Brain Nuclear Extracts Research Articles

Fructose-2,6-bisphosphate restores DNA repair activity of PNKP and ameliorates neurodegenerative symptoms in Huntington's disease.

Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3) are the two most prevalent polyglutamine (polyQ) neurodegenerative diseases, caused by CAG (encoding glutamine) repeat expansion in the coding region of the huntingtin (HTT) and ataxin-3 (ATXN3) proteins, respectively. We have earlier reported that the activity, but not the protein level, of an essential DNA repair enzyme, polynucleotide kinase 3'-phosphatase (PNKP), is severely abrogated in both HD and SCA3 resulting in accumulation of double-strand breaks in patients' brain genome. While investigating the mechanistic basis for the loss of PNKP activity and accumulation of DNA double-strand breaks leading to neuronal death, we observed that PNKP interacts with the nuclear isoform of 6-phosphofructo-2-kinase fructose-2,6-bisphosphatase 3 (PFKFB3). Depletion of PFKFB3 markedly abrogates PNKP activity without changing its protein level. Notably, the levels of both PFKFB3 and its product fructose-2,6 bisphosphate (F2,6BP), an allosteric modulator of glycolysis, are significantly lower in the nuclear extracts of post-mortem brain tissues of HD and SCA3 patients. Supplementation of F2,6BP restored PNKP activity in the nuclear extracts of patients' brain. Moreover, intracellular delivery of F2,6BP restored both the activity of PNKP and the integrity of transcribed genome in neuronal cells derived from striatum of HD mouse. Importantly, supplementing F2,6BP rescued the HD phenotype in Drosophila, suggesting F2,6BP to serve in vivo as a cofactor for the proper functionality of PNKP and thereby, of brain health. Our results thus provide a compelling rationale for exploring the therapeutic use of F2,6BP and structurally related compounds for treating polyQ diseases.

Quantifying Activity for Repair of the DNA Lesion 8-Oxoguanine by Oxoguanine Glycosylase 1 (OGG1) in Mouse Adult and Fetal Brain Nuclear Extracts Using Biotin-Labeled DNA.

The reactive oxygen species (ROS)-initiated DNA lesion 8-oxoguanine (8-oxoG) is commonly used as a biomarker to measure oxidative stress levels in tissue samples from animals and humans. This lesion also can play a pathogenic role in cancer, birth defects, and neurodegeneration, among other disorders. The level of 8-oxoG may be enhanced due to ROS-initiating environmental factors (e.g., drugs, gamma radiation, microbial infection) or due to a decrease in the activity of oxoguanine glycosylase 1 (OGG1), an enzyme that repairs this lesion. Measurement of the activity of OGG1 can be useful in elucidating mechanisms and complements measurements of 8-oxoG levels in tissues of interest. This protocol describes an assay for measuring the activity of 8-oxoG in mouse adult and fetal brain tissues. Briefly, a synthetic duplex containing the 8-oxoG residue in one of the nucleotides (49-mer), labeled with biotin at the 3'-end, is incubated with protein extract from the tissue of interest containing OGG1, which cleaves the 8-oxoG residue producing a cleavage product of ~27-mer. The percent cleavage quantifies the activity of OGG1 in that tissue. The biotin tag allows rapid and sensitive detection of the cleavage product via chemiluminescence, avoiding the problems of safety and short half-lives of radionuclides encountered in assays employing a radioactively-labeled substrate.

