Gail A. M. Breen

Early dysregulation of the mitochondrial proteome in a mouse model of Alzheimer's disease

Mitochondrial structural and functional alterations appear to play to an important role in the pathogenesis of Alzheimers disease (AD). In the present study, we used a quantitative comparative proteomic profiling approach to analyze changes in the mitochondrial proteome in AD. A triple transgenic mouse model of AD (3xTg-AD) which harbors mutations in three human transgenes, APP(Swe), PS1(M146V) and Tau(P301L), was used in these experiments. Quantitative differences in the mitochondrial proteome between the cerebral cortices of 6-month-old male 3xTg-AD and non-transgenic mice were determined by using two-dimensional difference gel electrophoresis (2D-DIGE) and tandem mass spectrometry. We identified 23 different proteins whose expression levels differed significantly between triple transgenic and non-transgenic mitochondria. Both down-regulated and up-regulated mitochondrial proteins were observed in transgenic AD cortices. Proteins which were dysregulated in 3xTg-AD cortices functioned in a wide variety of metabolic pathways, including the citric acid cycle, oxidative phosphorylation, pyruvate metabolism, glycolysis, oxidative stress, fatty acid oxidation, ketone body metabolism, ion transport, apoptosis, and mitochondrial protein synthesis. These alterations in the mitochondrial proteome of the cerebral cortices of triple transgenic AD mice occurred before the development of significant amyloid plaque and neurofibrillary tangles, indicating that mitochondrial dysregulation is an early event in AD.

Transcriptional activation of the F1F0 ATP synthase α-subunit initiator element by USF2 is mediated by p300

Abstract We have been studying the transcriptional regulation of the mammalian F 1 F 0 ATP synthase α-subunit gene ( ATPA ), a TATA-less initiator-containing gene. We have previously determined that the transcription factor, upstream stimulatory factor 2 (USF2), can activate the ATPA gene through an initiator element in the core promoter. Here, we demonstrate that the coactivator p300 interacts functionally with USF2 proteins to potentiate the activation of the ATPA initiator element by USF2. The physiological relevance of this interaction was shown in vivo by expression of the adenovirus E1A oncoprotein. Wild-type E1A, but not E1A mutants that lacked p300-binding sites, inhibited the USF2-dependent transactivation of the ATPA initiator element. Furthermore, overexpression of p300 could reverse the inhibitory effect of E1A. Collectively, our results indicate that the USF2-dependent transcriptional activation of the ATPA initiator element is mediated by p300.

Upstream stimulatory factor 2 stimulates transcription through an initiator element in the mouse cytochrome c oxidase subunit Vb promoter.

Upstream stimulatory factor (USF) is a basic helix-loop-helix-leucine zipper transcription factor that plays an important role in transcriptional activation and cell proliferation. In this article, we demonstrate that the mouse cytochrome c oxidase subunit Vb gene (Cox5b) can be transactivated by ectopic expression of USF2 through an initiator (Inr) element in the core promoter. Importantly, using a dominant-negative mutant of USF2, we demonstrate the role of endogenous USF2 proteins in the transcriptional activation of the Cox5b Inr. Domains of USF2 encoded by exon 4, exon 5 and the USF-specific region are important for maximum activation of the Cox5b Inr. Using the adenovirus E1A oncoprotein, we show that p300/CBP acts as a coactivator in the USF2-dependent activation of the Cox5b Inr. We also demonstrate that although expression of multifunctional regulatory factor, Yin Yang 1 (YY1), can stimulate transcription of the Cox5b Inr to a modest extent, expression of YY1 together with USF2 greatly reduces the level of activation of the Cox5b Inr. Furthermore, we show that the transcription factor, Sp1, represses both the YY1- and the USF2-dependent activation of the Cox5b Inr, indicating competition among Sp1, YY1, and USF2.

