p53. Furthermore, the levels of p202 also decrease after exposure of cells to ultra violet light, which correlate with increase in the levels of p53. We report that the sequence-specific DNA binding of p53 to the 5'-regulatory region of the 202 gene contributes to the transcriptional repression of the 202 gene. Interestingly, overexpression of p202 in cells induced to undergo p53-dependent apoptosis significantly delays this process, indicating that the negative regulation of the 202 gene by wild-type p53 is important to potentiate apoptosis.

p202, an interferon-inducible murine protein, is a member of the "200 family" of proteins and is primarily nuclear. p202 is a modulator of transcription; it binds several transcription factors, including NF-kappaB p50 and p65, AP-1 c-Fos and c-Jun, and E2F1, and inhibits their transcriptional activity. p202 also binds pRb, the retinoblastoma protein, and if overexpressed it retards cell proliferation. Here we report that using the yeast two-hybrid assay we found that p202 bound the murine homolog of the human p53-binding protein 1 (53BP1), a protein shown to interact with the DNA binding domain of the p53 tumor suppressor protein. p202 bound a 98amino acid segment from 53BP1. This binding was inhibited by the replacement in p202 of a histidine (from the M(F/L)HATVA(T/S) sequence that is conserved among all of the 200 family proteins) by phenylalanine. We also report that overexpression of p202 inhibited the p53-dependent expression of reporter genes containing p53-activable segments from the mdm2 and p21 genes, whereas a decrease in the p202 level (in consequence of the expression of 202 antisense RNA) resulted in an increase in the p53-dependent expression of these reporters. Expression of the 53BP1 segment binding to p202 overcame the inhibition by overexpressed p202 of the transcription of reporters mediated by the p53 or the AP-1 transcription factors and of the proliferation of yeast.

Many of the antimicrobial, immunomodulatory, and cell growth regulatory activities of the interferons are mediated by interferon-inducible proteins. One family of such murine proteins is encoded by six or more adjacent and structurally related genes (gene 200 cluster). Two homologous human genes have also been reported. p202, encoded by the Ifi202 gene in the gene 200 cluster, is a 52-kDa nuclear phosphoprotein. Constitutive overexpression of p202 in transfected cells is growth-inhibitory. We report here that p202 binds the cell growth regulatory retinoblastoma protein (pRb) in vitro and in vivo. The binding is due to direct interaction between the two proteins. p202 has two nonoverlapping segments for binding pRb, and pRb has two nonoverlapping segments (one of them including the pocket region) for binding p202. The hypophosphorylated form of pRb binds to p202, p202 is the first interferon-inducible protein found to bind pRb.

We have described earlier a gene cluster, including at least six interferon-activatable genes closely linked to the erythroid α spectrin locus and the serum amyloid P-component locus on murine chromosome 1. Here, we report that sequences of three genes from the cluster (the 201, 202, and 204 genes) are very similar in a segment extending from at least 550 nucleotides upstream of the 3′ end of the transcription initiation region to beyond the first exon intron border (96% similarity between the 202 and 204 genes and 89% similarity between the 201 and 204 genes). This region contains the following two types of interferon-responsive enhancers: a GA box and a Friedman Stark sequence. The proteins coded for by the 202 gene (51 kDa) and the 204 gene (72 kDa) are hydrophilic. The amino acids have been conserved in the two proteins in 47% of the sequence. Each protein includes two apparently contiguous, approximately 200-amino acid long segments with much sequence similarity (27% in the 202 protein and 34% in the 204 protein). These segments are preceded in the 204 protein only by a segment including four perfect and three imperfect repeats of a 7 amino acid sequence. These and other data suggest that the evolution of the gene cluster involved the duplication of a DNA segment generating a double length transcription unit and subsequent divergence and duplication of this unit giving rise to at least two interferon-activatable genes (the 202 and 204 genes).