The Many Faces of MeCP2

Methyl-CpG-binding protein 2 (MeCP2) is an X-linked transcription factor that binds to methylated CpG dinucleotides in the genome. Its primary function was originally thought to be limited to repression of gene transcription in keeping with the transcriptional silence of methylated genes. CpG methylation is a hallmark of epigenetic regulation of genome function because it is copied to replicating DNA strands during cell division and is maintained across development as a form of epigenetic memory. Cytosine methylation in CpG clusters (islands) in the genome can modify chromatin structure by inhibiting activator DNA-binding proteins from binding to recognition sequences in DNA while allowing access by repressor proteins to methyl-CpP binding domains (MBD) that prevent the activation of gene transcription. MeCP2 is a member of the MBD family of proteins that associate with histone deacetylases (HDACs) and histone metyltransferases (HMTs) to form stable repressor complexes. MeCP2 point mutations cause Rett syndrome, the most common inherited form of mental retardation in girls. However, exactly which protein partners are affected by different MeCP2 mutations is controversial. For example, MeCP2 has been shown to associate with the Sin3a–HDAC chromatin remodeling complex in the brain-derived neurotrophic factor (Bdnf) gene to modulate its expression, and loss of MeCP2 from the Bdnf promoter causes loss of local histone methylation (Chen et al, 2003; Martinowich et al, 2003). However, the function of MeCP2 may be determined by its multifaceted interactions with many different protein partners leading to different functional outcomes. Indeed, MeCP2 appears to associate with many different complexes in brain nuclear extracts some of which repress, but others, like CREB, activate, gene transcription (Chahrour et al, 2008).

β‐N‐Oxalyl‐l‐α,β‐diaminopropionic acid regulates mitogen‐activated protein kinase signaling by down‐regulation of phosphatidylethanolamine‐binding protein 1

β-N-Oxalyl-L-α,β-diaminopropionic acid (l-ODAP) an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor agonist activates protein kinase C in white leghorn chick brain. The current study focuses on the protein kinase C downstream signaling targets associated with L-ODAP excitotoxicity in SK-N-MC human neuroblastoma cells and white leghorn male chick (Gallus domesticus) brain extracts. L-ODAP treatment in SK-N-MC cells (1.5 mM) and chicks (0.5 mg/g body weight) results in a decreased expression and increased phosphorylation of phosphatidylehthanolamine-binding protein 1 (PEBP1) up to 4 h which however, returns to normal by 8 h. D-ODAP, the non-toxic enantiomer however, did not affect PEBP1 levels in either chick brain or SK-N-MC cells. Decreased PEBP1 expression correlated with subsequent activation of Raf-1, MEK and ERK signaling components of the mitogen-activated protein kinase cascade and nuclear translocation of hypoxia inducible factor-1α (HIF-1α) in chick brain nuclear extracts and SK-N-MC cells. SK-N-MC cells over-expressing PEBP1 inhibited nuclear translocation of HIF-1α when treated with l-ODAP, indicating that down-regulation of PEBP1 is responsible for HIF-1α stabilization and nuclear localization. Excitotoxicity of L-ODAP may thus be the result of phosphorylation and down-regulation of PEBP1, a crucial signaling protein regulating diverse signaling cascades. L-ODAP induced convulsions and seizures in chicks could be the result of a hypoxic insult to brain.

Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes

TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative diseases. Although TDP-43 resembles heterogeneous nuclear ribonucleoproteins, its RNA targets and physiological protein partners remain unknown. Here we identify RNA targets of TDP-43 from cortical neurons by RNA immunoprecipitation followed by deep sequencing (RIP-seq). The canonical TDP-43 binding site (TG)n is 55.1-fold enriched, and moreover, a variant with adenine in the middle, (TG)nTA(TG)m, is highly abundant among reads in our TDP-43 RIP-seq library. TDP-43 RNA targets can be divided into three different groups: those primarily binding in introns, in exons, and across both introns and exons. TDP-43 RNA targets are particularly enriched for Gene Ontology terms related to synaptic function, RNA metabolism, and neuronal development. Furthermore, TDP-43 binds to a number of RNAs encoding for proteins implicated in neurodegeneration, including TDP-43 itself, FUS/TLS, progranulin, Tau, and ataxin 1 and -2. We also identify 25 proteins that co-purify with TDP-43 from rodent brain nuclear extracts. Prominent among them are nuclear proteins involved in pre-mRNA splicing and RNA stability and transport. Also notable are two neuron-enriched proteins, methyl CpG-binding protein 2 and polypyrimidine tract-binding protein 2 (PTBP2). A PTBP2 consensus RNA binding motif is enriched in the TDP-43 RIP-seq library, suggesting that PTBP2 may co-regulate TDP-43 RNA targets. This work thus reveals the protein and RNA components of the TDP-43-containing ribonucleoprotein complexes and provides a framework for understanding how dysregulation of TDP-43 in RNA metabolism contributes to neurodegeneration.