Neuron-selective toxicity of tau peptide in a cell culture model of neurodegenerative tauopathy: Essential role for aggregation in neurotoxicity

Intracellular aggregation of tau is a pathological hallmark in Alzheimers disease and other tauopathies. The mechanisms underlying tau aggregation and the role that these aggregates play in neuronal death have remained controversial. To study these issues, we established a cell culture model of tauopathy using a hexameric peptide with the sequence 306VQIVYK311 located within the third microtubule‐binding repeat of tau, rendered cell‐permeable by a tag of nine arginine residues (R9). This peptide (VQIVYK‐R9), designated as T‐peptide, self‐assembles in vitro into paired helical filament‐like aggregates. Primary neuronal cells treated with T‐peptide die within 24 hr. Neurodegeneration correlates with the ability of the peptide to aggregate. Two peptides with mutations in the hexameric core, K‐peptide (VQIVKK) and VV‐peptide (VQVVVK), that are incapable of aggregating are not toxic, whereas two other mutant peptides, V‐peptide (VQVVYK) and F‐peptide (VQIVFK), which aggregate, are also neurotoxic. Two other peptides that aggregate in vitro, but are not derived from tau, are not neurotoxic suggesting sequence dependence. Although localizing to the nucleus, T‐peptide induces aggregation of cellular proteins in the cytoplasm. These aggregates are not caused by disruption of endogenous tau localization, although endogenous tau is reduced in neurons exposed to T‐peptide. Interestingly, nonneuronal cells are less sensitive to T‐peptide toxicity, recapitulating in part the selective lossof neurons in tauopathies. Moreover, T‐peptide treatment leads to mitochondrial dysfunction, a common feature of neurodegenerative disorders. The model system described here represents a convenient paradigm for studying the mechanisms underlying tau aggregation and neurotoxicity and for identifying compounds that can prevent these effects.

Regulation of the Nuclear Gene That Encodes the α-Subunit of the Mitochondrial F0F1-ATP Synthase Complex ACTIVATION BY UPSTREAM STIMULATORY FACTOR 2

We have previously identified several positivecis-acting regulatory regions in the promoters of the bovine and human nuclear-encoded mitochondrial F0F1-ATP synthase α-subunit genes (ATPA). One of these cis-acting regions contains the sequence 5′-CACGTG-3′ (an E-box), to which a number of transcription factors containing a basic helix-loop-helix motif can bind. This E-box element is required for maximum activity of theATPA promoter in HeLa cells. The present study identifies the human transcription factor, upstream stimulatory factor 2 (USF2), as a nuclear factor that binds to the ATPA E-box and demonstrates that USF2 plays a critical role in the activation of theATPA gene in vivo. Evidence includes the following. Antiserum directed against USF2 recognized factors present in HeLa nuclear extracts that interact with the ATPApromoter in mobility shift assays. Wild-type USF2 proteins synthesized from expression vectors trans-activated theATPA promoter through the E-box, whereas truncated USF2 proteins devoid of the amino-terminal activation domains did not. Importantly, expression of a dominant-negative mutant of USF2 lacking the basic DNA binding domain but able to dimerize with endogenous USF proteins significantly reduced the level of activation of theATPA promoter caused by ectopically coexpressed USF2, demonstrating the importance of endogenous USF2 in activation of theATPA gene.

Bovine liver cDNA clones encoding a precursor of the α-subunit of the mitochondrial ATP synthase complex

cDNA clones encoding a precursor of the alpha-subunit of the mitochondrial ATP synthase complex have been isolated from a bovine liver cDNA library using the alpha-subunit gene from Saccharomyces cerevisiae as a probe. Analyses of the nucleotide sequence of these cDNA clones reveal that the bovine liver ATP synthase alpha-subunit is initially synthesized as a precursor with an aminoterminal extension 43 amino acids in length. This aminoterminal presequence contains seven basic residues, no acidic residues, and seven polar uncharged serine and threonine residues. A single mRNA species, approximately 1.85 kb in length, was detected for the ATP synthase alpha-subunit precursor in both bovine liver and heart.