Differential regulation of HIF‐1 alpha and VEGF in an anoxia tolerant brain

We examined vascular endothelial growth factor (VEGF) expression during anoxia and related it to the binding activity and expression of HIF‐1alpha in the anoxia tolerant turtle Trachemys scripta. Whole animals and neuronal cultures were exposed to anoxia and reoxygenation to determine changes in mRNA and protein levels; immunohistochemistry showed VEGF in brain slices while EMSA was performed for HIF binding. High constitutive VEGF transcript and protein levels increased 2‐fold during anoxia, returning to basal upon reoxygenation. Brain slice VEGF‐positive cells also increased following 24h anoxia; VEGF colabelled with NeuN in the cortex while ependymal cells were positive for VEGF only. By contrast, high basal HIF protein levels did not increase in anoxia or reoxygenation, and decreased in whole brain nuclear extracts, as did DNA binding activity. As the results suggested a suppression of HIF in anoxia, we exposed cells to a HIF stimulator, which significantly increased cell death over 4h anoxia. Thus while VEGF is upregulated in this anoxia tolerant model, it is unlikely to be regulated by HIF. As HIF binding in hypoxic conditions promotes many functions known to be actively downregulated in the anoxic turtle, it is not surprising that nuclear levels and HIF binding decrease in this model, to the point where increased HIF activity decreases cell survival. This work was funded by NIH grant R15 S048909–01 to SLM.

Identification of Adjacent Binding Sites for the YY1 and E4BP4 Transcription Factors in the Ovine PrP (Prion) Gene Promoter

The PrP gene encodes the cellular isoform of the prion protein (PrP(c)) which has been shown to be crucial to the development of transmissible spongiform encephalopathies (TSEs). PrP knock-out mice, which do not express endogenous PrP(c), exhibit resistance to TSE disease. The regulation of PrP gene expression represents, therefore, a crucial factor in the development of TSEs. Two sequence motifs in the PrP promoter (positions -287 to -263 from transcriptional start) were previously reported as being highly conserved, and it was suggested that they represent binding sites for as yet unidentified transcription factors. To test this hypothesis, binding of nuclear proteins was analyzed by electrophoretic mobility shift assays using ovine or murine cells and tissues with radiolabeled DNA probes containing the conserved motif sequences. Specific binding was observed to both motifs, and polymorphic variants of these motifs exhibited differential binding. Two proteins bound to these motifs were identified as the Yin Yang 1 (YY1) (motif 1) and E4BP4 (motif 2) transcription factors. Functional promoter analysis of four different promoter variants revealed that motif 1 (YY1) was associated with inhibitory activity in the context of the PrP promoter, whereas motif 2 (E4BP4) was linked to a slight enhancing activity. This represents the first demonstration of binding of nuclear factors to two highly conserved DNA sequence motifs within mammalian PrP promoters. The action of these factors on the PrP promoter is haplotype-specific, leading us to propose that the prion protein expression pattern and, with it, the distribution of TSE infectivity may be associated with PrP promoter genotype.

Hu Proteins Regulate Polyadenylation by Blocking Sites Containing U-rich Sequences

A recent genome-wide bioinformatic analysis indicated that 54% of human genes undergo alternative polyadenylation. Although it is clear that differential selection of poly(A) sites can alter gene expression, resulting in significant biological consequences, the mechanisms that regulate polyadenylation are poorly understood. Here we report that the neuron-specific members of a family of RNA-binding proteins, Hu proteins, known to regulate mRNA stability and translation in the cytoplasm, play an important role in polyadenylation regulation. Hu proteins are homologs of the Drosophila embryonic lethal abnormal visual protein and contain three RNA recognition motifs. Using an in vitro polyadenylation assay with HeLa cell nuclear extract and recombinant Hu proteins, we have shown that Hu proteins selectively block both cleavage and poly(A) addition at sites containing U-rich sequences. Hu proteins have no effect on poly(A) sites that do not contain U-rich sequences or sites in which the U-rich sequences are mutated. All three RNA recognition motifs of Hu proteins are required for this activity. Overexpression of HuR in HeLa cells also blocks polyadenylation at a poly(A) signal that contains U-rich sequences. Hu proteins block the interaction between the polyadenylation cleavage stimulation factor 64-kDa subunit and RNA most likely through direct interaction with poly(A) cleavage stimulation factor 64-kDa subunit and cleavage and polyadenylation specificity factor 160-kDa subunit. These studies identify a novel group of mammalian polyadenylation regulators. Furthermore, they define a previously unknown nuclear function of Hu proteins.

Selective Inhibition of Nuclear Factor-κB Activation After Hypoxia/Ischemia in Neonatal Rats Is Not Neuroprotective

Activated nuclear factor-kappaB (NFkappaB) has been shown to increase transcription of several genes that could potentially contribute to neuronal damage, such as proinflammatory cytokines, chemokines, and inducible nitric oxide synthase. The aim of our study was to investigate whether inhibition of NFkappaB activation could prevent hypoxia/ischemia (HI)-induced cerebral damage in neonatal rats. We used a cell permeable peptide (NEMO binding domain [NBD] peptide) that is known to prevent the association of the regulatory protein NEMO with IKK, the kinase that activates NFkappaB. Via this mechanism, the NBD peptide can specifically block the activation of NFkappaB, without inhibiting basal NFkappaB activity. Cerebral HI was induced in neonatal rats by occlusion of the right carotid artery followed by 90 min of hypoxia (Fio(2) = 0.08). Immediately upon reoxygenation, as well as 6 and 12 h later, rats were treated with vehicle or NBD peptide (20 mg/kg i.p.). Histologic analysis of brain damage was performed at 6 wk after HI. To assess NFkappaB activation, electromobility shift assays (EMSAs) were performed on brain nuclear extracts obtained 6 h after reoxygenation. Increased NFkappaB activity could be shown at 6 h after HI in both hemispheres. Peripheral administration of NBD peptide prevented this HI-induced increase in NFkappaB activity in both hemispheres. Histologic analysis of long-term cerebral damage revealed that inhibition of NFkappaB activation by administration of NBD peptide at 0, 6, and 12 h after HI resulted in an increment of neuronal damage. In conclusion, our data suggest that inhibition of NFkappaB activation using NBD peptide early after HI increases brain damage in neonatal rats.

Neuronal cell type-specific promoter of the α CaM kinase II gene is activated by Zic2, a Zic family zinc finger protein

To understand the neuronal cell type-specific expression of Ca 2+/calmodulin-dependent protein kinase II (CaM kinase II), we investigated binding proteins that specifically activated the promoter of the α isoform of CaM kinase II (α CaM kinase II). Proteins that bind the promoter sequence were found in rat brain nuclear extract by electrophoretic mobility shift assay. Then, we screened for binding proteins in a mouse brain cDNA library using the yeast one-hybrid system. Zic2, a Zic family zinc finger transcription factor, was identified as one of the binding proteins. To investigate the effect of Zic2 on the promoter activity, Zic2 cDNA was expressed with a luciferase reporter gene containing a neuronal cell type-specific promoter of α CaM kinase II in neuronal and non-neuronal cells. The promoter activity of α CaM kinase II was enhanced 1.3–5-fold in cultured neuronal cells by Zic2. The activation was varied among neuronal cell types. Zic2 also increased the promoter activity in non-neuronal cells, although the relative luciferase activites in non-neuronal cells were lower than those in neuronal cell lines. These results indicated that Zic2 was one of the proteins binding to, and regulating the activity of, the promoter of α CaM kinase II.