Two cytoplasmically inherited oligomycin-resistant Chinese hamster cell lines exhibit an altered mitochondrial translation product

Mitochondria from two different cytoplasmically inherited oligomycin-resistant Chinese hamster ovary cell lines synthesize an altered polypeptide compared to mitochondria from wild-type cells. For example, mitochondria from both oligomycin-resistant cell lines synthesize a polypeptide with a molecular weight of approximately 20,500, which is present in very low amounts in wild-type cells. In contrast, mitochondria from wild-type cells synthesize a polypeptide with a molecular weight of approximately 19,500, which is present in very low amounts in one of the oligomycin-resistant mutants and in reduced amounts in the other mutant. The gene which encodes this altered polypeptide is cytoplasmically transferred together with the oligomycin-resistant phenotype. This is the first example in mammalian cells where an altered mitochondrial gene product is shown to be associated with the cytoplasmic transfer of oligomycin resistance.

Biochemical genetics of the mammalian oxidative phosphorylation system: Analysis of the difference in the sensitivity of various Chinese hamster cell lines to inhibitors of the mitochondrial ATP synthase complex

Seven different Chinese hamster cell lines were found to vary greatly in their sensitivity to inhibitors of the mitochondrial ATPase. In plating-efficiency experiments, Chinese hamster lung V79 and bone marrow M3-1 cells were approximately 10,000-fold more resistant to oligomycin, 100-fold more resistant to efrapeptin, and 10-fold more resistant to ossamycin and leucinostatin than were ovary CHO or peritoneal B14 cells. In vitro experiments indicated that the increased resistance of V79 versus CHO cells to these inhibitors was due to an increased resistance of the mitochondrial ATPase. Heat-inactivation experiments indicated that there was a difference in the structure of the mitochondrial ATPase of V79 and CHO cells. Genetic experiments indicated that the difference in the sensitivity of V79 and CHO cells to inhibitors of the ATPase and the difference in the structure of the mitochondrial ATPase of V79 and CHO cells was due to a difference in both a nuclear and a cytoplasmic gene.

Novel Cell Model for Tauopathy Induced by a Cell-Permeable Tau-Related Peptide

In the present study, a cell penetrating peptide (CPP)-amyloid conjugate was prepared (T-peptide), where the amyloid-forming sequence was homologous to a nucleating sequence from human Tau protein (306VQIVYK311). Kinetic and biophysical studies showed the peptide formed long-lived oligomers which were taken up by endocytosis and localized in perinuclear vesicles and in the cytoplasm of murine hippocampal neuroblastoma cells and human HeLa cells. Thioflavin S (ThS) staining of amyloid colocalized with pathological phosphorylated Tau, suggesting that the peptide was able to seed endogenous wild-type Tau. Subsequent experiments showed that aggregates present in the lysosomes mediated lysosome membrane permeability (LMP). We observed a decrease in total Tau, irrespective of phosphorylation state, consistent with Tau fragmentation by lysosomal proteases. We found cytotoxicity of T-peptide could be abrogated by inhibitors of lysosomal hydrolases and caspases, consistent with a model where Tau fragments processed by the lysosome leak into the cytoplasm and induce toxicity in subsequent downstream steps. It is our hope that the T-peptide system may prove amenable to the evaluation of small molecule inhibitors of cytotoxicity, especially those which target either Tau aggregation or the lysosomal/autophagy system.

Altered form of subunit 6 of mitochondrial ATP synthase complex in oligomycin-resistant mutants of Chinese hamster ovary cells.

Using an antiserum generated against a synthetic peptide predicted from the DNA sequence of the ATPase 6 gene of the mitochondrial DNA, we demonstrate that mitochondria from two oligomycin-resistant Chinese hamster ovary cell lines with a defined mutation in the ATPase 6 gene synthesize an altered ATPase 6 gene product. This altered gene product migrates in sodium dodecyl sulfate-polyacrylamide gels as if it has a molecular mass that is larger by 1000 daltons than the wild-type ATPase 6 gene product. We also demonstrate that mitochondria from four other independently isolated oligomycin-resistant Chinese hamster ovary mutant cell lines contain a similar altered ATPase 6 gene product. These results suggest that all six oligomycin-resistant cell lines have a similar mutation in the ATPase 6 gene of the mitochondrial DNA that encodes subunit 6 of the ATP synthase complex.