Estradiol increases signal transducer and activator of transcription (STAT3) phosphorylation and DNA binding in brain nuclear extract after MCAO occlusion

Estradiol increases signal transducer and activator of transcription (STAT3) phosphorylation and DNA binding in brain nuclear extract after MCAO occlusion

Evolutionarily conserved and divergent regulatory sequences in the fish rod opsin promoter

Fish have multiple types and subtypes of opsin genes that are expressed in a highly regulated manner in retinal photoreceptor cells. In the rod opsin proximal promoter region (RPPR) of zebrafish ( Danio rerio), the BAT 1 regulatory region contains highly conserved OTX (GATTA) and OTX-like (TATTA) sequences that can be recognized by the mammalian cone–rod homeobox (CRX) protein. However, binding of zebrafish crx to the OTX sequence has remained elusive. In contrast to the BAT 1 region, the Ret 1 region, located approximately 20 bp upstream of the BAT 1 region in mammals, is not conserved in zebrafish. In the Ret 1 region, even the core OTX-like sequence (AATTA sequence in mammals) is destructed. We show in this study that a region between Ret 1 and BAT 1 (denoted IRB, Inter- Ret 1- BAT 1) is highly conserved among fish species. Using electrophoretic mobility shift assay (EMSA), we show that zebrafish crx binds to the conserved OTX sequence and that the fish-specific IRB region specifically binds elements present in both retinal and brain nuclear extracts of zebrafish. These results imply that the regulatory mechanisms of opsin gene expression consist not only of evolutionarily conserved but also of divergent machinery among different animal taxa.

Trans-activators Regulating Neuronal Glucose Transporter Isoform-3 Gene Expression in Mammalian Neurons

The murine facilitative glucose transporter isoform 3 is developmentally regulated and is predominantly expressed in neurons. By employing the primer extension assay, the transcription start site of the murine Glut 3 gene in the brain was localized to -305 bp 5' to the ATG translation start codon. Transient transfection assays in N2A neuroblasts using murine GLUT3-luciferase reporter constructs mapped enhancer activities to two regions located at -203 to -177 and -104 to -29 bp flanking a previously described repressor element (-137 to -130 bp). Dephosphorylated Sp1 and Sp3 proteins from the 1- and 21-day-old mouse brain nuclear extracts bound the repressor elements, whereas both dephosphorylated and phosphorylated cAMP-response element-binding protein (CREB) in N2A, 1- and 21-day-old mouse brain nuclear extracts bound the 5'-enhancer cis-elements (-187 to -180 bp) of the Glut 3 gene, and the Y box protein MSY-1 bound the sense strand of the -83- to -69-bp region. Sp3, CREB, and MSY-1 binding to the GLUT 3 DNA was confirmed by the chromatin immunoprecipitation assay, whereas CREB and MSY-1 interaction was detected by the co-immunoprecipitation assay. Furthermore, small interference RNA targeted at CREB in N2A cells decreased endogenous CREB concentrations, and CREB mediated GLUT 3 transcription. Thus, in the murine brain similar to the N2A cells, phosphorylated CREB and MSY-1 bound the Glut 3 gene trans-activating the expression in neurons, whereas Sp1/Sp3 bound the repressor elements. We speculate that phosphorylated CREB and Sp3 also interacted to bring about GLUT 3 expression in response to development/cell differentiation and neurotransmission.

Paradoxical Role of Methyl-CpG-Binding Protein 2 in Rett Syndrome

Publisher Summary This chapter introduces Rett Syndrome (RTT), which is a neurodevelopmental disorder representing one of the most frequent causes of severe mental retardation in females. It describes the paradoxical role of methyl-CpG-binding protein 2 (MeCP2) in rett syndrome. —C—phosphate—G— (CpG) is the least frequently represented dinucleotide in the mammalian genome because of deamination reactions that convert methylated cytosines to uracils. MeCP2 was originally isolated from rat brain nuclear extracts in a screen to identify novel proteins that selectively bound probes containing methylated CpGs. Unmethylated CpG sites are conserved in CpG islands that tend to be clustered at the 5’ end of many mammalian genes. Methylation of CpG sites at the 5’ end of a gene is usually associated with transcriptional silencing. MeCP2 localization to transcriptionally inactive heterochromatic regions of the nucleus depended on DNA methylation and the presence of an 85-amino acid methyl-CpG-binding domain (MBD) toward the N-terminal end of the protein.

Jun localization in cytosolic and nuclear compartments in brain–pituitary system of the frog, Rana esculenta: an analysis carried out in parallel with GnRH molecular forms during the annual reproductive cycle

The presence of c-jun like mRNA was assessed in the brain of the frog, Rana esculenta, during the annual sexual cycle. In parallel, Jun protein and GnRH molecular form (mammalian and chicken II also indicated as GnRH1 and GnRH2, respectively) activity was studied in order to establish possible relationships. Northern blot analysis of total RNA reveals the presence of a 2.7 kb c-jun-like mRNA. Western blots, carried out on cytoplasmic and nuclear protein extracts, show the presence of Jun immunoreactive band of 39 kDa in brain and pituitary. Fluctuations of c- jun-like mRNA and Jun immunoreactive protein (cytoplasmic and nuclear) levels in brains during the year indicate relationships among transcription, translation, and nuclear activity. In particular, mRNA levels increase gradually from September until November when Jun protein concentration peaks in cytosolic extracts. Conversely, the nuclear protein reaches highest concentration in July when the cytosolic level shows low values. Immunocytochemical studies confirm the presence of Jun immunoreactivity in both cytoplasmic and nuclear compartments of several brain areas, including those primarily involved in gonadotropin discharge (e.g., anterior preoptic area and preoptic nucleus). GnRH molecular forms and Jun are colocalized in anterior preoptic area and preoptic nucleus. Moreover, during the period characterized by GnRH release, Jun levels strongly decrease in nuclei. Finally, we show that treatments with a GnRH analog (buserelin, Hoechst, Frankfurt) increase Jun levels in brain nuclear extracts.

Requirement of Dimerization for RNA Editing Activity of Adenosine Deaminases Acting on RNA

Adenosine deaminases acting on RNA (ADAR) convert adenosine residues into inosines in double-stranded RNA. Three vertebrate ADAR gene family members, ADAR1, ADAR2, and ADAR3, have been identified. The catalytic domain of all three ADAR gene family members is very similar to that of Escherichia coli cytidine deaminase and APOBEC-1. Homodimerization is essential for the enzyme activity of those cytidine deaminases. In this study, we investigated the formation of complexes between differentially epitope-tagged ADAR monomers by sequential affinity chromatography and size exclusion column chromatography. Both ADAR1 and ADAR2 form a stable enzymatically active homodimer complex, whereas ADAR3 remains as a monomeric, enzymatically inactive form. No heterodimer complex formation among different ADAR gene family members was detected. Analysis of HeLa and mouse brain nuclear extracts suggested that endogenous ADAR1 and ADAR2 both form a homodimer complex. Interestingly, endogenous ADAR3 also appears to form a homodimer complex, indicating the presence of a brain-specific mechanism for ADAR3 dimerization. Homodimer formation may be necessary for ADAR to act as active deaminases. Analysis of dimer complexes consisting of one wild-type and one mutant monomer suggests functional interactions between the two subunits during site-selective RNA editing.

Age-related base excision repair activity in mouse brain and liver nuclear extracts.

To assess DNA repair activity relative to age, in vitro base excision repair assays were performed using brain and liver nuclear extracts prepared from mice of various ages. An 85% decline in repair activity was observed in brain nuclear extracts and a 50% decrease in liver nuclear extracts prepared from old mice compared with 6-day-old mice. Brain nuclear extracts prepared from old mice showed a decreased abundance of DNA polymerase-beta, but the addition of purified protein did not restore base excision repair activity. Abundances of other tested base excision repair proteins did not change relative to age. The conclusion is that, during aging, a decline in DNA repair could contribute to increased levels of DNA damage and mutagenesis.

AP2-like cis element is required for calretinin gene promoter activity in cells of neuronal phenotype differentiated from multipotent human cell line DEV

Calretinin (CR) is an EF-hand calcium binding protein expressed at high level in neurons. To identify regulatory elements in CR gene promoter, cultured rat cortical cells were transfected with constructs containing its 5′-end deletion mutants and the luciferase reporter gene. A fragment ending at −115 bp upstream of the transcription start site had high promoter activity and was able to induce expression of luciferase specifically in neuronal cells of cortical cultures. The wild type sequence of −115/+54 CR promoter fragment preferentially drove the expression of green fluorescent protein analog in cells of neuronal phenotype differentiated from multipotent human cell line DEV. Electrophoretic mobility shift assays (EMSA) revealed that the −115/−71 CR gene promoter region contains a binding site for a factor present in brain nuclear extract. Among oligonucleotides containing consensus binding sites for transcription factors within this region, the one representing AP2 binding site was able to compete formation of a protein complex. Mutations of this site prevented the binding between brain protein(s) and the −115/+54 CR gene promoter region and abolished the preferential expression of reporter gene in neuronal cells of DEV line. Thus, the AP2-like element seems to be essential for the neuron-specific activity of the CR gene promoter.

Geldanamycin induces heat shock proteins in brain and protects against focal cerebral ischemia.

Geldanamycin (GA), a benzoquinone ansamycin, binds Hsp90 in vitro, releases heat shock factor (HSF1) and induces heat shock proteins (Hsps). Because viral and transgenic overexpression of Hsps protects cells against ischemia in vitro, we hypothesized that GA would protect brain from focal ischemia by inducing Hsps in vivo. Adult male Sprague-Dawley rats were subjected to 2-hour middle cerebral artery occlusions (MCAO) using the suture technique followed by 22-h reperfusions. GA or vehicle was injected into the lateral cerebral ventricles (i.c.v) 24 h before ischemia. Geldanamycin at 1 microg/kg decreased infarct volumes by 55.7% (p < 0.01) and TUNEL-positive cells by 30% in cerebral cortex. GA also improved behavioral outcomes (p < 0.01) and reduced brain edema (p < 0.05). Western blots showed that the 1 microg/kg GA dose induced Hsp70 and Hsp25 protein 8.2-fold and 2.7-fold, respectively, by 48 h following administration. Immunocytochemistry showed that GA induced Hsp70 in neurons and Hsp25 in glia and arteries in cortex, hippocampus, hypothalamus, and other brain regions. GA reduced co-immunoprecipitation of HSF1 with Hsp90 in brain tissue homogenates, promoted HSE-binding of HSF in brain nuclear extracts using gel shift assays, and increased luciferase reporter gene transcription for the Hsp70 promoter in PC12 cells. The data show that geldanamycin protects brain from focal ischemia and that this may be due, at least in part, to geldanamycin stimulation of heat shock gene transcription.

Nuclear Localization of G Protein β5 and Regulator of G Protein Signaling 7 in Neurons and Brain

The role that Gbeta(5) regulator of G protein signaling (RGS) complexes play in signal transduction in brain remains unknown. The subcellular localization of Gbeta(5) and RGS7 was examined in rat PC12 pheochromocytoma cells and mouse brain. Both nuclear and cytosolic localization of Gbeta(5) and RGS7 was evident in PC12 cells by immunocytochemical staining. Subcellular fractionation of PC12 cells demonstrated Gbeta(5) immunoreactivity in the membrane, cytosolic, and nuclear fractions. Analysis by limited proteolysis confirmed the identity of Gbeta(5) in the nuclear fraction. Subcellular fractionation of mouse brain demonstrated Gbeta(5) and RGS7 but not Ggamma(2/3) immunoreactivity in the nuclear fraction. RGS7 and Gbeta(5) were tightly complexed in the brain nuclear extract as evidenced by their coimmunoprecipitation with anti-RGS7 antibodies. Chimeric protein constructs containing green fluorescent protein fused to wild-type Gbeta(5) but not green fluorescent fusion proteins with Gbeta(1) or a mutant Gbeta(5) impaired in its ability to bind to RGS7 demonstrated nuclear localization in transfected PC12 cells. These findings suggest that Gbeta(5) undergoes nuclear translocation in neurons via an RGS-dependent mechanism. The novel intracellular distribution of Gbeta(5).RGS protein complexes suggests a potential role in neurons communicating between classical heterotrimeric G protein subunits and/or their effectors at the plasma membrane and the cell nucleus.